Thomas' Hematopoietic Cell Transplantation

Thomas’ Hematopoietic Cell Transplantation Stem Cell Transplantation Fourth Edition

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Dedication This book is dedicated to our patients and their families whose courage and trust enabled them to choose a difficult, dangerous, and sometimes unproven therapy which offered the only chance in their fight against a fatal disease. Today, many thousands of patients are alive after a successful hematopoietic cell transplantation which would have been impossible without the fortitude of our patients, who took risks and thereby paved the way for the increasing application of this therapy. The progress in the area of clinical transplantation of hematopoietic cells would not have been possible without the dedicated, tireless work of the nurses and the supporting staff in the many transplant units around the world.

Thomas’

Hematopoietic Cell Transplantation Stem Cell Transplantation

Edited by

Frederick R. Appelbaum,

MD

Member and Director, Clinical Research Division Fred Hutchinson Cancer Research Center Professor and Head, Division of Medical Oncology University of Washington School of Medicine Seattle, Washington

Stephen J. Forman,

MD

Director, Department of Hematology and Hematopoietic Cell Transplantation Staff Physician, Department of Medical Oncology and Therapeutics Research City of Hope Comprehensive Cancer Center Duarte, California

Robert S. Negrin,

MD

Professor of Medicine Chief, Division of Blood and Marrow Transplantation Stanford University Stanford, California

Karl G. Blume,

MD

Emeritus Professor of Medicine Division of Blood and Marrow Transplantation Stanford University Stanford, California

Fourth Edition

A John Wiley & Sons, Ltd., Publication

This edition first published 1994, © 1994, 1999, 2004 by Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical and Medical business to form WileyBlackwell. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wileyblackwell The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by physicians for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Thomas’ hematopoietic cell transplantation / edited by Frederick R. Appelbaum . . . [et al.]. – 4th ed. p. ; cm. Includes bibliographical references and index. 1. Hematopoietic stem cells–Transplantation. I. Appelbaum, Frederick R. II. Thomas, E. Donnall. III. Title: Hematopoietic cell transplantation. [DNLM: 1. Hematopoietic Stem Cell Transplantation. 2. Transplantation Immunology. WH 380 T457 2008] RD123.5.B652 2008 617.4′4 – dc22 2008030325 ISBN: 978-1-4051-5348-5 A catalogue record for this book is available from the British Library. Set in 8.75 on 11 pt Times by SNP Best-set Typesetter Ltd., Hong Kong Printed & bound in Singapore by Fabulous Printers Pte Ltd 1

2009

Contents

Contributors, ix Preface to the First Edition, xvii Preface to the Second Edition, xviii Preface to the Third Edition, xix Preface to the Fourth Edition, xx Thomas HCT Fourth Edition, xxi List of Abbreviations, xxiii

9 Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation, 102 Edwin M. Horwitz 10 Genetic Manipulation of Hematopoietic Stem Cells, 116 Grant D. Trobridge & Hans-Peter Kiem Section 2b Immunology

SECTION 1 History and Use of Hematopoietic Cell Transplantation 1 A History of Allogeneic Hematopoietic Cell Transplantation, 3 E. Donnall Thomas 2 The History of Autologous Hematopoietic Cell Transplantation, 8 James O. Armitage 3 Uses and Growth of Hematopoietic Cell Transplantation, 15 Mary M. Horowitz SECTION 2 Scientific Basis for Hematopoietic Cell Transplantation Section 2a Hematopoiesis and Stem Cell Biology

4 Generation of Definitive Engraftable Hematopoietic Stem Cells from Human Embryonic Stem Cells, 27 Laurence Dahéron & David T. Scadden 5 Biology of Hematopoietic Stem and Progenitor Cells, 36 Susan Prohaska & Irving Weissman 6 Molecular Biology of Stem Cell Renewal, 64 Peter M. Lansdorp 7 Cellular Biology of Hematopoiesis, 72 Catherine M. Flynn & Catherine M. Verfaillie 8 Expansion of Hematopoietic Stem Cells, 88 Colleen Delaney & Irwin Bernstein

11 Overview of Hematopoietic Cell Transplantation Immunology, 131 Paul J. Martin 12 Histocompatibility, 145 Eric Mickelson & Effie W. Petersdorf 13 Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation, 163 Michael R. Verneris & Jeffrey S. Miller 14 Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect, 176 Robert Korngold & Thea M. Friedman 15 Mechanisms of Tolerance, 188 Megan Sykes 16 The Pathophysiology of Graft-Versus-Host Disease, 208 James L.M. Ferrara & Joseph H. Antin 17 Immune Reconstitution Following Hematopoietic Cell Transplantation, 222 Robertson Parkman & Kenneth I. Weinberg 18 The Human Graft-versus-Tumor Response – and How to Exploit It, 232 Edus H. Warren 19 Dendritic Cells in Hematopoietic Cell Transplantation, 248 Miriam Merad, Matthew P. Collin & Edgar G. Engleman 20 The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases, 264 Judith A. Shizuru Section 2c Technical Aspects

21 Pharmacologic Basis for High-dose Chemotherapy, 289 James H. Doroshow & Timothy W. Synold v

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Contents

22 High-dose Preparatory Regimens, 316 William I. Bensinger 23 Radiotherapeutic Principles of Hematopoietic Cell Transplantation, 333 Jeffrey Y.C. Wong & Timothy Schultheiss 24 Radioimmunotherapy and Hematopoietic Cell Transplantation, 351 Damian J. Green & Oliver W. Press 25 Documentation of Engraftment and Characterization of Chimerism Following Hematopoietic Cell Transplantation, 365 Paul J. Martin 26 The Detection and Significance of Minimal Residual Disease, 376 Jerald P. Radich & Marilyn L. Slovak 27 Pathology of Hematopoietic Cell Transplantation, 390 Howard M. Shulman, Robert C. Hackman & George E. Sale 28 Biostatistical Methods in Hematopoietic Cell Transplantation, 406 Joyce C. Niland & Paul Frankel 29 Outcomes Research in Hematopoietic Cell Transplantation, 428 Stephanie J. Lee

SECTION 3 Patient-oriented Issues in Hematopoietic Cell Transplantation 30 The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation, 445 Karl G. Blume & Robert A. Krance 31 Nursing Role in Hematopoietic Cell Transplantation, 461 Rosemary C. Ford & Mihkaila M. Wickline 32 Ethical Issues in Hematopoietic Cell Transplantation, 478 David S. Snyder 33 Psychosocial Issues in Hematopoietic Cell Transplantation, 488 Richard P. McQuellon & Michael Andrykowski 34 Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients, 502 Karen L. Syrjala & Samantha Burns Artherholt 35 Sexuality Following Hematopoietic Cell Transplantation: An Important Health-related Quality of Life Issue, 515 D. Kathryn Tierney 36 Hematopoietic Cell Transplantation: The Patient’s Perspective, 526 Susan K. Stewart

SECTION 4 Sources of Hematopoietic Cells for Hematopoietic Cell Transplantation 37 Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation, 535 Phyllis I. Warkentin & Elizabeth J. Shpall 38 Bone Marrow and Peripheral Blood Cell Donors and Donor Registries, 544 Dennis L. Confer, John P. Miller & Jeffrey W. Chell 39 Cord Blood Hematopoietic Cell Transplantation, 559 Hal E. Broxmeyer & Franklin O. Smith 40 In Utero Transplantation, 577 Alan W. Flake & Esmail D. Zanjani 41 Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy, 590 Thomas C. Shea & John F. DiPersio 42 Removal of Tumor Cells from the Hematopoietic Graft, 605 John G. Gribben 43 Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation, 618 Norbert Schmitz 44 Cryopreservation of Hematopoietic Cells, 631 Scott D. Rowley 45 Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation, 645 Jürgen Finke & Roland Mertelsmann 46 Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors, 657 Claudio Anasetti, Franco Aversa & Andrea Velardi 47 Hematopoietic Cell Transplantation from Unrelated Donors, 675 Effie W. Petersdorf 48 Donor Selection for Hematopoietic Cell Transplantation, 692 Ann E. Woolfrey SECTION 5 Hematopoietic Cell Transplantation for Acquired Diseases 49 Hematopoietic Cell Transplantation for Aplastic Anemia, 707 George E. Georges & Rainer Storb 50 Hematopoietic Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria, 727 Robert P. Witherspoon 51 Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia, 734 Jerald P. Radich & Ravi Bhatia

Contents

52 Hematopoietic Cell Transplantation for Juvenile Myelomonocytic Leukemia, 751 Charlotte M. Niemeyer & Franco Locatelli 53 Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia, 761 Frederick R. Appelbaum 54 Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia, 775 Julie-An M. Talano, James T. Casper & David A. Margolis 55 Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults, 791 Stephen J. Forman 56 Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children, 806 Parinda A. Mehta & Stella M. Davies 57 Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders, 827 H. Joachim Deeg 58 Hematopoietic Cell Transplantation for Multiple Myeloma, 845 Muzaffar H. Qazilbash & Sergio A. Giralt 59 Hematopoietic Cell Transplantation for Hodgkin’s Disease, 860 Philip J. Bierman & Auayporn Nademanee 60 Non-Hodgkin’s Lymphoma, 878 Laura J. Johnston & Sandra J. Horning 61 Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia, 897 David B. Miklos 62 Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis, 914 Raymond L. Comenzo & Morie A. Gertz 63 Hematopoietic Cell Transplantation for Breast Cancer, 931 Yago Nieto & Elizabeth J. Shpall 64 Hematopoietic Cell Transplantation in Germ Cell Tumors, 948 Christie J. Moore, Brandon Hayes-Lattin & Craig R. Nichols 65 Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors, 958 Richard W. Childs & Ramaprasad Srinivasan 66 Hematopoietic Cell Transplantation for Neuroblastoma, 970 Jason Law & Katherine K. Matthay 67 Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors, 985 David M. Loeb & Allen R. Chen 68 Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection, 1001 John A. Zaia, Amrita Krishnan, & John J. Rossi 69 Hematopoietic Cell Transplantation for Autoimmune Diseases, 1014 Richard A. Nash

vii

70 Hematopoietic Cell Transplantation for Rare Hematologic Malignancies, 1030 Vinod Pullarkat & Stephen J. Forman 71 Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies, 1043 Brenda M. Sandmaier & Rainer Storb 72 Management of Relapse after Hematopoietic Cell Transplantation, 1059 Ginna G. Laport & Robert S. Negrin

SECTION 6 Hematopoietic Cell Transplantation for Inherited Diseases 73 Hematopoietic Cell Transplantation for Thalassemia, 1079 Guido Lucarelli & Javid Gaziev 74 Hematopoietic Cell Transplantation for Sickle Cell Disease, 1090 Mark C. Walters 75 Hematopoietic Cell Transplantation for Immunodeficiency Diseases, 1105 Trudy N. Small, Wilhelm Friedrich, & Richard J. O’Reilly 76 Hematopoietic Cell Transplantation for Osteopetrosis, 1125 Peter F. Coccia 77 Hematopoietic Cell Transplantation for Storage Diseases, 1136 Charles Peters 78 Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders, 1163 Rajni Agarwal 79 Hematopoietic Cell Transplantation for Fanconi’s Anemia, 1178 John E. Wagner, Margaret L. MacMillan, & Arleen D. Auerbach SECTION 7 Complications of Hematopoietic Cell Transplantation 80 Mechanisms and Treatment of Graft Failure, 1203 Robert Lowsky & Hans Messner 81 Blood Group Incompatibilities and Hemolytic Complications of Hematopoietic Cell Transplantation, 1219 Margaret R. O’Donnell 82 Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation, 1226 Jeffrey McCullough

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Contents

83 Vascular Access and Complications, 1244 I. Benjamin Paz 84 Pharmacologic Prevention of Acute Graft-Versus-Host Disease, 1257 Nelson J. Chao & Keith M. Sullivan 85 T-Cell Depletion to Prevent Graft-versus-Host Disease, 1275 Robert J. Soiffer 86 Manifestations and Treatment of Acute Graft-Versus-Host Disease, 1287 Corey Cutler & Joseph H. Antin 87 Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy, 1304 Steven Z. Pavletic & Georgia B. Vogelsang 88 Bacterial Infections, 1325 Helen L. Leather & John R. Wingard 89 Fungal Infections after Hematopoietic Cell Transplantation, 1346 Janice (Wes) M.Y. Brown 90 Cytomegalovirus Infection, 1367 John A. Zaia 91 Herpes Simplex Virus Infections, 1382 James I. Ito 92 Varicella-zoster Virus Infections, 1388 Dora Y. Ho & Ann M. Arvin 93 Epstein–Barr Virus Infection, 1410 Wen-son Hsieh & Richard F. Ambinder 94 Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation, 1419 Michael Boeckh 95 Gastrointestinal and Hepatic Complications, 1434 Simone I. Strasser & George B. McDonald 96 Lung Injury Following Hematopoietic Cell Transplantation, 1456 Kenneth R. Cooke & Gregory A. Yanik 97 Kidney and Bladder Complications of Hematopoietic Cell Transplantation, 1473 Sangeeta Hingorani 98 Endocrine Complications Following Hematopoietic Cell Transplantation, 1487 Fouad R. Kandeel

99 Common Potential Drug Interactions Following Hematopoietic Cell Transplantation, 1523 Anne Poon & Lowan Ly 100 Critical Care of the Hematopoietic Cell Transplant Recipient, 1539 Gundeep S. Dhillon & Norman W. Rizk 101 Nutrition Support of the Hematopoietic Cell Transplant Recipient, 1551 Polly Lenssen & Saundra Aker 102 Pain Management, 1570 Noelle V. Frey & Jonathan R. Gavrin 103 Oral Complications of Hematopoietic Cell Transplantation, 1589 Mark M. Schubert & Douglas E. Peterson 104 Growth and Development after Hematopoietic Cell Transplantation, 1608 Jean E. Sanders 105 Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation, 1620 Mary E.D. Flowers & H. Joachim Deeg 106 Secondary Malignancies after Hematopoietic Cell Transplantation, 1638 Smita Bhatia & Ravi Bhatia 107 Neurologic Complications of Hematopoietic Cell Transplantation, 1653 Harry Openshaw 108 Vaccination of Allogeneic and Autologous Hematopoietic Cell Recipients, 1664 Trudy N. Small

SECTION 8 Looking Forward 109 Hematopoietic Cell Transplantation in the Future, 1673 Ernest Beutler Colorplates, facing page 514 Robert C. Hackman Index, 1677

Companion CD-ROM A companion CD-ROM is included with the book, containing: • The complete text • A database of figures from the book for downloading • A Search feature The CD-ROM is suitable for PC and Mac computers.

Contributor list

Rajni Agarwal, MD Assistant Professor of Pediatrics, Division of Stem Cell Transplantation, Department of Pediatrics, Stanford University School of Medicine, Stanford, California Saundra Aker, RD, CD Consultant, Nutrition and Patient Food Services, Seattle Cancer Care Alliance, Seattle, Washington Richard F. Ambinder, MD, PhD Professor in the Departments of Oncology, Pathology, Pharmacology and Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland

Samantha Burns Artherholt, PhD Research Fellow, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington Ann M. Arvin, MD Professor of Pediatrics, Microbiology and Immunology, Department of Pediatrics, Infectious Disease Division, Stanford University School of Medicine, Stanford, California Arleen D. Auerbach, PhD Director of the Laboratory of Human Genetics and Hematology, The Rockefeller University New York, New York

Claudio Anasetti, MD Chair, Department of Blood and Marrow Transplantation, Moffitt Cancer Center Professor of Medicine and Oncology, University of South Florida, Tampa, Florida

Franco Aversa, MD Head, Hematopoietic Stem Cell Transplant Unit Professor, Department of Clinical and Experimental Medicine Sections of Hematology and Immunology Perugia University, School of Medicine, Perugia, Italy

Michael A. Andrykowski, PhD Department of Behavioural Science, University of Kentucky College of Medicine, Seattle, Washington

William I. Bensinger, MD Professor of Medicine, University of Washington Fred Hutchinson Cancer Research Center, Seattle, Washington

Joseph H. Antin, MD Professor of Medicine, Harvard Medical School, Dana Farber Cancer Institute, Boston, MA

Irwin Bernstein, MD Member, Fred Hutchinson Cancer Research Center; and Professor of Medicine, University of Washington School of Medicine, Seattle, Washington

Frederick R. Appelbaum, MD Member and Director, Clinical Research Division, Fred Hutchinson Cancer Research Center, Professor and Head, Division of Medical Oncology, University of Washington School of Medicine, Seattle, Washington James O. Armitage, MD The Joe Shapiro Professor of Medicine Section of Oncology/ Hematology, University of Nebraska, Medical Center, Nebraska Medical Center, Omaha, Nebraska

Ernest Beutler, MD Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California Ravi Bhatia, MD Director, Stem Cell Biology Program, Division of Hematology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, California ix

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Contributor list

Smita Bhatia, MD, MPH Staff Physician; Director, Epidemiology and Outcomes Research, Division of Pediatric Hematology/Oncology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, California Philip J. Bierman, MD Associate Professor of Medicine, Department of Internal Medicine, Section of Oncology and Department Hematology, University of Nebraska Medical Center, Omaha, Nebraska Karl G. Blume, MD, FACP Emeritus Professor of Medicine, Division of Bone Marrow Transplantation, Department of Medicine, Stanford University, School of Medicine, Stanford, California Michael Boeckh, MD Assistant Member, Program in Infectious Diseases Fred Hutchinson Cancer Research Center, Assistant Professor, University of Washington School of Medicine, Seattle, Washington Janice M. Y. Brown, MD Associate Professor Divisions of Blood and Marrow Transplantation and Infectious Diseases, Stanford University School of Medicine, Stanford, California Hal E. Broxmeyer, PhD Chairman and Mary Margaret Walther Professor of Microbiology and Immunology, Professor of Medicine Scientific Director, the Walther Oncology Center Indiana University School of Medicine Cancer Research Institute, Indianapolis, Indiana James A. Casper, MD Professor of Pediatrics, Section of Pediatric HematologyOncology-Transplantation Department of Pediatrics Medical College of Wisconsin Children’s Hospital of Wisconsin, Milwaukee, Wisconsin Nelson J. Chao, MD Professor of Medicine and Imunology, Duke University Medical Center, Durham, North Carolina Jeffrey W. Chell, MD Chief Executive Officer, National Marrow Donor Program, Minneapolis, Minnesota Allen R. Chen, MD, PhD, MHS Associate Professor, Oncology and Pediatrics, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Pediatrics, Baltimore, Maryland

Richard Childs, MD Senior Investigator, National Heart, Lung and Blood Institute, National Institutes of Health, Bethseda, Maryland Peter F. Coccia, MD Ittner Professor of Pediatrics and Chief Section Pediatric Hematology/Oncology; and Director, Pediatric Bone Marrow Transplantation Program; and Vice-Chairperson, Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska Matthew Collin, MD, PhD Department of Gene and Cell Medicine, Mount Sinai Medical School, New York, New York Raymond L. Comenzo, MD Associate Attending, Hematology Service Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York Dennis L. Confer, MD Clinical Professor of Medicine, University of Minnesota; and Chief Medical Officer, National Marrow Donor Program, Minneapolis, Minnesota Kenneth Robert Cooke, MD Associate Professor of Pediatrics, Division of Hematology/ Oncology, Blood and Marrow Transplant Program, Case Western Reserve School of Medicine, Rainbow Babies and Children’s Hospital, Cleveland, Ohio Corey S. Cutler, MD MPH FRCPC Assistant Professor of Medicine, Harvard Medical School, Dana Farber Cancer Institute, Boston, Massachusetts Laurence Daheron, PhD Massachusetts General Hospital, Center for Regenerative Medicine, Boston, Massachusetts Stella Davies, MB, BS, PhD, MRCP Jacob G. Schmirdlapp Endowed Chair and Professor of Pediatrics; and Director, Blood and Marrow Transplantation Program, Division of Hematology/Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio H. Joachim Deeg, MD Member, FHCRC Professor of Medicine, University of Washington School of Medicine, Seattle, Washington Colleen Delaney, MD, MSc Assistant Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Assistant Professor, Department of Pediatrics, University of Washington, School of Medicine, Seattle, Washington

Contributor list

Gundeep S. Dhillon, MD, MPH Assistant Professor of Medicine, Stanford University School of Medicine, Stanford, California John Dipersio, MD, PhD Chief, Division of Oncology, Deputy Director, Siteman Cancer Research, Washington University School of Medicine, St Louis, Missouri

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Stephen J. Forman, MD Director, Department of Hematology and Hematopoietic Cell Transplantation, Staff Physician, Department of Medical Oncology and Therapeutics Research, City of Hope Comprehensive Cancer Center, Duarte, California Paul Frankel, PhD Statistician, City of Hope National Medical Center, Duarte, California

James H. Doroshow, MD, FACP Chairman, Department of Medical Oncology and Therapeutics Research, Associate Director for Clinical Investigation, City of Hope Comprehensive Cancer Care Center, Duarte, California

Noelle V. Frey, MD Instructor of Medicine Division of Hematology-Oncology Abramson Cancer Center University of Pennsylvania, Philadelphia, Pennsylvania

Edgar G. Engleman, MD Professor of Pathology and Medicine, Stanford University, School of Medicine, Stanford Blood Center, Stanford, California

Thea M. Friedman, PhD Director of Research Laboratory Services, The Cancer Center at Hackensack Medical University, Hackensack, New Jersey

James L. M. Ferrara, MD Professor of Medicine and Pediatrics; and Director, Blood and Marrow Transplantation Program, University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan

Wilhelm Frierich, MD Professor of Pediatrics, Department of Pediatrics, University Children’s Hospital Ulm, University of Ulm, Ulm, Germany

Jürgen Finke, MD Head, Division of Allogeneic Stem Cell Transplantation, Dept Hematology and Oncology, Medizinische Klinik Univesity, Medical Center, Frieberg, Germany Alan W. Flake, MD Professor of Surgery and Obstetrics and Gynecology, University of Pennsylvania; and Ruth and Tristram Colket Professor of Pediatric Surgery, Director, Institute for Surgical Science, Children’s Hospital of Philadelphia Abramson Research Center, Philadelphia, Pennsylvania Mary E. D. Flowers, MD Assistant Member, Fred Hutchinson Cancer Research Center; and Assistant Professor, University of Washington School of Medicine, Seattle, Washington Catherine Flynn, MB BCH BAO Faculty of Medicine, Stem Cell Institute Leuven, Catholic University Leuven, Belgium Rosemary C. Ford, RN, BSN, OCN Nurse Manager, Transplant Clinic, Seattle Cancer Care Alliance, Seattle, Washington

Jonathan R. Gavrin, MD Director, Symptom Management and Palliative Care Interim Director, Pain Management Services, Physician Co-chair HUP Ethics Committee Department of Anesthesiology and Critical Care Department of Medicine Hospital of the University of Pennsylvania Javid Gaziev, MD Senior Hematologist, Mediterranean Institute of Hematology International Centre for Transplantation in Thalassemia and Sickle Cell Anaemia, Rome, Italy George Earl Georges, MD Associate Member, Fred Hutchinson Cancer Center Associate Professor of Medicine, University of Washington Seattle, Washington Morie Gertz, MD Professor of Medicine, Mayo Medical School; and Chair, Division of Hematology, Mayo Clinic, Rochester, Minnesota Sergio A. Giralt, MD Professor of Medicine, Department of Stem Cell Transplantation and Cellular Therapy, University of Texas, MD Anderson Cancer Center, Houston, Texas Damian J. Green, MD Research Associate, Fred Hutchinson Cancer Research Center and The University of Washington, Seattle, Washington

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Contributor list

John G. Gribben MD, DSc Professor of Experimental Cancer Medicine, St. Bartholomew’s Hospital, Barts and The London NHS Trust, London, UK Robert C. Hackman, MD Member and Director of Pathology, Fred Hutchinson Cancer Research Center, Seattle, Washington Brandon Hayes-Lattin, MD Associate Professor of Medicine, Division of Hematology/ Medical Oncology Director, Adolescent and Young Adult Oncology Program, Oregon Health and Science University Portland, Oregon

Fouad R. Kandeel, MD, PhD Director, Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center, Associate Clinical Professor, UCLA School of Medicine, Duarte, California Hans Peter Kiem, MD Member, Fred Hutchinson Cancer Research Center Professor of Medicine, University of Washington, Seattle, Washington Robert Korngold, PhD Chief, Division of Research, The Cancer Center Hackensack University Medical Center, Hackensack, New Jersey

Sangeeta Ram Hingorani, MD, MPH Assistant Professor, Pediatrics University of Washington Children’s Hospital and Regional Medical Center, Division of Nephrology, Seattle, Washington

Robert A. Krance, MD Professor of Pediatric and Medicine, Baylor College of Medicine; and Director of Pediatric Stem Cell Transplantation Program, Texas Children’s Hospital, Houston, Texas

Dora Y. Ho, MD Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California

Peter M. Lansdorp, MD, PhD Senior Scientist, Terry Fox Laboratory, BC Cancer Research Centre; Professor, Division of Hematology, University of British Columbia, Vancouver, BC

Sandra J. Horning, MD Professor of Medicine, Stanford University Medical Center, Stanford Cancer Center, Stanford, California

Ginna G. Laport, MD Assistant Professor of Medicine, Stanford University Medical Center, Division of Blood and Marrow Transplantation, Stanford, California

Mary M. Horowitz, MD, MS Chief Scientific Director, Center for Blood and Marrow Transplant Registry (CIBMTR), Medical College of Wisconsin, Milwaukee, Wisconsin Edwin M. Horwitz, MD, MS Children’s Hospital of Philadelphia, Abramson Research Center, Philadelphia, Pennsylvania Wen-son Hsieh, MD Assistant Professor in the Department of Oncology, Johns Hopkins School of Medicine, Baltimore, Maryland James I. Ito, MD Director, Department of Infectious Diseases, City of Hope National Medical Center, Duarte, California Laura J. Johnston, MD Assistant Professor of Medicine, Stanford University Medical Center, Division of Bone Marrow Transplantation, Stanford, California

Helen L. Leather, B. Pharm, BCPS Clinical Pharmacy Specialist BMT/Leukemia, Shands at the University of Florida; and Clinical Associate Professor, College of Pharmacy, University of Florida, Gainesville, Florida Stephanie J. Lee, MD, MPH Associate Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Polly Lenssen, MD Manager, Clinical Nutrition Children’s Hospital and Regional Medical Center, Seattle, Washington David M. Loeb, MD, PhD Assistant Professor, Oncology and Pediatrics, Director, Musculoskeletal Tumor Program, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University Robert Lowsky, MD Assistant Professor of Medicine, Stanford University Medical Center, Stanford, California

Contributor list

Guido Lucarelli, MD Director, Mediteranean Institute of Hematology, Policlinic of the University of Roma Tor Vergata, Roma, Italy Lowan L. Ly, Pharm D Clinical Pharmacist, Hematopoietic Stem Cell Transplant, University of Washington Medical Center/Seattle Cancer Care Alliance, Seattle, Washington David A. Margolis, MD Associate Professor, Medical College of Wisconsin, Milwaukee, Wisconsin Paul J. Martin, MD Member, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington, School of Medicine, Seattle, Washington Kathryn K. Matthay, MD Professor of Pediatrics, University of California School of Medicine, Chief, Pediatric Hematology-Oncology Department UCSF, San Francisco, CA Jeffrey McCullough, MD Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota George B. McDonald, MD Professor of Medicine, University of Washington; and Head Gastroenterology/Hepatology Section, Fred Hutchinson Cancer Research Center, Seattle, Washington Margaret L. MacMillan, MD Assistant Professor of Pediatrics, Division of Hematology/ Oncology and Blood and Marrow Transplantation, University of Minnesota Medical School, Minneapolis, Minnesota Richard P. McQuellon, PhD Comprehensive Cancer Center of Wake Forrest University, Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina Parinda A. Mehta, MD Director in the Blood Disease Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio Miraim Merad, MD Department of Gene and Cell Medicine, Mount Sinai Medical School New York, New York Roland Mertelsmann, MD, PhD Medical Director, University Medical Center, Department Medicine I Hematology/Oncology, Frieberg, Germany

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Hans Messner, MD, PhD Director of Bone Marrow Transplant Program, Princess Margaret Hospital, and Senior Scientist, Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Canada Eric Mickelson, BSc Associate Staff Scientist, Fred Hutchinson Cancer Research Center, Seattle, Washington David B. Miklos, MD, PhD Assistant Professor of Medicine, Division of Blood and Marrow Transplantation Department of Medicine, Stanford University, Stanford, California Jeffrey S. Miller, MD Professor of Medicine, Division of Hematology, Oncology & Transplantation, University of Minnesota, Minneapolis, Minnesota Christie J. Moore, DO Providence Cancer Center Division of Medical Oncology Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Portland, Oregon Auayporn P. Nademanee, MD Associate Clinical Director, Division of Hematology and Bone Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, California Richard A. Nash, MD Member, Program in Transplantation Biology Clinical Research Division Fred Hutchinson Cancer Research Center; and Associate Professor, University of Washington, Seattle, Washington Robert S. Negrin, MD Professor of Medicine and Chief, Division of Blood and Marrow Transplantation, Stanford University, Stanford, CA Craig R. Nichols, MD Director, Oregon Testicular Cancer Program, Division of Medical Oncology, Robert W. Franz Cancer Research Center Earle A. Chiles Research Institute, Portland, Oregon Lewis Nick, RN, BSN, MT(ASCP) Technical Director, Accreditation Program, Foundation for the Accreditation of Cellular Therapy, University of Nebraska Medical Center, Omaha, Nebraska Charlotte Niemeyer, MD Professor of Pediatrics, Medicical Director, Division of Hematology and Oncology, University Children’s Hospital Frieberg, Germany

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Contributor list

Yago Nieto, MD, PhD Department of Stem Cell Transplantation and Cellular Therapy, the University of Texas, M. D. Anderson Cancer Center, Houston, Texas

Anne Poon, Pharm D Clinical Pharmacist, Hematopoietic Stem Cell Transplant University of Washington Medical Center/Seattle Cancer Care Alliance, Seattle, Washington

Joyce C. Niland, PhD Chair, Division of Information Sciences Edward and Estelle Alexander Chaired Professor, Beckman Research Center Associate Director for Informtation Sciences, City of Hope Cancer Center, Duarte, California

Oliver W. Press, MD, PhD Member, Fred Hutchinson Cancer Research Center, Penney E. Petersen Chair for Lymphoma, Research, Professor of Medicine and Biological Structure, University of Washington, Seattle, Washington

Margaret R. O’Donnell, MD, FRCPC Associate Clinical Director Division of Hematology/ Hematopoietic Cell Transplantation, City of Hope, Duarte, California

Susan Prohaska, PhD Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, California

Richard O’Reilly, MD Chairman, Department of Pediatrics, Chief, Marrow Transplantation Service, Memorial Sloan-Kettering Center, New York, New York Harry Openshaw, MD City of Hope National Medical Center, Duarte, California Robertson Parkman, MD Professor of Pediatrics, Children’s Hospital of Los Angeles Division of Research Immunology and Bone Marrow Transplantation, Keck School of Medicine, University of Southern California, Children’s Hospital of Los Angeles, Los Angeles, California Steven Z. Pavletic, MD Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland I. Ben Paz, MD Associate Professor, Division of Surgery, Director, Department of General Oncologic Surgery, City of Hope, Duarte, California Charles Peters, MD Professor of Pediatrics, University of Missouri – Kansas City, School of Medicine, The Children’s Mercy Hospital, Kansas City, Missouri Effie W. Petersdorf, MD Member, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington School of Medicine, Seattle, WA Douglas E. Peterson, DMD, PhD Professor and Interim Head, University of Connecticut Health Center, Department of Oral Health and Diagnostic Sciences Farmington, Connecticut

Vinod Pullarkat, MB, BS, MRCP Assistant Professor Division of Hematology and Hematopoietic Cell Transplantation, City of Hope Medical Center, Duarte, California Muzaffar H. Qazilbash, MD Associate Professor of Medicine, Department of Stem Cell Transplantation and Cellular Therapy, M.D. Anderson Cancer Center, Houston, Texas Jerald P. Radich, MD Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington Norman Rizk, MD Guggenhime Professor of Medicine, Stanford University School of Medicine, Stanford, California Scott D. Rowley, MD, FACP Chief, Adult Blood and Marrow Transplant Program Hackensack University Medical Center, Hackensack, NJ; Clinical Associate Professor of Medicine, University of New Jersey School of Medicine, Newark, NJ George E. Sale, MD Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Jean E. Sanders, MD Full Member, Clinical Research Division Director, Pediatric Hematopoietic Stem Cell Transplantation, Fred Hutchinson Cancer Research Center, Professor of Pediatrics, University of Washington, Seattle, Washington Brenda Sandmaier, MD Member, Fred Hutchinson Cancer Center, Professor of Medicine, University of Washington, Seattle, Washington

Contributor list

Dave Scadden, MD Gerald and Darlene Jordan Professor of Medicine, CoChair, Department of Stem Cell and Regenerative Biology, Co-Director, Harvard Stem Cell Institute, Harvard University Director, MGH Center for Regenerative Medicine Chief, Hematologic Malignancies, MGH Cancer Center Massachusetts General Hospital, Boston, Massachusetts Norbert Schmitz, MD Director, Department of Hematology and Stem Cell Transplantation, Lohmiehlenstr. Germany

David Snyder, MD Chair, City of Hope Bioethics Committee Associate Director, Division of Hematology and Hematopoietic Cell Transplantation, City of Hope, Duarte, Calitornia Robert J. Soiffer, MD Dana Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA Rampprasad Srinivasan, MD, PhD Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Mark M. Schubert, DDS, MSD Professor of Oral Medicine School of Dentistry, University of Washington; and Director of Oral Medicine, Seattle Cancer Care Alliance and Fred Hutchinson Cancer Research Center, Seattle, Washington

Susan K. Stewart Blood & Marrow Transplant Information Network (BMT InfoNet), Highland Park, Illinois

Timothy Schulteiss, PhD Department Director and Professor, Division of Radiation Oncology, City of Hope Cancer Center, Duarte, California

Simone I. Strasser, MBBS, MD, FRACP A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia

Thomas Shea, MD University of North Carolina at Chapel Hill, School of Medicine, Division of Hematology/Oncology, Chapel Hill, North Carolina Judith A. Shizuru, PhD, MD Associate Professor of Medicine, Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University, School of Medicine, Stanford, California E. J. Shpall, MD Professor of Medicine, Baylor College of Medicine, Houston, Texas Howard M. Shulman, MD Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Marilyn L. Slovak, PhD, FACMG Director, Department of Cytogenics, City of Hope, Duarte, California Trudy N. Small, MD Associate Attending, Department of Pediatrics Bone Marrow Transplantation Service, Memorial Sloan-Kettering Cancer Center, New York, New York Franklin O. Smith, MD Director, Division of Hematology/Oncology Professor of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio

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Rainer Storb, MD Head & Member, Transplantation Biology Program, Fred Hutchinson Cancer Research Center, Professor of Medicine University of Washington School of Medicine, Seattle, Washington Simone I. Strasser, MBBS, MD, FRACP Clinical Associate Professor, Senior Staff Specialist, A.W. Morrow Gastroenterology and Liver Center, Royal Prince Alfred Hospital, Australia Keith M. Sullivan, MD James B Wingaarden Professor of Medicine, Director, LongTerm Follow-up and Information Research Division of Cellular Therapy, Department of Medicine, Durham, North Carolina Megan Sykes, MD Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts Timothy W. Synold, Pharm.D. Associate Professor, Department of Clinical and Molecular Pharmacology, Co-Director, Analytical Pharmacology Core Facility, City of Hope Comprehensive Cancer Center, Duarte, California Karen Syrjala, PhD Director of Biobehavioral Sciences and Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington

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Contributor list

Julie Talano, MD Assistant Professor of Pediatrics, Department of Pediatric Hematology and Oncology, Medical College of Wisconsin, MaccFund Research Center, Milwaukee, Wisconsin E. Donnall Thomas, MD Member, Fred Hutchinson Cancer Research Center, Professor Emeritus, University of Washington School of Medicine, Nobel Laureate Medicine/Physiology, 1990, Seattle, WA D. Kathryn Tierney, RN, PhD Oncology Clinical Nurse Specialist, Division of Blood and Marrow Transplantation, Stanford University Medical Center, Stanford, California Grant D. Trobridge, PhD Fred Hutchinson Cancer Research Center, Seattle, WA Andrea Velardi, MD Professor, Department of Clinical and Experimental Medicine, Sections of Hematology and Clinical Immunology Perugia University, School of Medicine, Perugia, Italy Catherine M. Verfaillie, MD Professor of Medicine, Director, Stem Cell Institute, Director, Stamcelinstituut, Leuven, Belgium Michael R. Verneris, MD Assistant Professor Department of Pediatrics – BMT University of Minnesota, Minneapolis, Minnesota Georgia B. Vogelsang, MD The Johns Hopkins University, Sidney Kimmel Cancer Center, Baltimore, Maryland John E. Wagner, MD Professor of Pediatrics, Director, Division of Hematology/ Oncology and Blood and Marrow Transplantation CoDirector, Center for Translational Medicine, University of Minnesota Medical School, Minneapolis, Minnesota Mark C. Walters, MD Blood and Marrow Transplantation Program, Children’s Hospital & Research Center, Oakland, CA and the Dept. of Pediatrics, University of California, San Francisco School of Medicine, San Francisco, California Phyllis I. Warkentin, MD Professor of Pathology and Pediatrics, University of Nebraska Medical Center, Department of Pediatrics, Omaha, Nebraska

Edus H. Warren, III, MD, PhD Associate Member, Fred Hutchinson Cancer Research Center, Seattle, Washington Kenneth I. Weinberg, MD Department of Pediatrics, Division of Pediatric Stem Cell Transplantation, Anne T. and Robert M. Bass Professor in Pediatric Cancer and Blood Diseases, Stanford University, Stanford, California Irving Weissman, MD Director, Stanford Institute for Stem Cell Biology and Regenerative Medicine Director, Director, Ludwig Center at Stanford, University of Stanford, California Mihkaila Wickline, MN, RN, AOCN HSCT Clinical Nurse Specialist, Seattle Cancer Care Alliance, Seattle, Washington John R. Wingard, MD Price Eminent Scholar and Professor of Medicine, Director, Bone Marrow Transplant Program, Division of Hematology/ Oncology, Deputy Director of the University of Florida, Gainesville, Florida Robert R. Witherspoon, MD Member, Clinical Research Division, Fred Hutchinson Cancer Research Center, Professor of Medicine, University of Washington, Medical Director, Transplant Clinic, Seattle Cancer Care Alliance, Seattle, Washington Jeffrey Y. C. Wong, MD Professor & Chair, Division of Radiation, Oncology, City of Hope Cancer Center, Duarte, California Ann E. Woolfrey, MD Director, Unrelated Donor Program, Associate Member, Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington Gregory A. Yanik, MD Clinical Associate Professor of Pediatrics and Communicable Diseases, Division of Hematology/Oncology, Blood and Marrow Transplantation Program, University of Michigan Cancer Center, Ann Arbor, Michigan John A. Zaia, MD Chair, Division of Virology, Beckman Research Institute of City of Hope, Duarte, California Esmail D. Zanjani, PhD Professor, Department of Medicine, University of NevadaReno, Reno, Nevada

Preface to the First Edition

The widespread application of bone marrow transplantation (BMT) to the treatment of a steadily increasing number of life-threatening hematological, oncological, hereditary, and immunological disorders is the culmination of more than four decades of research by many investigators. Early attempts in the 1950s to transplant living cells from one individual to another were carried out in the face of considerable skepticism. It was generally accepted as axiomatic that the immunological barrier to “foreign tissue” could never be overcome. The horrors of Nagasaki and Hiroshima spurred interest in studies of the lethal effects of irradiation. It was discovered that mice given total body irradiation in doses lethal to the marrow could be protected from death by shielding the spleen or by an infusion of marrow, and that the marrow of such animals contained living cells of donor origin. These observations suggested that patients with leukemia might be given a lethal exposure of total body irradiation, which would destroy the malignant cells along with remaining normal marrow. The exposure would also destroy the immune system, making it possible to protect against lethality by a transplant of normal marrow cells. The theory was correct, but results were disappointing. Because the procedure was both unproved and dangerous, only those patients who had no other options were considered. Except for a few patients with an identical twin donor, there were no survivors beyond a few months. Understanding of the human leukocyte antigen (HLA) system was not yet available, and little was known about the complication we now call graft-versus-host disease (GVHD). Thus, after a brief period of enthusiasm, most investigators abandoned this seemingly hopeless pursuit. Fortunately, work in animal models continued. Studies in inbred rodents defined the genetics of the major histocompatibility system and the fundamental rules of transplantation biology. Immunosuppressive drugs were developed to limit the severity of the immune reactions between donor and host. Demonstration of successful marrow transplants in the canine model using littermates matched for the major histocompatibility complex set the stage for successful transplantation of marrow between human siblings. Thus, it is clear that a long series of experimental studies in animals ultimately made human marrow transplantation possible. By the late 1960s, much was known about the HLA system, more effective antibiotics were available, and platelet transfusions were becoming routine. Thus began the modern era of human BMT. THe past 25 years have witnessed an almost exponential growth in the number of transplants being performed and the number of diseases being considered for BMT. Initially, most grafts employed marrow from an HLAidentical sibling. Autologous marrow, long known to be effective in animal systems, is now being used with increasing frequency following intensive cancer chemotherapy. Hematopoietic progenitor cells from the peripheral blood are now being used for BMT, either alone or to supplement marrow. As a result of increasing national and international cooperation, large panels of volunteer marrow donors of known HLA type are becoming available to patients whose own marrow cannot be used or who do not have a family donor.

Currently, thousands of transplants are being performed each year world-wide. With the demonstration that marrow could be transplanted and that the cure rate would be substantial, the logical step was taken to treat patients early during the course of their respective disease (i.e. in leukemia when the burden of malignant cells was relatively low and when the patient was in excellent clinical condition). With improved patient selection, development of improved tissue typing methods, availability of potent antimicrobial agents, advances in supportive care, and improved prevention of GVHD, the results of BMT have continued to improve. Marrow transplantation is now being applied to a long list of diseases with a wide range of results depending on the disease, the type of transplant, and the stage of the disease. For some of the diseases, BMT has already proven to be the most effective therapy (e.g., some leukemias and severe aplastic anemia), whereas for others it is the only available curative treatment (e.g., thalassemia). In very rare genetic disorders, one successful BMT may establish the success of the treatment. For other more common disorders, controlled trials are necessary to define the proper role of allogeneic or autologous BMT, or therapy not involving BMT. Only through rigorous study and long-term follow-up can novel approaches be confirmed as effective (or ineffective). For those working in the field of marrow transplantation, a source of intellectual satisfaction has been the interdisciplinary nature of the studies. A view of the wide-ranging disciplines involved can be gleaned by reading the chapter titles for this book. A successful BMT program is always a team effort. There must be cooperation between blood banks, referring physicians, radiation oncologists, immunologists, and physicians from many subspecialties. A dedicated support staff of technicians, data managers, and, above all, nurses, is crucial. The nursing team in particular is responsible for the day-to-day care of patients. Nurses not only provide the bedside management of complex protocol studies, but also bear the burden of emotional support through the difficult hospital period. They are the most readily available source of information for the patients and families day and night. Without a strong nursing team, the entire BMT program is jeopardized. Most important are the patients who come to the transplant center with the courage to accept days, weeks, and sometimes months of discomfort in the hope of surviving a fatal disease. We must ensure that we acknowledge and respect the dignity and individuality of each patient, that we provide adequate information for informed decision making and then include patients and families in the decision process. The greatest reward for clinical investigators is to see patients reintegrated into their personal, social, and professional lives, free of their disease and its complications. Stephen J. Forman Karl G. Blume E. Donnall Thomas Summer, 1993

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Preface to the Second Edition

During the five-year period since the first edition of this book was published, an enormous amount of new scientific information has been generated through experimental research and through clinical trials. This growth in knowledge is reflected by the doubling in the volume of this book and a 50 percent increase in the number of new chapters. The selection of a new title Hematopoietic Cell Transplantation became necessary because bone marrow is no longer the major source of allogeneic and autologous hematopoietic cells. Hematopoietic stem cells from peripheral blood collections and from umbilical cord blood are rapidly gaining clinical importance for hematologic and immunologic reconstitution following myeloablative, high-dose therapies for malignant and non-malignant, hereditary or acquired life-threatening disease. It would be impossible to identify a single scientific observation as the leading contribution to the progress made during the past five years. Exciting new data have been reported in all areas of preclinical and clinical research: New histocompatibility antigens for optimal donor selection have been described, novel concepts have been developed to eradicate the underlying malignancies, engineering of allogeneic and autologous grfts has been further developed, previously unavailable drugs for post-transplant immunosuppression and for the prevention or therapy of infectious complications have been tested. The indication list

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for high-dose therapy and hematopoietic cell transplantation has been extended and the long-term evaluation and management of transplant recipients has been further defined. Still, the editors would caution the young investigator that “breakthroughs” do not occur. Each step towards a cure has to be earned through thoughtfully designed and carefully conducted pre-clinical experiments and clinical trials. Single institution studies require confirmation through prospective randomized trials, each needing several years before a new concept can be fully accepted as a proven advance. The editors hope that the new edition will serve as a resource for physicians and many other members of health care teams at transplant centers and for new and established investigators in the area of hematopoietic cell transplantation and related fields of research; in brief, for everyone who wishes to learn about this exciting field which is now at the beginning of its fifth decade of development as a curative treatment modality for disorders with an otherwise poor prognosis. E. Donnall Thomas Karl G. Blume Stephen J. Forman Summer, 1998

Preface to the Third Edition

Ten years after the first and five years following the publication of the second edition of this textbook, the editors and the publisher, Blackwell Publishing, present now the third edition. Because of the continued impressive growth of knowledge in the field of hematopoietic cell transplantation, a new and completely revised book was needed to document and critically review the scientific progress made during the past five years. The editors wish to emphasize several changes in the third edition compared to the second edition. First, the title of the book has been changed to honor the pioneering work of E. Donnall Thomas, the 1990 Nobel Laureate in Physiology or Medicine (see Tribute). Second, the reader will find ten new chapters which deal with new experimental or clinical research observations while four prior chapters have been deleted. Topics for the new chapters include stem cell expansion, the theory and application of non-myeloablative regimens followed by allogeneic hematopoietic cell transplantation, the experimental basis for transplantation for autoimmune diseases, long-term complications and

others. Third, we have again tried to avoid overlap and contradiction between chapters, however, following academic principles, we have allowed the expression of opposing views and opinions in certain areas, for example, on the topic of plasticity of embryonic and adult stem cells. The editors are indebted to the publishing house which again has produced a high quality product in a timely fashion for the international community of investigators at all professional levels and disciplines in the field of hematopoietic cell transplantation. Finally, this book would not have been possible without the dedicated work of our assistants Sara E. Clark, Stephen J. Loy and Kristine A. Logan. Karl G. Blume Stephen J. Forman Frederick R. Appelbaum Spring 2003

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Preface to the Fourth Edition

Fifteen years after the first, and five years following the third edition of this textbook, the editors and the publisher, Blackwell Publishing, are proud to present the fourth edition of Thomas’ Hematopoietic Cell Transplantation. The impetus for the development of this fourth edition came from the rapid expansion in our understanding of the biologic basis of hematopoietic cell transplantation coupled with its rapidly expanding clinical application. When compared to the last edition, readers will find fourteen entirely new chapters. These new chapters focus on the scientific basis of transplantation (including chapters on embryonic stem cells, mesenchymal stem cells, NK cell biology, and dendritic cell biology), on emerging applications of hematopoietic cell transplantation (including chapters on hematopoietic cell transplantation for chronic lymphocytic leukemia and other less common indications), and on complications of transplantation (including chapters on graft failure, vascular access and complications, interstitial pneumonia, nephro-urologic complications, endocrine complications and common potential drug interactions). While new chapters have been added, others have been dropped or their materials combined in an effort to avoid redundancies. The remaining chapters have been revised and updated to reflect the current state of the science.

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For the first time, a CD-ROM accompanies this textbook. The CDROM contains the entire text of the book in easily searchable form, as well as all figures in a convenient downloadable format. We are hopeful that our readers will find this addition useful. The editors greatly appreciate the assistance of Blackwell Publishing, which has been extremely helpful in the process of creating this volume and in producing a finished product of the highest quality. Also, this book would not have been possible without the dedicated work of our assistants, Kimberly Laramie, Sara E. Clark-Fuentes, and especially, Tanya Chiatovich. Finally, this edition honors the memory of Dr. Ernest Beutler whose laboratory and clinical contributions helped begin the field of transplantation as a curative therapy for patients suffering from leukemia. He was a colleague and mentor for many people who have made significant contributions to the field of hematopoietic cell transplantation. Frederick R. Appelbaum, MD Stephen J. Forman, MD Robert S. Negrin, MD Karl G. Blume, MD

Tribute

Thomas HCT Fourth Edition

Dr. E. Donnall Thomas and King Carl Gustaf of Sweden. Dr. Thomas and Dr. Joseph Murray (not shown in the picture) shared the Nobel Prize for Physiology or Medicine, 1990, recognizing the field of organ transplantation. Dr. Thomas was honored for endeavors in experimental and clinical bone marrow transplantation. Their prizes emphasize the importance of patient-related research.

Our textbook title honors Dr. E. Donnall Thomas, the single individual most responsible for creating the subject of this book. Don was born on March 15, 1920, the son of a solo general practitioner in a small Texas village. He recalls as a child accompanying his father to his small office and to patients’ homes. As Don has mentioned, between he and his father, they span the period from horse and buggy house calls to our modern high-tech medicine. He received his B.A. and M.D. from the University of Texas and that is where he met his wife Dottie. Besides raising three children and eight grandchildren together, Dottie has been Don’s partner in every aspect of his professional life, from working in the laboratory, to editing manuscripts and administering grants. Anyone who has been lucky enough to work with Don knows that if he is the father of marrow transplantation, then Dottie is, without question, the mother.

Don graduated from Harvard Medical School in 1947 and completed his internship and residency at Peter Brent Brigham Hospital in Boston. It was while in medical school that Don first became interested in normal and malignant hematopoiesis. During those years, Sydney Farber was initiating his first studies of the use of antifolates to treat children with acute leukemia and Don witnessed the very first patient to achieve a remission with this approach. He was exposed to the pioneering work of Allan Erslev and his search for erythropoietin. Most importantly, he learned of Leon Jacobsen and his studies showing that shielding the spleen protected mice from the otherwise lethal effects of total body irradiation. As data emerged that a similar irradiation protection effect could be achieved by transferring bone marrow from a non-irradiated to an irradiated mouse, Don became convinced of the clinical potential of marrow transplantation.

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Thomas HCT Fourth Edition

In 1955, Don moved to Cooperstown, New York and the Imogene Basset Hospital, a Columbia University affiliate, where he began working on marrow transplantation both in the canine model and in humans with Dr. Joseph Farrebee. In 1957, Don published the first report in human patients showing that complete remissions of leukemia could be achieved using total body irradiation followed by infusion of marrow from an identical twin. At that time, there was little understanding of the principles of human histocompatibility and so attempts to expand these studies to patients without identical twins were uniformly unsuccessful. These failures were the stimulus for a long series of experiments conducted by Don in the canine model showing it was possible to expose dogs to supralethal doses of irradiation and rescue them by reinfusing their own marrow, that the marrow could be cryopreserved, and that large doses of peripheral blood could substitute for marrow. But attempts at allogeneic transplantation in this outbred species continued to fail because of graft-versus-host disease or graft rejection. In 1963, Don moved to Seattle and the University of Washington to become the first head of the Division of Oncology. There he developed techniques for rudimentary histocompatibility typing in the dog and by the mid-1960s showed that by selecting matched donors and using methotrexate post-transplant, it was possible to successfully transplant marrow between matched littermates in almost every case. At the same time, based on the work of Dausset, Payne, Amos and others, the understanding of human histocompatibility also dramatically increased and so, in the late 1960s, Don made the decision to return to the subject of allogeneic transplantation in humans. He began to assemble a team of physicians, nurses and support personnel (many of whom are still part of the current Seattle Transplant Program) and obtained a program project grant from the National Cancer Institute. In November 1968, Dr. Robert Good and his colleagues carried out the first marrow transplant from a matched sibling for an infant with immunodeficiency and in March 1969, Don performed the first matched sibling donor transplant for a patient with leukemia. The Seattle Transplant Program was originally housed at the Seattle Public Health Hospital but in 1972, when the hospital was faced with closure by the federal government, the Program moved to Providence Hospital. In 1975, Don and his team moved to the newly created Fred Hutchinson Cancer Research Center, a move that provided Don with increased space, resources and scientific collaborations. That same year, he and his colleagues published their classic New England Journal of Medicine paper summarizing the field of allogeneic transplantation and particularly the early Seattle experience. These results demonstrated not

only the feasibility of the procedure, but also that there was a plateau on the survival curves following transplantation, suggesting that some of these patients were cured with this novel technique. Don continued to lead the Clinical Research Division of the Center and its transplant program until his partial retirement in 1989. Yet he continues to work writing manuscripts, delivering lectures and participating in research discussions at the Center. Appropriately, Don has received almost every possible award for his work, including the American Society of Hematology’s Henry M. Stratton Award, the General Motor’s Kettering Prize, the American Society of Oncology’s Karnofsky Award, the Presidential Medal of Science, and of course, the 1990 Nobel Prize in Medicine which he shared with Joseph Murray. With each award, Don has always emphasized how much his work was a team effort. He invariably mentions the contributions of Rainer Storb, Dean Buckner, Reg Clift, Paul Neiman, Alex Fefer, and Bob Epstein, who helped form the original Seattle Transplant Team, and of Ted Graham, who moved with Don from Cooperstown to help in the animal research. Don never fails to credit the nursing and support staff who played such a critical role in these efforts, and he always acknowledges the patients and their families who have been true partners in his work. Although Don is most noted for his pioneering scientific achievements, for those of us who have been fortunate enough to work with him, Don is equally admired for the way in which he achieved his success. He has always been focused, hardworking and uncompromising in his laboratory and clinical research. He can be a demanding critic and holds others accountable for their actions, yet at the same time he has always been generous with his ideas, loyal to his employees and quick to deflect praise to his coworkers. Although he has dedicated an enormous amount of himself to a life in medical science, he has managed to maintain a great deal of balance. He has traveled widely as a visiting lecturer, donated time to professional organizations and supported local and international charities. He and Dottie are avid and expert hunters and fishers and have a close and loving extended family. It is difficult to think of many other fields of modern medicine that are so much the result of a single man’s work. Don’s vision and dedication have changed the lives of literally hundreds of thousands of patients. By naming this textbook for him, we are joining those patients, and the many nurses, physicians and staff privileged to work in the field he created, in thanking Don both for what he has accomplished and for the way he has done it.

List of Abbreviations

AABB AAV ABMTR ABW ACC ACEI ACIF ACIP ACP ACTH ACV AD ADA ADCC ADL ADP ADR AEL AF AFO AFP AGM AGU AH AHCT AHRQ AIBW AICD AIDS AIHA AITD AITL AKI ALC ALCL ALD ALG ALI ALL ALT AMKL AML AMM

= American Association of Blood Banks = Adeno-Associated Virus = Autologous Blood and Marrow Transplant Registry – North America = Actual Body Weight = Acute Chest Syndrome = Angiotensin Converting Enzyme Inhibitor = Anticomplement Immunofluorescence = Advisory Committee on Immunization Practices = Advanced Care Planning = Adrenocorticotropic Hormone = Acyclovir = Autoimmune Disease = Adenosine Deaminase = Antibody Dependent Cell Mediated Cytotoxicity = Activities of Daily Living = Adenosine Diphosphate = Adverse Drug Reaction = Acute Erythroleukemia = Amniotic Fluid = Air Flow Obstruction = α-Feto Protein = Aorta-Gonad-Mesonephros (Region) = Aspartylglucosaminuria = Ancestral Haplotype = Autologous Hematopoietic Cell Transplantation = Agency for Health Care Research and Quality = Adjusted Ideal Body Weight = Activation Induced Cell Death = Acquired Immune Deficiency Syndrome = Auto-Immune Hemolytic Anemia = Autoimmune Thyroid Disease = Angioimmunoblastic T-Cell Lymphoma = Acute Kidney Injury = Absolute Lymphocyte Count = Anaplastic Large Cell Lymphoma = Adrenoleukodystrophy = Antilymphocyte Globulin = Acute Lung Injury = Acute Lymphoblastic Leukemia = Alanine Aminotransferase = Acute Megakaryoblastic Leukemia = Acute Myeloid Leukemia = Agnogenic Myeloid Metaplasia

AMN ANA ANC ANRC AP APC APL ARAC ARB ARDS ARF ARL ARSA ASBMT ASHI AST ATG ATL ATP ATRA ATTL AUC AVN AYA AZT B2M BACT BAL BAVC BCAA BCMA BCNU BCG BCR BCR BCRP BDNF BDP BEAC BEAM

= Adrenomyeloneuropathy = Anti-Nuclear Antibody = Absolute Neutrophil Count = Anthony Nolan Research Center = Alkaline Phosphatase = Antigen Presenting Cell = Acute Promyelocytic Leukemia = Cytosine Arabinoside = Angiotensin Receptor Blocker = Adult Respiratory Distress Syndrome = Acute Renal Failure = AIDS Related Lymphoma = Arylsulfatase A = American Society for Blood and Marrow Transplantation = American Society for Histocompatibility and Immunogenetics = Aspartate Aminotransferase = Antithymocyte Globulin = Adult T-Cell Leukemia = Autoimmune Thrombocytopenia = All-Transretinoic Acid = Acute T-Cell Leukemia/Lymphoma = Area under the Curve = Avascular Necrosis = Adolescent and Young Adult = Zidovudine = Beta-2-Microglobulin = BCNU, Cytosine-Arabinoside, Cyclophosphamide, Thioguanine = Bronchoalveolar Lavage = BCNU, Amsacrine, Etoposide, Cytosine-Arabinoside = Branched Chain Amino Acid = B Cell Maturation Antigen = 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (Carmustine) = Bacillus Calmette-Guerin = B Cell Receptor = Breakpoint Cluster Region = Breast Cancer Resistance Protein = Brain Derived Neurotropic Factor = Beclomethasine-17, 21-Dipropionate = BCNU, Etoposide, ARA-C, Cyclophosphamide = BCNU, Etoposide, Cytosine Arabinoside, Melphalan

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List of Abbreviations

BED BEE BEP BES BFU-E BL-CFC BLS BM BMC BMD BMDW BMI BMP BMSC BMT BMTSS BNLI BO BOOP BOS BSA BSE BSI BU BUN

= = = = = = = = = = = = = = = = = = = = = = = = =

CA11 CAFC CALGB CA-MRSA CAR CARES CBC CBER CBSC CBV CC CCI CCI CCNU CCR CCSG CD CDAD CDC CDC CDK CDRH CEH CFU-B CFU-BLAST CFU-F CFU-GEMM

= Carbonic Anhydrase Isoenzyme 11 = Cobblestone Area Forming Colony = Cancer and Acute Leukemia Group B = Community Acquired MRSA = Chimeric Antigen Receptor = Cancer Rehabilitation Evaluation System = Cord Blood Cell = Center for Biologics Evaluation and Research = Cord Blood Stem Cell = Cyclophosphamide, BCNU, Etoposide = Complete Chimerism = Charlson Comorbidity Index = Corrected Count Increment = 1-(2-Chloroethyl)-3-Cyclohexyl-1-Nitrosurea = Complete Cytogenetic Response = Children’s Cancer Study Group = Cluster Designation = Clostridium Difficile Associated Diarrhea = Complement Dependent Cytotoxicity = Center for Disease Control = Cyclin/Cyclin Dependent Kinase = Center for Devices and Radiological Health = Conserved Extended Haplotype = Colony Forming Units-B-Lymphocyte = Blast Forming Colony Units = Colony Forming Unit – Fibrolast = Colony Forming Unit – Granulocyte/Erythroid/ Macrophage/Megakaryocytic = Colony Forming Unit – Granulocyte Macrophage = Colony Forming Unit – Megakaryocytic = Colony Forming Unit – Mixed = Colony Forming Unit – Spleen = Colony Forming Unit-T-Lymphocyte = Chronic Granulomatous Disease = Comparative Genomic Hybridization = Current Good Tissue Practice

CFU-GM CFU-MEG CFU-Mix CFU-S CFU-T CGD CGH CGTP

Biologically Effective Dose Basic Energy Expenditure Bleomycin, Etoposide, Cisplatin BMT Events Scale Burst Forming Units – Erythroid Blast-Colony-Forming Cell Bare Lymphocyte Syndrome Bone Marrow Bone Marrow Cell Bone Mineral Density Bone Marrow Donors Worldwide Body Mass Index Bone Morphogenic Protein Bone Marrow Stem Cell Bone Marrow Transplantation Bone Marrow Transplant Survivor Study British National Lymphoma Investigation Bronchiolitis Obliterans Bronchiolitis Obliterans Organizing Pneumonia Bronchiolitis Obliterans Syndrome Body Surface Area Bovine Spongiform Encephalopathy Brief Symptom Inventory Busulfan (Myleran) Blood Urea Nitrogen

CMV-IP CNI CNL CNS CNS CNV COG COHNMC Con A CONSORT COX-2 CP CPR CR CREG CRH CSF CSP CSSCD CT CTC CTL CTLP CTN CTZ CVB CVC CVS CY CYP

= Coronary Heart Disease = Congestive Heart Failure = Cyclophosphamide, Hydroxydaunomycin, Vincristin, Prednisone = Chediak-Higashi Syndrome = Confidence Interval = Center for International Blood and Marrow Transplant Research = Chlorine Channel Pump 7 = Chronic Idiopathic Myelofibrosis = Combined Immunodeficiency Syndrome = Chemical Inducers of Dimerization = Critical Illness Polyneuropathy = Ciprofloxacin = Creutzfeldt-Jakob Disease = Chronic Kidney Disease = Cutaneous Lymphocyte Antigen = Common Lymphocyte Progenitor = Clinical and Laboratory Standards Institute = Chronic Myelogenous Leukemia = Chronic Myelomonocytic Leukemia = Common Myeloid Progenitor = Cytomegalovirus = CMV-Antibody Enriched Intravenous Immunoglobulin = Cytomegalovirus-Associated Interstitial Pneumonia = Calcineurin Inhibitor = Chronic Neutrophilic Leukemia = Central Nervous System = Clinical Nurse Specialist = Copy Number Variation = Children’s Oncology Group = City of Hope National Medical Center = Concanavilin A = Consolidated Standards of Reporting Trials = Cyclo-oxygenase-2 (inhibitor) = Chronic Phase = Cardio-Pulmonary Resuscitation = Complete Remission = Cross-Reactive Group = Corticotropin Releasing Hormone = Colony Stimulating Factor = Cyclosporine = Cooperative Study of Sickle Cell Disease = Computerized Tomography = Cyclophosphamide, Thiotepa, Carboplatin = Cytotoxic T-Lymphocyte = Cytotoxic T-Lymphocyte Precursor = Clinical Transplant Network = Chemotactic Trigger Zone = Cyclophosphamide, Etoposide, BCNU = Central Venous Catheter = Chorionic Villous Sampling = Cyclophosphamide = Cytochrome P

DAD DAG DAH DAT DC DCC

= Diffuse Alveolar Damage = Diacylglycerol = Diffuse Alveolar Hemorrhage = Direct Agglutinin Test = Dendritic Cells = Data Coordinating Center

CHD CHF CHOP CHS CI CIBMTR CICN7 CIMF CID CID CIPN CIPRO CJD CKD CLA CLP CLSI CML CMML CMP CMV CMV-IG

List of Abbreviations

DEB DEXA DEXA DFA DFCI DFO DFP DFS DHAP DHFR DHTR DIC DISF DKA DLA DLCL DLCO DLI DLT DM DMSO DNA DNAX DNR DPC DPT DRG DRS DSMB DSRCT DST DTH DTPA

= Diepoxybutane = Dual Energy X-ray Absorptiometry = Dual Energy X-ray Absorptiometry = Direct Fluorescent Antibody (Test) = Dana-Farber Cancer Institute = Desferrioxamine = Deferiprone = Disease-Free Survival = Decadron, High dose Cytarabine, Cisplatinum = Dihydrofolate Reductase = Delayed Hemolytic Transfusion Reaction = Disseminated Intravascular Coagulation = Derogatis Interview of Sexual Functioning = Diabetic Ketoacidosis = Dog Leukocyte Antigen = Diffuse Large Cell Lymphoma = Diffusion Capacity = Donor Lymphocyte Infusion = Dose Limiting Toxicity = Diabetes Mellitus = Dimethylsulfoxide = Deoxyribonucleic Acid = DNA Accessory Molecule = Do Not Resuscitate = Days Post Conception = Diphtheria/Pertussis/Tetanus = Dorsal Root Ganglion = Disability Rating Scale = Data and Safety Monitoring Board = Desmoplastic Small Round Cell Tumor = Donor Specific Transfusion = Delayed Type Hypersensitivity = Diethylenetriaminepentaacetic Acid

EACA EAE EB EBMT

= Epsilon Amino Caproic Acid = Experimental Autoimmune Encephalomyelitis = Embryoid Body = European Group for Blood and Marrow Transplantation = Epstein-Barr (Virus) Nuclear Antigen = Epstein-Barr Virus = Epstein-Barr Virus Associated Lymphoproliferative Disorder = Extracorporal Adsorption Therapy = Electrocardiogram = Extracellular Matrix = Eastern Cooperative Oncology Group = Extra Corporal Phototherapy = Early Death = Erectile Dysfunction = Etoposide, Dexamethasone, Cytosine Arabinoside, Cisplatin = Expanded Disability Status Scale = European Federation for Immunogenetics = Event-Free Survival = Enzyme-Linked Immunosorbent Assay = Eutectic Mixture of Local Anesthetic = European Neuroblastoma Study Group = European Organization for Research and Treatment of Cancer = Erythropoietin = Endoplasmic Reticulum

EBNA EBV EBV-LDP ECAT ECG ECM ECOG ECP ED ED EDAP EDSS EFI EFS ELISA EMLA ENSG EORTC EPO ER

xxv

ERCP ERT ESA ESBL ESC ESFT ESRD ET ETP EWOG

= Endoscopic Retrograde Cholangiopancreatography = Enzyme Replacement Therapy = Erythropoiesis Stimulating Agent = Extended-Spectrum beta-Lactamase = Embryonic Stem Cell = Ewing Sarcoma Family of Tumors = End Stage Renal Disease = Essential Thrombocythemia = Early Thymic Progenitor = European Working Group

FA FAB FACS FACT

FLC FLIC FLIPI FLU FLV FMF FNA FSCL FSH 5-FU FUO FVC

= Fanconi Anemia = French American British Classification = Fluorescence Activated Cell Sorter = Foundation for the Accreditation of Cellular Therapy = Foundation for the Accreditation of Hematopoietic Cell Therapy = Fluorescent Antibody to Membrane Antigen = Fas Ligand = Facilitator Cell = Famciclovir = Food and Drug Administration = Forced Expiratory Ventilation in 1 Second = Fresh Frozen Plasma = Fred Hutchinson Cancer Research Center = Familial Hemophagocytic Lymphohistiocytosis = Fluorescence in-situ Hybridization = Tacrolimus = Tacrolimus (FK) Binding Protein = Follicular Lymphoma = Face, Legs, Activity, Chest X-ray, Consolability (Pain Assessment Tool) = Free Light Chain = Functional Living Index – Cancer = Follicular Lymphoma International Prognostic Index = Fludarabine = Friend Murine Leukemia Virus = Flow Microfluorometry = Fine Needle Aspiration = Follicular Small Cleaved Cell Lymphoma = Follicle Stimulating Hormone = 5-Fluorouracil = Fever of Unknown Origin = Forced Volume Capacity

GAD GAD65 GAG GALC GALT GALV GAP GAVE G-CSF GCV GDP GELA GEP GF GFJ GFP

= Glutamic Acid Decarboxylase = Glutamic Acid Decarboxylase 65 = Glycosaminoglycan = Galactoceribrosidase = Gut Associated Lymphoid Tissue = Gibbon Ape Leukemia Virus = GTP-ase Activating Protein = Gastric Antral Vascular Ectasia = Granulocyte-Colony Stimulating Factor = Ganciclovir = Guanosine Diphosphate = Groupe d’Etude des Lymphomes d’Adulte = Gene Expression Profiling = Graft Failure = Grapefruit Juice = Green Fluorescent Protein

FAHCT FAMA FasL FC FCV FDA FEV1 FFP FHCRC FHL FISH FK506 FKBP FL FLACC

xxvi

List of Abbreviations

GITMO GLD GLR GLSG GMBP GM-CSF GMP GMP GnRH GOG G-6-PD GRE GRH GSH GSSG GST GTP GTP GU GvA GVHD GVLE

= Glomerular Filtration Rate = Growth Hormone = Growth Hormone Releasing Hormone = German Hodgkin’s Lymphoma Study Group = Gastro-Intestinal (Tract) = Gruppo Italiano Malattie Ematologiche Maligne dell’ Adulto = Gruppo Italiano Trapianti di Midolio Osseo = Globoid Cell Leukodystrophy = Glomerular Filtration Rate = German Lymphoma Study Group = Guinea Pig Myelin Basic Protein = Granulocyte/Macrophage-Colony Stimulating Factor = Good Manufacturing Practices = Granulocyte Macrophage Progenitor = Gonadotropin Releasing Hormone = Gynecologic Oncology Group = Glucose-6-Phosphate Dehydrogenase = Glucocorticoid Response Elements = Gonadotropin-Releasing Hormone = Glutathione (Reduced) = Glutathione (Oxidized) = Glutathione-S-Transferase = Good Tissue Practice = Guanosine Triphosphate = Genitourinary = Graft-versus-Autoimmunity = Graft-versus-Host Disease = Graft-versus-Leukemia Effect

4-HC HA HAART HAMA HBV HC HC HCFA HCG HCoV HCT HCV HD HD-AC HDC HDC HD-CY HDIT HDR HDT HES HES HHV6 HIPAA HIV HL HLA HLH HMPV HPC HPLC HPP

= 4-Hydroperoxycyclophosphamide = Hemagglutinins = Highly Active Anti-Retroviral Therapy = Human Antimouse Antibody = Hepatitis B Virus = Hematopoietic Cell = Hemorrhagic Cystitis = Health Care Financing Administration = Human Chorion Gonadotropin = Human Coronavirus = Hematopoietic Cell Transplantation = Hepatitis C Virus = Hodgkin Disease = High Dose ARAC = High Dose Chemotherapy = High Dose Conditioning = High Dose Cyclophosphamide = High Dose Immunosuppressive Therapy = Hypersensitivity Delayed Reaction = High Dose Therapy = Hydroxyethyl Starch = Hypereosinophilic Syndrome = Human Herpes Virus 6 = Health Insurance Portability and Accountability Act = Human Immunodeficiency Virus = Hodgkin Lymphoma = Human Leukocyte Antigen = Hemophagocytic Lymphohistiocytosis = Human Metapneumovirus = Hematopoietic Progenitor Cells = High-Pressure Liquid Chromatography = High Proliferative Potential

GFR GH GHRH GHSG GI GIGEMA

HPRT HPFH HPV HRPBC HRQOL HRSA HRT HSA HSC HSP HSV HSV-tk HTC HTLP HTLV HU HUVEC HUS HVEM HVG HVR

= Hypoxanthine-Guanine Phosphoribosyltransferase = Hereditary Persistence of Fetal Hemoglobin = Human Papillomavirus = High Risk Primary Breast Cancer = Health Related Quality of Life = Health Resources and Services Administration = Hormone Replacement Therapy = Heat Stable Antigen = Hematopoietic Stem Cell = Heat Shock Protein = Herpes Simplex Virus = Herpes Simplex Virus Thymidine Kinase = Homozygous Typing Cells = Helper T-Lymphocyte Precursor = Human T-cell Lymphotropic Virus = Hydroxyurea = Human Umbilical Cord Endothelial Cells = Hemolytic Uremic Syndrome = Herpesvirus Entry Mediator = Host-versus-Graft (Reaction) = Hyper-Variable Region

IA IAA IBD IBMTR IBW ICA ICAM ICH ICL 670 ICOS ICU iDC IDDM IDE IDSA IE IEF IFAR IFI IFN IFRT IG IGF-1 IHC IIP IL IL-1 IM IMPDH IMUST

= Invasive Aspergillus (Infection) = Insulin Autoantibody = Inflammatory Bowel Disease = International Bone Marrow Transplant Registry = Ideal Body Weight = Islet Cell Antibody = Intracellular Adhesion Molecule = International Conference on Harmonization = Deferasirox = Inducible Costimulator (Protein) = Intensive Care Unit = Immature Dendritic Cells = Insulin Dependent Diabetes Mellitus = Investigational Device Exemption = Infectious Diseases Society of America = Immediate Early (Antigen Expression) = Isoelectric Focusing = International Fanconi Anemia Registry = Invasive Fungal Infection = Interferon = Involved Field Radiotherapy = Immunoglobulin = Insulinlike Growth Factor-1 = International Conference on Harmonization = Idiopathic Interstitial Pneumonia = Interleukin = Interleukin-1 = Infectious Mononucleosis = Inosine Monophosphate Dehydrogenase = International Marrow Unrelated Search and Transplant = Investigational New Drug = International Neuroblastoma Research Group = International Neuroblastoma Staging System = Intraoperative Radiation Therapy = Interstitial Pneumonia = Intraperitoneal = Inositol-l, 4, 5-Triphosphate = Inherited Paternal Antigen = Invasive Pulmonary Aspergillosis

IND INRG INSS IORT IP IP IP3 IPA IPA

List of Abbreviations

IPI IPS IPS IPSS IRB IS IS ISCT ISO ISS IST IT ITD ITIM ITR IU-HCT IV IVF IVIG

= = = = = = = = = = = = = = = = = = =

JACIE

= Joint Accreditation Committee of ISCT-Europe and EBMT = Joint Commission of Health Care Organizations = Juvenile Idiopathic Arthritis = Juvenile Myelomonocytic Leukemia

JAHCO JIA JMML

International Prognostic Index Idiopathic Pneumonia Syndrome Interstitial Pneumonia Syndrome International Prognostic Scoring System Institutional Review Board Immunosuppressive Immune Synapse International Society for Cellular Therapy Isohemagglutinin International Staging System Immunosuppressive Therapy Intrinsic Timetable (Model) Internal Tandem Duplication Immunoreceptor Tyrosine Binding Inhibition Motif Inverted Terminal Repeats Intrauterine-Hematopoietic Cell Transplantation Intravenous In Vitro Fertilization Intravenous Immunoglobulin

KLH KPC KPS

= Keratoconjunctivitis Sicca = Killer-Cell Inhibitory Receptor or Killer Ig-like Receptor = Keyhole Limpet Hemocyanin = Klebsiella Pneumoniae Carbapenemase = Karnofsky Performance Score

LA LAD LAK LAMP LAT LC LCH LCL LCR LCT LD LDA LDH LET LFA LFR LFS LH LHRH LIF LIN LMP LMWH LOH L-PAM LPC LPD LPHD LPR

= Latex Agglutination = Leukocyte Adhesion Deficiency = Lymphokine Activated Killer (Cells) = Loop Mediated Isothermal Amplification = Latency Associated Factor = Langerhans Cell = Langerhans Cell Histiocytosis = Lymphoblastic Cell Lines = Locus Control Region = Long Chain Triglyceride = Linkage Disequilibrium = Limiting Dilution Assay = Lactate Dehydrogenase = Linear Energy Transfer = Leukocyte Function-Associated Antigen = Limited Field Radiation = Leukemia-free Survival = Luteinizing Hormone = Luteinizing Hormone Releasing Hormone = Leukemia Inhibitory Factor = Lineage = Latency Membrane Protein = Low Molecular Weight Heparin = Loss of Heterozygosity = L-Phenylalanine Mustard = Leukemic Progenitor Cell = Lymphoproliferative Disease = Lymphocyte Predominant Hodgkin Disease = Lipoprotein Receptor-Related Protein

KCS KiR

xxvii

LPS LTA LTC-IC LTR LUTS LV LVEF LY

= = = = = = = =

MAB MACS MALT MAOI MAPC MAPC MAPK MBC MBL MBP MCA MCD MCL MCL MCP MCR M-CSF MCT mDC MDR MDS MEAC MEC MEG-CSA MEG-GPA MEL MEP mESC MF MG MGDF MGF MGUS

= Monoclonal Antibody = Multipotent Adult Stem Cell = Mucosa Associated Lymphoid Tissue = Monoamine Oxidase Inhibitor = Mesenchyme Associated Progenitor Cell = Multipotent Adult Progenitor Cell = Mitogen Activated Protein Kinase = Metastatic Breast Cancer = Metallo-beta-Lactamase = Myelin Basic Protein = Middle Cerebral Artery = Minimal Change Disease = Mantle Cell Lymphoma = Mast Cell Leukemia = Monocyte Chemoattractant Protein = Major Cytogenetic Response = Macrophage-Colony Stimulating Factor = Median Chain Triglyceride = Mature Dendritic Cells = Multi Drug Resistance = Myelodysplastic Syndrome = Minimum Effective Analgesic Concentration = Medullary Epithelial Cell = Megakaryocyte-Colony Stimulating Activity = Megakaryocyte-Growth Promoting Activity = Melphalan = Megakaryocytic Erythrocyte Progenitor = Mouse Embryonic Stem Cell = Mycosis Fungoides = Myasthenia Gravis = Megakaryocyte Growth and Development Factor = Mast Cell Growth Factor = Monoclonal Gammopathy of Undetermined Significance = Minor Histocompatibility Antigen = Modified Health Assessment Questionnaire = Major Histocompatibility Complex = Metaiodobenzylguanidine = Minor Histocompatibility Antigens = Medical Internal Radiation Dose = Mucolipidosis = Mixed Leukocyte Culture = Metachromatic Leukodystrophy = Myeloid-Lymphoid-Initiating Cell = Mixed Lineage Leukemia = Mixed Leukocyte Reaction = Maroteaux-Lamy Syndrome = Moloney Leukemia Virus = Multiple Myeloma = Mitomycin C = Mycophenolate Mofetil = Matrix Metalloprotease = Measles/Mumps/Rubella

mHA MHAQ MHC MIBG miHA MIRD ML MLC MLD ML-IC MLL MLR MLS MLV MM MMC MMF MMP MMR

Lipopolysaccharide Latency Associated Transcripts Long Term Culture Initiating Cell Long Terminal Repeat Lower Urinary Tract Syndrome Lenti Virus Left Ventricular Ejection Fraction Life Years

xxviii

List of Abbreviations

MMR MN MODS MOG MOPP MP MP MPA MPB MPO MPP MPS MPSV MPT MPV MR MRC MRD MRI m-RNA MRP MRS MRSA mRSS MS MSC MSC MSCH MSD MSFC MSI MSKCC MS-LCH MSOF MTD MTOR MTX MUGA

= Mannose Macrophage Receptor = Membranous Nephropathy = Multiple Organ Dysfunction Syndrome = Myelin Oligodendrocyte Glycoprotein = Mustargen, Vincristine, Prednisone, Procarbazine = Melphalan Prednisone = Methylprednisolone = Mycophenolic Acid = Mobilized Peripheral Blood (Cells) = Myeloperoxidase = Multipotent Progenitor = Mucopolysaccharidosis = Myeloproliferative Sarcoma Virus = Melphalan, Prednisone, Thalidomide = Metapneumovirus = Molecular Remission = Medical Research Council (United Kingdom) = Minimal Residual Disease = Magnetic Resonance Imaging = Messenger RNA = Multidrug Resistance-Associated Protein = Magnetic Resonance Spectroscopy = Methicillin Resistant Staphylococcus Aureus = Modified Rodman Skin Score = Multiple Sclerosis = Mesenchymal Stem Cell = Mesenchymal Stromal Cells = Mouse Spinal Cord Homogenate = Multiple Sulfatase Deficiency = Multiple Sclerosis Functional Composite = Microsatellite Instability = Memorial Sloan Kettering Cancer Center = Multisystem Langerhans Cell Histiocytosis = Multi-System Organ Failure = Maximum Tolerated Dose = Mammalian Target of Rapamycin = Methotrexate = Multiple Gated Acquisition (Scan)

NAB NANT NAPI NASBA NAT NB NCCN NCI NCIC NCM nCR NCR NEO Nf1 NF-AT NGFR NGVL NHL NIH NIMA NIPA NK NK-T

= Natural Antibody = New Approaches to Neuroblastoma Therapy = North American Pulsed Field Type 1 = Nucleic Acid Sequence-Based Amplification = Nucleic Acid Amplification Test = Neuroblastoma = National Comprehensive Cancer Network = National Cancer Institute = National Cancer Institute of Canada = Nurse Case Manager = Near Complete Response = Natural Cytotoxicity Receptor = Neomycin = Neurofibromatosis type 1 = Nuclear Factor of Activated T Cells = Nerve Growth Factor Receptor = National Gene Vector Laboratory = Non-Hodgkin Lymphoma = National Institutes of Health = Non-Inherited Maternal Antigen = Non-Inherited Paternal Antigen = Natural Killer (Cells) = NK-T cells

NO NOD NPC NRH NRM NS NS NSAID NYBC NZW

= Non-Myeloablative = National Marrow Donor Program = Non-Myeloablative Hematopoietic Cell Transplantation = Nitric Oxide = Non-Obese Diabetic (Mouse) = Non-Protein Calories = Nodular Regenerative Hyperplasia = Non-Relapse Mortality = Nephrotic Syndrome = Natural Suppressor (Cells) = Non-Steroidal Anti-Inflammatory Drug = New York Blood Center = New Zealand White (Mouse)

OCTGT ODN OLD OMIM ONJ ONS OPA OPG OPM OR OS OSTM1

= = = = = = = = = = = =

PACT PAIgG PAIS PB PBHC PBHCT PBMC PBP PBPC PBS PCA PCNSL PCR PD PD PDC PDQ PDT PEC PET PFA PFS PFT PGD PGF PGK Ph PH PHA PHN PHQ PHS PIC PICC

= Psychological Assessment for Transplantation Scale = Platelet Associated Immunoglobulin = Psychological Adjustment to Illness Scale = Peripheral Blood = Peripheral Blood Hematopoietic Cells = Peripheral Blood Hematopoietic Cell Transplantation = Peripheral Blood Mononuclear Cells = Penicillin-Binding Protein = Peripheral Blood Progenitor Cells = Primer Binding Site = Patient Controlled Analgesia = Primary Central Nervous System Lymphoma = Polymerase Chain Reaction = Programmed Death = Protective Dose = Plasmacytoid Dendritic Cell = Physician Data Query = Photodynamic Therapy = Cisplatin, Etoposide, Cyclophosphamide = Positron Emission Tomography = Phosphonoformic Acid (Foscarnet) = Progression-Free Survival = Pulmonary Function Test = Preimplantation Genetic Diagnosis = Poor Graft Function = Phosphoglycerokinase = Philadelphia (Chromosome) = Pulmonary Hypertension = Phytohemagglutinin A = Post-Herpetic Neuralgia = Perceived Health Questionnaire = Public Health Service = Pre-Integration Complex = Peripherally Inserted Central Venous Catheter

NM NMDP NMHCT

Office of Cellular, Tissue and Gene Therapies Oligodeoxynucleotide Obstructive Lung Disease Online Mendelian Inheritance in Man Osteonecrosis of the Jaw Oncology Nursing Society Office of Patient Advocacy Osteoprotegerin Oropharyngeal Mucositis Odds Ratio Overall Survival Osteopetrosis Membrane Protein 1

List of Abbreviations

PIG-A PIH PIQ PIXY 321 P-gp PK PKC PLL PLP PLPHA PLS PLT PMC PML PMR PMRD PN PNET PNH POG POMS PORN PORT POV PR PRA PRCA PRO PRP PSA PSE PTA PTCL PTH PTLD PTT PTX PUVA PV PVP PVR

= Phosphatidyl Inositol Glycan-A = Prolactin Inhibiting Hormone = Performance IQ = Fusion Protein: IL-3/GM-CSF = P-Glycoprotein = Pharmacokinetic = Protein Kinase C = Prolymphocytic Leukemia = Proteolipid Protein = Post-Lumbar Puncture Headache = Psychosocial Level System = Platelet = Persistent Mixed Chimerism = Progressive Multifocal Leukoencephalopathy = Progressive Muscle Relaxation = Partially Matched Related Donor = Parenteral Nutrition = Primitive Neuroectodermal Tumor = Paroxysmal Nocturnal Hemoglobinuria = Pediatric Oncology Group = Profile of Mood Status = Progressive Outer Retinal Necrosis = Patient Outcomes Assessment Research Team = Premature Ovarian Failure = Partial Remission or Partial Response = Panel Reactive Antibody = Pure Red Cell Aplasia = Patient Reported Outcome = Polyribosylphosphate = Prostate Specific Antigen = Prednisone = Peripheral Tissue Antigen = Peripheral T Cell Lymphoma = Parathyroid Hormone = Post-Transplant Lymphoproliferative Disorder = Partial Thromboplastin Time = Pentoxifylline = Psoralen-Ultraviolet A Irradiation (Treatment) = Polycythemia Vera = Polyvinylpyrrolidone = Polio Virus Receptor

QALY QLQ QOL QTWIST

= = = =

RA RA RAC RAEB RAEB-T

= Refractory Anemia = Rheumatoid Arthritis = Recombinant DNA Advisory Committee = Refractory Anemia with Excess Blasts = Refractory Anemia with Excess Blasts in Transformation = Radioimmunotherapy = Receptor Activator of NF-Kb Ligand = Refractory Anemia with Ringed Sideroblasts = Red Blood Cell = Relative Biological Effectiveness = Renal Cell Cancer = Ratio of Cost and Charges = Replication-Competent Lentivirus = Refractory Cytopenia with Multilineage Dysplasia

RAIT RANKL RARS RBC RBE RCC RCC RCL RCMD

Quality-Adjusted Life Years Quality of Life Questionnaire Quality of Life Quality Time Without Symptoms of Toxicity

xxix

RCT RDA REE RFS RHC RIA RIC RIFLE RIR RISC RIT RFLP RLD RNA ROI ROS RPM RQ RR RRT RRT RSCH RSV RT RT3U RTOG

= Randomized Controlled Trial = Recommended Daily Allowance = Resting Energy Expenditure = Relapse-Free Survival = Residual Host Cells = Radioimmune Assay = Reduced Intensity Conditioning = Risk, Injury, Failure, Loss, Endstage = Reduced Intensity Regimen = RNAi Induced Silencing Complex = Radioimmunotherapy = Restriction Frequent Length Polymorphism = Restrictive Lung Disease = Ribonucleic Acid = Region of Interest = Reactive Oxygen Species = Rapamycin (Sirolimus) = Respiratory Quotient = Relative Risk = Renal Replacement Therapy = Regimen-Related Toxicity = Rat Spinal Cord Homogenate = Respiratory Syncytial Virus = Reverse Transcriptase = Resin T3 Uptake = Radiation Therapy Oncology Group

SAA SAE SAP SAS-SR SBA SCC SCD SCF SCID SCID-hu

= Severe Aplastic Anemia = Severe Adverse Event = Serum Amyloid Protein P = Social Adjustment Scale – Self Report = Soy Bean Agglutinin = Squamous Cell Carcinoma = Sickle Cell Disease = Stem Cell Factor = Severe Combined Immunodeficiency Syndrome = Severe Combined Immunodeficiency Syndrome/ Human (Mouse) = Symptoms Check List = Stem Cell Therapeutics Outcomes Database = Standard Deviation = Standard Dose Chemotherapy = Stroma Cell Derived Factor = Shwachman Diamond Syndrome = Standard Error = Sleep, Energy, Appetite Scale = Surveillance Epidemiology and End Result = Symptom Experience Report = Serological Identification of Antigens by Recombinant Expression Cloning = Spleen Focus-Forming Virus = Societe Francaise de Greffe de Moelle = French Society of Pediatric Oncology = Sex Hormone Binding Globulin = Stanford Hospital and Clinics = Sonic Hedgehog = Sickness Impact Profile = Systemic Inflammatory Response Syndrome = Signaling Lymphocyte Activation Molecule = Systemic Lupus Erythematosus = SLE Disease Activity Index = Small Lymphocytic Leukemia

SCL SCTOD SD SDC SDF SDS SE SEAS SEER SER SEREX SFFV SFGM SFOP SHBG SHC Shh SIP SIRS SLAM SLE SLEDAI SLL

xxx

List of Abbreviations

SM SMP SNP SNRI SOP SOS SPA SPECT sPLA2 SRBC SRC SS SSC SSOP SSP SSRI STAMP-I STAMP-V SUMC SVC SWOG

= Systemic Mastocytosis = Sympathetically Mediated Pain = Single Nucleotide Polymorphism = Serotonin-Norepinephrine Reuptake Inhibitor = Standard Operating Procedure = Sinusoidal Obstruction Syndrome = Staphylococcal Protein A = Single Photon Emission Computed Tomography = Secretory Phospholipase A2 = Sheep Red Blood Cell = SCID Repopulating Cells = Sézary Syndrome = Systemic Sclerosis = Sequence Specific Oligonucleotide Probes = Sequence Specific Primer = Selective Serotonin Reuptake Inhibitor = BCNU-Cisplatinum-Cyclophosphamide = Thiotepa-Carboplatin-Cyclophosphamide = Stanford University Medical Center = Superior Vena Cava = Southwest Oncology Group

α-TIF T1DM T3 T4 TAA TAI TAM TAP TBI TC TCA TCD TCD TCIRG1 TCR TDM TdT TERS TERT TFO TGF-β TGN T-GVHD TH TIL TIP TIPS TK TLC TLI TLR TM TMA TMC TMD TMP-SMX

= α-Transinducing Factor = Type 1 Diabetes Mellitus = Triiodothyronine = Thyroxine = Tumor Associated Antigen = Thoraco Abdominal Irradiation = Transplant-Associated Microangiopathy = Transporter-Associated Antigen Processing = Total Body Irradiation = Cytotoxic T cells = Tricyclic Antidepressant = T Cell Depletion = Transcranial Doppler (Ultra-sonography) = T Cell Immune Regulator 1 = T Cell Receptor = Therapeutic Drug Monitoring = Terminal Deoxynucleotidyl Transferase = Transplant Evaluation Rating Scale = Telomerase Reverse Transcriptase = Triple Helix Forming Oligodeoxynucleotide = Transforming Growth Factor Beta = Transgolgi Network = Transfusion Associated Graft-versus-Host Disease = Helper T Lymphocytes = Tumor Infiltrating Lymphocytes = Paclitaxel, Ifosfamide, Cisplatin = Transjugular Intrahepatic Portosystemic Shunt = Thymidine Kinase = Total Lung Capacity = Total Lymphoid Irradiation = Toll Like Receptor = Thrombotic Microangiopathy = Thrombotic Microangiopathy = Transient Mixed Chimerism = Temporomandibular Dysfunction = Trimethoprim-Sulfamethoxazole

TNC TNF TNF-α TNFR TPA TPMT TPN TPO TPO TRAIL TRALJ TREC TREG TRH TRIM TRM TSH TSP TTP TTR

= Total Nucleated Cells = Tumor Necrosis Factor = Tumor Necrosis Factor α = Tumor Necrosis Factor Receptor = Tissue Plasminogen Activator = Thiopurine S-Methyltransferase = Total Parenteral Nutrition = Thrombopoietin = Thyroid Peroxidase = TNF-Related Apoptosis-Inducing Ligand = Transfusion-Related Acute Lung Injury = T Cell Receptor Excision Circles = Regulatory T Cell = Thyrotropin-Releasing Hormone = Transfusion-Related Immune Modulation = Treatment Related Mortality = Thyroid Stimulating Hormone = Tropical Spastic Paraparesis = Thrombotic Thrombocytopenic Purpura = Transthyretin

UAMS UCB UCB UGD URD URI UV

= = = = = = =

VAD VATS VCAM VEGF VGPR VIP VLA VLCFA VNTR VOD VP-16 VRE VSV VSV-G VWF VWMD VZIG VZV

= Vincristine, Adriamycin, Decadron = Video-Assisted Thoracoscopic Surgery = Vascular Cell Adhesion Molecule = Vascular Endothelial Growth Factor = Very Good Partial Remission = Etoposide, Ifosfamide, Cisplatin = Very Late Activation Antigen = Very Long Chain Fatty Acid = Variable Number of Tandem Repeat = Venocclusive Disease = Etoposide = Vancomycin Resistant Enterococcus = Vesicular Stomatitis Virus = Vesicular Stomatitis Rhabdovirus = von Willebrand Factor = Vanishing White Matter Disease = Varicella Zoster Immune Globulin = Varicella Zoster Virus

WAS WBC WBM WBRT WHI WMDA WNV

= Wiskott-Aldrich Syndrome = White Blood Cell = Whole Bone Marrow = Whole Brain Radiotherapy = Women’s Health Initiative = World Marrow Donor Association = West Nile Virus

X-AND

X-Linked Adrenoleukodystrophy

University of Arkansas Medical Science Center Umbilical Cord Blood Unrelated Cord Blood Uridine 5’ Diphosphate Glucoronosyl Transferase Unrelated Donor Upper Respiratory Infection Ultraviolet (Light)

Plate 5.1 Chimeric analysis of yolk sac blood islands with fluorescent embryonic stem cell clones. Tetrachimeric mice were generated by the injection of four distinctly colored single cells into developing blastocysts. The resulting embryos were then analyzed for color combinations observed in the yolk sac, and revealed endothelial and hematopoietic cells of different colors in all individual blood islands, indicating that they developed from more than one cell. (a) A chimeric embryo at the early neural plate stage (EB). A white rectangle indicates an immature blood island. The arrow indicates the direction of tissue extension. al, allantois; am, amnion; ch, chorion; ec, embryonic ectoderm; me, intraembryonic mesoderm; ve, visceral endoderm. (b) Experimental scheme indicating the possible derivations of blood islands and the percentage of each type observed. (c) A type IV chimeric blood island with EGFP, ECFP, and mRFP1 endothelial cells, and ECFP, EGFP, and nonfluorescent hematopoietic cells. (Adapted from [172].)

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Progression to acute myelogenous leukemia (AML) or myeloid blast crisis, CML LT-HSC

ST-HSC

MPP

CLP

Progenitors (GMP)

CMP Blood cells

Self-renewal (regulated)

EVENTS ACCUMULATED: 1. Bcr-Abl or Aml-1–Eto, or jun-b, etc. 2. Antisenescence (e.g. tert) 3. Antiapoptosis 1 3. Antiapoptosis 2 5. Evasion of immune cells (1–4 events) 6. Activation/overexpression self-renewal genes

Self-renewal (Unregulaed)

blasts

blasts

blasts Leukemic Stem Cell

Plate 5.2 Comparison of stem cell self-renewal during normal hematopoiesis and leukemic transformation. During normal hematopoiesis, the signaling pathways that regulate self-renewal are tightly controlled, allowing proper differentiation to each of the mature blood cell lineages (top). Accumulation of mutations in the self-renewing hematopoietic stem cells (HSCs) over time until dysregulated self-renewal ability is attained, and leukemia propagation is initiated. Dysregulation of self-renewal mechanisms in transformed cancer stem cells leads to uncontrolled self-renewal and the production of leukemic blast cells. The leukemic population capable of sustaining disease is often distinct from the blast population, and may reside in the stem cell or progenitor cell compartment. Importantly, if the transformation event occurs in a progenitor cell, it must endow the progenitor cell with the self-renewal, because the progenitors would otherwise differentiate. See text for abbreviations.

5‘ 3‘

W W‘ C

-

W C C‘

+

a1A2 b 1b 2 c 1c 2

a

-

A

c A a

D a A

b B

c C

3‘ 5‘

+

b B

A1a2 B1B2 C1C2

C c

S asymmetric division

maintenance

A1a2 b 1B2 C1c 2

a1A2 B1b 2 c 1C2

S symmetric division

S self-renewal

stem cell niche

Cell Counts

Plate 6.1 The “silent sister” hypothesis [32]. Top panel: According to this hypothesis the two sister chromatids in metaphase chromosomes carry distinct epigenetic marks (indicated by + and − signs) at centromeric DNA and at certain “stem cell” genes (shown here is a single gene expressed as “A” or silent as “a”). Epigenetic differences at centromeres between sister chromatids are proposed to enable chromatid-specific segregation of chromosomes during mitosis. Such nonrandom partition of chromatids is proposed to regulate the expression of certain genes present on those chromatids in the daughter cells following mitosis. Bottom panel: Selective attachment of microtubules (MT) coming from the “mother” centrosome [96] (indicated by the red dot close to the stem cell niche) to sister chromatids containing the Watson (W) template strand (here defined as the 3′ to 5′ template strand indicated by the solid red line) during a polar, asymmetric stem cell division. Shown are three metaphase chromosomes (e.g. out of the 46 chromosomes in a human cell). The preference for one of the chromatids could result from a gradient of MT guiding proteins (red) that regulate preferential MT binding to the kinetochore at the sister chromatid containing the Watson template strand. Selective partition of sister chromatids results in one stem cell (S) with two active copies of “self-renewal” genes (the two parental copies of these genes are indicated by 1 and 2) ready to be transcribed (A, B, and C) and one cell committed to differentiate (D) in which expression of the two copies of the “stem cell” genes is suppressed (a, b, and c). Note that an occasional sister chromatid exchange event between a centromere and a relevant “stem cell” gene (illustrated as a2 in cell S and A2 in cell D) is not incompatible with the “silent sister” hypothesis. In stem cells (S) expression of silenced “stem cell” genes is predicted to be restored by default (bottom panel, right-hand side), and aberrant expression of a single “stem cell” gene in a differentiating cell (A2 in cell D) is not predicted to prevent differentiation altogether. However, inappropriate expression of multiple “self-renewal” genes (perhaps in combination with inappropriate suppression of tumor suppressor genes) in differentiating cells could result in abnormal cell proliferation. Such defects could result from high levels of sister chromatid exchange, defects in cell polarity, failure to establish or recognize epigenetic marks at centromeres or other factors involved in the proposed strand-specific chromatid segregation pathways. (c) Random segregation of sister chromatids restores expression of “silent” stem cell genes and results in self-renewal of stem cells. Without a polar distribution of guiding proteins (even red color), both daughter cells inherit a random mixture of active and inactive “stem cell” genes, which is predicted to restore the expression of the “stem cell” genes in both daughter cells if both cells are in the proper microenvironment (the stem cell “niche”). Epigenetic differences between sister chromatids are restored following expression during replication. Note that only one out of eight possible expression patterns for the three genes in the two daughter cells following random sister chromatid segregation is shown.

MSCs

Plate 9.1 A panel of data demonstrating the defining characteristics of mesenchymal stromal cells (MSCs): adherence, immunophenotype, and in vitro differentiation. Top left: Photomicrograph of undifferentiated MSCs showing the characteristic spindle shape and adherent properties of the cells (original magnification ×40). Top right: Flow cytometry histograms demonstrating the typical expression pattern of surface antigens (—) and isotype control (- - -), as indicated. Bottom: Immunocytochemical staining demonstrating the differentiation of MSCs into osteoblasts (Alizarin red stain), adipocytes (oil red O stain), and chondroblasts (Alcian blue stain). (Reproduced from [43], with permission.)

Osteoblasts

CD105

CD45

CD34

CD73

CD11b

CD19

CD90

CD3

HLA-DR

Log Fluoresence Intensity

Adipocytes

Chondroblasts

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Plate 14.1 Evaluation of murine graft-versus-host disease (GVHD) lingual tissue for apoptosis, T-cell infiltration, and cytokeratin 15 expression. (a) Light microscopy of hematoxylin and eosin-stained tissue from GVHD at day 11 after bone marrow transplantation reveals lymphocytic infiltration and apparent apoptosis in the rete-like prominences (RLPs) (arrow). (b) Apoptosis restricted to RLPs was confirmed by TUNEL immunohistochemistry (arrows). (c) Transmission electron microscopy demonstrates RLP infiltrated by a lymphocyte (left arrow) in apposition to an epithelial cell undergoing mitotic division (right arrow). (d,e) Immunohistochemistry for CD4 at day 4 (d) and day 7 (e) after bone marrow transplantation with progressive infiltration of RLPs by CD4+ T cells. Note the focus where CD4+ T cells ((e); arrow) surround a centrally located nonreactive cell ((e) inset). (f,g) K15 expression in sections adjacent to (d) and (e), respectively, reveals focal loss of K15 immunoreactivity ((f); arrow). Note the cell strongly positive for K15 and surrounded by nonreactive cells ((g); arrow and inset), a pattern reciprocal to the inset in (e) and suggestive of satellitosis. (Adapted from [98], with permission.)

Plate 21.1 Theoretical models of drug resistance. In the conventional model of drug resistance (a) a subpopulation of cells (yellow) have pre-existing genetic variations that confer multidrug resistance (MDR). Following chemotherapy, the resistant cells survive and proliferate, forming a recurrent tumour that is composed of the drug-resistant clone. In the cancer stem-cell model (b), tumors have a small population of tumor stem cells (red) and their differentiated progeny (blue). Following chemotherapy, the differentiated cells are killed, but the stem cells survive. In the “acquired resistance” stem-cell model (c), the tumor stem cells (red) survive therapy, and differentiated cells are killed. Mutations in the surviving stem cells (yellow) and their progeny (purple) may also display the drug-resistant phenotype. In the “intrinsic resistance” model (d), both the stem cells (yellow) and the differentiated cells (purple) are inherently drug resistant. (From [34], with permission.)

Plate 23.1 Dose distribution through T5–6 showing 6, 8, 10, 12, and 13.2 Gy isodose lines. The prescription dose was 12 Gy, with 50% transmission lung blocks.

Plate 23.2 Color wash demonstrating the typical dose distribution of a patient treated with targeted total body irradiation using tomotherapy. The target structure is skeletal bone. A, anterior; L, left; R, right. (Reproduced from [4], with permission from the American Society for Blood and Marrow Transplantation.)

Plate 23.3 Color wash demonstrating dose distribution of 30 Gy to the site of a chloroma in the maxillary sinus and high neck, combined with 12 Gy total marrow irradiation, in a patient with acute myeloid leukemia.

(a)

(b)

Plate 25.1 Assessment of chimerism in sorted granulocytes (a) and leukemic blasts (b) by dual-color fluorescence in situ hybridization (FISH). Hybridization was carried out with a biotin-labeled Y chromosome-specific DNA probe and a digoxigenin-labeled X chromosome-specific DNA probe. Hybridization was detected with Texas Red-conjugated avidin and fluorescein-conjugated antibody against digoxigenin. Cells were counterstained with 4′,6-diamidino-2phenylindole API. Male cells contain a single red fluorescent spot and a single green fluorescent spot, while female cells contain two green fluorescent spots and no red fluorescent spots. In this case, granulocytes were derived from a male donor, and leukemic blasts were derived from the female recipient.

14 14

der(4) der ?

4

(b)

(a)

(c)

(d)

(e)

Targeted scanning with CD19 monoclonal antibody CD19

+

lymphoblast

der(9)

9

22 BCR/ABL (f)

(g)

CD10 positive

BCR–ABL positive

(i)

(h) CD10 negative

BCR–ABL negative

Plate 26.1 Plasma cell-specific fluorescence in situ hybridization (FISH) analysis. (a–e) Cytospin slides are made from the bone marrow aliquot and stained with May-Grünwald stain. After the cells have been mapped to the slide, the slide is destained and hybridized with the selected DNA probe. For details, see [5]. (a) A plasma cell that has been mapped. This same cell is seen in panel (b) showing a variant immunoglobulin H (IGH) pattern using a dual-color 5′IGH/3′IGH break apart probe. Once an IGH gene rearrangement is found in a patient with multiple myeloma, the same slide is rehybridized to determine its partner chromosome in the IGH translocation. (c) A fusion signal is detected with the FGFR3 (fibroblast growth factor receptor-3)/IGH probe indicating the derivative chromosome 4 of the t(4;14) or der(4)t(4;14). The second fusion that would have indicated the der(14)t(4;14) is not present in this patient, the result of loss of the derivative 14 chromosome. In another slide from this patient (d and e), monosomy 13 is shown in the neoplastic plasma cells, but the segmented neutrophil adjacent to the plasma cells shows both chromosome 13 signals (one each in each of the segments of the nucleus). The chromosome 13 probe set used was composed of D13S319 probe (red), which maps to 13q14.3, and the LAMP1(lysosomal-associated membrane protein1)/13q34 reference probe labeled in green. Sequential immunohistochemistry (IHC)/FISH is shown in (f–i). (f) A CD19+ lymphoblast that is BCR/ABL1 fusion gene positive (g), indicating residual disease and not a hematogone. Similarly, (h) and (i) show the cell that is CD10+ is positive for the BCR/ABL1 fusion gene, and the CD− cell is negative for the BCR/ABL1 fusion gene. The BCR/ ABL1 probe used for the sequential IHC/FISH studies was the single fusion probe spiked with 9q34/argininosuccinate synthetase gene probe labeled in Spectrum aqua. All probes were obtained from Abbott Molecular Inc. (Des Plaines, IL).

Plate 26.2 Flow cytometry assays for detection of minimal residual disease. Leukemia samples can be labeled with multiple fluorescent antibodies, and cells can be distinguished by the size of the cell and the types of antibody that bind the cell surface. Normal hematopoiesis has very stereotypical cell antigen expression linked to differentiation; leukemia cells are distinguished by inappropriate expression patterns of these antibodies. In the example, a residual acute monocytic leukemia is analyzed with multiple antigens. In the bottom left panel, the sample shows an inappropriate expression of CD56 and CD4 consistent with residual disease. In this example, these cells made up 0.9% of the total nucleated cells. (Courtesy of Dr Brent Wood, University of Washington.)

Plate 26.3 Quantitative polymerase chain reaction (PCR). The cartoon shows the principle behind the two most popular quantitative approaches: the Taqman (ABI) and the LightCycler (Roche). In the Taqman (I) reaction, the probe 5′ reporter dye and a 3′ quencher molecule. When the probe is intact, the quencher blocks laser excitation of the reporter. During PCR, the 5′ exonuclease activity of Taq polymerase releases the reporter, which, free from the quencher, is excited by the laser detection system. In the LightCycler system (II), annealing of juxtaposition probes during PCR synthesis allows for the transference of resonance energy that can be stimulated by the laser and detected. In both systems, a standard curve is generated that plots dilutions of the standards versus the PCR cycle number at which a threshold signal is obtained (middle panel). The starting amount of target in unknown samples is thus detected by a backcalculation of cycle threshold in the unknown (Ct) against the standard curve. (Courtesy of Dr John Goldman.)

Plate 27.1 Fatal Mylotarg-associated sinusoidal obstruction syndrome. The obliterated central venule is surrounded by focal sinusoidal fibrosis. (Trichrome stain)

Plate 27.2 Markedly hypocellular marrow without evidence of engraftment, day 7. The interstitial fluid accumulation, fat necrosis, and iron deposition are consistent with chemoirradiation damage.

Plate 27.3 Post-transplant Epstein–Barr virus-associated lymphoma infiltrating the intestinal mucosa in a boy treated for graft-versus-host disease (GVHD) with cyclosporine and a monoclonal antibody to T lymphocytes. Although the GVHD improved, large bizarre cells with atypical nuclei characteristic of a high-grade post-transplant lymphoproliferative disorder rapidly formed tumor nodules in liver, spleen, lymph nodes, and mesentery.

(a)

(b)

Plate 27.4 (a) Unsuspected Epstein–Barr virus post-transplant lymphoproliferative disorder. After the first hematopoietic cell transplantation (HCT), the patient developed severe liver graft-versus-host disease. Donor lymphocyte infusion was followed by loss of engraftment. A second HCT was carried out following conditioning with antithymocyte globulin, Campath, and steroids. Jaundice developed 35 days later. At low power, the portal space contains a prominent infiltrate that extends outward into the periportal zone. (b) In the same case, higher magnification shows the large atypical immunoblastic cells that were positive for CD20 and EBER staining.

Plate 27.5 Skin biopsy from the recipient of identical twin marrow who had received a single 15 mg/kg dose of dimethylbusulfan 15 days previously. The epidermal separation, keratinocyte atypia, and scanty lymphoid dermal infiltrate can also be seen in graft-versus-host disease.

Plate 27.7 Severe mucositis of the lips and tongue with pseudomembranous exudate 20 days after hematopoietic cell transplantation following conditioning with total body irradiation and chemotherapy. Similar changes may be present with severe acute graft-versus-host disease or secondary infection such as with a herpes virus. Plate 27.6 Acute glossitis 1 day post hematopoietic cell transplantation after a conditioning regime of 60 mg/kg of cyclophosphamide followed by 12 Gy of fractionated total body irradiation. There is early mucosal damage with severe erythema and focal atrophy.

Plate 27.8 Effect of high-dose therapy on colonic mucosa, day 7. This biopsy specimen was taken following therapy with busulfan, cyclophosphamide, and total body irradiation (12 Gy). There has been obliteration of almost all epithelial cells, leaving cystic crypt remnants. (Hematoxylin and eosin with alcian blue counterstain)

Plate 27.9 Recovery from the effects of high-dose therapy on colonic mucosa, day 16. This repeat biopsy from the same patient as in Plate 27.8 indicates that regeneration is occurring, although some cystic crypts remain. (Hematoxylin and eosin with alcian blue counterstain)

Plate 27.10 Hepatic sinusoidal obstructive syndrome (SOS) 95 days post hematopoietic cell transplantation for malignancy. The trichrome stain shows the blue outline of an hepatic venule with marked luminal narrowing (arrow shows lumen diameter) caused by subendothelial trapping of red cells. The hepatocytes of acinar zone 3 are severely disrupted. When the clinical diagnosis of hepatic SOS is uncertain, it may be confirmed by a transvenous liver biopsy with measurement of an elevated gradient between wedged and free hepatic venous pressures.

Plate 27.13 Fatal hepatic sinusoidal obstructive syndrome (SOS) at autopsy 12 days post bone marrow transplantation for leukemia. At this low magnification, the hematoxylin and eosin-stained section demonstrates striking congestion, hemorrhage, and hepatocyte disruption in zone 3 (centrilobular) of the liver acinus toward the right. The portal space and surrounding acinar zone 1 on the left are well preserved. These early severe hepatic SOS lesions at times can be identified with the hematoxylin and eosin stain after tissue fixation with B5 or methyl Carnoy’s. However, stains for connective tissue are more effective.

Plate 27.11 Early hepatic sinusoidal obstructive syndrome (SOS) showing perivenular staining for procoagulants. Immunohistochemical staining for factor VIII (von Willebrand) demonstrates postsinusoidal obstruction corresponding to the endothelium-lined pores through which the sinusoids drain into the venules. Immunostaining for fibrin localizes in the same regions. These observations provide some of the rationale for using anticoagulation and antithrombotic therapy for hepatic SOS; however, these therapies are ineffective.

Plate 27.14 CD31 immunostaining of liver with fatal sinusoidal obstructive syndrome. There has been partial to complete loss of sinusoidal endothelial staining of the hepatic cords in zone 3 (arrows) which surround the subtotally occluded venule lacking an endothelium (arrowhead).

Plate 27.12 Late hepatic sinusoidal obstructive syndrome, day 63. The trichrome stain demonstrates fibrotic obliteration of the venous lumen with extensive fibrosis in the surrounding sinusoids. The resulting vascular obstruction produced intractable ascites. Immunohistochemical stains at this stage reveal extensive deposition of collagen, while fibrin and other blood procoagulants are no longer identifiable.

Plate 27.15 Hepatic sinusoidal obstructive syndrome (SOS) and coexistent graft-versus-host disease (GVHD), day 26. On this trichrome stain, the two most common liver problems after hematopoietic cell transplantation can be differentiated by the zone of the liver acinus they affect. The expanded portal space in the lower right demonstrates changes from GVHD, including fibrosis, proliferation of atypical cholangioles along the limiting plate, and destruction of small interlobular bile ducts. The SOS lesion along the left margin shows striking collections of embolized hepatocytes beneath the endothelial basement membrane.

Plate 27.16 Fatal sinusoidal obstructive syndrome. (a) Hematoxylin and eosin staining demonstrates a large perivenular zone of hemorrhagic necrosis with destruction of hepatocyte cords surrounding the damaged venule (V); the areas around the portal spaces (PS) are spared. (b) Trichrome staining demonstrates the subtotal occlusion of the venular lumen by collagen and extracellular matrix. (c) Immunostain Mac 387 for macrophage/ monocytes demonstrates a zone of cells at the interface of the viable and necrotic hepatocytes. (d) Smooth muscle actin immunostain demonstrates marked expansion of hepatic stellate cell processes within the damaged sinusoids surrounding the venule.

Plate 27.18 Acute skin graft-versus-host disease, day 28 post mismatched allogeneic hematopoietic cell transplantation. The epidermis demonstrates a prominent lichenoid reaction involving the tip of the rete ridge in the center of the specimen, a location of epithelial stem cells within the epidermis.

Plate 27.17 Hepatic phlebosclerosis, day 96. Eccentric thickening of the venular wall without striking luminal narrowing was associated with an early liver toxicity syndrome. The patient had received a 5-day infusion of highdose arabinoside-C followed by cyclophosphamide and total body irradiation. Liver enzyme and bilirubin elevations developed prior to the infusion of marrow. Changes of phlebosclerosis were widespread, but there were no identifiable intravenular hepatic sinusoidal obstructive syndrome lesions at autopsy. (Trichrome stain)

Plate 27.19 Early skin graft-versus-host disease involving the parafollicular bulge region of the pilar unit. Note the small numbers of mononuclear inflammatory cells which infiltrate the parafollicular bulge area (*) near the arrector pilorum muscle in association with apoptotic bodies.

Plate 27.20 Gastric biopsy with graft-versus-host disease (GVHD), day 35. Several studies have shown that of all upper endoscopic biopsy sites, the stomach most frequently shows histologic changes of GVHD. In the area shown here, large numbers of lymphocytes infiltrate the lamina propria and the basilar portions of the gastric crypts. There is apoptosis of crypt epithelial cells and frank early crypt destruction. Inflammation and apoptosis of this intensity are not required for the diagnosis of gastric GVHD. More subtle yet still diagnostic alterations are illustrated in Plates 27.27 and 27.28.

Plate 27.21 Erythema and desquamation before treatment for acute graftversus-host disease, day 27 (see also Plate 27.24).

Plate 27.22 Maculopapular rash of skin graft-versus-host disease without a notable decrease in inflammation, 1 week after institution of PUVA therapy (see also Plate 27.25).

Plate 27.23 Painful red-to-violaceous maculopapular rash involving the sole of the foot consistent with acute graft-versus-host disease in a 2-year-old girl 11 days after human leukocyte antigen-nonidentical hematopoietic cell transplantation.

Plate 27.25 Marked improvement in skin graft-versus-host disease following 6 weeks of PUVA treatment (same patient as in Plate 27.22).

Plate 27.24 Virtually normal skin with focal depigmentation after treatment for acute graft-versus-host disease, day 74 (same patient as in Plate 27.21).

Plate 27.26 Endoscopic appearance of acute graft-versus-host disease in the stomach, day 32. The pyloric channel is in the center of the picture. The mucosa of the gastric antrum is edematous, reddened, and friable.

Plate 27.27 Gastric graft-versus-host disease in which the severe mucosal erythema, edema, and erosion seen endoscopically on the left are more striking than the focal, mild, epithelial cell apoptosis (arrow) in the histologic section on the right. Although a lymphocytic infiltrate is absent, apoptosis in multiple crypts is consistent with graft-versus-host activity. Inflammation may have been partially controlled by immunosuppressive therapy. (Reproduced from [42], with permission.)

Plate 27.28 Gastric graft-versus-host disease (GVHD) in which the antral mucosa is endoscopically normal aside from edema (left). With air insufflation through the endoscope, the antrum did not distend and there was no motor activity. Biopsy of such a bland area can be diagnostic. In this case, as shown in the photomicrograph on the right, there was epithelial cell apoptosis (arrow) and moderate lymphocytic infiltration diagnostic for GVHD. (Reproduced from [42], with permission.)

Plate 27.29 Gastric graft-versus-host disease. Within the edematous antral mucosa, numerous lymphocytes infiltrate the glands. Apoptosis is widespread, along with segmental disruption of the glands.

Plate 27.30 Rectal biopsy with graft-versus-host disease, day 53. Three mucosal crypts display varying degrees of damage. The crypt on the right shows many apoptotic cells. More extensive cell damage is present in the crypt on the lower left. The upper crypt has lost all enterocytes. Note infiltrating lymphocytes in the lower left crypt associated with nuclear debris characteristic of apoptosis.

Plate 27.31 Surgical resection of severe longstanding intestinal graft-versushost disease (GVHD), day 207. Scarred, ulcerated mucosa alternates with dilated areas of mucosal regeneration. The ulcerated portions demonstrate complete loss of epithelium and severe submucosal fibrosis. Very few patients have responded to intestinal resection for localized GVHD.

Plate 27.34 Fatal pseudomembranous colitis. An acute abdomen developed 15 days post transplant. The degenerative colonic crypts were covered by a cap of mucin admixed with sloughed cellular debris containing large colonies of bacteria consistent with Clostridium spp.

Plate 27.32 Severe graft-versus-host disease (GVHD) of ileum and cecum, day 80. The mucosal lining and folds are replaced by a friable, beefy red, diffusely ulcerated lamina propria. The numerous exposed capillaries ooze blood and serum, a major cause of morbidity from intestinal GVHD. The endothelial cells and fibroblasts within the lamina propria are often infected with cytomegalovirus, as was true in this patient.

Plate 27.35 Endoscopic appearance of cytomegalovirus esophagitis, day 61. A large shallow ulcer with serpiginous, reddened borders is seen in the distal esophagus. The ulcer base (arrows) has a yellow, reticulated appearance.

Plate 27.33 Collagen deposition in the colon with longstanding refractory extensive chronic graft-versus-host disease. The thickened bowel has extensive deposition of collagen in the submucosa, serosa, and lamina propria of the mucosa.

Plate 27.36 Esophageal biopsy showing cytomegalovirus (CMV) infection, day 61. Beneath the epithelium are two cytomegalic cells (large arrows), each containing a single, large, oval intranuclear inclusion surrounded by a clear halo (Cowdry type A). A third cell (small arrow) is probably also infected but is morphologically nondiagnostic. It would presumably be positive by immunocytochemistry and in situ hybridization techniques. Studies of cell morphology as well as viral DNA and antigen expression indicate that the esophageal squamous epithelium is never infected with CMV.

Plate 27.37 Autologous gastric graft-versus-host disease (GVHD) or lymphocytic gastritis. The focal mononuclear inflammatory cell infiltration of the lamina propria and mucosal crypt associated with crypt epithelial cell apoptosis is histologically indistinguishable from GVHD in an allograft recipient.

Plate 27.38 Hepatic graft-versus-host disease. Periodic acid–Schiff-stained section highlights a damaged interlobular bile duct that is infiltrated by lymphocytes. The nuclei vary in size and are irregularly arranged. The syncytium-like segment reflects nuclear loss.

Plate 27.39 Acute hepatitic onset of graft-versus-host disease at 10 months post transplant. Three months after tapering off immunosuppression, the aminotransferases rose to 2000 U, and alkaline phosphatase was three times normal, with normal bilirubin. This biopsy (1) was done 26 days from the onset and shows an extensive lobular hepatitis with intense lymphocytic inflammation of sinusoids and portal spaces, and hepatocellular necrosis.

Plate 27.40 This biopsy (2) was done 1 month later when the total bilirubin was 30 mg/dL. (a,b) Lymphoplasmacytic portal infiltrate which obscures the damaged interlobular bile ducts. (c) Immunostaining with cytokeratin 7 demonstrates that damaged bile ducts are still present. (d) Perivenular (zone 3) cholestasis. Hepatocytes show ballooning, and there is focal dropout associated with a patchy lymphocytic infiltrate. The patient made a full recovery after immunosuppressive treatment for graft-versus-host disease was initiated.

Plate 27.42 Lichen planus-like chronic graft-versus-host disease of the forearm of a 24-year-old African-American male 220 days after bone marrow transplantation of human leukocyte antigen-identical marrow (see also Plates 27.44 and 27.45). Plate 27.41 Hepatic graft-versus-host disease with severe cholangitis lentalike cholangiolar hepatocellular cholestasis. The interlobular bile duct is no longer recognizable. A ductular reaction with large bile-filled cholangioles lies along the periphery of the enlarged irregular portal space. The adjacent hepatocyte cords display marked unrest, disarray, and cytoplasmic ballooning.

Plate 27.43 Periungual erythema and onychodystrophy 100 days after allogeneic bone marrow transplantation in a patient with ocular sicca and oral lesions of chronic graft-versus-host disease.

Plate 27.44 Lichen planus-like chronic graft-versus-host disease before treatment, day 220 (see also Plates 27.42 and 27.45).

Plate 27.45 Same patient shown in Plates 27.42 and 27.44 after treatment, day 770.

Plate 27.46 Poikiloderma in late chronic graft-versus-host disease in a 2year-old girl, day 450. Thinning of the epidermis and dermis is present, along with telangiectasia and reticulated pigmentation.

Plate 27.47 Localized late phase of chronic graft-versus-host disease resembling morphea with atrophic plaque formation, induration, and peripheral hyperpigmentation, day 684.

Plate 27.48 Generalized scleroderma with atrophy, sclerodactyly, and joint contracture, day 540 after human leukocyte antigen-identical bone marrow transplantation.

Plate 27.50 Late chronic graft-versus-host disease (GVHD) of skin, day 959. As this patient’s GVHD progressed, the epidermis became atrophic, with straightening of the dermal-epidermal junction. All dermal appendages were destroyed. The reticular dermal collagen became increasingly sclerotic. A panniculitis seen at the base of the specimen resulted in fibrosis of the subcutaneous fat and large vessels. This histologic picture corresponds to the dense hidebound and sclerodermatous changes of late chronic GVHD.

Plate 27.49 Oral labial biopsy with early chronic graft-versus-host disease (GVHD), day 92. The minor salivary gland exhibits a periductal mononuclear inflammatory infiltrate involving the duct epithelium, in which there is focal epithelial apoptosis. There is inflammation of adjacent acini. This process leads to fibrosis with acinar atrophy in lobules drained by the affected ducts. The squamous surface epithelium (not present here) showed grade II lesions similar to those previously illustrated in skin (see Plate 27.18). The lip biopsy is valuable in the diagnosis and staging of chronic GVHD because it frequently mirrors destructive inflammatory changes in the lacrimal, tracheal, and esophageal glands, as well as bile ducts.

Plate 27.51 Atrophy and fibrosis of the thymus associated with chronic graft-versus-host disease (GVHD) in a 13-year-old, day 350. The patient had not received immunosuppressive drugs, so the destruction was presumably the result of chronic GVHD activity.

Plate 27.52 Poikiloderma, day 719. The patient was treated vigorously for extensive chronic graft-versus-host disease with corticosteroids plus several other immunosuppressive agents. Although he had extensive dyspigmentation, telangiectasia, and atrophy of the skin, there were no contractures or scleroderma. Histologically, the epidermis is atrophic, with loss of rete ridges. The fibrotic papillary dermis contains many telangiectatic vessels, and there is intracellular melanin pigment. The deep dermal sweat glands remain despite earlier inflammatory involvement, and the reticular dermal collagen is normal.

Plate 27.54 Polymyositis in deltoid muscle biopsy associated with chronic graft-versus-host disease, day 1129. There is extensive endomysial chronic inflammation associated with myocyte destruction. The condition responded to immunosuppressive therapy.

Plate 27.55 Coronary arteriopathy associated with chronic graft-versus-host disease in a 15-year-old who died of myocardial infarction preceded by severe anginal symptoms. The lumen and muscular wall of all the major coronary arteries were subtotally to completely obliterated by a cellular infiltrate of lymphoid and monocytoid cells admixed with extracellular matrix. Plate 27.53 Lichenoid early chronic graft-versus-host disease (GVHD) of the skin, day 233. Biopsy of a papulosquamous lesion from the posterior neck demonstrates the characteristic features of the lichen planus-like type of chronic cutaneous GVHD. The thickened epidermis displays hyper- and parakeratosis, hypergranulosis, and acanthosis. The extensive destruction along the dermal-epidermal junction results in sawtooth-like changes of the rete ridges, mimicking lichen planus. The papillary dermis has considerable perivascular inflammation. The lichenoid reaction also involves the hair follicles and eccrine glands seen deep in the reticular dermis. At this stage, treatment can prevent progression to fibrosis (see also Plate 27.50).

Plate 27.56 Two Toxoplasma tachyzoites are present near the ciliated surface of a bronchial epithelial cell. Diagnosis of Toxoplasma pneumonia was established in less than 2 hours after bronchoalveolar lavage. (Wright–Giemsa stain of cytospin preparation)

Plate 27.57 Respiratory syncytial virus pneumonia in a lung biopsy, day 48. The presence of dark purple cytoplasmic inclusions in multinucleated epithelial cells (arrows) suggests the diagnosis but may be found with other viruses such as parainfluenza. During severe community outbreaks, infections may spread to hospitalized bone marrow recipients.

Plate 27.58 Varicella-zoster virus (VZV) hepatitis in a percutaneous needle biopsy, day 79. On the right, a periodic acid–Schiff stain demonstrates a pale necrotic area (*) bordered by cells containing nuclear inclusions (large arrows) visible by hematoxylin and eosin staining on the lower left. Immunofluorescence confirmed the presence of VZV (upper left), and intravenous acyclovir was begun within hours of the biopsy.

Plate 27.60 Fusarium hyphae invade the dermis in pretransplant skin biopsy.

Plate 27.59 Overwhelming adenovirus hepatitis at autopsy 92 days after allogeneic hematopoietic cell transplantation for acute myeloid leukemia. Dark basophilic nuclear inclusions produce “smudge cells” (large, lower arrows) with blurred nuclear margins. Occasional cells with a halo around the inclusion (small, upper arrow) suggestive of cytomegalovirus (CMV) infection are sometimes seen when adenovirus infection is severe. In this case, immunohistology studies for CMV were negative.

Plate 27.61 Pulmonary sinusoidal obstructive syndrome in a lung biopsy from a patient with pulmonary hypertension 48 days post second allogeneic bone marrow transplantation for acute lymphoblastic leukemia. Loose intimal fibrosis partially occludes the interlobular vein. This complication probably results from chemotherapy and may be more frequent than previously suspected. (Verhoeff–Van Gieson elastin stain)

Plate 27.62 Bronchiolitis with evolving subepithelial fibrosis and lymphohistiocytic infiltration. There is marked epithelial damage. In this patient with chronic graft-versus-host disease (GVHD) and obstructive pulmonary function abnormalities, the alterations are consistent with pulmonary GVHD. Similar changes could be seen in respiratory virus infection, which was absent in this biopsy. The ongoing inflammation and subtotal constrictive obliteration of this airway suggest that immunosuppressive therapy may be beneficial.

Plate 27.63 In a patient with a history similar to the preceding case, the bronchiolitis obliterans of chronic pulmonary graft-versus-host disease has progressed. This bronchiole is completely occluded by granulation tissue, and scattered mononuclear inflammatory cells are present. Without successful intervention with immunosuppressive therapy, this airway will finally be replaced by probably permanent scar tissue. The arrows indicate residual smooth muscle. The elastin fibers are black with this Verhoeff–Van Gieson stain. (a)

(c)

(bi)

(bii)

(biii)

(biv)

(d)

Plate 39.1 Proliferative capacity of human cord blood hematopoietic progenitor and stem cells. (a) Colony formation by multipotential (CFU-GEMM) (lower-left) and granulocyte–macrophage (CFUGM) (upper-right) progenitor cells from cord blood cells thawed after 21 years in a frozen cryopreserved state. (b) Colony formation from a high proliferative potential (i and ii) and a smaller proliferative potential (iii and iv) progenitor cell arising from a single high-CD34-expressing single cell sorted into a single microtiter plate (i and iii: magnification × 20; ii and iv: magnification × 80). (Reproduced from [102] with permission.) (c) A secondary umbilical cord blood CFU-GEMM colony derived from replating of a single CFU-GEMM colony onto a secondary plate, a measure of self-renewal of CFUGEMM. See [104] for more information. (d) Immunohistochemical staining of CD34+ cord blood cells that engrafted NOD/SCID mouse bone marrow using human Ki67, a marker of cell proliferation. The conclusion is that cord blood progenitors and stem cells have extensive proliferative capacity. See [238] for more information.

1.0

Probability of survival

0.8

0.6

0.4 Early match, CP (n= 16) Late match, CP (n= 40) Late mismatch, CP (n= 23) Early mismatch, CP (n= 81) Late match, beyond CP (n= 13) Late mismatch, beyond CP (n= 33)

0.2

0.0 0

2

4 6 8 Years after transplant

10

12

Plate 47.1 Impact of disease phase and human leukocyte antigen matching for chronic myeloid leukemia. CP, chronic phase. (Reproduced from [2].)

Plate 62.1 Renal biopsy showing amyloidosis. These images show three views of a renal biopsy: with periodic-acid Schiff (PAS) staining showing amorphous material in glomeruli (top), Congo red staining showing apple-green birefringence in polarized light (middle) and electron microscopy showing nonbranched linear fibrils about 10 nm in length. (See also Chapter 62.)

Plate 70.1 (a, b) Hands of a patient with Sézary syndrome show fissuring, cracking, and nail destruction. (c, d) One year after allogeneic hematopoietic cell transplantation (HCT), there is regression of skin involvement and normalization of the nails. (Reproduced from [94], with permission.)

Plate 79.1 Progression of dysplasia to refractory anemia with excess blasts in a single patient with Fanconi’s anemia over a 9-month period. (a) Marrow aspirate on February 21, 1996 demonstrating pancytopenia and early dysplastic changes in neutrophils. (b) Marrow aspirate on July 3, 1996 demonstrating progressive dysplasia and 3% blasts. (c) Marrow aspirate on September 24, 1996 demonstrating progressive dysplasia and 12% blasts. (d) Marrow biopsy on September 24, 1996 demonstrating severe aplasia. (See Chapter 79.)

Plate 79.2 Above: G-banded karyotypes from an abnormal clone in the marrow of a patient with Fanconi’s anemia. This is a hyperdiploid metaphase cell with 48 chromosomes including two extra derivative chromosomes (designated by arrows); one derivative chromosome is composed of two copies of the long arm of chromosome 1, and the other contains two copies of a small portion of the long arm of chromosome 3. This cell contains a total of four copies of portions of chromosomes 1 and 3. Below: A karyotype of the same abnormal clone as above, as characterized by fluorescent in situ hybridization (FISH) using a multicolor paint probe (Vysis spectral vision M-FISH). MFISH confirmed that the two derivative chromosomes detected by G-banding represented extra copies of portions of chromosomes 1 and 3. (Courtesy of Betsy Hirsch, PhD, Director of the Cytogenetics Laboratory of the University of Minnesota Medical Center, Fairview, Minneapolis, MN.) (See Chapter 79.)

Plate 79.3 Model of the Fanconi’s anemia (FA) pathway. The model shows the sequential assembly of the FA core complex in the cytoplasm and nucleus. DNA replication or DNA damage activates the complex, either directly or indirectly, leading to the activation of FANCD2 by monoubiquination and its targeting to nuclear foci. The association of FANCD2 with BRCA1, FANCD1/BRCA2, FANCN/PALB2, and FANCI, leads to as yet incompletely defined downstream events, such as DNA repair or the activation of DNA synthesis (S) checkpoints. In this model, the core complex is the sensor for DNA damage and participates by activating FANCD2 through ubiquination. FANCD2 and its associates facilitate repair. (Reproduced from [65], with permission.) (See Chapter 79.)

Plate 79.4 Hepatic adenoma. Pathologic specimen obtained at autopsy from a patient with Fanconi’s anemia with multiple adenomata (arrow in upper panel pointing to an adenoma) not revealed by ultrasound prior to hematopoietic cell transplantation. The lower specimen shows area of massive intrahepatic hemorrhage (arrowed) in one previously unidentified adenoma.

Plate 79.5 Before and after blastomere biopsy of the six to eight-cell embryo to diagnose Fanconi’s anemia and assess human leukocyte antigen type. (Electron micrograph provided by Mark Hughes, M.D., Genesis Genetics Institute, Detroit, Michigan.)

Plate 87.1 Poikiloderma, a diagnostic manifestation of chronic graft-versushost disease, which includes both atrophic and pigmentary changes. Note the combination of reticulated pattern, epidermal atrophy (cigarette-paper wrinkling of the skin surface), erythema, and brown pigment forming the borders of skin-colored or depigmented skin.

Plate 87.2 Lichen planus-like eruption involving the palmar surfaces, a diagnostic manifestation of chronic graft-versus-host disease. Note the erythematous/violaceous flat-topped papules or plaques with or without surface reticulations or a silvery or shiny appearance on direct light.

Plate 87.4 Alternative manifestation of deep sclerosis common in areas of excess subcutaneous tissue such as the abdomen, upper arms, and buttocks. Tissue is not hidebound but demonstrates prominent rippling, dimpling or a cellulite-like appearance.

Plate 87.5 Morphea-like superficial sclerotic skin changes, a diagnostic feature of chronic graft-versus-host disease. Note the localized, patchy areas of movable smooth or shiny skin with a leathery-like consistency, often with dyspigmentation. In this picture, there is a fibrotic, hypopigmented area in the center of the plaque with a slightly hyperpigmented border.

Plate 87.3 Diffuse deep sclerotic features involving the lower extremities, a diagnostic feature of chronic graft-versus-host disease. Note the nonmovable/ hidebound subcutaneous tissues, erosions and ulcerations, loss of hair follicles, and toenail dystrophy.

(a)

(b)

Plate 87.6 Lichen sclerosis-like lesion, a diagnostic manifestation of chronic graft-versus-host disease. Note the discrete to coalescent gray to white movable papules or plaques, with a shiny appearance and leathery consistency (a) or cigarette paper-like textural changes (b).

Plate 87.7 Nail dystrophy involving multiple digits is a distinctive feature of chronic graft-versus-host disease (GVHD), although it is insufficient alone to establish the diagnosis of chronic GVHD. Note the longitudinal ridging, splitting and brittle nail features, as well as periungual erythema.

Plate 87.8 New onset of patchy alopecia after recovery from chemoradiotherapy is another distinctive manifestation of chronic graftversus-host disease (but is insufficient alone to establish the diagnosis). Patchy alopecia may involve loss of scalp or body hair.

Plate 87.9 Lichenoid lesions of oral chronic GVHD showing crythema, white striae, plaque, and ulcer formation, day 394. (See also Chapter 23, p. 294; Chapter 50, p. 647; Chapter 67, pp. 920, 922, 923.)

Plate 87.11 Numerous vesicle-like mucoceles (a distinct manifestation of chronic graft-versus-host disease [GVHD]) are seen along the center of the soft palate. Patchy white lichenoid hyperkeratosis (diagnostic of chronic GVHD) and interspersed moderate erythematous changes are also evident across the soft palate. Additionally, note the foamy mucus saliva consistent with GVHD salivary gland dysfunction.

Plate 87.10 Decreased oral range of motion and microstomia in a patient with sclerotic features of chronic graft-versus-host disease. This is a diagnostic manifestation. Note the erythema, edema, and atrophy of the vermillion lip and labial mucosa.

Plate 87.12 Keratoconjunctivitis sicca with blepharitis. The eyelid margins are thickened, edematous, and erythematous. Also note plugging of the meibomian gland orifices (along the eyelid margin) and significant conjunctival hyperemia/injection. A punctal plug can be seen at the lower left margin of the photo.

Plate 87.13 Chronic graft-versus-host disease (GVHD) involving the vulvovaginal tissues. Note lichen planus-like manifestations, which are diagnostic of chronic GVHD. Also seen are erythema and agglutination of the labial tissues.

Plate 87.14 Chronic GVHD of liver, day 166. This biopsy shows a prominent plasmacytic infiltrate in the portal area and a paucity of interlobular bile ducts. The histological dichotomy between acute and chronic GVHD is less clear in liver than in skin. However, increased portal inflammation and loss of bile ducts are more common in chronic GVHD. Despite the frequency of chronic hepatic GVHD, cirrhosis and liver failure are rare and may reflect superinfection with hepatitis C virus. (See also Chapter 23, p. 292.)

Plate 87.15 Bronchiolitis obliterans in a lung biopsy, day 396. The patient had chronic GVHD with severe obstructive pulmonary disease. Similar small airway inflammation with fibrosis is seen in lung allograft rejection. (See also Chapter 23, p. 297; Chapter 50, p. 647.)

(a)

Plate 87.16 Edema in the extremities can be bilateral or, as shown, unilateral. Edema may be an early sign of evolving subcutaneous sclerosis and fasciitis.

(b)

Plate 87.17 (a,b) Manifestations suggestive of fascial involvement by chronic graft-versus-host disease (a diagnostic feature) can include rippling or the groove sign.

Plate 92.1 Modeling varicella zoster virus (VZV) pathogenesis in human tissue xenografts in vivo. Human tissue xenografts are maintained in mice with severe combined immunodeficiency. VZV infection of cells within their tissue microenvironments can be evaluated in mice with skin, T-cell (thymus/liver) or dorsal root ganglia xenografts.

Chronic lymophocytic bronchiolitis

Obstructive bronchiolitis obliterans (OLD)

Acute pneumonitis (IPS)

OLD b

c

Chronic interstitial pneumonitis

Restrictive interstitial fibrosis (RLD)

RLD

a

d Phase one

e Phase two

Phase three

Plate 96.1. A triphasic model of both obstructive lung disease (OLD) and restrictive lung disease (RLD) following allogeneic stem cell transplantation (SCT). Immune-mediated injury to the lung after allogeneic SCT can be conceptualized in three phases. In phase one, acute lung injury develops as a consequence of an allogeneic immune response, and results in the sequential influx of lymphocytes, macrophages, and neutrophils into an inflamed pulmonary parenchyma (a). Persistence of an inflammatory signal in the setting of dysregulated repair mechanisms promotes the transition from acute to chronic injury in phase two (b and d). As chronic inflammation proceeds to phase three, lung fibroblasts increase dramatically in number and contribute to the enhanced deposition of collagen and granulation tissue in and around bronchial structures, ultimately resulting in complete obliteration of the small airways and fixed OLD (c). If, by contrast, the principal target of early damage is the alveolar epithelium, fibroblast proliferation and intraseptal collagen deposition ultimately results in interstitial fibrosis and RLD (e).

(a)

(b)

Plate 103.1 Severe oral mucositis occurring 8 days after hematopoietic cell transplantation following conditioning with VP-16, cyclophosphamide, and total body irradiation (World Health Organization mucositis score of 4). Bleeding ulcerations are noted on the anterior ventral area of the tongue (a) in this patient, with simultaneous pseudomembranous fibrin exudate-covered ulcers evident on the lower labial mucosa (b).

Plate 103.2 Oral chronic graft-versus-host disease in a patient 1 year post hematopoietic cell transplantation, with lichenoid hyperkeratotic changes (stria-, papule-, and plaque-like changes) associated with generalized atrophic and erythematous mucosal changes involving the right buccal mucosa. Small initial pseudomembranous fibrin exudate-covered ulcers are indicated by arrows.

Plate 103.3 Severe oral chronic graft-versus-host disease showing distinct hyperkeratotic lichenoid striae and a pseudomembranous fibrin exudatecovered ulceration with intense surrounding inflammation in a patient approximately 18 months post hematopoietic cell transplantation.

Plate 103.4 A 45-year-old male with a history of multiple myeloma who had been treated with intravenous zoledronate for 2 years prior to having two lower right molars extracted. He presented with three areas of bisphosphonate-associated osteonecrosis of the lower right mandible 6 months following the extractions. Lesions were nonpainful and stable. Arrows indicate areas of bone exposure.

Plate 103.5 Bisphosphonate-associated osteonecrosis of the jaw involving the lower right lingual ridge in a patient with multiple myeloma treated with intravenous zoledronate monthly for 20 months. The patient’s second bicuspid and first molar had been extracted 3 months earlier due to what was thought to be severe peridontal disease; the primary extraction site healed, but an extensive area of osteonecrosis developed along the mandibular lingual torus that was present lingual to the original extraction site.

Section 1 History and Use of Hematopoietic Cell Transplantation

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

1

E. Donnall Thomas

A History of Allogeneic Hematopoietic Cell Transplantation

In the broad sweep of history, it seems strange that our knowledge of the functions of the bone marrow is less than two centuries old, and the knowledge of transplantation of bone marrow occupies little more than half a century. History, as defined in this chapter, will be restricted to those findings which led to human bone marrow transplantation. It will not deal with solid organ transplantation, extensive studies in mice or advances in immunology and immunosuppressive drugs except as related to clinical marrow transplantation. Attempts to employ marrow for therapeutic purposes began in 1939 and 1940 [1,2]. However, infusions of a few milliliters of marrow were not attempts at transplantation, and no useful results were seen. During World War II, some very interesting experiments were carried out by Rekers and colleagues in the then classified laboratories of the Atomic Energy Commission at Rochester, New York. When these studies were published in 1950 [3], they were found to be carefully conducted trials of infusion of bone marrow from normal dogs into dogs exposed to 350 R. The investigators found no significant effect on pancytopenia or on survival. They were not aware that at least twice this exposure is necessary to provide sufficient immunosuppression to achieve engraftment. Thus, they failed in their attempts to achieve marrow recovery by intravenous marrow infusion.

The beginning The seminal studies, first published in 1949, were those of Jacobson and colleagues, who found that a mouse would survive otherwise lethal irradiation if the spleen were exteriorized and protected from the irradiation [4]. They found that an intraperitoneal injection of cells from the spleen (a hematopoietic organ in the mouse) would achieve the same effect. Lorenz et al. [5] extended these observations by demonstrating a similar protective effect by an infusion of bone marrow cells. At first, it was hypothesized that this “irradiation protection” effect was due to some kind of hormone or growth factor contained in the infusion. Most of these early studies involved measuring death at 30 days after lethal irradiation. Infusions of preparations from a donor of different H2 type were effective when survival was measured at 30 days. Such a result favored a humoral mechanism since it would not be expected if cells were involved. However, in 1954, Barnes and Loutit reported that mice protected with syngeneic marrow survived beyond

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

100 days. Mice given allogeneic marrow (A into CBA) survived at 30 days (“academic survivors”) but died thereafter of a “secondary disease” [6]. Another argument in favor of a hormonal effect was that “cell-free” extracts contained the protective effect. The investigators did not yet appreciate the very small number of cells required for transplantation in the syngeneic situation. Barnes and Loutit [7] sounded a warning against the hormonal hypothesis in which they noted that the “cellular hypothesis” had not been excluded as an explanation of the irradiationprotective effect. The demise of the humoral hypothesis became inevitable in 1955 when Main and Prehn published an article in the Journal of the National Cancer Institute [8]. They studied mice given lethal irradiation and marrow from an H2-incompatible (BALB/cAnN into DBA/2JN) strain. Subsequently, the surviving recipient mice given a skin graft from the donor strain accepted the skin graft for the rest of their life. Trentin was able to show that the tolerance of the graft was specific for the marrow donor strain [9]. Survival of the graft could only be explained by the persistence of cells of the donor strain leading to “tolerance.” Proof that these animals protected against lethal irradiation by marrow infusion were true radiation chimeras came from several sources. In 1956, Nowell et al. described the growth and continued function of rat marrow cells in X-irradiated mice [10]. Most convincingly, Ford et al. showed that the marrow of lethally irradiated mice given infusions of syngeneic marrow marked by the T6 chromosome was made up of cells of the cytogenetic type of the donor [11].

Early clinical studies At this point, it became evident that marrow transplantation might be of use not only in irradiation protection, but also in therapeutic application to marrow aplasia, leukemia, and other diseases of the marrow and lymphoid system. In 1956, Barnes et al. described the treatment of mice with leukemia by lethal irradiation and marrow transplantation [12]. In Cooperstown, NY, Thomas and Ferrebee and their colleagues had already begun comparable studies in terminal patients with hematological malignancies. In 1957, they reported six patients treated with irradiation and intravenous infusion of marrow from a normal individual [13]. Only one patient showed a transient marrow graft. These failures were to be duplicated by many investigators, so that in 1970 Bortin was able to compile a list of approximately 200 attempts at allogeneic marrow grafting, all of which had failed [14]. In 1959, Thomas et al. [15] reported an identical twin with terminal leukemia who was given 850 R (748 rad) total body irradiation from

3

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Chapter 1

Fig. 1.1 A symposium on bone marrow transplantation in Paris in 1971. From left to right: George Mathé, Dirk van Bekkum, George Santos, Don Thomas, Charles Congdon, Delta Uphoff.

opposing cobalt-60 sources and an intravenous infusion of marrow from the healthy twin (a syngeneic transplant). This dose of irradiation would be expected to produce prolonged pancytopenia and death [16]. However, the patient showed prompt recovery and disappearance of the leukemia for 4 months. This study demonstrated that lethal irradiation followed by compatible marrow could have an antileukemic effect even in advanced leukemia. Most importantly, it showed that compatible marrow infused intravenously could restore marrow function in human beings after lethal irradiation. Also in 1959, Mathé et al. reported the infusion of marrow into patients exposed to potentially lethal irradiation in a reactor accident [17]. Subsequent analysis raised doubt about whether there had been a therapeutic effect [18]. Nevertheless, this experience gave a great boost to interest in marrow transplantation and attracted many investigators to the field. One of the driving spirits of the early studies was Charles Congdon of the Oak Ridge National Laboratory, TN. In 1957, he organized an informal series of conferences at Oak Ridge. The early results of these conferences were published in abstract form in small leaflets called Fundamental and Clinical Aspects of Radiation Protection and Recovery. From 1964 to 1970, the abstracts appeared under the title Experimental Hematology. These small booklets may be difficult to find but are well worth the effort. They list the authors and investigations of that early time: there was life before Medline. In 1971, these meetings were organized into the International Society for Experimental Hematology, which publishes the modern journal Experimental Hematology (Fig. 1.1).

Advances from animal studies Thus, it was soon recognized that marrow grafting in man would be very difficult. Many investigators therefore turned to animal studies for definition and resolution of the problems. Much early information came from the work of Brent, Billingham, Medawar, and their colleagues [19]. They studied the induction of tolerance in newborn mice. In the course of these studies, they described the syndrome of “runt disease” in newborn mice given allogeneic cells. The principles they established were to prove the same as for “secondary disease” in irradiated mice and, eventually, for graft-versus-host disease (GVHD) in man. Although there had been great concern about the possibility of runt disease in the early studies of human marrow transplantation [13], it was apparent that this would be a problem only with a successful graft.

In 1959, Billingham and Brent published a landmark study of runt disease in newborn mice [20], and in 1966, Billingham described in detail the biology of graft-versus-host reactions [21]. These authors noted the following: • Syngeneic cells did not result in runt disease. • Persistence of allogeneic cells in the recipient was necessary for the development of runt disease. • The severity and incidence of runt disease were determined by antigenic differences between donor and host. • Tolerance could occur in the absence of runt disease. • The severity of runt disease was enhanced by cells already sensitized by exposure to host strain cells. The principles they elaborated were applicable to all studies of hematopoietic cell transplantation (HCT). The mouse has been used widely in studies of transplantation biology. Numerous studies in the murine system are cited in the book by van Bekkum and de Vries [22]. The availability of inbred strains has permitted extensive studies of genetic factors in transplantation. Uphoff [23] showed that the severity of the secondary disease after irradiation and marrow grafting was controlled by genetic factors. Uphoff [24] and Lochte and colleagues [25] showed that methotrexate (MTX) could prevent or ameliorate the secondary disease now known as GVHD. The dog has been frequently used in transplantation and surgical studies. Dogs are outbred animals comparable to humans. They come in large families, permitting a study of inherited factors. Studies in dogs first demonstrated the utility of autologous marrow grafts [26,27]. Marrow from one group of dogs was collected and set aside. The marrow donor or control animals were then given lethal or supralethal irradiation. The control animals invariably died whereas those treated with their own marrow promptly recovered. Obviously, this type of study could not be done with human beings. Successful cryopreservation of marrow had been accomplished by freezing in glycerol [28] using the technique of Polge and Smith. A careful step-wise removal of the glycerol before infusion of the cells was essential. Cavins et al. showed that canine marrow could be cryopreserved by freezing in dimethylsulfoxide (DMSO), thawed and injected intravenously without removal of the DMSO [29]. This technique was to become standard for marrow transplantation. Goodman and Hodgson had shown that mice could be protected against lethal irradiation by the infusion of blood cells [30]. Cavins et al. showed that autologous canine buffy coat cells from the peripheral blood could be

A History of Allogeneic Hematopoietic Cell Transplantation

collected, cryopreserved in DMSO, and infused after lethal irradiation with subsequent recovery of marrow function [31]. The practical application of peripheral blood cells for transplantation had to await better methods of centrifugation for separation of the buffy coat cells and techniques for mobilizing hematopoietic cells into the blood. The dog was most informative in extensive studies of allogeneic marrow grafting in an outbred species. Ferrebee et al. [32] reported the first allogeneic marrow graft in an irradiated dog. Four dogs were given 400 R on each of 3 days. One control and two dogs given marrow from unrelated dogs died of the acute irradiation syndrome. The male dog given marrow from a female littermate engrafted, and the leukocyte nuclei showed female drumsticks characteristic of the donor. Thomas et al. carried out a series of studies of irradiation and allogeneic marrow grafting in the dog [33,34]. Allogeneic graft recipients showed all of the problems that were soon to be recognized in human patients. The problems were graft failure, graft rejection, GVHD and/or death from opportunistic infections. A few animals did not develop these problems and became long-term survivors with marrow cells of donor type. Presumably these survivors fortuitously had donors of sufficient histocompatibility to permit long-term survival. In 1960, methods for selecting compatible donors were unknown. Early studies of marrow grafting after irradiation were carried out with non-human primates. Autologous marrow grafts were effective in protecting monkeys against lethal irradiation [35]. After allogeneic marrow grafting, they developed early severe secondary disease [35] that was somewhat reduced by treatment with antilymphocyte serum [36]. Rhesus monkeys given blood transfusions before irradiation rarely accepted an allogeneic graft [37]. In the dog, prior transfusions from histocompatible donors also prevented successful engraftment [38]. Unfortunately, marrow-grafting studies in non-human primates were not very useful for application to human patients. The non-human primates were expensive and difficult to work with. Therefore, the number of subjects studied was small. They were also not available in families. In addition, survival studies were difficult because of infection and parasitism in wild-caught animals [39]. In 1960, Medawar and Burnett received the Nobel Prize for their discovery “of acquired immunological tolerance.” When the prize was announced, my wife Dottie and I were attending a transplantation meeting in Switzerland. The joy and celebration knew no bounds. We were not only honoring the winners, but also acknowledging that transplantation biology had become a recognized field of scientific endeavor.

Advances in knowledge of histocompatibility Techniques for defining tissue antigens in humans were crucial to the development of HCT. In 1954, Miescher and Fauconnet recognized antibodies induced by transfusion or pregnancy that reacted with antigens on white blood cells [40]. In 1958, Dausset and van Rood and their colleagues recognized that human leukocyte antigens (HLAs) followed the genetic principles of inheritance [41,42]. There followed numerous studies designed to elucidate the role of these antigens in transplantation. HLA proved, however, to be of minimal value in the prediction of the outcome of skin grafts [43,44]. Epstein et al. [45] developed typing sera for dog leukocyte antigens (DLAs). Storb and colleagues studied dogs given lethal irradiation and a marrow graft from a littermate [46,47]. They showed that those marrow grafts between mismatched littermates always failed. Grafts between DLA-matched donors and recipients showed much improved survival. The administration of a short course of MTX for immunosuppression after grafting improved the long-term survival of matched recipients to 90% or better [48]. Thus, studies in dogs illustrated the importance of

5

DLA matching. At about the same time, Terasaki showed that the outcome of kidney grafts between siblings was highly dependent on HLA matching [49]. Taken together, these studies showed that matching for leukocyte antigens would be essential in human marrow grafting.

Renewed clinical studies After all the disappointments of clinical marrow transplants in the late 1950s and early 1960s, there was general pessimism about the field, and many of the early investigators had moved on to other studies. Nevertheless, improvements in transfusion medicine and the treatment of infections, and especially an improved understanding of the importance of HLA typing, encouraged a renewed attack on the clinical application of marrow grafts. The first good news came from studies of children with an immunological deficiency. Because of their disease, these children could not reject a foreign graft, and therefore immunosuppression before grafting should not be necessary. In November 1968, Gatti et al. performed the first successful allogeneic graft in a patient with severe combined immunodeficiency [50]. Two similar successes were reported immediately thereafter [51,52]. All three patients were alive and well 25 years later [53]. In late 1967, the Seattle marrow transplantation team received a grant to support clinical marrow transplantation. The year 1968 was spent in assembling a team of nurses, technicians, dietitians, etc. who were to be dedicated to patients undergoing intensive therapy and marrow transplantation with a special emphasis on post-transplant clinical care. The first transplant by this team was carried out in March 1969 [54]. The patient was a 46-year-old man with the blastic crisis of chronic myelogenous leukemia. An initial typing showed the patient’s sister to be an HLA match. After an irradiation exposure of a calculated midline dose of 954 rad, marrow was infused intravenously. Engraftment by donor cells was evident in 13 days. The patient developed mild GVHD controlled by MTX. Subsequently, he developed fever and died after 56 days. Autopsy showed cytomegalovirus pneumonia, but there was no evidence of leukemia or GVHD. This patient showed successful engraftment and control of GVHD but illustrated the problem of opportunistic infection. Thus began the Seattle marrow transplant team’s long series of patients given marrow grafts from HLA-matched siblings. In 1972, Thomas et al. reported the first experience with allografting for severe aplastic anemia [55]. The first four patients were referred for marrow grafting after a failure of conventional therapy with steroids and multiple transfusions. Since these patients did not involve the problem of eradication of a malignant disease, they were prepared with immunosuppression by four large doses of cyclophosphamide following the regimen of Santos et al. [56]. All four grafts were initially successful. One patient died of GVHD, and one died of graft rejection. Two patients were long-term survivors. In 1975, the Seattle team published a landmark Medical Progress review in the New England Journal of Medicine that was to become a highly cited article [57]. That article reviewed the rationale and experimental background for marrow transplantation. It emphasized the importance of histocompatibility and the possibility of using unrelated donors. It described the preparation of the patient, the technique of marrow transplantation, and the importance of supportive care. It also described the use of HLA-matched siblings for 37 patients with aplastic anemia and 73 with leukemia who had reached an advanced stage of their disease before transplantation. Death from recurrent disease, opportunistic infection, and GVHD were analysed. Most importantly, the article described a number of survivors. In 1977, the Seattle team reported 100 patients with end-stage acute leukemia treated by chemotherapy, total body irradiation and allogeneic

6

Chapter 1

marrow transplantation [58]. Thirteen of these patients became longterm survivors. Also, by 1977, the time of follow-up of transplanted patients was long enough to show a change in the shape of the survival curve indicating a plateau, which suggested that some patients were cured of their disease [59]. The survival of some patients transplanted in a terminal stage of their disease raised the possibility of transplantation earlier in the course of the disease. Transplantation before the development of drug resistance and before the complications of advanced disease might be expected to improve survival. In 1979, two reports described marrow grafts for patients with acute myeloid leukemia transplanted in first remission [60,61]. As expected, survival was greatly improved, and approximately 50% of the patients became long-term survivors. Mathé had coined the term “adoptive immunotherapy” to indicate the possibility that lymphoid elements in the engrafted marrow might react against malignant cells in the patient to aid in eradication of the remaining malignant cells [62]. Initial attempts to evaluate the possibility that GVHD might include graft-versus-leukemia effects were unrewarding [57]. However, when enough cases had been accumulated, Weiden et al. were able to demonstrate that the more severe the GVHD, the less likely was the recurrence of leukemia [63]. In the 1970s, a major concern was the limitation of allogeneic grafting to HLA-matched sibling pairs. Obviously, only about one-fourth of the patients would have a suitable marrow donor. Because of the complexity of the HLA system, finding a matched unrelated donor seemed a formidable task. In 1979, Hansen et al. performed the first successful marrow graft from an unrelated donor for a patient with leukemia [64].

A young patient with refractory acute leukemia did not have a matched sibling. Quite by chance, it was observed that one of the hematology technicians had an HLA type that matched the patient. The graft was successful without GVHD until the leukemia recurred 2 years later. This patient’s experience stimulated the formation of the National Marrow Donor Program.

History is now The use of donor peripheral blood leukocytes or cord blood cells for HCT has become a routine part of transplantation. The use of donor lymphocytes to cure recurrent leukemia and the use of non-myeloablative preparative regimens are but a few of the advances that are current subjects of research. Molecular biological techniques have greatly improved the accuracy of tissue typing. We are just beginning to appreciate the importance of the microenvironment, the cytokines, and the multitude of genes regulating cell division and senescence. Each advance with new agents, such as imatinib mesylate, forces us to redefine the role of HCT in therapy of disease. Thus, at the end of the 20th century, grafting of hematopoietic cells had become widely employed for research and as a form of therapy for patients with a variety of diseases. Many investigators have been attracted to the field, and many institutions have established HCT programs. Progress in the clinical application of HCT and in new avenues of research is the subject of the remaining chapters of this book and is described by the investigators who carried out much of the work.

References 1. Osgood EE, Riddle MC, Mathews TJ. Aplastic anaemia treated with daily transfusions and intravenous marrow. Ann Intern Med 1939; 13: 357– 67. 2. Morrison M, Samwick AA. Intramedullary (sternal) transfusion of human bone marrow. JAMA 1940; 115: 1708–11. 3. Rekers PE, Coulter MP, Warren S. Effect of transplantation of bone marrow into irradiated animals. Arch Surg 1950; 60: 635–67. 4. Jacobson LO, Marks EK, Robson MJ, Gaston EO, Zirkle RE. Effect of spleen protection on mortality following X-irradiation. J Lab Clin Med 1949; 34: 1538–43. 5. Lorenz E, Uphoff D, Reid TR, Shelton E. Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Natl Cancer Inst 1951; 12: 197–201. 6. Barnes DWH, Loutit JF. What is the recovery factor in spleen [Letter]? Nucleonics 1954; 12: 68–71. 7. Barnes DWH, Loutit JF. Spleen protection: The cellular hypothesis. In Bacq ZM, editor. Radiobiology symposium. London: Butterworth; 1955. 8. Main JM, Prehn RT. Successful skin homografts after the administration of high dosage X radiation and homologous bone marrow. J Natl Cancer Inst 1955; 15: 1023–9. 9. Trentin JJ. Mortality and skin transplantability in X-irradiated mice receiving isologous or heterologous bone marrow. Proc Soc Exp Biol Med 1956; 92: 688–93. 10. Nowell PC, Cole LJ, Habermeyer JG, Roan PL. Growth and continued function of rat marrow cells in X-radiated mice. Cancer Res 1956; 16: 258– 61.

11. Ford CE, Hamerton JL, Barnes DWH, Loutit JF. Cytological identification of radiation-chimaeras. Nature 1956; 177: 452–4. 12. Barnes DWH, Corp MJ, Loutit JF, Neal FE. Treatment of murine leukaemia with X-rays and homologous bone marrow. Preliminary communication. Br Med J 1956; 2: 626–7. 13. Thomas ED, Lochte HL, Jr., Lu WC, Ferrebee JW. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med 1957; 257: 491–6. 14. Bortin MM. A compendium of reported human bone marrow transplants. Transplantation 1970; 9: 571–87. 15. Thomas ED, Lochte HL, Jr., Cannon JH, Sahler OD, Ferrebee JW. Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 1959; 38: 1709–16. 16. Cronkite EP, Bond VP. Radiation injury in man. Oxford, UK: C.C. Thomas/Blackwell Scientific Publications; 1960. 17. Mathé G, Jammet H, Pendic B et al. Transfusions et greffes de moelle osseuse homologue chez des humains irradiés a haute dose accidentellement. Rev Franc Etudes Clin et Biol 1959; IV: 226–38. 18. Andrews GA. Criticality accidents in Vinca, Yugoslavia, and Oak Ridge Tennessee. Am J Roentgenol Radium Ther Nucl Med 1965; 93: 56–74. 19. Billingham RE, Brent L, Medawar PB. “Actively acquired tolerance” of foreign cells. Nature 1953; 172: 603–6. 20. Billingham RE, Brent L. Quantitative studies on tissue transplantation immunity. IV. Induction of tolerance in newborn mice and studies on the phenomenon of runt disease. Philos Trans R Soc Lond B Biol Sci 1959; 242: 477.

21. Billingham RE. The biology of graft-versus-host reactions. In: The Harvey lectures. New York: Academic Press; 1966. pp. 21–78. 22. van Bekkum DW, de Vries MJ. Radiation chimaeras. London: Logos Press; 1967. 23. Uphoff DE. Genetic factors influencing irradiation protection by bone marrow. I. The F1 hybrid effect. J Natl Cancer Inst 1957; 19: 123–5. 24. Uphoff DE. Alteration of homograft reaction by A-methopterin in lethally irradiated mice treated with homologous marrow. Proc Soc Exp Biol Med 1958; 99: 651–3. 25. Lochte HL, Jr., Levy AS, Guenther D, Thomas ED, Ferrebee JW. Prevention of delayed foreign marrow reaction in lethally irradiated mice by early administration of methotrexate. Nature 1962; 196: 1110–11. 26. Alpen EL, Baum SJ. Modification of X-radiation lethality by autologous marrow infusion in dogs. Blood 1958; 13: 1168–75. 27. Mannick JA, Lochte HL, Jr., Ashley CA, Thomas ED, Ferrebee JW. Autografts of bone marrow in dogs after lethal total-body radiation. Blood 1960; 15: 255–66. 28. Barnes DWH, Loutit JF. The radiation recovery factor: Preservation by the Polge–Smith– Parkes technique. J Natl Cancer Inst 1955; 15: 901–5. 29. Cavins JA, Kasakura S, Thomas ED, Ferrebee JW. Recovery of lethally irradiated dogs following infusion of autologous marrow stored at low temperature in dimethyl-sulphoxide. Blood 1962; 20: 730–4. 30. Goodman JW, Hodgson GS. Evidence for stem cells in the peripheral blood of mice. Blood 1962; 19: 702–14.

A History of Allogeneic Hematopoietic Cell Transplantation

31. Cavins JA, Scheer SC, Thomas ED, Ferrebee JW. The recovery of lethally irradiated dogs given infusions of autologous leukocytes preserved at −80°C. Blood 1964; 23: 38–43. 32. Ferrebee JW, Lochte HL, Jr., Jaretzki A, III, Sahler OD, Thomas ED. Successful marrow homograft in the dog after radiation. Surgery 1958; 43: 516– 20. 33. Thomas ED, Ashley CA, Lochte HL, Jr., Jaretzki A, III, Sahler OD, Ferrebee JW. Homografts of bone marrow in dogs after lethal total-body radiation. Blood 1959; 14: 720–36. 34. Thomas ED, Collins JA, Herman EC, Jr., Ferrebee JW. Marrow transplants in lethally irradiated dogs given methotrexate. Blood 1962; 19: 217–28. 35. Crouch BG, van Putten LM, van Bekkum DW, de Vries MJ. Treatment of total-body X-irradiated monkeys with autologous and homologous bone marrow. J Natl Cancer Inst 1961; 27: 53–65. 36. Merritt CB, Darrow CC, II, Vaal L, Rogentine GN, Jr. Bone marrow transplantation in rhesus monkeys following irradiation. Modification of acute graftversus-host disease with antilymphocyte serum. Transplantation 1972; 14: 9–20. 37. van Putten LM, van Bekkum DW, de Vries MJ, Balner H. The effect of preceding blood transfusions on the fate of homologous bone marrow grafts in lethally irradiated monkeys. Blood 1967; 30: 749–57. 38. Storb R, Epstein RB, Rudolph RH, Thomas ED. The effect of prior transfusion on marrow grafts between histocompatible canine siblings. J Immunol 1970; 105: 627–33. 39. van der Waay D, Zimmerman WMTh. Problems in the sanitation of monkeys for whole-body irradiation experiments. Proceedings of the International Symposium on Bone Marrow Therapy and Chemical Protection in Irradiated Primates 1962. 1962. pp. 231–40. 40. Miescher PP, Fauconnet M. Mise en évidence de différents groupes leucocytaires chez l’homme. Schweiz Med Wochenschr 1954; 84: 597–9. 41. Dausset J. Iso-leuco-anticorps. Acta Haematol 1958; 20: 156–66.

42. van Rood JJ, Eernisse JG, van Leeuwen A. Leukocyte antibodies in sera from pregnant women. Nature 1958; 181: 1735–6. 43. Rapaport FT, Lawrence HS, Thomas L et al. Crossreactions to skin homografts in man. J Clin Invest 1962; 41: 2166–72. 44. Dausset J, Rapaport FT, Legrand L et al. Studies on transplantation antigens (HL-A) by means of skin grafts from 90 children onto their fathers [French]. Nouv Rev Fr Hematol 1969; 9: 215–29. 45. Epstein RB, Storb R, Ragde H, Thomas ED. Cytotoxic typing antisera for marrow grafting in littermate dogs. Transplantation 1968; 6: 45–58. 46. Storb R, Epstein RB, Bryant J, Radge H, Thomas ED. Marrow grafts by combined marrow and leukocyte infusions in unrelated dogs selected by histocompatibility typing. Transplantation 1968; 6: 587–93. 47. Storb R, Rudolph RH, Thomas ED. Marrow grafts between canine siblings matched by serotyping and mixed leukocyte culture. J Clin Invest 1971; 50: 1272–5. 48. Storb R, Epstein RB, Graham TC, Thomas ED. Methotrexate regimens for control of graft-versushost disease in dogs with allogeneic marrow grafts. Transplantation 1970; 9: 240–6. 49. Singal DP, Mickey MR, Terasaki PI. Serotyping for homotransplantation: XXIII analysis of kidney transplants from parental versus sibling donors. Transplantation 1969; 7: 246–58. 50. Gatti RA, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological reconstitution of sexlinked lymphopenic immunological deficiency. Lancet 1968; 2: 1366–9. 51. Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bone-marrow transplantation in a patient with the Wiskott–Aldrich syndrome. Lancet 1968; 2: 1364–6. 52. deKoning J, van Bekkum DW, Dicke KA, Dooren LJ, Rádl J, Van Rood JJ. Transplantation of bonemarrow cells and fetal thymus in an infant with lymphopenic immunological deficiency. Lancet 1969; 1: 1223–7.

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53. Bortin MM, Bach FH, van Bekkum DW, Good RA, van Rood JJ. 25th anniversary of the first successful allogeneic bone marrow transplants. Bone Marrow Transplant 1994; 14: 211–12. 54. Buckner CD, Epstein RB, Rudolph RH, Clift RA, Storb R, Thomas ED. Allogeneic marrow engraftment following whole body irradiation in a patient with leukemia. Blood 1970; 35: 741–50. 55. Thomas ED, Storb R, Fefer A et al. Aplastic anaemia treated by marrow transplantation. Lancet 1972; 1: 284–9. 56. Santos GW, Sensenbrenner LL, Burke PJ et al. Marrow transplantation in man following cyclophosphamide. Transplant Proc 1971; 3: 400–4. 57. Thomas ED, Storb R, Clift RA et al. Bone-marrow transplantation. I and II. N Engl J Med 1975; 292: 832–43, 895–902. 58. Thomas ED, Buckner CD, Banaji M et al. One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 1977; 49: 511–33. 59. Thomas ED, Flournoy N, Buckner CD et al. Cure of leukemia by marrow transplantation. Leuk Res 1977; 1: 67–70. 60. Thomas ED, Buckner CD, Clift RA et al. Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 1979; 301: 597–9. 61. Blume KG, Beutler E. Allogeneic bone marrow transplantation for acute leukemia [Letter]. JAMA 1979; 241: 1686. 62. Mathé G, Amiel JL, Schwarzenberg L, Catton A, Schneider M. Adoptive immunotherapy of acute leukemia: Experimental and clinical results. Cancer Res 1965; 25: 1525–31. 63. Weiden PL, Flournoy N, Thomas ED et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979; 300: 1068–73. 64. Hansen JA, Clift RA, Thomas ED, Buckner CD, Storb R, Giblett ER. Transplantation of marrow from an unrelated donor to a patient with acute leukemia. N Engl J Med 1980; 303: 565–7.

2

James O. Armitage

The History of Autologous Hematopoietic Cell Transplantation

Introduction The procedure known variously as autologous bone marrow transplantation, autologous peripheral blood cell transplantation, autologous stem cell transplantation, and autologous hematopoietic cell transplantation involves the intravenous infusion of a patient’s own hematopoietic stem cells to rescue the patient from severe bone marrow injury. Usually, the bone marrow injury has been caused by high-dose chemotherapy and/or radiotherapy as part of a treatment for cancer. Usually, but not always, the patient’s hematopoietic stem cells have been cryopreserved and thawed before reinfusion. Autologous hematopoietic cell transplantation tests the hypothesis that the resistance of cancer cells to chemotherapy and/or radiotherapy can be relative rather than absolute, and that the resistance might be overcome by dose escalation. The importance of dose in cancer therapy has been known for some time [1]. While it has been clear that arbitrary dose reductions can reduce the chance of a good outcome with potentially curative cancer chemotherapy, autologous hematopoietic cell transplantation tests the hypothesis that dose escalation to a point that would cause irreversible myelotoxicity (but tolerable because of replacing myeloid function with the infused autologous hematopoietic stem cells) might be able to cure otherwise incurable patients. This hypothesis is illustrated in Fig. 2.1. Both chemotherapeutic agents and total body radiotherapy injure organs other than the bone marrow. Thus, for effective autologous hematopoietic cell transplantation, drugs whose initial life-threatening toxicity is myeloid injury need to be chosen, and radiation doses must be kept below that which would cause lethal pulmonary, gastrointestinal or central nervous system toxicity.

Early attempts at autologous hematopoietic cell transplantation The use of bone marrow to treat cancer has been considered by healthcare providers for a very long time. The first reports of the use of bone marrow as a treatment for cancer appeared in the 1890s [2]. The theoretical basis for the use of hematopoietic stem cells to rescue patients from irreversible myeloid injury was provided by canine studies [3,4] showing that dogs could survive otherwise lethal total body radiotherapy after the infusion of their own autologous hematopoietic cells. A report

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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of the first attempts at autologous hematopoietic cell transplantation in humans appeared in 1958 [5] and reported two patients, one suffering from teratocarcinoma and one from renal cell carcinoma, who received wide-field “intensive” radiotherapy followed by the infusion of glycerolpreserved hematopoietic cells derived from the bone marrow. A subsequent report by the same investigators [6] described four patients managed in a similar way who seemed to have hematopoietic recovery after infusion of the autologous bone marrow cells. McFarland et al. in 1959 described five patients with non-Hodgkin’s lymphoma or Hodgkin’s disease who received high doses of mechlorethamine followed by the infusion of noncryopreserved autologous bone marrow cells [7]. Three of the five patients had hematopoietic recovery, but there was only transient improvement in clinical status in a few. Clifford et al., working in Kenya in 1961, reported three patients with lymphoma or anaplastic nasopharyngeal carcinoma who received highdose mechlorethamine followed by an infusion of glycerol-preserved bone marrow cells [8]. All three patients demonstrated tumor regression and clinical improvement in addition to hematopoietic recovery. In 1962, Pegg et al. described 29 patients with cancer who received intensive, large-volume radiotherapy or chemotherapy with one of several alkylating agents followed by autologous bone marrow cell infusion. Most patients showed hematopoietic recovery, and a few had significant tumor regression and clinical improvement [9]. In 1962, Kurnick described 82 patients (presumably including some from his previous reports) who received high-dose chemotherapy and/or radiotherapy for a variety of malignancies including germ-cell tumors, lymphomas, leukemias, carcinomas, and sarcomas. The report expanded his previous observations and described hematopoietic recovery in most patients with, apparently, modest clinical benefit [10]. In many of these early reports, the intensity of chemotherapy would not necessarily have caused extremely prolonged marrow suppression, and it is difficult to be certain of the contribution of the reinfused hematopoietic cells. In the case of cryopreserved cells, it is not certain that viable cells were reinfused. The first patients apparently cured of otherwise incurable malignancy using high-dose therapy and autologous hematopoietic cell transplantation were reported by investigators from the National Cancer Institute in 1978. The authors reported 22 patients with resistant malignant lymphoma who were treated with high-dose chemotherapy utilizing carmustine, cytarabine, cyclophosphamide, and thioguanine [11]. Twelve patients received an infusion of their own cryopreserved autologous bone marrow, and 10 patients served as controls without reinfused cryopreserved marrow. Recovery of peripheral blood counts occurred with a median of 13 days in the patients who received autologous

The History of Autologous Hematopoietic Cell Transplantation

Increasing dose

3

Death secondary to other organ toxicity 2

1

Death secondary to marrow toxicity

Dose level necessary to cure three hypothetical patients

Fig. 2.1 Potential for cure with autologous hematopoietic stem-cell transplantation. The patient represented by column 1 could be cured with doses that would not cause permanent marrow injury. The patient represented by column 2 could be uniquely cured by doses requiring autotransplantation. The patient represented by column 3 would die of nonhematologic toxicity with the doses necessary to cure the cancer.

hematopoietic cells, and at a median of 23 days in those patients who did not receive marrow infusion. In a separate report, the same authors described 14 patients with Burkitt’s lymphoma resistant to conventional therapy who received the same high-dose chemotherapy regimen and autologous hematopoietic cells when these were available. Eight of the 14 patients received autologous hematopoietic cell transplantation; three of those were apparently cured, whereas none of the six patients who did not receive autologous hematopoietic cell transplantation had a long-term, disease-free survival [12]. The success in these patients ushered in the modern era of autologous hematopoietic cell transplantation and was made possible by the reproducible freezing and thawing of autologous hematopoietic cells using dimethylsulfoxide, resulting in viable and transplantable stem cells after thawing. This was initially reported by Cavins et al. [13] in dogs, and the work by Appelbaum et al. at the National Cancer Institute [12] demonstrated that the same approach worked in humans. Although almost all autologous hematopoietic cell transplantations are carried out with cryopreserved cells, some investigators have utilized noncryopreserved hematopoietic progenitor cells for this procedure. For this to be practical, the treatment has to be completed very rapidly, and the rescue product has been reinfused in most studies within 48 hours. Early studies in the United States and Italy showed the feasibility of this approach [14–16]. Carella subsequently reported 10 patients with Hodgkin’s disease and non-Hodgkin’s lymphoma who received highdose therapy with carmustine or vinblastine, cyclophosphamide, and carmustine. Seven of 10 patients with resistant, refractory disease achieved remissions, and hematopoietic recovery occurred in nine of the 10 patients [17]. A subsequent report by the same authors described 13 patients with Hodgkin’s disease and showed similar results, with all but one of the patients having hematopoietic recovery [18]. However, despite occasional other reports, this approach has been rarely used, with almost all patients receiving cryopreserved products to re-establish hematopoiesis.

Autologous hematopoietic cell transplantation utilizing peripheral blood-derived cells The existence of circulating hematopoietic stem cells capable of reestablishing hematopoiesis was established in mice in 1962 [19] and subsequently in other species [20,21]. Initial attempts to utilize circulat-

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ing autologous hematopoietic stem cells for autologous transplantation in humans failed [22,23], probably related to the infusion of an inadequate number of hematopoietic stem cells. Although an adequate number of hematopoietic stem cells for transplantation can be achieved utilizing multiple apheresis procedures [24], the practical application of this treatment required methods to increase the number of circulating hematopoietic stem cells. This increase in circulating hematopoietic stem cells can be achieved utilizing endotoxin [25] and recovery from chemotherapy [26,27]. However, it required the availability of hematopoietic growth factors, as well as the demonstration that their administration could dramatically increase the number of circulating hematopoietic progenitor cells and decrease the number of apheresis procedures required for an adequate dose [28] to make this approach the standard method for autologous transplantation. The hematopoietic progenitor cells collected after the administration of hematopoietic growth factors altered the kinetics of recovery of hematopoiesis. Both granulocyte colony-stimulating factor (G-CSF) [29] and granulocyte–macrophage colony-stimulating factor (GM-CSF) [30] were found to expand the number of circulating hematopoietic progenitor cells. Sheridan et al. [31] showed that hematopoietic stem cells collected after the administration of G-CSF not only shortened neutrophil recovery time, but also dramatically shortened platelet recovery. This does not appear to be the case using autologous peripheral blood stem cells collected after “priming” with GM-CSF [24,32]. Other hematopoietic growth factors, including erythropoietin [33], for mobilizing circulating progenitor cells have been used, but once again platelet recovery was delayed. However, whatever method of mobilization of circulating hematopoietic progenitor cells is utilized, extensive preceding therapy decreases the ease of collection of an adequate number of hematopoietic progenitor cells and delays recovery of hematopoiesis [34,35]. Peripheral blood-derived hematopoietic progenitor cells and bone marrow-derived hematopoietic progenitor cells have been compared in two randomized trials [36,37]. Both studies showed that autologous peripheral blood hematopoietic progenitor cells were associated with more rapid engraftment and had a similar treatment outcome. The relative ease of collecting peripheral blood-derived hematopoietic progenitor cells has made this the most frequent approach utilized for autologous hematopoietic cell transplantation in the world today.

In vitro treatment of autologous hematopoietic blood or marrow collections The risk of reinfusing cancer cells during autologous hematopoietic cell transplantation has been understood to be a risk for decades. The presence of tumor cells in rescue products has been shown using immunohistochemistry [38], bone marrow culture [39], and the polymerase chain reaction [40,41]. Some studies have found a poorer outcome in patients in whom residual disease in the graft could be identified [41,42], whereas others have questioned the significance of this finding [40]. The description of patients with early pulmonary recurrence after autologous bone marrow infusion in patients with lymphoma suggested that reinfusion of tumor cells may have been the cause [43,44]. Schultz et al., however, used a mathematical model to suggest that, in leukemia, reinfusion of tumor cells was unlikely to be the cause of relapse after autologous hematopoietic cell transplantation [45]. Brenner et al., using gene-marking techniques, showed that in some patients who relapsed after autologous hematopoietic cell transplantation for acute myeloid leukemia, the tumor cells at relapse were derived from the reinfusion product [46]. Attempts at removing tumor cells have used multiple approaches. Nadler et al. first reported the use of antibody treatment of autologous hematopoietic cells in an attempt to eliminate tumor cells [47]. De Fabritiis et al. showed that clonogenic Burkitt’s lymphoma cells could

Chapter 2

be eliminated from human bone marrow using a combination of monoclonal antibodies, compliment, and 4-hydroperoxycyclophosphamide [48]. Hagenbeek and Martens suggested that cryopreservation of the hematopoietic cells was more toxic to leukemic blasts than normal progenitor cells, and that freezing might reduce the risk of reinfusing of clonogenic tumor cells [49]. Attempts to reduce the risk of reinfusion of tumor cells has also been addressed by attempts to concentrate CD34positive hematopoietic stem cells and “leave behind” the tumor cells [50,51]. The use of antibodies directed against some tumor cells administered systemically at the time of peripheral blood cell collection might also reduce the number of tumor cells being collected.

100 Percent surviving disease-free

10

80

60 Sensitive relapse

40

Resistant relapse

20 No remission

0

Diseases treated by autologous hematopoietic cell transplantation Non-Hodgkin’s lymphoma The first malignancy successfully treated by high-dose therapy and autologous hematopoietic cell transplantation was non-Hodgkin’s lymphoma [11] in the late 1970s. In the early 1980s, as techniques of cryopreservation of hematopoietic cells improved, numerous singlecenter publications appeared demonstrating the curative potential of high-dose therapy and autologous hematopoietic cell transplantation in patients who had failed standard chemotherapy for aggressive nonHodgkin’s lymphoma [52–57]. Several of those centers pooled data and reported on the results on autologous hematopoietic cell transplantation in 100 patients with relapsed or refractory aggressive non-Hodgkin’s lymphoma [58]. Observations from that paper have affected the way in which autologous hematopoietic cell transplantation has been applied to patients with lymphoma and other diseases. Patients who had once achieved a complete remission, relapsed, and responded again to salvage chemotherapy before undergoing autologous hematopoietic cell transplantation had a higher complete remission rate following the autotransplant and were more likely to remain in remission than those who had relapsed from complete remission but were no longer chemotherapy sensitive. Both groups had a better result than patients who were primarily refractory to treatment and never achieved a complete remission (Fig. 2.2). These observations were tested in a phase II trial of 50 patients with aggressive non-Hodgkin’s lymphoma who relapsed from complete remission. All patients received dexamethasone, high-dose cytarabine, and cisplatin to test for chemotherapy sensitivity. The actuarial 2-year event-free survival for patients who were chemotherapy sensitive and underwent autotransplantation was 40% [59], confirming the retrospective study and forming the basis for a prospective trial comparing autotransplantation with standard-dose chemotherapy [60]. In that subsequent trial, 215 patients were treated and 109 responded to standard-dose salvage chemotherapy. They were then randomly assigned to continuing standard-dose chemotherapy followed by radiation or autologous hematopoietic cell transplantation. At 5 years, the event-free survival was 46% in the group undergoing transplantation and 12% in the standard therapy group – a difference that was highly significant. In addition, overall survival significantly favored the transplant (53% versus 32%), despite the fact that patients from the standard chemotherapy arm were able to be transplanted at a later time. This study established high-dose therapy and autologous hematopoietic cell transplantation as the standard treatment for patients with relapsed aggressive non-Hodgkin’s lymphoma. The history of the use of high-dose therapy and autologous hematopoietic cell transplantation in other types of non-Hodgkin’s lymphoma has not led to the same consensus as for the common aggressive nonHodgkin’s lymphomas. A number of single centers had reported encour-

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2

3

4

5

6 Years

7

8

9

10

11

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Fig. 2.2 Results of a retrospective study of 100 patients with diffuse, aggressive lymphoma showing that patients with chemotherapy-sensitive relapse do better with autologous transplantation than those who are chemotherapy resistant. Both groups have a better outcome than patients never achieving an initial complete remission [58].

aging results with the use of high-dose therapy and autologous hematopoietic cell transplantation in patients with relapsed follicular lymphoma [61–64]. One prospective trial has been completed and showed an advantage in disease-free and overall survival to patients undergoing hematopoietic stem cell transplantation [65]. However, the application of autologous hematopoietic cell transplantation in patients with follicular lymphoma has been much more controversial, and the development of a consensus has been much slower. Patients with Burkitt’s lymphoma were the first to be treated successfully with high-dose therapy and autologous hematopoietic cell transplantation [11]. The advent of high-dose chemotherapy regimens not requiring hematopoietic stem cell support has been so successful in curing patients with Burkitt’s lymphoma that this treatment is rarely utilized today. Several early reports of autologous hematopoietic cell transplantation in the treatment of lymphoblastic lymphoma [66,67] led to an international randomized trial [68]. However, the results did not reach statistical significance, and the use of autotransplantation in lymphoblastic lymphoma has remained controversial. Hodgkin’s disease High-dose therapy and autologous hematopoietic cell transplantation as a successful treatment for patients with relapsed Hodgkin’s disease was first reported in the 1980s [18,69–72]. By the early 1990s. Hodgkin’s disease had become one of the most frequent diagnoses for which autologous transplantation was applied. Subsequently, the British National Lymphoma Investigation carried out a randomized trial of high-dose therapy and autologous hematopoietic cell transplantation for patients with relapsed or refractory Hodgkin’s disease, the control arm being the same drugs used in the high-dose regimen but at lower doses [73]. Another randomized trial carried out by the German Hodgkin’s Lymphoma Study Group compared autologous hematopoietic stem cell transplantation with further conventional therapy in patients with chemotherapy-sensitive relapsed Hodgkin’s disease [74]. Both studies showed an improvement in failure-free survival but no difference in overall survival. Multiple myeloma Although currently one of the diseases most frequently treated using high-dose therapy and autologous transplantation, the fact that visible

The History of Autologous Hematopoietic Cell Transplantation

tumor cells were being reintroduced with the stem cell infusion probably reduced early enthusiasm for this treatment. Multiple myeloma has been treated with alkylating agents for several decades. McElwain and Powles showed in the early 1980s that high doses of melphalan could produce responses in patients refractory to the drug given orally at standard doses [75], but with prolonged and severe myelosuppression. In 1988, Barlogie et al. reproduced the high response rate with high-dose melphalan and showed that the intravenous infusion of autologous hematopoietic stem cells could reduce the duration of myelosuppression [76]. Subsequent studies tested the value of ex vivo treatment of the rescue product to attempt to eliminate tumor cells [77,78], and peripheral blood-derived hematopoietic cells [79,80]. The combination of blood- and marrow-derived hematopoietic cells [81] and the use of CD34-positive enriched hematopoietic stem cells [82] were both tested without any clear advantage being seen to either approach. These studies reflected the increasing interest in autotransplantation for multiple myeloma. However, the widespread acceptance of this treatment followed the publication of the first randomized trial demonstrating the superiority of high-dose therapy and autologous transplantation over standard chemotherapy [83].

Acute leukemia Perhaps surprisingly, autologous hematopoietic cell transplantation has been extensively pursued in the treatment of patients with acute leukemia. This was originally based on the lack of matched, allogeneic donors for most patients, the apparent responsiveness of the leukemia to very high doses of therapy, and the hope that small numbers of reinfused leukemia cells in patients in remission, or the infusion of no or very low numbers of cells after ex vivo treatment of the reinfusion product, might lead to an improved treatment outcome. This treatment was particularly popular in Europe. The first report of autologous hematopoietic stem cell transplantation in acute myeloid leukemia appeared in the late 1970s [84]. Treatment carried out for patients in relapse yielded remission in most, but was usually followed by rapid relapse. The use of ex vivo marrow treatment of the autograft to reduce leukemia cell reinfusion [85,86] and the treatment of patients in remission improved the duration of remission. Similar or somewhat less good results were seen with early attempts of autologous hematopoietic cell transplantation in acute lymphoblastic leukemia [87]. The early prospective trials of autologous hematopoietic cell transplantation in acute myeloid leukemia [88] and acute lymphoid leukemia [89] showed a higher relapse rate with autologous transplantation compared with allogeneic transplantation, but no clear advantage over intensive treatments not involving hematopoietic cell transplantation.

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remissions but most patients relapsing [94,95]. No prospective randomized trials have been completed.

Carcinomas and sarcomas Probably the carcinoma most frequently treated using high-dose therapy and autologous hematopoietic cell transplantation has been breast cancer. The rationale was a dose response in metastatic breast cancer [96] and the ability to collect autologous cells apparently free of tumor. Early attempts with high-dose therapy and autologous hematopoietic cell transplantation in breast cancer yielded primarily partial responses [97]. There was some evidence of a higher response rate with high-dose alkylating agents as opposed to other cytotoxic agents [98]. A number of high-dose combination chemotherapy regimens were developed in phase II trials, and several randomized trials were subsequently carried out [99]. In the United States, a high level of antipathy developed between physicians using high-dose therapy and autologous hematopoietic cell transplantation, and insurance companies that were reticent to fund this procedure. The borderline or negative results of most randomized trials and the revelation of fraud in one positive trial [100,101] dramatically reduced the frequency of utilization of this treatment approach. High-dose therapy and autologous hematopoietic cell transplantation has been studied in numerous carcinomas and sarcomas, generally with discouraging results. These studies include the treatment of patients with malignant glioma [102,103], malignant melanoma [104–106], neuroblastoma [107], ovarian cancer [108], lung cancer [109–112], and germcell tumors [113]. Of these diseases, only the treatment of germ-cell tumors has continued as a standard therapy [114].

Nonmalignant disorders Allogeneic bone marrow transplantation has been known for some time to have the potential to cure autoimmune disease [115]. It was much less clear that autologous transplantation could be beneficial since cells of the original immune system were being reinfused [116]. Two patients who underwent autologous bone marrow transplantation for lymphoma seemed to show an improvement in their markers for an autoimmune disorder (myasthenia gravis in one case and systemic lupus in another) [117,118]. These results led to attempts at autologous transplantation aimed at treating autoimmune diseases. The first studies focused on rheumatoid arthritis [119–121]. Studies in this and other autoimmune diseases continue.

Conclusion Chronic leukemia Autologous hematopoietic cell transplantation has been utilized to treat patients with chronic myeloid and with chronic lymphocytic leukemia. The rationale for autotransplantation in chronic myeloid leukemia was the existence of surviving normal hematopoietic stem cells [90] and the occasional durable remissions seen in patients who received toxic doses of busulfan [91]. Treatment of patients with chronic myeloid leukemia that had transformed to the acute phase using autologous transplantation of cells stored during the chronic phase of the disease yielded remissions but rapid relapse [92]. Treatment of patients in chronic phase occasionally produced durable remissions [93]. Autologous hematopoietic cell transplantation in chronic lymphocytic leukemia has also been tested. However, early results showed frequent

The history of autologous hematopoietic cell transplantation is a clear example of how clinical advances reflect advances in basic science and come as the result of innovative, but sometimes painstaking, clinical studies. Autologous hematopoietic cell transplantation was developed to test the hypothesis that dose escalation could affect cure in patients who had otherwise incurable malignancies. This turned out to be true for groups of patients with lymphoma, and the treatment continues to be widely utilized today. Studies in most patients with carcinoma and sarcoma showed minimal benefit and, with the exception of germ-cell tumors and certain pediatric malignancies, the treatment is rarely utilized. Although not curative, prolonged survival in patients with multiple myeloma has made the treatment widely utilized, and the results of autologous hematopoietic cell transplantation in nonmalignant disease have been sufficiently interesting to lead to ongoing studies.

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Chapter 2

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18. Carella AM, Santini G, Santoro A et al. Massive chemotherapy with non-frozen autologous bone marrow transplantation in 13 cases of refractory Hodgkin’s disease. Eur J Cancer Clin Oncol 1985; 21: 607–13. 19. Goodman JW, Hodgson GS. Evidence for stem cells in the peripheral blood of mice. Blood 1962; 19: 702–14. 20. Storb R, Graham TC, Epstein RB, Sale GE, Thomas ED. Demonstration of hemopoietic stem cells in the peripheral blood of baboons by cross circulation. Blood 1977; 50: 537–42. 21. Appelbaum FR. Hemopoietic reconstitution following autologous bone marrow and peripheral blood mononuclear cell infusions. Exp Hematol 1979; 7 Suppl 5: 7–11. 22. Hershko C, Gale RP, Ho WG, Cline MJ. Cure of aplastic anaemia in paroxysmal nocturnal haemoglobinuria by marrow transfusion from identical twin: Failure of peripheral-leucocyte transfusion to correct marrow aplasia. Lancet 1979; 1: 945–7. 23. Abrams RA, Glaubiger D, Appelbaum FR, Deisseroth AB. Result of attempted hematopoietic reconstitution using isologous, peripheral blood mononuclear cells: A case report. Blood 1980; 56: 516–20. 24. Kessinger A, Armitage JO, Landmark JD, Weisenburger DD. Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells. Exp Hematol 1986; 14: 192–6. 25. Cline MJ, Golde DW. Mobilization of hematopoietic stem cells (CFU-C) into the peripheral blood of man by endotoxin. Exp Hematol 1977; 5: 186– 90. 26. Richman CM, Weiner RS, Yankee RA. Increase in circulating stem cells following chemotherapy in man. Blood 1976; 47: 1031–9. 27. Abrams RA, McCormack K, Bowles C, Deisseroth AB. Cyclophosphamide treatment expands the circulating hematopoietic stem cell pool in dogs. J Clin Invest 1981; 67: 1392–9. 28. Gianni AM, Siena S, Bregni M et al. Granulocytemacrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet 1989; 2: 580–5. 29. Duhrsen U, Villeval JL, Boyd J, Kannourakis G, Morstyn G, Metcalf D. Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 1988; 72: 2074–81. 30. Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD. Granulocytemacrophage colony stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1988; 1: 1194–8. 31. Sheridan WP, Begley CG, Juttner CA et al. Effect of peripheral-blood progenitor cells mobilised by filgrastim (G- CSF) on platelet recovery after high-dose chemotherapy [see comments]. Lancet 1992; 339: 640–4. 32. Haas R, Ho AD, Bredthauer U et al. Successful autologous transplantation of blood stem cells mobilized with recombinant human granulocytemacrophage colony-stimulating factor. Exp Hematol 1990; 18: 94–8. 33. Kessinger A, Bishop MR, Jackson JD et al. Erythropoietin for mobilization of circulating progeni-

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The History of Autologous Hematopoietic Cell Transplantation 47. Nadler LM, Takvorian T, Botnick L et al. Anti-B1 monoclonal antibody and complement treatment in autologous bone-marrow transplantation for relapsed B-cell non-Hodgkin’s lymphoma. Lancet 1984; 2: 427–31. 48. De Fabritiis P, Bregni M, Lipton J et al. Elimination of clonogenic Burkitt’s lymphoma cells from human bone marrow using 4-hydroperoxycyclophosphamide in combination with monoclonal antibodies and complement. Blood 1985; 65: 1064–70. 49. Hagenbeek A, Martens AC. Cryopreservation of autologous marrow grafts in acute leukemia: Survival of in vivo clonogenic leukemic cells and normal hemopoietic stem cells. Leukemia 1989; 3: 535–7. 50. Gorin NC, Lopez M, Laporte JP et al. Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with nonHodgkin’s lymphoma. Blood 1995; 85: 1647–54. 51. Berenson RJ, Bensinger WI, Hill RS et al. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 1991; 77: 1717–22. 52. Philip T, Biron P, Herve P et al. Massive BACT chemotherapy with autologous bone marrow transplantation in 17 cases of non-Hodgkin’s malignant lymphoma with a very bad prognosis. Eur J Cancer Clin Oncol 1983; 19: 1371–9. 53. Gorin NC, Najman A, Douay L et al. Autologous bone marrow transplantation in the treatment of poor prognosis non-Hodgkin’s lymphomas. Eur J Cancer Clin Oncol 1984; 20: 217–25. 54. Appelbaum FR, Sullivan KM, Thomas ED et al. Experimental hematology today. New York: Springer-Verlag; 1985. 55. Verdonck LF, Dekker AW, van Kempen ML et al. Intensive cytotoxic therapy followed by autologous bone marrow transplantation for nonHodgkin’s lymphoma of high-grade malignancy. Blood 1985; 65: 984–9. 56. Armitage JO, Jagannath S, Spitzer G et al. High dose therapy and autologous marrow transplantation as salvage treatment for patients with diffuse large cell lymphoma. Eur J Cancer Clin Oncol 1986; 22: 871–7. 57. Armitage JO, Gingrich RD, Klassen LW et al. Trial of high-dose cytarabine, cyclophosphamide, total-body irradiation, and autologous marrow transplantation for refractory lymphoma. Cancer Treat Rep 1986; 70: 871–5. 58. Philip T, Armitage JO, Spitzer G et al. High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate-grade or high-grade nonHodgkin’s lymphoma. N Engl J Med 1987; 316: 1493–8. 59. Philip T, Chauvin F, Armitage J et al. Parma international protocol: Pilot study of DHAP followed by involved-field radiotherapy and BEAC with autologous bone marrow transplantation. Blood 1991; 77: 1587–92. 60. Philip T, Guglielmi C, Hagenbeek A et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkin’s lymphoma [see comments]. N Engl J Med 1995; 333: 1540–5. 61. Liang R, Chen F, Lee CK et al. Autologous bone marrow transplantation for primary nasal T/NK cell lymphoma. Bone Marrow Transplant 1997; 19: 91–3.

62. Colombat P, Binet C, Linassier C et al. High dose chemotherapy with autologous marrow transplantation in follicular lymphomas. Leuk Lymphoma 1992; 7 Suppl: 3–6. 63. Rohatiner AZ, Johnson PW, Price CG et al. Myeloablative therapy with autologous bone marrow transplantation as consolidation therapy for recurrent follicular lymphoma. J Clin Oncol 1994; 12: 1177–84. 64. Schouten HC, Bierman PJ, Vaughan WP et al. Autologous bone marrow transplantation in follicular non-Hodgkin’s lymphoma before and after histologic transformation. Blood 1989; 74: 2579– 84. 65. Schouten HC, Qian W, Kvaloy S et al. High-dose therapy improves progression-free survival and survival in relapsed follicular non-Hodgkin’s lymphoma: Results from the randomized European CUP trial. J Clin Oncol 2003; 21: 3918– 27. 66. Milpied N, Ifrah N, Kuentz M et al. Bone marrow transplantation for adult poor prognosis lymphoblastic lymphoma in first complete remission. Br J Haematol 1989; 73: 82–7. 67. Santini G, Coser P, Chisesi T et al. Autologous bone marrow transplantation for advanced stage adult lymphoblastic lymphoma in first complete remission. A pilot study of the non-Hodgkin’s Lymphoma Co-operative Study Group (NHLCSG). Bone Marrow Transplant 1989; 4: 399–404. 68. Sweetenham JW, Santini G, Qian W et al. High-dose therapy and autologous stem-cell transplantation versus conventional-dose consolidation/maintenance therapy as postremission therapy for adult patients with lymphoblastic lymphoma: Results of a randomized trial of the European Group for Blood and Marrow Transplantation and the United Kingdom Lymphoma Group. J Clin Oncol 2001; 19: 2927– 36. 69. Jagannath S, Dicke KA, Armitage JO et al. Highdose cyclophosphamide, carmustine, and etoposide and autologous bone marrow transplantation for relapsed Hodgkin’s disease. Ann Intern Med 1986; 104: 163–8. 70. Philip T, Dumont J, Teillet F et al. High dose chemotherapy and autologous bone marrow transplantation in refractory Hodgkin’s disease. Br J Cancer 1986; 53: 737–42. 71. Carella AM, Congiu AM, Gaozza E et al. High-dose chemotherapy with autologous bone marrow transplantation in 50 advanced resistant Hodgkin’s disease patients: An Italian study group report. J Clin Oncol 1988; 6: 1411–6. 72. Phillips GL, Wolff SN, Herzig RH et al. Treatment of progressive Hodgkin’s disease with intensive chemoradiotherapy and autologous bone marrow transplantation. Blood 1989; 73: 2086–92. 73. Linch DC, Winfield D, Goldstone AH et al. Dose intensification with autologous bone-marrow transplantation in relapsed and resistant Hodgkin’s disease: Results of a BNLI randomised trial. Lancet 1993; 341: 1051–4. 74. Schmitz N, Pfistner B, Sextro M et al. Aggressive conventional chemotherapy compared with highdose chemotherapy with autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Hodgkin’s disease: A randomised trial. Lancet 2002; 359: 2065–71.

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75. McElwain TJ, Powles RL. High-dose intravenous melphalan for plasma-cell leukaemia and myeloma. Lancet 1983; 2: 822–4. 76. Barlogie B, Alexanian R, Smallwood L et al. Prognostic factors with high-dose melphalan for refractory multiple myeloma. Blood 1988; 72: 2015–19. 77. Anderson KC, Andersen J, Soiffer R et al. Monoclonal antibody-purged bone marrow transplantation therapy for multiple myeloma. Blood 1993; 82: 2568–76. 78. Reece DE, Barnett MJ, Connors JM et al. Treatment of multiple myeloma with intensive chemotherapy followed by autologous BMT using marrow purged with 4-hydroperoxycyclophosphamide. Bone Marrow Transplant 1993; 11: 139–46. 79. Dimopoulos MA, Alexanian R, Przepiorka D et al. Thiotepa, busulfan, and cyclophosphamide: A new preparative regimen for autologous marrow or blood stem cell transplantation in high-risk multiple myeloma. Blood 1993; 82: 2324–8. 80. Fermand JP, Chevret S, Ravaud P et al. High-dose chemoradiotherapy and autologous blood stem cell transplantation in multiple myeloma: Results of a phase II trial involving 63 patients. Blood 1993; 82: 2005–9. 81. Jagannath S, Vesole DH, Glenn L, Crowley J, Barlogie B. Low-risk intensive therapy for multiple myeloma with combined autologous bone marrow and blood stem cell support. Blood 1992; 80: 1666–72. 82. Schiller G, Vescio R, Freytes C et al. Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma. Blood 1995; 86: 390–7. 83. Attal M, Harousseau JL, Stoppa AM et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 1996; 335: 91–7. 84. Gorin NC, Najman A, Duhamel G. Autologous bone-marrow transplantation in acute myelocytic leukaemia. Lancet 1977; 1: 1050. 85. Gorin NC, Douay L, Laporte JP et al. Autologous bone marrow transplantation using marrow incubated with Asta Z 7557 in adult acute leukemia. Blood 1986; 67: 1367–76. 86. Yeager AM, Kaizer H, Santos GW et al. Autologous bone marrow transplantation in patients with acute nonlymphocytic leukemia, using ex vivo marrow treatment with 4-hydroperoxycyclophosphamide. N Engl J Med 1986; 315: 141– 7. 87. Carella AM, Martinengo M, Santini G et al. Autologous bone marrow transplantation for acute leukemia in remission. The Genoa experience. Haematologica 1988; 73: 119–24. 88. Zittoun RA, Mandelli F, Willemze R et al. Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia. European Organization for Research and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA) Leukemia Cooperative Groups. N Engl J Med 1995; 332: 217–23. 89. Kersey JH, Weisdorf D, Nesbit ME et al. Comparison of autologous and allogeneic bone marrow transplantation for treatment of high-risk

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refractory acute lymphoblastic leukemia. N Engl J Med 1987; 317: 461–7. Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC. Long-term marrow culture reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosome-positive chronic myelogenous leukemia. N Engl J Med 1983; 308: 1493–8. Finney R, McDonald GA, Baikie AG, Douglas AS. Chronic granulocytic leukaemia with Ph 1 negative cells in bone marrow and a ten year remission after busulphan hypoplasia. Br J Haematol 1972; 23: 283–8. Buckner CD, Stewart P, Clift RA et al. Treatment of blastic transformation of chronic granulocytic leukemia by chemotherapy, total body irradiation and infusion of cryopreserved autologous marrow. Exp Hematol 1978; 6: 96–109. Brito-Babapulle F, Apperley JF, Rassool F, Guo AP, Dowding C, Goldman JM. Complete remission after autografting for chronic myeloid leukaemia. Leuk Res 1987; 11: 1115–17. Rabinowe SN, Soiffer RJ, Gribben JG et al. Autologous and allogeneic bone marrow transplantation for poor prognosis patients with B-cell chronic lymphocytic leukemia. Blood 1993; 82: 1366–76. Khouri IF, Keating MJ, Vriesendorp HM et al. Autologous and allogeneic bone marrow transplantation for chronic lymphocytic leukemia: Preliminary results. J Clin Oncol 1994; 12: 748–58. Hryniuk W, Bush H. The importance of dose intensity in chemotherapy of metastatic breast cancer. J Clin Oncol 1984; 2: 1281–8. Tannir N, Spitzer G, Schell F, Legha S, Zander A, Blumenschein G. Phase II study of high-dose amsacrine (AMSA) and autologous bone marrow transplantation in patients with refractory metastatic breast cancer. Cancer Treat Rep 1983; 67: 599–600. Antman KH. Dose-intensive therapy in breast cancer. In: Armitage JO, Antman KH, editors. High-dose cancer therapy – pharmacology, hematopoietins, stem cells. Baltimore, MD: Williams & Wilkins; 1992. pp. 701–718. Rodenhuis S, Richel DJ, van der Wall E et al. Randomised trial of high-dose chemotherapy and haemopoietic progenitor-cell support in operable breast cancer with extensive axillary lymph-node involvement. Lancet 1998; 352: 515–21. Weiss RB, Gill GG, Hudis CA. An on-site audit of the South African trial of high-dose chemo-

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3

Mary M. Horowitz

Uses and Growth of Hematopoietic Cell Transplantation

Introduction The first successful transplantations of allogeneic hematopoietic cells were performed in 1968 in three children with congenital immune deficiency diseases [1–4]. In each instance, hematopoietic cells were collected from the bone marrow of sibling donors who were genotypically identical or closely matched to the recipient for human leukocyte antigens (HLAs). Since then, more than 800,000 patients have received hematopoietic cell transplantations (HCTs) to treat life-threatening malignant and nonmalignant diseases. Current estimates of annual number of HCTs are 55,000–60,000, worldwide (Fig. 3.1). Reasons for widespread use include proven and potential efficacy in many diseases, better understanding of the appropriate timing of transplantation and patient selection, greater availability of donors, greater ease of hematopoietic progenitor cell collection, and improved transplantation strategies and supportive care, leading to less transplantation-related morbidity and mortality and an increased ability to perform the procedure in older and sicker patients.

Changing indications for HCT HCT has efficacy in many diseases (Table 3.1). In some, transplantation corrects congenital or acquired defects in blood cell production and/or immune function. In others, it restores hematopoiesis after high-dose (myeloablative) cytotoxic therapy for malignancy and/or provides potent anticancer adoptive immunotherapy. In the 1970s, more than half of the diseases for which HCT was performed were nonmalignant disorders: 40% were for aplastic anemia and 15% for immune deficiencies. Fewer than half were for cancers, and these were mostly for advanced acute leukemia. In the 1970s, Thomas and colleagues showed convincingly that some patients with refractory acute leukemia could achieve longterm leukemia-free survival with high-dose therapy and HLA-identical sibling marrow transplantation (see Chapter 1) [5,6]. Better outcome was subsequently demonstrated in patients transplanted in first or second remission [7–9]. Syngeneic (identical twin) and allogeneic HCTs were shown to produce cytogenetic remissions and long-term leukemia-free survival in chronic myeloid leukemia (CML) in the late 1970s and early 1980s [10–12].

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Use of allogeneic HCT to treat leukemia increased dramatically in the 1980s. By 1985, about 75% of allogeneic transplantations were for leukemia, with approximately equal numbers for CML, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL); more than 90% were from HLA-identical sibling donors. Introduction of alternative targeted therapies for CML in the early 2000s led to decreased use of HCT, with current guidelines reserving HCT for patients who fail to respond to nontransplant therapy or who lose their response [13]. However, leukemia treatment still accounts for about 65% of allogeneic HCT procedures (Fig. 3.2) (Center for International Blood and Marrow Transplant Research [CIBMTR] Statistical Center, unpublished data). Experimental and clinical evidence for a dose–response effect of drugs used in lymphoma therapy led to trials of autologous HCT to allow dose intensification in non-Hodgkin’s lymphoma in the middle 1980s (see Chapter 2) [14–16]. Results were promising, and there was rapid acceptance of autologous transplantation as salvage therapy in persons failing conventional chemotherapy for lymphoma. This was followed by increasing use of autologous transplantation as consolidation of primary therapy in patients with high-risk disease. There is now more frequent use of allogeneic transplantation in lymphoma to harness immunemediated graft-versus-lymphoma effects, especially in patients whose disease recurs after autologous transplantation, and in patients with follicular histology who are rarely cured by autologous transplantation [17]. Lymphoma accounted for 12% of allogeneic HCTs in 2006 (CIBMTR Statistical Center, unpublished data). The rationale of dose intensification led to the application of autologous transplants to many other hematologic and nonhematologic cancers over the past 10 years. One striking development was a dramatic increase in their use for breast cancer in the early 1990s. Breast cancer accounted for 16% of autologous transplants done in 1989–90 and 40% in 1994–95 [18]. However, results of randomized clinical trials in early and advanced breast cancer were disappointing (reviewed in [19–21]). In 1999, the use of HCT for breast cancer declined dramatically, and in 2006 breast cancer accounted for fewer than 5% of autologous transplants in North America. Treatment of solid tumors still accounts for about 10% of autologous transplants (Fig. 3.2). In 1996, results of a randomized trial comparing high-dose therapy and autologous HCT with conventional therapy for multiple myeloma were published; this study found a significant survival benefit with autologous transplantation [22]. Autologous HCT for myeloma increased dramatically after this report. Subsequent data suggested improved results with sequential autologous transplantations or autologous transplantation followed by allogeneic transplantation [23,24]. Multiple myeloma is now the single most common indication for HCT, account-

15

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Chapter 3

ing for 45% of autologous transplants but fewer than 5% of allogeneic transplants (Fig. 3.2). Thirty percent of HCTs for myeloma in 2006 involved a planned sequential autologous–autologous or autologous– allogeneic transplant approach (CIBMTR Statistical Center, unpublished data). The most common indications for allogeneic and autologous HCT in North America in 2006 are shown in Fig. 3.2. About 70% of allogeneic HCTs are for leukemia or preleukemia: 34% for AML, 16% for ALL, 6% CML, 10% for myelodysplastic or myeloproliferative syndromes, and less than 5% for other leukemias. About 15% are for other cancers, including non-Hodgkin’s lymphoma (11%), multiple myeloma (<5%), Hodgkin’s disease (<5%), and other cancers (<5%). The remainder are for aplastic anemia (5%), immune deficiencies (<5%), and other diverse nonmalignant disorders. The most common indications for autologous transplants are multiple myeloma (48%), non-Hodgkin’s lymphoma

(28%), Hodgkin’s disease (12%), leukemia (<5%), neuroblastoma (<5%), and other cancers (CIBMTR Statistical Center, unpublished data). There is increasing interest in using HCT in several diseases where transplantation was not or rarely used in the past, some with promising results in either anecdotal reports or phase II studies. These include sickle cell disease (see Chapter 74), inborn errors of metabolism (see Chapter 77), chronic lymphocytic leukemia (see Chapter 61), solid tumors such as ovarian, renal cell, and small cell lung cancer (see Chapters 64 and 65), and autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus, and severe rheumatoid arthritis (see Chapter 69). These diseases currently account for less than 5% of HCTs, whether allogeneic or autologous. However, their prevalence is high, and if subsequent trials confirm efficacy, the numbers of persons treated with HCT could increase dramatically. Several large trials of HCT for

5,500

35,000 Autologous

30,000 25,000 20,000 15,000

Allogeneic

10,000 5,000 0 1970

1975

1980

1985

1990

1995

2000

2005

Year

Fig. 3.1 Numbers of allogeneic and autologous hematopoietic cell transplantations performed yearly worldwide. (Reproduced with permission of the CIBMTR Statistical Center.)

Number of Transplants

Number of Transplants

40,000

5,000

Allogeneic (Total N=8,000)

4,500

Autologous (Total N=11,000)

4,000 3,500 3,000 2,500 2,000 1,500 1,000 500 0 Multiple Myeloma

NHL

AML

Hodgkin Disease

ALL

MDS/MPS

CML

Aplastic Anemia

Other Leukemia

Other Cancer

NonMalignnant Disease

Fig. 3.2 Indications for hematopoietic cell transplantation in North America, 2006. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; MDS/MPS, myeloproliferative disorder/ myelodysplastic syndrome; NHL, non-Hodgkin’s lymphoma. (Reproduced with permission of the CIBMTR Statistical Center.)

Table 3.1 Diseases in which autologous and/or allogeneic hematopoietic cell transplants may be used Malignant

Nonmalignant

Leukemia/preleukemia Chronic myeloid leukemia Myeloproliferative syndromes (other than chronic myeloid leukemia) Acute myeloid leukemia Acute lymphoblastic leukemia Juvenile chronic myeloid leukemia Myelodysplastic syndromes Therapy-related myelodysplasia/leukemia Kostmann’s agranulocytosis Chronic lymphocytic leukemia Non-Hodgkin’s and Hodgkin’s lymphoma Multiple myeloma Solid tumors Breast cancer Neuroblastoma Sarcomas Ovarian cancer Small cell lung cancer Testicular cancer

Severe aplastic anemia Paroxysmal nocturnal hemoglobinuria Hemoglobinopathies Thalassemia major Sickle cell disease Congenital disorders of hematopoiesis Fanconi anemia Diamond–Blackfan syndrome Familial erythrophagocytic histiocytosis Dyskeratosis congenita Shwachman–Diamond syndrome Severe combined immune deficiency and related disorders Wiskott–Aldrich syndrome Inborn errors of metabolism Acquired autoimmune diseases

Uses and Growth of Hematopoietic Cell Transplantation

autoimmune disease are being conducted in Europe and in the United States. Hematopoietic cells are also ideal candidates as vehicles for gene therapy, and their use in this capacity is being explored in several settings (see Chapter 10). Finally, hematopoietic stem cells are now being used for the regeneration of nonhematopoietic tissues, including myocardium and nerves, in some phase I and II studies [25]. Changes in patient selection In the 1970s, marrow transplants were often applied as desperate measures for desperate situations. Not infrequently, patients came to transplantation with longstanding, refractory cancers, with active infection, after receiving multiple transfusions, and with poor performance status. The available drugs for prophylaxis of graft-versus-host disease (GVHD) were not optimal, nor were supportive care measures, especially antiviral and antifungal therapy. Not surprisingly, these procedures were associated with high risks for graft failure, GVHD, infectious and noninfectious pneumonitis, nonpulmonary infections, and other complications, all leading to high transplant-related mortality. Much of the acceptance and growth of transplantation is attributable to a better understanding of factors leading to improved transplantation outcomes, especially selection of appropriate patients for transplantation at a point in their disease course when transplantation is most likely to be of benefit. Many studies in diverse diseases demonstrate that HCTs done earlier are associated with lower risks of both transplant-related mortality and disease recurrence. Transplantation is increasingly applied as first- or second-line rather than “last chance” therapy. In the 1970s, only 20% of HLA-identical sibling HCTs for acute leukemia were carried out in first remission, while 60% were done for advanced disease (not in remission or in a third or subsequent remission). In contrast, in 2006, about 60% of HCTs for acute leukemia were done for patients in first remission, 20% in second remission, and only 20% for advanced disease (CIBMTR Statistical Center, unpublished data). Although delay beyond first remission may be appropriate for patients with standard-risk ALL or good-risk AML (who may have good outcomes with conventional therapy), most young patients with AML are probably best served by allogeneic transplantation carried out soon after diagnosis if a suitably matched donor is available. Such a strategy is not only associated with better outcome, but also decreases the chance that patients will develop an intervening complication (refractory relapse, life-threatening infection or organ toxicity) that will preclude transplantation. There has also been a trend for earlier use of autologous transplants. In 1989, the median interval between diagnosis and autologous transplantation for non-Hodgkin’s lymphoma was 23 months; in 2006, it was 15 months (CIBMTR Statistical Center, unpublished data). Similarly, in 1990, the median interval between diagnosis and autologous transplantation for multiple myeloma was 18 months; in 2006, it was 8 months. This change is attributable largely to the demonstration of efficacy of autologous transplantation as part of primary treatment or as first salvage therapy (see Chapters 58–60) for myeloma and lymphoma, leading to use earlier in the course of these diseases. Earlier treatment accounts, in part, for the fact that patients in 2006 were less likely to have poor performance status at the time of transplantation than were patients in the 1970s. About 40% of patients transplanted in 1974–79 had pretransplant Karnofsky scores of less than 80%, compared with 10% in 2006 (CIBMTR Statistical Center, unpublished data). HCT is now used in much older patients than in the 1970s, when the median age of transplant recipients was 17 years and fewer than 5% were older than 40 years. In 2006, the median age of allograft recipients was 39 years, and of autograft recipients 56 years (CIBMTR Statistical

17

Center, unpublished data). This is important since the diseases for which HCT is most frequently applied have their onset in older adulthood, often in the sixth decade. The ability to apply HCT in older patients makes it a useful treatment for many more patients. Autologous transplants have been successfully used in patients older than 70 years. Eighty-four percent of autologous transplant recipients in 2006 were over the age of 40 years, and 37% were older than 60 years. Allogeneic HCT is also being used in older patients, with the introduction of better GVHD prophylaxis, less toxic conditioning, and improved supportive care. Forty-nine percent of allograft recipients in 2006 were older than 40 years, in contrast to only 28% in 1995. Thirty-one percent of the allogeneic HCTs carried out in 2006 were in patients older than 50, and 10% were in patients older than 60 years. Several studies suggest that, among adults over the age of 30, increasing age has only modest effects on transplantation outcome, at least up to the age of 50 [26–28]. One recent approach to increase use of allografts in older adults is reduced-intensity or nonmyeloablative pretransplant conditioning. This strategy uses conditioning as immunosuppressive therapy to allow donor cell engraftment rather than as high-dose anticancer therapy. The approach relies on immune-mediated graft-versus-tumor effects for long-term disease control (see Chapter 71). First reported in the late 1990s, transplants using reduced-intensity conditioning now account for about 40% of allografts registered with the CIBMTR and 60% of allografts in patients over the age of 55 years. Data on long-term outcome or efficacy relative to conventional HCT are still lacking.

Hematopoietic cell sources In the 1970s and early 1980s, essentially all HCTs used cells collected from the marrow of closely HLA-matched related donors. The few transplantations done with HLA-mismatched related donors were associated with high risks for graft failure, GVHD, and poor outcome, except, in some series, when the disparity was limited to a single HLA antigen [29,30]. This limited application of HCT to the 25–30% of patients with an HLA-matched relative. Several developments dramatically increased the applicability of HCT to patients without HLA-identical relatives, including the use of autologous cells, collection of cells from blood rather than marrow, and use of unrelated donors. Autologous transplantation Although a few autologous transplantations were carried out before the 1970s, the approach of collecting cells from a patient before high-dose therapy with reinfusion afterwards did not generate much enthusiasm until the middle to late 1980s (see Chapter 2) [31]. The appeal of the approach rested in its applicability, since it did not require a donor, allowing dose intensification with hematopoietic cell support in many patients with chemotherapy-sensitive cancers. Autologous transplants were also associated with lower transplant-related mortality since GVHD did not occur and immune recovery was more rapid than after allografting. Although immune-mediated antitumor effects were also absent, and there was some concern about reinfusing cancer cells with the graft, early trials showed good results in persons with lymphomas failing other therapies. The technology diffused rapidly in the late 1980s, becoming the treatment of choice for those with relapsed lymphoma and increasingly used for other chemotherapy-sensitive but infrequently cured cancers, including acute leukemia, multiple myeloma, and selected solid tumors. An important development allowing diffusion of the technology was the demonstration that hematopoietic cell grafts could be collected from peripheral blood in a limited number of leukaphereses after mobilization with chemotherapy or growth factors such as granulocyte- or

18

Chapter 3

granulocyte – macrophage colony-stimulating factor [32–34]. The resulting hematopoietic cell products contained large numbers of progenitor cells and led to faster hematopoietic recovery after transplantation. It also eliminated the need for a marrow harvest in an operating room. Whereas in 1989–90, 85% of autologous transplants used cells collected from marrow, by 2000 fewer than 5% used marrow cells alone (CIBMTR Statistical Center, unpublished data). In contrast to allogeneic transplantations, many of which are still done in academic, tertiary care medical centers, autologous transplants were soon used in the community setting, partly because of the ease with which the hematopoietic cell product could be obtained, and partly because of the more rapid hematopoietic and immune recovery and lower frequency of transplant-related complications compared with allografting. This likely increased the numbers of patients offered HCT.

Allogeneic transplantation There were anecdotal reports of successful HCTs using HLA-matched unrelated donors as early as 1973 [35–37], but the polymorphism of human HLA made the feasibility of finding such a donor for an individual patient low. In the middle 1980s, several national and international groups organized marrow donor registries with panels of HLA-typed volunteers agreeing to serve as donors for unrelated patients. There are now more than 11 million HLA-typed volunteer donors worldwide (see Chapter 38). Accessing these donors is facilitated by their listing in a compendium by Bone Marrow Donors Worldwide (www. bmdw.org), a collaborative effort of many national registries coordinated by the Europdonor group (www.europdonor.org), and by rapid computerized searching made available by several national and international registries. The largest donor registry is the US National Marrow Donor Program (NMDP; www.marrow.org), which has about 7 million donors on its file. The NMDP recently made a web-based search application available for patients to perform a preliminary search (MatchViewsm). Currently, Caucasian patients have an 80% or greater chance of finding a donor that is matched at intermediate resolution typing for HLA-A and -B, and high-resolution typing for HLA-DR, through existing donor panels. Because of greater HLA polymorphism and fewer ethnically similar donors, patients in other groups have lower probabilities of finding an HLA-matched unrelated donor. Additionally, there is a growing body of data demonstrating superior outcomes of transplants where donor and recipient are matched at the allele level for not only HLA-A, -B, and -DR, but also HLA-C and, in some studies, DQ and DP [38,39]. The likelihood of finding such a highly matched donor is considerably less than 80% even for Caucasians. The establishment of large donor panels dramatically increased the use of unrelated donor transplantation. While in 1985 fewer than 10% of allogeneic HCTs were from unrelated donors, in 2006 over 40% were from unrelated donors. In some settings, survival after unrelated donor transplantation is similar to that after HLA-identical sibling transplantation, although the risks of graft failure and GVHD are higher (see Chapter 47). Unrelated donor HCT is associated with longer delays between diagnosis and transplantation, partly because of the time needed for the donor search and evaluation process, and partly because of reluctance to use this more difficult transplantation approach early in disease. As recently as 2002, most allogeneic HCTs used cells collected from marrow. For many years, there was reluctance to use cells collected from peripheral blood because of the large numbers of mature T lymphocytes, the cells that mediate GVHD, in blood-derived grafts. However, in 1995, three centers reported rapid hematopoietic recovery and acceptable acute

GVHD after HLA-identical related peripheral blood allografts in a small number of patients [40–42]. A rapid increase in allogeneic peripheral blood HCTs followed. About 70% of related donor and 60% of unrelated donor HCTs now use cells obtained by leukapheresis. Several randomized trials have compared the results of peripheral blood and marrow HCTs in the related donor setting [43–46]. Some, but not all, suggest an early survival advantage with peripheral blood HCT in patients with advanced disease. However, most studies also indicate an increased risk of chronic GVHD, associated in some studies with higher late mortality (see Chapter 43). Data about the relative efficacy of marrow and peripheral blood transplants in the unrelated donor setting are limited. An observational study of unrelated donor transplant recipients conducted by the CIBMTR and the NMDP suggests that survival is similar, but chronic GVHD is more frequent with peripheral blood versus bone marrow grafts [47]. A large randomized trial is being conducted by the US Blood and Marrow Transplant Clinical Trial Network (BMT CTN) to address this issue. Recent experimental and clinical studies suggest that umbilical cord blood (UCB), which is rich in hematopoietic progenitors, may be a good source of allogeneic hematopoietic cells for persons without a suitable marrow donor (see Chapter 39). To be useful, there must be a large supply of cord blood units readily available. Several cord blood banks are now established, with more than 300,000 units cryopreserved worldwide. The advantage of UCB transplantation is rapid availability of a graft without need for screening, typing and collecting cells from live donors, and the potential to target underrepresented ethnic groups for collection and storage. Most clinical data indicate that UCB transplantation has less potential for severe GVHD, even with some degree of donor –recipient HLA mismatch, than adult donor transplants. UCB transplants now account for about 55% of the unrelated donor transplants carried out in children younger than 10 years old. Hematopoietic recovery after UCB transplantation may be slow, particularly in adults and larger adolescents, in whom the cell dose (cells/kg of recipient weight) may be limiting. Consequently, UCB accounts for only 20% of the unrelated donor transplants done in children 10–19 years of age and only 5% of those in adults 20 years of age and older. However, a growing number of studies in adults have demonstrated the potential success of UCB transplantation [48–50]. Several approaches are being pursued to increase the speed and certainty of engraftment. These include use of double UCB grafts, co-infusion of mesenchymal cells, co-infusion of T-cell-depleted haploidentical peripheral blood stem cells, injection of cord blood into the bone marrow directly, and cell expansion using a variety of techniques. Use of relatives partially matched for HLAs is another way of offering HCTs to more people (see Chapter 46). About 10% of allografts are from relatives sharing one HLA haplotype and mismatched for one or more antigens on the unshared haplotype. Results with more than one antigen disparity on the unshared haplotype have been disappointing, and there has been little increase in the number of these transplants over the past 10 years. Recent studies, however, suggest that the approach may be successful if T-cell-depleted grafts containing high doses of CD34 cells are administered and if donors are selected to maximize NK cell alloreactivity [51,52]. If alternative-donor HCTs, whether from unrelated volunteers, unrelated cord blood or HLA-mismatched relatives, were to be applied in all or most indications currently considered appropriate for HLA-identical sibling transplantation, there would be about 12,000–15,000 allogeneic transplants yearly in the United States. This is far more than the estimated 6000–8000 now being done. It remains to be seen whether increasing awareness of the availability of unrelated donor marrow transplantation or the use of UCB transplants will increase these numbers.

Uses and Growth of Hematopoietic Cell Transplantation

Transplantation regimens and supportive care Historically, with the exception of some transplantations for immune deficiencies, infusion of hematopoietic cells was preceded by intensive immunosuppressive and/or cytotoxic therapy. This treatment is aimed at eliminating malignant cells and, in the case of allografts, host immune cells that mediate rejection. In the 1970s and 80s, most HCTs for malignant disease (primarily leukemia) used a combination of high-dose cyclophosphamide and total body irradiation (TBI), with or without other drugs, for pretransplant conditioning. According to data reported to the CIBMTR, there has been a trend away from the high-dose radiation regimens used in the 1980s and early 1990s. About 30% of allogeneic transplants for leukemia are now done using reduced-intensity regimens. About 25% use high-dose busulfan, usually with cyclophosphamide with or without other drugs, and another 25% use high-dose TBI with cyclophosphamide or other high-dose chemotherapy. The remainder are carried out with a variety of high-dose chemotherapy regimens. High-dose therapy regimens for autologous transplants vary according to the underlying disease, but only a minority includes TBI. Another important change in allogeneic HCT strategy has occurred in the approach to post-transplant immune suppression. In the 1970s and early 1980s, methotrexate (MTX) was used. In the middle 1980s, the calcineurin inhibitor cyclosporine (CSP) was substituted for MTX in many centers. In the middle 1980s, a regimen combining MTX and CSP was introduced, which proved to be superior to either MTX or CSP alone for preventing GVHD [53,54]. Tacrolimus is now the more commonly used calcineurin inhibitor, and the most common post-transplant immune suppressive regimen in 2006 was tacrolimus plus MTX. The risk of grade III–IV acute GVHD after HLA-identical sibling HCT decreased from more than 20% to less than 15% between the 1970s and the 1990s, and is less frequently fatal, accounting in part for the substantial decrease in early post-transplant mortality over the past three decades (CIBMTR Statistical Center, unpublished data) (Fig. 3.3). Also contributing to this decrease was better prevention of cytomegalovirus (CMV) disease through use of CMV-negative or filtered blood products for CMV-negative patients and prophylaxis, or early treatment with ganciclovir in CMV-positive patients, which reduced the incidence of CMV pneumonia, a lethal complication of allogeneic transplantation, from about 10% to 2% (Fig. 3.3). Noninfectious pneumonitis (idiopathic pneumonia syndrome) also decreased, possibly from decreased use of MTX and unfractionated radiation. Use of peripheral blood hematopoietic cell and post-transplant growth factors has hastened hematopoietic recovery and decreased hospital stays, particularly after autologous grafts. Many of the latter are now carried out, at least partly, in the outpatient setting. The net effect of these and other changes has been a decrease in transplant-related mortality in the first year after HLA-identical sibling HCT, according to CIBMTR data, from about 50% in 1970–79 to about 15% in 2002–06 (Fig. 3.3). Transplant-related mortality after autologous transplants is even lower: about 10% after autologous transplants for leukemia, and 5–10% after autologous transplants for lymphoma and solid tumors (CIBMTR Statistical Center, unpublished data). Causes of death after allogeneic and autologous transplantation are shown in Fig. 3.4. Transplant-related toxicities predominate after allogeneic HCT; recurrent malignancy predominates after autologous HCT.

Long-term survivors The increasing use of HCT and better outcome of recipients means increasing numbers of long-term transplant survivors. There are now about 150,000 persons surviving 5 years or more after transplantation, and that number will grow rapidly. Most 5-year survivors are well, off

19

all immune suppression, and leading normal lives. Some data suggest that, at least in patients receiving transplants for AML and aplastic anemia, mortality rates return to that of an age- and sex-matched general population by 5–8 years post-transplant [55]. However, transplant recipients remain at risk for late complications long after HCT (see Chapters 104–106) [55,56]. These include late infections, cataracts, abnormalities of growth and development, thyroid disorders, chronic lung disease, and avascular necrosis. All of these are more frequent in patients with chronic GVHD. There is also an increased incidence of leukemias, myelodysplasias, and solid tumors in transplant recipients compared with the general population (see Chapter 106).

100 1970-79

90

1980-89

80

1990-99

70

2000-05

60 50 40 30 20 10 0 Gr III-IV AGVHD

CMV Pneumonia

100-day mortality

1-year TRM

1-year survival

Fig. 3.3 Incidence of grade III–IV acute graft-versus-host disease (AGVHD), cytomegalovirus (CMV) pneumonia, and 100-day overall mortality, 1-year transplant-related mortality (TRM), and 1-year survival after human leukocyte antigen-identical sibling hematopoietic cell transplantation (HCT) for leukemia among patients reported to the Center for International Blood and Marrow Transplant Research by year of HCT. (Reproduced with permission of the CIBMTR Statistical Center.)

HLAHLA-identical Sibling

Autologous Relapse (70%)

GVHD (13%) Other (16%)

Organ toxicity (6%) IPn (1%)

Relapse (41%)

Infection (8%)

Other (15%) Infection (17%) IPn (3%) Organ toxicity (10%)

Unrelated Donor Relapse (34%)

GVHD (14%)

Organ toxicity (10%) Other (16%) IPn (6%) Infection (20%)

Fig. 3.4 Causes of death after HLA-identical sibling, unrelated donor, and autologous hematopoietic stem cell transplantations done in 2004–05. GVHD, graft-versus-host disease; IPn, interstitial pneumonitis.

20

Chapter 3

Second cancers and other complications may not all be due to HCT per se but to the chemotherapy and/or radiation preceding the HCT. Regardless, lifelong surveillance is necessary, as is increased awareness of late complications among the many nontransplant physicians who will care for these patients. The CIBMTR and the European Group for Blood and Marrow Transplantation (EBMT) recently published guidelines for following transplant survivors [57].

Assessing and improving results of HCT The field of transplantation has been well served by international collaboration to assess outcomes through international outcomes registries such as the CIBMTR (www.cibmtr.org) and the EBMTR (www.ebmt. org). Hundreds of transplant centers have contributed clinical data on hundreds of thousands of patients, allowing observational studies to be

carried out addressing many important issues in HCT (see Chapter 29). The past 10 years have seen an increasing number of multicenter prospective clinical trials related to HCT and, importantly, the establishment of networks dedicated to addressing issues in HCT. The EBMT has also established a clinical trials office to facilitate multicenter, multinational trials. In the United States, the BMT CTN (www.bmtctn.net), a large multicenter network sponsored by the US National Heart Lung and Blood Institute and the National Cancer Institute, has accrued more than 2200 patients to trials since 2003. A recent State of the Science symposium, organized by the BMT CTN in June 2006 and including many international participants, has identified the most critical trials to be undertaken over the next 5 years [58]. These initiatives have significantly increased the infrastructure available to translate our rapidly advancing knowledge in of cell biology, immunology, and genetics into treatment strategies that will improve outcomes for our patients.

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Française de Greffe de Moelle. J Clin Oncol 2000; 18: 537–71. Bensinger WI, Martin PJ, Storer B et al. Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 2001; 344: 175–81. Schmitz N, Beksac M, Hasenclever D et al. Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard-risk leukemia. Blood 2002; 100: 761–7. Couban S, Simpson DR, Barnett MJ et al. A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 2002; 100: 1525–31. Eapen M, Logan B, Confer D et al. Peripheral blood grafts from unrelated donors are associated with increased acute and chronic graft-versus-host disease without improved survival. Biol Blood Marrow Transplant 2007; 13: 1461–8. Laughlin MJ, Eapen M, Rubinstein P et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 2004; 351: 2265–75. Rocha V, Labopin M, Sanz G et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004; 351: 2276–85. Takahashi S, Iseki T, Ooi J et al. Single-institute comparative analysis of unrelated bone marrow transplantation and cord blood transplantation for adult patients with hematologic malignancies. Blood 2004; 104: 3813–20. Aversa F, Tabilio A, Velardi A et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med 1998; 339: 1186– 93.

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52. Ruggeri L, Capanni M, Urbani E et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: 2097–100. 53. Storb R, Deeg HJ, Whitehead J et al. Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft-versus-host disease after marrow transplantation for leukemia. N Engl J Med 1986; 314: 729–35. 54. Storb R, Deeg HJ, Pepe M et al. Methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versus-host disease in patients given HLA-identical marrow grafts for leukemia: longterm follow-up of a controlled trial. Blood 1989; 73: 1729–34. 55. Socié G, Veum-Stone J, Wingard JR et al. Longterm survival and late deaths after allogeneic bone marrow transplantation. New Engl J Med 1999; 341: 14–21. 56. Duell T, van Lint MT, Ljungman P et al. Health and functional status of long-term survivors of bone marrow transplantation. EBMT Working Party on Late Effects and EULEP Study Group on Late Effects, European Group for Blood and Marrow Transplantation. Ann Intern Med 1997; 126: 184–92. 57. Rizzo JD, Wingard JR, Tichelli A, Lee SJ, Van Lint MT, Burns LJ, Davies SM, Ferrara JLM, Socié G. Recommended screening and preventive practices for long-term survivors after hematopoietic cell transplantation: joint recommendations of the European Bone Marrow Transplant Group, Center for International Blood and Marrow Transplant Research, and the American Society of Blood and Marrow Transplantation. Biol Blood Marrow Transplant 2006; 12: 138–51. 58. Ferrara JLM, Anasetti C, Stadtmauer E et al. Blood and Marrow Transplant Clinical Trials Network State of the Science Symposium 2007. Biol Blood Marrow Transplant 2007; 13: 1268–85.

Section 2 Scientific Basis for Hematopoietic Cell Transplantation

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Section 2a Hematopoiesis and Stem Cell Biology

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

4

Laurence Dahéron & David T. Scadden

Generation of Definitive Engraftable Hematopoietic Stem Cells from Human Embryonic Stem Cells

Introduction In 1998, Thomson et al. described the first derivation of human embryonic stem cells (hESCs) from the inner cell mass of human blastocysts [1]. hESCs are characterized by their unique self-renewal and pluripotent capacity: they can proliferate almost indefinitely in vitro and have the capacity to differentiate into every cell type of the body. This landmark finding generated great expectations: the possibility of limitless supplies of engraftable tissues of all types, including hematopoietic tissues. Moreover, these cells represent a powerful tool to study embryonic development and lineage specification. They can also provide an unlimited resource of specific cell types for drug screening and toxicity testing. Stem cells are divided into three categories based on their differentiation potential: totipotent, pluripotent, and multipotent (Table 4.1). Fertilized eggs and each cell of the embryo up to the four-cell stage are totipotent. In a suitable environment, they can form an entire organism including all embryonic, extra-embryonic and adult cell types. In contrast, pluripotent cells are not able to independently form a placenta, or to organize the formation of a proper primitive streak. ESCs are pluripotent cells. They can self-renew and differentiate into any cell type of the body. Somatic stem cells that are found in the tissues of adult body or in the cord blood are multipotent. They have a restricted differentiation potential. Pluripotent cells are transiently present during embryonic development, from the late morula-stage embryo to the primitive ectoderm of the preimplantation blastocyst. True pluripotent cells have not been found in adult organisms. Even though ESCs are considered the counterpart of the pluripotent cells from the embryo, it has been argued that ESCs were just a culture “artifact.” Of note, some differences between these two types of cell have been shown, including gene expression, proliferation rate, cytokine dependence, fidelity of imprinting, and epigenetic changes [2]. Several groups have demonstrated the capacity of hESCs to differentiate into blood cells [3–7]. Thus, hESCs could offer a new source of hematopoietic cells for transplantation. Since many patients remain on the waiting list for HCT, it is essential to evaluate the potential of sources of hematopoietic stem cells (HSCs) other than bone marrow and cord blood. Due to the relatively low number of HSCs in cord blood, this

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

source is typically only used in the treatment of children. Given that hESCs can proliferate widely, they could potentially provide a large number of HSCs. However, the process of HSC derivation from hESCs is inefficient, many other cell types are generally produced, and the risk of teratoma from residual undifferentiated hESCs cannot be dismissed. Therefore, a long list of issues remains: (1) how can we efficiently derive HSCs from hESCs?; (2) how can we isolate HSCs from the heterogenous population of differentiated hESCs?; (3) are these cells capable of hematopoietic repopulation in vivo?; and (4) can we generate immunologically compatible HSCs? In this chapter, we present the progress that has been made and the obstacles along the path to the practical use of hESC-derived HSCs in clinical transplantation.

Generation of HSCs from ESCs What have we learned from murine ESCs? Mouse ESCs (mESCs) possess a robust capacity for hematopoietic specification in vitro. Factors involved in the different steps of differentiation from ESC to HSC have been described (Fig. 4.1). Two main procedures have been used to differentiate ESCs into blood: embryoid body formation and co-culture with stromal cell lines. Embryoid body formation is routinely used to induce the differentiation of ESCs. Embryoid bodies can be generated by culturing ESCs in hanging drops for several days, by culturing the cells in 1% methylcellulose or by plating ESCs in low-attachment dishes, allowing the cells to form aggregates. In the absence of factors that maintain pluripotency, these cells will spontaneously differentiate into all three germ layers. Observing the expression of several specific markers, Gordon Keller first showed that the sequence of events occurring in vitro, from primitive ectoderm to mesoderm to hematopoietic cells, parallels the different steps of the onset of hematopoiesis in vivo [8]. The emergence of hematopoietic progenitors can be detected by the colony-forming unit (CFU) assay. During mESC differentiation, CFU numbers increased from day 3 to day 6, reaching 1% of the total population of embryoid body cells at day 6. At this stage, the majority of precursors were primitive erythroid – small nucleated cells expressing embryonic globin. At day 10, the population of primitive erythroid cells decreased while macrophages (CFU-M), adult erythroid (CFU-E), neutrophil/macrophage (CFU-GM), and mixed precursors (CFU-GEEM) were found in larger numbers. Mast cells and lymphoid lineages were detected at a later stage (day 12–14), but to a lesser extent, suggesting that this in vitro differentiation system is not optimal for the generation of lymphoid lineages.

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Table 4.1 Sources of stem cells and their differentiation potential Potential

Sources

Examples

Totipotent cells

All embryonic, extraembryonic, and adult cells

Early embryo before the four-cell stage

Zygote Each cell from the two- or four-cell stage embryo

Pluripotent cells

All embryonic and adult cells Some extraembryonic

Early embryo: morula and blastocyst stage Epiblast

Embryonic stem cells Induced pluripotent stem cells

Multipotent cells

Restricted to specific lineages

Umbilical cord Fetus Adult body

Hematopoietic stem cells Mesenchymal stem cells Neuronal stem cells Skin stem cells

ESCs

Endoderm Ectoderm

+Activin A +BMP-4

Cardiac cells, lymphatic tissue

Mesoderm

Bone, kidney, muscle, cartilage

+VEGF +TPO +SCF

Hemangioblast +SCF +FGF2 +Flt3L

Self-renewal

+VEGF

HSC

Vascular progenitor cells

Differentiation CMP

CLP

Fig. 4.1 Cascade of differentiation from embryonic stem cells (ESCs) to blood cells. CLP, common lymphoid progenitor; CMP, common myeloid progenitor. For other abbreviations, see text.

Hematopoietic development occurred even in the absence of supplemental hematopoietic growth factors. In order to provide a supportive microenvironment, however, ESC differentiation was often performed directly on stromal cells such as OP9, a stromal cell line derived from newborn op/op mouse calvaria (that does not produce macrophage colony-stimulating factor [M-CSF]), S17 and MS-5 mouse bone marrow stromal cell lines, or AM20–1B4, a stromal cell line derived from the aorta–gonad–mesonephros (AGM) region. Both protocols, embryoid body formation and culture on stromal cell lines, permitted the derivation of primitive and definitive hematopoietic cells with a large predominance of primitive blood cells. Derivation of HSCs from hESCs Several laboratories have derived hematopoietic precursors from hESCs using the two approaches mentioned above for mESCs: embryoid body formation (Fig. 4.2) and co-culture with stromal cells. The first evidence for blood cell derivation from hESCs was described by Kaufman et al. [3]. They co-cultured undifferentiated hESCs on irradiated S17 or C166 (a yolk sac endothelial cell line) for 17 days and showed the emergence of a small population (1–2%) of CD34+CD38− cells, consistent with the phenotype of early hematopoietic precursors. This co-culture gave rise to CFUs with a peak at day 17–18 and colonies from multiple hematopoietic lineages. Other groups have co-cultured hESCs on OP9, showing a rapid and great efficiency for the derivation of CD34+ cells [5,6]. Subsequent co-culture of isolated CD34+ cells on

the MS-5 stromal cell line induced the production of macrophages, mature granulocytes, natural killer (NK) cells, and B lymphocytes. Despite the relative effectiveness of this co-culture system, reproducibility might be a problem since OP9 cells are very sensitive to culture conditions (including density, serum lot, and passage number). Stromalfree approaches, typically using an embryoid body system, have also been exploited to test the potential of hESCs to differentiate into hematopoietic cells. The influence of bone marrow protein-4 (BMP-4) and cytokines on the hematopoietic differentiation within embryoid bodies has been assessed and the successful generation of CD45+ cells shown when combinations of cytokines (stem cell factor [SCF], FMS-like tyrosine kinase-3 ligand [Flt3L], interleukin-3, interleukin-6, granulocyte CSF and BMP-4) were used [7]. Later, vascular endothelial growth factor-A165 (VEGF-A165) was shown to enhance selective induction of erythropoietic development [9]. These results indicate that hematopoietic commitment can be induced by exogenous factors, an important step to improve the derivation of blood cells from hESCs. In an effort to eliminate high variability in the differentiation systems, efforts have been made to eliminate co-culturing or the use of bovine serum. It has now been shown that four recombinant proteins (BMP-4, VEGF, SCF and fibroblast growth factor-2 [FGF2]) alone can induce the hematopoietic differentiation of hESCs [10]. These systems are all highly empiric and remain fairly primitive. It is anticipated that better understanding of the molecular events involved may permit a more targeted and robust system. Derivation of hemangioblasts from hESCs It is established in the murine system that both hematopoietic and endothelial cells develop from a common transient precursor, the hemangioblast. Initial evidence came from an in vitro mouse ESC differentiation system [11]. Within 3 days of differentiation into embryoid bodies, a population of cells with hemangioblastic properties could be detected. When cultured in methylcellulose, hemangioblasts could form blast colonies that had both endothelial and hematopoietic potential. The blast-colony-forming cells expressed genes, such as Scl, Flk1, and CD34, that are common to both hematopoietic and endothelial cells. These blast colonies contained endothelial progenitors as well as primitive and definitive hematopoietic cells. Importantly, clonal analysis demonstrated that a single cell could result in both blood vessel and blood cells. Since this seminal report, the existence of the hemangioblast has been demonstrated in both embryonic and adult murine tissues [12,13]. Demonstration of a comparable human cell has been more complicated. However, a number of reports now support that concept [14–16]. In particular, a method by which human ESCs were first differentiated

Generation of Definitive Engraftable Hematopoietic Stem Cells from Human Embryonic Stem Cells

29

Trypsin EB media

hESCs on MEFs

EBs

Collagenase

RTRT-PCR

Fig. 4.2 Differentiation of human embryonic stem cells (hESCs) by embryoid body (EB) formation and subsequent analysis of blood progenitor cells appearance via reverse transcriptase polymerase chain reaction (RT-PCR), fluorescence activated cell sorter (FACS) or colony-forming unit (CFU) assay. MEF, mouse embryonic fibroblast.

FACS

CFU assay

into embryoid bodies (by BMP-4, VEGF, SCF, thrombopoietin, and Flt3L) and, after 3.5 days, dissociated and plated in semisolid medium, has been shown to improve several aspect of hemangioblast generation. Specifically, the system represented several improvements: (1) it is serum free (and therefore more reproducible than protocols using serum, with variation between lots); (2) it allows the generation of a high number of blast cells (20–50 million blast cells can be generated in a week from 1.2 million undifferentiated hESCs); (3) these blast cells can be cryopreserved; and (4) they can differentiate into endothelial cell and multiple hematopoietic lineages. This represents a significant advance toward the derivation of a high number of hematopoietic cells from hESCs for clinical use. Derivation of blood-specific lineages from hESCs The development of culture conditions that induce specific lineages is of great interest for both studying the events resulting in lineage differentiation and generating cells that might be useful in adoptive transfer for therapy. T cells In 2006, a system enabling the derivation of T cells from hESCs was established [17]. This two-step protocol, combining preliminary differentiation on OP9 followed by secondary culture into human thymic tissue in immunodeficient mice, showed that hESCs could yield mature T cells when placed in a supportive environment. Resultant thymocytes were able to respond to T-cell receptor-mediated signals. Although these findings could have therapeutic implications, for instance for patients

with X-linked severe combined immunodeficiency disease, or infected with human immunodeficiency virus, the in vivo secondary culture is cumbersome and the number of T cells attained is limited. Another system was developed to efficiently induce T-cell differentiation from mouse ESCs [18], where after an initial differentiation on OP9, the cells were transferred on OP9 expressing the Notch receptor ligand Delta-like 1. This in vitro approach is of considerable interest, but remains untested for hESCs. Dendritic cells Dendritic cells (DCs) are potent antigen-presenting cells with an ability to elicit primary T-cell responses. Immature DCs can be isolated from peripheral blood or derived from bone marrow CD34+ cells [19,20]; however, hESCs could potentially provide an unlimited number of DCs. A three-step approach was used to generate DC from hESCs [21]. These cells expressed similar markers to HSC-derived DCs, such as CD1a, CD9, CD68, and CD86. Moreover, these cells were functional, as shown by their ability to induce the proliferation of adult and cord blood T cells and their capacity to present antigens through the major histocompatibility complex (MHC) class I pathway. Another group recently reported the derivation of genetically manipulated DCs from hESCs [22]. Thus, the generation of genetically engineered hESC DCs could offer a promising tool for antigen-specific immune therapy. NK cells hESCs have been shown to differentiate into mature functional NK cells [23]. CD34+/CD45+ cells were obtained by differentiating hESCs on the stromal cell line S17 and by directing the differentiation of these cells

30

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into NK cells by co-culture on the murine fetal liver AFT024 cell line. The hESC-derived NK cells displayed a normal phenotypic profile with expression of CD56, killer immunoglobulin-like receptor, and CD94/ NKG2, markers of mature NK cells. More importantly, these cells exhibited cytolytic activity against human tumor cells by direct cell-mediated cytotoxicity and antigen-dependent cellular cytotoxicity. Thus, these cells are not only a valuable means to study NK development, but could also provide a novel source for cellular immune therapy. Macrophages Using a related strategy, macrophages were derived from hESCs [24]. After 14–17 days of initial differentiation on the S17 stromal cell line, the hESC-derived CD34+ cells were grown in semisolid methylcellulose medium to obtain myeloid colonies. They were then cultured for an additional 2 weeks in liquid culture with cytokines (granulocyte–macrophage CSF and macrophage colony-stimulating factor). The resulting cells expressed CD14, human leukocyte antigen-DR (HLA-DR), CD4, CCR5, and CXCR4, and exhibited normal functions, as measured by phagocytosis, upregulation of B7.1 and cytokine secretion in response to lipopolysaccharide stimulation. The cells were also able to be genetically modified by lentiviral transduction, suggesting potential opportunities for their use as protein-secreting immune cells. Erythroid cells ESCs as self-replenishing units for the production of large numbers of cells has been long considered, particularly as a mode of generating red blood cells ex vivo. One consideration, however, is that the red cells produced may represent those of early embryonic development. The primitive erythroid cells found in the yolk sac during human development have a characteristic morphology: they are nucleated and express mostly embryonic globin (ε, ζ, and α). In the fetal liver, macrocytic erythrocytes are enucleated and express fetal globin (α2γ2), while mature, marrow-derived erythrocytes are characterized by adult globin (α2β2). Reports have varied as to whether hESCs generate embryonic or adult erythrocytes [25–27]. A five-step method using sequential exposure to fetal liver or adult stromal cell lines and sequential cytokines provided evidence of red blood cell production, but the cells were large and nucleated, and expressed exclusively embryonic and fetal globins [25]. Since mESCs can produce mature enucleated erythrocytes with adult globin [28], it may be a matter of time before similar success can be achieved with human cell sources.

Isolation and/or expansion of hESC-derived HSCs It is clear that we can differentiate hESCs into blood cells at various stages of commitment. Although the protocols cited in the first paragraph of this chapter allowed for the enrichment of blood cells in the differentiated population, the cells were not homogeneous, and the isolation of the cells of interest is likely necessary before transplantation. Isolation would address the major concern that some undifferentiated ESCs could remain in the population of cells being transplanted. Since undifferentiated ESCs have the potential to form teratomas (benign tumor containing cells from the three germ layers) when injected into SCID mice, it is crucial to eliminate any residual undifferentiated ESCs prior to transplantation. A negative selection could be performed using well-known surface markers specifically expressed in hESCs, such as TRA-1-60, stage-specific embryonic antigen-3 (SSEA-3) or SSEA-4. Another strategy was proposed involving the generation of a human ESC cell line that expressed the herpes simplex virus thymidine kinase gene [29]. This gene confers sensitivity to the drug ganciclovir. As a result, this system would allow the specific elimination of transplanted hESC-

derived cells in case of tumor development, with the use of a common pharmacologic agent. Another issue is the presence of cells from other lineages in the heterogeneous population of differentiated cells. Thus, an attractive approach is to positively isolate HSCs or specific blood lineages before transplantation. A limitation to this approach is the lack of defined surface markers for the prospective isolation of long-term HSCs. Mouse long-term repopulating HSCs are well characterized, and a panel of surface markers is commonly used to isolate these cells, such as Lin−, Kit+, Sca+, and Thy1lo [30]. In 2005, a family of cell surface receptors, the signaling lymphocyte activation molecule family, that distinguished long-term HSCs (LT-HSCs) from multipotent progenitors (MPPs), was identified [31]. While CD150 was present exclusively at the surface of LT-HSCs, CD48 was specifically expressed in MPPs. The combination of these two markers (CD150/CD48) was proven to be very efficient for the isolation of LT-HSCs. Both LT-HSCs and MPPs can give rise to all blood lineages, but only LT-HSCs can maintain lifelong production of blood cells. Thus, the development of this simple and direct method to isolate LT-HSCs was a major step forward to improve transplantation efficiency. If the signaling lymphocyte activation molecule family is conserved between species, the same approach could be used to isolate LT-HSCs from peripheral blood, bone marrow or differentiated hESCs. To date, these markers have been described only in mice. The potential for mESC-derived hematopoietic progenitor cells to engraft lethally irradiated mice and contribute to long-term, multilineage hematopoiesis has been demonstrated [32,33]. In this system, day 6 embryoid bodies were dissociated, and the cells were plated on OP9. Only the primitive hematopoietic cells expressing Hoxb4 or the combination Hoxb4/Cdx4 were able to expand in this culture condition. After an additional 2 weeks of co-culture, all of the cells were transplanted into irradiated mice. Engraftment and multilineage repopulation was demonstrated, but this is clearly a very cumbersome system. Isolation of only the cells of interest is a common problem in cells derived from ESCs. The need for isolation derives from the potential for teratoma formation if cells that are too immature are used. The problem of isolating appropriate cell population comes from the unclear validity of using markers that have generally been validated in adult tissues. Several markers known to be expressed on hematopoietic progenitors, such as CD90, CD133, and CD117, are also expressed in undifferentiated hESCs, limiting the use of these markers for HSC isolation. Whether the HSC-enriched CD34+/Thy1+/CD38− pattern applies to cells derived from human ESCs remains unclear [34].

Are ESC-derived hematopoietic progenitors capable of engraftment and hematopoietic repopulation? Transplantation of HSCs derived from mESCs Although the derivation of hematopoietic progenitor cells from mESCs has been well documented in the last 25 years, only a few groups have reported a robust long-term repopulation from HSCs derived from mESCs. Moreover, the most successful repopulations were obtained from ESC-derived HSCs that overexpressed the transcriptional regulators Hoxb4 and Cdx4 [32,33] – a system that would not be safe for therapeutic applications due to the risk of malignant transformation. Thus, the generation of functional and engraftable HSCs from ESCs remains extremely challenging. The lack of engraftment of ESC-derived HSCs is thought to be due to the primitive nature of the cells generated. In mammals, hematopoietic development follows a complex process with two successive waves of primitive and definitive hematopoiesis occurring in distinct anatomical sites [35]. Primitive hematopoieis arises in the blood islands of the

Generation of Definitive Engraftable Hematopoietic Stem Cells from Human Embryonic Stem Cells

yolk sac by day 18 of gestation in humans. It is transient and allows for the production of nucleated erythroblasts, as well as macrophages and megakaryocytes. These primitive red blood cells are critical for the survival of the early embryo, which develops in a highly hypoxic environment. The second wave of hematopoiesis is located in the AGM region in the embryo proper. It is thought that definitive HSCs migrate from the AGM to the fetal liver, where they expand extensively before reaching their final niche in the bone marrow during the second trimester. These definitive HSCs are mostly quiescent but can give rise to all blood cell lineages. In the 1970s, it was thought that yolk sac HSCs colonized the fetal liver, and later the adult bone marrow, giving rise to definitive HSCs. Studies in the 1990s, however, demonstrated that the first definitive HSCs originated from the AGM, independent of the yolk sac blood cells [36,37]. For a decade, this theory of two independent waves of hematopoietic onset, one from the yolk sac that supplies the embryo and one from the AGM that provides definitive adult HSCs, was accepted by many scientists in the field. However, this theory was recently challenged by an in vivo cell-tracing approach to follow the fate of yolk sac hematopoietic cells during mouse development [38]. Using time–genetic labeling of yolk sac hematopoietic cells, it was demonstrated that yolk sac-derived cells can indeed migrate into sites of definitive hematopoiesis and contribute to blood cell production in adults. Work from another group further demonstrated that definitive HSC could be derived from yolk sac if the cells were co-cultured with an AGM stromal cell line [39]. This observation implied that the AGM provided the specific microenvironment allowing the maturation of the yolk sac primitive HSCs into mature definitive HSCs. It may therefore be possible to instruct primitive cells derived from ESCs to provide engraftable HSCs.

Transplantation of HSCs derived from hESCs The repopulating potential of hESC-derived HSCs has been reported to be quite poor [40]. However, when hESC-derived HSCs were injected directly into the marrow cavity, rather than the bloodstream, of irradiated immunodeficient mice, human blood cells were found at the site. There was even evidence that similar cells could be secondarily transplanted [41]. However, in all cases the level of engraftment was very low (<1%) and inferior to that of somatic sources of HSCs.

Can we generate immunologically compatible HSCs? Another major concern for the use of hESC-derived blood cells in transplantation settings is their potential for being rejected by the recipient in the same manner that any conventional transplanted organ is rejected. Several strategies have been proposed to limit the likelihood of tissue rejection if hESC-derived cells ever become available for transplantation: (1) the creation of a global bank of hESCs; (2) the use of the potential immune privileged status of hESC derivatives; (3) the generation of tailor-made hESCs by reprogramming patients’ fibroblastic cells (nuclear transfer, fusion, and induced pluripotent stem [iPS]); and (4) the generation of parthenogenetic ESCs. In the following paragraphs, we review these different approaches.

Creation of a global hESC bank Attempts to define the HLA complexity of an hESC bank needed to cover a reasonable segment of the population have been performed. One analyzed the blood group and HLA types of 10,000 UK cadaveric organ

31

donors for compatibility with 6577 patients registered on the UK kidney transplant waiting list, and used it as a simulation model for the potential requirement for hESC-derived cell transplantation [42]. This study was based on a six-antigen match. The authors reported that as few as 150 hESC cell lines would provide a full match for a minority (less than 20%) of patients, but a beneficial match (meaning one HLA-A or one HLA-B mismatch only) for 38% of the recipients and an HLA-DR match for 84%. They suggested that only 10 cell lines homozygous for common HLA types would provide a complete match for 38% of the patients and a beneficial match for 67%. One caveat of this study is the underrepresentation of particular ethnic groups. Another study carried out on the Japanese population showed similar results [43], estimating that a bank of human ESC cell lines derived from 170 randomly selected embryos would provide 80% of patients with, at worst, a single mismatch at one HLA locus. We have to keep in mind that these data were obtained from a relatively homogeneous population. A much larger number of cell lines will be required to offer a good match to ethnic minorities. The ethical aspects associated with the creation of an hESC bank are complex and have been well articulated elsewhere [44].

Immune privileged? Several studies have suggested that hESCs and their differentiated derivatives are immune privileged and may circumvent rejection [45,46]. Supporting this claim, hESCs have been shown to express low levels of MHC class I and to lack expression of MHC class II antigens [47]. However, MHC class I expression increased during differentiation and could be rapidly induced by interferon treatment. Since NK cells target low-expressing MHC class I cells through a process called “missing self,” the expression of NK receptors on the surface of hESCs was checked [47]. hESCs did not express the NK receptor and thus were not subjected to NK-mediated lysis. This suggested that engrafted hESCs could escape the innate immune response. In another study, the same group further studied the immune response against hESCs and derivatives in vivo [46]. Although teratomas could develop into NOD/SCID mice after 1 month, no tumors were found in NK-cell-deficient, B-cell-deficient or immune-competent mice, implying that xenorejection of hESCs was T-cell dependent. In order to mimic the clinically relevant transplantation settings, they used a humanized (Trimera) mouse model [48] to evaluate allorejection of hESCs. Both hESCs and their derivatives could escape rejection and form teratomas when injected into the mice reconstituted with human peripheral blood monocytes. In contrast, transplanted human skin fibroblasts and B lymphocytes induced a donor leukocyte response and were completely eliminated. The alloantigen-specific immune response against hESCs was also examined in vivo [45]. An intramuscular injection of hESCs was performed into immunocompetent mice. Unlike the human MBA-1 control cells (megakaryocytic cell line), hESCs did not induce an inflammatory response in these mice. Moreover, hESCs and their derivatives had a limited ability to induce T-cell-mediated immune responses. In fact, their data suggested a direct inhibitory effect of hESCs on T-cell proliferation in response to allogeneic DCs. These immune-privileged properties of hESCs are reminiscent of the tolerance of maternal lymphocytes for embryonic cells. However, these properties were challenged in a recent study [49]. The authors demonstrated that hESCs induced a similar response to human fibroblasts on naïve and immunized T cells, both directly and in the presence of syngeneic DCs. More data need to be collected to confirm and understand the partial immune privilege revealed in these reports. The hESC derivatives used in these studies

32

Chapter 4

Patient’s fibroblasts

Transplantation

Induced pluripotent Fusion with ESC cells (iPS) Blood cells

Nuclear transfer

Reprogramming

Patient’s specific pluripotent cells

Differentiation

were the result of random differentiation through teratoma formation. No MHC class II expression was detected in this population of cells, which suggests a poor efficiency for hematopoietic differentiation or the presence of immature blood cells only. Blood cells being particularly immunogenic, further work needs to be performed on the immune response of this specific lineage. hESCs tailor made by reprogramming patients’ fibroblasts cells (nuclear transfer, fusion, iPS) One strategy to avoid graft rejection is to generate cell lines with the genomic material of the patient. In this case, the match would be perfect and the risk of rejection minimal. Three methods to generate patientspecific pluripotent cells have been described (Fig. 4.3). Somatic cell nuclear transfer is a promising technology that may generate such patient-specific hESCs [50]. In this method, the nucleus is isolated from the patient’s own cells, such as skin fibroblastic cells, and transferred into an enucleated oocyte. Through this process, the somatic cell nucleus is reprogrammed. The oocyte is then induced to divide and develop to the blastocyst stage, where ESCs can be generated. These ESCs are a nearly perfect match with the patient as they also include mitochondria from the oocytes. This technique, although difficult and labor intensive, has proven effective for somatic cell reprogramming in several species, such as mouse, rabbit, pig, cow, and recently monkey [51,52]. The best illustration of the success of this method is the birth of the famous sheep “Dolly” in 1996, the first mammal to be cloned from an adult somatic cell [53]. Despite the report from a Korean group in 2005 [54], to date nuclear transfer ESCs have not been established from human somatic cells. After an investigation from Seoul National University, it was revealed that Hwang et al. had falsified their data. This retraction was accompanied by a massive controversy concerning egg sources and the ethics of paying for egg donations. Given the likely low efficiency of the nuclear transfer method, there remain considerable practical and policy barriers to successful use of this approach. An alternative approach for the reprogramming of human somatic nuclei, without the use of oocytes [55], involves using hESCs to

Fig. 4.3 Generation of patient-specific human pluripotent cells.

reprogram the adult somatic nucleus after cell fusion. The resulting product of this fusion is a hybrid cell with tetraploid DNA content. Thus, the genome of the hESCs needs to be removed from the hybrid in order to obtain diploid customized cells suitable for transplantation. In the absence of technology to selectively eliminate the nucleus of the hESCs from the hybrid cells, this approach is not clinically applicable. A third strategy to generate patient-specific pluripotent cells has been reported recently [56–58]. The authors introduced four genes (Oct4, Sox2, KLF4 and c-Myc) into embryonic or adult fibroblastic cells and selected the cells that reactivate ESC-specific markers. Through this method, pluripotent cells that resembled mESCs were derived. These cells, called iPS for “induced pluripotent stem,” could self-renew and differentiate into all three germ layers. Their pluripotency was confirmed by the generation of chimeric mice and germline transmission after blastocyst injection. Furthermore, the therapeutic potential of these iPS cells was demonstrated in a humanized mouse model for sickle cell anemia [59]. In this report, mutant donor fibroblasts were reprogrammed into iPS and the genetic defect repaired by homologous recombination. Then the repaired iPS were differentiated into hematopoietic progenitors, and these cells were transplanted into the donor mice (Fig. 4.4). All hematologic and systemic parameters of sickle cell anemia significantly improved after transplantation, providing proof of principle for using iPS in therapeutic settings. Recently, two separate teams demonstrated the reprogramming of human fibroblastic cells using two sets of slightly different four genes [60,61]. This remarkable finding opens a road to generate patient- and disease-specific pluripotent cells. Thus, the reprogramming of adult fibroblasts to ESC-like cells by overexpression of four factors is a very attractive strategy to generate patient-specific pluripotent cells, but many hurdles remain. Most notable among these is the potential for the introduced genes to cause cancer. In addition, the method used to introduce the genes, retroviral transduction, has implicit risks associated with integration into the host genome. Intense effort is now underway to reduce these risks both by eliminating genes such as the Myc oncogene from the four-gene formula [62] and to replace gene transfer with small molecule stimulation.

Generation of Definitive Engraftable Hematopoietic Stem Cells from Human Embryonic Stem Cells

33

Mouse model of sickle cell anemia

Transplantation

Tail tip fibroblasts

Expansion of hematopoietic progenitors on OP9 Transduction with Hox-B4

Reprogramming Transduction with four factors

iPS

Fig. 4.4 Scheme for sickle cell anemia correction in mice combining reprogramming, gene transfer and cell therapy. EB, erythroid body; iPS, induced pluripotent stem (cell).

Differentiation EB formation iPS with human βA wildtype globin gene

Repair Gene correction by homologous recombination

Generation of parthenogenetic ESCs

Conclusion

Parthenogenesis is another method to create histocompatible hESCs. Parthenogenetic mESCs were isolated from blastocysts developed from unfertilized oocytes; thus they contained the genetic material from the oocytes donor only. The histocompatibility of differentiated mouse parthenogenetic ESCs was demonstrated by transplantation into immunecompetent recipient mice that shared genetic identity at the MHC loci [63]. Recently, six ESC cell lines were derived from human parthenogenetic blastocysts [64]. These parthenogenetic ESCs had the characteristics of ESCs, i.e. self-renewal and differentiation into three germ layers, and their MHC matched with that of the oocyte donors. Thus, these cells may provide a source of histocompatible cells for transplantation into the oocyte donors or their siblings. Surprisingly, it has been shown that one of the Korean cell lines generated by Hwang et al. was a parthenogenetic cell line [65], derived accidentally while attempting somatic cell nuclear transfer. This anecdote underlines the difference in technical difficulties between somatic cell nuclear transfer and parthenogenesis. To date, parthenogenesis offers the easiest method to generate histocompatible hESCs. Using an appropriate model, such as the humanized mice described above, the potential of these cells should be further analyzed in a transplantation setting.

The ability to use pluripotent cells derived either from embryos (hESCs) or reprogramming (iPS) as a source of HSCs remains a distant possibility. Much needs to be learned about how cells normally mature to acquire the features of adult HSCs, and how these steps in ontogeny may be controlled and accelerated in laboratory settings. It is worth recalling that approximately 20 years divided the first derivation of mESCs [66,67] and definitive proof of long-term multilineage engraftment of HSCs derived from mESCs [68]. hESCs were derived less than a decade ago, and human iPS cells less than a year ago; research on the derivation of definitive engraftable HSCs from such cells is in its infancy. Nevertheless, stunning progress has been accomplished in recent years. Hemangioblasts and hematopoietic progenitors, as well as fully mature blood cells, have been derived. Concurrently, use of pluripotent cells to reverse the pathology of mouse models of blood diseases has been demonstrated and points the way forward. We cannot yet know whether pluripotent cells will provide a clinically useful source for hematopoietic cell transplant in humans. However, these cells have tremendous potential as both a research tool and a possible therapeutic agent, a potential that can only be defined by ongoing intense investigation.

References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145–7. 2. Smith AG. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 2001; 17: 435– 62. 3. Kaufman DS, Hanson ET, Lewis RL et al. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 2001; 98: 10716–21. 4. Vodyanik MA, Bork JA, Thomson JA et al. Human embryonic stem cell-derived CD34+ cells: efficient

production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 2005; 105: 617–26. 5. Vodyanik MA, Thomson JA, Slukvin II. Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures. Blood 2006; 108: 2095–105. 6. Trivedi P, Hematti P. Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells. Exp Hematol 2007; 35: 146–54.

7. Chadwick K, Wang L, Li L et al. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 2003; 102: 906–15. 8. Keller G, Kennedy M, Papayannopoulou T et al. Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol Cell Biol 1993; 13: 473–86. 9. Cerdan C, Rouleau A, Bhatia M. VEGF-A165 augments erythropoietic development from human embryonic stem cells. Blood 2004; 103: 2504– 12.

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Chapter 4

10. Pick M, Azzola L, Mossman A et al. Differentiation of human embryonic stem cells in serum free medium reveals distinct roles for BMP4, VEGF, SCF and FGF2 in hematopoiesis. Stem Cells 2007; 25: 2206–14. 11. Choi K, Kennedy M, Kazarov A et al. A common precursor for hematopoietic and endothelial cells. Development 1998; 125: 725–32. 12. Huber TL, Kouskoff V, Fehling HJ et al. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 2004; 432: 625–30. 13. Pelosi E, Valtieri M, Coppola S et al. Identification of the hemangioblast in postnatal life. Blood 2002; 100: 3203–8. 14. Wang L, Li L, Shojaei F et al. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity 2004; 21: 31–41. 15. Kennedy M, D’Souza SL, Lynch-Kattman M et al. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 2007; 109: 2679– 87. 16. Lu SJ, Feng Q, Caballero S et al. Generation of functional hemangioblasts from human embryonic stem cells. Nat Methods 2007; 4: 501–9. 17. Galic Z, Kitchen SG, Kacena A et al. T lineage differentiation from human embryonic stem cells. Proc Natl Acad Sci U S A 2006; 103: 11742–7. 18. Schmitt TM, de Pooter RF, Gronski MA et al. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat Immunol 2004; 5: 410– 17. 19. Thurner B, Roder C, Dieckmann D et al. Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. J Immunol Methods 1999; 223: 1–15. 20. Caux C, Dezutter-Dambuyant C, Schmitt D et al. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 1992; 360: 258–61. 21. Slukvin II, Vodyanik MA, Thomson JA et al. Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway. J Immunol 2006; 176: 2924– 32. 22. Senju S, Suemori H, Zembutsu H et al. Genetically manipulated human embryonic stem cell-derived dendritic cells with immune regulatory function. Stem Cells 2007; 25: 2720–9. 23. Woll PS, Martin CH, Miller JS et al. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity. J Immunol 2005; 175: 5095–103. 24. Anderson JS, Bandi S, Kaufman DS et al. Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy. Retrovirology 2006; 3: 24. 25. Olivier EN, Qiu C, Velho M et al. Large-scale production of embryonic red blood cells from human embryonic stem cells. Exp Hematol 2006; 34: 1635–42. 26. Chang KH, Nelson AM, Cao H et al. Definitivelike erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin. Blood 2006; 108: 1515–23.

27. Qiu C, Hanson E, Olivier E et al. Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development. Exp Hematol 2005; 33: 1450– 8. 28. Carotta S, Pilat S, Mairhofer A et al. Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells. Blood 2004; 104: 1873–80. 29. Schuldiner M, Itskovitz-Eldor J, Benvenisty N. Selective ablation of human embryonic stem cells expressing a “suicide” gene. Stem Cells 2003; 21: 257–65. 30. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994; 1: 661–73. 31. Kiel MJ, Yilmaz OH, Iwashita T et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005; 121: 1109–21. 32. Kyba M, Perlingeiro RC, Daley GQ. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 2002; 109: 29– 37. 33. Wang Y, Yates F, Naveiras O et al. Embryonic stem cell-derived hematopoietic stem cells. Proc Natl Acad Sci U S A 2005; 102: 19081–6. 34. Baum CM, Weissman IL, Tsukamoto AS et al. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A 1992; 89: 2804–8. 35. Mikkola HK, Orkin SH. The journey of developing hematopoietic stem cells. Development 2006; 133: 3733–44. 36. Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 1996; 86: 897–906. 37. Cumano A, Dieterlen-Lievre F, Godin I. Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 1996; 86: 907–16. 38. Samokhvalov IM, Samokhvalova NI, Nishikawa S. Cell tracing shows the contribution of the yolk sac to adult haematopoiesis. Nature 2007; 446: 1056– 61. 39. Matsuoka S, Tsuji K, Hisakawa H et al. Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleures by aorta-gonad-mesonephros region-derived stromal cells. Blood 2001; 98: 6–12. 40. Wang L, Menendez P, Shojaei F et al. Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression. J Exp Med 2005; 201: 1603– 14. 41. Tian X, Woll PS, Morris JK et al. Hematopoietic engraftment of human embryonic stem cell-derived cells is regulated by recipient innate immunity. Stem Cells 2006; 24: 1370–80. 42. Taylor CJ, Bolton EM, Pocock S et al. Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching. Lancet 2005; 366: 2019–25. 43. Nakajima F, Tokunaga K, Nakatsuji N. Human leukocyte antigen matching estimations in a hypothetical bank of human embryonic stem cell lines in the Japanese population for use in cell transplantation therapy. Stem Cells 2007; 25: 983–5.

44. Lott JP, Savulescu J. Towards a global human embryonic stem cell bank. Am J Bioeth 2007; 7: 37–44. 45. Li L, Baroja ML, Majumdar A et al. Human embryonic stem cells possess immune-privileged properties. Stem Cells 2004; 22: 448–56. 46. Drukker M, Katchman H, Katz G et al. Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 2006; 24: 221–9. 47. Drukker M, Katz G, Urbach A et al. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc Natl Acad Sci U S A 2002; 99: 9864–9. 48. Lubin I, Segall H, Marcus H et al. Engraftment of human peripheral blood lymphocytes in normal strains of mice. Blood 1994; 83: 2368–81. 49. Grinnemo KH, Kumagai-Braesch M, ManssonBroberg A et al. Human embryonic stem cells are immunogenic in allogeneic and xenogeneic settings. Reprod Biomed Online 2006; 13: 712– 24. 50. Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N Engl J Med 2003; 349: 275– 86. 51. Meissner A, Jaenisch R. Mammalian nuclear transfer. Dev Dyn 2006; 235: 2460–9. 52. Byrne J, Pedersen D, Clepper L et al. Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 2007; 450: 497– 502. 53. Wilmut I, Schnieke AE, McWhir J et al. Viable offspring derived from fetal and adult mammalian cells. Nature 1997; 385: 810–13. 54. Hwang WS, Roh SI, Lee BC et al. Patient-specific embryonic stem cells derived from human SCNT blastocysts. Science 2005; 308: 1777–83. 55. Cowan CA, Atienza J, Melton DA et al. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 2005; 309: 1369–73. 56. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126: 663–76. 57. Wernig M, Meissner A, Foreman R et al. In vitro reprogramming of fibroblasts into a pluripotent EScell-like state. Nature 2007; 448: 318–24. 58. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448: 313–17. 59. Hanna J, Wernig M, Markoulaki S et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 2007; 318: 1920–3. 60. Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131: 861– 72. 61. Yu J, Vodyanik MA, Smuga-Otto K et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318: 1917– 20. 62. Nakagawa M, Koyanagi M, Tanabe K et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 2008; 26: 101–6. 63. Kim K, Lerou P, Yabuuchi A et al. Histocompatible embryonic stem cells by parthenogenesis. Science 2007; 315: 482–6.

Generation of Definitive Engraftable Hematopoietic Stem Cells from Human Embryonic Stem Cells 64. Revazova ES, Turovets NA, Kochetkova OD et al. Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells 2007; 9: 432–49. 65. Kitai Kim, Kitwa Ng, Peter J et al. Recombination signatures distinguish embryonic stem cells derived by parthenogenesis and somatic cell

nuclear transfer. Cell Stem Cell 2007; 1: 346– 52. 66. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154–6. 67. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium condi-

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tioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981; 78: 7634–8. 68. Perlingeiro RC, Kyba M, Daley GQ. Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential. Development 2001; 128: 4597–604.

5

Susan Prohaska & Irving Weissman

Biology of Hematopoietic Stem and Progenitor Cells

Introduction Stem cells are single cells that are capable of both self-renewal and differentiation [1–3]. In essence, self-renewal means that the cells can divide without undergoing discernible differentiation, and that they possess properties that allow large numbers of self-renewing cell divisions to occur in a regulated fashion over the life span of a host. Hematopoietic stem cells (HSCs) are therefore blood-forming cells that, at the single cell level, can duplicate by self-renewal as well as produce all types of differentiated blood cells [2,4,5]. There are three theoretical and two practical ways to define HSCs: (1) prospectively isolate single cells that can self-renew and differentiate in vivo [4–8]; (2) introduce chromosomal or genetic lineage markers into cells, and demonstrate the presence of that marker in all blood cell lineages, as well as in the cells that can generate them long term [2,9]; and (3) establish a culture system wherein prospectively isolated single cells can self-renew and give rise to all blood cell types. This last method remains theoretical because conditions have not yet been discovered that allow all myeloid and lymphoid cell outcomes to develop in the same culture system. A word on nomenclature: Although the founders of this field Till, McCulloch and their colleagues, called their first candidate stem cell a pluripotent stem cell [10–13], recent revisions in the nomenclature now distinguish stem cell hierarchies. • Totipotent stem cells are single cells that can give rise to every tissue of the embryo, the extraembryonic tissues, and the developing adult; so far, only the zygote has that property [14,15]. • Pluripotent stem cells are cells that can give rise to stem and progenitor cells of all tissues, but lack the capacity to give rise to extraembryonic tissues; cells of the inner cell mass of the blastocyst stage of embryogenesis (the latest preimplantation step) appear to be pluripotent, and successful culture of these cells results in pluripotent embryonic stem (ES) cell lines [14–16]. • Multipotent stem cells are single cells, like the HSC, that can selfrenew and differentiate into more than one cell type in a particular tissue lineage: HSCs, central nervous system (CNS) stem cells, peripheral nervous system stem cells and perhaps liver stem cells, epidermal/hair follicle stem cells, and muscle stem cells are leading examples of purified and enriched populations of these multipotent stem cell types [1,17– 24]. • Unipotent stem cells exist in each germline, where self-renewal and differentiation lead only to one or another type of gamete; however,

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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there exists in every chordate embryo/fetus a class of germline stem cells that are bipotent prior to colonization of the genital ridges, taking their unipotent specificity from the sex of the genital ridge in which they make their unipotent commitment. In this terminology, there can also be multipotent, oligopotent, and unipotent progenitor cells, which are differentiated from stem cells by their inability to undergo extended self-renewal [1,5,17,25–27]. Many investigators misuse the term “stem cells” when they transfer other populations of cells. In this volume, and hopefully in the journals that serve this field, “hematopoietic cell transplant” (HCT) means that the inoculum contains hematopoietic cells. Bone marrow transplantation, MPB (mobilized peripheral blood) transplants, and umbilical cord blood are common sources of cells for HCT. Transplantation of cells enriched for hematopoietic stem and progenitor cells that express CD34 is CD34+ HCT. Transplantation of putative stem cells is HSC, but the phenotype used should be specified in each paper, e.g., CD34+38loCD90+Lin1 HSC, or ALDH+ HSC, or dye exclusion cells in the side population, or the more restrictive lowest 10% of these, called side population tip cells. Hematopoietic progenitors may be called either by their function, if enriched/purified, for example CLP (common lymphoid progenitor), or by their marker phenotype, for example CD34+38+CD123+CD45RA−Lin− CMP (common myeloid progenitor).

History of HSC isolation Following the demonstration that just-lethal doses of whole-body irradiation caused death by hematopoietic failure, and that hematopoietic failure could be averted either by shielding hematopoietic organs or by transplantation of unirradiated bone marrow [28–30], the critical study that set the stage for the concept of HSCs was the demonstration that the injected bone marrow reconstituted the hematopoietic system rather than provided radiation repair factors [31–33]. In our view, the seminal study in the HSC field began with the observation by Till and McCulloch that transplantation of limiting numbers of bone marrow cells into lethally irradiated mice gave rise to colonies in the spleen that contained all elements of the myeloerythroid (but not lymphoid) lineages, and that the number of colonies was proportional to the number of bone marrow cells injected [12]. Two experiments followed these observations that opened the field to stem cell biology and changed the thinking of the hematology community. First, preirradiation of the marrow donor at doses that would allow the survival of some cells that had random chromosome translocations or inversions resulted in the finding that each spleen colony was the product of a clonogenic precursor [2]. Second, at least some of these colonies contained cells that also contained spleen colony-forming cells (CFCs), as well as cells that could be radioprotective [2,13,34]. Later, it was shown

Biology of Hematopoietic Stem and Progenitor Cells

that the daughter cells of some of these spleen CFCs included lymphocytes [10]. From these experiments, it was likely that there existed in the bone marrow single cells capable of self-renewal and full differentiation to hematopoietic fates. It was subsequently demonstrated that at least two classes of spleen colony-forming cell existed: those that peaked at days 8–10 and were the progeny, mainly, of nonstem cell oligopotent progenitors; and those that peaked in size at days 12–14, which included much more primitive progenitors and stem cells [35,36]. Recently, following the isolation of both stem and progenitor cells, it became clear that most of the day 8–10 spleen colonies are derived from committed myeloid progenitors [37] (see below), whereas the day 12–14 colonies are derived from HSCs, multipotent but self-renewing progenitors, and some latepeaking committed myeloid progenitors [37–41] (see below). Nevertheless, the impact of these elegant studies by the Till, McCulloch, Becker, Wu, and Siminovitch group established the field of hematopoietic stem and progenitor cell biology by in vivo assessment and first introduced genetic marking to show the existence of HSCs [2]. These important experiments were carried out mainly in the 1960s, before the discovery of monoclonal antibodies [42] or the development of high-speed multiparameter fluorescence-activated cell sorters [43]. The eventual isolation of HSCs required not only these innovations, but also the development of in vitro and in vivo assays for the identification and quantification of clonogenic precursors of the T-cell [44], B-cell [45], and myeloerythroid [46–49] lineages. While many important enrichments of these precursors were accomplished using cell sorting with monoclonal antibodies and lectins [50], it was not until a simultaneous analysis of all lineages was coupled with quantitative reconstitution of irradiated mice by limiting numbers of candidate HSCs that one could show that HSCs could be prospectively isolated rather than retrospectively inferred by chromosome marking [4,6,7,38,40,45]. We have isolated both mouse and human HSCs, and their surface phenotypes are shown in Fig. 5.1 [38,51,52]. They share the properties of expressing Thy-1 (Thy1.1+ or CD90+) and lacking the expression of a collection of mature blood cell lineage markers (for T cells, B cells, red blood cells [RBCs], granulocytes, monocytes, platelets, natural killer [NK] cells, etc) (Lin−) and both express c-kit, albeit mouse at higher levels than human. In addition, mouse HSCs express the stem cell antigen-1 (Sca-1) marker [38,40,53,54] and do not express surface Flk2/ Flt3, the receptor for the Flt3 ligand (Lft3L) [51,52], whereas their downstream multipotent progeny that increasingly lose self-renewal

Mouse Lin–c-Kit+Sca1+Thy1.1lo Flk2–CD34–CD150+

Human

HSC

Lin–CD34+CD38– Thy1(CD90)+

Lin–CD34+CD38– Thy1(CD90)–CD45RA–

Lin–c-Kit+Sca1+Thy1.1lo Flk2–CD34+CD150+ Multipotent Lin–c-Kit+Sca1+Thy1.1lo Flk2–CD34+CD150–

?

progenitors

?

Lin–c-Kit+Sca1+Thy1.1– Flk2+CD34+CD150–

Lineage committed progenitors

Fig. 5.1 Clonogenic multipotent progenitors have a distinctive marker profile. Shown are surface phenotypes of hematopoietic stem cells (HSCs) in human and mouse.

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capacity do express Flk2 [52]. Recently, long-term HSCs (LT-HSCs) were also found to express CD150 (Slamf1) [55,56], and other Slam family members, CD244 and CD48, selectively mark downstream multipotent and oligopotent progenitors. We isolated human HSCs of the phenotype CD34+ [57] Thy-1+Lin− [58] that were enriched for T-, B-, and myeloid cell potential in clonogenic assays, and were efficient at restoration of the human hematolymphoid organs in SCID-hu mice [58]. Human LT-HSCs are, in addition, CD38− to CD38lo [58–62]. Recently, we further characterized LT-HSCs to be contained within the Lin− CD34+CD38−CD90+CD45RA− fraction of cord blood. Transplantation of as few as 10 of these cells resulted in long-term repopulation in immunocompromised mice. In addition, we found the Lin−CD34+CD38− CD90−CD45RA− population to contain candidate human multipotent progenitors (MPPs), cells with limited self-renewal potential capable of transient multilineage blood reconstitution [63]. In both species, a search for other HSCs in the hematopoietic tissues was carried out. In mice, the Sca-1−, Lin+, c-kit −, Thy-1−, and Thy-1hi subsets did not, in the context of transplantation, contain any long-term multilineage hematopoietic potential [54]. In the human, CD34−, Lin+, and Thy-1− cells also did not contain any in vitro or in vivo long-term multilineage hematopoietic precursor potential [57,58,62,64], although some groups believe CD34−CD38− human HSCs exist [65–67]. In mice transplantation of limit dilution or a single LT-HSC results reproducibly in a high frequency of hosts (5–40%) showing long-term multilineage reconstitution, with self-renewal of the HSC, including its expansion to full normal levels, for the life of the host [4,5,7,8,68]. The bone marrow of allogeneic human HCT transplants contains self-renewed HSCs that also work in serial transplantation in SCID-hu mice [58]. Thus, in both mouse and human, HSCs that self-renew as well as differentiate to all blood cell lineages can be prospectively isolated as single clonogenic precursors.

Properties of mouse HSCs and other MPPs We have shown that transplantation of single, limit dilution or small numbers of LT-HSCs results in both HSC expansion and lifelong selfrenewal, through at least four or five serial transplant generations [68]. Transplantation of downstream short-term, self-renewing (ST)-HSCs or MPPs results in robust but transient multilineage reconstitution after which residual host HSCs recover [7,38,52]. It is important to point out that no matter how many ST-HSCs or MPPs are transplanted, at no time is there any measurable dedifferentiation from the short-term cells into the long-term pool [40]. In the normal steady state, about 1.5% and up to 8% of LT-HSCs randomly enter the cell cycle each day, whereas higher percentages of ST-HSCs and MPPs are in the cell cycle at any time [69–71]. Interestingly, when steady-state bone marrow LT-HSCs are isolated according to their position in the cell cycle, those cells with 2N DNA content are highly efficient, at both limit dilution and higher numbers of cells, at providing radioprotection and long-term multilineage reconstitution, whereas the dividing subsets (>2N DNA content) are less efficient at reconstitution, and are similarly less efficient at homing directly to the bone marrow rather than the spleen or liver [69,70,72]. Within the 2N DNA pool of LT HSCs, the G0 cells transplant much more readily than those in G1 [70]. Following transplantation, a much higher fraction of LT-HSCs remain in cell cycle in the radioprotected host, and this period can extend for at least 4–5 months after transplantation [40,68,69,73]. Given the poor transplantability of cycling HSCs, it is conceivable that protocols that test cells for “stemness” by serial transplantation could be confounded by these lingering effects of mobilization and radiation on cell-cycle status and reconstitution ability [68,69,73]. Therefore, experiments that are designed to measure the life span of expanding and

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Chapter 5 Kinetics of mobilization of LT-HSC following Cy/G-CSF

Cell-cycle status of LT-HSC in blood 298/307 0 40 80 120 100 200

1,000,000

Total HSC

100,000

10,000 Bone marrow Spleen Blood

1000

100 –1

0

1

2

3

4

5

6

7

8

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self-renewing HSCs by transplantation should be carried out in ways that remove the induced cell-cycle phenomenon from consideration. Although in the steady state only a small fraction of LT-HSCs is in the cell cycle, this ratio can be dramatically altered by a number of circumstances. For example, upon transplantation into irradiated hosts, probably every HSC that lands in a hematopoietic microenvironment undergoes, at least initially, symmetrical self-renewing cell divisions to expand the HSC pool. In addition, the techniques used to mobilize HSCs into the blood stream usually operate by first causing all HSCs to enter the cell cycle nonrandomly [72,74,75]. As shown in Fig. 5.2, treatment of a mouse with cyclophosphamide (which should kill no more than 4–8% of LT-HSCs, but should have a dramatic effect on both MPPs and oligolineage progenitors [see below] because they are largely in cell cycle), followed by granulocyte-colony-stimulating factor (G-CSF) treatment, results in every HSC entering the cell cycle, which leads to a 12–15-fold increase in the steady state number of LT-HSCs [74]. Even bleeding of mice, up to 25% or 50% of their blood volume two or three times in a week, results in a measurable increase in the fraction of HSCs in the cell cycle [76]. Replacement of RBCs lost due to bleeding suppressed the expansion, whereas replacement of fluid volume or white blood cells did not, indicating that HSCs are selectively responsive to decreases in RBC levels. Thus, positive and negative feedbacks on the regulation of HSC cell division must exist, but currently their nature and mechanisms are unknown. HSCs must go through many self-renewing cell divisions over the life of an animal, and it is reasonable to wonder if they are susceptible to a natural limit on the number of cell divisions they can undergo, often called the “Hayflick limit” [77], and whether HSCs have a special mechanism for maintaining the ends of their chromosomes, called telomeres, to avoid programmed cell senescence resulting from critical telomere shortening [78,79] (see Chapter 6). Both mouse and human HSCs have telomerase activity, but telomere length declines in the progeny of HSCs that have undergone a large number of cell divisions, in the context of normal blood development and in response to radiation and transplant recovery [80]. Therefore, the relatively high telomerase activity in HSCs [81] is not sufficient to completely prevent telomere shortening through several cell division cycles. The role of telomerase is complex. In the absence of a functional telomerase-reverse transcriptase (TERT) component of the telomerase complex, the progeny of dividing HSCs lose telomeres much more rapidly, confirming that it functions to maintain telomere length [27,80]. However, transgenic mouse strains that overexpress the TERT component at high levels in HSCs do not reduce the length of their telomeres, even following successive induced cell cycles by transplantation, and there is also evidence that TERT has a protective effect independent of its enzymatic function [82]. Nevertheless, these HSCs still lose trans-

5.2% S/G2/M

Hoechst 33342

Fig. 5.2 The total number of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow, spleen and blood of mice on successive days of cyclophosphamide/granulocyte colony-stimulating factor (CY/G-CSF) treatment. Total bone marrow hematopoietic stem cells (HSCs) were calculated by assuming that the femurs and tibias (less the epiphyses) contained 15% of all bone marrow in the mouse. Total HSC level in the blood was calculated by assuming that the total blood volume was 1.8 mL. Cell-cycle status of LT-HSCs in the blood was determined by Hoechst DNA staining [72].

plantation activity after the fourth or fifth serial HSC transplant generation [27]. This loss of transplantability with HSCs that retain telomeres points to other phenomena limiting the transplantation of these cells, including perhaps the persistent entry into the cell cycle, programmed cell life spans independent of telomere length, or random factors associated with transplantation and/or cell division [80,83]. Programmed cell death or senescence of HSCs need not occur only as a result of extensive numbers of HSC cell divisions. There are several genetic loci that regulate, in mice, both the frequency of HSCs and the frequency of HSCs that are in the cell cycle [84–86]. One genetic program that likely regulates HSC numbers is that leading to programmed cell death inhibitable by the antiapoptotic protein BCL-2 (and by extension, its antiapoptotic gene relatives such as BCL-x). In mice that have enforced high-level expression of human BCL-2 in HSCs, the steady-state levels of HSCs are increased four- to fivefold, and the competitive repopulation activity of these HSCs, cotransplanted with CD45 congenic HSCs into the same irradiated host, is high [87,88]. Therefore, it is likely that programmed cell death is one regulator of HSC numbers, and it is yet to be determined what stimuli to HSCs enact the programmed death pathway inhibitable by BCL-2. There are several genes whose expression is required to initiate hematopoiesis, hematopoiesis and angiogenesis, or angiogenesis resulting in failed hematopoiesis; these include AML-1/RUNX-1 [89], SCL-TAL [90], FLK1 [91,92]. It is unclear whether these genes act directly within HSCs, within their immediate precursors to generate HSCs or on other cell populations that have a role in the survival or stimulation of HSCs, or whether, as in the case of a developing vasculature, they regulate the movement of HSCs or their precursors from one part of the body to another [93]. Reverse transcription polymerase chain reaction (RT-PCR) analysis of highly purified HSCs, or the use of highly purified HSC messenger RNA probes to interrogate extensive mouse complementary DNA libraries has revealed that at least some of these genes are expressed intrinsically in HSCs [55,94–98]. It is important to point out that many factors could be playing a part in the movement of pre-HSCs or HSCs during embryonic and fetal development. Factors affecting the release of cells from a particular microenvironment, their movement through tissues or the blood stream, the recognition of endothelium in tissues, and their homing to stem cell niches could easily be read out as genes affecting HSCs without directly affecting either their development or their self-renewal capacity (see below). Genetic pathways for self-renewal of HSCs Self-renewal is the single distinguishing characteristic between LTHSCs and the rest of the multipotent and oligopotent progenitors in the hematopoietic system (see Chapter 6). While LT-HSCs can be expanded

Biology of Hematopoietic Stem and Progenitor Cells

in a variety of situations in vivo, the attempt to expand rigorously purified mouse or human HSCs in vitro with conventional cytokines such as steel factor (SLF), thrombopoietin (TPO), interleukin-11 (IL-11), IL-6, IL-3, and Flt3L, alone or in combination in serum-containing medium, has never resulted in more than a minor expansion in HSCs by phenotype, and little or no expansion by function [99–105]. Many investigators were led astray by the expansion of cells expressing CD34, but a broad variety of non-HSCs are also CD34+, and these cells were the result of proliferation of HSCs and progenitors coupled with cell differentiation. The introduction of the antiapoptosis protein BCL-2 into HSCs in mice allowed HSCs to survive serum-free culture conditions, so the response of LT-HSCs to factors in the absence of differentiating factors present in serum, such as monocyte/macrophage-CSF (M-CSF), G-CSF, granulocyte–macrophage CSF (GM-CSF), and others, could be studied [87,88]. Any combination of these cytokines and others when added to these BCL-2-containing HSCs led to a massive burst of proliferation and progenitor expansion by most HSCs, but no detectable HSC expansion [87,88]. These HSCs could respond to three factors as single factors in vitro: IL3, SLF, and TPO [88]. HSCs responding to IL-3 give large bursts of proliferation with concomitant maturation along the myeloid and mast cell lineages, without self-renewal of HSCs. One might have hoped that the HSC response to SLF alone might have been self-renewing divisions, as mutations in the SLF gene or its receptor, c-kit, lead to fetal or early neonatal lethality, usually because of profound anemia [106–111] (reviewed in [112]). However, the response of single highly purified Bcl-2-expressing LT-HSCs to SLF in serum-free medium was proliferation without detectable self-renewal, but with maturation through both CLP and CMP pathways [88]. Stimulation with TPO led to mild proliferation, and dedicated differentiation along the megakaryocytic lineage. Interestingly, RT-PCR analysis of messenger RNA from HSCs and highly purified myeloid progenitors revealed that HSCs might have the highest expression of the TPO receptor c-mpl of any of these cell populations [26,94,113]. Recent reports support a complex role for TPO in HSC biology. Neither TPO nor c-mpl is necessary for fetal HSC development, but HSCs in adult TPO- or c-mpl-deficient mice are cycling more and have impaired reconstitution ability compared with wild-type HSCs [114]. Further, TPO is produced by osteoblasts in the bone marrow, and blocking tpo antibody induces release from the niche and increased proliferation [115]. Taken together, these data suggest that TPO is important for HSC proliferation and self-renewal after transplantation, but also promotes quiescence by promoting retention in the niche with increased integrin expression and preventing cycling and subsequent HSC exhaustion. Some early experiments indicated that other factors might be involved in HSC expansion. For example, activation of the Notch-1 receptor, mimicked by retroviral transduction with the intracellular activated form of Notch-1, occasionally led to the generation of mouse cell lines with many of the characteristics of HSCs, although this response was neither general nor robust [116,117]. Provision of HoxB4 as a retroviral insert to semipurified mouse hematopoietic progenitors led to vigorous proliferation and at least some retention of hematopoietic multilineage and long-term reconstituting activity in vitro and in vivo [118–120], although a rigorous demonstration that this outcome was caused by expansion of HSCs alone, rather than provision of other signals that in addition enable their engraftment has not yet been established [119–121]. The Wnt/Fzd/Dsh/GSK-3β/β-catenin pathway is also implicated in HSC self-renewal (reviewed in [122,123]). Wnt, a highly hydrophobic protein, binds to surface frizzled (Fzd), a 7TM protein, in combination with a low-density lipoprotein-associated receptor, usually LRP5. Binding of Wnt to Fzd leads to activation of disheveled (Dsh). The action of Dsh prevents GSK-3 (glycogen synthase kinase) β-phosphory-

39

lation of β-catenin, and also allows β-catenin to dissociate from the complex and translocate to the nucleus, where it becomes available for binding to Lef/TCF factors and activation of transcription [122,123]. The addition of partially or fully purified Wnt3A to highly purified LT-HSCs in serum-free medium, with or without SLF, results in clonal proliferation of HSCs. In vitro, these HSCs go through a significant expansion, and up to 50% of the expanded progeny are cells of the LTHSC phenotype [124,125]. Transfection of nondegradable β-catenin lacking the N-terminal phosphorylation sites was reported to drive LTHSCs into massive and prolonged expanding cell divisions, with expansion of LT-HSCs both by phenotype and by function upon transplantation [124]. Inhibitors of Wnt signaling even blocked SLF and other cytokine-mediated proliferation of LT-HSCs [124]. When LTHSCs are transfected with a reporter of LEF/TCF + β-catenin activity in the nucleus and then transplanted into lethally irradiated hosts, LTHSCs reading out the reporter are retained in vivo long term, whereas myeloid progenitors derived from these LT-HSCs do not express the reporter [124]. Taken together, these results indicated that the Wnt/Fzd/ β-catenin pathway is likely to be an important pathway utilized by LTHSCs in self-renewing cell division. When activated by Wnt 3A, LTHSCs upregulate expression of both Notch-1 and HoxB4, genes also implicated in the self-renewal of HSCs. However, the regulated deletion of the β-catenin gene in transplanted bone marrow failed to affect the engraftment of hematopoiesis, or the retransplantation capacity of this marrow [126]. Contradictory results were recently reported with a different gene promoter encoding the cre recombinase required for βcatenin deletion [127]. The bmi-1 gene is a member of the polycomb family, which has a role in silencing gene expression by site-specific deacetylation of histones associated with those genes [128]. BMI-1 is expressed in high levels in mouse and human LT-HSCs, and mouse-made mutants for the BMI-1 gene have a profound defect in the transplantation and selfrenewal of LT-HSCs [97,128]. The two transcripts of the p16 locus, P16INK4A and P19ARF, are overexpressed selectively in the hematopoietic tissues in the BMI-mutant mice, thus implicating a role of BMI1 in the downregulation of genes whose expression is contrary to self-renewal of HSCs [129]. These could include decreased viability, enforced differentiation, or direct regulation or inhibition of genes involved in the self-renewing pathways such as those of the Wnt/Fzd/βcatenin and Notch pathways [116,117,122,124]. BMI-1 mutants do not show differences in expression in either HoxA9 or HoxB4 (Clarke et al., unpublished data), genes that also have some role in HSC functions [121,130]. JunB negatively regulates mouse LT-HSCs, and in the absence of JunB the HSC pool expands; in the expanded pool, p16ink4a and p19arf are expressed at lower levels than wild-type HSC, and Bcl-2 and Bcl-xl are increased in expression in JunB knockout compared with wild-type HSCs [131]. A number of genes have recently been reported to play a role in HSC self-renewal including junb, sox17, cited2, Mcl1, Tel/Etv6, Gfi1, Pten, and Stat5. Chapter 6 covers self-renewal in more detail, but taken together these results indicate that the control of self-renewal is a complex, multipathway process, much of which is probably controlled by the HSC niche. Migration of HSCs Hematopoietic capacity is known to be present in the adult mouse at highest levels in bone marrow, at about one-tenth that level in spleen, and at one hundredth or less of the level of bone marrow in blood [132]. In those early experiments, the ability of marrow or tissue transplants to retain erythropoiesis could not be assigned directly to actions of HSCs. There was no clear explanation as to why hematopoietic activity might

40

Chapter 5

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be present in organs other than the bone marrow, and certainly not in the blood. It was only when clinical HCT groups described the phenomenon of hematopoietic activity increasing in the blood of cytotoxic drug-treated individuals that the possibility of HSC mobilization into the blood was considered [133,134]. Empirical protocols using cyclophosphamide plus cytokines such as G-CSF, GM-CSF, SLF alone or in various combinations with G-CSF, IL-1, and IL-3, led to efficient mechanisms to mobilize hematopoietic cells into the blood stream [135–139]. While at the early stages, this activity was measured in terms of in vitro tests for hematopoietic CFCs, but CFC assays alone are not specific for HSC versus oligolineage progenitor activities [37,58]. However, there could be no doubt that HSCs were involved when MPB transplants were used in hosts conditioned with high-dose chemotherapy, resulting in both early and sustained hematopoiesis. When cyclophosphamide and G-CSF are used in the mouse model, wherein one can analyze directly the daily changes in marrow, spleen, and blood, it is clear that nearly every HSC and MPP enters the cell cycle rapidly [74]. These reach a peak in the marrow, at which time the cells appear rapidly in blood and spleen (Fig. 5.2). At all times, the HSCs appearing in the blood have a 2N amount of DNA, indicating that they are not proliferating in the blood; in fact, the fold increase in their number would have ruled out a blood-only expansion of the HSC pool [74]. Bromodeoxyuridine incorporation into their chromosomal DNA during the expansion phase of HSC in the “mobilized” bone marrow results in the labeling of virtually every HSC, and, interestingly, just 1 or 2 days later the 2N HSCs found in the blood are all labeled with bromodeoxyuridine [74]. Thus, mobilization appears to result as a consequence of marrow HSC proliferation, and accumulation of emigrant HSCs in extramedullary sites such as the spleen, liver, and blood. The proliferation that precedes mobilization has been described above, and it is notable that the cytokines (e.g. G-CSF) that show high efficiency in the mobilization process do not have receptors on native HSCs [26,113,140]. In the early phases of mobilization, there is a rise in cells of the myelomonocytic series, and it has been shown that the elaboration locally by myeloid cells of matrix metalloproteinases such as MMP-9 can serve to cleave cell–cell interaction molecules known to be active between HSCs and marrow stroma [141]. Similarly, elaboration of the chemokine IL-8 occurs, and infusion of IL-8 alone can cause a significant and rapid mobilization of HSCs [142,143]. The actual nature of the molecules involved in HSC release is not clear, although it is known that HSCs express integrin α4β1, which allows them to attach to hematopoietic stromal cell vascular cell adhe-

Fig. 5.3 Rapid clearance of mobilized hematopoietic progenitor cells from the blood stream. Clearance from the blood of eGFP+ progenitors and PKH-26+ red blood cells (RBCs), and predicted progenitor and RBC frequencies, respectively. In vivo homing to different organs of mobilized and consecutively transplanted hematopoietic progenitor and stem cells 3 hours after injection. (From [72] with permission.)

sion molecule [144–148]. Also, HSCs express the c-kit receptor that allows binding to, at least, cell surface SLF on stroma. Although HSCs have a number of other members of the adhesion and integrin family members, the role of each of these in cell proliferation, detachment from stroma, local emigration into sinusoids, movement through the blood stream, margination on distant blood vessels, transendothelial migration at these vessel sites, and localization to microniches that support LTHSCs are still unclear. In the adult mouse, homing of infused HSCs appears to involve both cell surface integrin α4β1 and cell surface CXCR4, a receptor for the chemokine stromal cell-derived factor-1 (SDF-1) [149–151]. Of all known chemokines, adult mouse HSCs migrate in vitro only in response to SDF-1, and this is chemotactic movement [151]. However released, co-infusion of labeled HSCs and labeled RBCs from mobilized blood into the blood of same-stage mobilized animals results in the rapid emigration of HSCs out of the blood, with retention of RBCs in the vessels [72]. In fact, most HSCs are gone by 1–5 minutes, and they do not reappear in the blood stream within the next several hours (Fig. 5.3). These HSCs nonrandomly home to hematopoietic sites in mobilized animals: bone marrow, spleen, and liver, all tissues with sinusoids lined by macrophages. This observation fits with previous studies on the rapid egress of infused lymphocytes out of the blood stream into tissues via recognition of particular vascular addressins for which they have cognate homing receptors [152,153], as well as the rapid egress from the blood stream of other nucleated cells such as monocytes [154]. There are similar HSC, MPP, and myeloid progenitor fluxes in normal mice. To maintain the approximate 100 HSCs found in the blood of mice, given a residence time in the blood of 5 minutes or less, would require fluxes of well over tens of thousands of HSCs and MPPs per day. Recent data suggest that HSCs and progenitors circulate through the blood and reside in the tissues for up to 36 hours before returning to the blood via the lymphatics [155]. It is not yet clear whether these cells are passing through the blood in one pass or whether they form a specialized pool of HSCs and MPPs yet have a higher probability of re-entering the blood than cells resident in deep marrow niches. If HSCs found in the blood are also derived from dividing HSCs, nearly one out of every two daughter cells from a self-renewing HSC division would have to enter the blood stream. Consistent with this emigration rate, infusion of purified LT-HSCs into untreated immunodeficient mice (to avoid immune rejection of cells with antigenic markers) led to a dose–response engraftment of marrow sites of between 0 and 250 HSCs, while up to 5000 HSCs engrafted no

41

% granulocyte chimerism

Biology of Hematopoietic Stem and Progenitor Cells

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Fig. 5.4 Hematopoietic stem cell (HSC) niches can be transiently saturated in unconditioned animals [157]. (a) RAG2−/−γc−/− (CD45.2) mice were transplanted with 10, 50, 250, 500 or 1000 HSCs from green fluorescent protein (GFP) transgenic mice (shaded bars). Eight weeks after transplantation, mice were given a second transplant of 4000 non-GFP HSCs from CD45.1 donors (open bars). Peripheral blood granulocyte chimerism was analyzed at 16 weeks after the first transplantation. The dashed line indicates theoretical chimerism if endogenous HSCs were displaced from bone marrow niches. (b) Additive donor HSC engraftment following repetitive transplantation. Unconditioned CD45.2 RAG2−/−γc−/− mice were transplanted with saturating doses of GFP+ CD45.1 HSCs (250–1000) and retransplanted with 4000 GFP− CD45.1 HSCs 8 weeks after the first transplantation. Bone marrow was harvested and donor HSCs were quantified as Lin−c-kit+Sca-1+Slamf1+CD34−CD45.1+ cells that are either GFP+ (first transplant) or GFP− (second transplant).

more niches than 50 cells. This plateau was about 0.5% of marrow sites. However, 50 cells on day 1 and another 50 cells on day 2 gave about 0.5% of each population (Fig. 5.4) [156]. Thus, about 0.5% of the marrow niches for HSC appear to be available at any moment, and these appear to be occupied by the next interval. Preclearing of HSC with a cytoreductive anti-c-kit antibody alone in immunodeficient mice leads to up to 80% engraftment with purified HSCs given in large numbers at the point that the monoclonal antibody reaches its nadir concentration in the blood [157]. The migration of HSCs into hematopoietic tissues involves a step of crossing macrophage-lined sinusoids. We have recently found that, during the mobilization process, HSCs and progenitors upregulate the expression of the integrin-associated protein CD47 [158]. One function of CD47 is as a ligand for the signal regulatory protein-α macrophage receptor [159]. In the absence of CD47, macrophage-bound cells are phagocytosed, whereas cells expressing high levels of CD47 signal the macrophage through the receptor to block phagocytosis. We have found that leukemias transfected with mouse CD47 at levels approximating those found on mobilized HSCs are protected from phagocytosis in vitro and engraft in vivo, whereas the same leukemias lacking these levels of CD47 are phagocytosed in vitro and fail to engraft in vivo [158]. Further, HSCs from CD47 knockout mice fail to engraft wild-type congenic mice [158]. Taken together, these experiments provide evidence that, during

migration, itinerant HSCs express high levels of CD47 to gain entry to hematopoietic sites. This interpretation of the fate of recently self-renewed and also resting HSCs calls for a deeper examination of hematopoietic niches in the marrow; niches that commit recently entering HSCs into the myeloid or lymphoid pathways need not be adjacent to the HSC niche if the daughter cells of HSCs are constantly migrating. In addition, the existence of a large flux of recirculating HSCs likely provides for the continuous replacement of empty HSC niches with functional HSCs; in the absence of such a pool of migrating HSCs, the death of an HSC could mean the permanent loss of that functional niche. This finding also has relevance when one considers claims of plasticity of stem cells in one or more tissues [5]. If tens of thousands of HSCs are fluxing through tissues every day, finding HSC activity in those tissues might only result from these itinerant HSCs, rather than from transdifferentiation of local tissue-specific stem cells into hematopoietic fates [5]. Ontogeny and aging of HSCs It is tacitly assumed that HSCs derive from the embryonic mesoderm. In mammals, the first site of definable blood cells appears to be the yolk sac blood islands, in mice at about 7.5 dpc [160,161]. Although the terminology used for development from the time of conception is E

42

Chapter 5

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Fig. 5.5 The ontogeny of hematopoietic progenitors. The sites of and duration of hematopoiesis in the fetus and neonate are shown. (Adapted from [178], with permission.)

(embryonic day) 1–21 (for mouse), in fact the transition from the embryo to fetus, at organogenesis, occurs at about 8–9 days post conception or coitus (dpc), so we will hereafter use the “dpc” abbreviation. At dpc 7–8, mouse yolk sacs contain cells capable of responding in vitro to produce hematopoietic CFCs [160,161]. The yolk sac connects to the developing embryo/fetus [162] at about dpc 8.5 later via the development of the vitelline vessels, which feed into the developing fetal liver. At very nearly that dpc 8–8.5 time interval, hematopoietic cells appear in and around the dorsal aorta of the developing fetus (reviewed in [163]). Transplantation of dpc 8–9 yolk sac blood island cells into the yolk sac cavity of dpc 8 allogeneic hosts results in mice that have a lifelong presence of donor-derived day 10 CFU-spleen (CFU-S) in marrow as well as thymocytes [93]. At about dpc 10–11, hematopoiesis begins in the fetal liver in the mouse, and continues there throughout fetal life (Fig. 5.5). Where do the first HSCs develop [164]? At dpc 3.5, the blastocyst implants into the uterus, and its inner cell mass appears to be a population of pluripotent cells without defined commitments [162]. Between dpc 3.5 and dpc 5.5, gastrulation occurs with the formation of the three germ layers [162]. It has been a mystery whether the developing postgastrulation mesoderm cells emigrate separately to yolk sac and embryo to give rise to independent origins of hematopoiesis, or whether only one of these sites is the initial site of commitment to hematopoiesis, the later appearance of hematopoiesis at the other site resulting from migration [93,163,165,166]. The idea of separate origins for extraembryonic (yolk sac) hematopoiesis and intraembryonic hematopoiesis was supported by the fact that yolk sac erythropoiesis resulted in the production of embryonic and fetal hemoglobins, while lifelong hematopoiesis resulted in the production of mainly adult hemoglobin [165,167]. These were called, respectively, primitive and definitive hematopoiesis [167]. Until recently, no experiment directly assessed whether these two kinds of hematopoiesis have distinct or common origins at the cellular level, To address this question, Nishikawa’s group used an inducible Runx1/ aml1 reporter system to label extraembryonic HSCs [168]. When embryos were induced at dpc 7.5, the time that HSCs first appear in the yolk sac blood islands [93,169], labeled hematopoietic cells were observed in the fetus and adult. As circulation was not established at this time point, these data suggest that the labeled HSCs that persist and

give rise to ongoing hematopoiesis must have derived from yolk sac HSCs and migrated into the embryo once circulation was established [168]. Because the allantois at dpc 9 has some runx1+ cells, it is another possible site of HSC generation in these experiments if the tamoxifen inducer has a half-life in the embryo of greater than a day. There is a close association of hematopoiesis and angiogenesis in both the developing blood islands and the developing dorsal aorta, and there is good evidence that mutant mice lacking the cell surface receptors for angiogenic peptides fail to develop either hematopoietic or angiogenic cells [91,170]. While these experiments suggest that a bipotent precursor called the hemangioblast exists that can give rise to both angiogenic stem cells and HSCs, only a small number of hemangioblast cells capable of producing both outcomes in vitro can be prospectively isolated from the pre-yolk sac embryo [171]. Rather than there being a hemangioblast precursor, it is possible that mutant mice that cannot make blood vessels properly cannot make blood because primitive stem cells travel via the blood, or that blood vessels make factors important for hematopoiesis. A recent set of experiments supports the hypothesis that the majority of embryo blood cells derive from distinct progenitors for hematopoiesis and endothelium [172]. Tetrachimeric mice were generated by the injection of four distinctly colored single cells into developing blastocysts. Analysis of the color combinations observed in the yolk sac of the resulting embryos revealed endothelial and hematopoietic cells of different colors in all individual blood islands, indicating that they developed from more than one cell (Plate 5.1). If both hematopoietic and endothelial cells derived from a common hemangioblast progenitor, each blood island would be monochromatic in these mice. However, single-color blood islands were not observed. Instead, the majority of blood islands contained blood and endothelial cells of multiple but not necessarily the same colors, indicating that multiple progenitors of each type came together to form each blood island [172]. About 15–20% of the blood islands examined could have derived from hemangioblasts; when we lineage-marked cells with the Flk1.cre/floxed Lacz-GFP (green fluorescent protein) constructs in the same ES cells, about 20% of blood cells were GFP+, showing an activated flk1 locus, but 80% were lacz+GFP−. Taken together, these data support about 20% of early blood cells deriving from a possible hemangioblast progenitor, and about 80% being derived from a hematopoietic progenitor that has not gone through a hemagioblast pathway. Similar conclusions were drawn by Shalaby et al. [91]. There is an interesting point coming out of ES cell biology that might be relevant to these issues. ES cells are derived from the inner cell mass of blastocysts, and likely represent pluripotent stem cells in the mouse at approximately dpc 3.5 age. They are maintained as pluripotent cells by the addition of leukemia inhibitory factor to their cultures, at least in mouse ES lines [173]. Removal of leukemia inhibitory factor from these pluripotent ES lines results in the onset of hematopoiesis, and during this development, at about 4 days, cells capable of in vitro hematopoiesis emerge, which express some markers shared with HSCs (Yamane, Domen, and Weissman, unpublished data). This interval, 3.5–4.0 days, correlates in developmental time to about dpc 7.5, when HSCs first appear in yolk sac blood islands [93,169]. Thus, these two systems appear in parallel to produce embryonic HSCs. The major site of the next (fetal) stage of hematopoiesis appears to be the fetal liver [161]. The numbers of HSCs in the fetal liver increase logarithmically from dpc 11–15. After dpc 15, fetal liver HSC numbers level off, but hematopoiesis continues to expand in the liver [53,174,175]. At dpc 16 hematopoiesis appears in the fetal spleen in mice, and at dpc 18 in the fetal bone marrow. The initiation of fetal liver hematopoiesis is blocked in mice with mutations in integrin α4β1 (very late antigen-4 [VLA-4]) or integrin α5β1

Biology of Hematopoietic Stem and Progenitor Cells

(VLA-5) [176,177]. Because integrins are involved in cell migration, it is reasonable to propose that immigration of these cells into or beyond the fetal liver might require steps that include the expression of α4β1, or induce the expression of integrin α5β1 at this stage of the liver. Integrin α4β1 is used by trafficking HSCs throughout life to enter sites of hematopoiesis [148], so the endowment of expression of integrin α4β1 permits fetal liver HSCs to be assayed by transplantation in fetuses or adults. It is important to note that neither yolk sac blood island HSCs nor ESderived HSCs in their native state can be transplanted into adult irradiated mice. It has been reported that the transition of yolk sac HSCs and ES-derived HSCs from that embryonic stage to the fetal stage can be facilitated by enforced expression by HoxB4, or by HoxB4 and cdx4 in these cells [118]. Therefore, there are important clues to how embryonic HSCs, presumably capable of primitive hematopoiesis, can develop into fetal HSCs capable of definitive hematopoiesis. During the various stages of fetal HSC function, it seemed possible that waves of HSCs emerge from tissues such as the fetal liver at specific time points to be accepted by and to engraft in secondary sites such as fetal spleen, fetal bone marrow, and lymphoid sites such as the thymus [93]. However, HSCs are found constitutively in the blood throughout fetal life, and it must be the preparedness of the developing organs such as the spleen, marrow, and thymus that results in the movement of hematopoiesis to those sites [178]. A detailed examination of HSCs in the developing fetal liver with respect to T-cell developmental potentials has revealed that changes are occurring at the level of HSCs which show that HSCs develop in a quantal but dynamic process [179]. Developing T cells gain their specificity and function in a coordinated series of molecular events, including selection of which class of T-cell receptor (TCR) genes are rearranged and selected, and to which distant sites the T cells will migrate. Within the thymus during fetal development, the first T cells to emerge use products of the TCR Vγ3 and TCR δ gene families, to be followed by use of the TCR Vγ4, then Vγ2 and Vγ5 and TCR αβ gene families. In mice, the TCR V genes at the γ locus that must rearrange to be expressed are, extending from the most proximal site of potential rearrangements, Vγ3, Vγ4, Vγ2, and then Vγ5. The first T cells to emerge in the thymus are Vγ3 cells, which colonize the skin only during fetal life [180]. While Vγ3 T-cell development is shutting down in the thymus, Vγ4 T-cell development arises, and these T cells will migrate during their fetal life to the presumptive female genital tract epithelia and the tongue epithelium of both sexes [179,180]. The development of Vγ4 T cells decreases at about day 16 or 17 of fetal life, to be overtaken by the almost simultaneous onset of that will inhabit the entire gastrointestinal tract, and αβ T cells that are largely restricted to lymphoid tissues. The kinetics of T-cell development can be studied with the use of fetal thymic organ cultures, although it should be noted that these are not physiologic cultures. When single clonogenic dpc 12 fetal liver HSCs are added to fetal thymic organ cultures, those thymuses produce Vγ3, then Vγ2, then Vγ5, and TCR αβ T cells [179]. When dpc 14 fetal liver HSCs are placed at clonal levels into the same stage fetal thymic organ culture, Vγ3 T cells do not emerge, but the rest of the repertoire is developed. Finally, dpc 12 fetal liver HSCs added directly into the adult thymus cannot produce the fetal program of Vγ3 T cells, but can produce the mature program of Vγ2, Vγ5, and αβ TCR cells [179]. Taken together, these studies indicate that, probably with each cell division, the fetal liver HSCs are changing (usually reducing) their developmental fates that would only be read out when their progeny entered the fetal thymus, and the change in these fates and the readout of these fates is dependent upon factors within HSCs and within the thymus microenvironment. After birth, and at least in young adult mice, the numbers of LT-HSCs, ST-HSCs, and MPPs are regulated at a relatively constant level.

43

HSCs in the mouse go through cell autonomous changes in number and developmental potential during aging. We first reported that there is a gradual loss of cells of the MPP phenotype, and an increase in both the number of LT-HSCs and the fraction of LT-HSCs that are in the cell cycle [83], most dramatically seen in the marrow of geriatric mice. With a refinement of markers for LT-HSC versus all other MPPs, we found that the number of LT-HSCs increased as reported, but that they retained a low level of cell-cycle activity; the increase was found primarily in an MPP fraction [96]. In old mice, myelopoiesis is retained or even enhanced, while lymphopoiesis in both T and B lineages falls. These fates are cell intrinsic; co-transplantation of 2-month-old and 2-year-old LT-HSCs into irradiated young mice led to increased output of LT-HSCs from the old versus young stem cells, and decreased output of CLP and T and B cells from the old HSCs compared with the young stem cells [96]. These same trends were seen in the small number of aged hosts transplanted with young and old HSCs. The cell intrinsic biologic properties of old versus young HSCs were mirrored by a microarray analysis of these highly purified cells: young HSCs had both myeloid and lymphoid transcripts, but old HSCs overexpressed myeloid and underexpressed lymphoid transcripts [96]. Of the top 32 myeloid transcripts overexpressed in old HSCs, 13 have been identified as proto-oncogene or translocation partners of myelogenous leukemia proto-oncogenes in humans. As myelogenous leukemias increase in incidence with age, it is reasonable to propose that initiating events may occur in HSCs, and if so, it is possible that genomic events such as translocation, inversion, etc, occur preferentially at highly transcribed loci. Does hematopoiesis only derive from HSCs, and do HSCs only give rise to blood? Several years ago, there were reports that bone marrow hematopoietic cells could give rise to brain cells, that brain stem cell populations could give rise to blood, that fat cells could give rise to neurons and mesenchymal fates, that muscle stem cells could give rise to hematopoietic and myogenic outcomes, and a host of others (reviewed in [181]). What was lacking in most of these experiments was the demonstration that the transplanted population was in fact a stem cell purified to homogeneity that could give rise at the clonal level to two different tissue types, and that the tissue types that had been generated were robustly characterized as functional mature cells of that tissue without cell fusion. Attempts to repeat the experiments showing transdifferentiation of CNS stem cells to hematopoietic tissues have thus far failed. A reexamination of the cells in muscle that give rise to blood show them to be CD45+ committed HSCs, separable from muscle progenitors that, on their own, give rise to muscle [182,183]. In some circumstances, regenerating muscle following an injury can incorporate cells whose markers are expressed in the regenerated muscle tissue, and the most common transplantable source of these cells is bone marrow [184,185]. While some claims have been made that these are derived from HSCs, in fact single purified HSCs, even over long intervals, cannot contribute to muscle or any other tissue, except a rare set of liver cells in irradiated hosts [5]. In another example, liver regeneration of hepatocytes bearing markers from injected purified HSCs, following multiple rounds of selection through liver toxicity, can occur [186], but recent evidence [187,188] indicates that these are mainly a result of rare cell fusion events between myelomonoctic progeny of the HSCs and some precursor of liver cells. Furthermore, the marrow or HSC or circulating precursor cells that enter undamaged or kainite-damaged brains are limited to microglia and CD45+ cells, as well as rare binucleate Purkinje cells derived by cell fusion [189]. We had previously proposed that cases of transdifferentiation of one tissue-specific stem cell to another tissuespecific stem cell might not really be transdifferentiation but differen-

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tiation from a more pluripotent precursor [1]. That possibility is still viable. The many claims that transdifferentiation could occur reflect a problem in stem cell biology as a young field. Most new investigators to the field did not appreciate the importance of purifying the populations to homogeneity, populations of cells of apparent phenotypic and functional homogeneity, and did not mark cells retrovirally or with other genetic markers to follow the fate of clonogenic precursors. The transition from discovery to accepted scientific fact It is important to note that, in order for a reported discovery to become accepted as scientific fact, the following several criteria need to be met: 1. The initial discovery must be published in fully peer-reviewed journals. 2. The experiment as published must be repeatable in many independent laboratories. 3. The phenomenon described should be so robust that other experimental methods must reveal it. 4. In the case of transplantation, the tissue regeneration must be therapeutic in quantity and quality. Based on these criteria, we do not believe there is sufficient evidence for any of the transplant claims of transdifferentiation. In addition, bone marrow is not the same as HSCs, but is a population of mature or maturing cells, and certainly includes at least two or three stem and progenitor populations: HSCs, mesenchymal stem cells (MSCs), and endothelial precursors, which might really be MSCs [18]. Thus, it is possible that the transdifferentiation ability or “plasticity” ascribed to HSCs to interpret formerly unexpected outcomes from cell transplantations, if not the result of cell fusion, is more likely derived from a class of previously unappreciated adult pluripotent cells, or perhaps the normal developmental fates of MSCs are not yet appreciated. It is conceivable that some of these very rare cell fusions could be part of a regenerative process, but there is no evidence today that such is the case. Thus, it is inappropriate for such initial claims to be considered true enough to be the basis of clinical trials or care protocols or public funding and policy decisions.

Lineage-committed hematopoietic progenitor cells Considerations for the definition/isolation of hematopoietic progenitors The developmental process from HSCs to mature cells must involve intermediates that have lost stem cell potentials but are not yet terminally differentiated. An important question and active area of research is the sequence in which lineage commitment occurs. Several findings have suggested the existence of oligopotent progenitors. First, the lack of lymphocyte in patients with severe combined immunodeficiency syndromes was taken as evidence for the existence of oligopotent, lymphoid-committed progenitor cells or lymphoid stem cells. However, the loss of lymphocytes caused by adenosine deaminase deficiency [190,191] or by mutations of the common cytokine receptor γ-chain (γc) [192,193] does not necessarily imply a common progenitor. Rather, several lymphoid cell types or their progenitors might be most susceptible to the induced alterations [194] or might be dependent on the same signal transduction mechanisms. This same caution needs to be applied to the interpretation of hematopoietic phenotypes of genetically altered mice, as mutants of Ikaros [195] or Notch-1 [196] that could either be essential factors for a common or multiple different progenitors [197]. Second, leukemia cells can be viewed as cells that are arrested at early developmental stages but have gained self-renewal capacity. The occur-

rence of leukemias that either coexpress antigens normally associated with myeloid or lymphoid cell types (mixed-lineage leukemias) or that harbor two leukemia populations of clonal origin (bilineal leukemias) could be taken as evidence for the existence of bipotent B-myeloid or T-myeloid progenitors [198–203] (reviewed in [204–206]). However, leukemia clones could be derived from HSCs, from MPPs or from restricted progenitors that, because of their altered gene expression profile, display cell surface gene products which are normally not present at these developmental stages. Third, the fact that single cells in bone marrow give rise to colonies that contain all myeloid but no lymphoid progeny in vitro (colonyforming unit-granulocyte/erythrocyte/macrophage/megakaryocyte [CFUGEMM]) or in vivo (mixed CFU-S) is suggestive for the existence of oligopotent myeloid progenitors. However, cells that give myeloid readout could also be multipotent but did not find the conditions to read out all possible progeny [48,207,208]. In light of these problems, several prerequisites need to be met to define restricted progenitors in a developmental hierarchy. First, in order to prospectively identify candidate populations, they need to be isolated to highest possible homogeneity/purity. Second, if a population shows oligopotent differentiation activity, it must be demonstrated that at least some single cells within this population give oligopotent readout, i.e. that this population is not a mixture of different monopotent progenitors. Third, it further needs to be shown that single oligopotent progenitors are not HSCs or MPPs. These prerequisites are difficult to achieve. At any given time point, progenitor cells might undergo intrinsic “stochastic” random commitment events, might not find suitable microenvironments to read out in all their possibilities or might even not read out at all [209,210]. However, by applying the above criteria, we and others were able to identify oligopotent developmental intermediates in mice and humans. These data strongly support the hypothesis that multipotent hematopoietic progenitors first lose their self-renewal ability and consecutively commit to either the lymphoid or the myeloid developmental pathway. Although not formally proven, based on frequency, cycling status, and in vitro and in vivo expansion potential of bone marrow lymphoid and myeloid committed progenitors, it seems possible that all hematopoiesis develops through either a common lymphoid or common myeloid developmental stage. CLP cells and lymphoid development Lymphoid cell development from HSCs is dependent on externally provided differentiation, growth, and survival factors. Of those, IL-7 might be most important. Its cognate high-affinity receptor is a complex composed of the IL-7 receptor-α (IL-7Rα) chain [211] and the γc chain [193,212]. Neutralizing antibodies to IL-7 or genetic ablation of either IL-7 or IL-7Rα inhibits both T- and B-cell development in vivo [213,214]. Targeted deletion of the γc gene leads to the loss of T and B cells and the additional loss of NK cells [215–217], presumably because of the impaired formation not only of the IL-7R, but also the IL-2R and IL-15R that might be nonredundant cytokines for NK cell development [218– 220]. Further, mice that lack Jak3, a signal transduction molecule associated with γc [221,222], display a phenotype similar to the γc-deficient mice [223–225]. Also, patients with genetic defects in Jak3 show similar defects in lymphoid development to patients with γc gene disruptions (X-linked severe combined immunodeficiency syndrome) [226,227]. Based on these data, IL-7 is recognized as a nonredundant cytokine for both T- and B-cell development that could act as a proliferation factor or a survival factor and/or could initiate lineage-specific developmental programs. IL-7R is expressed in both developing T and B cells, and in mature T cells [228]. In genetically modified mice that are either IL-7Rα−/−,

Biology of Hematopoietic Stem and Progenitor Cells

45

HSC

Population

MPP

HSC

Lin–KSCD150+ Flk2–Thylo(CD105lo CD34–FcγRlo)

MPP

Lin–KSCD150–Flk2+ Thylo/–(CD105lo, CD34–/+ FcγRlo)

GMLP GMLP

MEP

GMP ProT

Lin–KitintScaintIL7Rα+Flk2+

MCP

Lin–Kit+Sca–Ly6c–FcεRIα–CD27–T1/ST2+β7+

CMP

Lin–Kit+ScaloCD150+CD34+FcγR+

GMP

Lin–Kit+Sca–CD150+ Flk2–/+FcγRlo

MEP

Lin–Kit+Sca–CD150+ Flk2–FcγRlo

MkP

Lin–Kit+Sca–CD150+ Flk2–CD41+CD9+

BLP

MCP

MkP

Lin–Kit+ScaloFlk2++

CLP CLP

CMP

Phenotype

EP T cells

Mast cells

B cells

EP

Lin–Kit+Sca–CD150+ Flk2–CD105+

Dendritic cells Platelets

RBC

Myelomonocytic cells

Fig. 5.6 Schematic of hematopoietic development. Composite of current data from mice indicating intermediates in the hierarchy of hematopoietic differentiation. Hematopoietic cells (HSC), long-term reconstituting, self-renewing; MPP multipotent progenitors with limited or no self-renewal leading to transient but multilineage reconstitution; GMLP; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; BLP, B-lymphocyte progenitor; ProT, T-cell progenitor; GMP, granulocyte–macrophage progenitor; MEP, megakaryocyte–erythroid progenitor; MCP, mast cell progenitor; MkP, megakaryocyte progenitor; EP, erythroid progenitor.

γc−/− or IL-7−/−, T-cell progenitors express low levels of Bcl-2, an antiapoptotic protein [229]. However, incubation of T cells from IL-7−/− mice with recombinant IL-7 results in the upregulation of Bcl-2 [230], and enforced expression of Bcl-2 in either IL-7Rα−/− or γc−/− mice leads to significant rescue of αβ T-cell development [25,230,231]. Therefore, a critical role of IL-7 in developing and mature αβ T cells is the promotion of survival via expression of Bcl-2 or other antiapoptotic proteins as possibly Bcl-Xl [232]. However, enforced expression of Bcl-2 was not sufficient to rescue B-cell development and αβ T-cell development [25,87,230]. Here, IL-7R-mediated signals are necessary for the rearrangement of immunoglobulin heavy chain V segments via Pax-5 gene activation [233,234] and for V–J recombination of αβ TCR genes via Stat5 [235,236]. Therefore IL-7R signaling can either lead to the transmission of “trophic” survival signals in the T-cell lineage or “mechanistic” differentiation and proliferation signals in the B and αβ T-cell lineages, respectively. Thus, it was reasonable to search for oligopotent CLPs within the Lin−IL-7Rα+ fraction in adult mouse bone marrow. We identified a population of Lin−IL-7Rα+ Thy-1−Sca-1loc-kit lo cells that also express γc, indicating that this population possesses functional IL-7R (Fig. 5.6) [25]. In contrast, both LT-HSC and ST-HSC populations are IL-7Rα−. These CLPs possess rapid and potent T-, B-, and NK-cell-restricted differentiation activity in reconstitution assays. In both in vitro and in vivo assays, Lin−IL-7Rα+ Thy-1−Sca-1loc-kit lo cells completely lack myeloid differentiation activity [25]. Also no day 8 and day 12 CFU-S activity could be detected [25,37]. Injection of 1000 CLPs could generate 0.6– 1.1 × 107 CD3+ spleen T cells by 4–6 weeks, and 1.4 × 107 B220+ spleen B cells by 2 weeks in vivo. CLP-derived T- and B-cell generation peaks

7–10 days earlier than similar numbers of T and B cells derived from the same number of HSCs [25]. However, in contrast to HSC-derived cells, numbers of CLP-derived T and B cells begin to decline after 4–6 weeks, indicating that this population has no or limited self-renewal activity. Using a two-step assay, we demonstrated that CLPs contain clonogenic progenitors for both T and B cells. Single cells were cultured in methylcellulose in the presence of SLF, IL-7, and Flt3L to expand cell numbers, and a portion of the day 3 colonies were picked up and injected directly into the thymus. Those injected into the thymus differentiated into all stages of T-cell development and, in some cases, differentiated into both T- and B-lineage cells. The further cultured cells formed Blineage colonies composed of pro-B and pre-B cells [25]. Therefore, the defined CLPs match the criteria for the definition of oligopotent progenitor cells. We have subsequently shown that the purity of CLPs is further refined based on their expression of Flk2, whereas the Flk2 fraction of the previously defined CLPs contains B-committed cells [237]. Accordingly, CLPs exist downstream of Flk2+ ST-HSCs and MPPs in normal hematopoiesis [51,52,238]. Based on their frequency in bone marrow, their cycling status and their in vivo T-, NK-, and B-cell generation potential, CLPs theoretically could be a developmental intermediate for most if not all mature lymphoid cells. The commitment of CLPs to either the T-, B- or dendritic cell lineage may be determined simply by the microenvironment and signals that CLPs encounter. However, it is still unclear whether CLPs are an indispensable stage of T- and B-cell development. Coffman and Weissman identified the B220 marker that is present on all B-lineage cells [239], and early B-cell progenitors were found to be

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B220+, CD43+, CD24-BP1/6C3− [240,241]. Hardy and his colleagues further subdivided B-cell progenitors into fractions: pre-pro-B cells (fraction A0: AA4.1+CD4loB220−HSA−; A1: AA4.1+CD4+B220+HSA−; A2: AA4.1+CD4−B220+HSA−) and pro-B cells (fraction B: B220+CD43+HSA+6C3/BP-1−; C: B220+CD43+HSA+6C3/BP-1+) [242– 244] (reviewed in [245]). Some fraction A0 cells express c-kit and Sca1 and can give rise not only to B cell, but also to myeloid and T-cell progeny. Therefore, they might contain MPPs. The majority of fraction A1 cells are c-kit − and lack myeloid potential, but minor T-cell potential can be detected after intrathymic injection. Pro-B cells are B-lineage committed, rearrange DH–JH genes (fraction B) and undergo V–DJ recombination (fraction C). In view of their T- and B-cell readout capacity, the A0 or A1 fraction could contain CLPs, but because different markers were used for their isolation, a direct comparison of data from these populations is not possible. However, a majority of A0 and A1 fraction cells do not express IL-7Ra, while IL-7Ra is expressed on fraction A2 cells. B-lineage development has subsequently been studied in detail by a variety of groups using different combinations of markers that prevented direct comparison of the data [242,244,246–252]. Recently, we performed analyses to clarify these data by incorporating the majority of these described markers and others in our experiments. These studies allowed us to identify a committed B-lineage progenitor downstream of the CLP with the surface phenotype B220+CD43+AA4.1+Flk2+CD27+IL 7Ra+c-kitloCD24−CD19−CD11c−Ly6c−CD4− [253]. This population is distinguished from CLPs by the expression of B220 and comprises only approximately 5% of fraction A/pre-pro B cells, while the remaining cells are dendritic cell (DC) progenitors. Using the same markers described above, we also identified heterogeneity within the fraction B cells, which are thought to lie immediately downstream of the fraction A cells. One subset, which we term fraction B1, is highly proliferative, while the second subset, fraction B2, is largely quiescent and smaller in size. In contrast to the refined pre-pro B cells, fraction B1 cells do not respond strongly to IL-1, suggesting that the sensitivity to inflammatory signals within B-committed progenitors is restricted to pre-pro B cells and is lost immediately upon differentiation to their immediate downstream progeny. The earliest thymic progenitors (double-negative-1 cells) are CD4loCD8−CD44+CD25−c-kit+ cells [254–257] and therefore closely resemble the phenotype of HSCs. Most of these early thymic progenitors are T-cell committed (pro-T cells); however, some cells within this population are capable of differentiating into B, NK, and dendritic cells and, at a low frequency, into myeloid cells [257], but their clonal origin has not been established [256–258]. Whether the earliest thymic progenitor population contains a small number of recently homed CLPs that, within this microenvironment, preferentially differentiate into T cells, or whether CLPs commit to the T-cell lineage while still in bone marrow and successively home to the thymus [259], has been controversial. Allman et al. [260] suggest that thymopoiesis is maintained through early thymic progenitors that might develop from bone marrow progenitors more closely related to HSCs and not via a CLP-dependent pathway. This is based on findings that some cells (early thymic progenitors) within the double-negative-1 fraction do not express IL-7Rα at high levels, have some myeloid potential, give rise to T and B cells with kinetics that resemble those of ST-HSCs [52] more closely than those of CLPs, and were present at near-normal frequencies in Ikaros−/− mice while CLPs were not detectable by phenotype. While these data add important information on possible alternative T-cell developmental pathways, they do not assess directly how many early thymic progenitors or mature T cells are CLP-derived or whether most thymopoiesis is independent of CLPs. Both HSCs and CLPs might home to the thymus,

where T-cell commitment might immediately be initiated and thymus homing receptors might be lost. The issue of progenitor commitment to the T-cell lineage in bone marrow versus thymus is further complicated by the observation that, at least in experimental settings, extrathymic T-cell development can occur [261–263]. Recent studies comparing (Flk2+CD27+) MPPs and CLPs for their ability to seed the thymus and generate T cells indicate that CLPs are indeed the major source of normal thymic development (Serwold, Erhlich, and Weissman, submitted for publication). Both MPPs and CLPs injected intravenously give rise to thymocytes, but the MPPs do so with delayed kinetics reflective of their homing to the bone marrow and giving rise to CLPs that then go on to seed the thymus directly. Additionally, thymic myeloid cells were observed to derive from transplanted MPPs and not CLPs, suggesting that these cells are the progeny of myeloid progenitors that also enters the thymus. Further, when Flk2+ CLPs were plated in conditions capable of supporting differentiation into multiple lymphoid and myeloid lineages, single CLPs gave rise to T, B, NK, and DC lineages [264]. Approximately 50% of single CLP clones gave rise to both B- and T-lineage cells in this assay, and none gave rise to any macrophages or neutrophils. Taken together, these data confirm the role of CLPs as potent progenitors of both B and T lymphocytes in vivo and in vitro. Also of note, CLPs co-transplanted in addition to HSCs in autologous or allogeneic transplantation models can rescue mice from otherwise lethal cytomegalovirus (murine CMV) infections [265], providing additional evidence for the robust and functional T- and/or NKcell reconstitution capacity of CLPs in vivo. In humans, evidence for a T/NK bipotential precursor population in fetal thymus [266] and a terminal deoxynucleotidyl transferase (TdT)positive candidate lymphoid precursor population in CD34+ bone marrow cells have been reported [267]. However, clonal analysis was not carried out. A subpopulation of the CD34+TdT+ cells expresses the neutral endopeptidase CD10 [268]. Later, it was shown that fetal and adult bone marrow Lin−CD34+Thy-1−CD38+CD10+ cells contain clonal progenitors of B, NK, and dendritic cells [269]. As a population, these cells give rise to T cells in the SCID-hu thymus assay but could not generate myeloid progeny. In another report, it was shown that cord blood CD34+CD38−CD7+ cells contain clonal B, NK, and dendritic cell precursors; however, T-cell readout was not evaluated [242]. Therefore, both studies suggest but do not formally demonstrate the existence of CLPs in humans. IL-7 signaling might not be essential for normal B-cell development in humans because B cells can be generated without IL-7 in vitro [270], and disruption of the IL-7R in vivo causes T-cell but not consistently B-cell deficiencies [271,272] (reviewed in [268]). Human early lymphoid progenitors, in analogy to mouse CLPs, express IL-7Rα. Indeed, Lin−CD34+CD38+CD10+ cells can be subdivided into a CD10+IL-7Rα− and a CD10loIL-7Rα+ fraction, with the latter being highly enriched in clonal B-cell progenitors [273]. It has also been shown that pro-B cells proliferate and differentiate in culture with IL7 and bone marrow stromal cells, whereas pre-B cells are no longer responsive to IL-7 [274]. It is important to determine whether CD10loIL-7Rα+ cells or CD10+IL-7Rα− cells contain both T- and B-cell progenitors and whether thymus seeding cells share some of these phenotypes (for a review of human early thymocyte development, see [275]). CMP cells and myeloid development The identification of CLPs in the IL-7Rα-expressing fraction of mouse bone marrow which holds no myeloid differentiation activity [25] suggested that complementary progenitors common to all myeloid cells might exist in the IL-7Rα-negative cell fraction. In addition, we had noted that the Sca-1− subset of Thy-1loLin− cells contained myeloery-

Biology of Hematopoietic Stem and Progenitor Cells

throid progenitors [40]. Therefore, to exclude HSCs and CLPs, we searched for myeloid progenitors within the Lin−IL-7Rα−Sca-1−c-kit + bone marrow fraction. We identified three myeloid progenitor populations [26]: CMPs, FcgRloCD34+ (now known to be Sca-1lo not Sca-1− [276]), and their lineal descendants; megakaryocyte/erythrocyte progenitor cells (MEPs; FcgRloCD34−); and granulocyte/macrophage progenitor cells (GMPs; FcgRhiCD34+). These three myeloid progenitor subsets likely represent the major pathways for myeloid cell differentiation because they contain the vast majority of myeloid progenitor activity in steady-state bone marrow. CMPs give rise to all myeloid colonies in vitro, including CFU-mixed; GMPs give rise to CFU-granulocytes (CFU-G) and CFU-macrophage (CFU-M) as well as CFU-granulocyte–macrophage (CFU-GM); and MEPs give rise to burst-forming unit-erythroid (BFU-E), CFU-megakaryocyte (CFU-Meg) as well as CFU-megakaryocyte/erythroid (CFUMegE), each with high cloning efficiency. We demonstrated that CMPs as a population differentiate in vitro into cells with MEP and GMP phenotype and function [26]. Further, the majority of single CMPs generate both GM- and MegE-related progeny. Upon in vivo transfer, the three cell populations give short-term but not long-term readout corresponding to their in vitro activities, indicating that they have only limited if any self-renewal activity [37]. MEPs provide radioprotective cells for lethally irradiated mice housed under infection control conditions [37]. Lethally irradiated mice injected with MEPs, CMPs, ST-HSCs or MPPs show transient donor-derived hematopoiesis sufficient to sustain survival while reconstitution with residual host HSCs occurs [37,38,87]. In addition, the majority of day 8 CFU-S activity resides within the MEP population but is absent from GMPs [37], supporting previous findings that day 8–9 CFU-S are largely erythroid [40,277]. Our data also confirm previous findings that the majority of day 12 CFU-S activity resides not within lineage-committed progenitors but within the more primitive HSC populations with some activity in CMPs and MEPs [35,38,40]. Neither B- nor T-cell differentiation activity was detectable in either MEPs or GMPs. CMPs could not generate T cells; however, a small number of B-cell progeny were detectable in vitro and in vivo [26]. We also isolated a pure population of megakaryocyte progenitors in mice. These Lin−c-kit+Sca-1− CD34+CD9+CD41+ cells are highly efficient at producing megakaryocyte CFCs in vitro, micromegakaryocyte loci in vivo, and donor-derived platelet production in vivo, while they are devoid of erythrogenic activity [278]. The subsequent identification of additional markers has enabled higher-resolution delineation of myeloid developmental pathways. First, we and others found that the CMP population, like the CLP, was heterogenous for Flk2 expression [237,279] and the Flk2+ fraction contained the CMP activity and could give rise to GMPs, MEPs, and all myeloerythroid progeny including DCs. More recently, the addition of CD150 (Slamf1), CD105 (endoglin), and CD41 to the analysis revealed further heterogeneity within the myeloid progenitor populations [280]. These markers revealed that the previously defined CMPs could be further subdivided into CD150+ and CD150− fractions, and that MEPs can be divided into three subpopulations based on endoglin and CD150 expression. Detailed analyses with these markers led to the identification of isolatable pre-GM, pre-MegE, and pre-CFU-erythrocyte populations that give appropriate lineage restricted readouts in vitro and in vivo [280]. These data are consistent with another study that used a Gata1-GFP reporter to identify a population that refines the CMP phenotype or precedes it in the developmental scheme [276] (Fig. 5.6). Specifically, this population is Lin−Kit+ScaintFlk2−Gata1lo and gives rise in vivo to all myeloid lineages but very low lymphoid readout. Using a PU.1 reporter, this study also described a granulocyte–lymphocyte–macrophage progenitor that could give rise to lymphocytic and myeloid lineage cells but

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not platelets, similar to results from Adolfsson et al. which indicated that those MPPs that express the highest levels of Flk2 have lost platelet-forming ability [281]. This is also consistent with data suggesting that PU.1 represses MegE development [282]. Alternative developmental pathways There are several recent in vivo studies as well as in vitro data suggesting that myeloid versus lymphoid differentiation may not always be the first decision made during hematopoietic development, and either bipotent B-cell/myeloid or T-cell/myeloid progenitors, or both, might exist. In support of a B/macrophage progenitor, it was demonstrated that several B-cell lines can be modified to produce cells with features of macrophages by in vitro manipulations [283–285]. Importantly, transfection of v-raf into B-cell lines established from Eμ-myc transgenic mice could convert them into macrophages that maintained the identical rearrangement of an immunoglobulin H (IgH) gene [284]. This observation suggests that latent developmental potentials that are not accessible under normal conditions might be regained through genetic alteration. Indeed, we have shown that CLPs, which under normal conditions never generate myeloid readout, produced granulocytes and macrophages after they were genetically engineered to express the human IL-2 receptor β chain (IL-2Rβ) or the granulocyte/macrophage colony-CSF receptor and were stimulated with the cognate ligands [286]. This was blocked by enforced expression of Pax-5 [287]. Also in vitro, single fetal liver cells could be demonstrated to give rise to both B/myeloid and T/myeloid progeny [207,288,289]. In these experiments using a fetal thymic organ culture system in the presence of 60% oxygen for single cell readout, Kawamoto et al. [289] detected B/T/myeloid, B/myeloid, T/myeloid, but never T/B-bipotent progenitors from fetal liver cell populations. However, in these high oxygen concentration cultures, only approximately 5% of single Lin−Sca-1+c-kit + HSCs gave rise to multilineage (T/B/myeloid) outcomes, suggesting that this system is not reproducible enough to demonstrate all differentiation capacities of HSCs. Also, differentiation into T/myeloid or B/myeloid lineages might simply be because of the “random” commitment of HSCs in these culture conditions. Alternatively, T/B-committed bipotent progenitors might not be contained in the Lin−Sca-1+c-kit + population but only in the Lin−Sca-1loc-kit lo CLP fraction [25,290]. Recently, bipotent B/macrophage progenitors were also described at a very low frequency (~0.02%) in adult hematopoiesis in vitro [291], supporting the hypothesis that a B/macrophage developmental line might be conserved beyond fetal hematopoiesis. As described above, recent data also suggest that HSCs and progenitors circulate through the blood and reside in the tissues before returning to the blood via the lymphatics [155]. Further, these cells were shown to differentiate in the tissue directly into dendritic, myeloid, and B220+ cells. This result, taken together with studies that HSCs respond to reduced hematocrit [76], in vitro data providing evidence for direct differentiation to megakaryocyte development [88], and studies suggesting oligo- and bipotent progenitors, suggest that HSCs percolate through the tissues as a means of directly assessing the specific hematopoietic and immune demands. It is reasonable to hypothesize that these cells reflect the responsiveness of the hematopoietic system and indicate that steadystate hematopoiesis when demand for each lineage is constant is distinct from hematopoieis that occurs during infection or blood loss, when there is a specific need for an increase in certain lineages. Being able to shut down production of lineages not in demand would also be a means of conserving resources such that white blood cells are not generated at the expense of RBCs during anemia and, conversely, RBCs and platelets are not generated at the expense of innate or specific immune cells when they are in demand.

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Human myeloid progenitors As in the case of CLPs, multiple studies suggested the existence of human oligopotent myeloid progenitors [292–296]. Recently, we were able to identify human bone marrow and cord blood cell populations that were counterparts of mouse CMPs, GMPs, and MEPs [273]. They are negative for multiple mature lineage markers (including early lymphoid markers that might define human CLPs as CD7, CD10 or IL-7Rα), and all are CD34+CD38+. They are distinguished by the expression of CD45RA, an isoform of the CD45 cell surface tyrosine phosphatase that can negatively regulate at least some classes of cytokine receptor signaling [297], and IL-3Rα, a receptor that upon activation supports the proliferation and differentiation of primitive progenitors [88,298,299]. CD45RA−IL-3Ralo (CMPs), CD45RA+IL-3Rαlo (GMPs), and CD45RAIL-3Rα− (MEPs) cells show high myeloid cloning efficacy but low long-term (stromal hematopoietic) culture-initiating cell capacity, indicating that they do not self-renew. Although FcγRII–III (CD16/CD32) expression distinguishes mouse CMPs and GMPs, it was not detectable on any of the human myeloid progenitor populations, and all human clonal myeloid progenitors express CD34 while mouse MEPs are CD34− [26]. Interestingly, in mouse myeloid progenitors, IL-3Rα as well as CD45RA expression were both negative to low in MEPs, intermediate in CMPs, and positive in GMPs, without providing sufficient discrimination to clearly set a cut-off level between the different cellular fractions. As with the mouse myeloid progenitors, CMPs give rise to MEPs and GMPs in vitro, and a significant proportion of CMPs have clonal granulocyte/macrophage and megakaryocyte/erythrocyte readout potential. We did not test T-cell differentiation ability; however, no in vitro B- or NK-cell readout could be detected. However, some B cells might develop from large numbers of transplanted CMPs. As in the case of mouse CMPs, it is important to clarify whether the CMP population contains oligopotent B/myeloid progenitors, or monopotent B-cell progenitors occurred because of small numbers of contaminating B-cell progenitors in the large number of transplanted cells (see below).

Lineage commitment in fetal hematopoiesis While fetal liver HSCs show major similarities to adult HSCs, some phenotypic and functional differences exist [175,300–302]. Remarkably, fetal liver HSCs possess some cell fate potentials such as the generation of Vγ3+ and Vγ4+ T cells [179] and B-1a lymphocytes [262,303] that are not detectable in adult HSCs. Therefore, it was important to determine the phenotype and developmental capacities of fetal liver CLPs and CMPs. We recently identified fetal liver CLPs (Lin−IL-7Rα+ B220− /lo Sca-1loc-kit lo) [290], and the myeloid progenitors fetal liver CMPs (Lin−IL-7Rα−Sca-1−c-kit+AA4.1−FcgRloCD34+), fetal liver GMPs (Lin− IL-7Rα−Sca-1−c-kit +AA4.1−FcgRhiCD34+), and fetal liver MEPs (Lin−IL7Rα−Sca-1−c-kit+AA4.1−FcgRloCD34−) [304] that show high similarities to their adult counterparts. However, some significant differences in proliferative potential colony-forming activity, and lineage differentiation capacities exist. Of special note, fetal liver CLPs generate CD45+CD4+ CD3−LTb+ cells that are candidate initiators of lymph node and Peyer’s patch formation [305–308]. About 5% of these IL-7R+ fetal liver CLPs differentiated into macrophages as well as into B cells, at least in vitro. Interestingly, the fetal liver CLPs fail to express Pax-5, a suppressor of macrophage development [306] while adult marrow CLPs that lack myeloid potential are Pax-5+ [290]. Conversely, although fetal liver CMP and downstream fetal liver MEPs or fetal liver GMPs do not give rise to T cells, fetal liver CMPs possess some in vitro and in vivo B-cell potential. A close relation between B-cell and macrophage development for adult and fetal hematopoiesis was suggested before (see below). It can therefore be speculated that, during commitment to the

lymphoid or myeloid lineage in fetal liver hematopoiesis, the developmental capacity for macrophages and B cells might be the last to be lost in commitment to either the lymphoid or myeloid lineage. DC development DCs were initially defined as bone marrow-derived nonmacrophage leukocyte populations with high antigen presentation capacities and the ability to prime naïve T cells [309–311]. To date, with their numbers still growing, multiple DC subpopulations that differ in tissue distribution, surface marker expression, and functional capacities have been described in humans and mice (reviewed in [312,313] and Chapter 19). From an evolutionary point of view, DCs might belong to the myeloid as well as to the lymphoid lineage because they link the innate and adaptive immune responses. Based on current data, two opposing models of DC ontogeny can be suggested: either the mature DC subpopulation is determined at the level of an early hematopoietic progenitor or, alternatively, immature DCs retain the capacity to mature to distinct DC subpopulations in response to different environmental instructions. In mice, two major DC subpopulations are distinguishable by their expression of the CD8α chain [314]. It was reported that early intrathymic progenitors (CD4loCD44+CD25−c-kit +) and thymic pro-T cells (CD44+CD25+c-kit +) contain precursors capable to differentiate into CD8α+ DCs in vivo [258,315]. Therefore, and because the majority of thymic DCs and only a minority of secondary lymphoid organ DCs express CD8α+, the CD8α+ DCs had been considered to be related to the T-lymphoid lineage and were therefore termed “lymphoid DCs” in contrast to the CD8α− “myeloid DCs.” This model seemed to be supported, but not directly demonstrated, in mice deficient for the transcription factors RelB [316], PU.1 [317], and Ikaros [318] that lack only CD8α− DCs. However, other mutants, as in c-kit −/− , γc−/− [319], and Notch1−/− [320] mice, showed a developmental dissociation of CD8α+ DC and T cells. We and others therefore tested DC developmental potentials in adult and fetal lymphoid and myeloid committed progenitors. Surprisingly, CD8α+ and CD8α− DCs were produced both by highly purified CLPs and CMPs with similar efficacy on a per cell basis, therefore disproving the concept of CD8α+ “lymphoid”- and CD8α− “myeloid”-derived DCs in mice [290,320–322]. Further, beyond CLPs and CMPs, DC developmental potential is conserved in the lymphoid lineage in pro-T cells and in the myeloid lineage in GMPs, but it is lost once B-cell or megakaryocyte/erythrocyte commitment occurs [320,321]. In contrast to mice wherein DCs or their precursors are usually isolated from the lymphoid organs or bone marrow, human DCs and their precursors have usually been isolated from peripheral blood and only in rare cases from blood-forming organs, making direct comparisons difficult (reviewed in [313]). However, DCs could be generated from total CD34+ progenitor populations, lymphoid-restricted progenitor populations, and peripheral blood monocytes, suggesting that, in analogy to mice, DC developmental potential is conserved in myeloid- and lymphoid-restricted lineages [269,323–328]. Recently, an immediate DC precursor population that upon stimulation can produce high amounts of interferon-α, displays a set of distinct pattern recognition receptors, and can mature into DCs in vitro, was identified in humans [329–332] and later in mice [333–335]. This DC subpopulation has alternatively been referred to as plasmacytoid DCs, DC2 cells, and interferon-α-producing cells [312]. These cells can derive from both myeloid and lymphoid progenitors (reviewed in [336]) but are predominantly of myeloid origin [337]. Taken together, DCs are developing along a lymphoid and myeloid pathway in vitro and in vivo. By using simple assays as immunophenotyping, or functional readout as mixed lymphocyte reaction and cytokine

Biology of Hematopoietic Stem and Progenitor Cells

production, we did not detect differences in lymphoid precursor- and myeloid precursor-derived CD8α+ DC fraction DCs [321]. This is an unexpected finding, because it suggests developmental redundancy for DCs. Of note, DC developmental capacities in CLPs and pro-T cells correlate with their latent myeloid developmental potential that can be “rescued” by artificial introduction and activation of the IL-2β or GMCSF receptor [286,338]. It would therefore be important to characterize the signals that are involved to retain DC developmental capacities in the otherwise exclusively lymphoid- or myeloid-committed progenitor cells. Activation of the Flt3 tyrosine kinase could be one of them [237].

Gene expression profiles of HSCs Several powerful technologies have allowed the characterization of transcripts expressed in defined cells, populations of cells, tissues, and organs. These technical advances allow for reductionist approaches to understand the complexity of RNA transcripts found in these cells or tissues, and how the transcriptional profile changes upon differentiation, self-renewal or other functional events. One of the most powerful technologies is microarray analysis, wherein the expression profile of transcripts for a high fraction of all known genes in the species can be ascertained for purified cell populations. Several groups have performed microarray expression profiling of hematopoietic stem and progenitor cells from fetal, adult, and aged mice [55,56,94,97,98,238,280,339,340]. These studies have identified several genes that have been subsequently validated as important for HSC function, such as Bmi1 [97], or useful for HSC purification, such as Slamf1 [55,56]. Gene subtractions or other differential expression techniques have revealed a subset of genes that appear to be expressed in HSCs but not broadly elsewhere. In fact, some genes have been found that are expressed in HSCs and neurosphere cultures (highly enriched for CNS stem cells) [341], or HSCs and ES cells, or all three [140,342]. These gene expression arrays cannot detect important events that are essentially post-translational, for example the phosphorylation and dephosphorylation of proteins in signal transduction pathways, or the changes in subcellular location of proteins such as β-catenin under conditions when they are held within the degradation complex, or attached to the cytoplasmic face of cadherins, or allowed to be free in the cytoplasm and appear in the nucleus [122]. Most of the oligolineage progenitors downstream of mouse HSCs [25,26] and human HSCs [63,269,273,323] have been isolated to homogeneity, and in some instances a comparison of their gene expression profiles reveals important clues as to genes responsible for particular commitments to differentiated fates [140,342] (see below). It is reasonable to expect that the reductionist approach to cataloging genes in this way will reveal many new clues about stem cell behaviors, but it will require a similar cataloging of cellular proteins (proteomics) to begin to reveal cell protein modifications that lead one to identify important pathways in stem cell behavior.

Gene expression profile of stem and progenitor cells An important question is how the progeny of multipotent cells adopt one fate from a choice of several. Lineage commitment and subsequent differentiation of multipotent cells should involve the selective activation and silencing of particular gene expression programs. There are single or pairs of transcription factors that have “master” roles in hematopoiesis in altering the phenotype of hematopoietic cells in defined circumstances. Combinatorial control is operated at the level of two or more lineage-specific regulators. This transcriptional control might be more complicated, considering protein–protein interactions mounted by the

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formation of multiprotein hematopoietic-specific protein complexes [343], and changes in the expression levels of transcription factors [344]. Furthermore, as in the case of cytokine signals, roles in transcription factors are also sometimes redundant. Finally, single MPPs appear to coexpress transcription factors and cytokine receptors related to multiple lineages [345]. Promiscuous expression of multiple myeloid or lymphoid genes in hematopoietic branchpoints Prospectively purified LT-HSCs, oligopotent progenitors, and lineagecommitted progenitors have been used for a definitive sampling of the transcriptional profiles of cells at these particular stages of physiologic hematopoiesis. The expression patterns of lineage-affiliated genes in each stage of hematopoiesis are largely consistent with their known functions. Using semiquantitative RT-PCR assay targeted for each purified population, SCL (stem cell leukemia) [346,347] is expressed in HSCs and all myeloid progenitors. GATA-2 [348] and c-mpl are expressed in HSCs, CMPs, and MEPs, but not in GMPs [26,94,113]. Other MegErelated genes such as NF-E2 [347], GATA-1 [349,350], and the erythropoietin receptor are expressed in CMPs and MEPs but not in GMPs, and their expression levels are highest in MEPs [113,340]. Granulocyteaffiliated transcription factor C/EBPα [351] is expressed in HSCs, CMPs, and GMPs but not in MEPs, and its expression level is highest in GMPs [113,340]. All of these genes are expressed in CMPs, but not in CLPs or other late lymphoid progenitors, suggesting potential roles for each in myeloid-specific cell fate decisions. In contrast, the lymphoid-related transcription factor Aiolos [352] is not expressed in any myeloid progenitors, nor in HSCs. Ikaros, an Aiolos partner nuclear factor, thought initially to be a lymphoid-specific transcription factor [195,353], is expressed in HSCs and all stages of hematopoiesis [354]. Aiolos first appears at the CLP stage and is highest in T- and B-cell progenitors [26,113]. Lymphoid-related GATA-3 [355] and Pax-5 [356] are expressed at a low level in adult marrow CLPs but not in myeloid progenitors and fetal liver CLPs [26,290]. These data provide a clearer view of the regulation of gene expression in hematopoietic development. First, the expression of many myeloid and lymphoid genes is mutually exclusive in each pathway. Second, CMPs express both MegE- and GM-affiliated genes, and CLPs express both T- and B-lymphoid genes, although CMPs and CLPs have not committed to myeloid and lymphoid sublineages, respectively [26,113]. These data are consistent both with the priming hypothesis [357] and a model wherein chromatin is open prior to commitment, but the transcripts from that open chromatin are not critical for maturation [113]. The view of myeloid or lymphoid promiscuity has been significantly extended by using an oligonucleotide microarray analysis targeted for purified CMPs and CLPs. Genome-wide profiling using microarray methods revealed that CMPs and CLPs coexpress a vast majority of GM- and MegE-affiliated genes, and T-, B-, and NK-lymphoid genes, respectively [94,340]. Furthermore, coexpression of both GM- and MegE-related genes in CMPs and of both T- and B-lymphoid genes in CLPs have been formally demonstrated at the level of single cells [94]. By using RT-PCR analysis targeted for single cells, CMPs coexpress MegE-related β-globin, erythropoietin receptor, NF-E2 and GM-related myeloperoxidase, G-CSF receptor and PU.1, while CLPs coexpress Bcell-related l5 and/or Pax-5 and T-cell-related CD3d and/or GATA-3 [113]. These results indicate that the expression of lineage-related genes can precede commitment, and suggest that the “promiscuous” expression of multiple lineage-related genes may allow a flexibility of commitment at these oligopotent stages: for differentiation towards T- and B-cell lineages in CLPs, and towards GM and MegE lineages in CMPs.

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It is still unclear whether the primary mechanism of hematopoietic commitment could be operated first by opening chromatin at programmed sites, or whether the “priming” seen in promiscuous transcription patterns results from the presence of open chromatin and transcription factors that can form “sterile” transcripts. Nonetheless, in priming stages, multiple differentiation programs might collaborate or compete with each other until one becomes dominant. These programs should include cross-talk among transcription factors or transcriptional complexes, because transcription factor activity can be potentiated [358] or suppressed [359,360] by interaction with other transcription factors. Changes in levels of key transcription factors [344,361] may also be critical for the lineage decision. Cross-talk between different cytokine signals may also be important, as single progenitors coexpress multiple cytokine receptors. Further, fluctuations of gene expression of multiple differentiation programs in progenitors may lead to different outcomes depending on the microenvironment the cell inhabits. In this context, both intrinsic and extrinsic factors could play a part in lineage determination. However, data comparing HSCs from young and old mice show an intrinsic change in their differentiation potential with decreasing lymphoid potential with age, even when transplanted into a young mouse [96]. These data suggest that at least some portion of lineage determination is independent of the environment or that aging HSCs irreversibly lose responsiveness to signals provided by it. One way to view the hematopoietic system is to assume that, at or around the stage of yolk sac hematopoiesis, a pool of HSCs are created that are the only source of hematopoiesis for life. Each early HSC then is a clonogenic precursor of other HSCs, which self-renew, and their progeny, which do not. Over time and perhaps with a cell divisioncounting developmental clock, the clones begin to diversify in terms of responsive potential; these intrinsic epigenetic, and rarely genetic, changes prime the clones for their responsiveness to normal cues, such as diffusible or membrane-bound factors, or to pathologic cues, such as bacterial endotoxins. At any point, the clones that are present represent clonal competitions up to that point. In this way, intrinsic programs can select certain clones over others, but within an inbred colony of pretty clean mice, the clones chosen tend to be consistent, mouse-to-mouse. We later discuss changes that not only enable clonal selection, but are also proto-oncogenic in nature, so that the long process of preleukemic clonal progression is initiated and spread through the HSC clones.

Downregulation of genes irrelevant to committed lineages as a critical mechanism of lineage restriction According to the “priming” model, commitment could be dependent on two independent molecular events: upregulation of differentiation programs related to selected lineages, and the transcriptional abrogation of master control genes in the unselected lineages. A striking example is Pax-5, a key molecule for restriction to B-cell lineage [356]. In normal hematopoiesis, Pax-5 expression initiates at the CLP stage, and is upregulated in pro-B cells, but undetectable in pro-T cells or myeloid cells. Expression of Pax-5 in pro-B cells is important not to differentiate into other lineage cells, because pro-B cells from Pax-5-deficient mice (Pax5−/− pro-B cells) can differentiate into T cells, NK cells, granulocytes, macrophages, and osteoclasts as well as B cells [356]. Pax-5−/− pro-B cells rearrange the D–J locus of IgH genes and express other pro-B related genes. Thus, even after a set of B lineage-associated genes begins to be transcribed, differentiation programs for other lineages are still accessible. Conversely, enforced Pax-5 expression in myeloid progenitors suppresses myeloid development, presumably blocking signals from myelomonocytic cytokine receptors such as G-CSF receptor and GMCSF receptor [362].

Similarly, enforced expression of PU.1 in pro-T cells can block T-cell differentiation [363]. Ectopic GATA-1 expression in GMPs also inhibits differentiation into granulocyte/monocyte lineages, but induces transdifferentiation into megakaryocytes and erythrocytes [364]. A similar phenomenon is observed in ectopic cytokine receptors. Ectopic expression of IL-2Rβ or GM-CSF receptor in CLPs can deliver signals that redirect cells to myelomonocytic differentiation [286]. Further characterization of this latent myeloid potential revealed that CCAAT/enhancerbinding protein (CEBPα) is upregulated in response to the ectopic IL2 signal and leads to GM-CSF receptor expression and further myeloid differentiation [365]. This study also revealed that enforced expression of CEBPα in CLPs is itself sufficient to induce the myeloid outcome and, conversely, enforced Pax-5 expression blocks the myeloid differentiation induced from IL2Rβ. Similarly, Laiosa et al. showed that committed T cell progenitors could be induced to develop into macrophages or DCs by enforced expression of CEBPα or PU.1, respectively. These data suggest that committed T-cell progenitors are also susceptible to the lineage-instructive effects of myeloid transcription factors, but that T-lineage development occurs by the normal role of Notch signaling downregulating CEBPα and PU.1 in multilineage precursors. These data indicate that termination of lineage-related gene expression is equally important for completion of lineage commitment. First, in committed progenitors, abrogated differentiation programs for unselected lineages are not totally erased immediately after commitment, but could be reactivated by “instructive” signals mediated by ectopic cytokine receptors and transcription factors. For example, GMPs might be restricted to GM lineages simply because GATA-1 is downregulated, and CLPs are likely lymphoid restricted because they downregulate myeloid cytokine receptors. Second, programs for differentiation into committed lineages can be operated on condition that transcription factors for unselected lineages are downregulated, as Pax-5 and PU.1 inhibit myeloid and T-cell differentiation programs, respectively. Thus, a hierarchy of transcription factors and cytokine receptors organized at the epigenetic level is critical to maintain hematopoietic homeostasis. Changes in chromatin structure, allowing access for RNA polymerase to initiate transcription, are essential for genetic programs to be transcribed [366,367]. The activation of chromatin structure can occur prior to significant expression of genes [368]. It has been hypothesized that a wide-open chromatin structure is maintained in early hematopoietic progenitors, enabling multilineage-affiliated programs to be accessible [369]. This phenomenon of open chromatin in early hematopoietic progenitors could be the mechanism by which lineage priming occurs in stem or progenitor cells prior to their lineage determination [140,358,370,371]. Standard methods for studying chromatin structure require large numbers of cells, which has made their application to stem cell biology difficult, but we and others have made recent technical advances that have allowed chromatin immunoprecipitation to be performed on rare cells [372–374] so that we have been able to determine whether the epigenetic marks in control regions of lineage-directing genes modulate during hematopoiesis. Specifically, we analyzed histone modifications and DNA methylation patterns within the regulatory regions of erythroid (Gata1 and β-globin), myeloid (c-fms), and lymphoid lineage-affiliated genes (Gata3 and Ptcrα) in 50,000 prospectively purified stem and progenitor cells. By performing bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides localized at defined regulatory regions of these lineage-affiliated genes in HSC. These histone modifications are epigenetic markers of active transcription and either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with the pattern of gene expression. We also observed bivalent

Biology of Hematopoietic Stem and Progenitor Cells

domains at lymphoid-affiliated genes in HSC and downstream transitamplifying progenitors. Bivalent domains described by Bernstein et al. contain both activating and silencing histone modifications [375], and are associated with genes such as transcription factors that are expressed at low levels and affect developmental decisions. These data support the role of epigenetic modifications as being reflective of HSC multipotency and suggest that the remodeling of chromatin is an important mechanism of driving gene expression changes associated with developmental decisions.

Transplantation of HSCs in mouse and humans It is fair to say that the field of bone marrow transplantation, now more accurately called HCT, has provided one of the most striking medical advances in our lifetime and, similarly, the desire to understand cells involved in such transplants has opened the field of stem cell biology, first to HSC but then to other tissue-specific stem cells. It is important at the outset, however, to make sure that the scientific and clinical community uses the appropriate nomenclature for the type of transplant used as misconceptions as to the cell type transplanted lead to misconceptions about the scientific or clinical results obtained [14,15]. Thus, the transplantation of complex mixtures of hematopoietic cells in bone marrow or MPB is not the same as transplanting purified HSCs. Clinical HCTs have been used for a variety of purposes, but their primary use is the ability to regenerate the hematopoietic tissues of a cancer patient who has received otherwise lethal doses of a high-dose regimen of chemoradiotherapy [376,377]. Other uses have been the correction of inherited or acquired hematopoietic cell defects and dysplasias, and in animals the induction of transplantation tolerance to other cells, tissues or organs co-transplanted from the HSC donor, the hematopoietic cell donor, and also the reversal of autoimmune disease [19] (see Chapter 20). It was not clear whether purified HSCs could replace HCT, as it was not clear whether a rapid and sustained regeneration of a particular hematopoietic outcome could be accomplished with the primitive HSCs alone, without having oligolineage progenitors in the transplant. Several experiments have demonstrated in mice and in humans that HSCs are the principal cells that are functional in the transplant setting, and that

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by varying cell dose one could achieve both rapid and sustained regeneration of hematopoietic tissues [378]. Prospective isolation of HSCs with multiple markers allows the possibility that grafts would consist solely of HSCs and of no other contaminating cells. There are two important advantages, at least theoretically, to the transplantation of HSCs rather than HCT removal of cancer cells and removal of T cells. Autologous HCT from cancer patients can be accompanied by transplantation of the patient’s cancer cells; in some diseases, such as stage 4 breast cancer and many lymphomas, the HCT populations to be transplanted that are contaminated with cancer cells vary from 20% to 40% of all transplants [379–381]. In patients with multiple myeloma, contamination of the graft is the rule and not the exception [382]. Therefore, it was important to test whether positive selection of HSCs would effectively eliminate or purge cancer cells from the transplant. Deliberate spiking of MPB with cell lines representing human breast cancer, human non-Hodgkin’s lymphoma and human myeloma was performed, and four methods of HCT/HSC isolation were carried out to determine the full reduction of contaminating cancer cells. Two independent methods to identify breast cancer cells were used: microscopy to detect cells carrying breast cell cytokeratins, and a flow-based method. Cells representative of myeloma and B-cell lymphoma were detected by PCR specific for the immunoglobin heavy chain rearrangements in these B-lineage cells. All three techniques have a sensitivity of between 10−5 and 10−6. Over 105-fold reductions of contaminating cancer cells were accomplished with the multiparameter cell sorting of CD34+Thy+ cells from MPB, whereas techniques based on CD34 alone were at least two to three orders of magnitude less efficient (Fig. 5.7). The time to reach engraftment with purified HSCs (measured by absolute neutrophil count >500/mL and platelets >20,000/mL of blood [379,380,383]) (Fig. 5.8) is very similar to the time obtained when unmanipulated MPB is used as the HCT transplant, and similar to the dose–response observed for mouse HSCs in syngeneic transplants (Fig. 5.9) [378]. Even in acute myeloid leukemia (AML), HSCs can be separated from leukemic stem cells (LSCs) by surface markers [384]. Therefore, the stage is set to test whether the transplantation of cancer-depleted HSCs will lead to clinical benefits when compared with the transplantation of cancer-containing unmanipulated HCT grafts.

Comparison of fold depletion of cancer cells from mobilized peripheral blood CD34+ Thy1.1+ (flow) CD34+ (flow) CD34+ (paramagnetic beads) CD34+ (magnetic beads)

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Fig. 5.7 Comparison between CD34 only and CD34+Thy-1 isolation of human stem and progenitor cells in terms of fold depletion of human cancer cells. MPB (1.6 × 1010) was spiked with 1.2 × 108 cancer cells (T47D breast cancer, SU DHL 6 non-Hodgkin’s lymphoma cells, or RPMI-LAV myeloma cells) prior to cell sorting. The CD34+Thy-1 cells were flow sorted with a modified high-flow cytometer, as were the first CD34 flow sort test. The other devices were a commercial paramagnetic bead-coupled monoclonal passed through magnetized steel mesh, and the last a large magnetic bead device. Breast cancer cells were analyzed by immunofluorescence microscopy for cytokeratin, the non-Hodgkin’s lymphoma by Igh-BCL-2 real-time polymerase chain reaction (PCR), and the myeloma by CDR III real-time PCR. The data are represented as fold reduction of cancer cells from the sorted CD34 or CD34+Thy-1 cells. (Hanania, unpublished data.)

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Fig. 5.8 Transplantation of highly purified human hematopoietic stem cells in patients with metastatic breast cancer. Shown are the times to engraftment of neutrophils (absolute neutrophil count [ANC] > 500/mL] ) following transplantation with purified CD34+Thy1-1+ hematopoietic stem cells. (Reproduced from [379].)

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Fig. 5.9 Hematopoietic recovery in lethally irradiated C57BL/Ka mice syngeneically transplanted with different doses of purified hematopoietic stem cells (HSCs). Irradiated mice were injected with 100 (solid circles), 1000 (triangles), 5000 (solid triangles) or 10,000 (squares) HSCs. Shown are the recovery kinetics for white blood cell (WBC), and platelet (plts) counts. (From [378] with permission.)

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Allogeneic HCT transplants have been plagued from the beginning by the appearance of graft-versus-host disease as a result of T cells contained within the graft [385]. Mice receiving allogeneic HSCs have few or no contaminating T cells, and never develop graft-versus-host disease [378,386]. Dose–response comparisons of purified HSC transplants in syngeneic, in matched unrelated donor allogeneic, and in fully allogeneic circumstances have been carried out, and, for the most part, doses of HSCs sufficient to allow rapid and sustained engraftment can be achieved without contaminating T cells (Fig. 5.10) (see Chapter 20) [386]. However, the number of HSCs required for engraftment in the allogeneic setting can be 10 times higher than that required for syngeneic

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Fig. 5.10 Engraftment kinetics of allogeneic versus congeneic purified hematopoietic stem cells (HSCs). Comparison of stem cell dose required to achieve early hematopoietic recovery in syngeneic versus allogeneic irradiated mice. Syngeneic or allogeneic mice were transplanted with purified Thy-1loSca-1+ Lin−/loc-kit + (KTLS) cells and bled on sequential days post transplant. The kinetics of white blood cell (WBC) recovery were categorized into delayed, intermediate, and rapid recovery. (Adapted from [378] with permission.)

HSC engraftment (Fig. 5.10) [378]. One can achieve nearly equal levels of engraftment at the same HSC dose with syngeneic and allogeneic models if the allogeneic hosts are preconditioned with high-dose conditioning regimens that include either facilitator cells [387–389] from the donor, or monoclonal antibodies that eliminate NK- and T-cell populations [386,390]. High-dose conditioning alone results in donor B-celland myeloid-lineage conversion [386–390], but persistent donor and host T-cell chimerism requires elimination of the host residual T and NK cells with facilitator cells or antibodies. There are two kinds of facilitator cell found in mouse bone marrow and lymphoid tissues: classic CD8α+TCR+CD3+ T cells and CD8α+ TCR−

Biology of Hematopoietic Stem and Progenitor Cells

CD3− facilitator cells [387–389]. The immunocompetent CD8α+ TCR+ T cells have some capacity for graft-versus-host disease, so there is great interest in understanding the nature and function of CD8α+ TCR−CD3− facilitator cells [387]. There are two other kinds of cell that share this phenotype: CD8α+ immature DCs, and veto cells [387,391,392]. We have proposed that all three cell types are in fact a single cell class, and that it is only the assays that makes one believe that they are different lymphoid cell subsets [387]. This view is strengthened by the recent findings of Steinman and Nussenzweig [393] that CD8α+ DCs that do not have active or appropriate co-stimulation can in fact lead to the inactivation or disappearance of T cells (and, we propose, NK cells) that have receptors which recognize them. Thus, a cell that facilitates engraftment of allogeneic HSCs might be a cell that presents major histocompatibility complex/peptide antigens to reactive T and NK cells without delivering a co-stimulatory signal, or induces the cells to undergo activation-induced cell death. These same cells might also be responsible for the veto of active CD4 and CD8 T cells that recognize and respond to them. It has been known since the pioneering work of Main and Prehn that bone marrow chimeras are usually transplantation tolerant to cells, tissues or organs from the hematopoietic cell donor [359]. This method of inducing transplantation tolerance has been substantiated with pure HSC transplants [387,390], in either the high-dose or reduced-intensity setting, and whether the transplant is concurrent with the HSC transplant or occurs months later [387,390]. Hematopoietic chimeras in humans are also usually specifically transplant tolerant of donor tissues, and new trials for inducing tolerance with bone marrow cells in a reducedintensity but lymphoablative setting along with organ allografts from the bone marrow donor are underway [394]. Partial or complete replacement of an autoimmune diabetes-prone hematopoietic system in mice with purified HSCs from diabetes-resistant strains of mice in the prediabetic period completely precludes progression to diabetes (see Chapter 20), whereas the same treatment of already-diabetic hosts along with donor strain islet cell transplants cures the small numbers of mice undergoing this protocol [395]. This too has been accomplished in both high-dose and reduced-intensity conditioning settings. Additionally, in a mouse model of systemic lupus erythematosus, Smith-Berdan was able to achieve greatly increased overall survival by treating mice with established disease with reduced-intensity conditioning and transplants of major histocompatibility complex haplomismatched allogeneic HSCs [396]. Mice that received transplants exhibited stabilization or reversal of their lupus symptoms, including proteinuria, and elevated circulating immune complexes or autoantibodies compared with control mice. These results indicate that induction of durable mixed chimerism by transplantation of purified allogeneic HSCs after reducedintensity conditioning has the potential to reverse symptoms of established autoimmune disease.

Co-transplantation of HSCs and hematopoietic progenitors to protect against opportunistic infections The bane of clinical HCT in terms of infectious disease is the often lethal infections with Aspergillus fumigatus, CMV, and Pseudomonas [397]. Small numbers of each of these infectious organisms given to HSC transplanted mice can result in the morbidity and mortality that occurs in patients [397]. With the isolation of clonal CLPs, CMPs, and GMPs, cellular therapies for each of these disorders have been investigated. Co-transplantation of HSCs and CLPs can completely preclude the lethality of murine CMV infection in both syngeneic and allogeneic protocols [265]. Co-transplantation of HSCs and CMPs and GMPs can prevent the lethal consequences of Aspergillus infections if the Aspergil-

53

lus is given on day 9, 11 or 14 after HSC transplantation, and addition of G-CSF to these HSC/CMP/GMP transplant protocols precludes Aspergillus infections given just 3 days after transplantation [397]. Similar CMP plus GMP protocols are sufficient to eliminate Pseudomonas infections, and in both cases these treatments work in both the syngeneic and allogeneic settings.

Origin of leukemia: LSCs Poorly regulated or unregulated self-renewal is a property that all cancers have, and we call those cells within a cancer that have self-renewal capacity “cancer stem cells” [122]. Therefore, it is important for the reader to keep in mind that the major distinguishing property of normal and neoplastic stem cells is their capability of self-renewal, and thus it becomes important to understand whether the pathways that lead to self-renewal are novel in normal HSCs, or might be shared between different classes of tissue-specific multipotent stem cells, and might be acquired by cancer cell progenitors as they emerge as cancer stem cells [122]. Pten, a tumor suppressor that negatively regulates proliferation, is required to sustain long-term hematopoiesis and self-renewing LSCs [398]. To understand the mechanisms of leukemic transformation, it is important to understand the life history of leukemic progression (Plate 5.2). If one views leukemia development as a series of steps in malignant progression, malignant self-renewal may be obtained through several transformation steps that include a block in differentiation, the retention or acquisition of self-renewal, and the avoidance of programmed cell death by several independent cell-intrinsic pathways and by intrinsic and adaptive immune system immunosurveillance. A set of early stem/progenitor cells with extensive proliferation, or with reduced programmed cell death, that are not yet malignant may form “preleukemic” clones, and these clones may receive additional oncogenic events that induce differentiation blocks at a progenitor stage to become leukemia. Accumulation of mutations as a precondition for malignant transformation in human acute leukemias Molecular markers for leukemia, including oncogenic fusion proteins resulting from leukemia-specific chromosomal translocations, have disclosed the multistep nature of acute leukemia development. A typical case is chronic myelogenous leukemia (CML). CML-specific t(9;22) Philadelphia chromosome and its resultant Bcr–Abl fusion gene has been shown to be present in all myeloid cells, B cells, and rare T cells. Additional mutation(s) and/or epigenetic changes in patterns of gene expression are required for blastic transformation into acute leukemia. There are many other examples of human adult and childhood leukemias that support the early acquisition of predisposing mutations occurring in HSCs that require additional events to result in leukemic transformation. AML1/ETO chimeric protein that is the product of the t(8;21) translocated genomic region is continuously detectable in patients with t(8;21) AML who maintain long-term remission for more than 10 years. Although these patients are clinically “cured,” AML1/ETO remains detectable in HSCs (Thy-1+CD34+Lin−CD38), B cells, and erythroid and megakaryocyte CFCs [384]. In all of these cells, only HSCs self-renew, setting up clones that can acquire additional proto-oncogenic events. In childhood t(8;21) AML, AML1–ETO fusions are retrospectively detected in neonatal Guthrie spots [399], further suggesting that AML1–ETO fusion is at the level of HSCs (in some cases in utero). Acquisition of chromosomal translocations in utero is also demonstrated in infant t(4;11) and childhood t(12;21) acute leukemias. Clonotypic MLL–AF4 and TEL–AML1 genomic fusion sequences were demonstrated to be present in neonatal blood spots from patients who were

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diagnosed with acute lymphoblastic leukemia (ALL) [399–401] and were also detected in identical twins, whose genomic fusion sequence was identical [369]. In these cases, t(12;21) ALL developed at approximately 10 years of age, indicating that cells with TEL–AML1 persisted more than 10 years before leukemic transformation. Further, TEL– AML1+ ALL cells in twins sometimes possess an identical IgH rearrangement, implicating in utero transfer of preleukemic cells. In childhood ALL, preleukemic B- or T-cell progenitors appear to persist in the long term; “leukemic clones” possessing clonal TCR or IgH rearrangements are reported to persist for more than 3 years after achieving complete remission [402]; these might involve the only other blood cells that self-renew – memory T and B cells. These data strongly suggest that leukemic transformation commonly occurs by multistep processes. In mice and in humans, the only cell in the myeloid lineage that self-renews over long periods of time is the LT-HSC [1]. The preleukemic clone should exist in the LT-HSC to sustain multiple mutational events over time. Therefore, it appears that the accumulation of mutations occur as a precondition for leukemia development at the level of HSCs, or in progenitors that have gained the capacity of self-renewal.

Final leukemic transformation can occur at the level of myeloid progenitors Several lines of evidence show that final myeloid leukemic transformation can only occur at the level of progenitors that have acquired the property of self-renewal. In contrast, lymphoid leukemias are made up of neoplastic cells with clonally rearranged TCR or immunoglobulin genes. Whereas myeloid progenitors and myelomonocytic cells are not self-renewing, lymphocytes that contribute to the memory pool are selfrenewing. Thus, the final transformation to lymphatic neoplasms can occur in cells that already have regulated self-renewal capacity. The search for LSCs involves the one property shared by all malignancies – the ability to initiate and spread the disease. Transfer of human AML to immunodeficient mice was first demonstrated to be limited to CD34+CD38− or CD34+CD71−HLA-DR− cells, and was attributed to early progenitors such as HSCs [403]. We [396] and others [404] have shown that, in at least some AMLs, the LSCs lack CD90 expression. In our study of AML1/ETO AML, LSCs were in the MPP phenotype (CD34+CD38lo90− Lin−). Surprisingly, in the same patients, 1–40% of HSCs were positive for the t(8;21) translocation, but these HSCs in vitro gave rise to normal myeloerythroid colonies, not leukemic blasts [396]. As shown in Plate 5.2, these represent preleukemic clones; thus, AML1/ETO may be necessary but not sufficient for full leukemic transformation. A mouse model that expresses AML1/ETO driven by multidrug resistance protein-8 (MRP8) (GMP, granulocyte, and monocyte specific) promoter did not develop leukemia. After the injection of a DNA alkylating mutagen, N-ethyl-N-nitrosourea, however, almost 55% of mice develop AML-M2 [405]. Similarly, promyelocytic leukemia/retinoic receptor-α (PML/RARα) is a product of the t(15;17) translocation, and MRP8–PML/RARα transgenic mice develop acute promyelocytic leukemia-like disorders [406]. The frequency and rapidity of PML-RARα leukemias increases when they are crossed to MPR8–Bcl-2 mice [407]. In these mice, the proto-oncogenes were present in all cells of the GMP lineage, allowing rare subsequent events to be fixed. Inhibition of apoptotic pathways alone can allow leukemia development at progenitor stages. MRP8–Bcl-2 mice display increased numbers of mature monocytes, resembling chronic myelomonocytic leukemia, but do not develop acute leukemia. However, an additional introduction of Fas (lpr/lpr) mutations into these mice results in transformation into AML in about 15% of progeny, but only if leukemia emerges by age 8 weeks [408]. The transplantable GMP-stage leukemias are tert++, over-

express CD47, and have acquired the property of self-renewal, implicating a multistep process. Even mpr8–bcr–abl mice have increased frequencies of conversions to acute blastic leukemias if crossed to mpr8– bcl-2 strains. The general rules so far are that mouse AML LSCs induced as described are at the stage of the GMP, as is human CML myeloid blast crisis [409–412]. Most human AMLs are at the level of the MPP. No HSC-stage LSC has been found. LSC markers The identification and characterization of LSCs is crucial to the development of more effective treatment strategies. Ideal therapies will be those that target the LSCs while sparing normal HSCs, so identifying LSCspecific markers is an important strategy. To date we have identified several candidate LSC-specific markers, including CD96 and CD47 [158,413]. Fluorescence-activated cell sorter analysis indicates that, in many cases, CD96 is expressed on the majority of CD34+CD38− AML cells, whereas few, if any, normal HSCs (Lin−CD34+CD38−CD90+) expressed CD96 [413]. Further, in four of five samples, only CD96+ cells were able to propagate the leukemia in recipient immunocompromised mice, supporting its expression on LSCs and its usefulness as a potential therapeutic target. We identified CD47 in the mouse AML microarray screen, and have verified it as specifically upregulated in human AML. Subsequently, we have found it to be expressed in all mouse models of myeloid leukemia we have examined, and in human AML and blast crisis CML (manuscript submitted). Recent data suggests that expression of mouse CD47, which inhibits phagocytosis, is sufficient to allow the human myeloid leukemia cell line (MOLM-13) to engraft hematopoietic organs efficiently and develop into large tumors, whereas control MOLM-13 cells fail to engraft in immunocompromised Rag2−/−γc−/− cells [158]. Thus, CD47 does seem to play a role in the spread and/or life span of AML LSCs. These data also suggest that macrophagemediated killing may be a significant player in leukemia surveillance. There are increasing descriptions of mutations that occur at the posttranslational and epigenetic levels, and would not be detected by gene expression profiling. A study of 100 CML blood and marrow samples has demonstrated that GMPs from CML blast crisis have been shown to have increased levels of the active β-catenin in the nucleus compared with normal GMPs and to exhibit increased self-renewal in in vitro replating assays [412]. Dysregulation of microRNAs, small RNAs that bind to specific transcripts and either suppress their translation or target them for deletion, is another potential leukemogenic mechanism [414–417]. The important point here, as outlined in Plate 5.2, is that the elucidation of the phenotypes of normal HSCs and hematopoietic progenitors allows the classification of the various types of LSC, and the comparison of genes and gene transcripts between the normal and the neoplastic counterparts has revealed a plethora of new, and perhaps useful diagnostic and therapeutic, targets undetected when whole blood or marrow populations are analyzed.

Conclusion With the development of a general method to prospectively isolate hematopoietic stem and progenitor cells [1,40,45], it has become possible to construct increasingly detailed lineage pathways from LT-HSCs to mature blood cells. With the exception of memory T and B lymphocytes in the entire hematopoietic lineage, only LT-HSCs self-renew, and this self-renewal capacity is tightly regulated [1]. The genetic regulation of HSC self-renewal includes regulation of programmed cell death, regulation of cell-division frequency, and regulation of transition to

Biology of Hematopoietic Stem and Progenitor Cells

multipotent and to oligopotent progenitors. Gene expression analyses of prospectively isolated HSCs and progenitors are revealing insights into the mechanisms of self-renewal and those in lineage commitment. These same isolated HSCs and progenitors are increasingly useful in clinical HCT, whether to regenerate the myeloablated hematopoietic system in cancer patients, to condition allogeneic hosts for donor tissue or organ grafts, or even to replace an autoimmune-prone hematolymphoid system

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with a nonautoreactive donor population. Finally, the concept of selfrenewal as the critical regulated event limited to HSCs in normal hematopoiesis has now been extended to the hypothesis that within leukemias and cancers are relatively infrequent populations of LSCs that undergo poorly regulated self-renewal. Prospective isolation of LSCs should allow more direct approaches for immune and drug therapies for target molecules restricted to these malignant stem cells.

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337. Karsunky H, Merad M, Mende I, Manz MG, Engleman EG, Weissman IL. Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors. Exp Hematol 2005; 33: 173–81. 338. King AG, Kondo M, Scherer DC, Weissman IL. Lineage infidelity in myeloid cells with TCR gene rearrangement: a latent developmental potential of proT cells revealed by ectopic cytokine receptor signaling. Proc Natl Acad Sci U S A 2002; 99: 4508–13. 339. Rossi DJ, Bryder D, Seita J, Nussenzweig A, Hoeijmakers J, Weissman IL. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 2007; 447: 725–9. 340. Prohaska SS, Weissman I. Gene expression profiling of highly purified myeloid progenitor populations (submitted for publication). 341. Geschwind DH, Ou J, Easterday MC et al. A genetic analysis of neural progenitor differentiation. Neuron 2001; 29: 325–39. 342. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR. A stem cell molecular signature. Science 2002; 298: 601–4. 343. Sieweke MH, Graf T. A transcription factor party during blood cell differentiation. Curr Opin Genet Dev 1998; 8: 545–51. 344. DeKoter RP, Singh H. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 2000; 288: 1439– 41. 345. Hu M, Krause D, Greaves M et al. Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev 1997; 11: 774– 85. 346. Porcher C, Swat W, Rockwell K, Fujiwara Y, Alt FW, Orkin SH. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 1996; 86: 47–57. 347. Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 1995; 373: 432–4. 348. Tsai FY, Keller G, Kuo FC et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 1994; 371: 221–6. 349. Fujiwara Y, Browne CP, Cunniff K, Goff SC, Orkin SH. Arrested development of embryonic red cell precursors in mouse embryos lacking transcription factor GATA-1. Proc Natl Acad Sci U S A 1996; 93: 12355–8. 350. Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH. A lineage-selective knockout establishes the critical role of transcription factor GATA-1 in megakaryocyte growth and platelet development. EMBO J 1997; 16: 3965–73. 351. Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc Natl Acad Sci U S A 1997; 94: 569–74. 352. Morgan B, Sun L, Avitahl N et al. Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J 1997; 16: 2004–13. 353. Klug CA, Morrison SJ, Masek M, Hahm K, Smale ST, Weissman IL. Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin

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372. Attema JL, Papathanasiou P, Forsberg EC, Xu J, Smale ST, Weissman IL. Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis. Proc Natl Acad Sci U S A 2007; 104: 12371– 6. 373. Dahl JA, Collas P. Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells 2007; 25: 1037–46. 374. O’Neill LP, VerMilyea MD, Turner BM. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat Genet 2006; 38: 835–41. 375. Bernstein BE, Mikkelsen TS, Xie X et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006; 125: 315–26. 376. Thomas ED. Bone marrow transplantation from bench to bedside. Ann N Y Acad Sci 1995; 770: 34–41. 377. Thomas ED. A history of haemopoietic cell transplantation. Br J Haematol 1999; 105: 330–9. 378. Uchida N, Tsukamoto A, He D, Friera AM, Scollay R, Weissman IL. High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts. J Clin Invest 1998; 101: 961–6. 379. Negrin RS, Atkinson K, Leemhuis T et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant 2000; 6: 262–71. 380. Vose JM, Bierman PJ, Lynch JC et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with recurrent indolent non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 2001; 7: 680–7. 381. Stadtmauer EA, O’Neill A, Goldstein LJ et al. Conventional-dose chemotherapy compared with high-dose chemotherapy plus autologous hematopoietic stem-cell transplantation for metastatic breast cancer. Philadelphia Bone Marrow Transplant Group. N Engl J Med 2000; 342: 1069–76. 382. Gazitt Y, Tian E, Barlogie B et al. Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and granulocyte-macrophage colony-stimulating factor. Blood 1996; 87: 805–11. 383. Michallet M, Philip T, Philip I et al. Transplantation with selected autologous peripheral blood CD34+Thy1+ hematopoietic stem cells (HSCs) in multiple myeloma: impact of HSC dose on engraftment, safety, and immune reconstitution. Exp Hematol 2000; 28: 858–70. 384. Miyamoto T, Weissman IL, Akashi K. AML1/ ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc Natl Acad Sci U S A 2000; 97: 7521–6. 385. Ferrara JL, Levy R, Chao NJ. Pathophysiologic mechanisms of acute graft-vs.-host disease. Biol Blood Marrow Transplant 1999; 5: 347–56. 386. Shizuru JA, Jerabek L, Edwards CT, Weissman IL. Transplantation of purified hematopoietic stem cells: requirements for overcoming the barriers of allogeneic engraftment. Biol Blood Marrow Transplant 1996; 2: 3–14.

387. Gandy KL, Domen J, Aguila H, Weissman IL. CD8+TCR+ and CD8+TCR− cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers. Immunity 1999; 11: 579–90. 388. Kaufman CL, Colson YL, Wren SM, Watkins S, Simmons RL, Ildstad ST. Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 1994; 84: 2436–46. 389. Martin PJ. Donor CD8 cells prevent allogeneic marrow graft rejection in mice: potential implications for marrow transplantation in humans. J Exp Med 1993; 178: 703–12. 390. Shizuru JA, Weissman IL, Kernoff R, Masek M, Scheffold YC. Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection. Proc Natl Acad Sci U S A 2000; 97: 9555–60. 391. Heeg K, Wagner H. Induction of peripheral tolerance to class I major histocompatibility complex (MHC) alloantigens in adult mice: transfused class I MHC-incompatible splenocytes veto clonal responses of antigen-reactive Lyt-2+ T cells. J Exp Med 1990; 172: 719–28. 392. Thomas JM, Verbanac KM, Carver FM et al. Veto cells in transplantation tolerance. Clin Transplant 1994; 8: 195–203. 393. Steinman RM, Nussenzweig MC. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc Natl Acad Sci U S A 2002; 99: 351–8. 394. Millan MT, Shizuru JA, Hoffmann P et al. Mixed chimerism and immunosuppressive drug withdrawal after HLA-mismatched kidney and hematopoietic progenitor transplantation. Transplantation 2002; 73: 1386–91. 395. Beilhack GF, Scheffold YC, Weissman IL et al. Purified allogeneic hematopoietic stem cell transplantation blocks diabetes pathogenesis in NOD mice. Diabetes 2003; 52: 59–68. 396. Smith-Berdan S, Gille D, Weissman IL, Christensen JL. Reversal of autoimmune disease in lupus-prone New Zealand black/New Zealand white mice by nonmyeloablative transplantation of purified allogeneic hematopoietic stem cells. Blood 2007; 110: 1370–8. 397. BitMansour A, Burns SM, Traver D et al. Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation. Blood 2002; 100: 4660–7. 398. Yilmaz OH, Valdez R, Theisen BK et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 2006; 441: 418–19. 399. Wiemels JL, Xiao Z, Buffler PA et al. In utero origin of t(8;21) AML1-ETO translocations in childhood acute myeloid leukemia. Blood 2002; 99: 3801–5. 400. Gale KB, Ford AM, Repp R et al. Backtracking leukemia to birth: identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci U S A 1997; 94: 13950–4. 401. Hjalgrim LL, Madsen HO, Melbye M et al. Presence of clone-specific markers at birth in children with acute lymphoblastic leukaemia. Br J Cancer 2002; 87: 994–9. 402. Cave H, van der Werff ten Bosch J, Suciu S et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. Euro-

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6

Peter M. Lansdorp

Molecular Biology of Stem Cell Renewal

Several factors have led to intensive investigations of the hematopoietic system over the past few decades. The hematopoietic system represents the prototype of a self-renewing biologic system in that large numbers of blood cells have to be produced daily in order to compensate for the loss of relatively short-lived mature blood cells. The relative ease with which blood and marrow samples can be obtained and single-cell suspensions prepared has greatly facilitated in vitro and in vivo studies of hematopoietic progenitor and stem cells. Techniques to purify increasingly rare cells on the basis of cell surface antigens or functional properties have been refined and are available to most researchers. A large number of purified recombinant proteins and other molecules with activity on various hematopoietic cells have been discovered and have become available as well. In addition, a variety of in vitro and in vivo assays to measure functional properties of hematopoietic cells have been developed over the years, and some of these are now available as standardized tests. Together, these advances have allowed various studies with purified cells and molecules, some of which have revealed unexpected characteristics of stem cell “candidates.” From such studies, it has become clear that hematopoietic stem cells (HSCs) cannot be considered a homogeneous population of cells. Instead, HSCs represent a heterogeneous population of cells with functional properties that vary with replicative history, developmental stage, and age. This is illustrated by the pronounced developmental changes in the functional properties of purified “candidate” stem cell populations, the marked change in stem cell turnover in the first few years of life, and the age-related loss of telomeric DNA in HSCs. The age-related decline in stem cell renewal and replicative potential is clinically relevant as limited engraftment and failure of stem cell transplants could result from properties of HSCs that are predictable and, increasingly, measurable. In this chapter, some aspects related to the “self-renewal” of HSCs are reviewed. No attempt is made to cover the extensive literature in this general area. Specifically, the large number of studies aimed at dissecting the role of various transcription factors in stem cell properties and function are only briefly mentioned here. Instead, the focus is on the role of telomerase and the loss of telomere repeats in stem cells relative to their “self-renewal” in vitro and in vivo.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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The stem cell hierarchy A recurrent theme in experimental hematology over the past 40 years has been the redefinition of what HSCs are, based on changes and improvements in assays. The trend has invariably been towards the identification of cells with a lower frequency than was previously reported or proposed [1]. The inevitable conclusion of this trend will no doubt be the identification of the fertilized egg as the “mother of all stem cells.” But what could be the explanation for the seemingly endless debate about HSC definition, HSC purification, and the apparent inability of an extensive field to agree on basic principles? The fact that the behavior of HSCs upon transplantation depends on many experimental variables, including the number and type of transplanted cells as well as host factors, has certainly contributed to the confusion. In animal models, only a limited number of blood- or bone marrow-derived HSCs are required to completely regenerate hematopoiesis in suitably conditioned recipients. Although the minimum number of HSCs required for sustained engraftment in human patients is not known, it has been shown that a single purified HSC in the mouse can reconstitute hematopoiesis [2]. This remarkable fact has supported the older definition of HSCs as “pluripotent cells with self-renewal potential” [3]. Implicit in the definition of “self-renewal” is that the two daughter cells, derived from the division of a single parental HSC, are identical to each other and indistinguishable from the parental cell. However, this is most likely an oversimplification as the behavior of HSCs is known to vary markedly with the developmental stage of the donor [4]. Thus, the source of HSCs is an important variable in transplant studies. Indeed, stem cell “candidates” purified from human [5] or murine [6] fetal liver versus adult bone marrow show remarkable functional differences that correlate with measurable molecular changes occurring with age [7] and upon transplantation [8]. Loss of telomere repeats in stem cells, developmental changes in HSC function, and the facultative, cell dosedependent symmetric versus asymmetric (self-renewal versus maintenance) cell divisions of HSCs [9] greatly complicate generalizations about the nature and function of HSCs. Nevertheless, the central dogma and current paradigm in experimental hematology has remained that perhaps the frequency of HSCs, but not HSCs themselves, are subject to significant change. In a way, it is understandable that experimental hematologists hate to part with simple notions about stem cells as “units” and “self-renewal” as a concept. After all, it is these notions that allow, in theory, an external control of HSC properties and the net “expansion” of HSCs in vitro. Although the inclusion of “relative” in the definition of “self-renewal”

Molecular Biology of Stem Cell Renewal

may seem annoying, the fact remains that hematopoietic tissues typically contain a spectrum of “stem” cells, each endowed with different replicative potential and functional properties. The relative frequency of various HSCs within the hierarchy is likely to vary with the age of the donor. The optimal clinical use of HSCs in transplantation settings will take not only the number and viability of HSCs in the transplant into account, but also their developmental stage and their estimated replicative potential.

Hematopoiesis: how many stem cell divisions? The realization that stem cells are subject to pronounced developmental changes has focused attention on the question, “How many times can stem cells divide?” Unfortunately, no techniques to address this important question are currently available, and possible answers range from fewer than 100 times [1] to over 5000 times [10,11]. The total number of blood cells required for the lifelong maintenance of normal levels of blood cells can be calculated to be in the order of 4.1016 cells (∼1012 cells per day × 365 days × 100 years). In theory, only 55 divisions of a single cell would satisfy this need (255 = 4.1016). Because many hundreds of different HSCs are contributing to hematopoiesis in a normal individual at any given time, an absolute replicative potential of fewer than 100 cell divisions per HSC could presumably easily accommodate a lifetime of blood cell production and allow for the known apoptosis of cytokinedeprived committed progenitor cells and the remarkable regenerative capacity of HSCs displayed in response to marrow injury and upon (serial) transplantation. An important related question is how many HSC clones are actually present in normal adults. Because genetic markers such as point mutations can occur at any given cell division, the presence of such a clonal marker does not a priori allow the conclusive identification of a single HSC clone. For example, in patients with paroxysmal nocturnal hemoglobinuria, the presence of a mutation in the PIG-A gene is often used to identify the abnormal HSC “clone” [12]. However, such a mutation could have occurred relatively early in development, for example before the major expansion of HSC numbers in early childhood [13]. As a result, many hundreds of adult HSC clones would be expected to show the mutation and “clonal” marker. Conversely, when a mutation takes place very late in the stem cell hierarchy, only committed progenitors could be affected, resulting in the transient appearance of a clonal marker in the peripheral blood. The transient presence of BCR–ABL fusion genes in the circulation of normal individuals could be an example of such a late occurrence [14]. Presumably the likelihood of PIG-A, BCR–ABL or other mutations will be directly related to the number of cells at risk: relatively few in early development and many in adult tissues. The notion that HSCs have a limited replicative potential is further supported by several observations. Both the in vivo regenerative capacity [15,16] and the in vitro expansion potential [4,5] of HSCs appear to be under developmental control (see also below). Most HSCs in adult tissue are quiescent cells in line with expectations for cells that divide very infrequently and have only a limited replicative potential that lack “self-renewal” properties in an absolute sense. The loss of telomere repeats in adult hematopoietic cells (including purified “candidate” HSCs) relative to fetal hematopoietic cells [7] also fits a model that postulates a finite and limited replicative potential of HSCs [1,17]. Recent longitudinal studies of telomere length in baboons provide further support for the idea that each cell division in HSC is accompanied by a measurable loss of telomere repeats [18]. In this study, it was found that the rate of telomere loss was greatly reduced after the first year of life in line with the rapid expansion of the HSC compartment in the first few years deduced from cross-sectional telomere length measurements in human leukocytes [13].

65

Development and organization of hematopoietic tissue The hematopoietic system has been subdivided into a hierarchy of three distinct populations [19,20]. In this model, the most mature cells are morphologically identifiable as belonging to a particular lineage and have very limited proliferative potential. The cells in this most mature compartment are derived from committed progenitor cells with a higher but finite proliferative potential. Committed progenitor cells are in turn produced by a population of multipotential HSCs with variable “selfrenewal” potential. Hematopoiesis in embryos is first observed in “blood islands” in the yolk sac [15]. Blood island precursor cells are mesenchymal cells derived from the primitive streak region of the early blastoderm. In the mouse, the yolk sac does not contain precursors of adult hematopoiesis, which can first be found in the aorta–gonad–mesonephros (AGM) region at day 10 of gestation [21]. From the AGM region, hematopoiesis appears to move to the fetal liver before reaching its final destination in the marrow. The concept of subsequent waves of progenitor cells seeding different tissues is compatible with various observations. However, the origin and number of cells seeding a particular hematopoietic niche still needs to be defined. Interestingly, some cells from yolk sac, fetal liver, and marrow are all capable of producing spleen colonies in irradiated adult recipients [15]. This observation, together with the questionable use of spleen colony assays to measure HSCs [22], is probably responsible for what appears to be an incomplete appreciation of the developmental changes in the biologic properties of primitive hematopoietic cells. Results showing that cells from early stages of development can function, to a large extent, in adult hosts and vice versa [23] illustrate a remarkable functional adaptive capacity or “plasticity” of hematopoietic cells at various stages of development. However, in the mouse, cells giving rise to spleen colonies upon transplantation into lethally irradiate recipients (colony-forming unit-spleen or CFU-S) from different tissues were found to be qualitatively different in that the number of CFU-S per individual spleen colony decreased during ontogeny [15]. In these experiments, yolk sac CFU-S could be transferred in vivo seven times, fetal liver CFU-S four to six times, and marrow CFU-S from young and adult mice up to three times [15]. These findings support a functional decline in stem cell self-renewal properties with each subsequent cell division and/or a decreasing and limited replicative potential of HSCs (see below). Several more recent observations provide further evidence of ontogeny-related differences between populations of primitive hematopoietic cells at various stages of development (reviewed in [4,24]). In general, fetal cells have a higher turnover rate and a higher replating potential than their functional counterparts from adult marrow. This observation is illustrated in Fig. 6.1 [5,25], which shows the production of CD34+ cells in culture from “candidate” HSCs with a CD34+CD45RAloCD71lo phenotype purified from, respectively, human bone marrow, cord blood, and fetal liver. Using identical growth factors and culture conditions, no significant production of CD34+ cells was observed in cultures initiated with adult bone marrow HSCs, whereas the number of CD34+ cells in cultures of fetal liver HSCs increased several thousand-fold. As the same number of cells was cultured under identical conditions, these observations point to intrinsic differences between the candidate HSCs themselves. Developmental switches have also been described in the immune system (reviewed in [26]). The switches from embryonic to fetal to adult hemoglobin in red cells reflect epigenetic changes in HSCs that could also affect their biologic properties upon transplantation. Because such epigenetic differences between HSCs at different developmental stages

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Fig. 6.1 Ontogeny-related differences in production of CD34+ cells in culture. Human candidate hematopoietic stem cells (HSCs) with a CD34+CD45RAloCD71lo phenotype were purified from bone marrow, umbilical cord blood and fetal liver, and cultured in serum-free culture medium supplemented with interleukin-6 (IL-6), IL-3, stem cell factor, and erythropoietin (Epo) as described in [25]. Cultures (1 mL each) were initiated with 104 sorted cells. At the indicated time interval, the total number of nucleated (䊐) and CD34+ (䊏) cells present in the cultures was calculated from the cell counts and the percentage of viable CD34+ cells measured by flow cytometry. All CD34+ cells from bone marrow cultures and fractions of the CD34+ cells from cord blood and fetal liver cultures were sorted and used for continuation of the cultures. Reproduced with permission from [5]. Copyright (1993), The Rockefeller University Press.

are likely, many assumptions about HSCs in adults need experimental verification. For example, it is currently not clear that adult HSCs have a similar potential to their fetal counterparts to produce lymphoid progenitors. In the mouse, the ability of HSCs to produce specific lymphocyte precursors is under developmental control [27], and the number of HSCs with full lymphoid potential in adults is expected to be limited. Similarly, the cells of the microenvironment are expected to show changes as a function of normal development and as a function of age. Uncertainties regarding the composition of the stem cell microenvironment and its function over time, combined with similar questions about the cells in the stem cell compartment, represent ongoing challenges in the basic science of HSCs and HSC transplantation.

Molecular control of stem cell fate Of fundamental interest to experimental hematologists and developmental biologists are the mechanisms that control the fate of HSCs. At any point in time, a “stem” cell has a choice to contribute to either the immediate future (by differentiating and producing committed progenitor cells) or the more distant requirements for mature cells (by undergoing a functional self-renewal division). It has been proposed that such decisions at the level of pluripotent HSCs can be depicted as stochastic processes that are intrinsic to the cells [20,28]. If this concept is correct, the distinction between HSCs with limited and extensive in vivo repopulation potential will likely remain elusive as the critical distinction between the cells, the number of actual self-renewal divisions executed in vivo, would be governed by chance. This possibility is certainly in agreement with the difficulties encountered by many investigators in purifying repopulating HSCs to homogeneity. However, as was outlined above, the situation is more complex as the self-renewal properties of HSCs are also subject to developmental control. Furthermore, it seems likely that the two daughter cells resulting from cell division of a single HSC may each differ in their functional properties. Such asymmetric cell divisions are key in animal development [29] and need to be taken into account in models of HSC biology and hematopoiesis [9,30]. The ability of a limited number of HSCs to reconstitute hematopoiesis in an irradiated recipient strongly suggests

that factors in the environment are also crucial in directing stem cell fate. Most likely, HSCs switch upon transplantation from symmetric “self-renewal” divisions (which increase the absolute number of stem cells, albeit at the expense of telomere loss) to asymmetric “maintenance” cell divisions as the bone marrow and its HSC “niches” become occupied with cells. Although the molecular details that govern this switch remain to be elucidated, it seems likely that the orientation of the mitotic spindle relative to the basal layer is involved [9,31]. One interesting possibility is that, following DNA replication but prior to mitosis, the two sister chromatids in the stem cells carry distinct epigenetic marks at the centromeric DNA as well as at the regulatory regions of specific genes [32]. The nonrandom segregation of sister chromatids could support asymmetric cell divisions and maintenance of stem cells, whereas the random segregation of sister chromatids could favor self-renewal and increased stem cell numbers (Plate 6.1).

Transcriptional control of stem cell function Despite intensive studies, the role of soluble factors involved in stem cell fate decisions is not clear. The dramatic clinical effect of cytokines such as erythropoietin, granulocyte colony-stimulating factor, stem cell factor, thrombopoietin, and others have underscored the important and critical role that cytokines play in the proliferation and differentiation of committed hematopoietic progenitor cells. However, attempts to modulate cell fate decisions in early hematopoietic cells using cytokines have invariably been unsuccessful [28,30,33,34]. Perhaps the primary role of cytokines and the microenvironment in HSC biology is to provide the essential but primarily permissive environment required for survival, proliferation, and differentiation. But if growth factors or factors in the microenvironment do not dictate stem cell fate decisions, what factors do? Important clues to this crucial question have been obtained by studies of the expression of homeobox genes in human hematopoietic cells [35]. It was found that expression of Hox genes is restricted to the most primitive hematopoietic progenitors and that the overexpression of HoxB4 in murine HSCs results in a remarkable expansion of cells with long-term lymphomyeloid repopulat-

Molecular Biology of Stem Cell Renewal

ing potential [36,37]. The involvement of Hox genes in stem cell biology is particularly striking because Hox genes are better known as key regulators of patterning along the body axis of developing embryos [38]. It is possible that Hox genes are part of the network that controls stem cell expansion during development. However, the precise mechanism by which overexpression of Hox (fusion) genes increases self-renewal is currently not understood. Another signaling pathway that is clearly very important for stem cell self-renewal is the WNT signaling pathway [39–41]. This evolutionary conserved signaling pathway spans all the way from the cell membrane to regulate transcription at several stages of B- and T-cell development, as well as HSC self-renewal. Unlike the Hox genes, for which the connection with extracellular regulation is not clear, WNT signaling provides a degree of extracellular control over the self-renewal of HSCs. How the WNT pathway regulates stem cell fate in relation to other regulatory factors, including HOX proteins and other transcription factors, remains to be established [42,43]. None of the current models of how stem cell fate is controlled has led to procedures that allow a manipulation of HSCs ex vivo in a way that is clinically meaningful. Given the known complexity of transcriptional regulation and the difficulty of assaying stem cell function, mentioned above, a detailed understanding of the factors that control HSC self-renewal is likely to remain elusive for the foreseeable future.

Telomere structure and function Without extremely reliable mechanisms to duplicate and segregate complete copies of genomes into daughter cells, life in any form could not exist. Although much progress has been made in deciphering the many factors and pathways involved in securing the fidelity of DNA replication and chromosome segregation [44], many questions in this general area remain. The ends of chromosomes are crucially important to maintain genome stability, and telomeres are of particular interest in relation to DNA damage responses and DNA repair pathways. The DNA component of telomeres in all vertebrates consists of (TTAGGG)n. Telomeres distinguish intact from broken chromosomes, stabilize chromosome ends, and provide protection against nuclear degradation and end-to-end fusion events [45]. Telomeres are furthermore involved in the positioning of chromosomes within the nucleus and are also important for chromosome segregation [46]. Almost 50 years ago, Hayflick suggested that most normal human cells are unable to divide indefinitely but are programmed for a given number of cell divisions [47]. In 1990, several papers described a loss of telomeres with replication and with age, and suggested that progressive telomere shortening could explain Hayflick’s original observation [48–50]. This model was confirmed by subsequent studies showing that transfer of the telomerase reverse transcriptase gene could prevent telomere erosion and resulted in immortalizing of the cells that Hayflick studied in most detail: normal diploid human fibroblasts [51,52]. Since then, many papers have appeared that are compatible with the notion that telomere shortening limits the number of times most normal diploid cells can divide (reviewed in [53]). Telomeric DNA is lost in human cells via several mechanisms that are related to DNA replication, remodeling, and repair. Causes of telomere loss include the “end replication problem” [54,55], the nucleolytic processing of 5′ template strands following DNA replication to create a 3′ single-strand overhang [56,57], and the failed repair of oxidative DNA damage to telomeric DNA [58–60]. The relative contribution of the different mechanisms of telomere shortening that have been proposed (reviewed in [61]) to the overall decline in telomere length with age is not known and most likely varies between cell types and individuals, and as a function of age.

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To compensate for the inevitable loss of telomere repeats with each cell division, most cells express telomerase. Telomerase is a ribonucleoprotein containing the reverse transcriptase telomerase protein (hTERT), the telomerase RNA template component (hTERC), and the dyskerin protein as essential elements [62]. In addition, a number of proteins have been described that are important for telomerase assembly (sub) nuclear localization [63] and stability (reviewed in [64]). Telomerase is capable of extending the 3′ ends of telomeres. Telomerase levels are typically high in immortal cells that maintain a constant telomere length, such as the stem cells of the germline in the testis and embryonic stem cells. For reasons that remain to be precisely defined, the telomerase activity that is readily detected in human candidate HSCs [65] (reviewed in [66]) appears to be incapable of maintaining telomere length in these cells. Nevertheless, existing telomerase levels in HSCs are functionally important, as is highlighted in patients with the disorder dyskeratosis congenita (DKC). One possibility is that only very short telomeres provide a suitable substrate for telomerase in somatic cells. Patients with the autosomal dominant form of DKC have one normal and one mutated copy of the telomerase RNA template gene [67]. As expected, such patients show a modest reduction in telomerase levels, yet they eventually typically suffer from marrow failure, immune deficiencies, pulmonary fibrosis or cancer and rarely live past the age of 50 [68–70]. These findings are in stark contrast to those in the mouse, where a complete lack of telomerase activity is tolerated for up to six generations [71]. An emerging consensus is that telomere shortening in long-lived mammals acts as a tumor suppressor mechanism that prevents an unlimited and life-threatening proliferation of organ-specific stem cells and lymphocytes [72]. Current data are compatible with a model in which telomerase levels in human HSCs are tightly regulated and sufficient to elongate only a limiting number of short telomeres. As a result, the telomere length in human HSCs declines with cell proliferation and with age, eventually triggering replicative senescence or apoptosis. In patients with inherited telomerase deficiencies, limitations in the proliferation of various stem cells imposed by telomere loss occur earlier in life than normal.

Telomeres in the hematopoietic system Since the important original observation that telomeres in adult blood leukocytes are significantly shorter compared with germline material (sperm) from the same donor [73], the decline in somatic telomeres has been documented in three ways. The original observation was confirmed [48,74], it was shown that telomeres in various tissues were shorter in older donors [49,50], and telomere shortening was documented during in vitro culture of human cells [49,75]. In 1994, it was reported that cells produced in culture by highly purified human “candidate” HSCs from fetal liver, cord blood, and adult bone marrow could be distinguished by reproducible differences in telomere length, and that these cells showed a measurable decline in telomere length upon culture in vitro (Fig. 6.2) [7]. In the years that followed these initial reports, a large number of papers have appeared that have greatly refined our understanding of telomere shortening in human nucleated blood cells (reviewed in [17]). Studies in this general area have been facilitated by the development of quantitative fluorescence in situ hybridization (FISH) techniques to measure the telomere length in suspension cells using flow cytometry (“flow FISH” [76, 77]). With this technique, it has been shown that the age-related decline in telomere length in lymphocytes is much more pronounced than in granulocytes, and that rapid telomere shortening early in life is followed by a much more gradual decline thereafter (Fig. 6.3) [13].

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Fig. 6.2 Loss of telomeric DNA in cells from cultures of purified human candidate hematopoietic stem cells (HSCs) with a CD34+CD45RA10 CD710 phenotype from fetal liver, cord blood, and bone marrow. The purified cells were cultured in serum-free medium supplemented with a mixture of cytokines to stimulate their proliferation and obtain sufficient numbers of cells for telomere restriction fragment (TRF) length measurements by Southern analysis. The mean ± SE of TRF measurements for two different donors of each tissue is shown for the cells produced in culture (䊐) as well as for the total nucleated cells from each donor before culture and cell purification ( ). Reproduced with permission from [7]. Copyright (1994), National Academy of Sciences, USA.

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If one assumes that the number of cell divisions between HSCs and granulocytes is relatively constant throughout life and that telomere shortening in HSCs is (i) primarily resulting from replication, and (ii) relatively constant with each cell division, the telomere length in granulocytes can be used as a surrogate marker for the average telomere length

Fig. 6.3 Age-related loss of telomere length in lymphocytes and granulocytes from peripheral blood of normal individuals measured by flow fluorescence in situ hybridization (FISH). The specific telomere fluorescence of lymphocytes and granulocytes was analyzed after gating on these cells based on light scatter properties and DNA fluorescence. Note the heterogeneity in telomere fluorescence values, the overall decline in telomere fluorescence with age in both cell types, and the higher rate of telomere attrition in lymphocytes. In this study, it was found that a bi-segmented line resulted in a significantly better fit with the data than the linear fit shown on the left. Inserts show the results of bi-segmented fit analysis for lymphocytes (top) and granulocytes (bottom). Arrows indicate the optimal intersection of calculated regression lines which for both cell types was before 2 years of age. Reproduced with permission from [13]. Copyright (1999), the Rockefeller University Press.

in the “average” HSC clone. While the telomere length in leukocytes shows an overall decline with age [13,78], the telomere length at any given age in humans is highly heterogeneous, as is illustrated in Fig. 6.3 below. This variation appears to be primarily genetically determined [13,79]. For example, monozygous twins of over 70 years of age were

Molecular Biology of Stem Cell Renewal

shown to have a very similar telomere length in both granulocytes and lymphocytes, whereas such values in dizygotic twins differed more but not as much as between unrelated individuals [13]. Using further refinements in the flow FISH method ([76]), it was recently shown that the rapid decline early in life is followed by a slow decline until the age of 50–60 years, after which the decline again accelerates [80]. The decline in both granulocytes and lymphocytes is nonlinear and fits a cubic curve. The pronounced decline in telomere length in specific T and natural killer cells could trigger apoptosis in these cells during a normal lifetime and compromise immune responses in the elderly. In contrast, granulocytes show a much more modest decline in telomere length with age, and critical telomere shortening in HSCs during normal hematopoiesis does not seem very plausible. More likely, the total production of blood cells from a single HSC is primarily determined by differentiation into committed progenitor cells and not by replicative senescence. Furthermore, the occasional loss of individual stem cells via telomere shortening is not expected to impact on overall hematopoiesis (or on the overall telomere length in granulocytes) as long as an excess of HSCs exists in the marrow. Indeed, studies in mice suggest that HSCs accumulate with age, be it that their functional properties in terms of replicative potential and production of B lymphocytes is diminished [81,82]. Nevertheless, the age-related loss of telomeres in granulocytes, the (modest) loss of telomeres following allogeneic transplantation [8,83], and the aplastic anemia that follows partial telomerase deficiency [84–86] all indicate that telomerase levels in normal HSCs are limiting and that the proliferation of HSCs is ultimately also limited by progressive telomere shortening. The number of mature “end” cells, such as granulocytes, that is produced by an individual HSC is expected to be highly variable as a result of the poorly understood regulation of symmetric versus asymmetric cell divisions. Even a very limited number of additional self-renewal divisions in a HSC will greatly increase cell output, and if endowed with a modest proliferative advantage, individual HSCs can produce a staggering number of cells. This phenomenon is illustrated in clonal proliferative disorders such as paroxysmal nocturnal hemoglobinuria and chronic myeloid leukemia. However, even in chronic myeloid leukemia, clonally expanded Philadelphia-positive stem cells eventually appear to encounter a telomere crisis [87]. Unfortunately, with a large number of cells to select from, the genetic instability triggered by the loss of functional telomeres appears to facilitate the emergence of subclones in blast crisis that typically show additional genetic abnormalities, more malignant properties, and higher levels of telomerase.

The telomere checkpoint Most human somatic cells, including HSCs, express limiting levels of telomerase, and as a result, telomere shortening effectively limits their proliferative potential. Most likely, telomere shortening evolved as a checkpoint function to suppress tumor growth in long-lived species. The function of this “telomere checkpoint” may help explain poorly understood aspects of stem cell biology, including stem cell “exhaustion” in aplastic anemia and other disorders. Cells may bypass the telomere checkpoint by expressing high levels of telomerase or by inactivating downstream signaling events, for example by loss of p53 function. Some cells, including subsets of B cells [88], appear to avoid the telomere checkpoints altogether, and this process could make these cells more vulnerable to tumor development. Loss of p53 function also inactivates the telomere checkpoint. This is expected to be a rare event as both copies of the normal p53 allele in a cell must typically be lost or mutated in order to continue proliferation in the presence of many short and dysfunctional telomeres [89]. Loss of p53 function allows survival of cells with dysfunctional telomeres. The resulting chromosome fusions result in chromosome breakage and, as a result,

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in the loss and amplification of genes [90]. This leaves us with a paradox: telomere shortening can act both as a tumor suppressor mechanism (by imposing limits to the replication of somatic cells) and as a tumor promoter mechanism (by favoring genetic instability once cells have reached their telomere-imposed limit in replicative potential). Whether tumor growth is suppressed or promoted by progressive telomere shortening may depend on the number of cells that encounter such a telomere crisis and the genetic changes acquired by such cells prior to this encounter.

Conclusion In this chapter, some molecular aspects related to stem cell renewal were discussed. Developmental changes in the functional properties of HSCs and loss of telomeric DNA in HSCs are important considerations in both laboratory studies and the clinical use of HSCs. It is clear that, despite much progress over the last several decades, we are still left with many questions. Some of the most urgent are: How many times can HSCs divide, and what is the actual replicative potential of HSCs at various stages of development? To what extent are factors in the microenvironment (i.e., cytokines) capable of modulating the self-renewal properties of HSCs? How does the microenvironment change during development and with age? What are the mechanisms controlling the turnover time of HSCs in steady state hematopoiesis and during hematopoietic regeneration? Are differences in the replicative history of HSCs reflected in their anatomic location, cytokine response, and mobilization properties? Hopefully, answers to these important questions will become available in the near future. Studies with cultured hematopoietic cells have shown that the formation of hematopoietic colonies, as well as the proliferation and survival of various hematopoietic cells, typically requires the presence of “hematopoietic growth factors,” many of which have been cloned over the last decade and produced for clinical trials (reviewed in Chapter 41). The availability of recombinant growth factors has led to frantic research efforts over the past two decades to achieve clinically useful manipulation of blood cell production in vitro. Two sets of observations that were discussed in this chapter cast doubt about the feasibility of some of the perceived applications of ex vivo “expansion” and should probably be taken into account when laboratory or clinical experiments involving limited numbers of HSCs are con-templated. First, there are the observations indicating that currently ill-defined, intrinsic (genetic) mechanisms with a developmental component control the fate of HSCs. Second, hematopoietic cells, including purified HSCs, appear to lose telomeric DNA with each cell division [7]. Recent studies indicate that inherited telomerase deficiencies can give rise to a large number of disorders including idiopathic pulmonary fibrosis [91], aplastic anemia [86,92], and dyskeratosis [93]. The expectation is that inherited defects in telomere maintenance pathways will prove to be involved in many more diseases. The studies of telomeres have focused attention on possible genetic limitations in the replicative potential of somatic cells, including HSCs, and such restrictions are compatible with most experimental evidence produced in clinical and laboratory studies. In view of these considerations, it seems prudent to use reasonably large numbers of HSCs for transplantation, especially from adult donors [83]. Although the number of HSCs in fetal liver or cord blood is small, such cells are likely to be superior in terms of replicative potential. This genetic superiority must be balanced against practical disadvantages including the small number of cells available for transplantation. The small size of fetal or neonatal stem cell grafts is expected to compromise clinical results, especially in the presence of histocompatibility differences between donor and recipient [94,95]. Possibly, this limitation could be addressed by more effective immunosuppression and conditioning regimens. In addition, the outcome of fetal liver and cord blood transplants could possibly be improved by increasing the number of cells in the transplant, for example by culture ex vivo.

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Chapter 6

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72. Maser RS, DePinho RA. Connecting chromosomes, crisis, and cancer. Science 2002; 297: 565– 9. 73. Cooke HJ, Smith BA. Variability at the telomeres of the human X/Y pseudoautosomal region. Cold Spring Harb Symp Quant Biol 1986; 51(Pt 1): 213–19. 74. Allshire RC, Gosden JR, Cross SH et al. Telomeric repeat from T. thermophila cross hybridizes with human telomeres. Nature 1988; 332: 656– 9. 75. Counter CM, Avilion AA, LeFeuvre CE et al. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. Embo J 1992; 11: 1921–9. 76. Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc 2006; 1: 2365–76. 77. Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol 1998; 16: 743– 7. 78. Frenck RW, Jr., Blackburn EH, Shannon KM. The rate of telomere sequence loss in human leukocytes varies with age. Proc Natl Acad Sci U S A 1998; 95: 5607–10. 79. Slagboom PE, Droog S, Boomsma DI. Genetic determination of telomere size in humans: a twin study of three age groups. Am J Hum Genet 1994; 55: 876–82. 80. Baerlocher GM, Rice K, Vulto I, Lansdorp PM. Longitudinal data on telomere length in leukocytes from newborn baboons support a marked drop in stem cell turnover around 1 year of age. Aging Cell 2007; 6: 121–3. 81. Bryder D, Rossi DJ, Weissman IL. Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am J Pathol 2006; 169: 338–46. 82. Linton PJ, Dorshkind K. Age-related changes in lymphocyte development and function. Nat Immunol 2004; 5: 133–9. 83. Awaya N, Baerlocher GM, Manley TJ et al. Telomere shortening in hematopoietic stem cell transplantation: a potential mechanism for late graft failure? Biol Blood Marrow Transplant 2002; 8: 597–600.

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84. Harrington L. Making the most of a little: dosage effects in eukaryotic telomere length maintenance. Chromosome Res 2005; 13: 493–504. 85. Vulliamy T, Dokal I. Dyskeratosis congenita. Semin Hematol 2006; 43: 157–66. 86. Yamaguchi H, Calado RT, Ly H et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med 2005; 352: 1413–24. 87. Brummendorf TH, Holyoake TL, Rufer N et al. Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry. Blood 2000; 95: 1883–90. 88. Martens UM, Brass V, Sedlacek L et al. Telomere maintenance in human B lymphocytes. Br J Haematol 2002; 119: 810–18. 89. Martens UM, Zijlmans JM, Poon SS et al. Short telomeres on human chromosome 17p. Nat Genet 1998; 18: 76–80. 90. de Lange T. Telomere dynamics and genome instability in human cancer. In: Blackburn EH, Greider CW, editors. Telomeres. Plainview, NY: Cold Spring Harbor Laboratory Press; 1995. pp. 265–93. 91. Armanios MY, Chen JJ, Cogan JD et al. Telomerase mutations in families with idiopathic pulmonary fibrosis. N Engl J Med 2007; 356: 1370–2. 92. Fogarty PF, Yamaguchi H, Wiestner A et al. Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA. Lancet 2003; 362: 1628–30. 93. Vulliamy TJ, Marrone A, Knight SW, Walne A, Mason PJ, Dokal I. Mutations in dyskeratosis congenita: their impact on telomere length and the diversity of clinical presentation. Blood 2006; 107: 2680–5. 94. Petersdorf EW, Malkki M. Genetics of risk factors for graft-versus-host disease. Semin Hematol 2006; 43: 11–23. 95. Sierra J, Storer B, Hansen JA et al. Transplantation of marrow cells from unrelated donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor HLA-matching, and marrow cell dose. Blood 1997; 89: 4226–35. 96. Yamashita YM, Mahowald AP, Perlin JR, Fuller MT. Asymmetric inheritance of mother versus daughter centrosome in stem cell division. Science 2007; 315: 518–21.

7

Catherine M. Flynn & Catherine M. Verfaillie

Cellular Biology of Hematopoiesis

Introduction

Stem cells

Hematopoiesis

Stem cells can be defined based on their obligatory characteristics of (1) self-renewal and (2) differentiation, as well as (3) functional repopulation of a tissue in vivo. Self-renewal is defined as the ability of a cell to divide without maturation and produce progeny identical to the parent cell. This process can be symmetric, wherein a single stem cell gives rise to two daughter stem cells, or asymmetric, generating a single new stem cell and a second daughter cell that has differentiated features. What governs asymmetric versus symmetric cell divisions is not yet known. All stem cells can differentiate, in response to the correct intrinsic and environmental cues, towards progeny of one or more lineages. Depending on the number of cell types that stem cells can generate, they are termed totipotent, pluripotent, and multipotent (Table 7.1). Totipotent stem cells, or the fertilized oocytes, can form a fully functional organism. Pluripotent stem cells differentiate into all cells of the embryo, i.e. the three germ layers – endoderm, ectoderm, and mesoderm – and the germline. Multipotent stem cells can be isolated from various tissues in fetal and adult animals. Such multipotent stem cells, sometimes referred to as adult stem cells or tissue-specific stem cells, differentiate into a limited range of cell types that are specific to the tissue from where the stem cell was obtained [1]. Stem cells are the key ingredient of hematopoietic cell grafts and have been used clinically since 1968 when the first successful BM transplant was performed at the University of Minnesota [2]. Since then, hematopoietic cell transplantation (HCT) has become a well-established treatment modality for malignant and nonmalignant diseases [3]. HSCs, the best-described stem cells, are categorized as multipotent stem cells because they self-renew, differentiate into all the cells of the blood lineage, and reconstitute the hematolymphoid system for the life of an individual.

“Hematopoiesis” is derived from the two words, “(h)aima” for blood and “poiein” meaning to make. Hematopoiesis involves generating an entire hematopoietic and immune system for the life of an individual. The cellular biology of hematopoiesis encompasses three components: the hematopoietic stem cell and its progeny, the environment in which they reside, and the signals and cytokines that regulate their fate. In adults, hematopoiesis occurs in the bone marrow (BM), which supports the lifelong maintenance of stem cells and their regulated trilineage differentiation. The earliest hematopoietic cells of the developing mammalian embryo are detectable in the blood islands in the extraembryonic yolk sac at 2–3 weeks. Subsequently, hematopoietic cells are found within the embryo proper in the gonad–mesonephros region, from where hematopoietic stem cells (HSCs) migrate to the fetal liver and later to the BM. The regulation of HSC fate is complex, and the exact mechanisms that govern HSC self-renewal are not yet fully understood. This process involves factors intrinsic to the HSC and cues from the surrounding microenvironment, which plays a critical role as a supporting structure for HSCs. The BM microenvironment is composed of, aside from HSC progeny, such as macrophages, cells of mesenchymal origin like osteoblasts and adipocytes, and endothelial cells. The mesenchymal lineage cells are purportedly derived from a stem cell different from the HSC, namely the mesenchymal stem cell (MSC), also termed the marrow stromal cell. These three cell types create the HSC niche wherein the HSCs reside, and from which HSCs are released or whereto they home in manipulations aimed at the mobilization and transplantation, respectively, of HSCs. Both HSCs and MSCs continue to be a subject of intense interest and excitement, particularly with the wealth of recent information outlining mechanisms of HSC homing and mobilization, as well as the concept of stem cell plasticity. The focus of this chapter is to discuss HSC biology, HSC regulation, and a critical assessment of the presumed greater potentiality or plasticity of BM stem cells, whether HSCs or MSCs.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Stem cell plasticity A number of studies during the last decade have suggested that the therapeutic potential of BM cells may be more extensive than originally thought, because investigators have noted that HSCs and other cells residing in the BM may have differentiation ability beyond their tissuespecific stem cell fate. While conventional dogma dictates that multipotent adult stem cells are irreversibly committed to the organ in which they reside, the cloning of mammals from the nucleus of an adult somatic cell refuted the notion of irreversible commitment, and observations from a number of additional experimental models have challenged this paradigm [4]. Likely more than 1000 reports have suggested that

Cellular Biology of Hematopoiesis

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Table 7.1 Stem cell hierarchy Stem cell

Example

Explanation

Totipotent

Fertilized oocyte

Pluripotent

Embryonic stem cells Embryonic germ cells Inner cell mass of the blastocyst Primordial germ cells of the early embryo Hematopoietic stem cells Skin stem cells Neural stem cells

Cells with the capacity to form mesoderm, endoderm, and ectoderm, including germ cells and extraembryonic tissue Cells with the capacity to form mesoderm, endoderm, ectoderm, and germ cells. Cannot give rise to trophoblast

Multipotent

Cells within various adult organs that have the capacity to self-renew and differentiate into all the organ-specific cell types

stem cells residing in adult tissues can generate, under certain environmental conditions, specialized cell types other than those of the tissue from where the stem cells were obtained, and this even irrespective of the particular germ layer designation (mesoderm, ectoderm, and endoderm). This concept has been termed “stem cell plasticity” and has led to considerable excitement as well as skepticism. If correct, this would defy the developmental biologic principles that lineage-specific stem cells generate only differentiated cells from the tissue of origin, and would signify that adult stem cells may hold much broader therapeutic potential. BM-derived cells have been central to this paradigm shift, as they have been reported to contribute to a number of seemingly unrelated tissues and organs, including the central nervous system [5], skeletal muscle [6], liver [7,8], and cardiac muscle [9], particularly in the context of tissue injury. While evaluating reported plasticity of BM-derived cells, it is important to recognize that fresh adult BM contains multiple stem cell populations that include HSCs, MSCs, and endothelial progenitor cells [10]. In addition, cells with molecular features of hepatic progenitors and other cell types have been described [11]. It is thus possible that somatic stem cells may circulate from their tissue of origin and reside or pass through the BM. Plasticity would then only represent appropriate differentiation of a passenger progenitor/stem cell population. As will be discussed further in the chapter, there is also mounting evidence that, in particular, macrophages derived from HSCs can fuse with a differentiated host cell, creating the false impression that HSCs transdifferentiate in a cell-autonomous fashion to cell lineages other than the hematopoietic lineage [12]. It will be important to further decipher what possible mechanisms underlie BM cell plasticity, whether these be fusion or true transdifferentiation, to determine the clinical implications of the apparent plasticity.

Stem cells in BM HSCs During ontogeny, HSCs can be found in different locations, including the blood islands in the placenta, aorta–gonad–mesonephos region, fetal liver, and BM. In postnatal life, HSCs are found in the BM at a low frequency (1 : 105 cells of mouse BM [13]). At birth, HSCs are also found in the placenta [14,15] and in the peripheral blood of the infant, and hence umbilical cord blood (UCB) [16]. Postnatally, HSCs can also be found at very low frequency in peripheral blood [17], and they circulate at higher frequency following administration of cytokines such as granulocyte colony-stimulating factor [18] or of chemotherapeutic agents like

cyclophosphamide [19]. The observation that HSCs can leave the BM environment and be found in the circulation is of importance when considering possible plasticity of stem cells from tissues other than BM. As HSCs can only be defined based on their functional attributes, i.e. repopulation of the hematopoietic system following transplantation, murine HSCs remain the best characterized at present, chiefly because very accurate functional assays for murine HSCs exist that do not exist for human HSCs. The most accurate enumeration of murine HSCs is done via competitive repopulation assays [20], wherein a population of test cells are co-transplanted with a fixed number of BM cells and repopulation by the test cells is evaluated at 4–6 months following transplantation. To assess the self-renewal ability of the test cells, the BM from the initial recipient is grafted into secondary animals, where again repopulation by the test cells is evaluated. Using such in vivo studies, murine HSCs were found to be highly enriched (1 : 10 cells) within the c-Kit receptor (c-Kit)-positive, T-cell antigen (Thy-1.1)-low, lineage (Lin)-negative, stem cell antigen (Sca-1)-positive (KTLS) fraction of murine BM [21]. Subsequent studies have further enriched murine HSCs by evaluating additional stem cell markers, including CD50 and Flk1 among others [22]. Additional tools to identify murine HSCs include evaluation of the efflux of a number of dyes such as rhodamine (via the multidrug resistance pump) [23] or the DNA-binding dye Hoechst-33342 (via the breast cancer resistance protein) used to define the side population cells, or Sp cells [24]). Isolation of human HSCs has been more challenging owing to the lack of accurate assays such as the reconstitution of lethally irradiated animals. Surrogate in vitro and in vivo assays have been used to screen for biologic activity, including a number of long-term stromal cultures (long-term culture-initiating cells [25], cobblestone area-forming cells [26], myeloid–lymphoid initiating cells [27]) or transplantation into immunodeficient hosts such as the nonobese diabetic severe combined immunodeficiency (NOD-SCID) mouse (SCID-repopulating cell) [28] or the preimmune fetal sheep [29]. However, due to the xenogeneic nature of such transplantation models, accurate assessment of HSC quantity and quality is not possible. As in the mouse, investigators have used fluorescence-activated cell sorter (FACS) separation to enrich for candidate human HSCs. A number of different sorting strategies have allowed enrichment of human HSCs to 1 : 100–500, including selecting for CD34+Thy-1+Lin− [30], CD34+Lin−CD38− [31], CD34+Lin−CD38−ckit+Rho− [32] or CD34+CD38−Lin−Sp [33] cells from BM or UCB [28,34]. Despite the best selection procedures for human HSCs based on cell-surface markers or their ability to extrude dyes, the resultant population remains heterogeneous and contains less than 1% SCID repopulating cells.

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Chapter 7

MSCs The BM stroma plays an important role in sustaining the survival, proliferation, and differentiation of mature blood cells from HSCs. Cells within the BM microenvironment that contribute to this support are, in addition to HSC-derived macrophages, endothelial cells and mesenchymal cells, including osteoblasts and adipocytes. The latter cells are progeny from a stem/progenitor cell, the MSCs. Like HSCs, MSCs are rare [35], comprising 0.01–0.001% of BM cells [36]. MSCs were first described in 1976 [37] as plastic adherent cells from murine BM that, at the clonal level, gave rise to colonies of fibroblast-like cells which could be induced to differentiate to adipocytes, osteoblasts, and chrondrocykes. Owing to their fibroblastic appearance, Friedenstein named these cells “colony-forming fibroblasts” or “CFU-Fs.” Since the initial identification of MSCs in BM, MSCs have also been isolated from a number of other tissues, including UCB, placental tissue, fetal blood and liver, adipose tissue, exfoliated deciduous teeth, amniotic fluid, and adult lung and pancreas [38–43]. MSCs are self-renewing clonal precursors of nonhematopoietic stromal tissues. They are isolated and expanded in culture based on their ability to adhere to plastic in response to factors present in fetal calf serum. However, cultures of MSC are highly heterogeneous with only rare multipotent, uncommitted cells between a sea of more differentiated precursors. MSCs differentiate into cells found in limb bud mesoderm, including osteoblasts, chondroblasts, adipocytes, fibroblasts, smooth muscle-like cells or myofibroblasts, and skeletal muscle cells. However, this differentiation potential is quickly lost from ex vivo expanded MSCs, likely due to the fact that – as is true for HSCs – self-renewing cell divisions of MSCs in vitro occur rarely [44]. Morphologically, cultured MSCs are spindle-like cells with a fibroblast-like appearance. The phenotype of cultured MSCs has been established by a number of investigators as positive for CD44, CD29, CD90, CD105, CD73 but negative for hematopoietic markers CD34, CD45, and CD14, and endothelial markers CD34, CD31, and von Willebrand factor. The phenotype of freshly isolated MSCs is less clear, although enrichment can be obtained by sorting for cells positive for CD105 (SH2) and CD73 (SH3), or for Stro-1brightVCAMbright cells [45–47]. Whether one should even consider MSCs to be “stem cells” depends on how one defines stem cells. If the definition includes the notion of in vivo repopulation of a given tissue, few if any studies have demonstrated that MSCs are true stem cells. What the role is of MSCs within BM is not clear, although they likely are responsible for replacing osteoblasts and adipocytes. MSCs express a wide variety of cell adhesion proteins, interact with endothelial cells, and possess the ability to respond to a wide variety of cytokines and chemokines. Their differentiated progeny secrete a multitude of cytokines and growth factors, which play a role in supporting hematopoiesis. The ability of MSCs to enhance HSC engraftment has been tested and proven in murine models [48,49]. Specifically, co-transplantation of murine MSCs with limiting doses of murine HSCs results in significantly more mice in which hematopoietic reconstitution is seen. Similar results have been observed when human MSCs and HSCs are co-transplanted into immunodeficient mice [50]. This has led to ongoing clinical trials wherein MSCs are co-transplanted with – for instance – haploidentical CD34+ cells or UCB grafts to enhance engraftment [51]. Although MSCs are present in BM harvests, it is unclear whether host mesenchymal cells are replaced by donor-derived cells. Horwitz et al. found that 1–2% of osteoblasts from a patient after HCT were of donor origin [52], and Cilloni et al. [53] showed that 7 of 41 patients who underwent a sex-mismatched leukodepleted transplant displayed donor stromal engraftment. By contrast, a significantly larger number of studies concluded that MSCs, adipocytes, osteoblasts or stromal cells were not

donor derived following HCT. The reason for these discrepancies is not known. It is also not known why MSCs do not engraft. One possible explanation is that, for at least culture-expanded MSCs, over 95% remain within the lung vascular bed and rarely move beyond this capillary bed [54]. As MSCs secrete many cytokines that support hematopoiesis, investigators have examined the possibility that defects in MSCs may contribute to the pathogenesis of hematologic malignancies, including leukemia and myelodysplastic syndrome (MDS). One question is whether the stromal compartment is clonally abnormal. Stromal cultures from patients with MDS, chronic myeloid leukemia (CML), and acute myeloid leukemia have been examined [55–58]. Some have found that stromal cells are devoid of any chromosomal abnormality and are functionally normal, while others have found chromosomal abnormalities in stromal cells. Contaminating HSC-derived macrophages may account for some of the aberrations seen, as, for instance, macrophages (CD14+) in stromal cultures established from CML BM were enriched for Philadelphia chromosome (Ph)-positive cells whereas mesenchymal (CD14−) cells in these same cultures were Ph− [58]. Some reports suggest that endothelial cells in CML patients may be clonally related to the Ph+ HSC clone [59]. However, as has become clear in recent studies, some apparent endothelial progenitors are monocytic in origin [60]. Two recent studies examined the karyotype of hematopoietic cells and stromal cells from patients with MDS [56,57]. In both studies, karyotypic abnormalities were detected in stromal cells and hematopoietic cells, but there was a striking lack of overlap between the karyotypic abnormalities of the hematopoietic and stromal cells. It is not known whether the stromal chromosomal abnormality predates the leukemic process or whether the stromal abnormalities are caused by the same event that caused the initial genetic damage to the HSCs. Whether the stromal cells are initiators, supporters or simply bystanders in the clonal process is not clear. One also needs to be cognizant of the fact that chromosomal alterations may be acquired in culture after several passages, which result in the development of sarcomas when such MSCs are transplanted [61]. The ability of MSCs to secrete cytokines and growth factors may also be exploited for tissue repair. A large number of studies has been published wherein the trophic effects of MSCs on damaged tissues, ranging from radiation-induced damage to the gastrointestinal epithelium, to ischemic renal, cardiac or central nervous system lesions among others, have been described [62–66]. This has led to the contemplation of clinical use of MSCs for therapy of such disorders. A word of caution, however, comes from recent reports that transplantation of MSCs in ischemic tissues may not only have beneficial effects, but also cause aberrant tissue formation. For instance, two recent reports demonstrated that encapsulated structures were found in the infarcted areas of 51% of hearts after injecting MSCs and to a lesser extent unfractionated BM cells [67–69]. An interesting observation is that cultured MSCs can modulate the immune system. Specifically, MSCs are poorly recognized by allogeneic T lymphocytes [70] or by natural killer cells [71], while at the same time inhibiting T-cell [72] and B-cell [73–75] responses. The mechanism underlying this immunomodulatory effect, specifically the inhibition of mixed lymphocyte reactions, is not totally clear, although cell–cell interactions as well as the secretion of soluble T-cell inhibiting factors play a role. This has led to several provoking clinical reports wherein dramatic graft-versus-host disease (GVHD) modulation has been described following MSC administration [51,76,77]. A number of prospective studies are now ongoing testing the efficacy of MSC-based prevention or treatment of GVHD. In addition, the possible usefulness of MSCbased therapies for autoimmune disorders such as inflammatory bowel disease is being evaluated.

Cellular Biology of Hematopoiesis

A number of reports have suggested that MSCs may generate differentiated progeny other than mesenchymal progeny, including endothelial cells, cardiomyocytes, hepatic cells, and neural cells. These studies will be further assessed below.

Plasticity of BM-derived stem cells That BM-derived cells reconstitute the hematopoietic system is well established. Since the late 1990s, numerous reports have appeared concluding that BM-derived cells may also contribute to other tissues, a phenomenon termed plasticity. Some of these surprising results are thought to be due to plasticity of hematopoietic cells, whereas others have been ascribed to plasticity of MSCs. These studies will be discussed in the following paragraphs. While evaluating these studies, we will critically determine to what extent real transdifferentiation has been demonstrated. For that reason, we will here first define criteria that should be fulfilled to demonstrate true plasticity or transdifferentiation. Full proof of plasticity requires the following: 1 A single cell that ordinarily is destined to differentiate towards hematopoietic or mesenchymal progeny can generate the expected progeny as well as cells of another lineage. 2 Differentiated cell types are defined based on morphologic and phenotypic criteria as well as expression of the expected transcripts and proteins, and the acquisition of functional attributes of the unexpected progeny. 3 Finally, the differentiated cell should also integrate into a tissue in vivo and perform the function of the unexpected lineage cell in vivo. When most studies are evaluated based on this set of criteria, relatively few studies have proven that transdifferentiation is occurring. A seminal paper demonstrating the plasticity of BM-derived HSCs was published by Krause et al. in 2001 [78]. In this study, murine, male donor BM cells were HSC enriched by fractionation and lineage depletion, labeled with the membrane intercalating dye PKH26 and injected into irradiated female recipient mice. After 2 days, BM was harvested, bright PKH26 cells were reisolated by FACS, single PKH26 cells were injected into 30 irradiated female hosts, and donor reconstitution was assessed at 5 and 11 months. The HSC-enrichment process, by homing to the BM within 48 hours, identified a population of HSCs capable of durable reconstitution. At 11 months, donor cells contributed 12–86% of the peripheral blood in five mice, and BM from 4 of 5 of these animals could be serially transplanted. Among the five mice who survived to 11 months, HSC-derived progeny could also be found in nonhematopoietic tissues, as proven by presence of the Y chromosome in epithelial cells in the lung, gastrointestinal tract, epidermis, and dermis. Differentiation to the unexpected phenotype was defined based on morphology and expression of cell-type specific antigens, such as cytokeratin 8, 18, and 19. As Y chromosome-positive pulmonary epithelial cells also expressed mRNA for surfactant, it was suggested that these cells had functional properties of alveolar type II pneumocytes. A number of reports subsequently have suggested that the plasticity seen by Krause et al. may not be as extensive. For instance, Wagers et al. [79] evaluated mice grafted with single HSCs (defined as KTLS cells) and found vanishingly rare cells in the brain, liver, and heart that were donor in origin. In this study, donor origin was defined based on the expression of green fluorescent protein (GFP) in the donor HSCs. In addition, they evaluated tissues of parabiotic animals, where wild-type (WT) and enhanced GFP (eGFP) transgenic mice were surgically joined such that they rapidly develop a common, anastomosed circulatory system. As HSCs spontaneously migrate in and out the BM and can be found circulating, one would expect to find unexpected lineage differentiation from HSCs in this model. Even though extensive hematopoietic chimerism was seen, analysis of multiple tissues failed to demonstrate

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significant engraftment of nonhematopoietic tissues by circulating GFPpositive cells. One possible explanation for the discrepancies between the studies was the differing method of identifying donor cells (in situ hybridization of the Y chromosome versus a transgenic marker gene). A subsequent study by Krause et al. reconfirmed the ability of HSCs to transdifferentiate into lung epithelial cells, now using transgenic gene markers [80]. Hence the debate remains whether HSCs in this setting can transdifferentiate into epithelial cells. Since this initial seminal report (and prior to that) a multitude of papers has been published wherein authors describe the possibility that BM-derived stem cells can transdifferentiate. Before the studies are reviewed, we will, however, address possible mechanisms that could explain the perceived and unexpected lineage switch, as well as the many methods that can or have been employed to address plasticity, each with their own caveats.

Possible explanations of plasticity (Fig. 7.1) If plasticity is real, the long-held view that, during development at gastrulation, cells are definitively committed to a single germ layer

(a) Multiple stem cells

=

+

(b) cell fusion

+

=

(c) Trans de-/re-differentiation

(d) Pluripotency

TRENDS in Cell Biology

Fig. 7.1. Possible explanations for the perceived plasticity. (a) Stem cells for a given tissue might exist in an unrelated organ. (b) Perceived plasticity might be caused by the transplanted cells fusing with a host cell of a different lineage, leading to transfer of the genetic information of the transplanted cell to the host-derived cell. (c) Plasticity might occur via de- and redifferentiation, as is seen in cloning or in limb regeneration in amphibians. (d) Cells with pluripotent characteristics might persist even after the initial steps of embryological development. Reproduced with permission from Verfaillie [192]. Copyright (2002), Elsevier.

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(mesoderm, endoderm, ectoderm) would not be totally correct as, in plasticity, tissue-specific stem cells would overcome their definitive commitment to a single germ layer when they were placed in a different and suitable environment. That this might be possible follows from studies as far back as the 1970s in which it was demonstrated that, by transferring the nucleus of Xenopus cells into enucleated, unfertilized Xenopus egg cytoplasm, frogs could be generated with the genetic information of the transferred nucleus. These observations were extended in the 1990s with the cloning of sheep and subsequently other mammals from the nucleus of an adult somatic cell [4]. In addition, dedifferentiation and redifferentiation is a phenomenon known in lower animal species such as newts [81]. How cell fate is altered is not fully understood, although recent studies have shown that overexpression of four transcription factors can reprogram fibroblasts to cells with embryonic stem cell (ESC)-like properties [82,83]. Hence, it is not inconceivable that, in certain circumstances, such reprogramming may occur. What role tissue injury plays in its initiation is controversial. Cells may be summoned to sites of injury and, on arrival in the “damaged” environment, cytokines and external factors may induce changes in gene expression patterns and result in an altered phenotype, and possibly function. Alternative explanations for the perceived plasticity include the fact that tissue-specific stem cells other than HSCs also circulate and home to tissues other than the tissue of origin. In this scenario, neural or liver progenitor/stem cells may be “hiding out’ among the heterogeneous populations of cells existing in the BM. Migration of HSCs from the fetal liver to the BM during development is mediated by a CXCR4– SDF1 gradient, and homing of HSCs to the BM following transplantation is mediated by the same CXCR4–SDF1 gradient [84]. As many stem cells from other tissues also express CXCR4, it is not inconceivable that, during embryogenesis or in postnatal life, stem/progenitor cells other than HSCs may home to the BM [11]. That such cells may act as reserves for tissue regeneration or repair in adult life is tempting to speculate; however, there is no proof for such a hypothesis. A third explanation for plasticity arose in 2002, namely that donor cells can fuse with host cells whether in intact tissue or damaged tissue. Such fusion leads to the generation of 4N cells, which express cell surface and cytoplasmic markers derived from both the donor cell and the cell it fuses with. Terada et al. [85] and Ying et al. [86] found that in co-culture experiments with BM cells or neurospheres and ESCs, tetraploid hybrids could be created that adopted ESC characteristics. Similar findings were subsequently documented in vivo. In an elegant study, Alvarez-Dolado et al. [87] used a Cre–Lox recombination system to detect cell fusion events. Following transplantation of BM cells into lethally irradiated mice, they found that BM-derived cells fused with Purkinje cells, hepatocytes, and cardiomyocytes. Camargo et al. [88] also used a single HSC transplant model to demonstrate that HSCderived hepatocytes occur as a result of cell fusion. Which cell fuses with target cells in vivo has not been addressed in all studies. Nevertheless, two independent groups have demonstrated that HSCs themselves do not fuse with hepatocytes, rather that monocytes generated from HSCs are responsible [12,88]. It was furthermore shown that, even though part of the genetic program of the host cell is preserved, reprogramming by both activating and silencing of genes occurs, so that the hybrid cell resembles the host cell with which the donor cell fused [89]. A final explanation may be that rare, more pluripotent precursors of postnatal stem cells persist. Although pluripotent stem cells are present throughout the body of planaria, such cells were not thought to exist in higher mammalian species. However, as will be discussed below, there is mounting evidence that cells expressing the ESC transcription factors Oct4, Sox2, and Nanog can be isolated from human and murine BM or UCB [90–93]. The frequency of such cells decreases very fast with age.

The natural role of such cells in postnatal tissues is not known. However, the possibility exists that when such cells are placed in a different environment, such as the brain or liver, they have the inherent ability, without need for significant reprogramming, to respond to cues from these new environments and differentiate in a tissue-appropriate manner.

Techniques to assess plasticity One of the most important technical considerations in demonstrating plasticity is to identify without any ambiguity the donor cell in the target organ. A robust, unambiguous molecular marker for the donor cell allows accurate assessment of the clonal relationship of donor cells and facilitates identification of the donor cells following transplantation. A variety of methods has been used to mark the donor cells. For all detection methods, proper fixation, particularly fixative concentration, and staining is essential to ensure maximal signal strength. Donor markers used include the Y chromosome (in sex-mismatched transplantations), membrane or cytoplasmic dyes, labels that incorporate in the DNA, and genetic labels such as β-galactosidase (β-gal) and GFP transgenes. More recently, the advent of conditional genetic marking using the Cre–Lox technology has provided another method of detection. Each of these approaches has advantages and limitations. A recent paper by Brazelton and Blau provides a comprehensive review of stem cell tracking methodology [94]. Equally important when trying to identify a cell whose fate has changed is to definitively prove acquisition of a new cell fate. It is essential to demonstrate that the donor marker and the lineage-specific marker are expressed in the same cell. Cellular morphology can be complex, and it may be difficult to discriminate between two closely associated cells. If tissue sections are examined in two dimensions with standard microscopy, a donor cell may overlay a host cell with the typical host-tissue specific marker, and this may be mistaken for a transdifferentiated donor cell. These events are rare, but the reported frequency of adult stem cells contributing to nonhematopoietic tissues is often equally rare. Methods that allow visualization of thin optical sections and three-dimensional reconstructions of cells in tissue sections are therefore required. The important aspect of the detection of any specific signal in a tissue is the use of appropriate negative controls to evaluate potential confounding factors, including autofluorescence. Careful experimental design and appropriate controls can avoid many of the common pitfalls.

Fluorescent in situ hybridization for the Y chromosome In sex-mismatched transplantation in which male donor cells are transplanted into female recipients, a Y chromosome is present in all intact donor cells. Typically, it is detected using fluorescent in situ hybridization (FISH) with fluorescent probes for specific regions of the Y chromosome. However, the Y chromosome cannot be visualized in all male cells if thin tissue sections that only partially sample the nuclei are assessed, and it is technically difficult to use Y chromosome FISH on thicker sections that contain the full thickness of most cells (greater than 10 μm). Nevertheless, the specificity and sensitivity of Y-chromosome FISH are very high. False-positive results can be caused by cell overlay and from nonspecific binding of the probe. Experience and evaluation of good negative controls in parallel can help to avoid these problems. As discussed earlier, it is critical to demonstrate that the Y chromosome is in the cell of interest, which can be accomplished by threedimensional reconstruction of double-labeled thin sections obtained by confocal microscopy.

Cellular Biology of Hematopoiesis

DNA or cell membrane labels 3

Bromo-deoxyuridine (BrdU) or H-thymidine labels dividing cells by incorporating into DNA during the S phase of cell division and has been extensively used to identify cells transplanted into the brain. However, two recent studies have shown that BrdU is present in the DNA of grafted cells, which can be taken up by actively dividing cells in the injected/injured area if the grafted cells die [95,96]. This then creates the incorrect impression that transplanted cells have adopted a neuronal fate. Membrane dyes such as PKH26 and CM-Dil (3,3′-dioactadecyl-5,5′di(4-sulfophenyl)-oxacarbocyanine, sodium salt, SP-DIOC18) have also been used to mark donor cells [78,97]. Both these dyes are nontoxic fluorescent lipophilic dyes that incorporate into the lipid bilayer of cytoplasmic membranes; hence, the amount of dye per cell diminishes as the cell undergoes cell division, which makes monitoring of proliferating cells difficult. Another pitfall of this marker is that fusion or phagocytosis of a labeled cell with an unlabeled cell results in transfer of the dye to the host cell, and many studies using membrane dyes have not controlled for this possibility [98]. Similarly to other markers, tissue preparation for evaluation is critical as the embedding procedure can influence the detection of fluorescence; for example, PKH26 does not withstand embedding in poly(methyl methacrylate). Another method, used in combination with magnetic resonance imaging, is passive labeling of donor cells with small particles of iron oxide [99]. The label can also be visualized by Prussian blue staining on histologic examination. As with membrane dyes, the label can be diluted so that it becomes no longer visible with cell division, and the possibility exists for false-positive results from microglia that uptake iron particles released from dead cells. Another downside of iron oxide contrast agents is that once cells home to a lesioned area, it becomes difficult to distinguish contrast originating from the stem cells or from bleeding in the damaged area.

Transgenic markers One of the earliest tracking markers was bacterial β-gal. The ROSA26 strain of mice constitutively expresses β-gal in most cells and can be used as a marker in transplantation studies. However, two problems arise with its use: the expression of β-gal as a marker is relatively weak, and it can be difficult to distinguish it from endogenous mammalian β-gal activity [100]. Therefore, when rare events are observed using ROSA cells, it is necessary to exclude unanticipated upregulation of endogenous β-gal in response to the injury itself. Several mammalian cell types, such as Purkinje cells, have substantially higher endogenous β-gal activity than others, and these normal variations can be misleading. Mammalian β-gal can be distinguished by its intracellular location and activity at optimal pH, and techniques on how to optimize β-gal-based tracking in the central nervous system have been recently reviewed [101]. eGFP is a modified protein originating from a naturally occurring fluorophore, and several lines of transgenic mice have been generated that ubiquitously express eGFP [102,103]. However, transgene expression varies among mice, may not be completely ubiquitous, and may weaken with time, age, and tissue differentiation [104]. One can use antibodies to GFP to detect lower levels of GFP expression; however, many anti-GFP antibodies are not totally specific, and several may need to be tested to ensure specificity for the tissue of interest. Other limitations of GFP include its rapid diffusion out of unfixed cells and apparent GFP positivity due to autofluorescence of (injured) tissues. However, careful evaluation of the emission spectrum of GFP compared with autofluorescence may circumvent the latter problem [105]. Some have

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suggested that the presence of GFP in itself may promote differentiation, although abundant evidence suggests that the fluorophore is biologically inert [106]. Others have suggested that decreased GFP expression is a function of cellular differentiation and that expression in “transdifferentiated cells” is decreased, making detection of transdifferentiation difficult [6]. If this were true, it might account for many publications in the literature stating that no donor cells were detected when using GFP as a reporter gene to label BM.

Cre–Lox technology Cre–Lox technology has been a useful addition to the armamentarium to understand cellular plasticity and has been used to address the likelihood of cell fusion as a contributing mechanism. Cre–Lox recombination is a technique used to conditionally turn gene expression on and off in specific cell types and tissues. Cells with Cre recombinase under the control of a ubiquitous or cell-specific promoter are grafted in conditional Cre reporter animals, in which a loxP-flanked stop cassette is located in front of a LacZ or eGFP reporter gene. Only when Cre recombinase excises the floxed stop cassette is the LacZ/eGFP gene expressed. Alvarez-Dolado et al. used this method to show that BM-derived cells could fuse with hepatocytes, cardiomyocytes, and Purkinje cells [87]. Oh et al. showed that intravenously infused progenitor cells from the adult heart could fuse with cardiomyocytes in the setting of myocardial infarction [107], and Reinecke et al. extended this by adding skeletal myoblasts as a clinically relevant cell type that can fuse with cardiac myocytes in vitro and in vivo [108]. This method has failed to detect cell fusion in the pancreas [109]. Keeping these possible explanations and the many methodological pitfalls in mind, the following sections will review papers suggesting differentiation of BM cells into mesodermal, endodermal, and ectodermal cells. We will first discuss studies wherein investigators describe transdifferentiation of HSCs or MSCs into unexpected lineages, and then focus briefly on studies that have suggested that more pluripotent adult stem cells may be responsible for the observed apparent transdifferentiation.

Transdifferentiation of HSCs or MSCs BM-derived cells – mesoderm BM-derived skeletal muscle Already in the late 1990s, some studies suggested that BM-derived cells could contribute to skeletal muscle [110–112]. Ferrari et al. noted that, following muscle damage, more myogenic precursors than expected from resident muscle stem cells, also termed satellite cells, could be found in muscle [110]. To elucidate the source of these additional cells, they chemically injured the tibialis anterior muscle and injected into the limb either whole BM cells or satellite cells containing a LacZ reporter driven by a myogenic promoter. β-gal+ cells were found in the BM cells injected into muscle, even though more βgal+ cells were detected in the satellite cell-injected muscle. The myogenic potential of BM cells appeared to be derived from adherent cells. They also injured hematopoietically reconstituted mice grafted with BM cells from C57/MlacZline (H-2b) mice and found, in 5 of 6 animals, β-gal+ cells in the muscle. Together, these studies suggest that cells present in BM cells can contribute to muscle, and this contribution is enhanced when the muscle is injured. Gussoni et al. [6,113] isolated murine HSCs from WT male mice based on the SP property and transplantated these into female mice with a defect in the dystrophin gene (mdx mice). As expected, hematopoietic reconstitution was seen. In addition, in the recipient muscle,

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Y chromosome-positive cells expressing dystrophin were detected. When Sp cells were isolated from muscle of WT male mice and grafted into mdx mice, radioprotection was seen with a contribution of donor cells to muscle. As studies were not done using single cells, it is not clear whether the same cell that gave rise to hematopoietic reconstitution also contributed to muscle. By contrast, Corbel et al. used single HSC transplantation to address whether HSCs or their progeny contributed to muscle. Single GFP+SPckit+Lin−Sca-1+ (SPKLS) cells from CD45.1 mice were injected into CD45.2 recipients [114]. After 4 weeks, over 30% of blood cells were donor in origin. In these animals, muscle, without and with toxin injury, was evaluated at after 16 weeks for the presence of donor contribution. Donor-derived muscle cells were defined as cells with muscle morphology, encased in laminin displaying the characteristic sarcomeric pattern following antimyosin staining that were GFP+. In the mice reconstituted from a single SPKLS cell, GFP+ cells were detected in the uninjured and injured tibialis anterior muscle of three mice. Damage promoted integration of HSC-derived cells into the regenerating muscle. When BM from the primary transplanted mice was grafted in secondary mice, hematopoietic reconstitution confirmed the self-renewal capability of the original single cell. Moreover, in 1 of the 3 secondary recipients, GFP+ myofibers were also seen, strongly suggesting that the progeny of a single HSC can contribute to the hematopoietic system as well as muscle. The mechanism of contribution, i.e. direct transdifferentiation versus fusion of HSC-derived progeny with myofibers, was not addressed. That human BM contains cells with myogenic potential was shown by identifying rare dystrophin-positive cells in muscle of a 12year-old boy who underwent a BM transplant for immunodeficiency [115]. BM-derived cardiomyocytes Several studies have suggested that cardiomyocytes can be generated from BM, although controversy exists as to the degree and mechanism via which BM cells contribute to improved cardiac function. The cardiomyogenic potential of ESCs from a variety of species including human is well established [116,117]. Whether any other stem cell, except perhaps stem/progenitor cells found in the heart itself [107], can differentiate into bona fide functional cardiomyocytes is still not clear. Fukuda et al. demonstrated that treatment of MSCs with 5-azacytidine gives rise to spontaneously contractile cells with some features consistent with cardiomyocytes [113]. Such MSC-derived cardiomyocyte-like cells formed myotubes with branching fibers and at 4 weeks stained positive for antimyosin, antiactinin and antidesmin. The cells had a cardiomyocyte-like ultrastructure and, by reverse transcription-polymerase chain reaction had cardiomyocyte-specific gene expression. More importantly, electrophysiological studies demonstrated the presence of action potentials consistent with sinus node ventricular action potentials. While the phenotype of these cells derived in vitro at a transcript, protein, and functional level is convincing, no in vivo studies were performed. Toma et al. injected human LacZ-labeled MSCs into the uninjured left ventricle of an adult SCID/beige mouse [118]. At 2 weeks, rare (0.4%) β-gal-, desmin-, cardiac troponin T-, and α-actinin-positive cardiomyocyte-like cells were found. Sarcomeric organization of the contractile proteins in the β-gal+ cells was indistinguishable from that of neighboring recipient cardiomyocytes. Dzau and colleagues performed a number of studies wherein MSCs were grafted into the borderzone surrounding the infarct in mice, and demonstrated functional improvement. The functional improvement was higher when MSCs were genetically modified to express Akt, which was associated with prolonged and more abundant persistence of the grafted cells [119]. However, in subsequent studies, they demonstrated that a

similar effect could be obtained when media conditioned by Akt-transduced MSCs was injected, suggesting strongly that the effect was trophic, i.e. secretion by the MSCs of factor(s), such as secreted frizzled2, chiefly protecting at-risk cardiomyocytes in the ischemic borderzone, and perhaps also inducing recruitment of endogenous progenitors or endothelial cells [120]. Others evaluated whether hematopoietic cells could differentiate into cardiomyocytes. Bittner et al. described that, following male BM cell transplantation into irradiated normal and mdx female mice, not only skeletal, but also cardiac myofibers appeared to be derived from donor BM cells 10 weeks following transplantation [111]. The transdifferentiated phenotype was determined to be skeletal and cardiac muscle based on coexpression of skeletal and cardiac muscle-specific proteins in Y chromosome-positive cells. Jackson et al. transplanted 2000 SP cells from male C57BL/6-Rosa-Ly-5.2 mice into lethally irradiated female C57BL/6-Ly-5.1 mice and after 10 weeks induced a myocardial infarct [121]. Evaluation 2–4 weeks later indicated that 0.02% of the cardiomyocytes were β-gal+α-actinin+CD45− and 3.3% of the endothelial cells were β-gal+Flt-1+CD45− and hence apparently donor derived. Neither study determined the mechanism underlying the apparent transdifferentiation. Orlic and colleagues injected murine GFP+Lin−ckithi BM cells into the borderzone myocardium of female mice several hours after coronary artery ligation [9]. Evaluation 9–14 days afterwards suggested that 68% of the cells in the infarct were GFP+Y chromosome+ cells that coexpressed the early cardiomyocyte transcription factors myocyte enhancer factor-2 and gata-4. In addition, some GFP+Y chromosome+ endothelial cells and smooth muscle cells were also detected. Significant functional improvement was also seen. When Lin−c-kitlo cells were injected, no functional improvement was detected. However, two studies performed subsequently by independent groups demonstrated significantly less contribution of murine BM Lin−ckithi cells to the injured cardiac tissue, and significantly less significant improvement in cardiac function than in the original Orlic et al. report [9]. Murry et al. failed to demonstrate generation of cardiomyocytes from Lin−ckithi cells grafted into the borderzone myocardium, even though some functional improvement was seen [122]. Similarly, following grafting of Lin−ckithi cells from myosin heavy chain (MHC)–LacZ transgenic animals into infarcted hearts of WT mice, no β-gal+ cells could be detected in the heart. Unfractionated BM cells from β-actin– eGFP transgenic mice were also transplanted into lethally irradiated, nontransgenic recipients. Two months after hematopoietic engraftment, a myocardial infarct was induced. Although some eGFP+αMHC+ cells were detected in the borderzone, the apparent transdifferentiated cells were infrequent. Balsam and colleagues transplanted both GFP+Lin− ckithi and GFP+KTLS cells into the infarcted heart of WT mice and could not find cells of donor origin with cardiac characteristics [123]. They also infarcted the heart of WT mice parabiosed with β-actin–EGFP transgenic mice and did not find donor-derived cardiomyocytes in the infarct zone [124]. The latter study did not assess the impact on the cardiac function. Both authors cautioned therefore that the data initially obtained by Orlic et al. [9] might not be sufficient to warrant clinical studies. Nevertheless, a now large number of clinical trials in humans have ensued in which different types of BM cell have been injected following balloon angioplasty into patients who have suffered from a cardiac infarct. Despite initial encouraging results in nonrandomized studies, four studies have been published in 2006 wherein patients were treated in a randomized blind fashion with BM cells or vehicle control, demonstrating little functional benefit for the majority of patients treated with cells [125–128]. Of note, a clinical study in which the hearts of patients were examined at autopsy after sex-mismatched BM transplantation showed that Y

Cellular Biology of Hematopoiesis

chromosome-positive cardiomyocytes contributed 0.23 ± 0.06% of the myocardium, and no Y chromosome-positive cells were seen in gendermatched controls. These data are consistent with the murine studies indicating that very rare human BM cells have the potential to transdifferentiate into cardiomyocytes. Although this has been rarely assessed, the study by Alvarez-Dolado et al. has definitively demonstrated that rare apparent transdifferentiation may be due to cell fusion between donor cells and cardiomyocytes [87]. BM-derived cells – endoderm A number of reports have suggested that BM cells have the potential to generate endodermal tissue. Even though, during embryonic development, the mesoderm and endoderm lie anatomically close to each other and there is considerable crosstalk between these two tissues that enables each to develop, the transdifferentiation between two germ layers would perhaps be even more astounding than that between mesodermal cell types themselves. Petersen et al. were the first to suggest that BM cells contribute to liver regeneration [123]. Hepatic oval cells are regarded as facultative hepatic stem cells that can generate both hepatocytes and cholangiocytes. Their precise origin is not clear, but it is likely that they are derived from canals of Herring or reside as blast-like cells next to the bile ducts. Hepatic oval cells express CD34, Thy-1 and c-kit, markers also present on HSCs. Previous studies had shown that oval cells proliferate when hepatocyte proliferation is suppressed following liver injury. To test whether BM cells could contribute to liver regeneration, female rats were transplanted with BM from syngeneic male rats, and liver injury was induced using a protocol to stimulate oval cell proliferation (2-acetylaminofluorene [2-AAF] and carbon tetrachloride [CCL4]). Thirteen days following transplantation, Y chromosomepositive hepatocytes were detected by in situ hybridization. In a second experiment, irradiated dipeptidyl peptidase-IV (DPPIV)-negative female rats received BM from DPPIV-positive male rats. Liver injury was generated using the 2-AAF–CCL4 protocol. DPPIV expression was observed in oval cells and hepatocytes in the recipient liver, that coexpressed proteins found in mature hepatocytes. Finally, a liver transplant was performed from L21.6 antigen-negative donor rats into allogeneic L21.6-positive rats and hepatic ductular structures containing L21.6+ cells were present in the transplanted liver, suggesting that oval cells were derived from extrahepatic cells. The authors concluded that BM cells can under certain conditions become hepatic oval cells. Similar findings were seen in a murine model [8], and together these results support the concept of a BM or extrahepatic stem cell generating hepatic oval cells. Neither study established the exact phenotype of the “plastic” BM cells or their use in the therapy of liver diseases. Lagasse et al. [7] used an animal model of fatal hereditary tyrosinemia type 1 (fuarylacetoacetate hydrolase [FAH]-deficient mice) in which mutant mice develop progressive liver failure and renal tubular damage unless treated with 2-(2-nitro-4-trifluro-methylebenzyol)-1,3-cyclohexanedione (NTBC). Unfractionated BM or 50 KTLS cells from male FAH–WT β-gal−transgenic mice were injected into lethally irradiated female FAH−/− mice treated with NTBC. Three weeks after transplantation, NTBC was discontinued intermittently to allow selection of the liver repopulating cells. Four out of nine mice grafted with unfractionated BM and 9 of 20 mice grafted with KTLS cells survived and had donor-derived β-gal+FAH+Y chromosome+ cells in the liver with an improvement in liver function. These donor-derived cells coexpressed albumin with β-gal when cultured on a mouse embryonic feeder layer. However, later studies [89,129] demonstrated that the differentiation of HSCs into hepatocytes did not occur in a cell-autonomous manner but following fusion of HSC-derived monocytes with hepatocytes.

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A number of studies have also addressed whether apparent hepatic transdifferentiation occurs in humans. Theise et al. [129] demonstrated that liver tissue examined from male recipients of female livers and female recipients of male BM contained marrow-derived hepatocytes and cholangiocytes using in situ hybridization for the Y chromosome with colabeling with the monoclonal antibody CAM5.2, which identifies hepatocytes and cholangiocytes but not Kuppfer cells, stromal cells or circulating blood cells. The frequency of Y chromosomepositive hepatocytes and cholangiocytes varied but appeared higher in patients with GVHD or liver rejection. Engrafted human cells were most often scattered throughout the parenchyma as isolated cells [130]. Microchimerism for the Y chromosome can occur in the human female liver as a result of transplacental passage of male fetal blood cells during pregnancy; this was excluded as a contributing factor in a similar study by Alison et al. [131]. Both studies conclude that human BM cells can differentiate into mature hepatocytes and cholangiocytes in the absence of severe injury. Korbling et al. published that, following mobilized peripheral blood transplantation, up to 7% of epithelial cells of the liver, gastrointestinal tract, and skin appear donor in origin [132]. The frequency was low, and GVHD did not affect apparent donor cell engraftment in epithelia. Similarly, human hematopoietic progenitor cells transplanted into fetal sheep have been reported to differentiate into hematopoietic cells and hepatocytes [133]. While many studies as outlined have suggested that BM-derived cells may, at low level, contribute to liver, gut or lung tissue, and that this contribution increases in the setting of organ failure, the cell responsible is not known, and whether these phenomena occur outside the setting of cell fusion is not clear. One exception perhaps is the study by Krause et al. [80] demonstrating that contribution to lung epithelium may happen, at least in part, outside the setting of cell fusion [134]. A final tissue in which possible transdifferentiation has been suggested is the pancreas. To address the question of whether a cell within the BM can contribute to pancreatic β cells, Ianus et al. used mice transgenic for the Cre recombinase expression under the mouse Insulin2 promoter crossed into mice ubiquitously expressing the Rosa–Flox–GFP (RosaGFP) transgene (Ins2cre–RosaGFP) [109]. β cells derived from this donor mouse would hence express GFP. Irradiated WT mice were transplanted with Ins2cre–RosaGFP BM and analyzed 4–6 weeks post transplant for pancreatic engraftment. Approximately 1.7–3% of all cells within the pancreas were GFP positive, expressed characteristic β-cell markers (including Hnf3β, Pdx1, Glut2, Insulin1, and Insulin2), and secreted insulin in a glucose-responsive manner at levels similar to that of mature β cells. To exclude fusion as a mechanism, they transplanted Ins2cre BM into irradiated mice with the RosaGFP reporter. As no GFP-expressing β cells were seen, Ianus et al. concluded that the presence of GFP expressing β-cells in the first transplant model was not caused by fusion. In support of these data, Wang et al. reported that neonatal immunocompromised mice transplanted with GFP+ BM cells acquired significant engraftment in pancreatic epithelium (up to 40% with a mean of 4.6%) as well as rare GFP+Ins+ cells (0.03%) [135]. Other studies have questioned these results. Hess et al. demonstrated that BM cells transplanted into a diabetic model could reverse hyperglycemia, but that the mechanism was not direct differentiation to β cells [136]. Within the transplanted BM, the ability to reduce hyperglycemia was accounted for by a c-Kit+ subpopulation [136]. A similar study utilizing the same diabetic model revealed that BM contribution and support during regeneration was likely due to BM-derived endothelial progenitor cells (EPCs) [134]. The donor-derived endothelial cells may have facilitated the proliferation and ultimate recovery of remaining endogenous β cells.

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BM-derived cells – ectoderm Ectoderm is generated from the outer layer of the embryo and produces a surface layer of skin, the epidermis, and the central nervous system. One of the most difficult lineage boundaries to cross is the bridge between mesoderm and ectoderm, yet there are studies suggesting that this too might be possible. Eglitis and Mezey in 1997 were the first to report that BM-derived cells could contribute to, as expected, microglia (F4/80 antigen) but also (unexpected) astroglial (glial fibrillary acidic protein [GFAP]-positive) cells in the adult murine brain [5]. Sublethally irradiated female mice were transplanted with genetically marked male donor cells (enriched for HSCs using 5-flurouracil), the mice were sacrificed at intervals post transplantation, and the brain was examined using a combination of in situ hybridization histochemistry and immunohistochemistry. Doublelabeling analyses revealed that some BM-derived cells acquired glial markers. BM-derived cells in several mice were localized to the subependymal zone, an area known to be neurogenic during development, which would provide cues for glial differentiation. The type of BM cell contributing to astroglial cells was not determined. Mezey et al. transplanted WT BM cells intraperitoneally into PU.1−/− mice to rescue the animal from the typical early postnatal mortality [137]. Brains were examined at 1 and 4 months following transplantation using a combination of in situ hybridization to detect the Y chromosome and immunohistochemistry to visualize the neuronal nuclear marker, NeuN. BM-derived cells were found in the brain of all the animals examined, and the Y chromosome was present in 0.3–2.3% of the NeuN-immunoreactive nuclei. Mezey et al. concluded that BM cells can enter the brain and differentiate into cells that express neuronal markers, supporting the idea that mesodermally derived cells can adopt a neuronal (ectodermal) cell fate. In the same issue of Science, Brazelton et al. documented that when GFP-transgenic BM was grafted in lethally irradiated syngeneic mice, both NF200+ neurons and GFAP+ astrocytes could be found that were GFP+, concluding that cells from BM could generate neuroectodermal cells in vivo [138]. However, as indicated earlier, Wagers et al. were unable to find significant contribution of HSC to cells within the brain, except for rare Purkinje cells [79]. Likewise, Castro et al. could not find the same degree of transdifferentiation from BM to neuronal cells [139]. In a second series of studies, the ability of MSC to differentiate into neurons in vitro or in vivo was evaluated. Agents used to induce neural differentiation in vitro included nonspecific inducers, whereas others used cytokines and growth factors known to induce differentiation of ESCs to neuronal cells or induce differentiation of neural stem cells [140–143]. In most studies, “transdifferentiation” was shown only by the acquisition of neural morphology and the expression of a few neuronor glia-specific proteins or transcripts, which can already be detected at low level in undifferentiated MSCs [144]. A recent study has also suggested that dimethylsulfoxide induces rapid disruption of the actin cytoskeleton, leading to an apparent change in morphology of fibroblasts to cells that appear to extend neurites [145]. A few studies have assessed whether MSC-derived neuron-like cells have the functional attributes of neurons. Kohyama et al. showed that single-cell-derived immortalized BM stromal cells acquired the morphological and surface phenotype of neurons when treated with 5-azacytidine and the BMP2/4 inhibitor Noggin [146]. The resting potential of such cells of −5.0 mV and acquisition of potassium voltage-gated channels suggested the acquisition of some functional features of neurons. Wislet-Gendebien et al. co-cultured MSC-derived nestin-positive cells with cerebellar granule neurons, which led to the expression of astrocyte- and neuron-specific proteins [147]. More importantly, the resting membrane potential increased to −57 mV, at which time the cells also

acquired functional sodium voltage-gated channels, a phenotype similar to that of cells at an intermediate phase of neural development. The fact that MSCs co-cultured with paraformaldehyde-fixed cerebellar granule neurons yielded similar results suggests directed differentiation and not cell fusion. Dezawa et al. expressed the intracellular domain of Notch in MSC [148]. This led to the expression of early neural transcripts, and upon addition of forskolin and basic fibroblast growth factor, more than 95% of MSCs expressed markers for postmitotic neurons, and 50% of the cells acquired outward rectified potassium ion currents, but no voltagegated fast sodium currents. Following exposure to ciliary neurotrophic factor, 40% of the cells expressed genes found in dopaminergic neurons and secreted dopamine following depolarization. When grafted in 6hydroxydopamine treated animals, rotational behavior improved, as is seen with fetal midbrain transplantations. These studies suggest therefore that, under certain conditions, MSCs may acquire not only phenotypic, but also functional characteristics of neuroectodermal cells. Several groups have grafted rodent or human MSCs in vivo in intact or injured brain [149,150]. Although some authors have stated that MSCs transdifferentiate into neuronal cells, these conclusions have in general been based solely on the identification of neuron- (nestin) or glia- (GFAP) specific markers in rare donor cells. Moreover, in many studies, donor origin was determined by evaluation of BrdU or 3Hthymidine incorporated in the donor cells prior to grafting. As discussed earlier, recent studies have shown that such labels, previously thought to be superior over, for instance, membrane dyes, can be transferred from dying grafted cells to endogenously proliferating cells such as astroglia or neural stem/progenitor cells [95]. In addition, no study has excluded conclusively that the presumed neural differentiation is not caused by fusion between MSCs and host brain cells. Finally, no study has demonstrated that presumed MSC-derived neurons are functional. Nevertheless, as is true for grafting of BM cells in the heart, grafting MSCs in the brain may have beneficial effects by secreting trophic factors that enhance vascularization, recruit endogenous progenitors, and/or induce mature neural cell plasticity [151–154]. Endothelial cells in the BM Another cell type present in BM is the endothelial cell. HSCs proliferate and differentiate, and their progeny migrate into the blood circulation lined by endothelium. The endothelium forms the vascular niche within the BM, and at least some HSCs lie in close proximity to it [155]. The vascular niche is thought to promote HSC proliferation and differentiation, whereas the osteoblastic niche may favor quiescence [156]. Explanted endothelial cells can maintain HSCs in culture [157]. Its location at the interface between the BM and the circulation allows the endothelium to regulate transendothelial migration, homing, and mobilization of HSCs [84,158]. It has also been hypothesized that endothelial cells outside the BM medullary cavity may provide a back-up niche when the BM is stressed [159]. The boundaries separating the hematopoietic and vascular developmental programs are somewhat indistinct. HSCs are an accepted entity with a defined hierarchy based on proliferative potential. There remains controversy over the definition of an endothelial progenitor, and a similar organization for endothelial precursors, while postulated, is not proven. There is, at least during development and using in vitro embryonic stem cultures, evidence of a common endothelial and hematopoietic progenitor called a “hemangioblast.” This concept was proposed over 80 years ago when Florence Sabin observed the formation of blood and blood vessels from primitive mesoderm in chick blastoderm [160]. The term “hemangioblast” was coined by P.D.F. Murray in 1932, who noted that cells from the primitive streak of chick embryos formed simultane-

Cellular Biology of Hematopoiesis

ously both endothelial cells and blood islands [161]. Hematopoietic and endothelial cells develop in close proximity within the mammalian yolk sac [162,163], and in the embryo proper, current information suggests the presence of “hemogenic” endothelium in the dorsal aorta as a source of definitive HSCs [164–169]. Investigations in humans have shown similar results [170]. Using murine ESCs, several investigators have isolated hemangioblasts in vitro, which has allowed more extensive evaluation than cells from the embryo proper. ESCs generate embryoid bodies that can be induced to differentiate into hematopoietic lineage [171]. They generate a transient precursor with genes in common with both the hematopoietic and endothelial cell lineages. These transient precursors, referred to as the blast colony-forming cell population, may be an in vitro equivalent of a hemangioblast [168]. Whether circulating endothelial cells originate as outgrowths from vascular endothelium or reside in the circulation or BM is not clear [169]. Whatever the origin (reviewed more extensively in [172]), the endothelium has an intimate relationship with hematopoiesis throughout development. It has been difficult to study this association as EPCs share many phenotypic and functional characteristics with HSCs: they exist in the same fluid microenvironment and respond to the same stimuli. EPCs express KDR (VEGFR1), CD31, and Tie-2 (angiopoietin-1 receptor), ingest acetylated low-density lipoprotein, and bind lectins (a feature of most macrophages). That EPCs are present in BM comes from murine

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and human BM transplantation studies demonstrating that at least some endothelial cells are of donor origin [173]. Whether such endothelial cells could be derived from HSCs rather than EPCs has not been fully addressed. Grant et al. showed that, following transplantation of a single long-term repopulating HSC, perfusable endothelial cells in the retina of a mouse developed following vascular injury [174]. However, recent studies have demonstrated that, aside from EPCs that generate selfrenewing colonies of endothelial cells in vitro, HSC-derived macrophages can also generate cells with features of endothelial cells, such as expression of CD31, von Willebrand factor, and ability to take up acetylated low-density lipoprotein [175]. The difference between the two endothelial cell-like cell progenitors is the time after which they start generating colonies in in vitro cultures, the ability to replate the colonies, and the ability to generate perfused vessels in vivo. The fact that the progeny of HSCs can acquire features of endothelial cells makes evaluation of the possible lineage relationship of HSCs and EPCs in in vivo studies as well as in vitro studies more difficult.

Persistence of more pluripotent adult stem cells? Adult pluripotent stem cells (Table 7.2) There are an increasing number of studies suggesting that cells with greater potency than classical tissue-specific stem cells can be isolated

Table 7.2 Adult pluripotent stem cells isolated from different murine and human sources, with their phenotype and “plasticity” towards alternative dermal lineages Name

Source

Phenotype

Differentiation capacity

MAPC [90]

Murine BM

Endothelium, hepatocytes, neuron-like cells

MIAMI cell [186]

Human BM

hBMSC [97]

Human BM

MASC [93]

Human BM, liver, heart

SSEA-1 cell [91]

Murine BM

VSEL cell [92]

Murine BM

USSCs [187]

Human umbilical cord blood

AFS [188]

Human amniotic fluid

maGSCs [189]

Murine testis

Oct4+, Rex1+ Thy-1.1lo, Sca-1+lo, Flk-1+lo, SSEA-1+, CD13+ CD34−, CD44−, CD45−, CD117−, MHC-I−MHC-II− Oct3a+ Rex-1+ Thy-1+, CD10+, CD29+, CD44+, CD49e+, CD103+ CD34−, CD36−, CD45−, CD54−, CD56−, CD117−, HLA-II− Oct3/4− Thy-1lo, CD105lo, Cd117lo CD29−, CD44−, CD73− Oct4+, Rex1+, Nanog+ Thy-1+, CD13+, CD49b+, CD73+, CD44+, HLA−I+, CD29+, CD105+, Flk-1+, CD49a+ CD14−, CD45−, CD38−, HLA-II−, CD133−, CD117−, CD34− Oct4+, Rex-1+, Nanog+, SSEA-1+ Sca-1+,Thy-1+ CD73+, CD105+, CD44+, CD34−, CD45− Oct4+, Rex1+, Nanog+, SSEA-1+ Sca-1+ CD45− Thy-1+, CD105+,CD13+,CD29+, CD44+, CD49e+ Class-II−, CD34−, CD45−, CD117−, CD33− Oct3/4+,SSEA-4+ Thy-1+, CD44+, CD73+, CD117+, Class-IIlo CD34−, CD45− Oct4+, Rex-1+, Nanog+, SSEA-1+ Thy-1+, Sca-1+lo, CD117+lo, Ter119−, CD34−

Osteoblasts, chondrocytes, adipocytes, pancreatic islets, neural cells

Cardiomyocytes, endothelium, smooth muscle

Osteoblasts, myocytes, endothelium, hepatocytes, neurons, glia

Osteoblasts, chrondrocytes, adipocytes, hematopoietic cells, endothelium, hepatocytes, astrocytes Cardiomyocytes, pancreas, neural cells

Osteoblasts, chrondrocytes, adipocytes, hematopoietic cells, neural cells Osteoblasts, hepatocytes, neural cells

Cardiomyocytes, smooth muscle, endothelium, hepatocyte-like cells, neurons

AFS, amniotic fluid-derived stem cell; BM, bone marrow; hBMSC, human BM-derived multipotent stem cell; maGSC, multipotent adult germline stem cell; MAPC, multipotent adult progenitor cell; MASC, multipotent adult stem cell; MIAMI, marrow-isolated adult multilineage inducible cell; SSEA-1, stage-specific embryonic antigen-1; USSC, unrestricted somatic stem cell; VSEL, very small embryonic-like cell.

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from BM, UCB, and other tissues. The first report was in 2002, when multipotent adult progenitor cells (MAPCs) were described. MAPCs are isolated from adherent BM cultures under conditions that may not support MSCs, specifically low concentrations of fetal bovine serum (FBS) (2%). Interestingly, isolation of MAPCs from rat and mouse BM requires leukemia inhibitory factor, a cytokine that is also a prerequisite for the isolation of murine ESCs [90]. MAPCs isolated from rodents, swine, and human BM are a clonal population of cells that can be expanded ex vivo without signs of cell senescence. They express the ESC transcription factor Oct4, but not appreciable levels of Nanog or Sox2. By FACS, human MAPCs are characterized as CD13−CD34− CD45−HLA-II−, Thy-1+, and CD45loHLA-Ilo. Under specific in vitro differentiation conditions, they can differentiate into typical mesenchymal cell types as well as endothelium, hepatocyte-like endodermal cells, and neuron and glia-like neuroectodermal cells [90,176–184]. Differentiated cell types including smooth muscle cells, osteoblasts, endothelium (arterial or venous specified endothelium), hepatocyte-like cells, and neuroectodermal-like cells were characterized based on morphology, phenotype, expressed protein, and gene profile, as well as extensive functional characterization. When transplanted in vivo, murine MAPCs can regenerate a complete lymphohematopoietic system [90,185], and some cell lines also contribute to endodermal tissues such as liver, gastrointestinal, and pulmonary endoderm [90]. Undifferentiated or endothelially committed MAPCs also contribute to neovascularization in tumors, or in Matrigel assays [178,183]. Some, but definitely not all, cell lines also contributed in variable degrees to multiple somatic tissues when injected in the blastocyst, although germline transmission has not been observed [90]. In 2004, D’Ippolito et al. described marrow-isolated adult multilineage inducible (MIAMI) cells [186]. These cells are derived from human BM from vertebral bodies by plating on fibronectin at 3% oxygen and 15% FBS for the first 2 weeks; FBS is then decreased to 2%. MIAMI cells, like MAPCs, express Oct3a and Rex1, and can be expanded for 30–50 cell doublings without evidence of senescence or loss of differentiation capacity. By FACS, MIAMI cells are negative for CD34, CD36, CD45, CD117 and HLA-II. In vitro, MIAMI cells can be induced to differentiate to mesodermal cell types as well as cells with phenotypic features of neuroectodermal-like cells and endodermal features (transcripts of pancreatic endoderm), although no functional studies have been done to prove differentiation to ectoderm and endoderm. Yoon et al. purified cells from human BM that could be expanded from single cells for greater than 140 population doublings without senescence and loss of differentiation, named human BM-derived multipotent stem cells (hBMSCs) [97]. By FACS, hBMSCs are negative for CD29, CD44, and CD73, and express low levels of CD105, CD90, and CD117, all surface markers used to characterize mesenchymal cells. In contrast to most cells with apparent greater potency, BMSCs do not express Oct4. They can differentiate into cells with phenotypic features of endothelium, smooth muscle, neural cells, and hepatocytes. However, the investigators did not evaluate the functional properties of the lineage differentiated progeny. When co-cultured with cardiomyocytes in vitro, cells appeared to differentiate into cardiomyocytes, although fusion is, at least in part, responsible for this observation. When hBMSCs labeled with the dye DiL were given as an intracardiac graft in an infarcted heart, multilineage differentiation (endothelium, smooth muscle and cardiac muscle) was seen, which resulted in favorable remodeling and improved cardiac function. Whether the functional improvement was due to apparent transdifferentiation versus trophic factors secreted by BMSCs, as has been seen for hematopoietic cells and MSCs, is not clear. As discussed earlier, the use of DiL for in vivo tracking is not ideal. The authors also did not conclusively demonstrate that the resulting differentiation was

cell autonomous versus fusion between hBMSCs and resident cells in the heart. Multipotent adult stem cells (MASCs) were isolated from adult human BM, liver or heart tissue [93]. These cells were cultured in Mesencult (a medium optimized for human MSC cultures) and subsequently on fibronectin-coated dishes and above stromal cells with 2% FBS. The cell-surface phenotype is that of MSCs, but the cells express Oct4, Rex1, and Nanog transcripts, as well as Oct4 and Nanog protein. Clonally isolated cells differentiate into cells with phenotypic and functional features of osteoblasts, myocytes, endothelial cells, hepatocytes, neurons and glia. Unrestricted somatic stem cells, isolated from human UCBs, are CD45−, HLA class II-negative mononuclear cells with fibroblastic morphology and extended telomere length that can be expanded for 30–50 population doublings [187]. The cells express Oct4 and Rex1 and, in vitro/in vivo, demonstrate differentiation potential to osteoblasts, chondroblasts, adipocytes, and hematopoietic, neural and hepatic cells. As is true for many studies, limited proof exists for acquisition of functional features of the unexpected endodermal and ectodermal cell types. Similarly potent cells have been found in amniotic fluid [188]. In addition, Guan et al. demonstrated that cells with pluripotent features, including teratoma formation and contribution to somatic and germline cells when injected in the blastocyst, could be generated from murine testicular tissue [189]. The latter cells are therefore the only ones that have capabilities similar to those of ESCs. One question that has not been answered is whether the cell populations described above (MAPCs, MIAMI cells, BSSCs, and MASCs) exist in vivo or are created in culture as the result of dedifferentiation. Two recent studies may start to shed light on this. AnjosAfonso and Bonnet have shown that cells expressing stage-specific embryonic antigen-1 (SSEA-1), found on mouse ESCs, can be used to isolate cells from MSC cultures following one passage in vitro [91]. These cells express high levels of Oct4 as well as Nanog and Sox2, and can differentiate following expansion into cells with phenotypic and in vitro functional characteristics of multiple mesodermal cell types, including endothelium, as well as endodermal hepatocyte-like cells and ectodermal lineage cells such as astrocytes. When grafted in the tibia in vivo, pre-MSCs contribute to endothelium, osteoblasts, adipocytes, chrondrocytes, and hematopoietic cells. They further demonstrated that when SSEA-1− cells are isolated that do not express Oct4 and Nanog, these cells cannot be induced to express SSEA-1 or the ESC transcription factors. Kucia et al. recently demonstrated that a homogeneous population of Sca-1+Lin−CD45−CXCR4+ cells can be selected directly from murine BM or human UCBs that express, like the cells identified by AnjosAfonso and like ESCs, SSEA-1, Oct4, Nanog and Rex-1, or SSEA-4 in human UCBs [92,190]. These cells are difficult to culture ex vivo, but by performing co-cultures, phenotypic changes were seen consistent with differentiation to endothelium and possibly pancreatic β cells. No functional confirmation of such differentiation was demonstrated, nor was fusion excluded. The latter two studies suggest that rare cells exist in murine BM with phenotypic features of MAPCs, MIAMI cells, hBMSCs, unrestricted somatic stem cells, and MASCs. It is unknown whether the differentiation ability ascribed to MAPCs, MIAMI cells, hBMSCs, unrestricted somatic stem cells, MASCs, pre-MSCs, multipotent adult germline stem cells or amniotic fluid-derived stem cells is already present in the freshly isolated cells, and hence these cells represent cells with greater potency persisting in vivo into postnatal life, or whether the multilineage differentiation ability is acquired once cells are cultured in vitro, and therefore represent the dedifferentiation of rare Oct4+ cells.

Cellular Biology of Hematopoiesis

Conclusions and future directions This chapter has summarized the significant progress that has been made in defining the properties of stem cells within BM, including HSCs, MSCs, and endothelial progenitor cells. There are continuing advances in understanding the unique biologic properties of these cells and their progeny. This information is leading to increasing numbers of clinical applications for these cells, not only with HSCs, but trials are being initiated with MSCs and EPCs as well. New information highlighting the importance of the BM microenvironment demonstrates that the beneficial effect of these cells extends beyond their direct contribution to the host tissue. The trophic effects of MSCs can be harnessed to increase the potential of existing pharmacologic therapies, especially in ischemic disorders. The immunomodulatory characteristics of MSCs are also being tested in the setting of GVHD, graft rejection, and other immune-mediated disorders. The limitations and potential toxicity of these treatments should, however, be carefully assessed in animal models and clinical trials as translation to humans may reveal unsuspected complications. Many studies have suggested that BM-derived stem cells may differentiate into not only cells of the tissue of origin, but also cells of other

83

tissues, a process known as stem cell plasticity. The evidence for transdifferentiation is often confusing, with some studies providing evidence of phenotypic switch and others failing to demonstrate such phenomena. There may be technical reasons contributing to the differences between studies. In addition, the mechanisms underlying the apparent lineage switch are not always clear. Undoubtedly, fusion occurs, especially between cells that are more “fusigenic” by nature. Aside from fusion, there is mounting evidence for rare events of cell intrinsic lineage switch. Studies aimed at understanding the conditions that allow such transdifferentiation may ultimately yield optimization of local signals to induce such fate change. There is also more and more evidence that cells can be cultivated that have (or acquire) greater potency. Further characterization of the potential of these cells to be used for treatment of not only hematopoietic disorders, but also diseases of the central nervous system and liver, among others, may demonstrate that BM cells could be used outside hematologic diseases. Stem cell potential as we know it presently is in its infancy. The huge strides that have been made since the pioneering use of HSCs in human HCT in the 1960s are small steps along the way to fulfilling the possibly much broader, but latent potential of BM-derived cells.

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8

Colleen Delaney & Irwin Bernstein

Expansion of Hematopoietic Stem Cells

Introduction Hematopoietic stem cells (HSCs) are useful in a variety of clinical settings and are routinely available in sufficient numbers from a variety of sources, including bone marrow and mobilized peripheral blood. However, there are instances when the number of available HSCs is insufficient for adequate and/or timely engraftment of the host, in particular when cord blood is used as the source of HSCs for transplantation or in the case of gene therapy, where the frequency of transduced cells is too low. One potential solution to the problem of low stem cell numbers is ex vivo proliferation of the cells prior to transplantation. Extensive research has been done to define the optimal conditions necessary for ex vivo expansion of HSCs, and various expansion techniques have been developed for this purpose [1–7]. However, while there is still a lack of convincing data that pluripotent stem cells increase in number and retain an ability to engraft and sustain multilineage hematopoiesis in a myeloablated recipient following ex vivo expansion, more recent advances in the field, particularly with respect to molecular mechanisms that control HSC amplification, make this goal more realistic. In order to maintain hematopoietic function throughout the life of the organism, pluripotent HSCs are thought to undergo self-renewal, where at least one, possibly identical, progeny remains undifferentiated and capable of long-term, multilineage hematopoietic repopulation, thus preserving the stem cell pool [8]. The differentiated progeny of longterm repopulating cells are thought to include pluripotent, short-term repopulating cells, which in turn generate precursors committed to lymphoid or myeloid differentiation [9,10]. Whereas maintenance of the HSC pool in vivo requires asymmetric division in which only one of the progeny maintains stem cell properties, HSC expansion requires symmetric divisions giving rise to two HSCs (Fig. 8.1). The ability of HSCs to undergo symmetric divisions and expand in numbers has been demonstrated in transplantation studies. However, serial transplant studies have also demonstrated that HSC life span is limited and self-renewal potential diminishes with the age of the animal [11–13]. While several recent studies challenge our concepts of HSC senescence [14–16], the central tenet remains that, in the absence of transforming events, HSCs have finite, though very lengthy, life spans in vivo [17–19]. These “aging” or senescence processes appear to be accelerated in ex vivo

cultures [20]. Nevertheless, studies suggest that HSC progeny formed after several divisions retain the ability to engraft recipients long term, but may actually be somewhat more differentiated with less proliferative capacity that is detectable only under stress conditions. Thus, cells with HSC properties may themselves be heterogeneous, and their self-renewal capacity may depend on the source from which they are derived, with fetal liver HSCs having greater self-renewal capacity than cord blood HSCs, which in turn have greater self-renewal capacity than cells from adult bone marrow [16,21–23]. Substantial effort has focused on the exogenous signals that may be used to favor stem cell self-renewal versus differentiation in order to develop optimal conditions for the ex vivo expansion of stem cells. Most studies have evaluated using combinations of cytokines and/or bone marrow stroma and have met with limited success (reviewed in [3,6,24,25]). The effect of cytokines that support hematopoietic cell survival, proliferation, and differentiation has been extensively studied in vitro, but a significant role for these cytokines in enhancing selfrenewal has not been shown. Consequently, a stochastic model of cellular determination has been suggested in which the fate of hematopoietic precursors is not instructed by soluble cytokines [26,27], but rather by specific interactions between stem and other cells within a particular microenvironment or “stem cell niche.” These interactions are mediated by extrinsic regulators of stem cell fate and are likely to play a key role maintaining numbers of stem cells by regulating their self-renewal and differentiation, now demonstrated by the work of several groups [28–30]. Thus, more recent studies are aimed at identifying intrinsic and extrinsic factors that regulate HSC fate. Clinical trials, which have also mainly evaluated cytokine-driven expansion systems, have not yet provided evidence for stem cell expansion, but have demonstrated the feasibility and safety of ex vivo culturing of stem cells. However, a newer generation of clinical trials, as discussed below, is currently underway evaluating the use of extrinsic regulators of stem cell fate (Notch) and co-culture systems utilizing nonhematopoietic components (mesenchymal stromal cells) of the stem cell niche. Here, we address studies of ex vivo HSC expansion in animals and humans, as well as promising approaches under development.

Preclinical studies: growth factor and stroma-induced expansion Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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In this section, we review work in mice and large-animal models designed to address the ex vivo expansion of HSCs. We will focus on studies that have used in vivo repopulating assays to evaluate stem cell

Expansion of Hematopoietic Stem Cells

HSC

HSC HSC

HSC STR

HSC

HSC

HSC

HSC

STR STR

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STR HSC

Symmetric division

STR Asymmetric division

Fig. 8.1 Maintenance versus expansion of hematopoietic stem cell (HSC) numbers. Symmetric divisions will expand stem cell numbers, whereas asymmetric divisions maintain numbers. The number of HSCs generated may also be affected by cell loss due to apoptosis and, for transplantable stem cells, loss of homing properties. STR, short-term repopulating cell.

expansion as in vitro assays have not always proven informative of HSC function. We then briefly review factors that regulate HSC fate in ex vivo expansion cultures. Murine studies The discovery of transplantable HSCs in long-term cultures of mouse marrow cells (LTMCs) initially described by Dexter [31–33] suggested that HSCs might possibly expand in ex vivo cultures. The HSCs in these LTMCs were dependent on the stromal cells for survival, and their cell cycle status was influenced both positively and negatively by growth factors [34,35], as well as by cellular and noncellular components of the stromal cell layers [36]. However, it was unclear whether HSCs in LTMCs divided or simply remained quiescent. Subsequent studies using gene marking to follow the progeny of individual HSCs demonstrated that HSCs were able to undergo several divisions and still maintain HSC function measured in vivo [37,38]. Evidence for a limited number of in vitro divisions before loss of HSC function also came from studies demonstrating that the progeny of isolated HSCs, formed after one to three in vitro divisions, maintained in vivo repopulating function [39]. However, none of these and other studies [40] demonstrated net gains of HSCs number in these cultures, indicating that divisions were mainly asymmetric. HSC cultured for purposes of ex vivo expansion are altered in obvious as well as subtle ways, including the expression of various cell surface antigens such as receptors that function in cell adhesion and motility. Research is ongoing to better define HSC markers on freshly isolated and subsequently cultured HSCs [41]. Moreover, the impact of stem cell death, loss of homing properties [42,43], and change of cell-cycle status [44–46] that occur during in vitro culture of HSCs and how these influence in vivo repopulating ability have not yet been fully elucidated. For example, both mouse HSCs and human hematopoietic precursors appear to have diminished repopulating function if transplanted during the G1 and S phases of the cell cycle rather than in G0 phase. The combination of cytokines is also critical, with specific hematopoietic growth factors involved in promoting or inhibiting HSC function during ex vivo expansion. Elegant single-cell manipulation studies have demonstrated that interleukin-3 (IL-3) and IL-1 can abrogate the selfrenewal of murine HSCs in vitro [47], in contrast to stem cell factor

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(SCF) and Flt3 ligand (Flt3L), which maintain HSCs [48]. Further, the addition of exogenous thrombopoietin (TPO) to murine LTMCs led to an expansion of marrow repopulating cells [49]. Members of the transforming growth factor (TGF) family and cooperating factors have been demonstrated to inhibit the cell-cycle progression of HSCs in vitro [50–52]. The type of culture medium used also can influence the ex vivo expansion of HSCs. Whereas numerous studies using serum-containing medium have shown maintenance or loss of HSC activity following culture with cytokines, more recent studies using serum-free medium, which allows for a more careful dissection of the interaction of HSCs with defined ligands and avoids the introduction of inhibitory molecules such as TGF-β, have demonstrated enhanced repopulating ability, with some studies documenting up to an eightfold increase of murine marrow HSCs [41].

Large-animal studies To date, human trials of gene therapy and HSC manipulation have not been nearly as successful as experiments in mice might have predicted, and important differences in HSC behavior in vivo in large animals are beginning to be appreciated [53–55]. Thus, studies of stem cell expansion using clinically feasible procedures in larger animals to better mimic human physiology that are now being carried out may provide important insights needed for human clinical application. Thus far, the use of ex vivo cultured cells has mainly led to an enhanced rate of engraftment, but this may have occurred at the expense of long-term repopulating cells. The possibility of enhancing short-term repopulating activity by ex vivo expansion was suggested by two studies of lethally irradiated baboons engrafted with ex vivo, expanded CD34enriched peripheral blood progenitor cells (PBPCs) collected after granulocyte colony-simulating factor (G-CSF) mobilization [56–58]. Significant expansion of precursor cells with in vitro colony-forming activities was documented in both studies, and in one study severe combined immunodeficiency disease (SCID) mouse repopulating cells were also expanded twofold. Animals transplanted with ex vivo expanded cells experienced significantly shorter periods of post-transplant neutropenia, but the recovery of platelet and red cell production was significantly slower, and administration of TPO had no effect on platelet recovery. Although these studies were not designed to assess effects on long-term reconstituting stem cells, the possibility that cytokine-driven expansion may deplete long-term repopulating HSCs in primate models has been suggested by gene-marking studies, in which prolonging the period of in vitro culture worsened rather than improved gene transfer efficiency [59,60]. An increase in the number of precursors at the expense of in vivo marrow reconstituting function has also been observed in cats following ex vivo expansion of marrow cells [61]. In dogs, HSCs and progenitors have been cultured ex vivo for brief periods to promote gene transfer into marrow repopulating cells. The detection of gene-marked progeny in vivo suggests that at least a limited number of cells with HSC function divided in the ex vivo culture [62].

Xenogeneic animal models: transplantation of human cells Immunodeficient mouse xenogeneic transplant models In preclinical studies, xenogeneic transplant models have been developed in which human hematopoietic cells can engraft in immunodeficient mice and then be serially passaged in vivo [21,22,63–69]. These engrafting cells are referred to as SCID repopulating cells (SRCs). At

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present, the relationship of SRCs to HSCs in humans remains to be clarified, but the SRC is generally accepted as the most reliable surrogate marker of an HSC currently available. Nevertheless, human SRC expansion has been assayed in various immune-deficient mouse strains. The necessity for in vivo assessment of ex vivo HSC expansion was emphasized by several studies in which ex vivo cultures of CD34+ cells have resulted in the dramatic increase in numbers of colony-forming cell (CFC) or even long-term culture-initiating cell activity, but with a concomitant decrease in SRCs [70–73]. In contrast, several other studies have suggested a modest increase in engraftment of immunodeficient mice following short-term culture, in which cells were cultured with multiple cytokines or in the presence of stromal cells or stromal cellconditioned medium [6,74–80]. Quantitative studies using limiting dilution SRC assays have confirmed modest two- to fourfold cytokine-induced increases in SRCs. For example, Bhatia et al. and Conneally et al. have cultured CD34+ CD38− cord blood cells in serum-free medium supplemented with SCF, Flt3L, G-CSF, IL-3, and IL-6 and showed expansion of SRCs [65,74]. The study by Bhatia et al., in which cells were cultured for 4 days, found a fourfold increase in CD34+ CD38− cord blood cells, a 10-fold increase in CFCs, and a two- to fourfold increase in SRCs [74] . However, all SRCs were lost after 9 days of culture, despite continued expansion in the total number of cells throughout the culture period. In a similar study, Conneally et al. confirmed that this culture system produced significant increases in CFCs (100-fold) and long-term culture-initiating cells (fourfold), as well as a modest twofold increase in SRCs. Similarly, studies using an IL-6/IL-6-receptor chimera induced significantly higher levels of CD34+CD38− cells and a fourfold increase in SRCs by limiting dilution methods [79,81–84]. Cytokine-induced expansion systems have also utilized several cloned stromal feeder cells and mesenchymal stem cell layers as sources of growth factors, and have been shown to support long-term repopulating cells [85,86]. However, because the cultured cells are in contact with the stromal feeder cells in this type of culture system, it is less advantageous to use this system for clinical application, and noncontact stromalbased culture systems are also being explored as HSC expansion systems [4,87–91]. An example of this type of expansion system was reported by Shih and colleagues [89], in which a murine bone marrow-derived stromal cell line, AC6.21, treated with leukemia inhibitory factor, secreted a factor that resulted in expansion of human HSCs when the HSCs were cultured with the conditioned medium. The expansion occurred in the absence of the stromal cells, and the magnitude of expansion was dependent on the level of activity of the secreted factor. In another example, Lewis et al. cultured umbilical cord blood (UCB) CD34+ with IL-7, SCF, Flt3L, and TPO in a Transwell (where stromal cells are separated from CD34+ cells by a transmembrane) above the murine fetal liver cell stromal feeder line, AFT024, for 7 days and demonstrated the maintenance of long-term repopulating cells in culture using secondary transplants into NOD/SCID mice, as well as secondary and tertiary transplants into fetal sheep [92]. Additionally, the use of conditioned medium from the HS-5 cell line, an immortalized stromal cell line derived from human marrow after transduction with a replication-defective retrovirus containing the human papilloma virus E6/E7 gene, has been shown to support the proliferation of hematopoietic progenitors and maintain CFCs for up to 8 weeks in culture [88,93]. While the above studies documented the expansion of SRCs in shortterm culture conditions only, Piacibello et al. [94] reported maintenance of cord blood CD34+ cell growth for up to 12 weeks in serum-containing medium supplemented with Flt3L, SCF, and IL-6. Additionally, limiting dilution transplants demonstrated a more than 70-fold expansion of SRCs. Although it is not clear whether this approach will be reproducible, Tanavde et al. recently reported the ability to expand and maintain

UCB SRCs for up to 4 weeks [95] using CD34+ from UCB and adult mobilized PBPCs cultured in serum-free medium supplemented with Flt3L, SCF, and TPO with or without IL-6/IL-6-receptor chimera. UCB cells demonstrated extensive in vitro expansion and maintained repopulating ability throughout the 4-week culture period, whereas mobilized PBPCs showed less expansion and no engrafting potential of cultured cells (only fresh cells being engrafted). While the ability to maintain and expand repopulating cells in vitro for extended periods would be beneficial for gene therapy, augmentation of clinical transplantation studies with expanded cells will require shorter expansion times, as time to transplant is critical in the clinical setting. Moreover, there is always the concern that long-term culture of cells may in fact induce transformation of the cultured cells. Fetal sheep xenogeneic transplant model When primitive hematopoietic cells isolated from both cord blood and adult bone marrow are expanded ex vivo and transplanted into fetal sheep, only short-term engraftment of the animals is seen. In their initial study attempting ex vivo expansion of enriched adult human bone marrow cells with in vivo engrafting capabilities, Shimizu and colleagues were able to document the maintenance of cells with engrafting capabilities in fetal sheep for up to 2 months when cultured with KIT ligand, Flt3L, IL-6, and erythropoietin with or without IL-3 [96]. Engraftment was noted in secondary recipients of fresh human cells, but not in recipients of cultured cells, suggesting that the culture conditions used were unable to support or expand long-term engrafting cells. Similar results were demonstrated by McNiece et al. using a two-step culture procedure in which cord blood progenitors cultured in the presence of SCF, megakaryocyte growth and development factor, and GCSF for 7 or 14 days prior to transplant provided no long-term engraftment of fetal sheep [97]. While the expanded cells were capable of early rapid engraftment (threefold over fresh cells), they lacked secondary and tertiary engrafting potential compared with fresh cells, again suggesting that the culture conditions described resulted in the loss of long-term repopulating cells and led to an increase of more mature shortterm repopulating cells. Overall, preclinical studies of human HSC expansion assayed based on repopulating ability in xenogeneic transplant models show promise but must be interpreted with caution, as human NOD/SCID or fetal sheep repopulating ability has not been correlated with non-human primate or human in vivo repopulating ability. In fact, studies that compared repopulation by gene-marked cells from non-human primates in autologous recipients and NOD/SCID mice revealed substantially greater repopulation in mice [98]. Although this finding might reflect detection in mice of a more mature precursor, such as a short-term repopulating cell, other studies have demonstrated serial repopulation by expanded human cells in murine recipients, a function consistent with self-renewing stem cells. Further, while these studies have shown in vitro expansion of SRCs, the results are highly variable, and include numerous reports of loss of stem cell activity after ex vivo culture [73,97,99–101]. Finally, while studies of cytokine-induced expansion suggest the potential for enhancement of short- and long-term repopulating activity, only a few cell divisions at most have been observed. Thus, current efforts are now focused on identifying and exploiting other extrinsic and intrinsic regulators of stem cell fate. Novel approaches for ex vivo HSC expansion using extrinsic and intrinsic regulators of stem cell fate Optimization of cytokine-driven expansion systems has not led to clinically significant HSC expansion, perhaps due to a predominantly permissive, rather than directed, role in determining stem cell fate. The

Expansion of Hematopoietic Stem Cells

search for extrinsic and intrinsic regulators that act directly on human HSCs in regulating cell fate and self-renewal has suggested a role for regulatory molecules active in early development that are important in HSC maintenance and regulation. More recent studies have focused on the regulation of intrinsic signaling pathways by retrovirus-mediated transduction of HSCs, for example expression of homeobox genes. However, culture of cells for clinical application requires the use of extrinsic regulators of cell development including bone morphogenic proteins (BMPs), angiopoietin-like proteins, and Sonic hedgehog (Shh), Wnt, and Notch ligands. Retrovirally mediated overexpression of Hox transcription factors, in particular HoxB4, has led to extensive ex vivo HSC expansion in vitro (3 logs over control cultures) without loss of full in vivo lymphomyeloid repopulating ability [102–105]. Although methods to alter homeobox gene expression in the absence of transducing cells are not available, the self-renewal induced by HoxB4 has suggested the exploration of extrinsic regulators of cell fate involved in embryonic development, such as BMP-4, a member of the TGF-β superfamily, and Shh of the Hedgehog family of proteins, both having been implicated in early hematopoietic development [106,107]. In ex vivo expansion of human cord blood, soluble human BMP-4 has been shown to increase the survival of repopulating blood cells in ex vivo culture [106], while Shh has been shown to induce a few-fold increase in repopulating cells via mechanisms that are dependent on downstream BMP signals [108]. Wnt proteins, which are involved in the growth and differentiation of a variety of primitive tissues, have also been implicated in the regulation of hematopoiesis, possibly exerting their effects through stromal cells [109], and have been shown to stimulate the proliferation of hematopoietic precursor cells [110]. There are also data supporting the presence of an interactive relationship between the various signaling pathways. For example, Notch-1 receptors have been shown to be upregulated in response to Wnt signaling in HSCs [111]. The most extensively studied and successfully utilized extrinsic regulators have been ligands that activate the Notch pathway, with growing data supporting a role of Notch signaling in maintenance and/or self-renewal of HSCs. All four Notch receptors (Notch-1, -2, -3, and -4) identified in vertebrates have been detected in hematopoietic cells [112], and several investigators have reported the expression of Notch-1 and Notch-2 in human CD34+ or CD34+ lineage-negative (Lin−) precursors [112–114], and of the Notch ligands Delta-1 and Jagged-1 in human bone marrow stromal cells and human hematopoietic precursors [115– 118] . Moreover, expression of a constitutively active, truncated form of Notch-1 in murine hematopoietic precursors inhibited differentiation and enhanced self-renewal, leading to the establishment of an immortal cell line that phenotypically resembles primitive hematopoietic precursors [119]. This cell line, depending upon the cytokine context, can differentiate along the lymphoid or myeloid lineage. More recently, expression of constitutively active Notch in murine precursors transplanted in vivo led to an increased stem cell number that was evident in secondary transplantation studies [120]. While these findings point to a role for Notch signaling in the regulation of stem cell self-renewal, expression of activated Notch-1 in human CD34+ cord blood precursors induced only a modest increase in the number of progenitors [121]. In order to affect nontransduced cells, exogenous Notch ligands have been used to induce endogenous Notch signaling in HSCs. Initial studies in mice and humans using soluble or cell-bound Notch ligand revealed limited increases in precursor cell numbers [116,117,121]. However, Varnum-Finney et al. and others [122,123] have demonstrated a requirement for ligand immobilization to induce Notch signaling. Studies performed with hematopoietic precursors cultured in the presence of immobilized Delta-1 demonstrated profound effects on the differentiation of isolated murine marrow precursors, with a multi-log increase in

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the number of stem cell antigen (Sca-1)+ Gr-1− cells with short-term lymphoid and myeloid repopulating ability [124]. Similarly, studies with human cord blood CD34+CD38− precursors cultured in serum-free conditions with immobilized ligand and cytokines resulted in an approximately 100–200-fold increase in the number of CD34+ cells generated compared with control cultures, and included cells capable of repopulating immunodeficient mice [125,126]. Moreover, more recent studies with both murine and human hematopoietic progenitors have demonstrated density-dependent effects of Delta-1 on fate decisions of hematopoietic progenitors, and suggest that optimal ligand densities are required to promote expansion of cells that retain repopulating ability [125,127]. Other investigators have shown similar outcomes. Kertesz et al. demonstrated that culture of murine Lin− hematopoietic progenitor cells with immobilized Jagged-1 resulted in expansion of serially transplantable HSCs, with superior expansion dependent on the combinatorial effect of Notch and cytokine-induced signaling pathways [128]. These data indicate that manipulation of Notch signaling can enhance stem cell self-renewal ex vivo and thereby increase HSC numbers for transplantation. Despite limited evidence of ex vivo HSC expansion with current methodologies, including stroma and stroma-free systems, clinical trials have been conducted with autologous marrow, cytokine-mobilized peripheral blood cells or cord blood cells, and their ability to shorten engraftment periods has been determined. Stroma-free systems provide a clear advantage for clinical use, since they are easier to standardize and maintain for cyclic guanosine monophosphate. However, some studies have used noncontact systems in which stromal cells serve to condition the medium. These trials will be discussed in the next section.

Clinical trials of ex vivo expanded HSCs The first report of a clinical trial using in vitro cultured hematopoietic progenitor cells appeared in 1992. Since then, a number of trials have been carried out with the primary goal of enhancing hematopoietic recovery after high-dose chemotherapy with the infusion of expanded stem cell grafts derived from bone marrow, mobilized peripheral blood or cord blood. Clinical trials using bone marrow and mobilized peripheral blood Table 8.1 summarizes select trials with autologous bone marrow or mobilized PBPCs cultured ex vivo with cytokines and/or stromal elements [129–138]. Studies of expanded mobilized PBPCs were all done in the autologous setting, and included only those patients with sufficient stem cell harvests to allow the use of an aliquot of the apheresis product for expansion. This excluded “poor mobilizers” who clearly stand to benefit from expansion procedures allowing them to undergo treatment with high-dose therapy. However, it is not known whether HSCs derived from such patients are equivalent to those derived from patients with adequate mobilization of HSCs. These trials, primarily in breast cancer patients, assessed enhanced short-term repopulation as a function of decreased time to neutrophil and/or platelet engraftment. With one exception, these studies were unable to address the expansion of longterm repopulating HSCs because patients received nonmyeloablative chemotherapy and/or infusion of noncultured cells. Overall, although the safety and feasibility of stem cell expansion approaches were demonstrated, more rapid engraftment was not observed in patients receiving reduced-intensity preparative regimens. There have, however, been suggestions of ex vivo expansion of short-term repopulating cells in patients who have received myeloablative therapy.

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Table 8.1 Selected bone marrow and mobilized peripheral blood clinical expansion trials

Author/ reference

Conditioning regimen

Infusion expanded cells

Patients

Stem cell source

Cytokines/serum

Notes

Williams et al. (1996) [138]

n=9 Metastatic breast cancer

MPBPC

PIXY321, human serum albumin 1% × 12 days

NM1

Day +1

↑ TNC 26-fold CD15+ post expansion average 29%

Alcorn et al. (1996) [129]

n = 10 Nonmyeloid malignant

MPBPC

SCF, IL-1β, IL-3, IL-6, EPO + autologous plasma × 8 days

M/NM2

Day 0

↑ TNC 21-fold, CFU-GM 139-fold, BFU-E 114-fold (mean) No change in engraftment compared with controls

Bertolini et al. (1997) [131]

n = 10 8 breast cancer, 2 NHL

MPBPC

MGDF, SCF, IL-3, IL-6, IL-11, Flt3L, MIP-1α × 7 days

NM3

Day 0

Study aimed at looking at generating megakaryocytic progenitors

Holyoake et al. (1997) [133]

n=4 2 NHL, 2 MM

MPBPC Expanded cells only

SCF, IL-1β, IL-3, IL-6, EPO + autologous plasma × 8 days

M4

Day 0

No long-term engraftment seen ↑ TNC 8–27-fold, CFU-GM 17–130-fold

Reiffers et al. (1999) [130]

n = 14 MM

MPBPC

SCF, G-CSF, MGDF

M5

Day 0

↑ TNC 34.4-fold, CD34+ cells 2.6-fold, CFU-GM 14.1-fold (median)

McNiece et al. (2000) [134]

2 cohorts 1) n = 10 2) n = 11 Breast CA

MPBPC Cohort 2 – expanded cells only

SCF, G-CSF, MGDF ×10 days

NM6

Day 0

Cohort 1:

Stiff et al. (2000) [137]

n = 19 Breast cancer

BM (~75 mL) – expanded cells only

AastromReplicell Bioreactor Flt3L, EPO, PIXY321, 10% FBS, 10% horse serum × 12 days

NM1

Day 0

TNC 4.8-fold, CFU-GM 4.2-fold, CD34+Lin− cells 0.9-fold, LTC-IC 1.2fold (median) Engraftment times similar to autologous bone marrow transplantation Correlation: cell dose and engraftment

Pecora et al. (2001) [136]

n = 34 Breast cancer

BM (50–100 ml) for expansion + low-dose MPBPC

AastromReplicell Bioreactor Flt3L, EPO, PIXY321 (+TPO in seven cases), 10% FBS, 10% horse serum × 12 days

NM1

Day 0

CD34+Lin− cell number and quantity of stromal progenitors contained in the expanded product correlated with engraftment outcome

Engelhardt et al. (2001) [132]

n = 10 Breast cancer

BM (median 97 mL) – expanded cells only 2 patients: irradiated PBPCs

AastromReplicell Bioreactor Flt3L, EPO, PIXY321, 10% FBS, 10% horse serum × 12 days

NM1

Day 0

↑ TNC 4.5-fold, CFU-GM 18-fold (median) Correlation: CD34+Lin− cells and engraftment

Paquette et al. (2002) [135]

n = 43 Breast cancer

MPBPC

GCSF, SCF, MGDF, 1% human albumin, transferrin × 9–14 days

NM7

Day 0

Varied duration of cultures and starting cell density

↑ TNC 20-fold (median) ↑ CD34+ cells 1.8-fold Cohort 2: ↑ TNC 14-fold (median) ↑ CD34+ cells 2.0-fold TNC/kg post expansion best predictor of time to neutrophil engraftment

BFU-E, burst-forming unit-erythroid; BM, bone marrow; CFU-GM, colony-forming unit-granulocyte–macrophage; EPO, erythropoietin; FBS, fetal bovine syndrome; LTC-IC, longterm culture-initiating cells; M, myeloablative; MGDF, megakaryocyte growth and development factor; MIP, macrophage inhibitory protein; MPBPC, mobilized peripheral blood progenitor cells; MM, multiple myeloma; NHL, non-Hodgkin’s lymphoma; NM, nonmyeloablative; TNC, total nucleated cells. See text for other abbreviations. 1 Cyclophosphamide/carbo/thiotepa (STAMP V). 2 Melphalan/total body irradiation, BEAM, thiotepa/cyclophosphamide, carboplatin/etoposide/melphalan, cyclophosphamide/TBI. 3 Thiotepa/melphalan (breast cancer); mitoxantrone/melphalan (NHL). 4 Cyclophosphamide/total body irradiation (NHL) and busulfan/melphalan (MM). 5 Melphalan ± total body irradiation. 6 Cyclophosphamide/cisplatin/BCNU or taxol/cyclophosphamide/cisplatin or taxotere/melphalan/carboplatin. 7 Cyclophosphamide/carmustine/cisplatin.

Expansion of Hematopoietic Stem Cells

For example, in a study by Holyoake et al. [133], the gain of short-term repopulating cells at the expense of long-term repopulating cells was suggested. In that study, mobilized PBPCs cultured ex vivo with SCF, IL-1β, IL-3, IL-6, erythropoietin, and autologous plasma for 8 days were infused in the absence of noncultured cells into patients that had received myeloablative chemotherapy. None of the four patients showed evidence of long-term hematopoietic recovery, and all required infusions of unmanipulated cryopreserved autologous back-up mobilized PBPCs, after which full hematopoietic recovery was seen. Three patients showed initial neutrophil engraftment that was unsustained, suggesting that short-term repopulating cells may have been generated ex vivo at the expense of long-term repopulating ones, possibly by inducing their differentiation. A shortening of the neutropenic period following high-dose chemotherapy in 21 patients with breast cancer was also shown in a study in which autologous mobilized PBPCs were cultured with SCF, G-CSF, and megakaryocyte growth and development factor for 10 days. The patients in this study were divided into two cohorts: those who received expanded cells only, and those who received both expanded and unmanipulated cells in tandem [134]. Expanded PBPCs resulted in a more rapid neutrophil engraftment (p = 0.02 for cohorts 1 and 2 versus the historical controls), the best predictor of time to neutrophil engraftment was the total number of cells harvested after expansion, and patients receiving more than 4 × 107 cells/kg engrafted by day 8. Reiffers et al. also reported a decreased time to neutrophil engraftment using the same culture conditions to expand autologous mobilized PBPCs for infusion with unmanipulated cells into patients with multiple myeloma after high-dose conditioning. The use of continuous perfusion methods for the expansion of bone marrow has recently been evaluated in three trials utilizing the automated perfusion bioreactor system AastromReplicell [132,136,137]. Notably, these are the only trials that used bone marrow (unselected for CD34+ cells) as the source of stem cells for ex vivo expansion, thereby perhaps including accessory or stromal cells of unclear significance in expansion cultures, and perhaps enhancing the engraftment ability of this expanded cell population. In these studies, breast cancer patients underwent the same conditioning regimen, and cells derived from autologous bone marrow were placed directly into the automated perfusion bioreactor system for expansion with Flt3L, erythropoietin, and PIXY321 cytokines, as well as fetal bovine serum and horse serum. In all trials, cultures were initiated with small amounts of harvested marrow (75– 100 mL). The trials by Stiff et al. and Engelhardt et al. used expanded cells only, while Pecora et al. also infused mobilized PBPCs in low doses. Engraftment was not enhanced but did occur, suggesting the maintenance of HSCs in the bone marrow cultures, and all trials reported a correlation between the time to neutrophil and platelet engraftment with CD34+Lin− cell dose/kg. Stiff et al. reported that only 2 × 105 CD34+ cells/kg of the expanded cells were needed to produce optimal platelet engraftment. This cell dose was lower than the number of cells required for predictable platelet engraftment by day 28 in breast cancer patients undergoing PBPC transplantation with unmanipulated PBPCs, in which the minimal number of CD34+ cells required was reported to be 2–5 × 106/kg [139]. Clinical trials using UCB cells Rationale for use of UCB stem cells for ex vivo expansion With greater than 6000 UCB transplants performed to date since 1988 [140], UCB has emerged as an alternative source of HSC for transplantation. This is especially important for minority patients and patients of mixed ethnicity, for whom UCB is a particularly attractive alternative

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donor stem cell source as it is readily available with no donor attrition and allows reduced stringency in human leukocyte antigen (HLA) matching without an increase in graft-versus-host disease (GVHD). Numerous clinical studies have consistently shown that the total nucleated cell and CD34+ cell doses in cord blood grafts are highly correlated with the rate of neutrophil and platelet engraftment, as well as the incidence of graft failure and early transplant-related complication [141– 149]. Based on these studies, critical cell dose thresholds have been established, and outcomes for patients receiving less than the generally accepted threshold of more than 2.5 × 107 total nucleated cells/kg are significantly inferior in terms of engraftment, transplant-related mortality and overall survival. For patients weighing greater than 35–40 kg, obtaining an adequate cell dose from a single UCB unit may be an impossible hurdle. Delayed engraftment has also been correlated with HLA mismatch at more than two loci, but recent studies have suggested that the impact of HLA disparity on survival can be partially overcome by increasing cell dose [148,150]. Efforts to overcome the obstacle of small cell numbers in UCB grafts by ex vivo cytokine-mediated expansion have not yet demonstrated improved time to engraftment. Infusion of multiple cord blood units as an alternative approach to increase cell numbers is also under investigation. The initial results from this approach are encouraging and have demonstrated the safety of this approach in adults, with improved engraftment and decreased transplant-related mortality, but the significant delay in engraftment has not been abrogated in these patients [151]. Thus, the collective data point to improved outcome following UCB transplantation in patients receiving a higher cell dose, in terms of both CD34+ and total nucleated cell doses, an observation leading to the hypotheses that ex vivo expansion of CD34+ cell numbers or the infusion of more than one unit of cord blood might improve the rate of cord blood engraftment and overall survival. Several lines of evidence suggest that UCB contains a higher frequency of primitive hematopoietic progenitor cells and early committed progenitors than adult bone marrow or peripheral blood [21,68]. There is also increasing evidence that the stem cells isolated from UCB survive longer in culture [95] and may be less mature and have greater proliferative capacity [78,152–154]. Phenotypic analyses of UCB have shown that the more primitive cell population which expresses the CD34 antigen, but not the CD38 antigen, is fourfold more prevalent than in bone marrow or PBPCs, and that this subpopulation has a higher in vitro cloning efficiency than the same population isolated from adult bone marrow [155]. These findings correlate with data from studies using in vitro with bone marrow or PBPCs [156–158]. Second, there are numerous reports of the increased proliferative potential of UCB cells in response to cytokine stimulation. For example, using IL-11, SCF, and G-CSF or granulocyte–macrophage colonystimulating factor, Cairo and colleagues demonstrated an 80-fold increase after a 14-day expansion of UCB versus adult bone marrow [159]. Moreover, compared with adult bone marrow, UCB has been shown to have increased serial in vitro replating efficiency [153] and increased culture life span with increased progenitor cell production [94,156]. Finally, in vivo assays of UCB versus bone marrow have shown that HSCs from UCB, but not adult bone marrow, can engraft NOD/SCID mice without the use of exogenous cytokines [67]. More recently, Rosler et al. showed that expanded cord blood had a competitive repopulating advantage compared with expanded adult bone marrow using an in vivo assay with NOD/SCID mice [22]. A potential contribution to the differences observed between UCB and adult HSCs may arise from the differential response of HSCs from different sources in cytokine-driven expansion systems, leading to variations in cell-cycle status and homing ability of the expanded cells. Other possible sources of stem cells may have even greater proliferative

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potential. For example, murine fetal liver cells have greater proliferation and repopulation potential than HSCs isolated from adult murine bone marrow or peripheral blood [160,161]. Overall, these results suggest that UCB progenitor cells are functionally superior to adult bone marrow, with greater proliferative potential and possibly greater self-renewal capacity. Thus, UCB may represent a more viable target for ex vivo stem cell expansion, a possibility that has led to several clinical studies on the ex vivo expansion of cord blood cells to augment conventional UCB transplantation. Clinical ex vivo expansion trials with UCB UCB expansion trials (summarized in Table 8.2) [162–166] were undertaken to determine whether the delayed engraftment associated with UCB transplantation could be overcome if a portion of the cells from the UCB unit were expanded ex vivo. Like the trials using PBPCs and bone marrow, these initial trials utilized cytokine-based expansion systems as well as newer automated perfusion systems. The choice of exogenous cytokines used for culture has varied, reflecting the still undefined optimal conditions for expansion of the stem/progenitor cell. In all of the initial studies, only a portion of the single cord blood graft was used for expansion, and the expanded cells were infused in addition to unmanipulated cells, as there is a lack of definitive data to suggest that cultured cells retain HSC properties and do not differentiate. There have been no adverse toxicities associated with infusion of the expanded cell product, nor has there been any change in engraftment kinetics. However, only two of the studies have enrolled more than a handful of patients, making it difficult to draw any definitive conclusions. More recently, novel culture systems for the ex vivo expansion of UCB progenitors have begun patient accrual, including a Notch-mediated ex vivo expansion trial at the Fred Hutchinson Cancer Research Center, a trial utilizing a copper-chelating agent and an alternative strategy of coculture of UCB cells with bone marrow mesenchymal stem cells. These will be discussed below. In one of the larger studies to date, Jaroscak et al. reported preliminary data from a phase I trial at Duke University Medical Center undertaken to assess augmentation of UCB transplantation with cells expanded ex vivo in the AastromReplicell Cell Production System [162]. This trial included 28 patients with both malignant and inherited disorders who were conditioned with one of three regimens (Table 8.2), depending on diagnosis. On day 0, a portion of a single unrelated UCB unit was expanded ex vivo in medium supplemented with fetal bovine serum, horse serum, and cytokines (erythropoietin, PIXY321, and Flt3L) for 12 days. The expanded cells were then infused to augment the conventional transplant on day 12 after transplantation. Although expansion of total nucleated cells and CFCs occurred in vitro in all cases, no increase in CD34+Lin− cell number was achieved. In vivo, significant effects on engraftment kinetics were not observed with median time to neutrophil engraftment (absolute neutrophil count [ANC] > 500/μL) of 22 (range 13–40) days. No adverse reactions were observed due to infusion of the cultured cells. This phase I trial was an important contribution to the concept and development of ex vivo expansion of UCB CD34+ cells for use in the clinical setting, since it demonstrates both the safety of reserving an aliquot of an already small cord blood unit for expansion and the feasibility of expansion in a clinical setting. It is also possible that, although the numbers of CD34+Lin- and CD3+ cells were not expanded with this culture system and may have explained the failure to improve engraftment, the delayed infusion (day 10–12 post transplant) of expanded cells may have masked or prevented the benefit of a more rapid engraftment. Newer-generation clinical cord blood expansion trials, discussed below, are currently underway based on a double cord blood model, in which one unit is infused unmanipulated on the day of transplant with a second unit that has been expanded ex vivo.

Shpall and colleagues are currently conducting a number of cord blood expansion trials at the MD Anderson Cancer Center: a cytokinemediated expansion trial and a triethylenephosphoramide (TEPA)-based ex vivo expansion trial. The cytokine-mediated expansion trial is the successor trial to an earlier published trial performed at the University of Colorado [165], with some important changes. In this currently accruing trial, patients are now being randomized to receive either two unmanipulated cord blood units or one unmanipulated unit and one ex vivo expanded unit following high-dose therapy, compared with the previous trial in which only a portion of a single cord blood unit was used for expansion. For those patients randomized to receive expanded cells, the unit identified for ex vivo expansion is thawed, CD133 selected, and placed into a cytokine-mediated expansion system 14 days prior to the day of transplant. Accrual on this trial continues, but analysis of the first cohort of 22 patients enrolled has been performed (Shpall, personal communication). The median time to neutrophil engraftment is 20 and 21 days for patients receiving a conventional unmanipulated double UCB transplant and those receiving an unmanipulated unit and ex vivo expanded unit, respectively. Overall, 30% of patients developed acute grade III–IV GVHD. Based on preclinical data in which Peled et al. demonstrated enhanced expansion of CD34+ UCB progenitors, an additional ex vivo clinical expansion trial is enrolling at the MD Anderson Cancer Center using the copper chelator TEPA (Shpall, personal communication) [167]. Similar to the cytokine-mediated expansion trial, patients with hematologic malignancies are eligible, but all patients in this trial must have identified a single unit of cord blood that has been cryopreserved in two fractions. The smaller of the two UCB fractions is CD133 selected, and expanded ex vivo in the presence of cytokines and TEPA starting 21 days prior to infusion. Ten patients with high-risk malignancies have been treated thus far. The average fold expansion of total nucleated cells placed in culture was 219 (range 2–616), and the median time to engraftment was 28 (range 16–46) days for neutrophils and 48 (range 27–96) days for platelets. Evidence of GVHD during the 180-day study period was found in five of the 10 evaluable patients. Three patients experienced acute GVHD, all grade II, with skin involvement, and two patients developed chronic GVHD, one chronic extensive and one mild limited chronic. One patient with acute GVHD persisted to chronic limited GVHD with skin involvement. All cases were resolved with steroid therapy. There were no incidences of engraftment failure or grades III–IV acute GVHD, and 100-day survival was 90%. Thirty percent of the patients are alive and free of disease at a median 18 months post transplant. At the Fred Hutchinson Cancer Research Center, a phase I trial assessing the potential efficacy of cord blood progenitors cultured in the presence of an engineered form of the Notch ligand Delta-1 in contributing to rapid early engraftment in patients undergoing a high-dose UCB transplant is currently accruing patients. As discussed above, there is accumulating evidence on the role of the Notch signaling pathway in hematopoietic cell fate decisions. This trial has accrued only three patients to date, and while conclusions cannot be made as to the efficacy of this approach, the early results are encouraging (Delaney, unpublished data). Three patients with acute myeloid leukemia have enrolled and received cord blood progenitors that were cultured with Delta-1 following infusion of a second noncultured and unrelated donor cord blood graft. Upon harvest of the cells at day 16 post culture initiation, there was an average fold expansion of CD34+ cells of 116 (±49) and an average fold expansion of total cell numbers of 565 (±179) (Fig. 8.2(a)). The infused CD34 cell dose (defined as the number of CD34+ cells/kg recipient body weight) derived from the ex vivo expanded unit ranged from 1 to 13 million CD34+ cells/kg. In the first two patients, early myeloid recovery was observed, occurring at a time when engraftment due to the infusion of two noncultured

Expansion of Hematopoietic Stem Cells

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Table 8.2 Selected umbilical cord blood clinical expansion trials Author/ reference

Conditioning regimen

Infusion expanded cells

AastromReplicell system Epo, Pixy321, Flt3L × 12 days

CY/TBI or BU/ CY/ATG

Day +12

No significant change in neutrophil engraftment No infusional toxicities 2 deaths prior to engraftment

UCB – sibling 12.5% expanded + 87.5% unmanipulated

G-CSF, TPO, Flt3L, 10% autologous cord blood plasma × 10 days

TBI/thiotepa/CY

Day +1

ANC 310/μL by day +8 Event-free survival now greater than 1 year

n=2 CML (blast crisis and accelerated phase)

UCB (unrelated) 11%, 17% expanded + 89%, 83% unmanipulated cells

AastromReplicell system Epo, Pixy321, Flt3L × 12 days

(1) BU/ATG CY (2) CY/TBI/ATG

Day +12

No infusional toxicities Improved platelet engraftment No change in neutrophil engraftment Lin−, CD34+ cells decreased in one patient, increased 1.7-fold in other patient

Shpall et al. (2002) [165]

n = 37 (25 adult) Stratum A = 25 Stratum B = 12 (split unit) Dx: hematologic malignancy (34), breast cancer (3)

UCB (unrelated) Expanded fraction Cohort A = 40% Cohort B = 60% + unmanipulated cells

G-CSF, SCF, MGDF expanded in Teflon bags × 10 days

Adults: High-dose melphalan/ ATG/TBI Or High-dose melphalan/ ATG/BU Children: CY/TBI/ arabinoside C/ ATG

Stratum (A) Day +10 (B) Day 0

No infusional toxicities No change in engraftment seen No neutrophil engraftment failures

Jaroscak et al. (2003) [162]

n = 28 (26 children) Varied Dx: both malignancies and inherited disorders

UCB (unrelated, one sibling) Expanded + unmanipulated cells

AastromReplicell system PIXY321, Epo, Flt3L, 10% horse serum, 10% FBS × 12 days

Hematologic malignancy TBI/melphalan/ ATG Or BU/melphalan/ ATG Inherited disorders BU/CY/ATG

Day +12

No infusional toxicities No alteration in time to engraftment ↑ TNC 2.4-fold, CFU-GM 82.7-fold, CD34+ 0.5-fold (median)

Shpall et al. (personal communication; see text)

n = 22 (accrual continues)

UCB – double unrelated Randomization: (A) Double unmanipulated versus (B) Expanded + unmanipulated

CD133+ cells G-CSF, SCF, MGDF × 14 days

Myeloid malignancy Flu/BU/ATG Lymphoid malignancy Flu/melphalan/ thiotepa/ATG

Day 0

No infusional toxicities Fold expansion N/A Average time ANC > 500/μL (A) 20 days (B) 21 days

Shpall et al. MDACC (personal communication; see text)

n = 10 (accrual continues)

Single cord blood unit cryopreserved in two fractions: 80%–20%, 60%–40% or 50%– 50% (smaller fraction ex vivo expanded)

CD133+ cells TEPA + Flt3L, TPO, SCF, IL6 × 21 days

Myeloid malignancy Flu/BU/ATG Lymphoid malignancy Flu/melphalan/ thiotepa/ATG

Day +1

No infusional toxicities Fold expansion TNC 219 (mean), range 2–616 Average time to ANC > 500/μL 28 days (16–46 days)

Delaney et al. (personal communication; see text)

n=3 (accrual continues)

UCB – two units Unmanipulated unit Ex vivo expanded unit

CD34+ cells TPO, SCF, Flt3L, IL-3, IL-6 Serum free

CY/TBI/Flu

Day 0

No infusional toxicities Fold expansion TNC 565 (mean), range 210–791 Fold expansion CD34 115 (mean), range 41–210 Average time to ANC > 500/μL 15 days (9–20 days)

Patients

Stem cell source

Cytokines/serum

Stiff et al. (1998) [166]

n=9 Dx: advanced hematologic disorders

UCB (unrelated) Expanded + unmanipulated

Kögler et al. (1999) [163]

n=1 High-risk leukemia

Pecora et al. (2000) [164]

Notes

ATG, antithymocyte globulin; BU, busulfan; CML, chronic myeloid leukemia; CY, cyclophosphamide; Dx, diagnosis; EFS, event-free survival; FBS, fetal bovine serum; TBI, total body irradiation. For other abbreviations, see Table 8.1 and text.

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Patient 1 Patient 2 Patient 3

09

1.0 × 10

08

1.0 × 10

1.0 × 1007 1000000 0

5

10

1.0 × 10 10 1.0 × 10 1.0 × 10

Fold expansion 800

Patient 1 Patient 2 Patient 3

09

700

Fold expansion

Absolute CD34 cell number

1.0 × 1010

Total number of cells

CD34 cell growth

Total cell growth

(a)

08

1.0 × 10 07

600

TNC fold expansion CD34 fold expansion

500 400 300 200 100 0

1000000

15

0

Days in culture

5

10

15

TNC

CD34

Days in culture

Peripheral blood chimerism

(b)

Patient 1

Patient 2

Patient 3

100

100

100

75

75

75

50

50

50

25

25

25

0

0

0 7

14 21 28 42 56 80 100

7

7

14 21 28 42 56 80 100

100

100

100

75

75

75

50

50

50

25

25

25

Not done

0 7

Not done

0

14 21 28 42 56 80 100

CD33

7

14

21

28

CD14

0 7

14 21 28 42 56 80 100

100

100

100

75

75

75

50

50

50

25

25

14

21

28

CD56

25 QNS

7

Percent

0

0

0 14 21 28 42 56 80 100

7

7

14 21 28 42 56 80 100

100

100

100

75

75

75

50

50

50

25

25

25

7

14 21 28 42 56 80 100

21

28

CD3

0

0

0

14

7

14 21 28 42 56 80 100

7

14

21

28

Days post transplant Fig. 8.2 Clinical grade culture of umbilical cord blood (UCB) progenitors with Delta-1ext-IgG for therapeutic application results in significant in vitro expansion of both total nucleated and CD34+ cells. CD34+ cord blood progenitors were selected using the clinical grade Isolex 300i and placed into culture with Delta-1ext-IgG as described. (a) In vitro growth characteristics are presented for three patients enrolled to date, including absolute growth of total nucleated cells (TNC), CD34+ cells, and the resultant fold expansion of both total nucleated and CD34+ cells. Data shown in the bar graph are the mean ± standard error of the mean for the three patients. (b) Peripheral blood was sorted by fluorescence-activated cell sorting into CD3+, CD33+, CD14+, and CD56+ cell fractions to assess for contribution to engraftment from the ex vivo expanded and unmanipulated units (chimerism). Chimerism testing was performed by amplified fragment length polymorphism analysis. The y-axis represents the percent engraftment as contributed by the ex vivo expanded cord blood unit (black), the unmanipulated unit (grey), and the host (white). The x-axis represents time post transplant.

Expansion of Hematopoietic Stem Cells

units would not be expected. Both patients achieved an ANC of 100/μL by day 7 post transplant, a threshold that has been shown to be strongly associated with a survival benefit post allogeneic HCT [168], and an ANC of 500/μL by days 9 and 16 post transplant. A third patient received a more limited CD34 cell dose (1 × 106 cells/kg) due to a less robust in vitro expansion and did not show engraftment before 2 weeks. The CD34 fold expansion (at 41-fold expansion) for this patient was equivalent to the one outlier out of 19 in our preclinical validation runs that resulted in a CD34 fold expansion of less than 50. It is anticipated that a portion of cord blood units will result in less than expected expansion of CD34+ cells based on experience to date. Prediction of units that will expand poorly is not possible at this point prior to ex vivo expansion and suggests a benefit in having pre-expanded units cryopreserved for future use. Nonetheless, the average time to engraftment for all patients (defined as the first of three consecutive days with an ANC > 500/μL) was 15 days, contrasting the expected time to engraftment following a double unmanipulated cord blood transplantation of 23 days, as reported by Barker and colleagues [151]. The kinetics of hematopoietic recovery and the relative contribution of the expanded and unmanipulated cord blood units to engraftment over time were determined by a DNA-based assay for short tandem repeat loci on cells obtained from peripheral blood sorted by fluorescenceactivated cell sorting for CD3+, CD33+, CD56+, CD14+, and CD19+ cell fractions, beginning on day 7 post transplant. These studies revealed that initial myelomonocytic engraftment in these patients was derived from the cultured cells, with subsequent sustained engraftment primarily from the noncultured unit. Analysis of peripheral blood sorted cell fractions revealed loss of contribution to engraftment in all cell populations from the expanded cells by day 21 in two out of three patients transplanted to date; however, there was persistent contribution to engraftment from both the cultured and noncultured cells in the CD14, CD56, and CD19 (data not shown for CD19) sorted cell fractions at day 100 post transplant in one patient (Fig. 8.2(b)). Accrual continues on this study. It is not yet known whether loss of the cultured cord blood unit in marrow and peripheral blood engraftment resulted from loss of stem cell self-renewal capacity due to maturation of the stem cell during culture or yet-to-be-defined immune-mediated mechanisms that allow only cells from a single unit to survive in a double cord blood transplant setting. Our findings are consistent with

97

results seen in most patients undergoing conventional cord blood transplant with two unmanipulated units, where only one unit contributes to sustained donor engraftment, and donor dominance is usually observed early in the majority of patients. However, a clear difference in the expansion setting versus the conventional double cord blood transplant setting is the infusion of two unmanipulated, immunocompetent units versus infusion of an immunocompetent, noncultured unit and a T-celldepleted ex vivo cultured unit, which may allow for rejection of the cultured cells by the noncultured unit.

Conclusion Although optimal conditions for expanding HSC numbers remain undefined, the collective results of studies outlined above suggest that enhanced short-term and long-term repopulating ability may be achievable with cytokine-induced expansion systems. However, these studies further suggest that cytokine-induced effects on HSC self-renewal and expansion are limited, and clinically significant expansion has not been achieved. It is likely that we have not yet identified critical factors and combinations required to induce symmetric HSC self-renewal, and thus current efforts are now focused on identifying and exploiting factors previously shown to regulate stem cell fate in other developing organ systems or during embryogenesis. This approach is beginning to yield promising results, and improved methods for HSC expansion are emerging. Identification of genes and regulatory elements that are responsible for preventing stem cell senescence and driving symmetric divisions of the HSC, and a better understanding of how culturing affects the homing ability of stem cells and of the complex cellular interactions in the bone marrow microenvironment, are all essential to the ultimate success of ex vivo expansion. These areas are discussed in more detail in other chapters. Clinically, ex vivo expansion studies of primitive cells from bone marrow, PBPCs, and UCB have demonstrated the feasibility and safety of ex vivo expansion, and have suggested the enhancement of short-term repopulating cells perhaps at the expense of long-term repopulating cells. Continued development of methods using novel stem cell regulators that are shown to expand HSCs in preclinical studies is necessary, and current trials are underway to assess the safety and efficacy of these approaches, which hold promise for the future of expanding HSC numbers ex vivo.

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bone marrow: implications for gene therapy. Nat Med 1996; 2: 1329–37. Vormoor J, Lapidot T, Pflumio F et al. Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 1994; 83: 2489–97. Wang JC, Doedens M, Dick JE. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood 1997; 89: 3919–24. Glimm H, Eisterer W, Lee K et al. Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice. J Clin Invest 2001; 107: 199–206. Dorrell C, Gan OI, Pereira DS et al. Expansion of human cord blood CD34(+)CD38(−) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function. Blood 2000; 95: 102–10. Gan OI, Murdoch B, Larochelle A, Dick JE. Differential maintenance of primitive human SCIDrepopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells. Blood 1997; 90: 641–50. Danet GH, Lee HW, Luongo JL et al. Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34(+) cells after ex vivo expansion. Exp Hematol 2001; 29: 1465–73. Mobest D, Goan SR, Junghahn I et al. Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells. Stem Cells 1999; 17: 152–61. Bhatia M, Bonnet D, Kapp U et al. Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. J Exp Med 1997; 186: 619–24. Gilmore GL, DePasquale DK, Lister J, Shadduck RK. Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells. Exp Hematol 2000; 28: 1297–305. Luens KM, Travis MA, Chen BP et al. Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy1+Lin– cells with preserved ability to engraft SCID-hu bone. Blood 1998; 91: 1206–15. Novelli EM, Cheng L, Yang Y et al. Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency. Hum Gene Ther 1999; 10: 2927–40. Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999; 93: 3736–49. Ueda T, Tsuji K, Yoshino H et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL6, and soluble IL-6 receptor. J Clin Invest 2000; 105: 1013–21.

80. da Silva CL, Goncalves R, Crapnell KB et al. A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/ progenitor cells. Exp Hematol 2005; 33: 828– 35. 81. Kimura T, Sakabe H, Tanimukai S et al. Simultaneous activation of signals through gp130, c-kit, and interleukin-3 receptor promotes a trilineage blood cell production in the absence of terminally acting lineage-specific factors. Blood 1997; 90: 4767–78. 82. Kimura T, Wang J, Minamiguchi H et al. Signal through gp130 activated by soluble interleukin (IL)-6 receptor (R) and IL-6 or IL-6R/IL-6 fusion protein enhances ex vivo expansion of human peripheral blood-derived hematopoietic progenitors. Stem Cells 2000; 18: 444–52. 83. Kollet O, Aviram R, Chebath J et al. The soluble interleukin-6 (IL-6) receptor/IL-6 fusion protein enhances in vitro maintenance and proliferation of human CD34(+)CD38(−/low) cells capable of repopulating severe combined immunodeficiency mice. Blood 1999; 94: 923–31. 84. Tajima S, Tsuji K, Ebihara Y et al. Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells. J Exp Med 1996; 184: 1357–64. 85. Emmons RV, Doren S, Zujewski J et al. Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo. Blood 1997; 89: 4040–6. 86. Hanania EG, Giles RE, Kavanagh J et al. Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy. Proc Natl Acad Sci U S A 1996; 93: 15346–51. 87. Bhatia R, McGlave PB, Miller JS et al. A clinically suitable ex vivo expansion culture system for LTC-IC and CFC using stroma-conditioned medium. Exp Hematol 1997; 25: 980–91. 88. Roecklein B, Almaida-Porada G, Torok-Storb B et al. Serial xenogeneic transplantation of ex vivo expanded human CD34+ cells. Blood 1997; 90: 1750a. 89. Shih CC, Hu MC, Hu J et al. A secreted and LIFmediated stromal cell-derived activity that promotes ex vivo expansion of human hematopoietic stem cells. Blood 2000; 95: 1957–66. 90. Verfaillie CM, Catanzarro PM, Li WN. Macrophage inflammatory protein 1 alpha, interleukin 3 and diffusible marrow stromal factors maintain human hematopoietic stem cells for at least eight weeks in vitro. J Exp Med 1994; 179: 643–9. 91. Yildirim S, Boehmler AM, Kanz L, Mohle R. Expansion of cord blood CD34+ hematopoietic progenitor cells in coculture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture. Bone Marrow Transplant 2005; 36: 71–9. 92. Lewis ID, Almeida-Porada G, Du J et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 2001; 97: 3441–9. 93. Roecklein BA, Torok-Storb B. Functionally distinct human marrow stromal cell lines immortal-

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of multilineage progenitor cells. Blood 1997; 89: 3624–35. Reya T, Duncan AW, Ailles L et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 2003; 423: 409–14. Kojika S, Griffin JD. Notch receptors and hematopoiesis. Exp Hematol 2001; 29: 1041–52. Milner LA, Kopan R, Martin DI, Bernstein ID. A human homologue of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors. Blood 1994; 83: 2057–62. Ohishi K, Varnum-Finney B, Flowers D et al. Monocytes express high amounts of Notch and undergo cytokine specific apoptosis following interaction with the Notch ligand, Delta-1. Blood 2000; 95: 2847–54. Jones P, May G, Healy L et al. Stromal expression of Jagged 1 promotes colony formation by fetal hematopoietic progenitor cells. Blood 1998; 92: 1505–11. Karanu FN, Murdoch B, Gallacher L et al. The notch ligand jagged-1 represents a novel growth factor of human hematopoietic stem cells. J Exp Med 2000; 192: 1365–72. Karanu FN, Murdoch B, Miyabayashi T et al. Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells. Blood 2001; 97: 1960–7. Li L, Milner LA, Deng Y et al. The human homolog of rat Jagged1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch1. Immunity 1998; 8: 43–55. Varnum-Finney B, Xu L, Brashem-Stein C et al. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 2000; 6: 1278–81. Stier S, Cheng T, Dombkowski D et al. Notch1 activation increases hematopoietic stem cell selfrenewal in vivo and favors lymphoid over myeloid lineage outcome. Blood 2002; 99: 2369–78. Carlesso N, Aster JC, Sklar J, Scadden DT. Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics. Blood 1999; 93: 838– 48. Varnum-Finney B, Wu L, Yu M et al. Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J Cell Sci 2000; 113(Pt 23): 4313–18. Vas V, Szilagyi L, Paloczi K, Uher F. Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay. J Leukoc Biol 2004; 75: 714–20. Varnum-Finney B, Brashem-Stein C, Bernstein ID. Combined effects of Notch signaling and cytokines induce a multiple log increase in precursors with lymphoid and myeloid reconstituting ability. Blood 2003; 101: 1784–9. Delaney C, Varnum-Finney B, Aoyama K et al. Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 2005; 106: 2693–9. Ohishi K, Varnum-Finney B, Bernstein ID. Delta1 enhances marrow and thymus repopulating ability of human CD34(+)CD38(−) cord blood cells. J Clin Invest 2002; 110: 1165–74. Dallas MH, Varnum-Finney B, Delaney C et al. Density of the Notch ligand Delta1 determines

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generation of B and T cell precursors from hematopoietic stem cells. J Exp Med 2005; 201: 1361– 6. Kertesz Z, Vas V, Kiss J et al. In vitro expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines. Cell Biol Int 2006; 30: 401–5. Alcorn MJ, Holyoake TL, Richmond L et al. CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity. J Clin Oncol 1996; 14: 1839–47. Reiffers J, Cailliot C, Dazey B et al. Abrogation of post-myeloablative chemotherapy neutropenia by ex-vivo expanded autologous CD34-positive cells. Lancet 1999; 354: 1092–3. Bertolini F, Battaglia M, Pedrazzoli P et al. Megakaryocytic progenitors can be generated ex vivo and safely administered to autologous peripheral blood progenitor cell transplant recipients. Blood 1997; 89: 2679–88. Engelhardt M, Douville J, Behringer D et al. Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myeloablative chemotherapy. Bone Marrow Transplant 2001; 27: 249–59. Holyoake TL, Alcorn MJ, Richmond L et al. CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens. Bone Marrow Transplant 1997; 19: 1095–101. McNiece I, Jones R, Bearman SI et al. Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer. Blood 2000; 96: 3001–7. Paquette RL, Dergham ST, Karpf E et al. Culture conditions affect the ability of ex vivo expanded peripheral blood progenitor cells to accelerate hematopoietic recovery. Exp Hematol 2002; 30: 374–80. Pecora AL, Stiff P, LeMaistre CF et al. A phase II trial evaluating the safety and effectiveness of the AastromReplicell system for augmentation of low-dose blood stem cell transplantation. Bone Marrow Transplant 2001; 28: 295–303. Stiff P, Chen B, Franklin W et al. Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. Blood 2000; 95: 2169–74. Williams SF, Lee WJ, Bender JG et al. Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer. Blood 1996; 87: 1687–91. Glaspy JA, Shpall EJ, LeMaistre CF et al. Peripheral blood progenitor cell mobilization using stem cell factor in combination with filgrastim in breast cancer patients. Blood 1997; 90: 2939–51. Gebbie K, Aubry W, Champlin RE et al. Cord Blood: Establishing a National Hematopoietic Stem Cell Bank Program. Washington, DC: National Academies Press; 2005. Barker JN. Who should get cord blood transplants? Biol Blood Marrow Transplant 2007; 13(Suppl 1): 78–82. Brunstein CG, Wagner JE. Umbilical cord blood transplantation and banking. Annu Rev Med 2006; 57: 403–17.

143. Gluckman E. Current status of umbilical cord blood hematopoietic stem cell transplantation. Exp Hematol 2000; 28: 1197–205. 144. Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997; 337: 373–81. 145. Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335: 157–66. 146. Laughlin MJ, Barker J, Bambach B et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001; 344: 1815–22. 147. Rocha V, Gluckman E. Clinical use of umbilical cord blood hematopoietic stem cells. Biol Blood Marrow Transplant 2006; 12(1 Suppl 1): 34– 41. 148. Rubinstein P, Carrier C, Scaradavou A et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339: 1565–77. 149. Wagner JE, Rosenthal J, Sweetman R et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996; 88: 795–802. 150. Wagner JE, Barker JN, DeFor TE et al. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002; 100: 1611–18. 151. Barker JN, Weisdorf DJ, DeFor TE et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005; 105: 1343–7. 152. Broxmeyer HE, Hangoc G, Cooper S et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 1992; 89: 4109–13. 153. Lu L, Xiao M, Shen RN et al. Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential. Blood 1993; 81: 41– 8. 154. Mayani H, Dragowska W, Lansdorp PM. Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free longterm cultures supplemented with hematopoietic cytokines. Blood 1993; 82: 2664–72. 155. Hao QL, Thiemann FT, Petersen D et al. Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population. Blood 1996; 88: 3306–13. 156. Hows JM, Bradley BA, Marsh JC et al. Growth of human umbilical-cord blood in longterm haemopoietic cultures. Lancet 1992; 340: 73–6. 157. Mayani H, Lansdorp PM. Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood. Blood 1994; 83: 2410–17. 158. Steen R, Tjonnfjord GE, Egeland T. Comparison of the phenotype and clonogenicity of normal

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bone marrow, mobilized peripheral blood, and fetal liver. Blood 2001; 98: 2108–15. 162. Jaroscak J, Goltry K, Smith A et al. Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells: results of a phase 1 trial using the AastromReplicell System. Blood 2003; 101: 5061–7. 163. Kögler G, Nurnberger W, Fischer J et al. Simultaneous cord blood transplantation of ex vivo expanded together with non-expanded cells for high risk leukemia. Bone Marrow Transplant 1999; 24: 397–403. 164. Pecora AL, Stiff P, Jennis A et al. Prompt and durable engraftment in two older adult patients with high risk chronic myelogenous leukemia (CML) using ex vivo expanded and unmanipulated unrelated umbilical cord blood. Bone Marrow Transplant 2000; 25: 797–9.

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9

Edwin M. Horwitz

Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation

Introduction Hematopoietic cell transplantation (HCT) is well established as therapy for hematologic malignancies as well as many nonmalignant disorders. Over the last several years, the spectrum of diseases that may be treated with HCT has dramatically expanded, increasing the importance of this therapeutic modality and extending HCT beyond the traditional bounds of hematology and oncology. HCT is founded on the principle that hematopoietic stem cells (HSCs) can be intravenously infused, will home to and engraft in the stem cell niche within the bone marrow microenvironment, and will then proliferate and differentiate to repopulate all lineages of the blood. The cells that comprise the microenvironment are thought to support, and possibly regulate, hematopoiesis. However, one cellular constituent of the microenvironment, the marrow mesenchymal stromal cells (MSCs), may have quite complex biologic functions that can be exploited in the development of primary or adjunct cell therapy with HCT or in regenerative medicine. MSCs are spindle-shaped, plastic-adherent cells isolated from bone marrow as well as other tissue sources [1]. First recognized nearly 40 years ago [2], MSCs have emerged as one of the most rapidly evolving areas of research in HCT. The trilineage differentiation of MSCs, capable of giving rise to osteoblasts, chondrocytes, and adipocytes in vitro, led investigators to hypothesize a vast differentiation potential and focus research on the regenerative capacity of MSCs in an effort to repair diseased or damaged tissue [3]. More recently, MSCs have been recognized to possess striking immunomodulatory properties, and it is this characteristic of MSCs that has generated the greatest interest among physicians and investigators in the field of HCT [4,5]. Indeed, MSCs produce an extensive array of immunomodulatory cytokines as well as a variety of other, known and likely undiscovered, biochemical mediators. The secretion of these biologic response modifiers may well prove to be the most biologically and clinically important property of MSCs. While MSC research continues along many avenues, this chapter will focus on the biology and early clinical applications of MSCs in close relation to HCT.

“MSC” [2]. He showed that these cells were precursors for osteoblasts [2] and comprised the hematopoietic microenvironment [6], and that a fraction of the population, cells he termed “CFU-F”, possessed a high proliferative potential demonstrated by forming clonal colonies when plated at low density [7]. Friedenstein went on to demonstrate the extensive expansion potential, showing that single cells could regenerate an entire population of cells, suggesting that individual cells were precursors for fibroblasts in vivo [8,9]. However, Friedenstein did not suggest that the marrow stromal fibroblasts that he identified were stem cells. The first formal presentation of the concept that a nonhematopoietic stem cell might reside within the bone marrow microenvironment was by Maureen Owen [10]. She proposed that the marrow stroma consisted of a lineage of cells analogous to the hematopoietic system. She then expanded this theory and proposed a stromal stem cell, with fibroblastic, osteogenic, and adipocytic cells being the terminally differentiated phenotypes [11,12]. The term “mesenchymal stem cell” was proposed by Arnold Caplan, who hypothesized that this cell could differentiate to a wide variety of mesenchymal tissues and proposed a broad lineage “tree” suggesting that the mesenchymal stem cell could give rise to bone, cartilage, muscle, hematopoietic-supportive stroma, tendon, ligament, adipose, and other connective tissues [13]. Data demonstrating the identification of MSCs by reactivity with the monoclonal antibodies SH2 and SH3, the considerable self-renewal capacity, and the multilineage differentiation potential in vitro supported the notion that these cells were a homogeneous population of stem cells [14]. However, more recent data demonstrated that the adherent cells isolated from marrow are a heterogeneous population of cells [15,16], although a nonhematopoietic stem cell may exist within this cell population [17]. Many other investigators, too numerous to recount here, have made important contributions to the scientific development of MSCs. At present, MSCs are generally accepted to be a heterogeneous population of cells with diverse biologic properties suggesting a remarkable potential as cell therapy. While the expansion and differentiation potential of MSCs has yet to make a substantial impact in regenerative medicine, the cytokine-generating and immunomodulatory properties promise to contribute to HCT.

Brief history of MSCs The notion that a mesenchymal progenitor cell with osteogenic differentiation potential resided within bone marrow was considered in the latter 19th century. However, it was the pioneering work of Alexander Friedenstein that first identified the cells that now bear the designation Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Nomenclature The nomenclature of these cells merits clarification. MSCs have been designed by a wide variety of terms including “marrow stromal cells,” “marrow stromal fibroblasts,” and most often “mesenchymal stem cells,” which was the basis for the abbreviation MSC. While the specific properties of MSCs studied in the laboratory may vary depending on the methods of isolation and culture expansion, the assortment of terms, in general, refer to the same heterogeneous population of cells.

Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation

The term “stem cell,” however, implies specific biologic properties. Our general concept of a stem cell evolved from our understanding of the HSC. Till and McCulloch suggested that the stem cell could be defined as a cell with extensive self-renewal capacity and the potential to terminally differentiate into two or more lineages [18]. While MSCs can, indeed, undergo extensive self-renewal and trilineage differentiation to osteoblasts, chondrocytes, and adipocytes in vitro, true “stemness” is certainly much more complex and may be best defined operationally. As this idea has become increasingly recognized, many investigators have suggested that convincing data are lacking to support the notion that the unfractionated population of plastic-adherent cells from bone marrow designated as MSCs are properly considered as stem cells [19]. These thoughts are not intended to dispel the notion of a mesenchymal stem cell. Such a stem cell most assuredly exists and may, indeed, reside within the adherent population of cells that we designate MSCs or some other cell population. Notably, the antibody STRO-1, which identifies CFU-Fs from freshly harvested human bone marrow, was generated from CD34+ nonadherent cells [20]. Recently, Anjos-Afonso et al. showed that a subset of murine MSCs that expressed stage-specific embryonic antigen-1 (SSEA-1) was capable of single-cell expansion followed by multilineage differentiation in vivo, suggesting that SSEA1+ MSCs may be primitive stem/progenitor cells [17]. These data notwithstanding, the entire unfractionated population of MSCs isolated by plastic adherence does not seem to meet the criteria of a homogeneous population of stem cells. Therefore, in 2005, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy recommended that the term “mesenchymal stromal cell” was a more appropriate designation for this heterogeneous population of cells, maintaining the abbreviation MSC while reserving the term “mesenchymal stem cell” for a subset of these (or other) cells that demonstrate stem cell activity in vivo by clearly stated criteria [1].

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Table 9.1 Summary of criteria to identify mesenchymal stromal cells 1. Adherence to plastic in standard culture conditions 2. Phenotype Positive (≥95% +) CD105 CD73 CD90

3. Differentiation: (by staining of in vitro cell culture)

Negative (<2% +) CD45 CD34 CD14 or CD11b CD79α or CD19 HLA-DR

Bone, Adipose, Cartilage

HLA, human leukocyte antigen. Reproduced with permission from [21].

outcomes likely reflect, in part, acquired properties that may not exist under other culture conditions. Thus, widely discrepant results are often reported. However, our overall knowledge of MSCs is rapidly advancing, and consensus among investigators in the field is being achieved. Morphology MSCs are spindle-shaped, plastic-adherent cells in vitro. Once cells are established in culture, the cells may assume a very narrow elongated morphology, a somewhat wider dimension, or even a very broad, “fried egg” appearance. The more slender cells tend to be the more rapidly dividing cells, while the very broad cells divide slowly if at all. While cells that meet the criteria of MSCs have been expanded in suspension culture [23], plastic adherence remains one of the fundamental characteristics defining the cells.

Working definition of MSCs MSCs are a heterogeneous population of cells that defy simple definition because, presently, no specific marker or combination of markers is accepted to uniquely define the cells. Moreover, we lack a single functional assay, analogous to the repopulation assay for HSCs, that distinguishes MSCs. Investigators report studies of MSCs that have been isolated and expanded by different methods and the preparation of the cells seems to affect the nature of the cells [16]. Moreover, investigators use different approaches to characterize the cells. Thus, it is becoming increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy sought, in 2006, to standardize a current working definition of MSCs by proposing a set of minimal criteria to denote MSCs [21]. First, MSCs must be plastic adherent when maintained in standard culture conditions. Second, MSCs must express CD105, CD73, and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79 or CD19, and human leukocyte antigen (HLA)-DR surface molecules. Third, MSCs must differentiate to osteoblasts, adipocytes, and chondroblasts in vitro (Plate 9.1 and Table 9.1). These criteria will certainly require modification as new knowledge unfolds. Indeed, several novel surface markers of MSCs have been described in the 2 years since these criteria were published [22].

Biology Our understanding of the cell biology of MSCs is largely derived from in vitro studies and defined animal models of MSC therapies. Since many of the observed biologic properties of MSCs are greatly influenced by the conditions of isolation and ex vivo expansion [16], the measured

Isolation MSCs are most often isolated by “adherence selection.” For bone marrow, mononuclear cells are placed in tissue culture, and the MSCs will adhere to the plastic surface of the tissue culture vessel. Non-adherent cells are easily removed by changing the tissue culture media. Similar protocols are used when MSCs are isolated from other tissue sources. Tissue sources MSCs are most commonly isolated from bone marrow [14], but have also been obtained from adipose tissue [24,25], placenta [26], amniotic fluid [27], umbilical cord blood [28,29], and fetal tissues such as lung and blood [30]. Mobilized peripheral blood cells have been reported as a source of MSCs [31]; however, current consensus is that MSCs do not circulate in cytokine-mobilized or steady-state peripheral blood. Reports of MSCs in umbilical cord blood have also been conflicting, but we now recognize that cord blood unit storage time, volume, and cell count are critical parameters affecting the efficiency of MSC isolation [29], and there is general agreement that MSCs do reside within cord blood. Heterogeneity Despite the rather uniform morphology and cell surface marker expression, gene expression studies have clearly demonstrated that populations of MSCs are highly heterogeneous [16,32]. Moreover, human MSCs show substantial variation among different cell preparations from the same tissue source using the same laboratory protocols [16]. While MSCs were regarded to be the same general cell type regardless of the tissue source, recent data suggest that MSC gene expression reflects the

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tissue from where the cells were isolated, indicating biologically relevant heterogeneity among MSCs in different tissues [15,33]. These data suggest that different tissue sources may generate MSC products especially suited for different clinical applications. Surface phenotype MSCs were originally defined by their reactivity with the monoclonal antibodies SH2 and SH3 [14]. Subsequently, SH2 was shown to recognize an epitope on CD105 [34], and SH3 an epitope on CD73 [35]. MSCs also express CD90 (Thy1) [36]. These three antigens, expressed on all MSCs, comprise the surface marker panel of the proposed defining criteria. Additionally, MSCs uniformly express CD49b, CD49e, CD54, and CD166, and subsets of MSCs express CD50, CD62L, and CD106. Ex vivo expanded MSCs lack expression of hematopoietic and endothelial antigens, CD34, CD45, CD3, CD19, CD14, CD11b, and CD31. MSCs also lack expression of the co-stimulatory molecules CD80, CD86, CD40, and CD40L [37,38]. MSCs express moderate levels of HLA class I molecules but lack expression of HLA class II molecules on the cell surface. Upon stimulation with interferon-gamma (IFN-γ), class II antigens are expressed on the cell surface [38]. Interestingly, substantial class II molecules are constitutively expressed in the cytoplasm of MSCs. IFN-γ stimulation does not increase overall expression but induces detectable surface expression [39]. STRO-1, an IgM monoclonal antibody generated by inoculating mice with human CD34+ cells, binds to human MSCs (or perhaps MSC precursors) in freshly isolated bone marrow, but does not bind to murine MSCs or human MSCs in tissue culture [20]. The antigen recognized by STRO-1 has not been identified. The low-affinity nerve growth factor receptor, or CD271, is also expressed on human MSCs in freshly harvested bone marrow but not culture-expanded cells [40]. Second-passage expanded MSCs homogeneously express a unique set of chemokine receptors including CCR1, CCR7, CXCR4, CXCR5, and the surface-bound chemokine CX3CL1. Approximately 40–60% of early-passage cells express CCR9 and CXCR6. Expression of all of these chemokine receptors is markedly downregulated in late-passage MSCs [41]. CXCR4 has been reported to be expressed at moderate levels on the cell surface of all cells [41]. In contrast, CXCR4 has also been reported to be expressed on the surface of a very small subset (<1%) of MSCs, but abundantly expressed in the cytoplasm of most (>80%) of the cells [42]. Stimuli to upregulate surface expression of CXCR4 have not been reported. These contradictory reports may result from differing culture conditions, which could provide insight into the regulation of CXCR4 expression by MSCs and biologic significance of CXCR4 expression on the cells. If CXCR4 is expressed on most cells, it might represent an autocrine feedback loop, since stromal cell-derived factor (SDF-1), the ligand for CXCR4, is highly expressed by MSCs. If CXCR4 is expressed on the surface by a minority of cells, it may represent a marker of a unique subpopulation of MSCs. There are several newly identified surface markers expressed on MSCs. The neuronal ganglioside GD2 is expressed on MSCs in freshly harvested bone marrow, and expression is stable during ex vivo expansion [43]. SSEA-4 is expressed on both human and murine MSCs [44]. Additionally, frizzled-9 (CD349), platelet-derived growth factor-β (CD140b), and HER-2/erbB2 (CD340) have all been recently identified as potentially important MSC markers [22,44]. Prospective isolation of MSCs The identification of new surface markers has led to the prospective isolation of MSCs using immunomagnetic beads or fluorescence-

activated cell sorting of freshly prepared mononuclear cell preparations. GD2, CD271, and frizzled-9 have been shown to effectively serve as markers for the isolation of MSCs [22,40,43,45]; however, in all cases, culture expansion of the selected cells was required to obtain a sufficient sample for further study. If prospective isolation of MSCs is to become a common practice, a substantial advantage must be demonstrated. Indeed, both GD2-selected (Horwitz, unpublished data) and CD271selected [40] MSCs seem to have a greater proliferative capacity and more robust differentiation potential compared with MSCs isolated by adherence selection. Expansion potential MSCs have a remarkable capacity for ex vivo expansion. In standard culture conditions, MSCs isolated by adherence selection demonstrate a population doubling time of about 2–4 days [46]. Early-passage cells typically divide more rapidly than later-passage cells. Additionally, plating cell density can impact the expansion potential [47]. Most MSC preparations can be maintained in continuous culture for 4–6 months before senescence, but there is considerable variability. Importantly, extensive expansion may adversely affect the differentiation potential and cytokine secretory repertoire of MSCs and, as discussed below, may increase the risk of spontaneous malignant transformation in vitro. MSCs isolated from different species exhibit differing growth potentials. While human MSCs rapidly expand in culture, murine MSCs expand very slowly. Moreover, different mouse strains show a marked difference in growth and differentiation [48]. All MSC cultures, at present, require some serum supplementation. The development of serum-free media is an active area of MSC research but has not yet been achieved. Fetal bovine serum is the most commonly used supplement, but the translation of MSCs to the clinics and the report of a clinically significant immune response against fetal bovine serum proteins in one patient [49] has led to the development of human serum supplementation strategies. Human serum containing a platelet lysate [50] or intact platelets [51] has been shown to effectively support the ex vivo expansion of human MSCs. In vitro differentiation potential MSCs readily differentiate to osteoblasts, chondrocytes, and adipocytes in vitro. This trilineage differentiation is one of the most defining characteristics of MSCs that distinguish these cells from other fibroblastoidappearing cells. MSCs are most readily differentiated to osteoblasts and adipocytes in confluent monolayer tissue cultures. For osteoblast differentiation, the cultures are supplemented with β-glycerophosphate, L-ascorbic acid 2-phosphate and dexamethasone. This cocktail induces the differentiation of MSCs to osteoblasts and the secretion of an extracellular matrix upon which minerals, primarily calcium, from the media are deposited. Then, the cultures can be assessed with Alizarin red S or von Kossa stains, which color the mineral nodules. For adipocyte differentiation, standard culture medium is supplemented with dexamethasone, indomethacin, insulin, and 3-isobutyl-1-methylxanthine. The differentiated adipocytes contain fat globules that are stained with oil red O, coloring the fat globule. Differentiation to chondrocytes is more challenging. Suspensions of MSCs must be spun in a centrifuge and maintained in culture as a pellet of cells. The standard culture medium is supplemented with bone morphogenetic protein-6, transforming growth factor-beta 3, ITS+ (insulin, transferring, selenium), dexamethasone, L-ascorbic acid 2phosphate, sodium pyruvate, and proline. Then, the cell pellet is fixed and embedded in paraffin, and sections are placed onto slides for

Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation

staining with Alcian blue or toluidine blue, which colors the secreted proteoglycan matrix. Alternatively, successful differentiation of MSCs can be demonstrated by gene expression. Frequently assayed genes to prove terminal differentiation include core binding factor 1 or osteocalcin for bone, peroxisome proliferator-activated receptor-gamma 2 or lipoprotein lipase for fat, and collagen II or aggrecan for cartilage. Some reports have suggested that MSCs can also differentiate to other tissues such as neurons [52,53], skeletal muscle [54], and hepatocytes [55]. However, most of these studies have been performed in nonphysiologic settings, and contradictory data have also been reported [56]. The capacity of MSCs to robustly differentiate to tissues other than bone, cartilage, and fat, whether physiologic or experimentally inducible, remains to be established. Cytokine secretion MSCs have an enormous capacity for secretion of soluble mediators. MSCs secrete SDF-1 [57], which plays a critical role in the homing of HSCs to the marrow niche [58]. In vitro, MSCs constitutively secrete interleukin-6 (IL-6), IL-7, IL-8, IL-11, IL-12, IL-14, IL-15, macrophage colony-simulating factor, Flt-3 ligand, and stem cell factor. Upon IL-1α stimulation, MSCs are induced to further express IL-1α, leukemia inhibitory factor, granulocyte colony-stimulating factor, and granulocyte–macrophage colony-stimulating factor [38,59]. Finally, MSCs can secrete several chemokine ligands, including CCL2, CCL4, CCL5, CCL20, CX3CL1, and CXCL8 [41]. MSCs are also thought to secrete biochemical mediators unrelated to the lymphohematopoietic system. For example, subsets of MSCs have been shown to secrete brain-derived neurotropic factor and nerve growth factor [60]. The search for other, and novel, mediators of biologic processes generated by MSCs is another area of active investigation. Homing and migration Homing refers to a specific biologic process in which cells localize to a defined tissue and is typically mediated by chemotactic factors or ligand–receptor interactions. Although the literature is controversial, MSCs do not seem to home to normal, healthy tissue. However, MSCs may randomly lodge in various tissues after intravenous infusion and proliferate in some tissue sites but not others, generating the appearance of specific homing. MSCs do seem to migrate to sites of tissue injury such as the kidney [61], heart [62], and skin [63], presumably due to the local production of inflammatory mediators. The cells also seem to home to multiple tissues after experimental radiation injury [64,65]. Migration to tumors Ex vivo expanded MSCs have been reported to migrate to solid tumors after intravenous or intra-arterial infusion in experimental animals [66]. Initially, MSCs seemed to localize in the tumor, but the issue of homing was unresolved. Recent evidence suggests that MSCs genuinely home to systemic and central nervous system tumors after infusion into the circulation [67,68]. Moreover, low-dose (2 Gy) radiation can increase the recruitment of circulating MSCs into the tumor microenvironment [67]. This homing capacity can be exploited therapeutically by genetically engineering MSCs to secrete antitumor molecules. For example, IFN-β, which can induce tumor regression through indirect immunomodulation or direct antiproliferative effects, has been expressed in MSCs as a novel biotherapy in models of breast carcinoma, melanoma, and glioma [66,68,69]. The success of these animal models has led to the several pilot clinical trials to determine the clinical feasibility of this strategy.

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Lifespan of MSCs The turnover rate of bone marrow MSCs in situ is unknown. More relevant to clinical applications of MSCs is the lifespan of ex vivoexpanded MSCs after intravenous infusion. While a definitive answer is not yet available, and may be difficult to conclusively prove, three lines of evidence suggest that MSCs persist about 6–9 months in the recipient. First, Nolta et al. infused human IL-3-secreting-MSCs into NODSCID mice to support human hematopoiesis and were able to detect human IL-3 in the murine serum for up to 9 months [70]. Second, Horwitz et al. infused human MSCs into children with the bone disorder osteogenesis imperfecta and observed a striking acceleration of growth velocity over the first 6 months, with a slower rate of growth, although greater than controls, over the subsequent 6 months [49]. After 1 year, the growth rate approximated that prior to the MSC infusion. Finally, Keating et al. infused autologous MSCs gene-marked with factor IX complementary DNA into three adult patients after autologous HCT for a hematologic malignancy. Gene-marked cells were identified in recipient culture-expanded MSCs obtained from bone marrow aspirations up to 8 months after the MSC infusion. Studies at 12 months were consistently negative (Keating, personal communication).

Malignant transformation of MSCs MSCs are invariably expanded in vitro prior to scientific study in the laboratory or in preparation for a clinical trial. This extensive cell replication raises the possibility of spontaneous transformation to a malignant phenotype. In principle, the likelihood of a transformation event increases proportionally with the number of population doublings. Murine MSCs seem to more readily undergo malignant transformation than human MSCs [71], possibly due to their significantly greater susceptibility to chromosomal aberrations and spontaneous mutations with prolonged culture [72]. However, transformation events may occur after a relatively short interval under some in vitro culture conditions [73]. The risk of malignant transformation of human MSCs is of far greater importance as the cells are increasingly being studied in a wide variety of clinical venues. Human adipose tissue-derived MSCs have recently been reported to undergo spontaneous transformation after 4–5 months in culture, unequivocally demonstrated by the formation of tumors after infusion into NOD-SCID mice [74]. However, transformation was never observed among MSCs maintained in culture for up to 4 months, which is a greater duration than that typically employed in MSC clinical trials. Another report described a small population of bone marrow-derived MSCs undergoing transformation at the third passage of ex vivo expansion, a time interval that may be used in clinical trials [75]. Again, the malignant phenotype was confirmed by the formation of tumors in NOD-SCID mice. In both cases, the MSCs demonstrated markedly abnormal karyotypes. It is noteworthy that both reports identified the transformation event during the ex vivo expansion; nontransformed human MSCs have never been reported to initiate a malignancy after infusion into NOD-SCID mice. Malignant transformation of MSCs after infusion into human subjects in a clinical trial has never been reported. This may be because it is an exceedingly rare event and there have not yet been sufficient numbers of patients for such an event to occur. Alternatively, the latency for such an event could be quite long. Since MSCs do not seem to survive in the patients for more than 1 year, the likelihood of malignant transformation of ex vivo-processed, transplanted MSCs in patients may be significantly less than envisioned according to the experience with HCT [76] or gene therapy [77], or predicted from in vitro studies [74,75].

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Ectopic tissue formation after systemic infusion MSCs readily differentiate to osteoblasts, chondrocytes, and adipocytes in vitro. Additionally, consistent cardiomyocyte differentiation has recently been reported [78]. This broad differentiation capacity is the basis for investigating MSCs for tissue repair in regenerative medicine; however, it also raises the possibility that MSCs may inappropriately differentiate to a mesenchymal tissue, such as bone, after intravenous infusion. Such ectopic tissue formation is thought to be unlikely as the local environmental cues seem to regulate the differentiation of MSCs. In countless experimental systems, ectopic tissue formation had never been reported. Horwitz et al. infused allogeneic MSCs into children with osteogenesis imperfecta and documented MSC engraftment in the bone, but did not observe ectopic bone formation in the patients. Similarly, ectopic bone formation has not been reported in any clinical trial. A recent single report of murine MSCs injected into experimentally infarcted myocardium demonstrated calcifications at the site of the cell injection [79]. Thus, the potential for ectopic tissue formation seems to exist, but the incidence and conditions that may favor or retard such inappropriate differentiation of MSCs have not been identified.

Immunobiology While the specific interactions of MSCs with the immune system and the mechanisms of those interactions remain to be fully elucidated, it is widely accepted that MSCs have a profound impact on immunity [4,5]. While the field is rapidly advancing, several overriding themes emerge. First, all arms of the immune system seem to interact with MSCs (Fig. 9.1). Second, there seems to be a dynamic cross-talk between the immune effector cells and MSCs, suggesting that MSCs may play a physiologic role in regulating the immune response. Third, and unfortunately, the data are often conflicting and many reports are diametrically opposed to the research of others indicating the need for additional studies to fully understand the MSC–immune system interaction. The lack of unequivocal cellular and molecular mechanisms notwithstanding, the immunomodulatory properties of MSCs may be exploited therapeutically to suppress graft rejection, treat severe graft-versus-host disease (GVHD), and possibly prevent clinical GVHD. Indeed, the rec-

B cells

+

↓ proliferation

T lymphocytes The first studies to suggest that MSCs may possess inherent immunomodulatory properties were based on the recognition that ex vivoexpanded third-party human MSCs inhibit the proliferation of allogeneic lymphocytes in a mixed lymphocyte reaction (Fig. 9.2) [80]. Subsequent to the initial report, several investigators have confirmed the principal observation [37,81–84]. The effect seems to be nonspecific as MSCs inhibit the proliferation of T cells in response to alloantigens [37,81,85,86] and mitogens [80], and is also major histocompatibility complex (MHC) unrestricted as both allogeneic and autologous MSCs generate a similar suppression [37,81,83,85,87]. MSCs have been reported to inhibit the cytotoxicity of T lymphocytes [85], although other data suggest that MSCs only inhibit the proliferation and not the cytolytic activity [88]. The underlying mechanism for this antiproliferative effect has not been defined. In vitro, it seems to be MSC dose-dependent (Fig. 9.3) [80–82] and requires a prolonged exposure to elicit the full effect [81]. Soluble mediators are implicated since experiments in which lymphocytes and MSCs are separated by a semipermeable membrane show a similar antiproliferative effect [37,80,88]. Conditioned media from MSC cultures do not replicate the antiproliferative response [85,89], but conditioned media from cultures of MSCs and lymphocytes do seem to induce suppression [86]. Thus, the presence of MSCs seems to be required to generate the soluble mediators, albeit not direct cell-to-cell contact, suggesting a paracrine signaling between the T cells and the MSCs. The soluble molecules proposed to mediate the antiproliferative effect include transforming growth factor-β [80,89], hepatocyte growth factor [80], prostaglandin E2 [37,90], indoleamine 2,3-dioxygenase [91], and HLA-G [92]; however, the data are inconsistent [37,80,89,90,93]. Conceivably, different mediators predominate under differing in vitro assay conditions, leading investigators to identify different molecular factors. The antiproliferative effect of MSCs has been reported to resemble the divisional arrest anergy of activated T cells based on evidence that

↓ proliferation ↓ differentiation to plasma cells ↓ proliferation

O PGE2

↓ cytotoxicity + ↓ IFN-γ

ognition that MSCs can suppress the immune response is what attracted the collective attention of the HCT community.

+

↓ CTL formation ↓ IFN-γ, ↑ IL-4

IFN-g

IFN-g

≠ IDO

≠ IDO

↑ IL-10

NK cells

MSC O HGF O PGE2 O TGF-b O M-CSF O PGE2 O IL-6 O IL-10 O TGF-b ↓ maturation ↓ differentiation

T cells Treg ↓ activation

O PGE2 O TGF-b

↑?

↓ activation

Monocytes

iDCs

mDCs

Fig. 9.1 A schematic representation of the proposed immunomodulatory effects of mesenchymal stromal cells (MSCs). CTL, cytotoxic T cell; HGF, hepatocyte growth factor; IDO, indoleamine 2,3-dioxygenase; IFN-γ, interferon-gamma; IL, interleukin; M-CSF, macrophage colony-stimulating factor; NK, natural killer; PGE2, prostaglandin E2; TGF-β, transforming growth factor-beta; Treg, regulatory T cell. (Reproduced from [4], with permission.)

Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation

Fig. 9.2 Mesenchymal stromal cell (MSC)-induced suppression of T-cell proliferation in response to allogeneic peripheral blood lymphocytes (PBLs), dendritic cells (DCs) or phytohemagglutinin (PHA) in a mixed lymphocyte reaction. Seven-day mixed cultures were performed with (black bars) or without (white bars) MSCs. Data are expressed as mean ± standard deviation (SD) of triplicates of six separate experiments. *Statistically significant (minimum p = 0.004). (Reproduced from [80], with permission.)

Fig. 9.3 The dose dependence of mesenchymal stromal cell (MSC) effects on T-cell proliferation in a mixed lymphocyte reaction. Irradiated third-party MSCs were added to the reaction on day 0 in the designated doses per well. The numbers of stimulating and responding peripheral blood lymphocytes were kept constant in all experiments. (Reproduced from [81], with permission.)

MSCs induce a cell-cycle arrest in the G1 phase that persists after removal of the MSCs from the in vitro culture [94]. Indeed, MSCs lack expression of co-stimulatory molecules. However, MSCs seem to be able to present antigens and stimulate an immune response as well [95], again indicating that the MSC–T-cell interaction may vary depending on the conditions of the assay. B lymphocytes MSCs also interact with B cells, although the effects of MSCs are unclear. Less attention has been directed toward B-cell interactions, and the two

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principal studies to date are somewhat contradictory. Corcione et al. reported that MSCs inhibit the proliferation of B cells isolated from peripheral blood when stimulated with anti-immunoglobulin (Ig) antibodies, soluble CD40L, and cytokines [96]. This antiproliferative effect seems to be due to an arrest in the G0/G1 phase of the cell cycle and is likely mediated by soluble factors. Moreover, B-cell differentiation was impaired as IgM, IgG, and IgA production were all reduced, and chemotaxis to a variety of chemokines was significantly downregulated. In contrast to these findings, Rasmusson et al. have reported that MSCs stimulate IgG production and induce low-level proliferation of mononuclear cells or enriched B cells obtained from the spleen or blood [97]. Interestingly, studies using a semipermeable membrane showed a similar effect for the mononuclear cells but not the enriched B cells, indicating soluble factors are sufficient to induce IgG production when B cells are a component of a population, but direct MSC B-cell contact is required when B cells are enriched. This observation suggests that a cell distinct from B cells but within the mononuclear cell population may contribute to the B-cell response. Natural killer cells MSCs seem to inhibit some natural killer (NK) cell activity in vitro. MSCs have been reported to inhibit IL-2- and IL-15-induced proliferation of freshly isolated, resting NK cells and prevent the induction of cytotoxic activity and cytokine production; however, proliferation of NK cells preactivated with IL-2 was only partially inhibited [98,99]. Other studies have shown that the cytolytic activity of freshly isolated NK cells is unaffected by MSCs, whereas NK cells cultured with IL-2 and MSCs seem to have reduced, but not absent, cytotoxicity against K562 target cells [88,100,101]. Interestingly, MSCs seem to be highly susceptible to NK-cellmediated lysis due to the expression of surface ligands involved in NK-cell activation, such as NKG2D and DNAX accessory molecule-1 ligands [98]. However, ligand expression varied considerably among different MSC preparations, which translated to widely discrepant results of NK cytotoxicity assays. Moreover, MSCs exposed to IFN-γ, which upregulates MHC class I expression on MSCs, led to reduced susceptibility to measured NK cytotoxicity. Similar to the other immune cell systems, the underlying mechanism of the NK–MSC interaction has not been determined and may be more complex as the response of NK cells to various target cells may be differentially affected by MSCs. Semipermeable membrane experiments suggest that the MSC-associated suppression of IL-15-induced NK proliferation and cytokine production is mediated by soluble factors including transforming growth factor-beta, indoleamine 2,3-dioxygenase, and prostaglandin E2 [99, 101]. In contrast, the MSC-associated inhibition of NK cytotoxicity seems to require cell-to-cell contact [101]. Dendritic cells MSCs can inhibit the differentiation of CD14+ monocytes and CD34+ cells into dendritic cells (DCs), as well as the maturation and function of DCs in vitro [102–104]. MSC co-culture inhibited the granulocyte– macrophage colony-stimulating factor-, IL-4-induced differentiation of monocytes to DCs, but the inhibition was reversible. MSC–DC coculture induced mature DCs to downregulate surface expression of CD83, CD1a, CD80, CD86, and HLA-DR as well as IL-12 secretion. Consistent with this antigen expression pattern, MSC-treated DCs showed an impaired capacity for allostimulation of allogeneic T cells. While these studies were performed in vitro, three independent reports yielded consistent findings supporting the concept that MSCs suppress DC activity.

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In vivo animal models In vitro studies are ideally suited to dissect cellular and molecular mechanisms, but are fraught with the potential for errant conclusions since many complex physiologic interactions may be absent from the in vitro system. In vivo studies are necessary to demonstrate legitimate immunosuppression. The first study to demonstrate an immunomodulatory effect of MSCs in vivo was prolongation of skin allograft survival in a baboon model [82]. Approximately 20 × 106 allogeneic bone marrow-derived MSCs per kilogram were intravenously infused immediately after three skin grafts – autologous, MSC donor-derived, and third party – were surgically placed on the dorsum of recipient baboons. The skin allograft survival was prolonged in the animals who received the MSCs compared with control animals (11.3 versus 7.0 days). These investigators did not report whether MSCs migrated, or homed, to the site of the allograft. Thus, we cannot determine whether the MSCs exerted a local effect at the allograft site or a systemic immunosuppressive effect. However, the effect does not appear to be MHC restricted as the third-party allografts showed survival equal to that of the allografts derived from the baboon MSC donor. A second important animal model of systemic MSC-induced immunosuppression is the capacity of an intravenous MSC infusion to ameliorate experimental autoimmune encephalitis, a model of multiple sclerosis [105]. Importantly, histologic examination revealed a decrease of inflammatory infiltrates in the central nervous system, but the donor MSCs were found in the lymphoid organs of the treated mice. These data suggest that MSCs induced a state of T-cell anergy by acting at secondary lymphoid organs. The most compelling evidence to support the notion that ex vivoexpanded, intravenously infused MSCs can potentially modulate the immune response is the capacity to ameliorate the symptoms of clinical GVHD described below.

Immune privilege MSCs are considered by many investigators to be immune privileged [5]. Ex vivo-expanded MSCs express low levels of MHC class I molecules, and class II molecules are not expressed on the cell surface unless stimulated with IFN-γ [39]. MSCs also lack expression of Fas ligand or co-stimulatory molecules such as CD80, CD86 or CD40 [37]. Several laboratories have independently demonstrated that MSCs do not elicit a T-cell proliferative response in vitro, which is the primary basis for the designation that MSCs can escape immune recognition. The capability of third-party MSCs to be safely infused into patients with severe GVHD and ameliorate the symptoms in some subjects [106] is taken as supportive in vivo evidence. However, it is not strictly correct to infer immune privilege from such data. First, immunosuppression and immune privilege are distinct biologic properties, and immunosuppression does not a priori indicate immune privilege. Second, MSCs suppress T-cell proliferation in vitro by an antigen-independent mechanism, and therefore proliferation should not be used to determine immunogenicity. Third, the mechanism of immunosuppression in vivo seems to be far more complex than simply antiproliferative activity; consequently, the relevance of this assay to any feature of MSC immunomodulatory activity is unclear. Finally, infusion of allogeneic MSCs in immunosuppressed hosts does not provide data to support (or refute) the immunogenicity of the cells. An early observation to suggest that MSCs can be recognized by the immune system, and may function as antigen-presenting cells, came from a clinical trial of intravenous infusions of gene-marked MSCs in children with osteogenesis imperfecta [49]. These children had

previously undergone bone marrow transplantation and then received allo-geneic MSCs from the original bone marrow donor. While this was the first trial of allogeneic MSC infusions in humans, the patients’ posttransplantation reconstituted immune system and donor-derived MSCs were genetically identically. The MSCs were ex vivo expanded in fetal bovine serum (10%)-containing medium and then divided into two fractions for retroviral transduction with either a vector expressing the neomycin phosphotransferase gene (neoR), a bacterial protein, or a nonexpressing vector. One fraction of MSCs was minimally cultured and the other substantially expanded. The gene-marking vectors were alternated between the minimally expanded MSCs and the substantially expanded MSCs among the patients in the study. Both cell populations were infused into the patients, approximately 2–3 weeks apart, without an immunosuppressive preparatory regimen. In all evaluable patients, cells containing the nonexpressing vector were identified in biopsy-obtained culture-expanded osteoblasts or marrow stromal cells, while cells expressing the bacterial protein neoR were never identified (Fig. 9.4). This observation suggests the possibility that the MSCs expressing neoR were immunologically recognized in the immunocompetent hosts. Additionally, an in vitro cytotoxicity assay using mononuclear cells obtained from patients after the cell infusions revealed lysis of autologous MSCs transduced with the neoR-expressing retroviral vector but not of MSCs transduced with the silent vector (Fig. 9.5), supporting the notion that the neoR-expressing cells were subject to immune recognition and elimination. Finally, neither fraction of gene-marked cells was identified in one patient. This child developed hives and urticaria promptly after the second MSC infusion. Assay of antifetal bovine serum antibodies in the patients revealed that this patient had a 160-fold increase in antibody titer in the post-MSC infusion serum compared with the preinfusion serum, and he was the only child to exhibit a rise in titer (Fig. 9.6, upper panel). Moreover, he was the only child that did not show a clinical response (Fig. 9.6, lower panel). Together with the data of Spees et al., who showed that MSCs internalize fetal bovine serum proteins from the culture media [107], the clinical data suggest that fetal bovine serum proteins were taken up from media and the processed peptide antigens were presented by the MSCs to elicit a systemic immune response in this patient. Murine data further substantiate the notion MSCs can function as antigen-presenting cells and activate immune responses under certain conditions. Chan et al. reported that MSCs were able to take up antigens and induce T-cell responses to recall antigens after stimulation with IFNγ [108]. Further, Stagg et al. showed that mice immunized with ovalbumin-loaded, IFN-γ-stimulated MSCs developed antigen-specific cytotoxic CD8+ T cells and rejected ovalbumin-expressing tumor cells [109]. There are additional recent data to support observations suggesting that MSCs are recognized by the immune system in immunocompetent hosts. First, intravenously infused allogeneic murine MSCs can prime naïve T cells in the host animal [110]. Second, subcutaneously implanted murine allogeneic MSCs were rejected by the host animal. Splenocytes from the hosts showed a significant IFN-γ response against the allogeneic MSCs in vitro [111]. Third, intracoronary injection of human MSCs in rat myocardium led to an inflammatory infiltrate and rejection in immunocompetent animals; in contrast, persistent engraftment of the human MSCs was observed in immunoincompetent rats [112]. The preponderance of current data suggest that MSCs are not intrinsically immune privileged and, in fact, may stimulate an immune response by antigen presentation in some conditions. However, MSCs may be hypoimmunogenic due to their low-level expression of MHC molecules and complete lack of co-stimulatory molecule expression, and this property may be exploited in a variety of clinical settings.

← G1PLII (E) 1 ← γ Globin

2

10

5

20:1 10:1

← G1PLII (MC) ← LNc8 (E)

0

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← γ Globin ← G1PLII (E) ← LNc8 (MC) ← γ Globin

4

← G1PLI (MC) ← LNc8 (E) ← γ Globin

5

← G1PLII (E) ← LNc8 (MC)

G1PLII

E:T

Fig. 9.5 The cytotoxic response of peripheral blood mononuclear cells against transduced mesenchymal stromal cells (MSCs) is depicted. The immune cell-mediated lysis of donor MSCs transduced with the LNc8 retroviral vector (expressing neoR) in contrast to MSCs transduced with the G1PLII vector (no transgene expression) or mock supernatant. E : T designates the effector-to-target cell ratio. Each bar represents the mean of triplicate determinations. (Reproduced from [49], with permission.)

1

Optical Density

3

109

15

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Patient

G1PLII (+) LNc8(+) Normal DNA Bone Stroma Skin

Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation

0.1

0.01

← γ Globin

6 ← γ Globin Fig. 9.4 A polymerase chain reaction analysis for the gene-marking vector sequences in DNA isolated from osteoblasts, stromal cells, and skin fibroblasts of each patient, at 4–6 weeks after two infusions of gene-marked mesenchymal stromal cells (MSCs). DNA isolated from human cells transduced with either the G1PLII or LNc8 vector served as a positive control; normal human DNA was the negative control. Analysis of the gamma-globin (γ Globin) gene controlled for the quality and quantity of DNA. MC and E designate whether the signal represents minimally cultured cells or expanded cells, respectively. The vectors were alternated among the patients to avoid vector bias. ND, Not determined. (Reproduced from [49], with permission.)

Growth Velocity (percent of normal median)

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P2

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P4

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100 75 50 25

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Physiologic role of MSCs

Fig. 9.6 (Upper panel) Enzyme-linked immunosorbent assay measuring antifetal bovine serum antibodies in the sera of patients before ( ) and after ( ) both MSC infusions. Each bar represents the mean of triplicate determinations. Pos, positive control; Neg, negative control. (Lower panel) Growth velocity of the patients during the 6 months immediately before ( ) and after ( ) the first MSC infusion. The values are percentages of the median growth of age- and sex-matched unaffected children (Reproduced from [49], with permission.)

The physiologic function of MSCs is generally accepted as supportive: the cells contribute to the maintenance of the parenchymal cell function in the resident tissue. In bone marrow, for example, MSCs are thought to support hematopoiesis. However, MSCs may serve a much broader role in tissue homeostasis.

The currently available data suggest four plausible functions of MSCs. First, MSCs may, indeed, serve as supportive elements. MSCs secrete many cytokines that stimulate hematopoiesis and secrete SDF-1, which fosters HSC homing to the marrow [57,59]. Gene expression of ex

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vivo-expanded MSCs varies according to the tissue source, suggesting that the cells specifically support the parenchyma of origin [15]. Second, MSCs have a substantial capacity to modulate the immune response, determined primarily from in vitro studies, and may contribute to immune homeostasis in vivo. Third, MSCs readily differentiate to osteoblasts, chondrocytes, and adipocytes in vitro and may serve as a reservoir of these mature cell types. This is an especially attractive hypothesis in the bone marrow as the cellular constituency of marrow becomes less hematopoietic and more adipocytic with age. MSCs, residing at the interface of bone and hematopoietic marrow, are ideally located to give rise to both tissues; however, convincing data demonstrating this differentiation pathway in vivo has not been reported. Finally, MSCs may play a role in tissue repair throughout the body. The in vitro capacity of MSCs to secrete (1) growth factors, which could stimulate tissue recovery, (2) immunosuppressive molecules, which could limit tissue damage, and (3) collagen, which could contribute to scar formation, as well as the propensity to migrate to sites of wounds and inflammation support this notion. Despite these credible hypotheses, the biologic function of MSCs in situ has yet to be conclusively proven.

Clinical applications MSCs to foster engraftment of HSCs following HCT MSCs, in vitro, are far less sensitive to the cytotoxic effects of chemotherapy used in preparatory regimens than are HSCs [113]. Nonetheless, the marrow microenvironment is damaged by the chemoradiotherapy preparatory regimen, which likely disrupts the HSC niche [114], thereby contributing to the delay of hematopoietic reconstitution after both autologous and allogeneic HCT [115–117]. Ex vivo-expanded MSCs may, in principle, be able to engraft and repair the microenvironment, fostering HSC engraftment and hematopoietic reconstitution. However, MSCs do not appreciably home to the marrow after intravenous infusion, and many studies have shown that MSCs remain of host origin after HCT [118–120]; thus, MSCs are unlikely to foster hematopoietic reconstitution by structurally repairing the microenvironment. MSCs produce many prohematopoietic cytokines as described above. Conceivably, infused MSCs could promote hematopoietic reconstitution by secreting cytokines into the circulation that would promote HSC engraftment and hematopoietic reconstitution but not require specific anatomic localization such as homing to the marrow space. Support for this hypothesis comes from two xenotransplantation models: cotransplantation of human MSCs with hematopoietic cells in NOD-SCID mice and in utero co-transplantation in fetal sheep. Preclinical data MSCs (106 cells/animal) co-transplanted with CD34+ umbilical cord cells (0.03–1.0 × 106 cells/animal) in NOD-SCID mice resulted in threeto four-fold more human CD45+ cells in the peripheral blood and bone marrow than when the CD34+ cells were transplanted alone [121]. Analysis of the bone marrow revealed a greater number of human hematopoietic (CD34+) progenitors and myeloid (CD33+) cells, but not B (CD19+ or CD20+) lymphocytes. Moreover, human MSCs were not detected in the marrow. These data support the notion that the effects of MSCs are mediated by cytokine secretion, especially myeloidresponsive cytokines, in contrast to contributing to the cellular makeup of the microenvironment. Interestingly, all of the effects were statistically significant (P ≤ 0.05) only at the lower doses of transplanted CD34+ cells. In another study, human CD34+ cells were isolated from mobilized peripheral blood and co-transplanted with human MSCs into NODSCID mice [122]. The MSCs were again found to enhance myelopoiesis,

and also megakaryopoiesis, but not B lymphopoiesis. Similar to the previous study, where the CD34+ cells were obtained from cord blood [121], these investigators only observed an MSC-mediated enhancement of hematopoiesis after transplantation of low (<106) CD34+ cell doses. Maitra et al. showed that, after transplantation of 4 × 106 human umbilical cord blood mononuclear cells into sublethally irradiated NODSCID mice, only two of 10 mice had detectable human CD45+ cells in their blood [123]. After co-transplantation of human MSCs (106 cells/ animal), eight of 10 mice engrafted with human hematopoiesis (p = 0.02). In contrast, none of four mice co-transplanted with murine MSCs engrafted with human cells. No effect was observed when higher doses of umbilical cord blood cells were used. This study confirmed that the MSC effect is only evident at limiting doses and further suggested that human cytokine secretion is responsible for these effects since murine MSCs would be expected to be able to repair the NOD-SCID microenvironment as effectively as, or perhaps more so, than human MSCs. In utero HCT has the potential to treat many genetic disorders without the toxicity of a chemotherapy conditioning regimen. However, this approach is hampered by exceedingly low levels of donor hematopoietic engraftment. In an effort to develop strategies to overcome this barrier, Zanjani and colleagues co-transplanted sheep MSCs with human hematopoietic cells into preimmune fetal sheep. They found that MSCs markedly increased the level of peripheral blood donor cells at 60 days and 30 months after transplantation relative to animals transplanted with hematopoietic cells alone [124]. These investigators then showed that co-transplantation of both autologous and allogeneic human MSCs led to greater long-term engraftment of human hematopoietic cells in the bone marrow, and earlier and higher levels of donor cell in the circulation during gestation and after birth. These strategies are currently being developed for study in pilot clinical trials. Clinical studies The first effort to determine whether MSCs could promote or impede engraftment and hematopoietic reconstitution in patients was reported by Koc et al. in 2000 [125]. Twenty-eight women undergoing autologous HCT with mobilized peripheral blood cells for advanced breast cancer were infused with 1–2.2 × 106 ex vivo-expanded MSCs/kg 4 hours before the hematopoietic graft. The MSCs were harvested about a month prior to infusion, before the patients underwent mobilization for hematopoietic cell collection, and expanded for two to six passages. The median time to attain a neutrophil count greater than 500/μl was 8 (range 6–11) days and a platelet count greater than 20,000/μl was 8.5 (range 4–19) days. Interestingly, MSCs were detected in the venous blood up to 1 hour after the infusion, indicating that the cells are not completely cleared from the circulation by the initial passage through the pulmonary capillaries. Definitive clinical conclusions cannot be drawn from this single-arm uncontrolled trial, but the rapid hematopoietic recovery demonstrated that the MSCs did not impede hematopoietic engraftment in patients after autologous HCT. Lazarus et al. assessed the capacity of ex vivo-expanded MSCs obtained from HLA-identical sibling donors to facilitate hematopoietic recovery after co-transplantation with either bone marrow or peripheral blood hematopoietic cells in patients with hematologic malignancies [126]. MSCs were isolated, expanded, and cryopreserved prior to the initiation of the conditioning regimen. The MSC units were thawed at the bedside and intravenously infused into the patients 4 hours before the hematopoietic cell graft. The median hematopoietic cell doses were 3.6 × 108 mononuclear cells/kg for bone marrow and 5.0 × 106 CD34+ cells/kg for peripheral blood grafts, while the MSC dose ranged from 1 to 5 × 106 cell/kg for both patient groups. Overall, the median time to attain a neutrophil count of 500/μl was 14 (range 11–26) days, and to a platelet count of 20,000/μl was 20 (range 15–36) days. The patients

Mesenchymal Stromal Cells and Hematopoietic Cell Transplantation

receiving peripheral blood grafts demonstrated a more rapid neutrophil recovery (median 13.5 days) compared with those receiving bone marrow (15.5 days). While this study also lacked a control cohort of patients, the time to neutrophil recovery of bone marrow may be somewhat faster than is often experienced. On the other hand, it is less clear whether the time to engraftment of PBPCs represents a hastening of hematopoietic recovery. Haploidentical HCT in children typically results in rapid hematopoietic reconstitution. For example, children undergoing haploidentical HCT at St Jude Children’s Research Hospital, Memphis, TN showed a median time to neutrophil recovery of 11 days (Hale, personal communication). However, primary graft failure/rejection occurs frequently with these highly T-cell-depleted grafts, and a second HSC infusion is often required. Ball et al. sought to determine whether MSCs could positively impact engraftment in pediatric haploidentical transplantation [127]. Fourteen children underwent transplantation with HLA-disparate CD34+ cells obtained from mobilized peripheral blood. A median of 21.5 × 106 CD34+ cells/kg were infused. MSCs were isolated from bone marrow about 5 weeks before HCT and expanded up to three passages. The ex vivo-expanded cells were infused, either cryopreserved or fresh, at a dose of 1–5 × 106 cells/kg 4 hours before the hematopoietic cell graft. Compared with 47 age-, sex-, and diagnosis-matched historical controls, there was no difference in the time to neutrophil recovery (12 versus 13 days) or the time to platelet (20,000/μl) recovery (10 versus 13 days). However, the time to attain a total leukocyte count of 1 × 106/μl was shorter in the MSC group (11.5 days) compared with controls (14.9 days; p = 0.009). More importantly, none of the children receiving MSCs (n = 14) experienced graft failure. In contrast, the graft failure rate among the controls was 15%, consistent with the experience of others. One of the consistent themes of the preclinical studies is that MSCs are most beneficial at limiting doses of HSCs. This observation suggests that umbilical cord blood transplantation may be the most useful setting for MSC co-transplantation. Umbilical cord units must have a threshold cell count for clinical use, as discussed elsewhere in this text, since low cell doses increases the risk of graft failure. Moreover, the time to neutrophil and platelet reconstitution tends to be longer when umbilical cord grafts are used, compared with other HSC sources. Based on this rationale, Wagner and colleagues have co-transplanted parental (haploidentical) MSCs with unrelated umbilical cord blood grafts. Eight patients underwent transplantation with an HLA-matched, unrelated umbilical cord graft for a hematologic malignancy. Haploidentical MSCs were isolated from parental bone marrow obtained at the time the patient was referred for transplantation, and the ex vivo-expanded cells were cryopreserved for storage. MSCs, thawed immediately prior to intravenous infusion, were transplanted at a median dose of 2.1 × 106 cells/kg 4 hours prior to the umbilical cord blood graft. The median umbilical cord blood total nucleated cell dose was 3.1 × 107 cell/kg. All patients attained a neutrophil count of 500/μl at a median of 19 (range 8–28) days compared with 86% of historical control patients at a median of 30 (range 10–59) days. Six of the eight patients achieved a platelet count of 20,000/μl at a median of 1.7 (range 1.1–3.2) months compared with 79% of historical controls at a median of 2.7 (range 1.5–6.6) months (MacMillan and Wagner, personal communication). This study, the first clinical trial of MSC co-transplantation with umbilical cord grafts, suggested that the MSCs may have the capacity to foster hematopoietic cell engraftment and promote hematopoietic reconstitution. HSC expansion Ex vivo expansion of HSCs is a long sought but elusive goal (see Chapter 8). Stem cell expansion would obviate the problem of low stem cell

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doses, which can be problematic in all graft sources. However, this strategy is especially relevant to umbilical cord blood transplantation, where low cell doses are quite often limiting and the overall quantity and HLA spectrum of available units is far less than what is currently available using matched, unrelated volunteer donors. Before MSCs became a cell therapeutic agent, “stromal cells” were used to generate long-term bone marrow cell cultures. This hematopoietic marrow cell culture methodology has been applied to the clinical setting by using MSCs as a “feeder” layer to culture-expand primitive hematopoietic progenitors, and conceivably the HSC, in anticipation of transplanting the entire cellular constituency of the culture providing both HSCs and MSCs to foster engraftment and promote hematopoietic reconstitution. McNiece et al. reported a preclinical study of umbilical cord blood cell expansion on MSCs. Cord blood mononuclear cells were seeded onto an established confluent MSC layer and maintained in media that contained hematopoietic growth factors. After 13 days, analysis of the cultures revealed a 10–20-fold increase in the total nucleated cells, a seven- to 18-fold increase in committed progenitors, a two- to fivefold increase of primitive progenitors, and a 16–37-fold increase in CD34+ cells. MSCs in each culture expansion were from a single donor; however, three different donors were tested without an identifiable difference [128]. The results, suggesting a potential of HSC expansion, were confirmed in a subsequent laboratory study [129], and preclinical models documented the feasibility of such a clinical strategy. These encouraging results led to a recently opened clinical trial at MD Anderson Cancer Center, Houston, TX to test the safety and feasibility of transplantation of a graft consisting of both umbilical cord blood cells that were ex vivo expanded on a supportive cell layer of MSCs, and the MSCs from the expansion culture. Three patients have been transplanted without complications, and all have attained a neutrophil count of 500/ μl at 7–20 days (Shpall, personal communication). While these results are preliminary, precluding any conclusions, the early observations are encouraging. If the observed rapid neutrophil recovery is confirmed, a similarly rapid platelet recovery is documented, and the long-term outcome of the patients (regimen-related toxicity and relapse) is equal to or better than conventional umbilical cord transplantation, then this therapeutic strategy could hold promise to overcome one of the major barriers to umbilical cord transplantation. MSCs to treat GVHD The immunomodulatory capacity of MSCs suggests that these cells may be effective agents for the treatment of GVHD. Based on limited preclinical in vitro data, and without animal models, a 9-year-old boy with severe refractory gut and liver GVHD after a matched unrelated donor peripheral blood HCT was treated with ex vivo MSCs derived from his haploidentical mother [106]. While continuing to receive prednisolone and cyclosporine, an infusion of 2 × 106 MSCs/kg led to a prompt reduction of his serum bilirubin concentration and stool output. His cyclosporine was then discontinued to enhance a graft-versus-leukemia effect, but this led to an acute exacerbation of his gut and liver GVHD. A second infusion of MSCs (1 × 106 cells/kg) again resulted in a rapid reduction of his serum bilirubin and stool output. About 18 months after the MSC infusions, the patient had recurrent GVHD, developed interstitial pneumonia, and died. Although the final outcome of the patient was regimen-related death, this landmark report indicates the potential of MSCs to effectively treat GVHD. An additional eight patients with steroid refractory grades III–IV GVHD and one patient with extensive chronic GVHD treated with MSC therapy have been reported [130]. A median of 1 × 106 MSCs/kg was intravenously infused without incident. Six patients were treated once, and three patients received two infusions, with MSCs obtained from

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HLA-identical siblings, haploidentical family members or unrelated mismatched donors. Of the patients with acute GVHD, six showed a complete response and two died with no response. The patient with chronic GVHD demonstrated a response in the liver but not in the skin and died of Epstein–Barr virus lymphoma. Several additional case reports have appeared supporting the capacity of MSCs to treat GVHD. Preliminary results of an ongoing multicenter European Group for Blood and Marrow Transplantation study to treat steroid-resistant GVHD were presented at the 2007 meeting of the American Society for Blood and Marrow Transplantation [131]. The outcome of 40 patients with grades III–IV acute GVHD were reported. The MSC dose was a median 1.0 × 106 cells/kg recipient body weight (range 0.4–9 × 106 cells/kg). Adverse effects were not seen after MSC infusions. Nineteen patients received one dose, 19 patients received two doses, and two patients received three and five doses, respectively. MSC donors were HLA-identical siblings in five cases, haploidentical donors in 19 cases, and 41 cases of third-party HLA-mismatched donors. Among the 40 patients treated for severe acute GVHD, 19 had a complete response, nine showed improvement, seven patients did not respond, four had stable disease and one patient was not evaluated due to short follow-up. Twenty-one patients were alive between 6 weeks and 3.5 years of follow-up after transplantation. Nine of these patients have extensive chronic GVHD. One patient with acute lymphoblastic leukemia developed recurrent leukemia, and one patient developed de novo acute myeloid leukemia of host origin. The final results of this trial are anticipated to be published in the near future. The worldwide clinical experience is early, and the data to date only suggestive; however in view of the dismal outcome in patients with grades III–IV acute GVHD, these preliminary reports are intriguing. The considerable interest in MSCs as therapy for GVHD originated from the report of a single patient treatment, which ensued without a supportive animal model. This sequence of events is quite fortunate for the field as the subsequently published animal data suggest that MSCs may not effectively treat established GVHD, in contradiction to the clinical findings [132,133]. While the role of MSC therapy for GVHD remains to be completely defined, the recognition of the possible clinical benefits warranting further study underscores the importance of pilot clinical trials, in distinction to relying solely on preclinical animal models, to investigate the therapeutic potential of a novel agent.

MSCs as adjunct therapy with HCT for inborn errors of metabolism HCT is the only curative therapy for a wide variety of inborn errors of metabolism, as detailed elsewhere in this text. MSCs may be used as adjunct cell therapy in the treatment of these disorders in several ways. First, for many disorders, such as osteopetrosis and Hurler’s syndrome, primary graft failure due to an abnormal microenvironment is relatively common. MSC co-transplantation may foster donor HSC engraftment

and promote hematopoietic recovery in these patients as in patients with hematologic malignancy. In this setting, the issue is not limiting HSC doses, but rather the abnormal host stem cell niche. However, umbilical cord blood grafts are often used to treat these children and may, in fact, offer some advantages over other graft sources. The potential benefits of MSC co-transplantation may prove vitally important for children with metabolic disorders since complete donor chimerism is the best outcome that the treating physician can offer. Second, MSCs may, in principle, engraft and differentiate in the target tissue, replacing the genetically defective cells with normal donorderived cells; however, such a mechanism has never been proven to underlie the observed functional benefit of MSC therapy. Third, MSCs may serve as a source of normal enzyme and can potentially crosscorrect defective cells. MSCs express many enzymes whose deficiency is the etiology of a genetic disorder treated with HCT [134], and normal fibroblasts have been shown to possess the potential for crosscorrection [135]. MSCs have been investigated clinically in osteogenesis imperfecta, Hurler’s syndrome, and metachromatic leukodystrophy. Horwitz et al. treated six children with severe osteogenesis imperfecta, a genetic disorder of bone characterized by growth retardation and bony deformities, with conventional allogeneic bone marrow transplantation followed by infusions of donor-derived MSCs (1–5 × 106 cells/kg) [49]. MSCs were documented to have engrafted in the bone in some patients, albeit at very low levels. Nonetheless, all evaluable patients showed a striking acceleration of growth as an unequivocal clinical response. Koc et al. infused 2–10 × 106 donor-derived MSCs/kg into five children with Hurler’s syndrome and six children with metachromatic leukodystrophy, two disorders characterized, in part, by skeletal and neurologic defects, who had previously undergone matched-sibling HCT [136]. There was a substantial improvement in nerve condition velocity, and the bone mineral density was either maintained or improved in all patients.

Future considerations We are now beginning to unravel the highly complex biology of MSCs that will undoubtedly foster clinical applications. MSCs hold great promise to facilitate HSC engraftment when the HSC dose is limiting. Additionally, MSCs clearly can modulate the immune response, despite our presently inadequate understanding of the MSC immune system interaction. MSCs will likely play a role as adjunct therapy with HCT and possibly as a single-cell agent for the treatment of genetic or rheumatologic disorders. Moreover, novel approaches using MSCs as cell therapy in regenerative medicine are emerging. Three great challenges lie ahead. First, we must better understand the surface markers of MSCs and define subsets of cells correlating the surface phenotype and the biologic function. Second, we must more clearly understand the immunomodulatory capacity of MSCs and how to most effectively apply MSCs clinically. Finally, we must conduct numerous pilot clinical trials followed by large, multicenter clinical trials to first demonstrate and then validate the clinical utility of MSCs.

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71. Aguilar S, Nye E, Chan J et al. Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung. Stem Cells 2007; 25: 1586–94. 72. Miura M, Miura Y, Padilla-Nash HM et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006; 24: 1095–103. 73. Tolar J, Nauta AJ, Osborn MJ et al. Sarcoma derived from cultured mesenchymal stem cells. Stem Cells 2007; 25: 371–9. 74. Rubio D, Garcia-Castro J, Martin MC et al. Spontaneous human adult stem cell transformation. Cancer Res 2005; 65: 3035–9. 75. Wang Y, Huso DL, Harrington J et al. Outgrowth of a transformed cell population derived from normal human BM mesenchymal stem cell culture. Cytotherapy 2005; 7: 509–19. 76. Fraser CJ, Hirsch BA, Dayton V et al. First report of donor cell-derived acute leukemia as a complication of umbilical cord blood transplantation. Blood 2005; 106: 4377–80. 77. Hacein-Bey-Abina S, von Kalle C, Schmidt M et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348: 255–6. 78. Zimmet JM, Hare JM. Emerging role for bone marrow derived mesenchymal stem cells in myocardial regenerative therapy. Basic Res Cardiol 2005; 100: 471–81. 79. Breitbach M, Bostani T, Roell W et al. Potential risks of bone marrow cell transplantation into infarcted hearts. Blood 2007; 110: 1362–9. 80. Di Nicola M, Carlo-Stella C, Magni M et al. Human bone marrow stromal cells suppress Tlymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002; 99: 3838–43. 81. Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003; 57: 11–20. 82. Bartholomew A, Sturgeon C, Siatskas M et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002; 30: 42–8. 83. Krampera M, Glennie S, Dyson J et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 2003; 101: 3722–9. 84. Augello A, Tasso R, Negrini SM et al. Bone marrow mesenchymal progenitor cells inhibit lymphocyte proliferation by activation of the programmed death 1 pathway. Eur J Immunol 2005; 35: 1482–90. 85. Potian JA, Aviv H, Ponzio NM, Harrison JS, Rameshwar P. Veto-like activity of mesenchymal stem cells: functional discrimination between cellular responses to alloantigens and recall antigens. J Immunol 2003; 171: 3426–34. 86. Djouad F, Plence P, Bony C et al. Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals. Blood 2003; 102: 3837–44. 87. Bentley SA, Knutsen T, Whang-Peng J. The origin of the hematopoietic microenvironment in continuous bone marrow culture. Exp Hematol 1982; 10: 367–72.

88. Rasmusson I, Ringden O, Sundberg B, Le BK. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation 2003; 76: 1208–13. 89. Le Blanc K, Rasmusson I, Gotherstrom C et al. Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes. Scand J Immunol 2004; 60: 307–15. 90. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005; 105: 1815–22. 91. Meisel R, Zibert A, Laryea M et al. Human bone marrow stromal cells inhibit allogeneic Tcell responses by indoleamine 2,3-dioxygenasemediated tryptophan degradation. Blood 2004; 103: 4619–21. 92. Nasef A, Mathieu N, Chapel A et al. Immunosuppressive effects of mesenchymal stem cells: involvement of HLA-G. Transplantation 2007; 84: 231–7. 93. Rasmusson I, Ringden O, Sundberg B, Le Blanc K. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp Cell Res 2005; 305: 33–41. 94. Glennie S, Soeiro I, Dyson PJ, Lam EW, Dazzi F. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood 2005; 105: 2821–7. 95. Anderlini P, Sheth S, Hicks K et al. Re: Imatinib mesylate administration in the first 100 days after stem cell transplantation. Biol Blood Marrow Transplant 2004; 10: 883–4. 96. Corcione A, Benvenuto F, Ferretti E et al. Human mesenchymal stem cells modulate B-cell functions. Blood 2006; 107: 367–72. 97. Rasmusson I, Le Blanc K, Sundberg B, Ringden O. Mesenchymal stem cells stimulate antibody secretion in human B cells. Scand J Immunol 2007; 65: 336–43. 98. Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchymal stem cellnatural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 2006; 107: 1484–90. 99. Spaggiari GM, Capobianco A, Abdelrazik H et al. Mesenchymal stem cells inhibit natural killer cell proliferation, cytotoxicity and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood 2008; 111: 1327–33. 100. Krampera M, Cosmi L, Angeli R et al. Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells 2006; 24: 386–98. 101. Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells 2006; 24: 74–85. 102. Jiang XX, Zhang Y, Liu B et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005; 105: 4120–6. 103. Zhang W, Ge W, Li C et al. Effects of mesenchymal stem cells on differentiation, maturation, and function of human monocyte-derived dendritic cells. Stem Cells Dev 2004; 13: 263–71. 104. Nauta AJ, Kruisselbrink AB, Lurvink E, Willemze R, Fibbe WE. Mesenchymal stem cells inhibit generation and function of both CD34+-

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derived and monocyte-derived dendritic cells. J Immunol 2006; 177: 2080–7. Zappia E, Casazza S, Pedemonte E et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 2005; 106: 1755–61. Le Blanc K, Rasmusson I, Sundberg B et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004; 363: 1439–41. Spees JL, Gregory CA, Singh H et al. Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther 2004; 9: 747–56. Chan JL, Tang KC, Patel AP et al. Antigenpresenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma. Blood 2006; 107: 4817–24. Stagg J, Pommey S, Eliopoulos N, Galipeau J. Interferon-gamma-stimulated marrow stromal cells: a new type of nonhematopoietic antigenpresenting cell. Blood 2006; 107: 2570–7. Nauta AJ, Westerhuis G, Kruisselbrink AB et al. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 2006; 108: 2114–20. Eliopoulos N, Stagg J, Lejeune L, Pommey S, Galipeau J. Allogeneic marrow stromal cells are immune rejected by MHC class I- and class IImismatched recipient mice. Blood 2005; 106: 4057–65. Grinnemo KH, Mansson A, Dellgren G et al. Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium. J Thorac Cardiovasc Surg 2004; 127: 1293–300. Kumagai M, Manabe A, Coustan-Smith E et al. Use of stroma-supported cultures of leukemic cells to assess antileukemic drugs. II. Potent cytotoxicity of 2-chloro-deoxyadenosine in acute lymphoblastic leukemia. Leukemia 1994; 8: 1116–23. Calvi LM, Adams GB, Weibrecht KW et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003; 425: 841–6. Galotto M, Berisso G, Delfino L et al. Stromal damage as consequence of high-dose chemo/ radiotherapy in bone marrow transplant recipients. Exp Hematol 1999; 27: 1460–6.

116. O’Flaherty E, Sparrow R, Szer J. Bone marrow stromal function from patients after bone marrow transplantation. Bone Marrow Transplant 1995; 15: 207–12. 117. Domenech J, Gihana E, Dayan A et al. Haemopoiesis of transplanted patients with autologous marrows assessed by long-term marrow culture. Br J Haematol 1994; 88: 488–96. 118. Simmons PJ, Przepiorka D, Thomas ED, TorokStorb B. Host origin of marrow stromal cells following allogeneic bone marrow transplantation. Nature 1987; 328: 429–32. 119. Agematsu K, Nakahori Y. Recipient origin of bone marrow-derived fibroblastic stromal cells during all periods following bone marrow transplantation in humans. Br J Haematol 1991; 79: 359–65. 120. Cilloni D, Carlo-Stella C, Falzetti F et al. Limited engraftment capacity of bone marrow-derived mesenchymal cells following T-cell-depleted hematopoietic stem cell transplantation. Blood 2000; 96: 3637–43. 121. Noort WA, Kruisselbrink AB, in’t Anker PS et al. Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34(+) cells in NOD/SCID mice. Exp Hematol 2002; 30: 870–8. 122. Angelopoulou M, Novelli E, Grove JE et al. Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice. Exp Hematol 2003; 31: 413–20. 123. Maitra B, Szekely E, Gjini K et al. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 2004; 33: 597– 604. 124. Almeida-Porada G, Flake AW, Glimp HA, Zanjani ED. Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Exp Hematol 1999; 27: 1569–75. 125. Koc ON, Gerson SL, Cooper BW et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol. 2000; 18: 307–16. 126. Lazarus HM, Koc ON, Devine SM et al. Cotransplantation of HLA-identical sibling culture-

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10

Grant D. Trobridge & Hans-Peter Kiem

Genetic Manipulation of Hematopoietic Stem Cells

Introduction and history Hematopoietic stem cells (HSCs) are particularly amenable to ex vivo manipulation and have long been considered an attractive target for gene therapy. There are many potential therapeutic applications, from curing genetic diseases to making the hematopoietic system resistant to alkylating agents to allow more effective chemotherapy, to protecting the hematopoietic system from human immunodeficiency virus (HIV) infection. Methods for the genetic manipulation of HSCs evolved from the pioneering efforts of investigators working to establish means for efficient gene transfer to mammalian cells. Initial studies of gene transfer into murine hematopoietic repopulating cells utilized calcium phosphate-mediated transfection of mouse DNA encoding drug-resistance genes into mouse bone marrow (BM) cells [1,2]. The gene transfer efficiency was low in these studies, but they established the potential for HSC gene transfer and also chemoprotection of HSCs. The development of retroviral vectors allowed for efficient gene transfer to murine hematopoietic progenitor cells [3] and pluripotent repopulating cells [4–6]. Transduction with retroviruses results in unique, well-defined proviral vector integrants that can be used to track the progeny of individual stem or progenitor cells. Retroviral proviruses were used as markers to demonstrate that self-renewing clones capable of both myeloid and lymphoid repopulation potential could be transduced [4–6]. Two additional prescient predictions were made in these early studies. First, the culture conditions that alter stem cell kinetics may also affect transduction efficiency [5]. Second, retroviral integrants near genes involved in hematopoietic proliferation could alter the phenotype of these stem cells, and retroviral markers might be used to identify nearby genes involved in hematopoiesis, analogous to the use of viruses to identify oncogenes [4]. These studies demonstrated the feasibility of gene therapy for hematopoietic disorders by demonstrating gene transfer to HSCs capable of expansion into multiple hematopoietic lineages with persistent transgene expression. Improvements were made to the retroviral vector systems to reduce the possibility of generating contaminating replication-competent retrovirus in vector preparations, and gene transfer to large-animal HSCs was attempted. Relatively efficient gene transfer to canine hematopoietic progenitor cells in vitro was achieved [7], but the transduction of repopulating HSCs in large-animal models was inefficient in early studies

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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[8,9]. The first clinical trials evaluated gene transfer to autologous BM collected from acute myeloid leukemia patients or neuroblastoma patients after intensive chemotherapy [10,11]. Long-term gene marking was significantly higher than observed in previous large animal studies, with G418-resistant colonies detected up to 18 months post-transplant at frequencies of approximately 5%. The presence of the retroviral marker in resurgent blast cells of two patients with acute myeloid leukemia demonstrated that infused autologous marrow could be a source of leukemic cells. These studies also demonstrated for the first time in humans that infused gene-modified autologous cells could contribute to long-term hematopoiesis. Subsequent clinical gene transfer studies resulted in lower transduction frequencies, but transduction protocols were refined and the first clearly curative HSC gene therapy was reported 7 years later by Cavazzana-Calvo et al. for X-linked severe combined immunodeficiency (SCID-X1) [12].

Gene transfer vectors Retroviral vectors For most HSC gene transfer applications, the goal is to transduce multipotential HSCs and obtain transgene expression in mature hematopoietic cells following expansion and differentiation. Retroviral vectors have been by far the most widely used vectors for gene transfer to HSCs, due in large part to their ability to integrate efficiently into target cell chromosomes. This allows the integrated vector provirus containing the transgene to be transmitted to all HSC progeny via mitosis during the extensive expansion of HSCs to form the entire hematopoietic system. All retroviruses contain gag, pol, and env genes, replicate using an RNA intermediate, and integrate into the host genome forming a defined provirus structure with two long terminal repeats (LTRs) (Fig. 10.1). Integration occurs throughout the host genome but is not random; gammaretroviruses, lentiviruses, and spumaretroviruses each have distinct integration site preferences. Gammaretroviral vectors Gammaretroviruses, formerly a subset of the Oncovirinae or oncoretrovirus subfamily, are “simple” retroviruses that do not contain accessory genes. Gammaretroviral vectors were the first vectors used for HSC gene transfer and continue to be widely used for many gene transfer applications. To generate vectors, most of the viral structural gene sequences are replaced by the desired transgene(s). LTR sequences and viral cisacting sequences required for efficient incorporation of vector genomes

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Fig. 10.1 Retroviral life cycle. The retroviral virion is composed of matrix, nucleocapsid, and capsid proteins from the gag gene that form the virion core containing two viral genomes, and is surrounded by a plasma membrane with incorporated envelope proteins produced from the env gene. (1) The envelope protein mediates attachment to the cellular receptor. (2) Fusion of the viral and cellular membranes allows entry of the retroviral core into the cytoplasm. (3) The RNA genome is converted into a double-stranded DNA genome by the viral reverse transcriptase (RT). For foamy retroviruses, this occurs before virus entry. (4) The viral genome and associated proteins, referred to as the preintegration complex (PIC), must transit to and enter the nucleus. (5) Once in the nucleus, the viral integrase mediates integration of the reverse-transcribed double-stranded DNA viral genome into the host genome. This produces a proviral vector structure with LTRs at each end of the vector genome that form a junction with the host chromosome. The LTRs contain enhancer and promoter sequences to drive viral gene expression. (6) After production of viral proteins and RNA genomes from the integrated provirus, viral cores bud through host cell membranes containing viral envelope to produce new virions.

into virions referred to as the Ψ region are retained (Fig. 10.2a). This results in a replication-incompetent vector where the internal transgene can be expressed either from the viral LTR promoter or from an internal promoter. Vector virions are produced by supplying the viral structural genes in trans. Gammaretroviral vectors have evolved from early vectors that contained complete LTR sequences, and could generate contaminating replication-competent viruses by recombination with helper plasmids during preparation, to highly engineered vectors with LTR fusion promoters and “self-inactivating” (SIN) deletions in the LTR (Fig. 10.2b). Gammaretroviral vector preparations are commonly produced using a packaging cell line where the helper proteins are produced from stably integrated gag, pol, and env genes. This allows facile production of vector-containing supernatants by introducing the desired vector into a packaging cell line (Fig. 10.3a). An important disadvantage of gammaretroviral vectors for gene transfer to HSCs is that the target cell must be actively dividing for efficient transduction [13]. This is presumably because the viral preintegration complex requires breakdown of the nuclear membrane during mitosis in order to enter the nucleus [14]. HSCs are quiescent, residing primarily in G0, so efficient transduction by gammaretroviral vectors requires stimulation into the cell cycle.

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Fig. 10.2 Retroviral vectors. (a) Gammaretrovirus, lentivirus, and foamy virus vectors. Schematics for Moloney murine leukemia virus (MLV), human immunodeficiency virus (HIV-1) and primate foamy virus (PFV) are shown, with an example vector schematic below. For each vector, only cis-acting regions required for efficient gene transfer are present in the vector, which include the long-terminal repeats (LTRs) and the packaging signal, Ψ. The vectors do not express viral proteins; the structural proteins gag, pol, and env are provided in trans (not shown) so the vectors are replication-incompetent. The MLV vector shown expresses a neomycin phosphotransferase reporter gene (neo) from the vector LTR. The HIV-1 vector shown expresses a therapeutic transgene (ther) from an internal phosphoglycerate kinase (pgk) promoter. Vector transcripts are driven by a fusion LTR promoter (f-LTR) that contains a Rous sarcoma virus promoter with HIV-1 LTR elements. This vector also contains a woodchuck post-transcriptional regulatory element (w) to enhance transgene expression. The foamy virus vector expresses a green fluorescent protein from an internal pgk promoter, and vector transcripts are expressed from a fusion LTR promoter that includes cytomegalovirus promoter elements. (b) Self-inactivating vector design. For the lentiviral vector shown, vector transcripts made during viral vector production are driven by a f-LTR promoter and the 3′ LTR contains a deletion in the U3 region. During reverse transcription, the deleted U3 region of the 3′ LTR is copied to the 5′ LTR of the vector provirus, resulting in an integrated vector with LTRs that are essentially inactive. In the integrated vector provirus, transcription is driven by the internal pgk promoter.

Lentiviral vectors Lentiviruses such as HIV-1 are “complex” retroviruses that encode accessory genes in addition to gag, pol, and env. Lentiviral vectors have several advantages over gammaretroviral vectors, including the ability to efficiently transduce quiescent cells [15]. The ability of lentiviral vectors to enter the nucleus independently of mitosis is thought to be responsible for efficient transduction of quiescent cells, but other differences may also be involved [16]. Lentiviral vectors efficiently transduce

aphidicolin-arrested cells, demonstrating that they do not require mitosis, but transduction of serum-starved G0 cells is less efficient. It is now generally accepted that lentiviral vectors require progression of the cell cycle to G1b, likely because reverse transcription is inefficient in G0 cells [17]. Several groups have shown that efficient lentiviral transduction of repopulating HSCs requires cytokine stimulation supporting this conclusion. SIN lentivirus vectors can be produced at high titers and can

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Fig. 10.3 Retroviral vector production. (a) Isolating high-titer gammaretroviral producer clones using packaging cell lines. Established packaging cell lines express the vector structural proteins Gag, Pol, and Env. (1) The vector of interest is introduced into the packaging cell line to generate cells with stably integrated vector. The vector can be introduced into the packaging cell lines by transfection of a vector plasmid, or alternately, if the vector contains a functional LTR, it can be introduced by infection of the packaging cell line with vector virions. (2) Individual “producer” clones are isolated using cloning rings or by limiting dilution. (3) Producer clones are tested to identify a clone that produces vector at high titer. (4) The producer clone is expanded and the producer cell culture medium containing vector virions is collected. (b) Lentiviral vector production by transient transfection. (1) Typically, human embryonic kidney 293 cells are transfected with a plasmid that produces the vector transcript and plasmids that express helper proteins required to form vector virions including Gag, Pol, and an Env pseudotype. (2) Following transfection, the cells are washed and incubated for 24–72 hours, and culture medium containing the vector virions is harvested up to three times during this period.

efficiently deliver large complex transgenes that reduce the titers of gammaretroviral vectors, such as locus control region-driven globin genes. Most lentiviral vector preparations used for HSC gene transfer have been produced by transient transfection of human embryonic kidney 293 cells. A typical four-plasmid transient vector production scheme is shown in Fig. 10.3b. A disadvantage of lentiviral vectors has been the concern that recombination between vector genomes and viral helper genes may lead to a contaminating replication-competent lentivirus that has some pathogenic properties of the parent virus. Most current lentiviral vector systems have deletions in the viral tat, vpr, vpu, and nef accessory genes and have extensive deletion of the viral env gene, but do require rev. They also have SIN deletions in the LTR including the TATA box. Additional safety measures have been undertaken to reduce the homology between helper and vector sequences by codon optimization, and to reduce the potential for replication-competent recombinants by splitting the Gag and Pol helper functions onto different plasmids [18]. It is thus impossible to generate the wild-type parent virus from the vector and helper plasmids, but concerns remain that a novel replication-competent virus could be generated. To address this, assays have been developed to detect a potential replication-competent lentivirus in vector preparations to be used for clinical applications [19]. Foamy virus vectors Foamy or spumaretrovirus-based vectors have been developed more recently and have several properties that make them well suited for HSC gene therapy. Foamy viruses have never been shown to cause disease in

any host, including humans. They are the largest of the retroviruses at approximately 13 kilobases (kb) and can carry large (at least 9 kb) transgene cassettes efficiently. Foamy viruses are complex retroviruses that contain the accessory genes tas (formerly called bel-1) and bet. They infect many host species but are not endemic in human populations, although zoonotic infections can occur. In current foamy virus vectors, there are deletions of the tas and bet accessory genes and deletions in the LTR, making these vectors SIN. Foamy virus vectors require mitosis for transduction, but they form a stable transduction intermediate capable of being maintained in quiescent cells [20]. This may explain their ability to efficiently transduce hematopoietic repopulating cells with minimal ex vivo stimulation [21,22]. Foamy virus vectors are typically produced by transient transfection of human embryonic kidney 293 cells. Retroviral pseudotypes for HSC gene transfer Retroviral vectors can be “pseudotyped” at varying efficiencies by substituting a heterologous envelope protein from another enveloped virus for their envelope. This can alter the tropism of the vector to allow transduction of the desired target cell type. The choice of pseudotype is critical for HSC gene transfer as the availability of the receptor on HSCs can affect the transduction efficiency. Early studies showed that the ecotropic envelope of murine leukemia virus-based gammaretroviral vectors allowed for highly efficient gene transfer to murine repopulating cells. However, the human homolog of the mouse ecotropic receptor does not allow for entry via the ecotropic envelope, and the amphotropic envelope receptor is not highly expressed on human progenitor cells [23]. Receptor expression levels can influence the efficiency of gene transfer [24,25] so alternative pseudotypes have been evaluated. The envelope glycoprotein from the vesicular stomatitis rhabdovirus (VSVG) has been commonly used, due to its broad tropism and its ability to allow for efficient concentration by ultracentrifugation. Lentiviral vectors pseudotyped with VSV-G can be concentrated 1000-fold to titers of over 109 transducing units/ml and retain most of their infectivity when frozen. Additionally, lentiviral vectors pseudotyped with VSV-G can mediate efficient gene transfer into large-animal long-term repopulating cells. However, there are drawbacks to using VSV-G. VSV-G is toxic, which has limited its use in packaging cells, and human sera can inactivate VSV-G-pseudotyped vectors, which limits in vivo delivery. The feline endogenous virus RD114 envelope has been used for pseudotyping both gammaretroviruses and lentiviral vectors and appears to be more efficient for the transduction of hematopoietic cells than VSV-G when compared at similar multiplicities of infection. The RD114 allows for efficient concentration of both lentiviral vectors and gammaretroviral vectors by centrifugation. Additionally, the gibbon ape leukemia virus (GALV) envelope has allowed for efficient transduction of long-term repopulating cells in primates [26]. In some cases, pseudotyping can be inefficient, for example when pseudotyping HIV-derived vectors with the RD114 or GALV envelopes. In these examples, creating chimeric envelopes by substituting the transmembrane or cytoplasmic domain with the corresponding domain(s) from the amphotropic envelope, which does efficiently pseudotype HIV vectors, increases titers. Foamy virus vectors do not pseudotype efficiently with VSV-G or with gammaretroviral envelopes, but the foamy virus envelope allows efficient transduction of large-animal HSCs and also allows concentration by ultracentrifugation. Envelope glycoproteins from many other viruses such as rhabdoviruses (Chandipura, rabies, and Mokola), filoviruses (Ebola, Marburg, and Lassa), and alphaviruses (Ross River virus and Semliki forest virus), have been used to pseudotype retroviral and lentiviral vectors, but efficient transduction of repopulating HSCs has not been demonstrated using these pseudotypes (reviewed in [27]).

Genetic Manipulation of Hematopoietic Stem Cells

Other vector systems

Gene transfer to HSCs

Adenoviruses are nonenveloped icosahedral viruses that have a doublestranded DNA genome of approximately 30–38 kb pairs and contain fiber projections that mediate attachment and entry. Adenoviral vectors can be produced at very high titers (>1012/mL), have a large transgene capacity, and are able to mediate highly efficient gene transfer and gene expression in nondividing cells. However, most adenoviral vectors have been developed from the Ad5 serotype, which utilizes a fiber that binds the Coxsackie-adenovirus receptor and requires alphav integrins, which are not abundant on hematopoietic cells. Additionally, adenoviruses do not integrate efficiently so gene expression is lost as transduced cells divide and expand. Modified adenoviral vectors have been developed for HSC gene therapy that contain fibers which allow transduction of hematopoietic cells, and contain inverted terminal repeats (ITRs) from adeno-associated viruses (AAVs), which increase the integration frequency [28]. Adenoviral vectors with AAV ITRs that have erythroid-specific transgene cassettes can deliver genes to human cord blood-derived CD34+ cells with transduction frequencies of 5% in erythroid colonies [29]. AAV vectors are derived from helper virus-dependent parvoviruses and can mediate efficient gene transfer to several tissues, particularly liver and muscle. AAV vectors require S phase for efficient transduction, and their transgene capacity is approximately 5 kb, but very high titers can be produced and several different capsid proteins are available to modify the tropism of these vectors. As discussed above, the AAV ITRs can mediate integration, but following transduction a large percentage of AAV vector genomes remain in complex episomal forms. There have been reports of transduction of hematopoietic cells with AAV2 vectors, the first AAV vectors developed and the most commonly used, but it is now generally accepted that transduction of hematopoietic cells with AAV2 vectors is inefficient (reviewed in [30]). The frequency of gene transfer to rhesus monkey repopulating cells with AAV2 vectors was estimated to be approximately 0.001% [31]. Improved transduction of mouse HSCs has been reported using AAV1 vectors [32], but large-animal studies have not yet been reported using this serotype. Herpesvirus vectors have large genomes (120–230 kb pairs) that can be engineered to carry large transgene cassettes and also have the ability to transduce nondividing cells. Herpesvirus genomes do not normally integrate but can be maintained episomally or can be engineered to integrate by introducing AAV ITRs. Replication-competent, incompetent, and herpes virus “amplicon” vectors, which are bacterial plasmids that contain only the necessary cis-acting regions necessary for replication and packaging, have been developed for various gene therapy applications. Transduction of CD34+ cells has been demonstrated [33]. Nonviral vectors are appealing for HSC gene transfer from the perspective of facile large-scale production and characterization for clinical use. There are many nonviral approaches (reviewed in [34]), but plasmid DNA is typically introduced by electroporation (or more recently nucleofection), by lipid formulation designed to effect delivery into the cytoplasm or by particle bombardment. However, HSCs are difficult to transfect. The main challenges for plasmid gene delivery are achieving efficient delivery of DNA into the nucleus in quiescent cells without significant toxicity from the procedure, and also achieving efficient integration or replication and segregation of episomes during the extensive expansion of HSCs. Efficient nonviral gene delivery to cord blood-derived CD34+ cells has been reported using nucleofection, and electroporation of plasmids that allow for integration via a transposon have been used to mark CD34+ cells, but gene transfer to human CD34+ cells capable of repopulating immunodeficient mice was inefficient [35].

Source of HSCs for gene transfer

119

It was realized early on that manipulating a mouse donor using chemotherapy with 5-fluoruracil could improve the transduction efficiency of murine BM-derived HSCs [4]. BM recovering from 5-fluoruracil treatment is enriched for cycling early progenitors and stem cells, which likely explains the efficient transduction with gammaretroviral vectors. In large-animal models, the cytokine granulocyte colony-stimulating factor (G-CSF) alone or in combination with stem cell factor (SCF) is commonly used to prime BM, or to mobilize primitive repopulating stem cells into the peripheral blood prior to collecting target cells for gene transfer. However, SCF is unavailable for clinical use in the United States due to anaphylactic reactions in some patients, so G-CSF alone has also been evaluated as a priming/mobilizing cytokine. Hematti et al. observed low transduction frequencies in rhesus macaques with gammaretroviral vectors using G-CSF-mobilized peripheral blood cells [36]; however, efficient gene transfer was observed in the baboon model using G-CSF-mobilized cells when gammaretroviral vectors were produced with a GALV pseudotype in human 293 packaging cells [26]. More recently, AMD3100, a CXCR4 antagonist, has been used for highly efficient mobilization. AMD3100 reversibly blocks the interaction between the chemokine receptor CXCR4 and its ligand SDF1α which plays an important role in regulating stem cell mobilization. In the rhesus macaque, cell-cycle analysis of AMD3100-mobilized CD34+ peripheral blood cells shows that these cells have significantly fewer cells in G0 (31%) compared with G-CSF-mobilized cells (79%), and significantly higher numbers in the G1 phase [37]. Additionally, a higher percentage of AMD3100-mobilized cells expressed CXCR4 and were able to migrate towards the CXCR4 ligand SDF-1α in vitro, suggesting they may have improved homing ability. In this study, in vivo gene-marking levels with a gammaretroviral vector were higher for AMD3100-mobilized cells than for G-CSF-mobilized cells. A comparison of the ability of AMD3100 and G-CSF-mobilized patient CD34+ cells to repopulate immunodeficient mice also showed improved engraftment. These studies serve to illustrate the importance in selecting the source of cells and the vector pseudotype for efficient HSC gene transfer, as the phenotypes of target cells can vary significantly. Cord blood is another promising source of HSCs for gene transfer, although the low number of repopulating cells that can be harvested has limited its utility for application in adults. Most gene transfer experiments in large animals and humans use CD34+ cells isolated using magnetic beads coupled to an anti-CD34 antibody. Ex vivo culture conditions During ex vivo culture, the objective is to stimulate the target cells into the cell cycle to increase gene transfer efficiency while maintaining their engraftment and long-term repopulating ability. Conditions for efficient transduction of murine HSCs were established over 20 years ago, but these conditions were not effective for large-animal studies or for clinical trials. Ex vivo conditions for efficient gene transfer to large-animal repopulating cells have more recently been developed by experimentation with different cytokine cocktails and by employing CH-296 fibronectin fragment [38]. CH-296 (RetroNectin) is a modified recombinant 639 kDa human fibronectin fragment that contains the fibronectin cellbinding and heparin-binding domains, and enhances transduction of HSCs with gammaretroviral, lentiviral, and foamy virus vectors. The enhancement is believed to occur as a result of co-localization of vectors and target cells, at least for gammaretroviral vectors, as well as from inhibiting apoptosis of repopulating cells during ex vivo culture [39] and increasing engraftment [40]. Many cytokine cocktails have been used

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(reviewed in [41]), and for large-animal studies SCF, G-CSF, megakaryocyte growth and development factor (MGDF), interleukin (IL)-3, and IL-6 are commonly used with CH-296. The use of IL-3 has been shown to reduce the engraftment capability of murine HSCs [42], but IL-3 has been used in cytokine cocktails for high-level gene transfer in primate models and in clinical studies. For lentiviral and foamy virus vectors, efficient gene transfer to long-term large-animal repopulating cells has been demonstrated with minimal ex vivo culture, as short as 18 hours. For gammaretroviral vectors, efficient transduction of largeanimal repopulating cells typically requires a prestimulation period of at least 24 hours, and a total ex vivo culture time of 72–96 hours, presumably due to the fact that gammaretroviral vectors require mitosis and are more labile than lentiviral or foamy virus vectors in quiescent cells [20]. In vitro assays for HSC gene transfer In vitro assays have limited use for predicting gene transfer efficiency to true long-term repopulating cells but are essential tools for vector development. Hematopoietic cell lines are useful for establishing the tropism of novel vectors and pseudotypes, and also for establishing the specificity of lineage-restricted promoters. Long-term culture initiating cell assays can be used as a surrogate assay for gene transfer to repopulating cells, but more commonly gene transfer into hematopoietic progenitors is determined using colony-forming unit assays. Cells enriched for hematopoietic progenitor cells are isolated and exposed to vector before being plated in semi-solid media with cytokines that allow for the development of myeloid and erythroid hematopoietic colonies. Colony-forming unit assays allow evaluation of the transduction efficiency of immature hematopoietic cells, but also indicate whether gene expression has been maintained during expansion of the individual progenitor cells into a colony. In some cases, mosaic transgene expression is observed, which can indicate delayed integration. Low-level gene expression in colonies, with loss of transgene expression over time in parallel liquid cultures, indicates transient gene expression, commonly observed when using nonintegrating vectors, or as a result of pseudotransduction. Pseudotransduction is the transfer of protein present in the vector preparation to target cells [43], which can be a problem, particularly when using concentrated vector preparations.

vectors due to insertional mutagenesis [46]. With the development of xenotransplantation mouse models to assay gene transfer into human hematopoietic cells, an increasing frequency of studies are now performed in immunodeficient mouse models. Immunodeficient xenotransplant mouse models The SCID-hu mouse [47], the SCID mouse [48], and the immunodeficient BNX (B is beige or reduced natural killer [NK] cell number, N is nude or athymic, and X is Xid, which reduces lymphokine activated killer cells) mouse [49] xenograft models set the stage for studies aimed at improving gene transfer to the human hematopoietic cells. The human cells that repopulate immunodeficient mice, now commonly referred to as SCID repopulating cells or SRCs, were found to be distinct from hematopoietic progenitors assayed in vitro and were relatively refractory to transduction with gammaretroviral vectors [50]. Efficient transduction of SRCs with gammaretroviral vectors was obtained by stimulating human CD34+ cells with cytokines and extending the ex vivo culture time [51]. Conversely, efficient transduction of SRCs with lentiviral and foamy virus vectors can be obtained with minimal ex vivo culture [21,52]. An early study by Ferrari et al. [53] used the BNX mouse to demonstrate functional correction of human adenosine deaminase (ADA)-deficient patient cells using a retroviral vector. Further refinements to immunodeficient mouse models, such as deletion of the IL-2 common γ chain have allowed for robust engraftment of both myeloid and lymphoid cells using low numbers of transplanted human cells [54]. However, mouse models are limited by a short life span and a limited proliferative demand placed on the transplanted cells. A comparison of baboon repopulating cells with baboon SRCs using the same source of retrovirally marked CD34+ cells showed that SRC engraftment did not correlate with long-term engraftment in the baboon [55]. In this study, a comparison of the individual clones that repopulated the baboons and the SRCs revealed common proviral vector integrants at 6 weeks but not at 6 months after transplantation. These results suggest that distinct hematopoietic stem/progenitor cells are responsible for hematopoietic reconstitution in immunodeficient mice compared with nonhuman primates. Thus, while immunodeficient mouse models are useful for improving gene transfer techniques to HSCs, transduction frequencies in SRCs may not correlate to frequencies observed in human and large-animal long-term repopulating cells.

Gene transfer to mouse HSCs Mice continue to be useful for developing methods for efficient gene transfer to HSCs due to their relatively low cost. Typically, BM cells are isolated from the femurs of donor mice, enriched for HSCs, exposed to vector, and injected by tail vein into lethally irradiated congenic recipients. Mouse repopulating cells that give rise to long-term multilineage reconstitution and self-renewal can be highly purified by fluorescence-activated cell sorter by removing cells with lineage (Lin) markers (CD3, CD4, CD5, CD8, Ter119, B220, Gr-1, and Mac-1 can be used) and collecting cells that express Sca-1 and c-kit markers. Purified mouse Lin−Sca-1+c-kit+ cells, or “LSK” cells, contain virtually all the repopulating ability, but selecting Thy1.1lo and Flk2− cells can be used to further enrich for stem cells. After transplantation, donor cells are commonly detected using the Ly5 allele, which is expressed on the cell surface (also known as CD45 or protein-tyrosine phosphatase receptor, type c) and can be detected by flow cytometry. Stable gene transfer to pluripotent HSCs with self-renewal capability can be demonstrated by analyzing integration sites in secondary recipients [5]. Mouse models are commonly used to develop improved methods for HSC purification and ex vivo transduction [44], to study methods for ex vivo expansion of HSCs [45], and more recently to compare the genotoxicity of viral

Gene transfer to large-animal repopulating cells Long-term marking frequencies obtained in gene transfer experiments in large-animal models such as canines and monkeys have more accurately predicted gene transfer frequencies obtained in clinical trials than have mouse studies. This is likely due to similar stem cell kinetics, as discussed above, and the fact that the cytokines used in these models during ex vivo transduction protocols have higher identity to human cytokines and thus also more similar function. Gene transfer to longterm repopulating cells in large animals models was initially difficult to achieve (reviewed in [56]), but more recently efficient gene transfer has been observed using improved protocols with CH-296 fibronectin fragment [57], typically used with a cytokine cocktail that includes SCF, MGDF, and Flt-3 ligand, and may also include G-CSF, IL-3, and IL-6. With sufficient ex vivo prestimulation and culture time, gammaretroviruses can now be used for stable polyclonal gene transfer to primate long-term repopulating cells at frequencies of 10–25% and in one case 60% in the peripheral blood. Gene transfer into baboons and rhesus macaques using HIV-derived lentiviral vectors has been inefficient, presumably due to post-entry restriction by Trim5α [58]. Hanawa et al. have achieved high levels of

Genetic Manipulation of Hematopoietic Stem Cells

multilineage gene marking (average 18%) into rhesus macaques using simian immunodeficiency virus-derived lentiviral vectors [59], which are not restricted by rhesus Trim5α. Similar gene transfer frequencies have been obtained using HIV-derived lentiviral vectors into pigtailed macaques (Macaca nemestrina), which are permissive for HIV-based lentiviral transduction ([60] and unpublished data by authors). In these two studies, amphotropic (simian immunodeficiency virus)- and VSV-G (HIV)-pseudotyped lentiviral vectors gave similar marking efficiencies with ex vivo culture times of 72 hours and 48 hours, respectively. The limiting factor(s) for HSC transduction is unclear in these largeanimal experiments, but it is interesting to note the similar transduction efficiencies using lentiviral vectors pseudotyped with the retroviral amphotropic envelope or with VSV-G. Transduction in the presence of CH-296 has been shown to allow saturation of the amphotropic vector in human CD34+ cells in vitro [61], so it will be of interest to determine whether transduction efficiencies can be further increased by combining vectors with different pseudotypes. Efficient polyclonal multilineage gene transfer to canine repopulating cells has been achieved using lentiviral vectors [62] and foamy virus vectors [22] using an even shorter 18-hour ex vivo culture protocol. In summary, efficient gene transfer to large animal repopulating cells can be achieved with gammaretroviral, lentiviral or foamy retroviral vectors. However, for lentiviral and foamy virus vectors, rapid ex vivo transduction protocols can be employed, which may enhance engraftment.

121

would be especially important for genetic and infectious diseases, in which only a reduced-intensity conditioning may be possible. Although self-renewal has been demonstrated ex vivo, attempts to expand longterm repopulating cells with cytokine cocktails have only allowed for modest increases in HSCs. Another approach is to more directly manipulate pathways involved in stem cell maintenance or expansion such as homeobox proteins, or Notch and Wnt signaling proteins. Homeobox or Hox genes regulate differentiation during embryogenesis; HoxB3 and HoxB4 are preferentially expressed in BM cells that are highly enriched for primitive cells. Expression of HoxB4 from a gammaretroviral vector resulted in a 40fold increase in mouse HSCs [65], and a Tat–HoxB4 fusion protein has been developed for ex vivo applications to avoid potential genotoxicity from permanent HoxB4 expression. The Wnt and Notch pathways are involved in stem cell maintenance and self-renewal. Wnt proteins and Notch ligands have also been used to expand repopulating cells, but the level of expansion observed was less dramatic. Expansion of gene-marked cells in vivo

The ability of hematopoietic cells to efficiently home to the BM and provide lifelong hematopoiesis has helped to establish ex vivo transduction as the most common method for gene transfer to HSCs. However, direct injection of vector preparations into recipients may be particularly advantageous for diseases such as Fanconi anemia, in which ex vivo manipulation of HSCs is problematic due to their fragility. Additional advantages are that enrichment and ex vivo culture of HSCs, which are expensive for large-animal and clinical studies, are not required, and loss of engraftment ability due to ex vivo culture might be avoided. Direct intrafemoral injection of lentiviral vectors pseudotyped with VSV-G resulted in low but persistent transduction levels in mouse HSCs [63], but VSV-G pseudotyped vectors are inactivated by human sera, so for clinical in vivo applications, alternate pseudotypes will be important. Ravin et al. evaluated the intravenous delivery of RD114-pseudotyped gammaretroviral vectors into 3-day-old SCID-X1 dogs [64]. In this model, gene-modified repopulating T cells have a selective advantage, and therapeutic levels of corrected T cells were achieved in three of four dogs. Therapeutic levels of T cells were not achieved in the dog that was administered the lowest dose of vector; the level of corrected T cells was very low and a limited number of clones was present. Surprisingly, in this study, a significant number of gene-modified B lymphoid and myeloid cells were observed. Since these cells are not expected to have a selective advantage, this suggests that gene transfer was relatively efficient. It will be interesting to determine whether in vivo delivery can be used to efficiently transduce HSCs in large-animal models and to determine whether lentiviral vectors have an advantage for in vivo delivery to HSCs, which are considered to be quiescent.

Increasing the percentage of gene-modified repopulating cells post transplant using in vivo selection could expand the potential of HSC gene therapy to hematopoietic diseases such as thalassemias, which require relatively high levels of gene transfer [66]. Positive selection of cells post-transplantation has been demonstrated using a method in which drugs that bind to and dimerize a modified receptor molecule transgene transmit a growth signal to the cell [67]. The small-molecule drugs are called chemical inducers of dimerization, and the receptors are modified to remove their extracellular domain and to contain binding regions for their small-molecule ligands. To date, this approach has been limited in that receptors that can expand HSCs have not been identified. Negative selection using alkylating agents that are toxic to stem cells such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) or temozolomide can be performed to expand gene-modified stem cells that are protected by a mutant O6-methylguanine-DNA methyltransferase (MGMT) transgene [68]. In this approach, the drug O6-benzylguanine inactivates endogenous MGMT protein but not the mutant MGMT protein. Following ex vivo gene transfer of the mutant MGMT transgene to HSCs, treatment with BCNU and O6-benzylguanine is toxic to cells that did not receive the MGMT transgene and results in expansion of genemodified cells. This approach allows for the durable expansion of repopulating cells in large-animal models (Fig. 10.4), demonstrating that selection occurs in very primitive cells (reviewed in [69]). MGMT-mediated selection might be used for gene therapy of hematopoietic diseases such as thalassemias by including therapeutic globin and MGMT transgenes together in a bicistronic vector. Gene transfer of MGMT to HSCs can also be used to protect the hematopoietic system from high-dose chemotherapy during treatment for solid tumors or leukemias. Early clinical studies explored the use of the multidrug resistance gene MDR1 for this purpose, but the marking was very low and in vivo selection was not observed. More recently, a clinical study has started enrolling patients to evaluate chemoprotection of the hematopoietic system using MGMT-mediated selection. In this study, a gammaretroviral vector will be used to transfer a mutant MGMT to patient CD34+ cells.

Expansion of gene-modified cells prior to infusion

Immune responses to transgenes

Ex vivo expansion of long-term repopulating cells prior to or during transduction could increase the number of infused cells and thus increase the engraftment of gene-modified cells. Infusion of a higher number of stem cells would more efficiently compete with endogenous stem cells so less conditioning and potentially less toxicity would be required. This

In many situations, the transgene delivered to HSCs will be foreign, and there is the potential for an immune response to gene-modified hematopoietic repopulating cells. In gene-marking studies, a foreign protein such as neomycin phosphotransferase or green fluorescent protein has been expressed in HSCs and their progeny. In cells that express these

In vivo delivery of transgenes to HSCs

Chapter 10

% EGFP expressing cells

122

100 80 60 40 20 0

TMZ/O6BG Granulocytes Lymphocytes

3 1 2 Years after transplantation

Fig. 10.4 MGMT-mediated in vivo selection. A dog was transplanted with autologous CD34+ cells transduced with a gammaretroviral vector expressing the P140K mutant MGMT and enhanced green fluorescent protein (EGFP). After hematopoietic reconstitution, in vivo selection was achieved using a series of chemotherapy treatments with the alkylating agent temozolomide (TMZ) and the MGMT inhibitor O6-benzylguanine (O6BG). Stable increases in gene-modified granulocytes and lymphocytes indicate selection of primitive long-term repopulating cells.

proteins, they can be processed and presented through MHC class I and II pathways and can trigger cytotoxic, helper, and memory T-cell responses. Processed peptides from green fluorescent protein have been shown to elicit T-cell immune responses in transplanted mice, baboons, and rhesus macaques. Immune responses can also be a problem for clinical applications where a patient may express a truncated form of the therapeutic transgene or may not express the transgene at all. In these cases, the immune system may recognize the therapeutic transgene as foreign. This has been observed in a canine preclinical model for Hurler’s syndrome, where a strong humoral response was associated with a loss of in vivo repopulating cells [70]. In settings where a myeloablative pretransplant conditioning regimen will be used, pre-existing immune cells are eliminated and a state of tolerance to the foreign transgene can be established. However, even when a high-intensity conditioning has been used, cytotoxic T cells specific to a foreign antigen have been observed [71]. Cyclosporine has been used successfully to inhibit the development of immune responses in canines after transplantation of gene-modified cells. Immune responses are not expected to occur in clinical gene therapy situations where there are severe preexisting immunodeficiencies such as for SCID-X1.

Clinical trials of HSC gene transfer Early studies The first clinical HSC gene-marking studies demonstrated that persistent gene marking could be obtained in human repopulating cells [10,11]. Early therapeutic clinical trials attempting to transduce HSCs were performed using the multidrug resistance gene MDR-1 [72–74]. The product of the MDR-1 gene, P-glycoprotein, is an energy-dependent drug efflux pump that reduces the intracellular concentrations of a wide range of drugs and xenobiotics including chemotherapy agents such as etoposide. The approach was to protect the hematopoietic system from the doselimiting myelosuppressive effects of chemotherapeutic agents used to eradicate tumors. In these studies, the gene transfer rates were very low, less than 1 in 104 cells, similar to earlier studies in the canine and primate models. Additional early clinical HSC gene transfer studies demonstrated low-level but persistent gene marking for ADA-SCID [75,76] and Gaucher’s disease [77], but therapeutic benefit was not achieved. In 1995, a report commissioned by the National Institutes of Health concluded that the low efficiency of gene transfer suggested that more

emphasis be put on basic aspects of gene transfer, novel vector development, and the study of stem cell biology, but that there was a clear and legitimate need for clinical studies to evaluate various aspects of gene therapy approaches (http://www.nih.gov/news/panelrep.html). Improvements to HSC gene transfer were made and several clinical studies were undertaken, including trials for Fanconi anemia [78], leukocyte adhesion deficiency [79], and acquired immune deficiency syndrome [80], but with no clear therapeutic benefit. To date, clinical trials for gene transfer to HSCs have used gammaretroviral vectors, but lentiviral vectors have been approved for HSC gene therapy for β-thalassemia [81] and also for HIV gene therapy. Table 10.1 summarizes recent clinical trials for HSC gene transfer. SCID-X1 The first clearly therapeutic HSC gene therapy trial was reported for SCID-X1 [12], which is caused by mutations in the common cytokine receptor γ chain (γc, IL2RG locus), resulting in the absence of mature T lymphocytes and NK cells. Corrected SCID-X1 lymphoid progenitors have a strong selective advantage due to the restoration of survival and proliferation signals mediated by the γc receptor. Patients with no HLAidentical siblings were eligible for the study and, due to the immunodeficiency, no pretransplant conditioning was given. Autologous CD34+ cells transduced with a gammaretroviral vector pseudotyped with the amphotropic envelope were used. Of the initial 10 patients treated, T-cell development occurred in nine, and T-cell reconstitution correlated with the dose of CD34+ cells infused [12]. In these studies, small numbers (1%) of transduced myeloid cells were detected, and analysis of vector integration sites by linear amplification-mediated PCR demonstrated polyclonal T-cell repopulation and transduction of primitive multipotential progenitors with self-renewal capacity [82]. An additional four patients were successfully treated by Gaspar and colleagues in the UK using a gammaretroviral vector pseudotyped with the GALV envelope [83]. These authors recently reported that, of a total of 10 patients now treated, six had restoration of immune function with no requirement for prophylactic immunoglobulin replacement [84]. The success of these studies was attributed in part to the improved transduction methods for gene delivery into CD34+ cells, which included CH-296 and cytokine cocktails that included SCF, MGDF, IL-3, and Flt-3 ligand. For both studies, the T-cell repertoire was diverse, and T-cell receptor excision circle (TREC) analysis identified naïve T cells after transplantation. Thus, a gene therapy approach may allow for better long-term thymopoiesis than is observed with HLA-identical and haploidentical allogeneic transplantation, in which hematopoiesis remains of host origin in the absence of pretransplant conditioning [85]. In two older patients treated at 15 and 20 years of age, SCID-X1 gene therapy failed [86]. In both patients, there was efficient gene transfer to CD34+ cells in vitro, and in one patient there were similar marking levels in B, NK, and myeloid cells to patients less than 1 year old that were successfully treated. Chinen et al. investigated gene therapy as a salvage treatment for three patients aged 10, 11, and 14 years that had previously received T-cell-depleted BM transplants from a parent, but had incomplete immunological reconstitution with frequent infections and chronic diarrhea [87]. In this study, G-CSF mobilized peripheral blood CD34+ stem cells were transduced with a GALV-pseudotyped gammaretroviral vector expressing γc and infused without prior conditioning. All three patients had detectable retroviral marking in myeloid and lymphoid lineages to at least 12 months post-transplant. The 10-year-old patient had relatively high gene marking in T cells consistent with a strong selective advantage in this lineage. In this 10-year-old patient, CD4+ and CD8+ counts increased, and CD4+CD45RA+ naïve cells and TRECs persisted, indicating restoration of thymic function. Vβ spectratype

4

2

3

2

1

1

2

SCID-X1

SCID-X1

SCID-X1

ADA

ADA

ADA

CGD

Gamma

Gamma

Gamma

Gamma

Gamma

>38 months

25, 26 years

Gamma

3.2 years

7, 30 months Gamma

10–14 years

15, 20 years

4–33 months

1–11 months* Gamma

Vector

BM

BM

GALV

GALV

GALV

Ampho

PB

BM

BM

BM

Ampho/ BM GALV GALV PB

GALV

Ampho

Env

G-CSF

None

None

None

G-CSF

None

None

None

Mobilization

5 days

NR†

96 hours

4 days

4 days

3 days/96 hours

3 days/96 hours

3 days

Time

Ex vivo culture

3, F, S, T

3, F, M, S/3, F, S, T

Cytokines

None

NR†

None

4%

3, F, S, T

F, M, S

3, F, S, T

3, F, S, T

4%/none 3, F, M, S/3, F, S, T None 3, 6, F, S, T

None

4%

FBS

Liposomal busulfan

Busulfan

Melphalan

Busulfan

None

None

None

None

Conditioning

10.5§

1.4‡

0.9, 8.6

29–31

2.8, 35

8–26

14–38*

0/1 complicating pancytopenia 2/2 complete clonal expansion 1/2 loss of transgene function, death

1/1 complete

2/2 complete

1/3 partial

0/2

4/4 complete

7/10 complete 2/10 partial 4 leukemias

Cells infused Immune (/kg*106) restoration

39.5–45% 3.6–5.1¶

9%

25–30%

21–25%

39–45%

13–30%

27–58%

10–40%*

Marking before infusion

3, interleukin-3; 6, interleukin-6, ADA, adenosine deaminase; Ampho, amphotropic; BM, bone marrow; FBS, fetal bovine serum; F, Flt3 ligand; GALV, gibbon ape leukemia virus; G-CSF, granulocyte colony-stimulating factor; M, megakaryocyte growth and development factor; NR, not reported; PB, peripheral blood, S, stem cell factor; SCID-X1, X-linked severe combined immunodeficiency; T, thrombopoietin. * For patients that were reported. † Not reported. ‡ CD34, ADA+ cells infused. § Total number of cells infused. ¶ CD34, gp91+ cells infused.

Ott et al., 2006 [94]

10

SCID-X1

Cavazzana-Calvo et al., 2000 [12], Hacein-BeyAbina 2003 [113], Ginn et al., 2005 [114] Gaspar et al., 2004 [83] Thrasher et al., 2005 [86] Chinen et al., 2007 [87] Aiuti et al., 2002 [90] Gaspar et al., 2006 [92] Engel et al., 2007 [115]

Patients Ages

Disease

Reference

HSC source

Table 10.1 Recent hematopoietic stem cell (HSC) clinical gene therapy studies

Genetic Manipulation of Hematopoietic Stem Cells

123

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analysis showed increased T-cell receptor diversity. Thus, a clear therapeutic benefit could be established in this patient, and the authors noted that in addition to the younger age, this patient also had previous full T-cell reconstitution from a prior haploidentical BM transplant. For SCID-X1, the capacity to reinitiate thymopoiesis may be compromised in older patients with loss of thymic potential. Thus, gene therapy should be initiated as early as possible, and previous thymic activity may help predict success in older patients. A combined 17 of the 20 enrolled SCID-X1 patients have been successfully treated in the French and UK studies. Unfortunately, the finding of leukemia in four patients from the French SCID-X1 trial, all of which were vector mediated, has dampened enthusiasm for retroviral gene therapy. One patient has now died as a result of the leukemia. However, the morbidity and mortality of this disease combined with the limitations of current transplantation protocols support the continued use and refinement of this approach. Hopefully, improved vector systems designed to reduce the potential for malignancy from vector-mediated insertional mutagenesis (discussed below) will reduce the risk of this severe adverse side-effect of an otherwise highly successful therapy. ADA deficiency In ADA-SCID, an accumulation of deoxyadenosine triphosphate induces cell death by apoptosis in lymphocyte precursors, impairing lymphocyte development and function. ADA-SCID was one of the first diseases proposed for clinical trials because the gene was available and could be expressed from a retroviral vector, the level of protein required for therapeutic effect was estimated at 10–15% of normal levels, and corrected T cells would have a selective advantage. T cells were used initially as targets for the first clinical study of gene transfer to ADAdeficient patients in 1990 [88], and Aiuti et al. observed that, following transplantation of lymphocytes in one patient, discontinuation of polyethylene glycol (PEG)-ADA enzyme replacement therapy led to immune reconstitution [89]. Thus for ADA, the enzyme replacement therapy likely reduces the survival advantage provided to gene-modified cells, and thus limits the potential therapeutic effect from ADA gene transfer. Aiuti et al. treated two patients using CD34+ BM cells transduced with a gammaretroviral vector and observed sustained engraftment and clinical improvement with restoration of ADA activity in peripheral blood lymphocytes [90]. Unlike previous studies, these ADA patients received nonmyeloablative pretransplant conditioning with busulfan (2 mg/kg/ day on days 3 and 2 before infusion), and did not receive PEG-ADA enzyme replacement therapy. Within the lymphocyte subsets, increased cell numbers were first detected in B cells and NK cells, followed by T cells. Both patients had corrected cells in multiple lineages including granulocytes, erythrocytes, megakaryocytes, and lymphoid cells. The frequency of gene-modified cells in the lymphoid subsets was higher, suggesting a stronger selective advantage for these cells. Virtually all NK cells in the peripheral blood and BM were transduced. In B lymphocytes, the frequency of transduced B cells was higher in the peripheral blood than in the BM, suggesting a growth or differentiation advantage for corrected peripheral B lymphocytes. These investigators have now treated a total of 12 patients without any evidence of leukemia. Integration site analyses on some of these patients have recently been published [91]. Gaspar et al. [92] treated a patient with no HLA-matched donor who had a poor response to PEGADA replacement therapy using a gammaretroviral vector pseudotyped with the GALV envelope. PEG-ADA was stopped 1 month prior to treatment, and autologous BM CD34+ cells were transduced and infused after the patient had been conditioned with melphalan at 140 mg/m2. Following gene therapy, T-cell counts and NK-cell counts rose,

CD27+CD45RO− T cell numbers increased, and TREC activity was detected 1 year post-transplant, indicating successful re-establishment of thymopoiesis. The Vβ T-cell repertoire was equivalent to age-matched controls, and vector integration analysis showed polyclonal engraftment of myeloid and lymphoid cells. Substantial but not complete recovery of immune function was observed and may have been limited by reduced thymic function as a result of prolonged PEG-ADA enzyme therapy before the gene therapy. Chronic granulomatous disease Chronic granulomatous disease is caused by a defect in the nicotinamide dinucleotide phosphate phagocyte oxidase (phox) complex, and approximately 70% of patients have mutations in the X chromosome gene encoding the gp91phox subunit (X-linked chronic granulomatous disease). As a result, the antimicrobial activity of phagocytes is impaired due to an inability to generate reactive oxygen species, and patients have recurrent infections with granulomatous lesions. Unlike SCID-X1 or ADASCID, gene transfer to myeloid cells is required and the transgene does not confer a growth advantage to gene-modified cells, so high levels of gene transfer are likely necessary to observe a therapeutic effect. Supporting this, early attempts at gene therapy for chronic granulomatous disease in the absence of conditioning resulted in low levels of gene transfer without therapeutic benefit [93]. Ott et al. treated two patients after nonmyeloablative conditioning with liposomal busulfan at 4 mg/kg/day on days 3 and 2 before infusion [94]. In this study, G-CSF-mobilized CD34+ were transduced with a gammaretroviral vector pseudotyped with the GALV envelope. In both patients, high levels of gene marking were observed and gene marking in peripheral blood leukocytes from patients 1 and 2 increased to 46% and 53%, and then decreased to 27% and 30%, respectively. Biochemical evidence of respiratory burst activity was demonstrated, and although the level of superoxide production was lower than in wild-type cells, clinical benefit was established for both patients. Analysis of vector integration sites initially showed a highly polyclonal repopulation, but after 3 months, clones with insertions in the MDS1-EVI1, PRDM16 and SETBP1 loci began to dominate. Transactivation of these genes by the strong spleen focus-forming virus promoter in the vector likely contributed to the expansion of these clones. A third patient was treated, and to date no leukemia has resulted and granulocyte numbers have not exceeded those before gene therapy, suggesting that these cells have so far retained normal homeostasis. It is unclear if the clinical benefit can be attributed to this expansion, but activation of host genes that lead to clonal expansion and may later lead to malignancy is a significant safety concern. One patient died of a severe bacterial sepsis after colon perforation. There was still very high gene marking in this patient, but the function of the transduced cells was greatly decreased. In this setting of clonal expansion, silencing of a dominant clone likely led to loss of protection, which has not yet been a problem in other trials.

Vector-mediated insertional mutagenesis Leukemia in the SCID-X1 trial The ability of retroviruses to efficiently integrate into the genome is also a disadvantage; retroviral vectors are insertional mutagens and can cause leukemias, as observed in the French SCID-X1 gene therapy trial. It has long been known that many gammaretroviruses (formerly oncoretroviruses) cause leukemia, but it was previously thought that replicationincompetent vectors had a low risk of insertional mutation leading to malignancy. Leukemias were not observed as a result of vector integration in previous clinical T-cell gene transfer experiments, possibly in

Genetic Manipulation of Hematopoietic Stem Cells

part due to the biology of T cells [96], Leukemias were also not observed in many HSC large-animal gene transfer experiments (reviewed in [41]). This is in contrast to replication-competent viruses, where multiple rounds of infection in vivo would greatly increase the risk for malignancy. Leukemia was observed in three of 10 rhesus macaques when replication-competent retrovirus contaminated a vector preparation used for gene transfer to enriched BM cells [95]. In the French SCID-X1 trial, there was no evidence of replicationcompetent retrovirus, and studies were undertaken to characterize vector integration sites to understand the etiology of the leukemias. Vector integrants were observed near the LMO2 and CCND2 genes. The LMO2 gene encodes a transcription factor involved in early hematopoiesis that had previously been involved in leukemogenesis via chromosomal translocation (reviewed in [97]). So far, there has not been any leukemia in the UK SCID-X1 gene therapy trial, and it is currently unclear what differences between this study and the French study could account for this. The approach and vector in both studies were similar, but in the UK trial the protocol included culture in serum-free medium, and a lower concentration of IL-3 was used. Additionally, in the French trial the vector was pseudotyped with an amphotropic envelope, and in the UK trial a GALV pseudotype was used. It is also unclear if the therapeutic transgene for γc was involved in the leukemias, possibly through aberrant IL receptor signaling. Expression of γc from a lentiviral vector was found to cause lymphoma in a mouse model [98], and a database of viral integrations from mouse tumors identified a leukemia with an integrations at both the IL2RG and LMO2 loci [99], suggesting that they could cooperate to contribute to leukemia. Overexpression of γc alone in human CD34+ cells had no effect on T-cell development in vitro, and it has been suggested that integration at LMO2 may represent the primary oncogenic event and that restoring IL-7 signaling with the γc transgene creates a preleukemic condition of immature cells under intense proliferative pressure [100]. It is clear from these studies that specific diseases may have inherent risks from a perspective of vector-mediated insertional mutagenesis, and that preclinical models may yield insights into the potential adverse side-effects of proposed clinical trials. Deregulation of host genes by vector proviruses An area of active investigation towards safer HSC gene therapy is the development and testing of vectors with an improved safety profile. Gammaretroviral vectors have a preference to integrate close to the promoters of genes [101], lentiviral vectors have a preference to integrate within highly expressed transcripts [102], and foamy virus vectors integrate less frequently near promoters than gammaretroviral vectors and less frequently than both gammaretroviral and lentiviral vectors within transcripts [103]. Although the mechanisms of these integration preferences are not yet well understood, they appear to be defined in part by tethering interactions between the preintegration complex and host proteins, and also by the state of host chromatin (reviewed in [104]). Integration by retroviral vectors can deregulate host genes by several mechanisms (Fig. 10.5), with the potential to lead to clonal dominance, as observed in the chronic granulomatous disease trial, and to leukemia as observed in the French SCID-X1 trial. Gammaretroviral vector integrants can deregulate the expression of genes over 500 kb from the insertion site [105], and a comparison of the frequency of integrants in canine repopulating cells shows that a significant percentage of gammaretroviral, lentivirus and foamy virus vectors are observed near genes that have been associated with malignancy [106]. Thus, vector designs that reduce the ability of integrated vector proviruses to deregulate the expression of nearby genes should reduce the potential for malignancy.

(a) //

Increased transcription from LTR

(b) //

Enhancer activation

(c)

Expression of a novel isoform

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(d) //

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Insertional inactivation // pA

Fig. 10.5 Deregulation of cellular genes by integrated vector proviruses. There are several mechanisms whereby integrated vector proviruses can deregulate the gene expression of proto-oncogenes, tumor suppressors or genes involved in hematopoiesis. Four examples of deregulation are illustrated. The integrated vector provirus is indicated by two long-terminal repeats (LTRs) with an internal transgene indicated by a hatched box. The cellular gene promoter and exons are indicated by a black box and gray boxes, respectively. In (a), transcription initiated from the vector 3′ LTR transcribes the cellular gene at increased levels. In (b), enhancers in the vector LTRs activate transcription from the cellular promoter increasing transcription. In (c), transcription from the vector 5′ LTR results in a spliced mRNA that creates a novel truncated isoform of the cellular gene that may have distinct properties from the native form, including oncogenicity. In (d), the vector provirus is integrated between the second and third exons, thereby disrupting transcription of the cellular gene via premature polyadenylation using the 3′ LTR polyadenylation signal.

Approaches to reduce the potential for vector-mediated malignancy There are several modifications that can be made to improve vector safety, including using SIN vectors. Unlike the LTR-driven vectors used in the SCID-X1 trials, in SIN vectors the promoter–enhancer sequences are removed, greatly reducing the potential for transcriptional activation by the 3′ LTR and for enhancer activation by both LTRs. Inserting insulator sequences into the LTRs that interfere with enhancer activation of nearby genes has also been proposed as a safety feature, and experiments in a human T-cell line suggest that lentiviral vectors with a chicken HS4 insulator may have a reduced potential for clonal dominance [107]. A straightforward approach to improve safety is to use lineage-specific promoters in the context of a SIN vector to restrict gene expression to the desired hematopoietic lineage. This should reduce the possibility of deregulating proto-oncogenes in stem cells and in lineages where gene expression is not required (reviewed in [108]). Efforts to target the integration of retroviral vectors to defined locations by modifying components of the retroviral preintegration complex have also been explored. Yeast LTR retrotransposons provide a compelling precedent for targeted integration into a eukaryotic genome. Yeast retrotransposons have many similarities to retroviruses yet have a remarkable specificity for integration. The Ty3 retrotransposon integrates in a small window, two or three nucleotides upstream of Pol III transcripts. To date, targeting the integration of retroviral vectors has been much less efficient. In one study, a zinc finger protein with a unique address was fused to the lentiviral Pol [109]. The integration profile was modified, but titers were greatly reduced, limiting the potential of this approach.

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Another approach is to avoid integration by correcting HSC genes or introducing therapeutic genes at specific chromosomal locations using a gene targeting approach. The frequency of conventional gene targeting by transfection is low in human primary cells, but targeting frequencies can be greatly increased by cleaving the target site with a nuclease to stimulate homologous recombination. Modification of the γc locus was obtained in up to 18% of human hematopoietic cells using a zinc finger nuclease [110], and targeted gene addition using this approach is also remarkably efficient in hematopoietic cell lines [111]. Integrase-deficient lentiviral vectors have been developed for the efficient delivery of DNA donor templates and zinc finger nucleases to stem cells. Nonintegrating lentiviral vectors that express zinc finger nucleases have led to targeting in up to 30% of cells in hematopoietic cell lines, with targeted gene addition in up to 5% of human hematopoietic progenitor cells [112].

Summary and future directions Recent advances in the ability to enrich and transduce human repopulating cells have led to recent successes in HSC gene therapy. The unfortunate occurrence of leukemia in the French SCID-X1 trial has stimulated research into defining the relative safety of different gene transfer approaches and developing safer vectors. Innovative approaches to overcome this challenge are being explored, including targeted integration and gene-targeting approaches. As gene transfer efficiencies increase and the safety of gene transfer is improved, we should expect that additional diseases will be cured using HSC gene therapy. Note added in proof: One case of leukemia has now been reported in the UK SCID-X1 trial.

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80. Kohn DB, Bauer G, Rice CR et al. A clinical trial of retroviral-mediated transfer of a rev-responsive element decoy gene into CD34(+) cells from the bone marrow of human immunodeficiency virus1-infected children. Blood 1999; 94: 368–71. 81. Bank A, Dorazio R, Leboulch P. A phase I/II clinical trial of beta-globin gene therapy for betathalassemia. Ann NY Acad Sci 2005; 1054: 308– 16. 82. Schmidt M, Hacein-Bey-Abina S, Wissler M et al. Clonal evidence for the transduction of CD34+ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial. Blood 2005; 105: 2699–706. 83. Gaspar HB, Parsley KL, Howe S et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 2004; 364: 2181–7. 84. Schwarzwaelder K, Howe SJ, Schmidt M et al. Gammaretrovirus-mediated correction of SCIDX1 is associated with skewed vector integration site distribution in vivo. J Clin Invest 2007; 117: 2241–9. 85. Fischer A, Le Deist F, Hacein-Bey-Abina S et al. Severe combined immunodeficiency. A model disease for molecular immunology and therapy [Review]. Immunol Rev 2005; 203: 98–109. 86. Thrasher AJ, Hacein-Bey-Abina S, Gaspar HB et al. Failure of SCID-X1 gene therapy in older patients. Blood 2005; 105: 4255–7. 87. Chinen J, Davis J, De Ravin SS et al. Gene therapy improves immune function in preadolescents with X-linked severe combined immunodeficiency. Blood 2007; 110: 67–73. 88. Blaese RM, Culver KW, Miller AD et al. T lymphocyte-directed gene therapy for ADA-SCID; initial trial results after 4 years. Science 1995; 270: 475–80. 89. Aiuti A, Vai S, Mortellaro A et al. Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement [Letter]. Nat Med 2002; 8: 423–5. 90. Aiuti A, Slavin S, Aker M et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002; 296: 2410–13. 91. Aiuti A, Cassani B, Andolfi G et al. Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy. J Clin Invest 2007; 117: 2233–40. 92. Gaspar HB, Bjorkegren E, Parsley K et al. Successful reconstitution of immunity in ADA-SCID by stem cell gene therapy following cessation of

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PEG-ADA and use of mild preconditioning. Mol Ther 2006; 14: 505–13. Malech HL, Maples PB, Whiting-Theobald N et al. Prolonged production of NADPH oxidasecorrected granulocytes after gene therapy of chronic granulomatous disease. Proc Natl Acad Sci U S A 1997; 94: 12133–8. Ott MG, Schmidt M, Schwarzwaelder K et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006; 12: 401–9. Vanin EF, Kaloss M, Broscius C, Nienhuis AW. Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis. J Virol 1994; 68: 4241–50. Recchia A, Bonini C, Magnani Z et al. Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells. PNAS 2006; 103: 1457–62. Nam CH, Rabbitts TH. The role of LMO2 in development and in T cell leukemia after chromosomal translocation or retroviral insertion [Review]. Mol Ther 2006; 13: 15–25. Woods NB, Bottero V, Schmidt M, von Kalle C, Verma IM. Gene therapy: therapeutic gene causing lymphoma. Nature 2006; 440: 1123. Dave UP, Jenkins NA, Copeland NG. Gene therapy insertional mutagenesis insights. Science 2004; 303: 333. Pike-Overzet K, de Ridder D, Weerkamp F et al. Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy. Leukemia 2007; 21: 754–63. Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome are favored targets for MLV integration. Science 2003; 300: 1749–51. Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 2002; 110: 521–9. Trobridge GD, Miller DG, Jacobs MA et al. Foamy virus vector integration sites in normal human cells. PNAS 2006; 103: 1498–503. Lewinski MK, Bushman FD. Retroviral DNA integration – mechanism and consequences [Review]. Adv Genet 2005; 55: 147–81.

105. Kustikova O, Fehse B, Modlich U et al. Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking. Science 2005; 308: 1171–4. 106. Beard BC, Keyser KA, Trobridge GD et al. Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, and foamy virus. Hum Gene Ther 2007; 18: 423–34. 107. Evans-Galea MV, Wielgosz MM, Hanawa H, Srivastava DK, Nienhuis AW. Suppression of clonal dominance in cultured human lymphoid cells by addition of the cHS4 insulator to a lentiviral vector. Mol Ther 2007; 15: 801–9. 108. Chang AH, Sadelain M. The genetic engineering of hematopoietic stem cells: the rise of lentiviral vectors, the conundrum of the LTR, and the promise of lineage-restricted vectors. Mol Ther 2007; 15: 445–56. 109. Tan W, Dong Z, Wilkinson TA, Barbas CF III, Chow SA. Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells. J Virol 2006; 80: 1939–48. 110. Urnov FD, Miller JC, Lee YL et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005; 435: 646–51. 111. Moehle EA, Rock JM, Lee YL et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. PNAS 2007; 104: 3055–60. 112. Lombardo R, Beausejour C, Genovese P et al. Efficient gene correction and targeted gene addition in human stem cells using engineered zinc finger nucleases and integrase-defective lentiviral vectors. Mol Ther 2007; 15(Suppl 1): S121, #321. 113. Hacein-Bey-Abina S, von Kalle C, Schmidt M et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302: 415–19. Erratum in Science 2003; 302: 568. 114. Ginn SL, Curtin JA, Kramer B et al. Treatment of an infant with X-linked severe combined immunodeficiency (SCID-X1) by gene therapy in Australia. Med J Aust 2005; 182: 458–63. 115. Engel BC, Podsakoff GM, Ireland JL et al. Prolonged pancytopenia in a gene therapy patient with ADA-deficient SCID and trisomy 8 mosaicism: a case report. Blood 2007; 109: 503–6.

Section 2b Immunology

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Paul J. Martin

Overview of Hematopoietic Cell Transplantation Immunology

Introduction Immunology plays a central role in allogeneic hematopoietic cell transplantation (HCT). Any appreciation of the immunological mechanisms involved in engraftment, graft-versus-host disease (GVHD), control of malignancy, the development of tolerance, and immune reconstitution requires some understanding of the immunogenetic basis for immune reactions provoked by grafting tissue from one individual to another. Insight into the cellular basis of alloreactivity requires an understanding of immune recognition, the development of the immune system, and the nature of immune responses. This chapter will serve to introduce the reader to the immunology of HCT. Citations are made to selected reviews from recent literature and to other chapters that provide detailed information.

Fundamental differences between marrow transplantation and solid organ transplantation HCT differs fundamentally from grafting of most other organs. In solid organ transplantation, the graft generally contains only limited numbers of cells with immunologic function. The primary clinical concern rests with preventing rejection by the recipient immune system. Lifelong administration of immunosuppressive medication is generally required to prevent rejection not only by cellular and humoral mechanisms in the recipient at the time of transplantation, but also by those generated from recipient-derived immunologic precursors after transplantation. The preparative regimen administered before HCT (see Chapters 21–24) eliminates most, though not all, precursors and mature cells of the recipient immune system. The graft contains large numbers of precursors and mature cells that replace those of the recipient. Thus, the immune system in the recipient is generated by the graft and originates from the donor. The primary clinical concerns rest not only with preventing graft rejection by recipient cells that survive the conditioning regimen, but also with preventing donor cells from causing immunemediated injury (GVHD) in the recipient, while allowing immunologic reconstitution for recognition and control of pathogens. Immunosuppressive medications (see Chapter 84) are administered after transplantation primarily to prevent GVHD. Eventually it becomes possible to discontinue such treatment. The subsequent persistence of engraftment

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

and the absence of GVHD, together with recovery of host immune defenses, indicate that a state of immunological “tolerance” has been achieved between the donor and recipient.

Transplantation antigens Genes that encode major histocompatibility antigens Immune reactions provoked by grafting tissue from one individual to another are caused by transplantation or histocompatibility antigens (see Chapter 12). Genes encoding transplantation antigens are located both within and outside the major histocompatibility complex (MHC). The human MHC is located on the short arm of chromosome 6 and contains a series of genes encoding two distinct types of highly polymorphic cell surface glycoprotein termed human leukocyte antigen (HLA). HLA class I antigens contain a single polymorphic α chain noncovalently associated with β2-microglobulin. HLA class I antigens encoded by three loci termed HLA-A, HLA-B and HLA-C are known to provoke immune reactions in HCT. The role of antigens encoded by other class I loci has not yet been defined. As of May 2007, 405 HLA-A alleles, 729 HLA-B alleles, and 219 HLA-C alleles with protein-changing exon polymorphisms have been described. HLA class II antigens are encoded by three clusters of loci (HLA-DR, -DQ, and -DP) and contain a single α chain noncovalently associated with a β chain. The α chains of the HLA-DR, -DQ, and -DP antigens are encoded by the respective DRA, DQA1, and DPA1 genes, while the β chains are encoded by the respective DRB, DQB1, and DPB1 genes. Nine distinct DRB genes encode polymorphic HLA-DR β chains. Antigens encoded by the DRB1, -DQ, and -DP genes are known to provoke reactions in HCT. The role of antigens encoded by other DRB genes has not yet been defined. As of May 2007, 2 HLA-DRA alleles, 421 HLADRB1 alleles, 25 DQA1 alleles, 59 DQB1 alleles, 14 DPA1 alleles and 113 DPB1 alleles with protein-changing exon polymorphisms have been described. HLA matching The genes encoding HLA class I and class II antigens are tightly linked, such that they are inherited in families as “haplotypes” with low recombination frequencies. An HLA-matched sibling can be readily identified by an informative family study, especially if family members are HLAheterozygous and if the parental haplotypes can be identified by reliable markers and clearly assigned by segregation analysis. For a given patient there is a 0.25 probability that any one sibling inherited the same

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paternal haplotype and the same maternal haplotype, thereby being “HLA-genotypically identical.” Siblings identified this way are identical not only for the polymorphisms detected by HLA typing, but also for all other polymorphisms in the MHC. The term “HLA-matched” has a different significance when applied to unrelated individuals. In this context, matching can be defined only with respect to the polymorphisms encompassed by the testing. Unrelated individuals who are HLA-phenotypically identical can have MHC disparity that is not detected by the methods used for HLA typing. Because of the highly polymorphic nature of HLA antigens, the probability that two unrelated individuals will have the same HLA-A, -B, -C, -DR, -DQ and -DP alleles is extremely low. Structure of HLA and cell biology of antigen presentation MHC class I and class II antigens have similar structures [1]. The general structure consists of a floor formed by eight antiparallel β strands overlaid by two α helices in antiparallel orientation to each other, leaving a groove or cleft between them for binding of small peptides. The polymorphic residues of HLA molecules are located predominantly in the β-strand floor and in positions on the α helices that point into the groove between the two helices. MHC class I and class II molecules bind short peptides for display at the cell surface where they can be recognized by T cells (Fig. 11.1 [1,2]). Peptides that bind MHC class I molecules are generated either from exogenous proteins internalized by the endocytic “cross-presentation” pathway [3] or from endogenous proteins [4]. Endosomal proteases such as cathepsin S cleave internalized proteins into peptides that are then bound to MHC class molecules. Internalized proteins can also be transferred to the cytosol. Ubiquitin-modified proteins of exogenous or endogenous origin are degraded in the cytosol by proteasomes that have a preference for the generation of short peptides containing a hydrophobic or basic residue at the carboxy terminus. The resulting peptides are transferred into the lumen of the endoplasmic reticulum by an adenosine triphosphate-dependent transport mechanism. The transport mechanism functions most efficiently with peptides containing 8–12 amino acids with a hydrophobic residue at the carboxy terminus. Binding of peptides to MHC class I molecules is dictated by the way that the side chains of specific “anchor” residues of the peptide fit into “pockets” formed by polymorphic residues lining the groove between the α helices of the class I molecule. Thus, each specific MHC molecule displays a characteristic preference for peptides with distinctive anchor residues. For example, HLA-A2 molecules preferentially bind nine-mer peptides with leucine or methionine at position two and valine or leucine at the carboxy terminal position nine, while HLA-B35 molecules preferentially bind peptides with proline at position two and a hydrophobic residue at position nine. Peptides that bind MHC class II molecules generally originate from extracellular proteins that are taken into cells by endocytosis [2]. Acidification of endosomes activates proteases that degrade internalized proteins into peptides. Peptides bind MHC class II molecules when endosomes fuse with vesicles containing newly synthesized membrane proteins being transported to the cell surface. Binding of peptides to MHC class II molecules is dictated by the way that the side chains of specific residues of the peptide fit into pockets formed by polymorphic residues lining the groove between the α helices of the class II molecules. Minor histocompatibility antigens Minor histocompatibility antigens (mHAs) originate from genetic polymorphisms outside the MHC [5]. mHAs can be generated by a variety of mechanisms, including gene insertions and deletions, differences

between Y and X chromosome homologs, amino acid coding changes resulting from single nucleotide polymorphisms, and post-translational modifications of amino acids. Proteins that give rise to mHAs are processed either through the endosomal or cytosolic pathway to generate peptides that are presented at the cell surface by MHC class I molecules or through the endosomal pathway to generate peptides that are presented by MHC class II molecules. Recognition of a minor antigen by T cells is specified by both the polymorphic peptide and the MHC molecule to which it is bound. Polymorphisms producing a single amino acid substitution in a peptide presented by MHC molecules can be sufficient to generate a T-cell alloimmune response in the absence of any MHC disparity. Only a few mHAs have been defined biochemically. Genetic studies in mice have identified more than 40 loci that encode mHAs, each typically having two known alleles. Human genomes contain more than 15,000 protein-changing single nucleotide polymorphisms. Because these polymorphisms are distributed throughout the entire genome, siblings other than identical twins always have considerable disparity for mHAs, even when they are HLA-matched, but the immune system responds to only a small number of “immunodominant” antigen mismatches between the donor and recipient. Differences in responses against major and minor antigens Immune responses against major histocompatibility antigens differ greatly from responses against mHAs. Generation of an in vitro response against major histocompatibility antigens does not require in vivo priming, and the precursor frequency of T cells that respond to any given major histocompatibility antigen has been estimated to be as high as 1–10%. Major histocompatibility antigens also induce alloimmune antibody responses detectable by serologic testing. Generation of an optimal in vitro response against mHAs requires in vivo priming. The precursor frequency of T cells that respond to MHC-identical stimulators with disparity for multiple mHAs after priming has been estimated at 0.01– 0.10%. mHAs generally do not induce alloimmune antibody responses detectable by serologic testing. At least two mechanisms might account for differences in the strength of immune responses against major histocompatibility antigens compared with mHAs. First, MHC molecules encoded by any given allele contain a variety of different peptides, whereas mHAs generated by single nucleotide polymorphisms involve a single specific peptide. For this reason, the variety of epitopes and numbers of responding T cells are far greater for major histocompatibility antigens than for mHAs. Second, certain major histocompatibility determinants are present on all MHC molecules encoded by a given allele, independent of the peptides that are bound between the α helices, whereas any given minor histocompatibility epitope is present only on the small fraction of MHC molecules that contain a specific polymorphic peptide. For this reason, the cell surface density of epitopes can be much greater for major histocompatibility antigens than for mHAs.

Movement of cells in the immune system Development of an immune response requires highly orchestrated patterns of cellular migration throughout the body tissues [6]. These tissues include the thymus and marrow, representing the primary lymphoid organs where T and B cells are respectively produced, the spleen, lymph nodes and intestinal Peyer’s patches, representing secondary lymphoid organs where lymphoid cells mature and initiate immune responses, and other tissues, where immune defenses are brought to bear against pathogens. During the afferent phase of the immune response, lymph nodes function as central meeting points where highly dynamic processes of

Overview of Hematopoietic Cell Transplantation Immunology

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Virus Receptor Phagocytosis Cytoplasm Nucleus

Exocytosis Endoplasmic reticulum

Viral RNA or DNA

HLA–peptide complex

Replication Viral mRNA

Golgi apparatus Defective protein HLA class I molecule

Protein Viral mRNA

Fig. 11.1 Mechanisms of peptide presentation by major histocompatibility complex (MHC) class I and class II molecules. (a) Peptide presentation by MHC class I molecules occurs through degradation of endogenous and exogenous (e.g. viral) proteins in the cytosol. Peptides degraded by proteasomes are transported from the cytosol into the endoplasmic reticulum and inserted into the binding groove of MHC class I molecules, which are then transported through the Golgi apparatus to the cell surface. (b) Peptide presentation by MHC class II molecules occurs through degradation of extracellular proteins in primary lysosomes. MHC class II molecules are synthesized in the endoplasmic reticulum and delivered through the Golgi apparatus to the lysosomal compartment. HLA-DM molecules are delivered through transport vesicles to the same compartment where they facilitate loading of peptide into the binding groove of MHC class II molecules, which are then transported to the cell surface. Adapted from Klein and Sato [1] with permission. Copyright (2000), Massachusetts Medical Society. All rights reserved.

Proteasome Peptide Proteasome

(a) Extracellular self or foreign protein

Early endosome

Cytoplasm

Exocytosis

Nucleus

MHC class II compartment

Primary lysosome HLA class II molecule

Degradation of protein

HLA-DM

Exocytosis HLA–peptide complex HLA-DM

(b)

cell movement allow responding T and B cells to encounter antigenpresenting cells (APCs) that have migrated from the periphery [7,8]. During the efferent phase of the immune response, activated T cells leave the lymph nodes and migrate to the peripheral tissues, and activated B cells form germinal centers for production of antibody. Movement of T cells across vascular endothelium into lymph nodes during the afferent phase of the immune response and into peripheral tissues during the efferent phase is regulated by selectins [9], integrins [10], and chemokines [11–13]. Movement of T cells out of secondary lymphoid organs is regulated by sphingosine-1-phosphate receptors [14]. In addition, retinoic acid, a vitamin A metabolite, facilitates homing of T cells into the intestine, whereas activated vitamin D facilitates homing of T cells into the skin.

Innate and adaptive immune responses Transplantation evokes both innate and adaptive or acquired immune responses. In general, the innate immune system is poised for immediate

Endoplasmic reticulum HLA-DM

responses against pathogens, whereas acquired immune responses are activated more slowly over a period of days. The receptors involved in innate immunity are encoded directly in the germline and typically recognize Toll-like receptor family members and other invariant molecules involved in the pathogenic effects or survival of invading organisms [15]. The receptors involved in adaptive immunity are encoded by the rearrangement and selection of germline genes and typically recognize foreign “nonself” epitopes, regardless of whether or not they originate from a pathogenic organism. Innate immune responses are generally activated through a common signaling pathway involving an adapter molecule termed MyD88 [16]. Innate immune responses occur without proliferation of the effector cells, whereas adaptive immune responses are activated through the clonal expansion of precursors followed by differentiation into effectors. Innate immune responses regulate adaptive immune responses through the activation of dendritic cells that are primarily responsible for presentation of antigens. In this way, innate immunity serves as an alarm that signals the “danger” posed by an invading organism, thereby evoking an adaptive immune response

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against the organism [17]. Barrier organs such as the intestinal tract, skin, and lung have highly sophisticated mechanisms that regulate interactions between the innate and adaptive immune systems to control invading pathogens without causing local damage [18–21].

Regulation of natural killer cell responses Natural killer (NK) cells are a component of the innate immune system. These cells emerge from the marrow, develop in lymph nodes and tonsils [22], and are characterized by the expression of CD (cluster of differentiation) 56 and CD16, and by absence of CD3. NK cells mediate their effects through cytotoxic activity and production of cytokines such as interferon-gamma (IFN-γ) [23], tumor necrosis factor-alpha (TNF-α) [24], and granulocyte macrophage colony-stimulating factor. The importance of NK cells for transplantation comes from their ability to recognize MHC class I alloantigens. Human NK cells collectively express at least four different types of activating receptor and at least two types of inhibitory receptor. Killer immunoglobulin-like receptors (KIRs) with cytoplasmic tails containing immunoreceptor tyrosine-based activation motifs (ITAMs) and certain C-type lectins in the CD94 family activate NK cells after binding to MHC molecules. NKG2D is an NK-activating receptor that recognizes MHC class I-like molecules that are expressed in response to cellular stress [25]. KIRs with cytoplasmic tails containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and Ctype lectins other than NKG2C and NKG2D in the CD94 family inhibit activation of NK cells. Genes that encode both activating and inhibitory KIRs are located in a cluster on the short arm of chromosome 19, while genes that encode members of the CD94 family are located in a cluster on the short arm of chromosome 12. The balance of activation and inhibitory signals mediated through cell surface receptors regulates NK function, and inhibitory signals dominate the effect of activation signals [26] (Fig. 11.2 [27]). Mature NK cells must develop self-tolerance through expression of at least one inhibitory

Natural killer cell

Killeractivating receptor

Natural killer cell

Killerinhibitory receptor No attack

Ubiquitous molecule

Kill

MHC class I molecule

Normal cell

Abnormal cell lacking MHC class I molecules

Perforin and granzymes

Fig. 11.2 Activation and inhibition of natural killer (NK) cells. A variety of cell surface ligands can bind killer-activating receptors on NK cells. Normal cells express major histocompatibility complex (MHC) class I molecules that bind to killer inhibitory receptors on NK cells, thereby overriding the activation signal and preventing a cytotoxic attack. Abnormal cells that lack MHC class I molecules are susceptible to attack by NK cells because the activation signal is not blocked by inhibitory signals delivered through killer inhibitory receptors. Adapted from Delves and Roitt [27] with permission. Copyright (2000), Massachusetts Medical Society. All rights reserved.

receptor that recognizes a ligand on host cells [28]. The expression of this receptor prevents the NK cell from killing targets that have the inhibitory ligand, which is often an MHC class I molecule. In this way, self-MHC molecules shield potential targets from attack by NK cells. When the self-MHC molecule that binds the inhibitory receptor is absent, for example on a tumor cell or virally infected target, the NK cell is activated, and the target cell is killed.

Self-tolerance and alloantigen recognition by T cells Molecules used for recognition of antigens Adaptive immune responses are orchestrated and mediated by T and B lymphocytes [27]. In T cells, immune recognition is mediated by the T-cell receptor (TCR); in B cells, immune recognition is mediated by immunoglobulin molecules. In general, each T cell and B cell expresses only one type of receptor for recognizing antigen epitopes. Both types of cell utilize similar mechanisms for generating an enormous diversity of clonally distributed receptors. This diversity is generated initially by somatic rearrangements that link V (variable), D (diversity), and J (joining) segments in various combinations to encode a large repertoire of distinct clonally distributed receptors among T cells and B cells. Selection of T cells in the thymus Precursors destined to become T cells originate from stem cells in the bone marrow and migrate to the thymus (Fig. 11.3 [27,29]). Within the thymus, developing T cells that express receptors capable of recognizing foreign peptide antigens in association with the organism’s own “self”-MHC molecules survive and are selected for export to the peripheral blood, lymph nodes, spleen, and other organs [30]. This process of “positive selection” is mediated primarily by thymic cortical epithelial cells. Before export to the periphery, developing T cells that express receptors with high affinity for peptide–MHC complexes or “self”-antigens on adjacent cells are deleted by a process termed “negative selection” [30]. Negative selection is mediated most efficiently by marrow-derived dendritic cells and also by thymic medullary epithelium. In particular, thymic medullary epithelial cells mediate the negative selection of self-reactive T cells by promiscuous expression of proteins that are “tissue-specific” for organs outside the thymus [31]. Interactions with thymic epithelial cells can also cause developing T cells to become nonresponsive or “anergic” to self-MHC molecules, and cortical epithelium can induce the development of regulatory T cells that can suppress responses against self-MHC molecules [32]. Negative selection, induction of nonresponsiveness, and generation of regulatory T cells in the thymus represent the principal “central” mechanisms for establishing self-tolerance (see Chapter 15). Despite the redundancy of mechanisms for inducing central tolerance, some self-reactive T cells emerge from the thymus and migrate to the periphery. Presentation of self-antigens by resting or quiescent dendritic cells outside the thymus and by lymph node stromal cells in the absence of alarm signals from an innate immune response has been proposed as a major mechanism of “peripheral” tolerance to explain why self-reactive T cells that escape negative selection in the thymus do not cause immunologic injury under normal conditions [33–35]. After presentation of self-antigens by quiescent dendritic cells, T cells proliferate vigorously, but during persistent exposure to self-antigen, they are then deleted, become anergic or differentiate as regulatory T cells [6,35,36]. Alarm signals from an innate immune response induce activation of APCs and

Overview of Hematopoietic Cell Transplantation Immunology

CD8

Cortex T-cell receptor

135

Medulla Dendritic cell or macrophage

MHC

MHC T cell CD8 or CD4

Thymus gland

T-cell receptor

CD4 Positive selection Cortical epithelial cell

Cortex T cell

Medulla

T cell

Apoptosis

CD8 and CD4

T cell

Apoptosis Negative selection

Positive selection

T cell

CD8 or CD4 Surviving cells leave the thymus

Fig. 11.3 Positive and negative selection of T cells in the thymus. T cells must pass two tests as they develop the ability to express a receptor that recognizes major histocompatibility complex (MHC)–peptide complexes. First, immature T cells that express both CD4 and CD8 must have a receptor that recognizes an MHC–peptide complex on thymic cortical epithelial cells in order to survive. Those that fail this test die through apoptosis, as shown for the cell at the lower left. Those that pass this test are positively selected to travel to thymic medulla where expression of CD4 or CD8 is lost, as shown for the cells at the upper left. Maturing T cells that express CD4 or CD8 and have a receptor that binds too well with an MHC–peptide complex on medullary dendritic cells are negatively selected and die through apoptosis, as shown for the cell at the lower right. Maturing T cells that express CD4 or CD8 and have a receptor that binds weakly with an MHC–peptide complex on medullary dendritic cells escape negative selection and are exported to the periphery, as shown for the cell at the upper right. Adapted from Delves and Roitt [27] with permission. Copyright (2000), Massachusetts Medical Society. All rights reserved.

convert otherwise tolerogenic signals into a productive adaptive immune response.

Activation of T-cell responses by alloantigens Interactions between APCs and T cells Dendritic cells, B cells, and macrophages function as APCs. Among these, dendritic cells have by far the most potent activity as APCs. Different types of dendritic cell arise from marrow cells through distinct developmental pathways, and in many tissues, dendritic cells develop locally from a pool of resident precursors [37,38]. Dendritic cells collect antigen at sites of infection in peripheral tissues and then migrate to lymph nodes. Upon arrival in the lymph nodes, dendritic cells present antigen or transfer antigen to other cells for presentation to T cells and B cells [38]. Activation of an alloimmune response begins with binding between MHC–peptide complexes on APCs and the TCR on T cells [39]. Interaction between TCR and MHC–peptide complexes requires close apposition between T cells and APCs. Adhesive interactions exemplified by the respective binding of CD11a/CD18 (leukocyte functionassociated antigen-1 [LFA-1]) and CD2 on T cells with CD54 (intracellular adhesion molecule-1) and CD58 (LFA-3) on APCs help to overcome the repulsive forces created by the net negative surface charge of these cells [40,41]. Optimal activation of a T-cell immune response typically involves three-way interactions between APCs, CD4 cells, and CD8 cells [42,43]. CD4 cells are activated by dendritic cells that present MHC class II alloantigens and mHAs bound to self-MHC class II molecules. Direct

binding between MHC class II molecules on APCs and CD4 molecules on T cells facilitates this interaction. Activation of CD4 cells by antigen increases expression of CD154, also known as CD40 ligand or gp39. CD154 on activated CD4 cells binds to CD40 on APCs, which causes APCs to increase expression of CD86, also known as B7-2. CD86 molecules on APCs bind to CD28 on T cells, providing “costimulation” signals that augment cellular responses, especially under conditions of suboptimal TCR signaling or low precursor frequency [44,45]. Costimulatory signaling also results from binding of certain TNF family members to receptors on T cells [24,46]. CD8 cells are activated by dendritic cells that present MHC class I alloantigens and mHAs bound to self-MHC class I molecules. Direct binding between MHC class I molecules on APCs and CD8 molecules on T cells facilitates this interaction. CD4 cells help CD8 responses by increasing costimulation through APCs and by secretion of cytokines such as interleukin-2 (IL-2). In some situations, CD8 cells produce IL-2 and other cytokines with helper functions.

Signal transduction leading to activation of T cells Signal transduction in T cells leads to transcriptional activation of numerous genes by activator protein-1 (AP-1), nuclear factor-kappa B (NFκB), and nuclear factor of activated T cells (NF-AT) [47,48]. In broad outline, the process begins by the formation of “immunologic synapses” that concentrate TCR–CD3 complexes, CD2, and CD4 or CD8 within small regions of the membrane. Molecules associated with the cytoplasmic domains of TCR–CD3 complexes and CD4 or CD8

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assemble as a scaffold that initiates a cascade of protein tyrosine phosphorylation events leading to activation of p21 ras and phospholipase Cγ. The p21 ras pathway activates expression of Fos, which functions as a component in the AP-1–promoter complex for transcription of IL-2 and other genes involved in activation. Phospholipase Cγ cleaves phosphoinositide-4,5-diphosphate (PI4,5-P2) in the plasma membrane to form diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). DAG activates protein kinase C isoforms that regulate p21 ras and NFκB. In the nucleus, NFκB family members function as transcriptional regulators for numerous cytokine genes, including IL-2. IP3 releases Ca2+ from intracellular stores and allows influx of extracellular Ca2+, thereby activating calcineurin, an enzyme with serine/threonine phosphatase activity. Activated calcineurin dephosphorylates NF-AT, which is then translocated from the cytosol to the nucleus where it activates transcription in conjunction with AP-1. Cyclosporine (CSP) and tacrolimus bind to intracellular proteins, forming drug–protein complexes that inhibit the enzymatic activity of calcineurin, thereby interfering with signal transduction in T cells after stimulation with antigen. Signal transduction through costimulation has not been fully characterized, but appears to involve several pathways leading to increased production of cytokines through activation of Jun, which functions as a component in the AP-1–promoter complex. In addition, CD28 signaling increases production of IL-2 by stabilizing IL-2 messenger RNA. Outcomes after activation of T cells Activation of peripheral T cells can produce at least six qualitatively distinct results. TCR signaling together with appropriate costimulation signals results in production of IL-2, followed by differentiation into effectors and memory cells, as discussed below. TCR signaling during the S phase of the cell cycle, and interactions between Fas ligand (FasL) and Fas can cause apoptosis or activation-induced cell death, resulting in deletion [49,50]. With suboptimal TCR signaling or suboptimal costimulation, T-cell activation can induce a state of anergy characterized by lack of IL-2 production with persistence of effector functions and responsiveness to IL-2 [51,52]. Alternatively, suboptimal costimulation or strong signaling through inhibitory receptors can produce adaptive tolerance, characterized by a proximal block in tyrosine kinase activation and calcium mobilization, and by lack of response to IL-2 [51,53]. Finally, activation in the presence of IL-10 can result in the generation of T cells with regulatory activity [54]. Complex mechanisms regulate costimulation during an immune response [44,55]. Costimulation of resting T cells occurs through CD28, while costimulation of activated T cells occurs through the inducible costimulator (ICOS) protein. Activation of T cells induces the expression of CD152, also known as CTLA-4, a second ligand for binding CD80. The high avidity of binding between CD152 and CD80 competitively limits the degree of signaling through CD28 in activated T cells. In addition, signaling through CD152 inhibits activation of T cells through induction of phosphatase activity that counteracts the kinase cascade induced by signaling through the TCR. T-cell responses are also inhibited by ligation of other molecules that have homology with CD28, including PD-1 and B and T lymphocyte attenuator (BTLA) [44,55].

T-cell effector functions Activated T cells differentiate into effector cells that produce cytokines and mediate cytotoxic activity. Under in vitro conditions, both CD4 cells and CD8 cells can be induced to differentiate according to characteristic response patterns of cytokine production [56]. In the presence of IL-12 produced by monocytes or activated dendritic cells, T cells are stimulated to differentiate into effectors that produce “type 1” cytokines,

including IFN-γ and lymphotoxin, which activate proinflammatory macrophages. In the presence of IL-4, activated T cells are stimulated to differentiate into effectors that produce “type 2” cytokines, including IL-4 and IL-5, which activate B-cell proliferation and differentiation. Both CD4 cells and CD8 cells can differentiate into cytotoxic effectors. The cytotoxic effector function of T lymphocytes can be mediated by at least two mechanisms [57–59]. One of these involves cell surface interactions between FasL expressed on activated cytotoxic T lymphocytes (CTLs) and Fas expressed on cells targeted for lysis. Another involves exocytosis of granules containing perforin and granzymes from CTL effectors. Perforin facilitates transport of granzymes into the cytosol of cells recognized by CTLs. Both mechanisms induce apoptosis in target cells. T cells exhibit two general types of immune response in vivo (Fig. 11.4 [6]). “Primary” responses occur when immunologically “naive” cells first encounter a specific antigen. The immune response after an initial encounter with antigen develops during a period of approximately 1 week and results in the expansion of specific clones of effectors having receptors capable of recognizing the antigen. This initial expansion is followed by clonal contraction as the antigen is cleared [60]. After stimulation of naive T cells, asymmetric cell division differentiates effector and memory T-cell fates. Memory T cells can be divided into at least two distinct subsets. The “central” memory population resembles the antigen-inexperienced naive T-cell population, migrates preferentially through lymphoid tissue, and is long-lived, whereas the “effector” memory population resembles the antigenexperienced effector T-cell population, migrates preferentially in nonlymphoid tissues, and is short-lived [61–66]. Memory cells are generally characterized by selection for high TCR affinity, increased expression of adhesion molecules and altered migratory patterns with distinctive cytokine production and response profiles. Memory cells remain poised to respond quickly after a repeated encounter with antigen. “Secondary” responses by memory cells develop during a period of several days and show greater intensity than the original primary response. CD4 cells are required for maintenance of CD8 memory cell survival and function [67]. IL-7 has an important role in survival of memory CD8 cells, while IL-15 is necessary for proliferation of memory CD8 cells [49,68–71]. A variety of regulatory T-cell (Treg) populations have been defined in different experimental systems [54,72–75]. One extensively studied population of Treg is characterized by high expression of CD25 and CD39 and low expression of CD127. Generation of these cells is regulated by expression of a nuclear transcription factor termed FoxP3 [76]. These regulatory T cells are activated by specific antigens and are dependent on IL-2 [77], but they do not proliferate rapidly after stimulation [78]. They inhibit the response of other cells through nonspecific contact-dependent mechanisms or through the secretion of antiinflammatory cytokines such as transforming growth factor-beta (TGF-β) [79] and IL-10 [80].

Selection of donors for allogeneic HCT The immunogenetic relationship between the donor and recipient profoundly influences the outcome of HCT, much more so than with solid organ transplants. Within the constraints of current practice, however, these differences are most apparent when analyzing immunologic outcomes, such as graft rejection and the incidence and severity of GVHD, and are less apparent when analyzing disease-free survival (DFS). Best results are seen after transplantation from HLA-genotypically identical sibling donors, but only 30% of patients have such a donor. Some patients without an HLA-identical sibling have an HLA-haploidentical family member with limited disparity between the nonshared haplotypes (see Chapter 46). Previous studies have shown that patients receiving an

Overview of Hematopoietic Cell Transplantation Immunology

HEV

Normal tissue

Naive T cell

Afferent lymph vessel

Dendritic cell

Inflamed tissue

Constitutive homing Lymphoid organ

Inflammationinduced recruitment

Tissuespecific homing

Fig. 11.4 Migration of naive and memory T cells. Naive T cells (top center) adhere to highendothelial venules (HEV) in lymph nodes and then percolate through the lymph node to efferent lymphatics leading to the thoracic duct where they return to the blood for recirculation. Activated dendritic cells (upper right) migrate to lymph nodes through afferent lymph vessels, where they present antigen to naive T cells (bottom center). During activation and clonal expansion, naive T cells differentiate into short-lived effector T cells that migrate directly to inflamed tissues (upper right). The response to antigen also gives rise to long-lived effector memory T cells that patrol tissues through specific homing mechanisms (upper left). Activated T cells also give rise to long-lived central memory T cells with migratory behavior resembling that of naive T cells. Adapted from von Andrian and Mackay [6] with permission. Copyright (2000), Massachusetts Medical Society. All rights reserved.

Central memory T cell

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Postcapillary venule

Inflamed venule Antigen

Central memory T cell (long-lived)

Activated T cells

Effector memory T cell (long-lived)

HLA-haploidentical transplant incompatible for a single HLA-A, -B or -DR antigen can have a DFS similar to that of patients with marrow from HLA-genotypically identical sibling donors (see Chapter 46). Patients receiving transplants incompatible at two or three of these loci have a significantly lower probability of survival (see Chapter 46). HCT from HLA-phenotypically matched unrelated donors or cord blood donors is an alternative for patients who lack a donor in the family (see Chapters 47 and 39). Unrelated HCT has been made feasible by the development of large registries of HLA-typed individuals and cryopreserved cord blood units available to serve as a source of hematopoietic cells. With registries currently available in the United States and in other countries, it is now possible to identify an HLA-A, B-phenotypically matched DRB1 allele-matched unrelated donor for at least 70% of patients. If a single HLA-A, -B or -DRB1 mismatch were allowed, then it would be possible to identify donors for many more patients. In certain circumstances, DFS for patients with HLA-A, -B, -C, -DRB1, and -DQB1 allele-matched unrelated donors may be comparable to that for similar patients with HLA-genotypically identical sibling donors (see Chapter 47).

Outcomes influenced by genetic disparity between the donor and recipient Engraftment Graft rejection after HCT may be manifested as either the lack of initial engraftment or the development of pancytopenia and marrow aplasia

Dendritic cell Clonal expansion

Differentiation

Effector T cell (short-lived)

after initial engraftment. In humans, rejection, drug toxicity, sepsis, and certain viral infections can all cause graft failure. The key findings that support a diagnosis of rejection in a patient with graft failure are the absence of donor cells and the presence of recipient T cells. The durable persistence of donor cells makes rejection unlikely, even when recipient T cells can be detected. Experiments in animal models have demonstrated that both NK cells and T cells of the recipient can mediate allogeneic marrow graft rejection. Briefly summarized, NK-mediated rejection does not involve immunologic priming and occurs within 1–2 days after transplantation. The effectors have a life span of less than 1 week but are highly resistant to irradiation. In rodents, NK-mediated rejection can be overcome by treatment of the recipient with cyclophosphamide (CY) before the transplant. Because NK precursors are sensitive to irradiation, NK-mediated rejection can also be overcome by split-dose irradiation of the recipient, with an interval of 1 week between two irradiation exposures. Rejection mediated by unprimed T cells does not occur until 7–8 days after transplantation. Rejection by T cells can be enhanced by priming or transfusion-induced sensitization before the transplant. T-cell effectors have a longer life span than NK cells and are generally more sensitive to radiation than NK cells. Experiments in animal models can be established in ways that allow recipient NK cells, T cells or both to be involved in causing marrow graft rejection. The increased risk of rejection associated with transfusion-induced sensitization and the decreased risk of rejection associated with the use of higher levels of total body irradiation (TBI) before the transplant clearly implicate recipient T cells as effectors that can mediate HCT

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rejection in humans. In support of this hypothesis, recipient-derived T cells with antidonor HLA-specific cytotoxic activity have been isolated from the blood of patients with graft failure after HLA-mismatched HCT. A delayed onset of rejection and an occurrence of rejection after grafting from an HLA-identical donor would argue against a role for NK cells in causing rejection. NK-mediated rejection also seems unlikely among patients who have received high-dose CY before the transplant, if it is assumed that this agent has similar effects on human and murine NK cells. Studies have not yet determined whether NK cells can reject NK-incompatible grafts in humans when the conditioning regimen does not include CY. Mechanisms by which recipient NK cells or T cells might cause marrow graft rejection have not been fully elucidated. Although it seems intuitively obvious that cell-mediated cytotoxicity might be involved, recipients deficient in perforin, granzyme B or FasL function can reject an allogeneic marrow graft. These results indicate redundancy in the mechanisms by which recipient effectors can kill donor hematopoietic cells. This hypothesis was supported by observations that NK cells from certain strains of mice could not mediate marrow graft rejection when perforin- and FasL-mediated mechanisms of cytotoxicity were both absent. Because hematopoietic stem cells do not express Fas under steady-state conditions, any potential involvement of a FasL-mediated mechanism in marrow graft rejection would require the induction of Fas expression, possibly through exposure to IFN-γ or TNF-α. Alternatively, TNF-α released by CTLs or NK cells could have a direct suppressive or cytotoxic effect on hematopoietic stem cells. Graft rejection by CD8 cells after immunization of the recipient against donor alloantigens appears to be mediated by mechanisms other than perforin or FasL. The risk of graft failure depends on the degree of genetic disparity between the donor and recipient. In particular, graft failure has been associated with HLA class I disparity after unrelated marrow transplantation. The association between HLA class I disparity and graft failure has been observed primarily among patients with chronic myeloid leukemia (CML) and other diseases that are not treated with intensive chemotherapy before referral for transplant. The risk of graft rejection is also greatly increased by transfusion or pregnancy-induced alloimmunization of the recipient against the donor before the transplant. Alloimmunization presumably occurs when a fetus or transfusion donor happens to have transplantation antigens in common with the donor. Sensitization of the recipient against some alloantigens can be detected by the presence of antibodies that recognize donor T cells or B cells. After sensitization, rejection may be caused either by memory T cells that survive the conditioning regimen or by antibody-mediated destruction of donor cells. The use of more intensive conditioning regimens can reduce the risk of rejection by eliminating a greater proportion of recipient T cells before the transplant. Post-transplant administration of immunosuppressive agents such as methotrexate (MTX) and possibly CSP decreases the risk of rejection by interfering with the function of recipient cells that survive the conditioning regimen. Donor T cells can also play a critical role in preventing rejection by recognizing any surviving recipient T cells and eliminating them primarily through a perforin-mediated cytotoxic mechanism. Under certain experimental conditions, donor NK cells and T cells that do not cause GVHD can prevent rejection. Other experiments have shown that precursor-plasmacytoid dendritic cells of the donor can facilitate marrow engraftment. A variety of methods can be used to produce mixed reconstitution with both donor and recipient marrow cells after reduced-intensity conditioning in animals and humans. These methods generally include pretransplant conditioning with an amount of TBI or busulfan (BU) that is sufficient to ablate a significant fraction of hematopoietic stem cells in

the recipient, together with the administration of immunosuppressive agents to prevent rejection. In animals, rejection can be prevented by administration of agents that block costimulation of T cells after the transplant, resulting in durable mixed chimerism. In humans, a pretransplant regimen of low-dose BU or TBI and the immunosuppressive nucleoside analog fludarabine is sufficient to prevent rejection of an MHC-matched graft in nearly all patients who have previously received treatment with intensive chemotherapy, if the graft contains T cells and if certain immunosuppressive medications are administered after the transplant. After engraftment has been established, the immunosuppressive medications can be withdrawn, permitting donor T cells to eliminate residual hematopoietic cells and T cells of the recipient, thereby converting mixed chimerism to full donor chimerism. In some ways, this type of transplant resembles a controlled form of transfusion-induced GVHD, in which the primary targets of the disease are recipient hematopoietic cells, T cells, and malignant cells. In this case, hematopoietic cells in the graft prevent marrow failure and death. Acute GVHD Development of GVHD represents a major determinant of outcome after allogeneic HCT (see Chapters 86 and 87 and [81–86]). This complication is initiated by donor T cells that recognize recipient MHC class I alloantigens, class II alloantigens, and mHAs on recipient APCs that persist after the pretransplant conditioning regimen (see Chapters 14 and 16). Activation of donor CD8 cells occurs through recognition of recipient MHC class I alloantigens and endogenous peptide mHAs presented in the context of recipient class I MHC molecules. MHC class I molecules on dendritic cells can “cross-present” exogenous peptides acquired though phagocytosis of necrotic cells [3]. Results with one well-studied donor–recipient strain combination suggested, however, that crosspresentation of recipient mHAs by donor APCs to donor CD8 cells does not contribute to the initial pathogenesis of GVHD, although this indirect pathway for the presentation of recipient mHAs to donor CD8 cells may play a role in augmenting the disease. Activation of donor CD4 cells occurs through recognition of MHC class II alloantigens on recipient APCs. Activation of donor CD4 cells also occurs through recognition of recipient minor antigens processed by either recipient or donor APCs. Despite the large number of mHA mismatches between the donor and recipient, only a small number of “immunodominant” recipient alloantigens are prominently involved in the overall response. The pretransplant conditioning regimen greatly increases susceptibility of the recipient to GVHD. Within as little as 6 hours after irradiation, recipient dendritic cells increase their expression of CD86 as part of a response that resembles activation by the alarm signals of innate immunity. The initial activation of donor cells induced by alloantigens on recipient APCs occurs within the first 24 hours after the transplant and is followed by rapid, IL-2-dependent clonal proliferation and differentiation into effectors. During the afferent phase of GVHD, the initial activation of donor T cells by recipient APCs occurs in the spleen, lymph nodes or Peyer’s patches. Migration of donor T cells into secondary lymphoid organs requires either L-selectin (CD62L) or α4β7 integrin. Recent studies have shown that memory T cells do not cause GVHD in rodents. This unexpected result could reflect the absence of CD62L, a reduced repertoire of TCR receptors or a reduced proliferative potential among memory cells as compared with naive cells. During the subsequent afferent phase of the disease, activated donor T cells migrate to target organs where they cause tissue injury in the skin, liver, and gastrointestinal tract. Chemokines expressed in target tissues bind to chemokine receptors on T cells and help attract T cells into these tissues. Activated T cells from both the CD4 and CD8 subsets

Overview of Hematopoietic Cell Transplantation Immunology

produce proinflammatory cytokines, and both subsets generate CTLs. No single effector mechanism can account for the entire clinical spectrum of acute GVHD (see Chapters 14, 16 and 86). In some patients, the disease presents as a syndrome of high-grade fever, confluent erythematous rash, and vascular leak with pulmonary infiltrates and edema during the first 10 days after the transplant. In this situation, the disease can be viewed appropriately as a “cytokine storm” that generally shows striking improvement after treatment with high-dose glucocorticoids. In most patients, however, the disease presents in much less dramatic fashion with a slowly progressive morbilliform rash or with anorexia, nausea, and vomiting. Whether a cytokine storm plays a role in these more typical cases of acute GVHD without overt systemic toxicity remains to be determined. Events leading to a cytokine storm include epithelial damage to the gastrointestinal tract, which facilitates translocation of endotoxin into the blood stream. Macrophages that have been primed by alloactivated T cells show exquisite sensitivity to endotoxin, producing inflammatory cytokines such as TNF-α and IL-1, which contribute to tissue injury [24]. Administration of antibodies that neutralize TNF-α and IL-1 can prevent early death related to severe GVHD following very high TBI exposure in mice. In humans, however, attempts to neutralize these cytokines individually have not produced any major clinical benefit in preventing GVHD. Cytokines play complex roles in the pathogenesis of GVHD. For example, activated T cells produce IFN-γ, which makes macrophages highly sensitive to stimulation by endotoxin. On the other hand, GVHD is exacerbated by neutralization of IFN-γ and by the use of donor T cells that cannot produce IFN-γ. These results suggest that IFN-γ actually has a complex dual role to promote and control GVHD in mice. Some studies have suggested that GVHD is more severe when caused by T cells polarized to produce type 1 cytokines compared with type 2 cytokines. It is now clear that both cell types contribute to the pathogenesis of GVHD, although with different patterns of organ involvement in mice. Type 1 cells cause intestinal pathology, whereas type 2 cells cause pathologic changes in the skin, intestinal tract, and liver. The presence of eosinophils in duodenal biopsies from patients with GVHD supports the involvement of type 2 cells in the pathogenesis of the disease. Cell-mediated cytotoxic mechanisms clearly contribute to the pathogenesis of GVHD, particularly in recipients prepared with lower amounts of TBI. In mice, GVHD is less severe when induced by perforindeficient donor T cells compared with wild-type donor T cells. In a model of GVHD caused by disparity for mHAs, recipients transplanted with FasL-deficient donor T cells developed cachexia, but inflammatory lesions in the skin and liver were absent, and the profound B-lymphoid hypoplasia associated with GVHD did not occur. The severity of hepatic GVHD was reduced in Fas-deficient recipients, but the severity of GVHD in other organs was increased. Neutralization of FasL prevented hepatic lesions in wild-type recipients but had no effect on intestinal GVHD, while neutralization of TNF-α improved intestinal GVHD but had no effect on hepatic GVHD. Neutralization of both FasL and TNFα prevented GVHD in all organs. These results suggest that the mechanisms leading to tissue damage vary among different organs. At the same time, however, donor T cells that cause GVHD are susceptible to both FasL and perforin-mediated cytotoxicity, and in certain circumstances, interventions that block either of these regulatory mechanisms can exacerbate GVHD. Under certain conditions, activated donor NK cells have been shown to reduce the severity of GVHD. Mechanisms proposed to explain this effect include production of TGF-β or IL-4 by donor NK cells or rapid elimination of recipient dendritic cells by donor NK cells after the transplant [87]. Donor CD25+CD4+ regulatory T cells can also reduce

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the severity of GVHD in mice. GVHD can be ameliorated by adding large numbers of regulatory T cells to the graft and exacerbated by removal of these cells from the graft. The ability of regulatory T cells to prevent GVHD is partly mediated by their ability to produce IL-10. Clinical studies have shown that the risk of acute GVHD depends on the degree of genetic disparity between the recipient and donor. The risk of GVHD is greater with unrelated donors than with related donors, mostly because of unavoidable MHC disparity. The risk of GVHD is higher with multiple disparities than with a single disparity. The type of post-transplant immunosuppression and compliance with the prophylactic regimen influence the risk of GVHD. The most effective regimens currently in widespread use include a calcineurin inhibitor (CSP or tacrolimus) combined with MTX or another antimetabolite such as mycophenolate mofetil (see Chapter 84). Multidrug regimens are more effective than single-drug regimens. Acute GVHD can be prevented by removing mature T cells from the donor marrow (see Chapter 85). The benefit brought about by a reduced risk of GVHD, however, is offset by increased risks of graft failure and recurrent leukemia, and by delayed immune reconstitution (see below). For this reason, removal of T cells from the donor marrow to prevent GVHD has not appreciably improved DFS after the transplant. Research studies are attempting to determine whether the donor cells that cause GVHD can be distinguished from those needed to prevent rejection, eliminate malignant progenitors or initiate immune reconstitution. In the past, many clinical trials have employed methods aimed at global T-cell depletion, even though GVHD is initiated by the relatively small subset of donor cells that recognize recipient histocompatibility antigens. In the future, it may be possible to prevent GVHD by specific depletion of donor T cells that recognize recipient alloantigens, by interference with positive costimulatory signals needed for optimal activation of T cells, by interference with mechanisms involved in the migration of T cells in vivo, by administration of agents that increase activation-induced apoptosis in donor T cells that recognize recipient alloantigens, or by infusion of regulatory T cells [88]. In addition, the severity of GVHD might also be decreased with the use of agents that protect gastrointestinal integrity after the conditioning regimen or by neutralizing endotoxin and other agents that activate innate immunity. Chronic GVHD The biological mechanisms leading to chronic GVHD are not as well understood as those leading to acute GVHD, and the relationship between acute and chronic GVHD is not entirely clear. Although acute GVHD has been recognized as a risk factor for chronic GVHD, not all cases of acute GVHD evolve into chronic GVHD, and chronic GVHD can develop in the absence of any prior overt acute GVHD. In the skin, the initial phase of chronic GVHD is characterized by an intense mononuclear inflammatory infiltrate with destructive changes at the dermalepidermal junction, accompanied by irregular acanthosis, hyperkeratosis or atrophy. Evolution of the disease is characterized by increasing dermal fibrosis, ultimately resulting in dermal sclerosis (see Chapter 27). Other pathognomonic hallmarks of chronic GVHD include destruction of tubuloalveolar glands and ducts in the skin, salivary and lacrimal glands, and respiratory epithelium, and destruction of bile ducts in the liver. Defects in negative selection in the thymus can cause chronic GVHD in mice. A “chronic GVHD” syndrome resembling systemic lupus erythematosus in mice has been extensively studied, but the manifestations of this disease differ from those of chronic GVHD in humans. Chronic GVHD characterized by cutaneous scleroderma has been described with a limited number of donor–recipient strain combinations. In one carefully studied model, skin thickening and deposition of type 1 collagen

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was preceded by infiltration with donor T cells and activated macrophages that had increased expression of TGF-β1. In this model, the development of fibrosis could be prevented by administration of a neutralizing antibody against TGF-β. In humans, genes related to the TGF pathway have decreased expression in CD4 and CD8 cells from patients with chronic GVHD compared with controls without chronic GVHD. This apparent paradox might be explained by the ability of TGF-β to suppress immune responses early after transplantation, while contributing to the development of fibrosis at later time points. In humans and in mice, chronic GVHD has been associated with the development of autoantibodies, and phase II studies have demonstrated clinical improvement in manifestations of chronic GVHD in some patients after treatment with B-cell-specific monoclonal antibodies. In humans, the use of growth factor-mobilized blood cells is associated with an increased risk of chronic GVHD compared with marrow, but experimental studies with mice have indicated that T cells do not account for this effect. Clinical studies attempting to correlate the presence or absence of chronic GVHD with the numbers of Treg cells in the blood have not produced consistent results. Other studies have demonstrated increased numbers of IL-4-producing CD8 cells and a preponderance of CD4 effector memory cells relative to central memory cells in patients with chronic GVHD. Taken together, these results suggest that multiple pathways involving T cells, B cells, nonlymphoid cells, and Treg cells can lead to chronic GVHD. Graft-versus-leukemia effects Myelosuppression represents the dose-limiting toxicity of many agents used for the treatment of malignancy. By circumventing this limitation, HCT allows the administration of chemotherapy and irradiation at doses two-to-threefold higher than would otherwise be possible. It has been recognized, however, that the therapeutic advantage of allogeneic HCT results not only from the ability to deliver more intensive treatment, but also from antineoplastic effects mediated by the graft (see Chapters 14 and 18). Clinical evidence for a graft-versus-leukemia effect (GVLE) came initially from retrospective observations that leukemic relapse occurred less frequently in patients who developed GVHD compared with those who did not. Other clinical observations have supported the concept that certain cells in an allogeneic marrow graft can help to eliminate malignant cells that survive the conditioning regimen. Removal of T cells in the donor marrow has been associated with an increased risk of relapse. The risk of relapse after T-cell-depleted marrow transplantation for acute leukemia is comparable to the risk in patients who do not develop GVHD after transplantation with unmodified marrow. Thus, the increased risk of relapse associated with T-cell depletion in patients with acute leukemia could simply reflect a decreased incidence of GVHD. These data and results from certain experimental animal models suggest that the occurrence of GVHD per se can have an antileukemic effect. Removal of T cells from the graft causes a strikingly increased risk of recurrent malignancy among patients with a pretransplant diagnosis of CML. Multivariate analyses have shown that the increased relapse risk cannot be explained entirely by a decreased incidence of GVHD. In patients with CML, donor T cells may therefore exert an antileukemic effect that is independent of overt GVHD, either because CML targets express antigens that do not initiate GVHD or because sensitivity to attack by donor cells is higher for CML targets than for epithelial cells in GVHD target organs. Efforts to gain a therapeutic advantage from interventions designed to increase GVLEs have had mixed success. Among patients with highrisk malignancies, the administration of nonirradiated peripheral blood buffy coat cells from the marrow donor during the first 4 days after

transplantation caused an unacceptably high risk of GVHD and transplant-related mortality. Among patients who have recurrent malignancy after transplantation, remissions can occasionally be induced simply by stopping the administration of immunosuppressive medications. In patients who do not have GVHD, remissions can be induced by infusion of donor lymphocytes (see Chapter 72). Remissions are most often induced in patients with CML in chronic phase, myeloma or myelodysplasia. Remissions occur much less frequently in patients with acute leukemia. Donor lymphocyte infusions can cause acute or chronic GVHD and transient marrow aplasia, depending on the number of T cells administered and the degree of HLA-mismatching between the recipient and donor. Multiple mechanisms and cell types are likely to account for the antileukemic effects associated with GVHD. Thus, alloantigens expressed by malignant cells can serve as direct targets for GVHD effector cells, or GVHD may activate other effectors such as NK cells and lymphokine-activated killer cells that have cytotoxic activity against leukemic cells. Certain cytokines elaborated during GVHD may have effects on the proliferation and differentiation of malignant cells. Studies with mice have suggested that perforin and TNF-α-mediated cytotoxic mechanisms of CD8 cells might have particular importance for GVLEs. FasL-mediated cytotoxic mechanisms were neither necessary nor sufficient for activity against the tumors used for testing in mice. On the other hand, donor CD4 cells appear to have a prominent role in control of CML in humans, and similar findings have been reported in some experimental animal models. Since perforin does not play a dominant role in the cytotoxic activity of CD4 cells, these results suggest that perforin-independent mechanisms might be important for GVLEs in patients with CML. In order to mediate a GVLE, CD4 cells must be activated by MHC class II antigens in the recipient. In some cases, expression of MHC class II antigens on nonmalignant cells was sufficient to enable GVLE, demonstrating that CD4 cells can act indirectly. From a clinical standpoint, the central question is whether GVLEs can be separated from GVHD. With new insights gained from ongoing laboratory research, future clinical trials might demonstrate the feasibility of manipulating the immune system to prevent leukemic relapse without increasing the morbidity and mortality caused by GVHD. Donor NK cells or NK-T cells might be especially well suited for this purpose. Alternatively, it might be possible to produce GVLEs through the use of donor-derived T cells that recognize antigens selectively expressed by malignant cells of the recipient. Infusion of donor effector cells that recognize minor antigens with expression limited to lymphoid or hematopoietic cells of the recipient might also produce potent antitumor effects without causing GVHD (see Chapters 14, 18 and 72).

Development of tolerance GVHD is caused entirely by the progeny of mature donor T cells in the graft at the time of transplant, and rejection is caused entirely by the progeny of recipient T cells that survive the conditioning regimen. T cells that develop in the recipient thymus after HCT do not cause acute GVHD or rejection. Those that express receptors for recipient alloantigens are eliminated or induced into anergy through interactions with thymic epithelial cells. Any marrow-derived dendritic cells of the recipient that survive in the thymus after the transplant can also help to eliminate developing T cells that recognize recipient alloantigens. The presence of donor marrow-derived dendritic cells in the thymus prevents the development of T cells that could recognize and destroy the graft. In the absence of chronic GVHD, prophylactic immunosuppressive treatment can be discontinued without complications. Donor-derived T helper and T cytotoxic cells that recognize recipient alloantigens can be detected by in vitro assays in all patients after allo-

Overview of Hematopoietic Cell Transplantation Immunology

geneic HCT regardless of whether or not clinically evident GVHD has occurred. Characteristics that determine the extent of immunologic injury caused by these cells have not been defined. To some extent, donor T cells that recognize recipient alloantigens must be subject to the same mechanisms that induce peripheral tolerance in autoreactive T cells that emerge from the thymus. Induction of anergy, development of regulatory cells or suppressor cells, and activation-induced cell death or clonal exhaustion are possible mechanisms that prevent or limit the immunologic injury that would otherwise be caused by donor T cells that recognize recipient alloantigens [89]. Inflammatory mediators can interfere with induction of tolerance in donor T cells that recognize recipient alloantigens. Results from experiments with animal models suggest that calcineurin inhibitors may interfere with development of tolerance. On the other hand, clinical experience has shown that premature withdrawal of calcineurin inhibitor administration after HCT can precipitate acute or chronic GVHD. In the future, HCT may be developed as a method for facilitating transplantation of other tissues from the same donor (see Chapter 15). In one experimental approach, rejection is prevented by depletion of mature T cells in the recipient through the administration of antibodies before the transplant and through the effects of donor T cells to inactivate or eliminate any remaining recipient T cells that can recognize donor alloantigens. Donor-derived T cells that develop in the thymus after the transplant are tolerant of recipient histocompatibility antigens after negative selection by marrow-derived cells and epithelial cells of the recipient, and they are tolerant of donor histocompatibility antigens after negative selection by donor marrow-derived cells. Preservation of recipient marrow cells together with the donor marrow allows complete MHC incompatibility between the donor and recipient without jeopardizing immune function after the transplant, thereby considerably broadening the availability of donors.

Immune reconstitution Pathways of T-cell reconstitution After HCT, all patients develop profound immunodeficiency as a consequence of the preparative regimen, the effects of donor T cells, and the administration of immunosuppressive medications. Reconstitution with donor cells corrects this immunodeficiency (see Chapter 17 and [29,90,91]). Reconstitution of innate immunity with NK cells and natural IFN-α/β-producing cells occurs within the first month after transplantation, and reconstitution of adaptive immunity follows later. Risk factors for delayed reconstitution of adaptive immunity include the use of cord blood cells, unrelated donors or grafts containing low numbers of CD34 cells or T cells, older age of the recipient or donor, the occurrence of acute or chronic GVHD, and high-dose glucocorticoid treatment. Reconstitution of T cells can occur both through thymus-independent expansion of the mature T-cell population in the graft and through maturation of marrow-derived cells in the recipient thymus. Clinical and experimental studies have indicated that the initial reconstitution of T cells after transplantation occurs primarily through proliferation of mature donor T cells in the graft, and the contribution of new T cells from the thymus becomes apparent later. Production of new T cells in the thymus persists throughout life but declines gradually with age [92]. Irradiation and GVHD impair recovery of thymic function after the transplant, with more profound effects in adults than in children. The thymic pathway of T-cell reconstitution is more important for recovery of CD4 cells than for CD8 cells. For these reasons, the balance between contributions from the thymus-independent and thymus-dependent pathways of T-cell reconstitution depends on the age of the patient, the

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number of T cells in the graft, the time interval from transplant, and the expression of CD4 or CD8. Thymic production of T cells in humans can be assessed by an increase in the volume of the thymus by imaging studies, an increase in the percentage or number of cells with T cell receptor excision circles (TRECs), and an increase in the percentage or number of cells that express CD45RA as an indication of a naive phenotype. TRECs are formed during the process of TCR-a and -b gene rearrangement in T cells during development in the thymus. The presence of TRECs has been used to identify peripheral T cells that have recently emigrated from the thymus. Since TREC DNA is not copied during cell division, the percentage of TREC-containing cells decreases as recent thymic emigrants proliferate. For this reason, the most informative measurements of thymic output require assessment of both TREC content and T-cell proliferation. In lymphopenic mice, naive T cells emerging from the thymus proliferate as a result of homeostatic stimulation and convert from a naive phenotype to a “pseudomemory” phenotype in the absence of specific antigen stimulation. This process is driven by insufficiency of the peripheral T-cell repertoire. T cells that have recently emigrated from the thymus proliferate and convert to a pseudomemory phenotype unless the peripheral repertoire already contains T cells with the same TCR specificity. The presence of naive T cells in the periphery therefore indicates both thymic output and sufficiency of the T-cell repertoire. The time for restoration varies for different components and functions of the immune system. In recipients transplanted with unmodified marrow from an HLA-identical sibling, proliferative responses to phytohemagglutinin begin to reach the normal range after approximately 4–6 months, the number of peripheral blood CD4 cells begins to reach the normal range after 7–9 months, and production of immunoglobulin G as measured by in vitro assays recovers after 7–9 months. Recovery of immune responses is delayed by several months in recipients transplanted with T-cell-depleted marrow, and recovery of CD4 cells is accelerated when hematopoietic growth factor-mobilized stem cells are used for transplantation. Long-term reconstitution of immunity against certain pathogens probably represents the acquisition of new memory cells generated by exposure to antigens after the transplant. During the first 3 months after HCT, selective proliferation among different T cells in the thymus-independent pathway of reconstitution causes severe skewing in the distribution of TCRs among repopulating T cells. During this time, the spectrum of TCR gene rearrangements remains abnormally limited and unstable. Changes in the T-cell repertoire during this period of time most likely result from antigen-driven proliferation, especially in patients with GVHD. Naive T cells produced through the thymus-dependent pathway of reconstitution have a normal spectrum of TCR gene rearrangements. Throughout the first 3 years after the transplant, the progeny of mature T cells in the graft are gradually replaced by T cells that have originated through maturation of marrow cells in the thymus. With this replacement, the spectrum of TCR gene rearrangements gradually becomes normal. The rate at which the spectrum of TCR gene rearrangements returns to normal depends on the number of mature T cells in the graft and the level of thymic function. Thymus-dependent dilutional replacement of cells that originated from the thymus-independent pathway would be expected to occur more slowly with grafts that contain a large number of mature T cells and more rapidly with grafts that contain small numbers of mature T cells. Abnormalities in the spectrum of TCR gene rearrangements can persist for more than 3 years in patients with severely impaired thymic function. For reasons that are not yet clear, the spectrum of TCR gene rearrangements also remains abnormal in patients with persistent mixed chimerism, even in the absence of GVHD. The diversity of immunoglobulin gene rearrangements in B cells is also reduced after HCT. One mechanism for this effect is related to an

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abnormally low rate of somatic mutation in rearranged immunoglobulin genes during B-cell development after HCT. Effects of GVHD on immune reconstitution GVHD impairs T-cell immune reconstitution through at least four distinct mechanisms. First, GVHD causes injury to thymic epithelium, thereby reducing T-cell reconstitution and diversification of the TCR repertoire through the thymus-dependent pathway. Second, GVHD decreases the total number of T cells that can be accommodated within the periphery, perhaps by causing microenvironmental abnormalities that reduce the number of functional peripheral T-cell niches. Third, stimulation by recipient alloantigen induces expression of FasL and Fas, resulting in activation-induced apoptosis in a large fraction of donor T cells that recognize recipient alloantigens. Fourth, during GVHD, donor T cells that do not recognize recipient alloantigens are induced to express Fas and become susceptible to fratricidal or bystander apoptosis caused by donor T cells that express FasL. Both acute and chronic GVHD block B lymphopoiesis. Patients with chronic GVHD do not develop immunocompetence until chronic GVHD resolves. Opportunistic infections as an indicator of impaired immune reconstitution Opportunistic infections are a key clinical indicator of immunodeficiency after HCT. Pathogens involved in opportunistic infections prominently include the Epstein–Barr virus (EBV) (see Chapter 93), cytomegalovirus (CMV) (see Chapter 90), adenovirus (see Chapter 94), and fungal organisms (see Chapter 89 and [93]). The development of lymphoproliferative disorders caused by EBV infection is a particularly striking example of opportunistic infection associated with immunodeficiency after HCT. The risk of this often-fatal complication is increased among adults who receive a T-cell-depleted graft from an unrelated donor followed by immunosuppression with antithymocyte globulin and steroids after the transplant. Risk factors for CMV infection, fungal infection, and adenovirus infection are very similar. Prolonged lymphopenia represents a common thread among these risk factors. In particular, CD4 lymphopenia has been strongly correlated with the risk of opportunistic infections after HCT. In addition, more subtle abnormalities of T-cell function are likely to increase the risk of opportunistic infections. Examples include qualitative defects in T-cell signal transduction and reduced production of TNF-α by activated CMV-specific CD8 cells. Approaches for enhancement of immune reconstitution In certain conditions, EBV-specific immunity can be reconstituted and regression of EBV-associated lymphoproliferative disorders can be induced by infusion of a relatively small number of donor lymphocytes. Alternatively, immune responses against specific pathogens can be reconstituted by infusion of antigen-specific T-cell lines or T-cell clones (see Chapter 17). Immune reconstitution might also be accelerated through a variety of other strategies. Administration of keratinocyte growth factor has been shown to protect thymic epithelium from the effects of irradiation and GVHD, thereby improving immune reconstitution through the thymus-dependent pathway after the transplant. Thymic epithelium is a source of IL-7, a growth factor involved in production of T cells and maintenance of T-cell homeostasis in the periphery. Exogenous administration of this cytokine does not reverse the effects of GVHD on thymic stromal function but can enhance T-cell production through direct effects on T cells as they develop in the thymus. Exogenous administration of IL-7 also stimulates proliferation of mature T

cells, protects T cells from apoptosis, and enhances T-cell function [71]. Although exogenous administration of IL-7 exacerbated GVHD after HCT in nonirradiated mice, this effect was not observed in irradiated mice in which endogenous levels of IL-7 are already increased as a result of lymphopenia. Expression of the IL-7 receptor α chain is reduced in donor T cells that have proliferated in response to recipient alloantigens but not in other T cells. For this reason, IL-7 has little effect on T cells that recognize recipient alloantigens but stimulates proliferation of other T cells that may be important for immune reconstitution. Effects similar to those observed after exogenous administration of IL-7 also occur after exogenous administration of IL-15 after HCT. Under some conditions, however, exogenous administration of IL-15 causes GVHD in irradiated recipients. Other potential approaches for enhancing immune reconstitution after HCT include androgen blockade, administration of flt3 ligand, and administration of synthetic oligodeoxynucleotides containing unmethylated cytosine–guanine motifs that can expand naive and memory populations by preventing T-cell death. In most recipients without chronic GVHD, immunologic function has recovered sufficiently to allow responses after administration of diphtheria, pertussis, and tetanus vaccine and pneumococcal vaccine at 1 year after HCT (see Chapter 17). To some extent, antigen-experienced donor T cells contribute to this immune reconstitution, and responses to polysaccharide–protein conjugate vaccines in the recipient after the transplant can be enhanced by vaccination of the donor before the transplant. Administration of live viral vaccines, such as measles, mumps, rubella, and oral polio, is deferred until at least 2 years after transplantation, even in healthy recipients, as a precaution to avoid inadvertent infection with these attenuated strains. These vaccines should not be given to patients with chronic GVHD or to anyone taking immunosuppressive medications.

Summary and conclusions Although this overview of the immunology of HCT has focused on the individual outcomes of engraftment, GVHD, control of malignancy, tolerance, and immune reconstitution, these remain highly interconnected as different aspects of a single overall process. For this reason, the evaluation of interventions designed to influence one particular immunologic outcome of HCT will require equivalent scrutiny of other immunologic outcomes. Historically, clinical HCT has evolved from the biomedical knowledge and understanding gained from animal studies. Murine models have the advantages of well-defined immunogenetics and a wealth of reagents for dissecting the cellular and humoral mechanisms involved in HCT immunobiology, while canine and nonhuman-primate models more closely mimic the immunological and medical challenges of HCT in a large outbred species. The wide range of experimental manipulations afforded by animal models has enabled rapid progress in the biomedical understanding of HCT immunology in all of its interconnected complexity, although differences between species must be carefully considered in the interpretation of results [94]. Approaches that have produced tolerance to alloantigens in mice have generally not been successful in non-human primates or humans. In part, differences in memory T cells populations between laboratory rodents and other species might explain these results [95–98]. Memory T cells produced by prior infections can cross-react with alloantigens, because TCR recognition is degenerate. Memory T cells have a lower threshold of activation, are less susceptible to regulation, and have been more resistant to approaches that induce tolerance in naive T cells. Nonetheless, insights from fundamental studies of immunology provide a rational foundation for the further development of HCT as a therapeutic modality in humans.

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46. Watts TH. TNF/TNFR family members in costimulation of T cell responses [Review]. Annu Rev Immunol 2005; 23: 23–68. 47. Simeoni L, Smida M, Posevitz V, Schraven B, Lindquist JA. Right time, right place: the organization of membrane proximal signaling [Review]. Semin Immunol 2005; 17: 35–49. 48. Baniyash M. TCR zeta-chain downregulation: curtailing an excessive inflammatory immune response [Review]. Nat Rev Immunol 2004; 4: 675–87. 49. Marrack P, Kappler J. Control of T cell viability [Review]. Annu Rev Immunol 2004; 22: 765–87. 50. Green DR, Droin N, Pinkoski M. Activationinduced cell death in T cells [Review]. Immunol Rev 2003; 193: 70–81. 51. Macian F, Im SH, Garcia-Cozar FJ, Rao A. T-cell anergy [Review]. Curr Opin Immunol 2004; 16: 209–16. 52. Appleman LJ, Boussiotis VA. T cell anergy and costimulation [Review]. Immunol Rev 2003; 192: 161–80. 53. Schwartz RH. T cell anergy [Review]. Annu Rev Immunol 2003; 21: 305–34. 54. Roncarolo MG, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings MK. Interleukin-10secreting type 1 regulatory T cells in rodents and humans [Review]. Immunol Rev 2006; 212: 28–50. Erratum in: Immunol Rev 2006; 213: 257. 55. Leibson PJ. The regulation of lymphocyte activation by inhibitory receptors [Review]. Curr Opin Immunol 2004; 16: 328–36. 56. Stockinger B, Bourgeois C, Kassiotis G. CD4+ memory T cells: functional differentiation and homeostasis [Review]. Immunol Rev 2006; 211: 39–48. 57. Zimmerman Z, Jones M, Shatry A, Komatsu M, Mammolenti M, Levy R. Cytolytic pathways used by effector cells derived from recipient naive and memory T cells and natural killer cells in resistance to allogeneic hematopoietic cell transplantation [Review]. Biol Blood Marrow Transplant 2005; 11: 957–71. 58. van den Brink MR, Burakoff SJ. Cytolytic pathways in haematopoietic stem-cell transplantation [Review]. Nat Rev Immunol 2002; 2: 273–81. 59. Russell JH, Ley TJ. Lymphocyte-mediated cytotoxicity [Review]. Annu Rev Immunol 2002; 20: 323–70. 60. Badovinac VP, Harty JT. Programming, demarcating, and manipulating CD8+ T-cell memory [Review]. Immunol Rev 2006; 211: 67–80. 61. LeFrancois L. Development, trafficking, and function of memory T-cell subsets [Review]. Immunol Rev 2006; 211: 93–103. 62. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance [Review]. Annu Rev Immunol 2004; 22: 745–63. 63. Berard M, Tough DF. Qualitative differences between naive and memory T cells [Review]. Immunology 2002; 106: 127–38. 64. Weninger W, Manjunath N, von Andrian UH. Migration and differentiation of CD8+ T cells [Review]. Immunol Rev 2002; 186: 221–33. 65. Jenkins MK, Khoruts A, Ingulli E et al. In vivo activation of antigen-specific CD4 T cells [Review]. Annu Rev Immunol 2001; 19: 23–45. 66. Dooms H, Abbas AK. Control of CD4+ T-cell memory by cytokines and costimulators [Review]. Immunol Rev 2006; 211: 23–38.

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67. Williams MA, Holmes BJ, Sun JC, Bevan MJ. Developing and maintaining protective CD8+ memory T cells [Review]. Immunol Rev 2006; 211: 146–53. 68. Surh CD, Boyman O, Purton JF, Sprent J. Homeostasis of memory T cells [Review]. Immunol Rev 2006; 211: 154–63. 69. Ma A, Koka R, Burkett P. Diverse functions of IL-2, IL-15, and IL-7 in lymphoid homeostasis [Review]. Annu Rev Immunol 2006; 24: 657–79. 70. Surh CD, Sprent J. Regulation of mature T cell homeostasis [Review]. Semin Immunol 2005; 17: 183–91. 71. Fry TJ, Mackall CL. The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance [Review]. J Immunol 2005; 174: 6571–6. 72. Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses [Review]. Annu Rev Immunol 2004; 22: 531–62. 73. Nanno M, Shiohara T, Yamamoto H, Kawakami K, Ishikawa H. gammadelta T cells: firefighters or fire boosters in the front lines of inflammatory responses [Review]. Immunol Rev 2007; 215: 103– 13. 74. Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM. Th17: an effector CD4 T cell lineage with regulatory T cell ties [Review]. Immunity 2006; 24: 677–88. 75. Izcue A, Coombes JL, Powrie F. Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation [Review]. Immunol Rev 2006; 212: 256–71. 76. Ziegler SF. FOXP3: Of mice and men [Review]. Annu Rev Immunol 2006; 24: 209–26. 77. Malek TR, Bayer AL. Tolerance, not immunity, crucially depends on IL-2 [Review]. Nat Rev Immunol 2004; 4: 665–74.

78. Yamazaki S, Inaba K, Tarbell KV, Steinman RM. Dendritic cells expand antigen-specific Foxp3+ CD25+ CD4+ regulatory T cells including suppressors of alloreactivity [Review]. Immunol Rev 2006; 212: 314–29. 79. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-beta regulation of immune responses [Review]. Annu Rev Immunol 2006; 24: 99–146. 80. Pestka S, Krause CD, Sarkar D, Walter MR, Shi Y, Fisher PB. Interleukin-10 and related cytokines and receptors [Review]. Annu Rev Immunol 2004; 22: 929–79. 81. Negrin RS, Contag CH. In vivo imaging using bioluminescence: a tool for probing graft-versus-host disease [Review]. Nat Rev Immunol 2006; 6: 484– 90. 82. Wysocki CA, Panoskaltsis-Mortari A, Blazar BR, Serody JS. Leukocyte migration and graft-versushost disease [Review]. Blood 2005; 105: 4191– 9. 83. Blazar BR, Murphy WJ. Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) [Review]. Philos Trans R Soc Lond B Biol Sci 2005; 360: 1747–67. 84. Murphy GF, Korngold R. Significance of selectively targeted apoptotic rete cells in graftversus-host disease [Review]. Biol Blood Marrow Transplant 2004; 10: 357–65. 85. Ferrara JL, Reddy P. Pathophysiology of graftversus-host disease [Review]. Semin Hematol 2006; 43: 3–10. 86. Shlomchik WD. Graft-versus-host disease. Nat Rev Immunol 2007; 7: 340–52. 87. Ruggeri L, Aversa F, Martelli MF, Velardi A. Allogeneic hematopoietic transplantation and natural killer cell recognition of missing self [Review]. Immunol Rev 2006; 214: 202–18.

88. Nguyen VH, Zeiser R, Negrin RS. Role of naturally arising regulatory T cells in hematopoietic cell transplantation [Review]. Biol Blood Marrow Transplant 2006; 12: 995–1009. 89. Chiffoleau E, Walsh PT, Turka L. Apoptosis and transplantation tolerance [Review]. Immunol Rev 2003; 193: 124–45. 90. Auletta JJ, Lazarus HM. Immune restoration following hematopoietic stem cell transplantation: An evolving target [Review]. Bone Marrow Transplant 2005; 35: 835–57. 91. Moss P, Rickinson A. Cellular immunotherapy for viral infection after HSC transplantation [Review]. Nat Rev Immunol 2005; 5: 9–20. 92. Taub DD, Longo DL. Insights into thymic aging and regeneration [Review]. Immunol Rev 2005; 205: 72–93. 93. Romani L. Immunity to fungal infections [Review]. Nat Rev Immunol 2004; 4: 1–23. 94. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology [Review]. J Immunol 2004; 172: 2731–8. 95. Selin LK, Brehm MA, Naumov YN et al. Memory of mice and men: CD8+ T-cell cross-reactivity and heterologous immunity [Review]. Immunol Rev 2006; 211: 164–81. 96. Koehn B, Gangappa S, Miller JD, Ahmed R, Larsen CP. Patients, pathogens, and protective immunity: the relevance of virus-induced alloreactivity in transplantation [Review]. J Immunol 2006; 176: 2691–6. 97. Taylor DK, Neujahr D, Turka LA. Heterologous immunity and homeostatic proliferation as barriers to tolerance [Review]. Curr Opin Immunol 2004; 16: 558–64. 98. Adams AB, Pearson TC, Larsen CP. Heterologous immunity: an overlooked barrier to tolerance [Review]. Immunol Rev 2003; 196: 147–60.

12

Eric Mickelson & Effie W. Petersdorf

Histocompatibility

Introduction The human major histocompatibility complex (MHC), also termed the human leukocyte antigen (HLA) complex, encompasses a 4-megabase (Mb) span of DNA on the short arm of chromosome 6. The molecules encoded by genes within this region are of fundamental importance to the innate and antigen-specific immune systems. An MHC has been identified in all vertebrate species studied to date, and MHC genes have been shown to be highly conserved throughout vertebrate evolution [1]. The MHC is an extraordinarily gene-dense region that contains more than 300 genes, approximately 30% of which have immune-related function [1,2]. These functions include antigen processing and presentation (the classical HLA-A, B, C, DM, DO, DP, DQ, and DR genes; PRSS16; PSMB; AP; and UBD), inflammation (ABCF1, AIF1, DAXX, IER3, LST1, LTA, LTB, NCR3, and TNF), leukocyte maturation (DDAH2 and LY6G), the complement cascade (BF, C2, C4A, and C4B), maternal–fetal immunology (the nonclassical class I HLA-E, F, and G genes), stress response (MICA/MICB), immune regulation (NFKB, RXRB, and FKBPL), and the immunoglobulin superfamily (AGER, BTN, C6orf25, and MOG). HLA antigens encoded by MHC-resident genes are expressed on virtually all nucleated cells, and this ubiquitous expression contributes to their fundamental role in the acceptance and rejection of transplanted tissues. Evidence for an MHC in humans was first recognized in the early 1950s following observations that sera from patients with febrile transfusion reactions could cause the agglutination of leukocytes from their transfusion donors, as well as other individuals [3]. Subsequent studies showed that leukocyte antibodies could also be found in the sera of multiparous women. The term “HLA” was derived by combining the human-1 (HU-1) and leukocyte antigen (LA) designations used by Dausset and Payne, respectively, to describe the newly discovered leukocyte antigen system [3–7]. Since 1964, a series of 14 collaborative International Histocompatibility Workshops has greatly extended our knowledge of the HLA system and contributed to the standardization of HLA typing methods as well as HLA nomenclature (Table 12.1) [8–21]. During an immune response, cells of the immune system interact through recognition of cell surface molecules encoded by genes of the human MHC. T cells recognize foreign antigens presented as peptide

fragments in association with MHC molecules. Before T cells can recognize an antigen, the antigen must be processed by an antigen-presenting cell, bound to a “self” MHC molecule, and transported to the cell surface. Many different peptide antigens can be processed and presented to T cells, including those derived from bacteria, viruses, toxins, and foreign cells and tissues, as well as from autologous tissue and cellular products. Definition of the peptide-binding motifs that characterize HLA molecules provides important information about the role of the HLA system in peptide presentation, immune responsiveness, and disease susceptibility [22–24]. The role of the MHC in antigen presentation helps explain the extensive polymorphism of the MHC at the population level: the greater the polymorphism of the MHC, the greater the array of foreign peptides that can be presented to the immune system. In this way, MHC genes influence T-cell immune responsiveness by the selection of antigens that can be bound and presented for T-cell recognition. MHC molecules have a major effect in transplantation due to the fundamental role they play in T-cell activation and initiation of an alloresponse. With the advent of DNA typing methods, it is now possible to define each class of HLA molecule by its unique sequence (http://www.ebi. ac.uk/imgt/hla). The remarkable diversity among genes of the HLA system has greatly exceeded expectations and reflects the fundamental role the MHC plays in immune responsiveness and susceptibility and resistance to disease [25]. An appreciation of HLA polymorphism is also central to understanding how histocompatibility antigens function as transplantation determinants through their interaction with T cells (see Chapter 11) and natural killer (NK) cells (see Chapter 13) and by the presentation of minor histocompatibility antigens (see Chapter 18). Because the HLA system is highly polymorphic, most individuals are heterozygous for two different parentally derived alleles at each HLA locus. For a given HLA locus, the antigens encoded by the two HLA alleles are codominantly expressed, and a heterozygous individual will therefore possess two different antigens at that locus. The polymorphism displayed by HLA genes, coupled with their tendency to be strongly linked to one another, has important implications in donor–recipient histocompatibility matching for hematopoietic cell transplantation (HCT). In this chapter, the principles of genetic diversity and linkage disequilibrium (LD) and the practical ramifications of these two hallmarks of the MHC in relationship to HCT are reviewed.

HLA genes: structure and function Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

HLA antigens are encoded by a series of closely linked genes within the MHC at position p21.3 on the short arm of chromosome 6. This region

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Table 12.1 The International Histocompatibility Workshops Workshop

Year

Chairman

Venue

Advances

References

1st

1964

D.B. Amos

Durham, NC, USA

[8]

2nd 3rd 4th 5th 6th 7th 8th

1965 1967 1970 1972 1975 1977 1980

J.J. van Rood R. Ceppellini P. Terasaki J. Dausset F. Kissmeyer-Nielsen W. Bodmer P. Terasaki

Leiden, The Netherlands Turin, Italy Los Angeles, CA, USA Evian, France Aarhus, Denmark Oxford, UK Los Angeles, CA, USA

9th

1984

E. Albert, W. Mayr

10th

1987

B. Dupont

11th

1991

12th 13th

1996 2002

T. Sasazuki, K. Tsuji, M. Aizawa D. Charron J. Hansen

Munich, Germany; Vienna, Austria Scanticon, NJ; New York, NY, USA Yokohama, Japan

Study of the Hu-1, LA and FOUR antigens; MLC; microcytotoxicity serology technique Computerized data analysis; skin grafting Family studies; HLA in renal transplantation Definition of 27 HLA-A, -B, and -C specificities Worldwide typing of 49 populations Description of Dw specificities Definition of DR1-7 specificities; HTC testing Definition of HLA-MB (DQ) and MT (DR52/53); HLA in renal transplantation and disease association New class I and II specificities; transplantation HLA class II in renal Establishment of RFLP/T-cell clones and HTC methods; creation of standardized homozygous cell line panel HLA class I PCR typing; anthropology

14th

2005

J. McCluskey

St-Malo and Paris, France Victoria, BC, Canada; Seattle, WA, USA Melbourne, Australia

Sequencing; class I DNA typing; HLA in medicine Virtual DNA analysis; identification of SNP markers; HLA in anthropology, disease association, HCT MHC and anthropology, disease, infection, HCT, cancer, non-classical HLA genes

[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

HCT, hematopoietic cell transplantation; HLA, human leukocyte antigen; HTC, homozygous typing cell; MLC, mixed lymphocyte culture; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SNP, single nucleotide polymorphism.

CLASS II 1000 Kb

CLASS III 1000 Kb

CLASS I 2000 Kb

MICB DOB DP

DM

TAP

C4B DR

C4A C2

B C

E

A

HG F

Hfe

DQ CYP21 Bf

DNA

TNA αβ

HSP 70

MICA LT

Centromeric

Telomeric

Fig. 12.1 The human major histocompatibility complex (MHC) on the short arm of chromosome 6. The class II HLA-DP, DNA, DM, DOB, DQ, ad DR genes and the class I HLA-B, C, E, A, H, G, and F genes are shown in black; those encoding classical transplantation antigens are shown as solid bars, others are cross-hatched. Other HLA region-associated genes are shown in gray: transporter of antigenic peptides (TAP); 21-hydroxylase (CYP21; also termed 21-OH or P450-C21B); complement component C4; properdin factor B of the alternate complement pathway (Bf); complement component C2; heat-shock protein (HSP70); tumor necrosis factor (TNF) complex with TNF-α, TNF-β, and lymphotoxin-A (LTA); MICB; MICA; and the hemachromatosis gene (Hfe). The distance from the HLA-F gene telomeric to the Hfe gene is approximately 4000 kilobases.

was first sequenced in 1999 [26] and is now known as the classical MHC [27]. The classical MHC comprises approximately 4.0 Mb of DNA, equivalent to 0.1% of the human genome. With the more recent complete sequencing of chromosome 6, however, the human MHC is now considered to extend an additional 3.9 Mb telomeric to the classical MHC to the HIST1H2AA gene, and an additional 0.2 Mb centromeric to the RPL12P1 gene [28]. The extended MHC, or xMHC, thus comprises a total of 7.6 Mb. HLA genes of the xMHC are clustered in three distinct regions, designated class I, class II, and class III (Fig. 12.1). Genes within the class I and class II regions share structural and functional properties and are considered part of the immunoglobulin gene superfamily. Although distinct in sequence and structure, both class I and class II genes encode polypeptides that are critical in controlling T-cell recognition and determining histocompatibility in transplantation.

A characteristic feature of HLA genes is their extreme polymorphism (Tables 12.2 and 12.3). HLA diversity is a reflection of the primary immunologic function of MHC molecules, which is to bind and present antigenic, and potentially pathogenic, peptides for recognition by antigen-specific T-cell receptors (TCRs). The differing structural properties of HLA class I and class II molecules account for their respective roles in activating different populations of T lymphocytes. Cytotoxic T lymphocytes recognize antigenic peptides presented by HLA class I molecules, while helper T lymphocytes recognize antigenic peptides presented by HLA class II molecules. HLA class I and class II molecules are characterized by distinctive α and β polypeptide subunits that combine to form αβ heterodimers that characterize the mature molecule. Class I molecules were originally defined by typing with alloantisera and class II molecules defined by testing in

Histocompatibility

147

Table 12.2 Class I HLA antigens and alleles A locus

B locus

C locus

Antigens†

Allele(s)

Antigens

Allele(s)

Antigens

Allele(s)

Antigens

Allele(s)

A1 A2 A3 A23(9)‡ A24(9) A9 A25(10) A26(10) –§ A11 A29(19) A30(19) A31(19) A32(19) A33(19) A34(10) A36 A43 A66(10) A68(28) A69(28) A28 A74(19) A80

A*0101-10 A*0201-75 A*0301-14 A*2301-12 A*2402-49 A*2410; 19; 22 A*2501; 02 A*2601-17 A*2503; 04 A*1101-21 A*2901-04 A*3001-09 A*3101-05 A*3201-06 A*3301-06 A*3401-04 A*3601-02 A*4301 A*6601-04 A*6801-02; 6804-11; 6813-19 A*6901 A*6803; 12 A*7401-05 A*8001

B7 B703 B8 B13 B14 B15 B18 B27 B2708 B35 B37 B38(16) B39(16) B40 B41 B42 B44(12) B45(12) B46 B47 B48 B49(21) B50(21) B51(5)

B*0702; 0704-26 B*0703 B*0801-13 B*1301-07 B*1401-06 B*1501-64 B*1801-05 B*2701-07; 2709-23 B*2708 B*3501-37 B*3701-05 B*3801-07 B*3901-24 B*4001-35 B*4101-05 B*4201-02 B*4402-24 B*4501-04 B*4601-02 B*4701-03 B*4801-07 B*4901-03 B*5001-02; 5004 B*5101-24

B52(5) B53 B54(22) B55(22) B56(22) B57(17) B58(17) B59 B67 B73 B78 B81 –§ –

B*5201-03 B*5301-02 B*5401-02 B*5501-10 B*5601-07 B*5701-07 B*5801-06 B*5901 B*6701-02 B*7301 B*7801-05 B*8101 B*8201 B*8301

Cw1 Cw2 Cw10(w3) Cw9(w3) Cw3 Cw4 Cw5 Cw6 Cw7 Cw8 –§ – – – – – –

Cw*0102-04 Cw*0202-04 Cw*0302; 0304 Cw*0303 Cw*0305-12 Cw*0401-08 Cw*0501-04 Cw*0602-07 Cw*0701-14 Cw*0801-09 Cw*1202-07 Cw*1301 Cw*1402-04 Cw*1502-10 Cw*1601-02; 1604 Cw*1701-03 Cw*1801-02

† Antigens are defined by alloantisera. Antigens listed in parentheses are broadly defined public specificities subsequently split into two or more subtypic antigens. ‡ Allele(s) encoding the corresponding antigen expressed at the cell surface. § For some HLA antigens defined by DNA typing methods, no corresponding alloantigen has been defined by alloantisera or cellular typing.

functional assays such as the mixed lymphocyte culture (MLC) reaction [29,30]. The class I region Classical HLA genes At least 17 loci including several pseudogenes exist in the HLA class I region (Fig. 12.1). Three of these loci, termed class Ia, encode HLA-A, B, and C alloantigens that constitute the major class I determinants important for matching in tissue transplantation. Genes of the HLA-A, B, and C loci show a striking degree of sequence and structural homology with one another, and all are highly polymorphic. As of June 2007, for example, more than 830 alleles had been defined at the HLA-B locus, making HLA-B the most polymorphic locus in the human genome, as summarized in Table 12.2 [31]. Class I HLA-A, B, and C genes consist of eight exons and seven intervening introns. Exon 1 is a leader sequence; exons 2, 3 and 4 encode the α1, -2, and -3 domains of the class I molecule, respectively; exon 5 encodes the transmembrane portion, and exons 6, 7 and 8 encode the cytoplasmic tail. The expressed HLA class I molecule consists of a single polymorphic α (heavy) chain of approximately 338–341 residues in length that is noncovalently bound at the cell surface to a β2-microglobulin light chain (Fig. 12.2). The gene for β2-microglobulin is located outside of the HLA complex on chromosome 15. Structurally, class I molecules comprise two α-helical regions overlaying an eight-strand

antiparallel β-pleated sheet that together form the functional groove for peptide binding (Fig. 12.3a) [22]. Nucleotide substitutions within exons 2 and 3 of class I genes are not distributed randomly but are concentrated in discrete hypervariable regions (Fig. 12.4). The corresponding polymorphic sites within the α1 and α2 domains of the encoded proteins facilitate the binding of peptide fragments for presentation to the TCR [32]. These polymorphic sites determine the allospecificity of the molecule and form the basis for their classification as HLA alloantigens (Table 12.2). In addition, polymorphic residues 77–80 of HLA-C molecules and 77–83 of HLA-B molecules (the latter constituting the Bw4 epitope) define the specificity of NK receptor recognition of HLA-C and -B ligands. Nonclassical genes The class I region contains at least three nonclassical genes – HLA-E, F, and G [33,34] – and two class I-like genes, MICA and MICB [35,36]. Genes at the HLA-E, F, and G loci are termed class Ib genes and are very similar to class Ia genes in terms of overall organization and the relationship between exon sequence and function of the encoded molecule. Class Ib genes appear to serve specialized roles in immune responses, and the molecules they encode have a prominent role in both adaptive and innate immune pathways [37]. One difference between classical Ia and nonclassical Ib molecules is their tissue expression: whereas class Ia molecules are expressed on all nucleated cells and are therefore present in most tissues, class Ib molecules are more restricted

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Table 12.3 Class II HLA antigens and alleles DRB1 locus

DQB1 locus

DPB1 locus

Antigens†

Allele(s)

Antigens

Allele(s)

Antigens

Allele(s)

DR1 DR15(2)‡ DR16(2) DR17(3) DR18(3) DR3 DR4 DR11(5) DR12(5) DR13(6) DR14(6) DR7 DR8 DR9 DR10

DRB1*0101-07 DRB1*1501-11 DRB1*1601-08 DRB1*0301; 0304-05 DRB1*0302-03 DRB1*0306-18 DRB1*0401-38 DRB1*1101-41 DRB1*1201-07 DRB1*1301-47 DRB1*1401-40 DRB1*0701-04 DRB1*0801-23 DRB1*0901 DRB1*1001

DQ5(1) DQ6(1) DQ2 DQ7(3) DQ8(3) DQ9(3) DQ3 DQ4

DQB1*0501-04 DQB1*0601-17 DQB1*0201-03 DQB1*0301;0304 DQB1*0302;0305; 0310 DQB1*0303 DQB1*0306-09 DQB1*0401-02

DPw1 DPw2 DPw3 DPw4 DPw5 DPw6 –§

DPB1*0101 DPB1*0201-02 DPB1*0301 DPB1*0401-02 DPB1*0501 DPB1*0601 DPB1*0801-8901¶

DQA1 locus –§ – – – – –

DQA1*0101-06 DQA1*0201 DQA1*0301-03 DQA1*0401 DQA1*0501-05 DQA1*0601

DPA1 locus –§ – – –

DPA1*0103-07 DPA1*0201-03 DPA1*0301-02 DPA1*0401

† Antigens are defined by alloantisera. Antigens listed in parentheses are broadly defined public specificities subsequently split into two or more subtypic antigens. ‡ Allele(s) encoding the corresponding antigen expressed at the cell surface. § For some HLA antigens defined by DNA typing methods, no corresponding alloantigen has been defined by alloantisera or cellular typing. ¶ Eighty-one additional DPB1 alleles have been defined (DPB1*0801-8901) for which no corresponding alloantigens have been defined.

α2

α1

N N

C

C

β2m

α3

Fig. 12.2 Schematic representation of an HLA class I molecule. Ribbon diagram of a class I molecule showing the four domains. The α3- and β2microglobulin domains proximal to the cell membrane are shown at the bottom, and the polymorphic α1 and α2 domains are shown at the top. The β strands are indicated as wide arrows, while the α-helical portions are shown as coiled ribbons.

Histocompatibility

(a)

149

(b)

Fig. 12.3 The location of polymorphic sites in the antigen-binding cleft of HLA class I and class II molecules. Schematic representation of an HLA class I molecule (A) and an HLA class II molecule (B) as seen from the top surface. The α1 and α2 domains (class I) and α1 and β1 domains (class II) form the sides of the antigen-binding cleft. Polymorphic sites with maximum amino acid variation from allele to allele are shown as colored bars.

Amino acid position 5 *

10 *

20 *

90 *

Cw*0102

MKYFFTSVSRPGRPGRGEPRFIS

Cw*0202

R

Y

A

Cw*0302

R

Y

A

Cw*0408

R

S

Cw*0502

R

Y

A

Cw*0710

R

D

A

Cw*0701

R

Y

A

S

W

H

A

H

A

....

100 *

151 *

AGSHTLQWMCGCDLGPD

A

I

R Y

D

D

I

170 *

REAEQRRAYLEGTCVEWLRR

R Y

A

A

....

160 *

V

W

E

L

L

R F

L

R Y

M

II

R S

I

R Y

II

A

L L

E

G

III

Fig. 12.4 Variable sites within a class I molecule. Sequence alignment of seven HLA-C alleles illustrating the clustering of amino acid substitutions in hypervariable regions (indicated by roman numerals, bottom). The polymorphisms map to sites on the floor (I) and within the antigen binding groove (II, III) of the class I molecule (see Fig. 12.3a). The consensus sequence (the most common amino acids found at a given position) is shown for the Cw*0102 allele.

in distribution and tend to be tissue specific. Of the three, HLA-E is the most ubiquitously expressed [34,38]. HLA-G is expressed specifically in placental tissue [39,40], while HLA-F is expressed in the bladder, liver, and placenta and on lymphoblastoid cells [41]. The function of HLA-E, F, and G molecules is not fully known, but they appear at least in part to serve as ligands for NK cell receptors and thereby regulate NK function [42,43]. HLA-E molecules bind specifically to the conserved leader sequence peptides from class Ia MHC molecules to promote cell surface interactions with the CD94/NKG2 class of NK cell receptors. MICA and MICB genes share amino acid sequence homology with the classical class I proteins but are distinguished from HLA-A, B, and C by the lack of association to β2-microglobulin [44] and the absence of peptide-binding function [35] due to a shallow peptide-binding groove [36]. MIC proteins are expressed in the intestinal epithelium and play a fundamental role as cell stress response genes inducible by heat shock [35]. MICA and MICB products are ligands for the NKG2D-activating receptor involved in NK immunosurveillance and in co-stimulation of T-cell responses [45]. The clinical importance of MICA and MICB in transplantation is unknown, although preliminary data suggest that donor–recipient matching for the region inclusive of the MICA and MICB genes is associated with clinical outcome [46].

The class II region The HLA class II region is comprised of nine distinct genes: DRA, DRB1, DRB3, DRB4, DRB5, DQA, DQB, DPA, and DPB. The class II region also contains functionally related genes (TAP and LMP) that play a role in the loading of class I molecules with peptides. Six additional class II genes or gene fragments have been described, but these are either nonfunctional pseudogenes or do not encode proteins known to participate in transplant-related immune interactions. Class II genes have been collectively referred to as HLA-D region genes since they were initially described in the mid-1970s after the description of the HLA-A and B (1965) and C (1971) loci. Class II genes are divided into five families, designated DR, DQ, DO, DN, and DP, based on their degree of sequence homology and their location within the HLA-D region. Class II HLADR, DQ, and DP genes, like class I genes, are highly polymorphic, with more than 270 alleles thus far defined at the DRB1 locus [31]. Class II molecules consist of a single polymorphic (DQ and DP) or nonpolymorphic (DR) α chain noncovalently bound to a polymorphic β chain (Fig. 12.3b) [23]. The β chains of HLA-DR, DQ, and DP antigens are encoded by the DRB, DQB, and DPB genes, respectively. Polymorphic HLA-DR β chains are encoded by four distinct DRB genes termed

150

Chapter 12 DR haplotype

Variable presence of HLA-DR genes DRB1

ψ

DRB1

ψ

DRB1

ψ

ψ

DRA

ψ

DRA

ψ

DRA

ψ

DRA

ψ

DRA

DR1, 10, 103

DRB5

DR15, 16

DRB3

DR3, 11, 12 13, 14 DRB1 DR8

DRB1

ψ

ψ

DRB4

DR4, 7, 9

Fig. 12.5 Variable expression of DRB genes according to HLA haplotype. Open boxes: DRB genes; gray boxes: nonpolymorphic DRA gene; black boxes: pseudogenes (Ψ).

DRB1, DRB3, DRB4, and DRB5. HLA-DRB genes are inherited as a genetic unit, or haplotype (see “Characteristics of the MHC,” below). Within the HLA-DRB region, variable numbers of genes are expressed from one haplotype to another (Fig. 12.5). For example, DRB haplotypes that type as DR1 include four genes: a polymorphic DRB1 gene, a nonpolymorphic DRA gene, and two pseudogenes. In contrast, DRB haplotypes that type as DR3 include five genes: a polymorphic DRB1 gene, a nonpolymorphic DRA gene, a polymorphic DRB3 gene, and two pseudogenes. Further polymorphism within the HLA-D region can result from trans pairing of a polymorphic DQ α chain encoded by one parental chromosome with a polymorphic DQ β chain encoded by the other parental chromosome. Polymorphic sites within class II molecules are localized in specific regions of the α1 and β1 domains of the α and β chains, respectively, to enable binding of a large array of peptides [32,47]. The polymorphic α1 and β1 domains also determine the allospecificity of the class II molecule (summarized in Table 12.3). Antigens encoded by DRB1 and DQB genes are known to be potent stimulators of immune reactions in HCT (see Chapter 47).

Historical perspective: collaborative studies of the human MHC, development of an HLA nomenclature and evolution of laboratory typing methods Following the description of the first HLA group in 1958, there was a rapid growth of interest in and knowledge about the genetic structure and biologic function of the human MHC, or HLA system. The dramatic advance in knowledge was largely due to the early appreciation of the scope of work necessary to elucidate the HLA system and the broad collaboration beyond what would be possible within individual laboratories. This led to the organization of an international group of investigators willing to share reagents and unpublished data, first begun in 1964 (Table 12.1). The first HLA antigens had been defined by individual investigators using their own reagents, antisera, and cell panels, and employing a variety of locally developed laboratory assays. An exchange of reagents was necessary to compare antisera and standardize antigen definition, establish a common nomenclature, and develop standardized testing methods. The International HLA Workshops thus provided a critical mechanism for promoting this collaboration, as well as creating a forum for promoting new technology and disseminating reagents and technical skills worldwide.

The class III region The HLA class III region is located between the HLA class II (centromeric boundary) and class I (telomeric boundary) regions of the HLA complex (Fig. 12.1). The class III region comprises some 60 genes within approximately 700 kb of DNA, making it the most gene-dense region of the human genome. More than 14% of the sequence within this region is coding, with an average of 8.5 genes per 100 kb of DNA [48]. The class III region genes display LD with HLA loci, and haplospecific polymorphisms have been described for several common xMHC haplotypes [49]. The class III region has been associated with susceptibility to numerous diseases, including Graves’ disease, Crohn’s disease and systemic lupus erythematosus. Those with proven function include genes encoding the complement components C2, C4, and Bf, tumor necrosis factor, certain transport proteins, and heat shock proteins. The role of class III cytokine genes in modulating risk for graft-versus-host disease (GVHD) after allogeneic transplantation is under active investigation [50].

The early period: 1964–72 Between their initial discovery in the late 1950s and the first HLA Workshop in 1964, leukocyte (HLA) antigens were usually identified using agglutination assays employing leukoagglutinating (immunoglobulin M [IgM]) antibodies. During the first HLA Workshop at Duke University in 1964, Terasaki and colleagues introduced a complementdependent microdroplet assay that represented a significant improvement over the leukoagglutination method. The microdroplet assay required only microliter amounts of cytotoxic (IgG) antibodies that were of relatively high specificity and suitable reproducibility. Cytotoxic antisera were carefully selected for HLA specificity and were usually obtained from multiparous women who had been immunized to HLA alloantigens through pregnancy. Based on available data from the serotyping assays studied during the 1st Workshop, a preliminary analysis of the relationship between serologic matching of renal transplant donors and recipients and graft outcome was undertaken.

Histocompatibility

The in vitro MLC, recognized as a potential method for assessing histocompatibility, was also studied in the 1st Workshop. The MLC assay involved culturing responder leukocytes, later identified as T lymphocytes, with second-party stimulator cells and measuring the ensuing proliferative response. The magnitude of the response was considered an indication of the disparity between the two cell populations for certain HLA antigens, although the exact nature of these antigens was as yet undetermined. The 3rd HLA Workshop (1967) provided evidence that the genetic system under study consisted of two loci that were closely linked. Following this workshop, a nomenclature committee of geneticists and immunologists was organized to decide on a standardized nomenclature. The designation of “HL-A” (H standing for the Hu1 system of Dausset, L standing for leukocyte, and A for the first recognized locus within this system) was subsequently adopted. Since 1967, the naming of HLA genes and the alleles comprising each HLA locus has been delegated to the World Health Organization Nomenclature Committee for Factors of the HLA System [31]. The first report of this nomenclature committee was published in 1968 [51] and was the initial attempt to establish antigen specificity using serologically defined antibodies. By the 4th HLA Workshop (1970), 11 official HL-A specificities (HLA1, A2, A3, A5, A7, A8, A9, A10, A11, A12, and A13) had been defined, with numeric designations being assigned in sequential order of discovery and reporting. At least eight other specificities were given provisional “Workshop” designations (e.g. w27) indicating probable but not conclusive characterization. There was also evidence of a third sublocus within the HL-A system [52]. The following year, a landmark paper by Yunis and Amos suggested that the rejection time of skin and organ transplants was dependent on immunization against the products of at least two separate, but closely linked, genetic systems: HL-A (defined serologically), the mixed leukocyte reaction (defined by cellular typing) and possibly a third, the hypersensitivity delayed reaction [53]. Following the 5th Workshop (1972), the nomenclature committee confirmed the existence of at least two subloci within the HL-A system and assigned the known antigens to two distinct segregant series, the first, or LA, series, and the second, or FOUR, series. The identity of the locus encoding determinants causing stimulation in the MLC reaction remained to be elucidated. The middle period: 1975–84 This period of investigation of the HLA system was highlighted by the introduction and refinement of serologic methods for defining class II region determinants, the development of additional cellular assays for the definition of the class II region, and a dawning recognition of the enormity of HLA diversity which ultimately ushered in the DNA era of HLA testing. Four International Workshops were held between 1975 and 1984 (Table 12.1). By the 6th Workshop (1975), it was recognized that disparity for HLA-D region antigens led to the lymphocyte activation and proliferation measured in the MLC assay [54]. The 6th Workshop was the first to include the use of D-region homozygous typing cells to study determinants of the newly defined HLA-D locus [55]. Numerous reports of HLA antigens expressed on B cells but not on T cells were also presented at the 1975 Workshop. These antigens were termed “Ia-like” or “D region-associated B-cell antigens,” and their description paved the way for the future description and characterization of the HLA-DR locus and the serologically defined DR series of leukocyte antigens. Following the 6th Workshop, the World Health Organization nomenclature committee recommended that the term “HL-A” be changed to “HLA” in recognition of the fact that HLA was an entire genetic region that contained multiple loci designated A, B, C, and D.

151

Although the direct MLC had been used as a tool for donor selection in HCT since the late 1960s, its utility as a test of histocompatibility was largely restricted to confirming nonreactivity, or HLA-D region compatibility, between HLA-A, B, DR serologically matched donors and recipients. Data were ultimately published indicating that the MLC was not consistently predictive of the risk of clinically severe acute GVHD [56]. In the late 1980s, with the advent of precise class II molecular typing tools, DNA-based methods supplanted both the direct MLC and the use of D-region homozygous typing cells for the evaluation of donor– recipient compatibility in HCT. In contrast, the leukocyte antibody crossmatch continues to be used as an important indicator of histocompatibility in HCT. Historically, leukocyte antibody crossmatches were performed using a complement-dependent cytotoxicity assay testing recipient serum against donor lymphocytes. More recently, crossmatches have been performed utilizing flow cytometry, since this method provides both increased sensitivity and specificity, particularly when relatively weak HLA antibodies are present. By using fractionated T and B lymphocytes as target cells in the crossmatch assay, antibodies directed against both HLA class I and class II antigens can be demonstrated. A positive antidonor result in the complement-dependent cytotoxicity assay, indicative of the presence of donor-directed HLA antibodies in the HCT recipient, has been shown to be highly predictive of graft failure [57]. Similarly, a positive patient antidonor crossmatch by flow cytometry has also been shown to be predictive of graft failure [58]. The crossmatch test thus represents an important assay for donor pretransplant evaluation to predict the risk of graft rejection, particularly when HLA-mismatched donors are considered. In the mid-1980s, serologic typing became more refined as large numbers of highly specific HLA antisera were accumulated and tested against diverse panels of target cells during International Workshops. It was soon recognized that operationally monospecific antibodies could detect alloepitopes shared by more than one HLA class I gene product [59,60]. The term “crossreactive” was coined to describe antigenic epitopes shared by two or more distinct HLA antigens, “public specificities” to describe epitopes shared by more than one distinct antigen, and “private specificities” to describe epitopes that were unique to a single antigen (Tables 12.4 and 12.5). The identification of private and public specificities provided a means to cluster serologically crossreactive antigens into crossreactive groups (CREGs). In the early unrelated donor transplant experience, CREG-mismatched unrelated donors were considered when matched donors were not available [61]. During the 9th HLA Workshop (1984), several papers were presented describing the use of restriction fragment length polymorphism to study the HLA system at the DNA level. These early efforts demonstrated the power and utility of DNA technology for HLA typing and paved the way for its use in subsequent Workshops. At the completion of the 9th Workshop, more than 124 HLA specificities at the A, B, C, D (MLCdefined), DR, DQ, and DP loci had been defined [62]. The later period: 1987 to the present day The discovery of polymerase chain reaction (PCR) technology in the mid-1980s propelled the HLA field into the DNA era. At that time, the MHC was one of the most comprehensively characterized regions of the genome, and PCR-based technology development was feasible for the HLA class II region because of the availability of sequence information for HLA-DR antigens. The first International HLA Workshop to introduce DNA-based methods to define the extent of genetic variation of the MHC was the 10th Workshop (1987), chaired by Dr Bo Dupont. A major thrust of this workshop was to examine HLA class II sequence diversity using the restriction fragment length polymorphism technique.

152

Chapter 12

Table 12.4 HLA-A and -B cross-reactive antigen groups (CREGs)*

A

B

C

D

E

F

G

H

I

J

K

L

HLA-A locus A1, A3, A11, A36 A23, A24, A9 A25, A26, A34, A66, A43, A10 A19, A29, A30, A31, A32, A33, A74 A2, A28, A68, A69 HLA-B locus B5, B18, B35, B51, B52, B53, B70, B71, B72, B78 B12, B21, B44, B45, B49, B50, B4005 B14, B64, B65 B8, B59 B15, B46, B57, B58, B62, B63, B70, B71, B72, B75, B76, B77 B16, B38, B39, B67 B7, B27, B42, B73 B7, B22, B54, B55, B56, B67 B7, B40, B41, B48, B60, B61, B81 B13, B47 * Cross-reactive groups defined by the National Marrow Donor Program (NMDP). Antigens in each line are members of the same HLA-A or -B locus CREG and share a common public specificity (epitope); antigens in different lines belong to different CREGs. A donor–recipient incompatibility for antigens in the same line (e.g. B13 versus B47) represents a minor mismatch. A donor–recipient incompatibility for antigens from different lines (e.g. B18 versus B21) represents a major mismatch. Certain antigens (e.g. HLA-A80) are not part of any defined cross-reactive group. HLAB7 is a member of three different CREGs.

Table 12.5 Definition of Public and Private HLA-DR Specificities DR1 DR2 (DR15, DR16) DR3 (DR17, DR18) DR4 DR5 (DR11, DR12) DR6 (DR13, DR14) DR7 DR8 DR9 DR10 The public specificities, DR1-10, are given in the left-hand column. The private specificities associated with the DR2, 3, 5 and 6 public specificities are shown in parentheses.

Data from these studies revealed previously undetected diversity within this region. The development of different platforms for DNA typing was prolific. Three major technologies emerged and became integrated in both clinical and research applications, including sequence-specific primer (SSP)

Fig. 12.6 Sequence-specific primer (SSP) typing. In the SSP method, a panel of amplification primers is designed to detect all known polymorphisms encoded by an HLA locus or group of alleles. Polymerase chain reactions (PCRs) are first performed for each of the primer pairs. The PCR products are then electrophoresed on a gel, and the presence or absence of a PCR product of the appropriate size is scored. Assignment of an HLA type is made by examining the pattern of positive and negative PCR reactions. Advantages of the SSP method include its relatively low cost, its technical simplicity and the speed with which low-resolution typing can be achieved. High-resolution-level typing requires as many primers as there are polymorphisms, and large numbers of PCR reactions are therefore necessary, increasing both assay time and cost. This figure shows the results of a typical SSP assay in which genomic DNA from a single sample was amplified with a panel of HLA-C primer pairs, electrophoresed on an agarose gel, stained with ethidium bromide and visualized with ultraviolet translumination. All primer pair amplicons (lanes A through L) showed amplification of the 796base pair (bp) fragment from the internal control primers, demonstrating successful PCR conditions in each sample. The sample was typed as Cw*06 and Cw*07 by the identification of specific amplification products in lane F (1062 bp fragment specific for Cw*0701/0702/0703) and lane G (304 bp fragment for Cw*06).

methods (Fig. 12.6) [63], sequence-specific oligonucleotide probe (SSOP) hybridization (Figs 12.7 and 12.8) [64,65], and direct automated sequencing-based typing (SBT) (Fig. 12.9) [66,67]. SSP provided a simple and low-cost method for low-to-intermediate resolution of HLA alleles. The SSOP and SBT methods provided high resolution of alleles and the capacity for increased throughput. As the use of these methods in the research and clinical settings increased, so did the discovery of novel HLA alleles. As a result, the HLA nomenclature committee introduced new guidelines for the naming of HLA alleles following the 10th Workshop. In the revised nomenclature system, the allele (e.g. DRB1*0405) is defined by the genetic locus (DRB1) followed by an asterisk separating the locus name from the allele name. The first two digits of the allele name were meant to correspond to the broadly defined serologic specificity group to which the allele belongs (04), with the second two digits (05) uniquely identifying the allele. In 1989, 2 years following the conclusion of the 10th Workshop, the first HLA nomenclature committee meeting outside of the venue of an International Workshop was held. This was the first instance in which official designations were assigned to HLA alleles that had been defined by direct sequencing of the corresponding HLA gene. By this time, a total of 56 HLA class I and 78 HLA class II alleles had been defined by sequencing-based typing. In 1990, the discovery of “silent” (synonymous) nucleotide substitutions in HLA genes (i.e. those that do not result in changes in amino acid composition of the encoded HLA molecule) required the addition of a fifth (and later sixth) digit to the allele name. DNA-based methods were a major theme of the 11th Workshop (1991). This workshop explored the extent of diversity of HLA class II genes and was the first Workshop to introduce high-throughput strategies for HLA class II typing. In 1994, the first HLA null allele, DRB4*01012N, was recognized. To accommodate the growing number of polymorphisms within coding and noncoding regions of HLA genes, additional digits were subsequently added to the nomenclature system. Since 1995, monthly nomenclature updates in HLA journals and the

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A B

Fig. 12.7 Sequence-specific oligonucleotide probe (SSOP) “forward blot” typing. With this method, the HLA gene of interest is amplified by PCR and immobilized on nylon membranes. Oligonucleotide probes labeled with a reporter molecule (either radioactive or nonradioactive) are then allowed to hybridize to the membranes. Probes with sequences complementary to the membrane-bound DNA will hybridize, while those with as little as a single nucleotide mismatch will not. The pattern of positive and negative hybridization is used to deduce the HLA type of the target DNA. In this method, only the known polymorphic sites in an HLA gene are probed, and therefore no information is obtained for regions of the gene not defined by the sequence-specific probes. Although novel patterns of probe hybridization may indicate the possible presence of a new allele, alleles with polymorphisms outside of the probed regions may escape detection. Since many samples being examined for a given HLA gene can be tested on the same set of membranes, the SSOP method is particularly well suited to highvolume HLA typing. This figure shows the results of SSOP typing of genomic DNAs of 12 known control samples and 82 unknown samples that were amplified with HLA-A locus-specific primers, blotted on a series of nylon membranes and probed with a panel of HLA-A locus probes. Positive hybridization of probes labeled with alkaline phosphatase was detected by chemiluminescence and exposure to X-ray film. In this example, a probe specific to a sequence found only in A*29 and A*43 at codons 62 and 63 of exon 2 showed positive hybridization with two control samples (outlined in bold) and eight of the unknown test samples.

IMGT database (http://www.ebi.ac.uk/imgt/hla) have been necessary to accommodate the ongoing, relentless discovery of new HLA variants. Beginning in 2002, the expansion of HLA allele designations to eight digits has provided greater flexibility for cataloguing variation. Many of the alleles currently defined by DNA-based typing methods do not have a serologically defined equivalent, placing a further requirement for flexibility and adaptability on the HLA nomenclature system. Application of PCR-based methods in the mid-1990s carried through to the 12th HLA Workshop (1996) chaired by Dr Dominique Charron, in which methods for characterizing classical class I alleles provided a breakthrough for population-based analysis in this region of the MHC. The 12th Workshop included studies of human diversity, HLA in cancer, transplantation, and disease, as well as a thorough exploration of newly defined HLA genes, alleles, and haplotypes. With DNA-based typing technology solidly established in HLA research, the 13th and 14th HLA Workshops focused international collaborative efforts on furthering understanding of the functional significance of the MHC in disease models. In the 13th Workshop (2002), several new research initiatives were undertaken, including studies of cytokine gene polymorphism, HCT, HLA and disease (type 1 diabetes, rheumatoid arthritis, celiac disease, narcolepsy, and spondyloarthropa-

C

D E F G

Fig. 12.8 Sequence-specific oligonucleotide probe (SSOP) “reverse probe” format typing. In this assay, the sequence-specific probes are immobilized on a solid-phase support. Sample DNA is labeled during PCR amplification and allowed to hybridize to the immobilized panel of probes. As with forward SSOP, HLA alleles are deduced from the pattern of positive and negative probe reactions. In this figure, genomic DNA was amplified with DRB locusspecific biotinylated primers and hybridized to a panel of probes for DRB1, DRB3, DRB4, and DRB5 sequences immobilized on a nylon membrane strip. The pattern of positive hybridization reactions, detected by color formation, was interpreted with a computerized analysis program. The sample was typed as DRB1*15 or *16 by the presence of band A, DRB1*04 by bands B and C, DRB4*01 by band D, and DRB5*0101 or *0104 by bands E and F.

thy), HLA and cancer, HLA and reproduction, the HLA peptide repertoire, the killer immunoglobulin-like receptor (KIR) system, and minor histocompatibility antigens. A major contribution resulting from the 13th Workshop was the creation of a public database (dbMHC) in collaboration with the National Institutes of Health, the National Center for Biotechnology Information, and the National Library of Medicine. This database currently serves as an important resource for the scientific community, housing genotype and phenotype data for the international community. The 14th Workshop (2005) continued the tradition of collaborative research in HLA genetics. Extension of studies in disease, transplantation, minor histocompatibility antigens, and anthropology contributed to a significant expansion of research and the number of worldwide populations under analysis. Concurrent with the development of DNA-based typing methods during this period, several functional assays were studied as possible adjuncts for the identification of optimally matched HCT donors.

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Chapter 12 G A G T M C T G G A A C A G C C A G A A G G A C W T C C T G G 180 190 200

Fig. 12.9 Automated fluorescent sequencing-based typing (SBT). The HLA gene is first amplified in a polymerase chain reaction (PCR). Cycle sequencing of the PCR product is then performed using primers that are labeled with a fluorescent label (dye primer, usually 5′ primer). Alternatively, the dideoxynucleotides ddATP, ddTTP, ddGTP, and ddCTP can be individually labeled with fluorescent label (dye terminator). In dye primer sequencing the label is incorporated into the 5′ end of the HLA sequence, whereas in dye terminator sequencing the addition of a labeled dideoxynucleotide at the 3′ end terminates the sequencing reaction. In both approaches, the sequencing reaction is electrophoresed on a polyacrylamide gel using an automated sequencer. The fluorescent signals are captured and interpreted as a sequence of DNA bases by a computer program. Assignment of the HLA allele(s) is performed by comparing the derived sequence to all known sequences for a given gene. This figure shows the partial sequence chromatogram for polymorphic positions 179 and 199 (arrows) of DRB1 exon 2 (residues 60 and 67) from an HLA-DRB1*0801,1201-positive sample. The genomic DNA was sequenced using direct automated dye primer fluorescent chemistry on an Applied Biosystems Model 377 sequencer (ABI, Inc., Foster City, CA, USA). The laser-induced fluorescent signal emitted from each sequenced fragment is captured and interpreted by software and identified on the chromatogram above the peak as an A, C, G or T nucleotide base, except at heterozygous positions. Using the Inernational Union of Biochemistry codes, heterozygous position 179 has M, indicating signals from A (DRB1*0801) and C (DRB1*1201), while the W at position 199 indicates that T (DRB1*0801) and A (DRB1*1201) were incorporated.

Primary among these was the limiting dilution assay, a modification of the MLC in which patient and donor cells are co-cultured for an initial 3–6-day priming phase, followed by plating at limiting dilution which then generates a readout of patient-versus-donor and/or donor-versuspatient alloimmune reactivity. The frequency of both cytotoxic T-lymphocyte precursor and helper T-lymphocyte precursor cells can be measured in this manner [68–72]. The presence of elevated patient antidonor cytotoxic T-lymphocyte precursor and/or helper T-lymphocyte precursor frequencies might signal an increased risk of rejection of the stem cell graft, whereas the presence of elevated donor antipatient precursor frequencies might signal an increased risk of GVHD. Today, the use of cellular methods such as limiting dilution assay for the assessment of donor–recipient compatibility has largely been supplanted by DNA assays, although the former may have some utility in donor screening when several otherwise equally matched donors are available [73].

Characteristics of the MHC: LD and haplotypes LD As of April 2007, more than 469 HLA-A, 794 HLA-B, 244 HLA-C, 525 HLA-DRB1, 71 HLA-DQB1, and 124 HLA-DPB1 alleles had been defined (http://www.ebi.ac.uk/imgt/hla) (Tables 12.2 and 12.3) [31]. If each HLA allele occurred randomly, more than 3 × 1023 unique HLA-A, B, C, DRB1, DQB1 combinations (phenotypes) would be possible in the human population, and the prospects of identifying a matched unrelated donor would appear insurmountable. However, the HLA region is characterized by the phenomenon of LD, in which two or more alleles occur in nonrandom association and are hence observed at a higher frequency than would be predicted by chance alone, i.e. by the product of their individual allele frequencies. In studies of unrelated individuals where no family study is available, LD is often used as a mathematical prediction of the departure from expectation given the known frequencies of the phenotype or genotype in the population [74,75]. Strong positive LD between HLA-B and C and between HLA-DR and DQ increases the probability that a patient and donor matched for HLA-

A, B, and DR will also be matched for HLA-C and DQ. Conversely, mismatching at HLA-B or -DR increases the chance of HLA-C or DQ mismatching, respectively. HLA-A1, B8, DR3, the most common North American Caucasian haplotype, serves as a good example of how LD may aid in the identification of unrelated donors. Matching for two loci (e.g. A1 and B8) will almost always determine matching for the third (e.g. DR3), fourth (e.g. Cw7), and fifth (e.g. DQw2) loci. Moreover, an antigen (e.g. HLA-B8) that shows characteristic LD with another antigen is often found to be encoded by a specific and characteristic allele (e.g. HLA-B*0801). Using the example of the HLA-A1, Cw7, B8, DR3, DQw2 haplotype, the alleles that encode the phenotypically defined antigens of this group are HLA-A*0101, Cw*0702, B*0801, DRB1*0301, and DQB1*0201. The success of identifying suitable unrelated donors is a function of the haplotypic combination of alleles that the recipient has inherited. When patients possess HLA haplotypes with rarer combinations of alleles, it may be feasible to match for some but not all loci. LD phenomena also explain the highly complex patterns of multilocus HLA mismatching among unrelated donors and transplant recipients that have been uncovered in the retyping effort of legacy samples [76–78]. In a recent study of HLA-A, B, DRB1 serologically matched pairs by the National Marrow Donor Program, 29% encoded allele mismatches at HLA-A, B or DRB1 and 89% of HLA-A, B, DRB1 allele-identical pairs encoded additional mismatches at HLA-C and DP [79].

Haplotypes HLA alleles are inherited en bloc as a haplotype on the same strand of DNA. A rigorous identification of haplotypes can only be achieved by ascertainment of segregation of markers in a family study. The family study plays an important early role in planning for an allogeneic transplant. A family study provides the definitive means to validate the patient’s genotype and to determine the availability of HLA genotypically identical siblings who might be considered donors. Since the HLA system is inherited in classical Mendelian fashion, the probability that a patient and sibling will inherit the same two parental haplotypes is one

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Fig. 12.10 A family study. The paternal haplotypes are designated a/b and the maternal haplotypes c/d. Sibling 1 is the propositus. Compared with sibling 1, sibling 2 is a/c (HLA genotypically identical), sibling 3 is a/d (haploidentical), sibling 4 is b/c (haploidentical), sibling 5 is b/d (complete mismatch), and sibling 6 is a/c–d (maternal c–d recombinant). Siblings 1 and 4 are mismatched at HLA-B in the graft-versus-host (GVH) vector only. Siblings 1 and 6 are mismatched at only HLA-A for both GVH and host-versus-graft vectors; they are matched from HLA-B to DR due to the maternal recombinant haplotype.

Table 12.6 HLA haplotype frequencies in selected populations*

Population

HLA-A, -B, -DR haplotype

Haplotype frequency (%)

Caucasian American

A1, B8, DR3 A3, B7, DR2 A2, B44, DR4 A33, B58, DR3 A33, B44, DR6 A24, B52, DR2 A30, B42, DR3 A1, B8, DR3 A3, B7, DR2 A2, B35, DR8 A29, B44, DR7 A1, B8, DR3 A1, B8, DR3 A3, B7, DR2 A2, B44, DR2

5.2 2.6 2.2 1.6 1.5 1.4 1.7 1.2 0.8 1.8 1.7 1.7 4.7 2.7 2.0

Asian American

African American

Latin American

Native American

* Data taken from [82].

in four, or 25% (“HLA genotypically identical” or “identical by descent”) (Fig. 12.10). The probability that a patient and sibling have inherited one identical paternal or maternal haplotype and one nonidentical (nonshared) haplotype is 50% (“haploidentical”). The probability that a patient and sibling have inherited neither of their parental haplotypes in common is 25%. HLA genotypically identical siblings are identical by descent and therefore share not only the same HLA alleles and antigens, but all variation linked to the haplotypes. Haploidentical siblings are identical by descent only for the shared haplotype; they are variably mismatched for the nonshared haplotype, depending on fortuitous

sharing of maternal and paternal HLA alleles and antigens. In rare situations, a maternal or paternal recombination event gives rise to HLA mismatching between two otherwise matched siblings (Fig. 12.10). When a family study is not available, as is the case when unrelated volunteer transplant donors are considered, haplotypes can be estimated using statistical tools based on the known frequency of HLA alleles and antigens in the population being examined [80,81]. Table 12.6 shows estimated HLA haplotypes in an unrelated donor pool representing five different racial groups [82]. The HLA gene and haplotype frequencies of donor registries worldwide and the probability of identifying suitable

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donors have been extensively evaluated [83–90]. Information on HLA allele, gene, and haplotype frequencies from donor registries is important not only for estimating the probability of a successful search, but also for estimating optimal registry size and composition (http://www. allelefrequencies.net). Haplotypes include the known HLA alleles that are physically linked on the same DNA strand as well as undetected interlocus variation. The terms “ancestral haplotypes” (AHs) and “conserved extended haplotypes” refer to the definition of specific conserved long stretches of DNA sequences thought to be derived from a prototype ancestor [49,91]. AHs include HLA-B7, B8, B13, B35, B44, and B57, observed at high frequency in Caucasian populations [49,91]. New ethnically specific haplotypes are generated by the shuffling of interlocus (“frozen”) blocks of conserved regions at points of low LD. Recombinational events together with gene conversion, genetic drift, and balancing selection result in haplotypes that share not only the same HLA alleles, but also blocks of highly conserved sequences in strong positive LD with those HLA alleles [49,92–94]. In this way, HLA alleles are markers for untyped genetic variation including novel genes that are potentially important in transplantation (described in more detail in the section “Towards a new paradigm: haplotype matching in support of unrelated donor HCT”). A specific HLA allele may also be found on one or several different haplotype backgrounds [93,95–98]. Although haplotype-specific alleles and blocks have been well characterized for many of the common haplotypes, the extent of block sharing among other less common haplotypes on unrelated chromosomes is unknown. The association of certain haplotypes with transplant outcome has been described [46,99,100]. HLADR15-positive haplotypes are associated with a lower incidence of acute GVHD after allogeneic hematopoietic transplantation [100] and may indicate the presence of haplotype-specific determinants that affect transplant outcome.

DNA-based typing methods vary according to the level of discrimination they provide in defining the nucleotide sequence of an HLA gene. When the DNA typing method allows identification of a serologically defined antigen-equivalent (e.g. HLA-A2), the method is termed “lowresolution.” For example, SSOP hybridization methods employing a restricted number of probes may provide only limited sequence information about a particular HLA gene, equivalent to that achievable by serology. Typing methods that provide information beyond the serologic level but short of the allele level are termed “intermediate resolution.” For example, an SSOP method that employs a wider array of probes might identify the presence of either HLA-A*0201 or *0209 in an amplified DNA sample, but might be unable to discriminate one allele from the other. This intermediate-resolution result would be characterized as “HLA-A*02” or “HLA-A*0201/09”. Typing methods that generate nucleotide sequence information allowing precise identification of an HLA allele (e.g. HLA-A*0201) are termed “high resolution.” Highresolution typing results may be achieved by direct automated sequencing of an HLA gene (SBT) or by the use of large panels of oligonucleotide probes that test all known regions of variability within a gene. In order to interpret HLA-typing results and select donors for transplantation, it is necessary to know whether the typing was carried out at low-, intermediate- or high- resolution. A patient and donor who are “matched” for HLA-A and B antigens by low-resolution typing methods may be mismatched for HLA-A and/or B alleles (see Chapter 47). Histocompatibility guidelines for unrelated HCT have recently been revised to accommodate DNA-based typing methods [104]. Informatics systems to facilitate automated data analysis and reporting are being actively developed. These systems will not only enable the accurate interpretation of complex sets of HLA data, but also promote the donor search process within large volunteer registries [105]. Definition of a mismatch

Clinical applications Identification of donors for HCT in the DNA era Most HLA typing methods currently in use in clinical and research laboratories are based on the amplification of specific HLA genes from genomic DNA using PCR. PCR-based typing methods provide either direct determination of the entire coding region sequence of an allele (e.g. SBT) or partial sequence information allowing inference of the HLA allele (e.g. SSOP or SSP typing). These methods have clarified the relationship between HLA genes and their encoded antigens. For a given HLA locus (e.g. HLA-A), the gene variant at that locus is termed the allele (e.g. A*0201), and the alleles expressed by the two parental haplotypes constitute the genotype (e.g. A*0201, *0301). Each HLA allele, consisting of a unique nucleotide sequence, encodes the corresponding unique HLA molecule expressed at the cell surface. HLA molecules are characterized and classified by their reaction with HLA antibodies, and are hence referred to as HLA antigens. The combination of two antigens at a given locus, encoded by the alleles of the two parental chromosomes, is termed the phenotype (e.g. HLA-A2, A3). Because of the broadly reactive nature of HLA antibodies, two or more unique sequences (e.g. HLA-A*0201, *0205, and *0213) may have the same serologically defined phenotype (e.g. HLA-A2). An increasing number of class I and class II alleles are definable by DNA-based typing methods but not by serologic methods. To bridge the transition in nomenclature from serologic to DNA-based typing methods, “serologically equivalent” designations have been defined [101–103]. Each HLA allele is given a name, or search determinant, which encompasses a broader family of alleles [104]. This nomenclature facilitates donor identification as long as serologically typed donors are present in the registry.

As described above, the availability of DNA-based typing methods provides two major “levels” of definition of HLA genes. The “low” resolution level defines the serologic equivalent of the antigen (e.g. HLA-A2 versus A11), whereas the “high” resolution level defines nucleotide sequence disparity between individuals who are otherwise serologically matched (e.g. A*0201 versus A*0205). Traditionally, mismatching between serologically detectable HLA-A and B antigens has been termed “minor” (CREG) if the antigens share serologic crossreactive epitopes (Tables 12.4 and 12.5). Examples of CREG mismatches include HLA-A2 and A28, and HLA-B60 and B61. A “major” HLA-A or B antigen mismatch occurs when the antigens do not show serologic crossreactivity. Examples of major mismatches are HLA-A1 and A2, and HLA-B8 and B44. Serologically detectable mismatches at HLA-DR can be defined in two ways. The term “minor” mismatch has been used to describe mismatching between the antigen subtypes, or splits, of a given parent antigen. Examples of HLA-DR minor mismatches include DR15 and DR16 (splits of DR2), DR11 and DR12 (splits of DR5), DR13 and DR14 (splits of DR6), and DR17 and DR18 (splits of DR3). The term “major” HLA-DR mismatch has been used to describe mismatches between two parent antigens (or between their respective subtypes). Examples of major mismatches include DR1 and DR2, DR3 and DR4, and DR15 and DR17. Vector of incompatibility In 1994, Anasetti et al. [106] demonstrated the relevance of the vector of HLA compatibility in risk of graft failure and acute GVHD in related haploidentical transplants. The vector (sometimes referred to as the “direction” of the mismatch) can be defined for host-versus-graft (HVG) and graft-versus-host (GVH) alloreactivity. The presence of donor anti-

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Table 12.7 Vector of mismatch Examples Vector

Definition

Donor

Recipient

HVG

Presence of donor alleles or antigens not present in the recipient

GVH

Presence of recipient alleles or antigens not present in the donor

B*0801,4402† B*0801,4402§ B*0801,4402† B*0801,0801§

B*0801,4405 B*0801,0801 B*0801,4405 B*0801,4405

GVH, graft-versus-host; HVG, host-versus-graft. † These combinations contain bidirectional (both HVG and GVH disease) mismatch vectors. § Unidirectional mismatches.

gens or alleles not shared by the recipient determines HVG allorecognition. The presence of recipient antigen or alleles not shared by the donor provides the immunologic basis for GVH allorecognition. Examples of HVG and GVH vector mismatches are provided in Table 12.7. Mismatching between a donor and recipient can be described as “bidirectional” if both HVG and GVH vectors are present at a given HLA locus. Mismatching is called “unidirectional” if one but not the other vector is present. Unidirectional mismatching in the GVH vector occurs when the donor is homozygous and the recipient is heterozygous and shares one allele or antigen with the donor (e.g. patient A*0201, *0205 versus donor A*0201, *0201). Unidirectional mismatching in the HVG vector occurs when the patient is homozygous and the donor is heterozygous and shares one allele with the patient (e.g. patient A*0201, *0201 versus donor A*0201, *0205). Histocompatibility testing of related donors Donor selection begins with a full evaluation of available family members of the transplant recipient (Fig. 12.10). A family study includes HLA typing of the father, mother, full siblings, and additional relatives, where indicated. The family study confirms the recipient’s genotype and haplotypes, and provides information about the existence of genotypically matched siblings and/or haploidentical family members. When more than one equally suitable related donor is available, additional selection criteria including the number of HLA disparities on the mismatched maternal or paternal haplotype, and the presence or absence of disparity and of homozygosity for HLA KIR ligands, may be considered (Chapters 13 and 46). When several equally matched unrelated donors are available, non-HLA factors including donor age, parity, gender, and cytomegalovirus serostatus, may be considered in donor selection, as described in Chapters 47 and 48. Haploidentical siblings may be variably mismatched for the nonshared haplotype depending on whether the maternal and paternal HLA haplotypes fortuitously encode the same HLA antigen(s) or allele(s). The haplotypes of a child may be described in relation to the mother (“inherited maternal HLA antigens”) or father (“inherited paternal HLA antigens”). Noninherited maternal (NIMA) or paternal (NIPA) antigens refer to the HLA antigens expressed on the nonshared haplotypes. NIMA and NIPA have clinical significance. Exposure to NIMA in utero is tolerizing, whereas that to NIPA is immunizing. Donor-specific suppression of T-cell responses against NIMA is associated with lower risks of acute and chronic GVHD and transplant-related mortality after transplantation from mother to child compared with father to child [107,108]. In addition to maternal and paternal sharing of HLA antigens, selection of haploidentical family members can also include consideration for the presence of mismatched HLA KIR ligands encoded by HLA-B, C and

certain A alleles (see Chapter 46). For these purposes, the availability of high-resolution typing of HLA class I alleles of the recipient provides the necessary information to determine whether a patient is Bw4 positive or negative, and whether the recipient is C1/C2 heterozygous, C1/C1 homozygous or C2/C2 homozygous. Histocompatibility testing of unrelated donors Prior to 1990, donor selection criteria included typing and matching for HLA-A, -B and -DR antigens. The serologic definition of HLA antigens, however, was not adequate for detecting allelic variants that occur between the majority of serologically matched donors and recipients. As laboratory technology for discriminating HLA alleles became available in the early 1990s, a transition from serologically based phenotyping to DNA-based genotyping for DRB1 occurred. Application of DNA-based genotyping to class I HLA-A and B genes followed soon thereafter. As robust DNA methods were applied retrospectively to address research questions about the importance of genetic variation in transplant outcome, new information became available demonstrating that HLA-C [109–111] and HLA-DQB1 [112] function as classical transplantation antigens. From these data, the criteria for prospective donor evaluation and selection were expanded to include five loci: HLA-A, B, C, DRB1, and DQB1. Donor typing and matching for the two determinants at each locus gives rise to the 10 possible alleles considered relevant in HCT. Donors and recipients who have the same 10 alleles at the five loci are commonly described as “10/10 allele-matched.” When only three loci – HLA-A, B, and DRB1 – are considered, the term “6/6” refers to recipients and donors who share the same low-resolution-defined HLAA, B, and DR antigens. If the DRB1 typing was performed at high resolution, 6/6 refers to matching for HLA-A and B antigens and DRB1 alleles. The term “8/8” refers to high-resolution matching at the four loci HLA-A, B, C, and DRB1. In addition to HLA matching of unrelated donors, pretransplant assessment of the presence of anti-HLA antibodies in recipient serum is vital, as a positive crossmatch places the patient at high risk for nonengraftment [57,58,113]. When more than one equally matched unrelated donor is available, additional donor selection criteria include cytomegalovirus serostatus, donor age, donor gender, and ABO type [114]. Today, there are over 11 million registered unrelated donors available worldwide (http://www.worldmarrow.org), and the median time from the initiation of a formal unrelated donor search to a request for stem cell donation is 51 days (http://www.marrow.org). For any given patient, however, the probability of identifying a suitable donor within a reasonable timeframe ranges from 20 to 80% depending on the racial/ethnic background of the recipient and whether that recipient possesses relatively common or uncommon HLA haplotypes [89,90,115]. In the

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United States, over half of all patients for whom a search is initiated will have 10 or more suitably matched National Marrow Donor Program donors [114]. In general, Caucasian patients have a higher probability of donor matching than non-Caucasian patients (http://www.nmdp.org). In a study of 549 unrelated donor searches conducted in Europe between 1987 and 2000, nearly 60% of North-western European patients received a transplant within a median time of 4.4 months from the start of the search, compared with 32% of non-North-western European patients, while half of the latter patients failed to find a compatible donor [116].

Towards a new paradigm: haplotype matching in support of unrelated donor HCT When an HLA genotypically identical sibling is not available to serve as a donor for transplantation, a haploidentical related family member (see Chapter 46), unrelated volunteer donor (see Chapter 47) or cord blood unit (see Chapter 39) may be considered. As described in Chapter 47, the concept that the complete and precise HLA allele matching of the unrelated donor and recipient stems from the hypothesis that “8/8” or “10/10” matching might approximate the haplotype matching that is possible between sibling donors. Clinical experience demonstrates that this level of precision lowers complications after unrelated donor transplantation [117–120]; however, these risks are still higher than after sibling transplantation. Since HLA-matched unrelated donors and recipients are not identical by descent, novel undetected MHC resident variation encoded on the haplotypes could be responsible for post-transplant risks despite HLA allele matching. Indeed, early clinical observations suggest that MHC variation is organized into highly predictable segments of sequences [93,95,96], and although the specific content of these regions are still under investigation, donor–recipient identity in these regions has been associated with superior clinical outcome [46,121]. Haplotype variation better defines the full extent of genetic variability within individuals, and the use of haplotypes for disease mapping has been especially informative for association studies of complex human diseases [122]. Most often, haplotypes are used as proxies for ungenotyped markers because they may allow the identification of susceptibility markers that might escape detection when tested individually. Furthermore, because haplotypes define not one but many physically linked markers, they provide important information regarding traits or diseases resulting from the additive effects of genetic variation that may alter disease susceptibility or severity and response to therapy [123]. When family studies are not available, haplotypes can be inferred with statistical methods, although the limitations of these methods for individual haplotype estimation and for less common haplotypes preclude their use for unrelated donor–recipient pairwise analysis [80,81]. To address whether the HLA haplotype can serve as proxy of transplantassociated risks, and as a tool to identify potential undetected haplotypelinked variation, a novel method for phasing HLA alleles has recently been developed [124]. In this method, 2-Mb-long genomic DNA fragments are isolated which carry HLA-A linked to HLA-B (1.4 Mb) and HLA-B linked to HLA-DR (1.2 Mb). Each haplotype is separated using arrays of oligonucleotide probes specific for the known HLA-B alleles of the sample. The HLA-A and HLA-DR alleles are then determined using oligonucleotide arrays to link the HLA-A–B with the HLA-B–DR haplotype. This method has been applied to assess the degree of HLA haplotype matching among 10/10 allele-matched unrelated donors and recipients, and the post-transplant risks associated with haplotype mismatching [125]. As discussed in greater detail in Chapter 47, the probability of severe acute GVHD was significantly higher when the donor and recipient were haplotype mismatched. The increase in GVHD was

offset by lower relapse suggestive of a graft-versus-leukemia effect. These data indicate that undetected variation linked to haplotypes may be involved in GVHD.

Novel MHC resident transplantation determinants Many genes have been implicated in GVHD after matched unrelated HCT [126]. Haplotyping data point to the MHC as a potential rich source of susceptibility genes [125]. Mapping efforts are now greatly facilitated by the completion of a sequence map of chromosome 6 (http://www. hapmap.org/; http://www.sanger.ac.uk/HGP/Chr6/) [127]. Tools for mapping genes that cause MHC-associated disease include microsatellite (Msat) markers and more recently single nucleotide polymorphisms (SNPs) that define haplotype-associated polymorphisms (“tagSNPs”). MHC Msats Msats are di-, tri-, tetra-, and pentanucleotide repeats and complex repetitions, and have been instrumental in the construction of the secondgeneration human genome map [128]. Since 1996, over 400 Msats have been identified within the 3.8-Mb interval of the classical HLA region, of which 241 are polymorphic and occur on average one every 16 kb (112 di-, 21 tri-, 87 tetra-, and 21 pentanucleotide repeats) [129]. Wellknown recombinational hot spots include those associated with Duchenne muscular dystrophy, β-globin genes, and TCR β genes among others [130]. The maintenance of high levels of disequilibrium between Msat and HLA loci makes Msats effective markers for disease association and linkage analysis of genetic traits [131]. Although it is not likely that Msat markers themselves confer susceptibility to disease, Msats can serve as functional coding and regulatory elements. Msats located in the upstream promoter regions of coding sequences may provide enhanced function to expression vectors and binding sites for regulatory proteins, and affect transcription [130]. Msats have been powerful tools for mapping novel transplantation determinants within the MHC [132–134]. In a recent analysis of 31 Msats in 344 HLA-matched unrelated donor–recipient pairs, Msats were in strongly positive LD over much of the MHC [133]. Five common Caucasian haplotypes (HLA-A1, B8, DR3; A3, B7, DR2; A2, B44, DR4; A29, B44, DR7; and A2, B7, DR2) were examined using a new measure called “haplotype-specific heterozygosity,” which predicted informative Msats. The HLA-A1, B8, DR3 and HLA-A3, B7, DR2 haplotypes could be predicted with over 90% probability using three Msat loci (D6S265/ D6S2787/ D6S2894 and D6S510/ D6S2810/ D6S2876, respectively). These results demonstrate that Msats are informative markers for HLA haplotypes and that sequence variation exists outside of the classical HLA loci. The clinical importance of Msat-defined sequence variation in unrelated HCT has recently been evaluated [134]. Among a study population of 819 10/10 HLA-matched unrelated transplant pairs, donor–recipient mismatching for Msats residing within the extended class I, class III, and class II regions was associated with an increased risk of death. The presence of certain donor or recipient Msat genotypes was associated with increased risk of GVHD. These data point to class I, II, and III region variation that influences clinical outcome and highlights the utility of Msats for mapping putative susceptibility genes. Since Msats provide additional information over a larger span of the MHC compared with the evaluation of individual HLA genetic loci, a role for Msats in the selection of potential unrelated donors for transplantation has been proposed [135]. For larger population-based applications, a role for Msats in estimating ideal size of donor registries has been evaluated (http://www.euromado.org/) [136].

Histocompatibility

The future of genomics and hematopoietic cell transplantation Genes that cause disease are located within haplotypes and can be identified by testing the association of disease with informative SNPs and other genomic markers [137]. SNPs are the most abundant kind of inherited variation [138]. Over 11 million SNPs have been deposited in the dbSNP public repositories at a SNP density of 1 per 180 base pairs (bp) (http:// www.hapmap.org/; http://www.sanger.ac.uk/HGP/Chr6/) [127]. An estimated 4% of these SNPs occur in coding and 96% within non-coding regions of genes [122,123]. Intense efforts are in progress to define the linkage of SNPs in genomic DNA, to specify the organization of SNPs into haplotype blocks, and to identify tagSNPs that could serve as proxies for haplotype blocks. Most often, haplotypes are used as proxies for ungenotyped markers because they may allow the identification of susceptibility markers that might escape detection when tested individually. In this way, use of haplotypes for disease mapping is especially informative for association studies of complex human diseases wherein the full extent of genetic variability within individuals is unknown [123]. Importantly, since haplotypes define not one but many physically linked markers, they provide important information regarding traits or diseases resulting from the additive effects of genetic variation that may alter disease susceptibility or severity and response to therapy [139]. Analysis of the class II region has yielded important information on the extent of LD and genetic recombination within the MHC [98] and the sharing of conserved sequences among haplotypes that share the same alleles [93]. Regions of extensive high LD are interrupted by meiotic crossover “hot spots” in regions of lower LD. There is recent evidence that unique HLA alleles can be defined as part and parcel of these SNP haplotypes and that multiblock haplotypes may define antigens and alleles encoded at HLA-A, B, C, and DR loci [95,97]. In addition to their utility as a mapping tool, SNPs and SNP haplotypes

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provide insight into the evolutionary and recombinational history of the MHC [97]. In hematopoietic cell transplantation from unrelated donors, reduced alloreactivity based on haplotype-associated SNPs is an area of ongoing research (see Chapter 47).

Conclusion Genes of the HLA system encode a complex array of histocompatibility molecules that play a central role in immune responsiveness and in determining the outcome of tissue transplantation. The primary goal of histocompatibility testing for patients undergoing HCT is the identification of a suitable HLA-matched donor to reduce the risk of post-transplant complications resulting from HLA incompatibility. The recent advent of DNA-based methods that allow discrimination of HLA genes at the allele level has enhanced our ability to perform typing and matching with speed and precision. The extensive polymorphism of the HLA system, however, makes the selection of an optimally matched unrelated donor a challenging endeavor. Recent data demonstrate that the permissibility of an HLA mismatch is shaped by a complex interaction of qualitative and quantitative differences in the HLA sequences between the donor and recipient. Current experience indicates that it is possible to achieve excellent clinical results with a less than perfectly matched donor. Identification of the elements that define tolerable mismatches will permit increased availability of unrelated donor and mismatched family member donor HCT to patients who lack a genotypically identical sibling. The future of histocompatibility testing in support of allogeneic transplantation will incorporate not only consideration of the classical HLA genes, but also functional variation that is linked to HLA loci. Ultimately, the key to understanding the histocompatibility barrier in transplantation will require information on the content of the MHC region and the organization of that genetic variation on haplotypes.

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[Review]. Curr Opin Genet Dev 2003; 13: 569– 75. Guo Z, Hood L, Malkki M, Petersdorf EW. Longrange multilocus haplotype phasing of the MHC. PNAS 2006; 103: 6964–9. Erratum in: Proc Natl Acad Sci U S A 2006; 103: 9374. Petersdorf EW, Malkki M, Gooley TA et al. MHC haplotype matching for unrelated hematopoietic cell transplantation. PLoS Medicine 2007; 4: e8. Ferrara JL, Reddy P. Pathophysiology of graftversus-host disease [Review]. Semin Hematol 2006; 43: 3–10. International HapMap Consortium. A haplotype map of the human genome. Nature 2005; 437: 1299–320. Murray JC, Buetow KH, Weber JL et al. A comprehensive human linkage map with centimorgan density. Cooperative Human Linkage Center (CHLC). Science 1994; 265: 2049–54. Cullen M, Noble J, Erlich H et al. Characterization of recombination in the HLA class II region. Am J Hum Genet 1997; 60: 397–407.

130. Carrington M. Recombination within the human MHC [Review]. Immunol Rev 1999; 167: 245– 56. 131. Hearne CM, Ghosh S, Todd JA. Microsatellites for linkage analysis of genetic traits [Review]. Trends Genet 1992; 8: 288–94. 132. Li S, Kawata H, Katsuyama Y et al. Association of polymorphic MHC microsatellites with GVHD, survival, and leukemia relapse in unrelated hematopoietic stem cell transplant donor/recipient pairs matched at five HLA loci. Tissue Antigens 2004; 63: 362–8. 133. Malkki M, Single R, Carrington M et al. MHC microsatellite diversity and linkage disequilibrium among common HLA-A, HLA-B, DRB1 haplotypes: implications for unrelated donor hematopoietic transplantation and disease association studies. Tissue Antigens 2005; 66: 124. 134. Malkki M, Gooley TA, Horowitz MM et al. Mapping MHC-resident transplantation determinants. Biol Blood Marrow Transplant 2007; 13: 986–95.

135. Carrington M, Wade J. Selection of transplant donors based on MHC microsatellite data. Hum Immunol 1996; 50: 151–4. 136. Foissac A, Fort M, Clayton J et al. Microsatellites in the HLA region: HLA prediction and strategies for bone marrow donor registries. Transplant Proc 2001; 33: 491–2. 137. Kruglyak L. Prospects for whole-genome linkage disequilibrium mapping of common disease genes. Nat Genet 1999; 22: 139–44. 138. Wang DG, Fan JB, Siao CJ et al. Large-scale identification, mapping, and genotyping of singlenucleotide polymorphisms in the human genome. Science 1998; 280: 1077–82. 139. Nagel RL, Fabry ME, Pagnier J et al. Hematologically and genetically distinct forms of sickle cell anemia in Africa. The Senegal type and the Benin type. N Engl J Med 1985; 312: 880–4.

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Michael R. Verneris & Jeffrey S. Miller

Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation

Introduction Natural killer (NK) cells are large granular lymphocytes that express CD56 (neural adhesion protein) and lack the CD3 receptor (i.e. they are CD56+CD3−). In humans, such cells make up approximately 10–15% of the peripheral blood (PB) lymphocytes. NK cells are a critical component of the normal immune system, and in their absence or dysfunction patients are at risk for developing recurring and life-threatening viral infections [1–3]. NK cells have also been implicated in tumor surveillance since they preferentially kill malignant target cells while sparing normal, nontransformed cells. This is accomplished by displaying numerous nonrearranged, germline receptors on their surface. These receptors recognize the presence or absence of major histocompatibility complex (MHC) class I, as well as “stress” receptors, both of which are perturbed upon the surface of cells undergoing malignant transformation and/or viral infection. In addition to providing cytotoxic responses, NK cells are an important source of cytokines that feed back to components of the adaptive immune system, including T cells, B cells, and dendritic cells (DCs). NK cells also directly interact with these cell types and thereby shape adaptive immune responses. As one of the earliest lymphocyte populations to recover after allogeneic hematopoietic cell transplantation (allo-HCT), NK cells may be important in the early immune events that occur after allo-HCT. The purpose of this chapter is to provide the reader with up-to-date information on NK cell biology as it relates to allo-HCT. Since there are both similarities and differences between murine and human NK cells, we will mainly focus on human studies, but, where necessary, relevant murine information will be included.

NK cell subpopulations: CD56bright and CD56dim NK cells NK cells have been subdivided into two major subsets on the basis of CD56 staining intensity and the expression of the low-affinity immunoglobulin (Ig) receptor, CD16 (FcγRIII). Approximately 85–90% of all PB NK cells express CD16, and the majority of these cells also show dim CD56 expression. Consequently, the CD56dimCD16+ fraction of NK cells accounts for the bulk of the PB NK cells. A smaller fraction (∼10–15%) of PB NK cells shows high CD56 expression. CD56bright cells

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

have variable CD16 expression (i.e. CD56brightCD16−/+). These two NK cell subsets differ in a number of ways including response to exogenous cytokine stimulation, cytotoxicity, and cytokine production, suggesting that they may have distinct roles in immune responses (reviewed in [4]). Cytokines, such as interleukin-2 (IL-2) and IL-15, are essential for the growth, development, and activation of NK cells. For these cytokines to transmit intracellular signals, the expression of a trimeric protein complex is required. This complex is made up of the common. γ chain (CD132) and the β chain (CD122), as well as a “private” chain (IL-2α (CD25) or IL-15α). While CD56bright and CD56dim NK cells do not differ in expression of the common γ chain (CD132), there are differences between the NK cell subsets in the expression of the other two chains (α and β) of the IL-2 receptor. Specifically, only CD56bright NK cells display all three chains of the high-affinity IL-2 receptor (α, β, and γ) and are able to proliferate in response to minute (picogram) quantities of IL-2 [5–7]. In contrast, CD56dim NK cells express only the β and γ chains of the IL-2 receptor and show poor proliferation under these same conditions [5–7]. Further differences in responses to cytokines are that about one half of the CD56bright NK cells express the stem cell factor (SCF) receptor (CD117, c-KIT), while CD56dim cells lack this receptor. CD56bright NK cells do not proliferate in response to SCF alone, but the addition of SCF to IL-2 significantly augments in vitro NK cell proliferation of CD56bright cells [8]. This increased proliferation (of CD56bright cells in the presence of SCF) is at least partly due to the antiapoptotic activity of BCL-2, which is induced by this cytokine combination [9]. CD56bright and CD56dim NK cells are functionally different as well. Compared with CD56dim NK cells, CD56bright cells produce significantly more interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), TNF-β, granulocyte–macrophage colony-stimulating factor, IL-10, and IL-13 following activation with monocyte-derived cytokines such as IL-12, IL-15, and IL-18 [4,10,11]. In contrast, CD56dimCD16+ NK cells have approximately 10 times more cytotoxic molecules (perforin and granzyme B) than CD56bright cells [11]. Accordingly, freshly isolated CD56dim NK cells have higher cytotoxicity compared with CD56brightCD16− cells [6]. However, after short-term culture in cytokines, both NK cell subpopulations show similar killing ability [6]. As mentioned above, another major difference between CD56bright and CD56dim NK cells is the expression of the low-affinity Ig receptor, CD16. This translates into functional differences since CD56dimCD16+ NK cells have the ability to recognize and kill antibody-coated targets through antibody-dependent cellular cytotoxicity (ADCC). Antibody-coated B cells (Anti-CD20, rituximab) and breast cancer cells (Anti-Her2neu, trastuzumab) are more effectively killed by NK cells [12,13], suggesting that ADCC is an important mode

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of target cell killing in certain circumstances. The majority of CD56dim cells also express receptors for MHC class I molecules. These receptors, known as killer Ig-like receptors (KIRs), play a critical role in determining whether NK cell activation occurs (discussed below). In contrast, a significant proportion of CD56bright cells lack KIR receptor expression. Collectively, such studies suggest that these two NK cell subpopulations may have distinct immune functions, with CD56bright NK cells producing cytokines and CD56dim NK cells having more of a cytotoxic/effector role.

NK cell cytotoxicity A hallmark feature that distinguishes NK cells from most other cell types is the ability to directly kill malignant cell lines (K562) without prior antigen priming or cytokine stimulation. One of the first steps in NK cell cytotoxicity is the establishment of a stable conjugate between the NK cell and the target cell, known as an “immune synapse” (IS). Recent studies demonstrate that the early events in the creation of the IS involve the binding of CD2 and β2-intergrins (CD11a and CD11b) on the surface of NK cells with their cognate ligands on target cells. Elegant work using confocal microscopy has demonstrated that these proteins organize into a ring-like structure at the point of contact between the NK cell and the target cell, forming the periphery of the IS [14]. Following this, numerous NK cell surface receptors are recruited to the center of IS. Assuming that the balance of activating signals is greater than inhibitory receptor signals, actin polymerization facilitates the redistribution of cytotoxic granules to the center of the IS [14], for eventual release. In general, target killing by NK cells occurs through two separate mechanisms: (1) the granule release pathway, and (2) the membrane expression of TNF superfamily death receptors. With respect to the granule release pathway, NK cells contain lysosomal granules that store a number of cytotoxic proteins including serine proteases (granzymes A, B, H, K, and M), perforin, and granulysin. Following activation by a target cell, the contents of these granules are redistributed within the NK cell and released into the IS (described above). Once released, these serine proteases enter the target cell and induce apoptosis. Regarding the second method of killing (TNF superfamily receptors), the two bestcharacterized membrane receptors involved in this process are FasL and TNF-related apoptosis-inducing ligand (TRAIL) (reviewed in [15]). Both interact with their respective ligands present on the surface of the target cells. FasL binds to its receptor, Fas (CD95), which is broadly expressed, including on human hepatocytes. In the case of TRAIL, two receptors (DR4 and DR5) are selectively expressed by malignant cells [16]. Binding of either FasL or TRAIL to their receptors on the surface of malignant target cells results in activation of apoptotic cell death pathways (caspases), culminating in target cell apoptosis. Numerous malignant target cell lines, and samples from patients with acute leukemia and myelodysplastic syndrome, have been shown to express the Fas or TRAIL receptors [17–19].

response that is initially rapid and broad but transient, followed by immunity that is slower, more precise, and long lasting. Over the past 10–15 years, there has been significant progress in the understanding of the receptors that control NK cell triggering. Through the pioneering work of Karre and Ljunggren, it was noted that a subclone of a malignant cell line was more sensitive to NK killing than the parental line. Further experiments showed that the more sensitive subclone had lost MHC class I expression. This observation lead to “the missing self hypothesis,” which predicted that NK cells are inhibited by selfMHC [20]. Such a hypothesis reasonably explained how NK cells might be tolerant of normal tissues but could also kill malignant or virally infected targets (considering that MHC class I downregulation has been observed during both these processes [21,22]). Since this original observation, MHC class I-specific receptors have been identified on NK cells from a number of species (reviewed in [23,24]). Interestingly, some of these receptors are unique between mouse and man, suggesting their recent (and parallel) evolution, perhaps in response to infectious challenges [25]. In humans, two groups of MHC-specific receptor systems have been identified and characterized. These are: (1) the KIR family, and (2) a c-lectin receptor made up of a heterodimeric complex between NKG2A, C, and E molecules and CD94 (Fig. 13.1). KIR receptors The KIR gene locus is present on human chromosome 19q13.4 and comprises a multigene family of up to 15 individual cell surface receptors and two pseudogenes, which are not expressed (reviewed in [24– 26]). KIR genes are further subdivided based on the structure of both the extracellular and intracellular domains. These domains regulate ligand binding and signaling, respectively. In general, KIR genes are referred to as either KIR2D or KIR3D, depending upon the number of Ig domains in the extracellular portion. KIR receptors also vary with respect to the length of their intracellular signaling domain and are classified as either “long” or “short.” KIR with long intracellular signaling domains contain immunoreceptor tyrosine-binding inhibition motif (ITIM) regions that recruit inhibitory signaling proteins, such as SHP-1 and SHP-2 [27,28], resulting in a significant attenuation of NK cell

Inhibitory receptors NK cell

Target cell

KIR3DL2

HLA-A3/11

KIR3DL1

HLA-Bw4

KIR2DL1

HLA-C2

SHP-1 SHP-2

KIR2DL2 KIR2DL3

MHC-specific NK cell receptors: KIR and CD94/NKG2A While both B cells and T cells use a single (and unique) antigen receptor produced through recombination of germline DNA, NK cells express multiple receptors encoded by nonrearranged germline DNA that are used for target cell recognition. The implications of these differences are that NK cells can rapidly respond to antigenic challenges, but have no capacity for immunologic memory. In contrast, B cells and T cells take longer to initially respond, but are capable of amnestic responses following repeat antigenic challenges. Such a balanced system of antigen recognition provides the organism with the benefits of a total immune

Activating receptors

CD94 NKG2A SHP-1

(a)

HLA-C1

NK cell

FcεR1γ Syk/Zap70 NKp30 CD3ζ NKp44 Syk/Zap70 DAP12 FcεR1γ NKp46 Syk/Zap70 CD3ζ PLCγ 2 PKC

LFA-1 Fyn DNAM-1 ? FcεR1γ

PI3K

NKG2D DAP10

HLA-E

Target cell ?BAT3

???

??? PVR Nectin-2 MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4

(b)

Fig. 13.1 Natural killer (NK) cell receptors and their ligands. The various inhibitory receptors (a) and the activating receptors (b) and their ligands. Also shown are the associated adapter proteins and the intracellular signaling pathways that are activated.

Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation

activation (reviewed in [26]). In contrast, KIRs with short tails lack ITIMs and instead associate with adapter proteins, such as DAP12, to mediate signals that activate NK cells [29]. Thus, KIRs with short intracellular tails are activating, while those with long, ITIM-containing regions are inhibitory. A nomenclature committee has assigned a cluster designation (CD) number of CD158 for the KIR genes, with individual loci designated by a small letter with or without a number (e.g. KIR3DL1 = CD158e1) [30]. Given the vast polymorphism of the human MHC, it was difficult to conceive of how a single KIR could recognize multiple MHC alleles from different individuals. X-ray crystallography shows that, like the T-cell receptor, KIR interacts with the face of the MHC molecule, but more to the right-hand side and outside the peptide-binding groove [31]. This corresponds to a motif conferred by a conserved stretch of amino acids at positions 77–83 of HLA-B and at position 80 of HLA-C [24]. Thus, despite the vast polymorphism of MHC class I alleles, the alignment of this amino acid stretch falls into two different groupings, known as Bw4 and Bw6 for HLA-B. Likewise, depending upon whether there is an asparagine (asn) or a lysine (lys) at position 80, HLA-C can be divided into C1 and C2 groups, respectively. These dimorphisms in HLA-B and C convert the recognition of highly diverse, multiallelic MHC class I molecules into a biallelic system. However, differences do exist in the KIR recognition of HLA-B and C. For instance, only HLABw4 alleles are recognized by KIR3DL1. To date, HLA-Bw6 alleles do not appear to be recognized by KIR. In contrast, both HLA-C1 and C2 are ligands for different KIR receptors (KIR2DL2/KIR2DL3 and KIR2DL1, respectively). While the specificity of many of the inhibitory KIR receptors are known to be MHC class I, the KIR family also contains receptors for which the specificity is not known, including most of the activating (or short) KIRs. The KIR gene locus is a multigene family; however, individuals vary with respect to the number of KIR genes that they possess (Fig. 13.2). By sequencing the KIR region of 68 individuals, Uhrberg and coworkers found that all individuals contain a set of framework genes including KIR3DL3, KIR2DL4, and KIR3DL2. They could also discern common haplotypes with varying gene content, called haplotypes A and B [32]. While both haplotypes contained inhibitory and activating KIR genes, the A haplotype showed fewer genes, typically only one activating receptor (KIR2DS4). In contrast, the B haplotype contained the full complement of inhibitory genes, but also expressed multiple activating KIR genes [32]. These haplotypes are present in relatively equal frequencies in Caucasians [33]. To add further complexity to the KIR system, each gene shows considerable polymorphism between individuals, not unlike HLA genes [32]. Some of these polymorphisms are functionally important. For example, KIR3DL1*004 is not expressed on the cell surface so it cannot function to recognize its respective Bw4 ligand [34]. Still further complexity is appreciated since KIR and MHC exist on separate chromosomes and thus do not co-segregate during meioses. This leads to a situation in which some individuals express receptors for ligands they do not have. For example, the evaluation of a large population showed that the expression of KIR3DL1 on an individual’s NK cell population did not correlate with whether the individual did not possess the cognate ligand (Bw6/Bw6), or had one (Bw4/Bw6) or two (Bw4/ Bw4) copies [35]. Early on, it was believed that each NK cell appeared to express at least one MHC-specific inhibitory receptor. An extensive analysis of 100 NK clones from two separate individuals showed that each clone was inhibited by self-MHC. Interestingly, individual NK clones from the same donor expressed from between two and nine receptors in various combinations, both at the mRNA and the protein level, suggesting variegated acquisition of KIRs by individual NK cells [36]. Additional work showed that NK clones could also display KIRs for which

Gene frequency KIR2DL1 KIR2DL2 KIR2DL3 KIR2DL4 KIR2DL5 KIR2DS1 KIR2DS2 KIR2DS3 KIR2DS4 KIR2DS5 KIR3DL1 KIR3DL2 KIR3DL3 KIR3DS1

97.8 51.2 91.9 100 51.3 40.2 51.6 27.3 96.1 32.9 96.0 100 100 38.4

Individual gene polymorphisms

165

Variegated gene expression

A CC G G T G A

Fig. 13.2 The complexity of the killer immunoglobulin-like receptor (KIR). Individuals vary in the number and type of gene that they express, and data are shown (genograquency) for more than 450 patient types (unpublished observations) (left). There is also considerable diversity within an individual KIR gene at the nucleotide level (i.e. polymorphisms; middle) and at the level of cellular expression (right).

there were no MHCs in the host [32]. Thus, KIRs are clonally distributed on individual NK cells, creating a complex system of individual NK cells, each expressing a different complement of KIR receptors (Fig. 13.2). This creates a network of NK cells capable of recognizing subtle changes in a single MHC gene on target cells. While beyond the scope of this review, KIRs are not restricted to NK cells and have also been observed on mainly CD8+ T cells of memory phenotype, and may play a role in antigen specific responses. NKG2A NKG2A is the other MHC-specific inhibitory receptor expressed by the majority of NK cells. For NKG2A to be presented on the cell surface, it must covalently associate with the CD94, creating the heterodimeric protein complex CD94/NKG2A [37]. Like inhibitory KIR, NKG2A contains two ITIM regions in the cytoplasmic tail segments [37], and ligation of this complex results in a strong inhibition of NK cell cytotoxicity [38,39]. In contrast to KIRs that recognize the classical MHC class I proteins (HLA-A, B, and C), CD94/NKG2A recognizes HLA-E as its ligand [40–42]. Remarkably, CD94/NKG2A recognizes a small segment of the leader peptide of classical MHC class I (HLA-A, B, and C) (amino acids 3–11) complexed within HLA-E, and this in turn stabilizes HLA-E on the cell surface of target cells [38,40]. Thus, while KIR directly assesses the MHC class I expression the surface of target cells, CD94/NKG2A both directly assesses the HLA-E expression and also indirectly determines MHC class I expression since, if HLA-A, B, and C are not expressed, the leader sequence is not presented in the groove of HLA-E and the expression of HLA-E is reduced. NKG2A is one member of the NKG2 gene family found on chromosome 12, within the NK cell gene cluster [43]. Other members of this gene family have also been investigated, including NKG2C, NKG2E, and NKG2D (see below). Analogous to “long” and “short” KIRs, which have similar extracellular domains but differ in the intracellular signaling domains, NKG2C shares considerable homology with the extracellular portion of NKG2A and also recognizes HLA-E. In contrast to NKG2A, both NKG2C and NKG2E lack ITIMs and mediate NK cell activation through association with DAP12 [39]. However, despite the fact that both NKG2A and NKG2C can be expressed on the same cell, the system appears to have evolved to favor tolerance, since NKG2A has a considerably higher affinity for HLA-E than does NKG2C [44].

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NK cell activating receptors: NKG2D, natural cytotoxicity receptors, and DNAM-1 NKG2D As described above, much of the initial work on NK cell receptors focused on identifying and characterizing inhibitory KIRs and CD94/ NKG2A. However, the discovery of NKG2D as an NK cell-activating receptor generated considerable excitement in the NK cell field and was viewed as a major breakthrough. Unlike other NKG2 members (NKG2A, -C, -E, etc.) that form a heterocomplex with CD94, NKG2D exists on the surface of NK and T cells as a homodimer [45,46]. Interaction of NKG2D with its ligands (see below) or antibody crosslinking delivers signals that can potentially override inhibitory KIR receptor signaling, proving that NKG2D is a potent, activating receptor. Like most other NK cell surface proteins, NKG2D is not able to directly signal and uses the adaptor protein DAP10 to transmit intracellular signals (Fig. 13.1) [45]. DAP10 in turn recruits the p85 subunit of the phosphoinositol 3 kinase protein [45]. Additionally, NKG2D triggering leads to the production of a number of cytokines including TNF-α, granulocyte– macrophage colony-stimulating factor, and IFN-.γ [47]. NKG2D generated much interest, in part, due to the ligands it recognizes. NKG2D binds to two groups of self-proteins that appear to be preferentially expressed on distressed cells, such as malignant cells. The first group of NKG2D ligands are MHC class I polypeptide-related sequence A and B (MICA and MICB). These two closely related MHC-like proteins are within the MHC gene complex, approximately 46 kilobases centromeric to HLA-B. The promoter regions of MICA contain a heat shock protein-like element, similar to HSP70, and, accordingly, MICA and MICB are expressed during cell division, radiation-induced genotoxic stress, and malignant transformation, and following infections (viral and bacterial) [48–53]. Interestingly, MICA also shows limited expression on normal cells, with the highest level of expression in the gastrointestinal tract, a target tissue for graftversus-host disease (GVHD) [48]. Like MHC class I, MICA and MICB are polymorphic, with approximately 58 and 18 individual alleles, respectively. Some of these alleles show differing binding affinities for NKG2D [54], again reflecting the complexity of NK receptor–ligand interactions. The other group of ligands recognized by NKG2D is the UL16-binding proteins, ULBP1–4 [47,55], which have minimal homology to the MICA and MICB proteins. Like MICA and MICB, ULPB1– 4 are also expressed on malignant tissues and freshly isolated malignant cells [56,57]. Natural cytotoxicity receptors (NKp30, NKp44, NKp46) The natural cytotoxicity receptors (NCRs) were identified by the Moretta group after raising monoclonal antibodies (mAbs) against NK clones and then screening these antibodies for the ability to trigger NK cytotoxicity (using a redirected killing assay). Using this approach, these investigators identified three functionally similar, but structurally separate, NK-activating receptors. Collectively, these receptors have been referred to as NCRs since they play a major role in the NK cell recognition of malignant targets. The first of these receptors, NKp46, is identified as a type I transmembrane receptor of the Ig superfamily located within the leukocyte receptor complex on chromosome 19 (19q13.4) [58]. The second receptor, NKp30, is a V-type receptor found in the TNF cluster of the MHC gene complex on chromosome 6 [59]. Both NKp30 and NKp46 are expressed on nearly all resting and activated human NK cells. The third NCR, NKp44, is found only on IL-2activated NK cells [60,61]. Similar to NKp30, the NKp44 gene is also present within the TNF cluster of the MHC gene complex on human chromosome 6.

Like some of the other NK cell receptors, NKp30, NKp44, and NKp46 are not able to signal directly and rely upon adaptor proteins to transmit activation signals. NKp30 and NKp46 use CD3ζ and FcεR1γ, while NKp44 associates DAP12. Ligation of these receptors with agonist antibodies induced calcium ion flux, cytotoxicity, and cytokine release. Studies show that NCRs are critically important in the recognition and killing of numerous malignant targets (reviewed in [62]). Antibody blockade of a single receptor shows some attenuation in cytotoxicity, but the blockade of two or more of NCR receptors results in a significant reduction in cytotoxicity, suggesting that these receptors cooperate with one another in inducing NK cell activation. In fact, biochemical signaling studies support such observations [63]. While most individuals have NK cells with strong NKp30 and NKp46 expression, some show an NCRdim phenotype, and this correlates with a reduction in NK cell cytotoxicity [64,65]. The ligands that are recognized by the NCRs remain unknown. Some studies have suggested that NKp44 and NKp46 recognize viral products such as hemagglutinin [66]. Likewise, Chisholm and Reyburn showed that vaccinia virus-infected cells became susceptible to NK cytolysis. Using solubilized receptors, they further demonstrated that this was due to an increased expression of NKp30, NKp44, and NKp46 ligands [67]. Whether these ligands are self proteins induced by viral infection or viral proteins themselves remains to be determined. However, considering that NCRs play a critical role in the recognition of malignant cells, it is reasonable to also assume that, in addition to identifying viral proteins, NCRs also recognize self proteins. Such ligands may be increased on cells that have undergone malignant transformation, perhaps like the NKG2D ligands MICA and MICB. Further support for NCR recognition of self proteins is that NKp30 interacts with immature DCs through NKp30 [68], implying that DCs express a ligand for NKp30. More recently, it has been reported that HLA-B-associated transcript 3 (BAT3) is expressed on DCs and may be a ligand for NKp30 [69]. DNAM-1 DNAX accessory molecule-1 (DNAM-1, CD226) is a receptor found on the surface of both NK cells and T cells, and is also involved in cytotoxic triggering [70]. DNAM-1 signaling requires cis-association with leukocyte function-association antigen-1 (CD18/CD11a), which results in Fyn-induced phosphorylation of DNAM-1 [71]. The ligands recognized by DNAM-1 include the polio virus receptor (PVR, CD155) and Nectin2 (CD112). NK cell cytotoxicity of neuroblastoma and leukemia cells lines correlates with PVR and/or Nectin-2 expression [51,72,73]. Importantly, it appears that the DNAM-1 cooperates with NKp30 and/or NKp46 to induce leukemia cell killing [73]. Recently, Bryceson and coworkers have shown that for resting NK cells to become activated, DNAM-1 cooperates with other NK cell receptors, such as NKp46 and 2B4, to induce calcium ion flux, cytokine secretion (IFN-γ and TNF-γ), and cytotoxicity [74].

NK killing of leukemia targets NK cell recognition of malignant elements of the bone marrow, such as leukemia and myelodysplastic syndrome, is of particular importance to the field of allo-HCT, since understanding such processes may allow for augmentation of graft-versus-leukemia (GVL) responses. In general, most in vitro studies show that acute myeloid leukemia (AML) cell lines and patient samples are more sensitive than those from patients with acute lymphoblastic leukemia (ALL). Considering that NK cell cytotoxicity is a balance between inhibitory and activating NK cell receptor signaling, the relatively lower cytotoxicity of ALL lines could be accounted for by either high expression of inhibitory receptor ligands

Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation

(i.e. HLA class I), low expression of activating receptor ligands or a combination of both. In fact, compared with AML blasts, ALL patient samples express higher amounts of MHC class I (i.e. KIR ligand [KIRL]), which can bind inhibitory KIR and negatively modulate NK cell activation and killing [51]. In support of this, a recent study using 21 primary ALL patient samples showed a significant correlation between the density of HLA class I molecules and the sensitivity to NK cell killing (the more molecules, the less the killing) [75]. Moreover, when high amounts of HLA class I were present on ALL blasts and KIR-Lmismatched donor NK cells were used (see below), killing increased, suggesting that in instances where ALL blasts show high MHC class I expression, KIR–MHC interactions may be important [75]. Despite a higher amount of HLA class I on ALL blasts relative to AML blasts, most malignant cells show a reduction in HLA relative to normal cells, perhaps as a strategy to evade T cell recognition. Using a large panel of HLA-A and B allele-specific mAbs and a panel of 32 patient samples (chronic myeloid leukemia [CML], ALL, and AML), Demanet and coworkers showed that 65% of samples had a reduction in one or more HLA class I relative to normal lymphocytes. Interestingly, however, when they grouped the downregulated HLA-B alleles into the Bw4 and Bw6 allospecificities, they observed downregulation of significantly more Bw6 alleles relative to Bw4 [76]. The fact that the inhibitory KIR receptor KIR3DL1 recognizes Bw4, but not Bw6, alleles might suggest that such leukemia cells were selected for, escaping both NK-cell and T-cell surveillance. On a related note, Verheyden et al. tested whether patient KIR genes might be associated with leukemia risk. In their study of 96 patients with acute and chronic leukemia and 148 controls, they found that leukemia patients were more likely to express the KIR2DL2 gene and be heterozygous for the HLA-C-specific KIR haplotype. Such patients would express most if not all inhibitory KIRs (and their ligands), creating a situation of excessive NK-cell restraint that might favor a subsequent escape of leukemia from immune surveillance [77]. In another patient cohort, these same investigators found that chronic lymphocytic leukemia and AML patients were more likely to express a particular KIR and its corresponding ligand (MHC class I) compared with controls, supporting the concept that NK cells are involved in the surveillance of malignancy which is influenced by subtle host factors, such as the interaction between KIR and its corresponding MHC [78]. Similar observations have been found in the progression to human immunodeficiency virus [79]. NK cells also require signals from the activating receptors to lyse targets, and less is known about the expression of the ligands for NK cell activating receptors on either leukemia cell lines or patient samples. This is in part due to the fact that these receptors have recently been characterized and fewer studies have been performed to date. As mentioned above, only some of the ligands for NK cell-activating receptors have been identified (such as NKG2D and DNAM-1), while the ligands for other receptors (NKp30, NKp44, and NKp46) remain unknown. Regardless, indirect methods suggest that malignant cell lines and patient samples do express activating receptor ligands. Moreover, the sensitivity to NK cell killing appears to correlate with the presence or absence of these receptors and their corresponding ligands on blasts. Regarding the expression of NKG2D ligands on leukemia cell lines and patient samples, Pende et al. found that the NKG2D ligands ULBP1–3 were present on T-cell ALL lines and patient samples, but were essentially absent on pre-B ALL patient samples [50,51]. Likewise, these investigators showed that only a minority of AML samples (seven of 25) expressed NKG2D ligands and that, in selected targets, mAb masking of NKG2D had little effect on NK cell cytotoxicity [51]. In contrast, Salih et al. found that over 50% of AML, ALL, and CML patient samples expressed NKG2D ligands [57]. The majority of CML

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patient samples also showed NKG2D-L expression [80]. Importantly, MICA can be shed from the cell surface and can be found in patient sera [57,80]. Soluble MICA can act as a decoy by binding to NKG2D on the NK cell surface and inhibiting receptor function and target recognition. Accordingly, surface expression of NKG2D is lower in patients with newly diagnosed AML [81]. Only one study has addressed the expression of the DNAM-1 ligands (PVR and Nectin-2) on patient-derived samples. In this study, these receptors were present on most AML patient samples and to a lesser degree on ALL samples. Using individual patient samples, the involvement of DNAM-1 in NK cell cytotoxicity of these targets was confirmed [51]. As described above, most individuals express significant amounts of NCR on their NK cells, constituting an NCRbright phenotype. However, a minority (∼10–15%) of normal donors show low NCR expression with an NCRdull phenotype [82]. This NCRdull phenotype is associated with reduced in vitro natural cytotoxicity [82–84]. Recently, it was observed that, at the time of diagnosis, over 60% of individuals with AML showed an NCRdull phenotype [85]. As these patients were treated and entered hematologic remission, most reacquired an NCRbright phenotype, suggesting that leukemia in some way leads to a downmodulation of these receptors, perhaps due to the release of soluble NCR ligands or the elaboration of cytokines that downmodulate NCR expression. However, other investigators have observed that NKp46 was no different than controls at the time of diagnosis [81]. Using solubilized receptors as fluorescence-activated cell sorter reagents, investigators have attempted to indirectly assess the expression of NCR ligands on malignant cells. This study suggested that NCR ligands are found at low levels on AML blasts, but the expression is less than has been observed on normal developing myeloid cells [56]. Methods to overcome the resistance of leukemia to NK cell cytotoxicity have also been explored. Torelli and coworkers studied NK cells isolated from 11 adults and 15 children with ALL. They found poor baseline NK cell killing of autologous blasts. However, autologous NK cell cytotoxicity was enhanced following cytokine stimulation (IL-2, IL-12, and IL-15) and culture in the presence of lymphoblastoid cell lines [86]. To redirect NK cells to ALL, Imai and coworkers transduced NK cells with a chimeric receptor made of the external portion of a CD19 mAb and the internal signaling domains of CD3ζ and 4-1BBL. NK cells expressing this construct showed marked proliferation, activation, and killing of NK-resistant ALL cell lines and patient samples [87]. Treatment of HL60 cells and AML blasts with a cocktail of differentiation agents (5-aza-2-deoxycytidine, trichostatin, vitamin D3 bryostatin1, and all-trans-retinoic acid) led to an induction of the NKG2D ligand ULPB1, which in turn enhanced NK cell killing of AML targets [88]. Similarly, treatment of leukemia patient samples with the histone deacetylase inhibitor trichostatin A led to increase in expression of the NKG2D ligands (MICA and MICB) and an enhancement in NK cell killing [89]. As well, treatment of AML patient samples with IFN-γ led to a marginal increase in NCR ligands and increased cytotoxicity [56].

NK cell development from hematopoietic progenitor cells Like other hematopoietic cells, human NK cells are derived from CD34+ progenitor cells. Cytokines such as IL-2, IL-3, IL-7, IL-15, SCF, and FLT-3L facilitate the in vitro differentiation of such progenitor cells along the NK cell lineage [90]. Of these cytokines, only IL-15 is required for NK cell development, since IL-15−/− mice are devoid of NK cells [91]. In contrast, mice engineered to overexpress IL-15 (IL-15 transgenic mice) develop uncontrolled lymphoproliferation in both the CD8 T-cell and NK compartments [92]. Thus, while IL-15 does not act solely on NK cells, it plays a central role in NK cell homeostasis. In addition,

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NK cell development is critically dependent upon stromal cells. Uncommitted, CD34+CD38− cells cultured in the presence of cytokines show marginal NK cell differentiation; however, both NK cell maturation and expansion are significantly improved if the same progenitor cells are cultured in the presence of fetal liver stromal cell lines (and cytokines) [93]. Such augmentation of NK cell development occurs in a contactdependent manner [93], but the receptor–ligand pairs involved in this process remain to be identified. Thus, both the local environment and the cytokine milieu influence NK cell development. One of the recurring paradigms in hematopoietic cell development is the progressive acquisition and loss of cell surface receptors during development. The expression of such receptors can be used to functionally characterize distinct developmental stages of differentiation (Fig. 13.3). Such a schema for human NK cell development has recently been proposed (reviewed in [94]). The earliest antigens expressed on CD34+ progenitors that mark enhanced potential to develop into NK cells include CD7, CD10, CD45RA, CD122, and intergrin-β7 [94]. Such progenitors, referred to as stage I or pro-NK cells, show increased capacity to differentiate into NK cells under the influence of the abovementioned cytokines, with less of a need for stromal support, suggesting that cells that bear these receptors are committed to the NK cell lineage. Following this, NK cell progenitors transit through a pre-NK (stage 2) and immature NK cell (stage 3) steps, characterized by the progressive loss of antigens associated with immature progenitors (CD34 and CD38) and acquisition of cytokine receptors (IL-3R and IL-7R) and NK cellactivating receptors (NKp44). Finally, differentiating NK progenitors acquire additional activating receptors (NKp30, NKp44, NKp46, and NKG2D) as well as the inhibitory receptor CD94/NKG2A in the CD56bright stage of NK cell development and KIR expression by CD56dim NK cells. While a similar set of differentiation steps has been identified in the murine bone marrow, the location of NK cell differentiation in humans remained elusive until the recent studies by Freud and Caligiuri, which demonstrated the presence of these NK cell intermediates in secondary lymphoid tissue such as lymph nodes and tonsils [95,96]. Development of NK cell self-tolerance The developmental mechanism by which NK cells acquire self-tolerance has been referred to as NK cell education. This has been one of the most

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Fig. 13.3 Stages of natural killer (NK) cell development. As NK cells develop from progenitor cells, they lose and acquire surface receptors. Shown are some of the hallmark receptors and functional properties of developing NK cells (adapted from [94]). Not shown is the ontogeny of the CD94-NKG2A+ NK cells [101,102]. How these cells fit into the current “linear” schema of NK cell development is unknown.

widely debated topics in NK cell biology over the past few years. Several models have been proposed to explain the integration of inhibitory receptor expression with the acquisition of effector functions. These concepts differ in their implied mechanisms and whether the process is one of activation or loss of function. “Disarming” refers to the suppression of effector function in maturing NK cells that receive stimulatory signals unopposed by inhibitory signals via self-MHC receptors, analogous to the development of T-cell anergy [97,98]. “Licensing” describes a terminal differentiation step by which NK cells only acquire mature function when they receive an appropriate signal via an inhibitory receptor ligating with self-MHC [99,100]. What is agreed upon between these and other models is that human NK cells lacking inhibitory receptors are hyporesponsive [101,102]. Therefore, rather than being autoreactive, they are self-tolerant. In support of this, in vitro differentiation models show that NK cells acquire functionality (killing and cytokine production) only after the acquisition of CD94/NKG2A [103]. Thus, self-tolerance may be the result of a coordinated genetic developmental sequence during which mature NK function is synchronized with the acquisition of adequate expression of self-inhibitory molecules.

NK cell reconstitution after allo-HCT Reconstitution of immunity after allo-HCT can be influenced by a number of factors including intensity of conditioning, hematopoietic cell source, graft manipulation, GVHD prophylaxis, and host factors (i.e. age and thymic function). In general, many studies document that, regardless of the stem cell source, NK cells typically recover within the first month after transplant, and are thus one of the first lymphocytes to recover after allo-HCT. Comparing the NK cell reconstitution for recipients of reduced-intensity conditioning and PB progenitor cell transplant to those undergoing high-dose conditioning followed by PB progenitor cell or BM transplant, Petersen et al. found that NK cell reconstitution was rapid (by day 28) and no different between the groups [104]. Similarly rapid reconstitution has been reported for umbilical cord blood, BM, and haploidentical transplant [105–107]. Regarding the function of NK cells recovering after transplant, some have shown good cytotoxicity [108], while others have found impaired killing [109,110]. One intriguing question was whether the recovering NK cells would have the same KIR repertoire as was present in the donor. This question was addressed by Shilling et al. by following 18 patients undergoing matched-related donor or matched-unrelated donor transplant for up to 3 years following transplantation [111]. They found that, early after transplant, the majority of patients showed high expression of CD94/ NKG2A and low amounts of KIR that slowly increased over time after bone marrow transplantation. Based on KIR acquisition, patients fell into three groups. The first group, which made up the majority of patients, recovered a KIR pattern similar to that of the donor within 6–9 months after transplant. A second group took longer (up to 3 years) but eventually showed a KIR distribution similar to the donor. The last group showed poor KIR acquisition and had multiple medical problems. GVHD prophylaxis and treatment may also impact on NK cell reconstitution and function. Vitale et al. studied the NK cells from transplant recipients before, during, and after steroid treatment for acute GVHD. They found that such cells showed a sharp reduction in cytotoxicity and that this was associated with a reduction in NKp30 and NKp46 [112]. Wang et al. cultured NK cells with physiologic doses of cyclosporine A and found that it significantly inhibited the proliferation of CD56dim NK cells and only marginally influenced CD56bright cells [113]. After 1 week of culture in cyclosporine A, NK cells retained their cytotoxicity and their ability to secrete cytokines, and were no different from

Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation

controls. Such findings are interesting considering that CD56bright cells predominate early after transplant [114–116], perhaps suggesting that cyclosporine A may play a role in the phenotype and function of NK cells recovering early after transplant. Giebel et al. evaluated the number of NK cells in the PB of individuals at day 30 after transplant and showed that patients receiving prednisone for GVHD prophylaxis had fewer NK cells compared with those that did not receive prednisone. In contrast to prednisone-treated recipients, there was no difference in the number of NK cells in the PB of patients treated with or without antithymocyte globulin [117], suggesting that prednisone may have more long-lasting effects on NK cell recovery. Cooley et al. compared recipients of either T-cell-depleted or T-cellreplete transplants at day 100 to their donors and found that the percentage of NK cells did not differ. They did, however, find that at day 100, the recipients of T-cell-replete transplants had more KIRs and a higher percentage of IFN-γ. producing NK cells compared with T-cell-depleted recipients, suggesting that T cells within the graft affect NK cell reconstitution [116]. Freud et al. also demonstrated that T cells could enhance NK cell development from progenitor cells in vitro [96].

NK–DC interactions DCs are professional antigen-presenting cells that are critically important in a number of immune responses. In general, DCs exist in the circulation and peripheral tissues as immature DCs. Such cells are highly phagocytic but poor at presenting peptide antigens to T cells. However, following activation, mature DCs are highly efficient at stimulating naïve T-cell responses. Recently, it has been appreciated that NK cells and DCs interact with one another via soluble factors (i.e. cytokines) and cell-to-cell contact. The topic of NK–DC interaction has received considerable attention in recent years since it may have important implications for a number of immunologic processes, including those that take place after allo-HCT. However, the nature of NK and DC interactions is complex and not fully understood. NK–DC interaction, also known as “cross-talk,” can result in a number of outcomes, including reciprocal activation of both cell types. Following co-culture with either immature or mature DCs, NK cells proliferate, express activation markers (CD25), acquire cytotoxicity, and produce IFN-γ and TNF-α [68,118]. This is driven by DC-derived cytokines including type I IFNs (α and β), IL-12, and IL-18, and by the transpresentation of IL-15 to NK cells via the IL-15α receptor on DCs [119– 122]. These same conditions (NK–immature DC coculture) also lead to the maturation of immature DCs characterized by the acquisition of CD83 and CD86, and the ability to prime alloreactive T-cell responses [68,118,123]. NK cell-induced maturation of DCs (and vice versa) occurs in part through the ligation of NKp30 (on NK cells) by its ligand (on DCs), since antibody masking of NKp30 severely reduces the activation and/or maturation of both cell types [68,118]. Other studies show that the interaction between NKG2D on NK cells and MICA/MICAB on DCs is also important for these interactions [123]. Paradoxically, NK cells can also kill DCs. While it is not entirely understood, high NK:DC ratios result in DC killing, while low NK:DC ratios lead to reciprocal activation (described above). Additionally, the maturational status of DCs influences the sensitivity to NK cell killing. Compared with mature DCs, immature DCs display relatively lower amounts of MHC class I on the cell surface and are more sensitive to NK cell killing [68,118]. Higher amounts of MHC class I on mature DCs leads to their relative resistance to killing by autologous but not allogeneic NK cells. However, the killing of autologous mature DCs by NK cells can be enhanced with blocking mAb against MHC class I, suggesting the involvement of KIR receptors [118]. Likewise, NK cells that

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express KIR receptors that can not recognize MHC on mature DCs show better cytotoxicity (i.e. KIR-L mismatched) [118]. Similar to the killing of tumor targets, the killing of DCs by NK cells is through NKp30, NKG2D, and DNAM-1 [68,118,123,124]. Communication between NK cells and DCs is potentially important in the setting of HCT since recipient-derived DCs can present antigen to donor T cells and induce GVHD [125]. Recent studies using a murine model suggest that donorderived NK cells can eradicate these host-derived DCs and prevent GVHD [126], implying that NK cells can negatively modulate GVHD.

NK cells and viral infections Inherited deficiency and/or dysfunction of NK cells has been associated with severe and life-threatening viral infections, including infections with members of the herpesvirus family, such as varicella zoster virus [1,2], cytomegalovirus (CMV) [127], and Epstein–Barr virus [128]. Similarly, mutations in the perforin gene (in the case of hemophagocytic lymphohistiocytosis) in humans result in fatal and uncontrolled viral infections [129,130]. Clearly, following allo-HCT, reactivation of herpesviruses also occurs. Whether NK cells play a role in this process is not well defined. However, it is noteworthy that there is a clear association between the expression of NK cell-activating protein Ly49H in certain mouse strains and susceptibility to murine CMV [131]. In addition, viruses appear to have evolved to actively prevent the expression of NK cell-activating ligands, suggesting a dynamic interplay between viruses and NK cells. For instance, the CMV protein UL16 leads to a reduction in the expression of the NKG2D ligands ULBP1-2 and MICA (but not ULBP3 or MICB) [47]. Similarly, the CMV protein UL141 reduces the expression of the DNAM-1 ligand CD155 (PVR) [132]. Thus, through the expression of these proteins, viruses may evade NK cell detection. As mentioned above, there is evidence to suggest that at least some of the ligands for the NCRs are viral hemagglutins [133,134]. Still further evidence for NK cells in the control of viruses are the observations that CMV reactivation was less frequently observed in transplant recipients of donors who expressed more activating KIRs [135,136].

Clinical studies Allo-HCT functions through the combination of both high-dose conditioning therapy (chemotherapy and irradiation) and an immunemediated eradication of resistant leukemia cells. This latter point, referred to GVL, is mediated via the actions of alloreactive T cells and NK cells. NK cell alloreactivity has received considerable attention over the past 5–10 years, in part due to observations that allogeneic NK cells can induce GVL without GVHD [126,137]. Considering the importance of HLA matching in allo-HCT and the fact that each KIR receptor recognizes specific allelic determinants of HLA, such interactions are likely to be important in post-transplant GVL reactions. However, such interactions are complex and not entirely understood. For instance, the expression of KIR on individual NK cells occurs through a stochastic process with each NK cell expressing a varying number of KIR receptors of differing MHC class I specificity [32]. Thus, contained within the circulation of all potential donors is a heterogeneous pool of NK cells (with regard to KIR expression; Fig. 13.2), and in any individual only a minority of these cells may have alloreactive capacity (i.e. are potentially unrestrained by KIR–MHC interactions). The observation that NK cell alloreactivity is important in posttransplant GVL was established by the Perugia group in the context of T-cell-depleted and CD34-selected haploidentical transplantation [126,138]. In these transplants, donors (parents or siblings) share one

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Fig. 13.4 Potential outcomes following transplants depending upon donor and recipient killer immunoglobulin-like receptor ligand (KIR-L) compatibility. Shown are the possible outcomes depending upon donor and recipient HLA-C dimorphisms (C1/C2) (a–c) and HLA-B dimorphisms (Bw4/Bw6) (d–f) (adapted from [159]).

haplotype with the recipient, but are also mismatched at the other haplotype. This creates a situation where considerable alloreactivity is possible, especially if donor NK cells express KIR that can not recognize MHC on recipient leukemia cells, fulfilling the “missing self” hypothesis. As shown in Fig. 13.4, three possible outcomes could occur following haploidentical transplantation: (1) NK cell alloreactivity in the graft-versus-host direction, which would favor GVL reactions, (2) NK alloreactivity in the host-versus-graft vector, which could favor rejection, or (3) no alloreactivity due to compatibility between the donor and recipient with respect to HLA epitopes. In general, alloreactions between NK cells expressing receptors for HLA-C appear to be dominant over those for HLA-B [139]. This is perhaps due to the higher number of HLA-C-specific KIR receptors and possibly their greater affinity for ligand. Consequently, in the original report addressing NK cell alloreactivity after transplantation, Ruggeri et al. found that the majority of alloreactive NK cell clones generated after haploidentical transplantation were reactive to differences in HLA-C [138]. Such clones could be detected in patients for up to 1–3 months after transplantation. Following KIR-L-mismatched transplants (see below), these antirecipient NK cell clones could be detected in all patients, while in contrast they were essentially absent in KIR-L-matched transplant recipients [126]. AML patients transplanted with a KIR-Lmismatched donor showed a 0% probability of relapse at 5 years. In contrast, a similar population of AML patients, transplanted in the same manner with a KIR-L-matched donor, showed poor outcomes, with 75% relapsing by 5 years [126]. Importantly, these investigators found that the effect of KIR-L mismatching was restrictive to patients with myeloid leukemia. There was no improvement in outcome for those transplanted for ALL from a KIR-L mismatched donor. A recent updated analysis by

this group shows that patients with chemotherapy resistant relapses did not benefit from KIR-L-mismatched donors [140]. The above observations of NK alloreactivity have been referred to as the “ligand–ligand” model, where the prediction of NK cell alloreactivity is based on the presence or absence of various MHC class I alleles (HLA-B or C) in the recipient relative to the donor (i.e. whether the donor and recipient express the same or different HLA-Bw4 or HLAC1/C2 alleles). Thus, KIR-L mismatch analysis using the ligand–ligand model can be viewed as an analysis of donor versus recipient epitope similarity in the graft-versus-host direction for HLA-B and C alleles. Determination of KIR-L mismatch status using this model can be performed using an online tool, available at http://www.ebi.ac.uk/ipd/ kir/ligand.html. However, the above approach may be limited since it assumes that a particular donor expresses KIR receptors for the corresponding ligand. It is well established that individuals differ in the number of KIR genes contained in their genome, and thus not all individuals will express all KIR receptors [32]. Leung and coworkers extended the above method by not only taking into account whether donors and recipients differed with respect to HLA-B and C alleles, but also determining whether the donor expressed the KIR receptor for the mismatched HLA class I allele (i.e. KIR-L). These investigators found that, using their “receptor–ligand” model, patients with both AML and ALL experienced lower rates of leukemia relapse if transplanted with a KIR-L-mismatched donor that expressed the appropriate KIR [141,142]. Importantly, not all investigators were able to demonstrate a benefit for AML patients undergoing haploidentical transplantation using KIRL-mismatched donors. Nguyen et al. transplanted 10 AML patients with a haploidentical donor (eight of 10 being KIR-L mismatched) using the same regimen and PB progenitor cell selection techniques as the above studies. However, unlike the above studies, they found that all patients died due to either relapse (seven of 10) or infection (three of 10) [110]. The authors demonstrated that NK cells recovering after this procedure were immature in both by phenotype and function, and speculated that this might account for their clinical observations. Whether KIR-L mismatching impacts outcomes in the setting of unrelated donor transplantation has recently been the subject of numerous retrospective studies, with mixed results. Initially, a large registry analysis by Davies et al. showed that a KIR-L-mismatched donor had no impact on the incidence of AML relapse following T-cell replete transplantation [143]. Shortly after this report, Giebel et al. showed a protective effect of KIR-L mismatching for AML patients in the unrelated donor setting [144]. Interestingly, this latter study used antithymocyte globulin for in vivo T-cell depletion, leading to the speculation that T cells might influence NK alloreactivity. Similar results were obtained by Hsu et al. in a T-cell-deficient matched sibling cohort [145]. However, in a group of heterogeneously treated patients with myelodysplastic syndrome and myeloid leukemia, Bornhauser and coworkers found that KIR-L-mismatched unrelated donors actually resulted in a higher rate of relapse using post-transplant antithymocyte globulin [146]. In a population of T-cell-replete transplants, similarly negative outcomes were observed for KIR-L-mismatched donors (due to both relapse and nonrelapse mortality) [147]. However, in a subset of patients with myeloid disease (early myeloid leukemia), KIR-L mismatch imparted a protective effect, with patients experiencing fewer relapses [148]. Still other studies show no protection from relapse for KIR-L-mismatched donors in the T-cell-depleted [149] or the T-cell-replete setting [150]. The use of KIR-L-mismatched donors may also impact transplantrelated outcomes other than relapse. For instance, Schaffer et al. found an increase in treatment-related mortality in KIR-L-mismatched recipients due to a higher rate of infectious complications [151]. Similarly, Kroger et al. found that KIR-L-mismatched recipients had a higher rate

Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation

of treatment-related mortality [149]. Examining HLA-C mismatch, De Santis et al. found that in donors and recipients that were mismatched for C1 and C2 epitopes, there was more grade II–IV GVHD if the mismatch was in the graft-versus-host vector [152]. Conversely, if the mismatch was in the rejection vector (host-versus-graft), more rejection was observed. Regardless of the vector, these investigators found that KIR-L mismatching was associated with inferior outcomes and increased treatment-related mortality [152]. However, analyzing a large dataset (n = 1571), Farag et al. were not able to demonstrate any impact of non-relapse mortality in KIR-L-mismatched transplants in either vector [150]. Clearly, differences in hematopoietic cell source, conditioning regimen, and GVHD prophylaxis may impact such observations. In addition to KIR-L mismatching, some investigators have analyzed datasets based on the presence or absence of a specific KIR or the quantity of KIR receptors expressed by the donor. In the setting of fully HLA-matched transplant (HLA 8/8), KIR-L mismatch cannot occur; however, transplant recipients whose donor expressed both KIR2DS1 and KIR2DS2 had better outcomes due to a reduction in leukemia relapse [153]. Chen et al. found that more activating KIRs in the matched related donors resulted in lower treatment-related mortality and better overall survival [135]. An increase in the total number of KIRs (both activating and inhibitory) contained within the donor was associated with a reduction in GVHD and an improved overall survival [152]. In contrast, Kroger et al. found that, following unrelated transplant, the recipients of donors with less activating KIR had reduced relapse rates [149]. Still further studies show that the presence of KIR2DS2 in unrelated donors is associated with lower rates of overall survival and disease-free survival due to more severe GVHD [154].

Adoptive NK cell transfer Another way to promote antitumor activity with alloreactive NK cells is through adoptive transfer. This can be achieved using several strategies. One approach is based on ex vivo NK cell expansion, but there are several potential limitations to this. Most importantly, NK cells stimulated by supraphysiologic concentrations of cytokines tend to undergo apoptosis when removed from ongoing stimulation and may not persist or expand in vivo. In addition, marked changes in size and adhesion receptor expression occur with activation, and these may alter homing characteristics in vivo. Consequently, developing strategies for in vivo NK cell expansion may be optimal. The safety and success of this approach was established in a trial using in vivo expanded haploidentical, related-donor NK cell infusions to treat 43 patients with metastatic melanoma, metastatic renal cell carcinoma, refractory Hodgkin’s disease or refractory AML [137]. The trial, which tested three preparative chemotherapy regimens of differing intensity, confirmed that successful NK cell expansion was only seen in the AML cohort who received the fully lymphodepleting cyclophosphamide and fludarabine regimen used by Rosenberg. Patients received NK cell infusions on day 0 following one or two doses of intravenous cyclophosphamide (60 mg/kg) on days −5 and −4 and daily intravenous fludarabine (25 mg/m2) on days −5 to −1, followed by 10 million units of subcutaneous IL-2 administered every other day regimen over 2 weeks. Successful expansion was only seen after high-dose cyclophosphamide/fludarabine, which was the only regimen to induce pancytopenia. Additionally, it was the only to induce a surge of endogenous IL-15 after chemotherapy. A significant inverse correlation was seen between the IL-15 level and the absolute lymphocyte count, high levels correlating with successful NK cell expansion, which supports the importance of IL-15 for NK cell homeostasis.

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In vivo expansion of NK cells was assessed using a polymerase chain reaction-based chimerism assay, with successful expansion defined by the presence of measurable donor NK cells at 2 weeks, following the IL-2 therapy. Eight of 15 evaluable patients had successful in vivo NK cell expansion, and the circulating donor-derived NK cells were functional in standard cytotoxicity assays. Clinical efficacy correlated with in vivo NK expansion. Of the 19 patients with poor-prognosis AML, five achieved a complete remission. The remission patients had significantly higher proportions of circulating NK cells that were significantly more cytotoxic against K562 targets suggesting that the observed clinical efficacy was mediated in part by the in vivo-expanded allogeneic donor NK cells. Furthermore, in this small cohort, four of the 19 NK donors were predicted to exhibit alloreactivity based on KIR-L mismatch in the graft-versus-host direction. Complete remission was achieved in three of four (75%) KIR-L mismatches, and only two of 15 (13%) KIR-L matching patients, supporting a role for KIR-L mismatching in the treatment of AML.

Conclusion NK cells recover early after transplantation. These cells kill malignant target cells without prior priming, using a limited number of patternrecognition receptors that detect cells undergoing stress. By understanding these receptors and their ligands, we are developing a better understanding of how NK cells might function after allo-HCT to mediate GVL. However, we still have an incomplete understanding of both the receptors that they use and the ways to maximally activate such cells. At present, there are several limitations to the therapeutic potential of adoptively transferred allogeneic NK cells. The NK cell yield from lymphapheresis collections is limited. Although NK cells can be successfully expanded in vivo, the success is unpredictable, and the expanded cell population is transient. Additionally, the homing signals required to direct NK cells to tumor sites are not fully understood. Furthermore, the alloreactivity of in vivo expanded NK cell populations may be heterogeneous due to variable KIR receptor expression or to differences in NK cell or accessory cell subsets. The use of monoclonal antibodies that block NK cell inhibitory receptors may increase antitumor killing [155], and more sophisticated techniques for subset selection in NK cell products may affect the interaction between the innate and adaptive immune responses. For example by removing regulatory T cells (that can suppress NK cell proliferation and killing), may also improve the immune effector functions of the expanding NK cells [156]. NK cell expansion may be improved by refining the use of lymphodepleting chemotherapy or the use of concurrent exogenous cytokine therapy. Irradiated cell lines such as NK92 and KHYG-1 may provide an inexhaustible supply of highly cytotoxic NK cells, but their in vivo homing and survival is not well known [157,158]. Ex vivo expanded cells from any source can be genetically modified to express tumor-specific receptors. For example, the NK92 cell line has been transfected with a chimeric antigen receptor for HER2/neu, which conferred superior cytotoxicity against HER2/neu+ targets [158]. Lastly, the antitumor activity of NK cells depends not only on the KIR-L mismatch status, but also on the tumor expression of appropriate activating ligands. Future strategies to enhance activating ligand expression on tumor cells may increase their susceptibility to NK cell-mediated lysis. Other avenues of research for NK cell immunotherapy include engineering NK cells with transferred genes, incorporating NK cells into DC vaccine therapies, and combination therapy with immunomodulatory drugs such as thalidomide, toll-like receptor agonists, and mAbs to target ADCC.

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125. Shlomchik WD, Couzens MS, Tang CB et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 1999; 285: 412–15. 126. Ruggeri L, Capanni M, Urbani E et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: 2097–100. 127. Bernard F, Picard C, Cormier-Daire V et al. A novel developmental and immunodeficiency syndrome associated with intrauterine growth retardation and a lack of natural killer cells. Pediatrics 2004; 113(1 Pt 1): 136–41. 128. Seemayer TA, Gross TG, Egeler RM et al. Xlinked lymphoproliferative disease: twenty-five years after the discovery. Pediatr Res 1995; 38: 471–8. 129. Janka G, Zur Stadt U. Familial and acquired hemophagocytic lymphohistiocytosis. Hematology Am Soc Hematol Educ Program 2005: 82–8. 130. Janka GE. Hemophagocytic syndromes. Blood Rev 2007; 5: 245–53. 131. Lee SH, Girard S, Macina D et al. Susceptibility to mouse cytomegalovirus is associated with deletion of an activating natural killer cell receptor of the C-type lectin superfamily. Nat Genet 2001; 28: 42–5. 132. Tomasec P, Wang EC, Davison AJ et al. Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141. Nat Immunol 2005; 6: 181–8. 133. Arnon TI, Lev M, Katz G, Chernobrov Y, Porgador A, Mandelboim O. Recognition of viral hemagglutinins by NKp44 but not by NKp30. Eur J Immunol 2001; 31: 2680–9. 134. Mandelboim O, Lieberman N, Lev M et al. Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature 2001; 409: 1055–60. 135. Chen C, Busson M, Rocha V et al. Activating KIR genes are associated with CMV reactivation and survival after non-T-cell depleted HLA-identical sibling bone marrow transplantation for malignant disorders. Bone Marrow Transplant 2006; 38: 437–44. 136. Cook M, Briggs D, Craddock C et al. Donor KIR genotype has a major influence on the rate of cytomegalovirus reactivation following T-cell replete stem cell transplantation. Blood 2006; 107: 1230–2. 137. Miller JS, Soignier Y, Panoskaltsis-Mortari A et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005; 105: 3051–7. 138. Ruggeri L, Capanni M, Casucci M et al. Role of natural killer cell alloreactivity in HLAmismatched hematopoietic stem cell transplantation. Blood 1999; 94: 333–9. 139. Colonna M, Brooks EG, Falco M, Ferrara GB, Strominger JL. Generation of allospecific natural killer cells by stimulation across a polymorphism of HLA-C. Science 1993; 260: 1121–4. 140. Ruggeri L, Mancusi A, Capanni M et al. Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood 2007; 110: 433–40. 141. Leung W, Iyengar R, Triplett B et al. Comparison of killer Ig-like receptor genotyping and phenotyping for selection of allogeneic blood stem cell donors. J Immunol 2005; 174: 6540–5.

Natural Killer Cells and Allogeneic Hematopoietic Cell Transplantation 142. Triplett B, Handgretinger R, Pui CH, Leung W. KIR-incompatible hematopoietic-cell transplantation for poor prognosis infant acute lymphoblastic leukemia. Blood 2006; 107: 1238–9. 143. Davies SM, Ruggieri L, DeFor T et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor. Blood 2002; 100: 3825–7. 144. Giebel S, Locatelli F, Lamparelli T et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 2003; 102: 814–19. 145. Hsu KC, Keever-Taylor CA, Wilton A et al. Improved outcome in HLA-identical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by KIR and HLA genotypes. Blood 2005; 105: 4878–84. 146. Bornhauser M, Schwerdtfeger R, Martin H, Frank KH, Theuser C, Ehninger G. Role of KIR ligand incompatibility in hematopoietic stem cell transplantation using unrelated donors. Blood 2004; 103: 2860–1; author reply 2. 147. Sun JY, Dagis A, Gaidulis L et al. Detrimental effect of natural killer cell alloreactivity in Treplete hematopoietic cell transplantation (HCT) for leukemia patients. Biol Blood Marrow Transplant 2007; 13: 197–205. 148. Miller JS, Cooley S, Parham P et al. Missing KIR-ligands is associated with less relapse and

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14

Robert Korngold & Thea M. Friedman

Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect

Introduction Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) and is a manifestation of the alloreactive response to host histocompatibility (H) differences mediated by mature donor T cells in the inoculum [1–3] (see also Chapter 1). Before discussing the pathogenesis of GVHD and the cell types involved, it is important to consider the essential features of T-cell specificity and T-effector function. The specificity of typical T cells expressing αβ T-cell receptor (TCR) molecules is directed to peptide fragments of antigen bound to major histocompatibility complex (MHC) molecules, human leukocyte antigen (HLA) molecules in humans and H-2 molecules in the mouse [4–9]. A more detailed understanding of the MHC molecules can be found in Chapter 12. As the result of a complex process of selection in the thymus, αβ+ T cells are rendered tolerant to “self” MHC molecules (plus the various endogenous peptides bound to these molecules) but display reactivity to self-MHC molecules complexed to foreign peptides [8,9]. Minor histocompatibility antigens (mHAs) – one of the principal targets for GVHD – fall into this category. T-cell specificity also encompasses reactivity to allo (foreign, nonself) MHC molecules [4]. GVHD to MHC alloantigens is intense and reflects that the precursor frequency of T cells for allo-MHC antigens is very high, far higher than for typical foreign peptide antigens complexed to self-MHC molecules. The basis for the vigorous MHC alloreactive response lies in the additive effects of individual T cells recognizing the array of different self-epitopes presented by the foreign MHC molecules, as well as some interaction with the MHC molecules themselves, regardless of the antigens bound [10]. In addition, GVHD in the context of MHC differences could also be a reflection of increased antigen concentration on the target tissues where every MHC can elicit T-cell responses. There are two classes of MHC molecule, termed classes I and II [4,7]. Class I molecules are expressed on virtually all nucleated cells and are recognized by the CD8+ subset of T cells. Class II molecules show a more restricted tissue distribution and are recognized by CD4+ T cells. The CD4 and CD8 molecules which define the two major subsets of T cell act as adhesion molecules and bind to nonpolymorphic regions of MHC class II and class I molecules, respectively [6]. Such binding

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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increases the avidity of TCR–MHC interaction and causes each T-cell subset to display MHC class specificity. Thus, the CD8+ subset of T cells reacts much more effectively with class I than class II MHC molecules, whereas CD4+ cells show the reverse specificity. As discussed later, this MHC class specificity of CD4+ and CD8+ cells applies to GVHD. In the case of unprimed cells, T-cell activation depends on contact with MHC-associated peptide (or MHC alloantigens) expressed on specialized antigen-presenting cells (APCs), such as macrophages and dendritic cells [4,8]. These cells reside in the T-dependent areas of the lymphoid tissues, i.e. the periarteriolar lymphocyte sheaths of the splenic white pulp and the paracortical areas of lymph nodes. Recognition of antigen in these sites also applies when T cells are transferred to allogeneic hosts, where they initially encounter host alloantigens expressed constitutively on host APCs in the T-dependent areas of the recipient. In the case of MHC differences, contact with host APCs is the key interaction for developing a response [11,12]; however, in MHCmatched transplantation settings, involving mHA differences, donorderived APCs may also play a role in stimulating donor T cells [13]. After contact with antigen on APCs, T cells proliferate extensively, release various lymphokines, and differentiate into T-effector cells; in allogeneic hosts, this chain of events constitutes a graft-versus-host reaction (which may or may not progress to overt GVHD). Whereas resting T cells are confined to the recirculating lymphocyte pool (blood, lymphoid tissues, and lymph), antigen-activated T cells have the capacity to penetrate the walls of capillary blood vessels and can thus disseminate throughout the body. Activated T cells show a particular propensity for homing to the gut, liver, lung, and skin, sites commonly affected by GVHD. When activated T cells re-encounter antigen in these sites, the cells express various effector functions that lead to target tissue injury. It should also be noted that for MHC differences, APC presentation of alloantigen alone can induce GVHD, particularly when mediated by donor CD4+ T cells [14]. Host nonhematopoietic target tissue cells, mainly the endothelial and epithelial cells, do not need to express the allo-MHC antigens. In contrast, with mHA differences for both CD4+ and CD8+ T-cell responses, development of GVHD not only requires APCs, but also depends upon antigen expression in the nonhematopoietic compartment [15,16]. The effector functions of T cells are complex and difficult to categorize [4]. Direct destruction of target cells by cytotoxic T lymphocytes (CTLs) is the simplest type of effector function and is probably a major cause of the protean pathology seen in GVHD. Other T cells have limited CTL activity but, after re-encountering antigen on local APCs in the tissue concerned, are able to release large quantities of various lymphokines. By attracting a spectrum of mononuclear cells from the

Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect

blood and also by causing direct tissue destruction (in the case of toxic lymphokines such as tumor necrosis factor-alpha [TNF-α]), these lymphokines elicit the typical lesions seen in delayed-type hypersensitivity. In many textbooks, it is stated that the effector functions of CD4+ and CD8+ cells are quite distinct, with CD8+ cells functioning as CTLs and CD4+ cells accounting for delayed-type hypersensitivity. However, this is an oversimplification of effector function because some CD8+ cells can release lymphokines and cause delayed-type hypersensitivity, and some CD4+ cells exhibit CTL activity [4].

Susceptibility to GVHD: the problem of host-versusgraft reactions As mentioned earlier, GVHD is directed to two broad categories of alloantigens – major (MHC) antigens and mHAs – but these antigens also provide the main targets for allograft rejection. The vast availability of congenic strains makes mice the species of choice for studying GVHD directed to major versus minor histocompatibility antigens, and for determining the relative contributions of CD4+ and CD8+ cells to GVHD. However, it is important to first consider the issue of host resistance to GVHD, since this disease is not an inevitable consequence of transferring T cells across histocompatibility barriers. For instance, injecting normal adult mice with even large doses of allogeneic lymphoid cells generally causes no pathology; the host mouse mounts a powerful response to the donor alloantigens, and the injected T cells are rapidly destroyed. Host-versus-graft (HVG) reactions involve three cell types: T cells, B cells, and natural killer (NK) cells [17–20]. HVG reactions mediated by T and B cells take several days to develop and cause graft rejection by a combination of CTL activity and production of alloantibody. In adult mice, the simplest approach for inactivating host T and B cells is to expose the host to total body irradiation (TBI), resting T and B cells being highly radiosensitive. In contrast, activated or memory T and B cells may be very radioresistant, and can thus persist in the host and mediate HVG effects. Furthermore, in MHC-matched models of HCT, the mechanism of cytotoxic CD8+ T-cell resistance is more obscure and does not seem to involve conventional cytolytic pathways [21]. HVG reactions mediated by NK cells are often intense and occur within hours of donor cell transfer [18,19]. The target antigens for NK cells are still poorly defined, but there is accumulating evidence that the specificity of NK cells participating in HVG reactions is directed to cells that lack self-class I molecules [20]. NK-mediated HVG reactions do not apply when hypothetical MHC H-2-heterozygous (a × b)F1 cells are transferred to their parental homozygous strain a mice because the host strain a class I molecules are fully represented on the donor cells. In this situation, HVG reactions are mediated solely by alloreactive T and B cells, responding to the strain b class I molecules present on the donor cells. A different situation applies in a → b, a → (a × b)F1 and (a × b)F1 → (a × c)F1 strain combinations, where a, b, and c each represent a different hypothetical parental MHC H-2 haplotype. In each of these combinations, the donor cells do not express the complete set of class I molecules of the host. For example, in the a → (a × b)F1 combination, the host NK cells recognize self-class Ia on the donor cells but do not see self-class Ib: the failure to recognize self-class Ib on the donor cells causes the F1 hosts to display “hybrid resistance,” and their NK cells reject the parental strain cells. Similar lack of self-class I recognition applies in a → b and (a × b)F1→ (a × c)F1 combinations; here, the GVH reaction by the host NK cells is termed “allogeneic resistance” [22]. In the case of parent → F1 combinations, it should be pointed out that the intensity of hybrid resistance varies considerably according to the particular class I disparity involved. In practice, hybrid resistance is only a problem when there

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is heterozygosity for the H-2Db class I molecule, for example when C57BL (KbDb) cells are transferred to [C57BL × CBA (KkDk)]F1 hosts. NK-mediated HVG reactions are especially strong in MHC H-2different a → b combinations. Inactivating NK cells in situ is not easy because these cells are highly radioresistant; injection of anti-NK antibodies can be effective, but this is a cumbersome and expensive procedure. In practice, there are a number of ways to work around the problem of NK-mediated HVG reactions. For example, if it is essential to use fully H-2-different a → b combinations, the activity of host NK cells can usually be overcome simply by injecting the donor lymphoid cells in large doses. The easiest solution, however, is to use a → (a × b)F1 combinations; as mentioned above, hybrid resistance in parent → F1 combinations is generally insignificant unless H-2Db heterozygosity is involved. It is worth noting that NK-mediated HVG reactions do not operate in minor H-different combinations since the donor and host are H-2 identical. NK cells express inhibitory (e.g. killer immunoglobulinlike receptors in humans and the Ly49 family of molecules in mice) and activating receptors (e.g. NKGC in humans and NKG2D in mice) that engage MHC and other molecules on the surface of target cells, and control the cytolytic response against donor lymphocytes and hematopoietic cells [23]. In a clinical setting, GVHD seen after HCT is often complicated by concomitant HVG reactions, especially if the host has been presensitized to the donor as the result of blood transfusion. In the case of MHCincompatible strain combinations, the mouse models outlined below are deliberately designed to avoid the problem of HVG reactions by: (1) using nonimmunized F1 hybrid mice as hosts for parental strain T cells; (2) avoiding H-2Db heterozygosity; and (3) exposing the host mice to TBI. With this protocol, one can examine “pure” GVHD with little or no interference from host T, B or NK cells. It should be mentioned that all of the models discussed below involve intravenous transfer of cells.

GVHD directed to MHC antigens Because of the high precursor frequency of T cells for allo-MHC antigens, these antigens elicit a very intense form of GVHD [3,24–28]. Nevertheless, with proper dosaging of cells, some a → b HCT models utilizing transplantation of parental bone marrow cells, with or without additional lymphocytes, into irradiated parental recipients (e.g. C57BL → B10.BR, H-2b → H-2k) have been very useful in testing a wide variety of agents for their effects upon GVHD development, despite the potential complications of HVG, mentioned above [29–31]. In a similar manner, transfer of even small numbers of parental strain T cells (<105) into irradiated F1 mice leads to a high incidence of lethal GVHD, and is especially severe when the host expresses combined H-2 class I and class II differences. GVHD directed to whole H-2 differences involves both CD4+ and CD8+ T cells, responding to class II and class I differences, respectively, and either population alone is able to induce lethal GVHD [25–27]. Assessing the relative importance of class I versus class II antigens as targets for GVHD necessitates using donor–host combinations differing solely at class I or class II loci.

GVHD to H-2 class II antigens Most mouse strains express two types of class II molecule, I-A and I-E; these molecules are the homologues of HLA-DQ and HLA-DR molecules, respectively. I-E alloantigens are much less immunogenic than I-A antigens, and GVHD directed selectively to I-E antigens tends to be weak and is generally nonlethal. I-A antigens, by contrast, are highly potent inducers of lethal GVHD. In general, the severity and intensity

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Chapter 14

of GVHD varies with the strength of antigenic differences between strain combinations. To study GVHD directed selectively to I-A antigens, the most convenient combination is C57BL/6 (B6) and B6.C-H2bm12 (bm12) [11]. These two strains are identical except for three amino acid differences in the b chain of the I-A molecule. Although seemingly small, this mutation is highly immunogenic for T cells. Indeed, the response of B6 T cells to bm12 and vice versa is as strong as with an allelic (nonmutant) I-A difference [32]. The key feature of GVHD developing in the B6–bm12 combination is that GVHD induction is strictly controlled by CD4+ cells, with little or no contribution from CD8+ cells [11,33]. Thus, whereas small numbers of B6 (or bm12) CD4+ cells cause close to 100% mortality in irradiated (B6 × bm12)F1 hosts under defined conditions (see below), even high doses of B6 (or bm12) CD8+ cells cause no mortality (provided that the CD8+ cells are thoroughly depleted of CD4+ cells). The failure of CD8+ cells to mediate anticlass II GVHD is to be expected because, as mentioned earlier, the specificity of CD8+ cells is strongly skewed to recognition of class I antigens. Proliferative responses of purified B6 CD8+ cells to bm12 APCs are extremely weak, both in vivo and in vitro [32]. When B6 CD4+ cells are transferred to irradiated (B6 × bm12)F1 mice, the donor T cells initially home to the T-dependent areas of the spleen and lymph nodes. Here, the T cells respond to host class II antigens expressed on host APCs (typical APCs being highly radioresistant) and then mount a powerful proliferative response [11]. Large numbers of donor-derived blast cells enter the circulation and then percolate throughout the body to reach the skin, gut, and liver, among other sites, where the cells mediate their effector functions. The type of GVHD which results from this GVH reaction depends critically on a number of different factors including: (1) the dose of TBI used to condition the host; (2) the dose of CD4+ cells injected; and (3) the source of marrow cells (donor or host) used for reconstitution [33].

Donor marrow plus donor CD4+ cells The simplest model for GVHD is to inject the F1 host mice with a mixture of donor CD4+ cells and donor marrow cells [33]; under these conditions, the donor CD4+ cells selectively attack the host and do not impair stem cell reconstitution. The severity of GVHD in this situation is quite variable and seems to be a reflection of the general health of the animal colony. If the mice are in excellent health and free from infection, GVHD tends to be quite mild when the conditioning dose of TBI is not above 8 Gy. Mortality rates are low, and, except for transient splenomegaly, the mice show minimal pathology. Raising the dose of TBI to 10 Gy, however, leads to acute GVHD and heavy mortality, with most deaths occurring within 2 weeks of T-cell injection. This acute pattern of GVHD is characterized by marked weight loss, mild atrophy of the lymphohemopoietic system and a distended small intestine. Exudative enteropathy is apparent, and death is probably largely a reflection of gut damage leading to dehydration and acute infection [28,34,35]. Toxic lymphokines play a key role in gut damage. The key role of toxic lymphokines is apparent from the finding that mice can be protected against gut damage (and death) by injecting anti-TNF-α antibodies [36]. This protective effect seems to be most effective in CD4+ T-cell-mediated GVHD [37]. Induction of acute lethal GVHD in heavily irradiated recipients requires surprisingly few CD4+ cells. Doses of 1 × 105 CD4+ cells elicit close to 100% mortality, and even 1 × 104 cells cause significant mortality. If the health of the colony is suboptimal, acute lethal GVHD is seen with much lower doses of TBI, for example 7 Gy. Chronic GVHD tends to be sporadic in the above model, and is generally seen only when very low numbers of CD4+ cells are transferred.

With higher numbers of CD4+ cells, the few mice that survive acute GVHD generally show rapid recovery. When chronic GVHD is seen, the hosts show prolonged weight loss, lymphoid atrophy, and evidence of infection, although skin lesions are rare.

Donor marrow plus high doses of donor CD4+ cells The acute lethal GVHD seen in heavily irradiated recipients applies when the donor CD4+ cells are injected in the range of 3 × 106 cells down to 1 × 104 cells [33]. Injection of higher doses of CD4+ cells paradoxically leads to protection. Thus, whereas doses of 1 × 106 CD4+ cells generally cause close to 100% mortality, increasing the number of CD4+ cells to 2 × 107 reduces the mortality rate to less than 40% [33]. Even lower mortality rates occur when bulk populations of CD4+ cells and B cells are injected. Thus, if 10 Gy-irradiated (B6 × bm12)F1 mice are injected with a dose of 1 × 108 unseparated B6 spleen cells (a mixture of CD4+ cells, CD8+ cells, B cells, and stem cells), mortality rates are less than 10%. In considering the mechanism of this protection, it should be pointed out that mice kept under “germ-free” conditions are relatively resistant to lethal GVHD [38]. It is quite likely therefore that lethal GVHD is largely a consequence of infection, the tissue damage elicited by the GVH reaction making the host susceptible to invasion by pathogens. The capacity of large doses of CD4+ cells and B cells to protect against mortality could thus be attributed to restoration of immunocompetence. Cellular and humoral immunity are restored, and the host repels pathogens entering through damaged mucosal surfaces. According to this interpretation, bulk populations of donor lymphoid cells do not limit the intensity of the initial GVH reaction but merely counteract the consequences of this reaction. Here, it is worth mentioning that the bm12 F1 recipients of bulk populations of donor B6 lymphoid cells do go through a “crisis” at about 2 weeks after transfer (animals show hunched posture and lethargy) but then go on to full recovery. The alternative explanation to this protective phenomenon of high-dose donor CD4+ T cells is that it provides sufficient regulatory T-cell (Treg) capacity to suppress the GVHD-mediating T cells.

Regulatory cells and GVHD Natural Tregs account for about 1–5% of the lymphoid population in mice and are most commonly defined phenotypically as CD4+CD25+ and express the FoxP3 transcription factor. They play an important role in controlling autoimmune reactivity [39,40], and their tolerance-inducing capability can have significant impact in allogeneic transplantation. Several murine models have demonstrated the ability of CD4+CD25+ Tregs to prevent the development of GVHD [41–43] or to control ongoing GVHD (Fig. 14.1) [44] and delayed donor lymphocyte infusion-initiated GVHD [45]. Donor-type Tregs appear to be capable of inhibiting GVHD while allowing graft-versus-leukemia (GVL) responses to occur, when co-transplanted with donor effector T cells [46] either at later time points [42] or after donor lymphocyte infusion [47]. Recipient-type Tregs also exhibit the same capability when co-transplanted [48], as do third-party sources of Tregs [49]. The most potent Treg activity for GVHD inhibition is exhibited by CD4+CD25+ T cells bearing the CD62L adhesion molecule [50,51], presumably allowing them to traverse into the lymph nodes, where much of the initial antihost allorecognition occurs. Treg administration during allogeneic bone marrow transplantation preserves the thymic and peripheral lymph node architecture and accelerates reconstitution of a diverse donor-derived T-cell repertoire [52]. In regard to chronic GVHD, Tregs have been able to block disease in nonlymphopenic hosts [53], and their absence allows for unregulated T helper type 1 and type 17 cell activity leading to autoimmune-mediated pathology [54]. Tregs are highly dependent upon

Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect

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Fig. 14.1 Regulation of ongoing graft-versus-host disease by delayed infusion of freshly isolated donor CD4+CD25+ T cells in a major histocompatibility complex-matched model. CBA mice were lethally irradiated (13 Gy, split dose) and were injected with 2 × 106 B10.BR antithy1-depleted bone marrow (ATBM) cells alone, or in combination with 3 × 106 B10.BR CD8+ T cells. On either day 4 or day 10 post transplant, CD8+ T-cell transplanted recipients were either left untreated or infused with 1 × 106 freshly isolated B10.BR CD4+CD25+ or CD25− T cells. HCT, hematopoietic cell transplantation. (Adapted from [42], with permission.)

interleukin-2 for continued viability, and it is interesting to note that immunosuppressive agents commonly used in bone marrow transplantation, such as cyclosporine, will also suppress Treg activity, whereas other noncalcineurin-related inhibitors, like rapamycin, will allow their survival and continued functional capabilities [55,56]. NK T cells, which express both CD8 and NK1.1 molecules, have also been recognized for their capacity to prevent GVHD development after bone marrow transplantation in murine models [57,58], while preserving antitumor cytolytic activity [57]. Host NK T cells can also function in these capacities [59,60], and NK T cells may also be involved in regulation of chronic GVHD [61].

Host marrow and donor CD4+ cells A very different pattern of GVHD occurs when donor CD4+ cells are transferred with host rather than donor marrow cells [28,33]. In this situation, the donor CD4+ cells attack the F1 host stem cells and cause death from hematopoietic failure within 3 weeks; stem cell engraftment is apparent at 1 week post transfer, but by day 14 the entire lymphohematopoietic system, including the marrow, shows near-total aplasia. Mortality rates approach 100% and are little influenced by either the dose of CD4+ cells injected (1 × 105–2 × 107) or the conditioning dose of TBI used (6–10 Gy). Even with a low dose of 6 Gy, as few as 105 CD4+ cells cause close to 100% mortality. It should be emphasized that the above syndrome of lethal marrow aplasia does not occur when the donor CD4+ cells are transferred with a mixture of donor and host bone marrow. Here, the CD4+ cells attack the host stem cells but do not prevent engraftment of the donor cells. Since semipurified CD4+ cells are often contaminated with stem cells, especially when derived from spleen cells, demonstrating marrow aplasia mediated by CD4+ cells depends critically on using a highly purified population of these cells. Lymph nodes are the best starting population for preparing stem cell-free CD4+ cells.

GVHD to H-2 class I antigens As for class II molecules, mice express two class I molecules, H-2K and H-2D. Both types of molecule are potent targets for lethal GVHD. Although a number of class I-different, class II-identical strain combinations are available, the simplest approach for studying anticlass I GVHD

179

is to use class I mutant mice, for example the series of “bm” mutant mice [62]. On the basis of skin graft rejection, investigators have isolated more than a dozen different bm mutant strains of mice exhibiting small mutations (1–4 amino acid substitutions) of the H-2K molecules of the B6 (H-2b) strain. The immunogenicity of these mutant molecules for “wild-type” B6 T cells is quite variable. Some mutants, for example bm1, are strongly stimulatory, whereas others, such as bm9, elicit only low proliferative- and CTL in vitro-generated responses [22]. Detailed information on the capacity of the various class I mutants to elicit GVHD is not yet available. The data discussed below apply to the B6–bm1 combination, using (B6 × bm1)F1 mice as hosts. Despite the dogma that CD8+ cells function poorly without exogenous help, purified B6 CD8+ cells give spectacularly high proliferative and CTL responses to bm1 stimulators in vitro in the absence of CD4+ cells or their products [32]. Helper-independent responses of CD8+ cells also apply in vivo [11,32]. Thus, when purified B6 CD8+ cells are transferred to irradiated bm1 F1 mice, the donor cells proliferate extensively in the lymphoid tissues in the absence of CD4+ cells, and then disseminate throughout the body to mediate their effector functions. The end result is GVHD. It should be mentioned that B6 CD4+ cells respond very poorly to bm1, and even large doses of B6 CD4+ cells fail to elicit GVHD in bm1 F1 hosts [63].

Donor marrow plus donor CD8+ cells The injection of B6 CD8+ cells into class I-different irradiated (B6 × bm1)F1 mice, along with B6 marrow cells, causes heavy mortality irrespective of whether the hosts are conditioned with heavy irradiation (10 Gy) or light (6 Gy) irradiation [63]. Mortality rates approaching 100% are observed with a wide range of T-cell numbers, from 2 × 107 down to 1 × 105 cells. The striking finding is that GVHD tends to be chronic rather than acute. Except for mild weight loss, most of the recipients appear reasonably healthy for the first 3–4 weeks after transfer. Then, often quite suddenly, the mice become obviously ill with hunched posture, diarrhea, and marked weight loss. The condition of the mice worsens progressively, and death occurs at around 5–8 weeks after transfer. At autopsy, the mice show the typical signs of chronic GVHD with marked weight loss, lymphohemopoietic atrophy, and lymphocytic infiltrations in various organs. Skin lesions can be severe, although this severity is variable. Gut damage is evident, but is much less severe than in class II-different combinations [34,35].

Effects of adding CD4+ cells Despite the evidence that CD8+ cells mediate helper-independent responses in vitro, one could argue that GVHD elicited to class I antigens in vivo reflects help from radioresistant host CD4+ cells. However, the observation that purified CD8+ cells cause lethal GVHD in hosts given multiple injections of anti-CD4 antibody [63] suggests that radioresistant host CD4+ cells do not provide help. Nevertheless, supplementing the injected CD8+ cells with small doses of donor CD4+ cells causes a marked alteration in the pattern of GVHD: instead of developing progressive chronic GVHD, the hosts develop acute GVHD and die at 2–3 weeks after transfer. This finding implies that although CD8+ cells function well in the absence of exogenous help in vivo, adding help significantly increases their potency. Interestingly, the capacity of CD4+ cells to augment GVHD elicited by CD8+ cells only applies when CD4+ cells are injected in small numbers (<1 × 106). When high numbers of CD4+ cells are transferred, marked protection occurs [62]. Thus, if an inoculum of 2 × 106 B6 CD8+ cells is supplemented with 2 × 107 B6 CD4+ cells, death rates in irradiated (B6 × bm1)F1 hosts drop from 100% to 0%. Mortality rates are also

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very low when a large dose of 1 × 108 unseparated B6 spleen cells is transferred. As for the B6 → bm12 combination, the protective effects of large doses of CD4+ cells in the B6 → bm1 combinations is probably a reflection of restoration of immunocompetence or, as mentioned above, increased Treg presence.

D2 (H-2d) T cells causes aplastic GVHD. Although the disparity in the effect mediated by B10.D2 and DBA/2 (both H-2d) T cells has yet to be resolved, the most likely possibility is that DBA/2 mice have a quantitative and qualitative deficiency of CD8+ cells [70].

GVHD to mHAs GVHD in nonirradiated hosts All of the patterns of anti-class II and anti-class I GVHD discussed above refer to experiments with irradiated hosts. What happens when nonirradiated hosts are used? The results vary according to the age of the recipients. If neonatal F1 hosts are used, the recipients of parental strain T cells develop a lethal form of GVHD characterized by prominent lymphocytic infiltrations in various organs, especially the liver, and enlargement of the spleen [11,64]. Splenomegaly is most pronounced at about 10 days post injection, and measuring the size of the spleen in neonates has long been a popular model for assessing the severity of GVHD [64]. Induction of splenomegaly is usually attributed to the action of CD4+ cells, but at least in the B6 → bm1 combination, purified CD8+ cells cause prominent spleen enlargement [11]. With large doses of either CD4+ or CD8+ T cells, the host mice usually die after a period of 2–3 weeks. When nonirradiated adult mice are used as hosts, two distinct patterns of GVHD are seen [65,66]. When GVHD is directed solely to class II MHC antigens, for example when purified B6 CD4+ cells are transferred to nonirradiated (B6 × bm12)F1 mice, the recipients develop a chronic “proliferative” form of sublethal GVHD associated with splenomegaly and prominent autoantibody production. In this situation, the donor CD4+ cells mount a prolonged response against host class II antigens and release large quantities of lymphokines. Autoantibody production is presumed to be a reflection of aberrant T–B interaction: the donor CD4+ cells respond to the alloantigens on the host B cells and drive the B cells to undergo polyclonal activation. This proliferative type of nonlethal GVHD is also seen when purified CD4+ cells are transferred across whole MHC barriers [67]. A quite different type of GVHD occurs when unseparated T cells are transferred to nonirradiated hosts expressing combined class I plus II differences, for example when B6 T cells are transferred to (bm1 × bm12)F1 mice [65]. Here, the graft-versus-host reaction involves both CD4+ and CD8+ cells. In the early stage of this reaction, the host mice exhibit the proliferative form of GVHD discussed above. After a few weeks, however, lymphoproliferation is succeeded by a phase of progressive chronic GVHD associated with lymphoid aplasia; many of the recipients eventually die. Although this aplastic form of GVHD is known to require the presence of donor CD8+ cells, the chain of events that lead to aplasia is still poorly understood. Some workers argue that the CD8+ cells act as suppressor cells [68,69]. The simplest possibility, however, is that the CD8+ cells act as CTLs and cause progressive tissue damage aided by the donor CD4+ cells. Such exogenous help seems to be essential because only minimal disease occurs when the donor cells are depleted of CD4+ cells (or unseparated T cells are transferred to hosts expressing only a class I difference rather than a combined class I and II difference). The inability of purified CD8+ cells to cause lethal GVHD applies only to nonirradiated hosts. As discussed earlier, CD8+ cells are highly potent at causing an aplastic form of lethal GVHD in irradiated hosts. Although transferring unseparated T cells to hosts expressing a combined class I and II difference generally causes an aplastic form of GVHD, this is by no means an invariable finding. For example, when (B6 × DBA/2)F1 mice (H-2b × H-2d) are injected with unseparated DBA/2 T cells, the recipients develop the proliferative type of GVHD rather than aplastic GVHD [69]. By contrast, injecting either B6 or B10.

mHAs are presented on both MHC class I and II molecules, and derive from processed proteins either from genes present in the recipient but not the donor, or from polymorphisms, most likely single nucleotide polymorphisms, differing between the donor and recipient. In humans, H-Y antigens, stemming from the Y chromosome SMCY gene, are an example of an mHA that is present only in male recipients of female donor hematopoietic stem cells [71], and HA-1 is an example of an mHA that involves a single nucleotide polymorphism difference [72]. Mouse models for GVHD directed to mHAs are of obvious clinical relevance because HCT in humans is restricted largely to MHC (HLA)-compatible donor–host combinations. As discussed in Chapter 1, GVHD in HLAcompatible combinations can be very severe. This disease is probably directed largely and perhaps entirely to mHAs. In this regard, a few human mHAs have been identified; some are ubiquitously expressed by host tissues in the context of appropriate MHC molecules, whereas others have more tissue-restricted expression, such as HA-1 on hematopoietic cells [73]. The first evidence that mHAs provide targets for GVHD in mice came from studies in which untreated marrow cells were transferred to irradiated H-2-compatible hosts expressing a variety of non-H-2 differences [74,75]. A high incidence of lethal GVHD was seen, but only when the donor and host differed at three or more minor H loci. Difference at other loci, for example Ly or Mls loci, failed to cause GVHD. Evidence that GVHD was caused by T cells came from the finding that depleting the marrow inoculum of contaminating mature T cells with anti-Thy1 antibody plus complement abolished GVHD [74]. It should be noted that, in contrast to human marrow, mouse marrow contains only small numbers (1–2%) of mature T cells.

mHA target antigens Although it is clear that lethal GVHD in non-H-2-different combinations requires mHA incompatibility, it is not clear which particular mHAs provide the targets for GVHD. Studies with available congenic strains of mice differing selectively at defined mHAs have shown that none of these isolated mHA differences elicits lethal GVHD [76,77]. In one strain combination, C3H.SW → B6, a single class I-restricted mHA that can cause GVHD has been identified [78]. In addition, as discussed later, in the B6 → BALB.B model, MHC class I and class II-restricted responses associated with single TCR Vβ families, i.e. Vβ14 and Vβ11, respectively, have been found to cause severe GVHD, and may involve the recognition of single mHAs [79,80]. It would thus appear that only a very limited number of immunodominant mHAs may be responsible for GVHD in particular strain combinations that are known to differ at dozens of minor H loci [76,81–84]. Why some mHAs are more potent than others in inducing GVHD is obscure. Experiments with B6 → BALB.B and related CXB recombinant inbred strains have indicated that the mHAs that act as targets for GVHD do not necessarily correspond with strong in vitro CTL responses [85]. In these strain combinations, several strong mHAs have now been fully characterized, molecularly and genetically [86–88], but they have proved inadequate when it comes to correlations with GVHD development [76,77,85]. In addition, the curious form of immunodominance, whereby in vitro CTL responses to weak antigens are suppressed by responses to stronger antigens [89], does not seem to apply to GVHD

Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect

(see Vβ analyses below). Instead, it appears that GVHD pathology is caused by a limited number of mHAs in each donor–recipient combination that satisfy the criteria of being expressed in appropriate target tissue and are capable of stimulating a large enough T-cell response. Theoretically, all of these mHAs may contribute to the development of disease, and given enough responding T cells, each would be capable of inducing GVHD pathology in its own right.

Features of anti-mHA GVHD Studies with six strain combinations expressing three or more mHA differences have shown that transferring a mixture of purified unprimed donor T cells plus T-depleted donor marrow cells to mice given an intermediate dose of irradiation (7.5–8 Gy) causes heavy mortality in each combination [90]. The most detailed information has come from the CBA → B10.BR combination [15,74,91], in which lethal GVHD approaches 100% and occurs with even very small numbers of T cells (<1 × 105). Based on experiments in which donor T cells were negatively selected to class I- or class II-restricted mHAs by blood to lymph passage through irradiated H-2-recombinant intermediate hosts, the T cells mediating GVHD in the CBA → B10.BR combination respond to mHAs presented by host class I rather than class II molecules [15,91]. These cells comprise a mixture of H-2D- and H-2K-restricted T cells. The patterns of GVHD elicited by mHAs depend on the dose of T cells injected. Large numbers of T cells produce acute GVHD and early deaths, whereas smaller numbers of T cells lead to a chronic form of GVHD with late mortality (Fig. 14.2) [74]. Histopathology is most prominent in mice with chronic GVHD and involves lymphoid atrophy, weight loss, and lymphocyte infiltration of the skin, liver, and lungs [92–96]; involvement of the gut is mild, although some mice develop chronic diarrhea. Symptoms of GVHD tend to be more severe in hosts conditioned with heavy (8–10 Gy) rather than light (6–8 Gy) irradiation, and when the general health of the colony is suboptimal. With regard to the marrow inoculum, using host rather than donor marrow has little effect in potentiating GVHD [97]. This contrasts with anti-H-2 GVHD, where reconstitution with host marrow leads to marked marrow aplasia

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(see above). It is worth mentioning that the capacity of large doses of CD4+ cells to protect against lethal GVHD does not seem to apply to GVHD directed to mHAs [97]. The reason for this difference is unknown.

Role of CD8+ cells The above finding that the effector cells in the CBA → B10.BR combination were H-2 class I restricted suggested that GVHD to mHAs is mediated largely, though not exclusively, by CD8+ cells (Table 14.1) [90]). In this and several other combinations, depleting the injected T cells of CD4+ cells generally has little or no effect in reducing the

Fig. 14.2 Lethal graft-versus-host disease (GVHD) in irradiated (750 cGy) CBA/J mice given graded doses of purified B10.BR LN T cells. The data show cumulative mortality after transferring 107 anti-Thy1.2-serum-treated B10.BR marrow cells (B10.BR anti-θ BM) supplemented with varying numbers of nylon-wool-purified B10.BR LN T cells or with anti-Thy1.2 serum-treated B10.BR LN cells (anti-θ LN). (Adapted from [56], with permission).

Table 14.1 Lethal graft-versus-host disease (GVHD) in six minor histocompatibility antigen-different H-2-compatible strain combinations: GVHD mediated by purified T cells and T-cell subsets % mortality (median survival time) after transfer of donor T cells plus donor marrow Donor → recipient

H-2

C3H.SW → B6

H-2b

DBA/2 → B10.D2

H-2d

B10.BR → CBA/J

H-2k

B10.S → SJL

H-2s

B10.D2 → DBA/2

H-2d

B10.D2 → BALB/C

H-2d

Some of the known genetic differences between donor and host

Whole T

CD4+

CD8+

Marrow alone

H-1, -3, -7, -8, -9, -13, Lyt-1, -2, Mlsb H-1, -3, -4, -8, -13 Lyt-1, -2, Mlsb H-1, -3, -7, -8, -9, -12 Tla, Mlsa, Lyt-1, -2 H-1, -3, -7, -8, -9 H-12, -13, Tla, Mlsc H-1, -3, -4, -8, -13, Mlsa Lyt-1, -2 H-1, -3, -4, -7, -8, -9, -13 Lyt-1, -2, Mlsc

48 (58) 26 (>80) 96 (38) 88 (34) 92 (38) 93 (20)

5 (>80) 12 (>80) 35 (73) 13 (>80) 85 (45) 78 (24)

77 (45) 73 (53) 96 (39) 88 (35) 42 (58) 75 (39)

0 (>80) 8 (>80) 8 (>80) 0 (>80) 7 (>80) 0 (>80)

Data were pooled from more than 20 experiments with totals of 15–39 mice for each strain combination. Donor marrow cells (4 × 106) were T-cell depleted and injected intravenously along with unseparated (whole) T cells or T-cell subsets (1 × 106 cells) into appropriate lethally irradiated (8–10 Gy) recipient mice. (Adapted from [56].)

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intensity of GVHD [91], nor does injecting the host mice with anti-CD4 antibody [97]. These findings imply that the capacity of CD8+ cells to mediate GVHD to mHAs does not necessarily require help from CD4+ cells. Yet, in some strain combinations, like B6 → BALB.B, the CD8+ cells will only mediate GVHD in the presence of CD4+ cells [98]. The criteria which dictate CD8+ cell dependency on exogenous help appear to involve the cytokines available to APCs and their ability to express the 4-1BB ligand molecule, which can bind to its counterpart (4-1BB; CD137) on CD8+ T cells and serve as a co-stimulator for activation and survival [99]. In an effort to investigate the heterogeneity of the T-cell response across a donor–recipient pair disparate at multiple mHA loci, the repertoire of responding CD8+ T cells in the B6 → BALB.B (8.25Gy) GVHD model was analyzed by TCR Vβ CDR3-size polymerase chain reaction spectratyping. This response was found to be oligoclonal in nature, involving T cells from seven Vβ families [79]. With the exception of a single Vβ family, these responses were also found after transplantation of B6 CD8+ T cells into irradiated CXBE mice, a recombinant inbred strain that is derived from a cross between B6 and BALB/c mice and therefore expresses a subset of the mHA found in the BALB.B strain. This overlap in the CD8+ T-cell response repertoire suggested that the phenomenon of competitive immunodominance was not a factor in the recognition of mHAs involved in the development of GVHD. In addition, it was demonstrated that lethal GVHD could be induced by the transplantation of T cells from a single purified B6 CD8+ Vβ family into either irradiated BALB.B or CXBE mice, supporting their involvement in disease pathogenesis and suggesting a possible hierarchy of GVHDmediating specificities within the responding T-cell population [79].

Role of CD4+ cells In the case of the CBA → B10.BR combination and certain other combinations, CD4+ cells play no obvious role in GVHD. The helper function of these cells is not required, and transferring even high doses of purified CD4+ cells generally fails to cause GVHD. Nevertheless, in two of six combinations tested from this panel, namely B10.D2 → BALB/c and B10.D2 → DBA/2, purified CD4+ cells do cause a high incidence of lethal GVHD [90,100] (Table 14.1). This is also true in the B6 → BALB.B combination, in which CD4+ T cells are required for CD8mediated GVHD and can cause GVHD on their own account [98]. Why CD4+ cells mediate GVHD in only a limited number of mHA-different strain combinations is obscure. Insight into this problem has been generated from studying the B6 CD4+-mediated GVHD response in BALB.B versus CXBE recipients, where BALB.B, but not CXBE, mice succumb to lethal GVHD despite sharing multiple mHAs. A comparison of the responding CD4+ TCR Vβ repertoires from the thoracic duct lymph of irradiated BALB.B and CXBE recipients was made at the early stages of GVHD development (5–6 days) [101]. The spectratype analysis revealed overlapping utilization of nine Vβ families. In addition, the unique skewing of two Vβ families in the B6 → BALB.B response suggested the recognition of BALB.B mHAs not expressed in the CXBE recipients. Interestingly, the B6 → CXBE strain combination also exhibited unique skewing of two other Vβ families, which may hint at “immunodominance effects,” since one would have expected them to also be present in the B6 → BALB.B response. Direct evidence that Vβ families found skewed by spectratype analysis were involved in the pathogenesis of GVHD came from immunohistochemical staining of lingual epithelial tissue from BALB.B recipients, which revealed a high correlation between several of the skewed Vβ families and those with increased representation in the epithelial tissue infiltrates [101]. Further proof that the skewed Vβ families were indeed involved with GVHD was provided upon transplantation of those CD4+

Vβ families into irradiated BALB.B recipients, which resulted in a significant level of lethal GVHD. In contrast, mice that received transplants of the unskewed Vβ families survived with minimal symptoms of disease [101]. Applying the above observations concerning differences in responding Vβ repertoires, the capacity of B6 CD4+ T cells to induce lethal GVHD in BALB.B but not CXBE recipients was further examined. Transplantation of highly enriched populations of the two B6 CD4+ Vβ families, that were uniquely skewed in the B6 → BALB.B response, i.e. Vβ2 and Vβ11, were indeed able to induce lethal GVHD in BALB.B recipients. These data suggested that the BALB.B unique responses contribute in a way that either qualitatively or quantitatively enhances the severity of GVHD. Subsequent data suggested that these cells might preferentially infiltrate the small bowel of BALB.B recipients, thus increasing their importance to the systemic severity of GVHD (Fig. 14.3) [102]. Further studies revealed that the B6 Vβ11 CD4+ T cells, alone, could mediate severe GVHD, and immunohistochemical staining of host lingual and intestinal epithelial tissues supported their capacity to infiltrate typical GVHD-associated target areas [80]. To further characterize the specific CD4+ Vβ11+ T cells involved in this anti-mHA response, TCR Vβ spectratype analysis was performed, and indicated that six Vβ chains were used by this reactive population [80]. Probing deeper into this limited Vβ response, using a nonpalindromic adaptor polymerase chain reaction method, it was found that there was dominant use of the TRAV13–TRAJ16 transcript combination [103]. Then, using laser capture microdissection, the identical TRAV– TRAJ nucleotide sequence was found in areas dominated by the infiltrating Vβ11(+) CD4(+) T cells during the development of GVHD, both in the rete-like prominences of the dorsal lingual epithelium and the ileal crypts of the small intestine [103]. These combined results provided evidence that a restricted repertoire of T-cell specificities, presumably recognizing a correspondingly low number of mHAs, was sufficient for the induction of severe GVHD. In order to investigate whether the scope of the mHA-driven alloresponse changes during the development of GVHD, the responding B6 T-cell repertoire was examined between days 7 and 40 post HCT in the B6 → BALB.B strain combination. The results indicated that for the CD8 response, eight Vβ families were consistently skewed throughout the period of observation. It is interesting that skewing in four of those Vβ families had not been detected earlier in the thoracic duct population when examining the single day 5 time point [79]. In addition, skewing of three other Vβ families appeared to develop at later stages post transplant, which could indicate weaker anti-mHA responses, responses to antigens revealed via tissue destruction, or responses to nonalloantigen, such as to infectious agents. Similar findings were obtained for the CD4 analysis. Eight Vβ families were skewed throughout the course of GVHD development, all of which had been detected earlier in the thoracic duct lymph at day 6 post transplant [101]. Four additional Vβ families were transiently skewed at later time points. Furthermore, elimination of the eight consistently skewed Vβ families from the CD4+ T-cell donor inoculum delayed the onset in the appearance of most of the remaining four responsive Vβ families. The reason for this delayed appearance is unclear but would be consistent with them resulting from tissue damage, which would likely be slowed in the absence of dominant GVHD effector cells. These complex relationships in the development of anti-mHA GVHD are in obvious need of further investigation. One further aspect of the Vβ repertoire studies that remains unclear is the redundant capacity of a single mHA to drive the response of multiple Vβ families via cross-reactive TCR-binding sites. Insight gained into this issue will provide clues as to the minimum number of mHAs required to elicit GVHD in an MHC-matched combination and

Murine Models of Graft-versus-Host Disease and Graft-versus-Tumor Effect A

183

100

100

80 % survival

% survival

80

60

40

ATBM – MMB3.19 CD4+ T cells – MMB3.19 Vβ7+ CD4+ T cells –MMB3.19

60 40 20

20

0 0

20

30

40

50

60

70

Day

0 0

10

20

30

40

50

60

70

80

90

Days post HCT

Unseparated CD4→BALB.B (5) Vβ2/11–→BALB.B (9) Vβ2/11Enr→BALB.B (10) ATBM alone→BALB.B (7) Vβ2/11Enr→CXB-2 (4) ATBM alone→CXB-2 (4) B 110 Mean % initial body weight

10

100 90

80 70 60 0

2

4

6 8 Weeks post HCT

10

12

Fig. 14.3 GVHD potential of B6 anti-BALB.B Vβ2+ and 11+ CD4+ T cell responses. BALB.B and CXB-2 mice were lethally irradiated and injected with 2 × 106 B6 anti-thy1-depleted bone marrow (ATBM) cells alone, or in combination with either 2 × 107 unseparated B6 CD4+ T cells, an equal number of Vβ2- and 11-depleted (Vβ2/11−) CD4+ T cells, or 1.1 × 106 Vβ2and 11-enriched (Vβ2/11+) CD4+ T cells (mean purity 91%). (a) Survival of transplanted recipients. (BALB.B CD4 versus BALB.B Vβ2/11+; p ≥ 0.14). (b) Body weights for each group normalized as the mean ± standard error of the mean % initial body weight during sequential 1-week periods. HCT, hematopoietic cell transplantation. (Adapted from [84], with permission.)

why some mHA dominate the responses. Overall, the study of Vβ representation in a heterogeneously responding T-cell population in this acute GVHD model has begun to provide valuable insight into the scope of the allogeneic anti-mHA T-cell response.

Approaches for the separation of GVHD and graft-versus-leukemia responses The particular goal of any effort in this area is to provide a mechanism for reducing GVHD potential without sacrificing GVL or the more generalized graft-versus-tumor capability of donor T cells. Various

Fig. 14.4 Vβ7-enriched CD4+ T cells mediate a graft-versus-leukemia response with minimal induction of graft-versus-host disease in an allogeneic bone marrow transplantation model. Lethally irradiated (B6 × DBA/2)F1 mice were injected with 2 × 106 B6 anti-thy1-depleted bone marrow (ATBM) cells and 2.5 × 105 MMB3.19-primed Vβ7-enriched (82%) B6 CD4+ T cells or an equal number of unfractionated CD4+ T cells. The mice received MMB3.19 challenge (2 × 104; intraperitoneally) 1 day post bone marrow transplantation. Spectratype analysis had previously indicated that B6 Vβ7+ T cells were reactive to the MMB3.19 tumor cells, but not to stimulation by host (B6 × DBA/2)F1 alloantigen. (Adapted from [86], with permission).

strategies have been utilized to minimize GVHD in murine models, such as focusing on providing only CD8+ [104–107] or CD4+ [108, 90] T-cell subsets, with or without retention of GVL activity. The subset approach for separation of the two processes has also been studied for donor lymphocyte infusion models [109]. Restricting donor T cells to effector/memory subpopulations, either CD62L− or CD44hi, has also proved capable of preventing GVHD and often without impairment of GVL responses [110–113]. Likewise, and possibly related to the induction of memory cells, allogeneic sensitization of donor T cells to either leukemia cells or host cells can amplify GVL responses with reduced GVHD [114]. Results for all of these investigations may vary, depending upon the histoincompatibility involved and the specific tumor type used. Other approaches involve ex vivo tolerization of alloreactive T cells [115,116], separation of donor T cells based on functional phenotypes [117,118], and cytolytic effector mechanisms [119,120]. Cytokines, their promoters or their inhibitors have also been administered to tip the balance of donor T-cell responses towards GVL [121–127]. Establishing mixed chimerism with nonmyeloablative conditioning regimens has also been used as a means of reducing GVHD effects and allowing GVL responses with either donor-derived [128] or recipient-derived [129] delayed donor lymphocyte infusion. The alloreactive specificity of the donor T cells, as well as tissuespecific expression of host mHA or tumor-specific antigens, can also lead to a more targeted immunotherapeutic approach for minimizing GVHD development [130–132]. Vβ spectratype analysis has also been used to identify antihost alloreactive and antileukemia-specific responses and guide selected immunotherapy for the latter with GVHD minimization (Fig. 14.4) [133]. The high inverse correlation between the clinical incidence of GVHD and the rate of leukemic relapse has long suggested that antihost alloreactivity may provide the dominant GVL effect [134]. Therefore, strategies that would involve permitting the initiation of GVHD, presumably with an associated concurrent GVL response that could then be brought under control to avoid full pathologic development of disease, would be highly desirable. As mentioned earlier, several studies using CD4+CD25+ Treg or other suppressor type cells have shown some promise in accomplishing this goal [44,46–48].

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Immunopathology As for anti-H-2 GVHD, the effector mechanisms involved in GVHD to mHAs are still not fully understood. The histopathology of GVHD mediated by CD4+ cells and CD8+ cells is quite similar in the major target tissues of the skin, liver, and intestinal tract, although gut pathology is more prominent in recipients of CD4+ cells [96]. In the skin, epidermal cytotoxicity is induced by both subsets, albeit by different pathways, but dermal fibrosis seems to be a unique aspect of CD8-mediated GVHD. Mast cell degranulation occurs in the skin early after transplantation of either subset, even before lymphocyte infiltration is detected [135]. In addition, TNF-α (from either mast cells or T cells) does not appear to play a role in initial keratinocyte damage associated with early GVHD stages, but is involved in later CD4-mediated pathology, once the cells have infiltrated into the epidermal layers. In the case of CD8+ cells, it would seem that tissue damage, at least in the skin, reflects primarily direct CTL activity, although cytokines other than TNF-α may contribute to pathology. It has also been established that apoptosis has a central role in epithelial injury in acute GVHD [136], and that the primary targets of attack are phenotypically and antigenically distinctive epithelial cells within the basal layer of the skin and squamous mucosa [137,138], potentially corresponding to cytokeratin-15-expressing follicular stem cells (Plate 14.1) [139]. These cells are confined to rete ridges in the skin and

rete-like prominences in the dorsal tongue, and their discovery raises questions about their central role in driving the GVHD response forward. In all, the course and kinetics of pathogenesis in GVHD to mHAs may depend on several variables, including the strength of the individual CD4+ and CD8+ cell responses, the levels of expression of antigens in target tissue and the fluctuations of cytokines, both locally and systemically.

Summary Studies in murine models have provided many important insights into the pathophysiology of GVHD directed across MHC and/or mHA disparities. We now have a workable understanding of the T-cell subsets that can be involved in any given situation of GVHD and clues as to their mechanisms of action. These and future studies in the mouse can help guide us in the development of novel approaches to selectively reduce the risk of GVHD while allowing for the generation of GVL responses that can counteract the relapse of leukemia.

Acknowledgment The writing of this chapter was supported by grants HL55593 and HL75622 from the United States Public Health Service.

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single minor histocompatibility antigen can cure solid tumors. Nat Med 2005; 11: 1222–9. Patterson AE, Korngold R. Infusion of select leukemia-reactive TCR Vb+ T cells provides graftversus-leukemia responses with minimization of graft-versus-host disease following murine hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2001; 7: 187–96. Kernan NA. T-cell depletion for the prevention of graft-versus-host disease. In: Thomas ED, Blume KG, Forman SJ, editors. Hematopoietic Cell Transplantation, 2nd edn. Malden, MA: Blackwell Science; 1999. pp. 186–96. Murphy GF, Sueki H, Teuscher C, Whitaker D, Korngold R. Role of mast cells in early epithelial target cell injury in experimental acute graftversus-host disease. J Invest Dermatol 1994; 102: 451–61. Gilliam AC, Whitaker-Menezes D, Korngold R, Murphy GF. Apoptosis is the predominant form of epithelial target cell injury in acute experimental graft-versus-host disease. J Invest Dermatol 1996; 107: 377–83. Murphy GF, Lavker RM, Whitaker D, Korngold R. Cytotoxic folliculitis in acute graft-versus-host disease. Evidence of follicular stem cell injury and recovery. J Cutan Pathol 1991; 18: 309– 14. Sale GE, Beauchamp M. The parafollicular hair bulge in human GVHD. A stem cell rich primary target. Bone Marrow Transplant 1993; 11: 223– 5. Whitaker-Menezes D, Jones SC, Friedman TM, Korngold R, Murphy GF. An epithelial target site in experimental graft-versus-host disease and cytokine-mediated cytotoxicity is defined by cytokeratin 15 expression. Biol Blood Marrow Transplant 2003; 9: 559–70.

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Megan Sykes

Mechanisms of Tolerance

Introduction Immune tolerance to a set of antigens can be defined as a state in which the immune system does not mount a destructive response to organs or tissues expressing those antigens but is capable of responding normally to foreign antigens. Tolerance may occur in several different immunologic states. Tolerance can be “operational,” in which, for example, a graft is accepted without immunosuppressive therapy but a second graft from the same donor is rejected. In a slightly more robust form of tolerance, a second graft of the same type from the same donor would be accepted, but a more immunogenic graft from that donor would be rejected. In an even more robust type of tolerance, any organ bearing the same set of alloantigens as the original graft would be accepted. Finally, tolerance may be systemic, meaning that the animal’s entire immune system is tolerant to that set of antigens, so that a response cannot be induced to them either in vivo or in vitro. In allogeneic hematopoietic cell transplantation (HCT), persistent graft survival and the absence of graft-versus-host disease (GVHD) depend on a state of mutual tolerance of the donor to the host (graftversus-host, or GVH, tolerance), and of the host to the donor (hostversus-graft, or HVG, tolerance). Unfortunately, this state is not always achieved, and GVHD or failure of engraftment, respectively, ensues. Development of tolerance in the HVG direction requires host conditioning that either eliminates mature host immune cells, creating an immunologic “clean slate,” or permits pre-existing T cells to be rendered tolerant by the donor hematopoietic cells. Since newly developing T and B lymphocytes have a unique capacity to be rendered tolerant by antigens they encounter during their maturation, especially on hematopoietic cells, these lymphocytes can be educated to recognize an engrafted donor and host antigens as “self,” so that a state of donor- and hostspecific tolerance results. The ability of hematopoietic stem cells (HSCs) to induce a state of donor-specific tolerance suggests an additional potential application for HCT, namely the induction of organ allograft acceptance without the need for chronic immunosuppressive therapy. Such a state of immune tolerance would circumvent several major limitations to the practice of organ and tissue transplantation. In particular, improvements in immunosuppressive therapy have greatly augmented early graft survival, but these have had little impact on late graft loss, which is due in large part

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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to chronic rejection. Moreover, a high incidence of malignancies and opportunistic infections as well as drug-specific metabolic and other toxicities severely limit the tolerability of long-term chronic immunosuppressive therapy. The induction of donor-specific immune tolerance would avoid these complications while also preventing chronic rejection. Reliable, nontoxic methods of achieving allogeneic HSC engraftment across major histocompatibility complex (MHC) barriers will be required before HCT can be routinely used for the induction of organ or tissue allograft tolerance. However, recent progress in experimental systems has brought this approach into pilot clinical trials. A shortage of allogeneic donors has become another major limitation to solid organ transplantation, resulting in considerable interest in xenotransplantation (i.e. transplantation of organs from other species) to augment organ and tissue availability. However, it has become increasingly clear that the immunologic barriers to xenografts are even stronger than those resisting allografts, making it unlikely that an acceptable amount of nonspecific immunosuppressive therapy could be used to prevent xenograft rejection. Thus, it will be especially critical to develop methods of inducing donor-specific tolerance across xenogeneic barriers in order to make xenotransplantation efficacious. Because of its demonstrated efficacy in experimental models, and because of recent developments in our understanding of the mechanisms by which HCT can induce allo- and xenotolerance, this approach may have the greatest potential to make tolerance induction a routine part of clinical organ transplantation. This chapter will first provide a general discussion of mechanisms of T-cell tolerance, and then discuss these mechanisms in the context of specific models associated with induction of HVG tolerance in the absence or presence of HCT. The mechanisms by which both mature T cells pre-existing in allogeneic HCT inocula as well as T cells developing de novo from donor hematopoietic cells can be rendered tolerant of host antigens will also be reviewed.

Mechanisms of T-cell tolerance Three major mechanisms have been proposed to explain the induction and/or maintenance of T-cell tolerance to self or alloantigens: clonal deletion, clonal anergy, and active suppression. For developing T cells, these processes take place in the thymus, which is the central organ for T-cell development. Hence, induction of tolerance among developing thymocytes is referred to as “central,” as distinguished from the “peripheral” tolerance that may develop among already mature T cells when they encounter antigen in the peripheral tissues. It will become apparent to the reader from the discussion that follows that overlapping

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mechanisms have been implicated in models involving both central and peripheral tolerance induction. Clonal deletion T-cell receptors (TCR), consisting of uniquely rearranged α and β chains, known as αβTCR, are the cell surface heterodimers that recognize complexes of MHC antigens plus peptide. The thymus plays a critical role in the development of tolerance by a clonal deletion mechanism. Deletion occurs during T-cell maturation when the avidity of an interaction between an immature thymocyte and an antigen-presenting cell (APC) in the thymus is sufficiently high to induce apoptotic cell death [1]. The concept of avidity includes variations in the intensity of T-cell signaling resulting from variations in TCR affinity and density and ligand density, and from interactions of accessory molecules with their ligands on APCs. High avidity of the overall T cell–APC interaction is due, at least in part, to a relatively high affinity interaction between a rearranged αβTCR and a self-peptide–MHC complex presented by an APC in the thymus. Several marrow-derived cell types, including dendritic cells (DCs), B cells, and thymocytes, as well as nonhematopoietic cells of the thymic stroma, have the capacity to induce intrathymic tolerance by both deletional and nondeletional mechanisms, but DCs may be the most potent deletional APCs in the thymus (reviewed in [2]). Evidence for clonal deletion as a mechanism of tolerance was originally obtained by using monoclonal antibodies (mAbs) that are specific for certain V-gene products contributing to the β chain of the αβTCR [3]. Certain Vβ gene products selectively recognize “superantigens” encoded by endogenous retroviruses in the genome of mammalian species. Superantigens bind to a unique site on the class II MHC molecule. If a particular Vβ is capable of recognizing a superantigen, developing thymocytes with TCR using these Vβ undergo deletion [3]. These observations allowed the original demonstration that self-tolerance occurs largely through an intrathymic deletional mechanism, and that tolerance to marrow donors also occurs through a similar mechanism when allogeneic bone marrow transplantation (BMT) is performed in mice [3]. Later data supporting clonal deletion as a major mechanism for induction of self-tolerance utilized TCR transgenic mice expressing TCRs specific for antigens expressed naturally in the animal or by bone marrow grafts. Deletion is not unique to immature stages of T-cell development. Peripheral deletion has been described for mature T cells upon exposure to antigen in vivo [4]. Activation-induced cell death, which leads to partial deletion of expanded T-cell clones, is a normal physiologic consequence of high levels of T-cell activation. Deletion is also the natural consequence of cross-presentation by lymph node DCs of self-antigen expressed in parenchymal tissues to CD8+ cytotoxic lymphocytes (CTLs), under noninflammatory conditions [5]. Peripheral CD8 cells may undergo clonal deletion through “exhaustion” in the presence of a large, persistent antigen load, which may explain the anergy and deletion observed for host-reactive CTLs in the context of GVHD [6]. As discussed below, exposure to alloantigens in the presence of co-stimulatory blockade can lead to peripheral deletion of alloreactive T cells by mechanisms that are incompletely understood. In addition, veto cells and “double-negative” T cells (see below) can delete alloreactive CTL precursors in the peripheral tissues. T-cell anergy Studies on mechanisms of tolerance often include functional data, such as limiting dilution analyses to quantify CTLs, helper T cells or T cells proliferating in response to antigen. However, the failure to detect T

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cells with a particular allospecificity using this approach does not distinguish between clonal deletion and functional inactivation, or so-called “anergy.” Anergy may result when T cells encounter antigens without receiving adequate accessory or co-stimulatory signals. Anergic lymphocytes cannot be fully activated by encounter with the antigen that its clonally distributed surface receptor (surface immunoglobulin [Ig] in the case of the B-cell receptor, and the TCR in the case of the T cell) specifically recognizes. In the case of T cells, anergy is associated with a lack of proliferation and interleukin-2 (IL-2) production, and can usually, but not always, be overcome by providing exogenous IL-2. T-cell anergy is associated with altered signaling and tyrosine phosphorylation patterns [7]. Numerous methods of inducing T-cell anergy in vitro have been described, many of which involve blocking of molecular interactions between T-cell surface molecules and their ligands on APCs. Some of these interactions are termed “co-stimulatory signals” because they provide signals to the responding T cell, other than those transmitted through the TCR–CD3 complex, that are essential to obtaining a full state of activation [7,8]. These molecules, including CD28, inducible co-stimulator, OX40, 4-1BB, and several additional co-stimulatory pathways, have been targets of attempts to induce tolerance by presenting allo- or xenoantigens without adequate co-stimulation (see below). T-cell anergy has, in some instances, been associated with TCR downregulation [4,9], and a similar phenomenon has been associated with B-cell anergy [10]. In addition to the encounter of antigen without co-stimulation, T cells may also be rendered anergic by an encounter with peptide ligands for which they have low affinity [1]. An overall low avidity interaction between a T cell and an APC may also result in anergy [11]. It appears that, in addition to mature peripheral T cells, thymocytes are also susceptible to anergy induction, and this can be induced by antigens presented on both hematopoietic and nonhematopoietic stromal [12] cells. Anergy can be reversed in vivo and can be overcome by infection or by removal of antigen. Anergy can be induced in vivo by nonhematopoietic cells. In hematopoietic chimeras prepared with recipient myeloablation followed by allogeneic BMT, all marrow-derived cells are of donor origin, so that clonal deletion of thymocytes recognizing host-specific antigens is incomplete [12]. T cells bearing these host-reactive TCRs can be anergized by thymic epithelial cells [13] or by antigens encountered extrathymically [14–16]. T-cell clones with specificity for particular alloantigens can exist without causing destruction of tissue bearing those antigens [11,13,14,16]. In some instances, T cells with receptors specific for the in vivo tolerated antigen can be stimulated to respond to the antigen in vitro [13,14,17] or may even induce a state of inflammation that does not lead to organ destruction in the absence of additional stimuli [11]. In other systems, the T cells are fully or partially refractory to stimulation through their TCRs in vitro [11,14]. However, anergy is not the only mechanism that can account for the failure of T cells with self-reactive or alloreactive TCRs to cause autoimmunity or graft rejection, respectively. In the situation where T cells respond to antigens in vitro but not in vivo, they seem to be able simply to ignore antigens presented in the periphery [9]. This may be due to the presentation of these antigens by “nonprofessional APCs” that are unable to activate T cells, or it may reflect a failure of recipient T cells to migrate to the antigen-bearing tissue [18]. The level of peripheral antigen expression, the recency of T-cell emergence from the thymus [9], and the presence or absence of proinflammatory cytokines and costimulatory molecules within peripheral tissues may all influence the decision of a T cell to ignore or respond to peripheral antigens. T-cell activation may even occur within a tissue without initiating the whole cascade of organ damage [11]. However, proinflammatory stimuli, as

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may be present due to infection, may overcome such precarious states [9]. Some experimental models in which transplantation tolerance has been induced include T-cell anergy in association with a persistent suppressive T-cell population [19]. However, in most of these models, including those involving co-stimulatory blockade and other approaches designed to render APCs non-costimulatory, anergy has not been shown clearly to be the sole mechanism maintaining tolerance in vivo. Suppression A third mechanism, active suppression, has also been implicated in the induction and maintenance of self- and allotolerance. Suppressive activity has been attributed to both T cells and non-T cells, and may be antigen-specific or nonspecific. In general, the models in which suppressor T cells have been implicated involve exposure to antigen in the presence of a pre-existing immune response to the antigen. When, on the other hand, all mature lymphocytes are eliminated prior to administration of the antigen, tolerance may be due mainly to clonal deletion or anergy mechanisms and not to active suppression [20]. In the ensuing discussion, we categorize mechanisms of suppression in terms of the degree of specificity of their ability to suppress immune responses. Specific suppression While functional evidence for the existence of specific suppressor cells had been obtained in several transplantation models [21,22], until the last decade there had been little success in identifying or cloning suppressive cells, or in understanding the mechanisms by which they suppressed. In early studies, anti-idiotypic recognition, which is the recognition of determinants specifically expressed by a rearranged T cell or Ig receptor, was implicated [21,22]. However, apparent idiotypic specificity can be difficult to distinguish from suppression by T cells with the same specificity as the T cell being suppressed. The importance of regulatory phenomena in self-tolerance and regulation of autoimmunity, and in certain transplantation models, has recently become very clear. Some of these suppressive cell types are briefly described in the ensuing paragraphs. T cells comprise several major subsets of regulatory cells. Regulatory T cells (Tregs) can be classified as “naturally arising” and “antigen driven” [23,24]. Naturally arising Tregs are generated in the postnatal thymus and constitutively express CD25 (IL-2 receptor α chain) as well as CTLA4 [25]. A deficiency in “Treg” numbers in neonatally thymectomized mice permits the development of multiorgan autoimmune disease in susceptible strains [25]. Tregs require nonhematopoietic thymic stromal cells for their selection, and expression of the forkhead winged helix transcription factor 3 (FoxP3) is essential for full differentiation and maintenance of their regulatory cell features, including failure to produce IL-2 and dependence on paracrine IL-2 [26]. While it has been suggested that Tregs are positively selected on cortical epithelial cells of the thymus [27], recent studies suggest that the thymic medulla may be the major site of their positive selection [28]. Once in the periphery, these Tregs can be expanded in the presence of the positively selecting peptide antigen [29]. While some studies have suggested that the TCR repertoire of Tregs leaving the thymus is skewed toward greater affinity for self-antigens than that of conventional T cells, recent studies challenge this conclusion and suggest that the repertoire and specificities of both subsets may be quite similar [30]. Tregs play an important role in preventing autoimmune disease throughout adult life, as illustrated by the development of severe autoimmunity when FoxP3+ cells are selectively depleted in vivo. Their importance in preventing autoimmunity in humans is illustrated by congenital FoxP3 deficiency, which results in immune dysregulation, polyendocri-

nopathy, enteropathy, X-linked (IPEX) syndrome [31]. The regulatory cells are themselves hyporesponsive to TCR-mediated stimulation alone, and require IL-2 for their development and expansion [32]. IL-15 can also promote their expansion [33]. Transforming growth factor-β (TGFβ) also plays an important role in maintenance of their function via maintenance of FoxP3 expression [26]. Suppression seems to require cell-to-cell contact [25]. CD25+CD4+ Tregs express the memory T cellassociated CD45 isoform (CD45RC+ in the rat, CD45RBlow in the mouse). Although CD4 cells have been the best-studied targets of regulation by these cells, they can also suppress CD8 T-cell reactivity. While the Tregs show specificity for the antigen to which they are inducing suppression, the effector mechanism of suppression is non-antigen specific [25]. The immunosuppressive drug rapamycin appears to allow Treg survival and expansion while suppressing effector T-cell responses [34]. Antigen-driven Tregs include murine and human CD4 cells stimulated through their TCRs in the presence of IL-10. These “Tr1” cells produce high levels of IL-10 and TGF-β without making IL-4, distinguishing them from T helper cells type 2 (Th2) [35]. These cells, which do not constitutively express FoxP3, suppress antigen-specific responses in vitro and can suppress inflammatory bowel disease syndrome induced by naïve CD4 cells in mice. Immature or specialized IL-10-secreting DCs promote Tr1 differentiation [35]. In addition to Tr1 cells, antigen exposure can also result in the differentiation of CD25−CD4 cells to FoxP3+ Tregs [36–38]. This differentiation appears to be critically dependent on TGF-β and is inhibited by the presence of IL-6, which, in combination with TGF-β, promotes the differentiation of proinflammatory IL-17-producing Th17 effector cells. Recent studies showing that the transcription factor nuclear factor of activated T cells (NFAT), which has long been known to interact with the activating transcription complex activator protein-1 upon TCR stimulation, can alternatively interact with FoxP3 to turn on the “Treg” program that includes CD25 and CTLA4 upregulation, suppression of IL-2 production and suppressive function [39]. Thus, the choice of transcription partner by NFAT may determine the regulatory versus effector differentiation pathway of a T cell. It has previously been suggested that T-cell polarization to the Th2 subset that secretes IL-4 and IL-10 could promote allograft acceptance [40]. For practical purposes, an immune response that is itself nondestructive and inhibits the development of destructive Th1 responses could provide a powerful means of ensuring graft acceptance. However, only limited data exist to implicate an active role for Th2 cells in tolerance induction [41,42]. It is clear that Th2 responses are not always benign, as Th2 cells and Th2-associated cytokines can mediate or contribute to allograft rejection [43,44]. The original T cells associated with a suppressive network were CD8+. Recently, several groups have reported that FoxP3+ CD8 cells, which may also express CD25, can mediate suppressive activities [37]. A subset of suppressive human CD8+CD28− T cells that upregulate the inhibitory ligands immuoglobulin-like transcript-3 and -4 expressed on APCs, rendering them tolerogenic, has also been described [45]. NKT cells (T cells that express natural killer [NK] cell-associated markers and may utilize an invariant TCR-α chain) are another subset of T cells with regulatory activity, which may be mediated in part by Th2-type cytokines. These cells have been reported to suppress autoimmune diseases, apparently by suppressing interferon-γ (IFN-γ) production via an IL-4-dependent mechanism. The role of these cells in various transplantation models is discussed below. A double-negative, NK1.1− T-cell population that suppresses skin graft rejection by CD8 T cells with the same TCR has been described in a TCR transgenic mouse model [46], but the importance of this cell population in the setting of polyclonal T-cell responses remains to be determined.

Mechanisms of Tolerance

Veto cells Alloresponses can be downregulated by “veto” activity, which may be mediated by several different T-cell types. “Veto” cells inactivate CTLs recognizing antigens expressed on the veto cell surface [47]. Several types of cell have been reported to have veto activity, including CTLs [47] and various bone marrow subpopulations, including CD34+ cells and their progeny [48]. CD34 cells mediate this activity through a tumor necrosis factor-α (TNF-α)-dependent mechanism, whereas CD8 T cells may act through a Fas–Fas ligand-dependent pathway [49]. Veto activity has also been ascribed to marrow cells with characteristics of NK or lymphokine-activated killer cells, which can be used to promote GVH tolerance and facilitate allogeneic marrow engraftment in murine BMT models [50]. Murine studies also suggest that veto cells might contribute to the donor-specific transfusion (DST) effect [51], wherein prior administration of donor blood induces subsequent hyporesponsiveness to that donor. One suggested mechanism for the veto effect involves triggering of a CTL through a surface class I MHC molecule while it is also activated through its TCR, resulting in apoptosis of the responding CTL [52]. This class I-mediated signal could result from binding of a T-cell’s class I molecules to CD8 molecules on the veto cell. However, not all veto cells express CD8, so this mechanism cannot explain all veto phenomena. Veto activity has been also suggested to involve the immunosuppressive cytokine TGF-β [53]. Other suppressive cell populations Non-antigen-specific suppressive activity has been previously attributed to “natural suppressor” cells, which were originally described as suppressive non-T, non-B cells that were detected in sites of hematopoiesis after BMT, in lymphoid tissues in the presence of GVHD, and in normal bone marrow [54]. Such suppressive populations proved to be heterogeneous, including NKT cells [55,56] and hematopoietic progenitors [57]. Overall, the abundance of suppressive populations results in a strong inhibitory effect of bone marrow on immune responses in vitro [54,58]. Suppressive populations found in lymphoid tissues in association with GVH responses include a myeloid progenitor cell population whose activity depends on IFN-γ and nitric oxide [59]. Suppressive myeloid progenitor cells have also been detected in the setting of sepsis and traumatic stress, and immature myeloid cells have been associated with cancer-induced immunosuppression in humans and mice. Other cell types with the capacity to downregulate T cell responses, such as mesenchymal stem cells and specially manipulated DCs, are discussed in more detail elsewhere in this textbook. Thus, while a plethora of phenotypes and activities for regulatory cells has been described in recent years, much remains to be learned about the relative importance of each of these and the circumstances in which they can be optimally generated and utilized to improve transplant outcomes.

Mechanisms of B-cell tolerance The above discussion has focused mainly on the induction of T-cell tolerance. If T-cell tolerance is achieved, T-cell-dependent antibody responses do not occur. In this setting, tolerance of mature B cells may occur via a deletional process associated with lack of T-cell help [60]. However, T-cell-independent B-cell responses are also important in settings where natural antibodies (NABs) are present (i.e. antibodies that are present in the absence of known antigen exposure), such as in ABO blood group-mismatched transplantation and xenotransplantation. Several mechanisms of B-cell tolerance have been described. Autoreactive developing B cells undergo developmental arrest, and recombination activating gene-dependent light-chain receptor editing then occurs. Formation of a non-autoreactive Ig receptor allows the B cell to survive;

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if such a rearrangement does not occur, the B cell undergoes apoptosis [61]. Encounter of immature B cells with soluble antigen expressed at relatively low levels leads to B-cell anergy [10]. The induction of B-cell anergy versus activation may be dependent on antigen concentration [10]. Antigen expressed only on cell membranes in the periphery has, in some instances, been associated with a deletion of B cells [10,62]. Anergy is a major mechanism of self-tolerance of newly formed B cells [63]. However, interaction with membrane-bound self-antigen can activate anergic B cells, breaking their tolerance [64]. Cells of the B-1 subset, which produce NABs responsible for xenograft hyperacute rejection [65], may undergo apoptosis when their surface Ig is crosslinked by cell-bound antigen [66].

Approaches to inducing HVG transplantation tolerance The potential benefits of inducing HVG tolerance in the setting of organ transplantation were described in the introductory section. To recapitulate briefly, these benefits include the avoidance of chronic immunosuppressive therapy, the prevention of chronic rejection, and facilitation of the ability to use xenogeneic (i.e. from other species) organ and tissue sources, such as the pig. Thus, induction of HVG tolerance has become a major goal of research in transplantation. In this section, we will discuss some experimental approaches to inducing tolerance and will divide the discussion into efforts to induce peripheral tolerance versus those aimed at achieving HSC engraftment and central T-cell tolerance. However, it should become apparent by the end of the discussion that tolerance cannot always be readily characterized as peripheral versus central, and that a combination of central and peripheral tolerance mechanisms may ultimately prove to be most practical and effective for the induction of HVG tolerance. Approaches to peripheral tolerance induction Tolerance of pre-existing, peripheral T cells has been induced in numerous animal models, often by mechanisms that are not fully understood. Evidence for anergy has been obtained in a few models [67], and recently, a role for regulatory cells has been found in many (see below). Peripheral deletion of donor-reactive T cells has been implicated [68] and directly demonstrated [69–72] in some models. It is important to note that many of the strategies for inducing tolerance to primarily vascularized rodent allografts such as hearts and kidneys rely heavily on the capacity of the organ graft itself to control the immune response, and that the tolerance thereby achieved is not systemic. Most of these tolerance protocols are not effective when tested with less “tolerogenic” grafts, such as primary skin allografts across full MHC barriers. Strategies that are effective for primarily vascularized allografts in rodents are often ineffective in large animals and man. Thus, a thorough understanding of the mechanisms involved in tolerance induction and successful extension to large-animal models are important criteria to fulfill before these approaches can be attempted in humans as a substitute for chronic immunosuppressive therapy. Some experimental approaches to tolerance induction are summarized below. DST, modified APCs or administration of autologous cells transduced with allogeneic MHC genes In rodents, infusion of donor leukocytes around the time of transplant can lead to graft prolongation and, in some cases, operational tolerance [73]. In other species, DST has been less effective as the only graftprolonging therapy, but it has shown beneficial effects, even in humans [74]. However, infusion of donor cells is also associated with the risk of sensitizing the recipient to donor alloantigens. Moreover, improve-

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ments in graft outcomes with modern immunosuppression, combined with the ability to treat uremia-associated anemia with erythropoietin instead of transfusion, have ended the practice of transfusion prior to transplantation. B cells and T cells in DST, both of which can present antigen in a non-costimulatory manner, may contribute to their immunomodulatory effect [75]. Administration of autologous bone marrow transduced with a single donor MHC antigen promotes tolerance to other antigens expressed by the cardiac or renal allograft donor in mice [76] and miniature swine models, respectively [77]. Several mechanisms of tolerance have been implicated in murine DST recipients, including veto activity leading to deletion of donor-reactive T cells [51], regulatory CD4 cells [78], and a role for the Th2 cytokines IL-4 [79] and IL-10 [78]. The extension of tolerance in these models from the antigens transduced into autologous cells to additional antigens expressed on the donor organ is a form of “linked suppression” (see below). The introduction of alloantigen on tolerogenic APCs can contribute to the acceptance of allografts [80]. Stimulation with immature DCs, or with DCs modified in certain ways, for example with cytokines [81], with transduced genes that promote T-cell death or tolerance [82] or with rapamycin [83], can favor the activation of Tregs over effector T cells. Attempts to intentionally immunize with “tolerogenic” DCs have been associated with prolonged heart, islet, and kidney allograft survival in rodent models, usually without permanent survival [84]. The combination of rapamycin-treated DCs and a short course of posttransplant rapamycin has recently permitted long-term survival of vascularized cardiac allografts in mice [83]. Thus, both mature and immature DCs, under various conditions, can favor graft acceptance. The mechanisms by which DCs promote unresponsiveness after differentiation under various conditions vary. Indoleamine 2,3 dioxygenease is an enzyme that catalyses tryptophan catabolism. Its production is induced by a signal transducer and activator of transcription-1 (STAT1)-dependent signal mediated by IFN-γ, which may, paradoxically, be produced by Tregs [85], which thereby promote DC-induced tryptophan depletion [86]. Plasmacytoid (type I IFN-producing) DCs have been implicated in the generation of IL-10-producing Tregs [87] via a mechanism associated with plasmacytoid DC expression of the costimulatory molecule inducible costimulator [88]. Costimulatory blockade, with or without infusion of donor lymphocytes Additional signals besides those resulting from TCR stimulation are required for T-cell activation. This costimulatory signal, or “signal 2,” must be provided to prevent the development of T-cell anergy [7]. While the B7 (CD80, CD86)–CD28 costimulatory interaction was the first to be defined, it is now known that multiple molecular pathways may costimulate T cells, many through Ig and TNF receptor family members [89]. Efforts to induce tolerance by blocking the B7–CD28 co-stimulatory pathway have enjoyed success in rodents receiving vascularized allografts under cover of CTLA4Ig [90], a fusion protein containing the B7-binding portion of CTL antigen-4 (CTLA4), a high-affinity T-cell ligand for B7. CTLA4Ig blockade of CD28 binding to both CD80 and CD86, combined with the tolerogenic capacity of primarily vascularized organ allografts in rodents, can lead to long-term graft acceptance. Signaling through the interaction of CD40 on APCs and B cells with its T-cell ligand (CD40L, CD154, which is expressed transiently on activated T cells) plays an important role in activating APCs so that they can optimally co-stimulate T cells. Signaling through CD40 leads to upregulation of B7 expression, as well as other costimulatory molecules, class II MHCs, and cytokines such as IL-12, and induces Ig class switching by B cells. This APC activation via CD40 is a major facet of CD4 T cell-mediated “help” for CTL generation [91]. Administration of

DST under cover of anti-CD154 mAb can prevent islet allograft rejection [92]. In thymectomized mice, the combination of DST and antiCD154 permits long-term skin graft survival in fully MHC-mismatched recipients, but this approach is less successful in euthymic mice [93]. DST in combination with anti-CD154 permits deletion of peripheral donor-reactive CD8 T cells [94]. Although CD4+ regulatory cells promote CD8 tolerance in this model [95], they are of insufficient potency or duration to prevent rejection by donor-reactive new thymic emigrants [94]. Xenogeneic skin graft prolongation is also observed with DST and anti-CD40L [96]. Cardiac allograft survival is markedly prolonged in the presence of anti-CD40L [97], but chronic graft arteriosclerosis still occurs [98]. Graft prolongation with anti-CD40L or CD28 blockade has been associated with a Th2-dominant immune response [99], which may promote graft arteriosclerosis [98]. The combination of CTLA4Ig and anti-CD40L antibody can markedly prolong the survival of primary skin allografts [100], renal allografts in large animals [101], and xenografts [102], but without inducing tolerance. Functional tolerance has been achieved with this combination only in rodent models of cardiac or islet allografts [103] or islet xenografts [104], but not in the more stringent models of primary skin grafting or large-animal vascularized organ or islet allografts [105–107]. Linked suppression and “infectious tolerance,” in which tolerant CD4+ T cells can render naïve T cells unresponsive in secondary recipients, have been demonstrated in several models involving co-stimulatory blockade. These phenomena are largely attributable to Tregs [108]. Costimulatory blockade more effectively suppresses CD4-mediated than CD8 cell-mediated alloreactivity [105,109], although the extent of CD8 cell-mediated resistance is genetically determined. A short course of rapamycin with anti-CD40L and CTLA4Ig, has, quite remarkably, permitted long-term acceptance of primary skin allografts across full MHC barriers in mice in certain strain combinations [110]. The combination of rapamycin, anti-CD154, and DST has also led to markedly prolonged islet allograft survival in non-human primates [111]. The beneficial effect of rapamycin may relate to its ability to allow the selective expansion of Tregs [34,112]. A recent protocol involving the combination of rapamycin, cytolytic IL-15–Fc fusion protein, and agonist IL-2 protein has been reported to lead to graft survival in stringent models by causing the death of activated effector T cells while sparing Tregs [113]. The mechanisms of hyporesponsiveness induced by organ transplantation with costimulatory blockade are only partly understood. The greater success in achieving acceptance of primarily vascularized allografts in rodents using a short course of costimulatory blockade or any of a number of different immunosuppressive agents suggests that these types of graft are highly tolerogenic. As noted above, comparable results usually have not been achieved in large-animal models of organ grafting, limiting the clinical applicability of these approaches. This disparity between results in large and small animals may reflect, in part, the inability of CD8 T cells to reject vascularized allografts in rodents without CD4 T-cell help, combined with the tendency of such grafts to activate Tregs, which then predominate over the suppressed effector T-cell response. Thus, many different forms of relatively mild and shortterm immunosuppression have successfully allowed the long-term survival of such grafts in rodents but not in large animals or man. Calcineurin inhibitors such as cyclosporine and FK506 can block graft prolongation induced by costimulatory blockade [100], possibly by preventing IL-2-induced priming for activation-induced cell death [110] and by preventing de novo T cell conversion to Tregs [112]. Consistently, IL-2 has been shown to play an essential role in the induction of tolerance with CTLA4Ig and cardiac allografts [114]. However, Fas and TNF-α receptors do not appear to be required for tolerance induction with this method.

Mechanisms of Tolerance

Despite the observation that Th1-associated cytokines are associated with graft rejection, IFN-γ plays a critical role in graft prolongation induced with combined CD40/CD28 co-stimulatory blockade [114], DST with CD28 blockade [114], and DST with CD40 blockade [95]. IFN-γ has several known mechanisms for downmodulating T-cell responses, including the promotion of activation-induced cell death, the blockade of T-cell differentiation to a proinflammatory Th17 phenotype, and the induction of indoleamine 2,3 dioxygenease production by DCs (see above). Antibodies to T cells and adhesion molecules Nondepleting anti-T cell mAbs, with or without T-cell-depleting mAbs, have been used to induce skin graft tolerance across minor or MHC barriers, and this has led to infectious tolerance mediated by Tregs [108]. Combination of such mAbs with DST can lead to permanent cardiac allograft survival in mice. Similar results have been achieved by blockade of 1eukocyte function-associated antigen-1 and intercellular adhesion molecule-1 (ICAM-1) interactions, which, in addition to promoting cell–cell adhesion, transmit costimulatory signals to T cells. LFA-1/ ICAM-1 blockade has also been combined with anti-CD154 or blockade of very late antigen-4 and vascular cell adhesion molecule (VCAM) interactions to achieve cardiac allograft prolongation. Other mAbs that have been used in rodent heart graft models include combinations of anti-CD2 and anti-CD3 mAbs [115], anti-CD2 alone [116], and antiCD2 plus anti-CD28 [117]. Various mechanisms, including Th2 deviation, have been implicated. Anti-CD45RB mAb, especially in combination with anti-CD154 or rapamycin, can promote allograft survival through a mechanism that is dependent on CTLA4 upregulation by T cells and Tregs [118]. Total lymphoid irradiation Total lymphoid irradiation (TLI) has also been evaluated as immunosuppression for organ transplantation and has been successful in about one third of baboons studied. In clinical transplantation, TLI has been reported to be beneficial, although it is cumbersome and toxic, especially in the case of cadaver-donor transplantation. Donor-specific tolerance has been demonstrated in a small number of patients in whom immunosuppressive therapy was terminated following kidney transplantation under cover of TLI. One of these patients was studied 12 years after immunosuppression withdrawal, and shown to have active anti-donor mixed lymphocyte reaction and no evidence for microchimerism [119]. TLI used in conjunction with antithymocyte globulin has been shown to induce tolerance to organs in several large-animal models [120]. The relationship between peripheral T-cell tolerance and central tolerance In addition to inducing central tolerance, the thymus can promote tolerance of T cells that are already in the periphery at the time of organ grafting. In a pig model in which class I MHC-mismatched kidneys are accepted under cover of a short course of cyclosporine, the thymus appears to play a role in the induction of tolerance among pre-existing peripheral T cells [121]. The thymus also promotes peripheral tolerance when soluble donor MHC antigens are injected intrathymically or intravenously in a rat model, as removal of the thymus before or within the first few days of allografting results in rejection of the allograft [122]. Migration of DCs from the periphery to the thymus can lead to deletion of thymocytes recognizing antigens these DCs present [123]. While this mechanism would explain the tolerization of newly developing T cells, it does not explain the role of the thymus in tolerizing pre-existing peripheral T cells. There are several possible explanations for the latter phenomenon:

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1 T cells that are activated in the periphery by the organ allograft may recirculate to the thymus, as has been described [124], and encounter donor antigen there, perhaps on graft-derived DCs, which may then inactivate the T cells. This mechanism has been implicated in the rat soluble antigen injection model described above [125]. 2 The migration of donor antigen to the thymus may result in the development of Tregs that specifically recognize the donor antigen, migrate to the periphery and downregulate the activity of destructive alloreactive T cells. The role of the thymus in the development of Tregs has already been discussed. However, recent evidence suggests that only antigens produced by thymic epithelial cells, and not those produced by hematopoietic cells, promote the production of Tregs recognizing them [28]. Nevertheless, active regulatory cell populations have been implicated in both the rat and pig models discussed above. 3 Recent thymic emigrants that encounter antigen in the periphery may differentiate into suppressive rather than rejecting T cells. The role of the allograft itself in inducing tolerance in the above models in which a pre-existing peripheral T-cell repertoire must be rendered tolerant should again be emphasized. Transferable tolerance is not induced by intrathymic marrow injection alone without an organ allograft in rats [126]. In contrast to models involving intrathymic injections or short-term immunosuppressive treatments and immediate solid organ transplantation, pure intrathymic deletional tolerance induced by mixed allogeneic chimerism (discussed below) is not dependent on the continued presence of an organ allograft [127]. In fact, this tolerance does not depend on the continual presence of any donor antigen in the periphery, as long as there is a continuous supply of donor antigen to the thymus [128].

Achieving HVG tolerance with HCT Resistance to engraftment of HSCs Two major factors influence the engraftment of HSCs. The first is sometimes referred to as “space” in the hematopoietic system. The mechanism by which myelosuppressive host treatment promotes marrow engraftment is not fully understood, and could include both the creation of physical niches and the upregulation of cytokines and other molecules that transmit signals to promote hematopoiesis. It is probably the species specificity of some of these interactions that accounts for the competitive advantage enjoyed by recipient marrow over xenogeneic marrow. An understanding of these physiologic barriers is likely to be critical to the success of the approach of using xenogeneic HCT to induce xenotolerance. Once these are understood, genetically modified donors of other species, such as pigs, could be engineered such that their HSCs would be better able to compete for hematopoietic function in a human marrow microenvironment. In syngeneic BMT recipients, a mild myelosuppressive treatment such as a low dose of total body irradiation (TBI) is required to make physiologic “space” for the engraftment of marrow cells given in numbers similar to those which could be obtained from marrow of living human allogeneic marrow donors [129]. However, this requirement can be overcome by the administration of very high doses of marrow [130,131]. While HSCs injected intravenously readily home to marrow niches [132], the efficiency of this process may be enhanced by direct injection into the bone marrow cavity [133]. The second factor limiting alloengraftment is the host immune system. Because of this immune resistance, allogeneic HCT can be performed successfully only in immunosuppressed recipients. Not surprisingly, T cells of the CD4 and CD8 subsets resist engraftment of class II- and class I-mismatched marrow grafts, respectively. It is also noteworthy that CD4 cells weakly resist engraftment of class I-disparate marrow and

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that CD8 cells quite markedly resist class II-disparate marrow grafts in mice [134,135]. NK cells, which may kill target cells due to the absence of self-MHC molecules on allogeneic marrow cells [136], resist allogeneic marrow engraftment in mice, and are responsible for the ability of A × B F1 recipients to resist engraftment of AA or BB parental marrow. NK cells of both humans and mice express clonally distributed surface receptors that recognize specific class I molecules. This recognition transmits an inhibitory signal to the NK cell, thereby preventing the NK cell from killing “self” class I-bearing targets [136]. While the ability of NK cells to resist the engraftment of pluripotent HSCs in mice is rather limited [137,138], NK cells might pose a more significant barrier to human leukocyte antigen (HLA)-mismatched human marrow engraftment, in which stem cell and progenitor cell numbers in the donor inoculum may be more limited. Such a role might become apparent in the future as more reduced-intensity conditioning regimens are used in the setting of HLA-mismatched transplantation. However, a clear role for NK cells in resisting alloengraftment has not yet been established for large animals or man. GVH-reactive NK cells have been reported to promote donor engraftment in mice and to mediate graft-versus-leukemia (GVL) effects against myeloid leukemias in man [139], and the presence or absence of NK cells in patients with severe combined immunodeficiency may determine whether or not cytotoxic host conditioning is needed in order to achieve engraftment of haploidentical HCT [140]. NK cells are tolerized by the induction of mixed chimerism [141], consistent with the observed long-term stability of the chimerism. Such tolerance requires that each functional NK cell receives adequate inhibitory signals from two completely disparate sets of MHC molecules on donor and host cells. This “tolerance” cannot be explained by changes in the expression of inhibitory or activating MHC-recognizing (Ly49) receptors [142]. Several observations suggest that anergy of NK cells chronically encountering cells lacking inhibitory ligands is responsible for their tolerance in mixed chimeras. Tolerance is broken when donor and host NK cells are separated in vitro [143]. Moreover, tolerance is specific for the donor and recipient in the allogeneic situation [142], but is associated with global unresponsiveness in the presence of mixed xenogeneic chimerism [144]. Inhibitory Ly49 receptors are quite broad in their class I allorecognition, and recognition of even fully allogeneic class I molecules can confer some protection from NK-mediated marrow destruction compared with that observed for cells lacking class I MHC. Because of the increased disparity of xenogeneic compared with allogeneic MHC molecules, NK cells likely receive less inhibitory signal from xenogeneic than allogeneic cells. Thus, “tolerance” that reflects anergy induction by repeated encounter with cells lacking inhibitory ligand would be associated in this situation with a global functional NK defect, as has been observed. Consistently, this global lack of NK function is similar to that observed in the presence of chimerism from class Ideficient marrow [145]. Humoral mechanisms can also mediate resistance to marrow engraftment. Antibodies that inhibit engraftment can exist without prior immunization (so-called “natural” antibodies, or NABs). For example, NABs that recognize ABO blood group determinants or xenoantigens, and that exist in normal sera, can resist the engraftment of hematopoietic cells that express the determinants recognized by the NAB. Antibodies against donor antigens can also be present due to presensitization, usually from prior blood product infusions in HCT recipients. Animal studies suggest that the effect of NABs on engraftment can be overcome by the administration of sufficiently high marrow doses [146], and in man, deleterious consequences of ABO-incompatible BMT can usually be avoided with adequate red blood cell depletion from the marrow product. Occasionally, plasmapheresis may be needed to remove such antibodies if they are present at particularly high titer [147]. The presence of antibodies

resulting from presensitization to donor alloantigens, on the other hand, is associated with a high incidence of marrow graft failure in humans and mice. A permissive environment for engraftment of allogeneic marrow can exist under several physiologic and artificially induced conditions, as outlined below. The mechanisms by which HCT induces tolerance in each of these models is discussed. HCT in developmentally immunodeficient recipients Hematopoietic engraftment has been achieved without conditioning in developmentally immunoincompetent recipients. This was first observed in Freemartin cattle (fraternal twins sharing a placental circulation) [148]. Since the prenatal diagnosis of a number of congenital diseases has become possible, the injection of HSCs to preimmune fetuses is an attractive approach to inducing tolerance for postnatal organ transplantation from the same allogeneic or xenogeneic donor. While renal allograft tolerance has recently been achieved with this approach in a largeanimal model [149], the timing and purity of HSC injection are critical in balancing the tendencies for rejection and GVHD, and chimerism and tolerance have been variable in other animal models [150]. Animals can also be rendered tolerant by injection of alloantigens shortly after birth. Several mechanisms have been invoked to explain the observed HVG tolerance (reviewed in [151]). Lasting microchimerism has been detected in some studies and may be essential for the maintenance of tolerance. Evidence to support an intrathymic deletional mechanism has been obtained in some, but not other, strain combinations. However, the presence of microchimerism does not always predict skin graft tolerance in recipients of allogeneic lymphocytes perinatally, and nontolerant animals can still maintain microchimerism following rejection of donor skin grafts. Thus, it is not surprising that several additional mechanisms have been implicated in rodents in which neonatal tolerance has been induced. These include the following: 1 The action of specific suppressor T cells [151]. Tolerance cannot be easily broken by the infusion of nontolerant host-type lymphocytes in animals rendered tolerant at birth, and this has been attributed to the presence of suppressive T-cell populations. Specific suppressor T cells have been detected, and Tregs have been implicated [152]. 2 Neonatal mice have a tendency to produce Th2 responses, which have been implicated in donor-specific skin graft acceptance [41,153]. 3 The ability of allogeneic spleen cell infusions to induce tolerance may reflect the high ratio of non-costimulatory APCs (resting T and B cells) in donor inocula to recipient T cells in the neonate, rather than any particular susceptibility to tolerance induction at this time of life [154]. HCT in immunocompetent recipients following high-dose conditioning Marrow chimerism and tolerance can be achieved in otherwise normal adult animals if immunodeficiency and “space” are artificially created by lethal TBI. While the toxicity associated with this approach makes it unusable for the purpose of organ allograft tolerance induction in humans, studies in animals have provided important information on the immunobiology of HCT and tolerance induction. In murine recipients of T-cell-depleted (TCD) allogeneic BMT, lymphohematopoietic repopulation is largely (95–99%) allogeneic; a small population of host cells (mainly T cells) survives permanently in such animals. If TCD allogeneic and TCD syngeneic marrow are co-administered, mixed allogeneic chimerism ensues, i.e. lymphohematopoietic cells of both donor and recipient type coexist permanently in the survivors [155]. These mixed chimeras show no evidence of GVHD and show specific tolerance, both

Mechanisms of Tolerance

in vivo and in vitro, to donor antigens [155], i.e. “systemic” tolerance is achieved. Animals reconstituted with fully MHC-mismatched TCD allogeneic marrow alone demonstrate a similar pattern of specific unresponsiveness but tend to show poorer immunocompetence both in vivo and in vitro, possibly due to a mismatch between the MHC restriction imposed by the host thymus and the MHCs of donor-type APCs in full chimeras across complete MHC barriers. Another potential advantage of mixed chimerism over full allogeneic chimerism is that hematopoietic cells are most efficient at inducing clonal deletion of T cells in the thymus [3]; thus, a continuous source of host-type hematopoietic cells assures that host-reactive T-cell clones will not emerge from the thymus [127], whereas such cells can emerge in animals reconstituted with TCD allogeneic marrow alone [12]. The importance of antigen on hematopoietic cells for the induction of tolerance among newly developing T cells in humans is illustrated by studies in patients with severe combined immunodeficiency receiving HLAmismatched BMT and in whom donor T-cell, but not B-cell or myeloid, engraftment develops initially. Newly developing donor T cells from these patients exhibit reactivity toward donor class II MHC antigens. This reactivity disappears if the engraftment of donor class II-bearing cells later occurs [156]. Clonal deletion is the predominant mechanism of HVG tolerance in lethal TBI-treated recipients of TCD allogeneic BMT [3,12]. In vivo evidence suggests that suppressor cells are not involved in the maintenance of tolerance to donor antigens, regardless of whether TCD allogeneic marrow cells are given alone or with TCD syngeneic cells. Tolerance can be readily broken in such chimeras by administering a relatively small number of nontolerant recipient-type spleen cells [157], indicating the absence of any potent suppressive activity. Results of in vitro co-culture studies supported this conclusion. As discussed earlier, one important difference between TBI/BMT recipients and neonatally tolerized animals is that TBI leads to the elimination of most pre-existing T-cells. Thus, the number of remaining alloreactive T cells may be too small to provide an adequate stimulus for the activation and expansion of regulatory T-cell populations. In other words, activation of Tregs may require the presence of alloreactive effector cells, which is not the case if there is complete T-cell ablation followed by the intrathymic deletion of donor-reactive cells. In peripheral tolerance models, in contrast, mature donor-reactive T cells already exist in the periphery, and the observed difficulty in breaking tolerance by the infusion of nontolerant host-type cells is consistent with the presence of potent suppressive mechanisms. When, on the other hand, “pure” deletional tolerance is induced in animals in which the pre-existing peripheral T-cell response has been fully ablated, the absence of suppressive cell populations makes it easy to abolish tolerance by the infusion of nontolerant host-type lymphocytes [128,157,158]. In contrast to the above chimeras prepared with lethal TBI followed by reconstitution with TCD allogeneic marrow, animals receiving nonTCD allogeneic cells are highly resistant to the breaking of tolerance by the administration of nontolerant host-type lymphocytes [157]. This resistance is dependent upon the GVH reactivity of those T cells [157]. Rather than implying the existence of regulatory mechanisms, this finding suggests that GVH-reactive T cells persist long-term in these chimeras and eliminate administered nontolerant host-type lymphocytes before they can “take” and eliminate donor cells. Studies in animal models have addressed the possibility that, following lethal irradiation and reconstitution with exhaustively TCD autologous marrow, tolerance might be induced by expression of antigen on nonhematopoietic organs grafted simultaneously with the TCD autologous marrow. This approach was, however, insufficient to induce tolerance to MHC mismatched skin allografts in rodents or to MHC-mismatched vascularized grafts in large-animal studies. Similarly, in vivo treatment

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with depleting anti-T-cell mAbs alone does not permit induction of skin allograft tolerance across MHC barriers in mice. Thus, alloantigen seems to be required within the lymphohematopoietic system for the most complete tolerization of newly developing T cells, probably reflecting the unique ability of alloantigen expressed on marrow-derived cells to induce clonal deletion in the thymus. An alternative to the induction of mixed chimerism in lethally irradiated recipients has been to reinfuse autologous marrow that is genetically modified to express an allogeneic MHC gene. This approach has led to donor-specific hyporesponsiveness in a class I-mismatched mouse model and with class II gene transduction in a pig model [77]. While it would be technically cumbersome to modify each individual transplant recipient’s marrow with all of the MHC genes of any given cadaveric organ donor, the tolerance-inducing capacity of the organ graft itself can induce spreading to other graft antigens of the tolerance that is induced by a single donor MHC gene introduced into the recipient marrow [77]. The discussion so far would suggest that induction of mixed chimerism may be the most straightforward way of ensuring donor- and hostspecific tolerance across MHC barriers. However, the original methods for producing mixed allogeneic chimeras in mice were not directly applicable to man, because of the toxicity of lethal TBI and the high risk of GVHD and graft failure encountered when HCT is attempted across MHC barriers in man. HCT following lethal TBI would not be justifiable as a means of inducing tolerance for the purpose of organ transplantation, for which less toxic chronic immunosuppressive therapy is available. If HCT, therefore, is to be used as a means of inducing donorspecific organ allograft or xenograft tolerance, it will be essential to develop less toxic and more effective methods of rendering recipients permissive for engraftment than those which are currently available. Conditioning regimens are needed which specifically eliminate the host elements that resist alloengraftment without producing generalized toxic effects. Some approaches to achieving this goal are discussed below. HCT in immunocompetent recipients following conditioning with immunosuppressive but nonmyeloablative protocols Recently, several HCT protocols have been developed that involve myelotoxic and/or immunosuppressive, but not myeloablative, conditioning regimens. The term “nonmyeloablative” is used in this chapter to describe regimens that are known to leave sufficient host hematopoiesis intact to avoid significant cytopenia without a bone marrow graft or if the donor graft fails. These criteria have been demonstrated for certain regimens used in animals and patients, but not for all reduced-intensity regimens. Since host HSCs survive after nonmyeloablative conditioning, mixed chimerism develops when allogeneic marrow is administered. Such regimens include TLI [159], sublethal TBI [160], administration of cyclophosphamide following sensitization with allogeneic donor antigens [161], and the use of mAbs against host T cells in combination with other modalities [162]. HCT has also been performed in unconditioned patients receiving organ transplantation under conventional immunosuppressive drug therapy. Low levels of chimerism have been achieved, but withdrawal of immunosuppressive therapy has not been made possible with this approach [163,164]. However, the incidence of chronic rejection may be decreased in this setting [164]. A brief description of some nonmyeloablative or reduced-intensity conditioning approaches follows. Sublethal TBI Induction of mixed chimerism using fractionated sublethal TBI and allogeneic BMT can lead to donor-specific tolerance across complete

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MHC barriers in mice [160]. Donor T cells that need not have alloreactivity against the recipient promote alloengraftment in this model, and donor T cells maintain tolerance [160]. In dogs, a low dose of TBI, in combination with post-transplant cyclosporine and mycophenolate mofetil, has been associated with mixed chimerism and tolerance across minor, but not major, histocompatibility barriers [165].

Recently, combinations of chemotherapeutic agents have been used as reduced-intensity conditioning in patients with hematologic malignancies who were considered to be at high risk from complications of conventional high-dose conditioning. Thus far, these approaches have not been combined with organ transplantation in man. Anti-T-cell antibodies

TLI Another early approach to achieving mixed hematopoietic chimerism and donor-specific tolerance involved the use of TLI plus BMT. The long bones are shielded during the radiation preparative regimen, so BMT results in mixed chimerism rather than full donor reconstitution, and recipient T cells are not fully ablated. Mice treated in this way have been shown to be both resistant to GVHD and tolerant to donor skin grafts (reviewed in [159]). NKT cells producing Th2-type responses predominate following TLI, and this Th1 to Th2 shift may play a role in permitting graft acceptance and preventing GVHD. TLI as the sole conditioning modality for BMT has only variably permitted the induction of mixed chimerism and tolerance in large animals, and this has often been associated with considerable toxicity. A trial of donor BMT with fractionated TLI in high-risk patients receiving renal allografts was not associated with chimerism, and significant toxicity was also observed [166]. A recent trial of HLA-mismatched allogeneic HCT in patients conditioned with TLI and antithymocyte globulin has demonstrated transient induction of chimerism, but withdrawal of immunosuppressive therapy was associated with rejection of donor kidneys grafted at the same time (reviewed in [120]). Nevertheless, remarkably low rates of acute GVHD have been observed in patients receiving HLA-identical HCT for treatment of hematologic malignancies using TLI plus antithymocyte globulin-based conditioning [167]. Pharmacologic agents Cyclophosphamide has long been a staple of high-dose conditioning regimens and of less-intense conditioning regimens for HCT to treat aplastic anemia. Pre-HCT cyclophosphamide has more recently been used in combination with other agents in reduced-intensity conditioning regimens for HCT to treat hematologic malignancies. As is discussed below, a nonmyeloablative HCT/kidney allograft regimen involving cyclophosphamide conditioning has been the first successful attempt at the intentional induction of organ allograft tolerance in humans [168]. In rodents, high doses of cyclophosphamide administered several days following inoculation with large numbers of allogeneic marrow and/or spleen cells permit microchimerism and donor-specific tolerance across multiple minor histoincompatibilities and selected MHC disparities. Additional host treatment with a monoclonal anti-T-cell antibody [162], with sublethal TBI or with fludarabine and low-dose TBI [169], permits the induction of tolerance across complete MHC barriers. Sensitization followed by administration of cyclophosphamide causes selective destruction of host T cells driven into cell cycle in response to alloantigen, so that pre-existing donor-reactive T-cell clones are eliminated [162], and newly developing T cells can mature in the presence of donor marrow-derived antigen in the thymus. While thymic and peripheral chimerism can be demonstrated in animals receiving the regimen involving cyclophosphamide alone, the levels of such chimerism are extremely low, reflecting the fact that cyclophosphamide is relatively ineffective at producing marrow “space.” Evidence has been obtained for intrathymic clonal deletion as a mechanism of tolerance in this model. However, chimerism was shown to disappear with time, and anergy was reported as a mechanism of longer-term tolerance. Post-transplant cyclophosphamide has recently been applied clinically in a reduced-intensity regimen for haploidentical HCT [170].

Early experimental efforts to combine BMT with T-cell depletion to eliminate host cells that mediate alloresistance involved polyclonal sera (antilymphocyte serum). A fraction of mice receiving antilymphocyte serum and skin allografts several days prior to administration of marrow from the same allogeneic donor could be rendered specifically tolerant of donor antigens in the presence of certain MHC disparities [171]. The addition of rapamycin to the regimen significantly increases the incidence of tolerance [172]. Complex mechanisms, including suppressor cells of donor origin, were implicated in this model, and more recent studies have demonstrated that T-cell depletion by antilymphocyte serum is associated with sparing of Tregs [172,173]. A similar approach has been reported to prolong renal allograft survival in large animals, with the best results being achieved when the donor and recipient share a DR class II MHC allele [174]. In the primate studies, a veto cell population has been implicated. Conventional marrow doses are administered in this model without host myelosuppression, so that high levels of donor HSC engraftment are not achieved. Moreover, T-cell depletion is incomplete, so the involvement of regulatory cells in tolerance is therefore not surprising. In vivo depletion of host CD4+ and CD8+ T cells along with TBI (at least 6 Gy) permitted engraftment of allogeneic marrow and induction of skin graft tolerance across complete MHC barriers [17]. Adding a high dose of thymic irradiation (7 Gy) to the regimen permitted engraftment of fully MHC-mismatched allogeneic marrow in animals receiving only 3 Gy TBI. Thymic irradiation is needed because thymocytes become coated with mAbs but, unlike T cells in the peripheral lymphoid compartment, are not eliminated. The low dose (3 Gy) of TBI is necessary for the creation of marrow “space.” Permanent mixed chimerism and donor- and host-specific tolerance are reliably induced across complete MHC barriers using this regimen (Fig. 15.1) (reviewed in [20]). This approach has been extended to a xenogeneic (rat→mouse) combination by adding anti-NK1.1 and anti-Thy1.2 mAbs to the conditioning regimen [175]. These mAbs are needed to deplete recipient NK cells and γδ T cells [176], both of which pose a much more significant barrier to engraftment of xenogeneic than allogeneic HCT. Allogeneic tolerance has been achieved in a non-human primate model using this nonmyeloablative approach to inducing mixed chimerism [177]. In both the allogeneic [128](reviewed in [20]) and the xenogeneic (reviewed in [179]) rodent systems, intrathymic clonal deletion, rather than peripheral suppression or anergy, is the major mechanism inducing and maintaining long-term donor-specific tolerance. Accordingly, donor class IIhigh cells are present in the thymuses of these animals at all times, including 10 days post-BMT, when the first wave of thymopoiesis is underway. Furthermore, tolerance can be broken by depleting donor cells with mAbs after stable chimerism has been established, and this loss of tolerance correlates with the de novo appearance in the periphery of T cells bearing donor-reactive TCRs [128]. However, if the host thymus is removed before depletion of donor hematopoietic cells, or if donor-depleted spleen cells from chimeras are transferred to syngeneic athymic mice, donor-specific tolerance is maintained, and cells with donor-reactive TCR do not appear in the periphery [128]. These results demonstrate that intrathymic chimerism is essential and sufficient to maintain ongoing deletional tolerance in long-term mixed allogeneic chimeras, whereas peripheral chimerism is not required for the maintenance of tolerance. Since persistent antigen is required to maintain

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anergy, tolerance cannot be explained by a peripheral anergy mechanism. Moreover, the ease with which new thymic emigrants break tolerance after donor cell depletion by mAbs or with infusion of nontolerant recipient lymphocytes [128] is strong evidence that suppression does not play a significant role in maintaining long-term tolerance. Thus, this is a relatively pure model of ablation of pre-existing peripheral and intrathymic mature T cells followed by lifelong central, deletional tolerance. The potential toxicity of the regimen shown in Fig. 15.1 can be further minimized by replacing thymic irradiation with a second injection of anti-donor mAbs on day –1, and deletional tolerance is again attained. These thymus-directed host treatments are needed to eliminate pre-existing donor-alloreactive thymocytes and also to create “thymic space.” Thymic engraftment is regulated independently of marrow engraftment, and specific measures are required to permit donor progenitors to repopulate the recipient thymus at high levels, even in syngeneic mice (reviewed in [180]). In this allogeneic BMT model, TBI can be eliminated from host conditioning by giving a very high dose of donor marrow cells, still achieving durable multilineage mixed chimerism and systemic donorspecific tolerance, all in association with the lasting presence of donor APCs in the thymus and intrathymic deletion of donor-reactive host thymocytes. Thus, high levels of allogeneic hematopoietic repopulation and central deletional tolerance may be achieved with a conditioning regimen that excludes myelosuppressive treatment. Hematopoietic cell engraftment and donor-specific tolerance have been achieved across a full haplotype barrier using a similar nonmyelosuppressive regimen plus high stem cell doses in a large animal porcine model [180]. Based on the mouse model in Fig. 15.1, a primate model for tolerance induction has recently been developed. Since effective T-cell-depleting mAbs are not available for use in primates, polyclonal antithymocyte globulin and a short (28-day) course of cyclosporine are used in their place. A high percentage of cynomolgus monkeys receiving class I- and II-mismatched marrow with this protocol develop transient mixed chimerism and donor kidney allograft acceptance [177]. Several other regimens involving various combinations of anti-T-cell antibodies, irradiation, and immunosuppressive drugs have also permitted the achievement of mixed chimerism in mice (reviewed in [181]). Depleting and nondepleting anti-T-cell mAbs can also be used to induce tolerance in association with conventional-dose BMT without irradiation, although tolerance is then only reliably achieved across minor

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Days Fig. 15.2 Renal function (measured by serum creatinine [creat]) and cyclosporine levels (measured by a monoclonal antibody assay) in two patients (Pt 1, Pt 2) with multiple myeloma who received combined kidney and bone marrow transplantation from human leukocyte antigen-identical sibling donors following nonmyeloablative conditioning with cyclophosphamide, pre- and post-transplant antithymocyte globulin, and thymic irradiation. Cyclosporine (CyA) was the only pharmacologic immunosuppressive agent given post-transplant, and it was discontinued in the third month post-transplant. Both patients have sustained normal creatinine levels (now at 7 years and 9 years) following discontinuation of cyclosporine, indicating the presence of tolerance to their kidney allografts. Partial and complete remissions of the multiple myeloma were achieved in the two patients, and no GVHD has occurred. (Reproduced from Buhler LH, Spitzer TR, Sykes M et al. Induction of kidney allograft tolerance after transient lymphohematopoietic chimerism in patients with multiple myeloma and end-stage renal disease Transplantation 2002; 74: 1405–9, with permission.)

histocompatibility barriers, and lasting high levels of chimerism are not achieved [17]. In this setting, clonal anergy rather than deletion may be the major mechanism of tolerance [17]. The successful induction of tolerance in a primate model using nonmyeloablative conditioning to induce mixed chimerism [177], combined with murine studies demonstrating the utility of mixed chimerism followed by delayed donor leukocyte infusions (DLIs) as an approach to achieving GVL effects without GVHD [182–184], has been used as the basis for a clinical trial of mixed chimerism induction followed by DLI for the treatment of patients with hematologic malignancies [185,186]. The experience with this relatively nontoxic protocol provided an opportunity to evaluate the potential of this approach to induce transplantation tolerance in patients with a hematologic malignancy, multiple myeloma, and consequent renal failure. Six patients received a simultaneous nonmyeloablative bone marrow transplant and renal allograft from an HLA-identical sibling, and have accepted their kidney graft without any immunosuppression for follow-ups as long as 9 years. This study describes the first intentional achievement of organ allograft tolerance in humans. Three of the six patients have had prolonged complete remissions of their myelomas [168] (Fig. 15.2). This is especially surprising since chimerism in four of these six patients (including two with prolonged complete remissions of their myelomas) was only transient [168]. These data raise the possibility that transient chimerism followed by marrow rejection, which was evidenced by sensitized anti-donor T-cell responses in some of these patients [168], could lead to antitumor responses, and this hypothesis has been supported by data in a mouse model [187]. The renal allograft tolerance achieved in patients who lost chimerism suggests that the kidney graft itself may participate in tolerance induc-

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tion and/or maintenance after chimerism has played its initial role. The detection of sensitized T-cell responses to donor minor histocompatibility antigens expressed on hematopoietic cells with unresponsiveness to donor renal tubular epithelial cells in these patients [168] raises the possibility that tolerance may be specific for minor antigens expressed on the kidney graft itself. In the primate model described above, chimerism is also transient, but both BMT and early renal transplantation have been shown to be essential for tolerance induction [177]. Because T-cell depletion is only partial in these models, it is clear that the pure central, deletional tolerance described above in murine models has not yet been achieved with nonmyeloablative conditioning in large animals or humans. Nevertheless, the promising results obtained in these patients have provided an important proof of principle. Recently, this approach has been extended to a pilot study in patients without malignant disease with renal failure from other causes, who receive HLA-mismatched combined kidney and BMT. A systemic state of donor-specific tolerance was achieved in 4 out of 5 patients. These four patients have been off all immunosuppression for periods of more than 1 year to almost 5 years, providing the first successful intentional achievement of organ allograft tolerance across HLA barriers (178). The mechanisms of T-cell elimination using antibody treatments are not entirely clear. Animals treated with T-cell-depleting mAbs after thymectomy show a partial but significant recovery of CD4+ and CD8+ T cells over a period of several weeks, suggesting that some T cells escape depletion and expand due to lymphopenia and/or antigenic stimuli. Many studies have shown that the initially recovering T cells in large animals and humans predominantly express the “memory” phenotype, consistent with lymphopenia-driven expansion, but also consistent with selective resistance of memory cells to antibody-mediated depletion. T-cell depletion with at least some mAbs is independent of complement, but this pathway seems to be involved in T- and B-cell depletion with other mAbs. Co-stimulatory blockade with HCT Both thymic irradiation and host T-cell depleting mAbs in the conditioning regimen shown in Fig. 15.1 can be replaced by co-stimulatory blockade [69]. All preconditioning can be eliminated by giving a high dose of fully MHC-mismatched donor marrow followed by a single injection of each of two costimulatory blockers [188] or repeated injections of anti-CD40 ligand [189]. This ability to replace recipient Tcell depletion with costimulatory blockade to allow bone marrow engraftment is encouraging for several reasons. First, it has been dif-ficult to achieve T-cell depletion with antibodies in large animals and humans that is as exhaustive as that achieved in the above rodent models, perhaps due to the use of inadequate doses or suboptimal reagents. Second, if sufficiently exhaustive T-cell depletion could be achieved in humans, T-cell recovery from the thymus might be dangerously slow, especially in older individuals, since thymic function diminishes with age (reviewed in [190]). The ability to minimize the degree and duration of T-cell depletion by replacing some [109] or all [69,188,189] of the T-cell depleting antibodies with co-stimulatory blockers is therefore encouraging. As in other protocols achieving sustained mixed chimerism, long-term tolerance is maintained by intrathymic deletion in mixed chimeras prepared with costimulatory blockade [69,109,188]. However, since alloreactive T cells are abundant in the peripheral repertoire at the time of BMT with these regimens, these peripheral T cells must be rendered tolerant by BMT under cover of costimulatory blockade. Initial tolerance of peripheral T cells involves specific deletion of donor-reactive CD4 [71] and CD8 [72] T cells. This specific peripheral deletion of CD4 cells only requires failure of the CD154–CD40 interaction and does not require antibody-mediated clearance of CD154+ cells [191]. In mice receiving BMT with anti-CD154 and a low dose of TBI, CD8 deletion

occurs within 1–2 weeks and requires CD4 cells that do not have characteristics of “natural” Tregs [72]. Unlike models involving “adaptive” Tregs, CD4 cells are not required for maintenance of tolerance after this initial 2-week period [72]. Thus, while CD4 cells are clearly required for CD8 tolerance in this model, there is currently no evidence that this regulatory function of CD4 cells involves a specific subset of cells that is differentiated to mediate suppression. Deletion of donor-reactive CD4 cells occurs more slowly, over 4–5 weeks, and is preceded by a state of anergy [71,191]. Regulatory cells do not appear to play a major role in maintaining the long-term tolerance induced by anti-CD154 with BMT, since tolerance and chimerism are obliterated by the infusion of relatively small numbers of nontolerant recipient-type spleen cells in this model and linked suppression is not observed [71]. Since HSC engraftment ensures complete central deletional tolerance in these long-term chimeras [69,70,109,188], and specific peripheral deletion is quite complete, there may be insufficient donor-reactive T cells present to induce the expansion and maintenance of specific regulatory cells. However, some models using CTLA4Ig and anti-CD154 in combination with allogeneic BMT may be associated with less-complete deletion of donor-reactive T cells and involve regulatory mechanisms [192,193]. GVHD does not occur in the rodent models discussed above, despite the use of unmodified donor bone marrow cells. This is most readily explained by the continued presence of the T-cell-depleting or costimulatory blocking antibodies in the serum of the hosts at the time of BMT. These levels are sufficient to prevent alloreactivity by the relatively small number of mature T cells in the donor marrow. There are similarities and differences in the mechanisms of tolerance induced by anti-CD154 in combination with BMT versus DST. Only the BMT model allows engraftment of HSCs and intrathymic deletion as a mechanism of long-term tolerance [109,188]. Thus, in contrast to BMT, DST and anti-CD40L allows long-term skin graft survival only in thymectomized mice [95]. The inability to maintain tolerance in the presence of a thymus indicates the failure to establish central tolerance in the absence of substantial chimerism. In addition, the inability to resist breaking of tolerance by new thymic emigrants in this model argues that powerful peripheral regulatory mechanisms are not operative in these animals, even though regulatory mechanisms may play a role in the initial suppression of CD4-mediated alloresponses [95]. Concurrent viral infections have a deleterious effect on the induction of mixed chimerism under cover of co-stimulatory blockade [194]. Another concern is that successful induction of allogeneic tolerance in the presence of viral infections may also lead to tolerance to the virus and failure to clear it. These results suggest that de novo systemic viral infections should be avoided during the period of BMT and tolerance induction, and have important implications for the practice of BMT with this approach. Application of mixed chimerism for the induction of xenogeneic tolerance Considerable difficulties have been encountered in achieving high levels of engraftment and hematopoietic function from highly disparate species. Xenogeneic hematopoiesis can be enhanced by the administration of donor-specific cytokines, and spontaneous seeding of the thymus with tolerance-inducing cells from the donor has been detected in pig cytokine-transgenic mice receiving porcine HCT, resulting in the achievement of donor-specific tolerance [195]. An alternative approach involves the removal of the recipient thymus and its replacement with a xenogeneic thymus. Donor-specific skin graft tolerance across the discordant pig to mouse species barrier has been achieved with this approach [196]. Normal, immunocompetent mice that are thymectomized and treated with T-cell-depleting mAbs before porcine thymus grafting recover T cells in the xenogeneic thymic grafts. These cells repopulate the periphery and are competent to resist infection [197]. Tolerance to both donor

Mechanisms of Tolerance

and host by intrathymic deletional mechanisms is observed, and this deletion reflects the presence of class IIhigh cells from both species within the graft [196,198]. Since MHC restriction is determined by the MHC of the thymus, and porcine MHC is entirely responsible for positive selection of murine T cells in this model [199], it was surprising that T cells that differentiated in xenogeneic thymus grafts were able to respond to peptide antigens presented by host MHC [197]. However, the excellent immune function achieved in humans receiving HLA-mismatched allogeneic thymic transplantation for the treatment of congenital thymic aplasia (DiGeorge syndrome) suggests that this “restriction incompatibility” may not be a major obstacle to the achievement of adequate immune function [200]. Perhaps this high level of crossreactivity, even between species, reflects the fact that MHC reactivity is inherent in unselected TCR sequences [201]. Importantly, it has been demonstrated that human T cells can also develop in porcine thymic grafts (in immunodeficient mice), and that these T cells show specific tolerance toward the MHC of the xenogeneic thymus donor [202]. Induction of B-cell tolerance using HCT As mentioned above, mixed chimerism can also induce tolerance across xenograft barriers. Pigs are widely believed to be the most suitable xenogeneic donor species for transplantation to humans, but transplantation from this species is impeded by the presence in human sera of NABs that cause hyperacute rejection of porcine vascularized xenografts. In humans, the major specificity recognized by NABs on porcine tissues is a ubiquitous carbohydrate epitope, Galα1-3Galβ1-4GlcNAc-R(αGal). Humans lack a functional α1-3Gal transferase (galactosyltransferase; GalT) enzyme, as do GalT knockout mice, which also make anti-αGal NABs. Both pre-existing and newly developing B cells producing anti-αGal antibodies are tolerized by the induction of mixed chimerism in GalT knockout mice receiving αGal-expressing allogeneic or xenogeneic marrow. The induction of mixed xenogeneic chimerism prevents hyperacute rejection, acute vascular rejection, and cell-mediated rejection of primarily vascularized cardiac xenografts. Long-term chimeras produced in GalT knockout mice lack anti-Gal surface Ig-bearing cells in the spleen, and show tolerance in enzyme-linked immunosorbent spot assays, suggesting that clonal deletion and/or receptor editing tolerizes B cells developing after BMT. However, tolerance develops by 2 weeks post-BMT, even in mice that are presensitized to Gal antigens, despite the fact that the conditioning regimen does not deplete antibodyproducing cells. In GalT−/− mice rendered tolerant by the induction of mixed chimerism 2 weeks earlier, cells with anti-Gal receptors are present in the spleen but do not produce anti-Gal (reviewed in [203]). This rapid tolerance of NAB-producing cells in mice rendered mixed chimeric with BMT is due to an anergy mechanism [204]. The cells producing anti-Gal and most IgM NABs in mice belong to the B-1b subset [65], and their tolerization requires the expression of complement receptors on recipient nonhematopoietic cells [205] by mechanisms that are incompletely understood. An alternative approach for tolerizing B cells toward the Gal epitope involves retroviral transduction of the GalT gene into autologous HSC. Administration of marrow transduced in this manner to lethally irradiated GalT knockout mice led to tolerance to the Gal epitope [206]. Establishing molecular chimerism involves modification of autologous BM stem cells, avoiding the difficulties in achieving engraftment of pig HSCs in primates. However, this approach has only been evaluated in lethally irradiated mice, and it is not yet known whether or not a transduced, transplanted BM population could compete sufficiently with host hematopoietic cells to give significant long-term Gal expression in recipients of a more clinically relevant, nonmyeloablative conditioning regimen. Even if this approach were to be successfully applied, non-

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Gal-reactive xenoreactive NABs and cell-mediated rejection would remain to be overcome. The significance of these non-Gal antibodies has become apparent since the recent development of αGal knockout pigs using nuclear transfer [207–209]. In contrast, all types of rejection can be avoided with a single treatment by induction of mixed xenogeneic hematopoietic chimerism.

Preventing GVHD by depleting or tolerizing mature donor T cells in the HCT inoculum: is GVH tolerance a desirable goal? In order to avoid GVHD, the de novo development of host-reactive T cells from progenitors in the marrow must be prevented, and the GVH reactivity of pre-existing mature T cells in the marrow inoculum must be inhibited. While avoidance of GVH alloreactivity is not always desirable (see below), many strategies for preventing GVHD induced by mature T cells in the HCT inoculum involve depletion or tolerization of pre-existing GVH-reactive T cells. Tolerization of mature T cells in donor marrow could have both beneficial and harmful effects. The beneficial effects of GVH alloreactivity include GVL/graft-versus-lymphoma effects and promotion of alloengraftment. However, T cells that lack GVH reactivity also have the potential, if given in sufficient numbers, to promote alloengraftment (see Chapter 11), possibly by the veto mechanism described above [210]. Additionally, non-GVH-reactive T-cell populations can confer immunity to pathogens, thereby avoiding the high risk of opportunistic infection associated with global T-cell depletion. Induction of global GVH tolerance is highly desirable in the use of HCT for the treatment of nonmalignant diseases, such as the induction of transplantation tolerance (discussed above), and the treatment of inborn hematologic, immunologic, and metabolic disorders. However, when hematologic malignancy is present, GVH tolerance can be associated with reduced GVL effects, since GVH alloresponses are potent and largely responsible for the GVL response. One way to circumvent this deleterious aspect of GVH tolerance would be to preserve tumor antigenspecific responses while responses to normal host antigens were inhibited. Several candidate tumor-specific antigens have recently been identified (see Chapter 18), and CTL activity can be generated against them, generating interest in the idea of tolerizing GVH-reactive T cells while maintaining tumor-specific responses. However, the frequency of tumor antigen-specific T cells pre-existing in a given T-cell repertoire is even lower than that against multiple minor histocompatibility antigens, and the generation of meaningful tumor-specific responses is likely to necessitate donor presensitization along with in vitro expansion of tumor-specific effector cells, which might limit the homing capacity of injected cells. Such prolonged cultures are impractical for routine use in the setting of HCT, in which leukemia-reactive cells must eliminate exponentially expanding leukemic cells. In theory, the less risky strategy of generating tumor-specific responses from autologous T cells could achieve similar outcomes. However, T-cell immunity may be markedly impaired in the tumor-bearing host, and the use of immunologically unimpaired allogeneic donors is therefore attractive. Minor histocompatibility alloantigens expressed by lymphohematopoietic cells (including leukemias and lymphomas) but not by the epithelial GVHD target tissues may also be targeted using in vitro expanded CTLs in order to achieve GVL without GVHD. Several human minor histocompatibility antigens have demonstrated this pattern of expression (see Chapters 12 and 18]. However, GVH disparities for some of these same minor antigens (e.g. HA-1) have been associated with an increased incidence of GVHD. Recently, it was shown that administration of primed T cells specific for a single immunodominant class I-restricted minor histocompatibility antigen could mediate GVL without GVHD.

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Avoidance of GVHD was dependent on the absence of GVH-reactive T cells with additional specificities in the donor inoculum [211]. If such T cells are present, the response to immunodominant minor histocompatability antigens may induce APC activation and cytokine production, which activates T-cell responses to additional minor histoincompatibilities that are shared by GVHD target organs. However, the potency of antitumor effects in HCT is greatest in the setting of MHC disparity [212–215], in which GVH alloresponses are most potent. The potent ability of the anti-MHC GVH alloresponse to mediate GVL effects reflects the extraordinarily high frequency of T cells recognizing MHC alloantigens that is present in the unprimed Tcell repertoire. T cells reactive with an allogeneic MHC determinant may represent as many as 2% or more of the total T-cell population, whereas T cells reactive with a peptide–self-MHC complex are several orders of magnitude less frequent. Several reasons for this high frequency of allogeneic MHC-reactive T cells have been identified. One is the large number of different “nonself” peptide–MHC complexes presented by an allogeneic MHC molecule (i.e. high number of different determinants), resulting in recognition by a large number of different T-cell clones. The second reason is the large number of MHC molecules on an allogeneic cell. This may lead to recognition by large numbers of TCRs that are sufficiently independent of the peptide component for their recognition of the MHC– peptide complex that they bind with relatively low affinity to the MHC molecules regardless of the peptides to which they are complexed. In these instances, the high density of MHC determinants compensates for the low affinity of the TCRs recognizing them. In contrast, since minor histocompatibility antigens are peptides, the number of minor antigen– MHC complexes on a given APC will be more limited, perhaps resulting in recognition only by TCRs with relatively high affinity. As is discussed later in this section, a number of experimental strategies have been developed that permit exploitation of the GVH alloresponse for GVL effects while avoiding GVHD. Specific depletion or tolerization of mature GVH-reactive T-cells present in the donor inoculum at the time of HCT Several approaches have been developed in efforts to tolerize GVHreactive mature T cells within hematopoietic cell grafts, including blocking of adhesive and co-stimulatory interactions of T cells with recipient APCs. One of these approaches was evaluated in a pilot clinical trial with some success at reducing acute GVHD [216], but insufficient numbers of patients have been transplanted with this approach to allow an assessment of protective immunity from infection or tumor relapse. Additional approaches involve the selective elimination, rather than tolerization, of GVH-reactive T cells. These approaches preserve T-cell populations that may react to tumor-specific antigens and pathogens and perhaps promote engraftment. This goal can be achieved by stimulating donor T cells with host antigens, then depleting the activated cells using antibodies or immunotoxins directed against cell surface markers associated with recent activation, such as CD25 and CD69. Early clinical results with this approach suggest that GVHD is reduced, though not eliminated, and that such inocula may confer some anti-infectious immunity to the recipients [217]. Use of suppressive T cell populations to induce GVH tolerance CD25+CD4+ T cells generated by in vitro culture with alloantigen and anti-CD154 have been shown to suppress GVHD and to tolerize naïve and presensitized T cells via a regulatory cell population [218]. Additionally, donor Tregs can inhibit GVHD while preserving some GVL activity [219], although it is unclear whether or not the Tregs reduce the magnitude of GVL effects. While several groups are now exploring the

possibility of infusing fresh or expanded Tregs clinically, expanding the cells ex vivo has proved to be difficult. One important benefit of Treg infusion in an experimental model is protection of the recipient lymphoid tissues from GVH-associated damage, resulting in improved antiinfectious immunity [220]. In mice, NKT cells in the marrow or in the lymphoid tissues after TLI treatment also have the ability to suppress GVHD, apparently via an IL-4-dependent mechanism [221], while GVL activity is preserved. As is discussed above, this approach has recently been applied in patients receiving HLA-identical HCT with strikingly low rates of acute GVHD [167]. However, it remains to be determined to what extent GVL effects are preserved with this approach. In addition to the NKT cells, T cells lacking NK-cell markers that are resident in murine bone marrow have a reduced capacity to produce GVHD, with preserved ability to mediate GVL [222]. Approaches to preventing GVHD that do not involve induction of tolerance Cytokine manipulation Considerable interest was generated in the 1990s by the notion that GVHD-inducing T cells had a Th1 (IFN-γ- and IL-2-producing) phenotype and that Th2 (IL-4-, IL-5-, and IL-13-producing cells) cells could suppress GVHD, and some evidence supported this possibility. Similarly, CD8 cells producing type 1 cytokines mediated severe GVHD in MHC class I-mismatched or parent into F1 hybrid combinations, whereas type 2 cytokines did not [223]. Meanwhile, Th2 cytokines mediated GVL and promoted donor marrow engraftment [223]. However, infusion of ex vivo polarized donor Th2 cells did not produce a measurable reduction in GVHD in a clinical trial [224]. In fact, the role of Th1- and Th2-associated cytokines in HCT and transplantation in general is rather complex. Systemic administration of Th1-type cytokines such as IL-2, IL-12 or IFN-γ shortly after BMT in mice can actually reduce the severity of GVHD [225–227] while preserving the GVL effect [228,229]. Furthermore, T cells from IFN-γ-deficient donors have an increased capacity to cause acute GVHD in lethally irradiated recipients (reviewed in [230]). While the mechanism by which IFN-γ inhibits graft rejection and GVHD is not fully understood, possible mechanisms have already been discussed above. Further studies have shown that Th2 and Th2-associated cytokines are also capable of contributing significantly to GVHD pathology of particular tissues. Other approaches A number of additional approaches to ameliorating GVHD do not involve initial tolerance induction of donor T cells in the HCT graft. One such approach is to transduce donor T cells with suicide genes, such as herpes simplex virus thymidine kinase, so that the alloresponse can be turned off at will, hopefully after tumor has been eradicated [231]. Initial clinical success with this approach has been somewhat limited, in part due to the fact that ganciclovir, the drug used to kill thymidine kinase-transduced T cells, is also needed clinically to treat cytomegalovirus reactivation, which is a common and serious complication of HCT. Furthermore, the transduction of GVH-reactive T cells may be incomplete, resulting in escape of some clones from suicide after treatment has been initiated. Finally, the manipulations involved in the transduction process itself might render the donor T cells less effective at eliminating leukemias when transferred back to the patient. The development of new vectors and the use of genes other than thymidine kinase may lead to further improvements in the utility of this approach. Another approach is to attempt to block the early inflammatory mediators of GVHD, such as IL-1 or TNF-α. While some of these approaches have been evaluated in pilot clinical trials, none has yet been shown to have

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a durable effect on GVHD. Recently, keratinocyte growth factor has been shown to ameliorate GVHD without blocking GVL, perhaps by reducing the disruption of the intestinal barrier associated with myeloablative conditioning and by preventing some of the thymic injury and consequent immune damage associated with GVHD (see Chapter 16). Delayed donor T-cell administration DLIs can mediate powerful GVL effects in patients with chronic myeloid leukemia, lymphomas, and multiple myeloma who have relapsed following allogeneic HCT (see Chapter 72). In established mixed allogeneic chimeras, which are immunologically tolerant of their original marrow donor’s antigens, a GVH reaction takes place after administration of DLI, resulting in conversion of the state of mixed hematopoietic chimerism to a state of full donor chimerism. This conversion to full chimerism is due to a GVH reaction directed primarily against recipient MHC alloantigens. Remarkably, this powerful GVH alloreaction is not associated with any clinically significant GVHD, even though recipient alloantigens are expressed on many cell types and donor T cells are given in numbers that would cause rapidly lethal GVHD in freshly irradiated recipients [182,183,215,232]. This demonstration that GVH reactions could be confined to the lymphohematopoietic system suggested an approach to separating GVHD from GVL effects. Since hematologic malignancies reside largely in the lymphohematopoietic system, the result suggested that GVH reactions might be confined to this system and eliminate tumor cells without entering the epithelial GVHD target tissues. Indeed, GVL is achieved without GVHD when delayed DLIs are given to established murine mixed allogeneic chimeras [184,215]. Recent studies have revealed mechanisms by which delayed DLI may mediate GVL without GVHD. Antihost MHC alloreactivity clearly mediates the most potent GVL effects [184,215]. Moreover, GVHreactive donor T cells become activated and proliferate markedly in mixed chimeric recipients of DLI [215,232]. However, these cells do not migrate to GVHD target tissues (epithelial tissues such as skin, intestines, and liver) unless there are inflammatory signals in those tissues [232]. The recipient’s recovery from conditioning-induced tissue injury markedly reduces the tendency of GVH-reactive donor T cells that are activated by host alloantigens in the lymphoid tissues to leave the lymphohematopoietic system and cause GVHD [232]. Provision of a local toll-like receptor stimulus promotes the entry of such cells into the skin and induces localized GVHD [232], indicating that tissue inflammation provides a critical checkpoint for T-cell recruitment to GVHD target tissues. Such inflammatory signals can be provided by the tissue injury induced by conditioning therapy and by toll-like receptor activation [232], which may be induced by infection or by conditioning itself. Both high-dose and reduced-intensity conditioning are associated with very early production of chemokines in the GVHD target tissues, which is followed by immigration of T cells that then elicit a further cascade of chemokines that amplifies the response [233]. Adhesion molecules that promote leukocyte infiltration through the microvasculature of these tissues are also likely to be upregulated by conditioning. Lethal TBI and cyclophosphamide are known to upregulate the proinflammatory cytokines IL-1, IL-6, and TNF-α [234–236], which can upregulate endothelial cell E-selectin, P-selectin, ICAM-1, and VCAM1 [237,238]. Proof of principle has been obtained that delayed DLI can mediate GVH responses confined to the lymphohematopoietic system in patients receiving nonmyeloablative HCT with in vivo or ex vivo T-cell depletion of the initial donor inoculum, who achieve mixed chimerism without GVHD [186,239]. However, some patients achieving initial mixed chimerism from a TCD donor inoculum nevertheless develop GVHD following administration of delayed DLI. In general, delayed DLI is somewhat variably associated with GVHD in patients, but to a lesser

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degree than would be expected in freshly conditioned recipients of similar cell numbers [240,241]. The greater ease in avoiding GVHD in mice compared with humans receiving delayed DLI after TCD HCT may reflect the more robust thymic T-cell recovery that occurs in mice than in heavily pretreated human adults receiving TCD HCT. This thymic recovery confers protection from infection, avoiding toll-like receptor stimuli, and also allows the recovery of regulatory T-cell populations that downregulate GVH reactions. The ability of Treg to inhibit DLI-induced GVHD has been demonstrated in mouse models [242– 244]. The potent antitumor responses seen in patients with advanced lymphoid malignancies who received the transplant approach described above (mixed chimerism induction using TCD HCT and nonmyeloablative conditioning, followed by delayed DLI [245]) led to the hypothesis that an initial state of mixed chimerism might permit more powerful graft-versus-tumor effects to be achieved from the GVH alloresponse than is possible in full allogeneic chimeras. To test this hypothesis, the GVL effects of DLI were compared in mixed versus full allogeneic chimeras inoculated with a host-type T-cell lymphoma in a murine model. Remarkably, DLI led to only a slight prolongation of survival in leukemic full allogeneic chimeras, whereas they led to almost complete protection, with 80–100% survival, in mixed chimeras receiving the same tumor inoculum (Fig. 15.3). This markedly more potent GVT effect of DLI in mixed compared with full chimeras was shown to be dependent on the expression of host class I and class II MHC molecules on recipient-type hematopoietic cells [184,215]. GVHD was not observed in the mixed chimeras with these potent GVL effects from DLI [184]. These data are not inconsistent with an earlier report showing a critical role for recipient hematopoietic APCs in initiating GVHD [246]; in that report, the donor T cells were given to freshly lethally irradiated mice, whereas the donor T cells were given as delayed DLI in the studies comparing mixed with full chimeras [184]. The complete absence of GVHD in mixed chimeras enjoying potent GVL effects of DLI dramatically illustrates the power to separate GVHD and GVT effects by confining the anti-MHC alloresponse to the lymphohematopoietic system. GVH tolerance of T-cells developing from progenitors in the marrow As has been discussed above, the mechanism of GVH tolerance in lethally irradiated recipients of TCD allogeneic marrow alone (i.e. “full” chimeras) appears mainly to involve T-cell anergy rather than clonal deletion [12]. In contrast, mixed chimeras show excellent clonal deletion of T cells reacting against host antigens [127,247], undoubtedly due to the intrathymic presence of host-type marrow-derived cells in mixed chimeras [127]. However, partial clonal deletion of host-reactive T cells has been described in lethally irradiated mice receiving allogeneic BMT alone [248,249], which may reflect the weaker ability of thymic epithelial cells (compared with hematopoietic cells) to induce intrathymic deletion [250–252]. Perturbations in the thymic environment, such as the destruction of thymic epithelial elements observed in GVHD [253,254], or thymic injury induced by treatment with cyclosporine [255,256], can impair processes leading to tolerance [257,258]. Defective production of Tregs may contribute to autoimmunity in this setting.

Summary and future directions It is clear that HCT has not yet met its full potential to provide a cure for lymphohematopoietic malignancies or to treat nonmalignant diseases. There is a need for improved methods of exploiting the immuno-

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Fig. 15.3 Superior donor leukocyte infusion (DLI)-mediated graft-versus-leukemia effects in mixed compared with full allogeneic chimeras. Mixed and full chimeras were prepared by lethally irradiating B6 mice and reconstituting them with either T-cell-depleted allogeneic (B10.A) bone marrow cells alone (to produce full chimeras), or a mixture of T-cell-depleted B6 plus B10.A marrow (to produce mixed [Mix] chimeras). DLIs were administered on day 56 postbone marrow transplantation, and this step was followed by intravenous injection of 500 EL4 (a B6 T-cell lymphoma) cells, on day 63 (↓). (a) Full chimeras receiving DLI and EL4 cells (䊏; n = 20) showed a slight but statistically significant prolongation of survival compared with full chimeras receiving tumor cells only (䊐; n = 12; p < 0.001). Full chimeras receiving (䉲; n = 14) or not receiving DLI (䉮; n = 7) showed no treatment-related mortality. (b) Mixed chimeras receiving (◊; n = 11) or not receiving (䉫; n = 8) DLI showed no treatment-related mortality. Mixed chimeras receiving DLI and EL4 cells (䊉; n = 25) had a significantly improved survival compared with mixed chimeras receiving EL4 alone (䊊; n = 14; p < 0.0001). (c) Mixed chimeras receiving DLI and EL4 cells (䊉; n = 25) had a significantly improved survival compared with full chimeras receiving EL4 +DLI (䊏; n = 20; p < 0.0001). (Reproduced from Mapara MY, Kim YM, Wang SP, Bronson R, Sachs DH, Sykes M. Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: a critical role for host antigen-presenting cells. Blood 2002; 100: 1903–9, with permission.)

therapeutic aspects of HCT without causing GVHD. In view of the new strategies and improved understanding of these phenomena that are developing, it seems likely that this challenge can be met and that HCT will ultimately be made available to all who could benefit from it, including those lacking an HLA-matched or closely matched donor. In addition, advances in the ability to achieve engraftment of hematopoietic and thymic tissue without ablative treatment of the host, and the demonstration of efficacy in primate models, have brought these approaches to initial clinical application for the induction of solid organ graft tolerance. Improvements in the ability to overcome HVG resistance to HLAmismatched HSC engraftment without increasing host toxicity should

further broaden the applicability of this approach. It will also be essential to extend this ability to xenogeneic marrow and organ transplantation, which presents additional immunologic and physiologic hurdles, to overcome the critical organ shortage that currently limits the number of life-saving organ transplants that can be performed. A major goal of transplant immunologists therefore now will be to extend new laboratory findings, first to additional preclinical models in large animals, and then to clinical protocols. In addition, it will be important to continue basic research into the mechanisms by which tolerance is induced and maintained in rodent model systems, so that these mechanisms can be further exploited.

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16

James L.M. Ferrara & Joseph H. Antin

The Pathophysiology of Graft-Versus-Host Disease

Acute GVHD pathophysiology: a three-step model Acute graft-versus-host disease (GVHD) results from complex interactions between donor T cells and host tissues in an inflammatory milieu. The pathophysiology of acute GVHD can be considered as a three-step process involving both the innate and adaptive immune systems (Fig. 16.1). The three steps are: (1) tissue damage to the recipient by the radiation/chemotherapy pretransplant conditioning regimen; (2) donor T-cell activation and clonal expansion; and (3) cellular and inflammatory factors. This schema underscores the importance of mononuclear phagocytes and other accessory cells to the development of GVHD after complex interactions with cytokines secreted by activated donor T cells. In step 1, the conditioning regimen (irradiation and/or chemotherapy) leads to damage and activation of host tissues throughout the body and the secretion of the inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1). These cytokines enhance donor T-cell recognition of host alloantigens by increasing the expression of major histocompatibility complex (MHC) antigens and other molecules on host antigen-presenting cells (APCs). An important aspect of the mucosal injury in the gastrointestinal tract is the resultant leakage of endotoxins into the systemic circulation. In step 2, host APCs present alloantigen in the form of a peptide– human leukocyte antigen (HLA) complex to the resting T cells. Costimulatory signals are required for T-cell activation, and these signals further activate APCs and further enhance T-cell stimulation. Donor T-cell activation is characterized by cellular proliferation and the secretion of cytokines, including IL-2 and interferon-gamma (IFN-γ). IL-2 expands the T-cell clones and induces cytotoxic T lymphocyte (CTL) responses, whereas IFN-γ primes mononuclear phagocytes to produce TNF-α and IL-1. Regulatory T cells (Tregs) that can act as a powerful brake on the GVH reaction also expand at this time. In step 3, effector functions of mononuclear phagocytes and neutrophils are triggered through a secondary signal provided by mediators, such as lipopolysaccharide (LPS), that leak through the intestinal mucosa damaged during step 1. Release of inflammatory cytokines stimulates host tissues to produce inflammatory chemokines that direct activated T cells and other effector cells such as mononuclear cells and granulocytes into target organs where they adhere due to increased adhesion molecule expression. This mechanism results in the amplification of local tissue injury, other cellular effectors and further promotion of a

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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proinflammatory response which, together with CTLs, leads to target tissue destruction. It should be noted from the outset that all these steps do not carry equal weight in the pathogenesis of acute GVHD. The pivotal interaction occurs in step 2, where host APCs activate allogeneic donor T cells. The subsequent cytokine cascade is clearly important, but blockade of individual cytokines may not reverse established GVHD when other cellular effectors such as CTLs are present. GVHD can also occur when no conditioning of the host has occurred (e.g. transfusion-associated GVHD).

Step 1: Effects of conditioning in hematopoietic cell transplantation The first step of acute GVHD starts before donor cells are infused. Prior to hematopoietic cell transplantation (HCT), a patient’s tissues have been damaged, sometimes profoundly, by underlying disease and its treatment, infection, and transplant conditioning. High-intensity chemoradiotherapy characteristic of HCT conditioning regimens activates host APCs that are critical to the stimulation of donor T cells infused in the stem cell inoculum. These important effects help explain a number of unique and seemingly unrelated aspects of GVHD. For example, a number of clinical reports have noted increased risks of GVHD associated with advanced stage leukemia, certain intensive conditioning regimens, and histories of viral infections [1]. Total body irradiation is particularly important in this process because it activates host tissues to secrete inflammatory cytokines, such as TNF-α and IL-1 [2], and it induces endothelial apoptosis that leads to epithelial cell damage in the gastrointestinal tract [3]. Injury to the gut is transient and self-limited after autologous HCT. However, after allogeneic HCT, further damage by GVHD effectors amplifies gastrointestinal tract and systemic GVHD by allowing the translocation of microbial products such as LPS into the systemic circulation. This scenario helps to explain the increased risk of GVHD associated with intensive conditioning regimens. The relationship between conditioning intensity, inflammatory cytokines, and GVHD severity has been confirmed by animal models [4] and clinical observations [1].

Step 2: Donor T-cell activation and cytokine secretion Donor T-cell activation Donor T-cell activation occurs during the second step of acute GVHD. Murine studies have demonstrated that host APCs alone are both

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Conditioning: Tissue Damage

(I) Host APC activation

Host tissues

Small intestine

TNF-a IL-I LPS

LPS

Mf Host APC

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TNF-g Donor T-cell

(II) Donor T-cell activation

Target cell apoptosis Thr

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CD8 CTL

(III) Cellular and inflammatory effectors

Fig. 16.1 The pathophysiology of acute graft-versus-host disease (GVHD). GVHD pathophysiology can be summarized in a three-step process. In step 1, the conditioning regimen (irradiation, chemotherapy or both) leads to the damage and activation of host tissues, especially the intestinal mucosa. This allows the translocation of lipopolysaccharide (LPS) and other inflammatory stimuli from the intestinal lumen to the circulation, stimulating the secretion of the inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) from host tissues. These mediators increase the expression of major histocompatibility complex (MHC) antigens and adhesion molecules on host antigen presenting cells (APCs), enhancing the recognition of MHC and minor histocompatibility antigens by mature donor T cells. In step 2, donor T-cell activation is characterized by a predominance of T helper type 1 (Th1) cells and the secretion of interferon-gamma (IFN-γ), which activates mononuclear phagocytes. Regulatory T cells (Tregs) limit the proliferation and clonal expansion of activated donor T cells. In step 3, effector functions of activated mononuclear phagocytes are triggered by the secondary signal provided by LPS and other stimulatory molecules that leak through the intestinal mucosa damaged during steps 1 and 2. Activated macrophages, along with cytotoxic T lymphocytes (CTL), secrete inflammatory cytokines that cause target cell apoptosis. CD8+ CTLs also lyse target cells directly. Damage to the gastrointestinal tract in this phase, principally by inflammatory cytokines, amplifies LPS release and leads to the “cytokine storm” characteristic of severe acute GVHD. This damage results in the amplification of local tissue injury and further promotes an inflammatory response.

necessary and sufficient to stimulate donor T cells [5,6]. Donor T cells migrate to secondary lymphoid organs, such as Peyer’s patches, lymph nodes, and the spleen, where they first encounter host APCs [7]. In murine models of GVHD to MHC differences between donor and host, robust donor T-cell proliferation is observed in the spleen as early as day 3 after HCT, preceding the engraftment of donor stem cells [5,7]. After allogeneic HCT, both host- and donor-derived APCs are present in secondary lymphoid organs. T-cell receptors (TCRs) of donor T cells can recognize alloantigens on either host APCs (direct presentation) or donor APCs (indirect presentation). During direct presentation, donor T cells recognize either the peptide bound to allogeneic MHC molecules or the foreign MHC molecules themselves [8]. During indirect presentation, T cells respond to the peptides generated by degradation of the allogeneic MHC molecules that are presented on self-MHCs [9]. In GVHD to minor histocompatibility antigens (mHAs), direct presentation is dominant because APCs derived from the host rather than from the donor are critical [6]. Several laboratories have identified that naïve (CD62L+) T cells cause experimental GVHD whereas memory (CD62L−) T cells do not, although memory stem cells may be involved [10,11]. In the majority of HLAidentical HCT, GVHD is induced by mHAs, which are peptides derived from polymorphic cellular proteins that are presented on the cell surface

by MHC molecules [12]. Because the genes for these proteins are located outside the MHC, two siblings often have many different peptides in the MHC groove. In this case, different peptides presented by the same MHCs are recognized by donor T cells and lead to GVHD. In mice, the actual number of so-called “major minor antigens” that can potentially induce GVHD is likely to be limited. In humans, of five previously characterized mHAs (HA-1, -2, -3, -4, and -5) recognized by T cells in association with HLA-A1 and -A2, mismatching of HA-1 alone was significantly correlated with acute grade II–IV GVHD. Theoretical models also predict substantial benefits if multiple minor loci could be typed [13]. Several mHAs are encoded on the male-specific Y chromosome and are associated with an increased risk of GVHD when male recipient cells are transplanted from female donors [14]. mHAs with tissue expression limited to the hematopoietic system are potential target antigens of graft-versus-leukemia (GVL) reactivity [15], and separation of GVHD and GVL by using CTLs specific for such antigens is an area of intense research [16]. Adhesion molecules mediate the initial binding of T cells to APCs. TCR signaling after antigen recognition induces a conformational change in adhesion molecules, resulting in higher-affinity binding to the APCs [17]. Full T-cell activation also requires co-stimulatory signals provided by APCs in addition to TCR signals. Two primary co-

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stimulatory pathways signal through either CD28 or TNF receptors. Currently, there are four known CD28 superfamily members expressed on T cells: CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), inducible co-stimulator, and programmed death-1. In addition, there are four TNF receptor family members: CD40 ligand (CD154), 4–1BB (CD137), OX40, and herpes simplex virus glycoprotein D or herpesvirus entry mediator. The best-characterized co-stimulatory molecules, CD80 and CD86, deliver positive signals through CD28 that lower the threshold for T-cell activation and promote T-cell differentiation and survival, while signaling through CTLA-4 is inhibitory [18]. The most potent APCs are dendritic cells (DCs); however, the relative contribution of DCs and other semiprofessional APCs, such as monocytes/macrophages and B cells, to the development of GVHD remains to be elucidated. Donor APCs may also amplify GVHD, but in the skin, Langerhans cells of host origin appear to be essential for the activation of donor T cells [18,19]. Signaling through Toll-like receptors (TLRs) and through other innate immune receptors such as NOD may act as “danger signals” and activate host APCs, amplifying the donor T-cell response [20,22]. DCs can be matured and activated during HCT by: (1) inflammatory cytokines; (2) microbial products such as LPS and the dinucleotide CpG entering the systemic circulation from intestinal mucosa damaged by conditioning; and (3) necrotic cells that are damaged by recipient conditioning. All of these stimuli may be considered “danger signals” [20] and may make the difference between an immune response and tolerance [23]. When T cells are exposed to antigens in the presence of adjuvant such as LPS, the migration and survival of T cells are dramatically enhanced in vivo [24]. The effect of age in enhancing the allostimulatory activity of host APCs may also help explain the increased incidence of acute GVHD in older recipients [25]. The elimination of host APCs by activated natural killer (NK) cells can prevent GVHD [26]. This suppressive effect of NK cells on GVHD may have relevance in humans: HLA class I differences driving donor NK-mediated alloreactions in the GVH direction mediate potent GVL effects and produce higher engraftment rates without causing severe acute GVHD [26,27].

Cytokine secretion by donor T cells T-cell activation involves multiple, rapidly occurring intracellular biochemical changes, including the rise of cytoplasmic free calcium and activation of protein kinase C and tyrosine kinases [28]. These pathways in turn activate the transcription of genes for cytokines such as IL-2, IFN-γ, and their receptors. Cytokines secreted by activated T cells are often classified as T-helper type 1 subset (Th1) (secreting IL-2 and IFNγ) or T-helper type 2 subset (Th2) (secreting IL-4, IL-5, IL-10, and IL-13) [29]. The role of Th17 cells, a recently described functional Tcell subset, has not yet been clarified in GVHD [30]. Several factors influence the ability of DCs to instruct naïve CD4+ T cells to secrete Th1 or Th2 cytokines. These factors include the type and duration of DC activation along with the DC : T-cell ratio and the proportions of DC subsets present during T-cell interactions [31]. Differential activation of Th1 or Th2 cells has been evoked in the immunopathogenesis of GVHD and the development of infectious and autoimmune diseases. Although this dichotomy has many exceptions, in both settings activated Th1 cells: 1 amplify T-cell proliferation by secreting IL-2; 2 lyse target cells by Fas–Fas ligand (FasL) interactions; 3 induce macrophage differentiation in the bone marrow by secreting IL-3 and granulocyte macrophage colony-stimulating factor; 4 activate macrophages by secreting IFN-γ and by their CD40–CD40 ligand interactions; 5 activate endothelium to induce macrophage binding and extravasation;

6 recruit macrophages by secreting monocyte chemoattractant protein-1 [32]. During step 2 of acute GVHD pathophysiology, IL-2 has a pivotal role in both controlling and amplifying the immune response against alloantigens. IL-2 induces the expression of its own receptor (autocrine effect) and stimulates proliferation of other cells expressing the receptor (paracrine effect). IL-2 is secreted by donor CD4+ T cells in the first several days after GVHD induction [33]. In some studies, the addition of low doses of IL-2 during the first week after allogeneic bone marrow transplantation enhanced the severity and mortality of GVHD [34]. The precursor frequency of host-specific IL-2 producing cells predicts the occurrence of clinical acute GVHD [35,36]. Monoclonal antibodies (mAbs) against IL-2 or its receptor can prevent GVHD when administered shortly after the infusion of T cells [37,38], but this strategy was only moderately successful in reducing the incidence of severe GVHD [39]. Cyclosporine (CSP) and tacrolimus dramatically reduce IL-2 production and effectively prevent GVHD. IL-15 is another critical cytokine in initiating allogeneic T-cell division in vivo [40], and elevated serum levels of IL-15 are associated with acute GVHD in humans [41]. IL-15 may, therefore, also be important in the clonal expansion of donor T cells in step 2. IFN-γ is another crucial cytokine that can be implicated in the second step of the pathophysiology of acute GVHD. Increased levels of IFN-γ are associated with acute GVHD [42,43], and a large proportion of Tcell clones isolated from GVHD patients produce IFN-γ [44]. In animals with GVHD, IFN-γ levels peak between days 4 and 7 after transplantation before clinical manifestations are apparent. CTLs that are specific for mHAs and produce IFN-γ also correlate with the severity of GVH reaction in a skin-explant assay of human disease [45]. Experimental data demonstrate that IFN-γ modulates several aspects of the pathophysiology of acute GVHD. First, IFN-γ increases the expression of numerous molecules involved in GVHD, including adhesion molecules, chemokines, MHC antigens, and Fas, resulting in enhanced antigen presentation and the recruitment of effector cells into target organs [46]. Second, IFN-γ alters target cells in the gastrointestinal tract and skin so that they are more vulnerable to damage during GVHD; the administration of anti-IFN-γ mAbs prevents gastrointestinal tract GVHD [47], and high levels of both IFN-γ and TNF-α correlate with the most intense cellular damage in the skin [45]. Third, IFN-γ mediates GVHD-associated immunosuppression seen in several experimental HCT systems in part by the induction of nitric oxide (NO) [48–50]. Fourth, IFN-γ primes macrophages to produce proinflammatory cytokines and NO in response to LPS [51]. At early time points after HCT, IFN-γ may paradoxically reduce GVHD by enhancing Fas-mediated apoptosis of activated donor T cells [52,53]. Both cell-mediated and inflammatory cytokine GVHD effector mechanisms can sometimes be inhibited if donor T cells produce fewer Th1 cytokines [54]. Furthermore, cell mixtures of Th2 donor cells with an otherwise lethal inoculum of allogeneic bone marrow and T cells also protect recipient mice from LPS-induced lethality, demonstrating the ability of Th2 cells to modulate Th1 responses after allogeneic transplantation [55]. Polarization of donor T cells toward a Th2 phenotype by pretreating HCT donors with granulocyte colony-stimulating factor also results in less severe GVHD [56]. It should be noted, however, that systemic administration of Th2 cytokines IL-4 or IL-10 as experimental prophylaxis of GVHD was either ineffective or toxic [57,58]. On the other hand, administration of Th1 cytokines can also reduce GVHD. High doses of exogenous IL-2 early after bone marrow transplantation protect animals from GVHD mortality [59]. It has been suggested that IL-2 mediates its protective effect via inhibition of IFNγ [42]. However, injection of IFN-γ itself can prevent experimental GVHD [60], and neutralization of IFN-γ results in accelerated GVHD

The Pathophysiology of Graft-Versus-Host Disease

in lethally irradiated recipients [61]. These paradoxes may be explained by the complex dynamics of donor T-cell activation, expansion, and contraction. Activation-induced cell death is a chief mechanism of clonal deletion and is largely responsible for the rapid contraction of activated T cells following an initial massive expansion [62]. Thus, the complete absence of IFN-γ may result in an unrestrained expansion of activated donor T cells, leading to accelerated GVHD, which may be of particular importance in recipients of intensified conditioning [4]. Similarly, administration of IFN-γ-inducing cytokines, such as IL-12 or IL-18, protects mice from GVHD in a Fas-dependent fashion [53]. Thus, moderate amounts of Th1 cytokines production after donor T-cell expansion may amplify GVHD; extremes in production (either low or high), particularly during T-cell expansion, may hasten the death of activated donor T cells, aborting T-cell expansion and reducing GVHD. Regulatory T cells In both humans and mice, Treg deficiency results in immune dysregulation and autoimmunity, as characterized by the human IPEX syndrome resulting from loss of function mutations of FOXP3 [63]. A similar condition occurs in scurfy mice, a strain that lacks the transcription factor FOXP3 [64]. FOXP3 appears to function as a master control gene for the development and function of natural Tregs, which normally constitute approximately 5–10% of the circulating CD4+ T cell population. Tregs constitutively express markers associated with activated/ memory T cells: CD25 (IL-2 receptor), glucocorticoid-induced TNF-α receptor, CD152 (CLTA-4), CD103, CD62L, and neuropilin-1; and downregulate the expression of CD45RB and CD127 [65,66]. Tregs suppress both innate and the adaptive immune functions [67–69] by producing inhibitory cytokines (IL-10 and transforming growth factorbeta) as well as by cell contact-dependent inhibition of APC function and direct cytotoxicity against antigen-presenting B cells [68,70]. Additional purified CD4+CD25+ Treg populations can suppress the proliferation of conventional T cells and prevent GVHD [71,72]. Tregs do not themselves induce GVHD, and the small numbers of Treg cells present in a graft appear to be overwhelmed by the large number of conventional donor T cells present. The use of calcineurin inhibitors and thymic injury from GVHD may also interfere with the development of adequate numbers of Treg cells that can control GVHD [71,73–75]. NK1.1+ T cells may also possess regulatory function, and both peripheral blood and marrow NK1.1+ T cells can prevent GVHD by their IL-4 secretion [76].

Step 3: Cellular and inflammatory effectors The pathophysiology of acute GVHD culminates in the generation of multiple cytotoxic effectors that contribute to target tissue injury. At a minimum, step 3 includes: (1) several inflammatory cytokines; (2) specific antihost CTL activity using Fas and perforin pathways; (3) large granular lymphocytes or NK cells; and (4) NO. Significant experimental and clinical data suggest that soluble inflammatory mediators act in conjunction with direct cell-mediated cytolysis by CTL and NK cells to cause the full spectrum of deleterious effects seen during acute GVHD. As such, the effector phase of GVHD involves aspects of both the innate and adaptive immune response, and the synergistic interactions of components generated during steps 1 and 2.

Cellular effectors The Fas/FasL and perforin/granzyme (or granule exocytosis) pathways are the principal effector mechanisms used by CTLs and NK cells to lyse their target cells [77,78]. Following recognition of a target cell through TCR–MHC interaction, CTLs secrete perforin and insert it into

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the target cell membrane, forming “perforin pores” that allow granzymes to enter the cell and induce apoptosis through various downstream effector pathways [79]. Ligation of Fas results in the formation of the deathinducing signaling complex and the subsequent activation of caspases [80]. A number of ligands on T cells also possess the capability to trimerize TNF-α receptor (TNFR)-like death receptors (DR) on their targets, such as TNF-related apoptosis-inducing ligand (TRAIL : DR4,5 ligand) and TNF-like weak inducer of apoptosis (TWEAK : DR3 ligand) [81,82]. The involvement of these pathways in GVHD has been tested by utilizing donor cells that are genetically deficient in each molecule. Lethal GVHD occurs even in the absence of perforin-dependent killing, demonstrating that the perforin/granzyme pathway plays a significant, but not exclusive, role in target organ damage. CD4+ CTLs preferentially use the Fas/FasL pathway during acute GVHD, while CD8+ CTLs primarily use the perforin/granzyme pathway, consistent with other conditions involving cell-mediated cytolysis [18]. Fas is a TNF-receptor family member that is expressed by many tissues, including GVHD target organs. Inflammatory cytokines such as IFN-γ and TNF-α can increase the expression of Fas during GVHD [83]. FasL expression on donor T cells is also increased during GVHD [84,85]. Elevated serum levels of soluble FasL and Fas have also been observed in some patients with acute GVHD [86,87]. FasLdefective donor T cells cause markedly reduced experimental GVHD in liver, skin, and lymphoid organs [88,89]. The Fas/FasL pathway is particularly important in hepatic GVHD, consistent with the marked sensitivity of hepatocytes to Fas-mediated cytotoxicity in models of murine hepatitis [90]. Fas-deficient recipients are protected from hepatic GVHD, but not from GVHD in other target organs [91]. Administration of anti-FasL (but not anti-TNF) mAbs significantly blocked hepatic GVHD damage occurring in one model [92], whereas the use of FasL-deficient donor T cells or the administration of neutralizing FasL mAbs had no effect on the development of intestinal GVHD in several studies [93,94].

Inflammatory effectors In the effector phase of acute GVHD, inflammatory cytokines synergize with CTLs, resulting in the amplification of local tissue injury and the development of target organ dysfunction in the transplant recipient. The cytokines TNF-α and IL-1 are produced by an abundance of cell types involved in innate and adoptive immune responses, and they have synergistic and redundant activities during several phases of acute GVHD. A central role for inflammatory cytokines in acute GVHD was confirmed by a recent murine study using bone marrow chimeras in which either MHC class I or MHC class II alloantigens were not expressed on target epithelium but on APCs alone [5]. GVHD target organ injury was induced in these chimeras even in the absence of epithelial alloantigens, and mortality and target organ injury were prevented by the neutralization of TNF-α and IL-1. These observations were particularly true for CD4-mediated acute GVHD but also applied, at least in part, for CD8mediated disease. A critical role for TNF-α in the pathophysiology of acute GVHD was first suggested almost 15 years ago because mice transplanted with mixtures of allogeneic bone marrow and T cells developed severe skin, gut, and lung lesions that were associated with high levels of TNF-α messenger RNA in these tissues [95]. Target organ damage could be inhibited by infusion of anti-TNF-α mAbs, and mortality could be reduced from 100% to 50% by the administration of the soluble form of the TNF-α receptor [2]. Extensive experimental data further suggest that TNF-α is involved in the multistep process of GVHD pathophysiology. TNF-α can:

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1 cause cachexia, a characteristic feature of GVHD; 2 induce activation of DCs, thus enhancing alloantigen presentation; 3 recruit effector T cells, neutrophils, and monocytes into target organs through the induction of inflammatory chemokines; 4 cause direct tissue damage by inducing apoptosis and necrosis [96]. TNF-α is also involved in donor T-cell activation directly through its signaling via TNFR1 and TNFR2 on T cells; TNF–TNFR1 interactions promote alloreactive T-cell responses [97], whereas TNF–TNFR2 interactions are critical for intestinal GVHD [98]. TNF-α plays a central role in intestinal GVHD in murine and human studies [92,95]. TNF-α also seems to be an important effector molecule in GVHD in skin and lymphoid tissue [94,95,99] and can contribute to hepatic GVHD, probably by enhancing effector cell migration to the liver via the induction of inflammatory chemokines. Studies in animals have demonstrated that neutralization of TNF-α alone or in combination with IL-1 resulted in a significant reduction of hepatic GVHD [5,100]. An important role for TNF-α in clinical acute GVHD has also been suggested by multiple studies. Elevations of TNF-α protein (serum) and messenger RNA (peripheral blood mononuclear cells) have been measured in patients with acute GVHD and other endothelial complications, such as hepatic veno-occlusive disease, now termed sinusoidal obstructive syndrome [101–103]. IL-1 is another proinflammatory cytokine that contributes to acute GVHD toxicity. Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD in the spleen and skin, two major GVHD target organs [104]. Experimental models implicated IL-2 in GVHD pathophysiology, and direct inhibition of IL-1 activity with IL-1 receptor antagonist looked promising in mice and in phase II clinical trials. However, an attempt to use IL-1 receptor antagonist to prevent acute GVHD in a randomized trial was not successful [105]. It is likely that redundancy of inflammatory pathways bypasses the direct inhibition of IL-1, reducing the clinical benefit of this approach. Finally, macrophages can also produce significant amounts of NO as a result of activation during GVHD. Several studies have shown that NO contributes to the deleterious effects of GVHD on target tissues and specifically to GVHD-induced immunosuppression [50,106]. NO also inhibits repair mechanisms in target tissue by inhibiting the proliferation of epithelial stem cells in the gut and skin [107]. In humans and rats, development of GVHD is preceded by an increase of NO oxidation products in the serum [108].

Toll-like receptors and innate immunity As alluded to above, components of the HCT conditioning regimen (in particular total body irradiation) and the secretion of type 1 cytokines (specifically IFN-γ) prime mononuclear phagocytes to produce inflammatory cytokines. However, the actual secretion of soluble cytokines occurs after primed macrophages are triggered or stimulated by a second signal. This stimulus may be provided through TLRs by LPS and other microbial products that have leaked through an intestinal mucosa damaged initially by HCT conditioning regimens during step 1. Since the gastrointestinal tract is known to be particularly sensitive to the injurious effects of cytokines [95,109], damage to the gastrointestinal tract during the effector phase can lead to a positive feedback loop wherein increased translocation of LPS results in further cytokine production and progressive intestinal injury. Thus, the gastrointestinal tract may be critical to propagating the “cytokine storm” characteristic of acute GVHD; increasing experimental and clinical evidence suggests that damage to the gastrointestinal tract during acute GVHD plays a major role in the amplification of systemic disease [97]. This conceptual framework underscores the role of LPS in the development of acute GVHD as suggested by several groups [51,109,110].

LPS is a major structural component of Gram-negative bacteria and is a potent stimulator of cellular activation and cytokine release [111]. LPS shed from bacteria that comprise normal bowel flora can elicit a broad range of inflammatory responses from macrophages, monocytes, and neutrophils. In particular, LPS may stimulate gut-associated lymphocytes and macrophages [51]. Following allogeneic HCT, LPS accumulates in both the liver and spleen of animals with GVHD prior to its appearance in the systemic circulation [110]. Elevated serum levels of LPS have been shown to correlate directly with the degree of intestinal histopathology occurring after allogeneic HCT [109,112]. A genetic mutation in the TLR-4 (Tlr4) gene makes mice resistant to LPS, and transplantation with LPS-resistant donor cells results in a significant reduction of TNF-α levels, gastrointestinal tract histopathology, and systemic GVHD compared with animals receiving LPSsensitive HCT [109]. In addition, transplantation from deficient (CD14−/−) donors that are insensitive to LPS stimulation in vitro resulted in a major reduction in gastrointestinal tract GVHD and improved longterm survival compared with normal controls [113]. Early animal studies showed that death from GVHD could be prevented if transplanted mice were given antibiotics to decontaminate the gut [114]. After clinical HCT, endotoxemia is associated with intestinal injury, hepatic toxicity, acute GVHD, and fever in the absence of bacteremia [112,115]. Accordingly, Gram-negative gut decontamination during HCT has also been shown to reduce GVHD [116], and the intensity of this decontamination can predict severity of GVHD [117–119]. Naturally occurring antibody titres to a rough-mutant strain of Escherichia coli J5, which protect humans and animals from septic shock, are also associated with a decreased incidence of acute GVHD after allogeneic SCT [120]. Molecules that act as competitive inhibitors to LPS at the cell surface and block nuclear factor-kappa B activation and nuclear translocation block the biologic response to LPS and result in decreased inflammatory cytokine release and reduced GVHD severity while preserving potent GVL effects and improved leukemia-free survival [113]. Traffic of cellular effectors Regulation of effector cell migration into target tissues during the development of GVHD occurs in a complex milieu of chemotactic signals where several chemokine receptors may be triggered simultaneously or successively. Inflammatory chemokines expressed in inflamed tissues are specialized to recruit effector cells, such as T cells, neutrophils, and monocytes [121]. Chemokine receptors are differentially expressed on subsets of activated/effector T cells. Upon stimulation, T cells can rapidly switch chemokine receptor expression, acquiring new migratory capacity [24,122]. The involvement of inflammatory chemokines and their receptors in GVHD has been recently investigated in mouse models of GVHD. Macrophage inhibitory protein-1 alpha recruits CCR5+CD8+ T cells into the liver, lung, and spleen during GVHD [123,124], and levels of several chemokines are elevated in GVHD-associated lung injury [125]. DCs in the lymph node activate lymphocytes and induce a profile of adhesion molecules and chemokine receptors that direct the activated cells to migrate back to the organ of DC origin. For example, activated T cells that leave a lymph node in the skin express cutaneous lymphocyte antigen [126]. T cells expressing cutaneous lymphocyte antigen only home back to the skin where they may mediate cutaneous GVHD, but they do not traffic to other organs. Similarly, T cells exiting mesenteric lymph nodes express α4β7 (LPAM-1), which is necessary for homing to gastrointestinal tract and gut-associated lymphoid tissues [127], but they do not express cutaneous lymphocyte antigen. Lymphocytes homing to the small intestine bear chemokine receptors for the chemokine thymus-expressed chemokine (CCL25) that is produced in the small intestine, but not in the skin. Thus, cells bearing the appropriate cell

The Pathophysiology of Graft-Versus-Host Disease

surface adhesion receptors for the gastrointestinal tract (e.g. LPAM-1) and chemokine receptors (e.g. CCR25) undergo firm adhesion followed by transmigration from the vessel into the appropriate organ.

GVHD prevention: from animal models to clinical practice The development of strategies for GVHD prophylaxis and therapy has suffered from a limited understanding of the underlying pathophysiology of GVHD. This conceptual limitation led to approaches that were based on an “oncologic” model of GVHD, i.e. the notion that since GVHD is dangerous and life-threatening, management requires the utilization of high-dose immunosuppressant regimens. High doses of multiple immunosuppressive agents have been used much like we use combination chemotherapy to treat malignancies. While there is often reasonable control of the clinical manifestations of GVHD, this “shotgun” approach lacks elegance and is usually associated with opportunistic infections, lymphoproliferative disorders or relapse of the underlying malignancy. Indeed, it is often infection while GVHD is in remission that results in mortality. The objective should be to reduce the nonspecific tissue damage associated with GVHD, while allowing effective immunologic recovery. The three-phase model described above can be simply summarized. GVHD is an exaggerated and dysregulated response of a normal immune system (that of the donor) to tissue damage that is intrinsic to transplantation. The donor’s immune system reacts as if there is a massive and uncontrolled infection, and its efforts to deal with this injury result in the clinical manifestations of GVHD. It is quite possible that the tissue injury intrinsic to the administration of high-dose chemoradiotherapy initiates the breakdown of mucosal barriers, allowing endotoxin into the tissues. TLRs on DCs bind to endotoxin and activate signal transduction pathways that lead to DC maturation and induce inflammation [128]. The upregulation of co-stimulatory molecules, MHC molecules, adhesion molecules, cytokines, chemokines, prostanoids, and other inflammatory mediators prime and trigger the attack on target tissues, fueling the fire. Thus, it is likely that components of cytokine storm [129], the danger hypothesis [20], and more traditional notions of adaptive and innate immunity [130] all apply. Moreover, with our improved understanding of inhibitory pathways that regulate and suppress inflammation, it may be possible to enhance regulatory mechanisms rather than attempt to engender immunologic paralysis.

Step 1: Reduced-intensity conditioning regimens The three cardinal organs affected by GVHD are the skin, intestinal tract, and liver. The common thread would seem to be exposure to the environment. Skin and gut have very obvious barrier functions and a well-developed reticuloendothelial system. DCs resident in these organs present antigens to naïve T cells, which then have alterations in cell surface characteristics that cause them to return to the organ that originated the specific DC. Similarly, the liver is the first line of defense downstream of the gut. One would expect the lung to be a similar target; however, its less intense exposure to organisms, particularly Gramnegative rods, probably reduces its primacy as a target organ. Nevertheless, recent studies clearly demonstrate pulmonary injury that is likely to reflect GVHD. All of these organs are rich in DCs, a probable prerequisite for generating the injurious cytokines as well as directing the attack of the adaptive immune system. They are all subject to injury from conditioning, and breaches of the barrier will allow organisms or endotoxin into the circulation. One prediction of this model is that less-intense conditioning regimens would be associated with less GVHD. This effect was initially

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observed after donor lymphocyte infusions. The T cells were administered without pharmacologic GVHD prophylaxis, and the resultant GVHD was less frequent and severe than would be expected in the absence of immunosuppressive drugs. It could be inferred that reducedintensity conditioning regimens might similarly be associated with less severe GVHD, both through less cellular injury per se and through a reduction in endotoxin exposure by virtue of less mucosal injury. Available data suggest that the rate of GVHD after reduced-intensity conditioning regimens is similar to the rate seen after conventional dose conditioning [131,132]. Interestingly, the onset of GVHD is delayed and an overlap syndrome of acute and chronic GVHD is more prevalent. This observation suggests that the initial tissue injury of high-dose regimens speeds the development of GVHD and reduced-intensity procedures seem to have little effect on chronic GVHD. Thus, while data on both reducing colonization with bacteria and reducing tissue injury with less-intense regimens are suggestive and support the basic concepts described above, the effect is insufficient to control GVHD adequately in human transplantation.

Step 2: Modulation of donor T cells Reduced T-cell numbers The advent of monoclonal technology in the 1980s led to the logical conclusion that if a pharmacologic reduction in T-cell numbers could partially control GVHD, albeit at the risk of drug-induced toxicity, then eliminating the cells with mAbs, immunotoxins, lectins, CD34 columns or physical techniques, might be more effective (reviewed by Ho and Soiffer in [133]). Furthermore, the ability to identify and target specific T-cell subsets opened the possibility that the graft could be engineered to control GVHD while allowing immunologic recovery as well as GVL. A typical unmanipulated marrow transplant entails the infusion of about 107 T cells/kg of recipient weight. Establishment of a clear dose–response relationship is difficult since the risk of acute GVHD depends on factors beyond simple dosing, for example minor histocompatibility disparities, virus exposure, disease and stage, donor age, recipient age, etc. Studies of counterflow elutriation showed that 106 T cells/kg plus CSP resulted in a GVHD rate similar to that of CSP alone, but 5 × 105 T cells/kg plus CSP resulted in an overall rate of 22%, and only limited skin involvement was observed [134]. After lectin depletion of T cells in the absence of supplemental immunosuppression, a T-cell dose of around 105/kg was associated with complete control of GVHD [135]. More recently, the combination of very high stem cell numbers and CD3 T-cell numbers of less than 3 × 104/kg allowed haploidentical transplantation without GVHD [136]. Global T-cell depletion (TCD) using a broad panel of mAbs has been largely abandoned because of the risk of graft failure [137]. However, it is clear that even depletion of T-cell subsets must be undertaken with caution. Depletion of CD4+ T cells or CD8+ T cells alone can be associated with a high graft failure rate [138]. However, depletion of CD5+ or CD6+ T cells seems to be associated with a low graft failure rate [139,140]. The presumption is that radioresistant T cells that survive the initial conditioning are responsible for graft rejection. When the stem cell source is rich in T cells, the GVH reaction will further reduce the residual population capable of alloreactivity, thus decreasing graft rejection. To some degree, the higher graft failure rates may be controlled by increasing the intensity of the conditioning regimen [141], adding back T cells [134], or with additional immunosuppressants [142]. Additional problems associated with T-cell depletion (TCD) include a higher incidence of Epstein–Barr virus-induced lymphoproliferative disorders, loss of the GVL effect with a consequent increase in relapses and slower immunologic recovery due to reduced passive transfer of

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immunocompetent donor T cells. These concerns also have been approached by increasing the conditioning intensity, delayed T-cell add-back [143], rituximab [144] or the infusion of specific T-cell lines [145]. However, in no case has there been an improvement in survival that can be definitively attributed to TCD. A large multicenter study of unrelated donor HCT similarly showed no improvement in outcomes after TCD [146]. Another interesting and novel approach to restore T-cell function without GVHD is to start by removing T cells from the initial graft using a technique such as CD34 selection. This step is followed by reacting donor and recipient lymphocytes in a mixed lymphocyte reaction in the presence of an anti-IL-2 receptor immunotoxin. This agent removes only activated T cells since it binds to CD25. The remaining resting cells are then infused to enhance immunologic recovery [147]. Alternatively, studies in mice have accomplished a similar effect, depleting donor cells activated in a mixed lymphocyte culture with antibodies to the activation antigen CD69 and immunomagnetic bead sorting [148]. Finally, both antithymocyte globulin and anti-CD52 therapy (alemtuzumab) deplete T cells in vivo. Antithymocyte globulin appears in some studies to reduce both acute and chronic GVHD [149]. Alemtuzumab also reduces acute and chronic GVHD but at the expense of high relapse rates and graft failure [150].

Reduced T-cell activation The introduction of CSP in the late 1970s was a significant advance in GVHD prevention. More recently a similar agent, tacrolimus, has been shown to provide similar control of GVHD. Both drugs inhibit T-lymphocyte activation, although the exact mechanism of action is not known. Both drugs bind to immunophilins: CSP binds cyclophilin, and tacrolimus binds FKBP12. The complex of drug and binding protein, calcium, calmodulin, and calcineurin is then formed, and the phosphatase activity of calcineurin is inhibited. This effect may prevent the dephosphorylation and translocation of nuclear factor of activated T cells, a nuclear component thought to initiate gene transcription for the formation of lymphokines (such as IL-2 and IFN-γ). The net result is the inhibition of T-lymphocyte activation. As a single agent, CSP was about as effective as methotrexate (MTX) [151]. However, in combination with MTX, there was a significant reduction in the incidence of GVHD and an improvement in survival [152]. Subsequent trials of tacrolimus and MTX compared with CSP and MTX showed no advantage for either combination [153,154]. The addition of prednisone to the conventional two-drug regimen resulted in similar rates of GVHD and no improvement in survival [155]. DCs appear to be critical to the development of acute GVHD [6]; therefore, interference with DC function would probably prevent GVHD. The goal would be to inhibit function selectively or temporarily until transplanted DC precursors had engrafted and become functional. Campath is an anti-CD52 mAb that has been extensively used for the prevention of GVHD, primarily in Europe. It is a promiscuous antibody, targeting B cells, T cells, monocytes, and other cells. Interestingly, it also appears to target DCs, and when the drug was administered prior to transplantation, host DCs were selectively depleted and the donor DCs repopulated the recipient [156], although the sensitivity of Langerhans cells to depletion is controversial [157]. These observations are difficult to separate from concomitant TCD, but they may be consistent with the theme that facilitation of DC turnover may ameliorate GVHD.

Reduced T-cell proliferation The first generally prescribed GVHD preventive regimen was the administration of intermittent low-dose MTX as developed in a dog

model by Thomas and colleagues [158]. Another regimen developed by Santos and based on cyclophosphamide [159] was never widely adopted. The principle of this approach is to administer a cell-cycle specific chemotherapeutic agent intermittently immediately after the transplant, when the T cells will have started to divide after exposure to allogeneic antigens. This was an effective approach compared with no prophylaxis [160]; however, GVHD control was incomplete. Subsequently, the addition of antithymocyte globulin or prednisone or both resulted in incremental improvement in the GVHD rate but no improvement in survival [161]. Long courses of MTX proved to be associated with a higher risk of interstitial pneumonitis and mucositis, and were abandoned with the advent of CSP. One concern regarding the use of MTX is that the enhanced mucositis might be associated with increased transfer of endotoxin across the mucosa. Ultimately, the course of MTX was abbreviated and combined with a T-cell activation inhibitor, such as CSP or tacrolimus. The use of cyclophosphamide immediately after HCT may also prevent both acute and chronic GVHD [162]. Mycophenolate mofetil (MMF) is another useful agent that may prevent GVHD with less mucositis and myelotoxicity than MTX. It is the prodrug of mycophenolic acid, a selective inhibitor of inosine monophosphate dehydrogenase, and inhibits the de novo pathway of guanosine nucleotide synthesis. Since T lymphocytes are dependent on the de novo synthesis of purines, mycophenolic acid inhibits the proliferative responses of T cells to both mitogenic and allogeneic stimulation. Myeloid and mucosal cells can utilize salvage pathways, so the drug is less toxic to mucosa and myeloid recovery. Mycophenolic acid also prevents the glycosylation of glycoproteins that are involved in adhesion to endothelium and may reduce recruitment of leukocytes into inflammatory sites. MMF does not inhibit the activation of T cells as such, but blocks the coupling of activation to DNA synthesis and proliferation [163]. Many trials of the combination of MMF with CSP or tacrolimus have shown reliable but incomplete control of GVHD when used in conjunction with a calcineurin inhibitor. Sirolimus is a macrocyclic lactone immunosuppressant that is similar in structure to tacrolimus and CSP. All three drugs bind to immunophilins; however, sirolimus complexed with FKBP12 inhibits T-cell proliferation by interfering with signal transduction and cell-cycle progression. Sirolimus (rapamycin) affects lymphocyte activation at a later stage than either CSP or tacrolimus, and activation stimuli that resist inhibition to the latter agents have been shown to be sensitive to sirolimus. Sirolimus has been shown to inhibit antibody-dependent cellular cytotoxicity and the cytolytic effects of NK cells and IL-2 lymphokine-activated killer cells. The sirolimus–FKBP12 complex binds to mammalian target of rapamycin (mTOR), which blocks IL-2-mediated signal transduction pathways that prevent G1/S phase transition. This effect is mediated through a complex pathway involving the inhibition of ribosomal protein synthesis at several levels, as well as effects on transcription and translation [164]. The drug has similar effects on proliferation of T cells induced by IL-2, IL-4, IL-7, IL-12, and IL-15. Interestingly, it may also interfere with co-stimulation [165] by inhibiting CD28-mediated blocking of inhibitor of nuclear factor kappa B and translocation of c-Rel to the nucleus [166]. Sirolimus contributes to massive T-cell apoptosis if signals 1 and 2 of T-cell activation are blocked, an effect that is not seen with CSP [167]. This effect may be related to its ability to inhibit the expression of bcl-2 and BAG-1 [168,169]. Sirolimus completely inhibits the kinase activity of the cdk4–cyclin B and cdk2–cyclin E complexes despite normal expression of these proteins and, as a consequence, blocks the hyperphosphorylation of the retinoblastoma gene product. Interestingly, sirolimus has inhibitory effects on integrin-mediated signal transduction [170], and dermal microvascular injury in a Hu-SCID transplantation model [171].

The Pathophysiology of Graft-Versus-Host Disease

Sirolimus has excellent antirejection activity in organ transplantation [170], and combination therapy with sirolimus and tacrolimus appears to be extremely effective in human organ allografting and murine models of marrow grafting. Since it acts through a separate mechanism from the tacrolimus–FKBP complex (and CSP–cyclophilin complex), which inhibits calcineurin, and thus T-cell activation, sirolimus is synergistic with both tacrolimus and CSP. While both tacrolimus and sirolimus bind to FKBP12, there appear to be adequate binding sites for both molecules. Therefore, in contrast to expectations, the drugs are not competitive, and in fact they appear to be synergistic. Since the sirolimus–mTOR complex does not bind to calcineurin, sirolimus also is free of nephrotoxicity and neurotoxicity, making combination therapy appealing. Clinical trials indicate that sirolimus is a promising addition to the GVHD armamentarium in both related and unrelated donor HCT [172].

Step 3: Blockade of inflammatory stimulation and effectors Reduced exposure to organisms Another hypothesis that flows from this model is that reduction of intestinal colonization with bacteria could prevent GVHD. The first indication that elimination of exposure to microorganisms could prevent GVHD was in germ-free experiments in mice, where GVHD was not observed until the mice were colonized with Gram-negative organisms [114]. Later, gut decontamination and use of a laminar airflow environment was associated with less GVHD and better survival in patients with severe aplastic anemia [116]. Similarly, studies of intestinal decontamination in patients with malignancies have shown less GVHD in some [118] but not all [173] studies. However, despite the power of a large number of patients in registry analyses [173], it is possible that the heterogeneity of care in the large number of participating centers obscured an important effect. Viral infections, particularly herpesviruses, may also be associated with GVHD [174]. Blockade of inflammatory effectors As alluded to above, an important role for TNF-α in clinical acute GVHD was suggested by studies demonstrating elevated levels of TNFα in the serum of patients with acute GVHD and other endothelial complications such as sinusoidal obstructive syndrome [101,102,175]. Importantly, the appearance of the increased levels was predictive of the severity of complications and overall survival. Patients with higher serum TNF-α levels during the conditioning regimen (pre-HCT) had a 90% incidence of grade II or greater acute GVHD and an overall mortality of 70%, compared with a mortality of 20% in patients without TNFα elevations [102]. Murine mAbs or F(ab)′2 fragments directed at TNF-α produced partial responses either as therapy for steroid-resistant GVHD [176] or as prophylaxis [177]. A phase II trial of etanercept, a chimeric fusion protein consisting of the extracellular ligand-binding portion of the human TNFR linked to the Fc portion of human immunoglobulin G1, has recently shown significant efficacy as primary treatment for acute GVHD when added to systemic steroids (Fig. 16.2). Almost 70% of patients had complete resolution of their GVHD within 1 month, compared with 33% of patients treated with steroids alone [178]. Blockade of chemokines may prevent the recruitment, localization, and activation of lymphocytes during GVHD. Prevention of lymphocyte entry into secondary lymphoid organs, acquisition of appropriate homing and adhesion molecules, or modification of their entry into target tissues could all reduce GVHD [179–181]. Such molecules as FTY720 [182,183] and alefacept [184–186] as well as new chemokine inhibitors may allow

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selective targeting of organ-specific lymphocytes, although as yet little clinical experience has accumulated.

Chronic GVHD It is important to recognize that chronic GVHD was originally defined temporally rather than clinically or pathophysiologically. The initial clinical reports of chronic GVHD used descriptions of clinical problems occurring at least 150 days after HCT [187]. By convention, many transplant clinicians and scientists use day 100 after stem cell infusion as a convenient divider between acute and chronic GVHD. However, it should be recognized that acute manifestations of GVHD may occur after day 100, and problems typically associated with chronic GVHD may occur before day 100. Thus, it is preferable to consider the symptom and signs per se rather than the timing of the onset of clinical findings. The National Institutes of Health consensus conference on chronic GVHD recognizes four categories of GVHD [188] (see Chapter 87). The lack of animal models that reproduce the critical aspects of chronic GVHD has limited our ability to address the pathophysiology of this complication. Chronic GVHD is often considered an autoimmune disease because of distinctive similarities to various autoimmune disorders, especially collagen vascular disease. The relationship is difficult to prove clinically, but experimental studies have demonstrated an autoimmune aspect of chronic GVHD pathophysiology [189]. Indeed, autoantibody production is commonly observed after transplantation, and some of the clinical manifestations of chronic GVHD are similar to those of scleroderma, lichen planus, and other autoimmune diseases. T cells from animals with chronic GVHD produce unusual patterns of cytokines, such as IL-4, or IFN-γ in the absence of IL-2 [190–192]. These cytokines can stimulate collagen production by fibroblasts, which can be further amplified by IL-4. Interestingly, there is often an eosinophilia as an early manifestation of chronic GVHD, further supporting the notion that cells with a Th2 phenotype are important mediators of this problem [193]. One mechanism to induce autoimmune attack might be failure of normal regulatory mechanisms in the setting of thymic damage or immunosuppressants or both. Failure of Tregs results in autoimmune phenomena increasingly implicated in both acute and chronic GVHD [191,192,194–197]. Moreover, Tregs are implicated in preventing autoantibody production [198]. The notion that autoimmune attack can be induced by calcineurin inhibitors after autologous or syngeneic HCT has been observed in animals by preventing the development of Tregs [199]. In clinical trials, the manipulation of CSP to engender autoimmunity produced limited GVHD and limited GVL responses [200,201]. However, thymic injury and GVHD in the context of allogeneic HCT is more ominous, and one randomized trial suggested that calcineurin inhibitors might increase the risk of chronic GVHD [152]. Another explanation of autoimmunity may be that the massive apoptosis of T cells early in acute GVHD overwhelms the macrophage/DC system, resulting in autoantigen presentation [202]. The autoreactive cells of chronic GVHD are associated with a damaged thymus, which may be injured by acute GVHD, by the conditioning regimen, or subject to age-related involution and atrophy. Thus, the normal ability of the thymus to delete autoreactive T cells and to induce tolerance may be impaired. In addition, data are accumulating to support the concept that B cells may contribute to chronic GVHD. The anti-CD20 mAb rituximab has been used clinically to treat severe autoimmune disorders including autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura, and now several series indicate that anti-B-cell therapy is an interesting approach to treating chronic GVHD [203–205]. Alloantibodies can facilitate the cross-presentation of antigens to effector T cells, thereby amplifying T-cell responses to mHAs [206,207]. Donor B

Chapter 16

B. Related donor recipients

C. Unrelated donor recipients

100 90 80 70 60 50 40 30 20 10 0

100 90 80 70 60 50 40 30 20 10 0

p<0.0001 0

1

2

3

4

5

6

7

8

9 10 11 12

p=0.001 0

1

2

3

4

WEEKS

30

60

90

6

7

8

0

9 10 11 12

1

2

3

4

120

150

180

5

6

7

8

E. Related donor recipients

F. Unrelated donor recipients

100 90 80 70 60 50 40 30 20 10 0

100 90 80 70 60 50 40 30 20 10 0

p=0.51 0

30

60

DAYS

90

9 10 11 12

WEEKS

Percent survival

D. All recipients

p=0.08 0

5

p=0.0005

WEEKS

Percent survival

Percent survival

Overall Survival 100 90 80 70 60 50 40 30 20 10 0

Cumulative incidence

Time to CR: A. All recipients 100 90 80 70 60 50 40 30 20 10 0

Cumulative incidence

Cumulative incidence

216

120

150

180

p=0.05 0

30

60

DAYS

90

120

150

180

DAYS

Fig. 16.2 Time to complete response (CR) for patients with graft-versus-host disease (GVHD) treated with steroids alone or etanercept plus steroids. Panel A shows the time to CR for all patients with GVHD treated with steroids alone (n = 99; dotted line) or etanercept plus steroids (n = 61; solid line). Panel B shows the time to CR for recipients of related donor HCT treated with steroids alone (n = 53) or with etanercept plus steroids (n = 42). Panel C shows the time to CR for recipients of unrelated donor HCT treated with steroids alone (n = 46) or with etanercept plus steroids (n = 19). The 95% confidence intervals for CR rate at 4 weeks are shown as error bars in panels A–C. Also shown are overall survival curves through 6 months from initiation of GVHD treatment by treatment group for all patients (panel D), recipients of related donor HCT (panel E), and recipients of unrelated donor HCT (panel F). The 95% confidence intervals for survival at 6 months are shown as error bars in panels D–F. (Reprinted from [178], with permission).

Table 16.1 National Institutes of Health consensus conference categories of graft-versushost disease

Time

Acute features

Chronic features

Acute Classic acute Persistent, recurrent or late onset

<100 days ≥100 days

Yes Yes

No No

Chronic Classic chronic Overlap

No limit No limit

No Yes

Yes Yes

cells are capable of generating antibody responses specific for recipient mHAs, and chronic GVHD is significantly associated with the presence of high-titer mHA antibodies [208]. The presence of antibodies to mHAs also suggests that donor B cells have productive interactions with donor CD4 cells [208,209]. B cells can act directly as APCs in their ability to process and present limiting amounts of antigen for presentation to T cells [210]. Recent data show that antibodies to the platelet-derived growth factor receptor are observed in both scleroderma and sclerodermatous chronic GVHD [211]. It has also recently been demonstrated in female-to-male transplants that donor-derived T cells can be isolated that recognize donor and recipient nonpolymorphic X chromosome-encoded proteins (e.g. DBX) [209] as well as the production of antibodies to epitopes on the polymorphic Y chromosome-encoded proteins, DBY [208]. In this

context, the allogeneic reaction may inactivate donor APCs that are essential for establishing chronic GVHD, through cross-presentation of host antigens to donor T cells [212]. Thus, during the course of chronic GVHD, a loss of tolerance to self may result in donor T-cell recognition of both self- and recipient antigens [213].

Conclusion Complications of HCT, particularly GVHD, remain major barriers to the wider application of allogeneic HCT for a variety of diseases. Recent advances in the understanding of cytokine networks, as well as the direct mediators of cellular cytotoxicity, have led to improved understanding of this complex disease process. GVHD can be considered to be an exaggerated, undesirable manifestation of a normal inflammatory

The Pathophysiology of Graft-Versus-Host Disease

mechanism in which donor lymphocytes encounter foreign antigens in a milieu that fosters inflammation. Tissue injury related to the conditioning regimen or infection is then amplified by direct cytotoxicity via perforin/granzyme and Fas/FasL pathways, through direct cytokineinduced damage, and by recruitment of secondary effectors such as granulocytes and monocytes. Blockade of TNF-α may represent a new

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treatment for acute GVHD. Thymic injury and loss of Treg function may enhance donor cell recognition of histocompatibility antigens by both T and B cells. Moreover the production of auto- and alloantibodies may further result in tissue injury as well as enhancing T-cell-related cellular injury. The net effects of this complex system are the severe inflammatory manifestations that we recognize as clinical GVHD.

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The Pathophysiology of Graft-Versus-Host Disease 175. Huang XJ, Wan J, Lu DP. Serum TNFalpha levels in patients with acute graft-versus-host disease after bone marrow transplantation. Leukemia 2001; 15: 1089–91. 176. Herve P, Flesch M, Tiberghien P et al. Phase I–II trial of a monoclonal anti-tumor necrosis factor alpha antibody for the treatment of refractory severe acute graft-versus-host disease. Blood 1992; 79: 3362–8. 177. Holler E, Kolb HJ, Mittermuller J et al. Modulation of acute graft-versus-host-disease after allogeneic bone marrow transplantation by tumor necrosis factor alpha (TNF alpha) release in the course of pretransplant conditioning: role of conditioning regimens and prophylactic application of a monoclonal antibody neutralizing human TNF alpha (MAK 195F). Blood 1995; 86: 890– 9. 178. Levine JE, Paczesny S, Mineishi S et al. Etanercept plus methylprednisolone as initial therapy for acute GVHD. Blood 2008; 111: 2470–5. 179. Sackstein R. A revision of Billingham’s tenets: the central role of lymphocyte migration in acute graft-versus-host disease. Biol Blood Marrow Transplant 2006; 12: 2–8. 180. Waldmann H, Chen TC, Graca L et al. Regulatory T cells in transplantation. Semin Immunol 2006; 18: 111–19. 181. Dutt S, Ermann J, Tseng D et al. L-selectin and beta7 integrin on donor CD4 T cells are required for the early migration to host mesenteric lymph nodes and acute colitis of graft-versus-host disease. Blood 2005; 106: 4009–15. 182. Hashimoto D, Asakura S, Matsuoka K et al. FTY720 enhances the activation-induced apoptosis of donor T cells and modulates graftversus-host disease. Eur J Immunol 2007; 37: 271–81. 183. Kim YM, Sachs T, Asavaroengchai W, Bronson R, Sykes M. Graft-versus-host disease can be separated from graft-versus-lymphoma effects by control of lymphocyte trafficking with FTY720. J Clin Invest 2003; 111: 659–69. 184. Shapira M, Adler SN, Jacob H, Resnick IB, Slavin S, Or R. New insights into the pathophysiology of gastrointestinal graft-versus-host disease using capsule endoscopy. Haematologica 2005; 90: 1003–4. 185. Shapira MY, Hirshfeld E, Weiss L et al. Mycophenolate mofetil does not suppress the graftversus-leukemia effect or the activity of lymphokine-activated killer (LAK) cells in a murine model. Cancer Immunol Immunother 2005; 54: 383–8. 186. Shapira MY, Resnick IB, Bitan M et al. Rapid response to alefacept given to patients with steroid resistant or steroid dependent acute graft-versushost disease: a preliminary report. Bone Marrow Transplant 2005; 36: 1097–101.

187. Shulman HM, Sale GE, Lerner KG et al. Chronic cutaneous graft-versus-host disease in man. Am J Pathol 1978; 91: 545–70. 188. Filipovich AH, Weisdorf D, Pavletic S et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease. I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 2005; 11: 945–6. 189. Parkman R. Clonal analysis of murine graft-vshost disease. I. Phenotypic and functional analysis of T lymphocyte clones. J Immunol 1986; 136: 3543–8. 190. Patriarca F, Skert C, Sperotto A et al. The development of autoantibodies after allogeneic stem cell transplantation is related with chronic graftvs-host disease and immune recovery. Exp Hematol 2006; 34: 389–96. 191. Zhang C, Todorov I, Zhang Z et al. Donor CD4+ T and B cells in transplants induce chronic graftversus-host disease with autoimmune manifestations. Blood 2006; 107: 2993–3001. 192. Zhang Y. Clinical study on pulmonary complications after allogeneic peripheral blood stem cell transplantation. Zhonghua Xue Ye Xue Za Zhi 2006; 27: 366–9. 193. Ellison CA, Bradley DS, Fischer JM, Hayglass KT, Gartner JG. Murine graft-versus-host disease induced using interferon-gamma-deficient grafts features antibodies to double-stranded DNA, T helper 2-type cytokines and hypereosinophilia. Immunology 2002; 105: 63–72. 194. Zhang W, Zeng Y-S, Zhang X-B, Wang J-M, Zhang W, Chen S-J. Combination of adenoviral vector-mediated neurotrophin-3 gene transfer and retinoic acid promotes adult bone marrow cells to differentiate into neuronal phenotypes. Neurosci Letts 2006; 408: 98. 195. Zorn E, Kim HT, Lee SJ et al. Reduced frequency of FOXP3+ CD4+CD25+ regulatory T cells in patients with chronic graft-versus-host disease. Blood 2005; 106: 2903–11. 196. Rezvani K, Mielke S, Ahmadzadeh M et al. High donor FOXP3-positive regulatory T-cell (Treg) content is associated with a low risk of GVHD following HLA-matched allogeneic SCT. Blood 2006; 108: 1291–7. 197. Shevach EM. From vanilla to 28 flavors: multiple varieties of T regulatory cells. Immunity 2006; 25: 195–201. 198. Bystry RS, Aluvihare V, Welch KA, Kallikourdis M, Betz AG. B cells and professional APCs recruit regulatory T cells via CCL4. Nat Immunol 2001; 2: 1126–32. 199. Sakaguchi S, Sakaguchi N. Thymus and autoimmunity. Transplantation of the thymus from cyclosporine A-treated mice causes organ-specific autoimmune disease in athymic nude mice. J Exp Med 1988; 167: 1479–85.

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200. Jones EY, Stuart DI, Walker NP. Structure of tumour necrosis factor. Nature 1989; 338: 225– 8. 201. Yeager AM, Wagner JE Jr, Graham ML, Jones RJ, Santos GW, Grochow LB. Optimization of busulfan dosage in children undergoing bone marrow transplantation: a pharmacokinetic study of dose escalation. Blood 1992; 80: 2425–8. 202. Brochu S, Rioux-Masse B, Roy J, Roy DC, Perreault C. Massive activation-induced cell death of alloreactive T cells with apoptosis of bystander postthymic T cells prevents immune reconstitution in mice with graft-versus-host disease. Blood 1999; 94: 390–400. 203. Cutler C, Antin JH. Chronic graft-versus-host disease. Curr Opin Oncol 2006; 18: 126–31. 204. Ratanatharathorn V, Ayash L, Reynolds C et al. Treatment of chronic graft-versus-host disease with anti-CD20 chimeric monoclonal antibody. Biol Blood Marrow Transplant 2003; 9: 505–11. 205. Zaja F, Bacigalupo A, Patriarca F et al. Treatment of refractory chronic GVHD with rituximab: a GITMO study. Bone Marrow Transplant. 2007; 40: 273–7. 206. Groh V, Li YQ, Cioca D et al. Efficient crosspriming of tumor antigen-specific T cells by dendritic cells sensitized with diverse anti-MICA opsonized tumor cells. Proc Natl Acad Sci U S A 2005; 102: 6461–6. 207. Hon H, Oran A, Brocker T, Jacob J. B lymphocytes participate in cross-presentation of antigen following gene gun vaccination. J Immunol 2005; 174: 5233–42. 208. Miklos DB, Kim HT, Miller KH et al. Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission. Blood 2005; 105: 2973–8. 209. Zorn E, Miklos DB, Floyd BH et al. Minor histocompatibility antigen DBY elicits a coordinated B and T cell response after allogeneic stem cell transplantation. J Exp Med 2004; 199: 1133–42. 210. von Bergwelt-Baildon MS, Vonderheide RH, Maecker B et al. Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application. Blood 2002; 99: 3319–25. 211. Svegliati S, Olivieri A, Campelli N et al. Stimulatory autoantibodies to PDGF receptor in patients with extensive chronic graft-versus-host disease. Blood 2007; 110: 237–41. 212. Harbers SO, Crocker A, Catalano G et al. Antibody-enhanced cross-presentation of self antigen breaks T cell tolerance. J Clin Invest 2007; 117: 1361–9. 213. Tivol E, Komorowski R, Drobyski WR. Emergent autoimmunity in graft-versus-host disease. Blood 2005; 105: 4885–91.

17

Robertson Parkman & Kenneth I. Weinberg

Immune Reconstitution Following Hematopoietic Cell Transplantation

Introduction The development of a competent immune system following hematopoietic cell transplantation (HCT) is necessary for a clinically successful transplant. Without immune reconstitution, HCT recipients are at an increased risk of infection with opportunistic organisms (viruses, bacteria, fungi, and protozoa) and possibly neoplastic relapse (Fig. 17.1) [1,2]. Regardless of the factors that influence immune reconstitution such as hematopoietic stem cell (HSC) source, graft-versus-host disease (GVHD) and its treatment, and the preparative regimen, immune reconstitution follows a general pattern. In the recipients of ablative therapy followed by HCT, the recipients’ pre-existing lymphoid immunity is eliminated by the immune-suppressive and chemotherapeutic agents used in the preparatory regimen. Lymphoid immunity following HCT comes from (1) a recapitulation of normal lymphoid immunity derived from the newly engrafted HSCs and (2) the mature lymphoid elements present in the graft (both T and B lymphocytes).

Normal lymphoid ontogeny Immunocompetence is mediated by T lymphocytes, B lymphocytes, and natural killer (NK) cells. Antigen-specific T lymphocytes have a central role in human immunity. They play a critical role in the control of both DNA and RNA viral infections; in the production of specific antibodies against respiratory bacteria in conjunction with B cells; and in the control of fungal infections with granulocytes. Thus, the development of normal antigen-specific T-lymphocyte function following HCT is required for the development of a competent immune system. Successful HSC engraftment results in the development of common lymphoid progenitors (CLPs) that give rise to both T and B lymphocytes and NK cells. The CLPs migrate to the thymus and undergo a recapitulation of normal T-lymphocyte ontogeny if adequate thymic function exists in the recipient thymus. In the thymus, the CLPs undergo sequential proliferation and differentiation under the control of both cell surface molecules and cytokines present on or produced by thymic stromal cells. Central to thymic proliferation and differentiation are interactions between notch-1 and delta as well as the cytokines interleukin-7 (IL-7) and IL-2. Recent thymic immigrants express the IL-7 receptor, but not

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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CD3, CD4 or CD8 (triple-negative thymocytes). Initially, CD3 is detected only cytoplasmically; surface CD3 expression coincides with the surface expression of rearranged T-cell receptor (TCR) genes, both TCR-αβ and TCR-γδ˜. The TCR-αβ. thymocytes initially express CD3 and TCR but not CD4 or CD8 (double-negative thymocytes). The expansion (positive selection) of T lymphocytes with specificity for peptides presented in the context of recipient major histocompatibility complex molecules expressed on thymic stromal cells, especially thymic epithelial cells, occurs at the double-negative stage. Following their positive selection, thymocytes capable of reacting with self-peptides undergo apoptosis (negative selection) after their TCR interacts with self-antigens presented by macrophages and dendritic cells at the doublepositive (CD4 and CD8 coexpressing) stage of thymocyte differentiation. If adequate CLPs and thymic function exist, single-positive CD3/ CD4- and CD3/CD8-expressing T lymphocytes derived from the newly engrafted HSCs can be found in the peripheral blood approximately 3 months following HCT, where they can undergo peripheral expansion after their TCR interacts with specific antigen. The factors that can have a negative impact on thymic function include aging, irradiation, chemotherapy, infection, and GVHD [3]. All of these factors can result in decreased thymic mass, as well as functional deficiencies in the capacity of the thymic stromal cells to produce cytokines, particularly IL-7 and Kit ligand, which are required for the proliferation and differentiation of thymocytes. In the non-HCT setting, thymic function after intensive chemotherapy is age dependent, with young patients having more new CD4 T lymphocytes than older patients [4]. Thus, older HCT recipients, who have received chemotherapy and total body irradiation and have GVHD, may have inadequate residual thymic function to produce adequate numbers of new T lymphocytes with the diverse TCR expression required for a competent immune system. Except for when a T-cell-depleted (TCD) HSC source is used, HSC products from normal donors contain antigen-specific T lymphocytes, naïve T lymphocytes, and CLPs. Based upon animal studies, both antigen-specific and naïve T lymphocytes can undergo significant peripheral expansion in a lymphopenic host primarily stimulated by IL7 [5]. Thus, recipients, who have reduced thymic function may develop relatively normal numbers of mature T lymphocytes that are derived not from the newly engrafted HSCs but from expansion of the infused T lymphocytes, and therefore develop limited TCR breadth. Since both T lymphocytes and CLPs do not have self-replicating capacity, there is only a finite homeostatic expansion of T lymphocytes that is possible, resulting in gaps in the TCR repertoire and making responses to some environmental pathogens impossible. This limitation is most clearly seen in severe combined immunodeficiency recipients

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produced by residual recipient plasma cells for as long as 1 year after HCT [10,11]. However, successful long-term antibody production requires the presence of both antigen-specific donor T lymphocytes and mature donor B cells when reimmunization or infection occurs. NK cells are the most frequent lymphoid cells seen early after HCT and reach normal levels by 3 months. NK cells express inhibitory receptors (killer Ig-like receptors [KIRs] and lectin-like CD94 : NKG2 heterodimers), as well as activating receptors. KIR and CD94 : NKG2 expression is genetically determined, and in normal individuals KIR expression is more frequent than CD94 : NKG2 expression. Initially following allogeneic HCT, the majority of NK cells express CD94 : NKG2, while only a minority express KIRs. Recipients then normalize their NK receptor expression (KIR > CD94 : NKG2) so that by 1–3 years after HCT, the recipient NK profiles resemble those of the donors [12]. The decrease in CD94 : NKG2 and increase in KIR expression parallels the changes that occur in normal postnatal NK ontogeny.

Months Post-Transplant

Fig. 17.1 Incidence of late infections. The effect of hematopoietic cell source and graft-versus-host disease (GVHD) in matched and unrelated hematopoietic cell transplant recipients was determined. (Reproduced from [1], with permission.)

transplanted without HSC ablation in whom the generation of new T lymphocytes is lost by 15 years after HCT [6]. Defects in either HSC engraftment or recipient thymic function will result in an HCT recipient that has defective or limited T-lymphocyte immunity. Thus, the broad repertoire of TCR expression required for successful immune reconstitution after HCT requires HSC engraftment. Assays have been developed to measure thymopoiesis; initially, immunophenotypic analyses (CD4, CD45RA expression, with or without CD62L or CD31) were used to quantify the generation of recent thymic emigrants (naïve T lymphocytes). A more specific assay is the molecular determination of the frequency of T lymphocytes containing TCR excision circles (TRECs) [7]. Recent thymic emigrants contain TRECs; as they undergo subsequent peripheral expansion, the frequency of TREC-containing cells decreases. There is an age-dependent decline in the frequency of TREC-positive T lymphocytes in normal individuals consistent with the normal involution of thymus, although the frequency of TREC-positive cells within the thymus remains constant. The breadth of TCR diversity can be quantified by spectrotyping. In addition to the production of single positive T lymphocytes with effector function necessary for protection against infectious agents, the thymus produces CD4+CD25+ T lymphocytes with regulatory function expressing FoxP3 [8]. The generation of regulatory T lymphocytes is under the control of the AIRE gene expressed by thymic medullary cells. The CD4+CD25+ regulatory T lymphocytes may play an important role in the prevention and control of both acute and chronic GVHD [9]. As in T-lymphocyte ontogeny, B cells are derived from CLPs and undergo differentiation in the bone marrow that can be monitored by the expression of surface molecules and the molecular analysis of the immunoglobulin (Ig) genes. Immature B cells have surface expression of IgM, and after interaction with specific antigen and cooperation with antigenspecific T lymphocytes, the IgM-expressing B cells mature into antigenspecific IgG-expressing B cells which then migrate to the peripheral lymphoid organs and become antibody-producing plasma cells. Thus, defects in antigen-specific T lymphocytes may also result in defects in specific antibody production. Since plasma cells are not part of the HSC inoculum, there is minimal transfer of antibody-producing cells at the time of transplantation. Studies have shown the persistence of antibodies

Assessment of immune reconstitution Immunophenotypic analyses can identify the presence of T and B lymphocytes and characterize their state of differentiation. However, the immunophenotypic analyses do not analyze their functional capacity. The response of T lymphocytes to mitogenic stimulation (phytohemagglutinin or anti-CD3 antibody) is the first function that can be quantified [13]. However, the function that predicts clinical protection against infection is the presence of antigen-specific T lymphocytes, which is most easily detected by using antigen-specific blastogenesis or cytokine capture assays. Antigens such as tetanus toxoid, Candida, and the herpes viruses (cytomegalovirus [CMV], herpes simplex virus [HSV], varicella zoster virus [VZV]) can be used. The presence of a positive blastogeneic response indicates that antigen-specific T lymphocytes are present and that adequate amounts of IL-2 are produced to support cell division. A more complete analysis, however, requires the addition of exogenous IL-2 to the cultures, since it is possible that antigen-specific T lymphocytes are present without the capacity to produce IL-2 early after HCT. Defects in effector mechanisms (γ-interferon and cytotoxic T lymphocytes) have been demonstrated. Therefore, the presence of a normal blastogeneic response to a specific antigen does not mean that all potential effector mechanisms are fully developed and is another example of a recapitulation immunologic ontogeny after transplantation [14,15]. Cytokine capture assays (IL-2, γ-interferon, etc.) after antigenic stimulation permit a further evaluation of T lymphocyte antigen-specific function. Most assessments of immune reconstitution have focused on immunophenotypic analyses and mitogenic responsiveness. Few studies have measured antigen-specific T-lymphocyte function. Specific antibody production can be determined after immunization with neoantigens (ΦX174), reimmunization with protein antigens (tetanus toxoid) or natural stimulation with bacterial polysaccharide antigens (polyribophosphate).

HSC sources A major determinant in the rapidity and breadth of immune reconstitution after HCT is the HSC product used. Autologous HCT Following immunoablative and high-dose conditioning, the recipients of autologous peripheral blood progenitor cells (PBPCs), either CD34 selected or unmanipulated, have severe lymphopenia. By 3 months following HCT, CD8 T-lymphocyte counts are higher than CD4 counts,

Chapter 17

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leading to an inverted CD4 : CD8 ratio with no difference between the selected and unselected products [16,17]. By 1 year, the CD4 T-lymphocyte counts have stabilized. In adults there is no correlation between recipient age and CD4 count, while in pediatric patients there is a correlation, with younger recipients having higher CD4 counts [18]. When thymic function is measured by TREC analysis, thymopoiesis is detected in most recipients, both adult and pediatric, regardless of age. In adult recipients, an increased frequency of TREC-positive cells correlated with a broader TCR repertoire, indicating better immune reconstitution [19]. Recipients without significant TREC-positive cells have normalization of their CD4 counts by the homeostatic expansion of the transferred mature T lymphocytes and therefore have a limited TCR repertoire. The presence of antigen-specific T lymphocytes with specificity for antigens to which the recipient had immunity prior to HCT can be detected in the peripheral blood following HCT. Immunization of recipients prior to transplantation with bacterial polysaccharide vaccines results in increased antibody production following reimmunization after HCT, indicating the transfer of both antigen-specific T lymphocytes and primed B cells as part of the PBPC product [20]. Given the lack of significant defects in immune reconstitution after the transplantation of autologous PBPC, recipients rarely have an increased risk of opportunistic infections.

200 100 0 Without GVHD With GVHD Control : 1 SD

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HCT from matched related donors For the first 20 years of HCT from matched related donors, bone marrow was exclusively used as the hematopoietic cell source. For the last 15 years, however, PBPCs, in addition to bone marrow, have been used. There are significant differences between the immune reconstitution of recipients receiving bone marrow and PBPC. In the recipients of unmanipulated bone marrow, there is a slight delay in the recovery of both CD4 and CD8 T lymphocytes compared with the recipients of autologous bone marrow [21,22]. CD8 T lymphocyte levels normalize more rapidly than CD4 counts, resulting in an inversion of the CD4:CD8 ratio, which does not normalize until 6–9 months following transplantation. CD8 T lymphocytes proliferate by homeostatic expansion, while CD4 expansion requires thymopoiesis. NK cells appear early after transplantation and are the predominant lymphoid cell in the first months following transplantation. Recipients of unmanipulated bone marrow have detectable mitogenic responses beginning 2–3 months following transplantation, but may require the addition of exogenous IL-2 for their detection since defects in IL-2 production can exist early after HCT [23]. Pre-existing donor-derived antigen-specific T lymphocytes cannot be detected until they are immunized or infected. No prospective studies have been undertaken yet to determine the time at which the T lymphocytes of matched recipients are able to respond to new antigenic stimuli. Antigen-specific T lymphocytes to infectious agents, including the herpesviruses, can be detected starting at 2 months following HCT. Responses to HSV are followed by responses to VZV and then CMV [24,25]. As in autologous recipients, TREC analysis has shown an age-dependent decline in thymic function. Pediatric recipients, regardless of age, have reproducible thymopoiesis, while the delayed and nonreproducible recovery of CD4 T lymphocyte counts is seen in adult recipients [26]. The recipients of peripheral blood hematopoietic cells show a more rapid recovery of CD4 T-lymphocyte counts, and, in comparison to bone marrow recipients, donor-derived antigenspecific T lymphocytes can be detected after HCT without immunization or infection, since 1–2 log more mature T lymphocytes are contained in the graft [27]. Initial analyses, however, have not shown a convincing decrease in opportunistic infections in the recipients of PBPCs compared with bone marrow [28]. The lack of clinical benefit associated with

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Fig. 17.2 Median values of serum immunoglobulin (Ig) G, IgA, and IgM in recipients after marrow transplantation. The shaded area represents values in marrow donors. GVHD, graft-versus-host disease (Reproduced from [45] with permission.)

peripheral blood hematopoietic cells may be due in part to the increased risk of chronic GVHD seen in PBPC recipients [29]. The number of B lymphocytes, as measured by surface IgG or CD20 expression, returns to normal levels by 1–2 months following HCT. The expression of variable heavy chain genes early after HCT demonstrates heavy chain gene usage similar to that seen in fetal B lymphocytes, suggesting a recapitulation of B-lymphocyte ontogeny [30]. Without replacement immune globulin, recipients have reduced levels of IgG, IgA, and IgM for the first 6 months after HCT (Fig. 17.2). Unrelated HCT The immunophenotypic recovery of the T lymphocytes in recipients of unrelated bone marrow or PBPCs is more variable than that of matched recipients, due in part to the increased use of immunosuppressive drugs [1]. Adult unrelated recipients have decreased thymic function when compared with children (Fig. 17.3). In children CD4 T-lymphocyte counts reach normal levels by 1 year, while at 2 years post HCT only some adult recipients have reached normal levels. The adult recipients have significantly less thymopoiesis, as measured by CD4, CD45RA or TREC assays, indicating that homeostatic expansion is a more important mechanism for post-HCT reconstitution in adult compared with pediatric recipients. When antigen-specific T-lymphocyte responses to opportunistic organisms are measured, results are variable. Increased TREC production correlates with the ability to develop antigen-specific CD4 T

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Fig. 17.3 Comparison of recovery of CD4 and CD4CD45RA T lymphocytes following T-cell depleted unrelated bone marrow transplant. The closed circles (•) represent recipients who developed life-threatening infections; the open circles (ο) represent those who did not. BMT, bone marrow transplantation. (Reproduced from [2] with permission.)

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lymphocytes and improved clinical outcome [31]. The most profound defect seen in unrelated recipients is an inability to produce protective levels of antipolysaccharide antibodies, which are necessary for protection against respiratory bacteria, a common cause of infection in unrelated recipients of either bone marrow or PBPCs [2,32]. TCD HCT Since T lymphocytes are central to the development of GVHD, their removal has the clinical benefit of reducing the frequency and severity of acute and chronic GVHD. In the absence of adequate pretransplantation conditioning, however, T-cell depletion can result in an increased risk of graft rejection. TCD HCT was initially used in the haploidentical setting for the treatment of infants with severe combined immunodeficiency [33]. Based upon its success, the use of T-cell depletion has been expanded, first to haploidentical HCT for neoplastic diseases and then, more recently, to both related and unrelated HCT [34,35]. The course of immune reconstitution following successful HSC engraftment of a TCD product is reproducible. No immunophenotypic T lymphocytes are seen for the first 3 months following HCT, at which time first CD3dim and then CD3bright T lymphocytes are seen. Proliferative responses to mitogenic stimuli may not initially be detected unless an exogenous source of IL-2 is provided, demonstrating that defects in cytokine production initially exist [23]. The comparison of adult and pediatric patients shows that the recovery of T lymphocytes in children receiving unrelated hematopoietic cells is similar to that with related recipients, while T lymphocyte recovery is slower in unrelated adults, indicating reduced thymic function in adult recipients [2]. Since there can be minimal homeostatic expansion of mature T lymphocytes contained in the hematopoietic cell product, all immune reconstitution has to be derived from the newly engrafted HSCs. Therefore, defects in thymic function may place adult recipients of TCD HCT at long-term risk for opportunistic infections due to a lack of broad-based immune reconstitution, which can be quantified by spectrotypic analysis of TCR

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9–12 12–18 18–24

Months post BMT

utilization [36]. Adult recipients of haploidentical TCD PBPCs following high-dose irradiation can have severe depression of CD4 T-lymphocyte counts as long as 1–2 years following HCT, which may explain their high likelihood of infection with DNA viruses (CMV) and fungi (Aspergillus) [37]. Cord blood HCT Cord blood is unique among hematopoietic cell sources in that the unmanipulated product does not contain any antigen-specific T lymphocytes, since the fetus exists in an antigenically null environment. Therefore, no transfer of antigen-specific T lymphocytes occurs, leading to concerns that recipients of cord blood HCT would be at an increased risk of opportunistic infections. Immunophenotypic analysis of pediatric recipients has shown normalization of NK cell counts by 3–6 months, and CD3 and CD4 T lymphocyte counts by 12 months, post HCT [38,39]. Normal TCR diversity is seen sooner in children than adult recipients. Functional analyses have revealed that recipients of unrelated cord blood hematopoietic cells can develop antigen-specific T lymphocytes within one month of HCT and this may explain why an increased risk of infections has not been detected [40]. Defects in the production of antipolysaccharide antibodies have been seen in the recipients of unrelated cord blood HCT, similar to those seen in recipients of unrelated bone marrow and peripheral blood hematopoietic cells.

Graft-versus-host disease Besides the HSC source, the other principal clinical variable that impacts the rapidity and breadth of immune reconstitution is the presence or absence of acute and/or chronic GVHD. Acute GVHD has little impact on the tempo of lymphoid reconstitution, as measured by immunophenotypic assessment [41]. The presence of acute GVHD is a major predictor of chronic GVHD, and both have been shown to have a negative impact on recipient thymic function, even after chronic GVHD is no longer present (Fig. 17.4) [42]. Chronic GVHD occurs more frequently in older

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individuals, who also have age-dependent decreases in thymic function, leading to a further compromise of their capacity to produce new T lymphocytes [43]. Recipients with ongoing chronic GVHD have no detectable TREC-containing cells, indicating an absence of thymic function, requiring that all their mature T lymphocytes are derived from homeostatic expansion and resulting in limited TCR breadth. The lack of adequate thymic function may explain why recipients with chronic GVHD demonstrate a reduced frequency of helper T-lymphocyte precursors [44]. The first immune defect reported in association with chronic GVHD was a reduction in the primary antibody response to a neoantigen (ΦX174) and a lack of class switching from IgM to IgG [45] (Fig. 17.5). Defects in antipolysaccharide antibody production are seen in matched recipients with a history of chronic GVHD [32]. Whether the defects in antibody production are due to the observed decrease in pre-B cells or to defective CD4 T-lymphocyte function is not clear [46,47]. The increased immunosuppression used to treat both acute and chronic GVHD may further contribute to recipient immunodeficiency, which explains the increased risk of both T-lymphocyte (DNA viral and fungal) and B-lymphocyte (respiratory bacterial) infections associated with GVHD.

Immunosuppressant therapy The use of immunosuppressive agents to prevent and/or treat acute and chronic GVHD can have an impact on post-transplant immune reconstitution. Methotrexate Methotrexate has been used for almost 40 years for prophylaxis against acute GVHD. Methotrexate mediates its activity through the destruction of T lymphocytes induced to divide by histocompatibility differences between the donor and recipient [48]. Unfortunately, donor-derived antigen-specific T lymphocytes present in the transplant inoculum with specificity for environmental pathogens may also be destroyed after stimulation by organisms or antigens present in the recipient. Since all dividing cells are equally susceptible, the use of methotrexate may explain why antigen-specific T lymphocytes can be detected in autologous HCT recipients but not allogeneic bone marrow T recipients.

Fig. 17.4 Cross-sectional analysis of T-cell receptor excision circle (TREC) levels in relationship to graft-versus-host disease (GVHD). (a) Individual CD41 and CD81 TREC levels are shown for a cross-section of patients through years of age with no history of GVHD. (b) Patients through 25 years of age with chronic GVHD. (c) Patients through 25 years of age with a history of GVHD. (d) A composite box plot for TREC levels is shown for all three groups. The top, bottom, and line through the middle of the box correspond to the 75th, 25th, and 50th percentile (median) respectively. The whiskers on the bottom and top extend from the 10th percentile and to the 90th percentile, respectively. (e) Individual CD41 (closed symbols) and CD81 (open symbols) TREC levels are shown for a cross-section of patients older than 25 years of age with no history of GVHD (squares), with chronic GVHD (triangles), and with a history of GVHD (circles). The normal range of TREC levels for individuals of ages in the groups studied are shown lying between the dashed lines. HSCT, hematopoietic stem cell transplantation. (Reproduced from [41] with permission.)

(a)

(b)

Cyclosporine Cyclosporine (CSP) has been in routine clinical use for more than 20 years for the prevention and treatment of GVHD [49]. Its primary mode of action is the inhibition of IL-2 production, resulting in a decreased proliferation of donor-derived T lymphocytes with specificity for

(c)

1000 No C-GVHD

No C-GVHD

K value

100 C-GVHD

No C-GVHD 10

C-GVHD

1

0.1

200

600 1000 1400 1800

C-GVHD

200

600 1000 1400 1800 Days

200

600 1000 1400 1800

Fig. 17.5 Maximum antibody activity following primary (a) and secondary (b) ΦX174 phage injection in normal subjects and allogeneic and syngeneic marrow transplant recipients. (c) Immunoglobulin G (IgG) antibody activity of the secondary response. The shaded area represents the 5–95th percentile response for normal subjects. The curves represent the estimated means for data from allogeneic recipients with or without chronic graft-versus-host disease (CGVHD). The dots represent individual data points from syngeneic recipients.

Immune Reconstitution Following Hematopoietic Cell Transplantation

recipient-restricted histocompatibility antigens. CSP may have a similar effect on the expansion of antigen-specific T lymphocytes with specificity for environmental pathogens, although HCT recipients on CSP are able to develop antigen-specific T lymphocytes, as detected by in vitro proliferative assays. Recently, however, another potential side-effect of CSP has been identified. In murine models, the regulatory effect of CD4+CD25+ T lymphocytes is abolished by the co-administration of CSP, suggesting that the clinical administration of CSP may prevent the termination of acute GVHD by regulatory T lymphocytes [50]. Also, CSP decreases class II major histocompatability complex expression in the thymus, which may decrease both positive and negative selection, resulting in less expansion of T lymphocytes with specificity for environmental pathogens, and reducing the elimination of autoreactive T lymphocytes potentially involved in chronic GVHD [51]. Antibody therapy The use of antibody therapy with relative specificity for T lymphocytes has been in routine clinical use for over 20 years: initially antithymocyte serum/antithymocyte globulin (ATG) and more recently antibodies to CD3, CD25, and CD52 [45,52,53]. The intravenous administration of antibodies results in the destruction of the target cell population by both complement-mediated lysis and antibody-dependent cell-mediated cytotoxicity. The effectiveness of the therapy can be monitored immunophenotypically, although an antibody to a different epitope is necessary to differentiate destruction from blocking. The administration of ATG decreases the primary antibody response to neoantigens even if the recipient does not develop chronic GVHD [45]. The post-HCT administration of anti-CD52 antibodies to recipient who had received in vivotreated anti-CD52 HSCs, was discontinued because of the increased frequency of and deaths due to CMV reactivation [52]. Antibodies that block the IL-2–IL-2 receptor interaction or kill CD25-expressing cells have been used in the treatment of GVHD, but also can block the expansion of antigen-specific T lymphocytes required for protection against environmental pathogens. Thus, antibody therapies that destroy circulating T lymphocytes may compromise immune reconstitution and eliminate both passively acquired donor-derived, antigen-specific T lymphocytes and new naïve T lymphocytes. Glucocorticoid steroids Glucocorticoid steroids have been used as second-line therapy for the treatment of acute GVHD and primary therapy for chronic GVHD for many years. Steroids have a generalized lympholytic effect. In addition, they have a selective action against the functional subpopulation (tumor necrosis factor-alpha production) of CD8+ antigen-specific T lymphocytes [54]. Thus, steroids may have a particularly detrimental effect in recipients with evolving immune systems who have fungal or viral infections, and this may explain the negative association between glucocorticoid administration and clinical outcome in recipients with fungal infections.

Preparative regimens Traditionally, HCT recipients are prepared with a combination of agents that have both immunoablative and myeloablative capacities, resulting in a recipient who has no effective immune system and negligible residual HSCs. Recently reduced-intensity conditioning (RIC) regimens have been evaluated which have immunosuppressive but not immunoablative capacity, and reduce but do not eliminate recipient HSCs. Such transplants rely upon the donor antihost reactivity of the donor T lymphocytes to eliminate the residual recipient immune system as well as the recipi-

227

ent HSCs. As a consequence, recipients may have mixed hematopoietic chimerism based upon the persistence of recipient HSCs, but in most cases the recipient T lymphocytes are 100% of donor origin by 3–4 months following transplantation [55]. Split T-lymphocyte chimerism is unusual, and significant persistence of recipient T lymphocytes raises the possibility of graft rejection. In many protocols, after the establishment of donor lymphoid and myeloid chimerism, recipients receive donor lymphocyte infusions (DLIs). The frequency of acute GVHD following the cessation of immunosuppression and/or the administration of DLI is in the range of 40–50%, with a significant proportion of recipients developing chronic GVHD. For this reason, many centers have stopped DLIs solely for attempting to improve chimerism. The immune reconstitution of the recipients of RIC HCT does not differ significantly from the recipients of high-dose HCT, but may be more rapid. There is the transfer of both antigen-specific and naïve donor T lymphocytes as part of the graft, as well as the requirement for both donor HSC engraftment and adequate thymic function for the development of a competent immune system. The nature of the graft has a significant impact on immune reconstitution in the RIC setting. Recipients receiving a TCD product will have delays in both immunophenotypic and functional T-lymphocyte recovery. The recipients of unmodified HSCs have more rapid immunophenotypic recovery and earlier detection of TREC-positive cells than do recipients receiving ablative conditioning, possibly due to the lack of methotrexate in the RIC setting [56,57]. A unique RIC regimen is the use of TLI and ATG which, in the matched related donor setting, results in reproducible donor engraftment without acute GVHD [58].

Graft-versus-tumor effect The presence of acute GVHD in recipients transplanted for lymphoid malignancies has been associated with a decreased incidence of relapse, and has suggested a graft-versus-leukemia effect [59]. Further work in animals and evaluation of HCT recipients have demonstrated that a graft-versus-tumor (GVT) effect can exist for other malignancies. Multiple cellular mechanisms are involved in the GVT effect. Donor-derived T lymphocytes with specificity for recipient-restricted histocompatibility antigens can produce GVHD, but are also capable of the destruction of residual recipient malignant cells, particularly lymphoid malignancies. T lymphocytes with specificity for antigens uniquely expressed (BCR/ABL) or overexpressed (PR1 and WT1) on tumor cells have been hypothesized to be targets for a GVT effect [60–62]. Recipients of haploidentical peripheral blood HCT for acute myeloid leukemia have a decreased relapse rate when the donor and recipient exhibit NK KIR mismatches, resulting in a lack of inhibition of the donor-derived NK cells [63]. Debates still exist as to the role of NK-mediated killing in the nonhaploidentical setting and against leukemias other than myeloid leukemias [64]. The development of a competent immune system after unrelated cord blood HCT, as measured by the presence of antiherpesvirus-specific T lymphocytes, was associated with a threefold decrease in leukemic relapse, suggesting that the presence of a competent immune system resulted in less leukemic relapse [39]. Based upon the clinical observations that a GVT effect existed, investigators have undertaken the administration of DLI containing both antigen-specific and naïve T lymphocytes to HCT recipients. Initially, DLI was administered to recipients with post-transplant Epstein–Barr virus-associated lymphomas [65]. Approximately 2–3 months were required before the clinical regression of lymphoma was observed. In most cases, acute GVHD developed, since the frequency of T lymphocytes capable of responding to histocompatibility antigens was greater than the frequency of cells capable of responding to Epstein–Barr virus antigens [66].

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Adoptive cellular therapy The success of DLI in treating virally induced lymphomas led to attempts to treat viral infections, particularly adenovirus and CMV. Because of the greater frequency of alloreactive T lymphocytes compared with virus-specific T lymphocytes, significant acute GVHD usually accompanied such attempts. Therefore, investigators generated virus- and fungus-specific lines in which the frequency of alloreactive T lymphocytes was reduced [67]. When nonalloreactive antigen-specific T lymphocyte clones were established, no GVHD occurred [68,69]. Their adoptive transfer was used for the successful prophylaxis or treatment of CMV, adenovirus, and Aspergillus. The increased use of adoptive cellular therapy, however, will require the development of techniques to produce antigen-specific T lymphocytes devoid of alloreactivity, to reduce the possibility of acute GVHD without the need for cloning. Based upon the successful use of donor-derived antigen-specific T lymphocytes for the treatment of viral and fungal infections and virusassociated lymphomas, investigators have now undertaken the evaluation of donor-derived T lymphocytes with specificity for tumor-associated antigens (WT1 and PR1) [70]. Two approaches have been proposed: first, the in vitro generation of nonalloreactive antigen-specific T lymphocytes, and second, the in vivo immunization of the HCT donor. Central to the successful use of in vitro-generated antigen-specific T lymphocytes is a lack of alloreactivity of the infused cells. Various techniques have been utilized to reduce the alloreactivity of the donor T lymphocytes, including preinfusion activation and the elimination of the alloactivated T lymphocytes by anti-CD25 immunotoxin or phototherapy, or anergenization by co-stimulatory (anti-B7.1 and anti-B.72) blockade [71–73]. The combination of nonalloreactivity and the in vitro expansion of antigen-specific T lymphocytes may provide the ability to prevent and treat infections during post-HCT immunodeficiency, reducing both infections and tumor relapse. Alternatively, nonalloreactive normal donor leukocytes could be infused to provide T lymphocytes with a broad TCR repertoire. When the target antigens for the GVT effect are identified, the immunization of the donor to the appropriate antigen followed by reimmunization of the recipient after HCT to expand the antigen-specific cells is appealing. Such strategies have already been shown to be successful for non-neoplastic antigens such as tetanus toxoid and CMV [11]. The administration of CD4+CD25+ regulatory T lymphocytes prevents acute GVHD in murine HCT models while not reducing the GVT effect. Improved immune reconstitution has also been observed, presumably due to reducing thymic damage due to acute GVHD [9,74]. Clinical

trials are underway to evaluate the infusion of in vitro-expanded CD4+CD25+ T lymphocytes to prevent acute GVHD [75].

Improving immune reconstitution after HCT Immune reconstitution after HCT could be improved by either increasing the rapidity by which antigen-specific T lymphocytes can be generated in vivo and/or broadening the repertoire of the newly generated T lymphocytes. Animal experiments have shown that thymic function can be protected from the deleterious effects of irradiation and busulfan by the administration of keratinocyte growth factor [76]. Clinical trials are now underway to demonstrate whether the peritransplant administration of keratinocyte growth factor will result in more rapid immune reconstitution. IL-7 is the major cytokine that is produced by the thymic epithelial cells, and its production is protected by the administration of keratinocyte growth factor. In animal experiments, post-transplant administration of IL-7 has resulted in improved immunophenotypic and functional immune reconstitution [77]. Androgen receptor antagonists have also been shown to enhance thymic function in aged animals [78]. Clinical trials are now underway to determine whether the peritransplant administration of either IL-7 or androgen receptor antagonists will result in improved immune reconstitution following HCT. Improved thymic function, besides increasing the rapidity of reconstitution, may also result in a broader TCR repertoire, resulting in the recipient being able to respond to more infectious pathogens or neoplastic cells. Since a minimum of 3 months is required following HCT to generate new T lymphocytes, the administration of large numbers of in vivoexpanded CLPs might result in the earlier appearance of new T lymphocytes, and therefore more rapid immune reconstitution [79,80]. In recipients with a history of chronic GVHD, no functional thymic tissue may exist. In such recipients, the transplantation of cultured postnatal thymus may support thymopoiesis, although the positive selection by the allogeneic thymic stromal cells may result in a limited TCR repertoire [81].

Prophylaxis against infection The prophylactic administration of antiherpesvirus drugs, particularly acyclovir, has reduced post-transplant infection with HSV and VZV and the incidence of CMV reactivation [25]. The reduction in viral reactivation reduces the antigenic stimulation that is necessary for the develop-

Table 17.1 Immunization following hematopoietic cell transplantation (Centers for Disease Control recommendations) Vaccine

Recipients without chronic GVHD

Recipients with chronic GVHD

Diphtheria–tetanus toxoid Oral polio virus (Sabin) Inactivated polio virus (Salk) Influenza Haemophilus influenzae type b (Hib) Pneumococcal polysaccharide conjugated 23-valent Hepatitis B Measles–mumps–rubella Varicella

6–12 months Not recommended 6–12 months Annually 6–12 months 12 months 6–12 months 1–2 years 2 years

12 months Not recommended Not indicated if on IVIG Annually [81] 6–12 months 12 months 6–12 months Not recommended Not recommended

GVHD, graft-versus-host disease; IVIG, intravenous immunoglobulin.

Immune Reconstitution Following Hematopoietic Cell Transplantation

ment of antigen-specific T lymphocytes. Therefore, there are delays in the appearance of antiherpes-specific T lymphocytes required for longterm protection following termination of the prophylactic drugs. In spite of prophylactic drugs, the recipients do develop antigen-specific T lymphocytes, which can be a criterion of discontinuation of the prophylactic drugs. Clinical experience indicates that the cessation of antiviral prophylaxis before the development of antigen-specific T lymphocytes increases the likelihood of clinical disease. When recipients, who have antiherpes-specific T lymphocytes develop herpes virus infections after the cessation of prophylactic drugs, defects in the production of γ-interferon and other effector mechanisms have been noted. The lack of production of antibacterial polysaccharide antibodies in related recipients with chronic GVHD and all unrelated recipients places them at increased risk for bacterial infection with respiratory bacteria

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[1]. Conjugated carbohydrate vaccinations are protective, but do not demonstrate that the recipient can respond to nonconjugated wild-type bacterial polysaccharide antigens.

Vaccination Since there is a lack of reproducible and clinically relevant transfer of antigen-specific T and B lymphocytes from normal donors to recipients, it is suggested by the Centers for Disease Control that HCT recipients be reimmunized with the normal childhood vaccines. Because of uncertainty of the immune response to live viruses, immunizations with live vaccines during the first 2 years following transplantation are not recommended, as is the use of live vaccines in any recipient who continues on significant immunosuppression usually due to the presence of chronic GVHD (Table 17.1) (see Chapter 12) [82].

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62. Rezvani K, Yong AS, Svani BN et al. Graftversus-leukemia effects associated with detectable Wilms tumor-1 specific T lymphocytes after allogeneic stem-cell transplantation for acute lymphoblastic leukemia. Blood 2007; 110: 1924– 32. 63. Ruggeri L, Capanni M, Urbani E et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: 2097–100. 64. Davies SM, Ruggieri L, DeFor T et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor. Blood 2002; 100: 3825–7. 65. Papadopoulos EB, Ladanyi M, Emanuel D et al. Infusions of donor leukocytes to treat Epstein–Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med 1994; 330: 1185–91. 66. Koehne G, Smith KM, Ferguson TL et al. Quantitation, selection, and functional characterization of Epstein–Barr virus-specific and alloreactive T cells detected by intracellular interferon-gamma production and growth of cytotoxic precursors. Blood 2002; 99: 1730–40. 67. Peggs KS, Mackinnon S. Augmentation of virusspecific immunity after hematopoietic stem cell transplantation by adoptive T-cell therapy. Hum Immunol 2004; 65: 550–7. 68. Perruccio K, Tosti A, Burchielli E et al. Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation. Blood 2005; 106: 4397–406. 69. Walter EA, Greenberg PD, Gilbert MJ et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med 1995; 333: 1038–44. 70. Doubrovina ES, Doubrovin MM, Lee S et al. In vitro stimulation with WT1 peptide-loaded Epstein–Barr virus-positive B cells elicits high frequencies of WT1 peptide-specific T cells with in vitro and in vivo tumoricidal activity. Clin Cancer Res 2004; 10: 7207–19. 71. Amrolia PJ, Cuccioli-Casadei G, Huls H et al. Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation. Blood 2006; 108: 1797–808. 72. Perruccio K, Topini F, Tosti A et al. Photodynamic purging of alloreactive T cells for adoptive immunotherapy after haploidentical stem cell transplantation. Blood Cells Mol Dis 2008; 40: 76–83. 73. Guinan EC, Boussiotis VA, Neuber D et al. Transplantation of anergic histoincompatible bone marrow allografts. N Engl J Med 1999; 340: 1704– 14. 74. Nguyen VH, Shashidhar S, Chang DS et al. The impact of regulatory T cell immunity following hematopoietic cell transplantation. Blood 2008; 111: 945–53. 75. June CH, Blazar BR. Clinical application of expanded CD4+25+ cells. Semin Immunol 2006; 18: 78–88. 76. Min D, Taylor PA, Panoskaltisis-Mortari A et al. Protection from thymic epithelial cell injury by keratinocyte growth factor: a new approach to improve thymic and peripheral T-cell reconstitution after bone marrow transplantation. Blood 2002; 99: 4592–94.

Immune Reconstitution Following Hematopoietic Cell Transplantation 77. Alpdogan O, Schmaltz C, Muriglan SJ et al. Administration of interleukin-7 after allogeneic bone marrow transplantation improves immune reconstitution without aggravating graft-versushost disease. Blood 2001; 98: 2256–65. 78. Goldberg GL, Alpdogan O, Muriglan SJ et al. Enhanced immune reconstitution by sex steroid ablation following allogeneic hemopoietic stem cell transplantation. J Immunol 2007; 178: 7473–84.

79. Arber C, BitMansour A, Sparer TE et al. Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation. Blood 2003; 102: 421–8. 80. Zakrzewski JL, Kochman AA, Lu SX et al. Adoptive transfer of T-cell precursors enhance T-cell reconstitution after allogeneic hematopoietic stem cell transplantation Nat Med. 2006; 12: 1039–47.

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81. Markert ML, Devlin BH, Alexieff MJ et al. Review of 54 patients with complete DiGeorge anomaly enrolled in protocols for thymus transplantation: outcome of 44 consecutive transplants. Blood 2007; 109: 4539– 47. 82. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. MMWR 2000; 49: 1–128.

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Edus H. Warren

The Human Graft-versus-Tumor Response – and How to Exploit It

Introduction The elimination of malignant disease after allogeneic hematopoietic cell transplantation (HCT) is in part due to an immunologic “graft-versustumor” (GVT) effect mediated by cells contained in or derived from the donor hematopoietic cell graft. GVT responses are closely associated with the development of graft-versus-host disease (GVHD), but GVHD is not required for GVT activity. Recognition of the critical role played by the GVT effect in allogeneic transplantation has provided cogent rationale for the development of HCT strategies with reduced-intensity conditioning regimens in which tumor eradication is mediated entirely by the donor graft. Despite the potency of the GVT effect, post-transplant persistence or recurrence of malignancy continues to be a major cause of treatment failure after allogeneic HCT. Cellular and molecular dissection of the GVT effect to facilitate the development of strategies for selectively enhancing GVT without inducing or aggravating GVHD is consequently the focus of considerable current research activity. This chapter will review (1) the evidence that supports the existence of and defines the characteristics of the GVT effect after allogeneic HCT, (2) the effector cells and target molecules of the GVT effect, and (3) potential strategies for exploiting the GVT effect to clinical advantage.

Defining characteristics of the GVT effect in human allogeneic HCT The principle that immune cells derived from the donor could contribute to the eradication of leukemia after allogeneic bone marrow transplantation was initially established in murine models [1,2]. A GVT effect operating in human allogeneic transplantation and associated with the development of acute and chronic GVHD was subsequently identified in retrospective analyses of relapse rates following allogeneic HCT with high-intensity conditioning regimens for advanced leukemia [3,4]. The existence of the GVT effect has subsequently been confirmed by three independent lines of evidence. First, complete regression of malignant disease after discontinuation of immune suppression has frequently been reported in patients whose tumors recurred after allogeneic HCT. Remissions induced by withdrawal of post-transplant immune suppression have been observed in patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia

(CML), chronic lymphocytic leukemia, Burkitt’s lymphoma, diffuse large B-cell and follicular non-Hodgkin’s lymphoma, multiple myeloma, ovarian cancer, and non-small cell lung cancer [5–12]. Second, infusions of lymphocytes harvested from the original hematopoietic cell donor have successfully been used to treat post-transplant persistence or recurrence of a comparably broad array of malignancies [13–17]. The antitumor activity of donor lymphocyte infusion (DLI) is most effective against CML that recurs in chronic phase after allogeneic HCT, but has also been observed in many patients with other kinds of leukemia, nonHodgkin’s lymphoma, and multiple myeloma. Third, allogeneic HCT with reduced-intensity conditioning regimens that permit stable engraftment of donor hematopoietic cells but have little, if any, direct tumoricidal activity has produced durable complete remissions of advanced hematologic malignancies [18–22] as well as selected solid tumors, including metastatic renal cell carcinoma [23], metastatic colon carcinoma [24], and metastatic breast carcinoma [25–27]. Three defining characteristics of the GVT effect were identified in retrospective analyses of relapse rates following HCT with bone marrow grafts from human leukocyte antigen (HLA)-identical sibling donors for early-stage or for advanced leukemia [3,4,28–32], and have subsequently been extensively confirmed in other transplant settings. By comparing the relapse rates seen in patients who developed no GVHD, acute GVHD, chronic GVHD, or both acute and chronic GVHD, it can be inferred that the GVT effect is strongly associated with the development of GVHD, particularly chronic GVHD (Fig. 18.1). However, comparison of the relapse rate observed in HLA-matched allogeneic HCT recipients who did not develop acute or chronic GVHD with that observed in recipients of HCT from syngeneic donors demonstrated that there is an antileukemic effect of allogeneic HCT per se that is independent of clinically significant GVHD. Finally, comparison of the relapse rate seen in HLA-matched allogeneic HCT recipients who received unmodified, that is, T-replete, bone marrow grafts with that observed in recipients of allogeneic grafts from which T cells had been depleted prior to infusion revealed that the antileukemic effect associated with allogeneic HCT is impaired by removal of T cells from the bone marrow graft.

Identifying the genetic determinants, effector cells, and target molecules of the GVT effect Autologous and syngeneic HCT

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Although the term “GVT effect” as commonly used tacitly implies an antitumor effect that develops in the setting of allogeneic HCT and is mediated by donor-derived cells, it is possible that a GVT effect might also occur in the setting of autologous or syngeneic HCT. Identification

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recognition of major histocompatibility (MHC) class II determinants, and can recognize and eliminate some MHC class II-expressing tumor cells. These preclinical studies consequently prompted clinical trials of cyclosporine administration after autologous or syngeneic HCT to induce syngeneic GVHD and enhance antitumor activity [42,43]. Although many patients in these trials did develop signs of GVHD, the induced GVHD was not associated with superior antitumor responses. In recent years, interest in potentiating GVT activity after autologous or syngeneic HCT has increasingly focused on stimulating T-cell responses against tumor-specific or tumor-associated antigens encoded by genes that are over or aberrantly expressed in the recipient’s tumor cells. In general, these antigens are also potential targets for immune therapy after allogeneic HCT, and they will therefore be discussed in that context. Allogeneic HCT Fig. 18.1 Actuarial probability of relapse among 2254 recipients of allogeneic bone marrow transplantation from human leukocyte antigenidentical sibling donors transplanted for chronic myeloid leukemia in first chronic phase, acute lymphoblastic leukemia in first remission, or acute myeloid leukemia in first remission, according to the type of graft and the development of acute (AGVHD) or chronic (CGVHD) graft-versus-host disease. (Reproduced from [28], with permission.)

of putative GVT activity associated with autologous or syngeneic HCT and mediated by cells contained in or derived from the autologous or syngeneic hematopoietic cell graft has been difficult due to the lack of appropriate controls. In theory, graft-derived immune cells directed at tumor-specific or tumor-associated antigens could mediate GVT responses in the autologous or syngeneic HCT setting. Examples of potential tumor-specific or -associated antigens include the BCR–ABL fusion protein expressed in CML, the PML–RARα fusion protein expressed in acute promyelocytic leukemia, and proteins such as WT1 and PR3 that are overexpressed in both myeloid and lymphoid leukemias. The demonstration that the relapse rate after identical twin transplantation is inversely correlated with the nucleated cell dose in the syngeneic graft has suggested, albeit weakly, that GVT activity may, indeed, occur after syngeneic HCT [33]. To date, however, little, if any, experimental evidence for a spontaneous antitumor effect mediated by graft-derived immune cells after either autologous or syngeneic HCT has been reported. Stimulation or potentiation of GVT activity in autologous or syngeneic hematopoietic cell grafts has been evaluated in numerous clinical trials. Post-transplant administration of interleukin-2 (IL-2) without or with ex vivo-activated autologous effector cells has been extensively evaluated in the autologous HCT setting [34–38], with inconsistent clinical results. An attempt to stimulate GVT activity in syngeneic HCT recipients based on adoptive transfer of donor lymphocytes and killed autologous leukemic cells, in order to immunize the infused donor lymphocytes against the autologous tumor, was associated with long-term disease-free survival in a small subset of treated patients [39,40]. The contribution of immunotherapy with donor cells and autologous tumor to the clinical outcome could not, however, be assessed. Studies in rodent models have demonstrated that administration of cyclosporine after autologous or syngeneic HCT can induce an autoimmune syndrome, termed syngeneic GVHD, with acute and chronic phases, that closely resembles GVHD seen after allogeneic HCT [41]. The autoreactive effector lymphocytes in rodent syngeneic GVHD show promiscuous

The critical genetic determinants, effector cells, and target molecules of the GVT effect associated with allogeneic HCT are strongly influenced by the degree and nature of genetic disparity between donor and recipient, the source, composition, and processing of the hematopoietic cell graft, and the recipient tumor type. A preponderance of evidence suggests that donor CD4+ and CD8+ T cells recognizing peptide–MHC complexes on the surface of recipient cells are the central mediators of GVT activity in HCT recipients who receive T-replete grafts from MHC-matched donors. Although there is no direct evidence as yet that donor natural killer (NK) cells contribute significantly to GVT in this setting, accumulating circumstantial evidence suggests the possibility of an NK contribution to GVT activity against myeloid leukemias. In contrast, numerous studies suggest that donor NK cells are likely to play a central role in GVT responses that occur in recipients of hematopoietic cell grafts from MHC-mismatched donors, particularly if the degree of MHC mismatch requires the in vivo or in vitro depletion of T cells from the donor hematopoietic cell graft in order to prevent lethal GVHD. The characteristics of GVT responses in the MHC-matched and MHCmismatched settings will therefore be discussed separately, below. GVT in haploidentical and MHC-mismatched HCT For those patients for whom no MHC-matched donor can be identified, hematopoietic cell grafts from haploidentical related donors can be considered. To lower the risk of severe GVHD, such grafts are usually extensively depleted of T cells prior to infusion (~1 × 104 CD3+ cells/kg), or the recipients are treated with agents such as cyclophosphamide or antithymocyte globulin in the early post-transplant period to deplete donor T cells in vivo. (HCT from haploidentical related donors is more extensively discussed in Chapter 46.) Analysis of disease response and donor–recipient genotypes at the MHC and at the killer-cell immunoglobulin-like receptor (KIR) locus on chromosome 19q in recipients of T-cell-depleted haploidentical related grafts revealed that donor– recipient incompatibility for the subset of KIR receptors with inhibitory function was strongly associated with potent GVT activity [44,45]. Functional analysis of donor NK clones from the subset of transplant pairs with incompatibility for inhibitory KIR – defined as the presence of donor NK cells that expressed as their sole inhibitory KIR a receptor for an MHC class I specificity that was expressed by the donor but absent in the recipient, termed “missing self” recognition – also revealed potent in vitro cytotoxicity against recipient lymphocytes and leukemic blasts. Moreover, the NK cytotoxicity was demonstrable primarily against lymphohematopoietic cells – including human leukemia engrafted in NOD/ SCID mice [45] – and not against nonhematopoietic cells, which was thought to explain the potent GVT activity observed in the absence of clinically significant GVHD. A surprising finding in these groundbreak-

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ing studies was the observation that GVT activity associated with donor NK alloreactivity in haploidentical HCT was far more effective against myeloid leukemia than against lymphoblastic leukemia, a finding which to date has not been entirely explained. It is possible that ALL cells do not express the cognate ligand for one or more activating KIRs expressed by alloreactive donor NK cells, appropriate ligation of which is also thought to be required for the activation of NK cytotoxicity [46]. The demonstration of a likely important role for donor NK alloreactivity in T-cell-depleted, haploidentical related HCT has prompted the formulation of clinical algorithms for predicting GVT activity, as defined by freedom from relapse, in this [47–49] as well as other [50,51] transplant settings. An alternative to the “missing self” NK alloreactivity model, the “missing ligand” model, was proposed, which more broadly defines donor–recipient KIR incompatibility as the absence in the recipient of any MHC class I specificity for which donor NK cells express inhibitory KIR, regardless of whether the donor expresses that MHC specificity [48]. This model has been used with some success to predict leukemic control – the proxy for GVT activity – in HCT recipients of T-cell-depleted grafts from MHC-matched siblings [50] and in recipients of grafts from unrelated donors [51], thus suggesting a possible contribution of donor NK alloreactivity to GVT responses in these transplant settings. Although it is still unclear whether the “missing self” or “missing ligand” model of donor NK alloreactivity more accurately predicts GVT activity in HCT recipients who receive T-cell-depleted grafts from matched or mismatched donors – an active debate continues [52] – substantial clinical and laboratory evidence does support an important role for alloreactive donor NK cells in GVT responses that occur in the T-depleted, mismatched HCT setting. GVT activity in HCT recipients of T-replete or partially T-depleted grafts from MHC-mismatched donors could theoretically be mediated by alloreactive donor T cells, alloreactive donor NK cells or both. Assessment of the relative contributions of donor T and NK cells to GVT activity in this setting is limited by the dearth of relevant clinical and laboratory data. It is likely that the contributions of donor T and NK cells are in part determined by the extent of T-cell depletion and the number and identity of MHC loci at which the donor and recipient are mismatched. In particular, donor T cells are likely to play the more important role in recipients mismatched for MHC class II but not class I alleles, because the available evidence suggests that the specificity of NK cell alloreactivity is determined primarily by the MHC class I, but not class II, molecules expressed on their target cells [46]. Limited experimental data have suggested that GVHD and GVT reactions in recipients of T-replete grafts from donors mismatched at a single MHC class II locus can be dominated by donor T cells that are heavily biased toward reactivity with the mismatched recipient allele [53]. GVT in MHC-matched HCT Donor–recipient disparity at genetic loci encoded within the MHC on chromosome 6p is the single most important risk factor for the development of severe, life-threatening GVHD after allogeneic HCT [54,55]. Consequently, most patients who undergo allogeneic HCT receive hematopoietic cell grafts from donors with whom they are either genotypically or phenotypically matched at the MHC. The increased relapse rate observed in recipients of T-depleted hematopoietic cell grafts from MHC-matched donors [28,30] indicates that donor T cells play a critical role in GVT responses after MHC-matched HCT. Studies in which CD8+depleted DLI from MHC-matched donors [56–58] was administered to patients with post-transplant relapse have demonstrated that CD4+ T cells can mediate GVT activity. A role for CD8+ T cells in human GVT has also been suggested by the observation of in vivo expansions of CD8+ T cells with in vitro reactivity against recipient tumor cells during GVT reactions after HCT [59] or DLI [60–66]. There is little direct evidence

that NK cells mediate GVT activity after T-replete, MHC-matched HCT, but a role for NK cells has been suggested by reports that the donor genotype at the KIR locus is associated with risk for relapse [67,68]. In vitro studies suggest that CD56+ NK cells could contribute to GVT activity after MHC-matched HCT for CML [69,70]. Minor histocompatibility antigens GVHD and GVT activity occur in the majority of allogeneic HCT recipients who receive T-replete hematopoietic cell grafts from genotypically or phenotypically MHC-matched donors. Thus, GVHD and GVT can also be triggered by donor–recipient genetic disparity at polymorphic loci outside the MHC, which are consequently referred to as minor histocompatibility loci. Studies in both mice [71,72] and humans [73,74] have demonstrated that many minor histocompatibility loci encode short peptides – termed minor histocompatibility antigens (mHAs) – that are encoded by polymorphic genes and are presented on the cell surface by MHC class I and class II molecules, where they can be recognized by donor CD8+ and CD4+ T lymphocytes, respectively. CD8+ T cells recognize peptides that are typically eight to 11 residues in length and are derived from the degradation of intracellular proteins by the proteasome, transported into the endoplasmic reticulum (ER) by the transporter associated with antigen processing, and are delivered to the cell surface as a trimolecular complex with MHC class I molecules and β2-microglobulin [75]. CD4+ T cells recognize peptides that are derived from lysosomal degradation of phagocytosed exogenous proteins or proteins produced endogenously within the cell, and are delivered to the cell surface as a complex with heterodimeric MHC class II molecules. Peptides associated with MHC class II molecules tend to be longer than those associated with MHC class I, due to the more “open” configuration of the ends of the peptide-binding groove in the class II heterodimer [76]. Donor T cell responses to mHAs presented on the surface of recipient cells are thought to be the central mediators of both GVHD and GVT activity. Molecular characterization of mHAs CD8+ and CD4+ T cell clones specific for mHAs presented by MHC class I and class II molecules, respectively, can be isolated from most HCT recipients of T-replete grafts [77]. Biochemical and genetic techniques have been used to identify the genes that encode a small but steadily increasing number of human mHAs recognized by T-cell clones from HCT recipients, and to define the nature of the polymorphism in these genes (reviewed in [78]). The majority of human mHAs that have been molecularly characterized to date are presented by MHC class I molecules and recognized by CD8+ T cells. This bias is primarily attributable to technical challenges posed by identification of MHC class IIrestricted antigens that have not yet been completely overcome, rather than to any fundamental difference in the nature of the CD4+ and CD8+ T-cell response to the polymorphic genes that encode mHAs. The first human mHAs to be identified [73,74] were characterized biochemically by purifying peptide/MHC class I molecules from large numbers of mHA-expressing cells, eluting the peptides from the MHC class I molecules through exposure to low pH, fractionating the resulting complex mixture of peptides using several rounds of reversed-phase high-partition liquid chromatography (HPLC), and testing the resulting fractions for their ability to enable T-cell recognition of target cells that do not natively express the mHA but do express the appropriate MHC molecule that “presents” the minor histocompatibility peptide to T cells. HPLC fractions that retain the ability to reconstitute T-cell recognition are characterized by mass spectrometry to determine the most likely sequences of the specific peptides in each fraction. Synthetic peptides corresponding to these candidate peptides can then be tested individually for the ability to reconstitute recognition.

The Human Graft-versus-Tumor Response – and How to Exploit It

A productive alternative approach to identification of genes encoding mHAs has been based on complementary DNA (cDNA) expression cloning [79,80]. This approach involves construction of a cDNA library from an mHA-positive cell line, creation of cDNA pools typically containing from 10 up to approximately 100 individual cDNA clones per pool, co-transfection of the cDNA pools in a parallel fashion along with a cDNA encoding the relevant MHC restricting molecule into mHAnegative host cells, and short-term co-culture of the transfectants with mHA-specific T cells. Aliquots of supernatant from the co-cultures are then tested for cytokine release (interferon-gamma [IFN-γ] or tumor necrosis factor) from the mHA-specific T cells to identify pools containing the cDNA that encodes the antigen. Positive cDNA pools are subcloned, and the subclones are screened in an analogous fashion until a single cDNA encoding the mHA of interest is identified. A third strategy for identification of genes encoding mHAs employs genetic linkage analysis to determine the chromosomal localization of the gene [81–83]. Candidate genes in the identified chromosomal region can then be evaluated directly, by testing cDNAs derived from all of the known protein-coding genes in that region for their ability to stimulate mHA-specific T cells in a transfection assay. Alternatively, bioinformatic analysis of the candidate genes in the region [82] or high-resolution linkage analysis [83] may permit focusing the search on a small subset of candidates, which can then be directly tested in a transfection assay. In one study, for example, analysis of the reported tissue expression profiles of each of the candidate genes in the chromosomal region with strong linkage was performed, in order to identify the subset whose expression profiles most closely resembled the tissue expression profile of the two mHAs under study [82].

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encoded by autosomal minor histocompatibility loci reveals that their polymorphism is generated by two fundamentally distinct genetic mechanisms: variation in human genome sequence, which creates most of the autosomal mHAs identified to date, and variation in human genome structure, which creates only two known mHAs, but likely creates other mHAs that have yet to be characterized. Single nucleotide polymorphisms (SNPs) represent the most common type of sequence variation in the human genome [86], and most mHAs are created by nonsynonymous SNPs that occur in the coding sequence of normal self proteins and create amino acid polymorphisms in the sequence of these proteins. Such amino acid polymorphisms can alter how peptides are generated from self-proteins inside the proteasome [87], are transported into the endoplasmic reticulum by the transporter associated with antigen processing [88], bind to MHC molecules [89], or are recognized by T cells when presented on the cell surface in a complex with MHC [79]. A single nucleotide deletion polymorphism

Classification of mHA-encoding genes The genetic loci encoding mHAs recognized by T cells after MHCmatched HCT can be divided into two broad categories that are distinguished by their chromosomal location. The first category comprises a small class of Y chromosome genes, 15 in total [84,85], that encode male-specific mHAs (H-Y) that are targets for female T cells in sexmismatched transplants (Fig. 18.2). The genes in this class are unique among Y chromosome genes in that they have X chromosome homologues that encode proteins that are 81–99% identical in sequence with the Y-encoded isoforms, and they are expressed both in and broadly outside the testis. They are likely derived from the ancestral autosome(s) from which the human X and Y chromosomes evolved [85]. The extensive sequence disparity between the protein products of these Y chromosome genes and the protein products of their X chromosome homologues creates a large number of male-specific peptides that could potentially serve as targets for female T cells in an MHC-matched HCT setting. Moreover, the extratesticular expression of these genes enables the presentation of these peptides to the female system outside the immunologic sanctuary of the testis. The immunologic significance of H-Y antigens is likely enhanced by the fact that the Y chromosome is inherited as a single functional genetic element, and therefore all H-Y antigens are in complete linkage disequilibrium. Twelve human H-Y antigens have been identified to date, and it is likely that many more remain to be identified. A heterogeneous group of autosomal loci widely distributed throughout the genome comprise the second major class of genes that have been shown to encode mHAs (Fig. 18.3). Although some of these loci encode well-characterized proteins with known functions, several were not previously known to generate a protein product. Indeed, for several molecularly characterized, autosomally encoded mHAs, the demonstration that they encode peptides presented on the cell surface and recognized by T cells constitutes the only experimental evidence that they are translated [79]. Molecular characterization of the antigenic peptides

Fig. 18.2 Location and characteristics of the 15 genes on the human Y chromosome with the potential to encode H-Y antigens. The size, in residues, of each gene’s largest predicted protein isoform is indicated, as is the percentage of sequence identity between this isoform and the protein encoded by the homologous X chromosome gene. The pseudoautosomal regions are indicated with solid black fill, the regions with homology to the X chromosome with stippling, and the centromeric region with crosshatching. No percentage identity figure is specified for the TMSB4Y, CYorf15A, and CYorf15B genes because the major protein products of these genes and of their X chromosome homologues have not yet been well defined.

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Fig. 18.3 Distribution by chromosome of genes, listed in italics, which encode molecularly defined human minor histocompatibility antigens. Multigene clusters that play important roles in histocompatibility (underlined) are listed alongside the chromosome on which they are encoded. KIR, killer cell immunoglobulin-like receptor locus; MHC, major histocompatibility complex; NKC, natural killer complex.

causing a frameshift in the coding sequence of the P2RX5 gene creates a recently identified mHA [83]. Larger-scale variation in human genome structure can also create mHAs through deletion of entire protein coding sequences. The UGT2B17 gene on chromosome 4q, for example, encodes an mHA that binds to two different MHC class I molecules – HLA-A*2902 and HLA-B*4403 – and is recognized by HLA-A*2902restricted and HLA-B*4403-restricted CD8+ T cells [80,90]. The antigenicity of this locus is attributable to a common, approximately 150 kilobase (kb) deletion polymorphism spanning the UGT2B17 locus on chromosome 4q for which a variable (2–71%, depending on race) but significant fraction of the human population is homozygous [91–94]. Hematopoietic cell donors who are homozygous for this deletion can recognize UGT2B17-derived peptides as mHAs in recipients who express the UGT2B17 protein. Expression of mHAs in normal tissues and leukemia Assessment of the expression profile of mHAs in normal and malignant tissues has provided insight into the potential contributions of mHAspecific T-cell responses to GVT activity. Many mHAs are expressed in both hematopoietic and nonhematopoietic cells in vitro [77,95], suggesting that T-cell responses to these antigens could contribute to both GVT and GVHD. However, a significant fraction of mHAs show expression that is limited to cells of hematopoietic origin [77,95,96], raising the prospect that T-cell responses against these minor antigens might con-

tribute selectively to GVT activity. In addition, CD4+ and CD8+ T-cell clones specific for mHAs that are selectively expressed in hematopoietic cells recognize lymphoid and myeloid leukemic cells in vitro [77,96– 99], and inhibit the growth of clonogenic leukemic cells in in vitro culture [98,99]. CD8+ mHA-specific cytotoxic T-lymphocyte (CTL) clones also inhibit the engraftment of human acute leukemia into NOD/ SCID mice [100], demonstrating that leukemic stem cells [101,102] express mHAs and can be targeted by mHA-specific CTLs. Contributions of mHA-specific T-cell responses to GVT Cellular and molecular dissection of immune responses occurring in HCT recipients after HCT or DLI has provided compelling evidence that mHAs expressed on recipient leukemic cells are targets of GVT responses in MHC-matched HCT. Serial flow cytometric analysis with MHC tetramers complexed with mHA peptides to detect and enumerate mHAspecific T cells in the blood of HCT recipients has demonstrated that mHA-specific T cells with in vitro reactivity against recipient leukemic cells increase in frequency in the peripheral blood before and during clinical regression of malignancy. A large in vivo expansion of CD8+, MHC/peptide tetramer+ T cells specific for an H-Y antigen encoded by the Y chromosome gene SMCY and presented by HLA-A*0201 [103] was observed in a male HLA-A*0201+ patient who had received a graft from his MHC-identical sister, and this expansion was temporally correlated with achievement of molecular remission of CML in blast crisis

The Human Graft-versus-Tumor Response – and How to Exploit It

[59]. At the peak of the response, tetramer+ cells comprised 15.9% of the CD8+ T cells in peripheral blood. MHC/peptide tetramer+ T cells isolated and purified by flow cytometry demonstrated in vitro cytotoxicity against the recipient’s leukemic blasts. Similar studies of GVT reactions occurring in patients with persistence or recurrence of malignancy after HCT who underwent DLI have identified expansions of MHC/ peptide tetramer+ cells specific for several other mHAs. In vivo expansions of CD8+ T cells recognizing the HLA-A*0201-restricted HA-1 and HA-2 mHAs encoded by the KIAA0223 [89] and MYO1G [73,104] genes, respectively, that were temporally correlated with clinical responses to DLI have been observed in HLA-A*0201+ patients with Philadelphia chromosome-positive ALL or CML [61,63–65]. A prominent CD8+ T-cell response to the mHA encoded by the P2RX5 gene was demonstrated in another patient with CML responding to DLI [83]. Cloned P2RX5 peptide-specific T cells from this patient specifically recognized CD34+ leukemic progenitor cells in vitro. mHA-specific T cells may also contribute to the regression of solid tumors after MHC-matched allogeneic HCT. Extensive regression of metastatic renal cell carcinoma was observed in one patient after three separate infusions of donor lymphocytes followed by initiation of IFNα [105]). CD8+ CTL clones recognizing multiple distinct mHAs presented by renal cell carcinoma cells were isolated from patient peripheral blood mononuclear cells obtained during and after tumor regression, including CD8+ T cells specific for the HA-1 minor antigen. The observation of HA-1 expression, which in nonmalignant cells is limited to the hematopoietic lineage [95], in primary renal cell carcinoma cells is consistent with previous studies that demonstrated aberrant expression of HA-1 on solid tumor cell lines of epithelial origin [106–108]. Although analysis with MHC/peptide tetramers of immune responses in patients responding to HCT or DLI has demonstrated that several different mHAs can serve as targets for GVT responses, it is likely that clinical GVT responses are directed at a significantly more diverse spectrum of antigens than tetramer-based analysis would suggest. Serial analysis of the peripheral blood T-cell repertoire in CML [109] or multiple myeloma [110] patients responding to DLI with CD4+-selected cells from MHC-matched sibling donors revealed the appearance of multiple distinct clonal T-cell populations that was temporally correlated with GVT activity. Moreover, complete responses observed in several CML patients after CD4+-selected DLI were associated with in vivo expansion of CD8+ T cells recognizing a number of novel, as yet uncharacterized mHAs expressed on recipient hematopoietic cells [60]. In vivo expansion of CD8+ T cells recognizing myeloma-specific antigens was observed in patients with multiple myeloma responding to CD4+-selected DLI [62]. Direct cloning of IFN-γ secreting peripheral blood T cells from three multiple myeloma or CML patients responding to unselected DLI led to the identification of CD8+ T-cell clones recognizing multiple distinct mHAs, including well-characterized antigens such as HA-1 as well as several other, as yet uncharacterized specificities [66]. Numerous retrospective analyses of MHC-matched transplant outcome have attempted to evaluate the contribution of mHA-specific T cells to GVT activity by correlating donor–recipient genetic disparity at specific minor histocompatibility loci with post-transplant relapse or remission of disease. The studies that focused on disparity at autosomal mHAs have had generally inconsistent results, but the largest of these showed no significant correlation between donor–recipient disparity at the loci encoding the HA-1 [111], HA-8 [112] or UGT2B17 [113] loci and control of leukemia after HCT. The failure to demonstrate any such correlation is most likely attributable to the fact that there are more than 50,000 polymorphic autosomal loci in the coding region of known genes that could potentially encode mHAs [114–116], and the clinical effect associated with disparity at any one locus will therefore account for a very small fraction of the total risk for relapse.

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In contrast, two large retrospective analyses of transplant outcome, however, have identified a lower incidence of relapse in male recipients of MHC-matched female hematopoietic cell grafts (F → M HCT) compared with all other donor–recipient gender combinations, suggesting that T-cell responses to H-Y antigens have a clinically measurable antileukemic effect [117,118]. The lower incidence of relapse observed after MHC-matched F → M HCT is seen in patients transplanted for CML, AML, and ALL, and is apparent even after controlling for GVHD [118]. The clinically measurable antileukemic effect associated with F → M HCT might, at first, be surprising, given the lack of an identifiable antileukemic effect associated with donor–recipient disparity at any single autosomal minor histocompatibility locus. However, this seeming paradox is readily resolved by the recognition that (1) the human Y chromosome contains at least 15 genes with the potential to encode mHAs (Fig. 18.2) [84,85], (2) each of these genes can potentially encode a significant number of distinct H-Y antigens, due to the extensive sequence disparity between their protein products and those of their X chromosome homologues, (3) the H-Y genes encode both MHC class I and class II-restricted H-Y antigens, and (4) all H-Y antigens are in complete linkage disequilibrium. Thus, in any female donor–male recipient HCT pair, the Y chromosome has the potential to stimulate a diverse repertoire of CD8+ and CD4+ T-cell responses to many distinct mHAs. T-cell responses to nonpolymorphic overexpressed antigens An increasing body of evidence indicates that nonpolymorphic antigens encoded by genes that are overexpressed in myeloid and lymphoid leukemic cells are also targets of T-cell responses that contribute to GVT after MHC-matched HCT. CD8+ CTLs specific for an HLA-A*0201restricted peptide termed PR1 that is derived from both proteinase-3 and elastase can be detected at increased frequency in the blood of many HLA-A*0201+ CML patients who achieve remission after MHCmatched HCT [119]. Proteinase-3, also known as myeloblastin, and elastase are both major constituents of the primary azurophil granules of normal promyelocytes as well as AML and CML blasts. PR1-specific CTLs recognize myeloid leukemia cells in vitro [120] and inhibit the growth of CML colony-forming units [121]. High levels of proteinase-3 or elastase expression in CD34+ myeloid progenitor cells in patients with advanced-phase CML are also associated with improved outcome after allogeneic HCT [122]. The protein product of the WT1 gene may also be a target for posttransplant GVT responses. Although early studies suggested that WT1specific T cells were not commonly generated in allogeneic HCT recipients [123], post-transplant detection of CD8+ WT1-specific CTL in five out of 10 ALL patients has recently been reported [124]. WT1-specific CTLs were only detected in those recipients in whom WT1 expression was detected pretransplant, and emergence of WT1-specific CTL post transplant was associated with a decrease in WT1 expression. Evidence from murine studies suggests that donor T-cell responses to tumor-associated antigens such as PR1 and WT1 may be driven by the alloresponse associated with GVHD that occurs in the early post-transplant period [125]. The identification of spontaneous T-cell responses to leukemia-associated proteins such as proteinase-3/elastase and WT1 has provided the rationale for development of strategies for exploiting immunity to PR1 and WT1 to enhance GVT activity post transplant. B-cell contributions to GVT Analysis of sera obtained from allogeneic HCT recipients has revealed that donor-derived humoral responses to polymorphic – particularly male-specific – and nonpolymorphic antigens expressed by recipient cells occur commonly in HCT recipients, and in some cases may contribute to GVT. The possibility of antibody responses to H-Y proteins was first suggested in 1979, when immunoglobulin (Ig) M antibodies

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were found in the serum of a heavily transfused female patient with aplastic anemia that were reactive mainly, but not exclusively, with cells from male donors [126]. Analysis of males undergoing F → M HCT has subsequently demonstrated that high-titer IgG responses against Ychromosome proteins can be detected in approximately 50% of cases [127,128]. Mapping the fine specificity of H-Y-specific antibodies has demonstrated a strong bias toward regions of Y-chromosome proteins where there is nonidentity with the products of their X chromosomeencoded homologues [127], consistent with the antigenic specificity of H-Y T-cell responses. In a study of 121 HCT patients and 134 healthy control individuals, antibodies reactive with the protein products of one or more of five Y chromosome genes – DBY, UTY, ZFY, RPS4Y, and EIF1AY – were detected in 52% of male patients with female donors but only 9% of male patients with male donors, and in 41% of healthy females but only 8% of healthy males [128]. The presence of antibody responses to one or more of the five Y chromosome genes was also strongly associated with the maintenance of disease remission, suggesting that humoral responses to Y chromosome gene products may be a marker for the selective graft-versus-leukemia effect observed in male recipients of MHC-matched female hematopoietic cell grafts [118]. SEREX (serological identification of antigens by recombinant cDNA expression cloning) analysis of allogeneic HCT recipients responding to DLI has demonstrated that GVT activity is associated with humoral responses to proteins expressed in recipient tumor cells. HCT recipients who achieve complete remission of CML [129–131] and multiple myeloma [132,133] after DLI administered for the treatment of persistent or recurrent disease make high-titer IgG responses to nonpolymorphic antigens that are over- or aberrantly expressed in CML or myeloma cells. Although a large fraction of the humoral response is directed at molecules that are not known to be expressed on the cell surface, antibodies to B-cell maturation antigen (BCMA), which is expressed on the cell surface, were found only in post-DLI responder sera from patients with multiple myeloma [133]. Serum containing anti-BCMA antibodies was able to induce complement-mediated lysis and antibody-dependent cellular cytotoxicity of a tumor cell line transfected with a BCMA cDNA,

as well as of primary myeloma cells expressing BCMA, suggesting that BCMA may be an important target of the GVT response in multiple myeloma. As has been demonstrated for other tumor antigens first identified by SEREX [134,135], it is likely that the targets of antibody responses after DLI are also specific targets of GVT-associated CD4+ or CD8+ T-cell responses, or both. Human genomic diversity and the number of mHAs The completion of the reference human genome sequence in 2003 has stimulated intense research activity focusing on how individual human genomes differ from this reference DNA sequence and from each other. It is now clear that a comparison of any two human genomes will show extensive differences not only in sequence [86], but also in structure [136,137] (Fig. 18.4 and Table 18.1). It is estimated that the human genome contains 10–15 × 106 SNPs, of which at least 5 × 104 occur in the coding sequence of known genes and are associated with amino acid sequence changes [86,114,116]. Variation in copy number, including deletion polymorphism, in the human genome is also extremely common, and one recent study found evidence for CNV involving 360 Mb of genomic sequence, or 12% of the genome [136]. Common deletion polymorphisms, including the UGT2B17 deletion polymorphism that generates two different mHAs [80,90], that span 267 known and predicted genes have already been identified [115], and it is likely that many more remain to be identified. The importance of non-SNP structural variation in the human genome was highlighted by a recent study that determined the diploid genome sequence of an individual human [138]. Although non-SNP DNA variants accounted for only 22% of the 4.1 × 106 genetic variants identified in this donor, they accounted for 74% of all variant bases. The enormous extent of human genomic diversity has profound implications for efforts to understand how polymorphism in the genome can elicit immune responses that contribute to GVT, and also for efforts to exploit these immune responses to decrease relapse and improve HCT outcome. Extensive sequence and structural polymorphism in the genome implies that there are a very large number of protein and peptide

Chromosome

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Fig. 18.4 Idealized representation of the different types of sequence and structural variation in the human genome. (Adapted from [194], with permission.)

The Human Graft-versus-Tumor Response – and How to Exploit It

structural variation in the human genome has stimulated the search for novel polymorphic genetic loci that could potentially be exploited to enhance GVT activity.

Table 18.1 The extent of human genetic variation Collective human genome Base pairs SNPs Validated HapMap phase 2 SNPs Validated HapMap phase 2 exonic SNPs Validated HapMap phase 2 nonsynonymous coding SNPs Estimated number of discrete segmental CNVs Base pairs affected by CNVs

3.3 × 1–1.5 × 4× 7× 3× >1,500 3.6 ×

109 107 106 104 104 108

Individual human genome SNP frequency Estimated number of CNVs per individual genome

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1/300 base pairs >100

CNV, copy number variant; SNP, single nucleotide polymorphism. Adapted with permission from Mullaly and Ritz [194].

sequences that, in the setting of MHC-matched allogeneic HCT, could potentially stimulate and serve as targets for donor T- and B-cell responses that could mediate GVT activity. Dissection of GVT responses at the cellular and molecular level is, indeed, beginning to show that GVT in any one HCT recipient is likely to be mediated by a broadly focused immune response directed at both polymorphic alloantigens and nonpolymorphic, over- or aberrantly expressed self-antigens on recipient tumor cells. The diversity of this response is likely to be one of the keys to the remarkable potency of the GVT effect. Nonetheless, posttransplant persistence or recurrence of disease remains a major cause of treatment failure after allogeneic HCT, and developing a more comprehensive understanding of the GVT response is an urgent research priority. Recognition of the enormous diversity in the genome demonstrates that there is abundant polymorphism to be exploited, but also suggests that whole-genome approaches to understanding GVT will likely provide the key to its successful exploitation.

Strategies for exploiting the GVT response Early attempts to manipulate the GVT effect and decrease the relapse rate after allogeneic HCT were motivated by the recognition that GVT is mediated by cells contained in or derived from the hematopoietic cell graft, and that exogenous immune suppression, although it suppresses GVHD, also suppresses GVT. Complete omission of GVHD prophylaxis in the MHC-matched sibling BMT setting led to severe and hyperacute GVHD, and the increase in GVHD-related mortality precluded the identification of any decrease in the relapse rate [139]. The prophylactic administration of donor buffy-coat cells after MHC-identical sibling BMT, with a standard course of methotrexate alone as GVHD prophylaxis, was also associated with an increased rate of severe, and often fatal, acute GVHD, and also prevented the detection of any effect on the relapse rate [140]. Characterization of T- and B-cell immune responses associated with GVT activity after HCT or DLI, and identification of their antigenic targets, has demonstrated that the targets of GVT and GVHD responses are not completely overlapping. Many current efforts to manipulate GVT are consequently focused on the development of therapeutic strategies for enhancing donor immune responses against defined antigens that are selectively or exclusively expressed on recipient tumor cells, and not widely expressed on tissues that are targets for GVHD. In addition, characterization of the extensive sequence and

Identifying genomic loci associated with GVT activity The identification of extensive sequence and structural variation in the human genome suggests exciting possibilities for exploiting genomic diversity to enhance GVT, but also poses several challenging new questions. What fraction of the polymorphic loci in the genome can elicit T- or B-cell responses that can contribute to GVT? Are there specific autosomal loci at which donor–recipient disparity makes a measurable contribution to GVT activity? Are there genetic variants in the donor or the recipient that influence GVT activity in a manner that is independent of donor–recipient matching or mismatching? The advent of microarraybased genotyping technologies that permit simultaneous determination of SNP genotype and of copy number at hundreds of thousands of unique sites distributed throughout the genome has raised the prospect of using whole-genome association analysis to address these questions in a comprehensive fashion. A genome-wide, case-control approach to the identification of loci associated with GVT could potentially facilitate the identification of polymorphic loci at which donor–recipient mismatching is significantly associated with decreased risk for relapse, and could identify genetic variants in the donor or in the recipient that, independent of donor–recipient matching or mismatching, are also associated with decreased relapse risk. MHC genotypically identical sibling pairs represent a transplant population that is uniquely suited to the application of whole-genome association analysis for the identification of loci associated with GVT activity. Full siblings share 50% of their genomes and, on average, are expected to be genetically identical for both alleles at 25% of their genomes. If risk of relapse is in fact measurably influenced by donor– recipient disparity at specific autosomal loci, it is reasonable to hypothesize that HCT recipients who relapse post transplant will, on average, show more genetic identity with their donors for both chromosomes at such putative “GVT loci” than recipients who do not relapse. Thus, in the case of MHC-identical siblings, where donors and recipients are selected for genetic identity for both alleles within the MHC, this hypothesis predicts that HCT recipients who relapse post transplant will show more genetic identity for both alleles with their donors than the expected 25% at GVT loci, and patients who do not relapse post transplant will show less genetic identity for both alleles with their donors than the expected 25% (Fig. 18.5). This hypothesis is currently being tested in a population of 1000 MHC-identical sibling donor–recipient pairs – 500 “cases” in which the recipient relapsed, and 500 “controls” in which no relapse occurred – who underwent T-replete allogeneic HCT for the treatment of a myeloid malignancy at a single transplant center. Donor selection For patients for whom several potential hematopoietic cell donors can be identified, deliberate selection of a donor who is mismatched with the recipient at one or more “GVT loci” represents one potential strategy for enhancing GVT. In the T-cell-depleted haploidentical transplant setting, for example, where alloreactive donor NK cells are primary mediators of GVT responses, selection of a donor with a specific KIR genotype and appropriate MHC class I mismatch with the recipient can take advantage of predicted donor KIR–recipient MHC incompatibilities [141]. Since most HCT recipients will have several potential haploidentical donor candidates from which to choose – including parents, siblings, and children – donor selection based on donor–recipient KIR–MHC

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Relapse (cases) No relapse (controls)

Fraction identical at both alleles

1.00

GVT locus Δ/2 0.25 Δ/2

6p

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Fig. 18.5 Identification of a putative “graft-versus-tumor (GVT) locus” through assessment of donor–recipient genetic identity for both alleles at a large number of polymorphic loci distributed across the entire genome. Major histocompatibility complex (MHC)-identical sibling donor–recipient pairs are genotyped at hundreds of thousands of polymorphic loci distributed across the genome using high-density oligonucleotide arrays. Although the average fraction of donor–recipient pairs who have identical genotypes at any given polymorphic locus outside the MHC is expected to be 0.25, the observed fraction will fluctuate around this mean value. At a hypothetical “GVT locus,” the observed fraction of donor–recipient pairs with identical genotypes in a population in which the recipient relapsed (“cases”) is expected to be significantly higher than the observed fraction of pairs with identical genotypes in a population of pairs in which the recipient did not relapse (“cases”), with an effect size of Δ.

MHC-matched sibling donors of patients with multiple myeloma have been safely vaccinated with purified idiotype protein harvested from their respective siblings, and cellular and humoral idiotype-specific immune responses were subsequently detected in the donors as well as in the sibling recipients to whom they donated bone marrow [145–147]. CML patients with post-transplant relapse have been safely vaccinated with BCR–ABL fusion peptides, and CD4+ BCR–ABL fusion peptidespecific T-cell responses were subsequently detected in several patients [148]. A vaccine containing the nonameric PR1 peptide derived from the proteinase-3 and elastase proteins in incomplete Freund’s adjuvant has been administered to patients with AML, CML, and myelodysplastic syndrome in the allogeneic HCT as well as nontransplant settings, and has been associated with durable complete remissions in several patients [149], and WT1-derived peptide vaccines administered to patients with AML in the nontransplant setting have elicited clinical responses [150,151]. Leukemic cells from CML [152] and AML [153– 155] patients acquire characteristics of dendritic cells and stimulate CTL responses when cultured in vitro with selected cytokines, suggesting that such cells could be used for a cellular leukemia vaccine. A cellular vaccine consisting of autologous leukemic blasts admixed with skin fibroblasts transduced with adenoviral vectors encoding human IL-2 and CD40L was administered to patients in remission after HCT, and stimulated donor CD8+ and CD4+ T cell responses to the recipient leukemic blasts [156]. Preliminary results of other vaccination trials with wholecell vaccines, including a trial in AML patients undergoing reducedintensity HCT of vaccination with autologous, irradiated AML cells transduced with the granulocyte–macrophage colony-stimulating factor (GM-CSF) gene, have appeared in abstract form, with final results expected in the near future. Adoptive cellular therapy – donor lymphocytes

genotypes to optimize GVT activity will likely become an increasingly important consideration in haploidentical, T-cell-depleted HCT. Donor selection based on donor–recipient KIR–MHC genotypes could also conceivably prove to be useful in mismatched unrelated donor HCT, but retrospective studies have not consistently identified an association between donor–recipient KIR ligand mismatching and relapse rate in this setting [51,142–144]. For the minority of patients for whom multiple related or unrelated MHC-matched donor candidates are identified, donor selection based on donor–recipient disparity at non-MHC loci awaits the unambiguous identification of loci at which mismatching has a statistically significant effect on post-transplant relapse. The subject of donor selection is more extensively reviewed in Chapter 48. Donor or recipient vaccination Active immunotherapeutic approaches to enhancing GVT based on pretransplant vaccination of HCT donors or post-transplant vaccination of recipients are currently being investigated at many transplant centers. Vaccination strategies can be employed to augment donor T-cell responses to specific recipient mHAs, or alternatively to tumor-specific or tumor-associated antigens expressed on recipient tumor cells. The immunogens currently being evaluated in clinical trials include whole proteins, specific peptides or peptide mixtures, DNA, and whole cells, including tumor cells and dendritic cells, some of which are genetically or biochemically modified to enhance their immunogenicity. A variety of adjuvants are also being administered with the immunogens. The results of published trials demonstrate that vaccination is in general safe and can elicit frequent humoral or cellular vaccine-specific immune responses, and occasional clinical responses.

The remarkable potency of DLI for the treatment of recurrent malignancy after HCT, and its frequent serious toxicities – most notably GVHD and myelosuppression [14,16,17] – have generated intense interest in the development of more selective adoptive cellular therapies that have greater antitumor efficacy with less toxicity to normal, nonmalignant tissues. Strategies for exploiting GVT that are based on the adoptive transfer of donor-derived cells permit ex vivo selection and manipulation of the cellular product, and potentially of the donor from whom it is derived, prior to infusion, as well as independent manipulation of the recipient in order to increase the safety and the efficacy of adoptive therapy. Adoptive transfer approaches also allow the genetic characterization or marking of cells prior to infusion to facilitate precise monitoring of the in vivo persistence and trafficking of infused cells. Administration of DLI in escalating doses and selective depletion of CD8+ cells from donor lymphocyte products prior to infusion have been performed in an attempt to harness the antileukemic effect of DLI while minimizing the toxicity attributable to GVHD. Sequential administration of escalating doses of CD3+ donor lymphocytes has proven to be an effective approach to the treatment of recurrent CML post transplant [157,158]. Doses in the range of 1–2 × 107 CD3+ cells/kg recipient weight have been associated with high response rates and low rates of clinically significant GVHD. Patients who do not respond to an initial dose at this level often respond to subsequent higher doses, at which the risk of GVHD is greater. Based on the hypothesis that the therapeutic efficacy of DLI for CML is attributable to the activity of CD4+ T cells, and that the GVHD associated with DLI is attributable to the activity of infused CD8+ T cells, donor lymphocyte products selectively depleted of CD8+ T cells have been used to treat recurrent CML and multiple myeloma post transplant [56,57]. Complete responses were observed in a majority of the patients with CML and many myeloma patients, with

The Human Graft-versus-Tumor Response – and How to Exploit It

rates of GVHD that were lower than would be expected based on historical controls. Whether the lower rates of GVHD were attributable to the depletion of GVHD-causing CD8+ T cells from the DLI products administered in these studies, or simply due to a lower mean dose of cells administered, cannot be determined from the available data. Concomitant administration of cytokines with DLI and ex vivo activation of DLI products prior to infusion have been explored as means to enhance the antitumor efficacy of adoptive therapy with donor lymphocytes. IL-2 administration during or after DLI has dose-dependent effects that range from prolonging the in vivo survival and persistence of adoptively transferred cells at low doses to nonspecific activation of both CD3+ T cells and NK cells at higher doses [15]. A reproducibly significant enhancement of the antitumor efficacy of DLI by administration of IL-2 has yet to be demonstrated. A retrospective analysis suggested that administration of IFN-α did not significantly improve the efficacy of DLI for treatment of CML recurrence [14], but DLI and IFN-α combined with GM-CSF has produced clinical responses in patients who were refractory to DLI or to DLI and IFN-α [152]. IFN-α can, however, elicit durable tumor regression in patients with persistence of metastatic renal cell carcinoma after reduced-intensity allogeneic HCT and DLI [23,105]. Whether the effect of IFN-α in this setting is attributable to a primary effect on activation or expansion of donorderived effector cells in the host, or alternatively to a direct antitumor effect on recipient tumor cells, is unknown. Infusions of donor lymphocytes activated and expanded ex vivo via CD3–CD28 co-stimulation has been used in combination with chemotherapy and conventional DLI to treat patients with a variety of aggressive malignancies whose tumors recurred or persisted post transplant. Complete remissions were seen in eight of 18 patients treated in one phase I trial [159], suggesting that further studies of ex vivo activated DLIs are warranted. Adoptive cell therapy with antigen-specific effector cells The low frequency of T cells in unselected donor lymphocyte products that are reactive with recipient tumor cells, combined with the presence in such products of alloreactive cells that contribute to GVHD, has stimulated interest in adoptive therapy with products manipulated ex vivo to selectively expand the subsets of effector cells reactive with recipient tumor cells. Infusion of donor-derived CTL lines generated by repetitive in vitro stimulation of donor peripheral blood mononuclear cells with recipient CML cells was followed by a complete remission in a CML patient with a post-transplant recurrence of accelerated-phase disease who had not responded to unselected DLI [160]. Although the contribution of the ex vivo manipulated cells to elimination of residual leukemia in this case could not be unambiguously established, the results demonstrate that adoptive therapy with products that are enriched for tumor-reactive cells will likely be more effective than infusion of unselected donor lymphocytes. Techniques for large-scale ex vivo expansion of antigen-specific T cell clones [161] are now being used to support clinical trials evaluating the safety and efficacy of adoptive cell therapy with homogeneous populations of cloned CD8+ and CD4+ T cells specific for antigens selectively or exclusively expressed on recipient tumor cells. Three broad categories of target antigens expressed on tumor cells are being investigated: polymorphic mHAs encoded by genes that are expressed on normal and malignant hematopoietic cells but not widely expressed in nonhematopoietic tissues, tumor-specific antigens, and nonpolymorphic tumorassociated antigens that are encoded by normal cellular genes and over- or aberrantly expressed in tumor cells. There are advantages and limitations associated with adoptive cell therapy directed at each of these three categories of target antigen, and thus they will be discussed separately.

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Defined mHAs that are expressed selectively in normal and malignant hematopoietic cells include those encoded by the HA-1 (KIAA0223) [89], MYO1G [73,104], HB-1 [79], UTY [162,163], BCL2A1 [82], CENPM [164], ECGF1 [165], P2RX5 [166], and HMSD [167] genes. It is likely that many more such mHAs with expression limited to hematopoietic cells remain to be identified [77]. The applicability of adoptive T-cell therapy with CD8+ T cells specific for MHC class I-restricted mHAs, however, is restricted to HCT recipients who (1) express the MHC class I allele that presents the mHA(s) to CD8+ T cells, and (2) are appropriately discordant with their donors for expression of the mHA (donor negative, recipient positive). Therefore, for mHAs that are presented by uncommon MHC class I alleles or are created by SNPs for which the minor allele frequency is low, these restrictions imply that eligibility for adoptive T-cell therapy targeting such antigens will be limited to a very small minority of HCT recipients. For any candidate hematopoietic-specific mHA, the proportion of HCT recipients who would be eligible for T-cell therapy targeting that antigen can be readily estimated from Internet resources which provide the allele frequencies in the four HapMap populations of most common (minor allele frequency ≥0.01) human SNPs (http://www.hapmap.org), as well as the frequency of various MHC alleles in several ethnic and racial groups (http://www.ashi-hla.org/publicationfiles/archives/prepr/motomi.htm). Tumor-specific antigens that could potentially be targeted with adoptive T-cell therapy to enhance GVT activity include fusion peptides such as BCR–ABL [168] and PML–RARα [169] that are created by tumorspecific chromosomal translocations, the Ig idiotype of B-lymphoid tumors [170], and novel peptide sequences created by the FLT3-internal tandem duplication mutation that is common in AML [171,172]. Many other potential tumor-specific antigens that could be targeted with adoptive cell therapy are likely to exist. Comprehensive genetic sequence analysis of breast and colorectal tumor cells, for example, has demonstrated that such tumors carry, on average, 90 mutations that generate amino acid substitutions and could therefore generate tumor-specific antigens [173]. The clinical utility of adoptive therapy targeting antigens created by tumor-specific mutations, however, will be highly dependent on the frequency with which those mutations recur in tumor cells from different patients. A recent comprehensive genetic analysis of pediatric ALL has shown that mutations causing nonsynonymous changes in the coding sequence of the PAX5 gene are common in tumors from different patients [174], suggesting that “shared” tumor-specific antigens that could be targeted in multiple different patients will, indeed, be identified. Antigens derived from nonpolymorphic proteins that are over- or aberrantly expressed in tumor cells and not widely expressed in nonhematopoietic tissues represent a third class of potential target for adoptive T-cell therapy to enhance GVT. The most extensively characterized antigens in this class include the PR1 peptide encoded by the proteinase3 and elastase genes and presented by HLA-A*0201 [120,121] and several different epitopes encoded by the WT1 gene [175,176]. Genes such as HOXA9 and BMI1 that are selectively expressed in normal hematopoietic and leukemic stem cells and whose products perform critical nonredundant functions in leukemic stem cells [177,178] also represent attractive candidates for T-cell therapy. A peptide encoded by BMI1 and recognized by CD8+ T cells has been identified [179], and is undergoing further evaluation as a potential target for GVT therapy. Adoptive cell therapy directed at nonpolymorphic antigens encoded by normal, unmutated genes that are overexpressed in tumor cells has potential limitations imposed by the expression of these genes, at some level, in normal tissues. Suppression of nonmalignant hematopoiesis might occur, for example, if normal hematopoietic progenitor cells also present the antigen to T cells, as has been suggested for an epitope encoded by WT1 [151]. Alternatively, expression of the antigen in

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normal tissues might lead to a deletion or functional inactivation of T cells with high avidity for the antigen, leaving only low-avidity cells with limited reactivity against tumor cells. Adoptive therapy with regulatory T cells Several subsets of human T cell with the capacity to suppress the in vitro activity or function of effector CD8+ or CD4+ T cells or of NK cells have been described, and there is evidence that these cells can regulate immune responses in vivo (reviewed in [180]) (see Chapter 15). The role of such cells in GVHD and GVT after allogeneic HCT in humans is poorly defined. Studies in murine systems, however, have suggested that adoptive transfer of CD4+CD25+ regulatory T cells can selectively inhibit GVHD but preserve GVT activity after MHC-mismatched HCT [181]. These studies have suggested that analogous strategies based on adoptive transfer of T cells with regulatory function to preserve GVT while inhibiting GVHD in human HCT could soon be developed. Adoptive therapy with genetically modified cells Genetic modification of donor lymphocytes to increase the safety and the efficacy of adoptive cell therapy is an active area of investigation in which significant progress has already been made. Donor lymphocytes transduced with multicomponent suicide genes combining a selectable marker with the Herpes simplex virus thymidine kinase gene, to permit in vivo ablation of adoptively transferred cells with acyclovir or ganciclovir, have been administered to HCT recipients in both the T-celldepleted, haploidentical [182,183] as well as T-replete, MHC-identical [184] transplant settings to treat post-transplant relapse or Epstein–Barr virus-related lymphoproliferative disease. The rationale for adoptive therapy with suicide gene-transduced cells is to permit administration of effector cells that are likely to have a potent antitumor effect, albeit with a risk for GVHD, and that can subsequently be ablated if clinically significant GVHD does, in fact, develop. The antitumor efficacy of suicide gene-transduced donor lymphocytes has been significantly greater in T-cell-depleted compared with T-replete settings due to the immunogenicity of the transgenes, which elicit CD8+ transgene-specific T-cell responses that dramatically limit the in vivo persistence of adoptively transferred, transgene-bearing cells in Treplete HCT recipients [182,184–186]. Although T-cell responses to transgene products have also been detected after transfer of transduced cells to recipients of T-cell-depleted HCT, GVT activity attributable to adoptively transferred cells has been observed in the majority of recipients [183,186]. Tumor regression was temporally correlated with the development of GVHD in most responding patients, and GVHD was largely controlled by the subsequent administration of ganciclovir. Novel suicide genes encoding human FAS or caspase death domains fused to a modified FK506-binding protein domain, which can be activated by a drug that dimerizes the FK506-binding protein domain, have been developed [187,188], and are currently being evaluated in clinical trials. Genetic modification of T cells is also being explored as a means to create GVT effector cells expressing high-affinity receptors for defined tumor-specific or tumor-associated antigens. Genes encoding chimeric antigen receptors comprising the antigen-binding domains of Igs fused to cytoplasmic signaling domains of the T-cell receptor (TCR)–CD3 complex, for example, have been constructed and introduced into polyclonal T cells from peripheral blood via retroviral transduction [189]. Transduced cells expressing the chimeric immunoreceptor demonstrate potent MHC-unrestricted cytotoxicity against cells expressing the target antigen, and can be expanded ex vivo with retention of chimeric receptor expression and antigenic specificity, for use in adoptive therapy.

Clinical trials of adoptive therapy with T cells transduced with a chimeric receptor specific for CD19 for the treatment of CD19-expressing B-lineage malignancies [190] are currently in progress. Effector T cells can also be rendered specific for tumor-specific or tumor-associated antigens through transfer of genes encoding the α- and β-chains of TCRs with defined specificity and affinity. T cells expressing transgenic TCRs retain the MHC restriction of the T cell from which the receptor was derived. The therapeutic potential of this approach was demonstrated in a recent clinical trial in patients with metastatic melanoma [191]. T cells expressing a transgenic αβ TCR specific for the melanoma-associated antigen encoded by the glycoprotein 100/PMEL17 (SILV) gene were adoptively transferred into patients with melanoma after the administration of lymphodepleting chemotherapy, followed by an infusion of IL-2. Durable engraftment of genetically engineered T cells at levels exceeding 10% of peripheral blood lymphocytes was seen in a majority of the patients for at least 2 months after T-cell transfer, and in two patients persistence of transduced cells was seen for 1 year after transfer, accompanied by objective regression of metastatic disease. TCR genes encoding a receptor specific for an HLA-B52-restricted H-Y antigen encoded by UTY have been transferred into peripheral blood T cells, and transduced cells exhibit HLA-B52-restricted UTY peptidespecific cytotoxicity [163]. Transduced cells can also be expanded ex vivo with retention of their antigenic specificity, and can potentially be used in adoptive therapy of male patients with leukemia or other tumors expressing UTY. In vivo persistence of adoptively transferred GVT effector cells will likely be required for optimal antitumor efficacy, and several different strategies for improving persistence and establishing durable antitumor immunity are currently being explored [192]. Genetic modification of human CD8+ T-cell clones to express chimeric cytokine receptors that deliver an IL-2 signal upon ligation of GM-CSF can endow them with GM-CSF-dependent growth, such that antigen recognition and consequent GM-CSF secretion will drive expansion of the T cells in an autocrine fashion [193]. Transfer into GVT effector cells of genes that enable resistance to immune suppressive agents commonly used in the allogeneic HCT setting, such as corticosteroids and mycophenolate mofetil, is also an active area of investigation, and such studies, if successful, will facilitate the use of adoptive cell therapy in patients suffering from or at risk for GVHD. Further development of T-cell-depleted transplant platforms in which alloreactive donor T cells are selectively removed from the hematopoietic cell graft prior to infusion will likewise facilitate the development of adoptive therapy in transplant settings where there is a high prevalence of GVHD with unmodified, T-replete grafts. It is anticipated that the development of T-cell-depletion strategies for selectively removing alloreactive donor cells that might potentially contribute to GVHD will alleviate, if not completely eliminate, the problem of poor immune reconstitution observed in recipients of nonselectively T-depleted grafts. Further characterization of the properties and function of the different subsets of human memory T cells, particularly the effector and central memory subsets, will likely provide valuable insight into the requirements for establishing sustained GVT activity via adoptive transfer.

Conclusion The GVT effect associated with allogeneic HCT is the clearest example of effective immunotherapy in humans. Genetic disparity between donor and recipient is the key to the therapeutic efficacy of HCT but is also the root of GVHD, its primary limitation. Cellular and molecular dissection of GVT responses has demonstrated that GVT after MHC-matched HCT is initiated by donor CD8+ and CD4+ T cells that recognize recipient mHAs encoded by polymorphic genes, and that this

The Human Graft-versus-Tumor Response – and How to Exploit It

alloresponse likely recruits additional effector cells recognizing tumorspecific or tumor-associated antigens encoded by nonpolymorphic genes that are over- or aberrantly expressed in recipient tumor cells. The GVT response in most HCT recipients is therefore likely to comprise a broadly focused immune response directed at a large number of polymorphic and nonpolymorphic target antigens. Selective expression of some GVT target antigens in recipient hematopoietic cells and recipient tumor cells,

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with limited expression in nonhematopoietic tissues, may allow the development of therapeutic strategies for enhancing GVT activity without inducing or aggravating GVHD. Characterization of the extensive sequence and structural variation in the human genome suggests that there are likely to be a large number of polymorphic protein and peptide sequences at which immunotherapy to augment GVT could potentially be directed.

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19

Miriam Merad, Matthew P. Collin & Edgar G. Engleman

Dendritic Cells in Hematopoietic Cell Transplantation

Introduction: the dendritic cell network Dendritic cells (DCs) are hematopoietic cells that belong to the antigenpresenting cell (APC) family, which also includes B cells and macrophages. The two main DC subsets are conventional DCs (DCs) and plasmacytoid DCs (PDCs). Although Langerhans cells (LCs) in the skin were described in 1868, their role as APCs was not appreciated until 1973 when Steinman and Cohn identified DCs in mouse spleen as potent stimulators of the primary immune response (reviewed in [1]). Shortly thereafter, several groups reported the presence of DCs in nonlymphoid tissues of rodents and humans, and demonstrated early evidence that these cells contribute to heart and kidney transplant rejection (reviewed in [2]). However, the low number of DCs in vivo, the paucity of markers that distinguish them from monocytes/macrophages, and the problems involved in purifying these cells made for slow progress in the understanding of DC biology. In the 1990s, the development of methods to isolate DCs from blood [3,4] and to generate DC-like cells from bone marrow (BM) or monocytes in vitro [5] led to an explosive growth of the DC field. DC heterogeneity DCs are characterized as cells that express the hematopoietic marker CD45 but lack most markers of T-cell, B-cell, monocyte, and natural killer (NK) cell lineages, and constitutively express major histocompatibility complex (MHC) class II molecules [6]. In mice, expression of the integrin and complement receptor CD11c/C3b is often used in association with the markers cited above to distinguish DCs. Several subsets of DC have been described in vivo based on their surface markers, their location in tissues or their progenitors [7]. Classification of DCs based solely on their surface phenotype is problematic as DC surface markers are modulated during their maturation, as discussed later. Instead, distinguishing DCs based on their anatomic location is likely to be more relevant as it reflects the nature of the antigenic milieu in which DCs develop. Tissue DCs can be divided into two major subsets: those present in lymphoid tissues including the spleen, lymph nodes (LNs), and thymus, and those present in peripheral nonlymphoid tissues. Among DCs in nonlymphoid tissues, those located at the inter-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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face with the external environment, such as the skin and the mucosa, can be distinguished from those located in internal organs such as the liver. Also, in vitro-derived DCs will be described as they have been widely used to study DC functions. It is important to note that the markers on human and mouse DCs overlap but are not identical (Table 19.1). DCs that occupy skin and mucosal tissues display distinct phenotypes and functions that are well adapted to the microenvironment in which they reside. Cutaneous DCs (Fig. 19.1) (reviewed in [8]) The skin represents the largest interface between the external environment and the internal tissues, and one of its major roles is to provide immune function at this critical site. The most superficial layer of the skin, the epidermis, provides the first immune barrier against foreign invasion. The dermis, separated from the epidermis by the basement membrane, supports the vascular network that supplies the avascular epidermis with nutrients. DCs are present on both sides of the basement membrane and play a critical role in the defense against pathogens. These include epidermal DCs, also called LCs, named after the Austrian medical student Paul Langerhans who first identified the cells in the human epidermis. The other population of DCs in the skin, located in the dermis, is called dermal or interstitial DCs. LCs account for 3–5% of total cells in mouse and human epidermis, with approximately 700 LCs/mm2. In the mouse epidermis, two populations of hematopoietic cells coexist, including LCs and a population of T cells that express the γδ T-cell receptor (γδ T cells). γδ T cells are absent from human epidermis, where LCs represent by far the largest population of epidermal hematopoietic cells. In both species, LCs form a tight network of cells constituting the first barrier to the external environment. Human and mouse LCs express the hematopoietic marker CD45, MHC class II molecules, the C-type lectin receptor langerin/CD207, and its associated pathognomonic Birbeck granules. LCs also express the adhesion molecule, E-cadherin, that is also expressed on keratinocytes and participates in homotypic adhesion. Similar to LCs, dermal DCs express the hematopoietic marker CD45 as well as MHC class II molecules, while they are negative for langerin and E-cadherin. Human, but not murine, dermal DCs also express the c-type lectin DC-SIGN and the coagulation factor XIIIa, although both markers can also be expressed on macrophages. LCs also differ from dermal DC in the factors necessary for their development, as LCs are the only DC population absent from mice deficient in transforming growth factor-β [8]. In contrast to all other DC populations, LCs are maintained by local radioresistant

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Dendritic Cells in Hematopoietic Cell Transplantation Table 19.1 Phenotype of dendritic cells in mice and human CD45

Lineage markers

CD11c

MHC class II

CD11b

CD8α

Langerin

Siglec-H

BDCA2

BDCA4

Mouse DC

+

−

++

+

CD8− subset: +

CD8+ subset: +

−

−

−

Human DC Mouse PDC Human PDC

+ + +

− B220low Ly6Clow CD4

+ +/− −

+ + +

+/− − −

− Activated PDC −

Epidermal LC: ++ CD8+ subset + Epidermal LC: ++ − −

+ −

+

+

DC, dendritic cell; LC, Langerhans cell; MHC, major histocompatability complex; PDC, plasmacytoid dendritic cell.

(a)

(b)

(c)

Murine skin cross-section

Human epidermal sheet

Epidermis LC

Migrating LC

Dermal DC MHC II Langerin

(d)

Dermis

CD11c/Langerin/DAPI

Human LCs

CD1a/DAPI

Human dermal DCs

Keratinocyte

T cell

Fig. 19.1 Cutaneous dendritic cells (DCs) in mice and man. (a) Epidermal sheet isolated from a C57BL/6 mice and stained for major histocompatibility complex (MHC) class II molecules and langerin. Langerhans cells (LCs) coexpress MHC class II and langerin, and form a very tight network of cells that contrast with dermal DCs, which are more sparsely distributed. (b) Cross-section of murine skin stained with anti-CD11c, antilangerin and DAPI (which stains nuclei) to reveal LCs and dermal DCs. LCs co-express CD11c and langerin, while dermal DCs express CD11c but not langerin. LCs are present exclusively in the epidermis, but migrating LCs on their way to the LNs can also be found in the dermis. (c) Human epidermal sheet stained with anti-CD1a antibody. Human LCs express CD1a; they represent the major hematopoietic cell population in the epidermis, where they form a tight network of cells constituting the first immunologic barrier with the environment. (d) Epidermal and dermal sheets were isolated from human skin maintained in culture for 2 days to allow the LCs and dermal DCs to migrate into the medium. Migrating cells are stained with Giemsa. Arrows show a contaminating keratinocyte in the epidermis and a contaminating T cell in the dermis. Figure includes material from M. Bogunovic and M. Merad (unpublished data) and Collin (unpublished data).

proliferative precursors that have taken up residence in the skin prior to birth [9], and we will discuss later in this chapter how these attributes may affect transplant immunity (Fig. 19.1). Mucosal DCs (Fig. 19.2) (reviewed in [10]) Type II mucosal surfaces are covered by stratified squamous epithelia, which share many common features with the skin and include those surfaces that cover the cornea, the oroesophageal cavity and the vaginal cavity. Mucosal DCs present in stratified epithelia other than skin are also called LCs. Corneal LCs express CD11c but little or no MHC class

II in the steady state, while oroesophageal LCs express MHC class II and langerin. Vaginal LCs express MHC class II, CD11c, F4/80, DEC205, and langerin but no Birbeck granules, whereas submucosal DCs, the counterpart to dermal DCs in the skin, reside beneath the basement membrane of type II mucosal epithelia and express CD11c and CD11b but no langerin. Type I mucosal surfaces are covered by simple epithelia including those that cover the small and large intestines, the upper female reproductive tract, and the pseudostratified epithelia of the respiratory tract. Type I mucosal surfaces contain mucus secreting cells called goblet

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(a)

Chapter 19

Murine gut cross-section

MHC class II-DAPI-CX3CR1

(b)

Murine gut serosa

MHC class II

cells, and mucosa-associated lymphoid tissue (MALT) that includes Peyer’s patches in the small intestine and isolated lymphoid follicles in the colon. Intestinal DCs, the most widely studied mucosal DCs, are enriched in the lamina propria (the tissue beneath the epithelium), in the MALT and in mesenteric LNs. DCs present in MALT and mesenteric draining LNs are very similar to those present in other lymphoid tissues. Lamina propria DCs include CD11b+F480+CD8α− and CD11b−F480− CD8α+ DCs and PDCs. CD8α+ DCs are usually absent from nonlymphoid tissues other than intestine, and some groups have suggested that the CD8α+ DCs present in the intestine reflect a contamination from lymphoid tissues as it is difficult to purify lamina propria DCs free of contaminating lymphoid tissue-resident DCs. In addition to lamina propria DCs, a large population of DCs is also present in the gut muscularis layer and the serosa at the interface with the peritoneum, and the exact role of these DCs in gut and peritoneal immunity, as well as their migration properties, remains to be examined [11] (Fig. 19.2). Lymphoid tissue DCs (Fig. 19.3) Lymphoid tissue-resident DCs are the most studied DC population in mice and include thymic, spleen, and LN DCs. Lymphoid tissue-resident DCs express MHC class II and CD11c, and can be further subdivided into two major subsets that include CD11b+CD8− DCs and CD11b−CD8+ DCs [7]. CD8+ DCs express low levels of langerin and are the only DC population able to cross-present cell-associated antigens to CD8+ T cells in mice, a process discussed later in this chapter. However, the clinical relevance of the CD8 marker on DCs in mice remains unclear, as it is absent on human DCs. Very similar types of DC exist in MALT and in the mucosa-draining LNs [10]. Although some investigators have tended to group these populations, different lymphoid organs are likely to contain DCs of different origin. In mice, the majority of thymic DCs express CD8, and initial studies suggested that thymic DCs are generated in situ from thymic lymphoid precursors [7]. Myeloid precursors can also give rise to CD8+ thymic DCs [12]. However, studies of DC ontogeny are based mainly on results obtained from the adoptive transfer of purified precursors into irradiated recipient mice, and it remains to be determined whether these results also apply in the steady state. Thymic DCs play a critical role in the induction of negative selection, while they seem dispensable for the induction of positive selection [13]. In the setting of allogeneic hematopoietic cell transplantation (HCT), it is likely that the host or donor nature of DCs that populate the thymus plays a critical

Fig. 19.2 Gut dendritic cells (DCs). (a) Crosssection of the colon isolated from transgenic CX3CR1–GFP mice. CX3CR1 is a marker of DCs in the gut. The section is stained with anti-major histocompatibility complex (MHC) class II antibody and DAPI. Gut DCs are MHC class II+ CX3CR1/GFP+. (b) Gut serosa whole mount stained with anti-MHC class II antibody. Serosal DCs form a network of cells that resembles the Langerhans network in the epidermis. Figure courtesy of M. Bogunovic and M. Merad (unpublished data).

role in the post-transplant immune response. Although DCs charged with peripheral antigens traditionally have been thought to end their life in draining LNs after only hours, a recent study showed that in vitroderived, systemically administered DCs distributed to the thymus, spleen, and LNs, where they survived for more than 40 days [14]. The spleen is populated mostly by blood DCs. Blood DCs transport blood-borne antigens such as plasma components, proteins expressed by circulating cells, and foreign antigens that breach the blood circulation. LN DCs are more heterogeneous in origin as they include blood DCs that enter the LNs through high endothelial venules as well as tissue DCs that enter the draining LNs through the lymphatics [1]. DCs present in the LNs should reflect the tissue environment from which DCs originate. For example, LCs and dermal DCs are present in skindraining LNs but are excluded from mesenteric LNs. Although careful studies are still needed to distinguish blood DCs from tissue-derived DCs in LNs, several groups use CD8 as a marker of blood-derived DCs based on the argument that CD8 is not expressed on peripheral nonlymphoid DCs [15]. Despite its absence from human DCs, the CD8 marker currently occupies a central stage in DC research as CD8+ DCs are very well equipped to cross-present antigens [15]. Based on these data, some groups have hypothesized that tissue DCs play mainly a ferrying role dedicated to the transport of tissue antigens to the draining LNs, while blood DCs are responsible for cross-presentation of cell-associated antigens to CD8+ T cells [15]. However, it can be misleading to use phenotypic expression as an indication of DC ontogeny as cell surface antigens are strongly modulated during the DC lifecycle and it remains possible that CD8 is induced during DC migration to lymphoid organs, which would explain why it is expressed only by lymphoid organ DCs. Another possibility is that tissue DCs and lymphoid CD8+ DCs derive from separate precursors, and that tissue DCs are dedicated to acquiring antigens, migrating to the draining LNs, and directly presenting processed antigens to T cells, while lymphoid DCs alone have the specialized machinery required to cross-present cell-associated antigens to T cells. PDCs (extensively reviewed in [16]) Similar to DCs, PDCs express MHC class II molecules constitutively and lack CD3, CD19, CD14, and CD56 lineage markers. Although human PDCs express very low-to-undetectable levels of CD11c, they express CD4 and CD45RA antigens, the c-type lectin receptor BDCA2, and BDCA4, a neuronal receptor often used to isolate PDCs in vivo.

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BONE MARROW

BLOOD

SPLEEN CD8+ DC

CLP Flk2+ pDC CMP Flk2+

CD8– DC

DCp

?

CDP Flk2+CD115+ Monocyle

Inflammatory CD8– DC DCp CD11c– MHCII+ Inflammation

DRAINING LYMPH NODES CD8+ DC

Blood monocyte During embryogenesis

CD8– DC

DCp

Embryonic precursor

Interstitial DC

UV

Interstitial DC

Langerhans cells

PPERIPHERAL TISSUES (GUT, LUNG, DERMIS?)

Via lymphatics

EPIDERMIS Radioresistant precursor

Langerhans cells

Fig. 19.3 Origin of dendritic cells (DCs) in mice. This cartoon illustrates a hypothetical view of DC differentiation pathways in mice. Embryonic hematopoietic precursors give rise to Langerhans cells (LCs) in the skin. LCs are maintained by radioresistant hematopoietic progenitors that have taken residence in the skin during steady-state conditions. After severe skin injury, local LC progenitors are eliminated and LCs are repopulated by circulating monocytes. Common myeloid precursor (CMP) and common lymphoid precursor (CLP) give rise to DCs, but the DC differentiation potential is restricted to the Flk2 (fms-like thyrosine kinase 2, the receptor for Flt3L)-positive populations in mice and humans. A common DC/plasmacytoid DC precursor (CDP) was recently identified in the bone marrow as an Flk2+CD115+MHC II+ cell. This precursor likely derives from Flk2+ CMP and Flk2+ CLP, although this has still not been confirmed. CDPs give rise to plasmacytoid DCs (pDC) and to DC precursors (DCp) in the blood (MHC II+CD11c−) that can further differentiate in lymphoid organs into CD8+ and CD8− DCs. Lymphoid organ DCp divide in situ for three to four cycles. In contrast to lymphoid organ DCs, it is still unclear if CDPs participate in DC homeostasis in nonlymphoid tissues. On the other hand, monocytes have been shown to give rise to DCs in nonlymphoid tissue including the gut and the lung, and potentially the dermis, while they do not give rise to splenic DCs in the steady state. However, in inflamed conditions, monocytes can be induced to differentiate into splenic DCs and into epidermal LCs. Lymph nodes (LNs) and mucosa-associated lymphoid tissue contain DCs that originate from the blood (i.e. are CDP progeny) in addition to DCs migrating from peripheral nonlymphoid tissue. For example, skin-draining LNs but not the mesenteric LNs contain LC-derived DCs, while both LNs contain CD8+ DCs. MHC, major histocompatibility complex; UV, ultraviolet.

Human PDCs also express high levels of the interleukin-3 (IL-3) receptor (CD123). Murine PDCs express low levels of the integrin CD11c, express low levels of the lineage markers CD45RA/B220+ and ly6C/GR1+ molecules, lack CD11b, and express plasmacytoid DC antigen-1 (PDCA1) and siglec-H, a member of the sialic acid binding immunoglobulin-like lectin (Siglec) family, recently identified as a specific surface marker of PDCs in mice. In the steady state, PDCs are generated in BM, circulate in the blood, enter LNs through high endothelial venules, and accumulate in the paracortical T-cell-rich areas of LNs. Thus, the migration pattern of PDCs closely resembles that of lymphocytes but is clearly distinct from

that of DCs. Migration of PDCs into LNs is greatly enhanced when the LNs drain a site of inflammation. PDCs are absent from nonlymphoid peripheral tissue such as the skin in the steady state, but they can be found in these tissues when they are inflamed. In vitro-derived DCs and PDCs In humans, CD34+ progenitor hematopoietic cells are commonly used to derive LC- and dermal DC-like cells [6]. Monocytes are also a common source of DC-like cells, referred to as monocyte-derived DCs. Granulocyte–macrophage colony stimulating factor is the most common cytokine used to obtain DCs in vitro, although it is dispensable for DC

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development in mice in the steady state [7]. Transforming growth factorβ is required to obtain LC-like cells in vitro in humans and epidermal LCs in vivo in mice [8]. Murine BM cells can also be used to obtain DC-like cells in the presence of granulocyte–macrophage colony stimulating factor either alone or in combination with tumor necrosis factor [6]. PDCs can be generated from BM cells in cultures enriched with the cytokine fms-like thyrosine kinase ligand-3 [7]. Although in vitroderived DCs differ from in vivo-generated DCs, these culture systems have been extremely valuable for analysing the molecular basis of DC function. DC functions In contrast to most cell populations, DCs are defined by a set of properties that include functional as well as phenotypic properties. The ability to take up, process, and present antigens, to migrate selectively through tissues, and to prime naïve T-cell responses are defining DC properties. The superior ability of DCs, particularly myeloid DCs, to stimulate T cells became clear in the late 1970s from studies that compared the ability of DCs, B cells, and macrophages to stimulate a primary mixed lymphocyte reaction, which could be used as a model for graft rejection if T cells were isolated from the donor graft and DCs from the recipient. Normally, mixed lymphocyte reactions are carried out with equal number of stimulator (APCs) and responder (T cells), but only one DC is necessary to activate 100–3000 T cells, consistent with the concept that DCs are specialized to initiate immunity [1]. Elegant studies have subsequently shown that mice deficient in DCs are defective in initiating adaptive T-cell responses [17], while mice that genetically lack macrophages (Csf-1op/op) are not [18]. Now it is clear that DCs form a specialized system to prime T cells not only to mismatched MHC proteins, but also to minor histocompatibility antigens (mHAs) and foreign proteins. More recently, several studies have demonstrated that, in addition to inducing immunity, DCs can play a critical role in the induction of central and peripheral tolerance, a function that might be interesting to utilize in the setting of allogeneic HCT. Although it seems counterintuitive that potent APCs induce tolerance, it is now clear that induction of tolerance as well as immunity requires two critical functions that are performed uniquely by DCs [1]. First, the antigen sampling and migratory capacities of DCs effectively allow naïve T cells to come into contact with peripheral antigens that they would otherwise not encounter. Indeed, cell-derived antigens located in diverse anatomic sites in the body must be found and recognized by T cells that circulate in the blood stream. This task is made more difficult by the low density of specific antigen–MHC complexes on the surface of most cells (<100 molecules per cell), which must be recognized by low-affinity T-cell receptors on T-cell clones with a frequency of 1/106 [1]. In addition, most somatic cells lack co-stimulatory molecules that drive the clonal expansion of T cells, production of cytokines, and development of cytotoxic T cells. DCs provide a means to overcome these challenges. Located in most tissues, DCs capture and process antigens and display large amounts of MHC–peptide complexes on their surface. DCs charged with antigens migrate to the LNs, where they locate in areas that are rich in T cells. The second major property of DCs is their capacity to translate environmental cues to T cells, which shape the fate of the T-cell response to such antigens. The capacity of DCs to translate the environment is based on their capacity to respond to environmental stimuli through a maturation process [19]. This suggests that DCs are present in immature and mature states, and that only mature DCs have the ability to either initiate an immune response or induce tolerance. It is important to recognize immature and mature DCs as they have different functional properties.

Immature DCs Immature DCs are well equipped to take up antigens through a wide range of mechanisms including phagocytosis, macropinocytosis, and antigen receptor-mediated endocytosis, using Fcγ receptors, Fcε receptors, C-type lectin-like receptors, scavenger receptors, and complement receptors [20]. The pattern of antigen receptor expression differs among DC subsets, and is likely influenced by the tissue in which the DCs reside. For example, epidermal LCs express the highest levels of the C-type lectin receptor, langerin, suggesting that the epidermis may require antigen sampling through langerin receptors. Other lectin-like receptors that participate in pathogen recognition and uptake include the mannose macrophage receptor, and DEC-205/CD205, blood dendritic cell antigen-2 (BDCA-2) and DC-SIGN [21]. An important difference between DCs and other APCs, in particular macrophages, is that macrophages mostly direct antigens to lysosomes, where they are degraded into amino acids. In contrast, DCs have the capacity to synthesize and target large amounts of MHC class II to late endosomes and lysosomes where they reside unproductively with internalized antigens, which can be expressed on the cell surface when needed [20]. Immature DCs express relatively low levels of surface MHC class II and co-stimulatory molecules [20], and instead accumulate abundant amounts of MHC class II within their lysosomal compartment together with antigens internalized by endocytic activity [20]. DC maturation One of the most complex and intriguing aspects of DCs is the process by which they mature, in situ, as first revealed in studies of LCs. Thus, freshly isolated LCs, which express low levels of MHC class II, are unable to present antigens to naïve T cells. However, upon culture in vitro, LCs increase their expression of MHC class II and T-cell costimulatory molecules, and are able to prime naïve T cells in vitro [1]. Subsequent studies showed that when LCs receive certain inflammatory signals, they can be induced to leave the epidermis and migrate to the draining LNs, where they localize in T-cell areas. These results establish the concept of DCs as immune sentinels, enabling naïve T cells that lack the ability to circulate through tissues to sample peripheral tissue antigens. More recent studies have also suggested that not only are DCs able to present and initiate an immune response against tissue antigens, but they are also able to instruct T cells to migrate back to the tissue from which they originated, which could be particularly helpful if DCs acquired antigens at sites of infection [22]. Stimuli such as Toll-like receptor signals, inflammatory cytokines, T-cell ligands such as CD40 ligands, and disruption of homotypic contacts between immature DCs have been shown to lead to a series of biochemical and phenotypic changes that result in DC maturation (reviewed in [20]). First, there is an increase in macropinocytic uptake, followed by downregulation of endocytic activity. Second, MHC molecules escape from the lysosomal compartment and are transported to the plasma membrane. Finally, the levels of MHC class II, co-stimulatory molecules, and MHC class I increase at the cell surface and extend into finger-like projections or dendrites, which dramatically enhance DC migration to the draining LNs and the ability of these cells to interact with other cells in their environment. Concomitant with these events, the cells also enhance their ability to form and accumulate peptide–MHC class II complexes, even using antigens internalized before exposure to the maturation stimulus. The endpoint of the maturation process leads to functionally competent DCs able to engage naïve T cells. Most studies of DC maturation have been performed in the context of injury signals, and therefore DC maturation has been wrongly thought to occur only in the context of inflammation. It now appears clear that

Dendritic Cells in Hematopoietic Cell Transplantation

mature DCs are present in LNs in the steady state, suggesting that maturation can occur independently of inflammatory signals [19]. The immunologic role of DCs in the steady state is becoming a focus of numerous laboratories. There is considerable evidence that DCs function not only to initiate immune responses, but also to maintain peripheral tolerance, by deleting self-reactive T lymphocytes and/or expanding regulatory T cells amid a constant steady state flow of mature tissuederived DCs to the T-cell area of the draining LN [23]. Antigen presentation (extensively reviewed in [20]) All DC populations constitutively express high levels of MHC class II molecules dedicated to presenting peptides derived from exogenous antigens. Mechanisms for capture of these antigens can be distinguished on the basis of the size of the cargo internalized and/or the mechanisms of internalization, which include phagocytosis, macropinocytosis, clathrin-dependent receptor-mediated endocytosis, and caveolin-mediated endocytosis. Phagocytosis involves the ingestion of large particles or cells greater than 1 μm in diameter. Macropinocytosis is constitutive in DCs and macrophages, and accounts for the uptake of large quantities of extracellular fluid and fluid-dissolved antigens. Receptors such as DEC-205 and FcRγ, upon ligation to their ligands, form complexes that are typically internalized by clathrin-coated vesicles. Proteins internalized by any of these mechanisms eventually reach the endosomal/lysosomal compartments, where they can be processed by resident proteases and loaded onto MHC class II molecules. Classically, the MHC class I pathway provides for the presentation of endogenous cellular antigens. For example, in virally infected cells, viral protein expressed in the cytosol is subject to proteosomal proteolysis, and the resulting peptides are translocated via transporters associated with antigen processing (TAP transporters) into the endoplasmic reticulum lumen and loaded onto MHC class I molecules. Although this process remains true for infected DCs, noninfected DCs can also take up, process, and present viral antigens to CD8 T cells through a process called cross-presentation. Soluble exogenous antigens can also be cross-presented to CD8 T cells. It is now clear that although cross-presentation occurs with greatest efficiency in DCs, other cells, such as macrophages and liver sinusoidal endothelial lining cells, also may have this potential. The mechanisms that regulate cross-presentation are currently an active area of research. Stimulation of allogeneic T cells Alloreactivity is the response of T cells to MHC complexes or mHAs not encountered during thymic development. The nature of the allogeneic T-cell response differs depending on whether the presenting MHC molecules of the recipient are matched or mismatched with those of the donor. The frequency of T cells specific for foreign antigens is thought to be approximately one in 106, while the frequency of T cells that recognize allogeneic MHC molecules is thought to be 1000-fold higher (103), which results in a very strong primary alloresponse [24]. The molecular basis underlying the specificity of alloreactivity has been a matter of debate, and several studies have suggested that the response of T cells to allogeneic MHC–peptide complexes is less peptide specific than the T-cell response to conventional antigens, and that this “degenerate” response explains the high frequency of alloreactive T cells [24]. A recent study in mice elegantly demonstrated that the high precursor frequency of alloreactive T cells is due to polyspecificity and not to “degeneracy” as T cells have a germline-encoded capacity to recognize multiple distinct peptide-mismatch MHC molecules [25]. In current clinical transplantation settings, the MHC molecules of donors and recipients are usually matched. In these situations, donor T cells recognize MHC-bound mHA peptides derived from the protein products of polymorphic genes that are present in the recipient but not

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in the donor [26]. In contrast to solid tumor-associated antigens, all mHAs should theoretically be able to elicit T-cell responses since the donor immune system is not tolerant to these antigens. The clinical manifestations of immune responses against mHAs are likely to be determined by the specific tissue expression of the proteins encoding these antigens. mHAs constitutively expressed in many tissues are likely to be targets for a combined alloreactive immune response directed against the host tissue (graft-versus-host disease; GVHD) and the tumor (graft-versus-tumor), while T cell responses directed against antigens that are restricted to the hematopoietic system, including the malignant hematopoietic cell clone, are likely to mediate GVT reactivity without severe GVHD. Numerous human mHAs have been described and include mHAs derived from autosomal genes such as HA-1, HA-2, HA3, HA-8, HB-1, and BCL2A1, and those that derive from the H-Y gene [27]. Most mHA-specific T-cell responses identified to date have been directed against mHAs expressed on hematopoietic cells, which favors the possibility that direct presentation by host professional APCs may be a more efficient way to induce tumor-specific CD8+ T cells than is cross-presentation by donor APCs, which requires uptake of exogenous antigens. Detailed genomic typing of the allelic variants of the targeted mHA in the patient and the donor, in addition to examining the expression and functional recognition of the target mHA on the malignant cells, has the potential to lead to novel immunotherapy strategies in clinical transplantation. Functions of PDCs One important function of human and mouse PDCs is to produce large amounts of interferon-α (IFN-α) in response to infectious pathogens. In addition to its antimicrobial properties, IFN-α can also promote T helper cell type 1 differentiation and has been shown to contribute to autoimmune disease [28]. The capacity of PDCs to capture and present antigens to T lymphocytes is still being examined in several settings. Freshly isolated human and mouse PDCs are very poor inducers of T-cell proliferation; however, upon activation, PDCs can differentiate into mature DCs with high levels of MHC class II and co-stimulatory molecules and T-cell stimulatory activity [17]. Recent studies indicate that PDCs exposed to immune complexes can use their surface Fc receptors to take up the antigens present in these complexes, leading to their processing and presentation to both CD4 and CD8 T cells (Bjorck, Engleman et al., in preparation). In humans, freshly isolated PDCs induce anergy in human CD4+ T-cell clones with various antigenic specificities. IL-2 and IFN-γ production by T cells is impaired, whereas IL-10 production is increased. Anergy is reversed by the addition of exogenous IL-2. In the mouse, freshly isolated antigen-pulsed spleen PDCs induce minimal proliferation and no cytokine polarization in antigen-specific T cells. Thus, PDCs, particularly in their immature state, or in certain anatomic localizations, may have tolerogenic functions, although the exact nature and mechanisms of these functions remain to be explored [17]. In allogeneic HCT, donor PDCs are enriched in granulocyte colonystimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells and are also present in donor leukocyte infusions (DLIs). Therefore, the immunomodulatory properties of PDCs should be examined in this context. Nonprofessional APCs Other cell types in addition to professional APCs can express MHC class II molecules, co-stimulatory molecules, and accessory factors including MHC invariant chains, HLA-DM and HLA-DO, under normal or pathologic conditions. These cells, sometimes referred to as nonprofessional APCs, include endothelial cells [29] and some epithelial cells [30]. These cells have a heterogeneous capacity for antigen processing and

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presentation of peptides that are often derived from endogenous proteins. Tissue inflammatory reactions such as those induced by GVHD may also increase the presentation capacity of nonprofessional APCs, enhancing local inflammation [31].

In murine experiments, radiation at myelosuppressive or lethal doses without postgrafting immunosuppression has been traditionally employed [37]. Reduced-intensity regimens have been developed [38,39], but the majority of murine studies continue to employ single-dose irradiation without immunosuppression.

DC homeostasis in the steady state Based on our current understanding of DC homeostasis, there is a continual recruitment of blood-borne DC precursors to peripheral tissue and lymphoid organs that gives rise to immature end-stage differentiated DCs. Peripheral tissue DCs migrate through the afferent lymphatics to draining LNs, where their life is limited to days [32]. Recent studies have led to a reappraisal of the importance of local renewal in DC homeostasis under quiescent conditions. Studies of parabiotic mice showed that, in contrast to other DC populations in the spleen and LNs, LCs were maintained exclusively by local hematopoietic precursors that had taken residence in the skin [9]. Local self-renewal of DCs has now been confirmed, although at much lower levels, in a number of tissues including spleen, LN, and dermis [33]. Short-pulse-labeling experiments label approximately 5% of tissue DCs without significant incorporation into monocytes or blood DCs, excluding a contribution from recent blood emigrants to labeling in the tissues [33]. In humans, 2–3% of LCs are in S/M2 phase by cell cycle analysis, and a similar proportion label with Ki-67 antibodies (Bogunovic and Merad, unpublished data). Recipient LCs can be identified unequivocally 1 year after human BM transplantation [34], and donor LCs persist in transplanted human limbs [33]. Importantly, in contrast to the steady state, severe skin injuries lead to the elimination of self-renewing LC progenitors followed by the recruitment of blood-borne monocytes and their differentiation into LCs in a csf-1-dependent manner [33]. These data are consistent with earlier studies suggesting that, in experimental inflammatory conditions, DCs are clearly derived from classic monocytes [35,36], and establish the concept that DC homeostasis is critically dependent upon conditions of quiescence or inflammation.

Response of DCs to conditioning therapy Conditioning A number of different modalities are used to condition the recipient for HCT. In humans, myeloablative radiotherapy and alkylator-based regimens are still practiced alongside a range of newer reduced-intensity and minimal or nonmyeloablative protocols. Conditioning often takes several days, and maintenance of postgrafting immunosuppression is the rule.

Pretransplant

Day 0

Effect of conditioning on blood DCs In common with other leukocytes, peripheral blood DCs are depleted by conditioning therapy. Clinical studies describe a rapid loss of both DCs and PDCs after myeloablative conditioning, which approximates the decline of neutrophils and monocytes [40]. Less is known about blood DC subsets following reduced-intensity conditioning, although the relative preservation of leukocyte counts in some patients suggests that DCs are also less depleted. One report suggests that PDCs are more labile than DCs after reduced-intensity regimens [41], and another study shows that monocyte-derived DCs are more easily isolated after reduced-intensity than high-dose regimens [42]. Alemtuzumab (monoclonal antiCD52 antibody) is a component of some reduced-intensity regimens and effects a swift reduction in the number of monocytes and blood DCs, which express CD52, unlike their tissue counterparts [43–46]. Patients often have lower pretransplant DC levels than normal controls, suggesting that BM hypoplasia associated with prior cytotoxic therapy or hematologic disease reduces peripheral DC counts [40]. Effect of conditioning on tissue DCs (Fig. 19.4) Surprisingly, in murine models, there are very few data on the immediate effects of radiation conditioning on DCs. An important study demonstrated rapid loss of recipient DCs from the spleen within 3–5 days; the peripheral blood, LNs, and liver were also reported to be similarly depleted [47]. Most experimental work has utilized chimerism analysis of congenic transplants following stable engraftment to infer the radiosensitivity of various DC populations. In these studies, the spleen and liver DCs are almost completely replaced. In contrast, epidermal LCs [9,48], a component of the dermal DC [49] and certain macrophage populations, including brain microglia [50] appear relatively radioresistant. LCs recover over several weeks almost exclusively from local host cells [9]; in the brain, about 70% of cells are recipient long term [50] and in the dermis, approximately 20% [49]. LNs contain some recipient cells depending on the tissue they drain; notably, skin-draining LNs may contain up to 30% recipient LC-derived DCs after transplantation [48]. Other organs such as the gut have not been evaluated in detail and may contain niche populations of resistant cells. The extent of depletion of

Day 14

Day 28

Day 100

Fig. 19.4 Cutaneous dendritic cell turnover in patients transplanted with allogeneic hematopoietic stem cells. Epidermal sheets isolated from patients at different times after transplant were stained with anti-CD1a. Langerhans cell numbers are markedly reduced around day 14 and return to normal around day 100 post transplantation. Scale bar: 40 μm. Figure courtesy of M. Collin (unpublished data).

Dendritic Cells in Hematopoietic Cell Transplantation

murine DCs after reduced-intensity conditioning is currently unknown and may be quite different since previous studies on tissue macrophage populations have demonstrated a significant sparing effect simply due to fractionation of irradiation [51]. The analysis of conditioning effects on human tissue DCs has thus far been restricted to the skin. Several studies have documented depletion of epidermal LCs in the first week of transplantation [52–55], and recent data illustrate a greater effect of myeloablative conditioning [34]. An important variable in human transplantation may be the intensity and proximity of prior therapy since patients often have lower numbers of leukocytes, including DCs, before transplantation than normal controls [34,56].

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DCs in G-CSF-mobilized peripheral blood progenitor cells (PBPCs), whereas myeloid DCs and PDCs are present in approximately equal numbers in BM [66,67]. In keeping with this, the proportion of T helper cells type 2 in G-CSF-mobilized PBPCs is much higher than in normal peripheral blood or BM [66,67]. These observations have been used to explain the equal incidence of acute GVHD following PBPC and BM transplant even though the total CD3+ cell content of PBPCs is 10-fold higher than that of BM. Among PBPC recipients, a higher frequency of PDCs has been associated with increased risk of death from relapse in one report [68], although in general total DC content is correlated with CD34 dose and a positive outcome [69]. The relatively low risk of GVHD after cord blood transplantation has also been linked to the apparently immature or tolerogenic status of cord blood DCs [70].

Mechanisms of conditioning-induced DC depletion The mechanisms that govern DC depletion by conditioning therapy are poorly understood. Direct cytotoxicity may occur with some agents, although most observers suggest that the secondary release of cytokines, chemokines, and endotoxin is likely to be responsible for the mobilization of DCs from the tissues [57–59]. The role of locally derived tumor necrosis factor-α and IL-1β in mobilizing LCs from skin explants is well described [60]. It should not be overlooked that post-transplant aplasia alone, which accounts for severe leukopenia in the peripheral blood, could lead to a significant depletion of DC populations with rapid turnover. The relative contributions of precursor depletion and enhanced mobilization are difficult to resolve in humans, whereas in mice parabiosis and congenic transplantation have allowed distinction between populations that are more or less dependent on blood-borne precursors [9,61]. Qualitative effects of conditioning on DCs Conditioning exerts qualitative effects on DCs. Irradiation induces a transient upregulation of co-stimulatory molecules and IL-12 expression [47]. As with the stimulation of migration, secondary stimulation by cytokines and microbial products is likely to be important. Although this has not been directly shown in the context of transplantation, circumstantial evidence comes from the ability of cytokine and lipopolysaccharide antagonists to diminish GVHD [58,62]. Recent work also demonstrates DC activation by NK cells and other effectors of innate immunity (reviewed in [63]). There are no data concerning the activation status of DCs as the intensity of conditioning is reduced, but the general assumption is that both the direct toxicity to organs and the immediate immunologic consequences are ameliorated. It is interesting to note that although more intensive conditioning should generate an increased inflammatory response, this may be offset to some degree by a greater depletion of recipient cells prior to transplant. Recipient DCs may “go quietly” after reduced-intensity conditioning, but there is also evidence of a high cumulative rate of late GVHD after some regimens, consistent with eventual recognition of a significant burden of recipient DCs [64,65].

Homeostasis after transplant Content of the graft The recovery of DC populations post allogeneic HSC transplantation is influenced by complex and interacting variables. The donor hematopoietic cell inoculum contains DC precursors and mature DCs [66,67]. Although practically undetectable after infusion, the relative proportions of DC subsets in different grafts have been linked to post-transplant outcomes. In particular, PDCs are five times more abundant than myeloid

Numerical recovery of DCs With the advent of accurate blood DC counting [40], many studies have analyzed the numerical recovery of blood DCs after allogeneic HCT [40,45,69,71–74]. In the first study, expression of DC activation antigens CD83 and CMRF-44 was used to confirm identity of lineage-negative, MHC class II-positive cells as DCs. Subsequent work has defined DCs as CD11c+ myeloid or CD123+ PDCs of the lineage-negative, MHC class II-positive fraction. Overall, myeloid DCs recover faster than PDCs [41], and brisk reconstitution is associated with improved outcomes [72–74]. This is perhaps not surprising given that normal blood DC counts are promoted by good BM function and the absence of GVHD. DC recovery is apparently not related to the dose of CD34 cells in the graft itself [69]. From earlier work on the skin, it was apparent that LC numbers were reduced for several months post transplant, although separation of conditioning from later effects was not clear [52–55]. Accurate blood DC counting has since revealed a profound depression of DC counts in association with GVHD [40,41,43,71,74]. In one study, temporal resolution was sufficient to reveal a transient increase in all blood DC subsets just at the onset of acute GVHD, and to demonstrate that treatment with corticosteroids was a major factor in DC depletion during GVHD [72]. A recent study has shown that activation antigens are expressed just prior to acute GVHD such that twice-weekly monitoring can be used to predict GVHD [75]. The mechanism of DC activation is not known, but activated DCs may be the source of IL-12, serum levels of which also have predictive value [76]. Blood DC chimerism Several studies focusing on the chimerism of human blood DCs have shown rapid repopulation by donor cells, in parallel with blood myeloid engraftment. In all of these studies, a minority of patients had a small percentage of persistent recipient blood DCs [77–80]. Despite the large number of patients reported in the last two studies, no significant correlations have been observed between DC engraftment and conditioning, donor type or GVHD. This is perhaps related to the robust conditioning regimens used by most investigators. Also, the potential of the studies to discriminate was limited by sampling different subgroups of patients relatively late, at 6 and 12 months post-transplantation. Taking all the data together, mixed DC chimerism appears slightly more frequent after reduced-intensity conditioning, although to date there are no analyses of DC chimerism following true “nonmyeloablative” or “minimal” conditioning [64,65]. Surprisingly, no clear relationship between GVHD and DC engraftment has been demonstrated, although prior studies on total myeloid engraftment predict that acute GVHD would favor complete donor chimerism in all peripheral blood leukocytes [81,82]. One study of monocyte-derived DCs showed a positive relationship between donor

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DC chimerism in the tissues is more difficult to examine in humans, and for this reason all studies of human transplant recipients completed to date are confined to the skin. An important early paper described the persistence of recipient LCs for up to 120 days after conventional transplantation, using quinacrine to identify the Y body of male CD1a+ (OKT6+) cells [84]. An interim report showed no clear engraftment of LCs with time, which may have reflected technical difficulties with combined immunofluorescence and fluorescence in situ hybridization prior to confocal microscopy [85]. More recent studies using emigrated LCs find that engraftment is largely complete by 100 days, although engraftment occurs more slowly after reduced-intensity conditioning [34,56]. The effect of prior GVHD in promoting LC engraftment is just discernible at 100 days, and patients without GVHD often harbor a small percentage of recipient LCs for at least 1 year after transplant [34]. Murine data also show persistence of recipient dermal DCs, especially after reduced conditioning [49]. It will be difficult to ascertain in humans whether other interstitial DCs, such as splenic or hepatic DCs, behave similarly to LCs or dermal DCs. Extrapolating from murine studies suggests that LCs have a unique mechanism of homeostasis and that other tissue DCs should turn over even more rapidly in parallel with the blood myeloid compartment. Given this assumption, the protracted survival of recipient tissue DCs appears unlikely in most human transplant recipients. Several factors may promote more rapid donor DC engraftment in humans than mice, including greater mHA disparity and higher T-cell content of the grafts. Prior cytotoxic therapy, which attenuates DC populations pretransplantation, may also lead to a more rapid depletion of recipient cells. Data are also lacking on DC turnover after minimally intensive transplants, which show gradual

engraftment, GVHD, and protection from relapse, but whether this simply reflects overall myeloid chimerism is unclear [42]. Although laborious, prospective studies of homogeneous patient groups with frequent and strict chronologic assessment will be required to clarify this in detail. Peripheral tissue DC chimerism (Fig. 19.5) In animal models, there exists a wealth of data on peripheral DC chimerism of the tissues following BM transplantation. Two factors play a role in determining donor chimerism of DCs in the tissues: the radiosensitivity or extent of depletion by conditioning, and the alloreactivity of donor T cells in the graft. Thus, in congenic transplantation, a number of tissues, such as the epidermis, dermis, and cutaneously draining LNs, retain host DCs as described above [9,49]. In addition, several studies show significant retention of host-derived macrophages in the brain, lungs, and liver [50,51]. However, the situation is radically different when host-reactive donor T cells are introduced in allogeneic transplantation. The effect of donor T cells is generally illustrated by experiments in which mixed myeloid chimeras are converted to complete donor chimerism by lymphocyte infusion [39]. The case for isolated persistent recipient DCs is most clearly demonstrated by the conversion of LCs to donor type by donor T cells [48]. Under these conditions, self-renewing local progenitors of LCs are depleted from the skin and replaced by inflammatory monocytes [83]. The fact that skin is a target organ of GVHD may be critical in this process, and it is perhaps less likely that donor T cells can also eliminate APCs in noninflamed organs such as the brain.

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Fig. 19.5 Fate of cutaneous dendritic cells (DCs) in patients after sex-mismatched allo-hematopoietic stem cell (HSC) transplant. (a–c) Persistence of human host cutaneous DCs despite complete donor-derived blood chimerism. Noninflamed skin samples were obtained from patients who underwent reduced intensity for sex-mismatched allo-HSC transplant prior to the development of graft-versus-host disease (GVHD). Cross-sections of 2 mm skin biopsies were subjected to fluorescence in situ hybridization of X (Cy3) and Y (Cy2) DNA probes followed by immunostaining of langerin (Cy3) and HLA-DR (Cy5). Nuclei were counterstained with DAPI. One representative image obtained from a female recipient transplanted with male donor hematopoietic cells. (a,b) Overlaid images of three of four color channels in indicated combinations show persistence of host female XX+langerin+ Langerhans cells (LCs) in the epidermis (a) and HLADR+langerin− DCs in the dermis (b). Magnification: ×600. (c) Dot graphs show the percentage of remaining host langerin+ LCs among total the LCs, and HLADR+ langerin− cells among the total dermal DCs (DDC), and the percentage of remaining host leukocytes among the total bone marrow (BM) leukocytes, in each patient 30 days after transplantation. (d) Absence of residual host DCs in advanced cutaneous GVHD lesions. GVHD-affected skin samples were isolated from four patients who underwent myeloablative conditioning for allo-HSC transplant and developed GVHD. The dot graph shows the percentage of remaining host LCs, host dermal DCs, and host BM nucleated cells in five separate patients. Figure includes material from [31], M. Merad and M. Bogunovic (unpublished data) and Bogunovic et al. [49].

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engraftment [64] and significantly delayed GVHD compared with more intensive regimens [65]. A notable exception to the relatively rapid turnover of human tissue APCs is the long-term survival of recipient microglia. Their persistence may be of questionable relevance to transplantation immunology, but a post mortem study has shown that more than 98% of recipient microglia survive up to 6 years after transplantation [86].

Acute GVHD Early work Acute GVHD is primarily a cell-mediated disorder. The interaction between donor T cells of the inoculum and BM-derived elements of the host lymphoid system was first demonstrated by the elegant experiments of Sprent and colleagues (reviewed in [87]). In these studies, recipient mice were infused with T cells that were later collected from the efferent lymph by cannulation of the thoracic duct, a process known as blood–lymph filtering. Alloreactive T cells are initially trapped in the lymphoid organs for 24–48 hours and then emerge primed to induce GVHD in secondary recipients. Filtering governs the responses of both mHA-reactive T cells, thus demonstrated to be H-2 “restricted” [88], and T cells primed across H-2 differences [89]. By transplanting recipients with donor BM 6 months beforehand, so that recipient DCs are replaced by donor cells at the time of T-cell infusion, it is possible to show that CD4 and CD8 T-cell priming is attenuated, although not completely abolished, and is therefore largely dependent on recipient BM-derived APCs [90]. Recent studies Blood–lymph filtering experiments heralded a series of tandem transplant studies that defined in detail the roles of recipient and donor DCs [91–93]. Using MHC class I-deficient BM grafts (from β2-microglobulin knockout mice) to prepare recipient animals, the seminal study of Shlomchik and colleagues proved that recipient APCs are required for the efficient induction of CD8 T-cell-mediated GVHD across an mHA mismatch [91]. The interpretation of this result is that recipient APCs present their endogenous mHAs in association with class I MHC much more effectively than donor APCs, which must cross-present the same antigen acquired exogenously. Complementary studies in which modified donor cells lack functional antigen-presenting capacity showed that donor DCs do in fact amplify CD8 T-cell-dependent acute GVHD but cannot induce it [92]. It is important to note that CD4 T cells are absent in these experiments. While this proves that CD8 T cells alone can mediate acute GVHD, donor APCs may be licensed much more efficiently to cross-present antigen in a full allogeneic response involving CD4 and CD8 T cells [94]. Another model has been used to demonstrate that donor DCs may also participate in the induction of acute GVHD indirectly by elaboration of IL-15 [95]. The contribution of recipient APCs to CD4 T-cell-mediated GVHD is less clear cut than in CD8 T-cell-mediated GVHD, partly because of the dual functions of CD4 T cells in GVHD: help for CD8-mediated cytotoxicity and direct cytokine-mediated organ damage. In turn, the removal of CD8 T and NK cells from an experimental system reduces IFN-γ-induced MHC class II expression by nonprofessional APCs and may artificially augment the role of professional APCs when CD4 T cells are put to the test [63]. The potential for direct CD4 T-cell-mediated pathology was clearly illustrated in one study in which a single MHC class II allele mutation (equivalent to a single mHA) expressed on recipient APCs alone was sufficient to induce lethal GVHD [93]. This

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finding, however, contrasts with the results of an earlier study in a multiple mHA-mismatched model, which demonstrated a strict requirement for alloantigen on target tissues, not only APCs [96]. Shlomchik and colleagues addressed the role of recipient and donor APCs also in a minor histocompatibility antigen-mismatched model, by using BM grafts or recipients deficient in co-stimulatory molecule expression [97]. In these experiments, both recipient and donor APCs were sufficient to initiate CD4 T-cell-mediated GVHD. Interestingly, there was a tissuespecific distribution of GVHD, with recipient DCs being more responsible for skin GVHD and donor DCs potentiating gut GVHD. The pattern of disease in these models was subacute with cutaneous and hepatic features of chronic GVHD.

Persistence of recipient LCs An important modification of these results was introduced by the demonstration that some populations of APCs, notably LCs of the epidermis, are not necessarily replaced by donor-derived cells following BM transplantation [9]. Under conditions of T-cell depletion, nearly all recipient LCs survive MHC-mismatched transplantation and are able to mediate acute GVHD upon reinfusion of donor T cells and a second inflammatory insult such as irradiation [48,98]. Cutaneous GVHD, in turn, leads to the rapid turnover of recipient LCs and complete donor chimerism [9,48,98]. Persistent recipient APCs may account in part for the breakthrough of T-cell priming and GVHD at high T-cell doses when BM transplantation is used as an experimental means to replace or inactivate APCs [90,91]. The observation that activated T cells are able to clear residual recipient APCs is important since it makes the induction of acute GVHD a self-limiting process. Studies by Sykes and colleagues have explored the conversion of mixed chimeras by donor T cells in detail, demonstrating the essential role of inflammation in controlling access to the tissues [99] and the combined requirement for CD4+ T cells and recipient APCs in mediating a robust graft-versus-leukemia (GVL) effect [100]. Under appropriate conditions, in which priming of alloreactive T cells is confined within the hematopoietic compartment, GVHD pathology can be prevented while allowing full expression of the GVL effect [39]. It remains to be determined whether a similar selective benefit can be obtained by targeted ablation of tissue resident DCs, particularly those self-renewing populations such as LCs, while sparing visceral DCs in the spleen and liver.

APCs that induce GVHD A limitation of using HCT to manipulate recipient APCs experimentally is that the identity of the APCs responsible for inducing acute GVHD remains obscure. While it is assumed that myeloid DCs play a significant role, PDCs, B cells or macrophages may also contribute. Recipient “addback” experiments have demonstrated that DCs are sufficient to induce GVHD in animal models [101,102], although removal of recipient macrophages with clondronate-loaded liposomes has also been shown to attenuate GVHD [103]. In animal models, recipient B cells have negligible or even negative effects on GVHD [101,104]. Alternative APCs are possibly more important in human transplantation, in which polyclonal memory T cells participate in the allogeneic response, than in model systems focusing on naïve T-cell priming to defined antigens. Murine models now exist in which the selective ablation of macrophages or DCs can be achieved by treatment with diphtheria toxin combined with lineage-restricted expression of diphtheria toxin receptor. These may permit further dissection of the APC subsets required for GVHD induction.

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GVHD kinetics From a clinical standpoint, it might be argued that the contribution of recipient APCs to GVHD is entirely predictable based on the simple fact that only recipient APCs are directly activated by conditioning and that classical acute GVHD occurs during or prior to engraftment when the presence of donor APCs is minimal. Although proof is likely to remain elusive in humans, a key role for recipient DCs was argued over 20 years ago in the pioneering studies of Perreault [52,84]. Intriguingly, a recent study showed that the pretransplant CD14 count is linked to GVHD risk [105] and, as described above, there is clear evidence that recipient tissue DCs survive conditioning therapy [34,49]. Donor T cells and GVHD promote DC engraftment, but the effect of alloreactivity on DC turnover is often ignored in attempts to show that persistent recipient APCs constitute a “risk factor” for developing GVHD. Pretransplant, every patient has 100% recipient DCs, and posttransplant the majority of patients convert to 100% donor-derived DCs. The occurrence of GVHD is linked to a period of rapid conversion from recipient- to donor-derived DC, which more than likely precedes the onset of clinical symptoms; thus, the faster recipient DCs are lost, the greater the risk of clinical GVHD. It is therefore very difficult to demonstrate a causal link between persistent recipient DCs and GVHD. For this reason, although it is possible that persistence of recipient APCs correlates with de novo acute GVHD following the withdrawal of immune suppression, it is perhaps just as likely that primed donor T cells, which have already effected complete donor DC engraftment, are the real risk factor. There is presently no evidence for or against either of these mechanisms. Perhaps a more promising approach to testing the role of persistent recipient DCs in humans is to examine correlations between DC chimerism and GVHD kinetics in cohorts of patients transplanted with regimens of varying intensity. There is no appreciable difference in LC chimerism at 100 days between high-dose and reduced-intensity fludarabine/melphalan transplants, but data are lacking on minimally intensive HCT that show significantly delayed GVHD kinetics [65]. It will be informative to determine whether DC engraftment is similarly retarded. Murine models, which incorporate the added refinement of elective donor T-cell infusion, broadly support this rationale. After a high-dose regimen, the transfer of donor T cells more than 1 month post transplantation does not generate GVHD [102,106], but after reducedintensity transplantation in which there is significant myeloid partial chimerism, GVHD can still be induced many months later. Even more directly relevant to these data is the clinical observation of progressive diminution of the toxicity and efficacy of donor lymphocyte infusions with time [82,107]. It has been suggested that the loss of recipient DCs is the cause as described in murine models [39,100], although this is not entirely consistent with human data. DLI is rarely given before 3 months, at which time all patients have at least 90% donor LCs [34] and probably complete engraftment of DCs in other organs. Unless a small percentage of recipient DCs are unusually potent or there is protracted survival of migratory cells in the LNs, other explanations for the reduction of DLI effect, such as active regulation by donor cells, should be considered [108].

GVHD tropism One of the major biologic questions concerning GVHD is its tropism for the skin, gut, and liver. It is likely that more than one mechanism explains this predilection. The skin and gut are both important epithelial barriers with commensal flora and dense APC networks. Simply from a numerical standpoint, these organs, together with the BM, spleen and

liver contain many more F4/80+ resident APC than the other viscera and skeletal tissues that are generally not affected by GVHD [109]. It is possible that DCs in these sites are also selectively primed by conditioning either because the epithelia themselves are more readily damaged and release inflammatory mediators (hair loss and mucositis being prominent features of chemoradiotherapy) or because there is greater ingression of microbial products from commensal organisms. In support of the latter argument, there is good evidence for a role of lipopolysaccharide in gut GVHD [57,62,110]. T cells primed in the skin and gut also recirculate to these sites with high efficiency through the induction of specific homing receptors, creating feedback circuits of tissue damage [111]. Studies using bioluminescence dramatically indicate the early involvement of gut and spleen in T-cell priming [112], although in other models the Peyer’s patch itself appears dispensable [113]. It is conceivable that local DC renewal could play some part in the tropism of GVHD for certain organs. Even though splenic and hepatic DCs rapidly prime donor T cells [47], this does not exclude roles for self-renewing DCs in prolonging the initial sensitization phase of GVHD or influencing the spectrum of GVH responses. However, such effects will tend to be subtle and self-limiting since donor T cells, once primed, initiate inflammation which promotes complete DC donor chimerism. In the skin, persistent recipient LCs are sufficient to generate cutaneous GVHD [48]. The radioresistant LC precursor may indeed be a dermal cell that gives rise to interstitial dermal DCs and LCs, both of which may persist after experimental transplantation [49,83]. Other target organs have not yet been closely scrutinized. The gut and associated Peyer’s patches may harbor radioresistant DC precursors, even though a rapid turnover of mesenteric LN DCs is apparent [9]. In the liver, the bulk of parenchymal DCs rapidly equilibrate with blood DCs [48], but this does not exclude a niche population of cycling DCs in focal GVHD targets such as the portal triad of the liver. The relative importance of persistent recipient DCs may be greater after reduced-intensity transplantation, and it is interesting to note that atypical patterns of single-organ GVHD are more frequently seen than in myeloablative transplant.

Chronic GVHD The pathogenesis of chronic GVHD has been difficult to study for several reasons. Clinical disease is very heterogeneous and animal models are less convincing than for acute GVHD. A number of fundamental features of human chronic GVHD must be explained by any successful model. First, chronic GVHD is delayed, classically to at least 100 days post transplant. Second, prior acute GVHD is a major risk factor, although chronic GVHD is less well controlled by T-cell depletion. Third, the tissue tropism is different from that of acute GVHD, although features of chronic GVHD may rapidly evolve and coexist with severe acute GVHD. Fourth, chronic GVHD has a very different response to therapy, being largely refractory to calcineurin inhibitors but responsive to corticosteroids and also rituximab, thalidomide, extracorporeal phototherapy, and other alternative agents. The features of chronic skin inflammation with sclerosis, mucosal ulceration, salivary and lacrimal gland dysfunction, and chronic hepatitis have parallels with human autoimmune disease. Whether this reflects the same induction process or simply a final common pathway of chronic inflammation is not clear. From a simple perspective, the delayed onset of chronic GVHD suggests a greater dependence on donor-derived APCs. It has long been held by clinicians and immunologists alike that engraftment of donor DCs might underlie the progression of acute to chronic GVHD on the grounds that DCs of different origins favor alternate routes of antigen presentation (reviewed in [87]). In mice, acute CD8 T-cell-mediated

Dendritic Cells in Hematopoietic Cell Transplantation

GVHD dependent on recipient APCs contrasts with subacute forms of CD4 T-cell-mediated GVHD, which occurs with donor APCs alone [96,97,114]. In the MHC matched setting, direct MHC class I presentation to CD8 cells is self-limiting and should decline as recipient APCs are removed. In contrast, MHC class II-restricted antigens continue to be presented to donor CD4 T cells indefinitely by donor APCs. This simple model accounts for the pathologic spectrum of acute and chronic disease by the selection of different minor antigens, potentially with tissue-specific distribution [115], and qualitative differences in CD8 and CD4 T-cell-mediated pathology [116,117]. It is easy to add that acute GVHD will accelerate the appearance of chronic GVHD through increased priming of donor CD4 T cells by activated donor DCs. More complex models of chronic GVHD invoke thymic dysfunction, principally in terms of defective negative selection [118,119]. Impaired negative selection as a result of acute GVHD has been documented in animals [120]. The merit of this explanation depends on the extent of thymus-dependent immune reconstitution following a transplant. In adult humans, thymic recovery attenuates with age, and mature alloreactive T cells may survive to propagate chronic GVHD completely independently of the thymus [121]. In a number of animal models, mature T cells also mediate chronic GVHD syndromes that are thymus independent [117]. The mechanism of negative selection in the thymus involves BM-derived elements, and the switch from recipient to donor could compromise the availability of recipient mHAs for negative selection, although recipient epithelial elements would still be present. If donor APCs populating the thymus are completely unable to present antigen, as in the case of transplantation with MHC class II–/– BM, a convincing picture of thymic-dependent chronic GVHD is seen [118]. Thymectomy is protective, although this only proves that a dysfunctional thymus can be worse than no thymus at all. This mechanism is perhaps relevant to de novo human chronic GVHD in the younger patient and cautions against attempts to boost thymic function indiscriminately in older patients. Studies of human DCs in chronic GVHD have revealed a relative depletion of tissue DCs [122] but a higher level of exclusively donorderived blood DCs, especially PDCs [79]. There are no data on DC chimerism in the tissues, and this makes it difficult to exclude a third hypothesis that chronic GVHD is the result of continuous immune activation by rarified long-term surviving recipient DCs. Although in humans all transplants attain at least 90% LC donor chimerism at day 100, just over a third of patients continue to yield occasional recipient LCs for at least 1 year post transplant [34]. Chronic allostimulation by these cells might explain the very frequent involvement of the skin and mucosa, often with a focal distribution. The main limitation of this idea is that it cannot account for the increased risk of chronic GVHD in patients with prior acute GVHD, which efficiently ablates recipient LCs. An interesting alternative suggested by recent results in a murine model is that persistent recipient B cells could contribute to chronic GVHD [123]. This must be tested in humans, and further experiments comparing lesional and nonlesional skin in cases of de novo chronic GVHD are required.

GVL effect Immunologic protection from relapse, or the GVL response, is likely to be a manifestation of the GVH response in hematopoietic tissue and therefore dependent on the same cellular process of induction [26]. Transient pancytopenia often accompanies DLI responses [124] and is the cardinal feature of transfusion-associated GVHD [125], indicating that BM is indeed a target organ of the engrafting immune system. Animal models confirm the importance of recipient APC in the GVL response. Functional recipient, but not donor, APCs are required for

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GVL mediated by CD4+ and CD8+ cells [92,126]. For CD8 T-cell-mediated GVL, donor alloantigens must be present on both recipient APCs and tumor; tumor lines expressing co-stimulatory molecules are also unable to substitute for professional APCs [126]. Of interest, stably engrafted donor APCs, while not required for GVL, are able to mediate some CD8 T-cell-dependent GVL activity at lower tumor burden while sparing the effect of GVHD [92,100,126]. GVL appears to occur at a lower threshold than GVHD in studies of escalated DLI in mice and humans, suggesting that access to a narrow therapeutic window between GVL and GVHD is possible [127,128]. Changing the density or location of host DCs in the recipient may influence the therapeutic advantage in favor of GVL. In murine models, recipient visceral DCs are sufficient to prime robust GVL responses and are readily accessible by donor T cells in the hematopoietic compartment [39,100,126]. In contrast, recipient peripheral tissues are not infiltrated by alloreactive donor T cells in the absence of inflammation [99]. The interaction of donor T cells with visceral, but not peripheral, DC populations may achieve a selective benefit in promoting a GVL effect without GVHD [39,99,100,126]. This argument underpins the logic of delayed T-cell add-back strategies [129] and pre-emptive DLI [82], which allow the inflammatory insult of conditioning to subside prior to the infusion of donor T cells. It is noteworthy that recent data indicate a more stringent requirement for priming GVL in MHC-matched chimeras than in mismatched transplants. In the matched animals, additional lymphodepletion or Toll-like receptor signaling is required to initiate alloresponses, whereas GVL in mismatched chimeras requires T-cell infusion alone [130]. These results may put new constraints on the efficacy of DLI in humans, although by comparison with mice, humans are significantly lymphopenic post transplant [122]. Finally, it has been proposed that, in certain malignancies, APCs may arise directly from the malignant clone, serving to prime donor T cells directly to tumor-associated antigens [81]. In chronic myelogenous leukemia, bcr-abl+ DCs can be derived in vitro from monocytes [131], but the spontaneous generation of these cells in vivo has never been examined. A similar argument applies to B cells in follicular lymphoma [132]. It is likely that the susceptibility of these diseases to GVL is due not only to efficient priming of donor T cells, but also to the slow kinetics of disease and relatively normal phenotype of the leukemic clone. In other diseases where the GVL response is less effective, relapse can easily occur during GVHD, suggesting that the target cells escape recognition or overwhelm the effectors rather than that there is a fault with T-cell priming.

DCs as potential therapeutic targets in transplant settings DCs are potential therapeutic targets in the peritransplant and post-transplant settings. Conventional drugs such as the calcineurin inhibitors, mycophenolate mofetil, and glucocorticoids are now known to mediate some of their actions through DC modulation [133–135] and, as described above, conditioning therapy and monoclonal antibodies have profound effects. A number of novel approaches involving DCs have also received recent interest. The first, suggested over 20 years ago, is that ablating recipient APCs might prevent GVHD [84]. The principal objection to this strategy, which parallels the problem of donor T-cell depletion, is that GVL might also be lost. The absence of appropriate APCs indeed diminishes the GVL response in a number of experimental models and provides further evidence that GVHD and GVL are facets of the same process [39,92,102,126]. There is also concern that regulatory T cells might not be formed, diminishing the potential therapeutic advantage [136]. A counterargument is that different regional populations of DCs

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may have more or less propensity to induce GVHD, as previously outlined. Prevention of hepatic GVHD by removal of hepatosplenic APCs with liposomal clodronate lends credence to this concept [103]. It is likely that prior cytotoxic therapy of human subjects attenuates GVH reactions, given that a single nonmyeloablative dose of cyclophosphamide pretreatment diminishes GVHD in mice [137]. This could explain the relatively high risk of GVHD seen historically in patients with chronic-phase chronic myeloid leukemia compared with patients given induction chemotherapy for acute leukemia, rather than biologic differences in the diseases themselves. Murine experiments have raised the profile of ultraviolet (UV) irradiation of the skin as a means to accelerate the depletion of recipient LCs [48]. The regional effects of UV may also be complemented by a general immunosuppressive effect [138,139]. However, reservations exist in view of the transient, cytokine-mediated nature of some responses [140] and subsequent recruitment of potent immunostimulatory APCs to the skin [141]. At least one early-phase clinical trial reported increased GVHD, although UV treatment was continued post transplant, making it difficult to evaluate selective effects of UV in the conditioning phase [142]. An alternative regional therapy is the pre-emptive use of topical corticosteroids [143]. Complementary to the attenuation of GVH responses by the removal of recipient DCs is the possibility of stimulating donor immunity by infusing recipient DCs post transplant. This is supported by experimental models [101,102] and could be an important adjunct to DLI, validating the concept of DC-based vaccination for malignancy. One trial of allogeneic HLA-matched DCs post transplant has already been reported [144]. There are logistical issues concerning the routine storage of recipient DCs prior to transplant since many will remain unused, but such a strategy might also be of specific use in cord blood transplantation, in which there is no recourse to the donor post transplant, or when matched

unrelated donors prove difficult to trace. Others have shown that regulatory or tolerogenic DCs from the recipient could also be given to treat GVHD in an MHC-mismatched model [145]. In general terms, therapeutic targeting of DC has the potential to control alloresponses beyond means currently available. Antibodies such as CMRF-44 recognize activated DCs in any site and are able to lyse blood DCs and LCs in the presence of complement [44]. Blockade of co-stimulatory receptors has been advocated for several years as a way of attenuating the initial encounter between DCs and T cells. Newer small molecules such as FTY720, a sphingosine receptor antagonist, have specific actions on DCs, preventing migration to the LNs, and a selective effect on GVHD has been reported with this drug [146]. Inhibitors of Fms-like tyrosine kinase-3 (Flt3) may also target DCs since the Flt-3 receptor, which is related to c-kit and CSF-1, appears to be required for the differentiation and maintenance of DC populations [147]. Flt-3 inhibitors have shown some activity in ameliorating experimental autoimmune encephalomyelitis [148]. Another novel compound, NK026680, has been reported to interfere with DC activation [149].

Conclusion The prominent role of recipient APCs in initiating GVH responses and the importance of GVL in eradicating human malignancy have galvanized efforts to understand the homeostasis of peripheral DC populations. Many questions remain, such as the identity and location of the principal APCs in the GVH response, especially in fully complex immune systems. Elegant results have been achieved in animals by isolating different components of the GVH reaction, and new methods to manipulate APCs in vivo are evolving. However, proof that DC-targeted therapy can improve the therapeutic benefit of the GVL response in relation to GVHD has not yet been obtained.

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20

Judith A. Shizuru

The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases

Introduction Autoimmune diseases (ADs) are a heterogeneous group of disorders that affect an estimated 3–5% of the United States population [1,2]. These diseases occur when there is a breakdown in the signals that mediate immune tolerance to normal tissues. The result of such breakdown is the activation of cellular effector mechanisms and subsequent tissue destruction. Theoretically, all tissue types can be targets of an immune response; however, it is not known why certain organs are involved more commonly than others. Six of the most common ADs are listed in Table 20.1; these are rheumatoid arthritis, systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1DM), Graves’ disease, multiple sclerosis, and pernicious anemia. Collectively, these diseases represent approximately 50% of ADs [3]. Autoimmune responses are generally sustained, persistent with manifestations of chronic tissue damage, presumably because self-antigens are continually produced on the targeted tissue, and, in severe cases, diminution of the response does not occur until the cells expressing the autoantigens are destroyed. More than 30 years ago, it was demonstrated that transfer of hematopoietic cells can alter the course of ADs in rodents. Bone marrow transplantation (BMT) experiments were shown to transfer disease from autoimmune prone rodents to unaffected ones [4–6] and, conversely, to prevent disease if the hematopoietic cells were transplanted from unaffected rodents to susceptible ones [5,7–9]. The goals of this chapter are to provide a basis for understanding how and why hematopoietic cell transplantation (HCT) may effectively treat severe ADs, and to describe the preclinical studies that have contributed to this understanding. Clinical updates are provided in Chapter 69. The chapter begins with an overview of how normal antigen-specific immune responses develop, followed by a discussion of why autoimmunity occurs. Thereafter, studies on preclinical models using HCT for the treatment of autoimmune syndromes are presented.

The immune response Induction and perpetuation of immune responses Induction of antigen-specific immune responses occurs as a complex cascade of events. T lymphocytes, specifically CD4+ T cells, are credited

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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with playing the pivotal role [10]. Activation of naïve CD4+ T cells forms the critical link between the recognition of foreign antigen and the induction and perpetuation of effector mechanisms that destroy the antigenic source. Most effector cells rely upon ancillary signals provided by activated CD4+ T cells in order to proliferate and differentiate. Because the consequences of nonspecific or inappropriate stimulation of naïve CD4+ T cells are potentially disastrous, a number of criteria must be met to activate these cells [10–12]. Only certain specialized cells designated professional antigen-presenting cells (APCs) fulfill these criteria. The APCs include B lymphocytes, macrophages, and dendritic cells (DCs). As part of their function, APCs take up foreign protein antigens by endocytosis. The antigens are then processed via an intracellular pathway into smaller peptide fragments, and the fragments are then bound to class II major histocompatibility complex (MHC) molecules that make their way to the surface of the APC. Among these professional APCs, DCs have emerged as the most influential in controlling immunity. The seminal event in the activation of a quiescent and circulating CD4+ T cell is an encounter with its antigen, which consists of the appropriate peptide bound to a self-MHC class II molecule. In addition to the binding of a T-cell receptor (TCR) to its cognate antigen, activation cannot be achieved unless this interaction is accompanied by the simultaneous delivery of a co-stimulatory signal(s). In the absence of co-stimulation, engagement of the antigen receptor can lead to T-cell anergy – a state of unresponsiveness to the antigen [13]. Only professional APCs both express high levels of class II MHC molecules on their surface and are capable of delivering the appropriate co-stimulatory signals. Co-stimulatory molecules expressed by APCs include B7-1 (CD80) and B7-2 (CD86) – the ligands for CD28, CD40, inducible costimulator ligand, and various adhesion molecules [11,12,14]. APCs themselves, and in particular DCs, demonstrate phenotypic and functional plasticity, and the expression of these differences when encountering naïve T cells determines whether or not the encounter will result in the induction of immune reactivity versus tolerance [15]. The environmental milieu in which a T cell encounters an APC also influences the outcome. Specifically, the cytokine environment can skew the commitment to certain effector pathways, resulting in the polarization of T-cell responses. Following TCR binding plus co-stimulatory signaling, naïve T cells respond by rapidly proliferating and differentiating. As part of this process, they begin to express new receptor molecules and synthesize and secrete a number of chemokines and cytokines. Antigen-specific T-cell clones expand and develop a distinct effector phenotype. At least three CD4+ effector phenotypes have been characterized based upon

The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases Table 20.1 Associations of human leukocyte antigen (HLA) serotype with susceptibility to autoimmune disease

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Disease

Affected organ

HLA association

Relative risk

Rheumatoid arthritis Systemic lupus erythematosus Type 1 diabetes mellitus Graves’ disease Multiple sclerosis Ankylosing spondylitis

Joints Systemic Pancreatic islets Thyroid Central nervous system Joints

DR4 DR3 DR3/DR4 heterozygote DR3 DR2 B27

4.2 5.8 25 3.7 4.8 87.4

their distinct cytokine-secretion patterns, which activate unique downstream cellular responses [16]. These subtypes are designated T helper type 1 (Th1), Th2, and Th17. Th1 cells secrete the cytokines interferongamma and tumor necrosis factor-beta (TNF-β), which permit these cells to be particularly effective in protection against intracellular pathogens, including viruses and bacteria, as well as tumor cells. Th2 cells secrete interleukin-4 (IL-4), IL-5, IL-10, and IL-13. Th2 cells activate B lymphocytes to become antibody-secreting plasma cells, a defense mechanism effective against extracellular bacteria and parasites. Th17 cells were recently discovered and secrete IL-17, IL-17F, IL-6, IL-22, and TNF-α. These latter cells appear to play an integral role in both tissue inflammation and activation of neutrophils to combat extracellular bacteria. A fourth subset of CD4 cells designated T regulatory (Treg) are noneffector cells that play an important role in the control of immune reactivity against self-antigens. Treg cells, first described by Sakaguchi in 1995 [17], constitute a minor population of CD4+ T cells that coexpress CD25. Although no specific surface marker is yet associated with Tregs, the forked/winged helix transcription factor (FoxP3) has been identified as a key regulatory gene in the development and function of Tregs [18]. CD4+CD25+FoxP3+ Tregs are discussed in more detail below. Differentiation of naïve CD4+ cells into Th1, Th2 or Th17 subtypes occurs within a few days of direct contact with APCs. A number of factors determine which effector subtype will predominate, and these factors include the nature and affinity of the antigen, the type of TCR signaling, the nature of the co-receptor signals that are involved, and most importantly the predominant cytokine environment. In broad terms, Th1 cells mediate the cellular immune response, turning on effector populations including activated cytotoxic T cells and other inflammatory cells such as natural killer (NK) and phagocytic cells. Th2 effectors mediate the humoral component, which includes antibodies and complement proteins. The actions of Th17 cells are just now being defined, but like Th1 cells they are proinflammatory. Control of immune reactivity To ensure that autoreactivity does not occur during the course of defending host tissues, activation of T lymphocytes is highly regulated. There are at least four ways that self-reactivity is controlled. These mechanisms are termed clonal deletion, immunologic ignorance, anergy, and regulation. Clonal deletion During development, T and B lymphocytes undergo a rigorous selection process to delete potentially self-reactive cells [19,20]. Hematopoietic stem cells (HSCs) give rise to all lymphoid progenitor cells. For T cells, the progenitors migrate to the thymus, which provides a specialized microenvironment for T-cell maturation and selection. Thymic medul-

lary epithelial cells are involved in the negative selection of self-reactive thymocytes [21] and are known to ectopically express peripheral tissue antigens [22]. Developing T cells that are potentially self-reactive – that is, those cells with TCRs that bind too strongly to self-peptides plus self-MHC molecules – are eliminated by the process termed negative selection or clonal deletion. Only those T-cells with receptors that have potential to recognize self-MHC molecules plus foreign peptides (positive selection) can leave the thymus and enter the blood stream. Immature B cells that express immunoglobulin receptors that bind too strongly to components of self either die within the bone marrow (BM) or become impaired in their ability to respond to antigen (anergic) [20]. Immunologic ignorance Most self-proteins are expressed at levels that are too low to serve as targets for T-cell recognition, and thus cannot serve as autoantigens. It is likely that only very few self-proteins contain peptides that are presented by a given MHC molecule at a level that is sufficient for effector T-cell recognition but too low to induce tolerance. T cells able to recognize these rare antigens will be present in the individual but will not normally be activated; they are said to be in a state of immunologic ignorance [23,24]. Most autoimmunity likely reflects the activation of such immunologically ignorant cells. Anergy A third level of control against nonspecific or self-reactive immune responses occurs if the requirements for lymphocyte activation fail. Quiescent lymphocytes traffic through the blood, lymphatics, and lymphoid organs in search of the cognate antigen that will bind their antigenspecific receptors. Engagement of these antigen receptors in the absence of appropriate co-stimulatory signals leads to a state of long-lived functional unresponsiveness known as anergy [12–14,25]. This cell-intrinsic state of unresponsiveness is associated with defects in cell-cycle progression and some effector functions, which is reversible with strong stimuli. Anergized T-cell clones produce negligible amounts of IL-2, which is crucial for clonal expansion following T-cell activation. Anergy can be overcome by elevated levels of exogenous IL-2 during restimulation [13]. Anergy has been observed in both T and B cells. Regulation or suppression A fourth way in which unwanted immune responses can be controlled is through populations that function to suppress lymphocyte activity. Experiments from the early 1970s [26] supported the existence of CD8+ cells that downregulated the reactivity of other T cells in an antigenspecific fashion. Although the phenomenon of cell-mediated immune suppression clearly exists, the identity of suppressor cells and their mechanisms of action was the subject of controversy for many years. The last decade has seen the re-emergence of the suppressor cell field; however, populations that mediate suppressive activity have been given the updated designation of regulatory cells. Among the most widely

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studied regulatory cells are the CD4+CD25+FoxP3+ cells introduced above [17,18,27] and another population designated NK-T-cells [28]. These regulatory cells have been identified in both rodents and humans. CD4+CD25+FoxP3+ Tregs have been shown to inhibit a variety of immune responses both in vitro and in vivo. Although Tregs require activation by antigen exposure to initiate suppressive functions, the effector (suppression) phase is independent of antigen specificity. Multiple mechanisms of suppression by Tregs have been described, with differences observed between in vitro and in vivo studies. In many experimental systems, Tregs function in vivo by production of the immunosuppressive cytokines IL-10 and transforming growth factor-beta. In contrast, Tregs suppress immune responses in vitro by a contact-dependent mechanism. In addition to the direct suppressive mechanisms, Tregs can inhibit immune responses through modulation of DC function. Cells that express both markers of NK cells (NK receptors) and TCRs qualify as belonging to a heterogeneous population termed NK-T cells. The most prominently studied subclass of NK-T cells has been termed invariant NK-T cells since their TCR diversity is very limited owing to their expression of a single α-chain (Vα14-Jα18 in mice, Vα24-Jα18 in humans) coupled with a β-chain rearranged with limited Vβ gene segments (Vβ8.2, Vβ2, and Vβ17 in mice, Vβ11 in humans). NK-T cells recognize glycolipid antigens presented by a monomorphic glycoprotein CD1d. Invariant NK-T-cells have been shown to suppress autoimmunity in animal models, as well as graft-versus-host disease (GVHD) responses [29–31]. Another form of regulation that has been observed in many autoimmune models involves preferential activation of CD4+ T-cell subsets (Th1 versus Th2) [32]. Several reports have shown that ADs are associated with activation of Th1 cells, which drive cellular responses mediated by activated macrophages and inflammatory processes. In certain animal models of AD, it has been shown that the relative activation of the CD4+ subsets can be manipulated to give either a Th1 response, which results in disease, or a Th2 response (humoral), which confers protection from disease. The preferential activation of Th1 and Th2 cells can be achieved by manipulation of the cytokine environment or by administration of an antigen by particular routes (such as feeding). Based on the observation that T-cell responses become progressively polarized towards one type or another, a hypothesis arose designated “Th1/Th2 paradigm” (which pre-dates the revival of regulatory T-cell subsets). The Th1/Th2 paradigm suggests that T cells reciprocally regulate their responses by producing autocrine factors to nurture their own development and activity while suppressing those of the other type. This hypothesis continues to evolve.

Autoimmune pathology ADs arise when self-antigens become targets for tissue destruction. The response may be directed against a single tissue type or a very limited number of tissues. Histologic studies have shown variability in the apparent causes of tissue destruction since predominance of antibodies, activated by T or B lymphocytes, or nonspecific inflammatory cells, can be found in the inflammatory lesions. On the basis of such studies, it was concluded that different diseases are predominantly mediated by either humoral (antibody) versus cellularly driven immune responses. A traditional categorization of immunologic diseases divides this syndrome into four types, designated as types I–IV hypersensitivity responses. Type I responses are caused by antibodies of the immunoglobulin E isotype and are considered to be allergic responses, not classical autoimmune responses. Types II–IV involve tissue damage. Type II responses are mediated by antibodies directed against the targeted tissue, type III responses by antibody–antigen complex deposition, and

type IV by cellular processes. It should be emphasized, however, that these classifications do not illuminate the more fundamental and important issue of what triggers autoimmune responses since, by the time an autoimmune process becomes clinically evident and classifiable by this scheme, the initiating events are obscured by the downstream effector mechanism causing the actual tissue damage. At least for the reactions of type II, III and IV, there appears to be a common pathway by which autoreactive lymphocyte clones develop and escape the controls that enforce self-tolerance. From our current understanding of how the immune system functions and from the cumulative experience in the study of autoimmune syndromes in animals, it is thought that loss of T-cell tolerance is the central pathogenic event. Genes and environment Both genetic and environmental factors appear to be required in the development of ADs. Family studies, animal models, and human epidemiologic studies all support the role of these factors in AD susceptibility. The importance of genetic predisposition was first identified by analyses of disease incidence in monozygotic twins. The concordance rates in twins ranges from approximately 15% for rheumatoid arthritis [3,33] to a robust around 57% for SLE [3,34]. Comparisons of these rates with disease incidence in the general population predict that genetic predisposition is a dominant factor. For example, the lifetime risk of developing T1DM in the general population in the United States is 0.4%, whereas for monozygotic twins the concordance rate is in the range of 30–50% [35,36]. For siblings, the rate is still significantly increased above that of the general population at around 6%, but it is lower than for twins. This decrease in sibling concordance rates compared with monozygotic twins suggest that multiple genes contribute to genetic predisposition. Thus, while genetic susceptibility is a dominant factor, the pattern of inheritance of ADs is complex [1,3]. The diseases are polygenic, meaning that they arise from several independently segregating genes, and while candidate genes have been identified for specific diseases, the only clearly defined consistent genetic markers for susceptibility to any ADs are certain alleles of the genes located within the MHC (Table 20.1). Inbred rodent strains exist that reliably develop spontaneous ADs (Table 20.2). These animals are highly inbred and thus genetically identical. Like human twins, many but not all animals in these inbred colonies develop disease. This lack of 100% concordance in genetically identical humans and rodents provides evidence for the essential role of environmental interactions on AD development. Observations made in rodents where environmental elements can be controlled reveal that some of the factors affecting disease incidence include exposure to infectious pathogens and diet [37–41]. For example, a germ-free environment has been shown to suppress or enhance autoreactivity in mice with spontaneously arising forms of multiple sclerosis [42] and diabetes [37], respectively. Furthermore, it is well known among investigators that raise nonobese diabetic (NOD) mice, a model for T1DM, that certain common mouse pathogens, such as pinworms, lead to a dramatically reduced incidence of diabetes. Oral ingestion of protein antigens has been shown to lead to marked suppression of systemic humoral and cell-mediated immune responses when animals are later immunized with the same antigen. This phenomenon is called oral tolerance [43]. A high-fat, high-protein diet has been shown to increase the rate and severity of diabetes in NOD mice [44]. Another factor contributing to autoreactivity is sexual dimorphism. Sexual dimorphism refers to a pattern of skewing of disease incidence and/or severity depending upon one’s sex. It has been observed in many human ADs, such as SLE and autoimmune thyroid disease, that human females demonstrate a disproportionately higher incidence compared

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Table 20.2 Animal models of autoimmune disease Strain or Designation

Disease model

Induction/manipulation

NOD mice BB rats (NZB/NZW)F1 mice MRL-lpr/lpr mice BXSB mice NZB/KN mice C57BL mice SJL mice Buffalo rats Mice – strains variable DBA1 mice Buffalo rats

Type 1 diabetes mellitus Type 1 diabetes mellitus Systemic lupus erythematosus Systemic lupus erythematosus Systemic lupus erythematosus Polyarthritis Multiple sclerosis (EAE in rodents) Multiple sclerosis (EAE) Multiple sclerosis (EAE) Multiple sclerosis (EAE) Rheumatoid arthritis Rheumatoid arthritis

Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous Spontaneous Induced – immunize with peptide of myelin oligodendrocyte glycoprotein Induced – immunize with mouse spinal cord homogenate Induced – immunize with rat spinal cord homogenate Induced – transfer of lymphocytes from myelin antigen-immunized syngeneic donors Induced – immunize with type II collagen Induced – administer Freund’s adjuvant

EAE, experimental autoimmune encephalomyelitis.

with males [45]. Rodents with ADs such as NOD mice show a similar pattern of sexual dimorphism. In NOD mice, castration studies [46] and administration of exogenous male hormones [47,48] have shown that sex-related hormones contribute significantly to the dimorphism. Data from human epidemiologic studies confirm the contributions of genetic and environmental factors in AD incidence. Such studies show clear associations with race, geography, and susceptibility to disease. Again using T1DM as an example, the incidence of disease is approximately 40 times higher in Finland than in Japan [36].

individuals comprise 2% of the United States population, but 40% of patients with T1DM. A similar evolution in the association of HLA type and disease susceptibility has occurred for Graves’ disease. Graves’ disease was among the first autoimmune disorders noted to have an association with HLA haplotype and the initial association was with the MHC class I genotype HLA-B8. Later, however, it became evident that stronger association was with HLA-DR3, which is tightly linked to HLA-B8 [52]. Function of MHC in AD pathogenesis

MHC genes and susceptibility to ADs The only established genetic association for predisposition to ADs is the genotype of the MHC [49]. This association was noted in the mid-1970s. Initially, correlations were made between class I MHC type and the spondyloarthropathies. Ankylosing spondylitis, an inflammatory and presumably autoimmune disease of vertebral joints, was found to be strongly associated with the class I human leukocyte antigen (HLA)-B27 allele. Individuals who are HLA-B27 positive have an approximate 90–100 times greater chance of developing ankylosing spondylitis than do individuals that lack B27. Later, the emphasis shifted to associations with class II rather than class I MHC molecules since frequent associations were found in subsequent studies with class II gene products and other ADs, such as Graves’ disease and T1DM (Table 20.1). In the last 30 years, the technology of HLA typing has advanced from serologic assays to the more sensitive molecular-based assays that detect variations of the nucleotide level (see Chapter 12). As this technology for HLA genotyping has become more precise, allowing examination of specific regions of the MHC, the associations have become stronger. For example, it has been observed that up to 95% of Caucasians developing T1DM express the HLA alleles DR3 or DR4, compared with about 40% of normal individuals, and that individuals heterozygous for both DR3 and DR4 have the highest risk of T1DM development [50]. Subsequent to these observations, it was shown that in fact the DQ rather than the DR genotype is a more specific marker for T1DM susceptibility, and that the previous correlation with HLA-DR was due to the fact that DR and DQ are products of closely linked genes (linkage disequilibrium) [51]. Thus, for T1DM, the highest-risk DQ alleles DQα1*0501/ DQβ1*0201 and DQα1*0301/DQβ1*0302 are invariably found in the DR3 and DR4 genotypes, respectively. Individuals heterozygous for these two DQ alleles are at greatest risk of T1DM development. Such

Although genetic associations are firmly established between ADs and defined MHC haplotypes, the way that these molecules contribute to autoimmune pathogenesis remains hypothetical. MHC molecules play central roles in both T-cell selection and during T-cell ontogeny, and in the presentation of antigen to T cells. Thus, it has been hypothesized that certain AD-associated MHC haplotypes permit the faulty selection of T cells during development and/or allow aberrant presentation of self-peptides to T cells that results in inappropriate T-cell activation [53–55]. MHC/peptide-restricted recognition of T cells results from the combined effects of the differences in peptide binding and of direct contact between allotypic portions of the MHC molecule. It is known that certain polymorphic amino acids that form the walls of the peptidebinding groove can result in profound differences in binding affinity of MHC molecules with self-peptides, as well as affect the confirmation of the MHC–peptide complexes seen by the antigen-specific TCR. Furthermore, other polymorphic residues of the MHC molecules can make direct contact with TCRs and thus affect antigen recognition. Therefore, it is possible that the disease-associated MHC haplotypes make certain self-antigens appear foreign and/or the haplotypes generate a strong enough immune response to self-antigen to induce T-cell activation. The above hypothesis presumes that the association of ADs with MHC haplotype derives directly from the function of the MHC gene products. While this hypothesis has been amply supported by data from both human and rodents, it is not conclusively proven. Disease association clearly maps to the MHC region; however, contained within this region are a number of other genes. Alternative hypotheses include that the MHC-haplotype serves only as a marker and that the true (and as yet undetermined) disease-associated genes are closely linked to the MHC alleles.

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Non-MHC genes and susceptibility to ADs The importance of genetic predisposition in AD susceptibility and the conclusion that several genes contribute to an AD phenotype has motivated the search for predominant non-MHC susceptibility genes. It is hoped that genome-wide analyses will enable the identification of such genes. To achieve this goal, international coalitions have been formed aimed at collecting large cohorts of families afflicted with specific ADs and employing state-of-the-art technologies to scan their genomes for the location of susceptibility genes. These analyses have confirmed the complex nature of genetic associations and supported the conclusion that defining the AD susceptibility genes is not easily achieved. The reason for the difficulty is that the inheritance of AD susceptibility is multifactorial – disease arises not only from a combination of multiple contributing susceptibility genes, but each gene also has the possibility to interact with a poorly defined array of environmental and/or stochastic factors. Furthermore, identification of AD susceptibility loci is complicated by two factors that commonly influence inheritance of multifactorial traits: genetic heterogeneity and epistasis. Genetic heterogeneity means that different combinations of individual abnormalities are capable of causing a similar disease phenotype. Examples of genetic heterogeneity are seen by comparison of the genomic locations of susceptibility genes in separate mouse models of ADs such as T1DM, SLE, and a rodent form of multiple sclerosis (designated experimental autoimmune encephalomyelitis [EAE]) wherein the genomic locations of many susceptibility alleles vary between models [3]. Even within the same AD syndrome such as different rodent models of SLE, susceptibility is mediated by a heterogeneous array of genes. Epistatic interactions classically refer to interactions in which the genotype at one locus affects the phenotypic expression of the genotype at another locus. Examples of epistatic interactions have been shown by Wakeland and colleagues who generated a series of congenic mice susceptible to SLE [56–59]. Those studies have shown that epistatic interactions between two susceptibility alleles can lead to a greater increase in disease severity than would be predicted by simply adding together their individual phenotypes. The studies have also shown that epistatic interactions can also diminish the autoimmune phenotype by interaction of susceptibility alleles with epistatic modifier genes. The initial approach taken to utilize the expanded human database was to apply linkage analysis. The principle of linkage studies is to determine if genetic markers can be identified in family members that share the same trait. The assumption is that the gene that causes the disease phenotype is either the one identified or in relative close proximity to the marker. Linkage analysis is most amenable to genes involved in recessive, and highly penetrant diseases. For ADs and other diseases caused by multifactorial traits, the best approach for linkage analysis has been to analyze affected sibling pairs in which allele sharing is analyzed using families with two affected siblings. Given the low incidence of AD, the collection of large cohorts of such families has been difficult, and, as a result, many of the linkage studies have had relatively limited sample sizes. Thus, although some linkage studies such as those in SLE have provided important insights into the characteristics of genetic predisposition to SLE, identification of disease alleles has been largely inconclusive. More recently, genome-wide association studies have been employed that take advantage of the detection and cataloging of single-nucleotide polymorphisms (SNPs) identified in the human genome and technologies that allow high-throughput SNP typing for large numbers of SNPs simultaneously. Association studies differ from linkage studies since they examine SNP typing in diseased versus control populations. This approach has led to the identity of a number of candidate susceptibility loci outside the MHC region for ADs, including multiple sclerosis [60],

inflammatory bowel disease [61–63], rheumatoid arthritis [64], T1DM [65,66], and SLE [67]. It is gratifying that many of the candidate susceptibility alleles map to genes that have key roles in immune responses. For example, the multiple sclerosis study identified alleles of the IL-2 receptor-alpha (IL-2RA) and IL-7RA genes as heritable risk factors [60]. Despite these promising findings, the results of association studies still need to be interpreted with caution, as linking the biology with allelic variation must take into account the complex interaction between disease alleles and modifiers, as well as environmental factors. What triggers autoreactivity? Several hypotheses exist to explain what triggers and perpetuates AD pathology. Based upon the cumulative data that link autoimmune responses with MHC type, and the central function of T cells in the induction and perpetuation of antigen-specific immune responses, these hypotheses have focused primarily on loss of T-cell tolerance, either through inappropriate presentation of antigens and stimulation to T cells via APCs, or through the failure to eliminate or silence self-reactive T-cell clones. Although T cells undergo rigorous selection processes during development to eliminate self-reactive clones, it is thought that such clonal deletion is imperfect, and that circulating, self-reactive naïve T cells exist that are controlled by the mechanisms of peripheral tolerance. The predominant view is that one or a few self-peptides trigger a cascade of cellular events that results in targeted tissue damage [54,68,69]. A typical immune response against a self- or foreign protein is usually focused on one or two peptide sequences (called epitopes) contained within that protein, which are termed dominant epitopes. Once a response is triggered against the dominant epitope, other peptide epitopes from the same protein become targets, thus expanding and perpetuating the immune response. This hierarchical extension of an immune response from a dominant epitope to a subdominant one is termed epitope spreading [69]. Most self-peptides cannot serve either as autoantigens simply because they are present at levels that are too low to be detectable by naïve T cells. However, a few self-peptides that have failed to induce tolerance may be present at high enough levels to be recognized by T cells. These peptides are likely to be the breakdown products of tissuespecific proteins, and it is thought that only certain proteins can act as autoantigens since there are relatively few distinct autoimmune syndromes, and individuals with a particular AD seem to recognize the same antigenic targets. Thus, autoimmunity can occur if an APC presents a dominant epitope of a self-protein in conjunction with the appropriate activating co-stimulatory molecules, resulting in activation of CD4+ T cells. Once autoantigen specific CD4+ T cells are triggered, barring intervention by suppressive or regulatory subsets, the pathway is set towards elimination of the inciting antigenic stimulus. Two predominant hypotheses of how spontaneous ADs may be induced are by molecular mimicry or tissue injury. The hypothesis of molecular mimicry (Fig. 20.1) suggests that immune responses directed against infectious agents can cross-react with self-antigens, causing autoimmune destruction. Thus, the inciting antigen could be a bacterium- or virus-derived protein that shares an amino acid sequence with a prevalent tissue-specific protein. Antibodies or cytotoxic T cells directed against the pathogen will also selectively destroy the normal tissue that expresses the cross-reactive protein. Relevant examples come from the studies in T1DM where correlations exist between congenital rubella and Coxsackie virus [70]. Mumps virus as well as cytomegalovirus have also been implicated in the pathogenesis of human T1DM. The best-documented information linking a preceding viral infection with the onset of diabetes comes from the study of children and young adults diagnosed with having congenital rubella

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Fig. 20.1 Induction of autoimmunity. Schematic illustration of events thought to cause an autoimmune response according to the hypothesis of molecular mimicry. An antigen-presenting cell (APC) internalizes a pathogen and presents a peptide derived from the pathogen to a CD4+ T cell by a disease-associated class II major histocompatibility molecule on its surface. The peptide sequence cross-reacts with a component on pancreatic islet β-cells. The CD4+ T cell is activated upon receipt of this first signal by its cognate antigen and co-stimulatory signals from the APC. Following activation, CD4+ T cells then activate downstream effector mechanisms through direct cell-to-cell contact and the secretion of cytokines. The pancreatic β cells are destroyed since they express a protein that contains the same peptide sequence as the inciting pathogen. CTL, cytotoxic lymphocyte; M, macrophage; NK, natural killer; PC, plasma cell.

syndrome. The incidence of T1DM in such patients has been estimated to be 10–12%, and up to 40% of congenital rubella syndrome patients show impaired glucose tolerance [71,72]. It has been shown that an immunogenic epitope for the viral capsid protein of rubella has structural similarities to an islet β-cell protein [73]. Furthermore, amino acid sequence homology with peptides from rubella virus and an enzyme found within β-cells called glutamic acid decarboxylase (GAD) has been identified [71]. GAD has been identified as an important autoantigen expressed in islet β-cells. Homology with Coxsackie virus and GAD have also been identified. These sequence homologies with virus-derived peptides and GAD, and the discovery of cross-reactive T cells and/or antibodies against these antigens from patients with T1DM, support the molecular mimicry mechanism of pathogenesis. The tissue injury hypothesis attributes activation of localized inflammatory mechanisms in response to organ injury as the inciting event. During inflammation, the release of chemoattractants and cytokines recruits macrophages, lymphocytes, and other effector cells. The result may be the release of tissue-specific antigens and uptake by APCs that can result in presentation of self-antigen at high enough levels to act as an immunogen. The two hypotheses are not mutually exclusive as virus infection and direct damage to islet β-cells could lead to localized inflammation and presentation of virus-derived antigens that cross-react with β-cell epitopes.

Animal models of AD Rodent models of AD have contributed significantly to the understanding of disease pathogenesis. Three major types of animal AD serve as models for study: (1) ADs that arise spontaneously; (2) ADs that are induced by antigen immunization or by adoptive transfer of autoreactive mature immune cells; and (3) ADs that are created by genetic engineering technology. Many of the concepts regarding the causes of autoimmunity have either originated or been confirmed by observations made in these animal models. Examples of these models and their homologous human diseases are shown in Table 20.2. The models rely on genetic

homogeneity so that recipients and donors are from highly inbred strains. Spontaneously arising ADs Animals that spontaneously develop autoimmune syndromes have been instrumental in revealing the complex nature of genetic susceptibility to ADs and in understanding the cellular events that lead to tissue destruction. The observations that, even among inbred animals, there is reliable but not 100% development of an autoimmune syndrome underscores the importance of the interaction of genetic plus environmental factors in autoimmune pathogenesis [3,74,75]. The essential role of certain MHC alleles as the primary susceptibility genes, the interactions between other minor susceptibility genes, and the role of T lymphocytes in driving autoimmune pathogenesis have all been confirmed by studies in these animals. The most extensively studied models of spontaneously arising ADs are mice that develop SLE-like syndromes and NOD mice that develop a disease resembling T1DM. The lupus-prone mice include the F1 hybrid of New Zealand black (NZB) and New Zealand white (NZW) designated (NZB × NZW)F1, MRL-lpr/lpr mice and BXSB mice [74,76]. As in human SLE, these animals develop autoantibodies to nuclear antigens and progressive severe glomerulonephritis. Extrarenal disease manifestations occur variably in the individual models and include lymphoproliferation with both splenomegaly and lymphadenopathy, hemolytic anemia, autoimmune thrombocytopenia, vasculitis, thrombosis, and arthritis. All of these lupus-prone strains exhibit premature thymic atrophy, the significance of which is unknown. In the (NZB × NXW)F1 model, heterozygosity at the MHC (MHC designation H-2d/z) has shown to directly impact on disease severity. Data on the non-MHC genes linked with murine lupus comes primarily from the New Zealand hybrid model for which genetic crosses have demonstrated confirmed linkage with approximately 12 loci from the NZB or NZW strains [3,74]. In MRL-strain mice, homozygosity for lpr or gld mutations results in acceleration of lupus autoimmunity [77]. Lpr is a spontaneous mutation

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of Fas (CD95), and gld is a mutation of Fas ligand. Binding of Fas ligand to Fas results in programmed cell death in the Fas-expressing cells. Although the role of these molecules in apoptosis is the subject of intense investigation, the mechanisms by which mutations in Fas lead to accelerated autoimmunity are not known. Regardless of this lack of understanding, the MRL background has been shown to significantly contribute to expression of the lupus-like disease, and neither the genes for Fas or Fas ligand appear to overlap with any of the New Zealand disease loci mapped thus far [77]. BXSB mice carry the Yaa (Y chromosome-linked autoimmune acceleration) gene [74,78], which results in more rapid and severe lupus-like disease in male versus female BXSB mice. The skewing towards higher disease severity in male contrasts the more common pattern of severity seen in female (NZB × NZW)F1 mice, NOD mice and in many human ADs, where disease severity is often skewed toward females. NOD mice develop a syndrome resembling human T1DM and are the most exhaustively studied model of an animal AD [75,79]. The disease pathogenesis begins with the infiltration of mononuclear cells into the insulin-producing islets of Langerhans at around 3–4 weeks of age. Infiltration of the islets progresses slowly over the course of several months until the islets are destroyed and the mice manifest symptoms of hyperglycemia at about 6–9 months of age. NOD mice express only one MHC class II gene product. The sequence of this class II molecule, designated I-Ag7, is unique to the NOD strain and binds peptides poorly – a characteristic that has been suggested to explain the association of this genotype with autoreactivity [53,80]. In addition, NOD mice are reported to have other immune defects [81,82], including differentiation and functional defects of macrophages, and an inability to generate effective numbers of Tregs. Progression to overt diabetes can be blocked in these mice by prolonged administration of antibodies directed against CD4+ T cells in the

MOG

prediabetic phase, demonstrating that NOD disease is CD4+ cell mediated [83,84]. Pathogenic T cells capable of transferring the disease have been cloned from these mice, and TCRs from such clones have been expressed in non-NOD background transgenic mice, causing diabetes [85–87]. Once hyperglycemia develops, NOD mice are difficult to cure since, like humans, physiologic normalization of hyperglycemia can only occur by islet or pancreas transplantation, and such transplantations are vulnerable to immune destruction by both anti-islet-specific immunity, and rejection on the basis of allogeneic differences.

Immunization and cell transfer models of ADs Conventional strain rodents can also be induced to develop autoimmune syndromes by immunization with proteins or peptides derived from defined tissues, or by transfer of pathogenic lymphocytes (Table 20.2). Genetic susceptibility seems to play a major role since certain strains are more prone to mount pathogenic responses than others. One of the best-studied examples of an antigen-induced AD is the rodent disease EAE [88,89]. EAE-affected mice or rats develop symptoms analogous to the human neurologic disease multiple sclerosis. The animals show symptoms of paralysis that, like those of multiple sclerosis, can be either progressive or fluctuating in severity. EAE is induced by subcutaneous immunization with components derived from the spinal cord (Fig. 20.2). These components range from the emulsified spinal cord itself, proteins derived from the spinal cord, or defined peptides from spinal cord proteins. The proteins that are known to induce EAE in mice include myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG). Defined peptides derived from these proteins can also induce EAE, and the exact sequence of such peptides depends upon the MHC haplotype of the

MOG

2 days

8–10 days

EAE affected MOG

Pertussis toxin Pertussis toxin

Encephalitogenic T cells 350 cGy

2 days

8–10 days

EAE affected Pertussis toxin

Pertussis toxin

Fig. 20.2 Experimental approach to causing an autoimmune disease (AD). Experimental autoimmune encephalomyelitis (EAE), a rodent disease resembling multiple sclerosis, can be induced by immunization of normal mice with components derived from mouse spinal cord. Top panel: Schematic for EAE induction using a peptide derived from myelin oligodendrocyte glycoprotein (MOG). The peptide is emulsified in Freund’s adjuvant. On day 0, the mixture is injected subcutaneously into the inguinal and bilateral axillary regions. Immunized mice receive an additional adjuvant injection (pertussis toxin) by intravenous route on days 0 and +2. Alternatively, encephalitogenic T-cell lines, T-cell clones or lymphocytes from syngeneic mice immunized with myelin components can be transferred into mice prepared with low-dose radiation plus pertussis toxin on days 0 and +2. EAE is graded on a scale of 0–5 as follows: 0, no clinical signs; 1, loss of tail tonicity; 2, flaccid tail and hind limb weakness; 3, hind limb paralysis; 4, complete hind limb paralysis; 5, moribund or dead. Bottom panel: hematoxylin and eosin-stained spinal cords from (left) a nonimmunized mouse compared with (right) an MOG-immunized mouse. Note that the intense mononuclear cell infiltrate is most prominent in the meningeal areas of the spinal cord of the immunized animal.

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phocyte receptors, and soluble molecules such as cytokines and chemokines. More recently, newer categories have arisen that include genes required for the survival and differentiation of regulatory subsets, such as FoxP3, and genes involved in the presentation of antigens for negative selection of T cells, such as AIRE (autoimmune regulator). While animals with these monogenic aberrations have been instrumental in revealing the basic biology of autoimmunity, they have not been used for purposes of clinical translation of HCT to the treatment of ADs, and therefore will not be further discussed.

mouse strain immunized. For any given mouse strain, only certain peptide sequences are pathogenic and are termed immunodominant. It was the study of the immunodominant peptide in EAE that led to the hypothesis of epitope spreading [68,69] as a mechanism by which immune responses can be perpetuated and expand to other antigenic specificities. In order to induce pathogenic autoimmune responses by immunization with peptides or proteins, it is critical that these antigens be administered as part of a mixture of adjuvants, such as Freund’s adjuvant and/or pertussis toxin. Complete Freund’s adjuvant is an oil-in-water emulsion containing dead mycobacteria that is thought to enhance immunogenicity in two ways. First, the bacterial products in adjuvant signal the APCs via the Toll pathway, resulting in the expression of co-stimulatory molecules and cytokines, and converting resting APCs to activated and migrating cells [90]. Second, emulsification of antigen in adjuvant serves to convert soluble protein antigens into particulate forms, which are more readily ingested by APCs. Another way to reproducibly induce ADs is by transfer of lymphoid cells from affected to unaffected immunocompetent recipients. Populations capable of transferring disease include cells taken from peripheral lymphoid organs, such as the spleen or lymph nodes [91,92], or cells extracted from autoimmune target tissues, such as infiltrative pancreatic islets in NOD mice. It is also possible to clone pathogenic T cells of a single antigen specificity that can transfer disease [85,86,93]. Cloned T cells are particularly valuable for tracking immune responses in vivo since they express a monotonous TCR that can be identified by labeled monoclonal antibodies (mAbs) that bind the variable region of the TCRβ chain (Vβ). Receptors from such clones can also be used to generate transgenic animals. Interestingly, adult T cells from normal mice that are depleted of CD4+CD25+ T cells can transfer an autoimmune syndrome with a wide spectrum of organ-specific manifestations including gastritis, oophoritis, orchitis, and thyroiditis [17]. Indeed, these transfer studies were one important way that the existence of regulatory CD4+CD25+FoxP3+ T cells was demonstrated [17,94,95]. Appropriate recipients for adoptive transfer studies are irradiated animals, or genetically immunodeficient strains that cannot produce T and/or B lymphocytes [96]. Two prominent examples of naturally occurring mutations that prevent normal lymphocyte development are a defect in DNA repair resulting in mice with severe combined immunodeficiency syndrome (SCID), and a defect in the Wnt signaling pathway, which results in mice that are both hairless and lack a thymus (nude mice). Mice with the nude defect cannot produce T cells. However, transfer of their hematopoietic progenitor cells to recipients with normal thymuses results in normal T-cell development. Hematopoietic progenitors from SCID mice cannot generate functional T or B cells even in a normal recipient. Genetically engineered knockouts of the recombination activating genes (RAG-1 or RAG-2) required for T- and B-cell receptor rearrangement results in defects phenotypically similar to the SCID mutation in that RAG-1 or RAG-2 knockout mice cannot generate functional T or B cells.

Preclinical studies and case reports in the human transplantation literature support the use of HCT for the treatment of severe ADs [98,99]. The clinical literature on this topic (see Chapter 69) contains several case reports demonstrating that patients undergoing allogeneic HCT for conventional indications (i.e. hematologic malignancy) with a coincidental AD experience long-term improved or even full remission of both disorders. Conversely, there have been case reports of transfer of ADs from AD-affected allogeneic HCT donors into previously unaffected recipients. In evaluating the preclinical literature for its relevance to the treatment of human ADs, it is important to bear in mind that heterogeneity exists among the experimental systems, and that often the studies were designed to answer basic questions about AD pathogenesis rather than to form a basis for direct translation to human therapy. A goal of the early investigators was to establish what cellular elements transfer or prevent disease. Thus, initial studies were directed towards the creation of allogeneic radiation BM chimeras to determine if transplantation of hematolymphoid elements could alter susceptibility to AD in rodents. As early as the 1960s, it was known that certain mouse strains, such as the NZB, develop a syndrome resembling human SLE. In 1969, Denman et al. [6] demonstrated that transfer of BM or spleen cells from NZB mice to MHC-matched nonautoimmune prone BALB/c mice (both H-2d) resulted in disease in the recipients. Later, in 1974, Morton and Siegel [4] demonstrated that, on the one hand, BM transferred into irradiated recipients could transfer disease from NZB donors into MHC-matched BALB/c or DBA/2 recipients, and, on the other hand, BM from BALB/c or DBA/2 donors could result in transient normalization of antinuclear antibody titers in NZB recipients. Donor chimerism was not measured in these studies, and one explanation proposed by the authors for the transient rather than persistent nature of antinuclear antibody depression was that perhaps only short-term chimerism was achieved in the NZB recipients. Later studies supported this latter idea since permanent allogeneic chimerism appears to be necessary to achieve long-term disease control [100,101]. Following the landmark studies of Morton and Siegel, other investigators confirmed in different SLE models that BM contains the cellular components capable of transferring disease [7,8].

Genetic engineering of ADs

ADs as “stem-cell disorders”

Genetically engineered mice that either express molecules at supraphysiologic levels or that have been knocked down for the expression of specific molecules have been generated in order to model and study defined aspects of AD pathogenesis. Numerous varieties of such genetically manipulated mouse strains now exist. There are at least three broad categories of mouse that have been manipulated at key regulatory molecules of the immune system [97]. These include the MHC, lym-

These seminal experiments led to the concept that the etiology of autoimmunity is determined by innate properties of the HSC and its differentiated lymphocytic progeny, and is independent of host environment. Indeed, it was later observed by other groups and in different animal systems that the genotypic origins of the BM (i.e. from susceptible or nonsusceptible strains) determined whether or not the animal developed or was protected from disease. However, not all studies have been con-

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sistent with this concept. For example, NOD disease (diabetes) has been transferred by parental NOD BM into F1 offspring of NOD mice crossed with different strains [91,102]. However, when NOD BM was transferred into genetically disparate recipients, the radiation chimeras engrafted with NOD BM and developed lymphocytic infiltrates of their islets, but most did not progress to overt diabetes [103]. These data show that while anti-islet reactivity can be transferred by BM, the host environment provides additional elements that permit the perpetuation of an immune response, which ultimately results in tissue destruction. (See also “Genotype and susceptibility” below.) The inconsistencies that exist in this literature are not surprising given that autoreactivity arises from a combination of interacting genetic and stochastic factors that affect hematolymphoid cells, as well as other tissues. Furthermore, BM grafts are complex mixtures of cells with different function. Among the population transferred and generated de novo in a recipient by BM are those that control antigen-specific immune responses – APCs and lymphocytes. APCs express the MHC-restricting elements that are instrumental in shaping the T-lymphocyte repertoire. Thus, it is logical that the BM genotype contributes significantly to autoimmune susceptibility. However, the concept that ADs are solely disorders of HSCs is overly simplistic. The other factors that modulate immune reactivity should be considered when interpreting the studies discussed in the next sections.

Rationale for HCT to treat ADs Both autologous and allogeneic HCT have been studied in preclinical models of AD. The rationale for efficacy differs between these two procedures. The use of autologous HCT is based on the idea that a nearcomplete ablation of autoreactive cells, primarily T cells, can be achieved by high-dose therapy followed by rescue with a hematopoietic graft that contains few or no pathogenic cells. Such an approach is analogous to the treatment of cancer wherein the conditioning regimen results in a cytoreduction of malignant cells and the patient is “rescued” with a hematopoietic graft that contains no or very few passenger cells. It is thought that the aberrant events that induce autoimmunity (such as infection with virus that cross-reacts with normal tissues) occur only rarely, and the manifestations of disease in AD patients reflect the perpetuation of effector responses that continue even though the inciting event has passed. Therefore, lymphoablation and reconstitution with autologous grafts that lack mature lymphocytes is thought to “reset” the immune system, and the disease will not recur assuming the likelihood that a second pathogenic event will trigger autoreactivity is extremely low. The rationale for allogeneic HCT is similarly based on the assumption that replacement of a defective immune system with a normal one will eliminate the autoreactive cells. Genetically distinct hematopoietic cells likely express genes that can modify recipient immune responses favoring tolerogenic rather than immunogenetic ones. In addition, mature donor cells (primarily T cells) confer an effect that has been termed “graft-versus-autoimmunity” (GVA), meaning that graft alloreactive cells eliminate host autoreactive cells. There is no expectation that GVA is specific for autoreactive cells, since it is a graft-versus-host effect that can result in broad host T-cell depletion. The majority of preclinical studies favor allogeneic HCT over autologous as the more efficacious approach to blocking autoreactivity. However, the current multicenter clinical trials of human disease have been exclusively directed to the use of autologous HCT (see Chapter 69) [104–107]. The reluctance to perform allogeneic compared with autologous HCT for ADs is based on concerns of unacceptable procedure-related morbidity and mortality in the former compared with the latter.

Definition of terms and experimental approaches Autologous, syngeneic, congenic Genuine autologous HCT has only rarely been performed in rodents because of the pragmatic limitations of harvesting autologous hematopoietic cells from small donors. Instead, syngeneic donors from the same inbred strain as the recipient, or congenic donors that differ from recipients by one or a limited number of nonhistocompatibility genes, are used. The advantage of using congenic donors is that the gene difference(s) permits determination of the origin of hematopoietic cells (residual host as opposed to graft derived) in the transplanted recipient. For example, mice that are identical except for a congenic difference at the CD45 allele are common laboratory tools. CD45 (previously designated Ly5) is expressed on all lineages of hematopoietic cells, and in mice there are two alleles, CD45.1 and CD45.2 [108,109]. mAbs exist that can distinguish between these two alleles. Thus, staining assays employing labeled mAbs, such as fluorescence-activated cell sorter analysis or immunohistochemistry, permit detection of donor versus host hematopoietic cells in the transplant recipients. Male into female transplantations or vice versa can also be used, in which case the chimerism analysis involves in situ hybridization looking for the presence or absence of the Y chromosome. Another type of donor has been termed pseudoautologous. Pseudoautologous means that, in models wherein the disease in recipients is induced by antigen immunization [110], the congenic or syngeneic donors undergo similar immunization. Grafts from such donors may contain contaminating mature autoreactive cells with the potential to cause disease, and therefore the use of such donors more accurately reproduces clinical autologous HCT. Grafts from congenic or syngeneic donor strains that develop spontaneous disease are not called pseudoautologous, although similar potential exists to transfer autoreactive cells in unmanipulated hematopoietic grafts. Here, the HCT studies are described as they were originally performed using a congenic, syngeneic or pseudoautologous donor with the understanding that all of these graft types serve as models for autologous HCT. Genetic differences and measurement of chimerism in allogeneic HCT By definition, allogeneic HCT uses donors that are genetically disparate from the recipients. Genetic disparity means that the donor and recipients are mismatched at multiple gene regions, and are distinct from congenic pairs in which the genetic differences are limited. Many of the studies were performed between donor and recipient pairs that were derived from distinct ancestral strains, and thus differ at both MHC and multiple other minor histocompatibility antigen genes. Determination of donor chimerism early on was possible using antisera or mAbs in cytotoxicity or staining assays. For transplantations involving MHC differences (MHC-mismatched and haploidentical), reagents that recognize MHC determinants have been used. Detection of chimerism between MHC-identical strains has been more difficult primarily because the reagents are more limited. In fact, studies that pre-date the late 1970s, when allele-specific mAbs were developed, did not evaluate chimerism. In order to use antibody-based assays to measure chimerism, allelic differences for defined gene products expressed at the cell surface must exist between donor–recipient strains. Furthermore, antibody reagents that distinguish the allele markers must be available, and allelic markers must be expressed on all or on subsets of hematopoietic cells. Examples of antibody reagents used for this purpose are those that recognize Lgp100 [111], a glycoprotein expressed on lymphocytes of certain strains, or CD45 [108,109], an allelic marker expressed on all hematopoietic cells. More recently, polymorphisms identified within the mouse

The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases

genome [112] can be used in polymerase chain reaction assays to differentiate hematopoietic cells between MHC-matched strains. Preparative regimens In the vast majority of rodent studies, recipients are prepared for transplantation with lethal radiation. Myeloablative radiation doses are strain specific and require titration studies to determine the dose(s) at which death occurs because of hematopoietic failure, and not other organ toxicities [7,113]. At such doses, mice that would otherwise expire are rescued by infusion of syngeneic BM cells. For any given strain, there is a range of doses that causes myeloablation without other toxicities [113,114], and thus some nonuniformity of radiation dose exists in the literature. However, such dose variation can affect the level of lymphoablation and the degree of resistance to engraftment of allogeneic hematopoietic cells. Therefore, comparisons of outcome between different experiments must take into consideration the potentially relevant effects of variability in radiation dose. The use of nonmyeloablative radiation-based approaches in rodent models in general has lagged behind studies in humans and large animals. Compared with the explosive development of reduced-intensity and nonmyeloablative conditioning protocols in humans, relatively few rodent studies have been performed. However, given the acknowledged concern of morbidity with conventional allogeneic HCT for the treatment of patients with ADs, the more recent rodent literature is testing the idea that reduced-intensity conditioning (RIC), with the consequent establishment of mixed donor chimerism rather than complete chimerism, may be sufficient to cure ADs (see the section below on reducedintensity allogeneic HCT). Chemotherapy that includes reagents used in human patients, such as cyclophosphamide (CY) and busulfan (BU), have been employed in studies whose goals were to explicitly model clinical transplantation [115,116]. CY is the best studied of the chemotherapeutic drugs in rodents, and reports from the early literature show that this agent alone without hematopoietic cell rescue is highly effective at ameliorating manifestations of ADs such as EAE in rats [117] and antibody production and immune abnormalities [118,119] in SLE-prone mice. Dimethyl myleran is an alkylating agent related to BU with profound marrow-suppressive activity and little immunosuppression that has been used for rodent HCT preparation [120,121]. Fludarabine, an agent that is widely used in human reduced-intensity regimens, has also been tested in animals. Unfortunately, mice are highly resistant to the lymphoablative effects of fludarabine and its congeners, making it difficult to model homologous reduced-intensity regimens in mice utilizing this drug. One attractive advantage of working with rodent systems is the availability of numerous antibody reagents that specifically target immune cell subsets and hematopoietic stem/progenitors. Although there is no study demonstrating that antibodies alone will effectively allow engraftment of allogeneic HSCs in wild-type animals, there are reports demonstrating that antibody treatment alone can permit engraftment in severely lymphoid deficient mice [122], and in NOD mice if high doses of CD8+ cells accompany the BM graft [123]. Cell targeting with mAbs has proven ability to augment engraftment when used in combination with nonmyeloablative radiation in NOD mice [124–126] and nonautoimmune strains. Hematopoietic graft types Unfractionated or T-cell-depleted (TCD) BM from wild-type donors or BM from mice with the nude defect have been the graft source in most studies. The reason for the use of nude mice as donors is that one manifestation of the nu/nu gene defect is the absence of a thymus [96]. Thus, BM grafts from nude mice are not contaminated with conventional T cells, but when engrafted into recipients with functional thymuses,

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their hematopoietic cells give rise to mature T lymphocytes. One group of investigators used positive selection by binding the plant lectin wheatgerm agglutinin [127]. The rationale for this approach was that wheatgerm agglutinin-positive BM cells were enriched for stem and progenitor cells, as well as an immunoregulatory “natural suppressor” population [128]. Our group [125,129] and others [130] have performed transplantations in autoimmune-prone mice with rigorously purified HSCs. HSCs were isolated using a combination of magnetic bead and fluorescenceactivated cell sorting with the positively selecting mAbs Thy1.1, c-Kit, and Sca-1 to yield a population with the composite phenotype of cKit+Thy1.1loLin−/loSca-1+ (KTLS) [131,132] (see also Chapter 5). The T-cell content of KTLS HSC grafts is reduced by more than 5 logs compared with BM. Of note, the use of KTLS HSCs or other manipulated BM populations in the allogeneic transplantation setting results in marked differences in the level of resistance to engraftment mounted by the host compared with unmanipulated BM. Furthermore, since KTLS HSCs lack mature immune populations, they cannot confer GVA activity. Consistent with the lack of the GVA is the observation that significant numbers of host T cells remain following KTLS HSC transplantations [125,129,130,133,134]. Thus, graft content has the potential to directly affect the outcome of autologous and allogeneic HCT in the treatment of ADs. Timing of the HCT procedure Since the progression of ADs in most rodent models is well characterized, it is possible to choose the time of HCT relative to the expected disease course. For example, in antigen-induced models, immunized animals have been transplanted either before or following the onset of overt manifestations. In spontaneously arising ADs, the transplantations can be performed during the phase when mice have documented abnormalities in immune function but few clinical signs of impairment, or at later disease stages. The studies show that animals are more consistently cured of the ADs if they undergo the HCT procedure at a very early stage of disease, even when there is measurable pathology such as lymphocytic infiltration of islets in NOD mice or evidence of glomerulonephritis in SLE-prone animals, but not at the point when they have suffered end-stage organ damage. If a single organ has been destroyed that is itself replaceable by transplantation, it is possible to perform simultaneous organ plus HCT transplantation (see Chapter 15). The best example of this approach in AD-affected animals has been in older NOD mice that have undergone simultaneous allogeneic HCT plus donormatched pancreatic islet transplantation in order to cure them of overt diabetes [124,125,135].

Autologous HCT Proof of concept The preclinical literature contains conflicting reports regarding the efficacy of syngeneic HCT in curing autoimmune syndromes [7]. Until the late 1980s, it was generally believed that syngeneic HCT would have no effect on AD pathogenesis. In fact, mice transplanted from syngeneic donors served as negative controls in allogeneic HCT studies to differentiate the effects of the preparative regimen alone from the effects of the allograft – a logical conclusion since syngeneic grafts were thought to merely perpetuate ongoing tissue destruction unless complete elimination of pathogenic cells was achieved. Indeed, in many studies [4,125,130,136–138], the animals in the syngeneic control group showed no amelioration of disease, whereas allogeneic HCTs were curative. However, a series of reports emerged, beginning in 1989 from van Bekkum and colleagues, demonstrating that significant remissions could

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be achieved with syngeneic transplantation in rats affected with antigen induced ADs [7]. In the original studies of arthritis caused by Freund’s adjuvant, the syngeneic BMT “control” group was unexpectedly noted to show equal resolution of disease as the allograft recipients [139]. Interestingly, rats that had undergone syngeneic BMT prior to adjuvant exposure developed disease equivalent in frequency and severity to that of naïve rats, whereas the reimmunization of rats that were disease affected at the time of syngeneic BMT did not reinduce disease. Later studies have reproduced this observation that syngeneic BMT is effective after antigen is administered but not before immunization [140]. These data suggest that one element in the effectiveness of HCT is disruption of an ongoing immune response, and that antigen exposure at the time of the procedure (and not before) is required for shifting the response from an immunogenic one to a tolerogenic one. The positive results from syngeneic BMT were repeated in a genuine autologous transplantation study wherein BM was harvested from arthritic rats by surgical removal of a femur, followed by preparation with myeloablative radiation and intravenous return of their own BM cells [141]. Thereafter, in order to reduce the suffering of the affected animals, transplantations were performed using pseudoautologous donors with the same stage of disease severity as the recipients at the time of transplantation. The curative effect of syngeneic and pseudoautologous BM transplantation was confirmed in a different AD rat model – EAE. For the EAE studies, rat spinal cord homogenate (RSCH) mixed with Freund’s adjuvant was used [116,142,143]. In the initial studies [143], the conditioning regimen of myeloablative radiation (850–1000 cGy) was begun shortly after the appearance of clinical symptoms, and a short period of exacerbated disease occurred with the radiation. It was shown that myeloablative radiation followed by transplantation of syngeneic BM from either nonimmunized or disease-affected immunized donors could lead to remission in most rats. However, a certain percentage of these animals spontaneously relapsed. Less intensive conditioning with a nonmyeloablative regimen was also performed using 750 cGy of total body irradiation plus CY. Although complete remissions were achieved in many rats, the rate of spontaneous relapse was much higher than that observed with the myeloablative treatment. Thus, EAE appeared to be more resistant to the curative effects of syngeneic BMT compared with adjuvantinduced arthritis. Success using syngeneic BMT in treating mice with EAE or spontaneously arising SLE (MRL-lpr/lpr) was also demonstrated by Karussis and colleagues [92,144,145]. In their studies of EAE, disease was induced by immunization with mouse spinal cord homogenate (MSCH) [144] or by adoptive transfer of lymph node cells from syngeneic donors immunized with guinea-pig myelin basic protein [92]. Experimental groups included mice that received different preparative regimens including two different doses of myeloablative radiation (900 versus 1100 cGy) or single dose CY (300 mg/kg). Treatment initiated at the early stage of disease or a few days following the onset of paralysis resulted in regression of clinical signs. Recipient preparation with CY yielded slightly better results than 1100 cGy of radiation, and both these treatments proved significantly better than 900 cGy. The inclusion of CY as a conditioning agent by this group and others was highly clinically relevant given the widespread use of CY in the treatment of human disease, and its use was based on several prior reports demonstrating the efficacy of CY alone in the treatment of rodent ADs [117–119]. Karussis et al. [144] tested the effect of high-dose CY with or without syngeneic BM rescue and showed that, while both treatments were equally effective in ameliorating disease symptoms, survival was superior in the BMT groups, suggesting that early rescue of hematopoietic function optimized the therapeutic outcome [144]. EAEaffected mice treated with the combination of CY plus syngeneic BMT

were also resistant to disease relapse when rechallenged with the encephalitogenic inoculum, leading to the conclusion that immunoablation followed by reconstitution induces immune tolerance to the autoantigen. Of note, mice that had paralysis for 14 days or greater experienced only a mild therapeutic effect. These investigators extended their studies on syngeneic BMT to the SLE-prone MRL-lpr/lpr mice (a spontaneously arising disease) [145]. Mice were prepared with either lethal total body irradiation or high-dose CY followed by rescue with either TCD or unmanipulated syngeneic BM. Improved survival and amelioration of serologic and pathologic evidence of disease occurred in all treatment groups, unlike untreated controls. However, long-term follow-up at longer than 20 weeks posttransplantation revealed a significant incidence of relapse. Under both preparative regimens, recipients of TCD BM grafts produced superior results compared with those receiving unmanipulated BM grafts. Autologous HCT in different disease phases Data from the above studies suggest that the timing of HCT relative to the disease phase is critical to the success of the procedure. Since it is expected that only patients with severe and longstanding ADs will be candidates for HCT, the relevance of this issue to clinical translation prompted studies comparing HCT at early versus late time points relative to disease induction. Most of the reports have focused on EAE. Burt et al. [146] showed, in a mouse model of EAE generated by adoptive transfer of lymphocytes reactive against a proteolipid protein peptide, that clinical improvement and prevention of glial scarring were achieved by syngeneic BMT if mice were treated in the acute but not the chronic phase (day +14 versus day +75 post lymphocyte transfer, respectively). Herrmann et al. [140] observed that disease symptoms in rats affected with MOG-induced EAE could be attenuated by transplantations from syngeneic, pseudoautologous, and allogeneic donors if rats were treated at the peak of acute disease after immunization (day +17) but not when the disease was in a chronic phase (day +140). A more recent study by Cassiani-Ingoni et al. [147] examined the effects of green fluorescent protein (GFP)-marked congenic BM cells in the central nervous system of EAE-affected mice. Lethally irradiated recipients were transplanted with these marked cells early in the disease course or during the chronic phase. The kinetics of reconstitution of the peripheral immune system was also measured. In agreement with other reports, transplantation was more effective when performed in the early stage of disease. However, changes that occurred in the peripheral immune system, that is, reconstitution by donor elements, seemed to have no influence in the central nervous system of mice with severe acute disease, or that were chronically affected. Rather, host glial responses to inflammatory injury appeared to perpetuate the disease process. Their results suggest that the underlying pathophysiology of an AD occurring within a target tissue may evolve over time such that even aggressive modulation of lymphoid responses will be insufficient to interrupt the process of tissue injury. Failures of autologous HCT: does T-cell depletion improve the outcome? Why some animal ADs respond to syngeneic HCT while others do not is a subject of debate. One hypothesis for the discrepancies is that antigen-induced diseases are more amenable to treatment with autologous HCT than spontaneously arising ones. Indeed, all of the reports showing successful long-term “cures” with syngeneic HCT have been in animals with antigen-induced diseases. Van Bekkum [7] has suggested that antigen-induced ADs are the more realistic models of human disease, since their etiology seems to more closely resemble events proposed to induce disease in human counterparts, that is, exposure to antigens cross-reactive to normal tissues. Although this point is not

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Fig. 20.3 Efficacy of allogeneic compared with congenic hematopoietic stem cell transplantation (HSC) in a spontaneously arising autoimmune disease reveals that the allograft confers protection beyond the effect of graft-versus-autoimmunity. (a) Non-obese diabetic (NOD) mice develop spontaneous autoimmune diabetes at around 4 months of age. Prediabetic young NOD mice were prepared for transplantation with lethal radiation plus antibodies to CD4+ and natural killer cells, and rescued with major histocompatibility complex-mismatched allogeneic or Thy1 congenic purified HSCs. Allografted mice did not develop diabetes, whereas congenic transplantations failed to block disease development. (b) Blood analysis showed that allogeneic HSC recipients were mixed T-cell chimeras compared with those undergoing bone marrow (BM) transplantation, which converted fully to donor type, yet both groups were protected from disease. (c) Prediabetic NOD mice prepared with lethal radiation and transplanted with HSCs from congenic T- and B-cell deficient NOD-SCID donors that cannot contribute to the recovering lymphocyte pool develop diabetes after a delay. (d) The NOD mice engrafted with NOD-SCID HSCs developed diabetes despite a marked reduction in their endogenous T-cell numbers. The figure shows that, even at 2 months post transplantation, T-cell levels were more than fourfold reduced. Taken together, these studies show that allogeneic HCT, in the absence of GVA, effectively blocks autoimmunity. Allo, allogeneic; Txp, tpx treatment.

proved, the successes described above have paved the way to the establishment of large clinical trials testing the use of autologous HCT for the treatment of severe and refractory human ADs [104–107] (see Chapter 69). One possible reason that spontaneously arising diseases did not respond to syngeneic BMT is because preformed autoreactive cells were not completely removed from the grafts. Indeed, syngeneic BMTs in the spontaneously arising lupus and MRL-lpr/lpr model showed better outcomes in recipients of TCD versus non-TCD BM [145]. Results were also different in an antigen-induced rat EAE model wherein comparisons were made between pseudoautologous, syngeneic, and TCD BM. Significantly higher incidences of spontaneous relapse were observed in rats that received BM plus peripheral lymphoid cells from pseudoautologous donors [142]. Given the newer technologies that permit the isolation of highly purified KTLS HSCs devoid of T-cell contamination, and the capability to more rigorously deplete endogenous host T cells with mAb targeting, studies have been done to re-evaluate whether, with such maneuvers, syngeneic HCT might be more successful in treating spon-

taneously autoimmune mice. In experiments from our laboratory [125], prediabetic NOD mice prepared with myeloablative radiation and antiT-cell antibodies were rescued with purified KTLS NOD mice from Thy1.1 congenic NOD donors. All HSC recipient mice were partial Tcell chimeras, although the absolute number of host NOD Thy1.2 cells was reduced to 25% of normal levels. Despite this treatment, most mice developed hyperglycemia within 6 months post transplant. In the same study, KTLS allogeneic HSC blocked development of diabetes in 100% of mice (Fig. 20.3) [125]. Lack of efficacy of syngeneic KTLS HSC transplantation was further shown in lupus-prone (NZB × NXW)F1 mice conditioned with highdose (1450 cGy) radiation [130]. Recipients of syngeneic transplants had circulating immune complexes and autoantibodies to doublestranded DNA, nuclear antigen, and histone equivalent to age-matched controls. Interestingly, recipients of either syngeneic HSCs or whole BM demonstrated accelerated mortality, which resulted in a death rate exceeding that of age-matched controls. Parallel studies using haploidentical HSC transplantations resulted in significant resolution of lupus

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parameters and prolonged survival. Thus, aggressive myelo- and lymphoablation, and rescue with genetically identical grafts devoid of mature lymphoid cells, was insufficient to block autoimmunity in at least two of the models of spontaneous AD. While these data seem to support the idea that autoimmunity arises as a result of “defective stem cells,” as previously discussed in this chapter, autoimmunity in animals such as NOD and (NZB × NXW)F1 mice is based upon multiple genetic defects that reside both within the hematopoietic lineage and outside it. Autologous transplantation augmented by gene therapy The “conventional” approach of using HCT to treat ADs relies upon the regeneration of a hematolymphoid system that follows a genetically scripted program driven by stochastic events encountered in the environment. Because experimental biologists now routinely produce genetically altered cell lines and rodent strains, the idea has emerged that BM can be engineered to express molecules capable of tipping the balance towards immune tolerance following transplantation. Several laboratories have reported success in blocking autoimmune pathology by the expression of selected molecules in hematolymphoid cells and transplantation into autoimmune-susceptible or affected hosts. For example, molecules known to be major targets in specific ADs have been expressed on APCs driven by an MHC class II promoter. The rationale for this approach is that antigens expressed on quiescent APCs in the thymus or periphery will lead to deletional and/or peripheral T-cell tolerance, respectively. Murphy et al. [148] used a double-transgenic model of autoimmune gastritis to demonstrate that the transfer of BM from disease-resistant transgenic mice that express a gastric antigen (H/K/ATPase) driven by an MHC class II promoter into mice that develop gastritis due to expression of the proinflammatory granulocyte–macrophage colony-stimulating factor established tolerance to the antigen. Steptoe and colleagues [149] tested a similar strategy in NOD mice. Proinsulin, the prohormonal precursor of insulin, is implicated as a key autoantigen that drives β-cell destruction in humans and mice. NOD transgenic mice were generated that expressed proinsulin under the control of an MHC class II promoter. Transplantation of these transgenic hematopoietic cells significantly inhibited the development of diabetes in prediabetic NOD mice [149]. While promising, this approach is applicable only to ADs in which the offending antigen is known (so that tolerance develops specifically to the autoantigen), which for most human ADs is generally not the case. Given the known role of disease susceptibility MHC class II alleles, the class II molecule itself has been a target for genetic manipulation. NOD mice express a single MHC class II molecule that is strongly associated with disease susceptibility (I-Ag7). Tian et al. [150] performed retroviral transduction of NOD BM cells with nonsusceptible I-Aβd–GFP or I-Aβk–GFP constructs. Expression of either of these retrovirally encoded MHC class II genes in NOD BM prevented islet inflammation and hyperglycemia when transplanted into young irradiated NOD mice. These investigators also showed that the MHC class II–GFP-transduced BM cells could protect against recurrent autoimmunity against islets since overtly hyperglycemic NOD mice could be cured of their disease if transplanted with transduced BM plus NOD-SCID islets, whereas controls that received GFP-transduced BM plus islets quickly destroyed their islets [151]. It is worth remembering, however, that MHC class II gene products are histocompatibility antigens. Therefore, for purposes of gene therapy and transplantation of the modified cells, expression of these molecules can result in untoward effects such as increased engraftment resistance and even potentially chronic GVHD [152,153]. A rapidly growing and related area of exploration is adoptive immunotherapy that targets gene expression to cells of specific hematolym-

phoid lineages. Primary T cells, T-cell hybridomas, and DCs (see Chapter 19) have been tested [154]. When transduced with regulatory proteins such as IL-4, IL-10, anti-TNF, and others, these populations have been shown to home preferentially to sites of inflammation and ameliorate disease in autoimmune animal models. While this form of cellular therapy has proven efficacy in vivo, its effectiveness would likely be potentiated in the context of autologous HCT. Autologous HCT results in significant cytoreduction of pathogenic cells, and provides a host environment that could promote greater expansion of genemodified populations (i.e. via mechanisms of homeostatic proliferation) compared with unconditioned recipients.

Allogeneic HCT In contrast to the conflicting preclinical literature supporting the use of autologous HCT for ADs, several studies have demonstrated that both spontaneous and induced forms of rodent ADs can be successfully treated by allogeneic HCT [7–9]. Indeed, the seminal studies by Morton and Siegel showing success with allogeneic but not syngeneic BMT motivated this area of research [5]. Variability exists among the experiments, including the models tested, the degree of donor–host genetic disparity, measurements of donor chimerism outcome, hematopoietic graft type, the preparative regimens, and timing of the HCT procedure relative to the onset of disease manifestations. Given these heterogeneities, it is therefore striking that rodents are consistently cured by an allogeneic HCT approach. Treatment of spontaneous ADs with allogeneic HCT In 1985, Ikehara and Good began a large series of studies demonstrating that allogeneic BMT could successfully treat spontaneously arising ADs in affected mice with overt clinical symptoms [8,9]. Similar to the prior reports, mice with spontaneously arising SLE-like syndromes were transplanted, including MRL-lpr/lpr (NZB × NBW)F1 and BXSB strain mice [155,156]. Resolution of disease was achieved even though treatment was initiated at relatively advanced stages of disease. These investigators went on to show protection from progressive insulitis and development of diabetes in young NOD mice [157]. Mice in their studies were prepared for transplantation with lethal (myeloablative) radiation, and in the initial reports recipients were rescued with either TCD BM from wild-type mice or BM from mice with the nu/nu defect. Thus, mature T cells were not transferred, but the grafts gave rise to functional T cells. Such grafts were incapable of causing GVHD, but were also limited in their ability to eliminate host T cells. Most of the experiments were performed between MHC-mismatched donor–recipient pairs. Donor chimerism was evaluated by H-2 specific antisera plus complement assays, and uniformly revealed that over 90% of spleen cells were of donor type. With the exception of the MRL-lpr/lpr SLE-affected mice [101] and NZB/KN mice that develop a spontaneous inflammatory polyarthritis [100], such allogeneic HCT was uniformly successful at not only blocking disease progression, but also resolving already established inflammatory lesions [155]. Competitive advantage of autoimmune HSC? The reasons why allogeneic BMT was unsuccessful in the MRL-lpr/lpr and NZB/KN mice was further studied by Ikehara and colleagues [100,101]. In these strains, HCT resulted in initial reversal of clinical manifestations and restoration of other immune aberrations; however, the effects of the transplants were transient, and mice regularly relapsed [155]. H-2 typing of the relapsed mice revealed correlation with the return of symptoms and loss of donor chimerism. These graft failures were attributed by the authors to abnormal radioresistant HSCs in the

The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases

host strains. In order to enhance engraftment of MHC-mismatched BM, MRL-lpr/lpr mice underwent an intensified regimen of increased radiation plus the chemotherapeutic agent CY. In addition, MRL-lpr/lpr recipients received both donor BM infusion and donor bone grafts [101]. The rationale for the added bone grafting was based upon the investigator’s prior studies, which showed better colonization and proliferation of donor BM on H-2-matched bone grafts [8]. They had concluded that such colonization in H-2-compatible BM stroma would enhance hematopoietic cell engraftment. MRL-lpr/lpr mice that received this regimen survived long term and were disease free. Further characterization of HSC from MRL-lpr/lpr mice suggested that HSCs from this strain have a competitive advantage over nonautoimmune strain HSCs based upon enhanced adhesion to stromal cells. The authors reported that this advantage was conferred by abnormally high levels of expression of the neural cell adhesion molecule [158], which they had previously found to support hematopoieis on stromal cell lines [159]. The principles of simultaneous TCD BM plus bone transplantation from MHC-mismatched donors were also applied to the arthritic NZB/KN mice [100]. The combined transplantations resulted in prevention of joint disease and long-term remissions. Studies characterizing hematopoietic transplantations in NOD mice have similarly suggested that HSCs/progenitors from this disease-prone strain exhibit enhanced engraftment potential compared with those derived from disease-resistant strains [160]. The NOD progenitors appeared to exhibit competitive advantage when co-administered with host-type HSCs in irradiated recipients. They produced more spleen colony-forming units in vivo in allogeneic recipients, and more granulocyte–macrophage colony-forming units in vitro compared with at least four disease-resistant control strains. Enhanced chemotaxis to stromal cell-derived factor-1 and adherence to BM stroma were proposed as potential mechanisms that permitted a superior NOD HSC engraftment. The observations that HSCs from certain autoimmune-prone strains dominate hematopoiesis are relevant to translation of the allogeneic HCT approach to the treatment of human ADs. If HSCs in AD-affected individuals do indeed have competitive advantage, designing the appropriate conditioning regimens and graft content that will permit permanent donor cell chimerism will be all the more challenging. However, it is yet to be definitely proven whether or not HSC engraftment potential and autoimmunity are linked. It is known that, among non-AD-prone strains, differences exist in the cell autonomous behavior of HSCs [161– 163]. Thus, it is possible that the observations made in the MRL-lpr/lpr, NZB/KN, and NOD mice are reflective of HSC characteristics that are separate from autoimmunity. Transplantation of purified HSCs Reports from our laboratory [125,129] and by Smith-Berdan et al. [130] (described above) demonstrated that AD in genetically prone mice was not altered by myelo- and lymphoablation and rescue with purified syngeneic or congenic HSCs. However, in those same studies, engraftment of allogeneic KTLS HSCs successfully blocked autoimmune pathogenesis. Our work focused on prediabetic NOD mice that were prepared for transplant with lethal radiation plus antibodies directed against NK and CD4+ cells followed by infusion of MHC-mismatched HSCs (Fig. 20.3) [125]. The antibodies were required in the preparative regimen because of the high level of resistance to engraftment encountered in NOD mice. Engraftment of purified MHC-mismatched HSCs compared with BM conferred equal protection, and resolution of islet inflammation was observed in both groups. Of note, the pattern of blood chimerism differed significantly between the HSC and BM groups. NOD mice transplanted with purified HSCs remained T-cell chimeras for an extended period of time post transplantation, whereas BM-transplanted

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animals converted promptly to complete donor T-cell type (Fig. 20.3b). In both groups, the other blood lineages (B cells, macrophages, and granulocytes) were 100% donor derived. The significance of persistent host T cells in animals with spontaneously arising ADs is that these host cells have the potential for autoreactivity (discussed in “Mechanisms by which allogeneic HCT abrogates autoreactivity” below). These data provide evidence that HSCs alone give rise to elements capable of blocking the radiation-resistant autoreactive cells, demonstrating that complete replacement of donor T cells is not a requirement to obtain curative benefit from allogeneic HSC transplantation. Stabilization or reversal of lupus signs was achieved by haploidentical transplantation of purified KTLS HSCs in the (NZB × NXW)F1 model. Donors in this study by Smith-Berdan et al. [130] were (DBA2 × C57BL/6)F1 (H-2d/b) that share the H-2d haplotype with the NZB parental strain. Myeloablation was achieved by radiation at a dose of 1450 cGy. Similar to the NOD studies, analysis of the peripheral blood revealed the recipients to be mixed T-cell chimeras, whereas the B-cell and myeloid compartments were donor derived. Haploidentical HSC transplantation resulted in improved survival, decreased proteinuria, and decreased circulating immune complexes and autoantibodies compared with untreated mice. In contrast no improvement was noted in recipients of syngeneic HSCs or BM, and, in fact, survival was worse in the syngeneic groups compared with untreated control (NZB × NXW)F1 mice. Allogeneic HCT and antigen-induced ADs Genotype and susceptibility The important distinction between induced ADs and spontaneous ones is that the latter generally arise in the context of a genetic background with multiple immune defects, whereas the former are induced in wildtype animals and require purposeful immunization to break T-cell tolerance. Nonetheless, susceptibility to develop an induced AD also appears to be strain specific since it is observed that, for any given antigen immunization protocol, certain rodent strains are more likely to develop autoimmunity, while others are resistant. Similar to the spontaneously arising ADs, much of the early literature on BM transfer experiments in autoantigen-immunized rodents sought to identify the cellular elements controlling autoreactivity. Thus, the studies were not designed to examine the curative potential of BMT that would involve BMT after disease induction; rather, they focused on the induction of disease in already established radiation-BM chimeras. In agreement with the studies in spontaneously arising ADs, the BM genotype seemed to dictate disease susceptibility or resistance [7]. Furthermore, there were at least two independent reports in 1981 [164,165] wherein the authors surmise that the mechanism by which the BM genotype exerts its autoreactive or protective effects is at the level of antigen presentation to T-cells – a hypothesis that remains compelling although yet to be proved. A notable exception to the results demonstrating that BM genotype is the sole determinant of induced AD development were published by Korngold et al. [138] in a model of acute EAE. In that study, MHCmatched hematopoietic chimeras were generated between SJL strain mice that were highly responsive to MSCH and the low-responder B10.S strain. Challenge of chimeras with MSCH derived from SJL spinal cord led to a high incidence of disease in the B10.S into SJL chimeras, but not in the SJL into B10.S mice. The outcome differed if chimeric mice were immunized with MSCH derived from B10.S mice in that both B10.S into SJL and SJL into B10.S chimeras developed severe disease. These data suggested that nonhematopoietic factors, such as elements within the central nervous system, control the development of EAE. This same group published a separate study in a relapsing EAE model [137].

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These experiments showed that immunization with MSCH derived from third-party MHC-disparate BALB/c or B10.S mice resulted in disease in B10.S into SJL chimeras but not SJL into B10.S chimeras, indicating once again that restriction in the development of EAE involves elements outside the hematopoietic system. Treatment of induced ADs with allogeneic HCT Success in curing advanced-stage induced ADs with allogeneic HCT was reported beginning in the late 1980s. Van Bekkum and co-workers established the adjuvant-induced arthritis and EAE models in rats, and tested the efficacy of MHC-mismatched transplantation in conjunction with the autologous HCT studies described above [7]. It was in the arthritis model that equivalence in the effect of syngeneic compared with allogeneic BMT was first described [139]. Susceptible strain Buffalo rats (MHC designation RT1Au) were lethally irradiated and transplanted with BM from nonsusceptible MHC-mismatched Wag/Rij (RT1Ai) or syngeneic BM at either weeks or many months following immunization with the adjuvant (Mycobacterium tuberculosis). The most effective results were obtained when treatment was initiated shortly after evidence of clinical manifestations at 4–7 weeks post immunization. Rats treated at the later stage had limited recovery with stabilization of disease, but not complete regression. Scarring and permanent joint destruction were thought to limit the therapeutic effect. Equivalent responses were seen in disease-affected recipients of allogeneic or syngeneic BM. Comparative studies of syngeneic versus MHC-mismatched allogeneic BMT have been performed in the rat EAE model [116,140]. In the studies by van Gelder and van Bekkum from 1995 [116], RSCH plus adjuvant was used to induce disease in rats, and recipients were prepared with lethal radiation or, for some groups of allografted rats, BU plus CY. Syngeneic transplantation was initially effective in inducing complete remissions in all recipients. However, as discussed earlier in this chapter, these results differed from the adjuvant-induced arthritis studies, since syngeneic and pseudoautologous recipients demonstrated significantly increased incidences of relapse that occurred spontaneously or following rechallenge with the spinal cord antigen. In contrast, allografted rats that were given TCD BM or BM from T-cell-deficient nude rats showed improved outcome, with complete remissions in all animals and markedly reduced spontaneous and induced relapses. The superior outcome of allogeneic HCT was attributed to a subclinical graft-versushost reaction (which would later be termed GVA). The rat EAE studies were extended to the use of MHC-matched donors using the same preparative regimen [166]. Allogeneic unfractionated BM was used in these studies and observed to induce complete remissions with low relapse rates compared with pseudoautologous recipients. Mixed chimeras were created by using grafts of TCD syngeneic plus TCD allogeneic BM. It was observed that the mixed chimeras relapsed more frequently than the complete chimeras, leading the investigators to conclude that clinical protocols for the treatment of multiple sclerosis should be designed to achieve full chimerism. The issue of whether or not a major goal of clinical allogeneic HCT for ADs should be mixed or full donor chimerism is still an open question. Animal studies now more often focus on reduced-intensity preparative regimens (see below), and there have been numerous reports that mixed hematopoietic cell chimerism results in successful amelioration of disease. In more recent years, Herrmann and colleagues [140] have reproduced and extended the observations made in the rat EAE model. MHCmatched, as well as syngeneic BM, transplantations were shown to confer significant clinical improvement in EAE-affected rats that had been immunized with MOG 1–125. In agreement with other reports, HCT was effective in the acute but not later phase of disease, and, importantly, allogeneic was no better than syngeneic transplantation in that regard. Rats that had been transplanted during the peak of EAE

disease and improved were protected from relapse when challenged again with the original MOG immunogen, but not when a different epitope of MOG was used for the rechallenge, a finding that supports the idea that the procedure induces antigen-specific tolerance. Furthermore, disease protection correlated with the presence of increased numbers of CD4+CD25+FoxP3+ cells. Van Wijmeersch et al. [167] have also recently studied amelioration of EAE in affected mice that underwent allogeneic HCT from susceptible versus nonsusceptible donors. Their data indicated that high levels of donor chimerism from a nonsuceptible donor were required in order to achieve the best therapeutic effect. Furthermore, alloreactivity induced by donor lymphocyte infusions enhanced the protective effect, thus supporting the idea that strong GVA is important for effectively treating ADs. MHC-mismatched versus matched HCT Most preclinical studies that have tested the efficacy of allogeneic HCT on AD pathogenesis utilized grafts mismatched at both MHC and minor histocompatibility loci. Surprisingly few have used transplantations of MHC-matched hematopoietic cells [4,127,129,166]. The importance of MHC identity or nonidentity relates both to the direct translation of the animal data to clinical protocols as HLA-matched donors will ideally be used, and to the mechanism by which allogeneic HCT mediates its ADprotective effect. Given the well-known association of MHC-genotype with autoimmunity (Table 20.1) and the importance of MHC molecules in shaping and maintaining the T-cell repertoire, the effect of MHCmatched transplantation on AD recipients should be a central issue. As a generalizable principle, it might be expected that donor grafts mismatched at the MHC would be more effective in blocking autoimmune pathogenesis compared with grafts that are MHC matched. In 2005, we examined the effect of MHC-matched HCT in the rodent disease with the strongest MHC association, namely diabetes in NOD mice [129]. Disease development in NOD mice has a well known linkage to the expression of a single unique class II MHC molecule, designated I-Ag7. In fact, NOD disease susceptibility appears to depend upon the presence of the I-Ag7 molecule. Transgene expression of nonI-Ag7 in the germline of NOD mice, as well as transplantation of transgenic BM-expressing nonsusceptibility class II alleles into young NOD mice, results in protection from the development of hyperglycemia [150,168]. It might therefore be predicted that the transplantation of MHC-matched hematopoietic cells would fail to confer disease protection. To address this question, C57BL/6 mice congenic for the entire NOD MHC region were used as donors of BM or purified KTLS HSCs [129]. These donors, designated B6.H-2g7, were matched at class I and class II loci of NOD but differed at minor histocompatibility loci. Prediabetic mice prepared with lethal radiation and engrafted with either BM or HSCs from B6.H-2g7 donors were 100% protected from diabetes. Similar to the chimerism levels observed in recipients of purified HSCs in the MHC-mismatched strains, the recipients were partial T-cell chimeras with significant residual host T cells remaining. These data give confidence that HLA-matched donor HCTs can be effective in the treatment of human ADs, since non-MHC “background” genes conferred protection even in the presence of a strong disease-linked MHC allele. Of course, a next important step is to identify and characterize the protective non-MHC genes. Reduced-intensity allogeneic HCT and establishment of mixed chimerism The major limiting factor in treating human ADs with allogeneic HCT is concern about the morbidity and mortality of the transplant procedure. Two ways in which the dangers of the procedure can be significantly decreased are by RIC and reduction of donor T cells to remove the risk

The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases

of GVHD. By performing these kinds of maneuver, the expectations are that recipients will develop mixed donor–host chimerism rather than convert to complete donor type. The establishment of mixed donor chimerism has not been an end-goal for clinical transplantations, since the majority of HCTs are done for hematolymphoid malignancies, and graft-versus-tumor responses with conversion to donor type are thought to be an essential element in the therapy. However, for ADs where a graft-versus-tumor response is not needed, the question remains of whether or not the establishment of mixed chimerism will be sufficient to block autoimmunity. The prevalence of RIC protocols in the human HCT literature is in no way matched by that in animal studies. There are relatively few preclinical reports on the topic of RIC transplantation for the treatment of ADs, and most of these have been published in the last 5 years. Li et al. [169] demonstrated that disease in NOD mice could be successfully blocked with reduced doses of radiation when delivered in conjunction with the infusion of high amounts of MHC-mismatched unmanipulated BM. They found that the radiation dose that permitted engraftment in NOD mice was significantly higher than for the nonautoimmune controls studied (750 versus 600 cGy, respectively). Engraftment correlated with high BM dose, and, despite the lower radiation dose, nearly all engrafted mice exhibited high levels of donor chimerism (>95%). Other studies have demonstrated that mixed chimerism, rather than full donor chimerism, is sufficient to protect NOD mice from progression to diabetes [170,171]. However, in those experiments, mixed chimerism was achieved by lethal radiation and infusion of grafts that contained both NOD and donor cells. Liang and colleagues [123] reported a novel nonradiation-based conditioning regimen to achieve mixed chimerism in prediabetic NOD mice. Anti-CD3 antibody was administered as a single dose, and 1 week

Fig. 20.4 Nonmyeloablative transplantation of major histocompatibility (MHC)-matched hematopoietic stem cells (HSCs) results in multilineage chimerism and blocks autoimmunity. (a) Prediabetic non-obese diabetic (NOD) mice were prepared for transplantation with sublethal radiation (500 and 700 cGy), which was sufficient to permit engraftment of MHC-matched purified HSCs from B6.H-2g7 donors. Recipients of B6.H2g7 HSC or whole bone marrow (WBM) were protected from development of diabetes. Recipients similarly prepared and transplanted with syngeneic HSCs progressed to hyperglycemia. (b) Testing of blood revealed the nonmyeloablated NOD recipients to be stable partial mixed chimeras. Shown are the chimerism studies for the different blood cell lineages from NOD mice that received 500 cGy and that were followed up to 1 year post transplantation.

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later recipients received high doses of unmanipulated MHC-mismatched donor BM in combination with high doses of CD8+ T cells. GVHD was not observed, and true multilineage chimerism was established. The NOD recipients showed reversal of islet inflammation and resistance to the development of hyperglycemia despite the presence of significant levels (~30%) of residual host T cells. In a follow-up study, these investigators demonstrated that, when this same treatment approach was used in new-onset hyperglycemic NOD mice, the inflammation in the islets resolved and the mice regenerated insulin-producing β-cells in the pancreas and recovery from the hyperglycemia [172]. These latter studies suggest that, by permanent interruption of the autoimmune process by allogeneic HCT in new-onset diabetics in whom some islet mass is preserved (a period which in humans with T1DM has been termed the “honeymoon” phase), it may be possible to prevent or delay the need for exogenous insulin. We have also performed reduced-intensity transplants in NOD mice using low-dose (500 cGy) radiation plus grafts of MHC-matched, B6.H2g7 purified KTLS HSCs [129]. NOD mice that received this treatment engrafted and all were protected from development of diabetes. This regimen resulted in stable multilineage mixed chimerism (Fig. 20.4). These studies address the clinical question raised above, as they show that reduced-intensity treatment and the establishment of mixed chimerism using grafts that have been rigorously depleted of T cells and come from MHC-matched donors can be curative of ADs. Cure of overtly diabetic NOD mice with combined nonmyeloablative BMT and donor-matched islet transplantation have been reported. In one study by Seung et al. [124], a preparative regimen of sublethal radiation (600 cGy) plus anti-CD40 ligand (CD40L) mAb permitted engraftment with a resultant high level of donor chimerism (>99%) in most mice that received MHC-mismatched BM. Diabetic chimeric NOD mice were

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transplanted with donor-matched islets and achieved long-term normoglycemia. In a more recent study, Sykes and colleagues [126] employed a conditioning regimen that combined the use of anti-CD4, anti-CD8, anti-Thy1.2, and anti-CD40L mAbs plus low-dose (400 cGy) radiation to achieve engraftment of MHC-mismatched BM in overtly diabetic NOD mice. This regimen resulted in the establishment of multilineage chimerism with amelioration of autoimmunity and the induction of tolerance to donor matched islets. RIC with allogeneic HCT has been tested in at least two nondiabetes animal models. Flierman et al. [173] studied MHC-mismatched transplantation in the collagen-induced arthritis model that manifests in affected mice as a systemic inflammatory B-cell mediated AD. Conditioning with sublethal radiation (600 cGy) and anti-CD40L led to high levels of chimerism. Interestingly, both syngeneic and allogeneic BMT led to the clinical suppression of arthritis. However, allogeneic BMT correlated with a superior reduction of pathogenic antibody. SmithBerdan et al. [130] used 1000 cGy in highly radiation-resistant, SLEprone (NZB × NZW)F1 mice to achieve durable multilineage mixed chimerism following transplantation of haploidentical KTLS HSCs. These stable chimeras had superior survival and exhibited stabilization or reversal of their lupus symptoms, which included proteinuria and circulating immune complexes or autoantibodies, compared with untreated controls and syngeneic recipients. In summary, these studies of reduced-intensity regimens show uniform success, although the regimens applied as well as the levels of donor cell engraftment achieved were highly variable. Those reports that show disease amelioration in the setting of significant persistent host T cells, and multilineage chimerism can and will be used as evidence that the establishment of mixed chimerism is sufficient to block autoimmunity. Mechanisms by which allogeneic HCT abrogates autoreactivity It is generally thought that allogeneic HCT interrupts AD pathogenesis by a combination of cytoreduction of host immune cells caused by the preparative regimen, and by alloreactivity against host T cells and other immune cell populations (GVA effect). This explanation, plus the concept that the allogeneic hematopoietic cell source replaces a defective HSC, leads to a formula that is pervasive in the literature and can be summarized as follows: Elimination of host cells + replacement of defective HSCs = AD cure.

While these two factors clearly play a prominent role in disease protection, the experiments reporting success in treating ADs with transplants of syngeneic cells, and more recently nonmyeloablative conditioning, argue that the formula is overly simplistic. The superior outcomes in allogeneic compared with autologous models demonstrate that the donor cells play a critical role in modifying recipient immune responses. However, the reported successes of autologous HCT underscores the fact that stochastic interactions occur between a regenerating immune system and the nonhematopoietic factors that drive autoreactivity. It is likely the shifts in these interactions will ultimately determine whether or not immune self-tolerance will be restored. In the broadest of terms, it can be concluded that allogeneic HCT demonstrates a higher success rate in spontaneous ADs over autologous HCT because certain hematopoiesis-specific susceptibility genes have been replaced by donor cells. Furthermore, given the dynamics of immune reactivity, there are likely a number of genes or genetic combinations that can favor a protective outcome. Moving from the more generalized view to focus on the specific ways allogeneic HCT alters autoreactivity reveals that a refined understanding of how HCT mediates protection lags far behind the basic science

of autoimmunity. The reductionist approach of examining immune responses by overexpression of antigen and/or lymphocyte receptors, or alternatively knockout of key regulatory molecules, has been highly fruitful for immunologists dissecting the mechanisms of tolerance induction. However, this strategy has not been widely applied to study the effects of HCT, and it is arguable as to whether or not such an approach will prove relevant to clinical transplantation. The studies examining the genetic differences between donor and recipient that make it more likely that allografting will be more efficacious than autologous HCT in the treatment of ADs will be reviewed. T-cell depletion: beyond conditioning and GVA Depletion of host T cells is the most extensively studied mechanism by which allogeneic HCT can alter immune function. There is ongoing discussion among clinical transplant professionals regarding the need to achieve full donor T-cell chimerism in order to obtain cures of human ADs. Separate from the effects of the conditioning regimen, unmanipulated allogeneic hematopoietic grafts replete with mature immune elements have the capacity to eliminate or reduce residual host hematolymphoid elements. GVA is the term that describes this pan-alloreactivity and conversion to complete donor type serves as a surrogate marker for GVA activity. While elimination of host T cells by this mechanism certainly increases the likelihood that autoreactivity will be reduced, GVA will be accompanied by significantly increased risk of GVHD. Animal studies show that, separate from GVA, hematopoietic allografts are capable of altering host immune responses and even complete blockade of autoreactivity. It should be noted that many of the animal studies on the topic of HCT for the treatment of ADs predate the technologies that allowed measurement of donor-versus-host hematopoiesis. Thus, T-cell chimerism was not assessed. There was an apparent assumption in the early reports that if animals survived lethal radiation following BMT, conversion to donor type must have occurred. In later studies, particularly by Ikehara and colleagues, wherein chimerism of the blood or spleen was assessed, the data consistently showed chimerism levels of over 90% [155]. However, lineage subset analyses were not performed. From more recent reports using purified HSCs [125,129,130,134], clinical studies examining chimerism in patients with TCD grafts [174], and analyses of chimerism following RIC transplantation [175], it is evident that the T-cell lineage is the most resistant to conversion to donor type. HSCs and TCD BM lack alloreactive graft-facilitating populations that mediate elimination of residual host immune cells [133,134]. Transplantation of allogeneic unfractionated BM into lethally irradiated mice results in near-complete conversion to donor type shortly after transplantation, whereas HSC transplantation consistently results in partial T-cell chimerism that persists for many months post transplantation [134,176]. Patients that have received TCD BM also demonstrate long-term mixed chimerism [174,177,178]. It is therefore reasonable to conclude that, in the many experiments wherein T-cell chimerism was not assessed, but in which TCD or BM from nude mice was used as the graft source, those autoimmune affected recipients remained partial T-cell chimeras for extended periods of time post transplantation. Studies shown in Fig. 20.3, from our laboratory, directly tested whether donor hematopoietic elements eliminate or modify residual autoreactivity in the absence of GVA. In those experiments, purified HSCs from allogeneic wild-type, compared with congenic NOD-SCID, donors were transplanted into NOD mice [125]. Recipients were prepared in an identical manner, and, following transplantation, both groups had similar levels of surviving host NOD T cells in their peripheral blood. HSC from NOD-SCID donors cannot contribute T cells to the regenerating immune system, yet recipients engrafted with NOD-SCID

The Experimental Basis for Hematopoietic Cell Transplantation for Autoimmune Diseases

HSCs developed diabetes, showing that the surviving recipient immune cells destroyed the islet tissue. In contrast, mice engrafted with allogeneic HSCs with comparable levels of surviving NOD T cells did not develop diabetes, and islet inflammation was reversed. We subsequently showed that nonmyeloablative conditioning with sublethal doses of radiation and transplantation of MHC-matched HSCs into NOD mice also prevented the development of diabetes (Fig. 20.4) [129]. In the latter studies, the recipients were multilineage partial chimeras, and even higher numbers of NOD T cells persisted compared with the NOD-SCID transplant studies. Taken together, these studies showed that the presence of active donor hematopoieisis without GVA was sufficient to alter the pathophysiology of an AD. Beyond overt alloreactivity against host immune cells, hematopoietic grafts can mediate deletion of autoreactive cells by negative selection. Negative selection occurs by both central thymic deletion and peripheral deletion of post-thymic cells [179]. BM-derived APCs are the most efficient mediators of negative selection. In a nontransgenic animal, it is virtually impossible to directly demonstrate negative selection of T cells for any particular self-antigen because, generally speaking, antigen-specific T-cells are too few in number to detect. However, this process can be measured for a class of nonconventional antigens called superantigens [180]. Superantigens are viral or bacterial proteins that interact with T cells via the class II MHC and Vβ segment of the TCR. T cells bearing the Vβ segment specific for the antigen die by apoptosis, resulting in near-complete elimination of all cells of the responding Vβ subclass. Some superantigens exist as endogenous genes in mice of certain strains, and during T-cell development the cells with receptors that have the Vβ capable of reacting to these “self-antigens” are deleted by negative selection. Thus, tracking T cells based upon Vβ staining in experimental systems in which exposure to superantigens can be manipulated (such as BM chimeras) serves as a surrogate assay for assessing negative selection. We followed the ability of purified HSC allografts to mediate negative selection of T cells reactive to endogenous superantigens by measuring Vβ subsets in chimeric NOD mice post HSC [125]. A donor strain (AKR/J) was selected that differed from NOD mice with regard to expression of superantigens, and thus donor and recipient demonstrated a differential pattern of Vβ deletion. NOD mice transplanted with AKR/J KTLS HSCs were mixed chimeras that retained significant numbers of host T cells. Results in the NOD chimeras revealed an absence of Vβ subsets reactive to superantigens derived from both donor and recipient. These data showed that the HSC graft mediated a negative selection of developing T cells, and, further, that the grafts eliminated potentially self-reactive post-thymic T cells of NOD origin.

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with MHC-mismatched BM leaves some room for doubt that similar consistent positive outcomes will be observed when AD-affected humans are treated using HLA-compatible HCT. There is a vast spectrum of non-MHC genes expressed in hematopoietic cells that can potentially alter the course of autoreactivity. These genes include those that affect overall immunoreactivity such as cytokines, cell proliferation, and lymphocyte or APC cell activation and apoptosis. Large consortiums have been assembled aimed at mapping these key genetic determinants of autoimmune susceptibility. An example that these efforts will be fruitful is the identification of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), a hematopoietically expressed molecule and critical regulator of T-cell activation, as a major non-MHC susceptibility gene associated with ADs in humans and mice, including NOD diabetes [181]. The studies implicate that common allelic variation in the expression of levels of alternative splice forms of CTLA-4 is a primary determinant of AD susceptibility. Another example is the genome-wide association study that identified alleles of IL-2RA and IL-7RA as heritable risk factors for human multiple sclerosis [60]. An approach that we have taken in NOD mice to identify the nonMHC genes expressed on hematopoietic cells that confer protection in the setting of HCT is to use as donors congenic mice generated by recombinant inbreeding. Extensive efforts by several groups led to the generation of NOD mice that contain congenic intervals in their genome, termed Idd, derived from the C57BL strain [75]. Depending on the introduced chromosomal interval, these congenic NOD mice demonstrate different degrees of diabetes protection. Among the known Idd loci, the most protective non-MHC loci introduced map to chromosome 3 (Idd3), chromosome 1 (Idd5), and chromosome 4 (Idd9). Our prior studies showed that HSC transplantation from MHC-matched B6.H-2g7 donors that express the full complement of C57BL background provided 100% disease protection [129]. Therefore, we sought to determine if one or more of these congenic intervals contained the protective gene(s). Purified KTLS HSC from NOD Idd3, Idd5, and/or Idd9 congenic donors were transplanted into lethally irradiated prediabetic wild-type NODs. Mice that received grafts carrying one or two protective loci developed diabetes. These results were somewhat disappointing, particularly because the mouse CTLA-4 gene maps within the Idd5 region [182]. However, significant diabetes protection was conferred when NOD mice that express multiple (Idd3, Idd9, Idd10, Idd17, and Idd18) protective alleles were used as donors. Thus, our NOD congenic studies show that, in this model of autoimmunity, protection by HCT depends upon multiple disease-resistant loci outside of the MHC. Progress on the genetic basis of T1DM supports this finding as several recent studies have described interactions of CTLA-4 with other susceptibility loci that regulate its expression and therefore contribute to pathogenesis [182,183].

Replacement of susceptibility genes of the host The expression of key regulatory genes on hematolymphoid cells that differ between donor and recipient is another way that allogeneic HCT can modify pathogenic immune responses. MHC genes are the prototypic genes, and indeed the only gene family identified to date, that fit this description. Experiments in NOD mice that directly addressed the effect of adding or replacing the MHC susceptibility allele by formal genetic crosses or transgenic technology showed that the expression of non-NOD MHC class II molecules provided a high degree of protection. HCT transplantation studies using BM from NOD MHC class II transgenic mice proved that coexpression of nonsusceptibility class II alleles on donor hematolymphoid cells could disrupt autoimmune pathology [150,168]. We [129] and others [4,127,166] have shown that transplantation of MHC-matched HCT can confer similar levels of protection in preclinical models. However, the fact that the majority of preclinical studies demonstrating successful treatment of advanced ADs were done

Regulatory cells Despite the prominence of Tregs in the basic and clinical immunology literature, few reports exist on the role of regulatory T cells as a mechanism by which allogeneic HCT blocks autoimmunity; and those that have examined the Treg question show disparate results. We studied the potential contribution of Tregs to disease amelioration in NOD chimeras in two different types of experiment [129]. First, by measuring the percentages and absolute numbers of Tregs (CD4+CD25+ cells) in the blood of control and transplanted mice, we observed that unmanipulated NOD mice had higher Treg levels compared with MHC-matched diseaseresistant B6.H-2g7 donors, and diabetes-protected chimeras had levels equivalent to wild-type NODs. Second, B6.H-2g7.SCID mice lack both CD4+CD25+FoxP3+ cells, and another subset of Tregs, NK-T-cells, were used as hematopoeitic cell donors. Despite the inability of the donated

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cells to generate these T-cell subsets, NOD recipients enjoyed nearly 100% disease protection. In contrast to our negative results, Herrmann et al. [140] reported, in an MOG-induced EAE rat model, that increased levels of CD4+CD25bright cells in lymph nodes and increased expression of Foxp3 in spleen were observed in EAE-protected transplanted rats compared with untransplanted controls. However, similar to our studies, they found that the EAE-susceptible rat strain had higher levels of CD4+CD25bright cells in their lymph nodes compared with disease-resistant rats. Because Tregs are a fundamentally important mechanism of immune control, there is no doubt that this question will be more exhaustively pursued in the future. Furthermore, negative results do not preclude the utilization of Tregs as adjunctive cellular therapy in HCT for autoimmunity since it is clear that shifting the balance in this way towards tolerogenic out-

comes is in line with “standard operating procedure” of the immune system.

Conclusion The knowledge that autologous and allogeneic HCT causes profound alterations in immune reactivity resulting in cures of ADs has been present in the scientific and clinical literature for decades. The animal studies provide a fundamental basis for understanding both the potential benefits and limitations of HCT to effectively treat these complex diseases. The experimental details discussed in this chapter are meant to provide a foundation for the thoughtful translation of these studies into clinical reality.

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158. Wang X, Hisha H, Cui W et al. The characteristics of hematopoietic stem cells from autoimmuneprone mice and the role of neural cell adhesion molecules in abnormal proliferation of these cells in MRL/lpr mice. Haematologica 2007; 92: 300– 7. 159. Wang X, Hisha H, Taketani S et al. Neural cell adhesion molecule contributes to hemopoiesissupporting capacity of stromal cell lines. Stem Cells 2005; 23: 1389–99. 160. Chilton PM, Rezzoug F, Ratajczak MZ et al. Hematopoietic stem cells from NOD mice exhibit autonomous behavior and a competitive advantage in allogeneic recipients. Blood 2005; 105: 2189–97. 161. Morrison SJ, Qian D, Jerabek L et al. A genetic determinant that specifically regulates the frequency of hematopoietic stem cells. J Immunol 2002; 168: 635–42. 162. Phillips RL, Reinhart AJ, Van Zant G. Genetic control of murine hematopoietic stem cell pool sizes and cycling kinetics. Proc Natl Acad Sci U S A 1992; 89: 11607–11. 163. Van Zant G, Holland BP, Eldridge PW, Chen JJ. Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice. J Exp Med 1990; 171: 1547–65. 164. Singer DE, Moore MJ, Williams RM. EAE in rat bone marrow chimeras: analysis of the cellular mechanism of BN resistance. J Immunol 1981; 126: 1553–7. 165. Ben-Nun A, Otmy H, Cohen IR. Genetic control of autoimmune encephalomyelitis and recognition of the critical nonapeptide moiety of myelin basic protein in guinea pigs are exerted through interaction of lymphocytes and macrophages. Eur J Immunol 1981; 11: 311–16. 166. van Gelder M, Mulder AH, van Bekkum DW. Treatment of relapsing experimental autoimmune encephalomyelitis with largely MHC-matched allogeneic bone marrow transplantation. Transplantation 1996; 62: 810–18. 167. Van Wijmeersch B, Sprangers B, Rutgeerts O et al. Allogeneic bone marrow transplantation in models of experimental autoimmune encephalomyelitis: evidence for a graft-versus-autoimmunity effect. Biol Blood Marrow Transplant 2007; 13: 627–37. 168. Slattery RM, Miller JF, Heath WR, Charlton B. Failure of a protective major histocompatibility complex class II molecule to delete autoreactive T cells in autoimmune diabetes. Proc Natl Acad Sci U S A 1993; 90: 10808–10. 169. Li H, Kaufman CL, Boggs SS, Johnson PC, Patrene KD, Ildstad ST. Mixed allogeneic chimerism induced by a sublethal approach prevents autoimmune diabetes and reverses insulitis in nonobese diabetic (NOD) mice. J Immunol 1996; 156: 380–8. 170. Kaufman CL, Li H, Ildstad ST. Patterns of hemopoietic reconstitution in nonobese diabetic mice: dichotomy of allogeneic resistance versus competitive advantage of disease-resistant marrow. J Immunol 1997; 158: 2435–42.

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Section 2c Technical Aspects

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

21

James H. Doroshow & Timothy W. Synold

Pharmacologic Basis for High-dose Chemotherapy

Introduction

Pharmacologic rationale for high-dose chemotherapy

The feasibility of utilizing high doses of chemotherapeutic drugs, rather than ionizing radiation, as conditioning agents prior to allogeneic bone marrow transplantation (BMT) was initially demonstrated in animal models nearly 40 years ago [1]. Successful human trials that employed high-dose cyclophosphamide (CY) with or without total body irradiation (TBI) for patients with aplastic anemia or acute leukemia undergoing BMT were performed soon thereafter [2,3]. A decade earlier, autologous BMT for patients with hematologic malignancies following TBI or highdose single-agent nitrogen mustard chemotherapy was reported, albeit with little success [4]. The outcome of allogeneic approaches to BMT depends, to a significant degree, on the immunosuppressive properties of the conditioning regimen. CY became a critical part of allogeneic transplant procedures 30 years ago because it combined significant antileukemic activity with profound effects on several different components of the immune system. In the course of the past 20 years, however, as the number of autologous marrow or peripheral blood hematopoietic cell transplants has increased, the specific therapeutic spectrum of action of the chemotherapeutic agents employed, the degree of enhanced tumor cell killing produced by the dose escalation made possible with hematopoietic cell support, and the extent to which specific drugs or drug combinations produce doselimiting extramedullary toxicities has become as important as their immunosuppressive nature. In this chapter, the rationale for the use of chemotherapeutic agents in higher than standard dose is examined from both a mechanistic perspective and with regard to the general pharmacodynamic features of high-dose chemotherapy that play a critical role in drug selection. In turn, each of the classes of antineoplastic agent currently employed in the high-dose treatment of both hematologic malignancies and solid tumors is reviewed with respect to their mechanism(s) of action and resistance, pharmacokinetics, pharmacodynamics, drug–drug interactions, and dose-limiting toxicities. Structural formulas are shown in Fig. 21.1 and kinetic parameters in Table 21.1. The pharmacologic underpinnings developed in this chapter will provide a basis from which to evaluate the rationale for the high-dose chemotherapy combinations described in Chapter 22.

Pharmacologic basis of dose intensity

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Pharmacologic definition of high-dose chemotherapy Although it has been estimated that over 25,000 patients worldwide received high-dose chemotherapy with hematopoietic cell support in 2004 (http://www.cibmtr.org/ABOUT/Annual_Report/DOCS/annual_ report.pdf), an adequate definition of the procedure, in many ways, remains elusive [5]. To define high-dose chemotherapy as the utilization of drug doses that produce bone marrow ablation would not be accurate in the setting of autologous hematopoietic cell support; furthermore, this definition would also be incorrect for patients receiving reduced-intensity conditioning and allogeneic BMT. Assuming that a level of drug dosage that requires hematopoietic cell support for safety reasons is, by itself, a sufficient definition of what constitutes “high-dose chemotherapy” is uninformative because it has no clear therapeutic implication. For many chemotherapeutic agents, an approximate threefold increase in area-under-the-curve (AUC) can be translated in vitro into a 10-fold increase in cytotoxicity (probably the smallest increment in cell kill that may be associated with clinical benefit). Thus, one possible definition of the minimum dose of a chemotherapeutic agent alone or in combination that qualifies as “high-dose chemotherapy” is a dose that increases systemic exposure by a factor of three compared with the standard chemotherapeutic dose. Establishing a pharmacokinetic definition of high-dose chemotherapy that takes into account individual systemic exposure would allow much more definitive pharmacodynamic comparisons of both treatment outcomes and toxicities for both currently available and future high-dose chemotherapy programs. Concentration–response effects in preclinical models Abundant in vitro evidence exists demonstrating various drug-specific, concentration-dependent patterns of induced cytotoxicity or apoptosis, or both, in human tumor cell model systems [5–9]. In general, the alkylating agents (such as melphalan [MEL], busulfan [BU], thiotepa, and BCNU [carmustine]) and platinum derivatives produce the most profound inhibition of tumor cell growth in vitro with the smallest increment in drug concentration. Antimetabolites (such as methotrexate, cytarabine, and fludarabine), on the other hand, frequently demonstrate a plateau effect in which cytotoxicity ceases to be log-linear within a more narrow range of drug concentration. The anthracyclines, the taxanes, and etoposide (VP16) are intermediate in this regard; however, for these latter classes of drug, prolonged exposure in vitro can markedly increase tumor cell killing out of proportion to the observed concentration × time product, suggesting that both the cellular pharmacology

289

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Chapter 21 Cl

H

NH3 Pt

O N

ClCH2CH2

NH3

Cl

N –– P ClCH2CH2

O

Cisplatin

Cyclophosphamide O C

CH2CH2 –– Cl

O

O N

ClCH2CH2

N –– P H

C

O

NH3 Pt

O

NH3

O Carboplatin

Ifosfamide H3C H O O

O

O

CH3 –– S –– O –– CH2CH2CH2CH2O –– S –– CH3 O

O

HO

O Busulfan

HO

O

O O O O

O N

OCH3

CH3O

ClCH2CH2 –– N –– C –– N –– CH2CH2Cl

OH Etoposide

H

O BCNU O

OH

O –OH

NH2

ClCH2CH2 N ––

–– CH2CH2

ClCH2CH2

OCH3 O

CH2OH

OH O CH3 NH2

C –– OH Doxorubicin

O

OH

Melphalan O O NH S

O

CH3

3′

2′

N –– P–– N

O 1′

O

12 13 14

OH

N

OH

O

10 9 11

8 3

1 2

OH O O

7 4

6 5

O

H O

CH3 O

Thio-TEPA

Paclitaxel

Fig. 21.1 Structural formulae for the principal drugs used in high-dose chemotherapy with hematopoietic cell support.

Table 21.1 Pharmacokinetic and pharmacodynamic features of drugs employed for high-dose chemotherapy Drug

Elimination

Pharmacokinetics

Dose intensity

Pharmacodynamics

Cyclophosphamide Melphalan Thiotepa Busulfan BCNU Cisplatin Carboplatin Etoposide Doxorubicin Paclitaxel

Renal Hydrolysis Hepatic Liver Hydrolysis/liver Renal Renal Renal Hepatic/biliary Hepatic/biliary

Nonlinear for active species Linear; 30–120 min Linear; 1–3 h Linear; 3 h Linear; 30 min Linear; 30–45 min Linear; 7–20 h Linear; 4–8 h Linear; 12–20 h Nonlinear; 15 h

8× 6× 8–10× 8× 3× 3× 4–5× 3–6× 3–4× 5× increase in dose yields 15× AUC

Cardiac toxicity and low AUC Mucositis Mucositis, CNS SOS Lung, SOS Kidney, neuropathy Ototoxicity, kidney Mucositis Mucositis Neuropathy and mucositis

AUC, area under the curve; CNS, central nervous system; SOS, sinusoidal obstructive syndrome.

Pharmacologic Basis for High-dose Chemotherapy

291

Table 21.2 Mechanisms of therapeutic resistance in high-dose chemotherapy 1. 2. 3. 4. 5. 6. 7. 8.

Decreased drug uptake or enhanced drug efflux producing a net reduction in the intracellular concentration of the cytotoxic agent Increased repair of DNA damage or damage to other critical targets Development of alternative synthetic pathways for metabolites critical for tumor cell growth Overexpression of the gene product targeted by the chemotherapeutic agent, or the production of an aberrant target that supports tumor cell growth but is not affected by the drug utilized Inherent insensitivity of tumor stem cells to cytotoxic agents Enhanced intracellular detoxification or metabolism of the high-dose therapy that limits tumor cell killing Overexpression of cytoprotective gene products that alter tumor cell sensitivity to the drug-induced cell death program Altered tumoral blood supply (oxygenation), or tissue factors that diminish drug activity or delivery

(drug uptake) and mechanism of growth arrest for these chemotherapeutic agents involve a variety of dynamic intracellular targets [8,10]. Tumor cell heterogeneity and growth kinetics While much has been made of the “steepness” of the slope of in vitro cytotoxicity curves in the selection of drugs for use in high-dose chemotherapy [11–13], it remains clear that a multiplicity of conditions, including the genotypic and phenotypic heterogeneity of tumor cells, variations in the perfusion or oxygenation of the tumor, as well as immunologic factors all come into play in determining the success or failure of intensive chemotherapy strategies in vivo [14–16]. None of these features can be strictly predicted by in vitro cytotoxicity assays; hence, in vivo preclinical models continue to play a critical role in the overall assessment of combination high-dose chemotherapy strategies, including those that employ high doses of alkylating agents [17–19]. Studies in murine systems established the ability of many classes of antineoplastic agent to kill tumor cells according to first-order kinetics; that is, a given concentration of drug utilized for a particular time period will kill a fixed number of tumor cells [17]. If each treatment course at a given dose is cytotoxic for a constant fraction of cells, the curability of the particular therapy depends on the number of courses delivered, the drug concentration applied, and the initial tumor burden [20]. While this concept has been unequivocally verified in many animal tumor models that undergo logarithmic tumor cell expansion, it is not clear how well it applies to more slowly growing and genetically heterogeneous tumors in man. A major factor that underlies the efficacy of all systemic cancer treatment is the genetic heterogeneity of human tumors [16]. Although most cancers may develop initially from a single malignant clone, all malignancies demonstrate genetic instability [21]. Thus, the enormous inherent variation in the expression of any one of the wide range of proteins that establish the innate sensitivity of malignant cells to systemic agents is probably responsible for both the unpredictability of the efficacy of treatment (from patient to patient but not in populations of patients) as well as the regrowth of clones of malignant cells after an initial response to treatment, as illustrated in Plate 21.1. Regrowth, thus, may represent the expansion of tumor cells that, before any systemic drug exposure, already expressed gene products capable of providing a growth advantage under the selective pressure of treatment with high-dose chemotherapy. The demonstration of heterogeneous populations of tumor cells in humans has provided a critical rationale for the use of combinations of high-dose systemic agents with different sites of action that might circumvent at least some mechanisms of inherent drug insensitivity. Acknowledgment of the extent of human tumor cell heterogeneity provides additional support for strategies that reduce tumor cell mass prior to high-dose chemotherapy administration, since it has been predicted that the probability of a tumor possessing random mutations leading to

insensitivity to systemic treatment is a function of the tumor cell burden [22]. Thus, reduction of tumor mass prior to systemic treatment may decrease not only the total body burden of cancer but also the number of tumor cells inherently resistant to dose-intensive treatment.

Tumor cell resistance to high-dose chemotherapy High-dose chemotherapy is not uniformly successful because many types of cancer in humans harbor small populations of tumor cells that are intrinsically resistant to treatment or acquire resistance during therapy. High-dose chemotherapy may also fail because the incremental increase in drug doses possible with hematopoietic cell support is insufficient either to produce lethal tumor cell damage or to result in sufficient concentrations at all sites of the tumor [22]. Where sufficient drug exposure is possible, however, even highly resistant tumor cells can be killed. Mechanisms by which resistance to high-dose chemotherapy develops are, at least in part, specific to the mechanism of action of the class of agents being utilized. However, many different biochemical and physiologic phenomena have been observed either in vitro or in vivo to modulate the effectiveness of several different antineoplastic drug classes utilized for high-dose chemotherapy (Table 21.2). These include: 1 reduced drug uptake by the tumor cell [23]; 2 enhanced drug efflux [24]; 3 increased repair of drug-induced damage to DNA or other critical tumor cell targets [15]; 4 development of alternative routes for the synthesis of biologically important molecules when the primary synthetic pathway is blocked by a given drug [25]; 5 overexpression of the target gene product for a specific drug [26]; 6 production of an aberrant drug target that does not interact with a given agent, yet retains biochemical function sufficient to support tumor cell growth [27]; 7 enhanced intracellular metabolism or detoxification of the chemotherapeutic agent which limits tumor cell toxicity [28]; 8 overexpression or modification of cytoprotective gene products which alter tumor cell sensitivity to the anticancer agent [29]; 9 variations in the oxygenation state or blood supply of the tumor, which can blunt the effectiveness of the drug directly or the delivery of the drug to the tumor itself [30]; 10 factors in the normal tissues of the host which can significantly change the concentration of drug delivered to the tumor [31]. Although the clinical relevance of each of these mechanisms is uncertain, samples of human tumors have been shown to express one or more of phenotypic markers of drug resistance [32,33]. The mutations that lead to the expression of these characteristics may occur prior to the “recognition” of a tumor and can become more prominent under the

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Chapter 21

selective pressure of therapy (Plate 21.1) [34]. A tumor cell does not start to express a resistance phenotype in the presence of a given drug; instead, cells with a particular phenotype emerge by a process of mutation/evolution [35]. Historically, tumor cell resistance has been viewed predominantly from the perspective of the emergence, through the selective pressure provided by cytotoxic therapy, of pre-existing, genetically resistant clones. However, an accumulating body of evidence has developed supporting the premise that, in both leukemias and solid tumors, populations of cancer stem cells exist that can both self-renew and differentiate (Plate 21.1). Genetic mutations in small numbers of these stem cells or their immediate progenitors may play a critical role in tumor growth [36]. Further, studies suggest that some tumor stem cells may express high levels of drug efflux pump proteins, making them particularly resistant to cytotoxic chemotherapy [37]. Whether related to the expression of drug transport proteins or not, leukemic stem cells, when compared with more differentiated blasts, appear to be substantially more resistant to the commonly employed antileukemic agents cytosine arabinoside and doxorubicin [38]. Thus, the use of high-dose chemotherapy may be viewed, in this current context, as one approach to overcoming the intrinsic insensitivity of the cells from which a tumor originates. Certain physiologic characteristics of tumors, distinct from their genotype, can impede the delivery of effective concentrations of drug to the entire tumor. A major impediment is size, and large tumor masses often contain areas of poorly vascularized tissue. Consequently, the delivery of cytotoxic drugs to these areas of tumor via the blood will be poor, even after high-dose chemotherapy [30,39]. For this reason, and because of the constraints of nonlinear dose–response relationships and growth kinetics that exist for large, heterogeneous tumors, the likelihood of benefit of high-dose chemotherapy in the clinic has regularly been shown to be greater in the presence of low-bulk disease. Distribution of drugs throughout the body is typically not uniform due to differences in normal tissue physiology. The blood–brain barrier is a critical impediment to penetration of drugs into the central nervous system [40]. The presence of tight junctions between cells and high levels of drug transport proteins in the blood–brain barrier protects the central nervous system from the cytotoxic effects of systemic therapy and can provide a pharmacologic sanctuary for tumor cells [41]. Similarly, the testes can be a sanctuary site for tumor cells due to high expression of drug transporters and the resulting limited drug penetration [42]. One strategy to overcome the poor distribution of cytotoxic drugs into sanctuary sites has been to increase total drug exposures through the use of high-dose chemotherapy regimens.

Genetic basis for variability in response to high-dose chemotherapy Contribution of pharmacogenomics to clinical response and toxicity Evidence for inherited differences in response to drug therapy dates to the 1950s. Early observations relating the extent of acetylation of isoniazid to peripheral neuropathy [43], and plasma cholinesterase activity to prolonged muscle relaxation in response to suxamethonium [44], are examples of inherited determinants of the variability in the clinical response to drugs that gave rise to the modern field of “pharmacogenomics.” Pharmacogenomics is the study of inherited genetic variations (polymorphisms) in the genes involved in drug metabolism, therapeutic response, and toxicity, and how these polymorphisms relate to inherited differences in pharmacokinetics and drug effects. The mechanistic basis for inherited pharmacologic traits first came to light in the 1980s with the cloning of a polymorphic human gene encod-

ing the drug metabolizing enzyme debrisoquine hydroxylase (CYP2D6) [45]. Large population studies of individuals given the antihypertensive debrisoquine, a drug that caused an unusual fainting response in some patients, revealed two distinct phenotypes (“extensive” and “poor” metabolizers), based upon the extent of urinary excretion of hydroxylated debrisoquine metabolites [46]. In 1988, the wild-type human CYP2D6 gene was cloned from “extensive” metabolizers along with three different variant transcripts from “poor” metabolizers, and expressed in mammalian cells [45]. The variant gene products arose from mutations that resulted in alternatively spliced proteins that were not functional, thereby providing a molecular explanation for the inherited differences seen in debrisoquin pharmacokinetics and hemodynamics. For a gene to be considered “polymorphic,” the genetic variants must exist stably in the population, and one or more of the variants must result in a protein with altered activity relative to the wild-type sequence. While, in most cases, polymorphisms are associated with decreased protein activity, there are also examples of where the genetic variation results in increased protein activity, usually due to an amplification of gene expression [47]. Since the initial cloning and characterization of the CYP2D6 gene, many more polymorphic human genes involved in drug metabolism and drug response pathways have been identified. Traditional approaches to pharmacogenomic investigations have been monogenic, focusing on the contribution of a single gene to phenotypic variability. Although in some cases the effects of alterations at a single gene locus are sufficient to produce major phenotypic changes, the clinical response to most pharmacologic agents is a polygenic process, with multiple genes playing a role in both the pharmacokinetics and the therapeutic mechanism of action of the agent. Two different study designs are currently being used to evaluate the relationship between genetics and drug response. Historically, pharmacogenomic investigations have employed the “target-gene” strategy, in which the study is limited to genetic variants in only those genes that are known to play a role in the pharmacology of the agent under investigation. Typical target genes include drug-metabolizing enzymes, drug transporters, drug targets, and drug-resistance genes. In contrast, the “genome-wide” approach makes no assumptions about the genes that may play a role in the pharmacology of a drug. Whole-genome studies take advantage of advances in gene array technology and the completion of the International HapMap Project [48]. The HapMap Project created a publicly available database that distills human genetic variability by identifying subsets of single nucleotide polymorphisms (SNPs) that exist in linkage disequilibrium (e.g. haplotypes). Over evolutionary time, genetic variants have been inherited as blocks of variants rather than as individual SNPs. When multiple SNPs are inherited together on the same allele, they are said to be in linkage disequilibrium. Therefore, although it is estimated that there are greater than 10 million individual SNPs, the HapMap has condensed individual variation down to around 300,000 alleles that, taken together, account for nearly all of the individual genetic variability, thus greatly simplifying the task of scanning the entire genome when attempting to explain individual differences in drug response. While nearly all of the pharmacogenomic relationships described in this chapter have been identified as the result of target-gene association studies, many more important pharmacogenomic discoveries will be made in the future using a genome-wide approach. Inherited variability in drug metabolism and transport Stable genetic variants have been identified for nearly every gene involved in drug metabolism, although such polymorphisms do not always have clear clinical significance. Depending upon the nature of the genetic change, the importance of the enzyme for the overall metab-

Pharmacologic Basis for High-dose Chemotherapy

olism of a given drug, and the expression of other drug-metabolizing enzymes, a genotype–phenotype relationship may not be obvious. While almost every gene is subject to genetic polymorphisms, the phenotypic consequences of the polymorphism may be subtle, such as placing an individual on one end of a normal distribution of drug metabolism phenotypes, rather than conferring a complete deficiency of the encoded enzyme. Thus, inactivating polymorphisms can be broadly categorized into two groups: those that confer complete or near-complete loss of activity, and those that confer more subtle changes in protein function. The molecular mechanisms that inactivate genes involved in drug metabolism include splice-site mutations resulting in exon skipping, microsatellite nucleotide repeats, gene duplication, point mutations resulting in early stop codons, enhanced proteolysis, altered promoter functions, critical amino acid substitutions, and large gene deletions [49]. Conversely, gene duplication has been associated with enhanced activity for some drug-metabolizing enzymes [50]. A number of clinically relevant polymorphisms have been identified in genes involved in both the detoxification and activation of anticancer agents used in the high-dose setting. Because these genetic variants typically encode proteins with decreased enzymatic activity, the impact of these inherited differences on in vivo drug biotransformation is amplified as doses are increased. Cytochrome P-450 2C subfamily. The members of the CYP2C group of proteins are an important subfamily of cytochrome P-450 enzymes consisting of four members: CYP2C8, CYP2C9, CYP2C18, and CYP2C19. In addition to involvement in the metabolism of arachidonic acid [51], CYP2Cs also metabolize many commonly used drugs, including the anticancer agents CY, ifosfamide, and paclitaxel employed in high-dose therapy. Inactivating polymorphisms have been identified for each member of this subfamily, the best described being those found in the gene encoding CYP2C19. All of the inactivating genetic changes are single-nucleotide transitions resulting in splicing defects, early stop codons, and amino acid substitutions. Poor metabolizers of substrates for CYP2C19 are homozygous for the variant alleles and represent approximately 3–5% of Caucasians, a similar percentage of AfricanAmericans, and 12–100% of Asian groups [52]. Inactivating polymorphisms in CYP2C19 have been shown to affect the metabolism of a number of drugs including omeprazole [53] and diazepam [54]. Toxic effects related to a decreased rate of clearance and increased systemic exposure occur in poor metabolizers given diazepam [52]. Each of the CYP2C subfamily members has been shown to catalyze the activation step of ifosfamide and CY to their 4-hydroxy metabolites in the following order of affinity: CYP2C19 < CYP2C18 < CYP2C9 < CYP2C8 [55]. Inactivating polymorphisms in these enzymes may result in decreased formation of the active oxazaphosphorine 4-hydroxylated metabolites in vivo, leading to decreased clinical efficacy of these important anticancer agents during high-dose therapy. CYP2C8 is present at relatively high levels in most human livers and plays a major role in the metabolism of the taxanes; CYP2C8 is the major cytochrome P-450 isoform catalyzing paclitaxel 6αhydroxylation, the primary inactivating step in paclitaxel metabolism in vivo [56]. Multiple polymorphisms have been identified in the gene encoding CYP2C8, resulting in protein variants with decreased enzymatic activity compared with the wild-type sequence. The existence of at least four CYP2C8 variant alleles, including two involving nonsynonymous mutations, has been confirmed [57]. Two of the genetic variants resulting from single amino acid substitutions, G416A (CYP2C8*3) and C792G (CYP2C8*4), exhibit decreased rates of paclitaxel 6αhydroxylation in vitro [57,58]. As a result, patients with one or more alleles carrying inactivating polymorphisms in CYP2C8 would be expected to have a decreased rate of metabolic clearance of paclitaxel.

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Cytochrome P-450 3A subfamily. Members of the CYP3A subfamily catalyze the oxidative and reductive metabolism of a structurally diverse set of exogenous and endogenous compounds [59]. The CYP3A members are the most abundant cytochrome P-450s in human liver and small intestine [60]. Substantial interindividual differences exist in CYP3A expression, exceeding 30-fold in some populations [61]. The wide variation in expression contributes greatly to the variation in oral bioavailability and systemic clearance of CYP3A substrates. Such variation in CYP3A can result in clinically significant differences in drug toxicities and response. Human CYP3A activities reflect the heterogeneous expression of at least three CYP3A subfamily members: CYP3A4, CYP3A5, and CYP3A7 [61]. Functional CYP3A4 is found in most adults, with a 10–40-fold variation in its expression. CYP3A7 is predominantly expressed in fetal life, and its expression seems to be silenced shortly after birth; however, some individuals express CYP3A7 messenger RNA into adulthood. CYP3A5 can also be detected in the liver and small intestine of some adults [62]. CYP3A4, the most abundant isoform of cytochrome P450 in adult human liver, is important for the metabolism of many clinically important anticancer compounds used in high-dose therapy, including the taxanes [63], vinca alkaloids [64], epipodophyllotoxins [65], and CY [66]. In addition to a high constitutive expression, CYP3A4 is inducible in response to certain exogenous substrates. Although several CYP3A4 variants have been identified, including five that result in coding sequence changes, none of the polymorphisms described affects enzyme catalytic activity [67]. However, a polymorphism in the promoter region for CYP3A4 has been described that can affect the extent to which CYP3A4 is inducible, rather than altering constitutive levels of the enzyme [68]. A polymorphism was discovered in intron 3 of the human CYP3A5 gene that creates an ectopic splice site, leading to a premature stop codon [69]. This common allelic variant in CYP3A5 is the principal genetic basis for polymorphic expression of this enzyme in humans, and explains why CYP3A5 expression is only detectable in a subset of the population. Because many CYP3A4 substrates are also substrates for CYP3A5, the CYP3A5 polymorphism influences overall CYP3A activity, and would be expected to shift subjects to the higher end of the phenotypic distribution for CYP3A activity. Thus, CYP3A5 can contribute significantly to the total metabolic clearance of many CYP3A substrates. Furthermore, because CYP3A4 and CYP3A5 have slightly differing catalytic activities and substrate specificities, polymorphic CYP3A5 expression may also contribute to differences in metabolite profiles and in susceptibility to inhibitory drug interactions. The polymorphism in CYP3A5 may be one important factor contributing to individual variation in the overall CYP3A-mediated metabolism of drugs. For example, multiple studies have demonstrated that the daily dosage requirement for the immunosuppressive agent tacrolimus in organ transplant patients is highly dependent on CYP3A5 genotype [70,71]. Simple DNA-based tests have now been developed that can be used to determine how individual differences in CYP3A5 contribute to the overall metabolic fate of CYP3A substrates, and to their pharmacokinetic and pharmacodynamic variability. Cytochrome P-450 2B subfamily. CYP2B6 belongs to the less wellcharacterized human CYP 2B subfamily of enzymes. Initially, the lack of knowledge regarding substrates and inhibitors slowed the phenotypic characterization of the enzyme and identification of the effects of genetic variants. Furthermore, like the CYP3A family, human CYP2B6 is strongly inducible [72], which likely masks genotype–phenotype relationships. With over 100 SNPs identified to date, CYP2B6 is one of the most polymorphic CYP genes in humans [73]. Sequence variations include nonsynonymous, silent, promoter, and intronic changes, many of them showing extensive linkage disequilibrium giving rise to distinct

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haplotypes with a wide array of functional consequences, including null alleles, partially reduced function/expression alleles, and alleles with increased activity. Many of the polymorphisms are rare, but others are common, with allele frequencies of 5% and up to over 50%, respectively, and with marked interethnic differences [74]. Although the true significance of CYP2B6 pharmacogenetics for drug treatment is still emerging, consistent genotype–phenotype relationships have been demonstrated for the anti-human immunodeficiency virus drug efavirenz [75], and associations have been reported for other drugs. As in the case for CYP2C19, CY also undergoes biotransformation to yield the active 4-hydroxy-metabolite via CYP2D6. The G516T SNP (also known as the *6 allele) is one of the most common variants in CYP2B6, with a frequency of around 20%. In a study of 29 patients with hematologic malignancies treated with conventional doses of CY (1 g/m2), the G516T variant allele was associated with a faster elimination of parent drug and a higher ratio of the 4OH-CY : CY AUC when compared with the wild-type allele [76], suggesting that the variant enzyme increases the rate of prodrug activation. A larger study of Japanese patients with either malignant lymphoma or breast cancer treated with CY has confirmed that individuals who are homozygous for the G516T variant have significantly faster CY elimination through metabolic activation when compared with patients who are either heterozygous or homozygous for the wild-type allele [77]. In addition, serious toxicity has been reported in patients who are G516T homozygous and receiving even modest doses of CY [78]. Other CYP2D6 variants have been associated with decreased activation of CY [77], and, given the complexity of CY metabolism, establishing the importance of CYP2D6 pharmacogenomics in the high-dose therapy setting will require large studies that include all possible genotypes. Glutathione S-transferases. The glutathione S-transferase (GST) family of enzymes is coded by a large supergene family located on several different chromosomes. In general, the GSTs detoxify strong electrophiles by catalyzing their conjugation to glutathione (GSH). A wide variety of substrates are detoxified by GSTs either by direct conjugation with GSH or by inactivation of the cellular byproducts of oxidative stress. As a result, the GST family of proteins plays a critical role in defense against environmental toxins (e.g. polycyclic aromatic hydrocarbons) and reactive oxygen species. Furthermore, many of the chemotherapeutic agents used in high-dose therapy are detoxified by GSTs, including BU [79], thiotepa [80], CY and ifosfamide [81,82], platinum-containing compounds, and anthracyclines [83]. Overexpression of GSTs results in cellular resistance to the cytotoxic effects of these chemotherapeutic drugs [84]; conversely, cells that have been depleted of GSH are further sensitized to the toxic effects of these agents [85]. Of the four main subfamilies of GST (A1, M1, T1, and P1), GSTM1 and GSTT1 forms are known to be highly polymorphic, with homozygous deletion of either or both genes at varying but significant frequencies in different ethnic groups [86]. Because GST genes are highly polymorphic, there has been considerable interest in determining whether particular allelic variants of each are associated with altered clinical phenotypes. The preponderance of evidence suggesting a role for GSTs in detoxifying reactive compounds comes from several molecular epidemiologic studies linking GST genotype with cancer risk [87,88]. However, much less is known about the importance of GST genotype as a determinant of either the pharmacokinetics of, or response to, highdose chemotherapy. Conjugation with GSH is the major route of biotransformation for BU, and is predominantly catalyzed by the GST isozyme GSTA1 with contributions from both GSTM1 and, to a lesser extent, GSTP1 [89]. At least seven polymorphisms of GSTA1 have been identified, and haplotype analysis has revealed the existence of five

unique alleles. None of the known polymorphisms or haplotypes appears to be associated with changes in hepatic GSTA1 expression or function. However, it has been suggested that the GSTM1-null genotype may predispose patients to BU-induced hepatic sinusoidal obstructive syndrome (SOS) through depletion of the cellular GSH pool in sinusoidal endothelial cells [90]. Multiple drug resistance (MDR) gene 1. Although passive diffusion accounts for the cellular distribution of some drugs and metabolites, more attention has been paid to the role of membrane transport proteins in critical physiologic functions such as oral drug absorption, biliary and renal excretion, and distribution of drugs into “therapeutic sanctuaries,” such as the brain and testes. The most widely studied drug transporter is the ATP-binding-cassette family member MDR1, which encodes the P-glycoprotein. To date, 29 SNPs have been reported to exist in the MDR1 gene, including 19 in exonic regions and 11 that result in an amino acid change (i.e. nonsynonymous SNPs) [91]. Interestingly, the SNP at exon 21 in position 2677 results in two distinct amino acid changes, Ala893Ser (G2677T) and Ala893Thr (G2677A) [92], a synonymous SNP in exon 26 (C3435T) was the first MDR1 variant to be associated with altered protein expression. Individuals who were homozygous for the 3435T allele were found to have fourfold lower P-glycoprotein levels in their livers and intestine compared with subjects who were homozygous for 3435C [93]. Although a large number of MDR1 genotype–phenotype studies have been carried out, reports of the clinical effects of individual MDR1 SNPs have been inconsistent and sometimes conflicting [91]. As discussed earlier in this chapter, SNPs are often inherited in blocks (i.e. haplotype) rather than as the individual polymorphisms. In the case of MDR1, studies have demonstrated a linkage disequilibrium between the synonymous polymorphism in exon 26 and other SNPs [94,95], suggesting that functional effects on P-glycoprotein may be haplotype dependent. Studies have demonstrated that analysis of haplotypes may be superior in predicting the pharmacokinetics of digoxin [95] and cyclosporine [96]. Given that the frequency of different haplotypes in MDR1 can vary by ethnicity [97], determination of haplotype may prove to be important when assessing the effects of MDR1 genotype on the outcome following high-dose therapy.

Drug metabolism, pharmacokinetics, and pharmacodynamics of high-dose chemotherapy Pharmacokinetic variability in high-dose chemotherapy The basic pharmacokinetic features that ultimately affect systemic exposure to a drug include the processes of absorption, tissue distribution, metabolism, and excretion. Although patient-to-patient pharmacokinetic variability is common for all of the chemotherapeutic agents in current oncologic practice, the extent of interpatient and intrapatient pharmacokinetic variability is accentuated in the high-dose chemotherapy setting [98–102]. It has been reported that, as a result of interpatient differences in pharmacokinetics, the systemic clearances of many of the most commonly used cytotoxic drugs vary over three- to 10-fold [103]. The wide range in the rate of drug elimination results in a correspondingly large variation in total drug exposures (e.g. AUC and steady-state drug concentration) in patients treated with equivalent doses. For drugs with steep dose–response curves and narrow therapeutic windows, the implication of such significant pharmacokinetic variability is the unpredictable probability of either clinical response or toxicity. The degree of pharmacokinetic variability that is routinely observed during high-dose chemotherapy may be due to pharmacogenetic variations in the population, as discussed previously [104], to the metabolic pattern of the

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Pharmacologic Basis for High-dose Chemotherapy H O

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Fig. 21.2 Activation and detoxification pathway of cyclophosphamide (CY). The drug is activated to 4-hydroxycyclophosphamide and aldophosphamide by cytochrome P-450 isoenzymes, and to phosphoramide mustard by exonucleases. P-450 isoenzymes appear to detoxify CY to the dechloroethyl derivative, aldehyde oxidase breaks down 4-hydroxycylophosphamide to ketocyclophosphamide, and aldehyde dehydrogenases detoxify aldophosphamide. (Reproduced from [110], with permission.)

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specific drug [105], to metabolic interactions among high-dose chemotherapeutic agents used in combination [106], to drug–drug interactions between the chemotherapeutic agents employed and other drugs required for symptom control or the treatment of associated infections [107], as well as to physiologic factors [108,109]. Perhaps the most important aspect of the pharmacokinetic variability of high-dose chemotherapy is the demonstration that the metabolism of several agents is saturable, leading to potentially large, unpredictable, and nonlinear changes in systemic exposure with modest changes in drug dose within the high-dose chemotherapy range. As described later for CY (Fig. 21.2), there appears to be saturation of CY activation pathways when the agent is used at high dose [110]. While the clearances of high-dose doxorubicin (Fig. 21.3) and etoposide increase lin-

Fig. 21.4 Peak plasma concentration of paclitaxel during a 24-hour continuous infusion as a function of paclitaxel dose. Nonlinear pharmacokinetics and greater interpatient variability of paclitaxel pharmacokinetics are seen starting at doses greater than 450 mg/m2.

early with dose, high-dose paclitaxel (Fig. 21.4) exhibits dramatic deviations from linearity as drug doses increase. The implications of nonlinear pharmacokinetics for agents employed in the high-dose chemotherapy setting are significant. Because the toxicities associated with high-dose chemotherapy are substantial, the margin for error (therapeutic ratio) is low. The inability to predict the pharmacokinetic behavior of a drug at high dose increases the potential risk of severe side-effects from that compound. Thus, the potential utility of therapeutic drug monitoring (TDM) that will allow targeted systemic exposures in the high-dose chemotherapy setting is greatly enhanced for agents that demonstrate nonlinear pharmacokinetics. Pharmacodynamics of high-dose chemotherapy and the individualization of chemotherapeutic drug exposures The first evidence that modification of drug dosing based on the pharmacokinetic behavior of a chemotherapeutic agent in an individual undergoing high-dose chemotherapy could positively affect the toxicity

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of treatment was published in 1989. The incidence of hepatic SOS after BU conditioning was demonstrated to be dramatically decreased in patients undergoing allogeneic BMT by prospective monitoring of BU levels and modification of drug dosing based on a targeted “safe” AUC [111]. Since then, the pulmonary toxicity of breast cancer patients receiving high-dose BCNU, cisplatin, and CY has been related to the AUC of BCNU [112], and the ototoxicity of patients treated with highdose thiotepa, CY, and carboplatin has been associated with the AUC for carboplatin [113]. The application of more detailed therapeutic drug monitoring (TDM) and adaptive control strategies in the high-dose chemotherapy setting may also allow the safer use of agents with saturable pharmacokinetics [114]. Ultimately, the demonstration that systemic exposure to one or more high-dose chemotherapy agents is correlated with therapeutic outcome, as has been shown for methotrexate in the treatment of childhood acute lymphoblastic leukemia [115], remains a critical goal for future high-dose chemotherapy trials. The considerable time and effort spent determining the extent and causes of pharmacokinetic variability for each of the cytotoxic agents described in this chapter has been undertaken with the ultimate goal of developing predictive models for drug disposition that will allow the clinician to administer an optimal dose based on each individual’s own physiologic, genetic or environmental profile. The fundamental approach to optimizing cytotoxic drug therapy in the past has been the assignment of doses based on a measure of patient size, while more intensive methods have involved near real-time monitoring of circulating drug concentrations in individual patients with subsequent dosage adjustment to achieve a predefined target drug level. The relative merits of any approach aimed at optimizing exposure based solely on the pharmacokinetic behavior of a drug is subject to debate because of the added contributions of cellular drug disposition and pharmacodynamic effects to the variability of drug response. However, in light of the significant patient-to-patient differences in the measured concentrations of most cytotoxic agents used in high-dose chemotherapy, optimizing drug exposures based on all currently available information regarding potential sources of pharmacokinetic variability is an appropriate initial strategy. Dosage adjustments based on body surface area. One of the earliest approaches to normalizing cytotoxic drug exposures was the use of body surface area (BSA) to correct for differences in individual patient size. This practice was based on the findings of Freireich and colleagues [116], who showed that the maximum tolerated doses of cytotoxic drugs in different animal species were most similar when scaled to surface area. During the initial stages of clinical drug development, BSA is used to guide dosage escalation to the point of defining the maximum tolerated dose. The recommended doses of drugs that are developed in this way are ultimately assigned based upon a BSA-based dosing algorithm. As a result, it has become common practice in clinical oncology to dose anticancer drugs on the basis of a patient’s BSA, with the aim of reducing interindividual variability in drug exposure due to factors related to patient size. The most commonly used formula to estimate BSA originates from 1916 [117], and was based on plaster of Paris models made of nine subjects ranging in weight from 25 to 90 kg. The surface area of the completed molds was calculated, and a formula derived based upon the mathematical relationship between each subject’s weight and height and their calculated surface area. Despite reports questioning the predictive performance of this formula [118], the original method of DuBois has been utilized in clinical oncologic practice for over 40 years. However, during the last decade, the use of BSA-based dosing has been criticized, and various alternative body-size measures have been proposed, such as lean body mass, that might be better predictors of drug clearance [119]. The skepticism toward BSA as an appropriate way to standardize drug

dosing corresponds with an increased understanding of the magnitude of pharmacokinetic variability and its many sources. A relationship between BSA and physiologic measures relevant to drug elimination such as hepatic and renal function has never been demonstrated beyond the contribution of body weight alone [120]. Furthermore, for several cytotoxic drugs, no relationship exists between the pharmacokinetic parameters of the drug and BSA [121]. That BSA cannot explain pharmacokinetic variability is not surprising since BSA varies over a fairly narrow range compared with the wider variations (three- to 10-fold) in drug clearance, volume of distribution, and excretion observed for essentially all antineoplastic agents. It has been suggested that the practice of BSA-based dosing should be performed only for those agents where a significant relationship between BSA and drug clearance has been proven [121]. For all other cytotoxic drugs, it has been proposed that dosing be fixed or adjusted based on other more meaningful predictors of pharmacokinetics such as indices of renal or hepatic function. Perhaps the strongest rationale for abandoning BSA-based dosing is that, by eliminating the need for BSA normalization, a significant source of dosage calculation errors could be eliminated without producing a significant impact on the probability of under- or overtreating patients based on differences in size, the net result of which might be increased patient safety [122]. Whether or not cytotoxic drug dosing based on BSA is abandoned, it is clear that better predictors of the pharmacokinetics of high-dose chemotherapy are required to optimize anticancer drug therapy. Dosing based on indices of renal or hepatic function. In contrast to BSA, the pharmacokinetics of anticancer agents have frequently been associated with the functional indices of the major drug-clearing organs, the liver and kidneys. Depending on the major route of elimination for a particular drug, it can be reliably predicted that significant changes in either hepatic or renal function will result in corresponding changes in drug clearance. For example, carboplatin is associated with an increased risk of severe toxicity in patients with creatinine clearances of less than 60 mL/min due to decreased drug clearance and greater total drug exposure [123]. The rationale for reducing the dose of cytotoxic drugs in the presence of hepatic impairment is also well documented for agents such as doxorubicin, vincristine, docetaxel, and paclitaxel [124]. In the case of hepatic impairment, the guidelines for dose adjustment are based on clinical measures of liver function such as bilirubin (e.g. doxorubicin and vincristine), aspartate aminotransferase (e.g. paclitaxel and docetaxel) or alkaline phosphatase (e.g. docetaxel). The association between drug-clearing organ function and pharmacokinetics has also led to the development of cytotoxic drug-dosing strategies based on indices of renal and hepatic function in patients with organ function tests within the normal limits. The best current example of dose adjustment guidelines for a cytotoxic agent based on organ function is for carboplatin. Calvert and colleagues first demonstrated that the carboplatin AUC could be accurately predicted using an equation that utilizes pretreatment renal function [108]. This a priori method for carboplatin dosing has led to a substantial reduction in pharmacokinetic variability, such that carboplatin is currently one of the few drugs of any class that is routinely dosed to achieve a target systemic exposure rather than on a mg/m2 or mg/kg basis. Although the original method of carboplatin dosage adjustment relied on measurement of glomerular filtration rate (GFR) by determining chromium-51-EDTA clearance, subsequent studies have shown that other measures of GFR can be substituted [125]. Therefore, the pharmacokinetics of carboplatin can be reliably predicted from routine clinical measurements, leading to improved dosing. Routinely available clinical indices of hepatic function are less helpful in predicting cytotoxic drug metabolism than is creatinine clearance in

Pharmacologic Basis for High-dose Chemotherapy

the case of kidney function. Liver function tests are not sufficiently sensitive to detect differences in drug-clearing capacity within the normal range of organ function, and are only correlated with drug clearance in the setting of clinically impaired hepatic function. As a result, the predictive value of any one clinical marker of liver function is usually limited with respect to the metabolism and transport of drugs. Composite scoring approaches based on multiple liver function tests, such as the Childs–Pugh classification [126], have been proposed as an alternative to single laboratory measures. However, as with individual liver function tests, such scoring systems have only demonstrated utility in patients with impaired hepatic function [127]. In addition, other standard clinical tests that measure the functional capacity of the liver, such as prothrombin time and albumin level, are of limited value as predictors of drug clearance due to the lag time required for a change in the synthetic function of the liver to be reflected in an alteration in drug clearance. Therapeutic drug level monitoring in high-dose chemotherapy. TDM has a long history of use in solid organ transplantation, as well as other areas of clinical practice. The widespread use of TDM is based on the principle of an optimal therapeutic window for the particular agent being monitored. TDM involves a near-real-time measurement of circulating drug levels followed by subsequent dose adjustment to achieve a predefined target drug exposure. For example, in the case of antibiotics, the goal of TDM is to assure that the drug concentration is maintained above the minimum inhibitory concentration for the infective agent being targeted, while staying below the concentrations associated with normal organ toxicity. The use of TDM in oncologic therapeutics, although promising, has been limited, in part because performing real-time pharmacokinetic monitoring of even a single cytotoxic agent requires dedicated laboratory personnel and resources typically available only at major medical centers. The first step in the process of developing TDM for high-dose chemotherapy is to demonstrate its value in a given clinical setting before subsequent work is done to make the process more practical. Once the role of individualization of a cytotoxic agent has been established, the use of limited pharmacokinetic sampling methods and automated assay techniques can be applied. Perhaps the most widely reported use of TDM in the area of high-dose chemotherapy is the practice of pharmacokinetic monitoring of BU in patients undergoing HCT. High-dose BU is an important component of many preparative regimens. The dose-limiting toxicity of high-dose BU is hepatic SOS, which occurs in approximately 20–40% of patients. BU is erratically absorbed after oral administration, and, at a given dose, there is considerable variability in its systemic exposure. Grochow first described the correlation between the AUC after the first dose of oral BU with the occurrence of SOS [128]. Subsequently, several investigators have confirmed the association between measured BU exposure and risk of SOS [129,130]. Generally, the risk of SOS has been shown to be increased with a steady-state BU concentration of more than 900 μg/L and a first-dose AUC of greater than 1500 μM × minute. In addition, lower rates of relapse in chronic myelogenous leukemia (CML) have been reported to occur in patients with higher BU exposures without an increased risk of toxicity [131]. Furthermore, measured BU exposure has been reported to be related to the rate of engraftment in children [132]. Therefore, it is now widely accepted that an optimal therapeutic window exists for BU dosing, leading many investigators to advocate the use of BU TDM to maximize the likelihood of engraftment and minimize the risk of toxicity and relapse in patients receiving the BU/CY preparative regimen. However, for each conditioning regimen that includes BU, as well as for each of the many different diseases for which hematopoietic cell

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transplantation (HCT) is a therapeutic option, the therapeutic window for BU is likely to be different. Therefore, more data are needed to define the optimal use of BU in each specific clinical situation. Complicating the issue of TDM for BU is the availability of an intravenous BU formulation that has led to a significant decrease in the incidence of SOS in the absence of TDM [133]. The intravenous route of BU administration avoids the hepatic first-pass effect, thereby decreasing the high local drug concentrations achieved in the hepatic sinusoids with oral dosing. Furthermore, the pharmacokinetic profile of BU is greatly improved by intravenous administration with respect to decreased inter- and intrapatient variability [134]. Moreover, studies investigating the feasibility of a once-daily administration of intravenous BU have demonstrated that, in addition to being much more convenient, the pharmacokinetic profiles and post-transplant complications are similar for once-daily BU compared with the traditional four-times-daily BU [135,136]. It is possible that the use of intravenous BU may obviate the need for TDM if it can be established that the therapeutic window is sufficiently large to allow for a fixed dose of drug that will be within the optimal range of systemic exposure for all patients. It is also possible that the therapeutic window for intravenous BU is different than that for the oral formulation, as well as for different schedules of administration (i.e. once a day versus four times a day). If once-daily intravenous BU is adopted more widely, the therapeutic range for optimization must be determined and validated prospectively before TDM becomes standard practice. Despite the remaining questions regarding the utility of TDM for BU, growing evidence for an optimal therapeutic window, along with significant interpatient pharmacokinetic variability, has made BU TDM the standard of practice in many HCT centers with access to analytic laboratories and clinical pharmacology resources. The challenge for further optimizing the use of high-dose BU will be to better define the therapeutic range in each particular clinical setting and to simplify the actual procedures for drug level monitoring. Efforts have also been made to optimize treatment with the triple alkylating agent regimen of CY, thiotepa, and carboplatin through the use of TDM. Carboplatin is an active high-dose regimen used for the treatment of relapsing germ cell cancer [137]. Significant intersubject variability exists in the pharmacokinetics of all three alkylators, resulting in marked differences in drug and metabolite exposures in patients treated with similar doses [138]. Because relationships have been identified between the pharmacokinetics of CY, thiotepa, and carboplatin and their associated toxicities [138,139], TDM has been employed with the ultimate the goal of decreasing variability in drug exposures and decreasing morbidity without comprising efficacy. Although TDM does lead to a marked reduction in the variability of exposures to 4OH-CY, thiotepa and tepa, and carboplatin, no clear benefit has been demonstrated for dose-individualized carboplatin compared with standard dosing with respect to toxicity [140]. However, the incidence of SOS does appear to be reduced in patients who have received an adjusted CY dose.

Therapeutic drug selection and drug development in the high-dose chemotherapy setting Drug selection in the high-dose chemotherapy setting Studies of new dose-escalation regimens supported by hematopoietic cells, as described previously, are frequently based on concentration– response relationships developed in vitro. While such studies may provide important new leads, it is remarkable how frequently investigators appear to utilize such data to the exclusion of the single most important criterion of drug selection in the high-dose chemotherapy setting: the degree of activity of the agent to be escalated for the disease

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Table 21.3 Guidelines for the development of new high-dose chemotherapy trials 1. Preclinical and clinical support required for the selection of drugs to dose-escalate: • Select agents with steep dose–response curves for the tumor of interest • Preference for drugs with myelosuppressive dose-limiting toxicities 2. Pharmacologic basis for the dose, schedule, and combination of drugs chosen: • Existing database supporting the schedule of administration • Additive or synergistic antitumor activity with other agents in the regimen; minimization of overlapping toxicities • Pharmacokinetic methodologies exist to measure plasma or serum levels of all agents employed in the regimen 3. Toxicity grading scale, definition of maximally tolerated dose, and assessment of late toxicities: • Select a scale validated for use in high-dose chemotherapy and employ it precisely to define the parameters for dose escalation and for the determination of dose-limiting toxicities • Distinguish modality-related adverse events from dose- or exposure-related toxicities in determining the maximally tolerated dose in any new regimen • Provide specific guidelines for the evaluation of dose escalation in tandem-cycle high-dose regimens • Assess late-occurring adverse effects (such as secondary leukemia) as well as persistent toxicities (peripheral neuropathy or pulmonary fibrosis) 4. Statistical considerations: • Select a design for dose levels and escalation procedures that will adequately characterize the dose–toxicity relationship and include pharmacologic correlates; this may require the accrual of more than the standard three to six patients per cohort • Apply the statistical plan throughout the study, avoiding exploratory or hypothesis-generating late analyses Adapted with permission from Margolin [141].

in question. Clinical evidence of therapeutic activity for the drug at standard dose against the disease to be treated in the high-dose chemotherapy setting should be the minimum starting point for the development of a high-dose chemotherapy program. The other important criteria that should be used for the development of high-dose chemotherapy programs include the absence of nonadditive extramedullary toxicities when the agents are used in combination (such as SOS), which can dramatically decrease the safe level of dose escalation; the presence of divergent mechanisms of cytotoxicity and lack of crossresistance for the agents being combined; and the extent of dose escalation possible for each drug in combination with hematopoietic cell support. None of these individual criteria, however, is more important than optimizing the use of the most therapeutically active chemotherapeutic agents. Conduct of drug development trials in high-dose chemotherapy The conduct of phase I trials in high-dose chemotherapy is a substantial investigational challenge [141]. Although the selection of drug combinations and doses for high-dose chemotherapy has often been based on many of the same dose-escalation principles used in studies performed at standard levels of dose intensity and without hematopoietic cell support, the modality of allogeneic transplantation itself is associated with specific morbidities that may confound attempts to attribute adverse events to the drug being escalated. Thus, the presence of underlying graft-versus-host disease and multiorgan compromise from infection can make the attribution of toxicity extraordinarily difficult. In both the allogeneic and autologous transplant settings, the tendency in the past has been to avoid formal, complete dose-escalation schemas for all of the drugs that comprise a specific combination, which leads to uncertainty about whether or not a particular regimen utilizes all of the agents at their maximally tolerated dose. One important aspect of the development of high-dose chemotherapy programs that is seldom considered is the remarkable lack of singleagent data conclusively demonstrating that a clinical dose–response actually exists for a particular agent in a specific disease. Close inspection of the data on the dose-intensive treatment of advanced breast cancer would indicate that only for the anthracyclines and perhaps CY does a definitive dose–response relationship clearly exist at the level of single-agent trials [142]. Thus, there is a critical need to examine

the clinical dose–response curve of important new therapeutic agents, such as the taxanes, in the diseases that may benefit from high-dose chemotherapy. The success of high-dose systemic therapies is also clearly linked to a number of factors specific to the host that bears the tumor. Among these factors are nutritional status, functional capabilities, and the integrity of major organ systems. The effect of these factors on the development of new high-dose chemotherapy programs cannot be overstated. Accurate assessment of organ system reserve prior to entry on novel high-dose chemotherapy treatment regimens is of fundamental importance, since the recognition of novel and potentially life-threatening side-effects produced by new high-dose chemotherapy combinations is a common event. Patients with dysfunction of an organ that is necessary for the metabolism or excretion of a high-dose cytotoxic agent are so likely to suffer untoward toxicity that they should not participate in early phase I high-dose chemotherapy trials. An outline of a proposed set of guidelines for the development of new high-dose chemotherapy trials is provided in Table 21.3.

Clinical pharmacology of drugs utilized for high-dose chemotherapy and HCT: pharmacokinetic, pharmacodynamic, and mechanistic considerations Alkylating agents The first chemotherapeutic agents to be utilized as preparatory regimens for HCT were the alkylating agents, a large group of drugs with the ability to covalently bond DNA and other biologically significant molecules through an alkyl group consisting of one or more saturated carbon atoms [143,144]. The common feature of these compounds is that they are composed of mono- or bifunctional alkyl groups linked to a core structure that confers pharmacologic and toxicologic differences on the alkylating moieties. The simplest backbone for a bifunctional alkylating agent is that of nitrogen mustard (HN2, mechlorethamine), which consists of a nitrogen atom (as NCH3) linked to two chloroethyl groups. As shown in Fig. 21.1, by contrast, L-phenylalanine mustard (MEL) consists of the same bifunctional alkylating groups attached to the Lphenylalanine molecule; CY and ifosfamide are two other clinically

Pharmacologic Basis for High-dose Chemotherapy

useful alkylating agents that have substituted other side chains for the methyl group in HN2. The common mechanistic feature of the alkylating agents is that, upon entering cells, the alkyl groups bind to electrophilic sites in DNA and other biologically active molecules; bifunctional alkylation of DNA can result in crosslinks between strands of DNA, which impedes replication. Other biochemically important molecules are also alkylated by such agents, but the dominant effect appears to be DNA crosslinking. Cyclophosphamide CY is a member of the oxazaphosphorine group of nitrogen mustard derivatives and is the most widely used alkylating agent in BMT preparatory regimens based, in part, on its broad range of antineoplastic activity as well as its immunomodulatory properties [145]. CY has significant activity against both acute and chronic leukemia, Hodgkin’s and non-Hodgkin’s lymphoma, and a variety of adult and pediatric solid tumors. With hematopoietic cell support, it can be escalated eight- to 10-fold from standard intermittent intravenous doses of 600–1000 mg/ m2 to 4000–7000 mg/m2 when used in combination high-dose chemotherapy programs [12,146]. High-dose CY is frequently used in combination with BU as a preparatory regimen for patients with acute or CML [147], and with high-dose etoposide and TBI for patients with non-Hodgkin’s lymphoma [148]. Mechanism of action. As described in detail in following sections, CY is a prodrug that must be activated by the hepatic cytochrome P-450 system to cytotoxic alkylating intermediates (Fig. 21.2). The presumed cytotoxic species, phosphoramide mustard, produces interstrand and intrastrand DNA crosslinks that are lethal, and that can induce apoptosis [149]. While little is known about the cellular pharmacology of CY and its active metabolites, a great deal is understood regarding cellular resistance mechanisms for this agent. The most clearly delineated pathway of CY resistance is the development of increased intracellular pools of reduced nonprotein sulfhydryls, including the tripeptide GSH [150]; alterations in intracellular GSH levels can significantly affect the level of phosphoramide mustard-induced DNA crosslinking [151]. Conjugation reactions between GSH, GSTs, and phosphoramide mustard provide an enzymatically enhanced detoxification pathway for CY degradation, decreasing tumor cell sensitivity [152]. In addition to glutathione transferases, increased intracellular levels of aldehyde dehydrogenases (particularly the 1A1 isoform), which convert aldophosphamide to the inactive carboxyphosphamide derivative (Fig. 21.2), lead to CY resistance [153]. Finally, it seems reasonable to conclude that overexpression of certain DNA repair genes capable of rendering tumor cells resistant to other nitrogen mustard derivatives is also likely to contribute to the clinical spectrum of tumor cell insensitivity to CY [154]. Metabolism, pharmacokinetics, and pharmacodynamics. The complex metabolism of CY is shown in Fig. 21.2; although the pharmacokinetics of the drug have been studied by several laboratories in the high-dose chemotherapy setting, investigations utilizing mass spectrometry-based analytic techniques have changed the interpretation of the CY metabolic pathway [110,155]. The initial bioactivation of CY is produced by hepatic, microsomal P-450-dependent hydroxylation to produce 4-hydroxycyclophosphamide, which is in equilibrium with the ring-opened aldehyde, aldophosphamide. 4-Hydroxycyclophosphamide or aldophosphamide, or both, can be detoxified by aldehyde oxidase or aldehyde dehydrogenase, respectively, to produce the inactive metabolites ketocyclophosphamide and carboxyphosphamide, which are excreted in the urine together with the parent compound and account for the majority of a delivered dose of CY. Aldophosphamide that is not

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detoxified enters tumor cells and, through the process of β-elimination, produces the active alkylating species phosphoramide mustard and acrolein, the toxic species responsible for CY-induced cystitis. Because of the multilayered metabolism of CY, it has been difficult to assign pharmacodynamic correlations for this drug. However, the incidence of CY-related cardiac toxicity after high-dose therapy has been associated with lower parent drug AUC and, presumably, increased activation to potentially toxic intermediates [156]. The effect of highdose drug administration on the metabolism of CY has been extensively studied using mass spectrometry [155,157,158]. It appears that while the overall pharmacokinetics of parent CY are similar over an eightfold range of drug concentration, there is substantial evidence that CY activation is saturated at a dose of 100 mg/kg. Increased renal clearance of the parent drug, and of the inactive carboxyphosphamide metabolite, parallels a decrease in the clearance of bioactivated species in the high-dose setting. These results bring into question the use of doses above 100 mg/ kg, which may only enhance toxicity, particularly cardiac toxicity, without improving exposure of the tumor to cytotoxic alkylating species [159]. Further, it appears that, with the more sensitive analytic techniques, important drug–drug interactions between CY and thiotepa (which appears to inhibit CY metabolism), BU or phenytoin (which may increase CY clearance) [106,160], and TBI (which has no effect on the bioactivation of CY) [161] have been defined. The pharmacokinetics of unchanged CY in the plasma reveals considerable interpatient variability, with a terminal half-life of 3–9 hours; however, the usefulness of monitoring the disappearance of the parent drug is unclear based on the saturable nature of the bioactivation pathways of CY at high dose. Approximately 10% of the parent drug is excreted unchanged in the urine; it has also been reported that the unchanged parent molecule can be sufficiently cleared by hemodialysis to allow the use of high-dose CY in patients with end-stage renal disease [162]. In addition to the drugs described above, other authors have found that inducers of microsomal metabolism, such as phenobarbital or dexamethasone, can enhance CY clearance. However, the dose of CY does not require alteration in the face of hepatic dysfunction. The major dose-limiting toxicity of high-dose CY is cardiac injury, which occurs much more frequently at doses above 150 mg/kg [159]. Endothelial damage has been associated with myocardial necrosis and the development of potentially lethal congestive heart failure. The etiology of CY cardiac toxicity appears to be related to a severe depletion of cardiac reduced thiol stores [150]. In addition, high-dose CY therapy has been associated with severe hemorrhagic cystitis; however, forced diuresis or co-administration, or both, of the thiol sodium-2-mercaptoethane sulfonate (mesna) has markedly decreased the incidence of this toxicity of acrolein. Pulmonary toxicity and SOS also occur more frequently in combination high-dose chemotherapy regimens that utilize CY. Finally, renal tubular injury may mimic the clinical picture of inappropriate antidiuretic hormone secretion [163]. Melphalan The development of the bifunctional alkylating agent MEL dates to its synthesis in 1953; that synthetic effort was based on the hypothesis that a substitution of phenylalanine for the methyl group of nitrogen mustard would produce an agent selectively concentrated by melanoma cells, since those cells utilize phenylalanine in several metabolic pathways [164]. MEL is, in fact, actively transported into tumor cells but not specifically into melanomas; however, its activity in the treatment of lymphoma, multiple myeloma, neuroblastoma, and breast cancer has led to the use of the drug in the high-dose chemotherapy setting [165]. The availability of an intravenous formulation in the United States in 1992 spurred the development of high-dose regimens utilizing MEL [166]. In comparison with the standard chemotherapy setting, the use of

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hematopoietic cell support allows the administration of MEL as a single agent in doses of 180–200 mg/m2, approximately sixfold higher than the standard dose range [165]. Mechanism of action. As an analog of nitrogen mustard, MEL hydrolyzes intracellularly, and produces interstrand and intrastrand crosslinking of DNA by way of its two chloroethyl groups, which spontaneously lose chloride ions to form reactive intermediates [167]. While a variety of different alkylation sites exist on DNA, it remains difficult to discern which of the various base oxygens or amino groups is the critical site underlying the cytotoxic properties of MEL. However, it is clear that DNA interstrand crosslinks correlate more closely with tumor cell killing than does DNA–protein crosslinking [167]. The cytotoxicity of MEL in vitro can be correlated with total drug exposure (concentration × time); further, the extent of cytotoxic DNA crosslinking increases with time, suggesting that critical bifunctional adducts develop from much more numerous monofunctional lesions [168]. The production of DNA adducts by MEL activates the poly-adenosine diphosphate (ADP)-ribose polymerase system in the nucleus, and may contribute to tumor cell killing through the depletion of intracellular pools of nicotinamide adenine dinucleotide [169]. MEL is transported into tumor cells by two different energy-dependent drug carriers [170,171]. A high-affinity L-amino acid transport system, which also carries leucine and glutamine, is augmented by a second, less efficient transporter at low MEL concentrations. In vitro, and potentially in vivo, high concentrations of leucine can protect cells from the cytotoxic effect of MEL by decreasing drug uptake in a competitive manner [172]; on the other hand, enhanced tumor tissue uptake of MEL occurs in experimental systems in vivo during exposure to an amino acid-lowering diet [173]. Resistance to MRL may occur along general lines common for the alkylating agents, which include increased intracellular thiol content [28,174], increased detoxification by way of the GST system [175], and enhanced DNA repair [176]. However, because of the specific transport pathways that exist for MEL, altered drug carrier proteins are also capable of producing significant degrees of resistance in vivo [177]. A specific mutation has been described in L1210 murine leukemia cells which results in lower affinity of the high-velocity transporter for MEL, with associated high-level drug resistance [178]. Metabolism, pharmacokinetics, and pharmacodynamics. MEL undergoes rapid hydrolysis in plasma to its inactive mono- and dihydroxy derivatives; it is also extensively (90%) protein bound [179]. From 5% to 13% of the delivered dose of MEL is excreted in the urine [180]. The penetration of MEL into tumor tissue demonstrates significant variability consistent with variations in perfusion [16]. Despite the development of the intravenous formulation, which is used for high-dose chemotherapy with MEL, inter- and intrapatient pharmacokinetic variability is substantial [99], often in excess of 10-fold with respect to systemic exposure [181]. MEL distribution follows a biexponential decay with a short distribution phase, α half-life, from 5 to 15 minutes, and a subsequent β half-life of 30–120 minutes [182]. There does not appear to be any difference in the clearance of MEL in children compared with adults [183]. Due to the short β phase of elimination, marrow or peripheral blood hematopoietic cell reinfusion following high-dose MEL can occur safely within 12–24 hours following drug treatment. The pharmacokinetics of MEL are linear over a very wide dose range [184]. This fact has allowed the use of a small test dose of MEL for the adaptive control of MEL exposure in individual patients. In this fashion, it has been possible to predict MEL clearance prior to high-dose treatment, and then to choose a dose that produces a target AUC level with less than 15% deviation from the desired systemic

exposure. Ongoing studies will determine whether adaptive control of high-dose MEL will prove essential from a pharmacodynamic perspective. Because of the limited renal excretion of MEL, high-dose therapy has been attempted in patients with multiple myeloma and significantly compromised kidney function (creatinine clearance <40 mL/min); no evidence of alterations in half-life or AUC were demonstrated, although the performance status of such patients may enhance their toxicity profile [181]. The lack of any major effect of renal dysfunction on the clearance of high-dose MEL has been confirmed [185]. The dose-limiting toxicities of high-dose MEL are mucositis and SOS [165]. Gastrointestinal mucosal injury both limits the escalation of drug dose above 200 mg/m2 as a single agent and its use at doses above 140 mg/m2 with other agents that produce severe gastrointestinal tract toxicity. SOS has been observed especially when high-dose MEL has been combined with other high-dose alkylating agents [186]. High-dose MEL, like other alkylating agents, can produce diffuse pulmonary alveolitis [187]; it also can stimulate inappropriate antidiuretic hormone secretion in the high-dose chemotherapy setting [188]. The current role of high-dose MEL therapy is in the treatment of hematopoietic malignancies [189–191], multiple myeloma [181], and neuroblastoma [192]. Its role in combination high-dose chemotherapy regimens for ovarian [193] and breast cancer [186] has also been investigated. Thiotepa The principal role for high-dose thiotepa is in combination with other alkylators for the treatment of patients with hematopoietic malignancies [194]. It has also been investigated in other high-dose combination regimens for patients with metastatic breast cancer [195,196]. The usual dose of thiotepa as part of such treatment programs is 500–800 mg/m2, which is an 8–12-fold increase over standard dose levels. Mechanism of action. Thiotepa (N,N′,N″-triethylenethiophosphoramide) was developed as an aziridine analog of nitrogen mustard [ClCH2CH2N(CH3)CH2CH2-Cl] because the loss of chlorine in the decomposition reaction of nitrogen mustard leads to the formation of a reactive cyclic aziridinium intermediate which produces the initial alkylation product. Alkylation by thiotepa, producing interstrand crosslinking in DNA, occurs after opening of the aziridine rings. While it seems clear that DNA crosslinks play a critical role in the cytotoxicity of thiotepa (or the parent drug’s immediate breakdown product), and that tumor cell killing is enhanced in an aerobic rather than an hypoxic environment [197], issues remain regarding the role of crosslinks versus the production of alkalilabile sites as cytotoxic features of tepa, its major metabolite [198]. Tepa is less cytotoxic and less stable chemically than the parent molecule, as well as being extensively protein bound [199]. The cellular pharmacology of thiotepa, which is highly lipid soluble, is characterized by an initial, rapid diffusion-mediated uptake into tumor cells [200] followed by irreversible binding to intracellular macromolecules. A secondary, much slower, but linear energy-dependent increase in intracellular thiotepa concentration reflects the balance between uptake and binding of a nonexchangeable pool of drug; cells lacking nuclei demonstrate a significantly reduced nondiffusion-mediated drug accumulation rate. Tumor cell resistance to thiotepa, where it has been investigated, appears to follow typical patterns of alkylating agent resistance. Alterations in intracellular thiol (GSH) pools and, in particular, the GST detoxification system may play an important role in the intracellular deactivation of thiotepa [201]. Thiotepa reacts directly with GSH at neutral pH; however, the reaction to form monoglutathionyl thiotepa is significantly increased by both GST α and π but not GST μ. Tepa also

Pharmacologic Basis for High-dose Chemotherapy

reacts readily with GST α and π, suggesting that the aziridine moieties of these molecules are the functional substrates. Since increased GST isoenzyme expression has been demonstrated previously to play an important role in the development of resistance to other alkylating agents that are GST substrates [202], it is likely that GST-mediated detoxification could contribute to the insensitivity of malignant cells to thiotepa. Experiments have also demonstrated that overexpression of a member of the glycosylase family of DNA repair proteins (formamidopyrimidine-DNA glycosylase) is specifically capable of removing the aziridine alkylation products of thiotepa in DNA (N7-aminoethyl guanine and aminoethyl adenine); repair of these lesions produces resistance to both the cytotoxic and mutagenic effects of thiotepa. In addition to providing a potential resistance mechanism, these studies suggest that ring-opened guanines contribute to the mechanism of action of the drug [203]. Other base excision-repair gene products also may contribute to thiotepa resistance [204]. Metabolism, pharmacokinetics, and pharmacodynamics. Thiotepa is extensively metabolized in the liver [205]; it is oxidatively desulfurated to tepa (triethylenephosphoramide) by the hepatic microsomal P450 system, in particular by CYP2B1 (the major phenobarbital-inducible P-450), CYP2C11 (a male-specific, constitutive isoenzyme), as well as CYP3A4 and, to a minor extent, CYP2B6 [206]. In murine species, phenobarbital pretreatment can block tumor growth delay after thiotepa treatment, consistent with increased conversion to the less cytotoxic tepa. The elimination of thiotepa occurs both by microsomal metabolism and tissue alkylation [207]. Experiments have demonstrated that thiotepa is a specific and potent inhibitor of CYP2B6, the P-450 isoform that plays an important role in the 4-hydroxylation of CY [208]. This observation is of real significance because clinical studies have shown that 4-hydroxycyclophosphamide levels are significantly diminished soon after the initiation of a thiotepa infusion if thiotepa and CY are administered concurrently [209]. Alterations in the activation of CY produced by thiotepa could substantially diminish the therapeutic benefit of highdose CY, suggesting that thiotepa should be delivered after the completion of any CY infusion. On the other hand, no clinically important effects on thiotepa metabolism have been demonstrated for CY or carboplatin [210]. The pharmacokinetics of high-dose thiotepa have been studied in detail [211,212]. The drug is cleared following a biexponential decay pattern with an α half-life of 10 minutes and a β half-life of 60–200 minutes. The apparent terminal elimination of tepa is in excess of 8 hours; however, because of the extensive protein binding of tepa, the importance of this prolonged elimination is unclear. It also remains uncertain whether tepa clearance is saturable. In children, thiotepa does not appear to present the problem of nonlinear pharmacokinetics; however, this observation has been reported by some laboratories in the adult population, but not others [212]. Both thiotepa and tepa have been detected in the urine after high-dose therapy; in the 48 hours following drug administration, approximately 10% of the administered dose was quantitated in urine as tepa or thiotepa mercapturic acid derivatives, and less than 1% as the parent drug [213]. Although a potential method for the adaptive control of thiotepa dosing has been proposed, it has not yet been reduced to clinical practice [214]. The two major dose-limiting toxicities of high-dose thiotepa are in the central nervous system and gastrointestinal tract [215]. High-dose thiotepa (≥900 mg/m2) as a single agent can produce somnolence, coma, and confusion. At single-agent doses of over 720 mg/m2, severe oral and esophageal mucositis as well as enterocolitis become significant problems. Other important side-effects include transient elevations of hepatic enzymes (related to the total systemic exposure to thiotepa and tepa, which may contribute to the development of SOS when thiotepa is used

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in combination with other high-dose alkylating agents), an acute erythroderma associated with maculopapular dissemination and desquamation, interstitial pneumonitis, and cardiac toxicity [138,199,216,217]. These side-effects are relatively specific for the high-dose setting and must be considered in the development of combination high-dose regimens, in particular with drugs that have the potential to produce overlapping extramedullary toxicities. Examples of thiotepa-containing combinations used in the high-dose setting include thiotepa and CY; thiotepa, CY, and TBI [218]; thiotepa, cisplatin, and CY [219]; thiotepa, MEL, and CY [99]; thiotepa, topotecan, and carboplatin [220]; and thiotepa, mitoxantrone, and etoposide [221]. Busulfan BU, which is one of the alkylating agents in longest use, continues to play an important role as a myeloablative agent in combination preparative regimens for patients with hematopoietic malignancies undergoing allogeneic or autologous BMT, where the dose employed (16 mg/kg) is increased eight- to ninefold higher than standard dose levels [222,223]. Mechanism of action. BU is an alkyl alkane sulfonate (Fig. 21.1); unlike nitrogen mustard, the alkylation pattern of BU involves a bimolecular displacement reaction rather than the initial formation of a reactive intermediate, and is thus less rapid. This difference in reaction sequence does not, however, explain the profound effect of BU on myeloid precursors as well as CML and certain myeloproliferative disorders compared with other alkylating agents [224]. BU clearly produces DNA–protein crosslinks, which may play a role in its mechanism of cytotoxicity; in some tumor cell models, DNA–protein crosslinking is much more prominent than DNA interstrand crosslink formation [225]. The drug is also profoundly mutagenic and can produce a wide range of chromosomal aberrations [226]. BU can be conjugated with GSH by GST α to a much greater extent than by the μ or π isoenzymes; overexpression of the α isoform may contribute to BU resistance both in vitro and in vivo [89]. Alterations in the repair of DNA crosslinks have also been associated with BU resistance, as might be expected from a member of the alkylator class [227]. Finally, BU resistance in human tumor cell xenografts has also been associated with alterations in drug transport [228]. Metabolism, pharmacokinetics, and pharmacodynamics. BU is highly lipophilic and demonstrates minimal protein binding; the parent molecule is eliminated principally by the liver and through tissue alkylation, with less than 5% of an administered dose excreted unchanged in the urine [229]. The tissue distribution of BU has been studied in primates, where drug levels are highest in liver; BU crosses the blood–brain barrier and reaches the substance of the brain at levels approximately 11% of those in the liver [230]. Although only a minor portion of BU is excreted into the urine, BU can be cleared in part by hemodialysis; however, multiple dialysis procedures are required to diminish systemic exposure to the drug [231]. The elimination half-life of BU in adults ranges from 1 to 7 (mean 3) hours; peak plasma levels are found after oral dosing in 1–3 hours [232]. Over the typical 4-day course of oral administration, however, steadystate concentrations after the last dose are significantly lower than those predicted from the AUC measurements following the first dose, suggesting that BU induces its own metabolism [229]. In children, the mean elimination half-life is 2 hours or less, and no significant change in clearance has been demonstrated from first to last dose [233]. Differences in the pharmacokinetics of BU in children undergoing high-dose chemotherapy compared with adults consistently include lower peak drug levels, faster elimination and clearance, and a larger apparent

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volume of distribution [234,235]. Wide inter- and intrapatient variability in BU disposition after oral administration has been observed in both adults and children [100,101], due in part to age, altered hepatic function, drug interactions, circadian rhythmicity, and bioavailability [236– 238]. In adults, oral bioavailability varies over a twofold range; the degree of variation is up to sixfold in children. Further, the acute emetic events that may occur during oral BU dosing further complicate the use of the oral route of administration [239]. Over the past decade, the importance of BU pharmacodynamics has been increasingly recognized. As discussed earlier in this chapter, the dose-limiting toxicity of BU in the setting of high-dose chemotherapy with hematopoietic cell support for the treatment of hematologic malignancies is SOS of the liver [111]. Adaptive control of BU dosing based on the plasma AUC or steady-state concentration of the drug after the initial dose(s) to produce a target level can significantly reduce the incidence of SOS. As a result, TDM of BU in the high-dose chemotherapy setting is now a standard part of treatment, even though the etiology of SOS is complex and is likely to involve other factors in addition to the AUC of BU [236]. In addition to SOS, the dose-limiting toxicities of high-dose BU include neurotoxicity characterized by seizures, mucositis, the potential for radiation recall phenomenon, and pulmonary fibrosis. The gonadal toxicity of high-dose BU, further, is extensive; essentially no return of ovarian function can be expected following typical high-dose BU/CY conditioning. Drug–drug interactions involving BU include the observation that the antifungal agent itraconazole can decrease the clearance of BU by up to 20%, probably through hepatic microsomal cytochrome P-450-mediated interactions [240]; in similar fashion, patients treated with diphenylhydantoin to prevent central nervous system toxicity from high-dose BU have significantly lower AUCs and shorter elimination half-lives for BU. Diazepam, used for the same purpose, does not alter BU disposition [107]. Finally, administration of high-dose BU prior to high-dose CY significantly enhances the conversion of CY to 4-hydroxycyclophosphamide; this interaction is especially important in light of the frequency with which BU and CY are combined as conditioning agents prior to HCT; it provides a further level of understanding for the degree of interpatient variability in the pharmacokinetics of both drugs in the highdose chemotherapy setting [106]. Studies have suggested that if CY administration is delayed until at least 24 hours after BU, at least some of the metabolic interactions between the two drugs may be eliminated [241].

produces interstrand and intrastrand DNA crosslinks as well as less cytotoxic DNA–protein crosslinks [243]. Breakdown products of BCNU produce isocyanates, which bind sulfhydryl and amino groups on proteins, inhibiting such enzymes as glutathione reductase and DNA ligase. The lipophilicity of BCNU allows it to enter tumor cells by passive diffusion. Resistance to BCNU follows several of the known pathways for alkylating agents; in particular, enhanced intracellular thiol levels [244] and glutathione transferase activity [152] have been associated with BCNU resistance. However, most efforts to modulate clinical resistance to BCNU have focused on the mammalian enzyme O6-alkylguanineDNA alkyltransferase, which is the specific DNA repair protein that removes alkylated guanines produced by BCNU in DNA [245,246]. A specific inhibitor of this enzyme, O6-benzyl guanine, has now become available for clinical testing; in vitro and in murine models, it has been demonstrated to markedly enhance the cytotoxicity of BCNU [247]. Metabolism, pharmacokinetics, and pharmacodynamics. In addition to spontaneous decomposition, BCNU may undergo denitrosation by hepatic microsomes, probably by glutathione transferases [152]. Phenobarbital-induced microsomes decrease the therapeutic effect of the nitrosoureas and increase drug clearance. BCNU undergoes a biexponential decay with an α half-life of 5–10 minutes and a β half-life of 10–30 minutes; it is highly (>75%) protein bound [248]. Significant interpatient variability in BCNU clearance has been observed. Approximately 80% of the administered dose is excreted in the urine as metabolites. Both CY and cisplatin, the two drugs with which BCNU is most frequently combined in the high-dose chemotherapy setting, increase the AUC and pharmacokinetic variability of BCNU in model systems [249]. This pharmacokinetic variability may help to explain the pulmonary toxicity of the CY, cisplatin, and BCNU high-dose regimen that has been correlated in the clinic with the AUC of BCNU [250]. This potentially severe pulmonary toxicity, which may be related to the depletion of pulmonary GSH levels, frequently requires corticosteroid management and may be fatal [251]. In addition to pulmonary toxicity, high-dose BCNU therapy has been associated with an increased risk of SOS [252], hypotension and myocardial ischemia [253,254], delayed renal toxicity after cumulative doses above 1200 mg/m2 [11], and rarely, encephalopathy. Platinating agents

BCNU

Cisplatin

BCNU (bis-chloroethyl-nitrosourea) is a lipophilic bifunctional alkylating agent linked to a nitrosourea moiety. It was originally developed in the early 1960s because of its ability to cross the blood–brain barrier in murine species. BCNU remains in active use for the treatment of primary central nervous system malignancies at standard doses of 200 mg/m2, which can be increased to 300 mg/m2 when combined with BCNU, etoposide, cytarabine, and CY (BEAC), and BCNU, etoposide, cytarabine, and MEL (BEAM) for the treatment of patients with relapsed or refractory lymphoma [242]. Doses up to 600 mg/m2 have been given when the drug has been used in combination with high-dose CY and cisplatin and hematopoietic cell support for patients with solid tumors [12].

The serendipitous finding that platinum salts, produced by the effect of an electric current passed through a platinum electrode in growth medium, were toxic to bacteria led to the discovery that the platinum complex which was produced had significant antineoplastic activity [255]. The prototype platinum coordination complex is the drug cisplatin (cis-diamminedichloroplatinum II), which continues to play a critical role in the curative therapy of testicular germ cell tumors [256] and ovarian cancer [257], as part of combination salvage therapy for relapsed and refractory Hodgkin’s and non-Hodgkin’s lymphoma [258,259], and in the palliative care of patients with breast, lung, and head and neck cancer [260–262]. Because of its therapeutic spectrum, the use of highdose cisplatin has primarily been investigated as part of the STAMP-I high-dose chemotherapy regimen (including CY, cisplatin, and BCNU) for patients with either responsive metastatic or high-risk primary breast cancer [12,13,263]; High-dose cisplatin has also been combined with high-dose CY and MEL [264], CY and thiotepa [265], and CY and etoposide [146] for a variety of solid tumors in conjunction with autologous bone marrow or stem cell support. The usual dose range for

Mechanism of action. BCNU undergoes spontaneous hydrolysis at physiologic pH in plasma or intravenous solutions, and thus must be utilized quickly after mixing. This spontaneous decomposition leads to the formation of the chloroethyl carbonium ion that is capable of alkylating macromolecules [11]. Bifunctional, stepwise alkylation of DNA

Pharmacologic Basis for High-dose Chemotherapy

cisplatin in these treatment programs is from 150 to 250 mg/m2 (approximately threefold standard levels), delivered either as a 72–96-hour continuous infusion or as a divided dose delivered over 1 week. Mechanism of action. The chloride groups on cisplatin, shown in Fig. 21.1, are labile [266], reacting in solution to form an aquated species in which the chloride molecules are replaced by water. Conversion to this more reactive species is facilitated by the significantly lower intracellular chloride concentration compared with that in plasma. The reactive aquated molecule can then undergo displacement reactions analogous to the alkylating agents to produce covalent crosslinks with a variety of macromolecules including DNA [267,268]. Tumor cell killing by cisplatin can be correlated with the formation of interstrand crosslinks as well as specific intrastrand lesions. DNA binding also decreases the efficiency of specific DNA repair enzymes [266]. The mechanisms that have been described to explain tumor cell resistance to cisplatin fall into three broad categories: decreased drug uptake, increased intracellular binding (and thus sequestration) of activated platinum species to thiols, and altered DNA interactions with or without changes in signal transduction [269]. In vitro, cell lines with acquired defects in cisplatin uptake have been well described; however, no clear evidence for a specific platinum efflux pump has been developed [270]. Several laboratories have demonstrated that alterations in intracellular thiol status significantly affect platinum cytotoxicity. Overexpression of the metallothionein gene [271], as well as its upregulation by heavy metal pre-exposure, decreases tumor cell sensitivity to cisplatin, probably by providing enhanced access to this sulfhydryl-rich protein. Further, acquired resistance to cisplatin has been associated with enhanced GSH synthesis in human ovarian and lung cancer cell lines [272,273] and can be at least partially reversed by GSH depletion [28]. In addition to altered drug accumulation or enhanced thiol-dependent sequestration (both leading to diminished nuclear platinum concentrations), resistance to cisplatin may also result from decreased binding of platinum to DNA or chromatin, or enhanced repair of platinum–DNA adducts [274–276]. The steady-state level of platinum–DNA adducts in peripheral blood mononuclear cells has been correlated with the response to platinum-containing chemotherapy regimens [277,278]. In addition to increased DNA repair rates, platinum resistance has also been associated with altered transcription factor activation, in particular diminished c-jun activation after cisplatin exposure, with a concomitant decrease in drug-related apoptosis in resistant cells [279]. Metabolism, pharmacokinetics, and pharmacodynamics. The clearance of cisplatin is primarily through endogenous inactivation via binding to biologic macromolecules, including protein sulfhydryls, and renal excretion. Cisplatin is extensively bound to plasma proteins. Its multiexponential clearance involves the initial disappearance of free or unbound drug in approximately 30–45 minutes, and the prolonged decay of protein-bound cisplatin over a terminal half-life in excess of 24 hours [280]; interpatient pharmacokinetic variability is substantial [102]. The excretion of cisplatin is almost exclusively by a process of renal tubular filtration, secretion, and reabsorption [281]; the clearance of cisplatin is proportional to the GFR. However, other data demonstrate clearly that, even with appropriately vigorous saline diuresis, there is extensive and long-term binding of cisplatin to the renal tubules; measurable platinum species have been detected in urine for as long as 8 years after treatment with the drug [282]. Hence, the use of high-dose cisplatin requires that patients enter treatment with normal renal function. Several interactions between cisplatin and other agents employed during high-dose chemotherapy have been described. Antiemetics employed during high-dose cisplatin therapy may affect drug clearance. Ondansetron appears to decrease the AUC of cisplatin, while prochlor-

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perazine may increase plasma levels of cisplatin in the high-dose chemotherapy setting [283,284]. In preclinical model systems, prior TBI has been demonstrated to diminish cisplatin renal clearance [285]. Inhibition of cisplatin secretion by the renal tubule with probenecid appears to protect the kidney from cisplatin toxicity after high-dose (up to 160 mg/m2) treatment [286]. High-dose cisplatin does not appear to alter the pharmacokinetics of high-dose MEL or high-dose etoposide [287,288]. The maximally tolerated intravenous dose of cisplatin during combination high-dose chemotherapy with hematopoietic cell support is a total dose of 250 mg/m2 administered in two fractions 1 week apart [146]. The dose-limiting toxicity of high-dose cisplatin is renal dysfunction, including significant electrolyte wasting; hence, great care must be taken to ensure that a continuing vigorous diuresis occurs before, during, and after high-dose platinum administration [289]. In addition, high-dose cisplatin is frequently associated with ototoxicity characterized by highfrequency hearing loss, especially above 2000 Hz [146,286,289]. Peripheral neuropathy, even at these high cisplatin doses, has been reversible and not dose-limiting in most studies, probably because the majority of patients treated (who suffered from breast cancer) had little or no prior cisplatin exposure. Nausea and vomiting from high-dose cisplatin-containing regimens can be managed well with 5-hydroxytryptamine-3 antagonists. The ability to deliver the full 250 mg/m2 of cisplatin during the HCT course was found to correlate favorably with relapse-free survival in women treated with high-dose cisplatin, CY, etoposide, and autologous stem cells for high-risk primary breast cancer [290]. Carboplatin Carboplatin (diammine 1,1-cyclobutanedicarboxylatoplatinum II) is an analog of cisplatin in which the two chloride ligands are replaced by the carboxylate moiety. Carboplatin has become a major component of high-dose chemotherapy regimens for the treatment of advanced testicular [291,292], ovarian [293], and breast [195] cancer. The DNA crosslinking species formed by carboplatin are identical to those formed with cisplatin, but the pharmacokinetics and spectrum of toxicity of this analogue are different. Metabolism, pharmacokinetics, and pharmacodynamics. Carboplatin disappearance is due, almost exclusively, to renal excretion; the drug is much less extensively protein bound (18–30%) than cisplatin [294]. The disappearance of carboplatin from plasma is characterized by a triexponential decay with an α half-life of approximately 20 minutes, a β half-life of about 1.5 hours, and a γ half-life of approximately 7.5–20 hours. Essentially all unbound platinum species have been cleared from the plasma within 24 hours after the completion of a high-dose carboplatin infusion [295]. Because there is minimal active tubular secretion of carboplatin, its clearance can be predicted from various formulae that utilize estimates of the GFR [296,297]. However, studies suggest that clearance estimates utilizing limited sampling schedules rather than measured creatinine clearance much more accurately reflect the actual, delivered AUC of carboplatin in the setting of high-dose chemotherapy with autologous hematopoietic cell reinfusion [298,299]. Over a substantial range of carboplatin dosage, clearance appears to be linear (50–70 mL/min), although there is significant interpatient variability, especially when high-dose carboplatin is used in combination high-dose regimens [300]. Further, the pharmacokinetic parameters defining highdose carboplatin disappearance from plasma are similar whether the drug is administered as a short (1-hour) or continuous (96-hour) infusion [294]. Several studies have also demonstrated that carboplatin species can be removed from the plasma by hemodialysis [301,302]. Potential drug–drug interactions have been defined for high-dose carboplatin; however, certain conflicting reports cloud interpretation of the

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data. High-dose carboplatin may affect clearance of MEL [303], but not that of paclitaxel [304], and paclitaxel does not impair the clearance of carboplatin [305]. High-dose carboplatin has been reported either to have no effect [306] or to significantly decrease [307] the clearance of high-dose etoposide. The maximally tolerated dose of carboplatin with hematopoietic cell support is 1600–2400 mg/m2 when the drug is used as a single, and 800–1200 mg/m2 in combination high-dose chemotherapy regimes [195,308,309]. While the drug produces minimal hepatic, renal, mucosal or ototoxicity or peripheral neuropathy when used at the standard dose, there is no question that dose-limiting liver toxicity, mucositis, and peripheral neuropathy are observed in the high-dose chemotherapy setting [310,311], where the AUC of the drug is correlated with these side-effects when it is used in combination with etoposide. In combination with high-dose thiotepa and CY, cumulative carboplatin AUC has been correlated with the development of ototoxicity [113]. Further, there is little doubt that when it is used at high dose, especially in patients with prior cisplatin exposure, carboplatin may produce significant nephrotoxicity. Despite these toxicities, the dose intensity of carboplatin in the setting of high-dose chemotherapy can be safely increased four to six times with hematopoietic cell support, compared with threefold for cisplatin, making it a valuable agent for the treatment of those diseases in which the therapeutic activity of carboplatin and cisplatin are equivalent. Topoisomerase II inhibitors and agents with pleiotropic mechanisms of action Etoposide Podophyllotoxin derivatives from the mandrake plant have been known for many years to possess antiproliferative effects [312]. Etoposide is the podophyllotoxin derivative most commonly used in both standard clinical oncologic practice and as a component of many high-dose chemotherapy regimens for hematopoietic malignancies [313,314], germ cell cancer [291,292], and other solid tumors [146,315,316]. The usual dose range for etoposide in the BMT setting is approximately 25–60 mg/ kg delivered either as a single dose or in divided doses, often over 3 days, an approximate three- to sixfold increase over standard therapy. Etoposide has been utilized extensively in the high-dose chemotherapy setting because of both its therapeutic activity and its relatively modest spectrum of extramedullary side-effects. Mechanism of action. The most thoroughly evaluated mechanism of tumor cell killing by etoposide has been its interaction with the nuclear enzyme topoisomerase II, which facilitates the uncoiling of DNA prior to DNA replication [317–319]. Topoisomerase IIα is a 170 kD dimer that is normally responsible for producing the transient DNA singlestrand breaks that are necessary for relieving torsional strain in DNA during transcription, replication, and repair. This is an ATP-dependent process [320] that is inhibited by the interaction of etoposide with the enzyme, preventing re-ligation of DNA. With the formation of a cleavable complex between topoisomerase II and etoposide, the enzyme is trapped prior to DNA ligation; in the presence of protein-digesting enzymes, protein-associated DNA strand breaks become apparent. When etoposide-related cleavable complex formation is decreased through competition with other catalytic inhibitors of topoisomerase II, cytotoxicity is diminished [321,322]. Although there is little question about the formation of topoisomerase II-mediated DNA damage after etoposide exposure in vitro, cleavable complex formation is clearly not sufficient in itself to explain the cytotoxicity of etoposide [323]. Since topoisomerase II-mediated DNA damage from etoposide is rapidly reversible, has not yet been convinc-

ingly demonstrated in vivo [324], and produces mainly single- rather than the more lethal double-stranded DNA scission, other mechanisms may contribute to the cytotoxic effects of etoposide. Etoposide can be metabolized to a free radical species [325] that enhances macromolecular binding of the drug [326] and may contribute to DNA damage and apoptosis [327,328]. Etoposide has been demonstrated to produce caspase activation leading to programmed cell death [329–332], as well as cleavage of poly-ADP ribose polymerase [333–335] that may, in part, be mediated by p53 [336]. It appears, further, that binding of etoposide to topoisomerase II is not itself critical for the induction of apoptosis [337]. Thus, the etoposide–topoisomerase II interaction may be viewed as producing only potentially lethal damage to the tumor cell. Tumor cell resistance to etoposide can be produced by several different mechanisms [338,339], including alterations in drug uptake and efflux, as well as changes in either the amount or binding affinity of topoisomerase II. Etoposide is a substrate for the P-glycoprotein, and overexpression of the MDR1 gene product leads to both decreased uptake and enhanced efflux of etoposide [340,341]. Inhibitors of P-glycoprotein have been employed to enhance the therapeutic activity of etoposide clinically [342,343]. However, acquired etoposide resistance may also be due to increased drug efflux that is related to the overexpression of the multidrug resistance-associated protein rather than MDR1 [344–346]. In certain subclasses of acute myelogenous leukemia, multidrug resistance-associated protein, rather than MDR1, expression has greater prognostic significance [347]. In addition to drug transport, the level of topoisomerase II protein or its specific activity significantly affects the tumor cell sensitivity of etoposide. Decreased drug-related cleavable complex formation may be due to lower absolute levels of topoisomerase II, to mutations in the enzymes that affect drug binding, or to the degree of topoisomerase II phosphorylation [27,346,348]. Further, evidence for the co-evolution of these resistance phenotypes (e.g. the overexpression of multidrug resistance-associated protein that occurs synchronously with alterations in topoisomerase II level) has been demonstrated [346]. Finally, as previously noted, etoposide exposure may initiate the programmed cell death cascade [330]; hence, intrinsic or acquired overexpression of specific genes participating in apoptosis may lead to etoposide resistance [349– 351]. Metabolism, pharmacokinetics, and pharmacodynamics. Etoposide is principally metabolized in the liver to its glucuronide [352]; approximately one-third of the drug is excreted by the kidney as the unchanged parent molecule, and another 10–20% as etoposide-glucuronide. Because renal clearance is increased in the presence of hyperbilirubinemia or liver metastases, no adjustment of etoposide dose is required in the presence of hepatic dysfunction [352–354]. However, the systemic clearance of etoposide is closely related to creatinine clearance [355]. Because etoposide dosage must be reduced in the presence of renal dysfunction, high-dose etoposide therapy should not routinely be considered in patients suffering from even a modest degree of kidney failure. Further, caution must be exercised when high-dose etoposide is delivered immediately after high-dose cisplatin, since acute cisplatin exposure can reduce the systemic clearance of etoposide by approximately 25%, leading to a substantially greater than expected AUC of etoposide and toxicity level [356]. High-dose carboplatin exposure, on the other hand, may not alter the disposition of high-dose etoposide [306]. The pharmacokinetics of etoposide have been studied over the entire range of intravenous dosing, from 0.1 to 3 g/m2. Etoposide, which is highly protein bound, has a biexponential decay with a mean terminal half-life after high-dose therapy of between 4 and 8 hours [357]; even when delivered at high dose (30–60 mg/kg), essentially all detectable drug has disappeared from the plasma in 36–48 hours [313]. Changes

Pharmacologic Basis for High-dose Chemotherapy

in etoposide dose over its entire therapeutic range produce linear changes in associated plasma AUC [306,357–359]. The linear pharmacokinetics of etoposide disposition, despite significant interpatient pharmacokinetic variability [98], make its toxicity profile predictable and potentially amenable to adaptive control strategies capable of targeting specific etoposide systemic exposures through the use of therapeutic drug level monitoring [114]. In addition to its potential pharmacodynamic interaction with cisplatin, the clearance of etoposide is increased by approximately one-third (with a concomitant decrease in AUC) in patients treated with BU and phenytoin before high-dose etoposide, compared with patients who are receiving TBI and etoposide [360]. Whether this alteration in clearance is due to the effect of phenytoin itself on the hepatic metabolism of etoposide or the combined interaction of phenytoin and BU on liver microsomal enzyme systems is unknown. Finally, it has been shown that the emulsifying agent Cremophor EL, used to solubilize paclitaxel, significantly decreases the total body clearance of etoposide [361]. In light of the pharmacokinetic interactions that have been shown to occur between paclitaxel and doxorubicin due to this emulsifier [362], significant care will be required in the development of the schedules of high-dose chemotherapy programs combining etoposide with paclitaxel to avoid potentially severe levels of toxicity. The toxicity profile of high-dose etoposide is, in part, related to the drugs with which it is combined during HCT [146,192,363]. However, even when etoposide is administered as a single agent [364], mucositis has clearly been demonstrated to be dose limiting, which has an important effect on the ability to combine high-dose etoposide with other drugs whose dose-limiting extramedullary toxicity is also focused on the gastrointestinal tract [221,363]. Anthracyclines The anthracycline antibiotic doxorubicin, isolated from Streptomyces species over 30 years ago [365], has a wide spectrum of antineoplastic action and is a major component of curative combination chemotherapy regimens for hematopoietic malignancies, as well as for breast and ovarian cancer and many childhood solid tumors [366,367]. Doxorubicin is one of the few antineoplastic agents with significant clinical utility for which a clear dose–response relationship (including doses severalfold greater than those traditionally used) has been demonstrated in humans [142,368,369]. High-dose doxorubicin has been used in combination with high-dose CY or high-dose etoposide, or both, and HCT for the treatment of patients with responsive metastatic or high-risk primary breast cancer [363,370–372]. Mechanism of action. Doxorubicin interacts pleiotropically with intracellular organelles and alters the biochemistry of the cell surface, the mitochondrion, many signal transduction pathways, as well as the nuclear replication apparatus [373–375]. Doxorubicin exerts its antiproliferative effects through each of the following mechanisms: binding to the nuclear enzyme topoisomerase II to form a cleavable complex, which interferes with the ability of this enzyme to reduce the torsional strain in DNA that occurs during mitosis; the generation of reactive oxygen species (including the hydroxyl radical), which damage the mitochondrial electron transport chain and hinder cellular energy production, as well as producing DNA base oxidation; and the activation of signal transduction pathways ultimately leading to programmed cell death [376–378]. As might be expected from an agent with such a wide repertoire of mechanisms of cell kill, drug resistance both in vitro and in vivo may involve alterations in a variety of cytotoxic pathways. Prominent among the resistance pathways that have been described are alterations in doxorubicin uptake and enhanced cellular efflux modulated by the overex-

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pression of one or more of several drug efflux pumps including P-glycoprotein, multidrug resistance-associated protein, lung-resistance protein or breast cancer resistance protein, the presence of which have all been described as conferring an adverse prognosis in anthracyclineresistant hematopoietic malignancies [33,347,379,380]. Metabolism, pharmacokinetics, and pharmacodynamics. Doxorubicin is principally metabolized in the liver either by flavin dehydrogenases, which catalyze the one-electron reduction of its quinone moiety with the ultimate formation of an aglycone species that is more easily conjugated for biliary export, or by aldo-keto reductases, which reduce the drug’s carbonyl side chain leading to a less cytotoxic species [381– 384]. Only about 60% of the elimination of doxorubicin can be accounted for even when the minor amount of renal clearance is included; it is likely that much of the remaining drug is bound to DNA and lipid membranes from which it is slowly released. It is not surprising, therefore, that the pharmacokinetics of doxorubicin demonstrate triexponential decay with a long terminal half-life in plasma of approximately 12–20 hours for both the parent drug and its side-chain alcohol metabolite doxorubicinol [385]. Because of the drug’s predominant hepatic metabolism, individuals with liver dysfunction may exhibit considerably enhanced anthracycline toxicity because of delayed drug clearance [386]. Of particular importance for the use of doxorubicin in high-dose chemotherapy, even doses between 150 and 165 mg/m2 delivered over 96 hours by continuous intravenous infusion (which are approximately threefold higher than standard treatment programs) produce steady-state plasma concentrations 10-fold lower than the peak drug levels observed after a bolus administration of 50–60 mg/m2 of doxorubicin [387]. Furthermore, as shown in Fig. 21.3, over a broad range of doses the pharmacokinetics and metabolism of doxorubicin are linear, suggesting that drug activation and detoxification pathways are not exceeded when the doxorubicin dose is escalated into the high-dose chemotherapy range. The toxicity of doxorubicin of particular relevance to the high-dose chemotherapy setting, other than cardiac toxicity, is dose-limiting damage to the oral and gastrointestinal mucosa, resulting in reversible but potentially severe stomatitis and diarrhea [363]. A reversible typhlitis, which is managed conservatively with parenteral nutrition, analgesics, and careful attention to fluid and electrolyte balance, may be observed in approximately 5% of patients receiving high-dose chemotherapy combinations including doxorubicin or etoposide, or both. This syndrome is self-limiting, should not require surgical intervention, and resolves rapidly as hematopoietic function recovers. As noted previously, the unique toxicity of doxorubicin is its cumulative, dose-dependent myocardial damage. Studies that have employed either gated cardiac blood pool scanning or endomyocardial biopsy as endpoints for functional or histologic confirmation of doxorubicin cardiac toxicity have demonstrated that the incidence of measurable heart damage begins to climb precipitously above a cumulative dose of 350–400 mg/m2 (rather than the traditional figure of 450–500 mg/m2) if the drug has been administered by short intravenous infusion. In patients for whom high-dose chemotherapy will include doxorubicin at a dose of 150 mg/m2 or higher administered by 96-hour continuous infusion, studies utilizing endomyocardial biopsy suggest that the maximum prior bolus doxorubicin exposure should not exceed 200 mg/m2 [370]. However, in individuals who have a long history of hypertensive heart disease or higher previous cumulative doxorubicin exposures, the safety of high-dose doxorubicin remains to be demonstrated. Taxanes Paclitaxel is the lead compound in the taxane class of antimicrotubule agents. Originally isolated from the bark of the Pacific yew, Taxus

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brevifolia, it is now available from semisynthetic and totally synthetic approaches [388,389]. Paclitaxel has a wide spectrum of antineoplastic activity, including breast, lung, and ovarian cancer, for which it is among the most active drugs currently available [390–393]. As in the case of doxorubicin, the use of high-dose paclitaxel followed by HCT has primarily been limited to the treatment of solid tumors. However, the nonlinear nature of paclitaxel pharmacokinetics provides an important illustration of the importance of understanding the pharmacology of an agent before significantly increasing the administered dose. Mechanism of action. Paclitaxel has a unique mechanism of tumor cell killing. After binding to the β subunit of tubulin, paclitaxel inhibits the dissolution of microtubules, upsetting the dynamic balance between microtubular formation and dissolution upon which many intracellular processes are dependent [394–397]. The net effect of enhanced tubulin polymerization is to produce a block in the metaphase of mitosis, leading to growth inhibition. However, paclitaxel has also been shown to diminish cell-cycle traverse in the nonmitotic phases of cell proliferation [396], which may be due to the effects of tubulin polymerization on both the cytoskeleton and signal transduction pathways. Paclitaxel also enhances the cytotoxic effects of ionizing radiation at concentrations that are achievable in the clinic [398,399]. Other important biochemical effects of paclitaxel in tumor cell systems include the activation of the nuclear factor-kappa B nuclear transcription factor and the phosphorylation of bcl-2, both of which may affect paclitaxel-induced programmed cell death [400,401]. By a sequence of events that remains to be completely characterized, paclitaxel exposure leads to apoptosis in a wide variety of tumor cell types [402–404]. Nonapoptotic cell killing also occurs [405]. One of the characteristic features of paclitaxel-related cytotoxicity is that it is independent of p53 status, which may help to explain the drug’s broad therapeutic range [406]. Two distinct mechanisms of paclitaxel resistance have been described. In vitro, paclitaxel-resistant tumor cells overexpress specific isoforms of β-tubulin [407]; further, in human ovarian tumors, increased expression of class III and class IVa β-tubulin isotypes has been demonstrated in ascites tumor cells from patients clinically resistant to paclitaxel compared with untreated patient samples [408]. Paclitaxel is also a substrate for P-glycoprotein; resistance in vitro mediated by enhanced drug efflux has been shown for both hematopoietic and solid tumors [397,404,409]. Similar to other known substrates for the MDR1 gene product, paclitaxel resistance can be modified by a variety of drug classes that decrease chemotherapeutic drug efflux [410,411]. The role of P-glycoprotein expression in modulating paclitaxel resistance in the clinic is under active investigation. Metabolism, pharmacokinetics, and pharmacodynamics. Paclitaxel is cleared primarily by the cytochrome P-450 system in the liver [412] and excreted, in part unchanged, in the bile. 6α-Hydroxypaclitaxel is the major hepatic microsomal metabolite found in both plasma and bile; it is formed through the action of the CYP2C isoform of cytochrome P450. Other, less abundant byproducts that result from hydroxylation of the lateral chain are due to metabolism by the CYP3A4 isoform of cytochrome P-450. Neither cimetidine, ranitidine, nor diphenylhydr-

amine affects paclitaxel metabolism; however, induction of CYP3A isoforms by barbiturates has been shown in vitro to affect the biotransformation of paclitaxel to less cytotoxically active species. Because of its extensive microsomal elimination, alterations in hepatic function play a major role in the elimination of paclitaxel [413,414]. Since even modest abnormalities in liver function can alter the pharmacokinetics of paclitaxel, patients receiving high-dose chemotherapy regimens containing paclitaxel must have normal hepatic function prior to treatment. The pharmacokinetics of paclitaxel when administered over 3 or 24 hours demonstrate a bi- or triexponential elimination with a prolonged γ phase of approximately 15 hours [415]. Several pharmacokinetic studies have detailed the saturability of paclitaxel elimination, which is most prominent with short administration times or with increasing dose levels [105,416,417]. The saturable distribution and elimination of paclitaxel is likely due to the effect of the Cremophor EL vehicle in which paclitaxel is solubilized [362,418]. The nonlinear pharmacokinetic profile of paclitaxel has important clinical ramifications. As shown in Fig. 21.4, for patients receiving high-dose paclitaxel over 24 hours, peak plasma concentrations clearly become nonlinear at doses above 450 mg/ m2; furthermore, the range of peak plasma concentrations observed at individual dose levels also widens as the dose is increased. Thus, a three-fold increase in dose from 250 to 750 mg/m2 leads to a 15-fold increase in peak plasma concentration and AUC. The disproportionate change in observed systemic exposure may lead to an unexpected degree of toxicity if it is not anticipated. The major, dose-limiting features of high-dose paclitaxel therapy are the development of significant peripheral neuropathy [419] and mucositis [420,421], the severity of which has been correlated with the observed AUC for paclitaxel [420]. At paclitaxel doses of 725–750 mg/ m2 administered over 24 hours, however, in combination with either high-dose CY and cisplatin or high-dose CY and doxorubicin and hematopoietic cell support, the level of these toxicities has been acceptable [420,422].

Conclusion Because the toxicity profile and pharmacokinetic properties of the commonly used antineoplastic agents frequently change when these drugs are used for high-dose chemotherapy, the development of novel treatment programs that utilize hematopoietic cell support depends on an intimate familiarity with the pharmacologic properties of the drugs employed in this setting. It is likely that better utilization of both currently available and investigational chemotherapeutic agents, either through a clearer understanding of the sources of patient-to-patient pharmacokinetic variability or through the use of TDM with adaptive control of drug exposure, will increase the efficacy and decrease the risk of high-dose chemotherapy, as well as improve our ability to combine drugs and overcome inherent or acquired resistance. A renewed focus on the therapeutic effects of the drugs employed with a special emphasis on disease-specific tumor cell killing and the critical effects played by pharmacogenetically related variations in drug exposure is likely to enhance the outcome of high-dose chemotherapy in the future for patients with hematologic malignancies.

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22

William I. Bensinger

High-dose Preparatory Regimens

Introduction

Phase I trials

Treatment regimens are administered directly prior to hematopoietic cell transplantation (HCT) with two goals, which vary according to the patient’s disease and the source of the graft. Since the majority of autologous and allogeneic transplant procedures are performed for the treatment of malignant diseases, the regimens must provide tumor cytoreduction and ideally disease eradication. The pharmacologic basis for this is discussed in Chapter 21. In the case of allogeneic transplants, the regimen must be sufficiently immunosuppressive to overcome host rejection of the graft. This second goal is discussed in detail in Chapter 11. This chapter will review high-dose chemotherapy and chemoradiotherapy regimens administered prior to autologous, syngeneic or allogeneic hematopoietic cell infusion. Most of the high-dose regimens described have been utilized for patients with hematologic malignancies. Some high-dose regimens, however, have been developed specifically for patients with solid tumors, including sarcomas and germ cell tumors. The overall results of HCT utilizing these high-dose treatment regimens are discussed in more detail in chapters dealing with specific diseases. The success of HCT as curative therapy for patients with malignancy is limited, in part, by transplant-related morbidity and mortality. Even if all transplant-related problems were solved, however, about one-half (range 10–90%) of patients receiving transplants for various stages of malignant diseases would die due to the inability of high-dose regimens to eradicate malignancy. This has led to intensive efforts to develop more effective high-dose regimens.

Dose-escalation trials to define maximum tolerated doses (MTDs) and phase II trials to define potential efficacy are, in general, performed in patients with advanced disease incurable by conventional therapy. Once a tolerable regimen has been developed, it can be evaluated in phase II and III trials in patients with less advanced disease. Conventional phase I trials of chemotherapeutic agents have beginning doses that are known to be nontoxic and are escalated to mild or moderate toxicity, which is usually hematologic. In the transplant setting, this is not a useful strategy where only nonhematologic toxicities are considered. An alternative approach is to make an estimate of the likely MTD, based on nonhematologic toxicities, for a combination of agents given in high doses [1,2]. This estimated MTD is then used as the starting dose with escalation and de-escalation depending on observed toxicities. The regimen-related toxicity (RRT) grading system, described below, is used to determine escalation or de-escalation of doses. In phase I studies, attempts are made to keep observed grade 3–4 toxicities to less than 25%, and patients are studied in groups of three to six as shown in Table 22.1. With this Fibonacci scheme, a maximum of 16–20 patients is required to make a first estimation of the MTDs of most new treatment regimens. Using the above methodology, several phase I trials have been successfully performed in the transplant community [3–8]. More recently, Storer has proposed a novel dose-escalation schema in which single patients are enrolled at increasing dose levels until a dose-limiting toxicity is observed. When this occurs, additional patients are enrolled at the next lower dose level in groups of four patients. Additional patients may be enrolled at this level, higher or lower levels depending on the incidences of dose-limiting toxicities [9,10]. This approach has the advantage of requiring fewer patients to reach the MTD.

Evaluation of new treatment regimens The development of new treatment regimens has, in general, required the empirical evaluation of a variety of drugs and radiation doses and schedules. Attempts have been made to systematize the development of new treatment regimens by performing phase I dose-escalation trials followed by phase II potential efficacy trials. Promising new regimens in ideal circumstances are then compared with established treatment regimens in randomized phase III trials. The methodology for trial design is discussed in more detail in Chapter 28.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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RRT grading system In order to carry out meaningful phase I and II studies of new high-dose treatment regimens in transplant patients, a toxicity grading system, excluding hematologic toxicities, has been designed. This grading system estimates the non-hematologic toxicities directly caused by a given transplant treatment regimen [11]. Morbidity is assessed in nine organ systems; heart, bladder, kidneys, lungs, liver, mucosa, central nervous system (CNS), gastrointestinal (GI) tract, and skin. Toxicity is graded on a 0 to 4 scale, with grade 4 being fatal and grade 3 being lifethreatening. Toxicities due to graft-versus-host-disease (GVHD), infection, and drugs administered post-transplant are excluded from this

317

High-dose Preparatory Regimens

grading system. Utilizing this grading system, one can begin to estimate the specific contribution the treatment regimen makes to overall morbidity and mortality of the transplant procedure [12–15]. The National Cancer Institute has developed a common toxicity grading system for chemotherapy treatments. This was modified in 1999 (version 2.0) to incorporate toxicities associated with marrow transplantation. In version 3.0, however (http://www.fda.gov/cder/cancer/toxicityframe.htm), the marrow transplant and leukemia adverse events were deleted. The rationales for this change were that grading should be independent of disease or treatment type, and to reduce inconsistencies in reporting related to type of treatment.

Marrow ablative agents – MTD when administered with HCT (Table 22.2) Total body irradiation (Tables 22.2–22.5) Total body irradiation (TBI) has been the primary therapeutic modality for autologous and allogeneic HCT for patients with hematologic malignancies. TBI has retained wide usage over the past 30 years because of excellent immunosuppressive properties, activity against a wide variety

Table 22.2 Maximum tolerated dose (MTD) of single-agent therapies used with hematopoietic cell transplantation

Reference

of malignancies even if resistant to chemotherapy, penetration of sanctuary sites such as the CNS and testicles, and the relative lack of nonmarrow toxicities when given at high doses. Only a single study has evaluated TBI alone [16], while other trials have involved the concomitant administration of cytotoxic agents, usually cyclophosphamide (CY). Extensive experience has been accumulated with 10–16 Gy of TBI given as a single dose or fractionated, following or preceding high doses of CY (Tables 22.2–22.4).

Table 22.1 Schema for conduct of phase I trials in hematopoietic cell transplantation Severe RRT/total

Dose level for next four patients

0/4 1/4 2/4

Next higher Same Next lower

RRT, regimen-related toxicity.

Agent

MTD

Dose-limiting toxicities

Marrow-ablative single agents used in high-dose regimens with stem cell support [17] Total body irradiation 10–16 Gy GI, hepatic, pulmonary [42] Busulfan 20 mg/kg GI, hepatic, pulmonary Pulmonary, hepatic [90] BCNU 1200 mg/m2 [116] Melphalan 200 mg/kg GI CNS, GI [127] Thiotepa 1135 mg/m2 Nonmarrow-ablative single agents used in high-dose regimens with stem cell support [132] [133] [136]

Cyclophosphamide Ifosfamide Etoposide

200 mg/kg 18–20 g/m2 2400 mg/m2

Cardiac Renal, bladder, neurologic GI

Agents used in high-dose regimens without determination of MTD with stem cell support [140] [142] [143] [151]

*90 mg/m2 *300 mg/m2 2000 mg/m2 *36 g/m2

Mitoxantrone Cisplatin Carboplatin Cytarabine

Cardiac Renal Hepatic, renal CNS

CNS, central nervous system; GI, gastrointestinal. * Maximum dose given with other agents with stem cell support.

Table 22.3 Maximum tolerated doses of total body irradiation (TBI) given with cyclophosphamide 120 mg/kg

Reference

Total dose (Gy)

Fraction size (Gy)

Fractionation interval (h)

Fraction number

Days TBI

[22] [30] [201] [3] [32] [4]

10.00 15.75 16.00 16.00 14.40 (children) 13.20 (adults)

10 2.25 2 2 1.2 1.2

– 24 24 6–8 4–6 4–6

1 7 8 8 12 11

1 7 8 4 4 4

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Chapter 22

Table 22.4 Randomized trials of total body irradiation regimens given with cyclophosphamide 120 mg/kg

Diagnosis

Number of patients

Fraction size (Gy)

Number of fractions

Total dose (Gy)

Probability of relapse

Probability of event-free survival

AML – first remission [22] AML – first remission [30] CML – chronic phase [31] Hematologic malignancies [23]

27 26 34 37 57 59 73 74

10.0 2.0 2.0 2.25 2.0 2.25 10.0 1.35

1 6 6 7 6 7 1 11

10.0 12.0 12.0 15.75 12.0 15.75 10.0 14.85

0.55 0.20 0.35 0.12 0.19 0 0.37 0.23

0.33 0.54 0.60 0.60 0.73 0.66 0.38 0.45

AML, acute myeloid leukemia; CML, chronic myeloid leukemia.

Table 22.5 Chemotherapeutic agents given with total body irradiation (TBI) and with or without cyclophosphamide Reference

Cyclophosphamide (mg/kg)

Total dose TBI (Gy)

Chemotherapy

Total dose

[151] [137] [123,124,157] [202]

– – – – – 60–120 50 60 100

10–12 13.2 9.5–11.5 5 or 12 Gy*

Cytarabine Etoposide Melphalan Melphalan Etoposide Cytarabine Busulfan

36 g/m2 60 mg/kg 110–140 mg/m2 140–180 mg/m2 60 mg/kg 36 g/m2 7 mg/kg (allografts) 8 mg/kg (autografts) 60 mg/kg × 1

[156] [2,68] [161]

5–12 12 12 12

Etoposide

* 5 Gy at 50 cGy/min × 1 or 12 Gy in six fractions.

A large body of experimental data has been developed concerning methods to improve the therapeutic index of TBI [17,18]. Larger doses of TBI that might reduce the likelihood of relapse are limited by GI and pulmonary toxicity, and in the long term by impaired growth and development, chronic pulmonary insufficiency, and second malignancies. In addition to the total dose, many factors, including radiation exposure rate, dose per fraction, interval between fractions, and radiation source (cobalt-60 or linear accelerator) have effects on efficacy and toxicity [17] (see Chapter 23). Many of the techniques of hyperfractionation were developed at Memorial Sloan Kettering Center [19–21]. Experimental studies and clinical trials indicate that TBI administered in fractions is more tolerable than single dose administration and that fractionation, if compensated for by increased total dose, can be given without compromising antitumor effects. Based on this principle, the use of fractionated TBI regimens has been explored in several centers, but there is a paucity of controlled studies of acute and delayed toxicities or of clinical efficacy compared with single-dose TBI. Two prospective randomized studies of fractionation versus single-dose TBI have been performed. One trial demonstrated clear superiority in eventfree survival (EFS) following a fractionated regimen in patients with acute myeloid leukemia (AML) transplanted in first remission [22] (Table 22.4). The second trial was performed in patients with a variety of hematologic malignancies undergoing autologous or allogenic HCT. Although the patients who received hyperfractionated TBI showed a trend toward better survival and a lower rate of relapse than patients receiving single-

fraction TBI, these differences were not statistically significant [22–24]. Other studies suggest that fractionation decreases the incidence of idiopathic interstitial pneumonia syndrome (IPS) (Chapter 96) and cataracts [25] (Chapter 105). A 1990 survey of TBI administration at 15 transplant centers found that 13 utilized some form of fractionation [17]. It is likely that almost all centers now utilize fractionated TBI, due to the decreased acute and delayed toxicities [24]. Not surprisingly, a meta-analysis found that the lung dose of irradiation correlated with the incidence of interstitial pneumonitis [26]. A baseline incidence of 3–4% without radiation increased to a 50% incidence of interstitial pneumonitis with a lung dose of 10.6 Gy. Other groups have utilized reduced yet ablative doses of TBI such as 8 Gy given in 4 fractions with fludarabine (120 mg/ kg) for patients with myeloid leukemias, with good reported efficacy and reduced transplant mortality [27]. Of interest, one center has reported recent results of high dose rate (30 cGy/min) single-fraction TBI (5.5 Gy) combined with CY for patients with AML undergoing allogeneic transplants from unrelated donors [28]. Transplant mortality was low (13%) for patients in first remission but 41% for more advanced disease. The leukemia-free survival at 3 years was 55% for patients in first remission, similar to other reports with higher total doses of irradiation. Although not proven, the antileukemic effectiveness of this lower dose appears to be a consequence of the relatively high dose rate. This same regimen, however, was associated with a high rate of graft rejection when used as a conditioning regimen for patients with chronic myeloid leukemia (CML) transplanted from unrelated donors [29].

High-dose Preparatory Regimens

Studies suggest that both the antileukemic effects and normal tissue toxicities of TBI have a relatively steep dose–response curve. In a study in patients with AML in first remission, patients receiving 15.75 Gy had a markedly decreased actuarial incidence of relapse compared with patients given 12 Gy. Survival was not improved, however, due to an increase in fatal toxicities of the lung and liver [30]. Another study comparing 12.0 Gy with 15.75 Gy TBI in patients with CML in the chronic phase demonstrated a markedly reduced relapse rate among patients receiving the higher TBI dose, but again a markedly increased transplant-related mortality that actually reduced relapse-free survival (RFS) compared with patients receiving 12 Gy TBI [31]. In studies examining the effect of escalating doses of hyperfractionated TBI at 2.0 Gy given twice daily, the MTD was 16.0 Gy [3]. When 1.2 Gy was given three times a day, however, the MTD was only 14.4 Gy [4], suggesting that the shorter interval between doses of TBI prevented DNA repair in normal tissues. In the past, most of the TBI regimens used in Seattle have utilized dual opposing Co60 sources [25]. This approach has the advantage of providing highly homogenous radiation exposure and allows the patient some freedom of movement. However, it is difficult to shield organs, and radiation can be delivered only at relatively low exposure rates (8 cGy/min). The majority of marrow transplant teams use linear accel-

319

erators to deliver TBI (see Chapter 23). With a linear accelerator, dose rates of 40 cGy/min or higher can be administered. The use of linear accelerators may have additional benefits as the radiation field can be shaped with the use of lung shielding to reduce pulmonary toxicity [32]. Electron beam radiation can also be delivered to the chest wall and spine to compensate for the lower dose of TBI to these shielded areas [32,33]. Shielding of other organs, such as the liver, may be useful if specific organ toxicity limits further increases in TBI dose [33]. Delivery of TBI with shielding of both lung and liver may be useful in patients with disease limited to bone, such as multiple myeloma, breast cancer, and Ewing’s sarcoma [33–35].

High-dose chemotherapy regimens (Tables 22.6–22.10) There have been extensive efforts to develop non-TBI-containing transplant regimens. One of the reasons for pursuing this line of research is that many patients with Hodgkin’s disease or lymphoma have received prior dose-limiting radiotherapy, especially to the mediastinum, which results in a high incidence of fatal IPS following TBI [36]. Chemotherapy regimens may also avoid the long-term sequelae of TBI, including cataracts, sterility, second malignancies, and growth and development problems in children. In addition, there has recently been concern about the development of myelodysplasia [37,38].

Table 22.6 Busulfan/treosulfan-based regimens Reference

Regimen

Chemotherapy

Total dose

[66]

BU/CY

[67]

BU/CY

[45]

BU/CY

[77] [79]

BU/Mel

[8]

BU/MEL/TT

[203] [204]

BU/CY/MEL

[84]

BU/CY/TT (autografts)

[85]

BU/CY/TT (allografts)

[73,205] [74] [86]

BU/E

[206]

BU/CY/E

[72]

BU/Flu

[87]

Treo/Flu

Busulfan Cyclophosphamide Busulfan Cyclophosphamide Busulfan Cyclophosphamide Busulfan Melphalan Busulfan Melphalan Thiotepa Busulfan Cyclophosphamide Melphalan Busulfan Cyclophosphamide Thiotepa Busulfan Cyclophosphamide Thiotepa Busulfan Etoposide Busulfan Cyclophosphamide Etoposide Busulfan Cyclophosphamide Etoposide Busulfan Fludarabine Treosulfan Fludarabine

16 mg/kg 120–200 mg/kg 16 mg/kg 120 mg/kg 14 mg/kg 150 mg/kg 16 mg/kg 140 mg/m2 12 mg/kg 100 mg/m2 500 mg/m2 16 mg/kg 120 mg/kg 90–140 mg/m2 10 mg/kg 120 mg/kg 750 mg/m2 12 mg/kg 120 mg/kg 750 mg/m2 16 mg/kg 60 mg/kg 16 mg/kg 150 mg/kg 30 mg/kg 16 mg/kg 120 mg/kg 5–40 mg/kg 16 mg/kg 200 mg/m2 30–42 g/m2 150 mg/m2

BU/CY/E

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Chapter 22

References

Regimen

Chemotherapy

Total dose

[207] [97] [96] [161]

*CBV

[102]

CBV + CPPD

BCNU Cyclophosphamide Etoposide BCNU Cyclophosphamide Etoposide BCNU Cyclophosphamide Etoposide Cisplatin Lomustine Cyclophosphamide Etoposide BCNU Etoposide Cytarabine Melphalan BCNU Etoposide Cytarabine Cyclophosphamide BCNU Cyclophosphamide Cisplatin BCNU AMSA Cytarabine Etoposide BCNU Melphalan Etoposide BCNU Melphalan BCNU Etoposide Cisplatin

300–600 mg/m2 6–7.2 g/m2 600–2400 mg/m2 15 mg/kg 100 mg/kg 60 mg/kg 500 mg/m2 7.2 g/m2 2.4 g/m2 150 mg/m2 15 mg/kg 100 mg/kg 60 mg/kg 300–600 mg/m2 400–800 mg/m2 800–1600 mg/m2 140 mg/m2 300 mg/m2 300 mg/m2 800 mg/m2 6 g/m2 600 mg/m2 5.6 g/m2 165 mg/m2 800 mg/m2 450 mg/m2 900 mg/m2 450 mg/m2 500 mg/m2 80–140 mg/m2 300 mg/m2 500 mg/m2 140 mg/m2 60–100 mg/m2 2400–3000 mg/m2 200 mg/m2

†CBV

[105]

[109] [110] [111] [80] [107] [108]

BEAM

[92]

STAMP-1

[208]

BAVC

[209]

[209] [210]

BEAC

Table 22.7 Nitrosourea-based high-dose chemotherapy regimens

BCNU, carmustine. * Drugs given simultaneously over 3 days. † Each drug given on a separate day with a day’s rest between.

There are potential advantages in terms of convenience and expense of moving away from TBI, in that TBI utilizes already overextended radiation resources, often for prolonged periods of time, and requires the skills of physicists and radiotherapists. Finally, it may be possible to develop chemotherapy regimens that are more effective than those containing TBI. It is unfortunate that there are at present few studies comparing outcomes of specific regimens used for HCT. Alkylating agents Alkylating agents are the major class of drug used in high-dose regimens with HCT support because they have several desirable characteristics. Many alkylating agents have marrow toxicity as the major dose-limiting factor, which allows for dose escalation when hematopoietic cells are utilized. Alkylators are not cell cycle-specific, making them capable

of killing nondividing, resting tumor cells. In in vitro testing, alkylators do not generally exhibit cross-resistance and have relatively steep log-linear dose–response curves [39]. Although it is not possible to combine alkylating agents at full dose when used alone, between 50% and 70% of the single-agent MTD can be used in two-to-three drug combinations. Increasingly, as regimens for the treatment of solid tumors evolve, there has been movement to incorporate tumor-specific drugs into the high-dose therapy setting [40,41]. It is important to point out, however, that drugs such as mitoxantrone or paclitaxel, which are highly active against tumors such as breast or ovarian cancer at conventional doses, are more difficult to deliver at higher doses due to nonhematologic toxicities, i.e. cardiac or neurologic. These characteristics limit the usefulness of such drugs in high-dose therapy regimens, and their value in comparison to alkylator-based regimens remains to be proven.

High-dose Preparatory Regimens Table 22.8 Melphalan-based high-dose regimens

321

Reference

Regimen

Chemotherapy

Total dose

[104]

MEL/E

[152]

MEL/A

[141]

MEL/MITO

[146]

MEL/MITO/CARBO

[211]

MEL/CY/P

[154]

MEL/MITO/TAX

[82]

Mel/flu

Melphalan Etoposide Melphalan Cytarabine Melphalan Mitoxantrone Melphalan Mitoxantrone Carboplatin Melphalan Cyclophosphamide Cisplatin Melphalan Mitoxantrone Paclitaxel Melphalan Fludarabine

140–180 mg/m2 60 mg/kg 140 mg/m2 12 g/m2 180 mg/m2 60 mg/m2 160 mg/m2 50 mg/m2 1400 mg/m2 80 mg/m2 5.6 g/m2 180 mg/m2 180 mg/m2 60–90 mg/m2 500–700 mg/m2 180 mg/m2 125 mg/m2

Table 22.9 Thiotepa-based high-dose regimens

Table 22.10 Etoposide-based high-dose regimens

Reference

Regimen

Chemotherapy

Total dose

Reference

Regimen

Chemotherapy

Total dose

[128]

STAMP V CTCb

ICE

TTCYMito

[140]

ICE

[140]

TTMit

[213]

CYTT

[214] [154]

CYTTP

500 mg/m2 6 g/m2 800 mg/m2 675 mg/m2 7.5 g/m2 60 mg/m2 1.2 g/m2 90 mg/m2 700 mg/m2 7 g/m2 600 mg/m2 40 mg/m2 3.75 g/m2

[215]

[212]

Thiotepa Cyclophosphamide Carboplatin Thiotepa Cyclophosphamide Mitoxantrone Thiotepa Mitoxantrone Thiotepa Cyclophosphamide Thiotepa Cisplatin Cyclophosphamide

Ifosfamide Carboplatin Etoposide Ifosfamide Carboplatin Etoposide Etoposide Cisplatin Carboplatin Etoposide Cyclophosphamide Cisplatin Cyclophosphamide Etoposide Cisplatin Etoposide Carboplatin Doxorubicin Etoposide Cyclophosphamide Cyclophosphamide Carboplatin Etoposide

16 g/m2 1.8 g/m2 1.5 g/m2 20 g/m2 1.8 g/m2 3 g/m2 650 mg/m2 100 mg/m2 1.8 g/m2 30 mg/kg 100 mg/kg 250 mg/m2 4.5–5.25 g/m2 750–1200 mg/m2 120–180 mg/m2 2250 mg/m2 2100 mg/m2 165 mg/m2 60 mg/kg 100 mg/kg 6 g/m2 2 g/m2 625 mg/m2

[216]

[142]

CEP

[217]

CEP

[122]

Busulfan (Tables 22.2 and 22.6) Busulfan (BU) is an alkylating agent with profound myeloablative properties, marked activity against nondividing marrow cells and possibly nondividing malignant cells as well. The maximum tolerated dose of BU, given as a single agent over 4 days followed by stem cell support, is approximately 20 mg/kg [42]. Single-agent testing to determine the spectrum of activity in patients with malignant disease has not been extensive [42–44]. However, BU is probably active in a variety of malignancies including multiple myeloma [43,45], lymphoma [46], acute lymphoblastic leukemia (ALL) [46], AML [47–49], myeloid metaplasia [50], testicular tumors [51], Ewing’s sarcoma [51], and breast cancer [52]. Although generally combined with other drugs, BU alone has been used as the conditioning regimen prior to first autologous transplants and for second allogeneic transplants in patients with CML [53]. One of the former problems with optimal utilization of BU had been the availability of only the oral form. Pharmacokinetic variability

[40]

[122]

between patients is high, with a two-to-threefold difference in plasma levels between patients [54]. Children have lower mean plasma levels of BU (650 ng/mL) than adults (1050 ng/mL) [55,56], which may be partially compensated for by dosing children by body surface area rather than weight [56]. Variation in BU plasma levels between individuals probably contributes to the significant differences observed in toxicity and clinical response in patients receiving the same mg/kg or mg/m2

322

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dose. Slattery et al. have demonstrated a relationship between low BU steady-state plasma concentrations and graft rejection in children [56]. There are also data suggesting a direct relationship between the severity of RRT, especially sinusoidal obstruction syndrome (SOS) of the liver, and high BU plasma levels [54,57]. This is discussed in more detail in the section on hepatic complications after transplantation in Chapter 95. In a patient accidentally overdosed with busulfan, hemodialysis resulted in accelerated clearance of the drug [58]. More recently, it has been shown that the steady-state BU concentration correlates with relapse in patients receiving HLA-compatible transplants for CML in chronic phase [59]. Seven relapses occurred among the 22 patients with BU state concentrations below the median of 918 ng/mL compared with none among the 23 patients at or above the median (p = 0.0003). Thus, the average steady-state concentration of BU over a 4-day period may be a more important determinant of toxicity and efficacy than the exact mg/kg or mg/m2 dose administered to any given patient [56,59]. Fortunately, within a given patient, BU is a pharmacokinetically predictable drug, and its absorption and elimination rate remain linear over a wide dose range and constant with time [56]. With repeated oral administration, steady-state BU plasma concentrations are achieved rapidly and can be predicted from first-dose kinetics. Following determination of first-dose kinetics, oral doses can be adjusted up or down to achieve the desired plasma levels. The strategy of targeted steadystate BU dosing has been used successfully to perform a dose-escalation trial in patients receiving fixed doses of CY and TBI [60]. Targeting of the steady-state plasma level of BU should increase the therapeutic index. Results of a phase 11 study indicate that targeting oral BU in patients with CML is associated with low transplant-related toxicity and a low relapse rate [61]. An intravenous preparation of BU has become available, and initial phamacokinetic data indicate considerably less individual variation in area under the curve (AUC) values than seen with the oral preparation [62]. Intravenous BU can be given as a single daily dose over 3 hours or twice daily rather than as the usual four oral doses per day [63,64]. Retrospective comparisons of intravenous BU with oral BU suggest less hepatic toxicity in patients receiving the intravenous form [65]. A regimen of BU combined with CY has been developed that has had a wide application for the treatment of a variety of malignant and nonmalignant diseases using autologous and allogeneic stem cell support [66]. The original regimen included BU 4 mg/kg/day for 4 days followed by CY 50 mg/kg/day for 4 days [66]. This regimen was subsequently modified by lowering the dose of CY to 120 mg/kg with an apparent decrease in toxicity without an increase in relapses [67]. It has been reported that 16 mg/kg combined with 120 mg/kg CY results in prohibitive toxicity in older patients with multiple myeloma and breast cancer undergoing autologous or allogeneic transplantation [35,45,52]. Although these reports could be due to small numbers of patients with advanced disease, lowering the dose of BU to 14 mg/kg decreased toxicity and allowed for an increase in the dose of CY to 150 mg/kg [45]. It has also been more recently shown that older patients with CML or myelodysplastic syndrome tolerate targeted BU and CY 120 g/kg quite well [61,68]. In patients with thalassemia, BU 14 mg/kg followed by CY 120–200 mg/kg has been a well-tolerated and effective regimen for allografting [69]. A randomized trial comparing BU dosed at 600 mg/m2 with BU dosed at 16 mg/kg for patients receiving transplant for thalassemia found no differences in outcome but, not surprisingly, that rejection occurred mainly in patients with low blood levels of BU [70]. Fludarabine, a nucleoside analog with significant immunosuppressive activity, has been substituted for CY in an effort to reduce RRT. In addition, fludarabine inhibits DNA repair and thus is synergistic with alkylators that cause breaks in the DNA strand. Several trials conducted

in patients with myeloid leukemias undergoing allogeneic HCT indicate lower toxicity profiles with fludarabine–BU combinations [71,72]. Etoposide (60 mg/kg) has also been utilized to replace CY in the BU–CY regimen for the treatment of patients with AML undergoing autologous bone marrow transplantation with encouraging disease-free survivals [48,73–75]. The major toxicities, SOS, mucositis, and skin breakdown, are similar to those described with BU + CY or BU + melphalan (MEL). BU (16 mg/kg) and MEL (140 mg/m2) have been combined and evaluated in patients undergoing autologous or allogeneic HCT for a variety of hematologic malignancies [76–81]. MEL (180 mg/m2) has also been combined with fludarabine (125 mg/m2) as an unrelated donor conditioning regimen for patients with hematologic malignancies [82]. A regimen of BU (12 mg/kg), MEL (100 mg/m2), and thiotepa (500 mg/m2) followed by autologous HCT has been evaluated in patients with a variety of malignant diseases [8,83]. This regimen produces profound marrow ablation with significant mucositis and is associated with an approximate 5% treatment-related mortality due to IPS. Regimens involving BU, thiotepa, and CY have been utilized for auto- [84] and allografting [85]. A regimen involving BU, CY, and etoposide has been utilized prior to an autologous and allogeneic HCT [86]. Treosulfan (dihydrobusulfan) (30–42 g/m2) has been substituted for BU in combination with fludarabine, CY or MEL as an allogeneic preparative regimen for patients with both myeloid and lymphoid malignancies [87–89]. Although studies are limited, this regimen may be associated with less RRT and a lower early transplant-related mortality. Nitrosoureas (Tables 22.2 and 22.7) Carmustine (BCNU) is a nitrosourea commonly used as a marrow ablative agent because it is active against a variety of tumors [90]. The active metabolite of BCNU is a chloroethyl carbonium ion produced by hydrolysis. It is highly lipid soluble and has been used extensively for the treatment of malignant brain tumors. In conventional doses, BCNU is limited by delayed marrow toxicity, pulmonary fibrosis, and renal dysfunction. The MTD, as a single agent, is 1200 mg/m2 when given with autologous stem cell infusions. The dose-limiting toxicities are pulmonary and hepatic [90]. When BCNU is used in combinations that include cisplatin and CY, there is a substantial increase in the risk of lung injury [91–93]. When carboplatin and CY are added to BCNU, a high rate of veno-occlusive disease has been reported [94]. BCNU and CY are often combined and may have synergistic activity. The first multidrug transplant regimen consisted of a combination of BCNU, cytarabine, CY, and thioguanine (BACT) [95]. Variations of these drugs have evolved into the three- and four-drug combinations in common use today involving BCNU. Regimens containing CY, BCNU, and etoposide (CBV) have been evaluated extensively in patients with malignant lymphoma receiving autologous transplants [96–99]. The doses of drugs and the schedules are shown in Table 22.6. These regimens are often used as substitutes for TBI-based regimens for patients with lymphoma who have received prior dose-limiting irradiation [100]. Doses of BCNU exceeding 450 mg/ m2 are associated with unacceptable pulmonary toxicity [101]. Early institution of steroid therapy at the first sign of dyspnea may prevent the more serious and often fatal complication of pulmonary fibrosis [91,102]. Patients who receive high-dose BCNU should receive close long-term follow-up for pulmonary complications, which have been noted in adults who received large doses of BCNU as children [103]. Less frequent severe toxicities include SOS, hemorrhagic cystitis, and nephrotoxicity. Although not extensively evaluated, all of these combinations are probably sufficiently immunosuppressive to achieve allogeneic engraftment [104]. Lomustine has been substituted for BCNU in an attempt to

High-dose Preparatory Regimens

decrease pulmonary toxicities [105], but without a clear reduction in lung complications [106]. BCNU, etoposide, cytarabine, and CY (BEAC), have been extensively evaluated for patients with malignant lymphoma [107,108]. MEL has been substituted for CY in the BEAC regimen to create BEAM and has also been extensively evaluated in patients with malignant lymphoma [80,109–111]. Since most patients with malignant lymphoma have PBHC collected after CY-containing regimens, the BEAM regimen may be more attractive than the BEAC regimen. Furthermore, the substitution of MEL for CY obviates the problem of cystitis and the need for mesna and/or bladder irrigation, making this a preferred outpatient regimen for patients with malignant lymphoma. More recently, allogeneic marrow transplants have been performed following the administration of BEAM [112]. A regimen of BCNU, cisplatin, and CY (STAMP-I) has been commonly used for patients with breast cancer [92]. However, even in patients with stage II–III breast cancer, treatment-related mortality has been reported to be 12%, predominantly due to pulmonary toxicities associated with the high dose of BCNU (600 mg/m2) [93], although in a more recent trial of STAMP-I for patients with metastatic disease, the treatment-related mortality was 8.6% [113]. The increase in pulmonary toxicities associated with this dose of BCNU has been reported to be as high as 59%, but it appears to respond to early intervention with corticosteroids [114]. Melphalan (Tables 22.2 and 22.8) One of the earliest chemotherapeutic agents developed, MEL is a bifunctional alkylating agent whose structure incorporates nitrogen mustard and phenylalanine [115]. Single doses of MEL, 150–240 mg/m2, followed by autologous stem cell infusion have been evaluated in patients with multiple myeloma and breast cancer with the dose-limiting toxicities being GI and hepatic [116–118]. MEL 240 mg/m2 has been utilized alone as a conditioning regimen for patients with hematologic malignancies receiving allogeneic transplants from HLA-matched siblings [119]. MEL has been widely used in high-dose regimens for patients with breast and ovarian cancer, multiple myeloma or lymphoma. MEL 100 mg/m2 has been used as a reduced-intensity conditioning regimen prior to allogeneic stem cell transplantation in patients with multiple myeloma [120]. More recently, fludarabine has been combined with MEL to form a reduced-intensity regimen for allografting [82,121]. MEL is included in several of the BU- and BNCU-based regimens listed in Tables 22.6 and 22.7, and Table 22.8 summarizes additional regimens that have included MEL for myeloablation. MEL is frequently combined with BCNU (BEAM), a regimen utilized for patients with malignant lymphoma [109–111]. MEL is utilized alone or with TBI to treat patients with multiple myeloma [122,123] or leukemia [124]. A randomized trial of single-agent MEL (200 mg/m2) compared with reduced-dose MEL (140 mg/m2) with TBI as a preparative regimen for patients with myeloma demonstrated a survival advantage for MEL alone [125]. As a result, single-agent MEL is by far the most commonly used conditioning regimen for patients with myeloma undergoing autologous peripheral blood HCT. Thiotepa (Tables 22.2 and 22.9) Thiotepa was first recognized for its antitumor properties in the 1950s, but because of severe myelosuppression it saw only limited use in breast and ovarian cancer. Both thiotepa and its major metabolite tepa are active against tumor cell lines in vitro. The plasma half-life of tepa is much longer than that of thiotepa and accounts for much of the clinical toxicity [126]. The MTD for thiotepa, as a single agent with stem cell support, is approximately 1100 mg/m2. Dose-limiting toxicities are of the CNS, GI tract, liver, and skin [127].

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Thiotepa is included in several of the above BU-, BCNU- and MELbased regimens outlined in Tables 22.6–22.8, and Table 22.9 summarizes additional regimens that include high doses of thiotepa. The most frequently used regimen for women with breast cancer is the combination of thiotepa, CY, and carboplatin (STAMP-V) [128]. When breast cancer was the most commonly treated disease with stem cell support, STAMP-V was likely the most widely used of all regimens. This is no longer the case, however, since there has been a marked decline in transplantation for breast cancer. Some investigators administer all three drugs by continuous infusion over 3 days [128], others by bolus on 3 consecutive days [129], and others give CY and thiotepa by bolus and carboplatin by continuous infusion. The superiority of any one of these methods of administration has not been documented.

Nonmarrow ablative agents and agents for which MTD with stem cell support is not known Cyclophosphamide (Table 22.2) CY alone was originally used to achieve allogeneic engraftment in patients with aplastic anemia and hematologic malignancies, but was modified in patients with malignant disease by the addition of TBI or combinations of chemotherapeutic agents. However, CY, with or without antithymocyte globulin, is still the most commonly used regimen for immunosuppression of patients with aplastic anemia prior to allogeneic transplantation [25]. Low doses of TBI (3–5 Gy) have been added to CY as a conditioning regimen for aplastic anemia in an effort to increase immunosuppression and reduce episodes of graft rejection [130]. CY is very immunosuppressive but is not marrow ablative as the dose-limiting toxicity is hemorrhagic myocarditis [131]. The MTD, as a single agent, is approximately 200 mg/kg, and infusion of marrow has no impact on survival at this or higher doses [132]. The major side-effect, at doses of 120–200 mg/kg, is hemorrhagic cystitis, which can be diminished by bladder irrigation or the administration of mesna. Both CY and its isomer ifosfamide have been extensively utilized with high-dose chemotherapy regimens for most tumor types treated with HCT.

Ifosfamide (Table 22.2) Ifosfamide, a structural isomer of CY, has been evaluated in high-dose regimens followed by autologous stem cells as a substitute for CY. Similar to CY, however, hematopoietic toxicity is not dose-limiting even when high doses of ifosfamide are administered [133]. The MTD is 18–20 g/m2 with the dose-limiting toxicities being renal, bladder, and neurologic, with lethargy, confusion, and seizures. Ifosfamide is always given with mesna for bladder protection. Ifosfamide is frequently combined with etoposide and carboplatin (ICE) for the treatment of lymphoma, but this regimen does not require HCT [134,135].

Etoposide (Tables 22.2 and 22.10) Etoposide is a semisynthetic derivative of podophyllin. It appears to act by stabilization of the topoisomerase II–DNA complex with subsequent DNA breaks and cell-cycle arrest. This mechanism of action may promote synergistic cell killing when combined with alkylators [104]. The MTD for etoposide, as a single agent with stem cell support, is approximately 2400 mg/m2. Although etoposide has substantial myelotoxicity, the dose limiting toxicity is to the GI tract. In a phase I study with marrow support, the tempo of recovery of peripheral counts was not related to the dose of etoposide and was more rapid than recovery after TBI regimens with marrow support [136].

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The use of etoposide in high-dose regimens has been reviewed [104]. Etoposide is commonly used with TBI [137] in the regimens listed in Tables 22.7 and 22.8, and additional regimens are outlined in Table 22.10. It is not clear whether any of the regimens in Table 22.10 requires HCT. BU has been combined with etoposide (Table 22.6) resulting in a unique HDT regimen that is particularly active in patients with acute leukemias [48,73]. The major dose-limiting toxicities of this regimen are GI and pulmonary. Mitoxantrone (Table 22.2 and 22.8) and doxorubicin (Table 22.10) Mitoxantrone has been utilized in a variety of high-dose regimens, but the MTD as a single agent with stem cell support has not been reported [138]. Single-agent dose-escalation trials in patients with metastatic breast cancer have been limited by cardiac toxicity and are disappointing in terms of response rates [139]. The highest dose of mitoxantrone administered in a high-dose combination regimen has been 90 mg/m2 given with thiotepa 1200 mg/m2 [140]. Regimens including mitoxantrone are included in Tables 22.8 and 22.9. In one study, CY 7 g/m2 and escalating doses of mitoxantrone led to unacceptable hemorrhagic cystitis despite mesna, leading the investigator to substitute MEL for CY [141]. In a similar fashion doxorubicin, a compound structurally related to mitoxantrone, has been combined with etoposide and CY as a high-dose regimen for patients with breast cancer [40]. The MTD of doxorubicin was 165 mg/m2, with the dose-limiting toxicity being mucositis. It is not clear that the use of mitoxantrone or doxorubicin in high-dose regimens requires stem cell support, and it remains to be determined whether such anthracycline-based regimens are more efficacious than alkylator-based combinations. At the present time, there is controversy as to whether high-dose regimens of any kind are useful for patients with high-risk or metastatic breast cancer [113]. Cisplatin (Table 22.2) The MTD for cisplatin as a single agent with HCT has not been reported, and the highest dose given in combination regimens has been 300 mg/m2 given with etoposide 60 mg/kg and CY 100 mg/kg [142]. Regimens including cisplatin are outlined in Tables 22.7–22.10. Although limited by nephrotoxicity when utilized as a single agent, when used with other drugs the doses of cisplatin do not exceed 200 mg/m2, and with adequate hydration, renal dysfunction is generally avoidable. Carboplatin (Table 22.2) The MTD for carboplatin with HCT is approximately 2000 mg/m2, with the dose-limiting toxicities being hepatic and renal [143]. Carboplatin in conventional doses is usually given in line with a formula based on renal function [144]. However, Shea et al. administered carboplatin in doses of 375–2400 mg/m2 and found a direct correlation between the mg/m2 and the measured AUC, suggesting that high doses could be based on body surface area [143]. Carboplatin has also been combined with etoposide, CY, and escalating doses of TBI (10–12.9 Gy) for the treatment of a variety of hematologic malignancies [145]. In one study, carboplatin was administered at doses of 1000–1600 mg/m2 in conjunction with high doses of MEL and mitoxantrone [146]. In a retrospective analysis of this study, dosing by the Calvert formula was more predictive of grade 3–4 toxicities than dosing by body surface area [146]. These data suggested that dosing of carboplatin to an AUC of 20 mg/mL/min rather than 1400 mg/m2 would decrease grade 3–4 RRT in patients

receiving MEL (160 mg/m2) and mitoxantrone (50 mg/m2) followed by autologous peripheral blood hematopoietic cell (PBHC) infusion [146]. High-dose chemotherapy regimens including carboplatin are outlined in Tables 22.8–22.10. A phase I–II trial determined that the MTDs of carboplatin and CY administered together are 1.8 and 6.0 g/m2, respectively [147]. Carboplatin (1500 mg/m2) with CY (120 mg/m2) and mitoxantrone (75 mg/m2) appears to be an active regimen for the treatment of patients with ovarian cancer [148]. Combinations of carboplatin, etoposide, and either paclitaxel or thiotepa are frequently used with HCT support for the treatment of germ cell tumors [149,150].

Cytarabine (Table 22.2) Cytarabine is a cell-cycle-specific antineoplastic agent that affects cells only in S-phase. Although its mechanism of action is not fully understood, its metabolite cytarabine triphosphate is believed to inhibit the action of DNA polymerase. It may also function as a false substrate when incorporated into DNA. The MTD of cytarabine when administered as a single agent with HCT has not been determined. However, in combination with other agents given in high doses, cytarabine is usually administered in doses of 3 g/m2 for 4–12 doses at 12-hour intervals [151,152]. Cytarabine 12–36 g/m2 is included in several regimens utilized for the treatment of patients with hematologic malignancies as outlined in Tables 22.7 and 22.8.

Paclitaxel Paclitaxel is an antimicrotubule drug that facilitates microtubule assembly from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stabilization inhibits microtubule reorganization, preventing progression to mitosis. Paclitaxel, 775 mg/m2, has been substituted for BCNU in a regimen including 5.6 mg/m2 CY and 165 mg/m2 cisplatin [153]. Paclitaxel 500–700 mg/m2 has been administered with MEL (180 mg/m2) and mitoxantrone (60–90 mg/m2) to patients with advanced ovarian cancer (Table 22.8) [154]. Paclitaxel 150–775 mg/m2 has also been combined with doxorubicin (165 mg/m2) and CY (100 mg/ kg) and utilized for patients with advanced breast carcinoma [41].

High-dose regimens with total body irradiation Total body irradiation with cyclophosphamide (Table 22.3) The use of CY with TBI is based on empirical observations. CY (120 mg/ kg) was originally given prior to TBI to a patient undergoing a syngeneic marrow transplant for advanced lymphoma in 1970 [155] in order to avoid tumor lysis and acute renal failure which had been previously observed with TBI given in a single dose of 10 Gy. This patient is alive without disease more than 30 years post-transplant (unpublished data). After this initial patient, CY was regularly given prior to TBI in patients receiving allogeneic or syngeneic marrow transplants with no apparent excess toxicity over that observed following TBI alone and thus become a “standard regimen” widely used in many transplant centers. Although several agents have been added to or substituted for CY (Table 22.5), most of the TBI regimens tested over the past 20 years have included CY before or after TBI. A variety of TBI regimens have been evaluated in attempts to define an optimal dose and schedule. Table 22.3 summarizes the MTD for several different schedules of TBI (delivered by cobalt-60 [60Co] sources at dose rates of 5–10 cGy/min) given after CY 120 mg/kg [24,30,31].

High-dose Preparatory Regimens

325

Randomized trials involving different TBI regimens (Table 22.4)

High-dose chemotherapy versus high-dose chemoradiotherapy (Table 22.11)

There has been a paucity of randomized controlled trials evaluating different TBI regimens [Chapter 23]. Three trials reported by the Seattle Marrow Transplant Team are summarized in Table 22.4 [22,24,30,31]. The first study demonstrated that 12.0 Gy TBI given in 2.0 Gy daily fractions was superior to 10.0 Gy TBI delivered as a single dose [22,24]. The second and third trials evaluated a higher dose of TBI (15.75 Gy) in patients with AML in first remission or CML in the chronic phase [30,31]. Both studies showed a decrease in the probability of relapse following the higher dose of TBI but no improvement in survival due to increased transplant-related mortality. Methods to decrease the toxicity of TBI or the use of an intermediate dose of TBI could possibly improve survival in these patients.

There are relatively few studies comparing chemotherapy regimens with TBI-based regimens. Two randomized trials have demonstrated the equivalency of BU/CY and CY/TBI in patients with CML in chronic phase receiving HLA-matched allografts [162,163]. One study in patients receiving HLA-compatible transplants for AML showed superiority for the CY/TBI regimen due to a lower relapse rate [164]. The Southwest Oncology Group compared BU/CY with etoposide/TBI in 122 patients with advanced acute leukemias or CML beyond first chronic phase undergoing matched allogeneic transplantation. There were no significant differences in toxicity, acute GVHD, survival or disease-free survival in patients entered on either regimen [158]. An analysis of nonrandomized trials reported by Horning et al. has suggested equivalency between patients with lymphoma receiving TBI and etoposide or CBV given prior to autologous transplantation [161]. There were no differences in outcomes seen in a retrospective analysis of a BCNU/ etoposide/CY regimen or TBI/etoposide/CY regimen for patients with Hodgkin’s disease undergoing autologous HCT [165]. A similar nonrandomized analysis comparing 381 patients with AML in first remission undergoing allogeneic HCT found no differences in survival between BU/CY and CY/TBI but a lower risk of relapse with CY/TBI [166]. In children with ALL undergoing allogeneic HCT, a small randomized trial demonstrated that BU/etoposide/CY resulted in an inferior EFS compared with a regimen of TBI/etoposide/CY [167]. In another trial of high-dose cytarabine, MEL and either BU or fractionated TBI in children with high-risk ALL undergoing allogeneic HCT, BU was associated with fewer long-term side-effects [168].

Total body irradiation with drugs other than cyclophosphamide (Table 22.5) Drugs other than CY have been administered with TBI, as shown in Table 22.5 [6,124,137,151,156–158]. At the present time, there is no evidence that any of these agents is superior to CY when given with TBI since randomized controlled trials have not been performed. When BU was substituted for CY given with 12 Gy TBI as allograft conditioning for patients with myelodysplastic syndrome, the toxicities were considerable with a day 100 nonrelapse mortality of 38% [159]. A large, retrospective analysis of patients reported to the International Bone Marrow Transplant Registry compared outcomes of two regimens used for allografting from HLA-identical siblings for patients with ALL in first or second remission [160]. All 502 patients received myeloablative doses of TBI with either CY (n = 298) or etoposide (n = 204). There were no differences in outcomes by regimen for patients in CR1 (complete remission 1), however, for patients in CR2, there was a lower risk of relapse and mortality when etoposide was used with any TBI dose when compared with CY and TBI doses below 13 Gy. Cyclophosphamide and total body irradiation with other drugs (Table 22.5) Other agents have been combined with CY and TBI, as shown in Table 22.6 [2,156,161]. Phase II trials suggest that many of these combinations may have better therapeutic efficacy than CY/TBI, but none of these regimens has been subjected to randomized comparison studies.

Modified total body irradiation regimens Because of the considerable toxicities to nontarget organs associated with external-beam TBI, there has been an interest in the application of technical modifications to TBI. In one early approach, external-beam TBI was given with 90% lung shielding followed by electron beam to the rib cage in order to deliver a uniform dose of radiation to all marrowbearing areas. This modified TBI was studied in a phase I trial as the second part of a tandem transplant for patients with bone-based malignancies [169]. The MTD in this trial was 13.5 Gy. Another trial combined modified TBI (9 Gy) with BU (12 mg/kg) and CY (120 mg/kg) to treat 89 patients with multiple myeloma [170]. The transplant mortality in this study was 2%, with a complete remission rate of 48%.

Table 22.11 Randomized trials of BU/CY versus TBI ± VP16 ± CY Reference

Diagnosis

Regimen

Number

Survival

Relapse

Event-free survival

[164]

AML

[163]

CML

[162]

CML

[158]

“Advanced” leukemias

[167]

ALL (pediatric)

BU/CY CY/TBI BU/CY CY/TBI BU/CY CY/TBI BU/CY VP-16/TBI TBI/VP16/CY BU/VP16/CY

51 50 65 55 73 69 61 61 22 21

0.51 0.75 0.61 0.63 0.80 0.80 0.30 0.30 0.45

0.34 0.14 0.44 0.11 0.13 0.13 NR NR NR NR

0.47 0.72 0.59 0.55 0.68 0.71 0.20 0.20 0.58 0.29

AML, acute myeloid leukemia; BU, busulfan; CY, cyclophosphamide; NR, not reported; TBI, total body irradiation.

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More recently, studies have been conducted using image-guided tomographic intensity-modulated radiation equipment that does not require organ shielding [171]. Simulations of dosimetry using this equipment indicate a 1.7–7.5-fold reduction in median doses to nontarget organs [172]. Furthermore, this approach may allow a significant escalation of doses of TBI of up to 20 Gy to target organs such as bone.

Marrow ablation using targeted radioisotopes Some alternatives to the external-beam delivery of radiation using radioimmunotherapy are discussed in more detail in Chapter 24. One approach to improving the cure rate of transplant conditioning regimens while simultaneously decreasing toxicities to normal organs is to replace or augment nonspecific external-beam TBI with targeted radiotherapy. Radiolabeled monoclonal antibodies have the potential to focus higher doses of radiation on tumor sites than is possible with external-beam TBI and expose normal organs to lower doses of radiation. Recent evaluations of these techniques have been performed in patients with multiple myeloma, malignant lymphoma, and AML [173–183].

Bone-seeking radioisotopes Isotopes that selectively bind to bone by themselves (yttrium-90) [184], or when complexed to bone-seeking compounds, are an attractive way to target radiation for patients with malignancies in which bone or bone marrow is the primary site of disease. Few studies have utilized radioisotopes alone for marrow ablation. Bayouth et al. performed a phase I trial of 166-holmium [166Ho] complexed to the bone-seeking isotope 1,4,7,10 tetraazacyclododecane-1,4,7,10-tetramethylene-phosphonic acid (DOTMP) in patients with multiple myeloma [178]. The total dose of radiation adsorbed that was delivered to the bone marrow for six patients ranged from 7.9 to 41.4 Gy, and marrow ablation requiring the infusion of stem cells was achieved in two patients. In several multicenter phase I–II trials, cohorts of patients with multiple myeloma received increasing doses of 166Ho-DOTMP designed to deliver 20, 30 or 40 Gy to the bone marrow. This was followed by high-dose MEL 140–200 mg/m2, external-beam TBI (some patients) and peripheral blood HTC [185]. There were no grade 3–4 nonhematologic toxicities within the first 60 days. After 60 days, significant hemorrhagic cystitis occurred at one center that did not use bladder irrigation. Seven patients experienced late thrombotic microangiopathy among the group receiving more than 30 Gy (or 2400 mCi dosed) to the marrow. The complete remission rate was 35%, with a median overall survival in excess of 24 months. Since two-thirds of the patients in these trials had relapsed or refractory disease, this remission rate was higher than would be expected with MEL alone. Another bone-seeking radioisotope samarium-153 (153Sm) with less beta energy than 166Ho, has been linked to EDTMP, a tetraphosphonate chelate, and studied in 18 patients with multiple myeloma who received MEL 200 mg/m2 following the isotope. The initial 12 patients received escalating doses of 6, 12, 19.8, and 30 mCi/kg [186]. Another six patients received targeted doses designed to deliver 40 Gy to red marrow based on an initial test dose. With a median of 31 months of follow-up, no cases of hemorrhagic cystitis, nephrotoxicity or thrombotic microangiopathy were observed. Five patients achieved a CR, with a very good PR in another seven, a response rate as good as or better than would be expected with MEL alone. The role of bone-seeking isotopes should be expanded over the next several years, and they may become a major therapeutic approach to the treatment of malignancies involving the bone marrow.

Sequential regimens with peripheral blood hematopoietic cell support The concept of administering tandem high-dose cycles or multiple, less-intensive treatments more frequently as an alternative to single high-dose marrow ablative therapy has only recently been explored [122,187–190]. Sequential cycles of high-dose carboplatin have been administered with growth factor and repeated PBHC infusions [187]. In a similar fashion, some groups have utilized tandem cycles of high-dose MEL 200 mg/m2 or MEL and TBI, followed by autologous stem cell infusion for the treatment of patients with multiple myeloma [189] or double transplants for breast cancer [122,190]. Tandem cycles of MEL produced complete response rates of 44% in patients with myeloma. Several prospective, randomized trials of single- versus double-intensive therapy for patients with multiple myeloma have been reported or are underway [191,192]. These trials employed MEL 140– 200 mg/m2 for the first cycle and MEL + TBI or BU for the second cycle. One trial found a small survival advantage for the tandem approach. Tandem cycles of CY, carboplatin, and etoposide used to treat 28 patients with untreated metastatic breast cancer resulted in a 50% CR rate and a 30% RFS at 24 months [122]. In another study, patients with stage IV breast cancer treated with a cycle of high-dose MEL followed by the STAMP-V regimen did not have a better outcome than when a single cycle of STAMP-V was used [190]. Tandem cycles of high-dose chemoradiotherapy with CY, etoposide, MEL, thiotepa, and TBI have been used to treat children with stage IV neuroblastoma [193]. Tandem cycles of paclitaxel, carboplatin, and either etoposide or ifosfamide have been utilized with autologous HCT for relapsed germ cell tumors [194]. Tandem autologous transplant, MEL (150 mg/m2) for cycle 1 and CBV or TBI/CY/etoposide for cycle 2, has been utilized to treat patients with primary refractory or relapsed Hodgkin’s lymphoma [195]. It remains to be determined whether or not this approach will be superior to single high-dose ablative regimens.

Allogeneic transplantation with nonmarrow-ablative regimens There is increasing evidence that stable donor chimerism can be achieved with sub-lethal doses of TBI and immunosuppressive drugs [196–198]. Partial, but stable, grafts would be of benefit for patients with nonmalignant diseases, such as thalassemia or sickle cell anemia, if they could be achieved without transplant-related mortality [199]. Patients with malignancies are often considered too old for high-dose regimens or, when young enough, have organ dysfunction that precludes administration of intensive regimens prior to allografting. In contrast to nonmalignant diseases, however, the successful treatment of cancers with allogeneic transplant almost certainly requires full donor engraftment in order to utilize the “graft-versus-leukemia” effect. It is of major interest to develop nontoxic methods of achieving donor chimerism that would then allow the evaluation of post-transplant immunotherapy with donor lymphocytes, which would then be relied on to produce the bulk of the antileukemic therapy [200]. Nonablative allografting strategies are discussed in detail in Chapter 71.

Summary Because of the relatively high probability of relapse in many patients receiving HCT, the further development of new treatment regimens is warranted. However, one can predict that it will continue to be difficult to identify more effective treatment regimens. Incremental

High-dose Preparatory Regimens

improvements in treatment regimens are expected to be small and difficult to measure, and will require the study of large numbers of patients. As HCT becomes more widely used, there will be a great need for controlled trials to evaluate the effectiveness of specific treatment regimens for specific groups of patients. Only centers with large numbers of patients or cooperative study groups can successfully perform the trials necessary to substantiate the effectiveness of a given treatment

327

regimen. In order to make high-dose therapies and PBHC support generally available, the procedure will have to be performed with low mortality and minimal morbidity in an outpatient setting. To achieve this goal without sacrificing efficacy, agents that protect normal, nonhematopoietic tissues from RRT may have to be utilized. In addition, studies utilizing targeted radioisotopes may allow further dose intensity to tumor-bearing tissues while sparing noninvolved organs.

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173. Scheinberg DA, Lovett D, Divgi CR et al. A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide. J Clin Oncol 1991; 9: 478–90. 174. Appelbaum FR, Matthews DC, Eary JF et al. Use of radiolabeled anti-CD33 antibody to augment marrow irradiation prior to marrow transplantation for acute myelogenous leukemia. Transplantation 1992; 54: 829–33. 175. Press OW, Eary JF, Appelbaum FR et al. Radiolabeled-antibody therapy of B-cell lymphoma with autologous bone marrow support. N Engl J Med 1993; 329: 1219–24. 176. Bierman PJ, Vose JM, Leichner PK et al. Yttrium 90-labeled antiferritin followed by high-dose chemotherapy and autologous bone marrow transplantation for poor-prognosis Hodgkin’s disease. J Clin Oncol 1993; 11: 698–703. 177. Papadopoulos EB, Caron P, Castro-Malaspina H et al. Results of allogeneic bone marrow transplant following 131I-M195/busulfan/cyclophosphamide (BU/CY) in patients with advanced/ refractory myeloid malignancies [Abstract]. Blood 1993; 82(Suppl 1): 80a. 178. Bayouth JE, Macey DJ, Kasi LP et al. Pharmacokinetics, dosimetry and toxicity of holmium-166DOTMP for bone marrow ablation in multiple myeloma. J Nucl Med 1995; 36: 730–7. 179. Press OW, Eary JF, Appelbaum FR, Bernstein ID. Myeloablative radiolabeled antibody therapy with autologous bone marrow transplantation for relapsed B cell lymphomas. In: Buckner CD, Clift R, editors. Technical and Biological Components of Marrow Transplantation. Boston: Academic Publishers; 1995. pp. 281–97. 180. Matthews DC, Appelbaum FR, Eary JF et al. Development of a marrow transplant regimen for acute leukemia using targeted hematopoietic irradiation delivered by 131I-labeled anti-CD45 antibody, combined with cyclophosphamide and total body irradiation. Blood 1995; 85: 1122–31. 181. Jurcic JG, Caron PC, Nikula TK et al. Radiolabeled anti-CD33 monoclonal antibody M195 for myeloid leukemias. Cancer Res 1995; 55: 5908s– 10s. 182. Gopal AK, Rajendran JG, Petersdorf SH et al. High-dose chemo-radioimmunotherapy with autologous stem cell support for relapsed mantle cell lymphoma. Blood 2002; 99: 3158–62. 183. Gopal AK, Gooley TA, Maloney DG et al. Highdose radioimmunotherapy versus conventional high-dose therapy and autologous hematopoietic stem cell transplantation for relapsed follicular non-Hodgkin lymphoma: a multivariable cohort analysis. Blood 2003; 102: 2351–7. 184. Rösch F, Herzog H, Plag C et al. Radiation doses of yttrium-90 citrate and yttrium-90 EDTMP as determined via analogous yttrium-86 complexes and position emission tomography. Eur J Nucl Med 1996; 23: 958–66. 185. Giralt S, Bensinger W, Goodman M et al. 166HODOTMP plus melphalan followed by peripheral blood stem cell transplantation in patients with multiple myeloma: results of two phase 1/2 trials. Blood 2003; 102: 2684–91. 186. Dispenzieri A, Wiseman GA, Lacy MQ et al. A phase I study of 153Sm-EDTMP with fixed highdose melphalan as a peripheral blood stem cell conditioning regimen in patients with multiple myeloma. Leukemia 2005; 19: 118–25.

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187. Shea TC, Flaherty M, Elias A et al. A phase I clinical and pharmacokinetic study of carboplatin and autologous bone marrow support. J Clin Oncol 1989; 7: 651–61. Erratum in: J Clin Oncol 1989; 7: 1177. 188. Fennelly D, Wasserheit C, Schneider J et al. Simultaneous dose escalation and schedule intensification of carboplatin-based chemotherapy using peripheral blood progenitor cells and filgrastim: a phase I trial. Cancer Res 1994; 54: 6137–42. 189. Vesole DH, Jagannath S, Glenn LD, Barlogie B. Allogeneic bone marrow transplantation (AlloBMT) in multiple myeloma (MM) [Abstract]. Proc Am Soc Clin Oncol 1993; 12: 405. 190. Ayash LJ, Elias A, Wheeler C et al. Double doseintensive chemotherapy with autologous marrow and peripheral-blood progenitor-cell support for metastatic breast cancer: a feasibility study. J Clin Oncol 1994; 12: 37–44. 191. Attal M, Harousseau J-L, Facon T et al. Double autologous transplantation improves survival of multiple myeloma patients: final analysis of a prospective randomized study of the “Intergroupe Francophone du Myelome” (IFM 94) [Abstract]. Blood 2002; 100(Part 1): 5a. 192. Cavo M, Tosi P, Zamagni E et al. Prospective, randomized study of single compared with double autologous stem-cell transplantation for multiple myeloma: Bologna 96 clinical study. J Clin Oncol 2007; 25: 2434–41. 193. Sung KW, Lee SH, Yoo KH et al. Tandem highdose chemotherapy and autologous stem cell resource in patients over 1 year of age with stage 4 neuroblastoma. Bone Marrow Transplant 2007; 40: 37–45. 194. Margolin KA, Doroshow JH, Frankel P et al. Paclitaxel-based high-dose chemotherapy with autologous stem cell rescue for relapsed germ cell cancer. Biol Blood Marrow Transplant 2005; 11: 903–11. 195. Fung HC, Stiff P, Schriber J et al. Tandem autologous stem cell transplantation for patients with primary refractory or poor risk recurrent Hodgkin lymphoma. Biol Blood Marrow Transplant 2007; 13: 594–600. 196. Storb R, Yu C, Wagner JL et al. Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood 1997; 89: 3048–54. 197. Storb R, McSweeney PA, Sandmaier BM et al. Non-myeloablative haematopoietic stem cell transplants. In: Schultze W, editor. Proceedings of the 4th Stem Cell Workshop: High-dose Therapy and Transplantation of Haematopoietic Stem Cells. Vienna: Blackwell WissenschaftsVerlag Berlin; 2000. pp. 170–4. 198. Storb R, Sandmaier BM, Forman S et al. What role for nonmyeloablative conditioning in allogeneic hematopoietic cell transplantation? Acta Haematolo Polonica 2003; 34(Suppl 1): 17–20. 199. McSweeney PA, Niederwieser D, Shizuru JA et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versustumor effects. Blood 2001; 97: 3390–400. 200. Mackinnon S, Papadopoulos EB, Carabasi MH et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of

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206. Crilley P, Lazarus H, Topolsky D et al. Comparison of preparative transplantation regimens using carmustine/etoposide/cisplatin or busulfan/ etoposide/cyclophosphamide in lymphoid malignancies. Semin Oncol 1993; 20: 50–4. 207. Gulati SC, Shank B, Black P et al. Autologous bone marrow transplantation for patients with poor-prognosis lymphoma. J Clin Oncol 1988; 6: 1303–13. 208. Meloni G, De Fabritiis P, Petti MC, Mandelli F. BAVC regimen and autologous bone marrow transplantation in patients with acute myelogenous leukemia in second remission. Blood 1990; 75: 2282–5. 209. Zulian GB, Selby P, Milan S et al. High dose melphalan, BCNU and etoposide with autologous bone marrow transplantation for Hodgkin’s disease. Br J Cancer 1989; 59: 631–5. 210. Lazarus HM, Crilley P, Ciobanu N et al. Highdose carmustine, etoposide, and cisplatin and autologous bone marrow transplantation for relapsed and refractory lymphoma. J Clin Oncol 1992; 10: 1682–9. 211. Peters WP, Stuart A, Klotman M et al. High-dose combination cyclophosphamide, cisplatin, and melphalan with autologous bone marrow support. A clinical and pharmacologic study. Cancer Chemother Pharmacol 1989; 23: 377–83. 212. Ellis ED, Williams SF, Moormeier JA et al. A phase I–II study of high-dose cyclophosphamide, thiotepa and escalating doses of mitoxantrone with autologous stem cell rescue in patients with

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23

Jeffrey Y.C. Wong & Timothy Schultheiss

Radiotherapeutic Principles of Hematopoietic Cell Transplantation

Introduction Since the initial pioneering efforts of Thomas and colleagues [1], radiation therapy continues to be an important part of conditioning regimens in patients undergoing hematopoietic cell transplantation (HCT). Irradiation is used primarily as a form of systemic therapy utilizing highenergy photons and large fields to deliver total body irradiation (TBI). In addition, total lymphoid irradiation (TLI), total abdominal irradiation, and locoregional irradiation directed to areas of higher tumor burden have also been combined in HCT conditioning regimens. A primary role of TBI is the eradication of malignant cells. In patients undergoing allogeneic HCT, TBI also provides a powerful means of immunosuppression to prevent rejection of donor hematopoietic cells. TBI is often combined with chemotherapy in conditioning regimens and offers distinct advantages compared with chemotherapy. Unlike chemotherapy, delivery of radiation therapy to the tumor site is not dependent on blood supply or influenced by interpatient variability of drug absorption, metabolism, biodistribution or clearance kinetics. Radiation therapy can reach potential sanctuary sites, such as testes and brain. In addition, chemotherapy-resistant clones that develop may still be sensitive to irradiation. Unlike chemotherapy, the dose of radiation can be preferentially distributed to areas of greatest tumor burden and restricted to critical normal organs. Past efforts utilized shielding to reduce the dose to critical organs such as the lung, kidneys, and liver, or delivered additional locoregional radiation therapy as boost treatment to areas of greatest tumor burden. Recent advances in the laboratory and in radiation therapy delivery systems now allow for new and novel opportunities to selectively target radiotherapy in the setting of HCT. Biologically targeted systemic radiotherapy, using radiolabeled antibodies [2] or bone-seeking radiopharmaceuticals such as 166Ho-DOTMP [3], have been evaluated as part of conditioning regimens in clinical trials. Rapid advances in the delivery of external beam radiotherapy using intensity-modulated radiation therapy coupled with image-guided radiation therapy now allow the radiation oncologist to “sculpt” the dose to tailor the dose distribution to the unique shape of each patient’s tumor. These advances are being translated to large-field applications and are being actively investigated as a means of image-guided targeted TBI or total marrow irradiation (TMI) [4,5].

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

This chapter will provide an overview of the principles and concepts in radiobiology and radiation physics that are important in optimizing TBI. An understanding of these principles is essential to minimize toxicities, maximize tumor cell eradication, and optimize the therapeutic ratio of this important treatment modality. Dose to tumor, dose to organs, volume of critical organ receiving a given dose, fraction size, number of fractions, time between fractions, dose rate, beam energy, uniformity of dose distribution within the target region, and reproducibility of patient alignment and set-up are all critical factors to consider in optimizing outcomes. The clinical results and normal tissue toxicities of TBI will be reviewed and will provide examples of how a rational application of radiobiology and radiation physics has led to improved outcomes. Finally, the rationale, methods, and clinical results of the new, targeted TBI strategies utilizing image-guided radiation therapy and intensitymodulated radiation therapy technologies will be described.

Radiobiologic principles It is now clear that fractionated TBI is superior to single-fraction TBI [6,7]. It is unclear why this was ever in question, since single-fraction treatments have never been found to be superior to fractionated radiation treatments when fractionation was an option. The issue of dose rate has not really been addressed systematically. In examining dose-rate effects, most studies have ignored the important influence of fractionation on the dose-rate effect, and have also ignored the fact that dose-rate effects will be different for every different cell type irradiated. Thus, the fundamental radiobiologic questions that remain in TBI are how should the treatment be fractionated and, given this fractionation, is there a doserate effect in the cell types of interest that is significant enough to be worth pursuing? The fundamental response to radiation is generally described by a cell survival function. This function gives the probability, S, that a given cell will survive a dose, d, of radiation. Of course, S depends upon the type of cell, its phase in the cell cycle, and the conditions under which it is irradiated, including the type of radiation. There are many mathematical models that describe the dependence of S on d [8]. If cells were killed as a result of a single event whose probability of occurrence was dose dependent, then the cell survival curve would be exponential. However, there is a shoulder on most mammalian cell survival curves, followed by an exponential fall-off at higher doses (Fig. 23.1). Two shape parameters are used to describe empirically the cell survival curve: the extrapolation number, n, and the inverse of the slope of the high-dose region, d0. If cell survival were purely exponential with no shoulder, d0 would

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Chapter 23

1

Log (cell survival)

0

–1 D

–2

C

–3

B A

–4 0

2

4

6

8

10

Dose (Gy) Fig. 23.1 Cell survival curve for multihit single target model. Curve A shows cell survival for a single dose. Curve C shows cell survival following a completely repaired single dose of 2 Gy. Curve B shows cell survival after incomplete repair of the initial 2 Gy fraction. Curve D shows cell survival for multiple 2 Gy fractions.

be the mean lethal dose; n is the value of the y-intercept of the extrapolated straight line portion of the curve. An alternative model of cell survival, and the one in more common clinical use today, is the linear-quadratic model [9]. In this model, cell survival is given by: S=e

− αd − β d 2

(1)

where the first term in the exponent can be interpreted as being the single-hit component of cell killing (e.g. double-strand breaks) and the second term is the two-hit component (e.g. single-strand breaks). Clearly, this model is incompatible with a model that has a linear high-dose behavior (in log S), but over the range of usual applicability (less than 10 Gy), the models are often indistinguishable. For N fractions, cell survival is given by: N

2 S = ⎡ e − αd −βd ⎤ = e − αD −βDd ⎣ ⎦

(2)

where D = d · N is the total dose given in N fractions of size d. The shoulder of the cell survival curve is a result of the repair of sublethal damage [10]. Sublethal damage consists of intracellular lesions that are not lethal by themselves, but may combine with other such lesions to produce a lesion that leads to cell death. For example, a singlestrand break on the DNA would not be lethal, but two single-strand breaks in close proximity would produce a lethal double-strand break. Repair of sublethal damage is thought to be complete in less than 24 hours, the normal interfraction interval in radiation oncology. However, mean repair times are generally estimated to be in the range of 1–4 hours,

a time that becomes important when considering treatments with multiple fractions per day (hyperfractionation). During this repair time, the shoulder on the cell survival curve is being restored, so that when repair is complete, an additional fraction has the same probability of killing a cell as the initial fraction of the same dose. If there is no repair, the two doses add arithmetically as if they were given in one single dose, producing a much lower cell survival. If the repair is partially complete, the cell survival is intermediate. This effect is depicted in Fig. 23.1. Curve A shows a single-dose cell survival curve; curve C shows the cell survival curve for a second dose following an initial dose of 2 Gy and complete repair. Curve B is the same as curve C except that the timing of the second dose is such that repair is not complete after the initial 2 Gy. Curve D shows the cell survival curve for a series of 2 Gy fractions with complete interfraction repair. If there is no shoulder on the cell survival curve, there is no advantage to fractionation in terms of cell survival. The same mechanism that gives rise to the shoulder on the cell survival curve is also responsible for the dose-rate effect. If the cell survival curve has no shoulder, it is generally believed that there will be no doserate effect and there will also be no fractionation effect. As opposed to most normal tissues, both leukemia and lymphoma cells are usually seen to have little or no shoulder. Therefore, in HCT, a therapeutic benefit is gained by lowering the dose rate or fractionating the dose, but not necessarily both. The more the dose is fractionated, the more cells that are killed by the single-hit mechanism, and the less impact the dose rate will have. In clinical radiation therapy, we must consider the cellular response in vivo. The response of hematologic malignancies to radiation is likely to be modeled appropriately by a cell survival model as discussed above. Solid tumors may demonstrate effects resulting from their organization, such as bystander effects and responses to the radiation damage and cell death of neighboring cells. Nonetheless, it is ultimately the response of the malignant clonogens that governs the therapeutic result, and therefore the cell survival model should hold. For normal tissues, the responses are more complex, especially for late effects. Even in this case, it is common to assume that there is a target cell type or a collection of target cells whose collective response may be described by: E = αD + βDd

(3)

where E is the level of effect necessary to elicit the response in the normal tissue or organ in question. (If the response in question is governed by the survival curve of a single cell type, then E = −ln S in equation (2); otherwise, E is representative of a level of effect that depends upon cell survival in an unspecified way.) The level of effect is assumed to be dependent upon the level of cell survival of the target cell. Equation (3) is an isoeffect equation, which means that different values of D and d that yield a similar value of E will produce similar effects in the organ whose radiobiologic response is determined by the values of α and β. The ratio of α and β, α : β, has clinical implications [10]. Higher values of α : β mean that the tissue will be less sensitive to a specific fractionation schedule and more dependent on the total dose. Lower values mean that the tissue’s response will be more sensitive to dose per fraction. Generally, late responses are characterized by lower values of α : β (1–3 Gy), whereas acute responses are associated with higher values (~10 Gy). To conclude, it is clear that fractionated TBI is less toxic than singledose TBI for the same level of malignant cell kill. Most TBI regimens are designed to be completed in a week, and therefore to increase fractionation beyond five fractions one must treat with multiple fractions per day. To maximize repair of sublethal damage, an interfraction interval of at least 6 hours should be used. Not only does this make three frac-

Radiotherapeutic Principles of Hematopoietic Cell Transplantation

tions per day logistically difficult, but the radiobiological impact of three fractions per day may be unpredictable. However, there are a number of standard fractionation regimens that employ three fractions per day successfully. Comprehensive statistical analysis of the comparative results from one, two, and three fractions per day is not yet available.

Radiation physics and physical considerations There are a large number of papers and reports that give details on the planning and delivery of TBI [11,12]. Discussed below are the principles that guide this treatment. TBI treatment is the least conformal treatment that is given in radiation oncology. Of course, the primary technical concern is to deliver the desired dose to the entire patient. The most straightforward way to accomplish this objective is to irradiate the entire patient in a single radiation beam. This is accomplished by using a large (approximately 4 m) source-to-skin distance and the largest practicable field size. The large source-to-skin distance reduces the dose rate to about 10% of that which is typically used in radiation therapy. The typical field arrangement is to have the patient standing on a specially designed stand, to rotate the collimator by 45o to utilize the diagonal dimension of the field, to use lead strips as compensating filters to achieve a uniform dose rate (at midline) from head to foot, to use a Plexiglas barrier close to the patient to defeat the skin-sparing effect of high-energy photons, and to block critical organs (usually lungs, sometimes kidneys, liver, and previously irradiated sites) to lower the dose to these tissues. The patient is treated with anterior and posterior fields, often given on alternating treatment sessions. Alternatively, the patient may be treated while lying on a gurney or a specially designed table at floor level. Treating on or near the floor is common in pediatric cases where the field size requirements are not as great. Because the source-to-skin distance is shorter, this technique will produce a higher dose rate unless the machine’s output rate can be electronically adjusted. On modern C-arm linear accelerators, the dose rate at isocenter is adjustable. This is often used to achieve a dose rate dictated by a particular clinical trial. The selection of beam energy is not critical, most treatment centers using photon energies between 6 and 10 MV. Photons with these energies have a skin-sparing effect produced by the build up of forward-scattered electrons until electronic equilibrium is established. (Electronic equilibrium is characterized by the same number and energy of electrons being scattered into a region as are scattered out of the region.) Because of this reduced skin dose, a beam spoiler of low atomic number material is placed between the patient and the source. Electrons scattered from this material to the patient increase the skin dose. This is done to achieve full dose up to the skin surface, but the necessity to treat to that level because of the presence of clonogens has not been demonstrated. The dose is usually measured at several points on the patient’s body with the goal of achieving a uniform dose within ±5%. To achieve this, a compensating filter is constructed of thin lead sheets on a Plexiglas tray inserted in the beam. This filter is designed to compensate for both the variations in patient thickness and the variation in beam intensity away from the center of the beam. Most treatment regimens now require the shielding of certain sensitive structures, with lung being the most common of these. One reason that the anteroposterior/posteroanterior fields are often preferred is that they allow the most accurate method for supplementing the dose to the chest wall when the lung is blocked. If no lung blocking is used, the dose to the lung is higher than the dose to midline by as much as 10%. This increase is a result of the reduced attenuation in the lung, whose density is about one-third of most soft tissues. Typically, when lung blocks are used, the blocks cover the central portion of the lung, with about 1–2 cm between the edge of the lung shadow on the film and the edge of the

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block. It is also typical not to use full-thickness lung blocks, which have a transmission factor of 3%, but rather to use blocks that transmit 50% of the primary beam. The areas of the chest wall that are shielded by the lung blocks are then treated with supplemental electron fields whose beam energy and dose contribution is calculated to bring the dose to the pleural surface to the prescribed dose. In Plate 23.1, we show the dose distribution at the level T5–6 for a TBI treatment of 12 Gy total dose with 50% transmission blocks over the lungs and supplemented with 9 MeV electrons. The effect of the beam spoiler is not included.

High-dose TBI-containing regimens: clinical results Most of the published clinical experience using TBI is in combination with high-dose systemic chemotherapy. Some of the agents frequently administered with TBI include cyclophosphamide (CY) [13], etoposide [14], melphalan [15], cytarabine [16], and thiotepa [17]. These agents are cytotoxic and result in acute and late toxicities that are often similar to those of TBI, including nausea, vomiting, mucositis, esophagitis, diarrhea, hepatic dysfunction, pneumonitis, gonadal dysfunction, and induction of second malignancies. Therefore, identifying toxicities and outcomes solely attributable to TBI is difficult, and the published literature more accurately represents the clinical results of TBI-containing regimens as opposed to TBI alone. Acute toxicities associated with high-dose TBI-containing regimens Fractionated (one fraction per day) or hyperfractionated (multiple fractions per day) TBI schedules, compared with single-fraction schedules, result in reduced acute toxicities and are better tolerated [18–20]. This is a primary reason why single-fraction TBI schedules are less utilized today. Nausea and vomiting are the most common side-effects observed, occurring in approximately 35–50% of patients [18,21,22]. In studies conducted prior to the use of modern antiemetics, Spitzer et al. [23] reported emesis in all patients after the first 1.2 Gy fraction of hyperfractionated TBI, but a decline with subsequent fractions. Buchali et al. [21], using a 12 Gy hyperfractionated regimen, reported an increasing incidence of emesis with each 1.5 Gy fraction up to the third fraction, with a constant incidence with subsequent fractions. Antiemetics such as ondansetron and granisetron can significantly reduce the incidence and severity of emesis [24,25]. Transient parotiditis occurs in approximately 10–30% of patients undergoing fractionated TBI, with a higher incidence (25–75%) associated with single-fraction schedules [21,22]. The onset is usually after the first fraction, with resolution over the next 24–48 hours. Xerostomia and xerophthalmia can occur in approximately 15–30% [21,22]. Mucositis is also observed in approximately 40–100% of patients in the days after completing TBI and can be influenced by the chemotherapy used. For example, mucositis is a primary acute toxicity with etoposidecontaining regimens [14]. Mucositis observed with TBI combined with high-dose melphalan limited the feasibility of this combination [26]. Diarrhea can also occur in the days after completing TBI in 10–35% of cases. Other acute side-effects include fatigue, decreased appetite, erythema, esophagitis, and alopecia. Recent studies provide some indication of the acute toxicities primarily related to TBI alone. Zaucha et al. [27], in a phase I dose-escalation trial, treated patients with a tandem autologous HCT approach, first with high-dose busulfan (BU), melphalan, and thiotepa, followed a median of 105 days later by TBI to a total dose of 12, 13.5 or 15 Gy at 1.5 Gy twice a day. Acute toxicities were primarily grade 1–2 (by the Bearman regimen-related toxicity scale [28]), with mucositis occurring in 100%. Gastrointestinal, skin, renal, and hepatic grade 1–2 acute toxicities

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occurred in 34%, 19%, 9%, and 9%, respectively. In a limited phase I dose-escalation study, McAfee et al. [29] reported mucositis in 88% and nausea in one-third of patients receiving 16, 18 or 20 Gy TBI alone at 2 Gy twice a day. High-dose TBI schedules utilized The first use of TBI reported by Thomas et al. [1] in a twin HCT study delivered the dose in a single fraction. Although engraftment was successful in both patients, relapse occurred within 12 weeks, suggesting that TBI alone was insufficient to prevent relapse. The same group subsequently added CY prior to TBI because of concerns of tumor lysis syndrome resulting from a single fraction of high-dose TBI. CY also provided additional immunosuppressive effects. Since then, high-dose chemotherapy, usually CY, has been combined with TBI. Typical TBI schedules deliver a total dose of 10–16 Gy at 1.2 Gy three times a day, 2 Gy twice daily or 3 Gy four times a day. A variety of TBI schedules have been evaluated to reduce toxicities, decrease relapse rates, and improve further the therapeutic index. Factors that are of radiobiologic importance include total dose, fraction schedule, fraction size, use of organ shielding, and dose rate. Only a few randomized trials have been performed, and most trials involve changes in multiple variables, making determination of the optimal TBI schedule difficult. Single-fraction versus multifraction schedules Early studies compared fractionated with single-fraction schedules. Fractionation offered the potential to reduce organ toxicities without compromising tumor control, given the greater ability of most normal tissues to repair radiation sublethal damage compared with leukemia cells [7]. Retrospective analyses from the groups at Johns Hopkins University [30] and Memorial Sloan Kettering Cancer Center [13] reported a reduction in interstitial pneumonitis (IP) with lung shielding and fractionated TBI (FTBI) (one fraction a day) or hyperfractionated (HFTBI) (multiple fractions a day) schedules compared with single-fraction TBI (STBI) and no lung shielding. An update of the Memorial Sloan Kettering Cancer Center experience by Shank et al. [19] reported a reduction of IP incidence from 50% with STBI to 4% with HFTBI and lung shielding. Subsequent studies comparing single versus multifraction schedules have reported mixed results [18,31,32]. Ozsahin et al. [18] found no significant differences in relapse rate, survival or incidence of IP or sinusoidal obstructive syndrome (SOS; formerly known as venoocclusive disease) in patients receiving 10 Gy STBI compared with 12 Gy (2 Gy twice daily) HFTBI. Lung dose was limited to 8 Gy in the SFTBI group and 9 Gy in the HFTBI group. A report analyzing the experience of multiple institutions in the Children’s Cancer Study Group [31] of patients with acute myeloid leukemia (AML) in first remission treated with 7.5–10 Gy STBI or 12–13.2 Gy FTBI (2 Gy twice a day or 1.2 Gy three times a day) reported a reduction in 2-year relapse rate from 23% to 0% (p = 0.07) in favor of HFTBI, but no significant differences in survival or incidence of IP. A single institution experience by Cosset et al. [33] observed no difference in relapse rate or overall survival, but documented a reduction in treatment-related mortality (55% versus 26%) and a statistically significant reduction in incidence of IP (45% versus 13%) and graftversus-host disease GVHD (77% versus 43%) with 13.2 Gy (1.2 Gy three times a day) HFTBI compared with 10 Gy STBI. Lung doses were comparable between the two groups (8–9 Gy). Finally, a large retrospective analysis of the experience from 21 institutions in France [32] also reported a significant reduction in incidence of IP (37.5% versus 1.7%) and GVHD (41% versus 22%) with a hyper-

fractionated schedule (10–13.2 Gy) compared with a single fraction of 10 Gy. Lung dose was 7.3 Gy in the hyperfractionated group and 8 Gy in the single-fraction group. However, no change in survival and an increase in relapse rate were observed with hyperfractionation (29% versus 16%; 0.5 < p < 0.1). Two randomized trials have compared single-fraction with multifraction TBI. A trial from Seattle [34] compared 10 Gy STBI with 12 Gy HFTBI at 2 Gy twice a day in patients with AML in first remission. HFTBI resulted in a reduction in idiopathic IP (4% versus 11%), a decrease in relapse rate (12% versus 22%) and a statistically significant increase in 5-year overall survival of 54% versus 33%. A more recent French study [35] randomized patients to receive 10 Gy STBI or 14.85 Gy HFTBI at 1.35 Gy three times a day and reported a statistically significant decrease in SOS (4% versus 14%) with HFTBI. No difference in incidence of IP was observed between the two groups. Eight-year cause specific and overall survival with HFTBI were 77% and 45%, respectively, compared with STBI, which had figures of 63.5% and 38%, respectively. TBI schedule was an independent prognostic factor by multivariate analysis if cause-specific survival was the endpoint. In summary, most studies document a reduction in toxicities with fractionated or hyperfractionated TBI schedules. This is consistent with the radiobiologic principles described earlier. Many studies, including the two randomized trials, also document improved relapse and survival rates with multifraction as opposed to single-fraction schedules. The majority of TBI schedules used today are fractionated or hyperfractionated. Total dose and dose escalation Fractionated schedules and the resultant reduction in toxicities allow for the possibilities of increasing total dose and improving outcomes. The Seattle group carried out two randomized trials comparing CY combined with 12 Gy at 2 Gy/day or 15.75 Gy at 2.25 Gy/day. In a trial of 116 patients with chronic myeloid leukemia (CML) in chronic phase, the higher dose resulted in a significantly lower relapse rate (0% versus 25%; p = 0.008) but a higher treatment-related mortality rate (24% 12 Gy and 34% 15.75 Gy; p = 0.13), and as a result no significant change in overall survival [36]. In a separate report of 71 patients with AML in first remission, relapse rate was also decreased with the higher dose (14% versus 39%; p = 0.06), but these gains were offset by an increase in nonrelapse mortality (38% versus 19%; p = 0.05), resulting in no difference in overall survival between the two arms [37]. Reports from other groups also suggest that higher doses have greater antileukemia effects. In several retrospective reports from the group in Genoa [38], relapse rate was significantly decreased if TBI dose was greater than 9.9 Gy (3.3 Gy/day). In an analysis of the Center for International Blood and Marrow Transplant Research and City of Hope Cancer Center databases, Marks et al. [39] recently reported that patients with acute lymphoblastic leukemia (ALL) beyond first remission receiving TBI/CY conditioning regimens had a lower relapse rate and increased disease-free survival if the TBI dose was 13 Gy or more. Phase I trials have been performed with increased toxicities but have no obvious benefit with increasing TBI dose [40–42]. Petersen et al. [41] determined a maximum tolerated dose of 16 Gy (2 Gy twice a day) when combined with CY 120 mg/kg. The primary dose-limiting toxicity was pneumonitis, followed by SOS, nephrotoxicity, and mucositis. Demirer et al. [40], also using a TBI/CY regimen, observed no additional benefit with higher total doses of 14.4 Gy at 1.2 Gy three times a day in patients with advanced ALL and non-Hodgkin’s lymphoma (NHL). Alyea et al. [42] escalated the dose in a TBI/CY regimen from 14 to 15.6 Gy. Acute GVHD and treatment-related mortality were higher at 15.6 Gy, but no improvement in relapse rate or overall survival was observed.

Radiotherapeutic Principles of Hematopoietic Cell Transplantation

Other phase I trials have escalated TBI doses without concomitant chemotherapy. Results of a limited phase I TBI dose-escalation trial without chemotherapy were recently reported in patients with NHL undergoing autologous HCT [29]. Fifty percent lung transmission blocks and blocks to limit the kidney dose to 15 Gy were utilized. The highest dose reached was 20 Gy (2 Gy twice a day), which was felt to be associated with acceptable toxicities. IP or SOS was seen in only one of nine patients. A dose-escalation trial of TBI without concomitant chemotherapy was also recently carried out by the Seattle group [27]. Patients with primarily bone-only metastatic disease from a variety of hematologic and solid tumors first received high-dose chemotherapy (BU, melphalan, and thiotepa) and then a median of 105 days later underwent TBI as part of a tandem autologous HCT trial. Although 90% transmission lung and liver blocks were utilized, the highest TBI dose achieved was only 15 Gy with grade 3 and 4 IP being dose-limiting. In summary, despite the use of fractionated schedules and organ shielding, escalation of TBI dose has been difficult due to dose-limiting normal tissue toxicities, which have primarily been pulmonary. This has limited total doses of most conditioning regimens to approximately 16 Gy or less. Trials attempting to demonstrate improved relapse and survival rates with increasing TBI dose have reported mixed results, perhaps due to the fact that the magnitude of dose escalation has been limited. Gains in disease control are associated with an increase in regimen-related toxicities and nonrelapse mortality, resulting in no improvement in overall survival. New strategies are clearly needed to allow further dose escalation without an associated increase in side-effects. Fractionated versus hyperfractionated schedules and fraction size Daily fractionated schedules have used fractions of approximately 2.25– 4.67 Gy. Hyperfractionated schedules are usually delivered at 1.2–2 Gy fractions a minimum of 5–6 hours apart. In general, larger fractions are predicted to be associated with greater normal organ effects, potentially reducing the therapeutic index. To date, no randomized trials have been performed specifically addressing these factors. The available clinical data suggest that larger daily fraction schedules are acceptable [43,44], although most schedules using fractions of 3 Gy or more have limited the total dose to 9.9–12 Gy. In a retrospective analysis of patients with high-risk NHL undergoing autologous HCT, Gopal et al. [45] compared groups treated with 10.2 Gy at 1.7 Gy twice a day with those treated to 12 Gy at 3 Gy/day. No differences in overall survival or in acute or late pulmonary complications were seen. The 3-year freedom from late pulmonary complications rate was 80% for the 10.2 Gy group and 70% for the 12 Gy group (p = 0.45). Three-year freedom from progression was significantly lower for the 12 Gy group (82% versus 31%; p < 0.001). Of note, lung shielding was not utilized for the 10.2 Gy group, but it was utilized in the 12 Gy group after the third fraction. In addition, the total dose of 10.2 Gy is lower than that delivered by other hyperfractionated schedules, raising the possibility that the two schedules did not deliver biologically equivalent doses. This may explain in part the differences observed. Consistent with this are the results of Corvo et al. [46], who retrospectively reviewed their experience with patients undergoing unrelated donor HCT. Comparisons were made between patients treated to 9.9 Gy at 3 Gy/day to those treated during an earlier period to 12 Gy at 2 Gy twice a day. Lung shields were used in both groups. The actuarial 5-year transplant-related mortality rate was 31% for the HFTBI group and 41% for the FTBI group (p = 0.1). For the HFTBI group, there was a statistically significant improvement in the 5-year leukemia relapse rate (13% versus 31%; p = 0.04) and overall survival rate (68% versus 51%; p =

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0.01). TBI schedule was an important predictor of overall survival and leukemia relapse rate by multivariate analysis. The authors concluded that the improved results were primarily related to the higher total doses delivered with the HFTBI schedule.

Dose rate TBI is delivered at relatively low dose rates usually in the range of 5– 30 cGy/min. This is lower than those used with standard external beam radiotherapy to more localized sites in the body, which are typically greater than 100 cGy/min. The use of lower dose rates is a result of the physical limitations encountered with TBI delivery, which require the patient to be treated at a much further distance from the beam source than conventional radiotherapy to be able to encompass the entire body in a large radiation field. Radiation delivered at a lower dose rate can potentially be more organ sparing than the same dose delivered at a higher dose rate, particularly for nonhematopoietic tissue. Therefore, the use of lower dose rates has the potential to further improve the therapeutic ratio of TBI. However, dose-rate effects may be greatest and most clinically important primarily at very low dose rates. Travis et al. [47] evaluated out to 1-year survival and histologic changes in mice receiving TBI at dose rates ranging from 1 to 25 cGy/min. A direct correlation was seen between dose rate and survival, as well as dose rate and histologic changes in lung and kidney. The dose-rate effects were greatest for dose rates ranging from 1 to 5 cGy/min. Consistent with this are the observations of Weiner et al. [48], who reported on 932 patients in the International Bone Marrow Transplant Registry database treated from 1978 to 1983 and observed a decrease in incidence of pneumonitis with lower dose rate, but only in patients treated at less than 6 cGy/min. The radiobiologic and potential clinical benefits of using lower dose rates are probably outweighed by other factors such as fractionation or organ shielding. Sampath et al. [49], in a multivariate logistic regression analysis of TBI in 1090 patients from 20 published studies, found an inverse correlation between incidence of pneumonitis and fractionation, but no dose-rate effect. A preclinical study by Tarbell et al. [50] reported a higher radiation-related mortality rate in mice receiving STBI at a high dose rate (80 cGy/min) compared with STBI at a low dose rate (5 cGy/ min). Dose-rate effects were not seen with twice or three times a day fractionation schedules, suggesting that fractionation was a greater factor in normal organ sparing than dose-rate effects. In a multi-institution randomized trial from France [18], 157 patients were randomized to either higher dose-rate or lower dose-rate groups. TBI was delivered either as a single fraction of 10 Gy or hyperfractionated to 12 Gy at 2 Gy twice a day, determined by physician preference rather than randomization. For the 10 Gy STBI group dose rate was 6 versus 15 cGy/min, with 3 versus 6 cGy/min for the 12 Gy HFTBI group. No statistically significant difference in survival or incidence of IP or SOS was seen with dose-rate or fractionation schedule. Of note, the highest estimated 4-year incidence of IP occurred in the singlefraction, high dose-rate group (49.1%), compared with 29.5% in the single fraction, low dose-rate group. The incidence of cataracts was reduced in the lower dose-rate groups (3.5%) compared with the higher dose-rate groups (14%). A subsequent retrospective analysis of 326 patients by Belkacemi et al. [20] compared patients treated at low (<4.8 cGy/min), medium (4.8–9 cGy/min), and high (>9 cGy/min) dose rates, and also reported a statistically significant reduction in cataract incidence of 0% with the lower dose rate, compared with 27% with the medium and 33% with the higher dose rates (p = 0.0001). A significant decrease in IP was also seen, with a 48% incidence for the higher dose-rate versus a 19%

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incidence for the medium dose-rate groups (p = 0.05). Survival and treatment-related mortality rates were not influenced by dose rate. More recently, Carruthers and Wallington [51] retrospectively analyzed their institution’s experience of 84 patients treated with 12 Gy TBI (2 Gy twice daily) at either 15 or 7.5 cGy/min. IP was observed in 43% of those treated at the higher dose rate compared with only 13% at the lower dose rate. A similar conclusion was reached by Beyzadeoglu et al. [52] in an analysis of 105 patients, where incidence of IP was greater in patients treated at a dose rate over 4 cGy/min. Higher dose rates in the order of 30–90 cGy/min have been used with acceptable toxicities, but total dose was limited to 5–5.5 Gy, making direct comparisons with other studies difficult [53,54]. Bredeson et al. [55] reported acceptable toxicity in 142 patients receiving TBI at 80 cGy/min. Although the majority received 5 Gy in a single fraction, 48 patients received 12 Gy at 2 Gy twice a day without lung shielding. In summary, although the range of dose rates utilized for TBI is limited, reducing dose rate within this range appears to result in a reduction in incidence of cataracts and, in some reports, pneumonitis. This appears to occur without a detrimental effect on survival or relapse rates in most studies. The clinical data suggest that dose-rate effects can be seen with single fraction as well as multifraction schedules, although data from animal models indicate a reduction in dose-rate effects with fractionation. Based on the available clinical data, the importance of dose-rate effects on TBI outcomes at levels greater than approximately 30 cGy/min is unknown. Organ shielding The primary dose-limiting toxicity of TBI is pulmonary. The use of shielding to reduce the total dose to the lungs has been shown to decrease the incidence of pneumonitis and is now routinely used at many centers. Shielding can be achieved by using full transmission blocks after a certain number of fractions, or through the use of “thinner” partial transmission blocks throughout the treatment course. Median lung doses with shielding have usually been limited to approximately 8–10 Gy. Shank et al. [19] reported a reduction of IP incidence from 50% with STBI and no lung shielding to 4% with HFTBI and lung shielding. Volpe et al. [56] reported a 3.8% incidence of lethal pulmonary complications if the lung dose was 9.4 Gy or less compared with 14.3% if it was over 9.4 Gy. Lawton et al. [57] noted lethal pulmonary events in six of 11 cases with 50% lung transmission blocks compared with 22 of 107 if lung shielding with 60% transmission blocks were used. A randomized trial was performed by Labar et al. [43] to assess the impact of lung shielding. Eighty-two patients received a TBI dose of 12 Gy (4 Gy/day), and lung blocks if used were placed after 9 Gy. The incidence of IP decreased from 15% to 5% with the use of lung blocks. No difference in leukemia-free survival at 3 years was seen between the two groups. Renal shielding has also reduced nephrotoxicity after TBI. The group at the Medical College of Wisconsin noted, at 18 months, a 29% incidence of nephropathy without shielding compared with 0% with kidney shielding in patients receiving 14 Gy HFTBI [58]. A recent study explored the use of extensive lung and liver shielding in patients with multiple myeloma undergoing autologous HCT [59]. Patients first received induction vincristine/adriamycin/dexamethasone, followed by TBI/BU/CY using 90% transmission lung and liver blocks with electron boosting to portions of the chest wall underlying the blocks. The total dose was 9 Gy at 1.5 Gy twice a day. A complete response rate of 48% and 41% was seen in patients with de novo and pretreated multiple myeloma, respectively. Transplant-related mortality rate was 2%. Grade 3/4 mucositis was observed in 75% of patients, and three patients developed reversible SOS. The authors conclude that, with

lung and liver shielding, TBI can be combined with BU and CY with acceptable toxicity and encouraging response rates. One group has reported a potential detrimental effect of shielding. A study from the Seattle group [60] found that shielding increased relapse rates in patients with refractory anemia undergoing allogeneic HCT. In this study, 14 patients were treated with CY and TBI to 12 Gy at 2 Gy twice a day (10 patients) or less than 12 Gy (four patients) utilizing 95% transmission lung and right hepatic lobe blocks. The underlying chest wall was then boosted with electrons using an approach similar to that reported by Shank et al. [19]. When compared with historical controls, there was an increase in relapse rate (34% versus 2%; p = 0.0004) and a decrease in disease-free survival (38% versus 61%; p = 0.16). No difference in nonrelapse mortality was seen. The authors hypothesized that 95% shielding may have shielded malignant cells or reduced immunosuppression and the graft-versus-leukemia effect.

High-dose TBI-containing regimens: normal tissue effects There are numerous side-effects and complications from HCT. It is often not possible to determine whether these normal tissue injuries are a result of the radiation, the chemotherapy or a combination of the two. In this section, we will address primarily those complications which limit the dose of radiation that can be given. Other complications that may be due at least in part to the radiation, but either occur at a low incidence for most transplant regimens, are not associated with sufficient morbidity to limit the dose, or do not appear to demonstrate a dose response will be enumerated but not discussed in detail. GVHD is known to increase the risk of some of these complications, and can occur with TBI as well as non-TBI-containing conditioning regimens. In some clinical studies, TBI does not appear to increase the risk of GVHD over non-TBI conditioning regimens [61–63], although one randomized trial reported a greater incidence of acute GVHD with TBI/CY versus BU/CY [64]. Development of GVHD is probably related more to the intensity of the regimen rather than to the TBI itself. In experimental models, the incidence of GVHD is increased with higher TBI doses [65] or with the addition of CY to TBI [66]. The initiating event is thought to involve damage to the intestinal mucosa by the conditioning regimen, resulting in a release of lipopolysaccharide into the circulation, release of tumor necrosis factor-alpha, and a subsequent increase in expression of host major histocompatibility antigens, enhancing recognition by donor T cells. In summaries of the HCT literature, it is very common to compare the outcomes for single-fraction versus fractionated dose schedules without regard to the total dose or dose per fraction. Perhaps implicit in all such comparisons is that the regimens being compared are assumed to have equal cancer or immunosuppressive effects. However, this is rarely, if ever, stated. In virtually every normal tissue at risk in radiation therapy, one can reduce the morbidity by increasing fractionation or, equivalently, decreasing the dose per fraction. Thus, it is naïve to ask simply whether fractionation ameliorates the side-effects. This can be assumed a priori. The more relevant questions are whether there is a demonstrable dose response, how much benefit there is to fractionation, whether there is added benefit to hyperfractionation, and what comorbidities or other treatments may impact the response. Lung: IP IP is a clinically important complication of TBI in patients undergoing HCT. Kim et al. define IP “as a condition in which a patient develops interstitial pulmonary infiltrates on chest X-ray with or without patchy alveolar infiltrate.” The patient produces scanty nonpurulent sputum

Radiotherapeutic Principles of Hematopoietic Cell Transplantation

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0.5

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Fig. 23.2 Dose–response for interstitial pneumonitis. Curve A is the single-fraction dose–response with data from Sampath et al. Curve B is the five-fraction dose–response. Curve C is the dose–response for 2 Gy per fraction.

which is negative in culture for bacterial and fungal infection [67]. Experimental and clinical data have confirmed that fractionated TBI is less toxic to the lung than single-dose TBI [19,30,33]. Fractionated doses above 15 Gy [68] are associated with higher incidences of IP. IP also occurs in conditioning regimens that do not include TBI at all. BU alone is associated with a crude incidence of IP of 4–10%. It is known that chemotherapy, particularly alkylating agents, given with TBI as part of the bone marrow transplant procedure contribute to pulmonary toxicity. This makes it difficult to distinguish TBI toxicity from other etiologies of lung damage [45]. To investigate the radiation dose response and the other factors that may affect the incidence of IP, Sampath et al. reviewed the literature, selecting papers that included sufficient data for a multivariate analysis, including dose, dose rate, chemotherapy regimen, and other variables [49]. The final number of articles included was 20, with a total of 26 TBI/chemotherapy conditioning regimens. Five regimens had no TBI, and four had no chemotherapy (upper-half body treatments). These figures reflect only those regimens in which patients received daily fractionation. The data for multiple fractions per day had too much dispersion to be fit by logistic regression; Fig. 23.2 shows the dose response obtained from multivariate analysis. Shown in this figure are the dose–response functions for one fraction, five fractions, and daily fractions at 2 Gy per fraction. These dose–response functions reflect the response to radiation only. The addition of CY and many other chemotherapeutic agents is associated with higher rates of IP. The findings of Sampath et al. indicated that there was no dose-rate effect for IP on multivariate analysis. The odds ratio for 120 mg/kg CY was 5.9, and the ratio α : β (between the coefficients of dose and dose squared, respectively, in the linear-quadratic model) was 2.8 Gy. The low value of the α : β ratio indicates that IP is significantly diminished with increasing fractionation. However, direct analysis of data from hyperfractionation regimens has not been possible. This could be due to the complex repair of sublethal damage to the lung. There appears to be a slow repair component that would at least partially offset the beneficial effects of hyperfractionation [69]. Other pulmonary side-effects include idiopathic pneumonia, radiation fibrosis, and diminished lung function.

Fig. 23.3 Dose–response for renal damage. The dose–response function is for 2 Gy per fraction. Data points are from the literature, with the dose being converted according to equivalent dose in 2 Gy fractions.

Renal toxicity Renal damage is expressed up to about 2 years after treatment. It is characterized by reduced filtration rates, increased blood urea nitrogen and creatinine, proteinuria, hypertension, and anemia. Ozsahin found no difference in renal toxicity among groups in a randomized trial comparing 10 Gy single fractions with 12 Gy in six fractions and using two different dose rates [18]. In a study combining literature reports of renal damage after TBI, Kal et al. reported their findings regarding the dose response after assuming an α : β ratio of 2.5 Gy and a repair half-time of 2 hours [70]. However, fractionation parameters were not analyzed, but rather assumed, and no accounting was made for regimens in which highly nephrotoxic drugs were used. We undertook a reanalysis of the data presented in that paper, adding a few studies not included by those investigators. Total dose, dose rate, dose per fraction, number of fractions, fractions per day, interfraction interval, age (pediatrics versus adults versus mixed), and chemotherapy were included in a multivariate analysis. We found that total dose, dose per fraction, and certain nephrotoxic drugs were the only variables predictive of renal injury. Dose rate was not significant. Figure 23.3 shows the dose response in 2 Gy fractions. The commonly used schedule of six times 2 Gy is associated with a renal toxicity rate of about 10%. Teniposide and cyclosporine were associated with relative risks of 5–6. For patients treated without these drugs, the α : β ratio was 4.5 Gy. This value of the α : β ratio would result in a substantial fractionation effect, but not as great as for the lung. Interestingly, reanalysis of Kal’s data did not show any benefit to hyperfractionation. Sinusoidal obstructive syndrome SOS is a dose-related and potentially fatal complication of the HCT conditioning regimen. It is much more common in regimens where the TBI is replaced with BU [61–63]. However, even with TBI regimens, the incidence can be up to 10%. Although a lower incidence is generally found with fractionated radiation [35], the dependence on radiation dose is modest. Only one case (11%) was seen in nine patients treated with doses of 16–20 Gy in 2 Gy fractions in a phase I dose-escalation study [29], and Ozsahin et al. found six cases in 100 patients treated with 12 Gy in six fractions [18]. There appears to be no dose-rate dependence for fractionated treatment, although there is some evidence for a singlefraction dose-rate effect below 7 cGy/min [71].

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Cataract formation Cataract formation occurs up to 10 years after HCT, and the cumulative incidence can reach 100% [72]. A dose–response effect for cataract formation has been shown by van Kempen-Harteveld et al. in a multiinstitutional study. (Although they assumed a dose-rate effect, statistical analysis presented in their paper argues against it.) They reported an α : β ratio of 0.75 Gy, indicating that cataractogenesis should be very sensitive to fractionation. In addition, treatment with steroids (for GVHD) and heparin shifted the dose–response curve to the left, but they did not determine relative risks associated with these drugs. This study has the advantage that the incidence of cataracts was determined using actuarial methods rather than crude incidence. Because of severe censoring, using crude incidence may not produce reliable results. In a different study, van Kempen-Harteveld et al. showed that shielding the lens in the anterior field reduced the incidence and severity of cataract formation while increasing the latency without increasing central nervous system recurrence [73]. For CML, Socie et al. reported a hazard ratio of 2.3 for TBI-associated cataractogenesis compared with BU regimens in a summary analysis of four randomized trials [63]. However, in AML, there was no difference in the two conditioning regimens. Also, chronic GVHD was associated with a hazard ratio of 2.6, but only for CML. Belkacemi et al. and Ozsahin et al. found a protective effect for dose rates lower than 4 and 6 cGy/min respectively [18,20]. Second malignancy induction There is no question that ionizing radiation can induce carcinogenic transformations. However, the risk of second solid tumors resulting from TBI has been overstressed in the literature. In a study of more than 19,000 HCT patients, an excess of 50 cancers were seen [74]. Even if every one of these were attributable to radiation, the absolute incidence is less than 0.3%. In fact, in multivariate analyses of these data, only fractionated radiation to TBI doses higher than 14 Gy and limited-field irradiation were associated with a significantly increased incidence of invasive solid tumors. In two separate single-institution analyses of over 4200 patients, TBI was not significantly related to induction of second solid tumors on multivariate analysis, despite the inclusion of basal cell cancers, accounting for nine out of the 46 solid malignancies [75,76]. It is clear that the incidence of radiation-induced solid malignancies from TBI, while occurring, is just barely above background, and can be detected only in populations in the order of 104 or higher. The risk is apparently higher among children than adults [77]. There is continued debate as to whether TBI contributes to the risk of developing myelodysplastic syndrome after autologous HCT. In addition to TBI, other possible contributing factors include the amount and type of pretransplant chemotherapy, pretransplant radiotherapy, the specific conditioning regimen, stem cell priming regimens, source of stem cells, selection criteria used for TBI, and length of follow-up between TBI and non-TBI treated cohorts. These factors are difficult to control for in any retrospective analysis. A number of retrospective studies have reported TBI as an independent risk factor for myelodysplastic syndrome in patients with NHL [78,79] and Hodgkin’s lymphoma [80]. However, Krishnan et al. [81], in a retrospective analysis of 218 patients at City of Hope Cancer Center, did not find TBI to be an independent risk factor. Only priming with etoposide was a risk factor. Harrison et al. [82], in a case-controlled study of 595 patients with Hodgkin’s disease, found extent of pretransplant therapy and pretransplant exposure to lomustine or the combination of mechlorethamine, vincristine, procarbazine, and prednisone to be risk factors, but not autologous HCT. Other late effects of TBI may include growth retardation, neurocognitive effects, gonadal dysfunction, and late cardiomyopathy. Because of

the highly censored population and the possibility of competing risks, analysis of the contribution of TBI to very late effects is extremely difficult.

High-dose TBI-containing versus non-TBI-containing regimens Multiple randomized trials and nonrandomized studies have been performed comparing TBI- with non-TBI-containing high-dose conditioning regimens. Most studies have compared TBI/CY to the BU/CY regimens initially developed by the groups in Baltimore [83] and Ohio [84]. Results of select randomized trials are summarized in Table 23.1. A number of randomized trials demonstrate an advantage for TBIcontaining regimens. Blaise et al. [85] randomized patients with AML in first complete remission to TBI/CY (120 mg/kg) or BU (16 mg/kg)/ CY (120 mg/kg). A statistically significant improvement in 2-year disease-free survival (72% versus 47%), overall survival (75% versus 51%), relapse rate (14% versus 34%), and transplant-related mortality (8% versus 27%) was observed in favor of TBI/CY. In multivariate analysis, BU/CY was associated with higher relapse rates and lower survival. A subsequent update with a median follow-up of 10.8 years continued to demonstrate an advantage for TBI/CY [86]. Dusenberry et al. [61] also observed a possible advantage to TBI/CY (120 mg/kg) compared with BU (16 mg/kg)/CY (200 mg/kg) in patients with AML. For patients in first complete remission, there was no difference in estimated 2-year disease-free survival. However, for patients beyond first complete remission, a trend towards improved disease-free survival was observed with TBI/CY (42% versus 9%; p = 0.06). Acute toxicities and incidence of IP were similar for the two regimens. None of the 18 TBI/CY patients developed SOS, compared with three of 17 receiving BU/CY. The Nordic Bone Marrow Transplantation Group also demonstrated an advantage for TBI/CY over BU/CY especially in patients with advanced disease [87,88]. Patients with AML, ALL, and CML in first remission, first chronic phase, and more advanced phases were entered. There was no significant difference in 3-year disease-free or overall survival between the two arms when looking at the entire population. However, for patients with high-risk disease (beyond first remission or first chronic phase), TBI/CY compared favorably with BU/CY resulting in a higher disease-free survival (61% versus 18%; p = 0.005) and overall survival (66% versus 21%; p = 0.002). In addition, TBI/CY was associated with a significantly lower incidence of SOS, obstructive bronchiolitis, hemorrhagic cystitis, seizures, and transplant-related mortality compared with BU/CY. The authors concluded that TBI/CY was the treatment of choice, especially in patients with advanced disease. A later update with 5–9 years’ follow-up reached similar conclusions [86]. Recently, Bunin and colleagues [89] randomized children with AML to TBI or BU (16 mg/kg) combined with CY (120 mg/kg) and etoposide (VP-16) (16 mg/kg). Event-free survival at 3 years was 58% for the TBI group and 29% for the BU group (p = 0.03). The differences were primarily seen in those receiving HCT from unrelated donors. Other randomized studies have not observed a significant advantage for TBI- over non-TBI containing regimens. The Seattle group [64,90] reported comparable efficacy and nonrelapse mortality between TBI/CY and BU/CY regimens for patients with CML in chronic phase. BU/CY was better tolerated, with a shorter length of hospital stay and a lower incidence of acute GVHD. A multi-institution randomized study from France also compared TBI/CY with BU/CY in patients with CML in chronic phase [91], observing comparable efficacy and transplant-related mortality between the two arms. Finally, a randomized trial performed by the Southwest Oncology Group compared TBI/etoposide with BU/

Radiotherapeutic Principles of Hematopoietic Cell Transplantation

341

Table 23.1 Select randomized trials comparing total body irradiation (TBI) versus non-TBI-containing high-dose hematopoietic cell transplantation (HCT) conditioning regimens Study (First author, year)

Patient number Disease type

Randomization

TBI dose and schedule

Relapse rate

Survival

Toxicities

Blaise, 1992 [85]

101 AML CR1 Allogeneic HCT

TBI/CY BU/CY

12 Gy 2 Gy BID Most patients

14% TBI/CY 34% BU/CY (p < 0.04)

2 year DFS: 72% TBI/CY; 47% BU/CY (p < 0.01) OS: 75% TBI/CY; 51% BU/CY (p < 0.02)

TRM: 8% TBI/CY; 27% BU/CY (p < 0.06)

Dusenbery, 1995 [61]

35 AML CR1 and > CR1 Autologous HCT

TBI/CY BU/CY

13.2 Gy 1.65 Gy BID

2 year 43% TBI-CY; 70% BU/CY (p = 0.17)

2 year DFS: 50% TBI/CY; 24% BU/CY (p = 0.12) If > CR1, DFS: 42% TBI/CY; 9% BU/CY (p = 0.06) OS: 46% TBI/CY; 35% BU/CY (p = 0.41)

IP no difference SOS: 0/18 TBI/CY; 3/17 BU/CY

Ringden, 1994 [87]

167 AML, ALL, CML, NHL CR1, CP1, advanced Allogeneic HCT

TBI/CY BU/CY

11.3–12 Gy 3–7 fractions

3 year DFS: 67% TBI/CY; 56% BU/CY Difference in advanced disease patients 49% TBI/CY; 17% BU/CY (p = 0.005)

TRM: 62% BU/CY; 12% TBI/CY (p = 0.04) IP no difference, but significant increase in SOS, cystitis, seizures, and obstructive bronchiolitis with BU/CY

Bunin, 2003 [89]

43 ALL CR1, CR2, CR3 Allogeneic HCT

TBI/CY/ etoposide BU/CY/etoposide

12 Gy 2 Gy BID

3 year DFS: 58% TBI/CY/etoposide; 29% BU/ CY/ etoposide (p = 0.03) OS: 67% TBI/CY/etoposide; 47% BU/CY/ etoposide (p = 0.04)

Clift, 1994 [64]

142 CML CP Allogeneic HCT

TBI/CY BU/CY

12 Gy 2 Gy QD

3 year 0.13 BU/CY; 0.13 TBI/CY (p = 0.43)

3 year EFS: 0.7 TBI/CY; 0.66 BU/CY (p = 0.36) OS: 0.8 TBI/CY; 0.8 BU/CY

Nonrelapse mortality no difference 0.24 TBI/CY; 0.18 BU/CY Hospital LOS, acute GVHD, elevated creatinine level greater with TBI/ CY

Devergie, 1995 [91]

120 CML CP Allogeneic HCT

TBI/CY BU/CY

10 Gy single fraction; 11–12 Gy Most 2 Gy BID

4.4% BU/CY 11.1% STBI 31.3% HFTBI (p = 0.039)

5-year DFS no difference 55% TBI/CY; 59.1% BU/CY 5-year OS no difference 62.9% TBI/CY; 60.6% BU/CY

TRM: 29% TBI/CY; 38% BU/CY

Blume, 1993 [93]

122 AML, ALL, CML >CR1 or >CP1 Allogeneic HCT

TBI/etoposide BU/CY

13.2 Gy 1.2 Gy TID

No difference between arms

No differences between arms

BID, twice a day; CP, chronic phase; CP1, first chronic phase; >CP1, beyond chronic phase; CR1, first complete remission; >CR1, beyond first complete remission; CR2, second complete remission; CR3, third complete remission; DFS, disease-free survival; EFS, event-free survival; LOS, length of stay; OS, overall survival; QD, four times a day; SOS, sinusoidal obstructive syndrome; TID, three times a day; TRM, treatment-related mortality. See text for other abbreviations.

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CY and found no difference in efficacy in patients with AML, ALL, and CML beyond first complete remission or chronic phase. Hartman et al. [92] performed a meta-analysis of five published randomized trials [64,85,87,91,93]. Survival and disease-free survival were better with TBI-based regimens compared with BU/CY, but the differences were not statistically significant. A significantly greater incidence of SOS was observed with the BU/CY regimens. The authors concluded that TBI regimens were at least as good as BU/CY regimens. Socie et al. [63] analyzed the long-term outcome of 316 patients with CML and 172 patients with AML originally entered on four of the randomized trials described above [64,85,87,91]. Mean follow-up on the study was over 7 years. For patients with CML, there was no significant difference in 10-year overall survival or disease-free survival. For patients with AML, a trend favoring TBI regimens was observed with projected 10-year overall survival (63% versus 51%; p = 0.068) and disease-free survival (57% v. 47%; p = 0.051). Disease-free survival rates were similar if analysis was limited to AML in first remission. Late complications were similar for the two regimens, with the exception of a higher incidence of cataracts with TBI/CY and permanent alopecia with BU/CY. Multiple nonrandomized studies in acute and chronic leukemias have also compared TBI and non-TBI regimens, with several trials demonstrating superior efficacy with TBI regimens. Table 23.2 summarizes results from selected trials. A limited number of studies have also compared TBI and non-TBI high-dose regimens in patients with multiple myeloma and have reported no advantage to TBI [26,94]. The French Myeloma Intergroup performed a prospective randomized trial [26]. Patients with newly diagnosed multiple myeloma were randomized after vincristine/adriamycin/ dexamethasone chemotherapy to 8 Gy TBI (2 Gy four times a day) and melphalan (140 mg/m2) versus melphalan (200 mg/m2). Median duration of event-free survival was similar (21 versus 20.5 months). In an analysis of the Spanish Registry for Transplantation in Multiple Myeloma [94], similar efficacy and treatment-related mortality rates were observed for the three most common regimens used: TBI (8–12.5 Gy)/melphalan (140 mg/m2), melphalan alone (200 mg/m2), and BU (12 mg/kg)/ melphalan (140 mg/m2). In summary, several reports, including at least four randomized studies, demonstrate or suggest increased efficacy and/or decreased toxicity with TBI-containing compared with non-TBI-containing high dose conditioning regimens, particularly in patients with acute leukemias. Other studies demonstrate comparability between the two approaches. With respect to toxicity and treatment-related mortality rates, high-dose chemotherapy-alone regimens, such as BU/CY, offer no clear advantage over TBI regimens and can be associated with a higher rate of SOS, seizures, obstructive bronchiolitis, and hemorrhagic cystitis [85,87].

TBI and reduced-intensity regimens Older patients or patients with comorbidities are often not able to tolerate standard myeloablative, full-intensity TBI containing as well as non-TBI-containing regimens. In some centers, full-intensity TBIcontaining regimens are limited to patients younger than approximately 50–55 years of age. As a result, reduced-intensity regimens are now being actively investigated. These regimens are usually associated with fewer acute toxicities [102,103], and are primarily used as a method of immunosuppression to allow engraftment of donor cells and the subsequent graft-versus-tumor effects. TBI-containing as well as non-TBI-containing regimens are being investigated. Low-to-intermediate doses of TBI have been used. McSweeney et al. [104], using 2 Gy TBI combined with cyclosporine and mycophenolate mofetil in 45 patients, reported reduced regimen-

related toxicities compared with standard high-dose regimens, a survival rate of 66.7% and a nonrelapse mortality of 6.7%. However, nonfatal graft rejection was seen in 20%. As a result the purine analog fludarabine was added to the regimen to increase immunosuppression and has significantly reduced the rate of graft rejection [105]. Subsequent reports, including those from other centers, using 2 Gy TBI-containing reducedintensity regimens, continue to show encouraging results [106,107]. Intermediate TBI doses of 4–9 Gy have also been evaluated as part of reduced-intensity regimens, with reported success [108–111]. For example, the group at Washington University in St Louis [112] evaluated a regimen of 5.5 Gy TBI given as a single fraction at 30 cGy/min combined with CY (120 mg/kg). In 80 patients with a variety of hematologic malignancies, graft failure was not observed, and all patients eventually demonstrated complete donor engraftment. Incidence of fatal organ toxicity was 2.5%, and treatment-related mortality at 2 years was 15%. Belkacemi et al. [113] retrospectively analyzed the European Group for Blood and Marrow Transplantation experience of 130 patients administered reduced-intensity regimens. A TBI dose of 2 Gy was administered in 78%, while 4–6 Gy was used in 22%. Median dose rate was 14.3 (range 6–16.4) cGy/min. TBI parameters did not have a statistically significant impact on outcome, possibly due to the small number of patients in each subset. Incidence of TRM was 43% for dose rates of 14.3 cGy/min and over, and 29% for less than 14.3 cGy/min (p = 0.09). The group at MD Anderson Hospital in Houston recently combined 9 Gy TBI (3 Gy four times a day) with the reduced-intensity regimen of fludarabine (120 mg/m2) and melphalan (140 mg/m2) in 29 patients with advanced pediatric hematologic malignancies [111]. The TBI dose used is close to that used in some high-dose regimens [38] and therefore begins to blur the distinction between myeloablative and reducedintensity regimens. However, assuming an α : β ratio of 2.8 Gy based on lung toxicity [49], this regimen is the biologic equivalent of only 10.9 Gy when compared with TBI schedules delivered at 2 Gy per fraction, which usually go to a total dose of 12–16 Gy. Grade 1–2 oral mucositis and diarrhea were the most common toxicities. Although 21 donors were unrelated, only one patient had graft failure. TRM rate was 24%. The authors conclude that this regimen was fairly well tolerated, but also point out that the same regimen was not tolerated in adults in a separate trial, forcing study closure. In summary, low-to-intermediate doses of TBI have been successfully integrated into reduced-intensity regimens with encouraging results and continue to be actively investigated. The optimal regimen and schedule remains to be determined.

Total lymphoid irradiation TLI has been used instead of TBI for patients undergoing allogeneic HCT with malignant conditions, and nonmalignant conditions such as aplastic anemia and thalassemia [114–116]. TLI provides the needed immunosuppression with a reduced dose to critical organs compared with TBI. However, Shank et al. [117] estimated that fractionated TLI was less immunosuppressive than an equivalent dose of fractionated TBI. Mantle and inverted-Y fields, which cover the major central lymphoid regions, are often utilized. As with TBI, TLI is often combined with chemotherapy such as CY or BU/CY. TLI (approximately 6–8 Gy) has also been combined with standard doses of TBI in an effort to further increase immunosuppression and engraftment, especially in patients receiving T-cell-depleted donor marrow [118,119]. Recently, the group from Stanford University evaluated the use of fractionated TLI and antithymocyte globulin to reduce the incidence of acute GVHD in patients undergoing allogeneic HCT [120]. This novel approach takes advantage of the immune system’s regulatory T cells and the relative radioresistance of regulatory natural killer T cells compared

351 Follicular lymphoma CR1, CR2, relapse Autologous HCT

GutierrezDelgado, 2001 [98]

TBI/CY/ etoposide 12 Gy 1.5 Gy BID

TBI/CY 12 Gy 3 Gy QD or 2 Gy BID

Two-institution retrospective

156 ALL CR1, CR2, >CR2 Allogeneic, autologous HCT

Granados, 2000 [97]

Singleinstitution retrospective

TBI/CY

IBMTR database analysis

627 Pediatric ALL CR1, CR2, >CR2 Allogeneic HCT

Davies, 2000 [96]

TBI/CY

Matched-pair analysis EBMT database

921 AML, ALL Most CR1 Allogeneic, autologous HCT

Ringden, 1996 [62]

TBI/CY ± other chemotherapy 10–13.2 Gy most 12 Gy at 2 Gy BID

TBI regimen

Japanese bone marrow transplant registry analysis

Study design

123 AML CR1 Allogeneic HCT

Patient number Disease type

Inoue, 1993 [95]

Study (First author, year)

BU Melphalan Thiotepa

BU/CY

BU/CY

BU/CY

BU/CY ± other chemotherapy

Non-TBI regimen

5 year 49% TBI/CY/etoposide 42% BU/melphalan/ thiotepa

3 year 47% TBI/CY; 71% BU/CY (p = 0.01)

35% TBI/CY; 41% BU/CY (p = 0.07)

2 year No difference except ALL intermediate risk Autologous HCT 82% BU/CY/;62% TBI/CY (p = 0.002)

2 year 16% TBI; 39% non-TBI (p = 0.197)

Relapse rate

5 year EFS 32% TBI/CY/etoposide 34% BU/melphalan/thiotepa

6 year EFS 43% TBI/CY; 22% BU/CY (p = 0.01)

3 year DFS 50% TBI/CY; 35% BU/CY (p = 0.005) 3 year OS 55% TBI/CY; 40% BU/CY (p = 0.003)

2 year DFS No difference except ALL intermediate risk Autologous HCT 14% BU/CY versus 34% TBI/CY (p = 0.002)

2 year OS 77% TBI 51% non-TBI (p = 0.001)

Survival

TRM 16% TBI/CY/etoposide 21% BU/melphalan/ thiotepa

TRM 17% TBI/CY; 22% BU/CY (p = 0.24)

No difference between arms

TBI/CY superior

TBI/CY superior

No difference except TBI better for intermediaterisk ALL

Increased SOS (p < 0.05) and cystitis (p < 0.001) with BU/CY

TRM 15% TBI/CY; 23% BU/CY (p = 0.02)

TBI regimens superior

Study conclusion

IP, no difference 21% TBI 24% non-TBI (p = 0.08)

Toxicities

Table 23.2 Select non-randomized studies comparing total body irradiation (TBI) versus non-TBI-containing high-dose hematopoietic cell transplantation (HCT) conditioning regimens

Radiotherapeutic Principles of Hematopoietic Cell Transplantation

343

180 AML, ALL, CML, MDS Allogeneic HCT

128 MDS, AML Allogeneic HCT

Mattsson, 2003 [100]

Scott, 2004 [101]

Singleinstitution retrospective

Singleinstitution retrospective

IBMTR analysis

Study design

TBI/BU 12 Gy 2 Gy BID

TBI/CY 10–12 Gy

TBI/CY 12 Gy

TBI regimen

BU/CY

BU/CY

BU/CY

Non-TBI regimen

3 year 26% TBI/BU 44% BU/CY

444% FTBI; 39% STBI; 16% BU/CY (p = 0.01)

5 year 12% TBI/CY; 19% BU/CY (p = 0.042)

Relapse rate

No difference DFS 24% TBI/BU 19% BU/CY

3-year OS 77% BU/CY 55% STBI 59% FTBI (p = 0.04)

No difference 5-year OS 60% TBI/CY; 55% BU/CY (p = 0.579) LFS 58% TBI/CY; 54% BU/CY (p = 0.438)

Survival

3-year NRM 50% TBI/BU 37% BU/CY

TRM no difference

CNS relapse 0% TBI/CY; 3% BU/CY SOS 6% TBI/CY; 13% BU/CY (p = 0.009) TRM no difference 30% TBI/CY; 27% BU/CY

Toxicities

BU/CY less toxic but with increased relapse rate

BU/CY superior

Decreased relapse with TBI/CY but no difference in LFS and OS

Study conclusion

BID, twice a day; CNS, central nervous system; CP1, first chronic phase; >CP1, beyond chronic phase; CR1, first complete remission; >CR1, beyond first complete remission; CR2, second complete remission; >CR2, beyond second complete remission; CR3, third complete remission; DFS, disease-free survival; EBMT, European Group for Blood and Marrow Transplantation; EFS, event-free survival; IBMTR, International Bone Marrow Transplant Registry; IP, interstitial pneumonitis; LFS, leukemia-free survival; LOS, length of stay; MDS, myelodysplastic syndrome; NRM, nonrelapse mortality; OS, overall survival; QD, four times a day; SOS, sinusoidal obstructive syndrome; TRM, treatment-related mortality. See text for other abbreviations.

581 AML CR1 Allogeneic HCT

Patient number Disease type

Litzow, 2002 [99]

Study (First author, year)

Table 23.2 (Continued)

344 Chapter 23

Radiotherapeutic Principles of Hematopoietic Cell Transplantation

with conventional T cells [121]. These regulatory T cells prevent acute GVHD by inhibiting the proliferation and cytokine secretion of CD4+ and CD8+ donor T cells without affecting antitumor activity [122]. Secretion of interleukin-4 by natural killer T cells appears to be involved [123]. Thirty-seven patients with lymphoid malignancies or acute leukemia received 8 Gy fractionated TLI (80 cGy × 10 fractions over 11 days) and antithymocyte globulin followed by human leukocyte antigenmatched mononuclear cells from related and unrelated donors. Only two of 37 had acute GVHD; seven of 33 had extensive chronic GVHD. Antitumor effects were observed. Of 18 patients with lymphoid malignancies and measurable disease at the time of transplant, 12 achieved complete remission without acute GVHD. An increase in the median percentage of natural killer T cells and a marked reduction in the response to alloantigenic stimulation of donor CD4+ T cells was also observed. The results are encouraging and are consistent with results observed in preclinical murine models.

Involved field radiotherapy Involved field radiation therapy (IFRT) to sites of initial involvement is often utilized in patients with Hodgkin’s and non-Hodgkin’s lymphoma undergoing HCT. This is based on the fact that lymphomas are radiosensitive and that locoregional disease, particularly if bulky on initial presentation, can often be a site of relapse after systemic therapies. Most patients who relapse after high-dose regimens show recurrence in sites of initial disease [124–126]. IFRT to involved sites will reduce relapse rates. For example, Poen et al. [124] reported on 100 patients with Hodgkin’s disease transplanted at Stanford. Of the 32 patients who relapsed, 22 relapsed at sites involved prior to transplant. In the 49 patients who received IFRT, 3-year freedom from relapse was 100% compared with 67% (p = 0.04) for the 13 who did not receive IFRT. Moskowitz et al. [127] combined 18 Gy IFRT and 18 Gy TLI prior to high-dose chemotherapy and autologous HCT in patients with refractory or relapsed Hodgkin’s disease. Event-free survival was 68%, and only 18% relapsed in previously irradiated areas. Similar findings were seen in patients with NHL undergoing HCT [126]. Radiotherapy doses used vary from approximately 18 to 40 Gy, usually delivered at 1.8–2.0 Gy/day. Field coverage and dose are influenced by multiple factors including tumor extent and tumor bulk, responsiveness to chemotherapy, previous radiotherapy, whether TBI is part of the conditioning regimen, and preference of the treating physician. Fields have covered all or most initial sites, primarily bulky sites defined on initial presentation, or residual sites remaining after systemic therapy. IFRT has been delivered either prior to or after high-dose conditioning regimens with acceptable toxicity [128]. Other studies have cautioned against combining TBI with IFRT to the mediastinum and thorax due to an increased risk of IP [129]. However, Song et al. [130], in a phase I study of 21 patients with lymphoma, saw no significant pulmonary toxicity with IFRT given 1 week prior to 12 Gy TBI. Others have successfully combined TLI with TBI with no reported significant additional toxicity [118,119].

Targeted TBI and future directions A dose–response relationship has long been recognized for many types of hematologic malignancy [131–133]. Many groups have therefore looked at escalating TBI dose to improve outcomes. TBI dose escalation in patients with AML and CML has resulted in lower relapse rates [36,37,134]. However, the number of treatment-related complications has increased – resulting in no change in overall survival. New strategies are clearly needed to allow further TBI dose escalation without an associated increase in short- and long-term side-effects. As a result, various

345

approaches are now being actively explored to deliver a more targeted form of TBI, one that would preferentially deposit more dose to areas of greatest tumor burden to maximize disease control, while simultaneously reducing dose to critical normal organs to reduce toxicities. Biologically guided systemic radiotherapy, using radiolabeled antibodies [2,135] or bone-seeking radiopharmaceuticals such as 166 Ho-DOTMP [3], are currently undergoing evaluation as part of HCT conditioning regimens. This area is covered in more detail in Chapter 24 and has been reviewed elsewhere [2,136,137]. Gopal et al. [138], in a multivariable cohort analysis, compared follicular lymphoma patients treated with high-dose anti-CD20 radioimmunotherapy with a matched cohort treated with conventional myeloablative conditioning regimens, usually containing TBI. This study demonstrated superior overall survival, progression-free survival, and treatment-related mortality with the radioimmunotherapy group. Since a significant fraction of the response rate with anti-CD20 radioimmunotherapy is due to the unlabeled antibody alone [139], perhaps a more interesting comparison will eventually be between an anti-CD20 radioimmunotherapy regimen and a TBIcontaining regimen that also includes unlabeled anti-CD20 antibody. Although encouraging, a limitation with these approaches is the inability to completely control the radiation dose to tumor or normal organ for a given patient. Once the dose has been administered, its distribution is dictated by multiple factors, such as tumor size, tumor burden, antigen expression, tumor microenvironment, clearance kinetics, and the molecular characteristics and in vivo stability of the agent. These factors vary from patient to patient and from tumor to tumor within a given patient. This can lead to low, unpredictable doses to a given tumor or higher than expected doses to critical organs. Recent technological advances in the delivery of external beam radiotherapy now allow for the delivery of a more targeted form of TBI. Helical tomotherapy (commercially available as the Tomotherapy HiArt System; Tomotherapy Inc., Madison, WI, USA) is a new delivery system that represents a radical departure from conventional linear accelerator design. Helical tomotherapy integrates CT image-guided radiotherapy and intensity modulated radiation therapy in a single device. Specifically, a 6 MV linear accelerator is mounted on a CT ring gantry and rotates around the patient as the patient translates through the ring. The treatment fan beam is segmented using a 64-leaf binary collimator, resulting in a minimum beamlet size of 5 × 6 mm. The maximum target size possible is approximately 60 cm in width by approximately 160 cm in length [140]. Helical tomotherapy therefore allows for the delivery of highly conforming dose distributions to large complex target shapes while simultaneously reducing the dose to critical normal organs [141], making it uniquely suited for the delivery of conformal targeted TBI. The group at City of Hope Cancer Center has recently reported the initial experience using helical tomotherapy to deliver targeted TBI or TMI in patients with multiple myeloma and acute leukemias [4,5,142]. Plate 23.2 shows the typical conformal dose distribution pattern that is achieved to the designated target structure (skeletal bone in this example), with simultaneous reduction of the dose to critical organs. Table 23.3 compares the median doses for various normal organs delivered through standard TBI to 12 Gy with lung shielding versus TMI to 12 Gy to skeletal bone. Figure 23.4 shows normal organ dose–volume histogram plots for standard TBI versus TMI in the same patient. A significant reduction in dose and volume of organ receiving the full dose is observed compared with standard TBI for all critical organs, predicting for reduced short-term and long-term toxicities. Finally, the system can be used to differentially reduce the dose to organs or any other user-defined avoidance structure, depending on the radiosensitivity of the organ in question, as well as to increase the dose to a particular target region depending on the clinical situation. Plate 23.3 shows the ability of the

346

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system to deliver a conformal boost dose of 30 Gy to a site of bulky disease along with 12 Gy TMI. Initial clinical results from the group at City of Hope Cancer Center usin TMI are encouraging and demonstrate reduced acute toxicities that are consistent with dosimetric predictions [143]. Of 15 multiple myeloma patients on a phase I tandem autologous HCT trial treated first with Table 23.3 Median dose to normal organs with total marrow irradiation (TMI) using tomotherapy compared with standard total body irradiation (TBI) to deliver 12 Gy

Organ at risk

Median dose (Gy) TMI

Median dose (Gy) TBI

TMI/TBI median dose

Bladder Brain Breast Esophagus Orbits Heart Lens Liver Left kidney Right kidney Left lung Right lung Optic nerve Oral cavity Ovary Parotids Rectum Small intestine Stomach Thyroid

7.5 7.1 7.7 4.9 6.0 6.1 2.3 6.9 7.4 6.9 6.3 6.4 6.4 2.5 7.0 4.6 4.8 5.0 4.6 4.4

12.3 12.2 12.4 11.7 12.0 11.5 10.5 11.7 11.9 11.9 9.0 9.7 12.3 12.5 12.5 13.1 12.6 12.5 11.5 12.6

0.61 0.58 0.62 0.42 0.50 0.53 0.22 0.59 0.62 0.58 0.70 0.66 0.52 0.20 0.56 0.35 0.38 0.40 0.40 0.35

Standard TBI utilized 10 MV photons to deliver 12 Gy. Fifty percent transmission blocks were used to shield the lungs. Electrons were used to deliver 6 Gy to the rib cage underlying the lung blocks. Data are an average of comparison plans from six patients. (Data from [5].)

melphalan, followed a minimum of 6 weeks later with TMI doses of 10–18 Gy (2 Gy twice a day), six reported no vomiting, six no mucositis, six no fatigue, 14 no erythema, and 10 no diarrhea. With a median follow-up of 12 (range 4–22) months no pulmonary toxicity has been observed. These results compare favorably to results from a phase I tandem autologous HCT trial of similar design reported by Zaucha et al. [27]. In this study, patients with multiple myeloma or other malignancies with primarily bone metastases were first treated with BU/melphalan/thiotepa followed a median of 105 days later by modified TBI to 12, 13.5 or 15 Gy using 5% transmission blocks over the lungs and liver. Grade 1–2 mucositis was seen in 100% and diarrhea in 34% of patients using the Bearman regimen-related toxicity scale. Death from pulmonary toxicity was observed in three of 17 patients treated at 15 Gy.

Summary TBI and large-field radiotherapy approaches are an important part of the conditioning regimen for many patients undergoing HCT because of the cytotoxic effects on malignant cells and potent immunosuppressive effects that aid in successful engraftment. Typical TBI schedules deliver a total dose of 10–16 Gy at 1.2 Gy three times a day, 2 Gy twice a day or 3 Gy four times daily. Although randomized trials are limited, the available data demonstrate that application of the same radiobiologic principles successfully employed for conventional field radiotherapy, such as fractionation, hyperfractionation, and organ shielding, have also helped to improve the therapeutic ratio of TBI, with reduced toxicities and improved outcomes. The effect of dose rate appears to be modest to negligible with fractionated TBI. Non-TBI-containing high-dose regimens offer no obvious advantage in reducing toxicities or improving control rates compared with TBIcontaining high-dose regimens, and have in some studies been shown to be inferior. Given the toxicities associated with myeloablative conditioning regimens, reduced-intensity regimens, often with low-dose TBI or TLI, are being explored, with encouraging results. Strategies to deliver a more targeted form of TBI are being actively investigated through the use of radioimmunotherapy, other biologically targeted radiotherapeutics, and newer image-guided intensity-modulated radiation therapy radiotherapy delivery systems. This offers the promise of further reducing the dose to normal organs, reducing toxicities, and allowing for dose escalation in an attempt to improve outcomes.

Fig. 23.4 Comparison of dose–volume histogram (DVH) plots for lungs, liver, and kidneys with total marrow irradiation (TMI) versus standard total body irradiation (TBI) to 12 Gy. TBI and TMI plans were performed on the same patient data set. Standard TBI utilized 10 MV photons to deliver 12 Gy. Fifty percent transmission blocks were used to shield the lungs. Electrons were used to deliver 6 Gy to the rib cage underlying the lung blocks. With standard TBI (dashed lines), close to 100% of the liver and kidneys receive the full dose of 12 Gy. Lung shielding results in reduced doses to the lung, although 100% of the lung still receives approximately 6 Gy. With TMI, DVH plots (solid lines) demonstrate reduced doses to a large percentage of each organ compared with TBI.

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antithymocyte globulin prevents graft-versus-host disease: the role of CD1-reactive natural killer T cells. Biol Blood Marrow Transplant 2003; 9: 355–63. Poen JC, Hoppe RT, Horning SJ. High-dose therapy and autologous bone marrow transplantation for relapsed/refractory Hodgkin’s disease: the impact of involved field radiotherapy on patterns of failure and survival. Int J Radiat Oncol Biol Phys 1996; 36: 3–12. Mundt AJ, Sibley G, Williams S et al. Patterns of failure following high-dose chemotherapy and autologous bone marrow transplantation with involved field radiotherapy for relapsed/refractory Hodgkin’s disease. Int J Radiat Oncol Biol Phys 1995; 33: 261–70. Mundt AJ, Williams SF, Hallahan D. High dose chemotherapy and stem cell rescue for aggressive non-Hodgkin’s lymphoma: pattern of failure and implications for involved-field radiotherapy. Int J Radiat Oncol Biol Phys 1997; 39: 617–25. Moskowitz CH, Nimer SD, Zelenetz AD et al. A 2-step comprehensive high-dose chemoradiotherapy second-line program for relapsed and refractory Hodgkin’s disease: analysis by intent to treat and development of a prognostic model. Blood 2001; 97: 616–23. Wirth A, Prince HM, Wolf M et al. Optimal scheduling to reduce morbidity of involved field radiotherapy with transplantation for lymphomas: a Prospective Australasian Leukaemia and Lymphoma Group Study. Bone Marrow Transplant 2005; 35: 291–8. Pezner RD, Nademanee A, Niland JC et al. Involved field radiation therapy for Hodgkin’s

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disease autologous bone marrow transplantation regimens. Radiother Oncol 1995; 34: 23–9. Song DY, Jones RJ, Welsh JS et al. Phase I study of escalating doses of low-dose-rate, locoregional irradiation preceding cytoxan-TBI for patients with chemotherapy-resistant non-Hodgkin’s or Hodgkin’s lymphoma. Int J Radiat Oncol Biol Phys 2003; 57: 166–71. Kaplan HS. Evidence for a tumoricidal dose level in the radiotherapy of Hodgkin’s disease. Cancer Res 1966; 26: 1221–4. Fuks Z, Kaplan HS. Recurrence rates following radiation therapy of nodular and diffuse malignant lymphomas. Radiology 1973; 108: 675–84. Chak LY, Sapozink MD, Cox RS. Extramedullary lesions in non-lymphocytic leukemia: results of radiation therapy. Int J Radiat Oncol Biol Phys 1983; 9: 1173–6. Clift R, Buckner CD, Appelbaum FR et al. Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: a randomized trial of two irradiation regimens. Blood 1990; 76: 1867–71. Nademanee A, Forman S, Molina A et al. A phase 1/2 trial of high-dose yttrium-90-ibritumomab tiuxetan in combination with high-dose etoposide and cyclophosphamide followed by autologous stem cell transplantation in patients with poor-risk or relapsed non-Hodgkin’s lymphoma. Blood 2005; 106: 2896–902. Wong JYC. Basic immunology of antibody targeted radiotherapy. Int J Radiat Oncol Biol Phys 2006; 66: S8–14. Meredith R, Wong JYC, Knox SJ. Targeted radionuclide therapy. In: Gunderson LL, Tepper JE,

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Damian J. Green & Oliver W. Press

Radioimmunotherapy and Hematopoietic Cell Transplantation A new scientific truth does not triumph by convincing its opponents and making them see the light, but rather because its opponents eventually die, and a new generation grow up that is familiar with it. Max Planck

Historical background For over half a century, minimal attention was paid to German pathologist Paul Ehrlich’s prescient notion for utilizing target specific antisera as a form of cancer therapy [1]. While in the late 1890s Hericourt and Richet reported temporary responses in patients treated with antisera raised against human osteogenic sarcomas, and Madame Marie Curie and colleagues identified a potential role for the α-particle-emitting radionuclide radium as an intravenous therapeutic agent [2], few other studies advanced the field of targeted radiation therapy for over 50 years. In 1949, Eisen’s report of a technique for linking radioactive iodine to antibodies without impairment of antibody binding [3] paved the way for Pressman and Korngold’s discovery that radioiodinated tumor-specific antibodies would preferentially accumulate in target tissues [4]. In the 1960s, Bale et al. demonstrated that iodine-131 (131I)-labeled antibodies could be effective as therapeutic agents [5]. Beierwaltes subsequently conducted one of the earliest trials in which 14 patients with metastatic melanoma received 131I-labeled rabbit antisera and one patient achieved a complete response (CR) [6]. In the 1960s, radioiodinated antifibrinogen was shown to provide some symptomatic improvement in a variety of tumor types [7]. In the 1970s, Ettinger et al. [8] demonstrated significant responses in patients with hepatoma treated with 131Ianti-carcinoembryonic antigen (CEA) antibodies or 131I-antiferritin antibodies in combination with external beam radiation and doxorubicin chemotherapy. Lenhard et al. [9] subsequently showed that 131I-polyclonal antiferritin antisera could produce objective remissions in patients with hepatomas, as well as up to 40% of patients with relapsed lymphoma. In 1975, Kohler and Milstein’s Nobel Prize-winning work made practical large-scale production of monoclonal antibodies (mAbs) with defined and reproducible specificity. Great excitement surrounding a new era of antibody-directed therapy ensued, but initial studies with unlabeled antibodies did not meet the expectations of many observers. Over the past decade, one chemically conjugated mAb and five unconjugated mAbs have been approved as cancer therapies in the United States (in addition two anti-CD20 radiolabeled mAbs, 131I-tositumomab and yttrium-90 [90Y]-ibritumomab tiuxetan have been approved; these will be given detailed consideration in a later section). The unconjugated

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

antibodies include anti-CD20 mAb (rituximab) for the treatment of βcell non-Hodgkin’s lymphoma (NHL); anti-HER-2/nu (trastuzumab) in breast carcinomas; anti-CD52 (alemtuzumab) for chronic lymphocytic leukemia; anti-epidermal growth factor receptor protein (cetuximab) for colorectal and subsequently head and neck malignancies; anti-vascular endothelial growth factor protein (bevacizumab) approved for colorectal followed by non-small cell lung cancer; and, most recently, another anti-epidermal growth factor receptor antigen mAb (panitumumab), also for colorectal malignancy. In addition, an anti-CD33 antibody chemically conjugated to the cytotoxic drug, calicheamicin, (gemtuzumab ozogamicin) mAb was approved for treatment of relapsed acute myelogenous leukemia (AML). Each of these unconjugated or chemically conjugated mAbs has shown some clinical promise, but most patients treated with these products as single agents subsequently relapse. As predicted in animal studies, the limitations of unmodified mAb therapy largely result from inadequate host effector mechanisms. In the absence of robust immune effector mechanisms, mAbs bound to antigen on the target cell surface usually do not diminish cell replication and survival sufficiently to eradicate tumors. In addition, antigenic modulation (internalization or shedding) after antibody–antigen complex formation limits the availability of a stable target in many clinical settings. A third impediment to effective cell killing results from variable expression of antigenic targets, including antigen-negative variants in most tumor types. Finally, antigen penetration into large tumors is limited by the vascular supply and the kinetics of intratumoral antibody convection and diffusion. Throughout the 1980s, interest in radioimmunotherapy (RIT) grew due to the recognition that radioimmunoconjugates could bypass many of the limitations enumerated above. By providing the mAbs with a cytotoxic mechanism independent of the host immune response, radiolabeled antibodies (RAbs) circumvent the major obstacle to antibodydirected therapy. Furthermore, the use of radionuclides with path lengths that span several cell diameters provides a “crossfire” effect that delivers toxic radiation from the surrounding antigen-bearing cells to tumor cells that are antigen negative or that lack direct access to antibody binding. In recognition of these advantages, initial preclinical studies with RIT sought to identify both appropriate antigen targets for optimal biodistribution, and factors necessary for tumor cell destruction. These early studies elucidated a number of key principles concerning the biodistribution of RAbs. In addition, they documented that four to 10 times more radiation could be delivered to tumors compared with normal organs by using radioimmunoconjugates. Unfortunately, myelotoxicity frequently prevented escalation of radiation dose to curative levels in early studies

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of RIT [10,11]. The understanding that marrow suppression was dose limiting inspired some groups to explore RIT in combination with stem cell support. The concept that hematopoietic stem cell rescue could facilitate dose escalation to curative levels was supported by the work of Thomas and colleagues in the early 1970s, who first defined a role for bone marrow transplantation (BMT) in the treatment of acute leukemia. Clinical trials conducted in Seattle demonstrated the antileukemic effect of total body irradiation (TBI) in combination with high-dose chemotherapy for BMT [12]. Importantly, higher doses of TBI correlated with a reduced risk of disease recurrence after transplant. In two randomized trials, patients with either AML [13] or chronic myelogenous leukemia (CML) [14] were given cyclophosphamide (CY) and either 12 or 15.75 Gy of TBI conditioning prior to allogeneic sibling-matched BMT. The rate of posttransplant relapse dropped from 35% to 13% in the AML population and from 30% to 7% in the CML group in those patients receiving the higher dose of TBI. These studies clearly demonstrated the potential for higher doses of radiation to generate more durable responses; however, those in the higher dose group experienced a significant increase in severe or fatal toxicities. Due to the increased frequency of toxicity (most often radiation-induced pneumonitis, mucositis or hepatic damage), the higher doses of TBI did not improve overall patient survival. Nonetheless, Thomas’ studies demonstrated a steep dose– response curve of the leukemia to radiation and supported the need for improved methods of selectively delivering radiation to malignant cells. With this background information, researchers in the late 1980s and 1990s finally developed effective clinical applications for Ehrlich’s initial concept of “magic bullets” as a form of cancer therapy. Animal models demonstrated that marrow ablation was feasible with radiolabeled mAbs and that subsequent stem cell infusion could restore marrow function reliably [15]. Nonmyeloablative doses of 90Y- and 131I-labeled anti-CD20 antibodies consistently produced remissions in 50–80% of patients with relapsed, refractory or transformed follicular NHL, and led to regulatory approval of both 90Y-ibritumomab tiuxetan (Zevalin) in 2002 and 131I-tositumomab (Bexxar) in 2003 by the United States Food and Drug Administration. Despite such high overall response rates, however, most patients treated with standard nonmyeloablative doses of radiolabeled anti-CD20 antibodies eventually relapse, indicating the desirability of further dose escalation.

Elements of RIT Specific antigen targeting with radiolabeled mAbs is designed to produce a biodistribution profile favoring radiation delivery to malignant cells; however, a portion of the radioimmunoconjugate remains unbound in the circulation and contributes to nonspecific irradiation of normal tissues. One unique aspect of RAb therapy is that tissue uptake of the therapeutic agent can be noninvasively assessed through imaging. The term “biodistribution” describes measured values of the relative levels of radionuclide throughout the body. In preclinical research models, biodistributions can be accurately measured by tissue sampling at serial time points after infusion of the radioimmunoconjugate, with quantitative measurements of the radionuclide content per gram of tissue [10]. In large-animal models and human subjects, biodistributions are usually determined using radionuclides that possess a γ-emission profile suitable for serial quantitative gamma camera imaging. Regions of interest including liver, lung, spleen, kidney, and marrow can be serially imaged, and activity curves representing counts per pixel generated. These data are calibrated using direct measurements of radionuclides in accessible tissues such as blood, marrow, and lymph nodes, allowing the construc-

Table 24.1 Factors that influence biodistribution of radiolabeled antibodies I. Nature of the targeted antigen a. Tissue distribution b. Cell surface stability II. Antibody a. Specificity b. Immunoreactivity c. Form d. Isotype III.

Antibody pharmacology a. Dose b. Infusion schedule c. Route

IV. Labeling procedure a. Choice of radionuclide b. Labeling chemistry

tion of time–activity curves and calculation of estimated absorbed radiation doses in tumor sites and normal organs (radiation dosimetry). These calculations are based on methods established by the Society of Nuclear Medicine Special Committee on Medical Internal Radiation Dose [16,17]. Factors which influence the biodistribution of RAbs have been delineated through both preclinical animal studies and patient trials (Table 24.1).

Target antigen selection Appropriate target antigen selection is crucial if a favorable biodistribution of RAb is to be achieved. Ideally, the target antigen will be expressed homogeneously on the cell surface of all target tumor cells at a high surface density, and will not be expressed on any normal cells. Unfortunately, such tumor-specific cell surface antigens are rare, and in practice the selection of lineage-specific hematopoietic antigens (e.g. CD20 and CD45) has proven to be highly successful in the setting of hematopoietic (stem) cell transplantation (HCT) where marrow rescue and hematopoietic reconstitution is anticipated. In fact, employing these more widely distributed lineage-specific antigenic targets offers an increased likelihood of eliminating both antigen-negative tumor cell variants and rare diseased cells within a “remission” marrow due to radioactive crossfire effects from adjacent antigen-positive hematopoietic cells [18]. Antigen density directly correlates with accumulation of antibody at tumor sites [19]. When the number of antigenic sites is less than 10,000, target saturation occurs at a relatively low antibody dose. This is a significant limitation, as each antibody molecule can be labeled with only a limited amount of radionuclide before immunoreactivity is impaired. Low antigen density theoretically poses less of a problem for studies involving α particles, which require only a single DNA “hit” to kill a cell. However, for the more commonly used β-emitting radionuclides, multiple hits are required to induce irreparable DNA strand breaks resulting in cell death. A second factor in antigen selection is the degree to which the receptor is internalized following binding. While some antigens remain stably on the cell surface after antibody binding, others are rapidly endocytosed and are transported to lysosomes for proteolytic degradation [20,21].

Radioimmunotherapy and Hematopoietic Cell Transplantation

After this process of internalization, the receptor density may remain low for several days. One of the best studied radionuclides in RIT, 131I, is not optimal for targeting internalizing receptors because rapid lysosomal metabolism of the conjugate leads to release of free 131I and 131Imonoiodotyrosine from cells [22,23]. This degradation significantly reduces the residence time of the radionuclide in tumor sites and therefore decreases the dose of cytotoxic radiation delivered to the malignant cells. When internalized antibodies are labeled with 111In or 90Y, however, small molecular weight cationic metabolites remain trapped within the lysosomes, and their impact is not significantly diminished [24]. Surface antigens that are not internalized following antigen–immunoconjugate complex formation exhibit minimal variation in cellular retention of 131I, indium-111 (111In), and 90Y [24,25]. CD20 is one of the best studied and most frequently targeted receptors for B-cell neoplasms and possesses many of the favorable attributes noted above. It has a high density of expression on more than 90% of B-cell tumors, and most investigators have demonstrated it to be minimally internalized and minimally shed from the cell surface of most B-cell malignancies.

Antibody Antibody specificity and binding kinetics also plays an important role in optimizing the biodistribution of RAb. Antibodies with a low immunoreactivity do not accumulate at sufficient levels in target tissues and result in a relatively nonspecific pattern of distribution. In the setting of HCT, accumulation at high levels in the marrow is anticipated and reversible; thus, nonspecific crossreactivity in radiosensitive organs like lung and liver is frequently dose limiting. In contrast, crossreactivity with marrow elements is of great significance in the non-HCT setting where myelosuppression frequently limits dose escalation. Another factor in antibody selection relates to stability, since mAbs susceptible to damage or agglutination during the labeling process often accumulate in the liver. In early studies, antibodies with high avidity were presumed to be optimal for RIT [26], but subsequent findings have suggested that very high avidity may promote a heterogeneity of antibody deposition through enhanced uptake at the most peripheral perivascular target sites. This so called “binding site barrier” [27] was subsequently demonstrated in a non-human primate study employing two different CD45 antibodies in which the antibody with slightly higher avidity preferentially localized to the most easily accessible antigen-positive cells and was rapidly cleared from the circulation (2 hours) [28]. Meanwhile the antibody with slightly lower avidity stayed in the circulation longer and demonstrated superior uptake in the lymph nodes, a less accessible target site. The use of antibody fragments has been investigated to improve the rate of penetration and overall distribution of RAbs. These smaller-sized fragments produced by enzyme digestion of intact antibodies (Fab, 50 kD; F[ab′]2, 100 kD) or through cloning and expression of the Fv portions of the immunoglobulin (Ig) light and heavy chains in Escherichia coli (single-chain Fv, 25 kD) permit more rapid diffusion into tumor sites [29,30]; however, their small size results in rapid clearance and short retention times compared with intact IgG antibody [31–35]. Due to the murine origin of most mAbs used for RIT, patients may develop a humoral immune response with formation of neutralizing human antimouse antibodies (HAMAs). The presence of HAMAs can result in an altered RAb biodistribution due to rapid circulatory clearance and increased hepatic uptake of the resultant immune complexes. HAMAs may also compromise diagnostic in vitro immunoassays employing analytes containing murine Igs (e.g. hormone assays) [36]. HAMAs usually develop 4–8 weeks after first exposure. They have

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posed less of a problem for patients with hematolymphoid malignancy in whom the capacity to mount a robust immune response to neoantigens is usually impaired. Moreover, HAMA-related complications are generally only relevant for patients treated with repeated doses of murine antibody; thus, HCT patients are at significantly lower risk because they complete RIT conditioning over a short time period. The HAMA response can be limited by the use of immunosuppressive agents (cyclosporine), the development of humanized or chimeric molecules, single infusions, and the use of less immunogenic antibody fragments. Antibody pharmacology Studies in murine models, non-human primates, and human subjects have documented the importance of appropriate antibody dosing to achieve optimal RAb biodistributions [10,11,37,38]. Each antibody possesses a specific dose range sufficient for achieving antigen saturation. Low doses result in relatively poor accretion of RAb in lymph nodes and tumor masses, resulting in unfavorable tumor-to-normal organ ratios. Excessive doses beyond target saturation simply increase the fraction of injected radionuclide that persists in the circulation and nonspecifically irradiates normal tissues. In the HCT setting, RAb infusions usually involve a trace labeled test dose (or “dosimetric infusion”), to assess the individualized biodistribution, followed by a therapeutic infusion. The rate of infusion is determined by patient tolerance, with fever, dyspnea, and hypotension limiting rapid infusion rates of some antibodies (e.g. BC8 antibody and rituximab). Based on the presence of a large number of antigenexpressing cells in the circulation of leukemic patients, alternate infusion schedules have been investigated. Animal studies have suggested that a small dose of unlabeled antibody to clear circulating cells expressing the target antigen may lead to an improved biodistribution of the subsequently administered labeled dose [39]. Preliminary patient trials have been inconsistent in this regard, with one group reporting no benefit of this approach in an anti-CD45 (BC8 [murine IgG1 mAb]) model [40], but another demonstrating a significant enhancement in bone marrow uptake when a low dose of unlabeled anti-CD45 antibody (YAML568 [rat IgG2a mAb]) was injected prior to therapy [41]. To allow for re-expression of an internalizing antigenic target (CD33), a model in which three divided doses are delivered at 48–72-hour intervals has been studied in patients with AML [42]. Labeling procedure Physical properties that are important in the selection of radionuclides for antibody conjugation include: types of emitted particles (α, β or γ), emission energies, half-life, availability, cost, ease of conjugation to proteins, and capacity for imaging (Table 24.2). 131I and 90Y are the best studied radionuclides for RIT, and both have been approved by the United States Food and Drug Administration for the treatment of patients with relapsed or refractory B-cell lymphoma in conjunction with murine anti-CD20 mAbs (131I-tositumomab and 90Y-ibritumomab). 131I has a half-life of 8 days and emits both β particles and γ rays. 90Y is a radiometal with a short half-life (2.7 days) that emits β particles which are four times as energetic as those from 131I, but virtually no γ rays; hence it cannot be easily imaged with a gamma camera. Dosimetry with 90Y is therefore reliant on the administration of a surrogate γ-emitting radionuclide, such as 111In. While the relative merits of 131I and 90Y have been debated extensively in the scientific literature on theoretical grounds, no randomized controlled trial comparing them has been performed. While 131I possesses a number of favorable attributes, including a high-energy gamma emission energy for imaging, low cost, relative abundance, and a well understood

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Table 24.2 Radionuclides for radioimmunotherapy

Radionuclide

Particles

Half-life

Median energy (MeV)

β-Emitters Iodine-131

β, γ

8.1 days

0.66 β

Yttrium-90

β

2.7 days

2.3 β

Lutetium-177

β, γ

6.7 days

0.50 β

Rhenium-188 Rhenium-186 Copper-67

β, γ β, γ β, γ

17 hours 89 hours 2.6 days

4.4 β 1.8 β 0.57 β

α-Emitters Bismuth-213

1 α, 2 β

46 minutes 5.84 α

Astatine-211

1α

7.2 hours 5.87 α

Actinium-225 4 α, 2 β

10 days

5.75–8.7 α

Comments

Well-defined chemistry, inexpensive, dehalogenation a problem, long half-life High energy, no γ radiation, accumulates in liver and bone Long half-life, low energy, stable when internalized, lower γ irradiation than I131 Limited access Lower γ radiation than I-131, can image with PET, limited access High energy, short path length, very short half-life Requires cyclotron for production Daughters a potential problem

PET, positron emission tomography.

chemistry for protein labeling, it also possesses several limitations, including rapid release of 131I-tyrosine and free 131I from tumor cells after endocytosis and proteolysis of the radioiodinated mAbs, thus reducing the residence time of the radionuclide at its target [23,25]. In addition, the γ-rays emitted by 131I are of sufficiently high energy (364 KeV) that they pose a potential safety risk to health-care workers and require radiation isolation for patients receiving the high doses required for HCT. 90Y has limitations as well. It is less abundant and more expensive than 131I, and has been shown to accumulate nonspecifically in liver and bone [43]. In addition, some groups have raised concerns about the use of 111In as a surrogate for 90Y dosimetry. Recently, there has been increased interest in the lanthanide radiometal 177lutetium, which possesses a β energy that is slightly less than that of 131I, emits γ rays suitable for gamma camera imaging, and possesses a half-life of 6.7 days. The lower-energy γ rays emitted by 177Lu reduce the potential radiation exposure of family members and healthcare workers compared with 131I. Copper-67 (67Cu), like 90Y and 177Lu, is retained intracellularly following internalization. It has both γ and β particle emissions and a half-life suitable for RIT. In addition, 67Cu emits technetium 99m-like photons that are suitable for imaging via positron

emission tomography. Expense and availability have been a major limitation to use of this radionuclide in the past, but there has recently been renewed interest in increasing the scale of production. Rhenium-186 (186Re) and rhenium-188 (188Re) have also been used in some studies. Both have γ and β emissions and well-described chelation chemistries; however, the use of 188Re is limited by its short half-life. α-Emitting radionuclides have considerable promise for RIT. Recent advances in the production and availability of α-emitting isotopes has sparked renewed interest in their use. These particles have a mass that is on average 7000 times that of β particles, and energies that range from three to 13 times greater than those of the β-emitters conventionally employed in RIT. This translates into significantly higher linear energy transfer for α emitters (approximately 100 keV/μm) compared with βemitters (0.2 keV/μm). Linear energy transfer is defined by the number of ionizations per distance traveled. Since the number of ionizations directly correlates with the potential to produce irreparable doublestranded DNA breaks, α particles exhibit a high relative biological effectiveness for cell killing. α-Emitters possess a number of theoretical advantages when compared with β-emitters. α-Emitters deliver higher energies over a much shorter range (path length <0.1 mm) [44], cause extensive cell damage that is independent of the dose rate, and exhibit no functional impairment in hypoxic environments, in contrast to βemitters [45]. In addition, α-emitters generally pose a low risk to healthcare workers due to their short path length. When conjugated to antibodies, the combination of high energy and short path length provides the capacity to kill only the antigen-expressing target cell while sparing surrounding normal cells; potentially reducing myelotoxicity. The absence of a significant “bystander effect” may not be optimal for larger-volume tumors, but is favorable for targeting small tumors, micrometastasis, and individual tumor cells (i.e. minimal residual disease in patients with leukemia) [46–48]. The α-emitting radionuclides best suited for clinical application include astatine-211 (211At) (half-life 7.2 hours), bismuth-212 (212Bi) (half-life 60.6 minutes) and 213Bi (half-life 45.6 minutes) [45,49]. While the short half-lives of α-emitting radionuclides complicate clinical trial design schemes, a number of groups have successfully conducted patient trials with α-emitting radionuclides conjugated to antibody (reviewed below). Retaining antibody immunoreactivity is essential during the physical process of radiolabeling, and great care must be taken to maintain sterility. The process can be exquisitely sensitive to external factors including fluctuations in temperature and pH. 131I can be covalently conjugated to antibodies on a tyrosine residue by either the chloramine T or Iodogen method. However, these simple approaches do not protect against rapid metabolism and release of free radioiodine when the antigen–antibody complex is internalized. Alternative processes have been developed to prevent intracellular degradation. The tyramine cellobiose method involves the formation of a polysaccharide bridge between the antigen and halogen radionuclide which allows for retention of the radioligand within the cell [50]. The nonhalogen radionuclides (90Y, 111In, and 177Lu) cannot be readily linked to antibodies by direct labeling and require a more complicated chemistry for attachment. This process requires the use of an intermediary molecule known as a chelate. Currently, the most commonly used chelation techniques include isothiocyanotobenzyl DTPA, or 1,4,7,10tetra-azacyclododecane-N,N1,N11,N111-tetracetic acid.

Dosimetry Optimal RIT treatment planning requires accurate individualized biodistribution data for calculating the predicted relative exposure of normal and target tissues from delivered radiation. In the clinical setting, this is

Radioimmunotherapy and Hematopoietic Cell Transplantation

achieved through the injection of a trace-labeled dose of antibody with subsequent serial single-photon emission computed tomography or planar imaging. Activity curves for specific regions of interest are generated and compared with curves derived from banked standard tissue biopsy specimens. This approach has been validated in animal models [38]. The accuracy of this approach is compromised for small deep-seated tumors or tissues not amenable to biopsy. Other factors limiting accuracy result from “signal averaging” that occurs when radionuclide content is assessed for whole organs. Current imaging techniques are incapable of identifying the heterogeneous distribution of antibody that occurs at a cellular level. This heterogeneity is most pronounced when evaluating large tumor volumes, for areas with poor vascular supply, and in cases when high-affinity antibodies accumulate at the periphery of tumors and in intravascular locations due to the “binding site barrier” [27]. Radionuclides with longer path lengths (e.g. 90Y) theoretically reduce the impact of cell-to-cell variation. In addition, the use of positron emission tomography in conjunction with photon-emitting radionuclides may improve the capacity to identify variable uptake within specific tissues. Based on the calculated biodistribution of the RAb, tables have been developed by the Committee on Medical Internal Radiation Dose that allow calculation of the mean absorbed dose over a specific organ volume for any radionuclide administered. This calculated dosimetry is based on formulas that weigh the contribution of the circulating radionuclide, radiation from neighboring organs to the organ of interest, and the direct contribution from the radionuclide delivered to the target site. This system for calculating dosimetry is not optimized for α-emitting particles because they deliver high energy over a very short range. Alternative methods involving microdosimetry have been developed for the α-emitters [51].

Radiation effects Conventional fractionated external beam therapy is capable of delivering high doses of radiation over a short period of time. Classically, fractionated doses of 1–2 Gy at 7–25 cGy/min up to a total dose of 10–15 Gy have been used in conditioning regimens for HCT. RAbs, on the other hand, deliver continuous, variable, exponentially decreasing, low doserate radiation. Although the peak rate of radiation delivery with RIT is substantially lower than with external beam TBI, there is continuous exposure of tumor cells to radiation, rather than intermittent exposure as with fractionated TBI. The exponential decline of radiation exposure seen with RIT is dependent on the chosen particle’s half-life of decay. There has been no systematic evaluation of the biologic impact of these different delivery mechanisms, but, based on strong evidence that the cytotoxic activity of radiation is proportional to its capacity to induce DNA strand breaks, some have suggested a potential advantage arising from continuous radiation exposure that prevents intervals during which cellular DNA repair can occur [52]. Consistent with this hypothesis, a number of human solid tumor xenograft experiments have demonstrated RAbs to have equivalent or even enhanced antitumor activity compared with external beam radiation [53,54]. Another potential explanation for this finding relates to what has been described as an “inverse dose-rate effect” [55]. It has been reported that, at some dose-rate ranges, a lower dose is more cytotoxic than a larger one. It is theorized that this seemingly paradoxical finding is related to induction of cell-cycle arrest in cells exposed to a continuous low dose-rate of radiation in the G2 phase of the cell cycle. Since the G2–M interface is the most radiosensitive phase of the cell cycle, the presence of a larger proportion of cells in this phase is believed to increase cytotoxicity.

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Leukemia The first studies integrating RIT into HCT targeted the CD33 myeloidassociated glycoprotein in patients with AML. These studies employed 131 I anti-CD33 (p67) followed by a standard cytoxan (CY)/TBI (12 Gy) preparative regimen. This regimen was selected because it provided adequate immunosuppression to ensure engraftment, while allowing the possibility for dose escalation of the RAb. CD33 was selected for initial targeting studies since this antigen is expressed on the leukemic cells in over 90% of cases of AML (as well as on normal myeloblasts, myelocytes, and metamyelocytes, but not on any nonhematopoietic cells). Identifying a “favorable biodistribution” of radioactivity, as defined by a trace-labeled test dose of antibody that distributes radiation preferentially to bone marrow and spleen, was considered essential prior to delivering an RIT therapy dose in these initial studies. The first small trial in Seattle enrolled nine patients, and only four met the biodistribution criterion required by the protocol for proceeding to the therapeutic 131 I-anti-CD33 infusion (estimated radiation doses to marrow and spleen that were greater than those to any normal organ) [56]. While all four patients tolerated both the therapeutic infusion of 131I-anti-CD33 and subsequent HCT without unexpected toxicities, three patients subsequently relapsed. Eradication of AML cells in this study was likely compromised by the relatively brief residence time of 131I-anti-CD33 in target tissues (9–41 hours). The rapid clearance of radioactivity from the marrow and spleen was thought to result from rapid internalization of the antibody–antigen complex followed by cellular metabolism of the radioimmunoconjugate, resulting in the release of 131I and 131 I-tyrosine from the marrow space. In addition, the relatively low density of CD33 per cell resulted in antigenic saturation at doses greater than 0.5 mg/kg. These considerations prevented dose escalation, and the study was halted. Despite these limitations, this initial study documented the feasibility of integrating RIT into HCT preparative regimens. In parallel with the studies in Seattle, investigators at Memorial Sloan Kettering Cancer Center (MSKCC) in New York also conducted trials targeting CD33 for myeloid leukemias. These studies employed a 131Ilabeled murine antibody called M195. While initial biodistribution studies revealed findings similar to the p67 experience in Seattle at the Fred Hutchinson Cancer Research Center (FHCRC) – with low concentrations of antibody administered prior to saturation – the reported halflife of 131I in the marrow was somewhat longer. Their first trial of 24 patients with myeloid malignancy involved a dose-escalation design. The labeled M195 was administered in divided doses, to allow for CD33 re-expression. Ninety percent of enrolled patients demonstrated a dramatic decrease in peripheral and marrow blasts. With doses of 131I of 5 GBq (135 mCi)/m2 or greater, profound pancytopenia ensued, and while HCT was not required by the study design, eight patients who received less than 5.9 GBq (160 mCi)/m2 received autologous or allogeneic HCT rescue. Among this group, one patient remained in remission at the time of study publication. No CRs were achieved in the patients receiving less than 5.9 GBq (160 mCi)/m2. In a subsequent study integrating radiolabeled M195 into a busulfan (BU)/CY allogeneic HCT preparative regimen for patients with relapsed or refractory AML, the MSKCC group reported 100% engraftment, and 18 of 19 study subjects achieving a remission, with three patients surviving disease free for 18–29 months post transplant. Ten patients died of transplant-related mortality, and six relapsed. As a refinement, the MSKCC group developed a humanized version of M195 (HuM195) with pharmacokinetic properties similar to murine M195, but without significant immunogenicity [57]. Cumulative longterm follow-up data from three phase I clinical trials investigating 131Ianti-CD33 antibodies in combination with BU/CY as a conditioning

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regimen for relapsed or refractory AML (n = 16), accelerated/myeloblastic CML (n = 14) or advanced myelodysplastic syndrome (MDS) (n = 1) revealed a median survival for all patients of 4.9 months (range 0.3–90+ months) after HCT. Three patients with AML were alive and disease free at over 59, over 87 and over 90 months. The intensified conditioning with 131I-anti-CD33 antibodies resulted in few toxicities beyond those seen with standard BU/CY preparative regimens. A number of limitations were associated with the M195 and HuM195 constructs. Radioiodination of these anti-CD-33 antibodies at high specific activity decreased the CD33-binding affinity, and, as a result, patients required multiple antibody infusions to deliver myeloablative doses to the marrow. In addition, the high doses of 131I required patient isolation to prevent high-energy (γ) emission exposure to hospital staff [42]. To avoid some of these limitations, the MSKCC group has investigated the role of 90Y as the therapeutic radionuclide; however, no data from these studies have been published to date. In the non-HCT setting, the MSKCC group has explored HuM195 conjugated to the α-particle emitter 213Bi. An initial imaging study in a leukemic patient population revealed 213Bi-labeled anti-CD33 to be visualized via gamma camera imaging as suitable for derived pharmacokinetic and dosimetry analysis [58]. A subsequent feasibility trial involving 18 patients with either relapsed/refractory AML or chronic myelomonocytic leukemia demonstrated the safety and feasibility of α-particle therapy [59]. All patients developed myelosuppression, and the absorbed dose ratios between sites of leukemic involvement (spleen, marrow, and liver) and whole body were reported to be 1000-fold greater than is seen with β-emitting constructs. Yet, while 93% of patients experienced a drop in peripheral blast counts, and 78% experienced reductions in marrow blast counts, no CRs were achieved. There were several limitations associated with this study. First, no maximum tolerated dose (MTD) was identified, because escalation beyond 37 MBq/kg was restricted by production capacity and cost. Second, the patient population studied carried a heavy tumor burden (average 1012 cells) that may not be well suited to the short-range and high-linear energy transfer properties of α particles. Nevertheless, incorporation of this approach into HCT conditioning regimens for patients in CR or near-CR remains a promising prospect, especially with alternative α-emitters such as actinium-225 (225Ac) [47,60]. Due to the limitations associated with CD33 targeting identified in their early studies, investigators at the FHCRC in Seattle selected an alternative target, the protein tyrosine phosphatase CD45, for subsequent investigations. Termed the “common leukocyte antigen,” CD45 is expressed on the surface of virtually all cells of hematopoietic origin except mature erythrocytes and platelets. CD45 possesses a number of potentially advantageous characteristics for RIT targeting. While the overall percentage of patients with AML and acute lymphoblastic leukemia (ALL) whose leukemic cells express CD45 is virtually equivalent to that of CD33 (85–95% versus 90%), the receptor density is 10-fold greater (100–200,000 versus 10,000–20,000 antigenic sites per leukemic cell). In contrast to CD33, which is rapidly internalized after antibody– receptor complex formation, the CD45 antigen remains stably fixed on the cell surface. After a series of murine and non-human primate studies revealing favorable biodistributions with the BC8 anti-CD45 antibody, a phase I trial for patients with AML or ALL was initiated employing an identical design to the FHCRC p67 trial [40,61]. The theoretical advantages of CD45 targeting were validated in this trial, which exhibited an improved biodistribution profile compared with the prior CD33 targeting experience. This was likely a function of improved hematopoietic tissue targeting due to a larger number of antigenic sites, and a period of red marrow retention that was more than double that seen in the p67 study (half-life 44.2 versus 21.4 hours). Of the 44 patients initially enrolled, 37 had

Fig. 24.1 Posterior gamma camera image revealing iodine-131 accumulation in the bone marrow, lumbar vertebrae, and pelvic axial skeleton in a patient with acute myeloblastic leukemia. Image obtained 40 hours post tracelabeled anti-CD45 antibody infusion. (Reproduced from [61], with permission. © The American Society of Hematology.)

favorable biodistributions (84%) with an average 2.3-fold more radiation delivered to the marrow than to the liver, which was the normal organ with the highest radiation exposure (Fig. 24.1). Patients with active leukemia experienced higher marrow-to-normal organ ratios than those treated in remission. Thirty-four patients were treated in a dose-escalation trial, receiving a conditioning regimen of 131I-labeled BC8 at doses ranging from 76 to 612 mCi combined with 12 Gy TBI, CY, and HCT. At each dose level, the activity of 131I delivered was predetermined by the estimated amount of radiation that would be delivered to the normal organ that was projected to receive the highest dose during the biodistribution step (usually the liver). 131I-BC8 was estimated to deliver a supplemental radiation dose of 50 Gy to the spleen and 24 Gy to the marrow before the MTD was reached. The dose-limiting toxicity was severe mucositis, which was encountered after the delivery of 10.5 Gy to dose-limiting normal organs. In the ALL subset, three of nine patients remained disease free 19, 45, and 56 months after transplantation. Seven of 25 AML/MDS patients remained without evidence of relapse 15–89 (median 85) months following transplantation. These promising findings led to a subsequent study for patients with AML in first CR (CR1) employing 131I-BC8 combined with a standard preparative regimen of targeted BU 600–900 ng/ml and CY 120 mg/kg. The antibody biodistribution was “favorable” in 88% of patients. Fortysix patients were treated with 102–298 mCi (3744–11026 MBq) of 131IBC8 antibody combined with BU/CY, with all but three receiving estimated radiation doses of 5.25 Gy to the liver, and average estimated absorbed radiation doses of 11.3 Gy to the bone marrow and 29.7 Gy to the spleen. Overall, 61% of patients were surviving disease free at a median of 3 years post transplant, with eight transplant-related deaths and eight relapses (Fig. 24.2). Based on an intention-to-treat analysis, the study patients were compared to an International Bone Marrow Transplant Registry cohort of 509 patients treated with BU/CY alone. Following adjustment for age

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nonrelapse mortality was 25%. Patients transplanted in complete or good partial (<15% blasts in the marrow) response had the best outcomes, with 59% disease-free at median follow-up. In contrast, only 8% of patients who had greater than 15% blasts in the marrow were alive at this time point [64]. Less promising results were reported in another study in which 188 Re-anti-CD66 was combined with a conventional preparative regimen. High-risk AML or ALL patients received primarily non-T-cell-depleted peripheral blood stem cells from either human leukocyte antigenmatched related or unrelated donors. A significantly higher rate of transplant-related toxicity and acute graft-versus-host disease was reported, with a transplant-related mortality of almost 50% (nine of the 19 patients enrolled) [65].

Non-Hodgkin’s lymphoma Fig. 24.2 Estimates of the probability of disease-free survival, nonrelapse mortality, and cumulative risk of relapse among 46 patients with acute myeloblastic leukemia in first complete remission who received a therapeutic dose of iodine-131-BC8 (102–298 mCi) followed by busulfan/ cyclophosphamide and allogeneic hematopoietic cell transplantation. (Reproduced from [62], with permission. © The American Society of Hematology.)

and cytogenetic risk, the hazard of mortality among the 131I-BC8 RITtreated patients was 0.65 (95% confidence interval [CI] 0.39–1.08; p = 0.09). Subgroup analysis revealed the overall 3-year survival to be 75% (95% CI 58–92%) among patients with intermediate-risk cytogenetics and 36% (95% CI 11–62%) for those with unfavorable cytogenetics. The risk of relapse in this study was found to be independent of the total absorbed radiation dose to both bone marrow and spleen [62]. There was no delay in engraftment associated with the RAb, and infusional toxicities were mild and manageable. The most frequent severe toxicity was grade 3 mucositis in two patients, and despite a planned delivery of 5.25 Gy to the liver, severe sinusoidal obstructive syndrome (SOS) was not seen. In Germany, a number of groups have explored CD66 (a, b, c, e) as an alternative target for patients with MDS or AML. CD66 is expressed at high levels on the surface of normal myeloid cells beyond the promyelocyte stage, but not on leukemic blasts. It is neither internalized nor shed. Targeting this receptor requires that the delivered radionuclide kill tumor cells entirely via “bystander” radiation delivered from adjacent nonleukemic cells in the marrow space. Fifty-five patients with AML or MDS who were in complete or partial remission but judged to be at high risk for relapse after standard HCT were enrolled in this phase I–II trial [63]. All patients enrolled had favorable initial biodistributions and received a therapeutic dose of 188Re-anti-CD66 antibody delivering a mean of 15.6 Gy of additional radiation to the marrow. Patients were then treated with a variety of full-dose conditioning regimens followed by allogeneic HCT. T-cell depletion for graft-versus-host disease prophylaxis was achieved by either CD34+ selection or administering alemtuzumab in the donor cell infusion bag. The kidney was the organ receiving the highest dose of supplemental radiation (mean 7.2 Gy), and late renal toxicity was a complication in 11% of patients, with an incidence correlating with the renal dose exposure. There was no increase in transplant-related mortality associated with the addition of RIT. Overall disease-free survival was 47% with a median follow-up of 61 months. Leukemic relapse accounted for 28% of patient deaths, and

NHL cells are ideal targets for RIT because of their exquisite radiosensitivity and their stable expression of well-characterized lineage-specific surface antigens. The direct cytotoxic effect of targeted radiation obviates the need for reliance on the host’s immune effector mechanisms, which are frequently impaired in NHL patients. The relatively recent approval of two CD20-targeted radioimmunotherapeutic agents by the United States Food and Drug Administration for the treatment of relapsed or refractory follicular and transformed follicular NHL – 131Itositumomab (Bexxar) and 90Y-ibritumomab tiuxetan (Zevalin) – generated excitement about the promising results seen with both standard and high-dose myelosuppressive treatment regimens. Some of the earliest NHL patient studies using mAbs employed patient-specific anti-idiotypic antibodies raised against the tumor-specific clonal idiotype of the Ig molecule expressed on B-cell malignancies [66,67]. These trials produced promising results with durable (>5 years) objective remissions in 60–70% of patients studied; however, the technical and financial limitations inherent in patient-specific production of anti-idiotypic antibodies rendered the widespread use of this approach unfeasible. Subsequently, most investigators focused on targeting lymphocyte differentiation antigens expressed on both normal and malignant lymphoid cell surfaces. The CD20 antigen was a particularly attractive target because of its high density (~150,000/cell) [68], and its presence on more than 90% of B-cell tumors. Further, most investigators believe it is minimally shed or internalized following antigen binding, and, while it is present on almost all B lymphocytes, the temporary elimination of normal B cells by anti-CD20 antibodies has not proven a significant limitation. Initial studies with unmodified murine anti-CD20 mAbs were of limited success due to the restricted capacity of murine Igs to fix human complement or mediate antibody-dependent cellular cytotoxicity with human effector cells. The first patient studies, conducted by Press et al. in Seattle, demonstrated that high doses of the murine anti-CD20 antibody 1F5 could be administered safely to patients, and, in individuals receiving the highest doses, dramatic albeit short-lived remissions could be achieved [69]. These initial studies helped to pave the way for the development of a human–mouse chimeric anti-CD20 antibody, rituximab, which ameliorated the murine antibody-associated limitations [70]. Early studies by Maloney et al. [71] at Stanford reported objective remissions in 46% of patients treated with rituximab in a multicenter phase II trial, with minimal toxicity. These results were confirmed in a phase III study in which 48% of patients with indolent lymphoma achieved objective responses, with 6% achieving CRs. The median duration of response was approximately 1 year [72]. While the initial studies with rituximab were encouraging, almost half of all patients with relapsed indolent lymphoma and 60–70% of patients

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with relapsed aggressive lymphomas failed to respond to single-agent rituximab. Further, only 5–10% of patients with indolent lymphomas achieved CR with rituximab, and the median response duration was less than a year. In light of these factors, a number of investigators continued to pursue mechanisms for amplifying the therapeutic promise of mAbs by conjugating radionuclides to them. The first studies to evaluate the potential efficacy of RAbs in lymphoma were conducted by DeNardo et al. (Davis, CA) in the mid-1980s [73]. In these studies, an antibody recognizing a DR variant antigen (Lym-1) was conjugated to 131I. The Davis group demonstrated both the feasibility and safety of RIT in a population of advanced-stage patients with relapsed B-cell malignancy; moreover, they documented an objective remission rate of approximately 50% in a population of patients with disease that had been resistant to standard therapy. Subsequently, a robust literature developed reporting clinical trial data that demonstrated the safety and efficacy of nonmyeloablative RIT in B-cell NHLs, a review of which is beyond the scope of this chapter. While most centers focused on CD20-targeted RIT at standard, nonmyeloablative doses, at the FHCRC in Seattle a series of studies in the mid-1980s was initiated to investigate the possibility of replacing external beam TBI with targeted RIT as a conditioning regimen for bone marrow or hematopoietic cell transplantation. Through hematopoietic cell rescue, Press and colleagues were able to bypass the dose-limiting toxicity to red marrow observed in standard RIT trials. While researchers in Seattle and elsewhere had made significant gains using high-dose chemoradiotherapy-based conditioning regimens for autologous HCT, cure rates of no more than 50% in patients with relapsed disease were achievable. Because further dose escalation was associated with unacceptable morbidity and mortality [74], integration of RIT into HCT conditioning regimens was tested to determine if it could maximize the tumor-to-normal organ ratios of delivered radiation, thereby improving cure rates while minimizing additional toxic effects. The first such study was designed to compare the biodistribution profiles for a number of different B-cell antibodies, to determine the impact of antibody dose on biodistribution, and to assess the toxicity and potential efficacy of increasing doses of 131I conjugated to the B-cell antibodies under study [37,75]. This phase I dose-escalation trial studied the biodistribution of anti-CD37 and anti-CD20 antibodies in 43 patients with relapsed B-cell NHL. Patients were given successive weekly doses of 0.5, 2.5, and 10 mg/kg of 131I trace-labeled (5–10 mCi) antibody. In the 24 patients with favorable biodistribution profiles, protein doses of 2.5 and 10 mg/kg were required to achieve favorable biodistributions for anti-CD20 and anti-CD37, respectively. The subset of patients who presented with tumor volumes of over 500 cm3 or splenomegaly, rarely met biodistribution cut-offs required for the therapy step. In the 19 patients who received therapeutic infusions, maximal doses to normal organs were calculated according to dose-escalation parameters allowing delivery of radiation at between 10 and 31 Gy to critical normal organs. The therapeutic doses of 131I ranged from 234 to 777 mCi delivered on 58–1168 mg of antibody. For the 15 patients who received autologous stem cell reinfusion, nonhematologic toxicities were mild and included nausea, fever, elevated thyroid-stimulating hormone level, and elevated hepatic transaminase levels at doses below 23 Gy. Pulmonary doses of 27 Gy defined the MTD, with two of four patients developing reversible cardiopulmonary toxicity at this level. Objective responses were seen in 95% of patients receiving therapeutic infusions of 131I-antibody, with 85% achieving a CR. Eight patients remained in CR 46–95 months following therapy. Patients assessed with anti-CD20 antibodies (tositumomab or 1F5) exhibited biodistributions demonstrating superior targeting compared with the other antibodies evaluated (anti-CD37 and anti-idiotype). Based on this finding, a phase II study with high-dose 131I-tositumomab (for-

merly known as “anti-B1”) was performed. In this trial, 22 of the initial 25 patients demonstrated favorable biodistribution profiles, and 21 patients received 345–785 mCi of 131I-tositumomab, delivering 25– 31 Gy to dose-limiting normal organs and higher doses (27–92 Gy) to tumor sites. All enrolled patients were reinfused with stem cells, and two patients died prior to engrafting: one with Gram-negative sepsis, and the other with progressive high-grade lymphoma. The regimen was well tolerated, with minimal toxicity when compared with standard TBIbased conditioning regimens. Four of the 25 patients developed a HAMA response. The overall response rate was 86%, and 76% achieved CR. With a median follow-up of 42 months, the estimated overall survival was 68%, and the progression-free survival was 42%. Eleven patients remained alive and free of disease recurrence for 5–10 years with no additional therapy administered. While the rates of CR and prolonged remissions in this population with multiply relapsed B-cell lymphomas were encouraging, maximal doses of RIT with stem cell support still only averted eventual relapse in approximately 50% of the patients studied. To further improve the fraction achieving durable CRs, Press and colleagues conducted a phase I–II study combining 131I-tositumomab with high-dose etoposide, CY, and autologous HCT [76]. Fifty-two patients were treated on this protocol, and the MTD was determined to be 25 Gy 131I-tositumomab (1.7 mg/kg), 60 mg/kg etoposide, and 100 mg/kg CY. Four patients died of opportunistic infections. The overall response rate was 87%, with 77% CRs and 10% partial remissions. At 2 years, the estimated overall survival for all 52 patients enrolled was 83%, and progression-free survival was 68%. When compared with a nonrandomized control group of 105 patients with NHL (Fig. 24.3) who were treated with TBI/CY/ etoposide and autologous HCT in a multivariate analysis that incorporated disease grade (indolent versus aggressive), stage, and chemosensitivity, the patients in the RIT-based conditioning regimen group

Fig. 24.3 Overall survival of patients with relapsed B-cell lymphomas who underwent high-dose chemoradiotherapy including either iodine-131tositumomab at myeloablative doses (52 patients; upper line) or a standard total body irradiation (TBI)-based preparative regimen (105 control patients; lower line) followed by autologous hematopoietic cell transplantation. Patients in the radioimmunotherapy group received up to 25 Gy to normal organs; the TBI group received 1.5 Gy twice a day for 4 days. Both groups were treated with identical doses of etoposide (VP16) and cyclophosphamide (Cy). (Reproduced from [76], with permission. © The American Society of Hematology.)

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demonstrated superior progression-free and overall survival (38% [p = 0.006] and 50% [p = 0.01], respectively). These findings are particularly encouraging in light of a subsequent report by Johnson and Press evaluating the ability of a variety of chemotherapeutics to synergize with 131I-based anti-CD20 RIT [77]. These studies, involving an in vitro lymphoma model and employing formal isobolographic and dose-modification factor analysis, demonstrated that the nucleoside analogs (fludarabine and cytarabine) synergize optimally with RIT, while CY and etoposide provide only minimal synergy. This suggests that more potent chemoradioimmunotherapy regimens containing a nucleoside analog may further improve the already impressive outcomes seen with RIT/CY/etoposide. Patients above age 60 comprise over a half of all new diagnoses of NHL in the United States, and are at particularly high risk for poor outcomes when diagnosed with NHL [78,79], yet individuals in this age group have been excluded from most HCT trials due to concerns about the toxicity of the conditioning regimens. Gopal et al. sought to exploit the reduced nonspecific normal organ toxicity seen with RIT-based conditioning regimens in a phase II safety and efficacy trial employing single-agent, high-dose 131I-tositumomab conditioning prior to autologous HCT. Twenty-four heavily pretreated NHL patients aged 60 years or over (median age 64) received 328–1154 mCi of 131I-anti-CD20 to deliver a maximum of 25–27 Gy to critical organs. One patient developed HAMA and was withdrawn from the study. Among the remaining 23 patients, there were no treatment-related deaths, and only two patients experienced a grade 4 non-hematologic toxicity (a pulmonary embolus and anorexia requiring parenteral nutrition). The overall response rate was 67%, with 13 patients attaining a CR or unconfirmed CR, and three patients achieving a PR. The estimated 3-year overall and progressionfree survival rates were 59% and 51%, respectively. Based on these promising findings, and the data suggesting a potent supra-additive effect for RIT combined with nucleoside analogs, a combined fludarabine/131Itositumomab trial for patients aged 60 years or over is ongoing. One of the limitations associated with high-dose 131I RIT conditioning regimens is the requirement that patients remain in inpatient isolation until their body radiation counts return to safe levels. To study the efficacy of an outpatient-based approach, Vose et al. at the University of Nebraska conducted a phase I trial combining conventional, nonmyeloablative doses of 131I-tositumomab with the BCNU (carmustine), etoposide, cytosine arabinoside, and melphalan (BEAM) high-dose chemotherapy transplant regimen followed by autologous HCT in 23 patients with aggressive, chemorefractory NHL [80]. The total body dose of radioimmunoconjugate was escalated up to the standard 75 cGy dose approved for this radiopharmaceutical. There was no significant difference in toxicity for patients receiving 75 cGy of 131I compared with patients receiving high-dose BEAM alone. The CR rate after HCT was 57%, and the overall response rate was 65%. At a median follow-up of 38 months, the event-free and overall survival rates were 39% and 55%, respectively. These data are encouraging in light of the limited toxicity associated with the addition of outpatient 131I to an outpatient autologous HCT regimen; in addition, the outcomes appear favorable since conventional HCT in chemotherapy-resistant NHL has previously demonstrated only 10–20% survival at comparable follow-up [81,82]. The Nebraska group has also explored the role of RIT in patients with relapsed NHL following high-dose chemotherapy and autologous HCT. In a phase I trial, 19 patients with progressive B-cell NHL after HCT were treated with a cohort-specific dose of 90Y-ibritumomab tiuxetan (0.10–0.20 mCi/kg) to determine appropriate dosing in this very highrisk population [83]. Dose-limiting or treatment grade 3 or 4 toxicities were seen only at the highest (0.2 mg/kg) dose level, with thrombocytopenia, neutropenia, and anemia occurring in 10, six, and four patients respectively. Responses were seen in nine patients, with five CRs, one

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unconfirmed CR, and three PRs. The median duration of CR/unconfirmed CR was 47 months. With a median follow-up of 37 months, the 1-year event-free and overall survival rates were 26% and 57%, respectively. Overall survival at 3 years was 33%, and the event-free survival was 13%. Nademanee and colleagues at City of Hope Comprehensive Cancer Center have reported on the role of high-dose 90Y-ibritumomab tiuxetan combined with high-dose etoposide and CY in the autologous HCT setting [84]. In a phase I–II trial, patients were given escalating doses of radiation based on dosimetry during phase I, and an MTD to critical organs was calculated to be 10 Gy. Subsequently, 31 patients with follicular (12 patients), diffuse large B-cell (14 patients), and mantle cell (five patients) lymphoma, including seven in first complete or partial remission, were treated with 10 Gy of 90Y-ibritumomab tiuxetan (36.6–105 mCi) in combination with either 40 mg/kg (cohort 1, six patients) or 60 mg/kg (cohort 2, 25 patients) of etoposide and a fixed dose of CY (100 mg/kg). There were five relapses, and 24 of the 31 patients were alive and in remission at a median follow-up of 22 months post therapy. The 2-year relapse-free and overall survival rates were 78% and 92%, respectively. These findings provide further data on the promise of integrating CD20-targeted RIT into conditioning regimens. While it is enticing to compare the results from this 90Y-based trial with 131I findings reported by Press, it should be reiterated that the patient profiles in these two studies were quite different in terms of inclusion criteria, differences in radiation dose, and number of prior therapies. A similar study of relapsed and refractory B-cell NHL patients has been conducted by Winter et al. at Northwestern University. In this phase I trial, 44 patients received dose-escalated 90Y-ibritumomab followed by high-dose BEAM therapy and autologous HCT. One patient developed transient SOS at the 700 cGy level, and two dose-limiting toxicities occurred at the 1700 cGy dose level; one patient developed grade 4 stomatitis on day 10, and a second patient developed septic pulmonary emboli on day 13. In addition, one heavily pretreated patient developed MDS on day 291. With a 21-month median follow-up, the 3-year overall and progression-free survival rates were 52% and 37%, respectively [85]. In a small trial conducted in Germany, Behr et al. have reported on seven patients treated with potentially myeloablative doses of either 131I-rituximab (five patients) or a humanized 131I-anti-CD22 mAb (two patients). CRs were reported in all five patients who received stem cell support [86]. A subsequent phase II trial from the City of Hope Center demonstrated encouraging results in adding 90Y-ibritumomab tiuxetan to highdose BEAM chemotherapy conditioning prior to autologous HCT. Forty-one patients with B-cell lymphoma whose age or prior radiotherapy deemed them ineligible for TBI or high-dose 90Y-ibritumomab tiuxetan were described. The patient population included diffuse large B-cell lymphoma (20 patients), mantle cell lymphoma (MCL) (13 patients), grade 2 or 3 follicular lymphoma (four patients), and transformed lymphoma (four patients). All patients received a standard nonmyeloablative dose of 90Y-ibritumomab (0.4 mCi/kg), with no dose adjustment for neutropenia or thrombocytopenia, followed by high-dose BEAM therapy 1 week later. The median age of the enrolled patients was 60 years, and the median number of prior therapies was two. After 18.4 months median follow-up, the estimated 2-year overall survival was 88.9%, and progression-free survival was 69.8%. The median times to white blood cell and platelet engraftment were 11 days and 12 days, respectively. No transplant-related deaths occurred in the first 100 days after transplant [87]. Overall, the rates of treatment-related toxicities were not different from those reported for patients receiving BEAM therapy alone. Grade 4 hepatotoxicity and pulmonary toxicity were seen in a single patient (in conjunction with sepsis).

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Shimoni and colleagues in Israel have also reported on the use of standard-dose 90Y-ibritumomab tiuxetan combined with high-dose BEAM chemotherapy as a conditioning regimen for aggressive NHL [88]. Their findings were similar to the previous studies, with an overall CR rate of 76% in the 21 evaluable patients (of the 16 patients in CR, five were initially in PR but reached CR after additional radiation). The 2-year progression-free and overall survivals were 52% and 67%, respectively. Patients with MCL comprise 4–10% of all cases of NHL and represent a special challenge as they exhibit the poorest long-term survival of any lymphoma subtype. There is no consensus regarding optimal first-line treatment for MCL patients, and the disease is generally considered incurable with chemotherapy regardless of the regimen employed (~2– 3-year survival) [89]. Furthermore, conventional HCT performed after relapse has generally been unsuccessful [90,91]. The Seattle group has performed a study evaluating responses in 16 patients with relapsed MCL with an autologous HCT conditioning regimen employing 131Itositumomab delivering 20 Gy (one patient), 23 Gy (one patient) or 25 Gy (14 patients) to the lung followed by CY and etoposide [92]. Based on prior experience demonstrating suboptimal biodistributions in patients with significant splenomegaly or bulky disease, patients in this trial were required to undergo debulking therapy prior to HCT if splenomegaly was present or the estimated tumor burden exceeded 500 cm3. This resulted in five patients without measurable disease prior to the 131 I-tositumomab infusion. In the remaining 11 patients evaluable for response, there were eight (73%) CRs, one (9%) PR, and two (18%) patients with stable disease. In the two patients found to have stable disease 1 month following transplant, both were subsequently documented to be in CR with no additional chemotherapy or radiation administered. As a result, the overall response rate for the 11 evaluable patients was 100%, with 91% achieving a CR. Limited data are available on the use of RIT in conditioning regimens for allogeneic HCT. Fietz et al. [93] have reported on two patients with relapsed lymphoma who received 90Y-ibritumomab tiuxetan combined with standard-dose fludarabine and CY prior to allogeneic HCT. One patient had previously exhibited a good response to standard 90Y therapy after failing autologous HCT and multiple standard chemotherapy regimens. The second patient had undergone both prior autologous HCT and, following relapse, an allogeneic human leukocyte antigen-identical sibling transplant. Both patients received standard 90Y dosing (0.4 mCi/ kg) without dosimetry. Both patients tolerated the procedure well, with no delay in engraftment. Only short-interval follow-up is available, with patient 1 showing slow progression of disease within the first 100 days and patient 2 achieving PR. The CD22 surface receptor has also been targeted by a number of groups. Goldenberg and colleagues in New Jersey reported on three patients treated with potentially myeloablative doses (90 mCi/m2) of 131 I-anti-CD22 (LL2 IgG) followed by autologous bone marrow support. Both evaluable patients had PRs lasting 2 and 8 months, respectively [94]. Vose et al. [95] have treated 21 patients on a phase I–II trial using multiple doses of 131I-LL2 antibody, with an overall CR rate of 24%. Five patients in this study required autologous HCT due to prolonged cytopenia. Based on patient experience suggesting a benefit from 131I-tositumomab or 90Y-ibritumomab therapy delivered shortly before standard reduced-intensity HCT, investigators in Seattle are currently accruing patients to a reduced-intensity allogeneic HCT trial in which standard dose 90Y-ibritumomab tiuxetan is combined with fludarabine (30 mg/m2 × 3 days) and TBI (2 Gy on day 0) with cyclosporine and mycophenolate mofetil immunosuppression. With safety as the primary endpoint, this study is open to a population of heavily pretreated, refractory/resistant patients whose age, comorbidities, and disease burden render them

ineligible for either standard reduced-intensity or traditional myeloablative allogeneic transplantation. In the group of six patients reported to date, none has shown disease progression, and three have demonstrated an objective response within 1 month of transplant. While clearly a limited subset, these findings are encouraging in this population of patients with chemotherapy-resistant and bulky disease (mean tumor bulk 6.5 cm at study entry) [96].

Hodgkin’s lymphoma The protein antigen ferritin is found at elevated concentrations in the Reed–Sternberg cells of Hodgkin’s lymphoma. Polyclonal antiferritin antibodies labeled with 131I were initially studied at nonmyeloablative doses, documenting the safety and efficacy of this approach. In a phase I–II study conducted at Johns Hopkins University [97], 19 patients with refractory Hodgkin’s disease were treated with 20–50 mCi of 90Ylabeled antiferritin antibody followed by autologous HCT, with an overall response rate of 65%. Bone marrow harvesting was unsuccessful in an additional 16 patients, and these patients were treated with a reduced activity (20 mCi), with a reported overall response rate of 58%. Good tumor responses correlated with smaller tumor size (<30 cm3 versus >500 cm3) and with higher levels of radioactivity 1 hour post infusion. In an updated report from this trial including 39 patients, 10 CRs and 10 PRs were reported, with only five patients alive at the time of the report all of whom had relapsed [98]. In a second study, Bierman et al. employed a high-dose chemotherapy regimen combined with 90Y-antiferritin conditioning prior to autologous transplant. Twelve patients with recurrent Hodgkin’s disease received 0.67–1.22 mCi of 90 Y-labeled antibody followed by cytoxan, BCNU, and etoposide. There were four transplant-related patient deaths, and three patients remained in CR for more than 2 years after therapy [99]. Schnell et al. [100] have reported a trial with 131I-anti-CD30 antibody in 22 patients with Hodgkin’s disease. Nineteen of the patients had advanced disease and were heavily pretreated with a median of four different prior regimens, including high-dose chemotherapy, followed by autologous HCT in 16 of the patients studied. The average dose to the red marrow was 0.29 ± 0.10 mGy/MBq. One patient experienced a CR lasting 5 months, and PR was seen in five patients, lasting up to 6 months. There were three mixed responses. Although the patient population in this study admittedly had highly resistant disease, with a majority having failed prior autologous HCT, the selection of CD30 as a target antigen may also have been a limitation to the success of this approach. The presence of CD30 is limited to Reed–Sternberg cells, which have a relatively sparse distribution; thus, only a small amount of radioactivity can be delivered to target sites using anti-CD30 antibody.

Solid tumors Solid tumors are generally less sensitive to the effects of radiation than hematologic malignancies, and are therefore less attractive for studies of RIT. Nevertheless, selected tumor types are sufficiently radiosensitive to merit evaluation with this approach. DeNardo and colleagues at the University of California (Davis) [101] have explored 131I-labeled chimeric antibody reactive with the L6 cell surface antigen, highly expressed on lung, breast, colon, and ovarian carcinomas. In a small study, three patients were given 131I-labeled antibody followed by autologous stem cell rescue, with one patient demonstrating a minimal response. Anti-CEA mAbs have been generated against a variety of CEA epitopes and used for therapy in a variety of tumors of epithelial origin. In

Radioimmunotherapy and Hematopoietic Cell Transplantation

breast cancer patients, a murine IgG1 mAb, T84.66, has been studied in six patients treated with 90Y-DTPA-cT84.66 followed by autologous HCT [102]. Patients received between 15 and 22.5 mCi/m2. All had temporary but measurable therapeutic responses, and the MTD was not reached. Another group has reported phase I dose-escalation data using high-dose 90Y conjugated to an mAb reactive with a human milk fatassociated antigen (BrE-3) [103]. In this study, nine patients with advanced breast cancer were treated with 15–20 mCi/m2 of 90Y-BrE-3. Four of eight patients had partial responses (PRs). Anti-mucin-1 (MUC-1) mAb 170H.82 is directed against an asialoGM1 antigen that is highly expressed in adenocarcinoma of the breast. This anti-MUC-1 mAb has been labeled with 90Y and given to three patients with metastatic breast cancer, with subsequent reinfusion of previously collected peripheral blood stem cells. A PR was reported in one patient [104]. A second study from the same group reported on three additional patients who received peripheral blood stem cell infusion to hasten hematopoietic recovery after 90Y-labeled anti-MUC-1 mAb therapy. Two patients experienced a minor reduction in soft tissue disease lasting less than 1 month [105]. Autologous stem cell support has also been used to enable dose escalation with 131I-anti-CEA antibody in patients with metastatic medullary thyroid cancer [106], and in children with neuroblastoma receiving 131I-labeled 3F8 anti-GD2 antibody [107].

Toxicity Toxicity, in the context of radiolabled antibodies in HCT, falls into three general categories: acute infusional reactions, short-term toxicities associated with radiation delivery, and long-term sequelae. Acute infusionassociated toxicities are antibody specific. The anti-CD20 antibodies (rituximab, ibritumomab tiuxetan, and tositumomab) are usually associated with only mild reactions, and, in the case of rituximab, tolerance improves with repeated infusions. In contrast, anti-CD45-BC8 causes almost universal fever, chills, and hypotension (when administered rapidly). The severity of these reactions can usually be reduced by premedication with diphenhydramine, meperidine hydrochloride or hydrocortisone. The short-term toxicities seen after high-dose RIT for HCT are usually related to myelosuppression. Pancytopenia is an expected consequence, and relatively prolonged periods of thrombocytopenia are not atypical. Residual radionuclide in the marrow can potentially pose a toxic risk to newly transplanted marrow cells. To prevent such damage, transplants are usually delayed until the residual radiation in the marrow space is of minimal risk. Press et al. [75] have performed the only study to date that delineates the dose-limiting nonhematopoietic toxicities from single-agent RAb therapy. In this study, 27 Gy to the lung was dose limiting; this represents a significantly higher dose than has been tolerated with external beam TBI (maximum dose 16 Gy). Other short-term toxicities seen with RIT are similar to those associated with external beam TBI. These include mucositis, nausea, and vomiting. Alopecia is uncommonly seen with high-dose RIT, in contrast to external beam TBI. Longer-term sequelae include hypothyroidism in almost 70% of patients receiving 131I therapy (even when prophylactic potassium iodide is given). Nephropathy has been reported in six of 93 patients treated with 188Re-anti-CD66 as part of an allogeneic HCT conditioning regimen [108]. In a multivariate comparison of 125 patients who received either high-dose RIT or conventional high-dose therapy with TBI followed by autologous HCT, the long-term risk of therapy related MDS/AML was between 6% and 7% for both groups [109], suggesting no increased risk associated with the RIT-based regimen.

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At standard (nonmyeloablative) doses, RIT in NHL has a CR rate of approximately 30%, with 20–25% of patients experiencing long-term disease-free survival; therefore, a majority of nonmyeloablative RIT patients will ultimately require additional treatment. The impact of RIT on subsequent autologous stem cell collection has been a subject of interest and concern. In a study by Ansell and colleagues designed to assess subsequent therapies after ibritumomab tiuxetan, a cohort of eight patients were described who underwent peripheral blood stem cell collection after RIT. One patient in this group required an additional bone marrow harvest to achieve adequate cell counts – all eight patients engrafted normally [110].

Future directions Clinical data have demonstrated that RAb therapy is capable of delivering between two- and fourfold more radiation to target sites than surrounding normal tissues. When patients with relapsed lymphoma receive myeloablative doses of anti-CD20 RIT followed by stem cell rescue, rates of response have improved dramatically. Objective remissions are seen in 85–90% of such patients, with 75–80% experiencing durable CR, many of which last 5–18 years [75,76,111,112]. Optimizing the therapeutic index of RIT further by improving biodistribution ratios in favor of target cells may improve outcomes in both hematologic malignancies and solid tumors. A number of pharmaceutical and academic research centers are focusing on the identification of new antigenic targets that are highly expressed on malignant cells and minimally expressed on normal tissues. The increased availability of β-emitting radionuclides with favorable physical characteristics, such as 177Lu and 67 Cu, may provide another avenue for future investigations. α-emitters, including those with longer half-lives (225Ac), also show great promise [51]. Already, investigators in Seattle and at the University of Tübingen have demonstrated in a canine model that TBI can be entirely replaced with 213Bi-anti-CD45 in allogeneic reduced-intensity HCT [113].

Fig. 24.4 Efficacy of pretargeting as demonstrated by planar gamma camera images of Burkitt’s lymphoma xenograft tumors in athymic mice. Animals were injected with either directly labeled indium-111 (111In)-anti-CD20 antibody (left) or pretargeted with anti-CD20–SA conjugate followed 24 hours later by clearing agent and then by 111In-DOTA-biotin (right). Arrows indicate radioactivity in tumors (T), and blood pool (B) 24 hours after injection of 111In-labeled reagents. Bladder activity is also seen in the conventional mouse. (Reprinted by permission of the Society of Nuclear Medicine from: Krishnan Subbiah, Don K. Hamlin, John M. Pagel, D. Scott Wilbur, Damon L. Meyer, Don B. Axworthy, Robert W. Mallett, Louis J. Theodore, Pat S. Stayton, and Oliver W. Press. Comparison of Immunoscintigraphy, Efficacy, and Toxicity of Conventional and Pretargeted Radioimmunotherapy in CD20-Expressing Human Lymphoma Xenografts. J Nucl Med. 2003 44(3):437–445. Figure 3.)

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A number of novel approaches are also being explored to improve the ratio of target to nontarget exposure. As noted previously, dose escalation in high-dose RIT for stem cell transplantation is limited by the nonspecific irradiation of normal tissues. The cause is both the slow distribution of the RAbs to target cells and the presence of unbound labeled antibody persisting in the circulation. One approach, pretargeting, is a process in which patients are initially treated with tumorreactive antibodies that are not bound to therapeutic radionuclides. This allows localization to tumor sites without subjecting the rest of the body to nonspecific irradiation. After maximal accumulation of antibody in the tumor, a small molecular weight radioactive moiety with high affinity for the tumor-reactive antibody is administered. Because of its small size, this second reagent penetrates tumors rapidly where it binds to the pretargeted antibody. The unbound molecules of the second (radioactive) reagent are small enough to be rapidly cleared from the blood and excreted in the urine within minutes. To further improve the targeted delivery of the radioimmunoconjugate, a clearing agent can be injected shortly before the radiolabeled small molecule to remove unbound antibody from the blood stream. The high-affinity binding that occurs between the pretargeting antibody and the subsequently administered radiolabeled small molecule can be achieved through a number of proposed mechanisms. One of the most promising exploits the exceptionally high affinity of avidin for biotin, while another approach employs bispecific antibodies. In animal models, pretargeting has generated therapeutic tumor-to-normal organ ratios of greater than 10 : 1 (Fig. 24.4), and cures have been achieved in 100% of

animals with lymphoma and solid tumor xenografts [114,115]. Early patient trials have exhibited similarly encouraging findings in both solid tumors and lymphoma [116–118]. Extracorporeal adsorption therapy offers another promising means for optimizing biodistribution. In this process, RAbs are injected and allowed 24–48 hours to circulate and distribute to antigen-bearing cells. Then patients undergo a hemodialysis-like procedure during which their blood volume is circulated across an avidin–agarose column. In this way, the biotin-conjugated RAbs that remain in the circulation are removed, thereby minimizing the nonspecific radiation to normal organs typically caused by perfusion with blood containing RAbs. In a small safety and efficacy trial conducted in Sweden [119], seven patients with relapsed or refractory B-cell lymphoma received biotin-conjugated 90Yrituximab, followed 24–48 hours later by extracorporeal filtration. The procedure was demonstrated to be safe and effective, and approximately 96% of the unbound 90Y-rituximab–biotin was cleared from the blood. It is anticipated that innovations such as those enumerated above will further enhance the feasibility, safety, and efficacy of radioimmunotherapy, with and without HCT. This work was supported by NIH/NCI grants P01 CA 44991, R01 CA 76287, R01 CA 109663, Leukemia & Lymphoma Society SCOR grant 7008, Lymphoma Research Foundation Career Development Award CICDG-007-010, ASCO Foundation Young Investigator’s Award 2007, and gifts from the Britton-Simmons Foundation, David & Patricia Giuliani, Hext Foundation, Edson Foundation, Wyner-Stokes Foundation, and James & Sherry Raisbeck.

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Lymphoma Prognostic Factors Project. N Engl J Med 1993; 329: 987–94. Solal-Celigny P, Roy P, Colombat P et al. Follicular lymphoma international prognostic index. Blood 2004; 104: 1258–65. Vose JM, Bierman PJ, Enke C et al. Phase I trial of iodine-131 tositumomab with high-dose chemotherapy and autologous stem-cell transplantation for relapsed non-Hodgkin’s lymphoma. J Clin Oncol 2005; 23: 461–7. Philip T, Armitage JO, Spitzer G et al. High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate-grade or high-grade nonHodgkin’s lymphoma. N Engl J Med 1987; 316: 1493–8. Vose JM, Zhang MJ, Rowlings PA et al. Autologous transplantation for diffuse aggressive nonHodgkin’s lymphoma in patients never achieving remission: a report from the autologous blood and marrow transplant Registry. J Clin Oncol 2001; 19: 406–13. Vose JM, Bierman PJ, Loberiza FR Jr, Bociek RG, Matso D, Armitage JO. Phase I trial of 90Yibritumomab tiuxetan in patients with relapsed B-cell non-Hodgkin’s lymphoma following highdose chemotherapy and autologous stem cell transplantation. Leuk Lymphoma 2007; 48: 683– 90. Nademanee A, Forman S, Molina A et al. A phase 1/2 trial of high-dose yttrium-90-ibritumomab tiuxetan in combination with high-dose etoposide and cyclophosphamide followed by autologous stem cell transplantation in patients with poor-risk or relapsed non-Hodgkin lymphoma. Blood 2005; 106: 2896–902. Winter JN et al. 90Y ibritumomab tiuxetan (Zevalin(R); 90YZ) doses calculated to deliver up to 1500 cGy to critical organs may be safely combined with high-dose BEAM and autotransplant in NHL. ASH Annual Meeting Abstracts 2006; 108: 330. Behr TM, Wörmann B, Gramatzki M et al. Lowversus high-dose radioimmunotherapy with humanized anti-CD22 or chimeric anti-CD20 antibodies in a broad spectrum of B cellassociated malignancies. Clin Cancer Res 1999; 5(10 Suppl): 3304s–14s. Krishnan A, Nademanee A, Fung HC et al. Phase II trial of a transplantation regimen of yttrium-90 ibritumomab tiuxetan and high-dose chemotherapy in patients with non-Hodgkin’s lymphoma. J Clin Oncol 2008; 26: 90–5. Shimoni A, Zwas ST, Oksman Y et al. Yttrium90-ibritumomab tiuxetan (Zevalin) combined with high-dose BEAM chemotherapy and autologous stem cell transplantation for chemorefractory aggressive non-Hodgkin’s lymphoma. Exp Hematol 2007; 35: 534–40. Witzig TE. Current treatment approaches for mantle-cell lymphoma. J Clin Oncol 2005; 23: 6409–14. Vose JM, Bierman PJ, Weisenburger DD et al. Autologous hematopoietic stem cell transplantation for mantle cell lymphoma. Biol Blood Marrow Transplant 2000; 6: 640–5. Jacobsen E, Freedman A. An update on the role of high-dose therapy with autologous or allogeneic stem cell transplantation in mantle cell lymphoma. Curr Opin Oncol 2004; 16: 106– 13.

92. Gopal AK, Rajendran JG, Petersdorf SH et al. High-dose chemo-radioimmunotherapy with autologous stem cell support for relapsed mantle cell lymphoma. Blood 2002; 99: 3158–62. 93. Fietz T, Uharek L, Gentilini C et al. Allogeneic hematopoietic cell transplantation following conditioning with 90Y-ibritumomab-tiuxetan. Leuk Lymphoma 2006; 47: 59–63. 94. Juweid M, Sharkey RM, Markowitz A et al. Treatment of non-Hodgkin’s lymphoma with radiolabeled murine, chimeric, or humanized LL2, an anti-CD22 monoclonal antibody. Cancer Res 1995; 55(23 Suppl): 5899s–907s. 95. Vose JM, Colcher D, Gobar L et al. Phase I/II trial of multiple dose 131Iodine-MAb LL2 (CD22) in patients with recurrent non-Hodgkin’s lymphoma. Leuk Lymphoma 2000; 38: 91–101. 96. Gopal AK, Pagel JM, Rajendram JG et al. Improving the efficacy of reduced intensity allogeneic transplantation for lymphoma using radioimmunotherapy. Biol Blood Marrow Transplant 2006; 12: 697–702. 97. Vriesendorp HM, Herpst JM, Germack MA et al. Phase I–II studies of yttrium-labeled antiferritin treatment for end-stage Hodgkin’s disease, including Radiation Therapy Oncology Group 87–01. J Clin Oncol 1991; 9: 918–28. 98. Herpst JM, Klein JL, Leichner PK, Quadri SM, Vriesendorp HM. Survival of patients with resistant Hodgkin’s disease after polyclonal yttrium 90-labeled antiferritin treatment. J Clin Oncol 1995; 13: 2394–400. 99. Bierman PJ, Vose JM, Leichner PK et al. Yttrium 90-labeled antiferritin followed by high-dose chemotherapy and autologous bone marrow transplantation for poor-prognosis Hodgkin’s disease. J Clin Oncol 1993; 11: 698–703. 100. Schnell R, Dietlein M, Staak JO et al. Treatment of refractory Hodgkin’s lymphoma patients with an iodine-131-labeled murine anti-CD30 monoclonal antibody. J Clin Oncol 2005; 23: 4669– 78. 101. Denardo SJ, O’Grady LF, Richman CM et al. Radioimmunotherapy for advanced breast cancer using I-131-ChL6 antibody. Anticancer Res 1997; 17: 1745–51. 102. Wong JY, Somlo G, Odom-Maryon T et al. Initial clinical experience evaluating yttrium-90chimeric T84.66 anticarcinoembryonic antigen antibody and autologous hematopoietic stem cell support in patients with carcinoembryonic antigen-producing metastatic breast cancer. Clin Cancer Res 1999; 5(10 Suppl): 3224s–31s. 103. Schrier DM, Stemmer SM, Johnson T et al. Highdose 90Y Mx-diethylenetriaminepentaacetic acid (DTPA)-BrE-3 and autologous hematopoietic stem cell support (AHSCS) for the treatment of advanced breast cancer: a phase I trial. Cancer Res 1995; 55(23 Suppl): 5921s–4s. 104. Richman CM, DeNardo SJ, O’Donnell RT et al. Dosimetry-based therapy in metastatic breast cancer patients using 90Y monoclonal antibody 170H.82 with autologous stem cell support and cyclosporine A. Clin Cancer Res 1999; 5(10 Suppl): 3243s–8s. 105. Richman CM, Denardo SJ, O’Donnell RT et al. High-dose radioimmunotherapy combined with fixed, low-dose paclitaxel in metastatic prostate and breast cancer by using a MUC-1 monoclonal antibody, m170, linked to indium-111/yttrium-90 via a cathepsin cleavable linker with cyclosporine

106.

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109.

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112.

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114.

115.

116.

117.

118.

119.

to prevent human anti-mouse antibody. Clin Cancer Res 2005; 11: 5920–7. Juweid ME, Hajjar G, Stein R et al. Initial experience with high-dose radioimmunotherapy of metastatic medullary thyroid cancer using 131IMN-14 F(ab)2 anti-carcinoembryonic antigen MAb and AHSCR. J Nucl Med 2000; 41: 93– 103. Cheung NK, Kushner BH, LaQuaglia M et al. N7: a novel multi-modality therapy of high risk neuroblastoma (NB) in children diagnosed over 1 year of age. Med Pediatr Oncol 2001; 36: 227– 30. Zenz T, Schlenk RE, Glatting G et al. Bone marrow transplantation nephropathy after an intensified conditioning regimen with radioimmunotherapy and allogeneic stem cell transplantation. J Nucl Med 2006; 47: 278–86. Gopal AK, Gooley TA, Maloney DG et al. Highdose radioimmunotherapy versus conventional high-dose therapy and autologous hematopoietic stem cell transplantation for relapsed follicular non-Hodgkin lymphoma: a multivariable cohort analysis. Blood 2003; 102: 2351–7. Ansell SM, Schilder RJ, Pieslov PC et al. Antilymphoma treatments given subsequent to yttrium 90 ibritumomab tiuxetan are feasible in patients with progressive non-Hodgkin’s lymphoma: a review of the literature. Clin Lymphoma 2004; 5: 202–4. Press OW, Eary JF, Appelbaum FR et al. Phase II trial of 131I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas. Lancet 1995; 346: 336–40. Liu SY, Eary JF, Petersdorf SH et al. Follow-up of relapsed B-cell lymphoma patients treated with iodine-131-labeled anti-CD20 antibody and autologous stem-cell rescue. J Clin Oncol 1998; 16: 3270–8. Bethge WA, Wilbur DS, Sandmaier BM. Radioimmunotherapy as non-myeloablative conditioning for allogeneic marrow transplantation. Leuk Lymphoma 2006; 47: 1205–14. Press OW, Corcoran M, Subbbiah K et al. A comparative evaluation of conventional and pretargeted radioimmunotherapy of CD20-expressing lymphoma xenografts. Blood 2001; 98: 2535–43. Axworthy DB, Reno JM, Hylarides MD et al. Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity. Proc Natl Acad Sci U S A 2000; 97: 1802–7. Forero A, Weiden PL, Vose JM et al. A Phase I trial of a novel anti-CD20 fusion protein in pretargeting radioimmunotherapy for B cell nonHodgkin’s lymphoma. Blood 2004; 104: 227–36. Forero-Torres A, Shen S, Breitz H et al. Pretargeted radioimmunotherapy (RIT) with a novel anti-TAG-72 fusion protein. Cancer Biother Radiopharm 2005; 20: 379–90. Knox SJ, Goris ML, Tempero M et al. Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer. Clin Cancer Res 2000; 6: 406–14. Linden O, Kurkus J, Garkavij M et al. A novel platform for radioimmunotherapy: extracorporeal depletion of biotinylated and 90Y-labeled rituximab in patients with refractory B-cell lymphoma. Cancer Biother Radiopharm 2005; 20: 457–66.

25

Paul J. Martin

Documentation of Engraftment and Characterization of Chimerism Following Hematopoietic Cell Transplantation

Genetic markers in hematopoietic cell transplantation Marrow, growth factor-mobilized peripheral blood cells, and cord blood cells used for hematopoietic cell transplantation (HCT) contain a wide variety of cell types that differ in function and life span. Hematopoietic stem cells (HSCs) in the graft are of central importance for recovery of marrow function after HCT. With the use of genetic markers, the survival, distribution, and differentiation of engrafted donor cells can be traced throughout the entire life of the recipient. When HSCs are obtained from a syngeneic donor, the only markers that might distinguish donor and recipient cells after HCT are those related to the underlying disease in the recipient. When autologous HSCs are used, genetic markers have sometimes been introduced into the graft in order to demonstrate that the graft contributes to long-term marrow function and to determine whether recurrent malignancy after transplantation originated from neoplastic cells that survived the pretransplant preparative regimen or from neoplastic cells infused with the graft (see Chapter 10). In allogeneic HCT, donor and recipient cells can be distinguished by testing informative genetic markers. Informative genetic markers in allogeneic HCT are those that distinguish cells of the donor from those of the recipient, and those that distinguish cells of the recipient from those of the donor. In many cases, the donor and recipient alleles at a single genetic locus will be informative in both directions. For a heterozygous donor with alleles “a” and “b” and a heterozygous recipient with alleles “a” and “c”, the “b” allele is informative for donor-derived cells, while the “c” allele is informative for recipient-derived cells. In some cases, the alleles at a single locus will not be informative in both directions. For a homozygous donor with the “a” allele and a heterozygous recipient with alleles “a” and “c”, the “c” allele is informative for recipient cells, but there is no informative donor allele at this locus. Evaluation of genetic markers in the donor and recipient before HCT allows loci that are informative to be identified for testing after the transplant. The probability of finding informative markers depends on the number of loci tested, the number of alleles at each locus, the allele distribution, and the relationship between the donor and recipient. Within members of a family, no more than four alleles can be defined at any single locus, while the number of alleles at any given locus in the population can be Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

much larger. For this reason, it is easier to find informative genetic markers with unrelated donors and recipients than with siblings.

Methods for evaluating chimerism Historical perspective In ancient Greek mythology, Chimera was a fearsome three-headed creature – lion at the front, goat in the middle and serpent at the rear. In today’s medical parlance, the term “chimerism” has been used to describe the presence of allogeneic hematopoietic or lymphoid cells in a transplant recipient. HCT recipients are sometimes described as having “full chimerism” when all hematopoietic cells and lymphoid cells are derived from an allogeneic donor. The terms “partial chimerism” and “mixed chimerism” are sometimes used when recipient hematopoietic or lymphoid cells persist together with donor cells after HCT. The term “split chimerism” is sometimes used when donor cells are present within some hematopoietic or lymphoid lineages but not in others. The claim that full chimerism has been established must always be qualified by understanding the limits of detection in the assay used to measure chimerism and by defining the hematopoietic and lymphoid lineages encompassed in the test. During the first few weeks after HCT, recipient-derived cells can be found in the blood or marrow of virtually all patients when sensitive tests are used [1,2]. The genetic markers and laboratory methods used for testing chimerism have evolved considerably during the history of HCT. Erythrocyte antigens were among the first markers widely used for confirmation of donor cell engraftment in HCT recipients. Antigens tested for analysis of chimerism after HCT have included those of the ABO, MN, and Rhesus systems and those of the Kell, Kidd, Duffy, Lutheran, Ss, and P systems. With assays for a wide variety of erythrocyte antigens, informative donor and recipient markers can be identified in more than 80% of sibling pairs. Mixed agglutination and flow cytometry assays can detect admixtures with a sensitivity of 0.1–0.5% [3]. Because erythrocytes have a relatively long life span in circulation, assay results can be confounded by transfusions before or after HCT unless precautions are taken to ensure that the HCT donor has a marker distinctive from those of the recipient and all transfusion donors. Although the long life span of erythrocytes makes it difficult to assess the function of the graft in an ongoing manner, this shortcoming can be circumvented by assays that define antigens on reticulocytes [3]. Finally, hemolysis caused by ABO incompatibility can delay the appearance of donor erythrocytes in patients who are well engrafted with donor myeloid cells, and results of chimerism studies limited to the erythroid lineage could be misleading.

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Chapter 25

Fig. 25.1 Assessment of chimerism in sorted granulocytes (a) and leukemic blasts (b) by dual-color fluorescence in situ hybridization (FISH). Hybridization was carried out with a biotin-labeled Y chromosome-specific DNA probe and a digoxigenin-labeled X chromosome-specific DNA probe. Hybridization was detected with Texas Red-conjugated avidin and fluorescein-conjugated antibody against digoxigenin. Cells were counterstained with 4′,6-diamidino-2phenylindole API. Male cells contain a single red fluorescent spot and a single green fluorescent spot, while female cells contain two green fluorescent spots and no red fluorescent spots. In this case, granulocytes were derived from a male donor, and leukemic blasts were derived from the female recipient. (See also Plate 25.1.)

Conventional cytogenetic analysis of metaphase chromosomes was historically well established as a method for evaluating chimerism in the blood and marrow after allogeneic HCT. Gender disparity between the donor and recipient allows sex chromosomes to be used as convenient genetic markers [4]. In some cases, a constitutional structural rearrangement or heteromorphism can be used as a genetic marker in same sex pairs [5]. Four major problems limit the applicability of cytogenetic assays for the assessment of chimerism after HCT. First, informative markers are generally not available for same-sex pairs except in cases where a disease-specific cytogenetic abnormality is present in the recipient. Second, techniques for cytogenetic analysis are highly laborintensive and cumbersome. Third, results are limited to cells in metaphase at the time of harvest. Finally, the number of metaphase cells that can be evaluated in any single assay is limited. During the late 1980s, techniques of molecular biology revolutionized the use of genetic markers in HCT [1,6–11]. The two tests most widely used today are in situ hybridization with sex chromosome-specific probes [1,10,11] and typing of variable number of tandem repeat (VNTR) or short tandem repeat (STR) polymorphisms by DNA amplification [9]. In situ hybridization permits examination at the level of single cells, but this method is applicable only when the donor and recipient are of opposite sex or when an informative autosomal marker is present [12]. Informative VNTR/STR polymorphisms can be identified in virtually all allogeneic donor–recipient pairs, but testing at the level of single cells is not possible. Both methods have high sensitivity and specificity, and both methods can provide quantitative results. Both methods can be adapted easily for use in clinical laboratories, and both can be used for evaluating archival specimens.

Molecular cytogenetics Molecular cytogenetic techniques have many advantages over conventional cytogenetic analysis of metaphase chromosomes. Techniques for in situ hybridization are simple and highly time-efficient, and the analysis can be applied equivalently for interphase and metaphase cells [13,14]. Techniques are sufficiently flexible to allow the simultaneous

determination of genotype and morphology or surface-marker expression in single cells [13–15]. Large numbers of cells can be analyzed, allowing high precision and sensitivity. Initial reports of molecular cytogenetic techniques for chimerism testing described the use of a human Y chromosome-specific repetitive DNA sequence probe for fluorescence in situ hybridization [1,10,11]. The upper limit of false-negative results in males was 5.6%, and the upper limit of false-positives in females was 2.7% [1]. Age-associated loss of the Y chromosome in cells from older males [16], Y chromosome loss associated with the tumor cell karyotype, and constitutional variation in the Y chromosome DNA probe target sequences are potential limitations of the assay. These limitations make it essential to test specimens either by conventional cytogenetics or by in situ hybridization before the transplant to ensure Y chromosome integrity. Cells that show no signal with the use of a Y chromosome-specific probe are inferred to be of female origin. The absence of signal with a Y chromosome-specific probe could result from technical errors that prevent hybridization. This potential problem can be avoided by the use of a mixture of X and Y chromosome-specific probes each labeled with a different chromophore to allow dual-color detection (Fig. 25.1 and Plate 25.1). With these reagents the upper limit of false-positive XX cells in males was 0.63% and the upper limit of false-positive XY cells in females was 0.30% [17]. The primary limitation of molecular cytogenetic testing with the use of sex chromosome markers is that this method can be used only when the donor and recipient are of opposite sex.

VNTR and STR polymorphisms Certain core DNA sequences are repeated in tandem in the genome, and the number of such tandem repeats at any given locus varies among different individuals (Figs 25.2 and 25.3 [18,19]). VNTR sequences are comprised of “minisatellite” cores 8–50 base pairs in length [20], while STR sequences are comprised of “microsatellite” cores 2–8 base pairs in length (e.g. CACACA and complementary GTGTGT) [21,22]. Polymorphisms in the number of tandem repeated core sequences within a locus are inherited as codominant Mendelian traits. A large number of

Documentation of Engraftment and Characterization of Chimerism Following Hematopoietic Cell Transplantation

loci are available for testing, and certain loci have more than 25 alleles [13,14,23]. Informative donor and recipient markers can be identified in virtually all allogeneic sibling pairs with a panel of as few as six loci. VNTR/STR polymorphisms can be identified most conveniently by using the polymerase chain reaction to amplify defined segments of DNA [24]. In these assays, oligonucleotides complementary to nonpolymorphic 5′ and 3′ sequences on opposite strands flanking the polymorphic VNTR/STR core (Fig. 25.2) are used to prime DNA synthesis through repeated cycles of denaturing, primer annealing, and heat-stable polymerase-mediated extension. When the two primer binding sites are spaced within a short distance of each other, each cycle of extension creates a primer-binding site for initiation of opposite strand synthesis in the next cycle. The 106–109-fold amplification of donor- and recipient-derived DNA during the reaction allows fragment length to be analyzed directly by staining with ethidium bromide staining or silver. With the use of fluorescent primers, reaction products can be visualized by a DNA sequencer or by capillary gel electrophoresis [13,14,25–28] (Figs 25.4 and 25.5 [29]). Efficiency can be improved by simultaneous amplification of different loci in a single “multiplex” reaction [13,14,26,27,30].

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The ability of a multiplex reaction to amplify several loci simultaneously circumvents the need to identify informative loci before testing is carried out, but donor and pretransplant recipient samples are still needed for the interpretation of results. The multiplex reaction should be designed to include loci that are most likely to be informative, which correlates with the frequency of heterozygous individuals in the population [31]. The use of DNA amplification enables chimerism testing with extremely small amounts of starting material, a major advantage when attempting to analyze samples from patients with graft failure and severe leukopenia. Depending on fragment length and the efficiency of

ApoB VNTR 5′ primer

TR core (TTTTATAATAAAATA)n 3′ primer

(a) D1S80 VNTR 5′ primer

Conserved 5′ flank

TR cores

Conserved 3′ flank

TR core (GAAGACCACAGGAAA)n

(b)

Fig. 25.2 Variable number of tandem repeat/short tandem repeat structure. Alleles reflecting differences in the number of tandem repeats can be distinguished by the length of fragments generated after DNA amplification with 5′ and 3′ primers specific for conserved sequences flanking the tandem repeat (TR) core region.

3′ primer

Fig. 25.3 Representative tandem repeat (TR) core sequences for (a) the apoB locus [19] and (b) the D1S80 locus [18]. Arrows show the position of primers complementary to conserved sequences flanking the TR regions. Core sequences at each locus show some heterogeneity resulting from base substitutions, insertions or deletions.

Recipient after HCT

Recipient before HCT

Donor (a)

Fig. 25.4 Assessment of chimerism by testing short tandem repeat (STR) polymorphisms. (a) Mixed chimerism in CD3+ blood cells tested at 1 year after hematopoietic cell transplantation (HCT) with a reduced-intensity conditioning regimen for treatment of acute myeloid leukemia. The figure shows capillary gel electrophoresis chromatograms of products after multiplex amplification of five loci (listed in the gray bars) with samples from the donor (bottom), recipient before HCT (middle), and recipient after HCT (top). Allele designations are numbered in boxes below each chromatogram. In this case, informative donor markers include D21S11 allele 29, D18S51 allele 11, and Penta E allele 12. Informative recipient markers include D3S1358 allele 16, TH01 allele 9.3, D21S11 allele 31.2, D18S51 allele 15, and Penta E allele 10. The percentage of donor DNA was calculated according to the height of informative donor peaks divided by the sum of the heights of all informative donor and recipient peaks at each locus. The percentage of donor DNA can also be calculated according to the areas under the respective peaks. The median percentage of donor-derived DNA among values for all informative loci in this specimen was 75%.

Donor 1

Donor 2

Recipient before HCT

Recipient after HCT (b)

Recipient before HCT

Donor

Recipient after HCT (c)

Fig. 25.4, cont’d (b) Engraftment with two different cord blood units. The sample obtained from the recipient after HCT represented CD14+ monocytes isolated by immunofluorescent staining and flow cytometry. The figure shows chromatograms from each of the two donors (top two profiles), the recipient before HCT (third from top), and the recipient after HCT (bottom). Informative recipient alleles (TH01 allele 6, D18S51 allele 19, and Penta E allele 12) were not detected in the sample from the recipient after HCT. The bottom profile shows that 20% of the sample was derived from one cord blood unit, and 80% was derived from the other cord blood unit. (c) Missing allele caused by chromosomal deletion in a patient with multiple myeloma. The figure shows chromatograms of products after amplification of the D16S539 locus from the donor (middle), recipient before HCT (top), and recipient after HCT (bottom). The sample from the recipient after HCT was a marrow aspirate obtained on day 497 after HCT. Both donor peaks (alleles 9 and 13) and both recipient peaks (alleles 10 and 11) are informative. Vertical gray bars across each profile indicate the expected positions of D16S539 alleles. The small peak corresponding to allele 9 in the recipient profile before HCT and the small peaks corresponding to alleles 12 and 8 in the donor profile are artifacts termed “stutter fragments” that contain one less repeat than the normal allele. The marrow sample from the recipient after HCT shows a reduced amount of allele 10 (arrow) compared with allele 11. This imbalance resulted from loss of one chromosome 16 in myeloma cells that persisted after HCT.

Documentation of Engraftment and Characterization of Chimerism Following Hematopoietic Cell Transplantation

amplification, the sensitivity for detecting admixtures is 1–5% and can approach 0.1% in certain cases. Methods involving DNA amplification can be confounded by contamination, which can be recognized when the DNA has originated from an individual other than the donor or recipient. Concurrent amplification of pretransplant samples from the donor and recipient together with the post-transplant sample generally allows unambiguous identification of donor-specific and recipientspecific reaction products and avoids confusion that could be caused by background bands. With the inclusion of standards, assays can be adapted for quantitative determination. Assay results can be affected when alleles have large differences in base pair length, since shorter alleles may be preferentially amplified [32]. Assay results can also be affected by technical artifacts such as stutter peaks that reflect amplification products containing one less repeat than the normal allele (Fig. 25.4) [32]. Accuracy may be improved by averaging results from all informative loci in a multiplex reaction [32]. Nomenclature for describing assay results has been proposed [33], and computer software is available for analyzing and summarizing the results [32]. Unlike molecular cytogenetic assays for sex chromosome markers, the multiplex DNA amplification assay for STR alleles can be used for all patients, regardless of the sex of the donor and recipient [34].

DNA amplification of other loci for assessment of chimerism With amplification of certain Y chromosome-specific sequences, sensitivity for detection of male cells in female recipients can approach 0.01% [35,36]. Loci with biallelic polymorphism not involving VNTR/STRs have also been used for quantitative and highly sensitive measurement of chimerism [29,37,38]. Two different approaches have been used for these assays. The first involves the use of oligonucleotide primers specific for single nucleotide polymorphisms or short 2-base pair insertions or deletions that differ between the donor and recipient [29,37]. Assay conditions are carefully controlled so that the primer binds to only one of the alleles, thereby allowing selective amplification of an informative donor or recipient allele. The second involves the use of oligonucleotide primers that are specific for longer insertion or deletion polymorphisms or null alleles that differ between the donor and recipient [38]. Informative single nucleotide markers or short insertion or deletion markers were identified for more than 90% of related donor–recipient pairs by testing 7–11 biallelic loci [29,37], and at least one recipient-informative marker was identified in approximately 80% of related donor–recipient pairs by testing 10 loci with longer insertions or deletions or null alleles [38]. Unlike assays with VNTR/STR loci in which amplification products from both donor and recipient DNA are measured simultaneously at the end of the reaction, assays with these loci measure amplification products from DNA of either the donor or the recipient in real time as they accumulate during the reaction in comparison to known standards. Realtime quantitative DNA amplification assays had 0.01–0.1% sensitivity (Fig. 25.5). Confidence intervals in these assays are within +30% to −25% of the measured values [37], which is acceptable with values below 50%. The accuracy of results can be improved by measuring the proportion of recipient cells in samples containing more than 50% donor cells or the proportion of donor cells in samples that contain more than 50% recipient cells. As an alternative to real-time assays, DNA segments can be amplified with the use of primers complementary to nonpolymorphic 5′ and 3′ sequences on opposite strands flanking a single nucleotide polymorphism that is informative for the donor or recipient. Amplified products can then be sequenced to determine the relative proportions of the informative donor or recipient nucleotide [39]. This method has approxi-

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mately 5% sensitivity, which is lower than that of other methods based on DNA amplification.

Clinical application of chimerism tests Samples used for chimerism testing Alleles that are informative for the donor and recipient must be identified before post-transplant samples from the recipient can be tested for chimerism. This identification can be easily accomplished by testing blood cells from the donor and recipient before the transplant. When a pretransplant recipient sample is not available, material from a buccal scraping, hair follicle or skin biopsy can be used after the transplant to identify recipient alleles [40]. Buccal scrapings and mouthwash specimens contain large numbers of cells that originate from the blood and cannot reliably be used to identify recipient alleles after the transplant [41]. Cells from blood or marrow are most frequently used for clinical chimerism testing. In most circumstances, results from testing blood cells can provide the necessary clinical information. One exception is the detection of minimal residual disease when malignant cells infiltrate the marrow but are not found in the circulation. Selected lineage-specific populations of interest can be isolated by flow cytometry in order to improve diagnostic accuracy and sensitivity for detecting small numbers of recipient or donor-derived cells [14,42–44]. For example, separation of blood samples into T lymphocyte and granulocyte fractions can be very helpful in interpreting results in patients who have received a reduced-intensity conditioning regimen [42,43,45]. Likewise, the diagnosis of recurrent malignancy can be confirmed by demonstrating that a small population of cells with an aberrant phenotype originated from the recipient [46,47]. Chimerism testing with isolated cell populations is particularly helpful in confirming the diagnosis of recurrent malignancy when the aberrant population represents less than 1% of the sample and when the aberrant phenotype detected after the transplant resembles a normal pattern of hematopoietic regeneration or differs from the pretransplant malignant phenotype.

General principles Chimerism tests have been used for a variety of purposes in human HCT (Table 25.1). With sufficiently sensitive assays, recipient cells have been shown to persist for longer than 2 years in most patients after treatment with a myeloablative conditioning regimen [2]. A complex interplay between prior intensive chemotherapy, the intensity of the pretransplant conditioning regimen, the number of CD34 cells in the graft, the posttransplant immunosuppressive regimen, and the effects of donor T cells in the graft governs the ability of recipient-derived lymphoid and hematopoietic cells to survive after HCT (see Chapter 11). In general, prior intensive chemotherapy, more intensive conditioning regimens, and administration of antithymocyte globulin as part of the conditioning regimen would be expected to decrease the persistence of recipient cells after transplantation, while higher numbers of CD34 cells in the graft and the occurrence of acute graft-versus-host disease (GVHD) would be expected to accelerate engraftment [11,48–52]. Given the critical role of donor T cells in helping to eliminate recipient lymphoid and hematopoietic cells that survive the conditioning regimen, depletion of donor T cells would generally be expected to increase the incidence of mixed chimerism after transplantation [1,23,51,53]. In some studies, disappearance of recipient cells within the first 100 days after HCT has been associated with an increased incidence of acute and chronic GVHD [54], while persistence of recipient cells beyond 90 days after HCT has been associated with an increased risk of recurrent malignancy in some studies [55].

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Chapter 25 STR-PCR

Real time PCR 40% R. cells

2 40% R. cells

3

Delta Rn

Peak height

6

R. genotype 31%

R. genotype 30% 0

0 (a)

1

220

230

240

(b)

10% R. cells

20

25

30

35

40

10% R. cells

2

Delta Rn

Peak height

6

3

1 R. genotype 8%

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0 220

(c)

230

240

(d)

1.25% R. cells

20

25

30

35

40

0.15% R. cells

2

Delta Rn

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6

3

1

R. genotype 0.14%

R. genotype undetected 0

0 220

(e)

230 Size

240

20

25

(f)

Table 25.1 Clinical applications of chimerism tests in marrow transplantation 1 Routine documentation of donor cell engraftment 2 Evaluate the persistence of donor cells in: (a) Patients with inadequate marrow function (b) Patients who are candidates for donor lymphocyte infusion (c) Patients who are candidates for a second allogeneic transplant (d) Long-term follow-up patients who are at increased risk of occult rejection 3 Assess the prognostic risks of graft-versus-host disease (GVHD), rejection or recurrent malignancy 4 Define whether recurrent malignancy or lymphoproliferative syndrome has originated from donor or recipient cells 5 Identify maternal cells in pretransplant patients with severe combined immunodeficiency 6 Correlate immune reconstitution with engraftment after transplantation for treatment of severe combined immunodeficiency 7 Determine whether cells from a transfusion donor can be implicated in causing GVHD 8 Verify the genetic identity of twins 9 Identify the origins of engraftment after HCT with multiple cord blood units

30 Cycle

35

40

Fig. 25.5 Comparison of short tandem repeat polymerase chain reaction (STR-PCR) and realtime PCR chimerism assays. Mixtures contained 40% (a and b), 10% (c and d), 1.25% (e) or 0.15% (f) “recipient” cells. In this case, the D21S11 STR alleles of the recipient (black peaks) were longer than those of the recipient (gray peaks) (a and c). This STR-PCR assay was able to detect the presence of 40% or 10% recipient cells (a and c), but 1.25% recipient cells could not be detected (e). Preferential amplification of shorter alleles is characteristic of STR-PCR assays, which explains the under-representation of recipient alleles (a and c). Donor (䊐) and recipient (䊏) alleles were tested individually in separate real-time assays (b, d, and f). The increase in fluorescence indicates the accumulation of amplification products as the reaction progresses from one cycle to the next. With decreasing amounts of the recipient S09a allele, fluorescence exceeds the threshold of detection (horizontal line) at progressively later cycles, shifting the curve to the right (䊏). With increasing amounts of the donor S 09b allele, fluorescence exceeds the threshold of detection earlier, shifting the curve slightly to the left (䊐). The real-time assay was easily able to detect 0.15% recipient DNA (f). Reproduced with permission from Alizadeh et al. [29]. Copyright, American Society of Hematology.

General recommendations for chimerism testing after allogeneic HSC transplantation have been published [43]. Before discussing individual applications of chimerism testing in detail, several points should be emphasized: 1 The clinical context weighs heavily in the interpretation of chimerism test results. For example, the absence of donor cells in patients with marrow aplasia could indicate rejection, but a similar absence of detectable donor cells in the nonfractionated marrow or blood of a patient with florid leukemia relapse could be caused by an overwhelming preponderance of recipient cells. 2 Test results are reported as a ratio in which changes in the numbers of donor and recipient cells can occur independently. Thus, an increase in the proportion of donor cells can be caused both by an increase in the number of donor cells and by a decrease in the number of recipient cells. 3 The interpretation of results depends on an awareness of the types of cell tested in the assay. For example, peripheral blood mononuclear cells can include T and B lymphocytes, monocytes, immature myeloid precursors, and leukemic blasts. More informative results can be obtained by analyzing purified cell populations. 4 The presence of a particular cell population does not allow direct inferences concerning its functional capabilities or effects. For example, the detection of recipient T cells after HCT does not necessarily indicate that rejection is likely to occur.

Documentation of Engraftment and Characterization of Chimerism Following Hematopoietic Cell Transplantation

5 The inability to detect a particular cell population does not necessarily indicate its absence. The interpretation of test results must always be made with an understanding of the assay sensitivity. 6 Time trends are often more informative than test results at a single time point. For example, the presence of 5% recipient cells at day 28 after HCT might have no clinical significance in and of itself, but a progressive increase in the proportion of recipient cells could herald rejection or recurrent leukemia. 7 Abnormalities within a single patient can have multiple causes. Thus, the inability to detect donor cells after HCT could indicate the occurrence of both rejection and recurrent malignancy. 8 The inability to detect recipient markers after HCT could result from the absence of recipient cells or from chromosome loss or deletion in malignant cells (Fig. 25.4).

Chimerism testing in patients with graft failure Although chimerism tests can be done for routine monitoring of donor cell engraftment after HCT [56], the results are of greatest importance in patients who have inadequate marrow function and in patients who might be candidates for donor lymphocyte infusion or for a second transplant from the same donor. Marrow graft rejection is usually defined by the absence of donor cells in a patient with pancytopenia and reduced marrow cellularity, but “occult” rejection and complete reconstitution with recipient cells can occur, especially in patients who have received a reduced-intensity conditioning regimen and in patients with a pretransplant diagnosis of chronic myeloid leukemia (CML) [57]. As noted, the presence of donor cells can be obscured by a florid leukemia relapse or by a malignant population with myelosuppressive effects. Testing the origin of mature T cells can be helpful to exclude rejection in this situation since these cells are not usually affected by recurrent malignancy. Diagnostic assessments of chimerism test results are especially difficult in patients with poor marrow function. The presence of recipient cells in the blood or marrow does not necessarily indicate that rejection is the cause of poor marrow function since recipient cells are frequently identified early after HCT in patients with normal marrow function [1,2]. Thus a patient with “benign” mixed chimerism [36] could have poor marrow function because of drug toxicity, viral infection or a possible marrow stromal defect. Likewise, the inability to detect recipient cells in any single test does not eliminate rejection or occult recurrent malignancy as a possible cause of poor marrow function. In these situations, serial testing can be highly informative. In patients who are candidates for donor lymphocyte infusions as treatment for recurrent malignancy (see Chapter 72), the detection of persisting donor cells by chimerism tests offers reassurance that rejection has not occurred. In addition, the risk of transient or irreversible aplasia after donor lymphocyte infusion may be much higher in patients with a low proportion of donor cells in the blood or marrow compared with those with a high proportion of donor cells in the blood or marrow [58]. In patients who are candidates for a second transplant from the original donor, the persistence of donor cells indicates that the recipient is still tolerant of donor cells. In this situation, the risk of rejection after a second transplant is negligible, and the preparative regimen can be designed for the sole purpose of eliminating malignant cells. Serial monitoring of chimerism can be used to monitor initial engraftment after HCT for the treatment of severe aplastic anemia. In some cases, infusion of donor lymphocytes might prevent rejection in patients with decreasing chimerism after HCT [59]. Late “occult” rejection and complete hematopoietic reconstitution with recipient cells has been observed in long-term follow-up after HCT for the treatment of aplastic anemia [60]. This distinctly unusual outcome is of biologic interest but

371

probably has no medical significance except for theoretical concerns that aplastic anemia might recur or that myelodysplasia might develop in the future.

Chimerism testing in patients at risk for recurrent malignancy In certain circumstances, chimerism tests can be useful in predicting recurrent malignancy after HCT. Findings that demonstrate an increasing proportion of recipient-derived cells in the blood or marrow after HCT can unquestionably herald relapse [23,44,61–66]. By itself, stable mixed chimerism in nonfractionated blood or marrow cells does not predict recurrent malignancy [67,68], although persistence of recipient cells within the CD19+ population has been correlated with an increased risk of relapse in patients with pre-B lineage acute lymphoblastic leukemia [69]. Chimerism testing to demonstrate the recipient origin of isolated cell populations with an aberrant expression of cell-surface antigens can facilitate the early detection of recurrent leukemia [46,47] (Table 25.2). In patients with malignant diseases that have aberrant expression of cell-surface antigens, a more direct and unambiguous measure of minimal residual disease can come from flow cytometry, as opposed to chimerism testing (see Chapter 26). Highly sensitive tests and very frequent monitoring would likely be necessary to gain early warning of impending relapse in patients with rapidly progressive diseases such as acute leukemia [56,70]. In several studies, selected patients with increasing levels of mixed chimerism have had immunosuppressive medications withdrawn or have received donor lymphocyte infusions in an effort to prevent recurrent acute lymphoblastic leukemia or acute myeloid leukemia after HCT [63–65,71]. In some cases, patients who had such interventions survived longer than those who did not have interventions, but bias in the selection of patients for intervention makes these results difficult to interpret. Serial monitoring of chimerism after HCT could offer greater benefit if interventions for treatment of recurrent acute leukemia were more effective [63]. Serial chimerism testing has been useful for early detection of recurrent malignancy in patients with less rapidly progressive diseases such as CML [72] or myeloma [73]. A correlation between persistence of

Table 25.2 Testing of isolated cell populations by single-color or dual-color fluorescence in situ hybridization (FISH) to detect recurrent leukemia Genotype (%) Sample

Percent in original sample

Unsorted marrow Granulocytes Lymphocytes Blasts

89.0 1.6 0.2

Y

XY

XX

X

100.0 97.7 2.0

0.0 0.3 86.9

11.1

98.5

Single-color FISH with a Y chromosome-specific probe on day 45 after the transplant did not detect an appreciable population of female recipient cells in unsorted marrow from this patient. Granulocytes were isolated by flow sorting of cells with high side scatter characteristics and low expression of CD45, and lymphocytes were isolated by sorting cells with low side scatter characteristics and high expression of CD45. A small population of blasts was isolated by sorting cells with high expression of CD34 and low expression of CD45. Two-color FISH demonstrated that the granulocyte and lymphocyte populations were derived from the male donor, while the blasts with aberrant antigen expression were derived from the female recipient. Some of the malignant cells in this patient had a single X chromosome.

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Chapter 25

recipient T cells after HCT and an increased risk of recurrent malignancy has been found in patients who received T-cell-depleted marrow for the treatment of CML [74]. The absence of donor T cells in the graft presumably allowed both T cells and malignant HSCs of the recipient to survive after HCT. With the use of a more intensive pretransplant conditioning regimen, persistence of recipient lymphocytes after T-celldepleted HCT was not associated with an increased risk of recurrent CML [75]. The persistence of recipient cells in the blood or marrow during the first 3 months after HCT has not been associated with an increased risk of relapse in patients who received unmodified (T-cellreplete) marrow for the treatment of CML [60]. This result is consistent with earlier findings that the detection of BCR–ABL rearrangements in the blood or marrow during the first 3 months after HCT is not associated with an increased risk of relapse [76].

Chimerism testing after reduced-intensity conditioning regimens A new two-stage strategy for HCT involves the initial establishment of mixed chimerism with the use of a reduced-intensity conditioning regimen, combined with potent post-transplant immunosuppression to prevent rejection and GVHD. After engraftment has been established, immunosuppression is withdrawn, allowing donor T cells to eliminate residual hematopoietic cells or malignant cells in the recipient through immunological mechanisms (see Chapter 71). In some situations, donor lymphocytes may be infused if withdrawal of immunosuppression does not have the desired clinical effect. Genetic marker studies and state-of-the-art chimerism tests play an essential part in clinical management with this approach to HCT and in the evaluation of results from these clinical trials. Results of serial monitoring have suggested that full donor T-cell engraftment precedes the development of full donor myeloid engraftment, GVHD, and antitumor effects [43,45,77]. Chimerism tests after reduced-intensity conditioning showed that low levels of donor T-cell and natural killer cell engraftment at early time points after the transplant were associated with an increased risk of rejection and the absence of antitumor effects, while high levels of donor T-cell engraftment were associated with an increased risk of GVHD, and earlier establishment of natural killer chimerism was associated with improved progression-free survival [78,79]. In some cases, a progressive decline in donor cell engraftment has been reversed by the administration of donor lymphocytes [43,71,80]. These results suggest that serial monitoring of donor cell engraftment can help to determine whether donor lymphocytes should be given pre-emptively in order to prevent rejection (see Chapter 71). In another study, recipient T cells persisted for a longer time after pretransplant conditioning with fludarabine and melphalan compared with fludarabine and busulfan, but no correlation was found between the onset of complete donor chimerism and the occurrence of acute GVHD or the risk of disease progression after the transplant [81]. The contrasting results among these studies emphasize that correlations between chimerism and clinical outcomes after HCT may vary according to the intensity of the conditioning regimen.

Other applications for genetic marker studies Genetic marker studies have found a variety of other useful applications in HCT. In rare cases, for example, it has been possible to demonstrate that hematologic malignancy after HCT originated in donor cells rather than recipient cells. Chimerism tests allow these unusual cases to be identified for further intensive study, although the mechanisms involved in malignant transformation of donor cells remain obscure. With chimerism tests, it has likewise been possible to assess the involvement of

donor or recipient-derived B lymphocytes in lymphoproliferative disorders caused by Epstein–Barr virus infection (see Chapter 93). In rare cases, genetic marker studies have demonstrated that GVHD was caused by the cells from a blood product transfusion or from a solid organ allograft. In HCT recipients, evidence implicating cells of a blood transfusion donor came from tests demonstrating the presence of genetic markers different from those of either the donor or the recipient. In some cases, it has been possible to trace the donor through further testing. The universal practice of irradiating all blood product transfusions should eliminate any risk of transfusion-induced GVHD in HCT recipients. Chimerism tests can be used to identify the origins of engraftment after HCT with multiple cord blood units (see Chapter 39). In this situation, testing must employ markers that are informative for both the recipient and each of the donors. STR markers are very useful for this purpose, since these loci typically have more than two alleles (Fig. 25.4). An extremely useful clinical application of genetic marker studies in HCT is the assessment of monozygosity in twins. With an identical twin donor, there is no risk of rejection or GVHD and hence no need for immunosuppressive treatment before or after HCT. In this situation, the pretransplant conditioning regimen is designed solely to eliminate malignant cells. The alleles of a genetic locus are fully informative in a family study when they allow unambiguous assessment of inheritance, which requires parental heterozygosity and the absence of allele matching between the parents. With human leukocyte antigen-identical twins of the same sex and with identity at three additional unlinked fully informative genetic loci, there is a less than 1 : 100 chance that the twins are not genetically identical. With identity at four unlinked fully informative loci in addition to HLA, there is a less than 1 : 500 chance that the twins are not genetically identical, and with identity at five unlinked fully informative loci in addition to HLA, there is a less than 1 : 1000 chance that the twins are not genetically identical [82].

Biologic insights from genetic marker studies Beyond the clinical applications described above, genetic marker studies have contributed valuable information towards understanding the biology of HCT. Genetic marker studies have recently emerged as powerful tools in studies of HCT for the treatment of congenital immunodeficiency. Infants with severe combined immunodeficiency syndromes (SCID) often have occult maternal T cells in the circulation. The presence of these cells might require immunosuppression to avoid rejection (see Chapter 75). Important information for understanding immune reconstitution after transplantation for the treatment of SCID has been obtained by analyzing the lineage-specific patterns of donor cell engraftment [83,84]. Results of genetic marker studies have made it clear that clinical resolution of nonmalignant diseases often can be achieved without complete donor replacement of the defective recipient lymphohematopoietic system [84,85]. For example, the need for red cell transfusions in patients with thalassemia can be averted by establishing mixed hematopoietic chimerism with only 25% normal donor cells in the marrow [85], and stable mixed chimerism can ameliorate the effects of sickle disease [86]. Genetic marker studies after HCT have been used to characterize the diversity of marrow-derived cells throughout the body. From these studies, it is now recognized that marrow-derived cells include not only hematopoietic and lymphoid populations, but also tissue macrophages, Kupffer cells in the liver, Langerhans cells in the skin, dendritic cells in the blood, and microglial cells in the brain [87–91]. The donor origin of tissue macrophages and microglial cells has given impetus to the application of HCT for treatment of certain congenital enzyme deficiency

Documentation of Engraftment and Characterization of Chimerism Following Hematopoietic Cell Transplantation

diseases (see Chapter 77). Most investigators agree that stromal cells in the marrow after transplantation are derived from the recipient, even when tested as long as 27 years after the transplant [92,93]. Some investigators have reported that marrow-derived cells can differentiate into mature hepatocytes and epithelial cells of the gastrointestinal tract [94– 96]. Rigorous experiments in mice reconstituted with single HSCs, however, did not show an appreciable contribution of donor cells in the brain, kidney, gut, liver or muscle, suggesting that transdifferentiation of HSCs is a rare event [97]. The small numbers of donor-derived, nonhematopoietic cells observed after HCT in human recipients could reflect cell fusion events or the progeny of other types of stem cells [98–100].

373

Summary The origin of marrow and blood cells after HCT can be identified by testing informative genetic markers that distinguish the donor and recipient. Molecular methods for in situ hybridization and DNA amplification have made chimerism tests widely accessible for a variety of clinical applications after HCT. Further work will be needed to develop standardized testing procedures for application to routine practice. Biologic insights gained from testing genetic markers will continue to guide future progress as new approaches are developed in using HCT for the treatment of malignant and nonmalignant diseases.

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patients with multiple myeloma. Exp Hematol 2006; 34: 688–94. Mackinnon S, Barnett L, Heller G, O’Reilly RJ. Minimal residual disease is more common in patients who have mixed T-cell chimerism after bone marrow transplantation for chronic myelogenous leukemia. Blood 1994; 83: 3409–416. Wiesneth M, Schreiner T, Bunjes D et al. Comparison of T-cell-depleted BMT and PBPCT with respect to chimerism, graft rejection, and leukemic relapse. J Hematother 1999; 8: 269–74. Radich JP, Gehly G, Gooley T et al. Polymerase chain reaction detection of the BCR–ABL fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: results and implications in 346 patients. Blood 1995; 85: 2632–8. Childs R, Clave E, Contentin N et al. Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses. Blood 1999; 94: 3234–41. Baron F, Baker JE, Storb R et al. Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2004; 104: 2254–62. Matthes-Martin S, Lion T, Haas OA et al. Lineage-specific chimaerism after stem cell transplantation in children following reduced intensity conditioning: potential predictive value of NK cell chimaerism for late graft rejection. Leukemia 2003; 17: 1934–42. Dey BR, McAfee S, Colby C et al. Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 2003; 9: 320–9. Valcarcel D, Martino R, Caballero D et al. Chimerism analysis following allogeneic peripheral

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blood stem cell transplantation with reducedintensity conditioning. Bone Marrow Transplant 2003; 31: 387–92. Smith SM, Penrose LS. Monozygotic and dizygotic twin diagnosis. Ann Hum Genet 1955; 19: 273–89. van Leeuwen JE, van Tol MJ, Joosten AM et al. Relationship between patterns of engraftment in peripheral blood and immune reconstitution after allogeneic bone marrow transplantation for (severe) combined immunodeficiency [Review]. Blood 1994; 84: 3936–47. Haddad E, Le Deist F, Aucouturier P et al. Longterm chimerism and B-cell function after bone marrow transplantation in patients with severe combined immunodeficiency with B cells: a single-center study of 22 patients. Blood 1999; 94: 2923–30. Andreani M, Manna M, Lucarelli G et al. Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia. Blood 1996; 87: 3494–9. Walters MC, Patience M, Leisenring W et al. Stable mixed hematopoietic chimerism after bone marrow transplantation for sickle cell anemia. Biol Blood Marrow Transplant 2001; 7: 665–73. Thomas ED, Ramberg RE, Sale GE et al. Direct evidence for a bone marrow origin of the alveolar macrophage in man. Science 1976; 192: 1016– 18. Gale RP, Sparkes RS, Golde DW. Bone marrow origin of hepatic macrophages (Kupffer cells) in humans. Science 1978; 201: 937–8. Kennedy DW, Abkowitz JL. Kinetics of central nervous system microglial and macrophage engraftment: analysis using a transgenic bone marrow transplantation model. Blood 1997; 90: 986–93. Auffermann-Gretzinger S, Lossos IS, Vayntrub TA et al. Rapid establishment of dendritic cell chimerism in allogeneic hematopoietic cell transplant recipients. Blood 2002; 99: 1442–8.

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26

Jerald P. Radich & Marilyn L. Slovak

The Detection and Significance of Minimal Residual Disease

Introduction Relapse remains the major obstacle to cure in leukemia. This problem persists despite ever-intensive chemotherapy regimens, new “targeted” molecular therapy, exhaustive study of mechanisms of drug resistance, and attempts to leverage immunotherapy against the leukemia clone. Unfortunately, once frank relapse occurs, the prospects for achieving a subsequent remission are low, and cure, dismal. One strategy to combat relapse is to detect and treat minimal residual disease (MRD) prior to overt relapse. Over the last decade, the technology to detect minuscule quantities of leukemia has developed and matured, and there are now ample clinical trials testifying to the power of MRD detection to predict relapse. The problem of defining remission and relapse is illustrated in Fig. 26.1. At diagnosis, patients may have a leukemia burden of up to 1010– 1012 cells. Thus, even after achieving the “gold standard” of a three-log depletion of leukemia following induction therapy, potentially millions of leukemia cells may persist despite the morphologic appearance of remission. The goal of MRD detection is to detect, quantify, and hopefully eliminate persistent and relapsing disease prior to frank hematologic relapse.

Methods of MRD detection The standard approach to evaluate residual leukemia by routine pathologic examination of bone marrow (BM) is limited by the sometimes subtle morphologic differences between malignant and normal cells. Fortunately, several techniques can be used to find the “needle in the haystack”; these are summarized in Table 26.1. Chromosomal assays Conventional metaphase cytogenetics can detect approximately one leukemia cell in 100 normal cells (denoted here as “10−1” sensitivity) if enough metaphases are analyzed [1,2]. Although cytogenetics is the method of choice for screening leukemic samples for risk stratification up front, these studies are also useful to define early response to induction therapy (cytogenetic remission) or to look for new, unanticipated

genomic changes during therapy. However, cytogenetics is limited by sampling only those few cells that divide in culture. Molecular cytogenetics is the analysis of genomic alterations using fluorescence in situ hybridization (FISH)-based technology. FISH uses chromosome-specific or locus-specific probes to target tumor-specific genetic aberrations in metaphase or interphase cells (as opposed to conventional metaphase cytogenetics) (Table 26.2 shows commonly used probes in hematology/oncology). FISH is useful for rapidly screening 200–10,000 cells for numerical chromosomal aberrations, even in samples insufficient for conventional cytogenetic analysis, and has the ability to identify minor abnormal clones that may be unrelated or associated with early clonal evolution of disease undetected by the conventional karyotypic study. Compared to conventional cytogenetics, the FISH sensitivity ranges from 10−1 to 10−3 (depending on the number of probes and number of nuclei scored). The sensitivity of FISH increases by combining it with other special techniques, that is, in combination with morphologic or immunohistochemical staining (Plate 26.1) [3–5], by using it on specific flow-sorted cell populations or by increasing the number of mitotic cells analyzed by hypermetaphase FISH [6–8]. Flow cytometry assays Cell surface antigen expression can distinguish malignant from normal cells. While truly tumor-specific antigens are rare, malignant cells often express cell surface antigens in subtly different patterns compared with normal cells [9]. By using combinations of multiple antibodies, “multiparametric” flow cytometric assays uses aberrant antigen expression patterns to “fingerprint” the malignant clone (Plate 26.2). If combinations of several antibodies are used to define the aberrant antigen expression, the sensitivity can reach as low as 10−2–10−3 in experienced hands. This technique has been very useful in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), where leukemia blasts often have evidence of abnormal antigen expression compared with normal hematopoietic cells. The current state-of-the-art of three- or four-color flow cytometry is quickly becoming displaced by techniques that allow seven colors or more. The increased sensitivity of flow cytometry may soon supplant the polymerase chain reaction (PCR; see below) for detection of MRD in many diseases. Polymerase chain reaction

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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The most sensitive approach to detect MRD involves nucleic acid amplification using PCR (Plate 26.3) [10]. In PCR, a specific genetic lesion target becomes the “fingerprint” of the malignancy in order for

The Detection and Significance of Minimal Residual Disease

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1012

Fig. 26.1 The measurement of minimal residual disease (MRD). At diagnosis, there may be as much as 1012 malignant cells present (red line). After an effective induction therapy that yields a “3-log kill,” a huge number of leukemia cells still remain despite morphologic remission. Various techniques can be used to test for MRD as the leukemia burden further lessens with continued therapy. The relative depth of remission plumbed by these techniques is illustrated.

Leukemia cells

109

Morphology Cytogenetics FISH

“Remission”

Flow 106

PCR

103

100 Time

Table 26.1 Common assays used for detection of minimal residual disease (MRD) Detection method

Target

Sensitivity

Pathologic (morphology) examination

Cellular morphology

Cytogenetics

Chromosome structure

FISH

Specific genetic marker(s)

Combined morphology/FISH or combined IHC/FISH

Specific cell type and specific genetic marker

~0.01–1%

Flow cytometry

Surface antigen (protein) expression

0.01–1%

PCR for chromosome aberrations

mRNA sequence (fusion gene transcripts)

PCR for immunoglobulin/ T-cell receptor genes

DNA sequence (junctional region)

5%

1–5%

0.08–5%

0.0001–0.1%

0.001–0.1%

Applicability

Comments

Standard clinical practice Defines CR

Determines additional testing that may prove useful for MRD testing

Standard clinical practice Upfront risk stratification Defines clonal evolution May assess rapidity of response at induction Screens for and may define therapy-related clonal changes

Labor-intensive with limited sensitivity With morphology data may suggest additional assays for MRD testing

Leukemia-specific target Rapid assay for known marker

Evaluates single cells Limited to probe availability

Targeting rare event tumor cells Screening stem cell harvests Assessing response in multiple myeloma

Time-consuming Requires automation Loss of antigen expression may result in false-negative results

Applicable for most patients Rapid test Newer 7–9 color flow assays should increase sensitivity and specificity

Measures single cells Immunophenotypes may shift over time Requires the use of two or more markers

Rapid, sensitive, and excellent patientspecific markers, especially for high-risk disease Limited to patients with known fusion gene transcripts

Positive test may not always be meaningful, especially with favorable-risk disease RNA degradation may result in false negatives Early t-MDS may be missed

Sensitive for patients with T- and B-cell disorders

Patient specificity means timeconsuming at diagnosis but rapid ASSAY at follow-up False negatives may result with clonal evolution or phenotype shifts

CR, complete remission; FISH, fluorescence in situ hybridization; IHC-FISH, sequential immunohistochemistry-FISH; PCR, polymerase chain reaction.

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Table 26.2 Common DNA fluorescence in situ hybridization (FISH) probes used in hematologic malignancy DNA probe HUGO/common name

Chromosomal location(s)

Common cytogenetic abnormality

Disease/utility

DXZ1 & DYZ1/DYZ3 MYCN ALK D3Z1 EVI1 BCL6

Xcen and Yα sat 2p24.1 2p23 3cen 3q26.2 3q27

Sex-mismatched HCT Double minutes/HSR t(2;5)(p23;q35) +3 inv(3)(q21q26.2)/t(3;3)(q21;q26.2) t(3q27)

Sex-mismatched HCT Neuroblastoma ALK lymphoma MM MDS/AML NHL

PDGFRA/CHIC

4q12

“cryptic” translocation or deletion

D5S23/D5S721/D9Z3/D15Z4 EGR1 (with D5S721/D5S23) D7Z1 D7S522/D7Z1 D8Z2 MYC/IGH MYC (MYCC or CMYC)

5p15.2/9cen/15cen 5q31 7cen 7q31/7cen 8cen 8q24/14q32 8q24

RUNX1/RUNXT1(AML1-ETO)

21q22/8q22

Hyperdiploidy by +5p, +9 or +15 −5/del(5)(q31) +7 −7/del(7)(q22) +8 t(8;14)(q24;q32) t(8;14)(q24;q32) t(2;8)(p11;q24) t(8;22)(q24;q11) t(8;21)(q22;q22)

Hypereosinophilia/eosinophilia leukemia, systemic mastocytosis MM MDS/AML MM MDS/AML AML, CML, MDS, MPD, ALL AML, NHL AML, NHL

CDKN2A/CEP9 (p16, INK4a, MTS1, CMM2, ARF, p19, p14, INK4, p16INK4a) BCR/ABL1 (BCR/ABL)

9p21/9cen

del(9)(p21)

ALL, many tumors

9q34/22q11.2

t(9;22)(q34.1;q11.2) and variants

CML, AML, ALL

11cen

+11

MM

D11Z1 ATM MLL D12Z3

11q22.3 11q23 12cen

del(11q) t(4;11) or t(11;*) +12

CLL MDS/AML, ALL CLL

ETV6/RUNX1 (TEL/AML1) D13S25 D13S319/13q34

12p13/21q22 13q14.3 13q14.3

t(12;21)(p13;q22) del(13q) −13/del(13q)

ALL CLL MM, CLL, MDS, MPD

RB-1 5’IGH-3’IGH IGH/FGFR3 IGH/CCND1(IGH/BCL1) IGH/BCL2 PML/RARAα CBFB (PEBP2B or MYH11/CBFβ) TP53 (P53)

13q14 14q32.3 14q32/4p16 14q32/11q13 14q32/18q22 15q22/17q12.1 16p13/16q22 17p13.1

del(13q) 14q32/IGH translocation Cryptic t(4;14)(p16;q32.3) t(11;14)(q13;q32) t(14;18)(q32;q22) t(15;17)(q22;q12.1) inv(16)(p13q22)/t(16;16)(p13;q22) del(17)(p13.1)

MDS, MPD NHL, CLL, MM, ALL MM Mantle cell lymphoma, MM NHL, ALL AML-M3 AML-M4Eο CML, CLL, AML, many tumors

ERBB2 (HER-2/neu) RARα MALT1 or BIRC/MALT1 (API2/MALT1)

17q11.2-q12 17q12.1 18q21

Double minutes/HSR Variant t(17q12.1) t(11;18)(q21;q21) or variants

Breast AML-M3 MALT lymphoma

D20S108/L3MBTL RUNX1 (AML1) EWSR1

20q12 21q22 22q12

del(20q) +21 and del/t(21q) t(11;22)(q24;q12) or variants/+22

MDS, AML, MPD AML, ALL Ewing sarcoma/PNET/AML

AML

ALK, anaplastic large cell lymphoma; HSR, homogeneously staining region; IGH, immunoglobulin heavy chain gene; MALT, mucosa-associated lymphoid tissue; NHL, nonHodgkin’s lymphoma; PNET, primitive neuroectodermal tumor. See text for other abbreviations.

The Detection and Significance of Minimal Residual Disease

PCR-driven reactions to have the desired sensitivity and specificity. Gene translocations, such as t(9;22) in chronic myeloid leukemia (CML), and t(15:17), t(8;21), and inv(16)/t(16;16) found in AML, are straightforward leukemia-specific markers for the detection of MRD [11–17]. In ALL, the most common translocation involves the Philadelphia (Ph+) chromosome; however, around 40–50% of cases of ALL (and AML) have some type of translocation that can be detected by PCR assays [18]. In ALL, the most used PCR markers involve the leukemia-specific “fingerprints” caused by the immunoglobulin heavy chain (IgH) and T-cell-receptor (TCR) rearrangement [19,20]. The advantages of PCR are the excellent sensitivity and specificity. Disadvantages include the potential for false-positive results from contamination of the amplified product, and false negatives due to RNA degradation and variant/complex rearrangements that fall outside of the selected primer sequences.

The clinical significance of MRD With the exception of CML, the vast majority of important studies of MRD have occurred in the setting of conventional chemotherapy. Moreover, the use of MRD in the conventional-therapy setting can bring patients to transplant in a timely manner. Therefore, for each of the specific leukemias, we will present first the data for MRD in conventional chemotherapy, followed by the use and significance of MRD in transplantation. MRD detection in ALL patients receiving chemotherapy In ALL, MRD detection is accomplished by either flow cytometry or PCR assays. Flow cytometry assays rely on the unique and aberrant constellation of surface antigens in the leukemia clone. PCR-based assays rely on the clonal IgH (V-D-J) or TCR gene rearrangements that can be used to follow MRD in approximately 70% of ALL cases. This PCR method takes advantage of the unique gene rearrangements that occur in these genes as cells develop into functional B and T cells. While all individual normal B and T cells should have unique gene rearrangements, a clonal outgrowth of malignant cells should all have identical gene rearrangements, and this “fingerprint” can later be used to distinguish malignant versus normal cells in “remission” samples. While both methods have excellent sensitivity, both are susceptible to clonal “shifts”

379

that occur with disease progression in 10–20% of cases, whereby the relapsing clone appears with a different gene rearrangement or surface antigen pattern than the presenting leukemia [21–24]. The recent combined use of these two methods should enable MRD monitoring in almost all patients and prevent false-negative results due to clonal evolution and phenotypic shift [25,26]. The clinical impact of MRD strongly depends on the time point at which it is assessed. Detection of MRD can be used to evaluate early treatment response, and thereby allows the identification of low-risk and high-risk patients who may profit from therapy reduction or therapy intensification, respectively. Early clearance of leukemia cells is a favorable prognostic indicator in childhood ALL [19,27,28]. Low or absent MRD in the BM after the completion of induction therapy predicts a favorable outcome, as MRD-negative patients have an overall relapse rate of only 2–10% (Table 26.3) [19,29–32]. On the other hand, high MRD levels at the end of induction treatment have been associated with high relapse rates of 70–100% [19,29–31,33]; these patients at risk for relapse may benefit from considering alternative treatment approaches such as transplantation [19,34–36]. MRD status after induction therapy may be the most significant prognostic factor in pediatric ALL, independent of other clinically relevant risk factors, such as age, blast count, immunophenotype, presence of chromosome aberrations at diagnosis, and response to induction therapy [19,31,37]. In pediatric ALL cases who relapse after achieving remission, MRD status after reinduction treatment is similarly important [35,38]. Eckert et al. [35] used a quantitative PCR assay to detect MRD in 30 relapsed patients. The event-free survival (EFS) between the two groups (MRD divided into <10−3 or ≥ 10−3 sensitivity levels) was significantly different (86% versus 0%, respectively; p < 0.0001). Coustan-Smith et al. [38] studied the clinical significance of MRD in ALL after first relapse using flow cytometry for MRD detection. Of the 35 children with morphologic remission after induction treatment, 19 (54%) had an MRD of 0.01% or more, and their subsequent relapse rate was 70%, compared with 28% for the 16 MRD-negative patients. The value of continuing MRD monitoring during maintenance therapy and off therapy is less clear, since frequent monitoring has been difficult to employ [28,31,39]. MRD studies in adult ALL have lagged behind pediatric studies. There is increasing evidence that in adult ALL, as in children, an early decrease of MRD level after induction therapy is an important predictor of prognosis and response to chemotherapy [40–42]. Thus, the assess-

Table 26.3 Comparison of minimal residual disease (MRD) studies in childhood acute lymphoblastic leukemia at the time point of induction

Study

EORTC (Cave et al.) [19]

Number of patients MRD method

178 Ig/TCR-PCR

MRD study time point

After induction therapy

iBFM (van Dongen et al.) [31]

St Jude (Coustan-Smith et al.) [29]

BFM-Austria (Dworzak et al.) [30]

NOPHO (Nyvold et al.) [32]

240 Ig/TCR-PCR and TAL1 deletion 5 weeks after diagnosis

195 Flow cytometry

108 Flow cytometry

104 Ig/TCR-PCR

End of induction

Day 33 after start of induction therapy

Day 29 after start of induction therapy

4/59 (7%) 1/5 (20%) 5/31 (16%) 3/3 (100%)

1/53 (2%)

Number of patients relapsing/number with given MRD result (relapse rate) MRD status Negative ≤10−4 <10−2, >10−4 ≥10−2

7/88 (8%) 5/30 (17%) 11/15 (73%)

2/71 (3%) 8/33 (25%) 12/38 (32%) 20/27 (67%)

Ig/TCR-PCR, immunoglobulin/T-cell receptor polymerase chain reaction. Reproduced with permission from Chung et al. [161].

9/123 (10%) −/33 (23, 43%) −/9 (72%)

9/32 (28%) 5/15 (33%)

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ment of MRD kinetics at different follow-up time points defined risk groups based on a specific threshold MRD level in standard-risk adult ALL patients [43]. Patients with a rapid MRD decline to lower than 10−4 or under the detection limit at day 11 (during first induction) and day 24 (at the end of induction) were at low risk, with a 3-year diseasefree survival (DFS) and overall survival (OS) of 100%. In contrast, patients with a persistent MRD of 10–4 until week 16 were at high risk, with a 3-year DFS of 5.8% and a 3-year OS of 45.1%. In adult Ph+ ALL, MRD kinetics can be followed by measuring Bcr-Abl transcript levels with quantitative real-time PCR [44,45]. Using this approach, 42 patients with complete hematologic response following induction therapy were divided into two prognostic groups: good molecular responders (with >2 log reduction of Bcr-Abl transcript levels after induction, and a >3 log reduction after consolidation therapy) and poor molecular responders, who had a higher MRD level at both time points [45]. The probability of 2-year survival was 48% for the good molecular responders compared with 0% for the poor molecular responders. Krampera et al. demonstrated that frequent immunophenotypic MRD measurement in the first year of treatment is a useful outcome predictor for adult T-cell-ALL patients [46]. MRD-positive patients prior to consolidation therapy had a probability of relapse at 2 years of 82%, compared with 39% in MRD-negative patients. In a larger study of 102 adolescent and adult ALL patients, MRD at day 35 was the most powerful independent prognostic parameter by multivariate analysis [47]. Patients in morphologic complete remission (CR) and a low MRD level (<0.05%) had a median relapse-free survival (RFS) of 42 months versus 16 months in case of higher MRD levels. An excellent prognosis with a projected 5-year RFS of 90% was observed when MRD levels at day 14 were very low (<0.03%). MRD in ALL post transplant In the past, allogeneic HCT was generally reserved for the treatment of high-risk patients in first remission and those who had relapsed after chemotherapy. As the risks of transplant are substantial, it is important to distinguish the patients who will benefit from hematopoietic cell transplantation (HCT) from those who will profit from a different therapeutic approach or further modification prior to or after HCT. MRD may be a method to accomplish this clinically valuable assessment.

MRD detection prior to and following transplantation is strongly predictive of outcome. Many reports have shown that MRD burden prior to conditioning is the most powerful single predictive factor for relapse post HCT (Table 26.4) [48–54]. In these studies, the EFS for the MRDnegative groups prior to HCT was often more than 70%, while for those in the MRD-positive group the EFS fell to around 17–50%. Both PCR and flow cytometry were used to detect MRD in these studies, and various criteria have been used to determine “low” from “high” levels of MRD burden. Taken together, however, the data are fairly consistent and logical, showing a very high risk of relapse (~75%) in cases with high levels of MRD prior to HCT, an intermediate risk of relapse in cases with low-level MRD (~50%), and a much smaller risk of relapse in those without MRD (~15%). MRD detection following transplant is also predictive of subsequent relapse. This is true for Ph+ ALL and non-Ph+ ALL cases. In the latter setting, studies have found the relative risk of relapse associated with MRD to be roughly five- to 10-fold, with relapse occurring within a few months of MRD detection [55,56]. The story with Ph+ ALL is slightly more complicated. In Ph+ ALL, PCR must be performed on both the “p210” Bcr-Abl variant transcript (found in CML and some ALL), and the “p190” Bcr-Abl transcript (found only rarely in CML but common in ALL). All studies of Ph+ ALL have demonstrated excess relapses in MRD-positive cases following transplant [57–59]. However, the data suggest that the risk of relapse associated with p190 Bcr-Abl MRD is stronger than with p210 Bcr-Abl. Thus, in 90 patients, the relative risk of relapse with any MRD post-transplant assay (compared with patients with all-negative MRD assays) was 4.4 [59]. For p190+ MRD, the relative risk was 8.7, while for p210 Bcr-Abl transcripts it was 2.2. Note that this difference in risk with different Bcr-Abl transcript variants was seen in the pretransplant MRD assessment as well. The median time from first MRD detection to relapse was 75 days. Likewise, Spinelli and colleagues [60] correlated MRD clearance immediately before, and 30 and 100 days after, allogeneic stem cell transplantation (SCT) with outcome in 43 adult “high-risk” ALL patients by real-time quantitative (RQ)-PCR using one or two molecular probes for BCR/ABL1, MLL/AFF1 (AF4/FMR2 family, member 1) (for t(9;22) and t(4;11), respectively; n = 22) or rearrangements of the T-cell receptor or immunoglobulin genes (n = 21). At 36 months, the OS of the 37 patients who underwent SCT in hematologic remission was 80% for

Table 26.4 A comparison of studies examining the relation between pre-hematopoietic cell transplantation minimal residual disease (MRD) levels and subsequent relapse Number of patients relapsing/number with given MRD result Positive Study

Number of patients

Negative

Low-level positive

High-level positive

Knechtli et al. [50] Uzunel et al. [51] Bader et al. [49] Krejci et al. [48] van der Velden et al. [53] Sanchez et al. [54] Sramkova et al. [52] Total

64 (TRM5) 30 41 140 17 24 25 341

8/38 0/5 2/14 12/77 2/11 3/18 1/17 28/180 (15.5%)

5/9 5/10 5/10 14/27

12/12 8/15 11/17 27/36 4/6 4/6 6/8

29/56 (51.7%)

58/80 (72.5%) 87/156 (55.7%)

Reproduced with permission from Chung et al. [161].

The Detection and Significance of Minimal Residual Disease

those who were PCR negative before transplantation (n = 12) compared with 49% for PCR-positive patients (n = 25; p = 0.17), and the cumulative incidence of relapse was 0% and 46%, respectively (p = 0.027). Importantly, the relapse rate of patients who were PCR negative at day 100 after transplantation was remarkably low (7%) compared with that among patients who were PCR positive (80%; p = 0.0006). Achieving a molecular CR before SCT is highly desirable and an apparent predictor for PCR negativity at day 100 post SCT. Taken together, “high-risk” ALL patients who do not achieve an early molecular remission post HCT should receive pre-emptive therapy such as donor leukocyte infusions, imatinib, interferon (IFN) or new experimental drugs. Most MRD studies have used BM samples rather than peripheral blood. In precursor B-cell and Ph+ ALL, the level of MRD is higher in BM than peripheral blood, which makes BM the preferred source for disease monitoring. However, the use of peripheral blood to detect MRD has several practical advantages over the use of marrow, especially in children, as peripheral blood sampling is simple and relatively painless, allowing more frequent monitoring, which may offset the sensitivity advantage of BM [61,62]. MRD detection in AML following chemotherapy In AML, the use of PCR-based detection of MRD is essentially limited to patients with AML containing unique breakpoint fusion regions, such as t(15;17), t(8;21), and inv(16). However, flow cytometry assays can be used to detect abnormal patterns of lineage and differentiationspecific markers suggestive of leukemia blasts in the majority of AML cases. Acute promyelocytic leukemia (APL) frequently harbors the t(15;17) translocation, which causes the promyelocytic leukemia/retinoic acid receptor-alpha or PML/RARα gene juxtaposition. APL patients who have residual disease after induction or consolidation therapy have a significant risk of relapse [63,64]. Serial quantitative MRD monitoring by PCR until completion of maintenance therapy might allow identifying patients at high risk of relapse in APL [65]. Thus, the study of 70 APL patients identified the MRD level after first consolidation as the most powerful predictor of relapse. Patients with a MRD level of 10−3 or more had a greater than 10-fold higher relapse rate at 5 years compared with a level of less than 10−3. In APL, there is evidence that the analysis of peripheral blood may be as informative as that of BM [66]. Disruptions of the core binding factor genes, runt related transcription factor-1 (RUNX1, AML1) and core binding factor subunit (CBFB), are involved in the t(8;21) and inv(16)/t(16;16) “good” risk translocations, found collectively in 15–20% of adult and pediatric AML cases. The monitoring of these translocations poses an interesting problem. The qualitative detection (yes or no) of the RUNX1/RUNX1T1 (previously known as AML1/MGT8 or AML1/ETO) and CBFB/MYH11 gene transcripts has a limited value in monitoring MRD and predicting relapse since these transcripts persist during long-term remission [67–75]. Therefore, quantification of MRD is essential in these AML variants [14,70]. Several groups have attempted to define the critical level of MRD that accurately segregates good and poor outcome. In general, a post-therapy MRD level of less than 1% has been associated with a relatively low risk of relapse [76–80]. MRD investigation at the end of treatment might have the best prognostic value in core binding factorleukemia [81]. Schnittger et al. created a score for risk stratification of AML by combining the transcription ratios at diagnosis and after 3–4 months during treatment [82]. Immunophenotypic detection of MRD by flow cytometry can be used for identifying risk groups after induction chemotherapy, and for providing important information for post-induction treatment strategies. In a study of 72 patients, repeat flow cytometry studies enabled the identifi-

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cation of specific cut-off values of MRD associated with significant survival impact after the induction and consolidation therapy [83]. A cut-off level of 1% after the first induction, of 0.14% after the second induction, and of 0.11% after the consolidation resulted in a 6.1-fold, 3.4-fold, and 7.2-fold higher relative risk of relapse, respectively. Similar conclusions can be drawn from other studies [84,85]. Coustan-Smith et al. [86] found MRD in 85% of children with a sensitivity of 10−3 or more by using flow cytometric detection of abnormal immunophenotypic combinations. Flow cytometric residual disease was found in 34.1% and 27.8% of patients after first and second induction, respectively, with a 2-year OS of approximately 30% compared with more than 70% in patients without detectable MRD. Sievers et al. [87] found immunophenotypic abnormalities in 41 (16%) of 252 children with AML who achieved morphologic CR with initial induction therapy, and MRD was highly associated with a poor outcome. The Wilms’ tumor gene (WT1) may be a molecular target for MRD detection, as it is overexpressed in around 80% of AML cases [88,89]. The ability to use this marker is complicated by the fact that it is also expressed in normal hematopoietic cells, although at a lesser degree; thus, the tricky issue is distinguishing low WT1 transcripts in leukemia versus background from normal cells. Thus, the most consistent use of WT1 transcript levels is as a prognostic tool at diagnosis. Several groups have reported that WT1 levels at diagnosis are associated with prognosis (low = good; high = bad) [90]. In some studies, the increase in WT1 transcripts during therapy is associated with subsequent relapse [88] and correlated with other disease markers, such as leukemia-specific fusion transcripts [89]. Other groups, however, have found WT1 transcript levels to be important at distinct intervals of treatment [91]. The most common genetic mutations found in AML involve fmsrelated tyrosine kinase-3 (FLT3) and nucleophosmin (NPM); both occur in approximately 25% or greater of cases. Theoretically, these may be targets of PCR-based assays, but not easily. The most common FLT3 gene alteration is an internal tandem duplication that causes a 5–100 base pair addition to the juxtamembrane domain. Primers can be made that allow for leukemia-specific amplification for a specific patient [92– 94]. However, the FLT3 mutation is not terribly stable, and around 10–20% of the time, patients relapse without the FLT3 mutation [95,96]. Mutation-specific PCR reactions have been constructed for the most common point mutations in NPM1, and many potential mutations exist [97]. It is unlikely that PCR mutation detection in these subsets of AML will replace the faster and near-equally sensitive flow cytometry. MRD in AML post transplant There are few studies documenting the prevalence and significance of MRD in AML following HCT. This in part is due to the fact that the most common translocations found in AML – for example t(15;17), t(8;21), and inv(16)/t(16;16) – are associated with intermediate or good outcomes with conventional chemotherapy, and thus are underrepresented in the transplant population. The significance of MRD following transplantation depends largely on the specific genetic subtype of AML, and generally correlates with the “rules” seen in the chemotherapy setting. Thus in t(15;17) AML, the detection of the PML-RARα transcript following HCT is highly correlated with relapse [98,99]. In t(8;21) AML, the detection of RUNX1RUNXITI transcripts remarkably often persists after allogeneic transplant, without subsequent relapse [69–71]. There are too few cases of inv(16)/t(16;16) studied in the transplant setting to make a firm conclusion about its association with relapse [68,100]. The tumor suppressor gene WT1 has been used as a marker of MRD following HCT [101]. The results have been mixed. Elmaagacli et al. studied 38 patients post HCT, of whom 14 of 38 were positive for ele-

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vated WT1 transcripts at least once post transplant. Of the 14 patients positive for MRD, seven relapsed, compared with five relapses in the 34 patients without MRD. Elevated WT1 transcript levels were in concordance with the persistence of translocation markers in 70% of cases. However, other studies [102] have not found WT1 transcript levels predictive of relapse following HCT. Another possibility is using WT1 transcript levels prior to transplant as a measure of disease burden to predict subsequent response. Osborne et al. [103] measured WT1 transcripts in BM harvests and correlated the result to the outcome after autologous transplantation. The median RFS for patients with high WT1 transcripts levels was 10.5 months, while patients with low levels have not reached the median RFS at a median follow-up of 94 months. WT1 transcripts are being investigated as a potential target for immunotherapy and WT1 transcript levels thus may indicate if a particular leukemia more susceptible to this therapeutic approach. Ogawa et al. [104] studied 72 patients (50 AML, 15 ALL, and 7 CML), post HCT, and all 20 patients who relapsed showed elevated WT1 transcripts at the time of relapse. After relapse, patients were treated with some form of immunomodulation (donor lymphocyte infusions or discontinuation of immunosuppressive therapy). The calculated WT1 transcript doubling time that occurred with relapse for patients was significantly longer for patients who responded to immunomodulation (a median doubling time of 26 days) compared with nonresponders (a median of 6 days). It is unclear, however, if the same information in regards to the kinetics could also be produced by serial flow cytometry studies. Lastly, the measurement of the MRD burden of transplantation products may also be used to guide patient-tailored purging procedures [105]. An MRD level in the stem cell products of less than 0.05%, as measured by flow cytometry, was found to be associated with an excellent RFS of 100% at 12 months. In an autologous transplant setting, the pretransplant MRD status, measured by multidimensional flow cytometry, predicted outcome reliably [106]. Patients (n = 19) with fewer than 3.5 × 10−4 residual leukemia cells were assigned as MRD of low risk, and those (n = 12) with 3.5 × 10−4 or more cells as MRD high risk; 100% of patients with high-risk disease relapsed versus 26% of the low-risk group. The median post-transplant RFS was 7 and 48 months for the high-risk and low-risk MRD group, respectively. Increasing MRD levels were shown in three of the five relapse patients by longitudinal flow cytometry monitoring. This report suggested that MRD can be used for predicting outcome in patients receiving autologous transplantation, and impending relapse can be detected by sequential MRD monitoring. MRD in CML following imatinib therapy The power and potential of MRD detection to predict relapse and guide therapy has been led by the studies in CML. The clinical utility of MRD detection of the chimeric Bcr-Abl transcript was first shown in the HCT setting, and has lately been utilized in studies of the tyrosine kinase inhibitors, especially imatinib. Quantitative PCR for Bcr-Abl transcripts defines a population of patients in cytogenetic remission who have a prolonged course of remission. Quantitative detection of CML patients treated with tyrosine kinase inhibitors may help in guiding therapy in regards to the depth of disease burden undetectable by cytogenetic testing. The landmark IRIS (International Randomized Study of Interferon versus STI-571) trial compared imatinib with the “standard” nontransplant therapy of IFN and cytarabine in newly diagnosed chronic-phase CML patients. At the time of study publication, more than 70% of imatinib-treated patients achieved a complete cytogenetic remission (CCR), compared with only 7% in the IFN and cytarabine arm [107,108]. Bcr-Abl transcripts detection by quantitative PCR was used to examine the patients who achieved CCR [109]. Patients in CCR after imatinib

therapy had a statistically significantly greater Bcr-Abl reduction than patients on the IFN/cytarabine arm. A 3-log reduction in Bcr-Abl copy number from baseline (a so-called “major molecular response” or MMR) was obtained in 39% of imatinib-treated patients after 12 months of therapy, compared with only 2% in the IFN/cytarabine arm. The depth of molecular response at 12 months was associated with progression-free survival. Thus, if patients did not achieve CCR, the risk for progression in patients without CCR was approximately 25%, compared with patients who achieved a CCR and had a less than 3-log reduction (~10%) or a 3-log or more reduction in Bcr-Abl transcripts by 12 months (~0%). Undetectable Bcr-Abl transcripts (defined as quantitatively undetectable Bcr-Abl transcripts, as well as a PCR-negative test by a more sensitive nested reverse transcriptase [RT]-PCR that was confirmed by another laboratory in the study) was unusual (<5% of cases at the 12-month mark). An important and often misunderstood feature of the IRIS molecular study was the use of log reduction as a measure of Bcr-Abl transcript level response, as opposed to actual Bcr-Abl transcript copy number, or the ratio of Bcr-Abl transcripts to a control gene transcript level. The analysis was not done using each patient’s baseline Bcr-Abl transcript value. Rather, each laboratory established its own baseline on the same set of 30 baseline samples. Subsequent individual samples had Bcr-Abl/Bcr transcript ratios calculated, and these values, compared with that laboratory’s baseline value, yielded the log reduction for that sample. This method had two major advantages. In regards to the IRIS study, it standardized the assay across the three laboratories and allowed other laboratories to compare results, as long as each had a baseline value, since the log reduction did not require the use of the same control gene from laboratory to laboratory. The recent expert consensus on Bcr-Abl transcript level advocates the use of an international scale based on the log reduction principle [110]. Notably, all Bcr-Abl molecular transcript testing in the IRIS trial was performed on peripheral blood samples. Similar to post-transplant MRD data, there is excellent correlation between RT-PCR Bcr-Abl transcript levels between BM and peripheral blood [108,111,112]. Thus, if a patient is in CCR, molecular testing for Bcr-Abl transcripts can be confidently performed on peripheral blood. Subsequent studies have validated the clinical utility of Bcr-Abl transcript testing after imatinib therapy [113,114]. The Hammersmith group studied a more heterogeneous group of chronic-phase CML patients who achieved CCR on imatinib [114]. Thirty-two of 106 patients studied had a persistent decline in Bcr-Abl transcript level, and predictably, none relapsed. Twenty-six patients demonstrated an increase in Bcr-Abl transcripts after the initial response, with subsequent relapse. However, 42 patients achieved a “plateau” Bcr-Abl transcript level without quite reaching an MMR, and remained in CCR. This study is in contrast with a study of 280 CML cases from the MD Anderson Center, which reported a 5% loss of CCR in MMR cases, compared with a 37% loss of CCR in cases that did not achieve a MMR [113]. However, in both studies, compared with the IRIS study, the CML cases included patients at various times from diagnosis as well as patients who had previously received IFN therapy. The rate of decline of Bcr-Abl messenger RNA transcripts after imatinib therapy predicts long-term response, as Bcr-Abl transcripts decline after the initial 2–3 months of imatinib predicts subsequent response [113,115–117]. Thus, patients who do not achieve at least a 1-log reduction in Bcr-Abl levels after 3 months of therapy have a lower probability of achieving CCR and MMR, or a higher probability of developing resistance [113,115]. Thus, CML patients treated with imatinib who achieve a less than 1-log reduction in Bcr-Abl transcripts after 3 months have a 13% chance of ever achieving a MMR, compared with greater than 70% in patients with a better response [115]. Similarly, a separate study found that only 80% of patients with less than 1-log Bcr-Abl

The Detection and Significance of Minimal Residual Disease

transcripts response at 3 months achieved CCR, compared with 100% for patients who achieved a greater reduction of Bcr-Abl levels [113]. Thus, frequent and early monitoring may help define patients who need early consideration for alternative therapies such as transplant or other experimental protocols. MRD detection post transplant A large number of studies have studied the interaction of MRD and relapse in CML post transplantation. A number of robust observations have been made that illustrate the complexity of interpreting MRD results. First, at least in CML the molecular detection and quantification of Bcr-Abl transcript levels may be performed on either peripheral blood or BM specimens [55]. While quantitative PCR is routine today, it should be noted that, in CML, the qualitative (yes or no) detection of Bcr-Abl transcripts post HCT is associated with relapse, and varies with the type of transplant and the time from transplant [55,118–123]. The highest risk of relapse associated with Bcr-Abl transcript detection appears to be associated “early” (≤12 months post HCT) after transplant [118,122,124]. In one study of 346 patients post transplant, a positive Bcr-Abl transcript assay 6–12 months post transplant was associated with a 42% risk of relapse as opposed to a 3% risk of relapse in PCR-negative patients. Tests earlier than 3 months post HCT were not strongly associated with relapse. Moreover, MRD can be detected by quantitative PCR for years after HCT. Bcr-Abl transcripts have been detected in 25–50% of patients 3 or more years post transplant, with subsequent relapse rates of around 10–20% [121,124–126]. Patients in CR for longer than 10 years post HCT have been found to be persistently MRD positive [127]. The type of transplant (allogeneic, T-cell depleted or unrelated) affects the association of MRD and relapse. This is presumably due to the immunologic effect associated with an allograft (“graft-versusleukemia” [GVL]). Transplants from unmanipulated unrelated donor grafts had a lower risk of relapse compared with matched related transplants. Patients undergoing T-cell depletion have a very strong association of MRD detection and subsequent relapse [121,128]. Thus while T-cell depletion decreases graft-versus-host disease, it clearly does so at the cost of the associated GVL effect. The quantification of Bcr-Abl transcripts by quantitative PCR further refines the predictive value of MRD detection in CML [126,129–134]. Thus, low residual Bcr-Abl transcripts post transplant was associated with a very low risk of relapse (1%), compared with a 75% relapse rate in patients with increasing or persistently high Bcr-Abl transcript levels [135]. Olavarria et al. studied 138 CML patients “early” (3–5 months) post transplant and found that Bcr-Abl transcript level was highly correlated with relapse [133]. Patients with undetectable Bcr-Abl transcripts had a 9% risk of relapse, compared with patients with a “low” (<100 Bcr-Abl transcripts/μg RNA) or “high” (>100 copies Bcr-Abl/μg) level of transcripts, who had a cumulative relapse rate of 30% and 74%, respectively. In addition, in a study of 379 CML patients “late” (>18 months) post HCT [126], 90 patients (24%) had at least one positive assay and 13 of 90 (14%) relapsed. Conversely, only three of 289 patients who were persistently Bcr-Abl transcript negative relapsed (hazard ratio of relapse = 19). A rising Bcr-Abl transcript level was a reliable portent of relapse.

MRD in chronic lymphocytic leukemia Other B-lineage hematologic malignancies – chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and non-Hodgkin’s lymphoma – can be studied by strategies similar to those employed to study MRD

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in ALL. Newer molecular determinants for risk-stratified management in CLL are being targeted as direct measurements of the malignant CLL cell rather than indirect measure of progression of disease. The most critical biologic parameters include molecular cytogenetics (FISH), 70 kD zeta-associated protein (ZAP-70), CD38 (measured by flow cytometry), and mutation status of the variable region of the Ig heavy chain gene (IgVH). Thus, IgH V-D-J rearrangements can be used in most lymphoid malignancies to detect MRD. Moreover, CLL has a phenotype that is detectable by flow cytometry with excellent sensitivity. In head-to-head comparisons of PCR and flow cytometry, PCR has been found to be slightly more sensitive, with approximately 10% of flow cytometry-negative cases being found to be PCR positive for clonal rearrangements. Obviously, however, this increased sensitivity must be weighed against the relative ease and speed of flow cytometry compared with the more complicated and costly PCR technique. Recent developments suggest that allogeneic HCT could play a larger role in the treatment of CLL (see Chapter 81), especially in patients with high-risk CLL prognostic factors of unmutated IgVH, del(11q), and del(17p), whereas the role of autologous HCT remains poorly defined and should only be considered in the context of a clinical trial [136–139]. Caballero et al. evaluated the efficacy of reduced-intensity conditioning allogeneic transplant in 30 poor-prognosis CLL patients. Twenty-five patients had active disease at time of transplant, including 14 of 23 with unmutated IgVH, eight of 25 with del(11q), with four unmutated IgVH, and six with del(17p), with five unmutated IgVH. MRD clearance, evaluated by the lack of CD79/CD5/CD19/CD23-positive cells in BM by flow cytometry in 21 patients, was observed in 68% and 94% of the patients at days 100 and 360, respectively. At 47.3 months (median follow-up), 22 patients were alive, with DFS, EFS, and OS at 5 years of 93%, 70%, and 72%, respectively. OS and EFS for unmutated CLL and/or with del(11q) aberrations (n = 13) (90% and 92%, respectively) were similar to those for the seven CLL patients who showed normal FISH, del(13q) positive, trisomy 12 or mutated IgVH CLL. Five of the six patients with 17p deletion transplanted with active and/or refractory disease reached CR after HCT. Thus, the clinical significance of MRD in CLL appears to be tied to the sensitivity of chemorefractory CLL to the allograft immune-mediated “GVL” effect [138,139]. In CLL cases treated with either anti-CD52 antibody-based therapy (Campath) or autologous transplant, the presence of MRD (by either flow cytometry or PCR) is very strongly associated with subsequent relapse. Rawstron et al. found a strong correlation of quantitative level of CLL in peripheral blood and BM [140]. In 25 cases who achieved CR after Campath1H antibody or autologous transplant, 19 were MRD negative at the time of CR, and their EFS was over 90%; in contrast, all six MRD positive patients at the time of CR subsequently relapsed. Similar findings were found by Esteve et al., who found that four of five patients who were MRD positive while in CR following autologous transplant eventually relapsed, compared with two of nine without MRD [141]. By contrast, MRD negativity following allogeneic transplantation is strongly associated with a very low relapse risk, but, interestingly, many patients who are MRD positive do not relapse, but remain low-level MRD positive. Of the 12 patients who had an allogeneic transplant, only one of eight cases was MRD positive at last contact. Some cases took up to 4 years to clear their MRD, and stayed MRD free for another year of follow-up. This long-term clearance of MRD has also been seen in other studies of MRD following allogeneic transplant, as well as after reduced-intensity transplants, where some patients became MRD negative after 6 months post “transplant” of an allogeneic related immune system [141,142]. This finding is reminiscent of the situation with CML, as noted above. It remains to be seen if the allogeneic effect has a time limit, that is, whether when the graft finally becomes tolerant of the host, the graft will also become tolerant of the residual disease.

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MRD testing in MM The significance of MRD testing in MM has been mired for several reasons. First, the definition of CR does not require the absence of monoclonal plasma cells but rather a reduction in plasma cells to less than 5% in BM samples [143]; thus, patients have a considerable tumor burden while in a “morphologic CR.” Second, PCR testing in MM is complicated by the need to develop a clonal specific probe due to the high IGH gene mutation rate in MM. Lastly, the standard for assessing prognosis in patients with MM proposed by the International Staging System [144] is based on the evaluation of serum levels of β2-microglobulin and albumin, and fails to incorporate the recent molecular pathology advances of MM. In an effort to correct this omission, the International Myeloma Working Group recently adopted a more stringent complete response definition, which includes the absence of clonal cells in BM by immunohistochemistry or immunofluorescence [145]. Recently, the Mayo Clinic further extended the International Staging System model to include the use of a genetic classification system of myeloma and risk-adapted therapy (mSMART) [146]. These recent International Staging System modifications imply a more methodical approach of MRD testing in future clinical trials on MM. The clinical outcomes for MM are highly heterogeneous, and the biology of the disease is defined by their pivotal genetic events. With the most significant advances in the treatment of MM, including the use of tandem autologous SCT in combination with the immunomodulatory drugs thalidomide and lenalidomide, as well as the proteasome inhibitor bortezomib, albeit none curative, the major challenge will be to incorporate genetic and other MRD prognostic parameters into the clinical trials that are designed to determine the best way, time, and sequence of administering these therapies. Genomic aberrations evaluated by interphase FISH have been shown to play a major role in the prognosis and evolution of patients with myeloma. The key to this approach is the requirement for plasma cell enrichment. At least three enrichment techniques followed by FISH analysis have been reported in the literature, namely, isolation of plasma cells by morphology (Plate 26.1), CD138+ enrichment, and κ/λ immunohistochemistry (also known as cIg-FISH) [3–5]. For example, three independent groups [3,147,148] independently reported that MM patients with t(4;14) or del(17p) fared worst when compared with the other genetic subgroups, especially when associated with high β2-microglobulin levels or more than 60% plasma cells in the marrow. The poor prognosis associated with −13/del(13q) was further attributed to the fact the t(4;14) and del(17p) patients showed the chromosome 13 aberrations simultaneously, with few patients benefiting from thalidomide maintenance therapy. However, emerging data suggest bortezomib may overcome the poor prognosis conferred by chromosome 13 deletions at relapse in MM patients with high serum albumin [149,150]. Taken together, these data have broad implications for patient counseling, choice of therapy, and innovative molecular approaches to clinical investigations for both younger and older MM patients [151]. Current data suggest that allogeneic HCT for MM results in CR rates of 22–67%, with molecular remissions in about one-third [152,153] and prolonged DFS in 25–33% of patients [154,155] (see Chapter 80). Martinelli and colleagues [153] noted that MM patients who achieved a molecular CR after allogeneic or autologous HCT had a significantly lower relapse rate (41% versus 16%; p < 0.05) and a longer RFS (35 versus 110 months, p < 0.005). Using a sensitive flow cytometry assay, Rawstron et al. [156] quantitated normal and neoplastic plasma cells and compared their results with standard immunofixation in 45 patients undergoing high-dose chemotherapy. By flow, neoplastic plasma cells were detected in 42% of the patients at 3 months after transplantation (compared with 27% by immunofixation). Patients with detectable MRD

showed a significantly shorter progression-free survival (median 20 months) compared with those with no detectable disease (median >35 months), and a significantly higher risk of early disease progression. Sarasquete and colleagues [157] compared the clinical utility of MRD in MM by allelic-specific oligonucleotide real-time quantitative (RQ)PCR and flow cytometry. Due to technical issues, allelic-specific oligonucleotide-RQ-PCR was only evaluable in 75% of patients compared with 90% for flow cytometry; however, PCR detected residual myelomatous cells (median 0.014%, range 0.001–0.11%) in 17 patients compared with just 11 patients by flow cytometry. This head-to-head comparison showed that the ability to predict outcome in MM is similar between PCR and flow cytometry (~0.01% level), with flow cytometry having the critical advantage of speed, possibly allowing for real-time clinical decisions, including dose escalation and de-escalation strategies or the timing of stem cell harvests based on MRD detected during therapy. Nevertheless, for a true statistical comparison, the same MRD technique should be utilized for all patients on a given protocol.

Problems and promise Lest the gentle reader be misled that the study of MRD is unblemished in clarity and precision, we alert said reader to pitfalls, both real and potential. The challenges of MRD are increasingly complex, and the rapidly changing landscape of molecular pathology encourages interactive dialog and standardization. Assays for MRD are labor-intensive, expensive, and not easily “exportable” The exquisite sensitivity of PCR assays comes with an exacting price – small variations in technique can lead to drastically wrong results. Moreover, contamination is relatively easy and can cripple a laboratory. Standard operating techniques can minimize these problems, but the fact remains that the successful laboratory is one that is careful, compulsive, vigilant, and perhaps somewhat neurotic. Moreover, the subtle nuances of a particular assay may not “travel” well, so standardization of assays is crucial, but difficult. Not all patients with detectable MRD progress to relapse This phenomenon has sometimes been referred to as “dormancy,” although it is not clear that the leukemia (if it is leukemia) is truly inactive. There are three clear demonstrations of dormancy. The first is in t(8;21) AML following conventional chemotherapy, as well as in CML following HCT [14,158]. In t(8;21) AML, the RUNX1-RUNX1T1 (AML1-ETO) messenger RNA can be detected in a minority of lymphoid and erythroid colonies taken from remission samples, suggesting that the disease involves a primitive leukemia progenitor cell [159]. As such, some MRD in this disease may arise from nonleukemia lymphoid cells. Furthermore, another example of dormancy is the observation that late relapses in pediatric ALL (>10 years after CR) have the same clonal IGH V-D-J gene rearrangement as at diagnosis [160]. How can such examples of dormancy be explained? Perhaps prolonged immune surveillance, most relevant after HCT, can control the expansion of a leukemia clone. Or, as in the example of t(8;21) noted above, the MRD assay may detect signal in a cell lineage not involved in the leukemia. Lastly, a residual leukemia cell may be “preleukemia,” not having all the mutations necessary for malignancy, or “postleukemia,” whereby additional mutations have rendered the clone relatively inert. Regardless, it is likely that the clinical definition of “cure” does not mean the elimination of all cells involved in the leukemia

The Detection and Significance of Minimal Residual Disease

process. The further study of dormancy may clarify what it takes to “cure” leukemia. Lack of standardization of the MRD assays, nomenclature, and clinical reporting The need for uniform or standardized MRD testing is critical to facilitate precise comparisons between new treatment strategies. This is particularly true in PCR-based assays. The problematic areas of gene nomenclature and clinical molecular reporting are being actively addressed by the pertinent international scientific professional societies in collaboration with the health-care professionals who utilize molecular testing that impact patient management. We need to be proactive and join in with this concerted effort to develop a uniform approach for quantifying and reporting MRD. To avoid future nomenclature issues, especially in reference to genes, their transcripts, and the resultant proteins, scientists are requested to follow the gene names and symbols provided the Human Genome Organization (HUGO) gene nomenclature (reader is referred to http://www.gene.ucl. ac.uk/nomenclature). Guidances for clinical molecular laboratory reporting, including FISH, PCR, mutations, deletions, and other genetic variations testing, have been proposed to assess the clinical relevance of MRD testing and facilitate precise comparisons among clinical trials.

Conclusion Today’s technologic advances in laboratory science are providing the tools to design therapeutic strategies according to the molecularly

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defined features of hematopoietic malignancies and the presence or absence of MRD. But much more work remains to be done in this area. The detection of MRD is strongly associated with relapse. MRD monitoring is being melded into the design of clinical trials, and is likely to improve the speed and efficacy of these trials. Many research avenues bear further investigation. The treatment of MRD in almost all settings is still under investigation, and cries out for large-scale studies. The recognition of “dormancy” may allow us to adopt therapies to achieve that state, rather than persist in high-dose therapy designed to eliminate leukemia, which may be impossible. In many leukemia subtypes, there are few good MRD markers. Further research (e.g. gene expression and proteomics studies) is expected to identify new markers for MRD assays, and provide insights into the cell signaling pathways that can be exploited as therapeutic targets. Recent genomic characterization studies of the hematopoietic disorders by highdensity microarrays (containing ~350,000 genetic loci) are unscrambling the recurrent theme between cancer and cellular differentiation. These assays have led to the discovery of deletions and loss-of-function mutations in genes known to be involved in regulating the differentiation of blood cells. It will be through these high-density molecular approaches applied to large patient cohorts that recurrent molecular aberrant patterns will lead to systematic strategies to tackle the underlying genetic causes of these disorders with the eradication of MRD. Without question, MRD detection is key in patient management. It is our responsibility to adopt standardized MRD techniques within clinical trials to clarify its role and greatly improve the pace at which therapeutic strategies are developed and tested.

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129. Branford S, Hughes TP, Rudzki Z. Monitoring chronic myeloid leukaemia therapy by real-time quantitative PCR in blood is a reliable alternative to bone marrow cytogenetics. Br J Haematol 1999; 107: 587–99. 130. Lin YT, Lin DT, Jou ST, Lin KS, Lin KH. Allogeneic bone marrow transplantation for Philadelphia chromosome-positive chronic myelogenous leukemia in childhood. J Formos Med Assoc 1997; 96: 320–4. 131. Mensink E, van de Locht A, Schattenberg A et al. Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR. Br J Haematol 1998; 102: 768–74. 132. Mughal TI, Yong A, Szydlo RM et al. Molecular studies in patients with chronic myeloid leukaemia in remission 5 years after allogeneic stem cell transplant define the risk of subsequent relapse. Br J Haematol 2001; 115: 569–74. 133. Olavarria E, Kanfer E, Szydlo R et al. Early detection of BCR-ABL transcripts by quantitative reverse transcriptase-polymerase chain reaction predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood 2001; 97: 1560–5. 134. Preudhomme C, Chams-Eddine L, Roumier C et al. Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay. Leukemia 1999; 13: 818–23. 135. Lin F, van Rhee F, Goldman JM, Cross NC. Kinetics of increasing BCR-ABL transcript numbers in chronic myeloid leukemia patients who relapse after bone marrow transplantation. Blood 1996; 87: 4473–8. 136. Caballero D, Garcia-Marco JA, Martino R et al. Allogeneic transplant with reduced intensity conditioning regimens may overcome the poor prognosis of B-cell chronic lymphocytic leukemia with unmutated immunoglobulin variable heavychain gene and chromosomal abnormalities (11q– and 17p–). Clin Cancer Res 2005; 11: 7757–63. 137. Gribben JG. Salvage therapy for CLL and the role of stem cell transplantation. Hematology Am Soc Hematol Educ Program 2005: 292–8. 138. Ritgen M, Stilgenbauer S, von Neuhoff N et al. Graft-versus-leukemia activity may overcome therapeutic resistance of chronic lymphocytic leukemia with unmutated immunoglobulin variable heavy-chain gene status: implications of minimal residual disease measurement with quantitative PCR. Blood 2004; 104: 2600–2. 139. Sorror ML, Maris MB, Sandmaier BM et al. Hematopoietic cell transplantation after nonmyeloablative conditioning for advanced chronic lymphocytic leukemia. J Clin Oncol 2005; 23: 3819–29. 140. Rawstron AC, Kennedy B, Evans PA et al. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy. Blood 2001; 98: 29–35. 141. Esteve J, Villamor N, Colomer D, Montserrat E. Different clinical value of minimal residual disease after autologous and allogeneic stem cell transplantation for chronic lymphocytic leukemia. Blood 2002; 99: 1873–4. 142. McSweeney PA, Niederwieser D, Shizuru JA et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replac-

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27

Howard M. Shulman, Robert C. Hackman & George E. Sale

Pathology of Hematopoietic Cell Transplantation

Introduction The hematopoietic cell transplantation (HCT) setting presents pathologists with four major types of complication: toxicities resulting from pretransplant cytoreductive conditioning regimens, immunologic problems (rejection or graft-versus-host disease [GVHD]), infections and malignancy. In the last few years, substantial changes have taken place in the practice of HCT, including the use of reduced-intensity conditioning regimens and the inclusion of older allogeneic recipients and those with autoimmune disorders or with substantial pretransplant comorbidities. There is also increased use of mismatched and/or unrelated HCT donors, different immunosuppression regimens, resulting in a change in the presentation of GVHD, and recognition of additional lateterm complications.

Pretransplant evaluation Lymphoreticular system Before a patient is accepted for HCT, the referral evaluation is used to confirm the diagnosis, review available protocols, and identify potential complications that may influence transplant decisions. The pathologist reviewing material from the referring institution will need relevant flow cytometric, immunohistologic, molecular, and cytogenetic data as well as details of recent therapy. Prior to undergoing HCT for hematologic malignancies, the marrow is evaluated to determine remission/relapse status. Of the various modalities utilized, multiparameter flow cytometry is among the most important because it samples thousands of cells and has the ability to detect as few as 0.01% malignant cells, even without having a prior immunophenotype signature for comparison [1–3]. In patients with lymphoma, review of the diagnostic material is especially important if the initial diagnosis was based on small needle biopsies. Following transplantation, the availability of slides showing the lymphoma immunohistology is very helpful when evaluating small lymphoid aggregates in the marrow or possible extramedullary relapse in surgical specimens. The pretransplant subclassification of myelodysplastic syndromes by World Health Organization criteria into aggressive compared with milder forms (e.g. refractory anemia with excess blasts versus refractive

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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anemia) relies predominantly on the marrow blast count. Post-transplant survival for both de novo and secondary myelodysplastic syndrome/ transformed acute myeloid leukemia is related to the disease stage (blast count), cytogenetics, conditioning regimen, and donor relationship [4]. Determining the pretransplant immunophenotypic signature of the malignant population is extremely valuable in separating some chronic myeloproliferative disorders from myelodysplastic disorders, monitoring minimal residual disease, and detecting early post-transplant relapse. Identification of the JAK2V617F and other novel mutations aids in the proper classification of myeloproliferative disorders [5]. Aplastic anemia requires precise evaluation of marrow cellularity and individual lineages to verify eligibility for HCT, and also to rule out myelodysplasia or paroxysmal nocturnal hemoglobinuria insidiously simulating primary aplasia. Lymphomas require review of previous diagnostic biopsies in order to evaluate possible marrow involvement. Detailed evaluation of myelofibrosis may be needed to distinguish chronic myeloproliferative disorders from acute panmyelosis, myelodysplastic syndromes, and metastatic disease. Studies may involve multiple marrow biopsies stained for reticulin and trichrome, as well as molecular testing of BCR–ABL1 fusion transcripts, JAK2V617F, and other novel mutations. Severe myelofibrosis portends slower engraftment but is not a contraindication to HCT [6]. Magnetic resonance imaging of the pelvis and femur helps assess the extent and distribution of myelofibrosis and other disease [7]. Several new agents have affected pretransplant evaluation. Most patients with chronic myeloid leukemia receive imatinib mesylate (Gleevec) or other more recent tyrosine kinase inhibitors which are effective in producing clinical or cytogenetic remissions. Their pretransplant marrows may be morphologically normal or even hypoplastic and display a normal myeloid-to-erythroid ratio with a low blast count. Granulocyte–macrophage colony-stimulating factor and other cytokines may cause confusing left shifts in hematopoietic cells. Bisphosphonates given for multiple myeloma may thicken trabecular bone in pretransplant marrow biopsies. Hepatic disease Pretransplant hepatic disease has a substantial influence on the choice of conditioning regimen and donor [8]. Patients at increased risk of developing sinusoidal obstructive syndrome (SOS), formerly called hepatic veno-occlusive disease, include those with inflammatory liver diseases demonstrating sinusoidal fibrosis, significant necroinflammatory activity or bridging fibrosis. Specific diseases include steatohepatitis, sinusoidal fibrosis associated with extramedullary hematopoiesis,

Pathology of Hematopoietic Cell Transplantation

prior toxicity from gemtuzumab ozogamicin (Mylotarg) (Plate 27.1) and rarely Gleevec [9], active chronic viral hepatitis, and amyloidosis. Poorly compensated cirrhosis is a contraindication for HCT because of the prohibitive risk of developing SOS after most high-dose regimens. Even compensated cirrhosis has a high likelihood of hepatic decompensation after reduced-intensity regimens [10].

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fibrosis), yet the associated clinicopathologic entity may be highly characteristic of toxicity. Other changes due to toxicity, particularly those in the mucous membranes [13], skin, gastrointestinal tract, and lung, cause diagnostic difficulties because of their clinical or histologic overlap with GVHD and infection (Plates 27.5–27.8) Studies of these organs with serial biopsies demonstrate the diffuse distribution of conditioning injury and marked lessening of these changes with time (Plate 27.9).

Post-transplant hematopoietic evaluations High-dose conditioning produces massive destruction of the host marrow, resulting in acute serous myelitis without necrosis of the bony trabeculae (Plate 27.2). The myelitis includes edema, hemorrhage, loss of most marrow elements except plasma and mast cells, and relative predominance of damaged and frequently necrotic fat associated with iron-rich macrophages. This damage gradually resolves over the first 4–6 weeks as colonies of donor myeloid, megakaryocytic, and erythroid precursors develop. Lymphoid cells are loosely scattered and sparse. Rarely, marrow necrosis occurs after intense cytoreduction or at the onset of leukemic relapse. Assessment of aspirated marrow cellularity should include particle sections because overall cellularity cannot be accurately judged from aspirate smears. Biopsies may be small or artifactually disrupted. Engraftment is usually simultaneous in the three major cell lines, although there can be considerable lag in one or more lineages. Failure or loss of donor engraftment may be manifest by the decline or absence of peripheral counts and marrow hypocellularity. In rare patients with GVHD and absence of marrow hematopoiesis, there is apparently only lymphoid engraftment. Graft rejection has no specific histologic features but may be defined as transient engraftment with proliferation of at least one cell line followed by its loss. Rejection following trilineage engraftment occasionally involves loss of only a single line, analogous to pure red cell aplasia. Although rejection usually results in hypocellularity, this may not be true in patients being treated for some nonmalignant conditions such as thalassemia or immunodeficiency. Proof of mixed chimerism or rejection may require confirmation by flow cytometric sorting of CD33 and CD3 cells followed by fluorescence in situ hybridization studies for X and Y chromosomes (if there is a host–donor gender disparity) or by amplified fragment-length polymorphisms. Despite numerous advances in the application of HCT, relapse remains the major cause of mortality. With the use of hematopoietic growth factors, it is not unusual to encounter rare circulating benign myeloblasts. Multiparameter flow cytometry can usually distinguish benign from malignant blasts and also identify rare aberrant blasts long before morphologic relapse is detectable [2,3]. Post-transplant lymphoproliferative disorder (PTLD) has a spectrum of characteristic plasmacytoid to frankly immunoblastic features associated with positive immunohistochemistry (IHC) stains for Epstein–Barr virus (EBV). PTLD lymphoma should be suspected in the setting of adenopathy or worsening of gastrointestinal symptoms despite treatment for acute GVHD (Plates 27.3 and 27.4) [11]. Serial monitoring of plasma EBV DNA by polymerase chain reaction (PCR) is used to detect increasing viral copy numbers, thus allowing early intervention [12].

Toxicity from pretransplant cytoreductive therapy The cytoreductive therapy given for treatment of underlying malignancy, marrow ablation, and immune suppression may cause widespread multiorgan dysfunction and atypical changes. The frequency, the organ systems involved, and the time of onset are related to both the type and the overall intensity of the conditioning protocol. The histopathology within some affected organs, such as the heart, may be disease “nonspecific” (edema, hemorrhage, necrosis of myocytes, and interstitial

Hepatic SOS (veno-occlusive disease) The most frequent life-threatening regimen-related toxicity involves a clinical syndrome of jaundice, weight gain, ascites, and painful hepatomegaly that typically develops in the first few weeks after HCT. In earlier editions, this syndrome was called hepatic veno-occlusive disease, referring specifically to the obliterative lesions within the lumina of small hepatic venules (Plate 27.10) [8,14]. However, based on more recent experimental and molecular studies, the authors have proposed renaming the syndrome “sinusoidal obstructive syndrome” because the primary site of injury occurs in the sinusoids before clinical signs appear. The term SOS reflects the proximate cause, toxic injury to the zone 3 sinusoidal and venular endothelium related to specific drug metabolites, and to intracellular depletion of glutathione stores. Secondary events are: a decrease in nitric oxide level associated with vasoconstriction; deposition of coagulants evinced by factor VIII staining within the widened venular subendothelial zone and the perivenular zone corresponding to the pores that drain the sinusoids into the small venules (Plate 27.11); and subsequently the deposition of extracellular matrix and collagens by activated and proliferated hepatic stellate cells (Plate 27.12). Liver biopsy and autopsy specimens reveal that the histology of injury to the sinusoids, hepatocytes, and venules may be rapidly progressive. The trichrome stain, which illuminates both parenchymal and connective tissue liver components, is critical for recognizing the diagnostic sinusoidal and venular injuries, while other connective tissue stains (e.g. Verhoeff-van Gieson, reticulin, and immunohistologic stains) are complementary. Examination of serial sections may be needed to identify characteristic histologic changes that may not be present in all levels. The sequence of damage derived from experimental and clinical studies consists of death and detachment of sinusoidal endothelial cells followed by their downstream embolization, leading to sinusoidal obstruction along with deposition of fibrin and procoagulants in the pores which drain into the venules, and hemorrhage in zone 3 and the space of Disse (Plates 27.10, 27.11, and 27.13). Immunostaining with CD31 demonstrates a loss of endothelium in the perivenular sinusoids and the central venule (Plate 27.14). Frank necrosis of perivenular hepatocytes, demonstrated by their absence of cytoplasmic cytokeratin immunostaining, is often more widespread and severe than the extent of venular injury. A consequence of the sinusoidal obstruction, elevated sinusoidal pressure, ischemia, and fragmentation of hepatocyte cords is dislodgement of clusters of liver cells, including hepatocytes, which may flow in a retrograde fashion into portal veins or embolize through disrupted pores into the damaged central venules (Plate 27.15). Within 3 weeks of high-dose conditioning, the deposition of sinusoidal reticulin is accompanied by a marked linear sinusoidal pattern of α smooth muscle actin staining for hepatic stellate cells and increases in monocytes/Kupffer cells at the interface of ischemic hepatocyte necrosis (Plate 27.16) [8,14]. Later changes include collagenous obliteration of the venules, thickening of the outer wall of the hepatic venules (phlebosclerosis) (Plate 27.17), extinction of zone 3 hepatocytes, and fibrotic replacement of the sinusoids adjacent to the terminal hepatic venules (zone 3) of the liver acinus (Plate 27.12), In prolonged and fatal SOS, the liver may take on a pattern of reversed cirrhosis with fibrotic linkage between destroyed perivenular zones.

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Correlation of histologic findings with clinical signs Several additional pericentral (zone 3) lesions that follow cytoreductive therapy are associated with SOS. Two studies have demonstrated that zone 3 sinusoidal fibrosis and phlebosclerosis, a nonocclusive perivenular fibrosis, are also associated with the same clinical features as SOS (Plate 27.17) [8]. An example of the histologic spectrum is a rapidly developing syndrome of SOS that has developed among some HCT patients treated with gemtuzumab ozogamicin (Mylotarg), a humanized anti-CD33 monoclonal antibody conjugated to calicheamicin. The associated histology of gemtuzumab ozogamicin-associated SOS differs slightly from SOS occurring after high-dose cytoreductive conditioning in displaying more sinusoidal fibrosis than venular occlusion (Plate 27.1). Late-developing nodular regenerative alterations are not associated with symptoms of early liver toxicity. They most likely are secondary to disturbances in hemodynamics and serve as an indicator of compensatory repair and regeneration of hepatocytes in different zones of the liver acinus [15]. Several other conditions that may result in an SOS-like picture from diminished sinusoidal blood flow with increased sinusoidal resistance are viral hepatitis with bridging fibrosis, occult cirrhosis, and steatohepatitis, both alcohol and nonalcohol-related.

cells of a lesion tends to favor GVHD, although it is by no means specific. A combined histologic, ultrastructural, IHC study of human upper endoscopic mucosal biopsies found both TNF-α and Fas expressed in the infiltrate and a correlation of the number of apoptotic cells with the day 90 transplant-related mortality [20]. Mucosal FOXP3 regulatory T cells are numerically reduced in gastrointestinal GVHD [21]. TUNEL and XY fluorescence in situ hybridization staining of skin biopsies from sex-mismatched HCT recipients has demonstrated both keratinocytes and dermal endothelial cells of donor origin, most numerous in the areas of severe GVHD damage, and only in the patients with GVHD. However, the implications for any subsequent repair of GVHD-damaged tissues are unclear since the switch from recipient to donor phenotype was present only in biopsies taken early and not later in the course of GVHD [22]. The use of IHC for routine diagnosis of GVHD is limited by the sparseness of the lymphoid infiltrates, additional delay and expense, lack of control data for other inflammatory conditions in the transplant milieu, and variations in the GVHD effector cell immunophenotype. There are limited data comparing the apoptosis marker caspase-3 to hematoxylin and eosin (H&E) stains on both GVHD and relevant non-GVHD inflammatory conditions [23]. The use of IHC with lymphoid, myeloid or EBV markers is indicated when deciding whether a post-transplant infiltrate is a recurrent hematologic malignancy or an EBV PTLD.

Graft-versus-host disease Pathogenetic studies

Surgical pathology of acute GVHD

A discussion of the pathogenesis and pathology of GVHD is detailed in several chapters of this edition and illustrated in several books devoted to this topic [16,17]. Pathologic studies have contributed to this body of knowledge by elucidating the selective epithelial targets in the afferent stage and cellular response in the efferent stages. In the afferent stage, cytokines released by tissue damage from conditioning upregulate the expression of minor or major histoincompatibilities on targeted cells which are presented to donor T cells. Preferential target cells reside in subregions of epithelium where cytokeratin 15 (K15) epithelial stem cells or their early progeny are located (e.g. the parafollicular bulge of the hair follicle and rete ridges of skin, and the laterobasal and neck regions in the crypt cells of gut) (Plates 27.18–27.20). In an experimental system using murine lingual epithelium, which resembles human cutaneous rete ridges, it has been shown that the genes that regulate apoptotic vulnerability are differentially expressed in a basal layer subpopulation distinguishable by K15 [18]. The classical concept for the efferent stage in GVHD is that donor cytotoxic lymphocytes attack host cells, including some epithelial cells of skin, intrahepatic bile ducts, and gut. Electron microscopic and immunohistologic data demonstrate lymphocyte epithelial attachments and activation of infiltrating cytotoxic lymphocytes in human biopsy material. Ultrastructural studies of human rectal biopsies of GVHD demonstrate cell-mediated cytotoxicity with close contact to targeted enterocytes. Additional nonlymphoid cells of the innate immune system also play a role in the effector stage of GVHD, as two French studies have demonstrated that activated eosinophils [19] and apoptotic granulocytes [20] (along with tumor necrosis factor-alpha [TNF-α] and Fas) are found in the lamina propria in gastrointestinal GVHD.

Tissue biopsies are used as a primary biomarker for establishing the diagnosis of GVHD, for assessment of activity to monitor therapy, and as a tool for prognosis. Preferably, the pathologists should be familiar with the changing histologic spectrum of GVHD [24,25], and have an awareness of relevant clinical data and questions surrounding a biopsy. Protocols for guiding histologic evaluation, and clinical information relevant for interpretation from skin, liver, gut, and mucosal biopsy sites, are available online at the American Society of Blood and Marrow Transplantation (http://www.asbmt.org/cGvHD_Guidelines.htm). The classification and criteria for the clinical and histologic diagnosis and staging of acute and chronic GVHD have undergone a number of modifications based upon the consensus of expert panels supplemented by evaluation of the available peer reviewed literature that was codified at the National Institutes of Health (NIH) consensus conference in 2005 [25,26]. This classification and the time of onset of acute GVHD have a bearing on the choice and duration of treatment for GVHD and the outcome [27,28]. In the current milieu, the artificial separation of acute from chronic GVHD based on the time of onset before or after day 100 is no longer tenable. Recipients of reduced-intensity conditioning regimens with a late taper of immunosuppression, and recipients of donor lymphocyte infusions at various intervals post HCT, may present with a clinical picture of acute GVHD several months after transplantation. Some patients may present with chronic features within 50–60 days post HCT. Lastly, there can be an overlap syndrome with acute and chronic features [24,26]. In the liver and gut (excluding cicatricial webs in the upper esophagus), there is no clear histologic demarcation between acute and chronic GVHD. The NIH pathology consensus committee’s minimal histologic criteria for GVHD by organ system are reproduced in Table 27.1 [25]. These guidelines may vary slightly within different institutions and could change with prospective clinicopathologic studies. While the basic pathology of GVHD is apoptosis and eventually destruction of selective targeted epithelia with or without fibrosis, there are a number of factors that can influence or cause difficulty in interpretation. These include residual toxicity from conditioning, immunosuppressive treatment that blunts inflammation, a key indicator of activity, co-existent infections, and drug reactions that can mimic GVHD. The timing of the biopsies,

Immunohistology In general, T-cell infiltrates, predominately CD8, have been found in IHC studies of skin biopsies. Those few studies that have studied blood and tissue ratios of T-cell subsets simultaneously have found parallel CD4:CD8 ratios. Later studies of GVHD in the skin and lip have shown that the infiltrates are cytotoxic T cells as defined by CD8, the cytotoxic marker TIA-1, and perforin. Similarly, DR-expression in the epithelial

Pathology of Hematopoietic Cell Transplantation

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Table 27.1 Histologic criteria for GVHD by organ system Organ or System

Minimal Criteria for Active GVHD*

Specific Criteria for Chronic GVHD†

Skin, any stage

Apoptoses in epidermal basal layer or lower malphigian layer or outer root sheath of hair follicle or acrosyringium ± lichenoid inflammation ± vacuolar change ± lymphocytic satellitosis

Skin lichen planus-like

Skin sclerotic Skin morpheic

Skin fasciitis Liver Gastrointestinal

Oral mucosa and conjunctiva Minor salivary or lacrimal gland

Global assessment of dysmorphic or destroyed small bile ducts ± cholestasis, lobular and/or portal inflammation Variable apoptotic criteria (≥ l/piece) in crypts

Combination of epidermal orthorkeratosis, hypergranulosis, and acanthosis with lichenoid changes ± syringitis of eccrine units ± panniculitis Collagenous deposition with thickening throughout the papillary dermis, or pan-dermal collagenosis ± panniculitis Clinically focal or localized lesion predominated by sclerosis in the lower reticular dermis or along the dermal-hypodermal border ± epidermal and appendigeal involvement Fibrous thickening of fascial septa with adjacent inflammation ± panniculitis Ductopenia, portal fibrosis, and chronic cholestasis reflect chronicity but are not specific for chronic GVHD Destruction of glands, ulceration, or submucosal fibrosis reflect long-standing disease but are not specific for chronic GVHD

Lymphocytic infiltration of mucosa with variable apoptosis‡

Lung

Infiltration and damaged intralobular ducts, fibroplasia in periductal stroma, and inflammation with destruction of acinar tissue§ Obliterative bronchiolitis: dense eosinophilic scarring beneath the respiratory epithelium, resulting in complete fibrous obliteration or some degree of luminal narrowing¶

* Conditions that result in lesser degrees of change include immunosuppressive treatment, biopsy very soon after the onset of signs, suboptimal or small tissue sample, insufficient scrial sectioning, confounding infection, drug reaction, or inflammatory conditions. † Once the diagnosis of chronic GVHD has been established or after immunosuppressive treatment, the histologic manifestations of active disease may meet only minimal diagnostic criteria for activity. ‡ Inflammation of the oral mucosa and within the minor salivary glands may persist from prior chemoirradiation or prior inflammation. The distinction between acute and chronic GVHD requires the addition of distinctive oral manifestations [9]. § The distinction of past acinar destruction and fibrosis from ongoing chronic GVHD activity can be difficult and relies on assessing lobules that are not completely fibrotic. Fibroplasia, with acinar and periductal inflammation and features of damage to ducts, such as vacuolar change, lymphocytic exocytosis, nuclear dropout, dyspolarity, or apoptosis, indicates chronic GVHD activity. ¶ Obliterative bronchiolitis [48] should be distinguished from bronchiolitis obliterans–organizing pneumonia [49], which is also associated with GVHD but has a different clinicopathologic presentation and a more favorable outcome. Reproduced from [25], with permission.

sampling error (especially with gut biopsies), and technical factors can cause a false-negative histologic assessment. Further, the diagnostic standards for minimal criteria for GVHD are inexact regarding the minimum required number of apoptotic epithelial cells, whether their presence is necessary in mucosal biopsies with inflammation, the number of serial sections needed to exclude a false-negative diagnosis, and the extent of dysmorphic change for a positive diagnosis in salivary and bile ducts. As a result, histologic examination may not always be the gold standard for the diagnosis. In reality, the histologic changes, especially in patients undergoing immunosuppressive treatment for GVHD, are often not pronounced; a biopsy can be interpreted as GVHD even though there may be confounding conditions and even though florid histologic alterations are absent. The pathology report can convey these issues by integrating the histologic findings with the relevant clinical details in a comment and by inserting qualifying phrases, whose implication is understood within that institution, into the final diagnosis (e.g. “possible,” “consistent with,” “favor,” etc.). In the final analysis, the decision

to treat for GVHD is made by the clinician based on a number of factors including the stage of a malignancy, desirability of a graft-versus-tumor effect, clinical stage of the target organ abnormality, rapidity of progression, toxicity of treatment, and histologic findings [28]. Grading of GVHD The initial histologic grading system by Lerner et al. was devised in 1974, with conditions far different from those of today. The scheme had four histologic grades for acute GVHD in the skin, liver, and gut, which attempted to use the chronologic progression of injury as a scale of severity based on the amount of inflammation, apoptosis, and degree of tissue damage [29]. In fact, GVHD histology is a snapshot in time such that mild (grade I) or severe (grades III–IV) changes may primarily be a reflection of timing and duration of activity (stage) and degree of immunosuppressive treatment. The Lerner scheme and other subsequent histologic grading schemata for GVHD were created without first being verified against useful clinical endpoints. Nonetheless, a histologic grade with a high level

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of inflammatory and/or apoptotic activity is highly relevant information that will impact the current treatment decisions [25]. Evidence-based observations indicate that time to recovery is proportionate to the severity (stage) of the destruction of the gut mucosa and/or bile ducts [8,25]. Whereas skin histologic grading schemata have limited independent prognostic value in the current milieu, the stage of cutaneous GVHD, acute or chronic, carries important therapeutic implications. Skin The cutaneous histologic grading system of Lerner et al. has provided a useful framework for discussing the minimal diagnostic criteria and the specificity of the histology for GVHD. Grade I is superficial perivascular dermatitis with epidermal basal cell vacuolization; grade II is an interface dermatitis with scattered apoptotic or dyskeratotic keratinocytes in the basilar or lower spinosum layers sometimes in close contact with the infiltrating lymphoid cells (lymphocytic satellitosis) (Fig. 27.1, Plate 27.19); grade III has extensive apoptosis with reticular degeneration, destruction of the basal layer with nuclear dyspolarity and atypia, and suprabasilar bulla formation (Plates 27.18 and 27.21); and grade IV is represented by ulceration of the epidermis (Plate 27.22) [29]. Distinction of GVHD from a variety of conditions is problematic and not always

Fig. 27.1 Skin, acute graft-versus-host disease, day 30. Marked lichenoid reaction involving the epidermis and acrosyringium with intraepidermal lymphocytes, apoptosis, and destruction of rete ridges. (Hematoxylin and eosin.)

resolved by using grade II change for the minimal diagnostic criteria [24,30–32]. The use and timing of the skin biopsy for diagnosing GVHD has wide institutional variation. At one extreme, some centers avoid using skin biopsies based on studies [30,31] that concluded that acute GVHD is histologically indistinguishable from other causes of rashes, viral infections, drug reactions, engraftment syndrome associated with lymphocyte recovery [33,34], and toxicity from the pretransplant cytoreductive conditioning [30–32]. The conclusion of Kohler et al. [30] that the skin biopsy has limited diagnostic utility cannot be easily corroborated because of differences in institutional practices, the basis for selection of patients for skin biopsy, and the uncertainty of clinically defining GVHD based on jaundice, diarrhea, and the extent of the rash. A second general criticism of these negative studies pertains to the relative prevalence of the putative non-GVHD causes and whether they can be distinguished from GVHD. The syndrome of painful cutaneous acral erythema involving the palms and soles is a distinct clinical entity caused by cytoreductive conditioning (Plate 27.23) [35]. Histologically, this toxic injury to the epithelium cannot be clearly distinguished from GVHD (Plates 27.5 and 27.18). Both may display varying degrees of epidermal keratinocyte atypia, vacuolar damage, dyskeratosis, apoptosis, and interface inflammation [31,32]. Depending on the particular high-dose conditioning regimen used, these authors [31,32] have advocated waiting 20–30 days before using the skin biopsy. The maculopapular rash attributed to engraftment with early lymphocyte recovery consists of scant superficial perivascular lymphocytic inflammation with little or no keratinocyte apoptosis that spontaneously improves without treatment or following withdrawal of current medications [33,34]. Although various viral etiologies, including HHV-6, have been suggested for some of these early rashes, there is little or no clinical or immunohistologic evidence to support this claim. Drug hypersensitivity, especially a rash with an atypical pattern or distribution for GVHD, should be considered a possibility. Tissue eosinophils in the infiltrate are not an indication of a drug eruption as they are often found in GVHD as well [36]. Drug eruption seems an unlikely explanation for most early rashes given the usual patient’s exposure to a multitude of drugs and the resolution of the rash without their discontinuation. There is no simple answer to this conundrum of when to use skin biopsies to diagnose early GVHD. The management of the early skin rashes is influenced by a set of variables including the sex and degree of donor match, whether conditioning is high dose, desirability of graftversus-tumor effect, extent of rash, rapidity of progression, and protocol requirements. In a recent informal survey of several tertiary centers, the experienced clinicians estimated that up to 60% of all patients undergoing myeloablative conditioning for an allogeneic HCT develop an early skin rash, of which they estimate 60% are from GVHD. In a study with a large cohort of 809 consecutive patients, 265 (33%) developed clinical grades II–IV GVHD. Twenty-seven percent of these had an early-onset acute GVHD manifested by day 14, referred to as hyperacute GVHD. Eighty-eight percent of the patients with hyperacute GVHD had severe skin involvement with a higher nonrelapse mortality, especially in recipients of mismatched unrelated or matched unrelated donors [28]. In previous editions, we advocated obtaining serial skin biopsies to aid in the interpretation of early skin rashes: GVHD was considered likely if the changes progressed without treatment or remained grade II or higher. Firoz et al. addressed this issue in a study using a decision analysis model to estimate the value of skin biopsy in the evaluation of a hypothetical case of a morbilliform rash covering 60% of the body surface post allogeneic stem cell transplant [37]. Ten experts provided their estimates of the prevalence of GVHD, the sensitivity and specificity of the skin biopsy, and the potential outcomes from treatment or observation. Only 25% of the experts chose an intervention consistent

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with their estimates of the prevalence, test characteristics (histologic results), and outcome evaluations. In this decision analysis modeling, the best patient outcomes occurred if biopsy was done when the prevalence of GVHD was less than 30% whereas treatment for GVHD was given without biopsy if the prevalence of GVHD was over 30%. When skin biopsies are obtained to monitor GVHD treatment, the cellular infiltrate may be minimal and apoptosis infrequent (Plates 27.21, 27.22, 27.24, and 27.25). It is important to review serial sections looking for apoptosis along the outer root sheath of hair follicles, the basilar layer of the epidermis, and at the tips of rete ridges. An eczematous pattern of spongiosis with abundant lymphocytes and little or no apoptosis suggests a drug reaction rather than GVHD. On histologic grounds alone, a severe drug eruption producing a clinical picture of Stevens– Johnson syndrome or toxic epidermal necrolysis with suprabasilar bullous formation and denudation can be difficult to distinguish from severe GVHD.

Gastrointestinal The initial sites of injury in the gut are focused on the regenerative or stem cell areas (i.e. the neck region of the gastric glands, the basal layer of the esophagus, and the lower lateral basilar portion of the crypts in the small and large bowel) (Plate 27.20). In humans, gut involvement is often diffuse, evinced by the gross and radiographic studies that demonstrate the simultaneous diffuse changes of mucosal edema with effacement of the normal mucosal folds involving the stomach and entire small and large bowel (Plates 27.26–27.28). In several studies of upper endoscopic biopsies, epithelial necrosis and crypt abscesses from the small intestines were always associated with diarrhea and usually with similar histologic changes in the rectal biopsy. On the other hand, some patients with positive gastric biopsies may present with only nausea and vomiting. Endoscopic biopsies are obtained to diagnose GVHD, monitor the response to immunosuppressive therapy, and to identify infections. The location, timing, and frequency of endoscopic biopsies are highly dependent on institutional bias and past experience with complications. In one study, the gastric biopsy provided the highest diagnostic yield even when the predominant symptom was diarrhea [38]. Based solely on histology, a recent study found that the combination of rectal biopsy and gastric biopsy provided the highest diagnostic yield for GVHD [39]. A recent pediatric study advocated first using the less invasive rectal biopsy alone to avoid the procedural risks of upper endoscopy [40]. In general, upper endoscopic biopsies are more difficult to interpret than rectal biopsies because of the disagreement on the minimal diagnostic threshold criteria for apoptosis and the overlap with reactive and inflammatory conditions. There are also differing opinions on whether a biopsy from the duodenum [19], gastric fundus [41] or antrum [38] provides the greatest sensitivity. Of greater importance is that the negative predictive value of an endoscopic biopsy is potentially affected by the endoscopist’s sampling and the number of serial sections examined. In acute GVHD, edema of the intestinal wall is readily apparent to the radiologist who performs imaging tests of the intestine and to the endoscopist who can also see mucosal erythema, friability, and microerosions. Edema and erythema are not often appreciated by the pathologist whose specimens are limited to tissue above the muscularis mucosal (Plates 27.26 and 27.27). Likewise, the diagnosis of GVHD by histology is based on findings that the endoscopist cannot see, and the diagnosis of GVHD by direct inspection of mucosa is based on findings that the pathologist cannot appreciate (Plate 27.28) [42]. Thus the gold standard for gastrointestinal GVHD may not be histology alone [25,38], but a composite of clinical signs and symptoms, endoscopic appearance, histologic findings, and absence of infection by culture and histology. That is, the

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inability of histology to demonstrate typical findings of GVHD does not mean that GVHD is not present in the gastrointestinal tract. Spectrum of gastrointestinal GVHD The sequence of damage in gut GVHD has been conceptualized into a four-tier grading system that is a modification of the original Lerner histologic grading schema [29]. 1 Grade I. Individual cell necrosis in basal and lateral crypts, with sparse lymphocytic infiltrate (Fig. 27.2, Plates 27.27–27.29). The minimal criterion is a single or rare apoptotic crypt cell, referred to as an exploding crypt cell. These alterations may involve only a few scattered crypts and can be missed unless the specimen contains properly oriented biopsies with multiple serial sections. In the grade I lesion, the exploding crypt cell may not have an accompanying lymphocytic infiltrate. Histologic grade I does not mean that the entire gut has the same grade; it is not uncommon for gastric and/or rectal biopsy histology to underestimate the severity of GVHD in the midgut, particularly the ileum. 2 Grade II. Apoptotic crypt abscess, in which the dilated walls of the apoptotically damaged crypt are lined by thin epithelium devoid of mucin, are infiltrated by lymphocytes. The crypt abscesses contain apoptotic debris. A scattering of lymphocytes, neutrophils, and eosinophils may be present in the interstitium, crypt walls, and crypt abscesses [13,17]. They may be present in considerable numbers, especially after withdrawal of immunosuppression in a fully engrafted recipient (Fig. 27.3, Plates 27.20 and 27.29). 3 Grade III. Crypt loss resulting in stretches of mucosa devoid of crypts with some focal ulceration (Plate 27.30) 4 Grade IV. There is mucosal denudation, and widespread ulceration with loss of the surface epithelium and crypts (Fig. 27.4, Plates 27.31

Fig. 27.2 Colonic graft-versus-host disease, grade I. A single crypt displays individual epithelial cell apoptosis (“exploding crypt cell”).

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Fig. 27.3 Colonic graft-versus-host disease, grade III. Destruction of crypts with formation of crypt abscesses containing apoptotic debris.

damage ranging from rare individual exploding crypt cells to widespread apoptosis, both single and confluent within a single crypt (Plate 27.29). In practice, identifying the minimal diagnostic threshold for grade I can require analyzing many serial sections. Focally enhanced gastritis, a focal mixed lymphohistiocytic infiltrate with or without neutrophils surrounding a small group of foveolae or glands without apoptosis, is associated with GVHD but, by itself, is not diagnostic [43]. The cumulative damage in grades II and III GVHD is typically widespread, whereas in grade IV there may be no crypts left to demonstrate the apoptotic destruction of the earlier grades (Fig. 27.4). All four grades may occur in different concurrent samples. In contrast to the colon and small bowel, the stomach rarely develops diffuse grade IV changes. Grade IV gut GVHD is slow to heal and often refractory, even with secondary immunosuppressive treatment or aggressive surgical management of obstruction (Plate 27.31). The inability to re-epithelialize, even with prolonged secondary treatment for gut GVHD, may result from a loss of the mucosal stem cells (Plate 27.32). Serial colonoscopic biopsies, obtained to assess response to treatment for severe GVHD of the lower gut, may show regenerating crypts without apoptosis lying in close proximity to grade II or III changes. A complete or partial resolution may take months. Rarely, refractory gut GVHD may develop collagenous deposits within the lamina propria, submucosa, and serosa, or a segmental intestinal stenosis with ulceration and fibrosis confined to the mucosa and submucosa (Plates 27.31 and 27.33). Differential diagnosis

Fig. 27.4 Grade IV graft-versus-host disease (GVHD) in a day 48 duodenal biopsy. Mucosal ulceration with loss of villous epithelium and subtotal destruction of crypts. The features that indicate the cause as GVHD are the two small remaining degenerative crypts showing apoptosis and infiltration by lymphocytes and eosinophils. Concurrent biopsies from the stomach and colon had less severe damage: focal grade III and grade II changes, respectively.

and 27.32). This results in an empty lamina propria devoid of surface epithelium and crypts. There may be granulation tissue and scattered areas of hemorrhage. In grades II–IV, the dilated small vessels in the lamina propria are lined by reactive-appearing endothelium and infiltrated by mixed cell populations. Multiple representative samples should be obtained and correlated with the endoscopic locations since areas of mucosal destruction and ulceration can be adjacent to large stretches of regenerative mucosa with little or no apoptotic activity. While the grading system is a useful descriptor, it is not intended to reflect severity since all four grades may be associated with extensive involvement and severe symptoms. Grade I covers a wide spectrum of

There is a lack of expert consensus on the minimal diagnostic criterion for gut GVHD because low levels of apoptosis also occur with various medications, including nonsteroidal inflammatory inhibitors, mycophenylate mofetil (MMF), and proton pump inhibitors [44], and with some infectious and/or reactive causes such as cryptosporidiosis, and phosphate enemas [41]. In general, reactive gastropathy does not produce apoptosis. The differential diagnosis of grade II–IV changes includes cytomegalovirus (CMV) enteritis, which produces a range of change from shallow circular ulcers to an inflammatory pseudotumor mass involving multiple layers of the gut (Plate 27.32). The viral inclusions typically involve the endothelium but may also affect the stromal cells and gut epithelium. There is a longstanding controversy as to whether the presence of CMV enteritis (a well known cause of apoptosis in gut biopsies) precludes a diagnosis of GVHD [25]. Studies have addressed this issue using IHC and in situ hybridization for CMV and caspase-3 IH for GVHD [23,45]. In CMV enteritis, apoptosis was largely confined to the cells with immunoreactive CMV antigens, whereas apoptotic cells were much more numerous in GVHD. These studies concur with the NIH chronic GVHD consensus [25] that multiple apoptotic crypt cells seen on H&E stain away from any CMV-infected cells identified by H&E or IHC indicate the copresence of GVHD. The immunosuppressive MMF can produce colitis with focal ulcerations with marked apoptosis and intense acute and chronic inflammation. Some reports of MMF colitis have noted diffuse gross changes as well. In practice, the distinction of MMF colitis from GVHD is often established by improvement in bloody diarrhea after withdrawal of the drug [46]. There are insufficient data on whether MMF can also produce lesions in the upper gut. Antibiotic related pseudomembranous colitis is related to the toxin produced within the colonic lumen by Clostridium species, especially Clostridium difficile. If severe, it can produce ischemic changes with apoptosis. Pseudomembranous colitis is distinguished from GVHD by the characteristic superficial pseudomembraneous cap of neutrophils, mucin, and necrotic cells with or without bacterial rods and by a positive assay for the Clostridium difficile toxin (Plate 27.34). However, occasional patients can have both GVHD and Clostridium difficile infection.

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Transplant-associated microangiopathy

Autologous GVHD

HCT transplant-associated microangiopathy (TAM) refers to a collection of microthrombotic complications, which includes the hemolytic– uremic syndrome [47] and possibly a thrombotic microangiopathy of the colon [48]. It is unclear whether TAM is a discrete clinical and pathologic entity that is distinct from other well-recognized post-transplant complications. Japanese investigators originally described TAM in 16 patients with steroid-refractory diarrhea and later in seven patients who received low-intensity conditioning with umbilical cord donor transplants. Their findings were ulcers, erosion, and diffuse exfoliation from the terminal ileum to the rectum, accompanied by swelling and myxoid change in the subendothelium of the damaged vessels, microthrombi, and fibrinoid necrosis of small arterioles. Many of their patients had concomitant GVHD and/or CMV enteritis. The Japanese investigators linked the genesis of the colonic lesions with exposure to tacrolimus because some patients had marked improvement after withdrawal of the drug [48]. However, colonic TAM should not be equated with tacrolimus toxicity since several viral infections and severe GVHD can produce a histologic picture with endothelial damage identical to TAM. In future studies, it would be useful to determine whether the histologic picture of TAM is caused by tacrolimus toxicity and whether it can be distinguished from that caused by GVHD, because the therapeutic approach involves either escalating or de-escalating the immunosuppressive regimen.

Thirteen percent of autografted recipients, particularly female patients treated for breast cancer or hematologic malignancy, develop a gastrointestinal syndrome that is both clinically and histologically indistinguishable from GVHD occurring in allogeneic recipients (Plate 27.37) [49]. These lesions may persist for several weeks to months following transplantation and rarely can be quite marked. Fortunately, nearly all have durable responses to one or two courses of prednisone. The genesis of this phenomenon is poorly understood. Possible mechanisms include GVHD manifest from maternal microchimerism, and autoreactive T cells resulting from in vitro depletion of selected T-cell subsets in the hemopoietic autograft.

Esophageal GVHD Like most squamous epithelia, that of the esophagus may be involved with GVHD. The diagnosis of acute GVHD presents considerable diagnostic difficulty since a variety of conditions, including acid peptic reflux and infections, can produce mucosal damage. Unless there is a combination of lichenoid interface changes with apoptosis along the basilar portion of the mucosa, the diagnosis of esophageal GVHD should be made with some qualification. Accurate diagnosis requires special stains and often IHC studies for microorganisms. Bacterial, viral, and fungal cultures should be obtained from lesions endoscopically suggestive of infection (Plates 27.35 and 27.36).

Fig. 27.5 Hepatic graft-versus-host disease, day 53. Despite the minimal inflammation within the portal space, the bile ducts (arrow) are reduced to degenerative cytoplasmic masses with a few remaining hyperchromatic nuclei.

Liver The liver is a major target of allogeneic GVHD [8], and occasionally autologous GVHD as well [50]. Liver involvement is heralded by a gradual rise in bilirubin and alkaline phosphatase and aminotransferases enzyme levels. It typically follows acute cutaneous or intestinal GVHD, but can present as the sole manifestation of GVHD. Hepatic GVHD is no longer classified as acute or chronic [25,26]. The histology is largely unrelated to the period when the GVHD arose, but is rather a reflection of the duration of active liver GVHD, the anti-inflammatory effects of immunosuppression, and the anticholestatic effect of ursodeoxycholic acid. In both the experimental and clinical settings, histopathologic and ultrastructural studies demonstrate that the small interlobular bile ducts are the preferential target of the alloimmune reaction (Fig. 27.5, Plate 27.38). In both controlled experimental studies and coded histopathologic studies, the diagnosis is based on the global assessment of characteristic dysmorphic, interlobular bile duct lesions, and cholestasis that is often marked. The histologic interpretation is directly affected by the quality, size, and timing of the sample. Thin-core needle biopsies may be difficult to interpret due to limited sampling of portal areas or crush artifact. The timing of the liver biopsy influences the histologic finding. When liver biopsies to diagnose GVHD are obtained soon after the onset of liver dysfunction, the characteristic bile duct changes may be absent or affect a minority of portal spaces [8].

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The amount and extent of the inflammatory component in liver GVHD is dependent on the degree of immunosuppressive treatment. The portal infiltrate is typically lymphocytic but may include a minor component of loosely scattered neutrophils, eosinophils, monocytes, and plasma cells. Inflammation is typically mild to scant with therapeutic levels of immunosuppressive agents even with extensive bile duct damage (Fig. 27.5). A clinical presentation of liver GVHD with an acute hepatitic onset may develop much later as the solitary manifestation of GVHD following cessation of immunosuppressive therapy or after donor lymphocyte infusion. The hepatic onset is characterized by marked elevation of aminotransferase enzymes with marked periportal inflammation that includes many plasma cells and a lobular hepatitic component with abundant necroinflammatory activity (pyknotic shrunken hepatocytes and acidophilic bodies) (Plates 27.39 and 27.40). The acidophilic body formation reflects the cytokine induction of the Fas/Fas ligand system with the hepatocytes being innocent bystanders. The characteristic dysmorphic interlobular bile ducts have a withered, shrunken appearance with a collapsed lumen, vacuolated cytoplasm, and irregular outlines. Individual or groups of epithelial cells are flattened and often missing nuclei, creating segments of anucleate eosinophilic cytoplasmic syncytia. Remaining nuclei display atypical features with anisonucleosis, hyperchromasia, and dyspolarity. Lymphocytic ductitis is frequently present in the damaged portions of the biliary epithelium, but apoptosis of bile duct epithelium is infrequent. Despite the similarities of GVHD to liver allograft rejection, endothelialitis of central venules is uncommon. With prolonged liver GVHD, bile duct destruction is best appreciated by stains other than H&E (e.g. cytokeratin 7 or 19, trichrome or periodic acid–Schiff [PAS]) (Plate 27.38). GVHD produces a variety of cholestatic changes including canalicular bile plugs, hepatocyte ballooning, and dropout of hepatocytes in the perivenular zone, along with a scattering of lymphocytes and collections of pigment-laden macrophages (Plate 27.40). Rarely, pseudoground-glass PAS, diastase-resistant hepatocytes develop from the accumulation of abnormal glycogen [51,52]. Frequently, there is some degree of ductular reaction, a proliferation of small ductules along the periphery of the portal space, that is common in inflammatory liver diseases. In addition to being a reparative response to duct injury, the proliferated ductules also appear to become a secondary target of liver GVHD because the destructive lymphocytic ductitis and cytologic changes in the ductules are similar to those in the damaged interlobular bile ducts. A particularly striking form of cholangiolar cholestasis with dilated periportal bile-filled ductules occurs in patients with concomitant intestinal GVHD (Fig. 27.6, Plate 27.41). This resembles cholangitis lenta associated with septicemia and results from the coexisting influence of gut GVHD, showering the liver with endotoxin, which in turn stimulates the production of TNF-α and interleukin-6, a promoter of bile ductule proliferation and cholestasis [53]. A consequence of the ductular reaction is the development of stellate fibrosis, periportal fibrous septae which extend out into the liver acinus, and rarely bridging fibrosis (Fig. 27.6) [8]. Yet, even in prolonged refractory liver GVHD with loss of bile ducts, cirrhosis rarely occurs. The few anecdotal case reports of cirrhosis related to GVHD were tainted by inadequate immunosuppression or a previous high prevalence of hepatitis C virus infection [8]. Differential diagnosis The timing, profile, and rate of rise of liver test abnormalities provide good indicators as to the likely diagnostic possibilities (see Chapter 95). The histologic differential diagnosis of liver GVHD includes drug liver injury. Most of the candidate antimicrobial drugs for causing liver injury do not produce the typical clinical or histologic patterns seen with GVHD. If the clinical situation permits, it is prudent to discontinue the

Fig. 27.6 Hepatic graft-versus-host disease, day 56. There is pronounced hepatocellular and cholangiolar cholestasis with portal-to-portal bridging and parenchymal collapse. Cholangioles have proliferated along the limiting plate.

candidate drug rather than trying to exclude it as a cause by histology. A common finding in patients biopsied after HCT is marked hepatic iron overload within both hepatocytes and Kupffer cells. The iron overload, which results from multiple transfusions and/or increased intestinal iron transport, has been associated with elevated aminotransferase levels [8,54]. Infections, especially viral, are an important diagnostic consideration because failure to treat them can lead to liver failure (see Chapter 95). CMV should not be considered in the work-up of severe hepatitis or jaundice since it results in mild anicteric hepatitis whose principal histologic findings in immunosuppressed patients are microabscesses that contain some neutrophils. The only exception is CMV papillitis of the ampulla of Vater leading to biliary obstruction. The life-threatening adenovirus and herpes group viral infections are discussed in the section on infection, below. If the portal spaces are greatly expanded by an infiltrate rich in plasma cells, the differential diagnosis and work-up should include a PTLD (Plates 27.4a and 27.4b) (see Chapter 56), the hepatitic onset of GVHD [55], and, rarely, autoimmune hepatitis [8]. The development of positive tests and/or immunohistologic stains for hepatitis B and C viruses does not exclude the coexistence of GVHD. It may be difficult to distinguish the bile duct changes in cases of severe reactivation of viral hepatitis B or C from prolonged GVHD with a ductular reaction. Nearly all cirrhosis developing after 10 years post HCT is caused by hepatitis C virus. The use of the liver biopsy in monitoring the therapeutic response and prognosis of patients with GVHD is unsettled. To validate the biopsy as a monitor of therapeutic response will require standardization of the biopsy findings in relation to the duration and type of immunosuppressive therapy through linkage to a large multi-institutional database, the use of uniform histologic criteria, and an adequate sample. Although there is no validated histologic grading system for liver GVHD, evidence-based observations of protracted GVHD indicate time to recovery from jaundice is proportionate to the extent of bile duct destruction. Chronic GVHD Since the earlier editions of this text, advances in HCT practice have caused major changes in the presentation and natural history of acute and chronic GVHD (Table 27.1) [24,26]. The clinical manifestations

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[24,26] and certain unique histologic changes [25] listed in Table 27.1 are the minimal criteria that define acute and chronic GVHD. The new clinical classification identifies an overlap syndrome, which is a composite of both acute and chronic features. The classification also includes acute GVHD that is persistent, recurrent or occurs after day 100 without meeting additional diagnostic criteria for chronic GVHD [56]. Since there is no clear histologic dichotomy between acute and chronic GVHD in the liver or gut, the histologic changes specific for chronic GVHD occur in the biopsies from skin and fascia, salivary and lacrimal glands, muscle, and lung. From the pathologist’s viewpoint, chronic GVHD is a multiorgan inflammatory disorder that resembles a mixture of GVHD and several autoimmune diseases. The importance of screening biopsies at day 80–100 of skin or lip in asymptomatic patients still on immunosuppression is controversial. A positive screening biopsy without diagnostic histologic features of chronic GVHD is no longer considered to be chronic GVHD. Likewise, a positive screening oral biopsy unaccompanied by distinctive gross oral features of chronic GVHD is no longer classified as chronic GVHD [25,26]. Accordingly, there is a wide spectrum of utilization of oral and skin biopsies in the diagnosis and management of late acute or chronic GVHD [57]. Biopsies are recommended to confirm active chronic GVHD when alternative diagnoses are entertained, the clinical signs are confined to internal organs or the clinical assessment of activity is obscured by prior changes. In one tertiary care center, 7% of patients sent there for consultation regarding chronic GVHD had been incorrectly diagnosed and treated for active chronic GVHD before referral [58]. Skin Chronic GVHD of the skin has a polymorphous gross appearance potentially involving all regions, including the nails (Plates 27.42–27.48). A detailed glossary of the gross cutaneous manifestations along with a standardized nomenclature and their likely underlying histopathology may prove useful in correlating appearances with the outcome and response to therapy [24,59]. The pathologist should be mindful of several caveats pertinent to the histopathology of chronic GVHD: 1 The histopathology changes over time. Usually, the predominant features of the early manifestations, referred to as lichenoid GVHD (Fig. 27.7), are lymphoplasmacytic inflammation involving the epithelium and tubuloalveolar glands in the eccrine, lacrimal, and aerodigestive tracts (Fig. 27.8). Unlike acute GVHD, chronic GVHD causes extensive destruction of these tubuloalveolar glands and ducts with corresponding clinical sicca syndromes (Fig. 27.8, Plate 27.49). 2 In later stages, referred to as sclerodermoid GVHD, there may be widespread fibrosis, stenosis, obliteration or atrophy of the involved tissues (Fig. 27.9, Plates 27.50 and 27.51). As a result, it may be difficult to distinguish active disease from residual fibrotic damage (Fig. 27.9, Plate 27.52) in the absence of chronic inflammation or serial biopsies showing progression of the fibrosis. However, the initial presentation of cutaneous chronic GVHD may be primarily with changes associated with the later stages of dyspigmentation and dermal sclerosis [59]. 3 Following a flare of chronic GVHD, the histologic changes may be more closely akin to those of acute GVHD. Lichenoid, sclerodermoid, and acute histologic features can coexist. The initial dermatopathologic descriptions of cutaneous chronic GVHD were of a biphasic disorder, initially resembling a combination of widespread lichen planus and lupus profundus, which later progressed to scleroderma-like gross and histologic changes (Figs 27.7 and 27.9, Plates 27.48 and 27.53). Smaller subsets of patients present with localized morphemic lesions or fasciitis (Plate 27.47) [60]. In the current milieu, one may see various combinations of these changes plus superimposed skin lesions that only satisfy criteria for acute GVHD [24,26,58].

Fig. 27.7 Skin, day 314, with the extensive or generalized type of early chronic graft-versus-host disease (GVHD). The so-called lichen planus-like chronic GVHD refers to the acanthotic and hyperkeratotic epidermis that has inflammatory and destructive changes along the dermal–epidermal junction. An eccrine unit at the base of the dermis (arrow) and a follicle deep in the dermis are also being destroyed. The dermal collagen is unaltered.

Full-thickness skin biopsies are essential to fully assess possible changes in the eccrine units, subcutaneous fat, and fascia, and to delineate the degree of dermal fibrosis (Plate 27.50). The specific criteria for the early lichen planus-like stage are the combination of hyperkeratosis, hypergranulosis, and irregular acanthosis with lichenoid basilar layer changes with or without syringitis (inflammation within eccrine units) or lobular panniculitis (inflammation of the subcutaneous fat) (Plate 27.53) [25]. Serial biopsies demonstrate progression of the fibrosis from the papillary dermis downward, consistent with an etiology related to the ongoing inflammation and release of cytokines (Plates 27.50 and 27.52). In contrast to chronic GVHD, the dermal fibrosis in systemic sclerosis (scleroderma) typically proceeds upward from the base of the dermis. Patients with cutaneous sclerodermatous chronic GVHD, like those with progressive systemic sclerosis, have an autoantibody to the platelet-derived growth factor receptor which stimulates the activation of skin fibroblasts in vitro, suggesting a direct role in the cutaneous sclerosis in both conditions [61]. The remodeling of the dermal collagen progresses from straightening of the dermal-epidermal border with loss of the rete ridges and dermal appendages, along with sclerosis of the papillary and adventitial dermis with variable degrees of fibroblastic stroma. If progressive, the reticular dermis becomes thickened by

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Fig. 27.8 Lip biopsy, minor salivary gland. The lymphoplasmacytic inflammatory cells are centered around small ducts. Signs of ductal epithelial cell destruction, irregularity of outline, nuclear stratification and hyperchromatism, and apoptosis (arrows) are similar to graft-versus-host disease changes in small bile ducts. The glandular interstitium is becoming fibrotic and the acini atrophic, resulting in a sicca syndrome.

Fig. 27.10 Lip biopsy, day 376. Fibrotic and destroyed minor salivary gland contains only ectatic ducts. These changes reflect previous damage from chronic graft-versus-host disease (GVHD). Unless they are accompanied by some inflammatory component in other glands or in the mucosa, they should not be taken as a sign of active chronic GVHD.

smudgy collagen bundles. Eventually, the dermal subcutaneous border becomes straightened, and fibrotic septae may form in the subcutaneous fat (Fig. 27.9, Plate 27.50). In patients receiving immunosuppressive treatment, the inflammatory changes may be minimal with evidence of active GVHD limited to epithelial vacuolar degeneration or apoptosis in the basilar layers of the skin and its appendages, oral mucosa or minor salivary glands of the lip. Morpheic GVHD presents as localized dyspigmented macules with variable degrees of induration. A full-thickness biopsy demonstrates nodular fibrous remodeling in the deeper reticular dermis with variable inflammation in the dermis and adjacent subcutaneous fat. The epidermis may have minimal apoptosis or vacuolar changes. Eosinophilic fasciitis of the skin presents with an acute painful deep groove or a rippling cellulite appearance. A deep incisional biopsy shows edema and fibrosis of the fascia and subcutaneous septae with a mixed infiltrate of lymphocytes, histiocytes, and eosinophils [25,60]. A lichen sclerosis atrophicus type of chronic GVHD leads to scarring and stricture when it affects the female genitalia. Histologically, there is epidermal or mucosal atrophy with basal vacuolar changes, a band-like infiltrate associated with small numbers of apoptotic cells, and a subepidermal zone of pale homogenized collagen. Several additional forms of cutaneous chronic GVHD include eruptive angiomas, vitiligo, hyperpigmentation, destructive nail dystrophies, and ichthyosiform change (Plates 27.46 and 27.48) [59]. Oral

Fig. 27.9 Late sclerodermatous chronic graft-versus-host disease (GVHD), day 910. The epidermis is atrophic. The dermal collagen is diffusely sclerotic with homogenization of the collagen, and the dermis–subcutis border is straightened. The dermis below the entrapped eccrine unit (arrow) represents acquired fibrous tissue resulting from chronic GVHD.

Oral chronic GVHD has mucosal changes similar to the skin with lichenoid interface inflammation, exocytosis, and apoptosis. The assessment of GVHD in minor salivary glands should only focus on lobules that are not completely fibrotic. Active GVHD changes include lymphocytic exocytosis into intralobular ducts and acini, and lymphocytic periductal inflammation with or without plasma cells (Fig. 27.8, Plate 27.49). Fibroblastic periductal and periacinar stroma is indicative of chronic GVHD activity, whereas dense fibrosis indicates only previous damage (Fig. 27.10). Because of residual nonallogeneic baseline inflammatory changes from high-dose conditioning [62], there are institutional variations in how the threshold for minimal activity is defined. One such proposal with more stringent criteria requires more than three apoptotic cells in the mucosa and a greater than 10% loss of acinar tissue or ductal

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epithelial necrosis [63]. The same criteria for oral biopsies also apply to the esophageal, vulvar, conjunctival, and lacrimal biopsies. Other sites involved in chronic GVHD Several infrequent manifestations associated with chronic GVHD resemble autoimmune disorders, including vitiligo, myositis (Plate 27.54), serositis, arthritis, synovitis, myasthenia gravis, and bullous pemphigoid. Rackley et al. published a series of 12 patients with a literature review of cardiac manifestations. Striking findings include an obliterative coronary artery vasculopathy resembling that of cardiac allograft rejection (Plate 27.55) and myocarditis, associated with bradycardia and, in some instances, sudden death [64]. A nephrotic syndrome is associated with membranous nephropathy and minimal change podocytopathy [65]. Kamble et al. reported on two cases of vasculitic neuropathy with a review of the literature of central nervous system lesions associated with chronic GVHD [66]. The etiologies of these changes have not been resolved. After thorough effort to eliminate an infectious component, the etiology could include some combination of late toxicity, alloreactivity, and immune dysregulation (autoimmunity).

Fig. 27.11 Lung, cytomegalovirus (CMV) pneumonia, day 68. Several large cells contain Cowdry type A nuclear inclusions (large arrows) as well as six to 12 smaller cytoplasmic inclusions (small arrows) diagnostic for CMV infection. (Hematoxylin and eosin.)

Infection Infection is a frequent complication of HCT and must be evaluated promptly and extensively in tissue and cytology preparations. The pathologist must be prepared to identify unexpected microorganisms, not just those suggested on the basis of nonspecific clinical findings. For example, although a nodular pulmonary infiltrate may indeed be the result of fungal infection, other treatable causes include nocardiosis, legionellosis, toxoplasmosis (Plate 27.56), and CMV, as well as other viral pneumonias. It is obvious, but bears repeating, that identifying microorganisms requires looking specifically for them. This must be done quickly in immunocompromised patients if effective therapeutic drug levels are to be established. Since pneumonia can progress with devastating speed in HCT patients, we immediately process, stain, and study lung tissue and lavaged cells at any hour of any day. The preliminary stains include Wright–Giemsa, Papanicolau, H&E, methenamine silver, PAS, modified Gimenez for Legionella, and Kinyoun for mycobacteria. Fite and Warthin-Starry stains as well as a large number of IHC antibodies and in situ hybridization probes for organisms are used as needed. The pathologist does a disservice to the patient, as well as to clinicians, by studying only H&E and silver-stained sections and reporting that there is no evidence of infection. Allograft recipients who received high-dose conditioning and have GVHD are at significant risk for reactivation of latent viruses, especially members of the herpesvirus group and adenovirus [67–69]. We now rarely see CMV or herpes simplex in bronchoalveolar lavages or tissue biopsies because seropositive patients receive prophylactic antiviral therapy, and patients who develop antigenemia or PCR positivity, are treated pre-emptively. However, treatment does not eradicate these viruses, so late infection may develop unnoticed after treatment is stopped. The incidence of late CMV pneumonia (Fig. 27.11) occurring at a median of approximately 24 weeks post transplant is increasing [70]. HCT recipients are at high risk for acquiring potentially fatal respiratory virus infections when the community prevalence of agents such as respiratory syncytial virus (Plate 27.57) is high. Occasionally, viral infection of a single organ will produce an ambiguous clinical presentation best resolved by prompt biopsy and rapid tissue evaluation. For example, severe abdominal pain may suggest impending bowel infarction from gut GVHD but may actually represent acute herpes simplex virus, varicella zoster virus (Plate 27.58) or adenovirus hepatitis (Plate 27.59), which can be diagnosed within a few hours

Fig. 27.12 Kidney, adenovirus (ADV) nephritis, day 77. Tubules lined by degenerating epithelial cells are strongly reactive for ADV antigen by indirect immunoperoxidase staining using a monoclonal antibody to the adenovirus hexon protein (Chemicon International, Inc., Temecula, CA, USA). ADV species 11 was isolated in culture. Possible ADV nephritis should be considered in patients with hematuria and positive urine culture [71].

by histologic studies of a transjugular liver biopsy. Similarly, hematuria accompanied by costovertebral angle tenderness may be secondary to adenovirus nephritis (Fig. 27.12), which can be diagnosed by renal biopsy [71]. Invasive mold infection develops in approximately 15% of patients undergoing either high-dose or reduced-intensity regimens. The most frequent cause is Aspergillus, followed by zygomycete and Fusarium species (Plate 27.60) [72]. The histologic demonstration of fungi in tissue is often crucial to the diagnosis and prompt treatment of invasive infection since culture isolation is slow and insensitive, fungemia is sporadic, and assay results for antigenemia are often inconclusive. Toxoplasma gondii (Plate 27.56), Pneumocystis jirovecii, mycobacteria, Nocardia, and Legionella are also organisms that can be identified

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rapidly by histologic and immunocytochemical staining and then specifically treated [73–75]. Although these organisms are unusual in most patient populations, their rapid identification can save lives. The incidence of toxoplasmosis over a 20-year period in Seattle was 0.3% [74], of nocardiosis over 25 years was 0.2% [75], and of legionellosis over 12 years was 0.6% [73].

Pulmonary complications Pulmonary complications continue to develop in HCT patients and are most frequent in those receiving allogeneic hematopoietic cells. In addition to the major role of infection already described, other mechanisms of injury may include drug toxicity, irradiation damage, GVHD, and malignant infiltration. The resulting histologic alterations are often nonspecific and may include diffuse alveolar damage, alveolar hemorrhage [76], interstitial and alveolar inflammation, alveolar proteinosis, and pulmonary venous occlusion (Plate 27.61) [77,78]. Several of these may coexist in the same patient and interact to cause significant tissue damage and symptoms. Diagnostic evaluation by the pathologist is closely coordinated with the clinical care team. Consideration is given to the underlying major disease and its treatment, possible exposure to infection or toxic damage, prior pulmonary complications, pretransplant conditioning regimen, post-transplant interval, degree of immunosuppression, whether GVHD is present, results of surveillance cultures and molecular tests for systemic infection, evolution of signs and symptoms, and results of imaging studies as well as pulmonary function tests. Typically, the first involvement for the pathologist is the immediate analysis of bronchoalveolar lavage cytospin preparations for possible infection (see the section on infection) or malignancy. Either negative or positive findings for these two complications will often allow clinicians to focus and thereby simplify therapy. Because the diagnostic yield for bronchoalveolar lavage is low, tissue is often needed to analyze histologic patterns and again carry out detailed morphologic, microbiologic, and molecular studies for infection. Transbronchial biopsies can usually be obtained safely and may fortuitously demonstrate infection, but they provide too little tissue to confidently interpret and categorize the histologic changes. Ideally, the patient is sufficiently stable to undergo video-assisted thoracoscopic surgery. This provides tissue which the pathologist can immediately dissect under sterile conditions so that focal lesions can be cultured for bacteria, fungi and viruses, imprints and postoperative frozen sections can be studied using special stains (see the section on infection), and the most informative areas can be submitted for fixation and permanent sections. Historically, pulmonary complications have developed in one-quarter to one-half of allograft recipients and have frequently been fatal. Improvements in antimicrobial prophylaxis and therapy have dramatically decreased the incidence of viral pneumonias, although these can still develop through reactivation of latent infection as well as through community exposure. A recently described paramyxovirus, human metapneumovirus, infects a significant proportion of transplant patients and can cause fatal pneumonia [79]. The exact nature and extent of its role in pneumonias previously classified as idiopathic are under study. Cytopathic tissue alterations diagnostic for human metapneumovirus have not been described, so antibodies for IHC need to be developed.

Interstitial pneumonia syndrome Approximately 5–15% of allogeneic HCT recipients develop the interstitial pneumonia syndrome (IPS) in which there are multilobar infiltrates by X-ray or scan, symptoms and signs of pneumonia with evidence

of abnormal physiology, and absence of active lower respiratory tract infection [80,81]. IPS is a generally severe and often fatal acute process, in contrast to the insidious chronic course of disorders such as usual interstitial pneumonia of Liebow. The histopathology of IPS is variable in pattern as well as severity and may include diffuse alveolar damage with hyaline membranes, interstitial lymphocytic infiltration, increased alveolar macrophages, and pulmonary hemorrhage. These cases probably have a multifactorial etiology, which may include cytokine-induced damage, toxicity from therapy, cell-mediated injury, and occult infection [82,83]. The decreased incidence of IPS after reduced-intensity regimens strongly suggests that conditioning therapy can play a crucial role [84]. Alkylating agents such as cyclophosphamide and busulfan have long been implicated in pulmonary toxicity. There is now increasing evidence that high-dose melphalan [85], as well as tyrosine kinase inhibitors such as dasatinib [86], may also cause lung damage. Acute GVHD is a risk factor for IPS [80,87]. However, IPS has not been firmly linked to an allogeneic reaction in humans, although there is evidence in mice that nonbacterial pneumonia is a combined effect of GVHD and radiation [88].

Pulmonary GVHD The lung is definitely a GVHD target organ. Bronchiolar epithelium is evidently the major target tissue, although cellular damage is often difficult to identify in the epithelial monolayer. There is no well-defined histologic pattern for acute pulmonary GVHD. Alloreactivity may contribute to the nonspecific features of damage and inflammation which characterize the IPS. The significant association of idiopathic bronchiolitis obliterans organizing pneumonia (also designated cryptogenic organizing pneumonia) with acute skin GVHD as well as chronic GVHD of the gut and oral cavity in a large, case-control study [89] indicates that this bronchiolocentric injury pattern with polypoid masses of granulation tissue obstructing the small airways (Fig. 27.13) could represent a transition between acute and chronic phases. In the 49 patients with bronchiolitis obliterans organizing pneumonia, day 108 was the median day of occurrence. It is well established that chronic pulmonary GVH activity is a major contributor to bronchiolar inflammation culminating in bronchiolitis obliterans. This relationship is supported by published reports of many patients with bronchiolitis obliterans (Fig. 27.14) and chronic GVHD [90], the strong association of airflow obstruction with chronic GVHD, and the symptomatic response to immunosuppressive therapy [82,83]. Because the clinical syndrome of obstructive pulmonary function in a GVHD patient with clear chest imaging studies is distinctive, a strong presumptive diagnosis of pulmonary involvement is usually established without obtaining a biopsy. However, lack of biopsy tissue has limited our understanding of this complication. In some of the few available biopsies, there has been evidence of differing stages of bronchiolar inflammation and fibrosis. In some bronchioles, lymphocytes infiltrate the mucosa and are associated with epithelial injury (Plate 27.62). Other airways show a presumably later stage of lymphocytic bronchiolitis in which there is constrictive narrowing of the lumen (Fig. 27.14) along with a few examples of complete fibrous effacement of the lumen (Plate 27.63). Stains for elastin such as the Verhoeff–Van Gieson are helpful in demonstrating the scarred bronchioles. An intriguing study of the expression of 15 genes in the innate immunity pathway suggests that a loss-of-function mutation in the bactericidal/permeability-increasing gene significantly increases the risk of developing airflow obstruction in human HCT patients [91]. Bronchiolitis obliterans is strongly associated with lung allograft rejection in both humans and animals [92,93], indicating that not only the morphology, but also the mechanisms of the two

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Fig. 27.14 Bronchiolitis obliterans with constrictive subepithelial fibrosis at 5 years post allograft transplantation. The patient had developed obstructive pulmonary dysfunction. This biopsy confirmed that chronic pulmonary graftversus-host disease had developed following a history of skin and gastrointestinal involvement. The arrows indicate smooth muscle bundles. There is also subintimal thickening of the accompanying artery.

Summary

Fig. 27.13 Lung, idiopathic bronchiolitis obliterans organizing pneumonia (BOOP; cryptogenic organizing pneumonia, COP), day 120 following marrow allograft transplantation. The patient developed dyspnea and a cough following tapering of corticosteroid therapy for chronic graft-versus-host disease (GVHD). A chest X-ray showed multifocal alveolar opacities clinically suggestive of fungal or viral pneumonia. The biopsy demonstrated areas of consolidation in which bronchioles were blocked by granulation tissue (arrow) which also extended into nearby alveoli and was associated with interstitial and alveolar infiltration by mononuclear inflammatory cells. In HCT recipients, BOOP is associated with GVHD in the skin, oral mucosa, and gut [88].

processes may be similar. There is vascular inflammatory involvement in lung rejection which may also be present in GVHD. Secondary infection may play a major role in chronic GVHD because patients have important deficiencies of immunoglobulins [94].

Among the future challenges in the pathobiology of GVHD is understanding why and how stem cells are targeted. The immunohistologyguided laser capture of the CK15 basal stem cells, coupled with real-time reverse transcription PCR, has demonstrated the heterogeneity of the basal cells regarding their pro- or antiapoptotic gene expression [18]. Future studies can be directed at events that influence this expression. Immunofluorescence studies on formalin-fixed tissues allow identification of the lymphoid subclasses in tissues and their composition in GVHD lesions. The role of the endothelium as a possible target of GVHD [95,96] and the microvascular changes associated with cutaneous sclerosis have not been well studied in clinical material. Multiparameter flow cytometry coupled with sorting for specific subpopulations of lymphocytes and dendritic cells aids in unraveling the complex immunobiology of GVHD. The influence of genes that are involved in the innate immunity pathway on gut and pulmonary GVHD and infection may provide intriguing clues into susceptibility to these complications [91]. Lastly, improved utilization of biopsies in the management of GVHD should be aided by the standardization of sampling and histologic reporting systems coupled with linkage to a database which includes the type and duration of treatment given before the biopsy.

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after nonmyeloablative and conventional conditioning regimens for allogeneic hematopoietic stem cell transplantation. Blood 2003; 102: 2777–85. Akasheh MS, Freytes CO, Vesole DH. Melphalanassociated pulmonary toxicity following high-dose therapy with autologous hematopoietic stem cell transplantation. Bone Marrow Transplant 2000; 26: 1107–9. Bergeron A, Rea D, Levy V et al. Lung abnormalities following dasatinib treatment for chronic myeloid leukemia: a case series. Am J Respir Crit Care Med 2007; 176: 814–18. Crawford SW, Longton G, Storb R. Acute graftversus-host disease and the risks for idiopathic pneumonia after marrow transplantation for severe aplastic anemia. Bone Marrow Transplant 1993; 12: 225–31. Lehnert S, Rybka WB, Seemayer TA. Amplification of the graft-versus-host reaction by partial body irradiation. Transplantation 1986; 41: 675–9. Freudenberger TD, Madtes DK, Curtis JR, Cummings P, Storer BE, Hackman RC. Association between acute and chronic graft-versus-host disease and bronchiolitis obliterans organizing pneumonia in recipients of hematopoietic stem cell transplants. Blood 2003; 102: 3822–8. Yoshihara S, Yanik G, Cooke KR, Mineishi S. Bronchiolitis obliterans syndrome (BOS), bronchiolitis obliterans organizing pneumonia (BOOP), and other late-onset noninfectious pulmonary complications following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2007; 13: 749–59. Chien JW, Zhao LP, Hansen JA, Fan WH, Parimon T, Clark JG. Genetic variation in bactericidal/permeability-increasing protein influences the risk of developing rapid airflow decline after hematopoietic cell transplantation. Blood 2006; 107: 2200– 7. Tazelaar HD, Prop J, Nieuwenhuis P, Billingham ME, Wildevuur CR. Airway pathology in the transplanted rat lung. Transplantation 1988; 45: 864– 9. Yousem SA, Burke CM, Billingham ME. Pathologic pulmonary alterations in long-term human heart–lung transplantation. Hum Pathol 1985; 16: 911–23. Sullivan KM. Intravenous immune globulin prophylaxis in recipients of a marrow transplant. J Allergy Clin Immunol 1989; 84(4 Pt 2): 632–8; discussion 638–9. Biedermann BC, Sahner S, Gregor M et al. Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease. Lancet 2002; 359: 2078–83. Ertault-Daneshpouy M, Leboeuf C, Lemann M et al. Pericapillary hemorrhage as criterion of severe human digestive graft-versus-host disease. Blood 2004; 103: 4681–4.

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Joyce C. Niland & Paul Frankel

Biostatistical Methods in Hematopoietic Cell Transplantation

Introduction With the tremendous strides and newly emerging science within the field of hematopoietic cell transplantation (HCT), advances in study design, biostatistical methodology, and biomedical informatics are required to keep pace. Clinical trial design and analysis methodology have been evolving over the past decade [1], providing the additional tools needed to obtain valid results and advances in evidence-based medicine based on HCT studies. This chapter provides an overview of the study cycle for new treatment discoveries, from observational studies through the phases of clinical trials as applied to HCT. The principles for designing such studies and the fundamentals regarding study conduct and monitoring are described. Subtleties and potential pitfalls of various study designs and analytic approaches are described, along with the requirements for full and complete reporting of study results. Special issues that must be considered during the conduct of human clinical trials are discussed, including appropriate monitoring through independent data and safety boards, along with international standards for the conduct and reporting of human research. With the post-genome era of biologic research underway, the clinical trial database, design, conduct, and analytic tools required to manage the huge amounts of genomic data must be considered. In addition, new methods report on dozens of immunologic parameters that are particularly relevant for HCT. Our greatest challenge will be understanding the meaning of these data, and applying this new understanding to develop targeted interventions and approaches to treatment. Because of the farreaching impact of genomics and related large-scale data on clinical research, for each section of this chapter the potential influence of these data on the study cycle is discussed. The need for evolving advanced biomedical informatics tools to support study design, data management, and analysis of these data is addressed as well. Throughout the chapter, formulas and calculations are avoided in favor of emphasizing the underlying concepts and theories of biostatistical methodology. It is hoped that this approach will provide physicians, fellows, medical students, and health-care professionals involved in HCT research with a basic understanding of, and guide to, appropriate approaches in study design, conduct, statistical analysis, and data computerization. Frequent references to related biostatistical/clinical trials articles and textbooks are included to promote better understanding. For

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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those seeking a more detailed treatment of the history, rationale, and methodology underlying clinical trials, the texts by Meinert and Tonascia [2], Pocock [3], and Friedman et al. [4] provide these underpinnings. For a comprehensive overview of clinical trial informatics applied to the field of oncology, the reader is referred to the text by Silva et al. [5].

The study cycle Figure 28.1 provides an overview of the typical study cycle for new treatment discoveries. While rational drug design through targeted efforts is becoming more common, it is still the case that often initial insights into the potential value of certain therapeutic approaches are gleaned through observational studies. Preclinical (animal and laboratory) studies also play a pivotal role in research hypotheses and in preparation for clinical trials. Often, both observational and preclinical investigations are conducted prior to launching experimentation via clinical trials, that is, planned interventional studies intended to ethically lead to an evaluation of the experimental therapy in people. The final phase of the study cycle is to observe the use of “successful” treatments stemming from the clinical trial results, now offered to the entire patient population, where extended follow-up of a larger patient population may demonstrate unintended and unforeseen consequences of therapy. The application of evidence-based medical approaches to the general population may lead to observational studies that cycle back into the next phase of research to advance medical discoveries. Often, disparate or diminished results will be observed when new treatments are first applied as the standard of care, as the careful selection process applied during clinical trial conduct is no longer in effect, and the patients being treated post clinical trials represent the gamut of stages, phases, and demographics. This field of study, known as outcomes research, will not be covered in this chapter, as this topic is discussed in detail in Chapter 32. The following sections focus on two of the key designs for observational studies, a frequent starting point of the study cycle.

Observational study designs Table 28.1, adapted from Green and Byar [6], suggests that there is a hierarchy of study designs, from the least convincing (anecdotal case reports) to the most convincing (the confirmed randomized control clinical trial). The key differential between observational studies and clinical trials is that the former do not involve any type of intervention with the subjects under study, while clinical trials by definition are experimental in nature and involve an investigator-controlled interven-

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Biological hypothesis Hematopoiesis RBC

Observational studies

BFU-E

CFU-E

Monocytes Macrophage

CFU-M

Sel f Renewal

Eosino phils Basoph ils

CFU-G M CFU-G

HSC

Neutrophils

LTCIC CFU-GEMM

Platele ts CFU-Mega

Megakaryocyte T Cell

Thymus Lymphoid Pro genitor ?

Outcomes research

Stem Cells CD34+/Lin -

Progenitor Cells CD34+/Lin +

B Cell

Bon e

Mature Blood Cells CD34-/Lin+

Animal studies

Fig. 28.1 The study cycle of new treatment discoveries.

Table 28.1 Hierarchy of strength of evidence concerning efficacy of treatment 1. 2. 3. 4. 5. 6. 7. 8.

Confirmed randomized controlled clinical trial Single randomized controlled clinical trial Case series with historical controls Retrospective case-control study Analyses of existing databases Series with literature controls Case series without controls Anecdotal case reports

Adapted with permission from Green and Byar [6].

tion. Much can be learned from conducting well-designed observational studies, and they may serve as a valuable precursor to the next novel treatment approach to be tested in clinical trials, particularly at the point when a randomized trial in HCT is not ethical or practical. While true experiments (i.e. clinical trials) allow an investigator to establish causal associations more conclusively, observational studies can provide major contributions to the understanding of disease processes. In general, observational studies examine the association between the outcome of interest (the “dependent” variable) and the patient’s background, treatment or pretreatment exposures, genetics, laboratory value or health-related behavior (the antecedent or “independent” variable). One difficulty with observational studies is that patients usually differ naturally in other characteristics beyond the specific factors under study. For example, people in various occupations differ not only with respect to their exposures to occupational hazards, but also with respect to their prior and current lifestyles. Such differences may come about by one or more mechanism such as self-selection, education level, fitness for a particular occupation or hiring practices. These potential influences on the outcome under study are known as “confounding factors.” While a robust study design will anticipate and measure confounding factors, many such factors are difficult, costly or impossible to measure fully. In this instance, the role of the primary factor under investigation is more difficult to demonstrate, and the conclusions will require caveats. In addition to such confounders, observational studies that examine

Clinical trials

survival endpoints can be misleading if follow-up is not standardized. For example, if standard follow-up is planned for 5 years, and passive follow-up occurs subsequently, the survival estimates will be unreliable, as passive follow-up gathers more information on the more at-risk patients. If HCT patients are compared with nontransplant patients, and follow-up is not uniform, this will also impact the validity of any conclusions. Researchers should recognize that the statistical analysis of observational studies can often be more difficult than prospective clinical trials. When observational studies are designed and analyzed carefully, one may be able to control some of the biases that are inherent in patient selection or outcome assessment. Two major types of design approach for observational studies, prospective and retrospective, can be applied in the area of HCT research. Each approach has attendant features, analytic methods, and advantages or disadvantages, as described in the subsequent sections. Prospective cohort studies In a prospective cohort design, groups or “cohorts” of subjects are selected based on an observed common exposure to some naturally occurring factor. For example, patients who have a matched sibling donor and receive an allogeneic HCT may be compared with patients with a similar disease status for whom no donor is available. Even through such a natural group assignment process there could be inherent selection bias between the two groups. As an example, donor availability is likely to correlate with family size, which in turn may correlate with the educational and economic levels of the study subjects. The decision to take a patient to transplant is not dictated solely by the availability of the donor; for example, a physician could withhold HCT because of the worsened clinical condition of a patient. Matching on as many potentially confounding factors as possible will help to reduce some of the bias. (However, care must then be taken to take the matching into account when analyzing the data via the appropriate statistical techniques.) There is also potential bias related to the length of time that a patient waits for an allogeneic HCT procedure. Transplant recipients may be at lower risk than all patients intended to go to HCT, as the recipients have not only responded to induction chemotherapy, but also survived relapse

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free for long enough to identify an appropriate donor. This “waiting time” to transplant causes considerable bias if comparing with nontransplant patients as the transplant group excludes those who would have undergone HCT had they survived long enough. To properly analyze data of this nature, patients should be followed from diagnosis, and in some settings it may prove useful to employ time-varying covariates in the model, via techniques such as the one described by Mantel and Byar [7]. An advantage of the prospective cohort design is that it permits the calculation of incidence rates among those “exposed” and those “not exposed” to the antecedent factor of interest, providing a direct estimate of the true relative risk (RR). With this study design, one also can calculate the difference in incidence rates between groups, known as the attributable risk. A primary disadvantage of prospective cohort studies is that they usually are quite expensive and require a long follow-up period to observe a sufficient number of outcome events. Just as with a controlled clinical trial, hypotheses for the observed difference of clinical importance between the groups, the alpha (type I) error that is allowable, and the desired power must be clearly established at the outset of the study, so that the requisite sample size can be calculated. Retrospective case-control studies When the outcome of interest is relatively rare, a retrospective casecontrol study may be the best design for evaluating potential factors associated with an outcome of interest [8]. In a case-control study, the cases are defined as subjects with a particular type and stage of disease. Controls who are free from the disease under study are chosen, matched to the cases based on certain known confounding factors such as age and gender. Care must be taken not to overmatch cases and controls on more than a few key factors, due to the increased difficulty of identifying appropriate matched controls, the increased complexity of the analyses, and the resultant inability to study any of the matching factors with respect to association with the outcome. A drawback of the retrospective case-control design is that incidence rates cannot be directly estimated, as no appropriate denominators are available (populations at risk). Instead, an odds ratio is calculated to estimate the RR between cases and controls, quantifying the odds of having the disease when the factor of interest is present versus when it is absent. The odds ratio is an accurate estimate of the true RR if, and only if, three assumptions hold true: (1) the controls are representative of the general population; (2) the assembled cases are representative of all cases; and (3) the frequency of the disease in the population is rare. Like the prospective cohort study design, case-control studies are subject to uncontrolled variation and potential biases. If a case-control study yields a modest difference in outcome between treatments, this must be interpreted cautiously, as it is difficult to conclude whether a true treatment effect actually exists, or whether the results are confounded by other factors. Case-control studies can be strengthened through several means, including larger sample sizes, adjustment or matching for potential confounding factors, careful testing of model assumptions, and adjustment for multiple comparisons. Systematic reviews and meta-analysis Reviews and meta-analyses of prior published studies will be considered here in the context of observational studies. While both systematic reviews and formal meta-analysis can be useful [9], we will focus on the more rigorous application of meta-analysis. The use of metaanalysis is most commonly employed when a large collection of studies can be said to have, in some manner, addressed the same question, and yet no single study has definitively answered the question, perhaps due

to small sample sizes. A typical analysis will include all relevant studies found in MEDLINE in a predefined time period with predefined study eligibility criteria, and will also search the Cochrane Database of randomized controlled studies. An exhaustive search of the literature is required to avoid selection bias, requiring reviews of studies referenced in the primary publications, but that is not sufficient. Additional effort must be expended to seek out negative trials that went unpublished, due to publication bias favoring positive results. While it may not be possible to eliminate all sources of bias, the recent trend requiring studies to be prospectively registered (http://www.clinicaltrials.gov) to qualify for publication in top journals is improving the landscape for meta-analysis. Recent examples of meta-analysis [10] concluded a positive effect for prophylactic antifungal therapy in patients undergoing allogeneic HCT and high-risk acute leukemia patients. Another recent meta-analysis outside of the transplant setting attracted widespread attention for reporting that high-dose vitamin E increases all-cause mortality [11]. Both studies have merits and limitations. The first paper included a conclusion based on combining allogeneic transplant studies with nontransplant studies, which is problematic in that prophylactic use in allogeneic transplants was already established as the standard of care. The vitamin E paper was criticized for a similar problem, that is, combining various forms of α-tocopherol, but it was also criticized for referring to αtocopherol as being synonymous with vitamin E, when it is simply one member of the vitamin E family. Meta-analysis requires various assumptions, resulting in potentially faulty conclusions when these assumptions do not hold. Nevertheless, both papers have significant merit that could impact clinical care. First, the antifungal paper provides a summary of the result of the one study on prophylactic antifungals in autologous HCT, and presents a valid subset analysis that shows that fungal-related mortality and the incidence of invasive fungal infections were reduced in nontransplant leukemia patients, with a marginally significant impact on all-cause mortality. Also, the second meta-analysis paper makes a good case for avoiding high-dose DL-α-tocopherol acetate, although this was not the primary conclusion of the paper as stated by the authors. Quality of life and cost-effectiveness studies Two burgeoning areas of observational study over the past several years include the study of quality of life (QOL) and cost-effectiveness. Many clinical trials now incorporate these endpoints into the protocol to corroborate the success of new interventions [12], particularly as the margin for improvement decreases as more successful treatment options become routinely available. A literature review has shown that citations for QOL studies grew from fewer than 30 in 1977 to more than 1000 some 20 years later [13]. Analysis of QOL associated with clinical trials may involve multiple scales assessed repeatedly over time [14], raising the challenge of appropriate statistical handling of multiple comparisons. To reduce the number of hypotheses and facilitate the interpretation of trial results for the primary question, “Does the overall QOL differ between treatment arms?,” summary measures collapsing over the multiple QOL items and scales often are used. With multiple QOL assessments over time, missing data can strongly influence the choice of methodology and analytic strategy. Methods have been developed to incorporate QOL into survival analysis, such as the Q-TWiST (Quality-Adjusted Time Without Symptoms of Disease or Toxicity of Treatment) method for quality-adjusted survival analysis using the Cox proportional hazards model [15]. The QTWiST model assumes that patients progress through a series of health states that differ in QOL. The Kaplan–Meier product limit estimate is

Biostatistical Methods in Hematopoietic Cell Transplantation

used to calculate the mean duration of each state, and quality-adjusted survival is estimated given a set of covariate values. (Kaplan–Meier/Cox models are described in more detail later in this chapter.) The results can be useful for investigating how prognostic factors might affect treatment benefits in terms of QOL. Further discussion of QOL and costeffectiveness studies is provided in Chapter 32. Impact of genetic data on observational studies Through the mapping and sequencing of the human genome, it has become possible to explore how genotypic differences lead to phenotypic variation, and to begin to understand the genetically influenced mechanisms underlying human disease [16]. Initial exploratory studies involve “data mining” of existing genetic databanks to look for correlations and begin to generate hypotheses. This initial data mining should be “unsupervised” in nature, employing methods that allow nonmathematical biologists to browse and manipulate the vast data already available in genetic databases such as GenBank and the Online Mendelian Inheritance in Man (OMIM) database. Investigators are beginning to use automated sequencing and analysis of DNA as an inexpensive screening of multiple loci for polymorphisms. Sharing of data across all research groups worldwide will be required to expedite discoveries of critical genetic patterns. As we move into the next phase of attempting to relate genetic information to clinical phenotypic data, DNA databanks oriented toward future hypotheses not framed at the outset will need to be created. Such databanks will consist of repositories of identified specimens, and will be able to serve many different “parent” clinical studies in future. To examine genotype–phenotype correlations, it will be critical that the genetic data are linked to repositories of detailed biologic, medical history, and treatment data on the same individuals. The standard endpoints used in clinical trials (e.g. efficacy and safety) will give way to less-clear outcomes such as “classification” and “correlation.” Epidemiologic trials will be increasingly important to tease out environmental versus genetic contributions to disease, and familial history data will become critical. This collection and exploration of complex phenotypic, intervention, and environmental data may prove to be a more daunting information management feat than the amassing of the genetic information, comprised of data elements with a very simple structure by comparison. Guidelines for the use of genomics in clinical research (and in clinical practice) will be required in the future, using an interdisciplinary approach involving clinical investigators, geneticists, biostatisticians, and bioinformaticists. All the newly available sources of information highlight that one key to innovation from observational studies is to observe without necessarily having a clear understanding of what might turn out to be important years later. The importance of observing unintended consequences and unforeseen predictors of outcome is often underestimated, but fortunately, with modern computerized laboratories, increased computerized data standards, growing efforts and advances in data mining, and the rigorous follow-up demanded in the HCT patient population, observational studies and data exploration of clinical data repositories are improving, and are often the leading motivation for future clinical trials for diseases where HCT is the current treatment of choice.

Clinical trial design in HCT research A clinical trial involves a true experimental design in which the investigator controls the intervention. The National Institutes of Health (NIH) defines a clinical trial as a prospective biomedical or behavioral research study of human subjects that is designed to answer specific questions about new interventions (drugs, treatments, devices or new ways of

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using known interventions). Clinical trials are used to determine whether the new interventions are safe and effective. The field of oncology is well suited to the application of clinical trials [17]. Outcomes generally are well defined and measurable, and the participation, compliance, and follow-up rates tend to be better in the face of such serious illness than they would be for less life-threatening diseases. Precursors of modern clinical trials date as far back as informal human experiments conducted in the 1700s, and modern trial methods began to appear in the medical literature in the 1930s. It is generally agreed that the earliest trial using today’s formal methods, such as patient selection and treatment assignment, was the UK Medical Research Council trial of streptomycin for the treatment of tuberculosis, designed by Austin Bradford Hill [18], in which two measures of success (endpoints) were defined as survival and improvement on chest X-rays, as interpreted by a radiologist who was unaware of (i.e. masked to) the type of treatment that the patients had received. There are five phases of clinical trial in the development of a new therapeutic intervention, including phase 0, the newest trial classification from the US Food and Drug Administration (FDA): • Phase 0: Does the therapy hit its target? (“Is the therapy biologically active?”) • Phase I: Determine the appropriate dose. (“How much therapy can be given safely?”) • Phase II: Demonstrate effectiveness. (“Does it work in a group of similar patients?”) • Phase III: Compare against the standard. (“Is it better than what we can offer now?”) • Phase IV: Evaluate the effects post marketing. (“Will the effect hold in the general population?”) A number of legal requirements must be in place before conducting a clinical trial, including review of the protocol by an independent body, determination that the participant’s risk–benefit ratio is reasonable, and a procedure for obtaining the participant’s informed consent prior to enrollment on the trial. (Note that “participant” has become the favored term for individuals enrolled on a clinical trial, rather than patients or subjects, emphasizing consideration of the enrollee’s human rights and expectation of an acceptable QOL, as well as acknowledging that some trials, such as vaccine development trials, enroll healthy individuals.) An in-depth treatment on human subject protections and regulatory requirements is beyond the scope of the current chapter, but a comprehensive overview can be found in the text by Amdur and Bankert [19]. Obviously, the fundamental core of a clinical trial must be based on the scientific question to be answered. However, the design also must incorporate an ethical concern for the participants, and a wise use of available financial, therapeutic, and human resources. The trials must be designed such that the results will ultimately be persuasive to the intended audience(s), including the healthcare and patient populations, and, when applicable, the regulatory bodies responsible for approval of new drugs/devices. The study design also must provide appropriate statistical control against potentially erroneous conclusions. By their nature, clinical trials tend to include lower-risk patients within a certain age group and with particular clinical characteristics. In the real world, patients are less well defined and do not fit the inclusion and exclusion criteria of the trial, such that the trial results will apply only to a proportion of all the patients with the same disease. As can be seen from Fig. 28.2, at least three major errors can occur within a clinical trial, leading to potentially spurious or misleading results. A type I error involves an incorrect conclusion of a statistically significant result when none is actually present. A type II error reflects an inability to detect a significant result when it truly is present. Bias is the third type of error, occurring when the data do not actually measure

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the intended results, but rather are influenced systematically by other existing factors. While type I errors can be diminished through the correct analytic approaches, avoidance of type II errors and bias must occur up front through the appropriate study design, sample size, and robust definition and collection of endpoints. Readers of published studies may wonder why several trials conducted to examine the same therapeutic regimen and endpoints in similar patients with a given disease entity often draw differing conclusions. Variation in clinical trial results can be explained by several factors that must be considered to ensure valid intertrial comparisons. These include, but are not limited to: the specific participant selection criteria; exact response criteria; interobserver variability in assessing the response; dosage modifications proscribed by the protocol; participant compliance with dosing rules; completeness of reporting of the results; and overall sample size of the trial. Prior to contrasting results across clinical trials, the astute reader should assess the comparability of the studies with respect to these critical points (provided that the report is complete enough to allow such an assessment, another frequent downfall in published studies). The primary tool to help control variability of these components within a clinical trial is strict specification of a detailed protocol, and measurement of protocol adherence during the conduct of the trial. The typical components to be specified when authoring a clinical trial protocol are shown in Table 28.2. Some items apply more in certain phases of clinical trials; for example, randomization typically occurs in phase

III trials (although randomized phase I and II designs also are possible). All of these protocol sections at least should be considered for inclusion when authoring a clinical trial protocol, and most are mandatory to create a valid protocol document. Table 28.3 provides a synopsis of the core elements of the first four phases of clinical trials typically conducted at research institutions. The table summarizes the primary aim, sample to be studied, general procedure, and pertinent notes relevant to each phase of study, as discussed in more detail in the following sections. Phase 0 clinical trials While the term “phase 0” is not new, and there is some confusion due to its use in chronobiology studies, this term is meant to reflect early phase I clinical trials. Generally, the goal is simply to evaluate whether the proposed targeted agent or therapy shows potential based on evidence that it “hits” the target. These studies are a subject of ongoing debate, but in 2006 the National Cancer Institute started its phase 0 program, based on FDA guidelines [20,21]. The first National Cancer Institute-sponsored phase 0 study investigated whether or not ABT-888 inhibited poly(ADP-ribose) polymerase (PARP) in the clinic. Two cohorts of three patients each received a single dose at 10 or 25 mg respectively. For the three patients in the second cohort, PARP was

Table 28.2 Key components for inclusion in a clinical trial protocol

Type I error

Type II error

Bias

Incorrectly concluding result is significant

Missing a truly significant result

Not truly measuring what you intend to measure

Must be controlled through design

Can be controlled through analysis

Fig. 28.2 Possible errors in clinical trials.

• • • • • • • • • • •

Objectives Background Hypotheses Drug information Staging criteria Patient eligibility Descriptive/stratification factors Randomization scheme Treatment plan Dosage modifications Toxicities monitored

• • • • • • • •

Study parameters Endpoint definitions Criteria for evaluation Special instructions Statistical considerations Registration guidelines Data submission schedule Inclusion of women, minority groups, and children • Ethical and regulatory considerations • Pathology review • References

Table 28.3 Core elements of phase 0–III clinical trials Phase 0 trials

Primary aim: Sample: Procedure: Notes:

Determine whether the new drug (or a new combination of drugs) is biologically active A small number of subjects (typically 3–15) to better understand the intended effect Observe biologic effects on single administration studies, at minimal doses, that could not be attributed to chance Helps demonstrate sufficient potential to pursue further study

Phase I trials

Primary aim: Sample: Procedure: Notes:

Identify the safe range and maximum tolerated dose for a new drug (or a new combination of drugs) Volunteers with advanced cancer no longer responding to standard therapy, or homogeneous subsets of high-risk patients Gradually increase the dose in successive groups of patients until “significant” toxicity occurs Response is rarely seen and is not a primary aim of a phase I trial

Phase II trials

Primary aim: Sample: Procedure: Notes:

Determine whether new drug has sufficient biologic activity to warrant further investigation Patients with a given type of cancer who have measurable disease Stop after first stage of accrual if insufficient response; otherwise continue to full accrual Biologic activity does not necessarily imply therapeutic benefit, e.g. if drug has serious side-effects

Phase III trials

Primary aim: Sample: Procedure: Notes:

Evaluate in a randomized trial the therapeutic role of a new drug with known biologic activity versus a standard Patients with a given type of cancer randomized to new versus standard Test for differences between groups in major endpoints reflecting benefit Usually expensive and requires a large number of patients

Biostatistical Methods in Hematopoietic Cell Transplantation

inhibited in the tumors by 92%, 99%, and 100%, which demonstrated that oral ABT-888 is bioactive and hits its target, as it would not be reasonable to consider such a change in PARP due to chance alone. More generally, phase 0 studies are intended to observe biologic effects on single administration studies (at minimal doses) that could not be attributed to chance alone, demonstrating sufficient potential to pursue further studies. The details of the statistical considerations of such small sample calculations are described in Kummar et al. [20], but, like most statistical methods, it is based simply on detecting an effect that would not reasonably be ascribed to chance alone. Theoretically, even a single patient can be sufficient where the observation could never have happened spontaneously. Nevertheless, most phase 0 studies use between three and 15 subjects to better understand the intended effect. Discussions regarding the risk–benefit ratio and related ethics of phase 0 studies are often contentious. In HCT, there are a variety of situations where phase 0 studies could apply, although they are most often called “pilot” studies due to the fact that often subtherapeutic doses are not given. For patients with acquired immune deficiency syndrome (AIDS) lymphoma, for example, lentivirus-transfected hematopoietic stem cells are genetically modified to code for multiple RNA decoys (small interfering RNA) to combat the AIDS virus (search http://clinicaltrials.coh.org/, keyword AIDS or 04047). While autologous transplant for lymphoma is a common standard second-line therapy, in this setting it has the added benefit of creating an environment that permits genetically modified stem cells to proliferate with limited competition from unmodified stem cells. Five patients are planned for the study, which will demonstrate whether there is successful engraftment of the genetically modified stem cells, and whether they retain their anti-human immunodeficiency virus activity. Another phase 0-type study in HCT involved testing for the persistence of adoptively transfected CD20-specific CD8+ T-cell clones. One unique opportunity afforded to the HCT environment is that genetically modified stem cells, or donor immunized stem cells (e.g. for cytomegalovirus), have an opportunity to proliferate and become the dominate stem cell phenotype. Such initial studies could be classified as phase 0, although some may prefer the term “pilot” as the FDA guidelines for phase 0 include lack of therapeutic intent and limited toxicity exposure (microdoses when possible), which is not particularly fitting for these HCT studies. Phase I clinical trials A phase I clinical trial is conducted to test a new intervention in a small group of people (e.g. < 80) to evaluate the toxicity profile, determine a safe dosage range, and begin to look at possible efficacy. This phase of trial is used to describe which organ systems are affected; to evaluate the extent, duration, and reversibility of toxicities; to observe any possible antitumor activity; and to better understand the pharmacokinetics and pharmacodynamics. Because of this emphasis on determining toxicities, some investigators see an ethical dilemma in enrolling patients in a phase I trial, as clinical benefit (e.g. response) is not necessarily the primary objective. However, while the probability of therapeutic benefit of a phase I intervention is unknown, especially in single-agent first-inhuman studies, the possibility of clinical benefit does exist, and the patient eligibility and physician referral often are predicated upon there being no better option than the phase I therapy. As such, a phase I trial often is the only means of continuing active therapy with the hope that the treatment will benefit some of the individual patients, even during this early testing. Standard phase I trial design For the typical phase I trial in solid tumors, the study sample consists of a heterogeneous group of volunteers with any type of advanced cancer

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that is no longer responsive to standard therapy. In contrast, phase I trials in diseases targeted for HCT often are performed on a homogeneous subset of high-risk patients who share the same disease type. Phase I trials for acute leukemia often are more aggressive with respect to hematologic toxicities, as it is generally accepted that antileukemic activity will be accompanied by myelosuppression. If the mortality in a subset of patients is quite high using an existing therapy, the potential gain may be great, even with accompanying high levels of toxicity. The recent success of new antileukemia drugs encourages patients refractory to standard therapy to try phase I agents, and patients have experienced a complete remission on a phase I study and proceeded to HCT, even in the more difficult to treat leukemias, as Kirschbaum et al. reported at the American Society of Hematology Conference in 2006 [22]. While less complex in terms of statistical analysis when compared with randomized phase III studies, phase I trials still require rigorous “statistical thinking” in terms of design and decision-making. Requirements for entry into a phase I study typically include: (1) a minimum estimated life expectancy of 8–12 weeks; (2) a reasonably good performance status (e.g. minimum Karnofsky Performance Status score of 80); (3) the cessation of all prior experimental therapy for the last 2–6 weeks; and (4) adequate major organ function to allow normal metabolism of the new agent. In addition, with molecularly or immunologically targeted therapies, phase I trials usually are restricted to patients with the specific target. Many new HCT therapies under investigation for the first time have specific molecular targets, such that a primary endpoint is the ability to shut down a specific enzyme or pathway. This is particularly the case in the testing of new biologic agents, where once a dose that saturates a target is reached, what happens at higher doses may not be of interest unless there are alternative pathways involved. Although response rates are examined within a phase I trial, because these trials generally consist of very small sample sizes, lack of response should not necessarily deter the investigator from proceeding to a phase II trial for estimation of therapeutic effect. Conversely, responses observed during the phase I trial should not be used as a substitute for performing a full phase II or phase III study. Dose-escalating phase I designs. For traditional interventions, the phase I trial will involve a dose-escalation scheme to determine the maximum tolerated dose (MTD) as a primary endpoint. This primary aim is carried out under the assumption that a larger dose or more aggressive regimen is more likely to provide therapeutic benefit to the patient, but will also increase the risk of adverse events. Because adverse events occurring at high doses may be irreversible and life-threatening, the MTD is a relative concept that weighs an acceptable level of toxicity against potential clinical gain. Establishment of the MTD is required not only for a new individual drug, but also for any new drug combination, as the cumulative effect of combining therapeutics with known toxicities becomes a critical unknown that must be established. The study plan must avoid overaccruing patients to subtherapeutic doses that are unlikely to be beneficial, while avoiding overexposure of patients to unacceptably high toxicity levels [23]. A dose-escalating study design should specify the starting dose, steps in the dose escalation, number of patients to be tested at each dose level, and criteria for moving to the next level and for specifying the estimated MTD. The dose range selected must be considered relatively safe yet likely to encompass the MTD. This requires a predefined level of toxicity considered to be unacceptable, referred to as a dose-limiting toxicity (DLT). A safe starting dose is selected based on preclinical pharmacology, animal toxicity or use in other types of patient [24]. For first-in-human use, one-tenth of the mouse equivalent dose that is lethal to 10% of nontumor-bearing animals (MELD 10) is often used, although arriving at a precise estimate can be difficult [25].

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Table 28.4 Examples of phase I dose-escalation schema

Enter 3 patients

Modified Fibonacci scheme*

Unit step size scheme†

Constant step size scheme‡

30 mg 60 mg 100 mg 150 mg 200 mg

30 mg 60 mg 90 mg 120 mg 150 mg

30 mg 40 mg 50 mg 60 mg 70 mg

For comparison, all three dose escalation schema examples were set at a starting dose of 30 mg. * Escalation factors: 100%, 67%, 50%, 33%, 33%, . . . † Escalation factors are set equal to the starting dose, e.g. 30 mg in this example. ‡ Escalation factors are set equal to an arbitrary constant, e.g. 10 mg in this example.

Grade III+ toxicity?

No

Step up 1 dose level No

Yes Any grade IV+

2/3 grade III

1/3 grade III

Add 3 more patients

Grade III+ toxicity? Yes

MTD = next lower dose

No 6 pts at MTD?

Yes

Accrue 6 pts

Phase I closed

Fig. 28.3 Traditional phase I trial schema. MTD, maximum tolerated dose; pts, patients.

The most common study procedure in a dose-escalating phase I trial is to increase the dose gradually in successive groups of three patients, until the predetermined level of unacceptable toxicity is observed. Typically, a dose ladder is prespecified, often the “modified Fibonacci scheme,” in which a sequence of incremental doses is established as the dose-escalation ladder. Escalation proceeds until the MTD is reached. Alternative dose-escalation schema include the unit step size scheme, in which a constant step size equal to the initial dose is applied, and the constant step size scheme, in which each dose is increased by a constant fixed increment over the previous dose (Table 28.4). Designs where the step size depends on the non-DLT toxicities are often used in first-inhuman phase I studies, but are also used more generally [26]. While the sample size necessary to progress to the MTD cannot be anticipated within a phase I plan, in our experience the typical number of levels ranges from three to 15, with fewer levels when testing a new combination of well-known drugs, and more levels for first-in-human use. A typical sample size might end up being 24, but studies with two patients (e.g. unacceptable toxicity at a dose where the blood concentration was below the level of effect seen in preclinical studies) and 60 patients have been conducted. In HCT trials, the study regimen often will have been used in other types of patient, so that the types of toxicities and a crude estimate of the MTD are available for planning purposes. However, patterns of toxicity can change markedly from one type of patient to another, such that caution is still required. A dose-escalation procedure commonly in use is shown in Fig. 28.3. Three patients are accrued to the first dose level. If no DLT is observed in those three patients (after one complete course plus any predesignated waiting period to observe drug-related toxicity), the dose is escalated to the next level specified by the protocol. If a single patient experiences DLT, three additional patients are treated at the same dose level; the dose may continue to be escalated only if no additional DLT is observed in the additional patients treated at that level. If a second DLT is detected, de-escalation ensues, with the initial estimate of the MTD being the preceding dose level. The trial is considered to be complete (MTD determined) when six patients have been accrued at the estimated MTD dose level, with the occurrence of at most one DLT out of six. In non-HCT studies, DLT is usually defined as treatment-related nonhematologic grade III or IV toxicity, with a few exceptions, and no hematologic DLT. Hematologic DLTs in a leukemia trial often can only be detected in the absence of blasts, and defining hematologic DLTs often requires a discussion of duration and results of a bone marrow aspiration. In HCT studies, DLTs are considerably more difficult to define, and no consensus has emerged. The most common rules today are still grade 3 toxicities by the Bearman Scale from 1988 [27].

One criticism of phase I trials is that “the recommended dose has no interpretation as an estimate of the dose which yields a specified rate of severe side effects” [28]. With the usual phase I escalation schema, only six patients are observed at the estimated MTD, and no measure of statistical precision (standard error or confidence interval) can be calculated. This is particularly troubling when the target is a dose with a specified amount of severe toxicity (the DLT rate), in an attempt to achieve a favorable risk–benefit ratio. Statisticians have examined the operating characteristics of phase I designs to try to improve upon the rather ad hoc design described above. Storer [29] provides an introduction to work in this area, showing how poorly the usual designs perform with respect to providing a reliable estimate of the MTD. He proposes some simple variations in the designs that may perform better, and methods for obtaining confidence limits for the MTD. The article by Korn et al. [30] provides a discussion of Storer’s approach along with other alternatives, and evaluates their performance via computer simulations. Ivanova et al. [31] have described an up-and-down design for the sequential allocation of subjects to dose levels, yielding a local estimate of the toxicity probability calculated from all previous responses. One point that helps mute criticism of the standard design is that generally there is no consensus regarding the target DLT rate. Most new phase I designs have rarely been implemented, partly due to concerns such as length of time to complete the trial, potential for excessive toxicity, and complexity of updating the models for dose escalation throughout the trial without an automated system in the clinic. As more institutions adopt electronic medical records, the capability of and access to computers at the point of care may change this last situation. Before implementation in the clinic, computer simulation studies are carried out to compare the performance of the proposed designs. Such comparisons traditionally use the performance criteria of proportion of patients treated at the recommended MTD (and the number above and below), and the average number of patients required to complete the trial, and base these simulations on a variety of assumptions regarding the true dose–toxicity profile. In future simulation studies, investigators may also prefer to incorporate the actual time to trial completion as a performance measure as well, as it is unethical to spend excessive time examining an unpromising regimen before moving on to the next therapeutic approach. Clarke et al. [32] have investigated a new phase I approach intended to minimize the time to trial completion by allowing new patients to enter the trial without waiting for toxicity evaluation on the full prior cohort of patients, yet conditioned on all available toxicity information to that point.

Biostatistical Methods in Hematopoietic Cell Transplantation

In HCT studies, there often are additional considerations to the phase I study besides concerns about DLTs. Specifically, some agents tested in HCT studies are designed to help reduce graft rejection. In such a setting, there is, hopefully, a therapeutic window below which the dose is too low to prevent rejection, and above which unacceptable toxicity occurs. Designs in this situation usually rely on extensive simulations to obtain rules that result in acceptable operating characteristics [33]. Regardless of the particular study design, uncertainty in estimating the MTD requires investigators to be cautious and monitor toxicities in a phase II trial very carefully, being prepared to adjust the phase II treatment dose as additional information accumulates. The phase I study result is a critical first step, for when the phase II treatment dose is modified mid-trial, the statisticians and the investigators or the data safety and monitoring board may decide that the study is essentially a “do-over,” and restart the patient count from the modification. This is less common in a phase III setting, when such modifications are generally minor. Phase II clinical trials Following establishment of the safety and toxicity profiles of a new intervention, a phase II clinical trial is conducted to determine whether the new therapy has sufficient evidence of biologic activity to warrant further investigation. Phase II trials are performed not only for antitumor activity, but also to prevent relapse, for prophylaxis and treatment of graft-versus-host disease (GVHD), for treatment of symptoms of other therapies (e.g. pain), for prophylaxis against and treatment of specific infections or for a combination endpoint (such as protocol-defined “clinical benefit”). The primary components of a phase II trial are summarized in Table 28.3. Standard phase II trial design Phase II trials typically are conducted in those patients who are most likely to respond favorably to a new regimen, so that potential activity of the new regimen is not missed. Therefore, participants with maximum performance status and a minimum amount of prior therapy should be selected whenever ethically possible. In addition, all patients in a phase II trial should be evaluable for the primary endpoint, which should be objective. Subjective methods of assessing treatment effect make the outcome of the phase II study more problematical and less convincing. One approach to phase II trials is the single-group design, with no control group. Like the phase I study, the single-group phase II trial is subject to the problems of any uncontrolled clinical trial. However, these studies are intended as low-cost screens for choosing safe doses and for eliminating treatments that do not have sufficient promise for further study. Similar to the phase I study, the uncontrolled phase II group study design depends on careful definition of the eligibility criteria, and uniform application of these criteria in admitting patients to the study throughout the accrual period. It is also extremely important to define clearly within the study protocol the response endpoint and its method of measurement. The number of patients to be studied in a phase II trial depends on the specified degree of disease activity required for the new treatment to be of therapeutic value, specified as the percentage of patients expected to respond to the treatment. The study should be large enough to furnish information that will rule out with high probability (e.g. 80–90%) a treatment that does not have this desired degree of activity, taking into account possible random variation in the limited sequence of patients studied. The simplest approach is to compute an upper confidence limit, and if this limit is less than the desired activity level, the treatment is rejected as of little interest. If the upper limit is greater than the desired level, investigation will proceed to determine just how active the regimen

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Table 28.5 Number of patients required for the phase II trial of an agent for given levels of therapeutic effectiveness and type II error Therapeutic effectiveness (%) Rejection error (beta) 5% 10%

5

10

15

20

25

30

35

40

45

50

59 45

29 22

19 15

14 11

11 9

9 7

7 6

6 5

6 4

5 4

Reproduced with permission from Gehan and Schneiderman [36].

might be, via a phase III trial. Of course, other factors might preclude further study, such as additional evidence of unacceptable toxicities or a higher MTD than indicated by the phase I study results. Statistical methods for computing the upper (one-sided) confidence limit are found in most elementary statistical texts, such as in Chapter 5 of Glantz [34] or Chapter 14 of Pagano and Gauvreau [35]. Commonly, a single-sample, two-stage design is employed in phase II trials, thereby allowing early termination of the study after the first accrual phase if the new regimen does not appear to be promising with some level of specified confidence. Table 28.5, from Gehan and Schneiderman [36], shows the number of patients required in the first stage of accrual if one desires either 95% confidence (rejection error or beta = 5%) or 90% confidence (beta = 10%) for efficacy levels ranging from 5% to 50%. The new treatment is rejected as not sufficiently active if no responses are seen among “n” patients treated. For example, if the study produced no responses among the first 11 patients, one would have 95% confidence in rejecting the new treatment as being active in 25% of patients. If one or more responses are seen among the first set of patients, accrual is continued into the second stage, until a large enough study group is reached to estimate the response rate with a prespecified level of precision that depends on the final sample size. As an example, with 35 subjects entered after concluding both stages of accrual, the response rate could be estimated with a maximum standard error of approximately 8% (95% confidence interval width of approximately 16%). The typical phase II study uses a binary endpoint – response or no response. Response could be defined to include only complete response, or to include both complete plus partial responses, as the outcome of interest. However, the counts themselves usually are based on quantitative measurements (e.g. estimates of the amount of reduction in tumor volume or counts of cutaneous lesions in herpes zoster). Generating waterfall plots can often provide additional information [37]. Variants of the phase II trial design Important work has been done on designs that control both risks of error (alpha and beta) and achieve significant savings in the number of patients required. Simon provides a thorough review of different approaches to phase II trials [38]. He has developed an optimized two-stage design with a smaller sample size in situations where the lack of efficacy becomes clear early in the accumulation of the data, allowing the possibility of earlier stopping at the end of the first stage of accrual [39]. Simon concludes that two-stage designs with a target sample size of 35–50 and a substantial probability of early termination usually are appropriate. Fleming [40] offers other commonly used procedures for two-stage designs. Since the relationship between type I and II errors, interim stopping points, and sample size are not always obvious or monotonic, we often explore around the “edges” of these designs to seek preferred designs, calculating the operating characteristics of alternative

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designs. Three-stage designs also are employed, especially when a quick “bail-out” option is needed for early lack of evidence [41]. Storer has proposed that designs for control of both alpha and beta risks be expanded to consider three possible decision outcomes: response rate unacceptable, response rate acceptable, and an intermediate region in which further study is required before a decision can be made [42]. Several statisticians have done work on combining decision-making risks with estimation, where the precision of the estimates is based on confidence limits. For example, Chang and O’Brien [43] have created a method and offer a computer program for obtaining confidence limits for the response rates from the results of multistage phase II studies. Another area of interest is Bayesian methods, based on the quantitative expression of prior beliefs about the treatment to be tested, and using this quantitative information and a measure of its precision in the study design and analysis. Phase II trials are a likely arena for using this approach, and Thall and Simon [44] provide a useful introduction to this topic. In a two-stage phase II trial, participant accrual typically must be suspended while the results of the first-stage accrual are examined to determine whether or not to continue into the second-stage accrual. This temporary suspension has drawbacks in that the momentum for patient accrual is lost, and the overall time to completion of the study is lengthened. In addition, there may be logistical problems in reactivating the trial if the data show that it is warranted to proceed to the second stage, particularly in a multicentered study setting. Herndon [45] has proposed an alternative “hybrid” design that allows accrual to continue while the results from the first stage of accrual are being examined to determine whether the study should continue to the next stage. Other variations on the standard phase II design include adding a phase II treatment arm to a phase III clinical trial [46], randomized phase II trials (or “active control phase III trials”) to select the most promising of several new agents [47], “calibrated” phase II trials, in which a subgroup of patients are randomized to a standard treatment to verify that the population under study is capable of responding to an active treatment [48], and randomized screening trials, often used when comparing a new agent combined with a known regimen versus the known regimen alone [49]. In these designs, one does not enroll typical phase III sample sizes. Typically, either the measure of statistical significance is reduced (e.g. p < 0.2), or a selection decision design is used, where the chance of choosing an inferior regimen is kept small. As a result, these randomized phase II studies can enroll conventional phase II sample sizes to each arm, and early stopping rules can apply both jointly and independently to the two arms. Often historical controls and phase II studies are suggested in lieu of random concurrent controls, that is, comparing patients treated earlier at the same institution(s) using a therapy that is different from the new therapy under consideration, or using historical control results from the literature. While it has been argued that historical controls are convincing in certain situations [50,51], most consider such comparisons to be suggestive of benefit only, rather than providing a valid, reliable basis for evaluation of a new therapy. A few medical advances have been so clear cut that historical controls would suffice (e.g. the discovery of penicillin or, more recently, the approval of PS-341 in second-line treatment for multiple myeloma); however, such quantum leaps in clinical discovery are rare. Phase II studies are also often used for decisionmaking prior to phase III studies or when phase III studies are unlikely, as is often the case in the transplant setting or in rare diseases such as T-cell chronic lymphocytic leukemia [52]. Accepting inferior decisionmaking as a long-term approach has drawbacks, and advances in oncology are more often observed through careful scientific evaluation through clinical experiments, and the phase III clinical trial is the accepted “gold standard” for establishing a benefit of a new therapy.

Phase III clinical trials After the efficacy of a new HCT drug or regimen has been established in a single group of participants through early therapeutic trials, generally the new therapy is tested against a standard (or a placebo, such as a placebo plus HCT versus HCT plus new agent) within a phase III trial. As shown in Table 28.3, in this phase of trial participants are assigned to therapeutic strategies by random, or probabilistic, assignment, to determine which therapy is superior. The phase III clinical trial requires an extremely detailed protocol to serve as a guide to the organization and conduct of the study, and to provide a plan for analysis of the results, allowing replication and confirmation of the study results. The protocol must specify the eligibility criteria for deciding whether the patient qualifies for the clinical trial, based on the scientific question asked. Hypothesis testing is used to determine whether any differences observed are significantly greater than would be seen by chance alone. The scientific credibility of a properly designed phase III study meets the international guidelines generally required as proof of the efficacy and safety of new drugs and therapeutic devices as the final step in approval for marketing. The International Conference on Harmonization (ICH) Technical Requirements for Registration of Pharmaceuticals for Human Use were developed by the FDA and the comparable regulatory bodies in Japan and the European Union, resulting in publication of the ICH Tripartite Guideline for General Considerations for Clinical Trials [53]. The ICH E9 guideline Statistical Principles for Clinical Trials was adopted by the Committee for Proprietary Medicinal Products in 1998. The principles within this document are scientifically sound, and are being followed by the three ICH regions, although at the current time some regions may be applying these guidelines more stringently than others [54]. To prevent delays in receiving marketing authorizations for new drugs, it is strongly recommended that the statistical guidelines provided in ICH E9 are consulted, and that planned statistical analysis strategies are modified to reflect these principles when designing drug development studies [55]. Standard phase III trial design The primary rationale for randomization within a phase III trial is to assure that the study arms can be compared in an unbiased fashion. Although the need to evaluate competing therapeutic strategies on comparable groups has been acknowledged for many years [56], the difficulty of assuring that groups of patients treated in different manners are truly comparable often has been underappreciated. The randomized trial balances treatment groups statistically, not only with respect to known and recorded factors, but also with respect to unknown patient factors. While the randomized groups will not be identical, any observed differences in therapeutic outcomes between them can be evaluated relative to differences expected through random chance alone. The group comparison is based on probability theory, and provides a measure of the statistical precision of the treatment difference estimate, accounting for random variation. This precision is measured by the standard error of the estimate, the confidence interval, and/or the p-value, as described in introductory statistical texts [34,35]. In addition to utilizing randomization to minimize patient variability that might confound the evaluation of treatment effects, HCT protocols also often attempt to minimize the variability in supportive treatments. However, strict requirements to a protocol-specified supportive care regimen may greatly impair participation among centers and accrual within centers, such that allowing for a more general supportive care regimen within wider guidelines may be necessary in HCT studies. The phase III study plan is based on information stemming from the preceding early phase studies, and calls for accrual and follow-up of

Biostatistical Methods in Hematopoietic Cell Transplantation

participants over a specified period of time. All participants are observed until the occurrence of a specified endpoint has occurred, such as death, progression of target disease or relapse, or to the administratively defined time for analysis at the end of the predefined observation period. HCT studies that include courses of nontransplant treatment as an integral part of the strategy must enroll and follow patients from the time that nontransplant treatment is begun. If the pretransplant treatment strategy differs between the two arms, care must be taken to ensure that patients are enrolled and followed from a similar time point for both arms. Randomization should occur immediately after ascertaining eligibility, as undue delay between randomization and transplantation can lead to high patient drop out, complicating valid interpretation of the trial results. Randomization process. The randomization procedures in a phase III clinical trial should be clearly described and justified in the protocol. To obtain valid trial results, it is crucial that the research team has no knowledge of the assignment at the time of decision for study entry for any given study participant, otherwise the advantage of the random assignment may be lost due to selection bias. A typical procedure is to screen the patient for eligibility based on already available information (disease, routine laboratory work, etc.), obtain patient consent, then perform any study specific eligibility tests (e.g. multiple-update gated acquisition scan), and then register the patient and obtain a random assignment to one of the treatment groups. These assignments are made according to a series of random numbers, with the sequence being computer generated in advance or as the patients are entered into the study. It is desirable to minimize heterogeneity in the participant group to avoid imbalances in patient characteristics. However, the benefits of a homogeneous study population must be balanced against the need to accrue sufficient numbers of patients in a timely manner, and the need to have the study sample reflect the population in which the treatment eventually will be used, to be able to generalize the study results. The randomization schedule often is balanced at successive points in accrual, by ensuring that the patients within a “block” (e.g. every six patients) are evenly assigned to the randomization groups as accrual progresses at each site. To protect the research team from being able to anticipate treatment group assignment, the block sizes can be varied, and block size information should not be specified in the protocol. If considerable variation in prognosis among the study patients is anticipated, more of the higher-risk patients may be assigned randomly to one treatment group and more lower-risk patients to the other group, making it difficult to detect a true treatment difference due to the unfair comparison. Heterogeneity among patients can be addressed in the design phase by using “stratified randomization,” in which patients are divided into risk groups (strata) based on a small number of known prognostic variables, such as age (younger/older) and remission status at transplant (first complete remission/beyond first remission). Randomization is carried out within each age/transplant status group. Because the number of strata grows rapidly with each new characteristic, stratification should be confined to only a few proven prognostic variables. For example, if there are four stratification factors, each of which takes on one of two possible values (e.g. male or female, disease stage III or IV, age young or old, and previous radiation therapy or not), this results in 16 possible strata. Therefore, designs that stratify on more than three factors generally are unnecessary and unwise. For additional prognostic variables, covariate adjustment can help account for imbalance in treatment arms. For the primary objective, these covariates should be specified a priori as part of the protocol. If the study is to be carried out at several sites (clinics, hospitals, and study centers), the variability among centers in transplant trials may be

415

quite considerable, even for tightly written protocols. In the case of such multicentered HCT trials, stratified randomization should be used to generate separate randomization assignments for each site, to ensure that the treatment arms are statistically balanced within each site. In their seminal 1976 paper on clinical trials design, Peto et al. recommended that, unless the trial size is quite small, there is generally little need for prerandomization stratification, other than for treatment center in the case of a multicenter trial [57]. Another approach to obtaining balance on participant covariates is to use Efron’s “biased coin” design or variations on this idea [58,59]. An example would be to balance a study adaptively on gender and age as new patients are accrued to the study, instead of randomizing within each of four gender–age strata. With this method, as each new participant is enrolled in the study, the degree of balance between the two treatment arms is examined separately for gender and by age. Randomization proceeds with a skewed probability favoring assignment to the treatment group that reduces any existing imbalance in prognostic factors between the two arms. One caution is that applying correct analytic methods within adaptive randomization can be more complex, as discussed by Lachin et al. [60] and Simon [61]. Study sample size. Planning the size of the study is crucial to determine the number of participants required to acquire sufficient information for comparing the treatment groups. While controlled trials tend to reduce the inherent bias in the data through the study design, extremely small sample sizes may limit the ability to avoid bias fully. Most treatments have relatively minor effects on disease, and it has become appreciated that trials can be heavily influenced by chance [62]. Establishing the reality of small differences between treatments often requires a very large sample size, making such studies quite expensive. Recruitment may have to occur from many hospitals, or even across several countries, making control of data quality quite difficult. Attaining a sufficient sample size for a phase III HCT trial requiring a human leukocyte antigen-identical sibling donor can be particularly problematic, as only about one-third of otherwise eligible transplant candidates have such a donor. For trials studying interventions designed to prevent later complications, for example trials to prevent chronic GVHD, relapse or late organ failure, early attrition from transplant-related mortality decreases the effective sample size available (and could bias the analysis of the longer-term endpoint if not handled appropriately). In calculating the desired sample size, several parameters must be specified by the investigator, based on prior data, past experience, and clinical relevance. The investigator must specify whether the statistical evaluation of the hypothesis is to test for no difference between the two arms, or to prespecify a desired direction of the test, for example that the new treatment is better than the standard treatment. These are known as two-sided and one-sided alternative hypotheses, respectively, with the two-sided approach being the more conservative method preferred by most peer-reviewed journals (unless strong biologic evidence or the use of a placebo comparison arm dictates that the direction of the test should be one-sided). The investigator also must determine the difference between treatment arms that is clinically important, generally based on his or her clinical experience and what the community has accepted as worthy of a change in practice in similar patients. The alpha error rate for the null hypothesis of no difference must be specified in advance, that is, the probability of declaring that the two treatments differ when they are really equivalent, with the apparent difference being due to random variation. (A traditional alpha error level is 5%.) The beta error rate also must be declared in calculating the study sample size, representing the probability of missing the specified clinically important treatment difference (often set at 20% or below, such that the power of the test [1 − beta] is 80% or higher).

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While this overview of the phases of clinical trials may make it seem relatively straightforward to move from phase I to phase II, and phase II to phase III, often this is not the case. Evidence for safety and dosing may be based on an accumulation of results from several trials, and, when appropriate and expedient, phases I and II may be combined in a single trial, particularly in the face of limited patient resources. At times, it may become necessary to study more than one intervention in the same patient, such as enrolling the same individual in both an experimental transplant trial and the follow-on trial of a new GVHD prophylaxis. In such instances, very careful planning of entry criteria, measurement of potential confounding factors, and adjustment for the primary treatment arm must be considered in the conduct and analysis of these companion trials.

Impact of genetic data on clinical trials design The genomics era has led to a rapidly improving understanding of germline polymorphisms and mutations, along with tumor genetics. Many specific oncogenes, tumor suppressor genes, genes related to DNA repair, and other genetic abnormalities found in cancer have been characterized for a variety of tumor types [63]. Germline mutations and heritable polymorphisms that increase the risk of cancer are better understood, and other polymorphisms are known to impact the metabolism of various drugs. Knowledge of these molecular markers can be exploited within clinical trials being conducted for the detection and treatment of cancer. Four avenues of expanding genomic research that impact the design of future clinical trials are made possible through the availability of the human genome mapping. First, genetic testing will provide earlier detection of the genetic risk of disease, and improved sensitivity and specificity in the diagnosis of disease. With the growing availability of genetic markers for presymptomatic conditions in hematologic malignancies, early intervention and prevention protocols will become more feasible and prevalent. Eligibility will be developed that includes disease specificity as determined by genetic testing. In addition, genetic testing may be used to refine detection of disease as the primary endpoint of a clinical trial [64]. For example, detection of minimal residual disease (MRD) can be approached through the detection of the unusual expression of a gene in leukemia cells, such as the aberrant expression of the WT1 gene in acute myeloid leukemia (AML), or the presence of very subtle genetic lesions, such as the detection of ras point mutations in AML [65,66]. However, it can be technically difficult to discriminate the rare aberrant expression or mutated allele from normal background expression or wild-type alleles, such that using genetic targets as a marker of MRD generally suffers from less sensitivity and specificity compared with the detection of translocations or gene rearrangements. Because of this concern, qualitative polymerase chain reaction (PCR) assays require controls to detect false-positive results from contamination or a suboptimal assay, false-negative results due to reaction failure, and/or contamination of the assay. The second new scientific area of pharmacogenomics is advancing greatly our knowledge of the human’s ability to metabolize, tolerate or respond to drugs based on the individual’s genetic make-up [16]. In this field, large-scale genomic technologies such as gene sequencing, statistical genetics, and gene expression analysis are systematically applied to speed the discovery of drug-response markers, whether they act at the level of the drug target, drug metabolism or disease pathways. Pharmacogenomics offers the prospect of predicting an individual’s susceptibility to side-effects when undergoing a new therapy, and to individualize treatment selection based on his or her genetic profile [67]. For further information, see Chapter 12.

Clinical trials are already using genetic prescreening and treatment “run-in” periods as part of the study eligibility, to ensure that the participants can metabolize and tolerate the drug, to individually calibrate the most potentially efficacious dose, and/or to stratify subjects by their genetic ability to respond to or tolerate the study drug. Defining patient populations genetically could improve greatly the outcomes, safety, and efficacy profiles achieved within therapeutic oncology research. As an example, a phase I study at City of Hope National Medical Center combining fixed-dose irinotecan with two other agents being escalated states that UGT1A1 7/7 patients are ineligible. As a note of warning, such laboratory-defined polymorphisms only scratch the surface of human genetic variability, so, when feasible, phenomenologic eligibility criteria (e.g. fasting bilirubin levels) may still need to be employed. The third emerging scientific area, pharmacogenetics, involves the development of new drugs based on information about genes and gene targets. Knowledge of molecular characteristics of certain cancers has made it possible to identify patients who could benefit from therapies that target those features [63]. For example, in the majority of patients with chronic myeloid leukemia (CML), leukemic cells have a “telltale” chromosomal abnormality involving “swapping” of genetic material between chromosomes 9 and 22, resulting in the development and proliferation of the leukemic cells. Effective pharmacologic intervention may be designed to target and specifically to inhibit this faulty enzyme. Based on a randomized phase III trial of 1106 patients, in December 2002 the FDA granted accelerated approval of a drug involving a genetic target, imatinib mesylate (Gleevec), for the initial treatment of patients newly diagnosed with Philadelphia (Ph)+ CML. The approval was granted in a record time of just under 6 months from receipt of the application, and was based on a statistically significant decrease in the risk of disease progression detected at the first interim analysis. In the fourth scientific avenue, clinical trials exploring novel gene therapies provide exciting possibilities for repairing or replacing defective genes, or inserting therapeutic genes, impacting study design and conduct. Gene therapy is predicated on the ability to clone and manipulate genes (see also Chapters 10 and 11) [63]. For some diseases, introducing a functional homolog of the defective gene that produces even small amounts of the missing gene product can have beneficial effects, making these diseases strong candidates for gene therapy. An example of a pilot study in gene therapy is a five-patient study at City of Hope to assess the feasibility and safety of cellular immunotherapy utilizing ex vivo-expanded autologous CD8+ T-cell clones genetically modified to express the interleukin-13 zetakine chimeric immunoreceptor in patients with advanced glioma. With the availability of genomic data, more exploratory and “datamining” studies will be conducted to help design the subsequent clinical trials, to find correlations, and to identify the constellation of genes that may determine the toxicity and efficacy of new therapies to be tested within a trial. Both randomized clinical trials and prospective observational studies that include storage of genetic tissue will provide opportunities to gain insights into the genetic basis of variation in response to treatments [68]. Ideally, all future clinical trials will include the banking of genetic samples, to continue to build extensive databases linking clinical and genetic data for future exploratory “data mining” and the discovery of new patterns and predictors. Future clinical practice may require that all trial results be coupled with genetic information to help determine treatment choices by genotype. By identifying those patients most likely to respond to novel drugs, it will be easier to demonstrate efficacy and safety in phase III studies, leading to smaller, more effective clinical trials with corresponding cost savings. In addition, this focused approach will be more ethical as it is more likely that a suitable efficacious drug will be administered to the right patients in a safe manner.

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While these new trials will be faster to complete, they will be more complex to analyze, as genetic information will be collected and correlated with clinical results within the trial plan. Trials will involve gene-based eligibility criteria and stratification by genetic risk/response data. The rapid pace of development of new therapies for experimentation will put rising pressure on standardization within the entire clinical trials arena, to support the requirement for even more efficient flexible study design tools. Already by the end of 2002, among 360 active breast cancer and CML trials identified via the NIH-sponsored website http:// clinicaltrials.gov, 61 (17%) included a genetic component. Of the 249 breast cancer trials, 26 were using hairy-related 2 (HER2) to characterize disease, four included testing for breast cancer 1/2 (BRCA1/BRCA2) for eligibility and stratification purposes, and three included microarray expression data for exploratory analyses. Among the 111 CML trials, 35 were utilizing the presence of the Ph chromosome as either an eligibility or a stratification factor.

Clinical trial conduct Single- versus multicenter studies While phase I and small phase II HCT trials usually can be completed within a single institution, randomized phase III clinical trials frequently require collaboration among several or many sites to achieve the targeted sample size. National cooperative groups such as the Southwest Oncology Group and the HCT Clinical Trials Network have been established to conduct randomized trials that may require thousands of patients to attain sufficient power. Alternatively, several transplant centers may join forces to achieve sufficient sample sizes to conduct trials within a smaller consortium setting, such as the City of Hope National Medical Center–Fred Hutchinson Cancer Research Center–Stanford University Medical Center consortium for the conduct of regional HCT trials. National and international registries such as the International Bone Marrow Transplant Registry are conducting large-scale observational studies based on the results that are routinely reported to them from hundreds of HCT centers. Each of these multicentered approaches creates new challenges and obstacles that must be conquered to conduct a valid study. One central location must be established as the Data Coordinating Center (DCC), responsible for final protocol documentation and dissemination, case report form development, database programming, and data storage. Data quality assurance, protocol adherence monitoring, and statistical analysis must also be addressed. Central registration of patients, review of eligibility and incoming data records by trained staff, insuring audit procedures for review of data records against the original source data, and ensuring appropriate handling of Institutional Review Board (IRB) approvals, amendments, and severe adverse events at all sites are responsibilities of the DCC in a multicenter model. In both single- and multi-center studies, an effective, convenient, and accurate mechanism for data capture and transmission needs to be developed and distributed to all participating sites. Implementation of a system for data collection ranges from paper-based data collection forms to online data entry systems. Mailing or faxing of paper forms for central data entry at the DCC has been the more traditional mode to date. However, electronic data transfer from distributed sites of data entry has been available since the 1980s [69], and is becoming more commonplace. In this case, a secure mode of data transfer with redundancy checking and audit trails must be established. At City of Hope, we have over 10 years of experience with directing a national DCC for cancer research that has established online data capture via the Internet using web-based forms [70]. Numerous additional considerations arise when transmitting data via the Internet, such as ensuring security of the data via encryption, and fault tolerance of the online system.

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Data and Safety Monitoring Boards Data and Safety Monitoring Boards (DSMBs) play an essential role in protecting the safety of cancer clinical trial participants, and in assuring the integrity of the research being conducted [71]. DSMBs serve a datamonitoring function above and beyond that traditionally served by IRBs, and as such are particularly important for studies that involve blinded or masked data, and cooperative clinical trials involving several centers. Generally, the DSMB monitors ongoing phase III trials; however, their purview also may include earlier-phase studies. DSMB members are charged with monitoring the accumulating trial data at regular intervals and making recommendations regarding appropriate protocol and operational changes based on the data. The DSMB helps ensure that trials do not continue beyond the point when the objective(s) have been met, and a clinically meaningful answer of importance to the scientific community and the public has been obtained. However, the decision to continue or to stop a trial, or to modify the protocol in a major way, rarely is straightforward. Such deliberations require considerable judgment and attention to numerous factors, to protect the safety of the research participants while ensuring the highest quality scientific research. The size and composition of a DSMB study panel are driven by the nature of the study involved, but it generally consists of five to eight members whose expertise encompasses the scientific area under study, ethics, regulatory affairs, and biostatistics. The committee should be multidisciplinary in nature, and made up of individuals who are free of any significant conflict of interest [72]. Once the DSMB membership has been established and charged with its responsibilities, the DSMB Chair, coordinator, and biostatistician typically agree to a basic format and schedule for the presentation of ongoing data, so that DSMB members will receive adequate reports within a sufficient time frame for the decision-making process. The study Principal Investigator may participate as an ex officio member of the DSMB; however, for phase III trials the data should remain “blinded” to the Principal Investigator during all discussions of the interim results. Although exceptions can be considered by the DSMB chair on a case-by-case basis, this principle is particularly important if the Principal Investigator is involved with patient care during the course of the study. The DCC will provide interim results to the DSMB on study progress, safety, and relative efficacy of the treatment arms, subject to restrictions of the protocol design. Methods such as group sequential designs satisfy the valid objectives of interim monitoring, while avoiding undesirable consequences such as increased alpha error [71]. After each meeting, the DSMB members will make a recommendation regarding the continuation of the trial(s) under review. Critical issues that should be incorporated in this assessment include increased morbidity/mortality related to the study intervention; severe or increased adverse reactions; unsatisfactory performance of the DCC or study sites; suspicion of fraud; inability to complete the study in a timely manner due to lack of patient enrollment; failure to comply satisfactorily with recruitment criteria; and any other issues concerning possible important protocol deviations or suggested changes. Depending on the findings and circumstances, actions to be taken by the DSMB include suggestions for increasing patient recruitment, extending the period of recruitment, stopping recruitment because of an inadequate rate of accrual, modifying the protocol because of safety or accrual issues, discontinuing the protocol because of poor protocol compliance, or terminating the study early following a planned interim analysis with highly significant results. An interim analysis that strongly suggests that the protocol cannot be successfully completed, for example if the initial study design estimates are found to be invalid or seriously inaccurate, also can lead to the recommendation for early study termina-

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tion. Other issues of concern in monitoring trial conduct include the problems of premature publication, and exaggerated estimation in trials that are stopped early [73].

Monogenic 11%

Impact of genetic data on clinical trials conduct The inclusion of genomic data in clinical trials will influence future organizational structures to provide the optimal infrastructure for management and analysis of the complex merging of genetic and clinical data. The application of industrial processing and quality improvement techniques may emerge more prominently to assist in decreasing the attendant possibility for errors with such complex data [74]. Clearly, the addition of genomic data to clinical trials raises new ethical questions regarding confidentiality, particularly in light of the enactment of the Health Insurance Portability and Accountability Act. In the past, there was relatively unrestricted access by third parties for secondary uses of data, and inadequate data anonymization, neither of which is tolerated under the Act’s regulations that became effective in April 2003. There are significant technologic and policy issues that must be addressed in protecting participant confidentiality and security of the data. Concerns about subjects’ rights, informed consent, privacy, and ownership of genetic material require strict attention in the development of DNA banks [16]. The biggest challenges to data access likely will stem from the complex organizational, social, political, and ethical issues that must be resolved to allow linkage of clinical and DNA information. Genetic data are more reliable, persistent, and specific than ordinary identifiers such as name, social security number, etc. As genetic research proceeds, such data may come to be highly predictive of current and future health status, making the data extremely sensitive in nature. It is likely that a separate consent process and/or wording for genetic tissue analysis will be required in the future. It may be necessary to move the consenting process earlier in the participant recruitment activities when genetic testing is a key component of eligibility screening for a clinical trial. Within the clinical trial setting, some test results may be noninterpretable with respect to the impact on health or treatment decisions, and should remain within the realm of the research team and database, at least until such interpretation becomes available. Safeguards will be critical when genetic data are incorporated into routine clinical practice as well. Privacy policies and procedures will be required to minimize the risk of inappropriate disclosure of private information for linked samples. The HIPAA mandates both high standards of data privacy, as well as the extent of patient access to their own data, and extends to clinical data generated by researchers who also are clinicians providing patient care. The use of genetic manipulations to create novel interventions dictates the need for greatly increased monitoring of protocol conduct, decision support for patient treatment, identification of adverse events in real time, and regulatory reporting of severe adverse events (SAEs). Figure 28.4 shows the number and type of gene transfer trials that were ongoing in 2002, with cancer being the predominant focal area and encompassing 62% of all such trials. As the number of gene transfer trials has grown, so has the number of SAE reports, from 285 reported from 1988–99, to 3263 reported in the period 1999–2002. In an attempt to enhance the safety of such highly experimental trials, the NIH has developed a system for the automated reporting of SAEs stemming from gene transfer trials, called the Genetic Modification Clinical Research Information System (GeMCRIS). This system is intended to facilitate the evaluation and analysis of safety information across all gene transfer clinical trials, and to provide database reports that will inform diverse user groups, including IRBs, bioethics committees, DSMBs, investigators, and research participants themselves. Secu-

Marking 8%

CVD 8%

Other 3%

Infectious disease 8%

Cancer 62%

Fig. 28.4 Gene transfer trials by type of application. CVD, cardiovascular disease.

rity features are in place to protect trade secrets and confidential commercial information contained in the database. GeMCRIS is web based and could provide the basis for continuous review of studies involving humans, even being suggested for extension to nongene-transfer trials in the future. It is hoped that the use of such systems will optimize patient safety, while providing useful data to inform the design and conduct of both ongoing and future trials. The critical nature of such SAE reporting was highlighted by the closure of a French trial studying human gene transfer as a possible treatment for X-linked severe combined immunodeficiency, after enrolling 11 patients. The strategy of the experiment was to correct the early block in T-cell and natural killer lymphocyte differentiation by transducing the subjects’ CD34+ cells ex vivo with a defective Moloney murine leukemia retroviral vector. This vector contains the common γ gene for the cytokine receptors responsible for the delivery of growth, survival, and differentiation signals to the early lymphoid progenitors. While nine of the 11 subjects experienced significant restoration of their immune system following the gene transfer intervention, two of the nine also experienced SAEs that appeared to be directly related to the experimental therapy. Both children developed a T-cell leukemia that appeared to be due to insertional mutagenesis after receiving the gene transfer product. Calls for tightening of government oversight of gene therapy research in humans also were heard following the death of an 18-yearold participant in a gene therapy experiment at the University of Pennsylvania involving transferring DNA to treat a genetic defect that reduces liver function. However, as of this writing, this single death is the only one directly attributable to gene therapy research. It is hoped that future government oversight will not restrict genetic research unreasonably, as it holds such great promise for the eradication of life-threatening disease.

Database management for clinical research The choice of software tools for research data management depends on the complexity of the study design and the volume and types of data to

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be collected. Options range from simple spreadsheets such as Excel for a very small straightforward study, to an Access database for a study of intermediate size and complexity, to MS SQL Server, SAS or Oracle databases for larger, more complex data collection efforts. For any approach chosen, however, some basic principles of database design hold true. Each entity (e.g. person) in the database requires a unique identifying code. More than one type of datum should not be mixed within the same data field, for example, entering both the value for weight and the code for kilograms as one data point within the field. Continuous values should always follow the same format and convention for data entry (e.g. white blood cells × 103), and categorical data should be entered using invariant coding schemes (e.g. M for male, F for female). Deviations from these basic rules make the ultimate data analysis and interpretation of the database difficult if not impossible without a major reworking of the entire dataset. Options for statistical analysis software also range widely, depending on the complexity of the required analyses and the expertise of the individual doing the programming. Data entered into an Excel spreadsheet can be manipulated directly within this system for simple statistics such as frequencies and means. Anything more complex than this is best run in an actual statistical analysis package, with the simplest being menu-driven systems such as Systat, and the more complex but powerful choice for biomedical research typically being S-Plus, R or, more typically, the SAS statistical package. Typically, few physicians have the time or inclination to learn to program these latter packages adequately, and would be better off consulting a biostatistician for analyses at this level of sophistication. In the future, the health-care institution within which clinical trials are being conducted is likely to be fully networked, facilitating the deployment of electronic data capture and decision-support tools, and allowing health-care providers and biomedical researchers ready access to electronic information for health care that could be used for biomedical research activities [75]. However, the real challenge in integrating data from electronic clinical care systems into a clinical trials data system has to do with the information access, data flow, coding schema (or lack of coded data in the case of free text information), compatibility of the rules and timing for data collection, comparability of field definitions, political and organizational boundaries, data security, and confidentiality. In large part because of all these challenges, the current clinical trials process is coordinated and monitored largely offline rather than dynamically, requiring duplicate data entry and resulting in inconsistencies, increased errors, and delays in reporting and analysis. Documentation of protocols is nonstandard, and there is no consistent means of determining eligibility for them. The health-care data network will need to be able to translate to and from any electronic medical record system to allow data to be collected at the point of use in a structured form to facilitate clinical trials and outcomes analysis. Further, unifying information “architecture” needs to be developed for cancer clinical trials, so that data can be exchanged across systems and institutions in a seamless fashion, based on protocols that are written in a standard format and structure. At City of Hope National Medical Center, we have developed a webbased system that delivers protocol information dynamically from our clinical trials database [76], and are working with the Association of American Cancer Institutes to achieve virtual exchange of protocol information nationwide. The National Cancer Institute’s Center for Biomedical Informatics also is working to harmonize the informatics approaches to cancer center clinical trials information management across the nation’s cancer centers through the caBIG initiative [77].

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Impact of genetic data on clinical trials information systems The rapid pace of future genetic discoveries and trials will result in extremely demanding data integration requirements. Much more flexible and responsive clinical trial data management systems will be needed to incorporate the high throughput of genomic data, while establishing the correct identifiers and appropriate key structure to be able to merge these data with incoming correlative clinical information. In addition to data from DNA or other genetic tissue from the participants in the dataset, a high-quality, large-sample clinical dataset with well-characterized participant and longitudinal follow-up for effects of treatments will be needed, both to store analyzable data for the clinical trials, and to provide the databanks for exploratory mining for future discoveries and new hypothesis generation [16]. A new generation of professionals with both biomedical and computer science skills will be required to provide advanced biomedical informatics and biostatistical expertise to build and use the critical tools for data retrieval, storage, classification retrieval, and correlative analysis capabilities for complex clinical and genetic data. With the advent of clinical trials that incorporate genomic components, the need for electronic data integration standards across systems and institutions becomes even greater. To facilitate sharing and pooling of genetic results worldwide, standards must be adopted for the coding and exchange of such data, and genetic protocol results will need to be published in a sharable electronic form, for merging into existing resources such as GenBank and OMIM.

Analysis of clinical research data Increasingly more sophisticated statistical techniques are required for HCT research, along with the application of sound basic biostatistical principles. The protocol needs to clearly define the primary and secondary endpoints to be analyzed, and the statistical plan for conducting the analyses. Certain biologic measurements may have more than one usage, and our understanding of the meaning of these endpoints may be evolving over time with new discoveries, such that definitions need to address how the information will be applied within the specific protocol. For example, the study of MRD generally aims to understand the biology and clinical significance of leukemia that persists in patients who are in complete pathologic remission [64]. Following marrow transplantation, the detection of MRD usually has been associated with subsequent relapse in childhood acute lymphoblastic leukemia, t(15;17)+ AML, and CML. However, MRD also has been detected in patients enjoying longterm remission. Therefore, the study of MRD has evolved from the objective of identifying patients at high risk for relapse, to explaining how an abnormality associated with leukemia can persist for years in an otherwise “cured” patient. This section on statistical analysis first covers some of the general principles and guidelines to be observed in the analysis of clinical research data, and then discusses more specific forms of analysis that frequently are applicable to HCT data. The intent-to-treat principle Biases and misinterpretations can be caused by careless use of the definitions of censoring in the statistical analysis, such as assuming that losses to follow-up due to withdrawal from the study are not related to differential risk of the endpoint. More obvious risks of bias can arise through the common practice of eliminating patients from the analysis after entry into the study by declaring them to be “inevaluable” for the primary study endpoint. The argument is that if the purpose is to deter-

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mine whether a new treatment is effective in forestalling or preventing disease, a patient who fails to go through the assigned cycle of treatment (e.g. because of failure to take the full course of drugs) should not be regarded as contributing valid information on the question. While the concept itself is valid, it is clear that deleting patients who are unwilling or unable to take the assigned regimen provides only one picture of the difference between treatment efficacies. Including in the comparison all patients thought to be candidates for the treatments at the time of randomization can provide quite a different view, and answer quite a different question. The inclusion of all patients in the analysis is called the “intent-totreat” approach, for obvious reasons. Both approaches are reasonable, but the plans and logic should appear in the protocol. Generally, the FDA will want an intent-to-treat analysis because the treatment difference expected in practice, taking valid account of drop-outs in actual practice, is most relevant in approving a new drug for the market.

Subgroup analyses Another common pathway to faulty conclusions lies in searching for possible treatment differences by analyzing multiple subgroups of patients, with the idea that although the new treatment may not be efficacious for all patients who are believed to be reasonable candidates for the study, it may be efficacious for certain subgroups (e.g. women versus men, younger persons versus more frail older persons, or patients with less serious disease versus more advanced disease). Planning such searches and the specific analyses at the writing of the protocol is wise. Unplanned searches at the end of the study will increase the possibility of uncovering differences that are due to random variation alone, and hence risk erroneously concluding that the treatment is effective in some subgroup. If such searches are planned beforehand, the size of the study can be enlarged to minimize the chance of such errors in looking at subgroups. Investigators should be clear in reporting the results of clinical trials to point out the degree to which conclusions may have arisen through unplanned searches for subgroup treatment differences, and readers should be aware of the possible dangers of undisclosed subgroup analysis.

Interim analyses The protocol should clearly state the plan for accruing patients to the trial, the target time and number of patients to be accrued, and the plan for stopping the trial and carrying out the target analysis. However, it is common to analyze the data at some preliminary points in the accrual and follow-up of the patients. Interim evaluations are conducted to protect patients against unexpected excessive or severe toxicities, and to allow earlier termination of the trial if that seems wise, either for unforeseen difficulties in execution of the trial, or for data suggesting a large and persuasive advantage in efficacy for the experimental treatment in a randomized study. Here it is important to protect both the patient and the study against overinterpretation of what may be chance variations in early data (see the section on DSMBs and also a comment in the next section, on statistical guidelines for early stopping of clinical trials). Group sequential methods call for analyzing accumulating responses in groups as they become available. The strategy is to identify a priori stopping boundaries at each interim analysis time point that preserve the desired type I and type II statistical errors. An adaptive design is one in which the data accumulating in a trial are examined periodically – or even continually – with the goal of modifying the trial’s design depending on what the data show about the unknown hypotheses. Among the

possible modifications are stopping early, extending the trial beyond its original sample size if some of the assumptions in the original design are not being met, reducing the power, dropping arms or doses, and adding arms or doses. Although adaptive designs can be conducted using the frequentist approach, in classical models flexibility is penalized and adaptation is difficult to effect, while the Bayesian approach appears more flexible, although most still require that the frequentist operating characteristics at least be simulated to provide adequate control of type I error. Berry gives a general introduction to the Bayesian approach and describes its role in clinical trials [78,79]. Returning to the example of the accelerated FDA approval of Gleevec, the protocol-specified analysis was to have occurred at 5 years. However, an interim analysis conducted after a median follow-up time of 14 months revealed a statistically significantly lower time to treatment failure (i.e. disease progression) in the Gleevec arm over the arm receiving interferon and cytarabine. It is important to note that because FDA approval for use of Gleevec in CML patients was based on an early interim analysis, the long-term effects of treatment with Gleevec were largely unknown initially.

Equivalency testing While the majority of HCT trials are interested in demonstrating a significant difference in efficacy, equivalency studies also are being reported more often in HCT, to demonstrate that there is a nonsignificant difference in efficacy. In such a setting, the classical test of significance can be inappropriately applied, with the danger of incorrectly accepting the null hypothesis of no difference solely because of inadequate sample size. If one wishes to use significance testing, the appropriate “null” hypothesis is that the standard therapy is more effective than the experimental therapy by at least some specified amount [80,81]. Since correct interpretation of the significance tests relies heavily on sample size, and statistical significance is often overemphasized as a binary decision rule, some investigators conduct equivalency trials using confidence intervals [82]. This approach is more intuitively appealing, and allows for sequential monitoring using repeat confidence intervals. It is impossible to demonstrate that a new treatment is exactly equivalent to a standard treatment. Therefore, to examine therapeutic equivalence, a maximum “acceptable” difference (delta) in the effectiveness of two treatments must be specified in advance. With a fatal outcome (e.g. overall survival), the new treatment can only be considered equivalent to the standard if no more than a very small decline in efficacy is allowed; with less toxic outcomes, larger differences may be clinically acceptable. Other parameters that must be specified are the confidence level (alpha) for the upper limit of the true difference between the new and the standard treatment, and the probability (1 − beta) that the confidence limit for the true difference will not exceed the specified value of delta [82]. Because the acceptable difference between two equivalent treatments usually is small (particularly in contrast to the larger differences desired when demonstrating superior therapeutic efficacy), the required sample sizes tend to be large. Whenever the common response rate is greater than the quantity (1 + delta)/2, sample sizes for equivalency testing are lower than those required if the same trial were to be (inappropriately) conducted using the traditional significance testing approach. Frequently in HCT studies, if the response rate is too low (e.g. less than 50%) investigators are more likely to conduct efficacy trials in an attempt to identify better treatments. It is only when response rates are satisfactorily high or toxicity is severe and the intervention is primarily intended to lower toxicity, that equivalency tests become of paramount importance.

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Analysis of recurrent states The statistical discussions so far apply with events that occur once during follow-up, for example the risk of first progression after HCT, death, or first occurrence of GVHD. However, often there is interest in not only the occurrence of a state (e.g. GVHD, pulmonary infection or hospitalization for any reason), but also how long it persists and how often it recurs. Describing such data for a group of transplant patients followed over time, with censoring, and comparing groups of patients, present statistical challenges. One measure of the importance of the state of interest (e.g. GVHD) is the proportion of surviving patients, uncensored by specific definition in the protocol, who are presently in that state at each time post transplant. This can be estimated by the prevalence curve [83], taken from Storb et al. [84] and shown in Fig. 28.5, which provides an estimate of the prevalence of chronic GVHD among disease-free surviving patients who received prednisone and in those that did not. The figure also indicates that chronic GVHD is higher among the patients who had earlier developed acute GVHD. However, as the investigators point out, one must be cautious in drawing causal conclusions, as the groups compared are defined by events that could be linked causally to the occurrence of chronic GVHD in ways that might obscure or complicate the interpretation. Examples would be the relationship between survival and the use of prednisone, or the preceding occurrence of acute GVHD and its concomitant implications for prednisone treatment and survival. As another technique for states that may recur and are sequential in nature, the probability that a patient is in a given state can be modeled by Markov or semi-Markov multistate models. These models have been

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used by Klein et al. [75] and Keiding et al. [85] to study the antileukemic effect of GVHD on relapse and death in remission following transplantation. Surrogate endpoints Cancer clinical trials often experience a problem in recruiting patients to studies, and/or sustaining the length of follow-up necessary to accumulate definitive endpoints, such as overall survival. In addition, subjects usually branch to alternative therapies upon progression, increasing patient variability, and new therapies arise, limiting conclusions. Studies of AIDS are even more difficult, as subjects are known to jump from one experimental study to another before disease progression. This prompts the statistical considerations of earlier endpoints as substitute or “surrogate” markers of later fatality, with the idea of drawing reliable conclusions about comparative risks of death from earlier results. Disease progression in cancer and in HCT has been used as a surrogate marker for length of life, but with some reservations about the reliability in translating progression comparisons to valid prediction of differences in mortality and RR. As more is learned about early markers in HCT, the interest in time-efficacy in research becomes more acute, and interest in the use, of early markers as surrogates to guide research grows. Two useful papers in this area, one by Prentice [86] that deals with statistical aspects of the definition and operational use of surrogate markers, and another by Ellenberg and Hamilton [87] on surrogate markers in cancer, provide good introductions to these concepts and related applications. Analysis of time to an event

1.0

Grade O Acute GVHD

Prednisone

0.5

No Prednisone 0 Grade I–IV Acute GVHD

1.0

Prednisone 0.5

No Prednisone 0

0

1

2

Years after Marrow Graft

Fig. 28.5 Prevalence curves for active chronic graft-versus-host disease. (Reproduced from [84], with permission.)

3

While most phase I and II studies focus on a binary end point, such as toxicity or response to therapy, phase III studies (and some phase II transplant studies) typically concentrate on longer-term events and the time to those events, such as time to progression of disease or survival time after transplant. The data for a given patient will include the time from HCT to occurrence of the event or to the time of data analysis, if the patient is treated successfully. Because the times of transplant vary over the study accrual period, each patient will have his or her own data point consisting of the time from HCT (or study entry) to event or end of the study at time of analysis. There also will be information as to whether or not the patient experienced the event of interest (e.g. relapse or death) by the time of analysis. The patient who avoids the event by the time of analysis is said to have a “censored” data point, as all that is known for that patient is that the event has not happened thus far. This form of censoring is called “administrative” censoring, because the incomplete information on the patient is due to the administration of the study, rather than to any irregularities in the data collection. More information will not be known about the censored patients unless the study is extended by further follow-up of all surviving patients and is reanalyzed. When allowing additional follow-up, it is, as noted earlier, critical to avoid passive follow-up due to the bias created. In comparing the results for two groups containing censored data points, standard methods for comparing means, such as the two-sample t-test, should not be used, as they do not take into account the censored aspect of the data. Methods for handling censored data are covered in several statistical texts [34,35], and texts that describe methods that are more advanced yet intended for the clinical scientist rather than the statistician also are available [88–90]. Often an extended time of follow-up is required to observe sufficient endpoints to allow a statistically precise determination of the relative superiority of one or the other treatment. Long periods of study will necessarily mean that some patients will be lost to follow-up due to other

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than “administrative” censoring, for example moving away from the study site or decision of the patient to leave the study for personal reasons. These losses to follow-up will also result in censored data points, and are usually treated in the same way as administratively censored data points in the analysis. However, losses to follow-up for reasons other than administrative censoring can be due to reasons that build biases into the study and the analysis. If the patients tend to drop out of one of the treatment arms for reasons that might be associated with the treatment itself and/or with the risks of the endpoint of interest (e.g. due to excessive toxicity combined with patient refusal for follow-up), an analysis that treats this as administrative censoring has these biases built into it. The analysis may then be misleading, particularly if such biased losses to follow-up occur relatively frequently. On the other hand, administrative censoring should not create a similar possibility of bias, as the patients are blindly and randomly assigned to the treatments over time. This form of censoring is considered to be “uninformative,” meaning that the reasons for censoring should not be expected to be related to the risk of event if the patient were to stay in the study. Kaplan–Meier survival analysis The familiar Kaplan–Meier graph is used for describing the typical censored clinical trial dataset, plotting estimates of the probability of “surviving” to time “t” post transplant (i.e. the probability of not experiencing the endpoint, e.g. progression, from transplant to time t). The graph indicates the times of death by dropping down a fraction of its value at each time of death (the event of interest). The surviving (censored) data points typically are denoted by hash marks on the survival function. In HCT studies, the survival pattern differs from more common survival distributions, with a high early mortality rate followed by a plateau in mortality after transplantation. Unfortunately, interest is often highest exactly where the data are least informative – at the end of the curve, where few patients are still at risk. This uncertainty should be quantified by reporting either pointwise confidence intervals or confidence bands. Standard errors can be computed for the Kaplan–Meier curve at any time point, and the estimated survival curve graphed with a plus and minus distance (confidence limits) around the curve at a few time points.

interval-censored data, and Kim et al. [95] for doubly-censored data. Even when technically incorrect, most HCT and cancer trials report survival analysis as if the event in question (e.g. progression or relapse) occurred on the last day of the interval. Fortunately, randomization protects against the influence of this approximation. In nonrandomized studies, the maximum influence is based on the surveillance interval. As long as clinically relevant differences are much larger than the surveillance window, the influence is limited, and as a result this is generally a bigger problem for nontransplant studies. Weighted Kaplan–Meier estimator for matched data. Studies on the comparison of transplantation with respect to standard therapy present a number of statistical challenges: they usually are not randomized, often are retrospective (based on registry data), and the treatment assignment is time dependent (waiting time to transplant). These challenges motivate the use of matching on a set of known prognostic factors and the waiting time to transplant, as an approach to identify a set of “controls” from the group of nontransplanted patients. Usually, the latter is a larger group than the transplanted one, and thus is likely to have more than one control for many of the cases. When a variable number of patients treated with conventional therapy matches each transplanted patient, the standard estimating and testing procedures need to be modified in order to account for the fact that matched data are highly stratified, with strata containing a few, possibly censored, observations. A weighted version of the Kaplan–Meier estimator, which accounts for a variable proportion of matching, has been proposed and its statistical properties studied [96]. The problem of the comparison of the survival experience in the two treatment groups was addressed, and two tests, based on the distance between the survival estimates calculated at a prefixed time point, were examined through simulations, the aim being that of obtaining an unbiased estimate of the effect of HCT. The procedures proposed were applied to data collected from an Italian study whose aim was the evaluation of HCT compared with intensive chemotherapy for pediatric patients with acute lymphoblastic leukemia. Although the focus was on a retrospective observational study, the methods proposed also are applicable to prospective studies. The log-rank test

Analysis of multivariate interval-censored survival data. Multivariate failure time data are observed in biomedical studies when subjects are followed for the occurrence of multiple events, such as recurrent infections or GVHD. In some studies, the multivariate events can be interval censored [91]. Interval-censored survival times occur when the outcomes are not directly observable but are detected from periodic clinical examinations or laboratory tests, such as periodic PCR testing for positivity for the Ph chromosome, or cytogenetic testing for signs of disease relapse. In interval censoring, the exact times of events are not known since the events could have happened at any time during the interval between the last visit when the subject was determined to be negative for the outcome and the first positive visit. Also, the timing and width of the interval often differ across subjects. Other examples of multivariate interval-censored data are from time to multiple tumor recurrences, which are usually diagnosed from periodic clinical examinations. If all study subjects were monitored according to the same examination schedule and no appointments were missed, this type of data could be treated as multivariate grouped survival data and analyzed using the approach of Guo and Lin [92], a grouped-data version of Wei et al.’s method [93]. However, the approach is not applicable to the more common situation when there are missed visits. In the proposed approach, the marginal distributions are based on a proportional hazards model for discrete survival data that has been utilized by Finkelstein [94] for singly

The analysis for comparing two curves that represent two treatment groups in a phase III study is usually carried out by a log-rank test of statistical significance (sometimes called the Cox actuarial or proportional rank analysis), and an RR is calculated, providing an estimate of the risk of failure in one group compared with the risk of failure in the other. This single number is based on the assumption that the RR between the two groups does not change over time. Several texts describe these calculations in detail [34,35,88–90]. If the RR is greater than 1, the data favor the control group; if it is less than 1, the experimental group is favored. The 95% confidence interval for the RR is preferred to simply the RR and the log-rank p-value. In the accelerated FDA approval of Gleevec mentioned earlier, the risk of progression was compared by the log-rank test. The RR was calculated to be 0.183, with a 95% confidence interval of 0.117–0.285, and a p-value much less than 0.001. The results from such a log-rank analysis might be represented by the layout as found in Table 28.6. The

Table 28.6 Results of log-rank analysis Treatment group

Relative risk

95% confidence interval

p-value

Gleevec

0.183

(0.117, 0.285)

<0.001

Biostatistical Methods in Hematopoietic Cell Transplantation

line of information refers to the experimental group receiving Gleevec in the phase III randomized study, with the RR of disease progression, expressed relative to the risk in the standard treatment group receiving interferon and cytarabine. The last three columns show the estimated risk of progression for the experimental treatment relative to the risk for the control treatment (0.183), suggesting that the Gleevec treatment group treatment will have 18% of the risk experienced by the interferontreated group. The confidence limits indicate a statistical precision of the RR estimate, ranging from approximately 12% to 28%. The p-value of <0.001 indicates that, in a study of this size, an estimate as far from the null value of equal risk (RR = 1.00) as the observed RR of 0.183 would occur by chance alone (no difference between the two groups) less than once in 1000 repetitions of the clinical trial. Many log-rank analyses adjust the estimate of the treatment effect, the interval estimate, and the p-value for baseline differences in the treatment groups. However, sometimes it is of interest to ask whether the RR of the experimental treatment compared with the standard treatment seems to change through the time of follow-up, when a change occurs in some covariate or simply based on a change in behavior of the curves at a certain time post transplant. For example, some marker of tumor burden might be monitored at specified time points, and the RR estimated as a function of the measured marker. Such an analysis is an extension of the log-rank approach and can be done with current readily accessible statistical software. However, as both the marker and the endpoint (e.g. clinical disease progression and mortality) are now allowed, and even expected, to differ in the two treatment groups, interpretation of the results with regard to causal linkage must be handled carefully, and such analysis is usually limited to exploratory work. Because of the high early mortality in HCT followed by a relative plateau, inferences based on the log-rank statistic are not well suited to studying long-term survival, potentially reporting a difference between study arms even though no long-term survival benefit exists. An alternative approach is to conduct a separate analysis of long-term survival, modeling the population as a mix of long-term survivors and patients undergoing an earlier failure process [97]. The failure distribution is used in a mixed model, where a probability of long-term survival is postulated. Such techniques are now implemented in S-Plus [98]. This parametric approach has its own set of limitations, and in particular model misspecification can inflate the type I error [99]. Cox proportional hazards model The Cox proportional hazards model can be used to detect multivariate effects and estimate effects in both a univariate and multivariate setting. Again, the validity of the estimate is dependent on the proportionality assumption, and a variety of techniques exist for testing this assumption (see Marubini and Valsecchi [88] for a discussion). If the violation is detected, time-dependent covariates can be used, although it makes interpretation more difficult. Generally, questions about the validity of the proportionality assumption arise when the statistical tests are inconclusive but the survival curves differ by a clinically important amount. In such a setting, it is often worthwhile to pursue either parametric survival methods or time-dependent covariates to better interpret the result. If the phase III study design has been stratified by gender, the analysis can be done for each gender separately, as above. However, the results can be averaged over the two gender strata to obtain one overall estimate of the RR, on the assumption that the RR is approximately the same across all characteristics and times within each gender and across the two gender strata as well. The analysis is simply an extension of the proportional hazards model, using the log-rank or Cox approach. If a baseline variable was used as a stratifying variable in the randomization, it is prudent to include it in the analysis, as presumably it was believed

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to be predictive of the endpoint, and care in balancing the groups within strata will provide more statistical precision in estimating the RR. Inclusion of this additional variable in the log-rank analysis reaps that benefit and also provides information on the degree to which the variable (e.g. gender) is indeed predictive of the endpoint. Other prognostic or predictive variables not even used in the randomization but measured at baseline (e.g. stage of disease before transplant or age of the patient at transplant) can also be used in the Cox proportional hazards analysis. It is important to evaluate the impact of center effects in case of either survival outcomes [100] or the Cox model [101]. Competing risks Transplant recipients face multiple competing risks for adverse events, especially in the early post-transplant period. While most HCT clinical trials target a single outcome, such as transplant complication, for example GVHD or sinusoidal obstructive syndrome, usually there is interest in several types of endpoint. One interest is in the risk of treatment failure due to the transplant itself, such as the toxicities associated with the transplant and possible engraftment failure. On the other hand, there is interest in the failure of the transplantation process in preventing progression of the target disease and enhanced survival of the patient. The protocol should provide clear-cut methods for defining and distinguishing the two types of failure as two types of endpoints, along with their times of occurrence. In this case, two different analyses can be carried out, using the same log-rank analysis. One can regard one of the events, for example death due to disease progression, as the event of interest, and regard the other event (death due to the transplant procedures) as a censoring of the data relative to knowledge of the risk of the treated target disease. Of course, the data also will have administratively censored data points, and perhaps a few other censored points due to other losses to follow-up (e.g. patients moving out of the range of observation). Then a second analysis can be performed for the other endpoint, this time considering death due to the target disease as a censoring event that prevents observation of an event attributable to the transplant itself. Here the attribution of risk to the types of failure is subject to questions of informative censoring, and definitions and procedures need careful consideration. Interpretation of the results can be problematic, even though the log-rank analyses themselves are well defined, as censoring for these competing risks creates an opportunity for confounding, and the resultant survival function is formally not a proper survival function. It is universally agreed that the Kaplan–Meier analysis overestimates the probability of the event of interest in the presence of competing risks [102]. Kalbfleisch and Prentice [103] recommend using the cumulative incidence as an estimate of the probability of an event of interest. The method of Fine and Gray [104] often is used to compare groups in the presence of competing risks. The Cox proportional hazards model gives valid results under modest assumptions when employed in testing the effect of a covariate on the hazard rate and when estimating the hazard ratio. A method to calculate the sample size for testing the effect of a covariate on outcome in the presence of competing risks is also provided. Impact of genetic data on clinical trials analyses The eventual goal of connecting the billions of pieces of correlative data available in the post-genomic era to occurences in the clinic requires the disciplines of bioinformatics, clinical research informatics, and biostatistics. It will be possible to begin to answer the question regarding why some people fail and some succeed within the same clinical trial, possibly explained by a specific genotype. Successful drugs have been withdrawn due to a few severe adverse reactions. With the advent of

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genetic information, we may be able to predict in advance who will be able to tolerate a new drug, to more fairly assess the probability of a successful impact on the disease. Clinical trials incorporating genomic data will lead to the capability of “personalizing” medicine. New biostatistical methods and novel applications of standard biostatistical techniques will be required for genetic trials. These new methods will involve “supervised analyses” including hierarchical clustering of tumors to look for differences in risks, drug updates, or outcomes. Future approaches will include stratification and testing of genetic subtypes related to survival, and use both microarray quantitative and reverse transcriptase-PCR data, and novel approaches to apply traditional principles to massive quantities of data. The amount of “noise” in microarray data often is ignored. Small changes in expression that are indistinguishable from random noise easily lead to false positives. Several such false-positive findings were inadvertently published in leading journals in 1999. In future, a semiautomated discovery of patterns, associations, and statistically significant genetic structures will be accomplished via “data mining.” This area borrows from the fields of artificial intelligence and neural networks, and allows the discovery of information hidden within huge datasets. Algorithms for analyzing genetic determinants of cancer susceptibility will need to be developed, sample size programs will need to be extended to determining power to detect genetic disease associations, interaction tests will be needed to detect genes involved in multilocus models, and association tests of observed versus predicted allele-sharing probabilities will be required.

Reporting of clinical trials results Increasingly more sophisticated statistical techniques are being utilized for HCT research, requiring more advanced expertise to apply them correctly. The omission of key elements from the design and conduct of such research studies limits their usefulness, as well as the ability of readers to interpret the results and draw informed conclusions. Accurate, complete reporting is critical to be persuasive to the audience at hand, and allow the critical reader to assess the validity, comparability, and generalizability of the study conclusions.

A survey article reviewed the transplantation literature to determine the quality and variety of analytic techniques being employed for 255 HCT articles included in several leading journals [105]. It was found that 20% of studies lacked some key elements within the analysis, 10% employed statistical methods that were inappropriate, and an additional 10% provided insufficient information to allow an assessment of the statistical methods. It is imperative to include sufficient detail to allow the reader to evaluate the appropriateness of the techniques employed and the validity of the results. There were 63 articles that reported nonsignificant results based on small sample sizes; however, 86% of these failed to provide an estimation of the study power, essential to interpreting the true meaning of these results. Although room for improvement remains, the past decade has seen substantial efforts to establish meaningful guidelines for reporting of clinical trials in the medical literature. Detailed publication guidelines have been given by George [106], Bailar and Mosteller [107], Zelen [108], Simon and Wittes [109], Mosteller et al. [110], and Simon and Altman [111]. Table 28.7 provides a suggested publications checklist for prospective authors of HCT studies, synthesized from several of the articles listed above. The Consolidated Standards of Reporting Trials (CONSORT) was developed to improve the suboptimal reporting of randomized controlled trials [112]. For 105 randomized clinical trials from 29 medical journals, the quality of reporting has been examined in relationship to whether a journal was a “CONSORT promoter,” as defined by inclusion of the CONSORT checklist in a journal’s “information to authors” section or a requirement that authors, manuscript reviewers or copy editors complete the CONSORT checklist. It was found that the number of methodologic factors reported was greater in CONSORT promoters than in journals not promoting CONSORT (6.4 versus 4.8 of 11 methodological factors, respectively; p-value = 0.0001) analyses. While journals that promote CONSORT demonstrated superior reporting of randomized controlled trials, persistent inadequacies in reporting remain. The items that fell below the 50% reporting level were: concealment of randomization (46%), data collector blinding status (37%), health-care provider blinding status (34%), co-interventions for each group during the study (30%), and data analyst blinding status (4%).

Table 28.7 Publications checklist 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

All important study-related dates should be given, such as the date the study opened, the closing date, and the approximate date of analysis (which could differ substantially from the date of publication) The population under study should be fully specified, including the eligibility requirements, exclusion criteria, and selection methods The type of study design should be specified, including whether it is a phase of a clinical trial, a prospective cohort study, a retrospective case-control study, a prognostic study, etc. The objectives and hypotheses should be clearly elucidated Details of the clinical trial or study design should be given based on the type of study, such as dose-escalation rules for a phase I trial, stages of accrual in a phase II trial, blinding techniques for a randomized phase III trial, matching criteria for an observational study, etc. In all cases, some justification of sample size and power considerations must be included The intervention involved should be described with sufficient detail that it could be reproduced The choice of controls should be elucidated, including potential biases for nonrandomized studies, patient or physician self-selection, and any known differences among patients, such as diagnosis, staging supportive care, evaluation, follow-up or prognostic factors All registered patients must be accounted for, including withdrawals or exclusions Mechanisms for ensuring data quality should be described, including quality control checks, review of response, toxicity assessments, etc. The follow-up for each patient should be summarized, including the distribution of time between the data analysis and the last patient accrual, and the last dates of contact for all patients who remain at risk of failure Sufficient detail should be provided to allow the reader to reproduce the analyses if the data were available. There should be a statement regarding adjustments made for multiple comparisons or multiple endpoints. Estimates of precision, i.e. confidence intervals or standard errors, should be provided. Modeling, stepwise procedures, and the creation of cut-off points need to be fully described

Biostatistical Methods in Hematopoietic Cell Transplantation

Other recommendations to help investigators improve their ability to appropriately interpret and apply medical statistics include additional medical school and postgraduate courses in statistics for physicians, increased recognition of the importance of medical statistics by the research community, and more attention to the statistical aspect of papers in medical journals. Further, an effective collaborating relationship between investigators and biostatisticians can be the key to the successful conduct of medical research. Central to such a successful collaboration is two-way communication, regarding both the scientific area of interest and the study design and analytical issues. Engaging the biostatistician solely to program analyses in isolation from the other stages of the study creation and conduct is suboptimal, and can lead to inappropriate models or misinterpretation of the data. Early interaction with the biostatistician during the study concept and design stage can help to avoid

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intractable problems with the study conduct and data collection. It is important to involve the biostatistician in writing the statistical methods and results of the final manuscript, and to give him or her an opportunity to review the manuscript in its entirety, to ensure that these sections have not been misinterpreted by the medical authors inadvertently. The biostatistician should take responsibility for verifying the consistency of results in the text and tables, and guarding that the conclusions drawn in the discussion section are supported by the analyses. If the recommended guidelines and principles included in this chapter are adhered to by investigators and biostatisticians, the quality of HCT research and literature should continually be further improved and enhanced, resulting in more rapid advancement in this critical scientific area, and more effective dissemination of high-quality HCT research findings.

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Stephanie J. Lee

Outcomes Research in Hematopoietic Cell Transplantation

Introduction

Definition

The field of hematopoietic cell transplantation (HCT) has grown dramatically since the first successful allogeneic transplant procedure was performed in 1968. However, as the subspecialty has matured, questions about costs and cost-effectiveness, quality of life (QOL), patient preferences, medical decision making, and aggregation of different data sources to guide treatment decisions have become relevant. These questions are best addressed by “outcomes” research, a field of study focused on obtaining the best results, broadly defined, given the available medical knowledge and limited health-care resources. A closely related discipline, health services research, is concerned with the social and political determinants of outcome such as access to health care and quality of care. There are several features of HCT that make outcomes studies especially relevant: (1) HCT involves high treatment-related risks compared with other medical interventions; (2) significant practice variation exists; (3) costs are high; and (4) the long-term results, considering both disease-free survival (DFS) and QOL, have much room for improvement. Issues of medical decision making, quality of care, resource allocation, and QOL are material to HCT, and all fall under the rubric of outcomes and health services research. On the other hand, several characteristics of HCT make outcomes studies challenging. For example, HCT patients are often not represented in large, administrative databases that collect standardized clinical, outcome, and resource utilization data. The most active transplant centers still perform only several hundred procedures per year, and smaller centers may do fewer than 10. Thus, the overall impact of HCT on the health of the general population and health-care finances is relatively small. Also, the field is changing rapidly, and adequate information on long-term results is available for few diseases and procedures. This chapter will present a brief history of outcomes research in American medicine to help frame the research topics and methods. The remainder of the chapter is organized around several specific questions in HCT that outcomes research is well suited to answer. Representative HCT studies are used whenever possible to illustrate the principles discussed. Readers are referred to Chapters 28, 34, and 38 for detailed information on related research methods and data sources.

It is difficult to state a precise definition of “outcomes research.” At some level, all results can be considered outcomes; thus, most scientific investigation is concerned with measurement and interpretation of outcomes. However, generally excluded from the definition of “outcomes research” are phase I, II, and III clinical studies addressing efficacy questions when the primary endpoints are toxicity, disease control, and survival. Similarly excluded are clinical epidemiology studies that describe an institutional experience with a disease or treatment. However, when the research question begins to consider how well a treatment works outside of a clinical trial or institutional setting (“effectiveness”), subjective endpoints (e.g. QOL or patient preferences), nonbiologic influences on outcomes (e.g. access, quality of care, physician–patient communication, and medical decision making), health-care policy (e.g. economic evaluation) or aggregation of data from multiple sources (e.g. decision analysis, meta-analysis, and use of administrative databases or registry data), the title of “outcomes research” is appropriate. A conceptual framework that distinguishes outcomes research from other types of clinical research is presented in Fig. 29.1 [1]. Major questions for outcomes research in HCT are:

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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• What are the costs of HCT and how can they be reduced? (Resource utilization, cost minimization.) • Are the clinical benefits of HCT worth the monetary cost? (Cost– benefit, cost-effectiveness, cost–utility analysis.) • What is the patient’s experience with HCT? (Qualitative research, QOL.) • How are new tools developed? (Prognostic models, patient-reported measures, severity of clinical syndromes.) • How can one combine information from several different data sources? (Registry studies, decision analysis, quality time without symptoms of toxicity [Q-TWiST], meta-analysis, evidence-based medicine.) • How can the practice of HCT be improved through health services research? (Access, quality of care, practice variation.)

History The ultimate goal of outcomes research is to improve the practice of medicine through the provision of data about the effectiveness, costs, risks, and benefits of treatment options, incorporating considerations at both the individual and societal level [2,3]. Approaches to achieve this goal in the United States have varied over the decades. Initially, it seemed that funding large-scale research projects would help establish which clinical practices worked and which did not, so that effective practices

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Clinical trials 1

429

Outcomes research Quality of care

2

Phase I, II, III clinical trials

Access Decision making

Treatment under study

Treatment options

Research

Prediction rules Efficacy

Effectiveness

3 OS

DFS

Response

Outcomes: Symptoms

HRQOL

Cost

2nd Analyses: Meta-analysis Decision analysis Applications

Fig. 29.1 Conceptual framework. Interaction is shown between research topics, endpoints, analytic techniques, and applications in defining outcomes research. In 1 are depicted the classic clinical trials and analytic techniques that are not outcomes research. 2 shows the study topics, endpoints, and analytic techniques that are considered to be outcomes research. Outcomes depicted in 3 may or may not constitute outcomes research, depending on the context. For example, overall survival as measured in a phase III trial is not an outcomes study (efficacy), whereas it is if observed in a large community cohort (effectiveness). Symptoms have both efficacy and outcomes influences. Applications are indicated in italic and may emanate from either clinical trials or outcomes research. DFS, disease-free survival; HRQOL, health-related quality-of-life OS, overall survival. (Reproduced from [1], with permission.)

Clinical decisions Examples: Clinical practice guidelines Treatment recommendations

could be promoted. However, it turns out that definitive conclusions about the most effective therapies are elusive because there are so many diverse factors (patient characteristics, patient preferences, and societal priorities) to consider. More recently, outcomes researchers recognized that, while it is important to know what works at a population level and to establish treatment guidelines, scientific methods to incorporate patient values and individualize treatment are also important. This more encompassing view of the situation recognizes the complexity of medical decisions and the often contradictory influences affecting patient outcomes. The “father” of the American outcomes movement was a surgeon named Ernst Codman. As early as 1914 he argued that the quality of hospitals could be judged only if procedure success rates were made available on a routine basis [4]. He advocated standardized measures of outcomes so that different institutions could be compared on a level playing field. In 1966, Avides Donabedian reintroduced the term “outcomes” when he developed his concept of quality assessment and its three components: structure, process, and outcome. He broadened the endpoints of interest: “Although some outcomes are generally unmistakable and easy to measure (death, for example), other outcomes, not so clearly defined, can be difficult to measure. These include patient attitudes and satisfactions, social restoration, and physical disability, and rehabilitation.” He echoed Codman in stating “Outcomes, by and large, remain the ultimate validators of the effectiveness and quality of medical care” [5]. In the 1970s and 80s, the looming crisis of health-care costs pushed outcomes research into the political spotlight. Archie Cochrane, after whom the Cochrane evidence-based database is named, warned that the medical system would become bankrupt if expensive technologies were routinely applied without evidence of benefit [6]. In 1973, Wennberg and Gittelsohn documented a surprising geographic variation in resource utilization, expenditures, and rates of hospitalization and procedures [7]. For example, rates of tonsillectomy varied dramatically within the state of Vermont although health outcomes seemed similar. This observation focused attention on practice variation and the possible cost savings that could be realized by eliminating unnecessary procedures. Several national databases were established to study practice variation in the United States. These included the Patterns of Care Study (focusing on radiation therapy practices), the National Cancer Data Base

Policy decisions Examples: Resource allocation Coverage decisions

(focusing on surgical practice), and the linkage of Medicare billing and Surveillance, Epidemiology and End Results (SEER) data (providing resource utilization and cancer-specific information on patients common to these databases). In addition, the Federal government established the Agency for Health Care Research and Quality to support outcomes research. Although this organization has been renamed and refocused several times, it is probably most famous for developing the Patient Outcomes Assessment Research Teams. The Patient Outcomes Assessment Research Teams program was designed to determine the relative effectiveness of treatments for common diseases, with an undertone of trying to standardize clinical practice. Discourse in the medical journals throughout this period reflected the outcomes movement. In 1988, Arnold Relman labeled “assessment and accountability” the “third revolution in medical care,” following the earlier revolutions of health-care expansion and the backlash of cost containment [8]. In 1990, Arnold Epstein further defined the “outcomes movement” as research efforts to address “the effectiveness of different interventions, the use of this information to make possible better decision making by physicians and patients, and the development of standards to guide physicians and aid third-party payers in optimizing the use of resources” [9]. The 1990s were a period of national economic growth in the United States, and concerns about health-care financing for specific procedures faded into the background behind debate about the overall structure of health-care coverage. Managed care and health maintenance organizations thrived, and physicians practiced in a more constrained setting with new concerns about financial risk. The incentive to save money may have replaced the original goal of outcomes research, which is to spend money wisely to improve overall health. As it became clear that major restructuring of health-care delivery and financing would fail, outcomes research faded from the political spotlight and returned to its origins, focusing on more applied questions. Treatment guidelines proliferated in the late 1990s, but these efforts grew out of a desire to standardize physician practice and improve patient outcomes rather than to contain costs. It is difficult to tell what the future holds for outcomes research, especially in HCT. HCT is a highly specialized practice that, for many, will fall outside of health-care policy and economic considerations.

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Table 29.1 Examples of cost-minimization studies Ref.

Less costly approach

More costly approach

[11,20–22] [23] [24] [25] [26,27,28,29,31] [30]

Peripheral blood progenitor cells for autologous HCT Delayed growth factor support in autologous HCT Growth factor support in T-cell-depleted BMT Acute GVHD prophylaxis with T-cell depletion in unrelated donor BMT Outpatient transplantation Hyperhydration to prevent hemorrhagic cystitis after cyclophosphamide conditioning

Bone marrow for autologous HCT Early growth factor support No growth factor support Methotrexate, cyclosporine Inpatient transplantation Mesna

BMT, bone marrow transplantation; GVHD, graft-versus-host disease; HCT, hematopoietic cell transplantation.

Nevertheless, there are many aspects of HCT that may make outcomes research more relevant. Attention to long-term outcomes, patient decision making and guidance based on what is known about short-term outcomes is especially critical for the field.

Specific questions for the field of HCT What are the costs of HCT, and how can they be reduced? On a per patient basis, the costs of HCT are high relative to other available medical interventions, ranging from approximately $30,000 for an uncomplicated autologous procedure to $200,000 for an allogeneic, myeloablative procedure using an unrelated donor [10–12]. The investment in infrastructure is immense, requiring support of the transplant centers and national resources such as the NMDP and the public cord blood banks. In the United States, insurance companies have tried to limit access to some HCT procedures by designating them “experimental,” but they are often forced by state law or by threat of patient lawsuits to acquiesce and finance the procedures. When it comes to HCT, society has shown itself to be quite willing to follow “the rule of rescue,” defined as the human imperative to help someone facing a preventable death, without regard for the resources consumed or the ultimate likelihood of success. Well-established research methods are available for quantifying monetary costs [13–15]. “Direct medical” cost refers to the monetary value of goods and services provided. These costs are usually captured through administrative billing systems or other itemized methods of determining resource utilization. The distinction between “charges” and “costs” is important in the United States. Charges are the amount billed to the patient or insurance company. Costs are usually lower and reflect the actual resources needed to provide a service. Because health-care organizations offset one expense against another and usually expect some amount of profit, charges are not directly linked to the resources needed to provide a service. Outcomes studies favor the use of costs since they reflect the actual resources expended. When costs are not available directly, conversion between charges and costs may be determined by “ratios of costs to charges,” a fraction recalculated on an annual basis. Institutions often aggregate logical groups such as clinical departments when setting their “ratios of costs to charges.” Fixed costs (such as physical space and personnel) and variable costs (supplies) are totaled and divided by the amount billed by the department during the same period of time. Direct nonmedical costs are expenditures related to health care but not directly used for goods and services (e.g. transportation to the hospital or hotel charges for family members accompanying patients). These costs have proven much more difficult to quantify since they must be captured directly from patients through cost diaries or receipts. Indirect nonmedical costs are even harder to quantify and include time off work and the loss of future earnings. Which costs to include in the analysis depends on the desired perspective. The “societal” perspective

includes all costs to the system regardless of who pays. Other perspectives can be imagined, such as the hospital, insurance company or patient, and would include only costs borne by that payer. One important feature of costs is that they vary by year because of inflation. Thus, it is important to consider the year in which the study was performed and the specific items that are included in the analysis. For example, health-care inflation is calculated from a “basket” of goods similar to the methodology used for the Consumer Price Index, and at 3–9% has outpaced general inflation. These conversion factors, published by the Bureau of Labor Statistics by month and year, help to “inflation-adjust” costs and allow comparability of studies performed at different times (http://stats.bls.gov). “Discounting” is distinct from inflation adjustment and is normally set at 3% per annum. Discounting reflects the fact that costs and benefits in the future are valued less than those that are immediately available, and allows the conversion of future dollars or future improvements in health to their current value. Once the relevant costs have been captured, they can be analyzed in a variety of ways. Some studies simply report the costs of an intervention. Others look for patterns of costs, predictors of costs or ways to decrease costs. Totals, breakdowns by specific categories, trends over time, and association with clinical characteristics or treatments have all been reported in HCT. For example, several studies have evaluated the costs or lengths of stay associated with specific complications or patient characteristics [16–19]. Cost-minimization studies compare the costs of treatment approaches that result in similar clinical patient outcomes. In these cases, adoption of the least costly approach does not compromise patient outcomes. Table 29.1 shows some examples of cost-minimization studies in HCT [11,20–31]. Are the clinical benefits of HCT worth the monetary costs? Deciding whether the clinical benefits of HCT are worth the monetary costs may seem to conflict with a physician’s duty as a patient’s advocate. However, in a society where health-care dollars are constrained, spending money for one person’s procedure ultimately means that another person may not receive some necessary treatment. As discussed below, the various forms of economic analysis (cost–benefit, costeffectiveness, and cost–utility) differ primarily in how they quantify clinical benefits. However, all are trying to provide information that may be used by policy makers to allocate resources and maximize the health and welfare of the entire population. Table 29.2 contrasts the types of economic analysis [32–34]. Cost–benefit analysis requires that clinical benefits be converted into monetary values to determine the net financial impact of an intervention. This is sometimes straightforward (inexpensive prophylactic antibiotics may prevent costly infections later), but it is often quite complicated and fraught with unpalatable value judgments. For example, what is the

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Table 29.2 Economic analyses Type

Equation

Illustration

Conclusion

Cost–benefit

Cost of providing treatment ($) minus benefits of treatment ($)

For treatment X: $100,000 − 0.50*$1,000,000 = −$400,000 For treatment Y: $50,000 − 0.30*$1,000,000 = −$250,000

Treatment X is the preferred approach, although society should support both treatments because they “save” money

Cost-effectiveness

(Cost of X minus cost of Y)/(benefit of X minus benefit of Y)

($100,000 − $50,000)/(0.5 lives − 0.3 lives)*20 years = $12,500/LY

Treatment X is cost-effective relative to other well-accepted medical procedures

Cost–utility

Same numerator as cost-effectiveness but denominator is (quality-adjusted benefit of X effectiveness minus quality-adjusted benefit of Y)

($100,000 − $50,000)/(0.5 lives*0.85 − 0.3 lives*1.0)*20 years = $20,000/QALY

Quality-adjustment raises the cost-effectiveness ratio of treatment X but it is still very favorable

For illustration purposes, consider treatments X and Y. Treatment X costs on average $100,000 per patient but cures 50% of patients, while Treatment Y costs $50,000 and cures 30%. Survivors live for another 20 years. However, patients undergoing treatment X suffer from long-term complications, so that their utility is 0.85 compared with patients undergoing treatment Y, who have a utility of 1.0. Separate studies suggest that, for the purposes of cost–benefit analysis, a life saved through medical intervention is worth $500,000–1,000,000 [32–34]. See text for details. LY, life year; QALY, quality-adjusted life year.

economic value of a life extended or saved? Attempts to use income as a surrogate lead to the uncomfortable conclusion that the lives of highwage earners are more valuable than those of homemakers or retired people [13]. Placing monetary values on goods that are not normally for sale (such as medical procedures or health) can be accomplished by creating a hypothetical market for that good, a technique called “contingent valuation” or “willingness to pay.” For example, one could ask patients, “If there were a treatment that would get rid of your chronic graft-versus-host disease (GVHD), how much would you pay for it?” This would give you an estimate of the “value” of successfully preventing or treating chronic GVHD. However, people have a hard time imagining such hypothetical situations and replying thoughtfully. Some may be offended by the concept of a market for health care, viewing medical treatment differently than houses, cars, food, etc. The amounts derived from such market exercises have been questioned because they seem too high [35], sometimes exceeding the net assets of the respondent. As a consequence, cost–benefit analysis is rarely performed in health care; it is much more common in business and environmental applications. Cost-effectiveness analysis avoids such value judgments by calculating a “cost-effectiveness ratio,” expressed as dollars per unit of clinical benefit [14]. To facilitate comparison across interventions, clinical benefit is usually measured in years of life gained (life years, but it may be any clinically recognized unit of benefit (e.g. cases of chronic GVHD prevented, days of hospitalization or lives saved). Costeffectiveness ratios are by definition comparisons of one treatment approach versus another (which may be “no treatment”) since they are calculated as: (cost of treatment X minus cost of treatment Y)/(benefit of X minus benefit of Y).

When several possible treatment options are available, one or more may be “dominated” (found to be both more costly and less effective than another option) and eliminated from further consideration. Because costeffectiveness analyses are intended for policy makers, it is important to specify the perspective (e.g. government program, hospital, health plan, etc.) and time horizon (e.g. 1 year, 100 years, etc.) of the analysis in order to reflect which costs and benefits have been included. The strategy most cost-effective for a health maintenance organization may not be the one that is most cost-effective from the viewpoint of a hospital, the patient or society.

Many people use the terms “cost-effectiveness analysis” and “cost– utility analysis” interchangeably. However, a cost–utility analysis specifically incorporates QOL considerations and usually has a denominator of quality-adjusted life years (QALYs). In these analyses, survival time is adjusted for the QOL associated with that survival. For example, for some people, a year of life in good health may be worth several years in poor health. These adjustment factors are called “patient utilities” and usually range from 0 (equivalent to being dead) to 1.0 (the year of life being fully valued). A utility less than 0 represents a health state worse than death. Utilities fulfill the mathematical condition of linearity so that 1 year of perfect health is considered equal in value to 2 years of life with a utility of 0.5. For example, some quoted patient utilities for health states are 0.98 for suffering the side-effects of β-blockers [36], 0.8 at 1 year after autologous transplantation for non-Hodgkin’s lymphoma (NHL) [37], and 0.5 following a stroke [38]. Patient utilities may be assessed using several techniques: standard gamble, time trade-off, and multiattribute utility theory. Standard gambles ask people what risk of death they would accept to reach perfect health, with one minus risk of death equal to patient utility. For example, if a patient is willing to assume a 15% chance of death to reach perfect health, the utility of their current, compromised health state is 0.85 (1.0 minus 15%). Assessment of patient utilities by standard gamble is limited by people’s ability to consider life and death risks hypothetically and rationally. However, the decision to undergo HCT is very much like a standard gamble. Patients may either opt for best supportive care or standard chemotherapy, or they accept some chance of treatment-related mortality from the transplant procedure in order to cure their diseases. Time trade-off questions ask people how much life expectancy they would trade for perfect health in their remaining time, with utility equal to time in perfect health divided by time in current compromised state of health. For example, let’s assume that a patient has a life expectancy of 10 years but has a painful, debilitating disease. If that patient were willing to trade-off (i.e. give up) 1.5 years of life so that the remaining 8.5 years would be in perfect health, his utility would be 8.5/10 or 0.85. Similar to utility assessment, time trade-off questions require people to consider hypothetical scenarios in which perfect health is guaranteed, but at a cost of some decrease in life expectancy. Multiattribute utility theory calculates utilities from QOL or functional status data. The conversion equations are derived from studies in which people complete QOL surveys and have their utilities assessed by standard gamble or time trade-off at the same time. Use of the equations

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Table 29.3 Examples of cost-effectiveness and cost–utility studies in hematopoietic cell transplantation (HCT) and medicine Ref.

Year of pub.

Treatment

Alternative

Cost/effectiveness ratio

[39] [40] [41] [12] [42] [43] [44] [45] [46]

1989 1992 1997 1998 1999 2001 1989 1987 1990

Allogeneic BMT for AML Autologous BMT for HD in 2nd CR Autologous BMT for relapsed NHL Unrelated donor BMT for stable phase CML Second allogeneic transplantation after relapse of acute leukemia Autologous PBSCT for MM Smoking cessation program Hemodialysis Captopril for hypertension

Conventional chemotherapy Conventional chemotherapy Conventional chemotherapy Interferon-α Conventional chemotherapy Conventional chemotherapy No intervention No dialysis No therapy

$10,000/LY $26,000/LY $9,200/LY $51,800/QALY $52,000/LY $23,300/LY* $1,300/LY $50,000/LY $72,000/LY

* £0.64 = $1.00. AML, acute myeloid leukemia; BMT, bone marrow transplantation; CML, chronic myeloid leukemia; CR, complete remission; HD, Hodgkin’s disease; LY, life year; MM, multiple myeloma; NHL, non-Hodgkin’s lymphoma; PBSCT, peripheral blood stem cell transplantation; QALY, quality-adjusted life year.

allows utility estimates to be mapped based on patient self-reported QOL data without the need for interviewers to administer standard gamble and time trade-off questions again. What makes cost-effectiveness and cost–utility analyses powerful is that diverse interventions may be compared and selected for their ability to provide maximal health benefit for money spent. For example, if health-care dollars are limited, these methods allow some rational basis for recommending whether society should routinely provide coverage for a patient with acute myeloid leukemia in third complete remission scheduled for an unrelated donor HCT, or instead cover patients with congestive heart failure and severe diabetes who need heart transplants. Similarly, one can compare the health-care gain of one high-cost treatment procedure, such as an autologous HCT for relapsed NHL, against the preventive strategy of providing statin therapy to several middle-age men. Based on the cost-effectiveness ratio for hemodialysis, a procedure covered separately by the federally funded Medicare program and thus available to all people, an acceptable cost-effectiveness ratio of less than $50,000 per QALY has been proposed. A ratio of over $100,000 is questionable because that money applied elsewhere may buy better health for the population. A ratio between $50,000 and $100,000 per QALY is in the gray zone. Some countries, such as Australia and Canada, require information on cost-effectiveness prior to drug approval. Table 29.3 shows examples of published cost-effectiveness and cost– utility studies in HCT and some comparisons in other fields [39–46]. League tables have been published to help put cost-effectiveness [47,48] and cost–utility ratios [49,50] into perspective. Online resources such as http://www.tufts-nemc.org/cearegistry contain comprehensive lists of cost analyses and patient utilities that may be downloaded [51]. It would be naïve to believe that costs do not affect access to HCT, although sometimes policies can be surprising. Gratwohl and colleagues found that countries with low or medium gross national incomes are performing the same number of HCTs for chronic myeloid leukemia (CML), whereas the transplantation rate has fallen substantially in highincome countries. The authors hypothesize that it may be less costly overall for poorer countries to perform an up-front HCT than pay for lifetime drug therapy or several years of drug therapy followed by eventual HCT [52].

able) and are referred to as “constructs.” For example, measurement of health-related QOL (the QOL related to health, disease, and medical treatment) and patient utilities are considered part of outcomes research. Measuring these endpoints relies heavily on methods developed for survey research, the collection of data directly from patients.

What is the patient’s experience with HCT?

Quality of life

Beyond the traditional biologic endpoints of survival and relapse reported in HCT studies, outcomes research tries to measure and put into perspective other factors that determine whether an intervention is ultimately judged a success or failure. Many of these factors are subjective (not directly observ-

QOL is composed of diverse determinants including physical abilities, symptoms, social wellbeing, psychoemotional status, and spiritual/existential experiences. It reflects how well people feel, what they can accomplish, how satisfied they are with their lives, and whether their

Qualitative methods The goal of qualitative studies is to capture the breadth of possible patient attitudes or experiences. One forum is a focus group, in which eight to 10 people are led by a moderator and discuss particular topics. Focus groups usually last about 2 hours, and participants may be paid a nominal amount for participation. They are often audio- or videotaped, with an additional researcher taking notes. The interactive nature of the communication process allows topics to be probed and ideas developed under the influence of group dynamics, which may lead to unexpected insights about the topic under discussion. Focus groups are often used for formative research to explore patient attitudes and opinions prior to launching a formal study. Qualitative information may also be collected through interviews or open-ended survey questions. In contrast to the majority of studies in which generalizability is critical, the goal in truly qualitative studies is not to obtain a representative sample. In fact, “purposive” or targeted sampling can be performed to ensure representation of the spectrum of possible patient experiences. For example, if 80% of the population has a typical experience, 5% has a less typical experience, and the remaining 15% all have unique experiences, the goal would be to interview 17 people, one from the majority, one from the minority and all 15 who had unique experiences. Usually, transcriptions are made of the interviews, and qualitative coding software is used to mark the transcripts for easier analysis. Transcripts are reviewed by a limited number of individuals who code them for themes, aggregate the concepts into broader groups if possible, and report on the range of patient experiences. Qualitative methods have been used in several studies to evaluate aspects of recovery following HCT [53–56]. These studies revealed several themes that are not particularly well covered in standardized instruments, for example strategies that patients use to compensate for limitations, multiple losses in all aspects of their lives, and the greater appreciation for life brought about by the HCT experience.

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Global QOL

Physical fatigue pain

Emotional anxiety depression

Social family friends

SF-36 (physical) Does your health now limit you in: • Vigorous activities, such as running, lifting heavy objects, participating in strenuous sports • Moderate activities, such as moving a table, pushing a vacuum cleaner, bowling or playing golf • Lifting or carrying groceries • Climbing several flights of stairs • Climbing one flight of stairs • Bending, kneeling, or stooping • Walking more than a mile • Walking several blocks • Walking one block • Bathing or dressing yourself

Fig. 29.2 Dimensions of quality of life and examples of the physical domain from validated questionnaires.

lives have meaning and purpose (for details regarding QOL after HCT, see Chapter 34). QOL studies in HCT have generally sought to: (1) describe the long-term QOL, adaptation, and recuperation of patients; (2) find predictors of better or worse QOL; and (3) compare populations treated with different procedures. There are numerous, multidimensional, validated instruments available for measuring QOL in HCT. When scored according to psychometrically tested methods, they provide standardized QOL information that may be compared with other populations. One commonly used instrument is the Medical Outcomes Study Short Form 36 Health Survey (SF-36) a generic multidimensional 36-item instrument that has been used on thousands of patients and healthy people to measure physical and mental health status [57]. Other cancer-specific instruments include the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 (30 items) [58–60] and the Functional Assessment of Chronic Illness Therapies (FACT; 27 items) [61]. Both cancer-specific instruments also offer HCT-specific modules that can be added to the core form to capture issues specific to HCT. Figure 29.2 shows an example of the physical functioning scales from the core instruments. Of note, comparative studies have shown that, despite similarly named subscales, instruments are actually measuring different constructs, and it is difficult to compare studies unless they use identical instruments [62,63]. Many QOL surveys have been translated into other languages, an arduous process that requires first translation and then back-translation to see if meaning is preserved. It is a common mistake to assume that interpreters can administer English instruments to a non-English speaker. Instead, the validated version in the subject’s native language should be used since interpreters may unintentionally change the meanings of items and responses. Ideally, any QOL report should include information about the instruments used, reasons for missing data, a comparison of respondents and those who choose not to participate, and response rates at each assessment point to assist in interpretation of study quality. Missing data are a great problem in QOL and survey research, for several reasons. First, 10–50% of data may be missing due to logistical problems and patient refusal to complete questionnaires. One barrier may be literacy, since studies have shown that approximately 25% of the United States popu-

Functional work sleep

Spiritual religion meaning

EORTC (physical) • Do you have any trouble with strenuous activities, like carrying a heavy shopping bag or a suitcase? • Do you have any trouble taking a long walk? • Do you have any trouble taking a short walk outside of the house? • Do you have to stay in bed or in a chair for most of the day? • Do you need help with eating, dressing, washing yourself or using the toilet? FACT (physical) • I have a lack of energy • I have nausea • Because of my physical condition, I have trouble meeting the needs of my family • I have pain • I am bothered by side-effects of treatment • I feel ill • I am forced to spend time in bed

lation is functionally illiterate [64]. Second, data are often not missing at random but rather reflect poor health or other characteristics that could influence QOL. Many validated scales are long, and ill HCT patients are more likely to refuse to complete them. This “participation bias” may be minimized by considering “respondent burden” when planning any survey-based study. Biostatistical methods for analyzing QOL data can be complicated because they must address issues of longitudinal data analysis, informatively missing data, and other problems exacerbated by the nature of QOL data [65,66]. Repeated-measures analysis and mixed models allow differences between populations and over time to be studied, but methods of reporting results might not be intuitive to physicians. Also, it is important to remember that QOL studies are often cross-sectional and represent only those patients surviving the procedure at a particular point [67,68]. Developing methods to place QOL differences into their clinical context is an active area of research. Although validated scales are psychometrically sound and allow a comparison of treatment groups by statistical testing, the results of QOL studies are not intuitive to patients and physicians [69,70]. For example, a survival difference of 10% is easily interpretable, but many find it harder to interpret a QOL difference of 50 versus 35 on a given scale and place such an observation in its clinical context. There is no intuitive feeling for what a person with a score of 50 feels like compared with someone with a score of 35. Thus, some researchers have embraced the concept of a “clinically meaningful difference,” defined as the difference in QOL that would prompt adoption of the intervention or a change in practice. There are two approaches to determine the clinically meaningful difference in a scale: anchor based and distribution based. Anchor-based methods rely on patient-reported differences to determine what is clinically meaningful. For example, patients are asked a global change question such as, “Overall, is your QOL a lot better, a little better, somewhat better, somewhat worse, a little worse or a lot worse?” This overall category is then compared with their QOL scores [71–73]. However, this method uses patient-perceived differences in QOL as the gold standard, raising the question of why we cannot just ask patients directly about changes in their QOL. The second approach is based on the statistical distribution of QOL scores. Generally, a difference of half a standard deviation is considered to be clinically meaningful.

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After HCT, patients generally rate their global QOL quite highly despite many specific physical and emotional difficulties. One possible explanation for this paradox is called “response shift,” the theory that perceptions of current QOL are more dependent on one’s perspective than on one’s actual health, and that this perspective changes with illness [74]. For example, consider two previously healthy patients with identical diseases, physical ailments, and social support systems. Upon diagnosis, the first patient believes he is afflicted with a terrible disease and embraces HCT as a life-saving procedure. Afterwards, he compares himself with others who died, and rates his overall QOL quite highly as a result of his response shift. The second patient considered himself healthy before the transplant despite his disease and reluctantly undergoes transplantation. After HCT, he pines for his previously healthy self and is very dissatisfied with his QOL. This patient did not have a response shift. Results and conclusions of specific QOL studies are discussed in more depth in Chapter 34. How are new tools developed? Prognostic models Prognostic models can help refine estimates of the risks and benefits of HCT for particular patients. Results can be used to counsel patients, compare study populations or adjust outcomes for clinical characteristics. Optimally, prognostic models are developed in one subset of patients and then validated in an independent population. The examples listed below all use variables known at the time of transplantation to predict mortality. The European Group for Blood and Marrow Transplantation identified five adverse prognostic factors for allogeneic HCT for CML: accelerated or blastic phase, disease duration greater than 12 months, patient age greater than 20 or 40 years, a female donor for a male patient, and transplantation from an unrelated donor [75]. Five-year survival in the most favorable group was 72% compared with 22% in the highest-risk group. The score was subsequently validated in independent cohorts of patients receiving high-dose chemotherapy [76] and reduced-intensity conditioning [77]. Several comorbidity or risk-adjustment models have been developed for HCT patients. Two are based on previously published comorbidity and performance scales used in other populations [78,79], although one was derived de novo [80]. While these prognostic models have been useful for physicians to conceptualize the degree of risk involved in HCT, the resulting mortality estimates have not been incorporated into clinical care. Anecdotally, physicians do not seem to calculate and formally discuss prognostic scores when counseling individual patients. Patient-reported measures Instrument (or survey) development is considered a facet of outcomes research because it establishes the validity of clinical tools to measure subjective endpoints. In order for an instrument to be useful, it needs to reflect what it purports to measure (validity), be an accurate measure (reliability), separate people into clinically meaningful groups (discrimination), and detect important changes (sensitivity). Instrument development from scratch is a demanding process. First, a list of relevant concepts should be created from prior literature, focus groups or other means of formative research. Then, a draft scale is created. Attention should be given to the wording of specific questions and response items to allow sufficient variability to capture the range of clinical conditions. Then, a pilot study is conducted with cognitive interviewing to ensure that patients understand the questions and are selecting response options consistent with their intent. Finally, a larger study is performed to document validity, reliability, and sensitivity. Many survey developers neglect the final step of confirming sensitivity to change. This is an

important feature since many instruments are intended to describe the experiences of population subgroups, compare one population with another or show changes over time. Reliability refers to whether a measure is consistently reflecting the true status of a subject. Internal reliability is usually reported as a Cronbach’s alpha with acceptable values greater than 0.7. Cronbach’s alpha measures whether items are correlated with each other and measure the same underlying construct. Stability of measurements is reported as “test–retest reliability,” the correlation between two measurements separated in time when an individual’s status has not changed. The range is from 0 to 1.0, with higher values reflecting greater stability and values over 0.5 generally considered acceptable. If test–retest reliability is less than 0.5 and the subject’s clinical situation has not changed, the scale is probably susceptible to influences unrelated to the clinical status of the person. Validity refers to whether an instrument is truly reflecting what it is supposed to measure, and is usually expressed as correlation coefficients or effect sizes. Content validity refers to how well the scale measures the different aspects of the construct. Convergent validity is demonstrated when the scale correlates highly with other scales measuring similar constructs, while discriminant validity means there is little correlation with scales measuring unrelated concepts. Discrimination refers to the ability of the scale to separate people into clinically meaningful groups, while sensitivity to change means that as a person’s clinical situation changes, they should score differently on the instrument. For example, Lee and colleagues have developed a chronic GVHD symptom scale [81]. This scale was designed to be self-administered and brief (5 minutes) and to follow patients with chronic GVHD over time to detect an improvement or worsening in their symptoms. It includes questions about bothersome eye, mouth, lung, skin, nutrition, emotional, and energy symptoms. Comparison with the SF-36 and FACT-BMT showed adequate convergent and discriminant validity, discrimination between patients with self-assessed mild, moderate or severe chronic GVHD, and sensitivity to change. Clinical syndromes Clinical syndromes such as acute and chronic GVHD have been notoriously difficult to measure, yet they are important endpoints in almost every allogeneic HCT report (for details, see Chapters 86 and 87). The greatest challenge arises from the heterogeneous clinical manifestations complicating standardization of severity grading. Martin et al. [82] showed that interobserver differences in acute GVHD grading from medical records were substantial, and suggested a more objective way of coding this complication. However, his approach has not been widely adopted. A second challenge to severity scale development is the need to validate the scale against a gold standard. Since a gold standard does not exist for GVHD severity, developers have used survival or nonrelapse mortality as objective endpoints. In acute GVHD, several grading systems have been proposed, including the Glucksberg scale [83], the modified Glucksberg scale [84], the Consensus Conference grading system [85], and the International Bone Marrow Transplant Registry (IBMTR) index [86]. Application of these grading systems first requires ascertainment of performance status and staging of skin, liver, and gastrointestinal involvement, followed by aggregation into five grades (0–IV or 0, A, B, C, and D). With the exception of the IBMTR index, all the grading systems were developed by observation and consensus. The IBMTR used one large set of patients undergoing human leukocyte antigen-matched sibling bone marrow transplantation for acute or chronic leukemia (“training set”; n = 2129 given cyclosporine and methotrexate) to develop the index, and then validated it in an independent dataset (“testing set”; n = 752 receiving T-cell depletion) using survival as the primary endpoint [86]. Additional validation studies in separate cohorts have yielded conflicting results [87,88].

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Several scales have also been proposed for grading the severity of chronic GVHD. Akpek and colleagues identified three dichotomous variables (extensive skin involvement, thrombocytopenia, and progressive onset) that could be combined to distinguish three groups at different risks of chronic GVHD-specific mortality [89], and validated the scale in four independent cohorts [90]. Lee and colleagues took a similar approach and developed a chronic GVHD severity score using IBMTR and National Marrow Donor Program (NMDP) data [91]. In 2006, a series of papers developed through a National Institutes of Health consensus process proposed new criteria for assessing chronic GVHD severity and response to therapy [92,93]. These tools await prospective validation. How can one combine information from several different data sources? Registry studies Several large transplant registries were established to provide national and international information on the outcomes of HCT. These include the Center for International Blood and Marrow Transplant Research (http://www.cibmtr.org), the NMDP (http://www.nmdp.org), European Group for Blood and Marrow Transplantation (http://www.ebmt.org), and Eurocord (http://eurocord.org). These registries collect, computerize, and make available data for analyses. While they suffer from limitations common to registries, including incomplete capture of all procedures, problems with data standardization, validation issues, and difficulty obtaining detailed clinical information, including long-term follow up, they also provide the only means for determining the effectiveness of HCT as practiced outside of clinical trials and single institutions. Many important research questions can only be answered by registry studies because large patient numbers are required. Rarer diseases and clinical situations for which no single institution has adequate experience are also best approached by registry studies. Large administrative databases have not been useful for HCT research in the past. A rare exception is the Nationwide In-patient Sample, which provides a representative sample of inpatient hospitalization lengths of stay, costs, and hospital codes for more than 33 states. This database has been used for some economic studies [94]. Administrative databases are powerful tools for addressing broad questions because of the sample size, but do not allow collection of additional data. In 2006, the C. W. Bill Young Cell Transplantation Program mandated development of a Stem Cell Therapeutics Outcomes Database (SCTOD) in the United States to collect outcomes data on all procedures using allogeneic hematopoietic cells. Hopefully, this important resource will provide a rich research database in the future since it will provide population-based data on HCT use. Decision analysis There is rarely a definitive clinical trial or report that provides all the data necessary to settle a clinical question. People believe they can weigh complicated decisions fairly, but research shows that this ad hoc approach is subject to serious cognitive biases and frequently results in suboptimal decisions. Decision analysis uses computer modeling to recommend the optimal treatment choice based on what is known about the probabilities and consequences of different treatment options [95]. Decision-analysis models are often depicted as “trees.” A square represents the decision to be made (choice node), while branches off of circles (chance node) represent possible clinical consequences. The analyst also decides which health states are relevant (e.g. dead, alive with disease, alive without disease or alive with chronic GVHD). Health states need to be broad enough to allow accurate estimation of the percentage of the population within them at any time but narrow enough to discriminate different clinical circumstances. A prudent analyst only includes health

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states material to the decision, or else a decision tree can quickly become “too bushy,” with small, perhaps inconsequential branches for which solid clinical data may be unavailable. The analyst also decides how often people can transition between different health states (cycle length) based on data available from clinical trials or observational studies. Advanced programming capabilities allow the probabilities of different outcomes to vary depending upon a patient’s characteristics, time from diagnosis or prior clinical course, assuming such data are available. The results of decision analysis provide a population-based approach to recommend an optimal treatment choice. Treatment options are compared based on the area under the survival curve (life years) or the quality-adjusted area under the survival curve (QALY). In practical terms, a decision analysis may not distinguish between one individual surviving an extra 10 years and five individuals surviving an extra 2 years, although patients may view these outcomes differently. Such value judgments are incorporated into the model using discounting functions that value early survival greater than distant years of life. While a decision analysis obviously cannot predict what will happen to any particular individual, results should, if the information put into the model is correct, accurately reflect what happens to the population on which it is based [96]. Comparison of life years or QALYs obtained from a decision analysis are not amenable to statistical testing in the classic sense, since p-values and confidence intervals reflect the uncertainty in measurements and likelihood of chance findings. In decision analysis, it is up to the reader to compare the gains in life years or QALYs and determine if one treatment is optimal. Obviously, such comparison is easier when the survival benefit associated with one option is several years and the other offers several weeks. Published league tables can help put gains in life expectancy into perspective [97]. Sometimes decision analysis can identify key pieces of information that should influence treatment decisions and, conversely, point out which considerations should not affect a rational decision. When assumptions and estimates have to be made because sufficient clinical data are not available, sensitivity analysis helps determine whether results hinge on those estimates. If conclusions are the same despite drastically changing an assumption, the analysis is “robust” and not dependent on that variable. Several assumptions may be tested at the same time to see if any combination of values would change conclusions. Several decision analyses have been performed in HCT. An analysis of autologous HCT versus conventional combination therapy for a 50year-old woman with progressively erosive, active rheumatoid arthritis after initial therapy suggests equivalent QALYs with either approach [98]. An analysis of allogeneic bone marrow transplantation versus periodic blood transfusion for patients with sickle cell anemia and elevated cerebral blood velocities suggested that either treatment approach was reasonable [99]. An analysis of the role of skin biopsy to confirm suspected acute GVHD suggested that treatment for acute GVHD without a biopsy would result in the best outcomes. Model inputs were derived from expert estimates for the prevalence of acute GVHD and the test characteristics of a skin biopsy given a patient with a rash 14 days post transplant [100]. A decision analysis of immediate versus delayed HCT in patients with myelodysplastic syndrome suggested that patients with intermediate- or high-risk myelodysplastic syndrome should undergo up-front HCT. In contrast, the population with lowerrisk MDS maximized their life years with delayed HCT [101]. Q-TWiST Q-TWiST stands for “quality time without symptoms of toxicity.” It is another method of integrating QOL and survival data [102,103]. Although concurrent data on QOL, survival, and DFS may be obtained in a single clinical trial, information on symptoms and QOL are often derived independently. Figure 29.3 shows a schematic of a Q-TWiST

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analysis in which patients are divided up into four mutually exclusive categories: alive without disease or treatment toxicity, alive with symptoms of toxicity, alive in relapse, and dead. Quality adjustment is applied to the different health states, and the areas under the curves are aggregated. If there are two available treatments, the option that provides the greatest quality-adjusted survival is judged superior. This type of analysis has been used to suggest that autologous HCT is better than chemotherapy for aggressive NHL in first complete remission [104] and that allogeneic transplantation is better than chemotherapy or autologous transplantation for pediatric acute myeloid leukemia in first complete remission [105]. Meta-analysis Meta-analysis allows the results of several clinical studies to be aggregated, increasing the power of the analysis and enhancing confidence in the results [106–108]. This statistical technique is particularly useful in detecting treatment differences if negative studies are due to small sample size and lack of power. Study-level meta-analyses use individual studies as the unit of analysis. Results are displayed as point estimates and confidence intervals for each study with some indicator (size of a dot or width of a box) to reflect relative study size. A diamond is often used to depict the aggregate estimate. Analyses can either use “fixed-

effect” or “random-effect” models. In fixed-effect models, one true effect of the treatment is assumed, and any differences in studies from that effect are considered part of variability. In random-effect models, each study estimate may be true because of differences in studies, and the analysis allows for a range of “true” values. Tests for homogeneity/heterogeneity can address whether the reported effect sizes from different studies could vary due to chance, although these tests lack power and should only be minimally reassuring. Both randomized controlled trials and observational studies are often included in metaanalyses. Sensitivity analyses that exclude lower-quality studies can be performed to increase confidence in the conclusions. Patient-level meta-analyses actually retrieve and analyze data on individual patients, although the source of each patient is incorporated into the analysis. Figure 29.4 shows the results of a patient-level analysis conducted by the Stem Cell Trialists’ Collaborative Group. Data from patients in nine randomized trials were aggregated. The study concluded that peripheral blood is associated with decreased relapse rates and better overall survival and DFS in patients with late-stage disease, but with the disadvantage of more extensive chronic GVHD [109]. All meta-analyses depend upon data available in the literature or otherwise attainable. Thus, publication bias may significantly affect results. A funnel plot is a graphic that can help determine if a metaanalysis is likely to suffer from publication bias. Effect size is plotted versus study size. If all studies are published and available regardless of their conclusions, the plot should result in a funnel shape with the apex centered on the true value. This pattern occurs because the ranges of effect sizes are wider for the smaller studies due to statistical factors, while larger studies should provide results closer to the actual truth.

Population (%)

Dead Relapsed

Alive Tox

Time

Fig. 29.3 Schematic of a Q-TWiST (quality time without symptoms of toxicity) analysis. The population is divided into dead, relapsed, alive (without disease or toxicity), and tox (alive with toxicity from treatment).

Evidence-based medicine Critical reviews of the literature summarize the available evidence for or against certain practices, and they are often translated into practice guidelines. The methodology for evidence-based reviews is very well established and rigorous, although the specific terminology used to reflect the levels of evidence and grades of recommendation have evolved. Several organizations, such as the Cochrane Collaboration, the Agency for Healthcare Research and Quality, Cancer Care of Ontario, the American Society of Clinical Oncology, the American Society of Hematology, and the American Society for Blood and Marrow Transplantation have performed critical reviews relevant to HCT. A typical grading system for critical reviews is shown in Table 29.4, and is based on the type, frequency, and consistency of evidence [110].

Fig. 29.4 Results of an individual patient-level meta-analysis of the outcomes associated with peripheral blood or bone marrow transplantation. aGVHD, acute graft-versus-host disease; BMT, bone marrow transplantation; cGVHD, chronic graft-versus-host disease; CI, confidence interval; O–E, observed/expected; OR, odds ratio; PBSCT, peripheral blood stem cell transplantation; SD, standard deviation. (Reproduced from [109], with permission.)

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Table 29.4 Levels of evidence and grades of recommendation Level

Types of evidence

1++ 1+ 1− 2++

High-quality meta-analyses, systematic reviews of RCTs, or RCTs with a very low risk of bias Well-conducted meta-analyses, systematic reviews of RCTs, or RCTs with a low risk of bias Meta-analyses, systematic reviews of RCTs, or RCTs with a high risk of bias High-quality systematic reviews of case-control or cohort studies; high-quality case-control or cohort studies with a very low risk of confounding, bias, or chance and a high probability that the relationship is causal Well-conducted case-control or cohort studies with a low risk of confounding, bias, or chance and a moderate probability that the relationship is causal Case-control or cohort studies with a high risk of confounding, bias, or chance and a significant risk that the relationship is not causal Nonanalytic studies, for example, case reports or case series Expert opinion

2+ 2− 3 4 Grade A

B C D

Grade of recommendation At least 1 meta-analysis, systematic review, or RCT rated as 1++ and directly applicable to the target population or a systematic review of RCTs or a body of evidence consisting principally of studies rated as 1+, directly applicable to the target population and demonstrating overall consistency of results A body of evidence including studies rated as 2++, directly applicable to the target population, and demonstrating overall consistency of results or extrapolated evidence from studies rated as 1++ or 1+ A body of evidence including studies rated as 2+, directly applicable to the target population, and demonstrating overall consistency of results or extrapolated evidence from studies rated as 2++ Evidence level 3 or 4 or extrapolated evidence from studies rated as 2+

RCT, randomized controlled trial. Reproduced from Jones et al. [110].

One practical limitation of evidence-based reviews compared with consensus statements or clinical reviews is that the published evidence is strictly interpreted [111]. Lack of randomized controlled trials in some areas can result in the frustrating conclusion that “evidence is insufficient to recommend for or against” a particular practice, even when widespread use seems to support its effectiveness [112]. However, the results of the Women’s Health Initiative, showing that routine hormone replacement after menopause can cause more harm than good [113], and lack of efficacy seen in trials of autologous HCT for high-risk or advanced breast cancer [114,115] reinforce the idea that randomized trials provide the highest level of evidence for a practice. Many organizations have produced evidence-based or consensus practice guidelines. A nonexhaustive list relevant to transplantable diseases includes the American Society for Blood and Marrow Transplantation (www.asbmt.org) [116–120], the National Comprehensive Cancer Network (NCCN; http://www.nccn.org), and the Physician Data Query (http://www.nci.nih.gov/cancer_information/pdq). However, relatively little research has evaluated the influence of practice guidelines on clinical practice and patient outcomes, and what has been published suggests less improvement than hoped [121–123].

How can the practice of HCT be improved through health services research? Access Studies seeking to understand the nonmedical barriers to appropriate health care are termed “access” studies. They generally focus on socioeconomic, political, cultural, and other nonbiologic factors. For example, internationally there is a strong association between transplant rate and team density, gross national product per capita, and health-care expenditures per capita, suggesting better access in some countries [124]. In the United States, ethnic and racial minorities have long been underrepresented in HCT statistics for unclear reasons. Survival varies by racial group even after controlling for disease and transplant characteristics [125]. Neither GVHD nor relapse appears to explain the higher mortality seen in certain racial groups [126]. While similar observations

have been made in other areas of medicine, issues of access and social determinants of outcome are only starting to be evaluated in HCT. Kollman and colleagues at the NMDP studied reasons why only a third of promising initial searches proceed to transplantation [127]. They identified death of the patient, worsening of the patient’s health, and length of the search process as major barriers accounting for up to a quarter of failures to proceed to transplantation. The importance of financial issues could not be adequately evaluated in this study, but 41% of coordinators listed insurance coverage as a potential barrier at the time of initial search. Importantly, 34% of white Americans compared with only 13% of African-Americans went on to transplantation. In another study at the Medical College of Virginia, 589 African-Americans were surveyed about barriers to participation in an unrelated donor program. The cost of donation, limited opportunities to donate, and lack of knowledge about the life-saving potential of HCT from an unrelated donor were important barriers. Importantly, with the introduction of an educational program, the African-American donor pool increased substantially [128]. Switzer et al. surveyed 1679 potential unrelated donors and found that Asian/ Pacific Islanders were more ambivalent about donation, more concerned about effects of donation on themselves and their families, and more anxious and depressed [129]. Unfortunately, one of the best data sources for access studies is not relevant for HCT. The linked SEER–Medicare database provides cancerspecific information and inpatient and outpatient billing data on approximately 14% of the United States population, but is limited to patients aged 65 or older [130]. The size and comprehensiveness of this database, along with the fact that it is population based, has made it a tremendous resource for studies elsewhere in medicine. For example, researchers have found that African-Americans are less likely than white Americans to receive screening exams for cancer, to be diagnosed with cancer in its early stages, and to receive adjuvant therapy and aggressive care [131–137]. AfricanAmericans are also less likely than white Americans to undergo some invasive procedures, such as renal transplantation, even after correcting for clinical characteristics [138] and patient preferences [139]. However, once access to treatment is controlled, survival and DFS are similar between African-American and white American patients, suggesting that the biologic response to treatment is comparable [140,141].

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Quality of care HCT has been relatively spared from scrutiny about quality of care. Significant practice variation has been tolerated and, in fact, encouraged, as a means of testing different approaches that could eventually improve the field of transplantation. The relatively low volume of procedures per center and the inevitable case mix differences have made it difficult to provide reliable standardized center statistics. Nevertheless, as part of its Health Resources and Services Administration contract, the NMDP has been reporting center-specific disease activity and risk-adjusted survival information for patients undergoing unrelated donor transplantation. The new SCTOD will have a public access interface that will also provide similar data on all allogeneic procedures performed at each center. There is every reason to believe that greater attention to institutional and programmatic factors contributing to patient outcome may identify ways to improve care and decrease costs [142]. Volume–outcome and experience–outcome relationships have been documented in solid organ transplantation and complicated surgical procedures [142–145]. A timeseries study of costs during autologous HCT for NHL suggested that technologic advances and learning curve effects (institutional familiarity with a procedure tends to improve outcomes) probably both contribute to falling costs, although the study was not designed to compare clinical outcomes [146]. From a regulatory perspective, accreditation by the Foundation for the Accreditation of Cellular Therapy ensures compliance with standards for clinical care and cell processing facilities. Programs are required to show evidence of documentation, standard operating procedures, and quality management programs. Increasing federal oversight of outcomes reporting through the SCTOD will provide both a tool for quality monitoring and improvement, as well as a wealth of research data that can address health services questions [147]. Practice variation The term “practice variation” refers to systematic heterogeneity in clinical practice unjustified by medical considerations. Undesirable practice variation results from external influences on physicians (e.g. training, practice environment and reimbursement structure) [148], patient characteristics (e.g. race, ethnicity, gender, and age, if these features should not influence treatment choice), and lack of definitive evidence for the effectiveness of various procedures [7]. In some contexts, practice variation is considered a measurement of poor-quality care. For example, a

study documented significant variation in vaccination practices following HCT [149], and recommendations for infectious disease prophylaxis have subsequently been published [150]. In the wake of a wellpublicized chemotherapy overdose, a survey found that 13% of HCT centers also admitted chemotherapy overdoses, with higher rates in newer centers. The American Society of Blood and Marrow Transplantation subsequently proposed safeguards for high-dose chemotherapy administration [151]. Within HCT, a wide practice variation has been noted in dimethylsulfoxide autologous cryopreservation protocols [152], approaches to prophylaxis and treatment of acute GVHD [153], definitions of steroidrefractory acute GVHD and salvage treatment regimen [154], infectious prophylaxis and compliance with published guidelines [155], and the diagnosis and management of chronic GVHD [156]. Ruutu and colleagues compared reported center practices for handling steroidrefractory acute GVHD with the actual outcomes of CML patients developing GVHD at the centers. They reported better outcomes when lower-dose steroids were used for initial acute GVHD treatment [157], consistent with findings from a randomized clinical trial [158]. Loberiza and colleagues found that lower 100-day mortality for allogeneic recipients was associated with a higher patient-per-physician ratio and centers where physicians answered calls after hours [159]. By studying outcomes associated with current heterogeneous approaches, it is hoped we can narrow variation towards more effective practices.

Summary Outcomes and health services research seeks to answer questions that are relevant as a procedure matures beyond the experimental phase: What are the costs of providing these services? Is society getting its money’s worth? What do patients experience with the procedure? Can we develop new or better tools to measure results of treatments? How do you pull together disparate sources of data (now that they are available)? Is the procedure equally available to all people and of the highest possible quality? Answers to these questions are moving targets as HCT evolves. Nevertheless, for people afflicted with diseases treated by HCT and societies trying to control health-care spending, decisions have to be made today. Outcomes research tries to provide the necessary data so that personal and societal decisions can be based on the best information available.

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among potential hematopoietic stem cell donors. Transplantation 2005; 80: 890–6. Potosky AL, Riley GF, Lubitz JD, Mentnech RM, Kessler LG. Potential for cancer related health services research using a linked Medicare-tumor registry database. Med Care 1993; 31: 732–48. Ayanian JZ, Udvarhelyi IS, Gatsonis CA, Pashos CL, Epstein AM. Racial differences in the use of revascularization procedures after coronary angiography. JAMA 1993; 269: 2642–6. Ball JK, Elixhauser A. Treatment differences between blacks and whites with colorectal cancer. Med Care 1996; 34: 970–84. Burns RB, McCarthy EP, Freund KM et al. Black women receive less mammography even with similar use of primary care. Ann Intern Med 1996; 125: 173–82. Klabunde CN, Potosky AL, Harlan LC, Kramer BS. Trends and black/white differences in treatment for nonmetastatic prostate cancer. Med Care 1998; 36: 1337–48. Bach PB, Cramer LD, Warren JL, Begg CB. Racial differences in the treatment of early-stage lung cancer. N Engl J Med 1999; 341: 1198– 205. Potosky AL, Harlan LC, Kaplan RS, Johnson KA, Lynch CF. Age, sex, and racial differences in the use of standard adjuvant therapy for colorectal cancer. J Clin Oncol 2002; 20: 1192– 202. Shavers VL, Brown ML. Racial and ethnic disparities in the receipt of cancer treatment. J Natl Cancer Inst 2002; 94: 334–57. Epstein AM, Ayanian JZ, Keogh JH et al. Racial disparities in access to renal transplantation – clinically appropriate or due to underuse or overuse? N Engl J Med 2000; 343: 1537–44. Ayanian JZ, Cleary PD, Weissman JS, Epstein AM. The effect of patients’ preferences on racial differences in access to renal transplantation. N Engl J Med 1999; 341: 1661–9. McCollum AD, Catalano PJ, Haller DG et al. Outcomes and toxicity in African-American and caucasian patients in a randomized adjuvant chemotherapy trial for colon cancer. J Natl Cancer Inst 2002; 94: 1160–7. Bach PB, Schrag D, Brawley OW, Galaznik A, Yakren S, Begg CB. Survival of blacks and whites after a cancer diagnosis. JAMA 2002; 287: 2106– 13. Showstack J, Katz PP, Lake JR et al. Resource utilization in liver transplantation: effects of patient characteristics and clinical practice. NIDDK Liver Transplantation Database Group. JAMA 1999; 281: 1381–6. Hosenpud JD, Breen TJ, Edwards EB, Daily OP, Hunsicker LG. The effect of transplant center volume on cardiac transplant outcome. A report of the United Network for Organ Sharing Scientific Registry. JAMA 1994; 271: 1844–9. Begg CB, Cramer LD, Hoskins WJ, Brennan MF. Impact of hospital volume on operative mortality for major cancer surgery. JAMA 1998; 280: 1747– 51. Edwards EB, Roberts JP, McBride MA, Schulak JA, Hunsicker LG. The effect of the volume of procedures at transplantation centers on mortality after liver transplantation. N Engl J Med 1999; 341: 2049–53. Freeman M, Vose J, Bennett C et al. Costs of care associated with high-dose therapy and autologous

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transplantation for non-Hodgkin’s lymphoma: results from the University of Nebraska Medical Center 1989 to 1995. Bone Marrow Transplant 1999; 24: 679–84. LeMaistre CF, Loberiza FR, Jr. What is quality in a transplant program? Biol Blood Marrow Transplant 2005; 11: 241–6. Kravitz RL, Greenfield S. Variations in resource utilization among medical specialties and systems of care. Annu Rev Public Health 1995; 16: 431– 45. Henning KJ, White MH, Sepkowitz KA, Armstrong D. A national survey of immunization practices following allogeneic bone marrow transplantation. JAMA 1997; 277: 1148–51. Dykewicz CA, Jaffe HW, Kaplan JE, in collaboration with the Guidelines Working Group Members for CDC, the Infectious Disease Society of America and the American Society of Blood and Marrow Transplantation. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. Biol Blood Marrow Transplant 2000; 6: 7–83. Chen CS, Seidel K, Armitage JO et al. Safeguarding the administration of high-dose chemotherapy: a national practice survey by the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant 1997; 3: 331–40. Windrum P, Morris TC, Drake MB, Niederwieser D, Ruutu T. Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of EBMT centres. Bone Marrow Transplant 2005; 36: 601–3. Ruutu T, Niederwieser D, Gratwohl A, Apperley JF. A survey of the prophylaxis and treatment of acute GVHD in Europe: a report of the European Group for Blood and Marrow, Transplantation (EBMT). Chronic Leukaemia Working Party of the EBMT. Bone Marrow Transplant 1997; 19: 759–64. Hsu B, May R, Carrum G, Krance R, Przepiorka D. Use of antithymocyte globulin for treatment of steroid-refractory acute graft-versus-host disease: an international practice survey. Bone Marrow Transplant 2001; 28: 945–50. Trifilio S, Verma A, Mehta J. Antimicrobial prophylaxis in hematopoietic stem cell transplant recipients: heterogeneity of current clinical practice. Bone Marrow Transplant 2004; 33: 735– 9. Lee SJ, Vogelsang G, Gilman A et al. A survey of diagnosis, management, and grading of chronic GVHD. Biol Blood Marrow Transplant 2002; 8: 32–9. Ruutu T, Hermans J, van Biezen A, Niederwieser D, Gratwohl A, Apperley JF. How should corticosteroids be used in the treatment of acute GVHD? EBMT Chronic Leukemia Working Party. European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 1998; 22: 614–15. Van Lint MT, Uderzo C, Locasciulli A et al. Early treatment of acute graft-versus-host disease with high- or low-dose 6-methylprednisolone: a multicenter randomized trial from the Italian Group for Bone Marrow Transplantation. Blood 1998; 92: 2288–93. Loberiza FR, Jr., Zhang MJ, Lee SJ et al. Association of transplant center and physician factors on mortality after hematopoietic stem cell transplantation in the United States. Blood 2005; 105: 2979–87.

Section 3 Patient-oriented Issues in Hematopoietic Cell Transplantation

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Karl G. Blume & Robert A. Krance

The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation Aegroti salus suprema lex

Introduction Most chapters of this book rely on scientific information that has been derived from laboratory studies or from controlled clinical trials. Not so this chapter. It refers mostly to the experiences and opinions of the two authors who have spent the better part of the past 30 years as physicians taking care of transplant recipients and carrying out clinical research studies. The recommendations the reader finds in this chapter are – at least on occasion – based on personal observations and preferences. We ask for your understanding as we know there are “many ways to Rome” and other physicians in other programs may choose approaches different from ours. Patients who are under consideration for high-dose chemotherapy (with or without total body irradiation [TBI]) or treatment with a reduced-intensity regimen followed by hematopoietic cell transplantation (HCT) require a careful clinical evaluation and in-depth counseling by experienced physicians, nurses, and a knowledgeable social worker at the respective transplant center. Most transplant candidates are referred by practicing hematologists or oncologists to the tertiary center where the transplant procedure will be performed. Information regarding the prior course, including initial diagnostic studies, previous drug and radiation treatments, and responses to these interventions, as well as psychosocial aspects of the transplant candidate are of utmost importance.

Written information for patients Prior to the time of the initial meeting with the medical and paramedical staff at the transplant center, patients should have received at least some written educational material regarding the rationale, principle, potential complications, and projected treatment results of HCT. An increasing number of patients are obtaining information from the Internet and may be quite knowledgeable and informed (or misinformed) based on the quality of the material that they are able to acquire. Many transplant centers have developed their own center-specific written information, as well as educational videotapes or compact discs, respectively, which are mailed to future patients and their families in advance of their first clinic visit. Another excellent source of general information is the Blood and Marrow Transplant Information Network.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

This organization was founded 17 years ago by a former HCT patient (the author of Chapter 36) who provides well-written handbooks to patients and publishes updated information concerning new aspects of HCT in regularly appearing newsletters. The Network also maintains a website (www.bmtinfonet.org) (for further details, see Chapter 36). It may be extremely helpful for transplant candidates to communicate with former transplant recipients who can share their experience with the future patient. Such contacts are much more valuable than the visit to so-called Internet “chat rooms” that the authors of this chapter consider “goldmines for misinformation” (although this statement about the “chat rooms” may reflect a generational prejudice).

The patient’s first visit to the transplant center The first meeting with the candidate and relevant family members is intended mainly for information gathering and not for decision making; i.e. the physician should form an impression as to whether the examination and the medical data identify the patient as a suitable transplant candidate. Conversely, the patient should be provided with well-organized general and also individualized information about the intended treatment course, potential complications, expected transplant outcome, and alternative procedures. With most patients, the communication should occur in lay language. Technical terms should be explained and abbreviations be avoided whenever possible. Simple illustrations can be particularly valuable, and a glossary may prove to be very helpful. Conversely, some patients are already very well informed, and time at the counseling meeting may be spent more effectively on discussion of specific issues. Until they become ill, most patients have never heard words like “allogeneic,” “autologous,” “haplotype,” “graft-versus-host disease” (GVHD), etc. The concept that a small number of donor stem cells can restore the entire bone marrow with all its production of blood cells needs to be explained. If one would ask 100 random people in Cleveland, Ohio, in Dortmund, Germany, or in Adelaide, Australia, what they know about the vital functions of their bone marrow, one would most likely get very few correct answers. It is therefore most important to take ample time to explain the rationale behind HCT. A well-informed patient is much more likely to participate actively in his or her future care. Finally, we need to be most careful with the terminology we introduce to our patients. A word like “mini-transplant” implies to most listeners that this must be a minor procedure and nothing can go wrong. Consider that, even at the most experienced centers, the day 100 nonrelapse mortality (NRM) after dose-reduced intensity therapy and allogeneic HCT is at least 5%, and at 2 years it reaches approximately 20%. What will

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be your response to the spouse, parent or child of a dying patient when the question is asked how a “mini”-procedure could directly or indirectly lead to a patient’s demise?

Advice on counseling new patients Each physician and clinical investigator should develop his or her own approach to the education of patients and the counseling process. Over the years, I (K.G.B.) have developed – for my own use – a simple four question–answer system (why, how, when, and what?) for my counseling meetings: 1 Why is transplantation being done?, i.e. the rationale for the procedure and discussion of alternative therapies. 2 How is the procedure performed?, including the explanation of sideeffects from the preparatory regimen, the transplant process itself (including the impact on an allogeneic donor), the description of the reason for and clinical presentation of GVHD, the efforts for the prevention of GVHD as well as the potential risks for other early or late sideeffects and relapse. 3 When should the procedure be performed?, for example very soon in case of relapsed acute leukemia, or electively for patients with multiple myeloma who may have several months or sometimes years until the preparation for transplantation is begun. 4 What is the projected treatment outcome for the individual patient who is being evaluated? It is advisable to quote a range of percentages for disease-free survival (DFS) and not a precise figure, which would be arbitrary at best. Patients may elect to tape the conversations in order to review the information presented to them at their leisure. The physician should also encourage the patient to take notes and to write down questions that may come up between meetings or at a later time. An atmosphere of trust between patients and their families and the transplant team is of greatest importance for the long weeks before, during, and after the transplant procedure. The more carefully patients are prepared for the difficult phases of transplantation and the more they understand the rationale for diagnostic and therapeutic procedures, the significance of expected (and sometimes unexpected) events, and the risk of the entire medical approach, the easier will be the task of the health care team to guide these often (or maybe always) frightened individuals through one of the physically and emotionally most challenging times of their lives. Issues such as the potential use of life support need to be addressed before the preparation for transplantation is begun. Some 10% of HCT recipients require invasive life support, most often during the first 2 months after transplantation, and only a few of such unfortunate patients recover (see Chapter 100) [1,2]. It may take years for physicians to gain the necessary experience, medical knowledge, and sound clinical judgment to “perfectly” counsel their patients. It obviously requires a high level of comfort to present a medical procedure that is offered with curative intent but is also associated with significant procedure-related morbidity and mortality. Even under optimal conditions, autologous HCT is associated with an approximately 5% early fatality rate, and still 10–25% of recipients of allogeneic HCT succumb sooner or later to GVHD or GVHD-associated complications such as overwhelming infections, sinusoidal obstruction syndrome, formerly called veno-occlusive disease, prolonged immunodeficiency or secondary malignancies. The patient and the family members need to hear from the counseling physician that the intended treatment planned to provide a cure of the life-threatening disease may – in the case of one of the potentially severe complications – lead to the patient’s early demise. Still, much of what is explained during the pretransplant meetings is frequently forgotten when devastating complications occur later on. The special situations of

desperate patients and their families explain why often little information from the counseling meeting is retained. End-of-life issues need to be addressed early. Many patients may hesitate to bring this issue up but are actually relieved to talk about it openly when encouraged. This complex problem is particularly important in families with strange relationships (in couples who are separated but are not divorced, and in couples who are not married; and sometimes, there are children, siblings, parents or step-parents who want to make decisions later on but are not entitled to do so by the patient). To avoid miscommunications or misinterpretation of information early after the first meeting or at a later time, it may be useful to provide the patient with a copy of the consultation letter directed to the referring physician. Table 30.1 lists issues and topics that should be addressed during the counseling meetings with transplant candidates. This list is only a guideline and should be reduced or expanded as appropriate by each transplant physician and for each patient under evaluation.

The problem of permanent infertility For many young transplant candidates, the high probability of permanent infertility after high-dose therapy and HCT represents a major problem. Modern technologies such as cryopreservation of sperm and fertilized eggs can be used to overcome this serious issue in only a limited number of patients. Unfortunately, the ejaculates of many younger men are aspermic or hypospermic due to the effects of prior combination chemotherapy used for remission induction treatment. In the case of many younger women, the procedures to harvest eggs for in vitro fertilization fail. Despite successful fertilization, the number of those in whom a viable post-transplant pregnancy occurs is still extremely low. A small number of patients, men and women, have spontaneously recovered reproductive function, with healthy children being born during the years after transplantation. These patients were usually children or adolescents at the time of transplantation. It cannot be predicted at the time of transplantation which patient will be able to regain fertility. However, it should be noted that patients conditioned with high-dose cyclophosphamide as therapy for aplastic anemia often are able to have children during the years following transplantation (see Chapter 49). For more on the psychologic and physiologic aspects of infertility, sexuality, quality of life, and delayed long-term effects after HCT, the reader is referred to Chapters 33, 34, 35, 36 and 105. The recent introduction of reduced-intensity regimens into the field of HCT may result in a higher number of patients from both genders who retain or recover fertility. Since such regimens have so far included mainly older individuals, the information regarding this important issue is still lacking; however, cautious optimism seems to be justified.

The choice of transplant procedure Most patients arriving at a transplant center have been told by their referring physician that they are in need of a transplant procedure. Many of them are surprised when they hear that there are many different types of transplantation: autologous HCT (hematopoietic cells from the patient’s own marrow or blood), allogeneic HCT (grafts from a donor’s marrow, blood or cord blood), and occasionally syngeneic HCT (using marrow or blood hematopoietic cells from an identical twin donor). The counseling physician has to use his or her best judgment and knowledge, as well as the experience at the respective transplant center to advise the patient concerning the type of procedure that has the greatest likelihood of success for the candidate’s particular condition. Not all centers can or should offer all the different procedures mentioned above. For example, HCT using cord blood cells for an adult

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Table 30.1 Issues and topics that should be addressed during counseling meetings with transplant candidates and their families I. Rationale (Why is HCT offered to you?) II. Principle (How is HCT performed?) A. Autologous versus allogeneic transplantation B. Preparatory regimen 1. Choice of a high-dose or reduced-intensity regimen (with or without irradiation) 2. Risks associated with high-dose therapy a. Nausea, emesis, diarrhea b. Fluid retention c. Veno-occlusive disease d. Pancytopenia, immunosuppression, and associated infectious complications e. Mucositis and pain in the oral cavity and throat f. Hemorrhagic cystitis after cyclophosphamide g. Cardiomyopathy after cyclophosphamide h. Dermatitis after etoposide i. Pneumonitis after nitrosoureas j. Need for transfusional support k. Temporary or permanent alopecia, especially after busulfan 1. Infertility m. Cataracts n. Second or secondary malignancies C. Hematopoietic cell grafting 1. Marrow versus peripheral blood cell graft 2. Risks associated with infusion of a hemopoietic cell graft a. Allergic reactions b. Discomfort from cryoprotectant c. Graft failure and graft rejection D. Risks for GVHD 1. Symptoms and prognosis of acute GVHD 2. Need for drugs to prevent/treat acute GVHD 3. Side-effects of immunosuppressive drugs 4. Symptoms and prognosis of chronic GVHD 5. Drugs to prevent/treat chronic GVHD

6. Immunodeficiency resulting from GVHD including bacterial, fungal, viral, and other infections E. Risk of relapse after HCT F. Risk of mortality after HCT 1. Financial affairs, a will 2. Durable Power of Attorney for Health Care 3. Use of life support III. Timing of transplant (When should the procedure be performed?) 1. Very soon, e.g. progressive acute leukemia 2. Within weeks or few months, e.g. acute leukemia in remission 3. Elective HCT, e.g. follicular small cleaved lymphoma in early stages IV. Projected result (What can the candidate expect from HCT?) 1. Underlying disease and remission status 2. Compatibility of donor/recipient pair 3. Performance status of candidate 4. Comorbid conditions 5. Prior therapies 6. Nutritional status 7. Age of HCT candidate 8. Previous infections 9. Transfusion history 10. Other individual aspects V. Other important issues 1. Banking of sperm, in vitro fertilized eggs 2. Habits such as nicotine, alcohol or drug addiction 3. Need for active participation by patient and caregiver during entire course 4. Rationale and need for clinical trials including randomization 5. Duration of stay in the area of the transplant center 6. Return to home and work 7. Sexual activities 8. Time to full recovery 9. Quality of life issues

GVHD, graft-versus-host disease; HCT, hematopoietic cell transplantation.

recipient should still be limited to clinical research centers with pertinent expertise and specific research protocols. Likewise, HCT from haplotype-matched donors requires a transplant team with a commitment to clinical research, including a trained group of specialists in the graft engineering laboratory.

Which outcomes data should one quote? The single most important issue for the patient is the expected overall survival. Information described below indicates that the remission status and patient age are the most predictive factors – with age losing some of its prognostic power in the days of reduced-intensity regimens. Ideally, each transplant center would have a rich database from which one could generate information pertaining to each disease, remission status, age, etc. In the absence of such a powerful tool, which data should be quoted by the counseling physician? Recent studies involving 163 HCT centers in the United States indicate a significant “center effect,” i.e. 100-day mortality after allogeneic HCT was lower in centers with a higher patient-per-physician ratio and in those centers where physicians answered calls after office hours [3]. The data from a less experienced center are likely to be less favorable.

The patient counseled at that center should either be quoted centerspecific results (if available) or the relatively representative data from the large national and international bone marrow transplant registries.

What does the patient hear and remember? The patient comes to the transplant center with the expectation of being cured. Much of the information provided during the counseling meetings is viewed and interpreted by the patient in a more favorable light than is actually realistic. An important analysis has previously been reported from the Dana Farber Cancer Institute indicating significant discrepancies between patient and physician estimates for the success of HCT [4]. In this prospective study, 313 autologous or allogeneic HCT recipients and their physicians responded to baseline and follow-up questionnaires concerning their expectations for cure with or without HCT and for treatment-related mortality. Information of actual treatment-related mortality and disease-free one-year survival was subsequently available for 263 patients. The data are shown in Table 30.2. Patients and their physicians had the most concordant and accurate expectations when the outcome of HCT was likely to be favorable. However, patients with more advanced diseases failed to recognize the higher risk associated

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Table 30.2 Discrepancies between patient and physician estimates for the success of hematopoietic cell transplantation [4] Estimate Cure without stem cell transplantation, %

Cure with stem cell transplantation, %

Type of Transplant

No. of Patients*

Patient

Physician

Patient

Physician

Actual Percentage with Disease-Free Survival (95% Confidence Interval)䉱

Autologous✦ Allogeneic Disease status Early Intermediate Advanced

94 169

20

7

70

32

44 (30–58)

78 71 20

20 13 19

8 7 7

80 73 80

62 42 31

52 (40–64) 32 (21–44) 10 (0–23)

* Five patients did not provide an estimate of cure without stem cell transplantation and 3 did not provide an estimate of cure with stem cell transplantation. 䉱 At 2 years. ✦ Ninety patients with intermediate and 4 patients with advanced disease.

with their situations. This important study illustrates the urgent need to further improve the communication with transplant candidates.

The second counseling meeting After the first visit to the transplant center, the patient should be allowed adequate time (several days) to consider the often overwhelming nature and amount of information. In a second meeting, the transplant physicians should assure themselves that the patient understands the whole impact of the proposed treatment, both in general and in its details. An adequate amount of time should be set aside to address all issues and questions concerning the planned treatment and its potential complications. During the second meeting with the patient, the issue of advanced care planning, including the need for arrangements of financial affairs, a will, Durable Power of Attorney of Health Care, and the possible use of life support (if indicated), are to be tactfully but distinctly presented and discussed. A recent study on 155 HCT recipients indicates that 69% reported having designated a health-care proxy, 61% had prepared an estate will, and 63% had discussed their wishes regarding life support with family and friends. In contrast, only 16% had communicated their choice of life support to their clinicians [5].

Unless innovative treatment concepts to overcome resistance are pursued in the form of exploratory research trials, these patients should be advised against HCT. Miracles are rare. Of course, the patient is expecting that everything possible is going to be done, and many transplant physicians forget – while counseling such desperate patients – that the long-term transplant outcomes data, for example for patients with acute leukemia in relapse, are extremely disappointing. Still, the fastest growing group of patients undergoing HCT from an unrelated donor is adult patients with acute leukemia beyond second remission, where DFS is consistently in single-digit figures. Finally, subjecting a volunteer unrelated donor to the physical and emotional stress of marrow donation for a patient with refractory disease and a very poor prognosis requires serious consideration. How often in our decision-making process before HCT do we think about the donor, and how often do we think about this volunteer once the graft is infused? Honestly, rarely. A negative outcome of a transplant effort affects many donors for months or even years. They may suffer feelings of loss, failure and – quite often – guilt, especially if the patient dies of GVHD. Needless to say, the same issues apply to all related donors; for details concerning HCT donors, see Chapter 38.

How to counsel patients seeking multiple opinions When to advise against transplantation No firm rules for a minimal success rate can be established that would be generally applicable. The patient and the entire health-care team need to keep in mind that a long-term 10% DFS rate implies that nine of 10 patients sooner or later succumb to either relapse or NRM. For example, most patients with active central nervous system (CNS) leukemia have already received CNS therapy including irradiation prior to arrival at the transplant center. They will require further CNS treatment (irradiation and intrathecal drug instillations), which put them at a high risk for leukoencephalopathy, a devastating, debilitating complication. Although not all such patients end up with extensive intellectual deficits, however, such long-term complications need to be considered carefully, especially because even less extensive neurological manifestations can severely affect the future quality of life of the transplant recipient. Patients whose disease has recurred several times represent a similar dilemma. The chances that their underlying disease will persist or recur after HCT are exceedingly high, especially after autologous HCT.

Receiving the diagnosis of cancer is one of the most devastating events in the life of any human being. It is certainly understandable that patients are very concerned about their future health and wish to obtain the best treatment that could restore their natural life expectancy. Therefore, many patients seek second or even more opinions, especially when it comes to risky procedures such as HCT. We all have met these “traveling” patients who arrive at the transplant center with stacks of computer printouts to obtain another opinion. They are clearly in need of very careful guidance. These consultations often take several hours. We need to explain that the HCT procedures performed at most centers differ only in one or the other detail. In principle, we employ the same concepts, be it high-dose or reduced-intensity regimens followed by infusion of hematopoietic cells. It is the experience and the care provided at the transplant center that makes the difference. Moreover, many academic centers may offer innovative approaches that are tested in the setting of clinical research trials and are not available at other institutions.

The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation

It is the task of the counseling physician to guide the concerned and often confused patient to a most important decision while objectively explaining the pros and cons of one versus the other method, for example T-cell depletion of the graft at the Memorial Sloan Kettering Cancer Center in New York versus post-transplant immunosuppression of the host at Fred Hutchinson Cancer Research Center or the Stanford Hospital and Clinics. In this situation, the patient–physician relationship returns to its basic meaning. There is no room for dogma. Ultimately, most patients will decide in favor of the center at which they feel most comfortable and where they understand the rationale for one or the other approach.

The new foreign patient The number of foreign transplant patients may vary considerably from center to center. Approximately 7% of transplant recipients treated at the Fred Hutchinson Cancer Research Center come from another country compared with less than 1% at the Stanford Hospital and Clinics. Many patients from other countries are unfamiliar with the American insistence of discussing all pertinent issues openly with them and their families. They may be more accustomed to having unpleasant facts glossed over. Also, there may be many different dietary preferences and regulations making it advisable to involve a dietitian early on. Rules on body invasion (endoscopy, biopsy, and autopsy) are important considerations requiring particular explanations and greatest sensitivity. No detailed rules can be provided in this chapter as the expectations and needs will vary with patients from different countries of origin.

The issue of clinical trials All progress made during the past 50 years in the field of HCT is based on well-designed preclinical experiments and carefully conducted prospective clinical trials. Almost every transplant center is engaged in one or, more commonly, several clinical trials. Without patient participation, such important investigations are never possible. Therefore, all patients should be informed about clinical research trials as early as possible and be encouraged to consider enrollment in studies as part of their HCT procedure. The concept that a patient simultaneously serves as a research subject and the physician accepts also the role and responsibility of a clinical investigator should be explained during the first and subsequent counseling meetings. The rationale for clinical research studies, including phase I or II trials, needs to be addressed. Most patients and some referring physicians do not have a clear understanding of the rationale of and need for randomized trials. These clinical research efforts are made to define how a new treatment compares with a previously utilized approach. It should be explained to the patient that the “new arm” might be better, the same or worse than the “old arm.” If the answer were known, the trial would be superfluous. During either the first or second meeting, the patient should receive a copy of the written informed consent for his or her perusal. NonEnglish speaking patients regularly need assistance from interpreters, and – depending on the region of the transplant center – informed consent documents are routinely provided also in Spanish, Chinese, and other languages. The consenting process itself should be completed only after all questions have been satisfactorily answered and should meet both institutional and federal standards. Investigators with a conflict of interest need to provide pertinent information to their patients.

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ing their candidacy for transplantation. These factors are described below. Disease and remission status This textbook will provide the most up-to-date information on the treatment outcomes of autologous or allogeneic HCT for the many different diseases during the various remission stages. The extent of the underlying disease and its sensitivity to chemotherapy strongly influence the treatment result for recipients of autologous and allogeneic hematopoietic transplants. The reader will find multiple examples of the effect of the pretransplant remission status on long-term DFS, for example in acute leukemia, non-Hodgkin’s lymphoma, breast cancer, and most other conditions. For example, the event of a first relapse terminating a first complete remission in an adult patient with acute lymphoblastic leukemia (ALL) has a devastating effect on life expectancy: 5-year survival for those patients who receive further chemotherapy is under 5%, and for those who proceed to HCT, the results – depending on the type of graft – range from 15% to 23% [6]. If a second remission is attained using reinduction chemotherapy, the long-term outcome following allogeneic HCT from a matched sibling donor in children, adolescents, and young adults may approach 30–50% (see Chapters 55 and 56). In order to evaluate a patient’s candidacy for HCT, the most recent clinical information regarding the extent of the underlying disorder must be reviewed or must be obtained newly if the previous information is no longer considered to be accurate. For example, a patient with acute leukemia referred during first complete remission should undergo another bone marrow examination if the previous study was performed more than one month earlier. Patients with extramedullary leukemia with involvement of the CNS or other manifestations (chloromas of skin, bone system or testicular involvement) have a considerably reduced chance for a successful transplant procedure compared with patients whose leukemia is limited to the bone marrow and blood system [7–9]. Appropriate diagnostic procedures (biopsies, and cytogenetic and molecular methods) must be selected to assess the extent of current disease activity. For some conditions, for example the myelodysplastic syndromes, recommendations concerning the timing of HCT have been proposed [10]. These decision analyses are based on a widely used International Prognostic Scoring System, which includes cytogenetic findings, number of blasts, and blood cell counts. Patients with acute bone marrow injury following exposure to toxic agents may require a new assessment of their marrow cellularity and function to detect whether there are signs of spontaneous recovery obviating the need for the transplant procedure. Candidates referred for autologous transplantation who have received extensive and prolonged therapy with drugs (especially alkylating agents) and irradiation require detailed cytologic, cytogenetic and – if feasible – molecular analyses of their bone marrow to rule out any clonal abnormalities that would predispose them to later myelodysplastic or leukemic conditions [11,12]. Patients with solid tumors, regardless of their assumed stage, should be restaged to rule out new metastatic disease to the brain, liver or elsewhere, conditions that are considered strong prognostic indicators for early relapse after HCT. Compatibility of the donor–recipient pair

Conditions affecting treatment outcomes A number of pretransplant factors and conditions strongly influence the treatment outcome and thus impact the advice to patients regard-

Many patients referred to a transplant center for allogeneic transplantation have been previously tissue-typed, and an intended donor has been identified at that time. The transplant center should, however, repeat the

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histocompatibility testing at its own laboratory to confirm the accuracy of the typing data. After all, the transplant team assumes responsibility for the future care of the transplant candidate, and thus the expense of repeated laboratory procedures is fully justified. Tissue-typing information influences the candidacy for allogeneic transplantation and therefore the discussion with future patients during the counseling meeting. Marrow grafting from a genotypically matched sibling donor is still the most widely used allogeneic transplant approach for patients with hematologic malignancies, bone marrow failure conditions, and certain inherited disorders. Highly reproducible clinical data from the matched sibling donor–recipient pair combination have become available during the past three decades. It is generally accepted that transplantation from related donors who are either phenotypically identical or matched at five of six loci results in outcomes similar to data observed after HCT from human leukocyte antigen (HLA)-identical siblings [13] (see also Chapter 46). Through successful preclinical and clinical efforts, HCT from HLA haplotype-matched donors has now become possible and has opened up an entirely new and exciting area of transplantation [14–18]. The majority of potential transplant candidates have a haploidentical family member and could proceed to HCT. However, this approach requires an experienced graft-engineering laboratory and is as yet limited to very few clinical research centers. Moreover, alloreactivity of donor natural killer cells derived from such mismatched grafts against the leukemic blasts seems to be most potent in transplant recipients with acute myeloid leukemia (AML), whereas there is little reactivity – and thus a high relapse rate – in patients with ALL [18,19]. Only transplantation from natural killer cellalloreactive donors is associated with a survival advantage [20]. The field of HCT from unrelated donors has made remarkable strides during the past two decades, in particular through the improved understanding of the major histocompatibility complex (see Chapter 12) and the introduction of molecular HLA typing methods for donor selection [21–25]. It is the transplant center that should assume the primary direction and the responsibility in the search for the unrelated donor. The success rate of HCT from unrelated donors is improving continuously, as demonstrated by large clinical trials [26,27]. Both types of graft – from marrow harvests and from apheresis – have yielded similar outcomes [28]. There is currently a trial underway, sponsored by the

Clinical Trials Network, in which the two sources of grafts from unrelated donors are being prospectively compared. More details are provided in Chapters 43 and 47. Performance status The invasive nature of high-dose cancer therapy, with or without irradiation, always causes some extent of regimen-related toxicity, sometimes resulting in little and at other times in extensive morbidity and NRM. Therefore, the transplant candidate must be in good-to-excellent condition when preparation for HCT is begun. The performance score system introduced more than 50 years ago by Karnofsky and colleagues is still a most useful tool for the clinician in the assessment of patients [29]. It has been generally accepted that a patient’s performance score should be 70% or above to qualify for transplantation. Most cooperative groups follow this requirement for patient enrollment, although some groups have introduced their own scoring system; for example the Eastern Cooperative Oncology Group uses a 0–3 score with 0 being asymptomatic and without any evidence of disease. The Karnofsky scoring system has been adapted for HCT recipients by Sullivan and Siadak [30] (Table 30.3). This grading system can also be used to describe the performance status following transplantation. By definition, the best score early after transplantation is 70% (on day 100 following allogeneic HCT). The previous need to be a young, vigorous patient even to be considered for HCT has changed considerably over the past 10 years. Reducedintensity conditioning regimens have been introduced and explored in rapidly growing clinical trials [31–38]. This novel approach relies heavily on the graft-versus-malignancy effect derived from donor T cells. Because of the less toxic side-effects of the low-dose preparatory regimens compared with the previously used high-dose combinations, the extensive morbidity of the first few weeks following HCT is not encountered in this setting, and it is possible to perform these kinds of procedures on an outpatient basis. With reduced-intensity regimens, it is possible to treat older patients (up to the age of 75 years) and others who would previously have been excluded because of serious comorbid conditions. This approach has been greeted with a great deal of enthusiasm, partially explaining the poorly chosen term “mini-transplant.”

Table 30.3 Karnofsky score [29], modified for hematopoietic cell transplantation [30] General

Score (%)

Description

Able to carry on normal activity, no special care needed

100

Normal, no complaints, no evidence of disease

Unable to work, able to live at home and care for most personal needs, varying amount of assistance needed

Unable to care for self, requires institutional or hospital care or equivalent, disease may be progressing rapidly

90

Able to carry on normal activity, minor signs or symptoms of disease

80

Normal activity with effort, some signs or symptoms of disease

70

Cares for self, unable to carry on normal activity or to do work

60

Requires occasional assistance from others but able to care for most needs

50

Requires considerable assistance from others and frequent medical care

40

Disabled, requires special care and assistance

30

Severely disabled, hospitalization indicated, death not imminent

20

Very sick, hospitalization necessary, active support treatment necessary

10

Moribund

0

Dead

The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation

However, we should realize that these HCT recipients still face all the serious clinical transplant-related problems of allografting for malignancy, namely GVHD, infections, and relapse. The only major aspect that has changed is the lack of toxicity previously observed during the first post-transplant month. Long-term survival data (beyond 10 years) are not yet available. We should also keep in mind that the comorbid conditions may continue to affect the patient’s quality of life and life expectancy after a reduced-intensity regimen and HCT procedure. For example, one of our 52-year-old patients with chronic myeloid leukemia (CML) underwent such a transplant from her histocompatible brother in spite of having serious coronary artery disease, type II diabetes mellitus, and morbid obesity. She is now, 7 years later, in hematologic, cytogenetic, and molecular remission of her disease but has suffered another myocardial infarct, and her future is much more compromised by her heart disease, diabetes, obesity, and some chronic GVHD rather than by her prior hematologic malignancy. Prior therapies for the underlying diseases Every drug and every exposure to radiation administered since the time of the original diagnosis of the underlying malignancy has a long-lasting cumulative effect on the patient. For example, the treatment with standard doses of radiation in the form of a mantle field for Hodgkin’s disease a decade earlier may pose a significant risk factor if such a patient becomes a transplant candidate and is considered for TBI or for treatment with high doses of nitrosourea drugs. The cumulative effect of anthracyclines, which are widely employed in the standard therapy of leukemias and lymphomas, is well known and may limit the further tolerance of cardiotoxic drugs that are under consideration for use in preparatory regimens. Fortunately, newer agents may have less untoward effects, as was recently demonstrated for imatinib when administered to patients with CML prior to allogeneic HCT [39], and this drug even seems useful in the prevention of post-HCT relapse of patients with Philadelphia chromosome-positive ALL or high-risk CML [40]. Also, rituximab, which is widely employed in the pre-HCT therapy of patients with all variants of B-cell lymphoma, can be given as a consolidation measure after autologous HCT [41]. Finally, the concept of “total therapy” for multiple myeloma revolves around extensive combination therapy, which is continued in a reduced intensity after autografting [42]. Comorbid conditions Although – or maybe even because – the number of patients treated with reduced-intensity regimens and HCT is growing rapidly, the need for a

Table 30.4 Ideal eligibility criteria for adult candidates for high-dose therapy followed by allogeneic or autologous hematopoietic cell transplantation (HCT)

careful history and a physical examination, as well as objective tests which allow for a determination of the patient’s organ functions, must be stressed. In addition, regimen-related toxicity remains an important issue for all candidates for autologous HCT who will be exposed to maximum tolerated doses of chemotherapeutic agents (with or without radiotherapy). Recently, a new pre-HCT evaluation system, the Charlson Comorbidity Index, has been introduced and clinically tested to evaluate the influence of comorbidities on HCT outcome [43,44]. Although not yet widely accepted, this new approach may turn out to be a very valuable tool in the risk assessment of transplant candidates. Ideal eligibility criteria for adult candidates for high-dose therapy followed by allogeneic or autologous HCT are described in Table 30.4. In the following paragraphs, some of the most important aspects of compromised organ function are discussed. Cardiac evaluation Patients over the age of 50 years or those with a history of extensive exposure to anthracycline drugs may be at risk for future cardiac complications, especially if high doses of cyclophosphamide are part of the planned conditioning regimen [45–47]. In our experience and that of other centers, life-threatening cardiotoxicity after HCT is rare, occurring in less than 2% of all transplant recipients [48]. An ejection fraction of less than 50% as measured by two-dimensional echocardiography and a prior history of congestive heart failure are significant independent correlates of clinical cardiotoxicity [49]. Many cooperative cancer research groups and also some insurance companies insist on the use of radionuclide tests of ventricular function as a requirement for the enrollment of patients to clinical transplant trials or for the authorization of financial coverage, respectively. There is little reason to insist that all transplant candidates be evaluated with multigated acquisition scans [50]. Considerable financial resources can be saved by relying on two-dimensional echocardiography (if indicated with a stressechocardiogram). Respiratory evaluation Pulmonary function tests such as the forced expiratory volume, forced vital capacity, and diffusion capacity have gained a firm place in the pretransplant evaluation of HCT candidates [51–54]. A decrease in these parameters before transplantation predicts for post-transplant respiratory impairment, especially if chronic GVHD occurs, both in children and adults [55–57]. The data are not as clear in recipients of autologous grafts. Exercise tolerance with pulmonary function tests did not predict later respiratory compromise in 191 patients who underwent autologous HCT for non-Hodgkin’s lymphoma [56].

Age Karnofsky performance score Left ventricular ejection fraction Pulmonary function test; forced vital capacity Diffusion capacity Serum creatinine Serum bilirubin Alanine aminotransferase Aspartate aminotransferase Body weight IBW, ideal body weight.

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Allogeneic HCT

Autologous HCT

0–60 70–100 ≥45% ≥60% ≥60% ≤1.5 mg/dL ≤2 mg/dL 1–2 × normal 1–2 × normal 95–145% of IBW

0–75 70–100 ≥45% ≥60% ≥60% ≤1.5 mg/dL ≤2 mg/dL 1–2 × normal 1–2 × normal 95–145% of IBW

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Hepatic evaluation Liver function tests are routinely obtained before and after marrow transplantation in an attempt to predict or detect hepatic compromise (for details, see Chapter 95). An elevation of transaminases at the time of admission for HCT was significantly associated with post-transplant hepatic sinusoidal obstruction syndrome. Other risk factors associated with this complication included vancomycin therapy during cytoreductive therapy, aciclovir therapy before transplantation, and radiation therapy to the abdomen at any time before transplantation [58]. The occurrence of sinusoidal obstruction syndrome after transplantation varies greatly between transplant centers, ranging from less than 2% to over 50% of patients [58,59]. Possibly, patient selection and donor– recipient matching, as well as a tight control of the patient’s fluid status, may play an important role in the occurrence or prevention of this syndrome. Renal evaluation Testing of renal function is a standard requirement at all HCT centers. Serum creatinine of under 1.5 mg/dL (for adult patients) and a creatinine clearance over 60 mL/min are considered adequate as eligibility criteria for transplant candidates. Considering the potential nephrotoxicity of several drugs that are used frequently after transplantation, such as cyclosporine, vancomycin, aminoglycosides, amphotericin, aciclovir, and other agents, adequate kidney function seems to be mandatory. However, in patients with multiple myeloma who are treated with highdose melphalan and autologous HCT, even highly abnormal kidney function may still permit a successful transplant outcome [60].

would be 105 kg. Similar calculations apply if the patient’s body surface is used to determine drug doses. For several drugs (busulfan, cyclophosphamide or others), pharmacodynamic monitoring is more meaningful for dose decisions than is body weight (see Chapter 21). Age of the transplant candidate Age of allogeneic HCT candidates During the early years of allogeneic transplantation, successful outcome was limited mostly to children, adolescents, and younger adults. Even 15 years ago, many transplant centers would not have accepted patients above the age of 50 years because of concerns regarding early mortality and the high incidence of chronic GVHD in older patients. In 1986, two groups reported encouraging results after high-dose therapy and allogeneic marrow transplantation in older adults, ages 32–54 years and 45–68 years, respectively [63,64]. Data for patients with CML in the age group of 50–60 years indicate excellent survival outcomes [65]. Likewise, the results of older patients with acute leukemia have improved compared with 15 or 20 years ago [66]. The arrival of less toxic, reduced-intensity regimens has allowed the extension of allogeneic HCT to patients in the age group 60–75 years [67–71]. In this – as in all other settings – the advice to proceed to HCT should be individualized and based upon physiologic rather than chronological age. After all, the 70-year-old with AML can be cured by HCT, but the comorbid conditions still persist. Age of autologous HCT candidates

Nutritional status A nutritional assessment by an experienced dietitian, including an exploration of the patient’s dietary habits, should be part of the evaluation process of each transplant candidate; for details see Chapter 101. Extreme nutritional conditions such as cachexia or morbid obesity require special considerations and counseling. Malnourished patients may require pretransplant enteral or parenteral nutrition to improve their general condition, and patients suffering from excessive obesity may be advised to reduce their body weight under the guidance of a dietitian – provided the underlying disease permits the delay of the transplant procedure. It has been demonstrated in a large clinical study of 2238 patients at the Fred Hutchinson Cancer Research Center that body weight impacts on NRM after allogeneic transplantation [61]. This analysis indicated that patients whose actual body weight (ABW) ranged between 95% and 145% of ideal body weight (IBW) experienced comparable NRM, whereas patients whose weight was less than 95% or above 145% had decreased survival. The treatment outcome was poorest in patients whose ABW was less than 85% of IBW. We have performed a similar analysis at Stanford University School of Medicine in 473 consecutive adult patients with hematologic malignancies who received high-dose combination therapy followed by autologous HCT. Again, those patients who were malnourished or those who were extremely obese fared the worst [62]. The dosing of drugs in the preparatory regimen is frequently adjusted to IBW, which may be considerably different from the patient’s ABW. In the case of very obese patients, this dosing policy would result in serious underdosing with all its inherent consequences, i.e. insufficient immunosuppression and decrease in tumor cell kill. To overcome this problem, some transplant centers have made provisions in their clinical protocols by using a formula to calculate an adjusted IBW, i.e. IBW plus 50% of the difference between ABW and IBW. For example, if a patient’s IBW is 80 kg and the ABW is 130 kg, then the adjusted IBW

Several transplant centers have analyzed the effect of age on the outcome of autologous transplantation [72–76]. Although there appears to be an increased risk of NRM during the early phase after transplantation in patients over the age of 50 years, it does not seem to be warranted to exclude older patients from autologous HCT. Over the past 20 years at the Stanford Hospital and Clinics, we have autografted 284 patients between the ages of 60 and 76 years with various forms of hematologic malignancies or selected solid tumors; 12 patients (4.2%) in this group of 284 patients died within the first 100 days from NRM compared with 106 patients (5.3%) from a cohort of 1996 who were younger than 60 years old (range 0–59 years). DFS of the 284 older patients was not statistically different from that of the 1996 patients who were under the age of 60 years and were treated during the same period with identical protocols. Ethnic and racial background Three studies have dealt with the effect of race on the outcome of autologous or allogeneic transplantation [77–79]. With respect to autologous HCT, race or ethnicity were not significantly associated with survival of 1366 patients [77]. Analyzing the outcome of 2221 recipients of allogeneic grafts from histocompatible siblings indicated that patients of African-American background had a significantly greater mortality than white patients [78]. HCT was more associated with severe acute GVHD, and NRM was higher among African-Americans than whites. Mortality among other racial and ethnic groups was not significantly different from that of white patients. In another study on 1752 recipients of grafts from histocompatible siblings, it was found that white Americans, African-Americans, and Irish cohorts were at significantly higher risk for acute GVHD than Japanese or Scandinavian cohorts, although overall survival rates after HCT were similar for all groups [79]. Obviously, considerably more information concerning the impact of race and ethnicity is needed.

The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation

Previous infections Beginning with the time of their initial presentation of a malignant disorder or a bone marrow failure condition, patients enter a phase in their lives when they become more susceptible to infections than healthy individuals. Their conditions are even more compromised by antineoplastic treatments with chemotherapy and/or irradiation or, in the case of marrow failure, exposure to immunosuppressive drugs such as cyclosporine, antithymocyte globulin or glucocorticosteroids. Many of the patients referred to transplant centers have a history of prior infections with bacteria, fungi or viruses [80]. Modern medicine has provided us with powerful antibacterial drugs and also with agents which are effective against viral infections. Recently, new azol compounds have become available for the prevention and/or treatment of fungal infections, especially infections with Candida and Aspergillus species [81,82]. The evaluation process of transplant candidates, in particular those who are scheduled to undergo allogeneic HCT, should include a meticulous search for any signs of subclinical infectious sources. Such studies include standard X-rays as well as computerized tomography scans and magnetic resonance imaging scans – if clinically indicated. In addition, a dental examination is generally recommended. Fortunately, much is changing to the better in the field of HCT through basic and clinical research. Ten years ago when the second edition of this book was published, it was stated in this very chapter that “patients infected with the human immunodeficiency virus (HIV) cannot be considered for HCT.” With combined use of powerful antiviral agents, patients with HIV-associated lymphoid malignancies can now be transplanted successfully, as demonstrated by a recent clinical trial reported from the City of Hope National Medical Center [83]. Concerning specific infections, the reader is referred to Chapters 88–94. The issue of hepatitis of donors and recipients is specifically dealt with in Chapter 95. This is probably the right place in this chapter to mention that the incidence of certain infectious complications in patients treated with reduced-intensity regimens followed by allografting is by no means different than the infectious complication rate of patients treated with myeloablative regimens (e.g. fungal infections or complications related to cytomegalovirus) [84,85]. For further details, see Chapter 71.

Transfusion history Most patients with acute leukemia receive multiple transfusions during their remission induction therapy, and some patients are refractory to platelet transfusions at the time of referral for HCT. Other patients may have never been transfused, namely those with CML, non-Hodgkin’s lymphoma, and multiple myeloma. Thirty years ago, patients with aplastic anemia used to be referred for transplantation after extensive transfusional support, sometimes even with blood products from their relatives, a circumstance which is associated with an increased risk for marrow graft rejection [86]. Consistent educational efforts have been successful, and it is now relatively rare that aplastic anemia patients are sensitized in this manner [87,88]. Conversely, it seems that transfusions from blood relatives and even from the future hematopoietic cell donor do not interfere with the subsequent engraftment process in patients transplanted for hematologic malignancies, probably because of the strong immunosuppressive effect of high doses of TBI [89]. It is not yet entirely clear how far the prior exposure to blood products influences the outcome of those transplant procedures which involve the use of reduced-intensity regimens. It could be that the rejection rate is increased in polytransfused patients. For those patients who have been transfused, a detailed transfusion history should be obtained, i.e. number of and response to red blood cell

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and platelet transfusions. It is also important to monitor the number of transfusions in order to assess cumulative iron load [90]. Elevated serum ferritin levels are strongly associated with lower overall survival and DFS in patients transplanted for AML or myelodysplastic syndromes, as reported from the Dana Farber Cancer Institute [91]. Recent studies indicate that many long-term transplant survivors have secondary hemosiderosis and may need iron mobilization therapy [92], especially patients with thalassemia [93].

Psychosocial assessment It is the goal of the psychosocial assessment to get to know the candidates’ personalities and related issues in order to maximize patients’ participation in their own care and to lay the foundation for a successful return to their personal and professional life after HCT. It is furthermore the goal to minimize problem areas that would interfere in any way with the transplant procedure and the recovery phase. Pretransplant family conflicts are among other factors associated with impaired medical and psychosocial recovery [94]. The patient’s psychosocial history and condition have the potential to enhance or detract from the transplant procedure. The assessment, including an evaluation of previous response to pain control, should be made by a qualified professional at the time the patient’s medical candidacy is being evaluated. In addition, appropriate insurance funding for HCT is part of the evaluation process. The purpose of the psychosocial assessment is summarized in Table 30.5.

Table 30.5 Goals of the psychosocial assessment of adult hematopoietic cell transplantation (HCT) candidates Purpose of psychosocial assessment: 1) Documentation of psychosocial stability 2) Identification of patient and family strengths and weaknesses in coping mechanisms 3) Assistance with preplanning for the HCT procedure, e.g. arrangement for childcare, transportation, state and federal program linkages, etc. 4) Coordination of any corrective action if necessary for the success of the transplant procedure, e.g. referral for psychiatric evaluation, compliance contract, drug testing, etc. Individual areas of patient assessment should include: 1) Previous methods of dealing with treatment issues and crises 2) Past and present drug, alcohol, and tobacco usage 3) Past and present psychiatric history 4) Understanding of diagnosis, treatment plan, and prognosis 5) Compliance with previous therapy needs 6) Motivation 7) Unique cultural, religious, literacy, and language needs 8) The ability to use information given 9) Previous activity and interest levels Other important areas for assessment are: 1) Emotional support for patient during all phases of transplantation 2) Caregiver availability, i.e. family members or friends 3) Employment issues 4) Advanced care directives, e.g. Durable Power of Attorney for Health Care, a will 5) Stability of the environment to which the patient is expected to return 6) Family adjustment to diagnosis, therapy, and prognosis 7) Ability to relocate geographically for transplantation 8) Other stressors, i.e. parents, children, insurance, transportation, etc.

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Substance abuse is associated with a poor outcome of HCT [95], and uncontrolled drug, nicotine, and alcohol dependence are exclusion criteria. The State Insurance System in California (Medi-Cal) does not provide coverage unless the transplant candidate has proven abstinence for an adequate length of time. A history of any substance dependence indicates the need for a formal psychiatric evaluation prior to acceptance for HCT. Noncompliance in the context of HCT is a major issue in the case of many adult patients [96]. Smoking during and at any time after transplantation is not at all acceptable. Several of the issues mentioned on psychosocial assessment overlap with what has been stated in earlier paragraphs describing the patient– physician interaction. It is actually desirable that some of the psychosocial issues be assessed by more than one person, i.e. the attending physician and the medical social worker. The impression of both examiners should be presented in a formal meeting in order to create an impression of the patient’s candidacy for HCT. During such a meeting, all medical and social issues should be presented to an institutional group of transplant physicians, social workers, research nurses, dietitians, etc., and the candidacy for HCT of the referred patient, including enrollment to institutional or national clinical trials, be determined.

Special considerations for pediatric transplant candidates The matter of consent Obviously, many of the medical and psychosocial concerns of the adult HCT candidate are pertinent to children, but because the patient is a child, the physician must attend to the concerns of the patient’s guardian(s) as well. Foremost among these concerns is the matter of consent. While for adult HCT candidates the impact of transplantation on a spouse and other family members must be considered, ultimately the patient, informed of the risks and benefits of transplantation, consents to treatment. Children and adolescents are not legally able to consent. Parents on behalf of the child must weigh the options and decide the best course. (I say the parents, but a child’s primary caregiver may of course be grandparents, other family members, court-appointed foster parents or representatives of children’s protective services. The consent process must be adapted to each child and caregiver relationship.) Providing parents with information and direction and helping them reach an appropriate decision for their child is the role of the physician. What follows will emphasize in my (R.A.K.) estimation the most important issues to be addressed when obtaining the consent for a child being considered for HCT. The reader is also referred to Chapter 32 on ethical issues. Initial visit by the patient and parents The patient, be it an infant, young child or adolescent, is the physician’s primary concern. Although in most circumstances it is the parents who sign the informed consent, the physician must be attuned to the patient’s capacity to understand the transplant process. Young children, precocious in the understanding of their disease and its implications, require an explanation of HCT rationale suitable to their level of comprehension. Alternatively, adolescents who do not choose to be involved in decisions regarding their treatment should be allowed to participate up to their level of comfort. The physician must grasp the child’s emotional maturity and provide an appropriate level of treatment discussion. Parents may be helpful in advising the physician of the patient’s desires, although some parents, irrespective of the child’s wants, seek to protect the patient from knowledge of the serious risks and consequences of treatment.

Children with malignant disease Whether a child is a candidate for an allogeneic or autologous HCT depends upon the disease diagnosis and status. HCT may be an integral component of the primary therapy plan, as autologous HCT is routinely performed for children with stage 4 neuroblastoma [97]. Alternatively, HCT may be used as salvage therapy, as allogeneic HCT can be recommended for children who have failed chemotherapy for ALL [98]. Depending upon the circumstances, the initial family conference must clarify the rationale for HCT. It happens that parents arrive at this meeting with preformed impressions and fears, which have arisen from conversations with other parents or with family and friends. Often what parents have heard is erroneous or exaggerated, and these notions must be corrected. The indications for HCT for children with malignant disease continue to evolve just as they do for adults. Where opinions differ regarding the necessity for HCT, it is important to discuss the alternative treatment with the parents. In centers where physicians limit their practice to HCT patients, the obligation regarding counseling families differs from centers where the physician is responsible for managing both the standard chemotherapy and HCT. In the former, a primary physician has determined that HCT should now be considered an option and refers the patient. Although the transplant physician must decide based upon his or her own understanding whether HCT is appropriate for the child, there is no inherent conflict between the treatment roles. In centers where roles overlap, the physician must be aware of potential bias and carefully explain the treatment alternatives, even offering the family the option of a second outside opinion. Children with nonmalignant disease Although children with malignant disease comprise the majority of transplant candidates, patients with genetic and immunologic disorders may benefit from transplantation, and these patients and their families require the consent discussion be attuned to the specific disease. Nonmalignant diseases eligible for HCT include conditions that are immediately life-threatening, for example aplastic anemia or severe combined immunodeficiency, and other conditions that, while not immediately life threatening, ultimately will lead to death or to devastation of the child’s quality of life, for example sickle cell anemia or adrenoleukodystrophy. When patients are referred for HCT for these indications, it is critical that the transplant physician work closely with the primary specialist. Ideally, a coordinated effort will accurately portray the intent for HCT and what might be achieved. It also lays the groundwork for the patient’s optimal care in the post-transplant setting. Even when transplantation is successful, there may be delayed toxicities from therapy, or patients may remain susceptible to elements of the primary disease, as children transplanted with Hurler’s disease may continue to accumulate glycosaminoglycans in joints and connective tissues [99]. Coordination of care is essential to maximize the transplant recipient’s quality of life. Patients with nonmalignant disease rarely receive chemotherapy prior to transplantation. Without this experience, patients and families lack a frame of reference to comprehend the side-effects, both expected and rare, that may occur during HCT conditioning therapy. The physician must carefully explain the necessity for conditioning therapy and reassure families that all efforts will be taken to minimize discomfort and risk for their child. Conditions affecting the child as a candidate for HCT The evaluation of the patient as a HCT candidate reconciles the patient’s current state of disease and overall physical condition. Both elements

The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation

influence treatment outcome and prognosis. For example, neither a child in otherwise good clinical health but with refractory acute lymphoid leukemia nor a child with adrenoleukodystrophy showing widespread demyelination is likely to benefit from HCT [100,101]. In such cases, outside a clinical investigation, the recommendation should be against HCT. Initially, for most families this will be unacceptable, but with a careful and compassionate explanation, the physician can bring the family to understand that the discomforts and toxicities of HCT are not justified for their child when there is no benefit to accrue. More difficult for the patient, family, and physician are those circumstances where the likely benefit from HCT is low but not altogether absent. Here too, candid discussion allows the family an opportunity to ask questions and discuss their concerns and expectations. Although it is rare that the family will decide not to proceed with HCT, knowing that refusing HCT is an option can be very important. It is possible that an older adolescent may refuse HCT or wish to delay hospital admission to fulfill an important life wish. These decisions, whichever way, should be respected and supported by the physician. Estimation of performance status is always subjective to a degree, and this is even more so for children compared with adults. The Karnofsky scale, intended for adults, can be used for older children; school serves as a surrogate for work. For younger children, the Lansky scale (Table 30.6) [102] is more appropriate, but this scale relies heavily on parental reports. Even more problematic, the scale includes developmental skills that are not applicable to toddlers and infants. Although it has been demonstrated that pretransplant performance measure may correlate with outcome, alone these scales lack sensitivity and specificity sufficient to counsel the family [103]. Combined with organ-specific evaluations, performance scale measures can identify patients at greater risk for regimen toxicity [104,105]. For children with malignant disease, the most likely explanation for a low performance status is the effects of the disease for which transplantation is being considered. Children have not lived long enough to suffer the morbidities that arise from aging and/or a lifetime of bad health habits. Organ-specific evaluation must anticipate the consequences of prior therapies, for example myocardial injury secondary to anthracyclines or mediastinal irradiation. Complications from prior therapy may be exacerbated during the transplant course, as a patient with previous pulmonary fungal disease and reduced respiratory reserve is at grave risk from further lung injury. The physician’s exposition of

Table 30.6 Lansky Play-Performance Scale [102] (for patients ages 1–14 years) Score (%)

Description

100 90 80 70

Fully active, normal Minor restrictions in physically strenuous activity Active, but tires more quickly Both greater restriction of, and less time spent in, play activities Up and around, but minimal active play; keeps busy with quieter activities Gets dressed but lies around much of the day, no active play; able to participate in all quiet play and activities Mostly in bed; participates in quiet activities In bed; needs assistance even for quiet play Often sleeping; play entirely limited to very passive activities Unresponsive Dead

60 50 40 30 20 10 0

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the performance status, organ evaluation, and prior morbidities enables the patient and family to better comprehend the risks undertaken during transplantation and to understand the rationale of the treatment plan. Organ system evaluation for the pediatric patient is similar to that for an adult but, depending upon the child’s age, may require modification. For the youngest patients, pulmonary function tests may not be available [106]. Some children’s hospitals can perform pulmonary function tests on infants, but their value in the transplant setting has not been established. If a child has experienced serious respiratory complications, imaging studies and consultation with a pediatric pulmonologist prior to transplant may be useful. Two potentially important conditions, obesity and iron overload, may increase the risk for regimen-related toxicity, particularly liver toxicity. The increase in morbidity and mortality associated with excess liver iron has been established for patients undergoing HCT for thalassemia, but undoubtedly this association is pertinent to any patient with iron overload [91,107]. Thus, any child coming to transplant having received frequent red cell transfusions, for example a patient with Diamond– Blackfan anemia or other marrow failure disorder, must be considered at heightened risk from regimen-related toxicities. Serum ferritin may screen for excess storage iron; however, liver biopsy combined with quantitative iron determination is necessary to identify those most susceptible patients [108]. No current assay for liver iron is totally satisfactory, and magnetic resonance imaging combined with liver biopsy may provide a more complete understanding of liver iron excess. Although iron accumulation by the heart can compromise cardiac function, adequate cardiac function determined by echocardiography diminishes the likelihood of clinically significant iron accumulation. Poorly nourished children coming to HCT can usually be managed successfully with parenteral nutrition; less apparent is the risk for toxicity that HCT imposes on the obese child. For obese patients, elevated liver transaminase may be indicative of liver steatosis, or of steatohepatitis when insulin resistance and hypertriglyceridemia are present as well [109,110]. The metabolic syndrome of obesity, hypertension, hyperlipidemia, and glucose intolerance is causally related to cardiovascular disease in adults, but the significance of these findings has not been established for children. It is to be noted that children, particularly adolescents, are more prone to the hyperglycemic effects of prednisone and L-asparaginase [111]. Parents and caregivers compound the problem by ignoring or even promoting obesity by allowing the child indiscriminate amounts of high-calorie food. Liver biopsy may be warranted to clarify the extent of liver injury, as the risk for hepatic toxicity, immediate and delayed, may be more frequent among these individuals [112]. The impact of HCT on the child’s neurocognitive development concerns parents and physicians alike. If possible, neurocognitive testing should be obtained for all children before the start of conditioning [113,114]. Baseline assessment provides a reference to measure future development, which becomes important if remediation efforts are needed later. This is most relevant for children who receive TBI and/or CNS irradiation for conditioning as such patients are most at risk for neurocognitive injury. Neurocognitive tests are absolutely essential to evaluate patients with metabolic storage disorders that cause neurocognitive deterioration. Poor neurocognitive performance index prior to HCT predicts an unsatisfactory outcome following HCT and should dictate against proceeding [115]. Counseling the patient and family Emotional distress for patients undergoing HCT often is most severe as treatment commences and tends to diminish thereafter [116,117]. Likewise, parents’ distress prior to HCT is greater than at any later time during the transplant course [118]. Thus, the initial meeting with the

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family should attempt to address the causes for their distress. Not knowing what to expect from HCT is a major source of anxiety and fear [119]. The physician, by providing details of the transplant course, allows the child and family to participate in and, to a degree, to control future events, which can help diminish stress. This said, it is important for the physician to tailor the discussion of medical matters in a manner that is appropriate to the cultural and social background of the family. While it is important to convey the rationale for HCT as well as its risks, benefits, and long-term consequences, the discussion must ultimately meet the needs of the patient and the family. Some families prefer very detailed information and may even question the necessity for particular elements of the treatment, for example the use of TBI. Yet other families become confused and anxious from too much detail and prefer to place their trust in the physician and be reassured that the physician will do the best for their child. If desired, other family members should be allowed to partake in the consultation process. Single parents, overwhelmed by the total responsibility for consenting, should be encouraged to bring a grandparent or other supportive person to this meeting [120–122]. If the treatment is a clinical trial, the physician must take care that the patient and family understand the experimental component, possible alternative treatments, and their right to refuse or withdraw [120]. The presence of a child life specialist at the consent discussion may be an effective means to assure understanding of the proposed treatment. Every family brings hope for the future of their child to the HCT process. HCT is the means to the future, and at that moment the parents’ focus is on the child safely passing through this treatment. Nonetheless, there are important long-term consequences arising from HCT which parents must be made aware of. Among these are the effects of HCT on intellectual development, growth, and fertility, and the risk for a second malignancy following HCT. Problems arising from school are not uncommon for children post transplantation. Undoubtedly, patients come to transplant already challenged academically because of the school absence related to the primary disease and its treatment. Certain diseases for which HCT is performed may directly impact the CNS, such as brain tumors or demyelinating diseases. For hematologic malignancies, previous treatment including intrathecal chemotherapy may have affected neurocognition. HCT conditioning therapy with TBI adds to the risk for future learning difficulties, although it has been shown that cognitive function does not invariably decline post HCT. Reassuringly, except among the youngest patients, many studies find preservation of pre-HCT neurocognitive function [114,123]. For children less than 3 years of age, most investigators document at least some fall-off in neurocognitive test performance. In relaying this information to parents, it is important to emphasize that a profound deterioration of intellect is rare and that coordinated efforts between neuropsychologists and educators may help the child compensate. Linear growth depends upon intact bone and endocrine function; radiation therapy disturbs both systems. As is true for neurocognitive function, the patient’s growth may have suffered prior to coming to HCT, and again it is the youngest patients who will be most seriously affected [124,125]. The physician must counsel the parents that while treatment with growth hormone may improve the child’s linear growth, it is unlikely that the child will achieve what would have been the predicted adult height (see Chapter 104). Although fertility has been discussed previously, there are several points unique to pediatric patients. Pubertal development may be delayed in up to half of children treated with fully ablative conditioning, and children receiving fully ablative HCT conditioning, particularly when the conditioning includes TBI, rarely are fertile [124]. For patients with malignancy and previously treated with intensive chemotherapy, fertility may have already been diminished. Often families indicate that they had

not been aware of this possibility. Even as parents learn of and accept the risks for regimen-related toxicity and the potential for unfavorable or even fatal outcome following HCT, it may be heartbreaking for some to have to accept the responsibility for their child’s infertility. For many cultures, the loss of the ability to conceive carries momentous impact, and the physician must be attuned to this. As already presented, the development of reduced-intensity conditioning therapy may allow more children to retain their fertility, and the potential for advances in reproductive medicine offers parents hope for the future [126–128]. Successful HCT allows children the chance to live a normal and meaningful life, but unfortunately not a life without persistent concerns. One undesirable consequence of surviving is the risk of secondary cancer. The risk is in part disease and HCT conditioning regimen specific [129]. Unfortunately, the risk of secondary cancer has not been adequately determined for every condition. HCT conditioning therapy that includes radiation has been associated with a higher incidence of new tumors, but heightened risk exists for patients receiving chemotherapy alone [130]. It may be important to note here that the estimate of developing solid tumors was 13% among patients transplanted for aplastic anemia and that, in the multivariate analysis, radiation, typically less than 10 Gy, was associated with that increased risk [131]. Although reduced-intensity conditioning regimens allow children with severe pretransplant morbidity to undergo HCT, the risk for secondary cancers from reduced-intensity regimens containing TBI has not been defined. Selection of stem cell source As previously noted, there is wide variation in the understanding of the origin of hematopoietic cells and how these are obtained. Some families arrive at the counseling session with a particular bias in this regard. It is the physician’s responsibility to clarify the advantages and disadvantages between alternative stem cell products and to explain the rationale behind the proposed treatment plan. For children with an HLA-genoidentical sibling donor, bone marrow will be the preferred stem cell product in almost every instance. The physician must explain to the parents and to the donor, suitable to the donor’s level of understanding, the side-effects and risks associated with the harvest procedure. A child life specialist can allay a young donor’s fear by rehearsing the process prior to the actual procedure. A sibling donor who grasps the gravity of the patient’s circumstance may assume responsibility or blame if the outcome of the transplant is not successful [132–134]. The donor must be reassured that the outcome of the transplant and the survival of the patient is not determined by the marrow and that the donor’s willingness is unequivocally a gift of tremendous portent. For patients without a sibling donor, either a mismatched related donor or an unrelated donor must be selected. Many factors go into this selection and will not be discussed further here. It is important that transplant centers with a particular approach to transplantation, which includes the stem cell source, acknowledge this during the discussion. Advanced directive Because HCT is an aggressive and intensive treatment and is intended to be life saving, the physician should establish during the initial sessions that other aggressive interventions, for example dialysis or mechanical ventilation, may be needed to support the patient during the treatment course [135]. Such discussion offers adolescents and some precocious younger children and their parents an opportunity to consider in what circumstances the decision would be to withhold or withdraw care. Rarely, in my experience, do adolescents provide an advanced directive; however, these matters may be discussed within the family, and parents

The Evaluation and Counseling of Candidates for Hematopoietic Cell Transplantation

will know of their child’s wishes. These conversations may become very important should care ultimately become futile.

Some final considerations HCT requires a cooperative, educated, and insightful patient. Through the educational efforts of the transplant team and the informed consent process, the patient should have acquired the high level of understanding and knowledge which is a prerequisite for participation throughout the long HCT process, both at the transplant center and subsequently in the domestic environment. It is sometimes forgotten or ignored that the transplant process is not completed with the return of the white blood cell count to the normal range. Deficiencies of the immune system persist for many months and sometimes years. Allogeneic marrow graft recipients especially are at risk for late infectious complications even if all in vitro tests are showing a return of

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normal immune function. The transplant patient should be instructed to seek medical advice for even relatively harmless-appearing symptoms. A well-functioning communication system between the transplant center, the patient, and the local physician has to be a permanent part of the post-transplant course. During the more than 30 years in the field of HCT, we can remember several sad circumstances which led to the demise of patients where a timely telephone call either from the patient to his local physician or from the local physician to the transplant center might have saved the patient’s life. It is our responsibility to educate and to educate again! This chapter can never be complete. There are simply too many medical or social circumstances that cannot be described in detail or even be anticipated. There are no rules for common sense and no substitute for professional judgment or rapid communication. Regarding many of the patient care issues before and after transplantation, we refer the reader to Chapters 31–36 of this book.

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109. Nanda K. Non-alcoholic steatohepatitis in children. Pediatr Transplant 2004; 8: 613–18. 110. Roberts EA. Pediatric nonalcoholic fatty liver disease (NAFLD): a “growing” problem? J Hepatol 2007; 46: 1133–42. 111. Pui CH, Burghen GA, Bowman WP, Aur RJ. Risk factors for hyperglycemia in children with leukemia receiving L-asparaginase and prednisone. J Pediatr 1981; 99: 46–50. 112. McDonald GB. Review article: Management of hepatic disease following haematopoietic cell transplant. Aliment Pharmacol Ther 2006; 24: 441–52. 113. Moore BD 3rd. Neurocognitive outcomes in survivors of childhood cancer. J Pediatr Psychol 2005; 30: 51–63. 114. Phipps S, Dunavant M, Srivastava DK, Bowman L, Mulhern RK. Cognitive and academic functioning in survivors of pediatric bone marrow transplantation. J Clin Oncol 2000; 18: 1004–11. 115. Peters C, Balthazor M, Shapiro EG et al. Outcome of unrelated donor bone marrow transplantation in 40 children with Hurler syndrome. Blood 1996; 87: 4894–902. 116. Fife BL, Huster GA, Cornetta KG, Kennedy VN, Akard LP, Broun ER. Longitudinal study of adaptation to the stress of bone marrow transplantation. J Clin Oncol 2000; 18: 1539–49. 117. Siston AK, List MA, Daugherty CK et al. Psychosocial adjustment of patients and caregivers prior to allogeneic bone marrow transplantation. Bone Marrow Transplant 2001; 27: 1181–8. 118. Phipps S, Dunavant M, Lensing S, Rai SN. Patterns of distress in parents of children undergoing stem cell transplantation. Pediatr Blood Cancer 2004; 43: 267–74. 119. Hummelinck A, Pollock K. Parents’ information needs about the treatment of their chronically ill child: a qualitative study. Patient Educ Couns 2006; 62: 228–34. 120. Dermatis H, Lesko LM. Psychological distress in parents consenting to child’s bone marrow transplantation. Bone Marrow Transplant 1990; 6: 411–17. 121. DuHamel KN, Manne S, Nereo N et al. Cognitive processing among mothers of children undergoing bone marrow/stem cell transplantation. Psychosom Med 2004; 66: 92–103. 122. Manne S, DuHamel K, Nereo N et al. Predictors of PTSD in mothers of children undergoing bone marrow transplantation: the role of cognitive and social processes. J Pediatr Psychol 2002; 27: 607–17. 123. Simms S, Kazak AE, Golomb V, Goldwein J, Bunin N. Cognitive, behavioral, and social outcome in survivors of childhood stem cell transplantation. J Pediatr Hematol Oncol 2002; 24: 115–19. 124. Sanders JE. Endocrine complications of highdose therapy with stem cell transplantation. Pediatr Transplant 2004; 8(Suppl 5): 39–50. 125. Sanders JE, Guthrie KA, Hoffmeister PA, Woolfrey AE, Carpenter PA, Appelbaum FR. Final adult height of patients who received hematopoietic cell transplantation in childhood. Blood 2005; 105: 1348–54. 126. Stein J, Dini G, Yaniv I. The hope and the reality of reduced intensity transplants in children with malignant diseases. Bone Marrow Transplant 2005; 35(Suppl 1): S39–43.

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127. Lobo RA. Potential options for preservation of fertility in women. N Engl J Med 2005; 353: 64–73. 128. Cheng YC, Saliba RM, Rondon G et al. Low prevalence of premature ovarian failure in women given reduced-intensity conditioning regimens for hematopoietic stem-cell transplantation. Haematologica 2005; 90: 1725–6. 129. Curtis RE, Rowlings PA, Deeg HJ et al. Solid cancers after bone marrow transplantation. N Engl J Med 1997; 336: 897–904. 130. Ades L, Guardiola P, Socie G. Second malignancies after allogeneic hematopoietic stem cell

transplantation: new insight and current problems. Blood Rev 2002; 16: 135–46. 131. Deeg HJ, Socie G, Schoch G et al. Malignancies after marrow transplantation for aplastic anemia and fanconi anemia: a joint Seattle and Paris analysis of results in 700 patients. Blood 1996; 87: 386–92. 132. Shama WI. The experience and preparation of pediatric sibling bone marrow donors. Soc Work Health Care 1998; 27: 89–99. 133. Packman W. Psychosocial impact of pediatric BMT on siblings. Bone Marrow Transplant 1999; 24: 701–6.

134. Packman WL, Crittenden MR, Schaeffer E, Bongar B, Fischer JB, Cowan MJ. Psychosocial consequences of bone marrow transplantation in donor and nondonor siblings. J Dev Behav Pediatr 1997; 18: 244–53. 135. Jacobe SJ, Hassan A, Veys P, Mok Q. Outcome of children requiring admission to an intensive care unit after bone marrow transplantation. Crit Care Med 2003; 31: 1299–305.

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Rosemary C. Ford & Mihkaila M. Wickline

Nursing Role in Hematopoietic Cell Transplantation

Introduction Caring for patients undergoing hematopoietic cell transplantation (HCT) is one of the most challenging and rewarding of all nursing subspecialties. These patients require nurses with expertise in assessing the acute, subtle, and unique clinical changes of HCT. In addition to the knowledge base of oncology nursing, the specialty of HCT nursing requires expertise in addressing unique diseases such as sinusoidal obstructive syndrome and graft-versus-host disease (GVHD). Most of nursing’s effort is focused on the post-transplant phase with managing side-effects and toxicities, and preventing complications caused by the high-dose chemoradiotherapy given to prepare for HCT and the consequences of GVHD [1]. Nurses are the members of the HCT multidisciplinary team that spend the most time with the patients, and often are the first to identify changes in clinical status. Nurses must also have psychosocial skills as they support the patients and their significant others through decisions and acute clinical changes encountered during the HCT process. Supporting medical research, the foundation of HCT care, is another factor that attracts nurses to this subspecialty. Integrating new research initiatives into clinical care and being on the front line as new therapies evolve to change standard practice is very exciting. Nurses feel they are working in a medical arena offering the best possible therapy to their patients. Those nurses who stay with this specialty over a number of years have witnessed therapies used in phase I research studies become the standards upon which current clinical research is based. These nurses can readily identify the improvements in patient outcomes that have occurred in the field of HCT over the past three decades in curing specific diseases, as well as improvements in the prophylaxis and treatment of GVHD and sinusoidal obstructive syndrome, infectious disease prevention and therapy, and symptom management. In addition, expanding HCT to treat autoimmune diseases and solid tumors has required nurses to become familiar with disease processes beyond hematologic malignancies. The recent efforts to treat patients using reduced-intensity conditioning regimens have presented new challenges to HCT nurses. Not only are these patients often older with more comorbidities than those eligible for myeloablative regimens, but also the patterns in their clinical courses in areas such as acute GVHD presentation can differ from those of myeloablative transplants. Nurses must be flexible and able to accom-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

modate changes in medical therapy such as extracorporeal photopheresis for the treatment of chronic GVHD [2]. There has been a major change in the site of HCT care from inpatient units to outpatient clinics with the goal of containing costs while maintaining high-quality care [3]. This change was made possible by major improvements in oral antiemetic efficacy, antibiotics with less frequent dosing, and improved ambulatory infusion devices [4]. Patients often receive all of their conditioning and hematopoietic stem cell (HSC) infusion in a clinic setting, and are admitted for clinical reasons (usually fever or mucositis pain) rather than to receive a therapy. Patients are also discharged from the hospital based on more liberal criteria than in the past. This change has resulted in increasingly complex ambulatory clinics where the focus of nursing care shifts from first-hand assessment and care of inpatients to teaching the caregivers to provide some of these services in the home setting [5]. This has caused the average acuity of the HCT inpatient populations to markedly increase, with patients acutely ill with complicated multisystem issues. It has also led to the majority of patients having multiple unscheduled admissions and discharges in the months after transplant. Nurses are responsible for ensuring continuity of care and patient safety as the responsibility for care passes from one site to another [6,7]. The intensity of therapy requires the further subspecialization of HCT nurses to meet the care needs of these patients. Transplant centers organize nursing support in a variety of ways depending on their larger operational structure. At most centers, specific nurses, or “coordinators,” have a role in the initial intake of patients into the program. Most outpatient clinics have a case manager structure, which allows for continuity of care coordination throughout the transplant process. HCT patients often spend large amounts of time in outpatient infusion areas. These patients require nursing expertise different from that of other oncology outpatients. Inpatient care is provided either on a specific transplant unit or on a designated portion of an oncology unit for HCT care with specific nurses assigned to this area. Each center needs to give serious consideration to whether to keep HCT patients requiring intensive care on the HCT unit or to transfer them to an intensive care unit (ICU). In either case, nurses with expertise in both HCT and intensive care are required. Discharging HCT patients from an inpatient unit to the outpatient setting is often complicated, and many centers have specific nurses with expertise in managing these transitions. Complex research protocols often require coordination by a specific nurse who works closely with the principal medical investigator. These nurses are responsible for ensuring documentation of protocol data and the reporting of adverse advents. They are key in ensuring that protocol coordination is seamless when patients transfer to and from the inpatient

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and outpatient settings. They are the primary resource for medical and nursing staff regarding protocol implementation. In addition, because of the relatively long length of stay in the medial centers with standardized treatments, HCT patient care provides ideal opportunities for nursing research. Nursing studies have been conducted in a number of areas including quality of life and symptom management [8–11]. The purpose of this chapter is to describe the nurse’s role at the various phases of the HCT process. Tables detail the key specific nursing responsibilities of assessment, teaching, care coordination, and carrying out the medical care plan at each phase. They do not list general nursing basics such as vital signs and weighing the patient. Assessment includes evaluating not only the basics of physical assessment of the patient, but also the ability of the patient and caregivers to cope with this intense experience. Teaching must be individualized to each specific patient and caregiver situation. There is no substitute for one-on-one assessment and teaching by the patient’s primary nurse [12]. One-on-one encounters allow for assessment of the patient’s assimilation of the required information and a time for the patient to ask whatever questions are of foremost concern. Teaching can be accomplished by a variety of methods, including written materials with photographs or illustrations and videotapes. Classes are also an effective method for centers that perform a large number of transplants. Coordination of care for these patients is extremely complex. Nurses assess the patient’s and family’s strengths and weaknesses, and plan interventions to assist them to integrate this intense process into their lives. Nurses must include clinical issues, operational resources, research requirements, and the patient’s needs and desires when planning care. Carrying out the medical care plan is an important role of the transplant nurse. Specialized understanding of the patient’s underlying illness, the transplant procedure, complications of transplant, and the typical treatments for each complication are important for successful implementation of the medical care plan.

Prework-up/prior to arrival at transplant center (Table 31.1) The first interface many patients have with an HCT transplant program occurs at the time they come for a second opinion. Often patients make

Table 31.1 Prework-up/prior to patient’s arrival at transplant center Assessment Understanding of the overall transplant process and time commitment Current symptoms from previous therapies or disease Current coping ability Current pain Current blood product requirements Sedation preference for procedures Teaching Length of time for work-up, mobilization, and transplant process Role of caregiver Care coordination Contact referring physician’s office to obtain report Confirm financial clearance Confirm housing plan Carrying out the medical care plan Maintain working knowledge of proposed transplant plan for patient

a consultation appointment to compare a particular program with other centers they are considering. These patients meet with a transplant physician to review their options, a financial counselor, and a nurse. The nurse’s role is to assist the patient with this decision by explaining the logistics of the program, including the usual length of stay on the inpatient unit and how much of the transplant process will be managed on an outpatient basis [13]. Many patients are interested in the nursing services at the program they are considering and ask questions about the nurse-to-patient ratio. The responsibilities of the patient’s caregiver are reviewed as well. At that time, many families are concerned with the logistics of relocating and incorporating this intense therapy into their lives. These patients present with a myriad of questions, and the nurse must be versatile in meeting the patients’ and families’ informational needs. Nurses often are also responsible for giving information regarding the transplant program to physicians and nurses from the referring center as well as to third-party payers.

Work-up (Table 31.2) Once a patient has met initial screening and has decided to undergo HCT at a specific center, the nurse’s primary responsibility becomes educa-

Table 31.2 Work-up Assessment Patient’s current fears and concerns Barriers to learning Current pain Knowledge of disease status Knowledge of transplant process Knowledge of patient’s rights and responsibilities when participating in medical research Usual coping strategies Level of fatigue and usual sleep patterns Patient’s experience in other health-care environments Identified caregiver(s)’ commitment Identified caregiver(s)’ barriers to learning Allergies Current medications and knowledge of purpose Compliance in taking medications Current central venous access History of central venous access Teaching Clinic logistics, including how to access care after hours Importance of having a caregiver during various phases of the transplant process Purpose of procedures, laboratory tests, and scans required for work-up Overall transplant process Usual complications of transplant Central venous catheter preoperative teaching Care coordination Assess ability to adhere to work-up schedule Confirm financial clearance for transplant Social work assessment Nutrition assessment Carrying out the medical care plan Maintain working knowledge of proposed transplant plan for patient Ensure all work-up studies are obtained in a timely manner

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Table 31.3 Preconditioning

Table 31.4 Donor work-up

Assessment Knowledge of need for compliance during conditioning regimen Knowledge of side-effects to report during conditioning Caregiver’s ability to report symptoms Caregiver’s ability to utilize ambulatory infusion pump if required History of antiemetics Assure appropriate patient central venous access Purpose of viral and/or fungal prophylaxis Purpose of prophylaxis for graft-versus-host disease if appropriate

Assessment Current fears and concerns Barriers to learning Donor screening per American Association of Blood Bank requirements Knowledge of donation process Knowledge of patient’s rights and responsibilities when participating in medical research Allergies

Teaching Treatment plan and schedule for chemotherapy and total body irradiation Antiemetic schedule and importance of compliance Care of central venous access Caregiver education for ambulatory infusion pump if hydration given in ambulatory setting Importance of laboratory monitoring Purpose of hydration Symptoms to report Differences between previous chemotherapy and conditioning chemotherapy Purpose of allopurinol if ordered Importance of compliance with Pneumocystis prophylaxis Care coordination Research consents Chemotherapy and total body irradiation order verification with protocol Dosimetry and simulation appointment with radiation oncology if applicable Plasma exchange if required Pager/cell phone for patient if conditioning to start in the clinic Carrying out the medical care plan Ensure central venous catheter is inserted in timely manner Ensure antivirals, antifungals, immunosuppressants, and antiemetic medications are initiated as ordered

tion. The patient must understand the specifics of the rigorous evaluation. The coordination of care during this phase is especially critical if the patient has an unrelated donor. The completion of work-up and the initiation of conditioning must start on an exact date to allow HSC infusion on the planned date of HSC procurement. The generosity of the volunteer donors must be respected and accommodated.

Preconditioning (Table 31.3) The nurse plays an important role in the informed consent process, supporting the medical staff’s explanations and plans to ensure, as much as possible, that the patient is making an informed decision regarding HCT.

Teaching Donation plan and schedule Importance of laboratory monitoring Preoperative teaching if applicable Symptom management Care coordination Assess ability to adhere to work-up schedule Research consents Storage of autologous red cells if marrow donation planned Carrying out the medical care plan Maintain working knowledge of proposed transplant plan for patient Ensure all work-up studies are obtained in a timely manner

Table 31.5 Mobilization and collection (autologous patients and allogeneic donors) Assessment Adequacy of veins for apheresis if applicable Knowledge of care of central venous access (if applicable) Teaching Purpose of monitoring CBC, CD34+ cells, and electrolytes Purpose and care of central venous catheter if applicable Specific medications used to mobilize, and usual side-effects if applicable Subcutaneous injections if applicable Temperature monitoring Importance of increasing calcium intake Medications to avoid (i.e. aspirin) Care coordination Coordinate home administration of subcutaneous medications Manage side-effect of growth factors Coordinate stem cell collection and storage with Cellular Therapy Laboratory Carrying out the medical care plan Administer subcutaneous injections in clinic Central catheter management Collect stem cells CBC, complete blood count.

Donor preparation (Table 31.4) Donors have been called the “forgotten patients” of transplant. The HCT recipient, appropriately, is the center of focus for the transplant team. However, donors also have concerns about their own health and the procedures they will undergo. It is ideal for donors to have a primary nurse with whom they can establish a relationship and who can prepare them for the hematopoietic cell collection and monitor them throughout the procedure.

Mobilization and collection (autologous patients and allogeneic donors) (Table 31.5) Nursing care during mobilization must be individualized to the specific medical protocols being utilized as well as the response of the donor or autologous patient. The nurse must stay in daily contact with the donor or autologous patient during this time as collection schedules can change based on cell yield. The nurse needs to assist in determining whether it

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is necessary to have a long-term central venous access catheter inserted before mobilization, if a short-term central venous access catheter is sufficient or if vein-to-vein access may be possible.

Conditioning (Table 31.6) Prior to the infusion of stem cells, the patient will receive conditioning therapy to eliminate residual disease (autologous and allogeneic transplants) and prepare the patient’s system to accept donor stem cells by creating “space” in the bone marrow and suppressing the patient’s immune system (allogeneic transplants) [14]. Conditioning can be chemotherapy (high-dose regimens or reduced-intensity regimens), biotherapy, total body irradiation, radioimmunotherapy or a combination of these modalities. Chemotherapy used for traditional myeloablative transplants is administered at higher doses than conventional chemotherapy to treat cancer. These high-dose regimens will lead to acute toxicities that may manifest more rapidly and intensely than with standard-dose therapy [15]. The nurse’s role is key in safe administration of high-dose therapy and in supporting the patient and caregiver in the management of acute toxicities from the high-dose regimen [4]. Toxicities associated with high-dose chemotherapy and/or radiation therapy

require vigilant monitoring with special emphasis on assessment of fluid, gastrointestinal tract, and renal status. Reduced-intensity regimens will be better tolerated, but will still require expert nursing monitoring to detect toxicities that need to be managed to reduce negative sequelae. Conditioning regimens may be administered in the outpatient or inpatient setting, based upon the type of preparatory regimen chosen, the availability of 24-hour triage and supportive services, and the available level of caregiver support. Care must be available to outpatients 24 hours a day, 7 days a week in order to safely provide for patients whose conditions may rapidly deteriorate. In general, successful management in the outpatient setting during the conditioning phase of transplant requires a high level of continuous caregiver support. Outpatient teaching places special emphasis on use of ambulatory infusion pumps, patient and caregiver ability to assess, report, and manage the side-effects of high-dose therapy, and access to support telephone numbers and 24-hour emergency care [12]. Printed patient and caregiver teaching reference materials containing outpatient guidelines and how to access emergency care are useful tools for the reinforcement of patient and caregiver teaching [4]. Patients and caregivers must understand symptoms that require immediate medical attention (Table 31.7).

Table 31.6 Conditioning Assessment Knowledge of potential side-effects of conditioning regimen Response to high-dose chemotherapy, immunotherapy, radioimmunotherapy, and/or total body irradiation Ability to perform self-care and infection control preventative measures (hand washing, personal hygiene, oral care, etc.) Renal and fluid status: intake and output; postural vital signs; daily or twice-daily weights; breath sounds, heart sounds; skin turgor; laboratory tests: serum electrolytes, blood urea nitrogen, creatinine Nutritional status: weight; amount, content, and patterns of nutritional intake Outpatients: • Patient/caregiver ability to assess, report, and manage side-effects of conditioning regimen • Knowledge of phone numbers to access support and emergency care 24 hours a day Teaching Treatment plan and schedule, including administration of intravenous hydration, antiemetics, uroprotectants, and other medications Potential side-effects of conditioning regimen (nausea, vomiting, mucositis, diarrhea, fever, etc.) Prevention and management of side-effects Level of caregiver support required Guidelines for preventing infection: • Hand washing • Personal hygiene • Nutrition/diet guidelines for immunosuppressed patients • Avoiding activities with high risk for infection Outpatients: • Signs/symptoms requiring inpatient hospitalization • Access to 24-hour triage and emergency care phone numbers Care coordination Clinic appointment schedules or admission to inpatient unit Total body irradiation schedule and coordination of care with radiation oncology department Patient and caregiver education: one-on-one instruction or attendance at caregiver classes Carrying out the medical care plan Administer pre- and postconditioning therapy, intravenous hydration, and medications (antiemetics, uroprotectants, etc.) Verification of chemotherapy orders Chemotherapy administration Drawing and monitoring laboratories

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Table 31.7 Quick reference for symptoms

Area of concern

Critical emergency DIAL 911

Call clinic now

Alertness, consciousness, activity

• Unconscious • Unable to arouse • Seizure

• • • • • • • • • • • • •

New or increased confusion Change in level of alertness Mood changes: irritable, tearful, agitated Change in vision Sleeplessness Falling down Numbness, tingling or loss of movement in limbs Dizziness Lethargic Change in energy level Tremors/shakiness Not able to get around Difficulty swallowing

Bleeding

• Uncontrollable, consistent bleeding • Patient is unconscious

• • • • • • • • •

New or increased bleeding Bloody urine New bruising Unable to stop nosebleed Bloody diarrhea Vomiting of blood Patient falls or is injured 1 or more feminine pad per hour is used Little red or purple spots on the skin

Breathing

• Not breathing • Choking – not moving air into chest

• • • • • • • •

Trouble breathing Gets “winded” more easily with minimal activity Feeling as if can’t get enough air Trouble breathing when laying flat Wheezing with breaths New or recurrent cough Persistent continuous cough Coughing blood or green/yellow material

Central venous catheter (Hickman Line)

• Line open to air and patient short of breath • Clamp line immediately

• • • • • • •

Line broken or leaking Face, neck, exit site swelling Inability to flush Line fell out Headache related to infusions Redness, swelling or tenderness at exit site Drainage from exit site

• • • • • • •

Constant or uncontrolled diarrhea New-onset diarrhea Diarrhea with fever and abdominal cramping Whole pills passed in stool Greater than five times each day Stool which is bloody, burgundy or black Mild abdominal cramping

• • • •

Dizziness Fatigue is getting worse Too tired to get out of bed or walk to the bathroom Staying in bed all day

• • • •

Fever greater than 1 degree above usual Cold symptoms (runny nose, watery eyes, sneezing, coughing) Shaking chills, temperature may be normal Temperature greater than 38.3° C or 100.9° F by mouth

Diarrhea

Fatigue

Fever/chills

• Unable to wake up

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Table 31.7 (Continued)

Area of concern

Critical emergency DIAL 911

Mouth pain/mucositis

• Not breathing

Call clinic now

Nausea/vomiting

Pain

• Severe chest/arm pain • Severe squeezing or pressure in chest

• • • • • • •

Having difficulty breathing Cannot swallow Choking Bright red blood in mouth Pain not controlled by medication White patches or sores appear on gums or mouth Difficulty swallowing food or fluid

• • • • • • •

Nausea persists without control from antinausea medications Vomit shoots out for a distance (projectile vomiting) Uncontrolled, constant nausea and vomiting Blood or “coffee ground” appearing material in the vomit Medicine not kept down because of vomiting Weakness or dizziness along with nausea/vomiting Severe stomach pain while vomiting

• • • • • • • • • • •

New or uncontrolled pain in body New headache Chest discomfort Pounding heart Heart “flip-flop” feeling Painful central line site or area of “tunnel” Burning in chest or stomach Pain or burning while urinating Strong stomach pain Pain with infusion of medications or fluids into central line New abdominal or back pain

Rash

• Sudden-onset body rash • Rash with severe pain

Swelling

• Sudden swelling with or without pain • Swollen legs, hands

Urination

• Unable to urinate for more than 8 hours • Bloody urine • Painful urination

Reproduced with permission from the Seattle Cancer Care Alliance Patient Education Program.

Bone marrow harvest (Table 31.8) The technique of harvesting marrow is essentially the same for allogeneic related donors, unrelated donors, and autologous patients. Education will prepare donors or autologous patients for anticipated side-effects, outlining those to report to health-care providers. Nurses educating donors or autologous patients should emphasize the expectations for the marrow harvest, including information about anesthesia and the aspiration procedure, admission to hospital or day-surgery settings, anticipated recovery period and discharge from the hospital or day-surgery to home, pain management plan for marrow aspiration site pain, site care post harvest, and follow-up visits. Patients receiving unrelated donor marrow will need instruction regarding when and how the donor marrow will arrive for infusion. Donor marrow is harvested at the medical center closest to the donor’s residence, and the marrow is flown to the patient’s location accompanied by an Unrelated Donor Program courier. Patients will also be told that

the identity of their unrelated donor must remain unknown to protect the donor. Autologous patients will have their marrow harvested and cryopreserved (frozen) for later use. The bone marrow will be thawed and given to them after they receive conditioning therapy.

Transplant phase (Table 31.9) The stem cell product infusion is very similar to a blood transfusion. The rate of infusion, method of administration, and potential side-effects depend on whether the product is cryopreserved or noncryopreserved. Dimethylsulfoxide is generally used as the preservative for cryopreserved autologous marrow, peripheral blood stem cells, and umbilical cord blood. Infusion of products cryopreserved with dimethylsulfoxide tends to cause a significant number of transient, self-limited side-effects amenable to premedication [16]. These side-effects are rarely severe

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Table 31.8 Bone marrow harvest

467

Assessment Results of preharvest donor work-up Preharvest physical reassessment Verification of informed consent Understanding of bone marrow harvest process and potential complications Understanding of signs/symptoms to report to health-care team post harvest Ability to access 24-hour triage and emergency care phone numbers Teaching Bone marrow harvest process and procedures: • Hospital admission location, schedule • Marrow aspiration sites • Autologous blood transfusions • Hospital discharge criteria • Site care post bone marrow harvest • Post-harvest iron supplements Potential complications: • Pain • Infection • Anemia related to blood loss • Nausea and vomiting (if general anesthesia used) • Dehydration Signs/symptoms to report to health-care team Access to 24-hour triage and emergency care phone numbers Patients receiving unrelated donor marrow: • Marrow harvested at donor residence (state/country) • Identity of donor remains unknown • Marrow arrives via courier • Anticipated time of unrelated donor marrow arrival Care coordination Operating room scheduling Type and crossmatch for autologous stored unit, if stored Time and location of hospital admission for day surgery Discharge teaching Discharge medications Post-harvest outpatient appointment and laboratory draws (generally, complete blood count with differential and platelets)

Table 31.9 Transplant Assessment Preinfusion: • Knowledge of stem cell infusion process • Renal and fluid status: intake and output; postural vital signs; weight; breath sounds; skin turgor; laboratory tests: serum electrolytes, blood urea nitrogen, creatinine • Response to prior red blood cell transfusions • Adequacy of venous access for infusion of stem cells (central venous access required for infusion of cryopreserved stem cell products due to DMSO; central venous access preferred for noncryopreserved products) • Availability of emergency medications During infusion: • Frequent vital sign monitoring and assessment to detect the early onset of potential infusion-related complications Post infusion: • Frequent vital sign monitoring and assessment to detect post-infusion-related complications • Tolerance of cell infusion • Renal function and fluid status: intake and output (general goal is to maintain urine output at 2–3 mL/kg/h); presence of hematuria due to red cell lysis Teaching Location for stem cell infusion (inpatient versus outpatient setting) Anticipated timing of stem cell product arrival on unit or in outpatient department

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Table 31.9 (Continued) Stem cell infusion procedure: Cryopreserved • Prehydration • Premedications (e.g. antiemetics, diphenhydramine, hydrocortisone, acetaminophen, etc.) • Transport of cryopreserved cells • Thawing of cells at bedside • Infusion of cells via central venous catheter Noncryopreserved • Premedication (if patient previously experienced transfusion reactions to red blood cell transfusions) • Transport of cells to patient care area • Infusion (generally via central venous catheter) Potential complications: Cryopreserved • Reactions to cryoprotectant DMSO including nausea/vomiting, tickle in throat, facial flushing, hypertension, hypotension, bradycardia, tachycardia, cardiac arrhythmia, chest tightness, cough, flushing, chills, fever, abdominal cramping, diarrhea, headache, transient taste and smell of DMSO, appearance of red urine post infusion • Prevention/treatment of throat tickle and nausea secondary to DMSO by antiemetics, sucking on hard candy, eating or smelling an orange • Transfusion reactions (if allogeneic) • Volume overload • Allergic reaction • Pulmonary microembolism (clumps of cell debris may occur after thawing): shortness of breath, dyspnea, cough Noncryopreserved • Fluid overload • Pulmonary microembolism secondary to fat emboli (may occur with infusions of bone marrow): shortness of breath, dyspnea, cough • Transfusion reactions (if allogeneic) • Allergic reactions • Excessive anticoagulation: may occur with rapid or large-volume infusions (marrow and blood cells anticoagulated with heparin and/or citrate solutions) Care coordination Initiation of graft-versus-host disease prophylaxis pre transplant Coordinate arrival of stem cell product with department or service handling and/or processing cells: • Apheresis unit (related allogeneic peripheral blood cells) • Cryopreservation laboratory (cryopreserved cell products [bone marrow, umbilical cord blood, peripheral blood cells]) • Operating room (related allogeneic bone marrow) • Unrelated donor program (unrelated donor peripheral blood cells or bone marrow) Carrying out the medical care plan Administer prehydration and premedications appropriately timed with arrival of stem cell product Administer stem cell product and monitor for complications DMSO, dimethylsulfoxide.

(less than 1% of all complications), although life-threatening complications have been reported [17]. Side-effects to be expected with cryopreserved cells are nausea/vomiting, tickle in the throat, facial flushing, hypertension or hypotension, bradycardia or tachycardia (can be delayed), cardiac dysrhythmia, chest tightness, cough, flushing, chills, fever, abdominal cramping, diarrhea, headache, transient taste and smell of DMSO (oyster, garlic or creamed corn odor), and/or appearance of red urine post infusion. Complications from infusion of noncryopreserved products may include volume overload and pulmonary abnormalities from fat or cellular debris emboli. In addition, patients may experience symptoms similar to blood transfusion reactions (chills, urticaria, fever, etc.) [12]. Patients and caregivers benefit from nursing education about the infusion procedure, potential side-effects, and premedications. Important care coordination considerations for nurses include ensuring initiation of GVHD prophylaxis prior to the transplant for allogeneic patients and

timing stem-cell product prehydration and premedications with the arrival of the products on the unit or the patient care area.

Pre-engraftment (Table 31.10) The majority of complications that occur prior to engraftment are the result of toxicities associated with high-dose conditioning regimen and/ or the administration of immunosuppressive medications [12]. Nursing care is directed at strategies to prevent potential complications, as well as early detection and prompt implementation of appropriate interventions if complications develop. Systematic and astute assessment skills are required to identify subtle clues indicative of multiple potential complications [1]. For the pancytopenic patient, transplant nurses play a critical role in assisting the patient in preventing infections and bleeding complications and in managing fatigue. Nurses are also critical to help the patient manage pain from mucositis and to monitor fluid, electrolyte, and nutritional status.

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Table 31.10 Pre-engraftment Assessment Assessments for potential conditioning-related toxicities: • Mucositis: color/integrity of oral mucosa; volume and consistency of oral mucus; airway patency; general level of consciousness; subjective complaints of pain and/or difficulty swallowing; ability to perform oral care and maintain fluid/nutritional needs • Nausea/vomiting: subjective reports of nausea; frequency and appearance of emesis, including gastroccult for presence of blood; abdominal assessment; fluid status, including intake and output, postural vital signs; effectiveness of antiemetic therapy • Diarrhea: amount, frequency, appearance of stool, presence of blood; abdominal assessment; fluid status, including intake and output; subjective complaints of fullness, cramping, pain • Infection: fever and/or chills unrelated to blood products or other medications; skin integrity, including oral and perineal areas; presence of adventitious breath sounds or cough; subjective reports of pain, weakness, fatigue; microbiology culture reports; hematologic laboratory values; chest X-ray report • Bleeding: appearance and integrity of skin, sclera, mucous membranes; appearance of oral secretions; presence of frank or occult blood in urine, stool and emesis; mentation and level of consciousness, vital signs noting hypotension or tachycardia; laboratory values (hematocrit, platelet count, coagulation studies, hepatic and renal function tests) • Sinusoidal obstructive syndrome: weight gain; increased abdominal girth; abdominal distention; right upper quadrant pain; hepatomegaly; mentation and level of consciousness; jaundice (skin and/or sclera); laboratory values (liver function tests including serum bilirubin, SGOT, alkaline phosphatase) • Pain: onset, location, description, intensity (using rating scale), aggravating and relieving factors; effectiveness of interventions; psychosocial assessment; effect of pain on activities of daily living • Renal insufficiency: strict intake and output; daily or twice-daily weight; postural blood pressure and heart rate; pulmonary assessment including respiratory rate, quality and presence of adventitious breath sounds; cardiac assessment, neck vein distention; presence of edema or ascites; administration of nephrotoxic drugs; laboratory values (serum blood urea nitrogen, creatinine, electrolytes; urine specific gravity) • Pulmonary: respiratory rate, rhythm, depth, quality; breath sounds; existence and quality of cough; sputum production; mentation and level of consciousness; presence of cyanosis or skin mottling; subjective complaints of shortness of breath, dyspnea, or pain; oxygen saturation Knowledge of patient/caregiver of potential complications Ability to participate in self-care measures to treat and/or prevent complications Ability of caregiver to participate in patient support Teaching Recognition, reporting and management of post-transplant complications: • Mucositis • Nausea/vomiting • Diarrhea • Infection • Bleeding • Sinusoidal obstructive syndrome • Pain • Renal insufficiency • Pulmonary complications Reinforcement of guidelines for preventing infections: • Hand washing • Personal hygiene • Nutrition/diet guidelines for immunosuppressed patients • Avoiding activities with high risk for infection Reinforcement of guidelines for preventing bleeding episodes: • Careful oral hygiene with a soft-bristled toothbrush • No rectal maneuvers • Avoid medications that increase risk of bleeding • Avoid falls • Use electric razors for shaving • Avoid nose blowing or picking • Women with menses should use pads, not tampons Rationale and procedures for administration of blood products, parenteral fluids and nutrition, medications Anticipated time to engraftment Signs and symptoms of engraftment Care coordination Coordinate multiple health-care provider appointments (outpatient) or health-care provider examinations and tests (inpatient) Provide patient and caregiver education: one-on-one instruction or attendance at caregiver classes

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Table 31.10 (Continued) Carrying out the medical care plan Blood product administration: • Platelet transfusions (often daily or multiple times per day) • Packed red blood cell transfusions Intravenous fluids: • Total parenteral nutrition/lipids • Hydration Medication administration: • Graft-versus-host disease prophylaxis (allogeneic patients) • Antibiotics, antivirals, antifungals • Antiemetics • Opioid/nonopioid analgesia (patient-controlled analgesia in inpatient setting) • Investigational drugs Multiple laboratory draws Frequent physical assessments and vital signs (inpatient: every 4 hours and as needed; outpatient: weekly clinic visit and as needed) Physical care: • Bath and skin care • Oral care • Central venous catheter dressing changes • Exercise/ambulation SGOT, serum glutamic-oxaloacetic transaminase.

Table 31.11 Early postengraftment Assessment Ongoing assessment for conditioning-related toxicities (see Table 31.10) Signs and symptoms of GVHD: • Skin: itching; redness or maculopapular rash on palms of hands, soles of feet, ears, trunk, extremities; erythroderma • Liver: right upper quadrant pain; hepatomegaly; jaundice; laboratory values (elevated serum bilirubin, SGOT, alkaline phosphatase) • Gut: nausea; vomiting; anorexia; diarrhea; abdominal cramping and pain Readiness for discharge from hospital: • Availability of caregiver on discharge • Competency of patient and caregiver to: (1) recognize, report, and manage post-transplant complications; (2) manage outpatient schedules and treatments; and (3) administer outpatient IV fluids and medications • Knowledge of phone numbers to access emergency care and support 24 hours a day on discharge Caregiver ability to provide support Teaching Signs and symptoms of acute GVHD Rationale and procedures for diagnostic testing for GVHD if signs and symptoms develop (e.g. skin biopsy, liver biopsy, and/or endoscopy) Management of GVHD (adding immunosuppressive medications to current GVHD prophylaxis), dietary restrictions Hospital discharge planning: • Criteria for discharge from inpatient to outpatient setting (e.g. nausea/vomiting/pain controlled; blood counts supportable; number and type of IV infusions or medications) • Level of caregiver support required on discharge (generally constant support required for first 2–3 weeks post discharge) • Outpatient administration of IV fluids and medications • Discharge medication schedule: immunosuppressant medications for GVHD prophylaxis and/or treatment (allogeneic patients), prophylaxis for Pneumocystis carinii pneumonia, antivirals, antibacterials, antifungals, electrolyte replacements, antiemetics, etc.) • Importance of reporting inability to take medications prescribed • Outpatient appointment schedules on discharge Recognition and management of post-transplant complications Likelihood of readmission to hospital post initial discharge Care coordination Coordinating multiple health-care provider appointments (outpatient) or health-care provider examinations and tests (inpatient) Scheduling procedures (marrow aspirations, chest-X-rays, etc.) Transitioning care from inpatient to outpatient setting

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Table 31.11 (Continued) Carrying out the medical care plan Blood product administration: • Platelet transfusions (often daily or multiple times per day) • Red blood cell or whole blood transfusions Intravenous fluids: • Total parenteral nutrition/lipids • Hydration fluids • Patient-controlled analgesia (inpatients) Medication administration: • GVHD prophylaxis • Antibiotics, antivirals, antifungals • Opioid/nonopioid analgesics • Electrolyte replacements • Investigational drugs • Antiemetics Multiple laboratory draws Frequent physical assessments and vital signs (inpatient: every 4 hours and as needed; outpatient: at each clinic visit and as needed) Physical care: • Bath and skin care • Oral care • Central venous catheter dressing changes • Exercise/ambulation GVHD, graft-versus-host disease; IV, intravenous; SGOT, serum glutamic-oxaloacetic transaminase.

Coordination of care can be very complex during this phase. Neutropenia and thrombocytopenia are perhaps the most dangerous complications during the pre-engraftment period. Nurses should follow evidence-based guidelines for the care of the neutropenic patient [18]. Patients who are outpatients during all or part of the pre-engraftment phase require intense patient education to ensure that they can manage the complications and promptly report symptoms requiring care. Patients often require administration of multiple red blood cell and platelet transfusions, intravenous fluids and parenteral nutrition, and multiple intravenous medications. Whether in the inpatient or ambulatory clinic setting, infusing all required intravenous medications and fluids presents a major challenge. Many transplant centers place double-lumen central venous catheters for use throughout the transplant. Patients may require additional intravenous access during the pre-engraftment phase in order to administer all of the required intravenous medications and fluids. Additionally, patients usually require frequent collections of blood specimens, diagnostic procedures, examinations or appointments with various members of the health-care team, coupled with ongoing needs for education, completing activities of daily living, and psychosocial support.

Early postengraftment (Table 31.11) Engraftment typically occurs between days 14 and 28 post transplant, depending on stem cell source. The most common issue in the early postengraftment period for allogeneic transplant patients is acute GVHD. Nurses play an important role in assessing patients for this problem and in teaching patients and caregivers how to monitor for its signs and symptoms. Transplant nurses must have a thorough understanding of the pathophysiology, clinical manifestations, and management of GVHD [12]. The nurse plays a key role in preparing the patient and family for discharge from the hospital. Criteria for transition from inpatient to outpatient settings will differ based on institutional policies, the patient’s condition, the availability of skilled outpatient teams, and family caregiver support. Caregivers must be knowledgeable in recognizing, man-

aging, and appropriately reporting complications, and be competent in performing aspects of the patient’s care. Detailed and structured discharge teaching is necessary to ensure adequate understanding of critical information by patients and caregivers [19]. Hospital readmissions after initial hospital discharge to the ambulatory care setting frequently occur during the early postengraftment phase [7]. Coordination and communication of patient care issues between the nursing staff of the inpatient unit and the ambulatory care setting allows for smooth transition and continuity of care. Particular attention needs to be given to caregivers’ ability to continuously cope with their multiple demands and roles.

Intensive care management of the transplant patient Most transplant patients require complex, high-acuity levels of care throughout their inpatient stay [20]. It is estimated that up to 40% of patients undergoing HCT will require nursing skills typical of most ICUs, including hemodynamic monitoring, noninvasive cardiac monitoring, administration of inotropic and vasoactive medications to support blood pressure, and mechanical ventilatory support [21,22]. The need for ICU support will vary based on the type of HCT performed and how ill the patient is prior to transplant. All transplant nurses, whether ICUtrained or not, need to be aware of early signs and symptoms of common complications of transplant so as to intervene early and prevent morbidity and mortality [23]. Management of critically ill transplant patients combines the complexity of oncology and critical care nursing skills, presenting significant challenges to the provision of expert nursing care. Oncology nurses generally are not trained or skilled in the management of critically ill patients. Conversely, critical care nurses generally do not have an oncology background. Patients with critical care complications may be transferred to a critical care unit. However, many institutions have worked towards merging the two specialties of oncology nursing and critical care nursing to meet the needs of these patients by training transplant

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Table 31.12 Discharge from transplant program

Assessment Knowledge of infectious risks Knowledge of expected follow-up office visits and laboratory testing Nutritional needs Need for continuance or removal of central venous catheter Allogeneic patients: • Current symptoms of GVHD • Visual examination of skin • Skin biopsy • Schirmer’s test • Knowledge of chronic GVHD Teaching Symptoms of chronic GVHD Recovery of immunity Recommendations for minimizing daily infectious risks (see Table 31.13) Recommendations for returning to work or school Management of fatigue Management of possible problems related to sexual function Management of cognitive and emotional issues When to report fever Necessity for consistent medical follow-up for life Care coordination Medication list Nursing care plan for local nursing care provider Home health-care agency if needed Special transfusion requirements and preparation of products (irradiation and CMV screening if CMV negative) Interface with health insurance carriers as needed Assist with research coordination Time after transplant CMV, cytomegalovirus; GVHD, graft-versus-host disease.

nurses to provide intensive care on the transplant unit or by crosstraining ICU staff to specialized transplant procedures [20]. Challenges to each approach include the maintenance of nurses’ competency. Transplant nurses may find it difficult to keep critical care skills up-to-date if the volume of transplant patients requiring critical care is low. Critical care nurses may face similar issues in maintaining transplant competencies and skills. Each institution must evaluate the available resources, outcomes of care, and cost-effectiveness of cross-training staff and maintaining clinical competencies when determining models for providing critical care to transplant patients [20,24]. Substantial progress has been made in treating the complications of HCT. Past reports of close to 100% mortality rates in HCT patients requiring ICU care are no longer the reality; the current mortality rate in ICUs is less than 50% [25–29]. Mortality remains highest for those HCT patients admitted to ICU for sepsis or respiratory failure [25,26].

Relapse post transplant Some patients may fail to respond to the HCT or, after initial response, relapse of underlying disease may occur [30]. For patients with disease relapse, options for care may include standard chemotherapy, donor lymphocyte infusion, discontinuation of immunosuppressive therapy, a second HCT or palliative care [31]. Nurses play a significant role in clarifying the treatment options presented by the medical staff. Nurses

also serve as advocates for the patient and family, helping to communicate the patient’s individual goals clearly to the health-care team.

Discharge (Table 31.12) Preparation for long-term recovery begins during the treatment phase and is part of the ongoing nursing care process. Discharge education is provided by nurses to review possible late effects of transplantation, such as chronic GVHD and infectious risks, and make recommendations related to activities of daily living for the first year after HCT (Table 31.13). It is also a good opportunity to discuss possible cognitive, sexual, and emotional concerns. Some large centers accomplish this educational goal through individual classes. This effort facilitates the transition from the transplant center to the care of the referring physician. Patients often express feelings of anxiety when leaving the security of the transplant program that they have relied on so heavily. If the patient is being discharged from the transplant center to their referring physician, identifying a designated contact person or group at the transplant center for both the patient and the referring physician for consultation on transplant-related problems is essential. This contact person can decrease the anxiety of the patient and caregiver and provide the referring physician access to transplant expertise and continuity of the medical plan. General guidelines should be provided by the transplant center for the referring physician that details the important aspects of long-term care of the transplant recipient [32].

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Table 31.13 Common questions asked by hematopoietic stem cell transplantation (HSCT) and long-term follow-up (LTFU) patients Questions often asked

Time after transplant

The general guidelines below may not apply to your case. You must discuss with your physician to assess if these rules apply to you.

All patients Less than 6 months

School Hot tubs (1) Swimming (1) (avoid head submersion and diving, use sun screen) Gardening (digging in soil); mowing the lawn; raking leaves Having plants in the home (not handling) Making/kneading yeast breads Carpenter work Occasional woodworking (sawdust) Animals, birds, reptiles, fish, other (not handling • New pets in patient’s household • Cats/dogs (not sleeping with pets) • Domestic birds (parakeets, parrots, etc.) (not with respiratory problems) • Poultry and wild birds (pigeons, chickens, ducks, geese, other wild birds, etc.) • Small cage rodents (gerbils, rabbits, hamsters, guinea pigs, hedge hogs, prairie dogs, etc.) (do not handle) • Reptiles (snakes, turtles, lizards, iguanas, etc.); ferrets • Farm animals (pigs, horses, cows, llamas, etc.) (do not handle; stay out of barns full of hay) • Wild and game animals (deer, elk, squirrels, bear, etc.); exotic animals (monkeys, etc.) (do not handle) • Zoos and petting zoos • Public aquariums (do not touch marine life in handling tanks) (2) • Animal trophy mounts in the house • Fishing (fresh and salt water) (OK to handle fish if wearing gloves; do not bait hooks) • Golfing (sun protection required; without venous catheter device) • Spectator events and crowds (2) (church, movies, games, etc.) (no hand shaking) • Sexual activity • Working with mechanical equipment (oil changes, working on cars and engines, etc.) • Camping and hiking • Down comforters (with cover) • Hunting (wild game and birds) and sport shooting (3) (wear latex gloves when handling game; do not clean game) • Horseback riding (stay out of barns full of hay)

Not receiving immunosuppression 6 months to 1 year

Receiving immunosuppression 6 months to 1 year

Receiving immunosuppression More than 1 year

No No No

No OK (1) OK (1)

No No No

OK No OK

No

No

No

No

OK OK No No

OK OK No OK

OK OK No OK

OK OK No OK

feces, litter boxes, cleaning utensils, cages/tanks, etc.) No No No OK OK OK No OK OK

No OK OK

No

No

No

No

No

OK

OK

OK

No

No

No

No

No

OK

OK

OK

No

No

No

No

No No (2)

No OK (2)

No OK (2)

No OK (2)

OK OK

OK OK

OK OK

OK OK

No

OK

OK

OK

No (2)

OK (2)

OK (2)

OK (2)

OK OK

OK OK

OK OK

OK OK

OK OK No

OK OK OK (3)

OK OK OK (3)

OK OK OK (3)

No

OK

OK

OK

(1) No swimming in pools with diapered or undiapered babies. No swimming if venous access device still in place (i.e. catheter). No patient history of sinusitis. Chlorinated and wellmaintained pools preferable over uncertain water conditions such as lakes, rivers and sea for swimming. Water aerobics OK. (2) Recommend caution and an understanding of the risk involved when participating in public events or going to locations with large crowds. (3) Shooting of rifles and shotguns not recommended if venous access devise still in place (i.e. catheter). Other reminders: • Minimize: exposure to dirt and aerosolized material. • Minimize: sun exposure and use of sunscreen (SPF > 15%). • Use: hat, long sleeve shirts and pants if outside for long period of time. • Avoid contact with people with respiratory illness or other transmissible diseases. • Autologous and teenage patients should be discussed separately. Discuss with your physician when restrictions may apply to your child’s care after one year post transplant. Reproduced with permission from the Fred Hutchinson Cancer Research Center Long-term Follow-up Program.

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Long-term recovery (Table 31.14) Nurses, under the guidance of the transplant center medical team, can successfully act as liaison between the referring physician and the transplant physicians. They can collect important and pertinent information

to triage transplant-related problems and, after discussion with the transplant physicians, relay possible solutions to the team providing care at home. One organized approach is to conduct long-term follow-up rounds on a regular basis to discuss nonurgent problems. Issues most frequently requiring the expertise of the transplant team are the diagnosis and treat-

Table 31.14 Long-term recovery Assessment Fatigue Pain Functional status Complete blood count Kidney function Liver function Magnesium level Drug levels Chronic GVHD symptoms (allogeneic patients): • Skin: erythema, dryness, macular or urticarial rash and pruritis, hyperpigmentation, vitiligo, mottling, lichenoid plaques, hyperkeratosis, exfoliation scleroderma or morphia • Mouth: dryness, mucositis, erythema, lichenoid changes, striae, tightness around the mouth, sensitivity to hot, cold or spicy foods, gingivitis • Eyes: dryness, grittiness, blurring, excessive tearing, photophobia and pain • Liver: elevated liver function tests not attributable to other causes and sometimes jaundice • Gastrointestinal system: anorexia, nausea, vomiting, diarrhea, dysphagia, malabsorption, weight loss • Lungs: cough, wheezing, dyspnea on exertion, history of recurrent bronchiolitis or sinusitis, bronchiolitis obliterans • Nails: ridging, onychodystrophy, onycholysis • Hair: premature graying of scalp hair, eyelashes, eyebrows, thinning of scalp hair, alopecia, decreased body hair • Vagina: dryness, dyspareunia, stricture or stenosis, erythema, atrophy or lichenoid changes not induced by ovarian failure or other causes • Myofascia: stiffness and tightness with restriction of movement, sometimes with swelling, pain, cramping, erythema, and induration, most commonly affecting the forearms, wrists and hands, ankles, legs, and feet, contractures • Muscles: proximal muscle weakness, cramping • Skeletal: arthralgia of large proximal girdle joints and sometimes smaller joints • Serosal: unexplained effusions involving the pleural, pericardial or peritoneal cavities • Thrombocytopenia, eosinophilia, hypogammaglobulinemia • Energy level: unusual fatigue Medication compliance Nutritional needs Symptoms of infection Hormonal issues Neurological symptoms Sexual dysfunction Teaching Symptoms to be reported: skin changes, nausea, anorexia, weight loss, diarrhea, dysphagia, dry or sensitive mouth, dry, gritty eyes or excessive tearing, coughing, wheezing or shortness of breath, thinning of scalp hair, ridged nails, loss of range of motion or stiffness, vaginal symptoms When to report fever How to access medical help after hours Review of infection prevention Importance of medication compliance Importance of reporting abnormal lesions or lumps, bowel changes or abnormal vaginal bleeding Importance of regular exercise Care coordination Health-care provider appointments Medication administration Laboratory draws Return visit to transplant center at 1-year anniversary of transplant and thereafter as needed Vaccinations after the first year Allogeneic patients: • Plans for work-up of chronic GVHD symptoms based on assessment • Plans for treatment of chronic GVHD GVHD, graft-versus-host disease.

Nursing Role in Hematopoietic Cell Transplantation

ment of chronic GVHD, pulmonary complications, gastrointestinal symptoms, engraftment issues, treatment of serious and life-threatening infections, booster immunization, recurrent disease, and the development of secondary malignancies [33]. The need for the transplant team to act as a resource may persist from a few months to several years. While patients who live at a distance from the transplant center benefit from periodic follow-up visits to the transplant center, the general oncologist or hematologist at home can provide the routine follow-up care. Timely and consistent communication between the transplant centers and community practices is essential for safe continuity of care [34]. Transplant nurses can also provide ongoing support in the outlying communities by offering educational programs and by sending out educational material when the need is recognized. Facilitation of support groups is another way for transplant nurses to provide ongoing education and support for local patients in the long-term recovery phase. If the transplant center is involved in ongoing research, the research nurse plays an integral role in continuing with the collection of data and following research protocols. In some research centers, patient health questionnaires are regularly mailed to post-transplant patients to update demographics, quality-of-life information and survival documentation. Research nurses can triage problem issues that are identified from the questionnaires.

475

The role of the nurses working with the HCT patients during longterm recovery requires patience and perseverance when considering the recurring nature of chronic GVHD and other long-term issues. As in other phases of HCT nursing, the work is rewarding and challenging, and it allows for the establishment of long-term and trusting relationships. While nurses working in the office setting may find the problems of this population to be daunting at times, the eventual recovery and return of patients to a satisfying and full life is a reward experienced by those who are fortunate enough to work with these patients.

Nursing practice issues in hematopoietic stem cell transplant Various nursing roles are needed to successfully care for transplant patients. The organization of nursing roles usually parallels that of the center in which the program resides and is based on that institution’s nursing philosophy and care delivery model. In the ambulatory setting, nursing models must complement the medical care delivery model for successful care. For example, primary care in the outpatient setting is often structured by having a specific RN work with a specific provider caring for one group or team of patients. See Table 31.15 for descriptions of the various nursing roles.

Table 31.15 Nursing role descriptions in hematopoietic stem cell transplantation Nursing title

Role description

Inpatient staff RN

Cares for patients requiring acute care. Includes assessment, patient teaching, and carrying out the medical care plan. Inpatient nurses may care for patients on a designated transplant unit or on an oncology unit

ICU RN

Cares for patients requiring critical care. Includes assessment, patient teaching, and carrying out the medical care plan. ICU nurses may care for patients in an ICU incorporated into the transplant unit or in a main hospital ICU

Nurse coordinator

Coordinates pretransplant preparations including donor identification. Initiates patient education regarding transplant process. May continue as case manager during patients’ transplant course. This position is required of National Marrow Donor Program designated centers

Discharge planning RN

Assists team in planning and coordinating patients’ discharge from hospital to ambulatory setting. Provides patient and caregiver education required to manage care at home

Outpatient clinic RN

Cares for patients requiring ambulatory care. Includes assessment, triage, care coordination, patient teaching, and carrying out the medical care plan

Outpatient infusion RN

Cares for patients receiving infusions and transfusions. In many centers the stem cell infusion occurs in the outpatient infusion unit

Home infusion RN

Cares for the patients receiving infusions in the home setting. Educates patients and care providers to administer intravenous solutions in the home

Apheresis RNs

Cares for patients and donors undergoing apheresis procedures

Long-term follow-up RNs

Cares for patients after the acute phase of transplant. Provides education and triage for ongoing complications or late effects of transplant

Research RNs

Partner with the principal investigator to implement research protocols. Coordinates research activities with primary care team

Nurse manager

Responsible for nursing care delivered in a specific unit or clinic. Ensures appropriate staffing levels for the safe delivery of care

Clinical nurse specialist

Responsible for advancing transplant nursing practice by promoting evidence-based practice, conducting nursing research, creating nursing care guidelines, and evaluating outcomes of nursing care at the population level

Nurse educator

Responsible for orientation and continuing nursing education for transplant nurses

Nurse practitioner/physician’s assistant

Provides medical care under the direction of the attending physician. Performs procedures such as lumbar punctures and bone marrow biopsies

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Achieving and maintaining an expert nursing staff is a constant challenge for institutions with HCT programs. Initial orientation of a nurse with oncology experience generally takes 6–8 weeks. After this time, the nurse can be counted in staffing patterns but is not considered fully competent until he or she has completed their first year. The nurse-topatient ratio is also an issue that must be considered when planning a HCT unit. Most inpatient units staff on a one nurse to two or three patient ratio. This is usually a much lower ratio than on other oncology units and may need to be justified to hospital administrators. Patients develop strong feelings of trust and confidence in specific nurses, and facilitating work schedules to maximize these relationships can be a challenge for nurse managers. Continuing education must be built into HCT nurses’ work schedules. This education includes updates and refreshers on nurse practice topics as well as education on new medical protocols. It is also important for the nurses to remain up to date with the results of medical research. Each unit should identify topics of high risk and high complexity that qualify for annual competency review and testing. Examples on an inpatient unit include “Administration of high-dose chemotherapy” and “Administration of stem cells.” Examples in an outpatient clinic include “Steroidinduced diabetes teaching” and “Donor screening.” It is important for HCT nursing leaders to have a proactive strategy to prevent “burnout” in their staff. These nurses are at high risk for experiencing job-related stress, moral distress, and compassion fatigue as they work in fast-paced units with acutely ill patients with uncertain outcomes. Flexible scheduling and supporting time off requests assist many nurses to keep their resiliency to continue to work with these patients. It is also important that the nurses have time to share their experiences with each other as they may not be understood by other support people in their lives, such as family members or even other health-care professionals. The need for HCT nurses to network on a national level had been identified since the 1980s, when many transplant units began. Nurses in

the initial transplant centers organized national conferences, including conferences in Seattle, Minneapolis, and Omaha. The conferences were very well attended but quite a stress on the resources of the individual centers. In 1989, the Oncology Nursing Society acknowledged the unique needs of several subspecialties and created “Special Interest Groups.” The Blood and Marrow Stem Cell Transplant (BMSCT) Special Interest Group of the Oncology Nursing Society is one of the largest. In 1998, the Center for International Bone Marrow Transplant Research/American Society for Blood and Marrow Transplantation agreed to have a concurrent nursing conference with their medical conference sponsored by the BMSCT Special Interest Group. This conference has been planned by this group for over a decade, and has grown from a half day to 3 full days.

Summary Expert nursing care is essential to the success of a HCT program. Dr E. Donnall Thomas, founding Medical Director of the Fred Hutchinson Cancer Research Center’s Transplant Program and recipient of the 1990 Nobel Prize in Physiology and Medicine, called transplant nurses “his secret weapons” [35]. Nurses who are drawn to this subspecialty within oncology, and are successful in mastering the required skills, are rewarded by intense, intimate, and long-term relationships with the patients during this time of crisis in their lives. There is great satisfaction in knowing that they have made a positive difference in the lives of these patients.

Acknowledgment The authors would like to acknowledge Judy Campbell and Juanita Madison for their contributions to the original chapter published in the third edition of this text.

References 1. Ford R, McDonald J, Mitchell-Supplee K, Jagels B. Marrow transplant and peripheral blood stem cell transplantation. In: McCorkle R, Grant M, Frank-Stromborg M, Baird S, editors. Cancer Nursing: A Comprehensive Textbook. Philadelphia: WB Saunders; 1996. pp. 504–30. 2. Woltz P, Castro K, Park BJ. Care of patients undergoing extracorporeal photopheresis to treat chronic graft-versus-host disease: review of the evidence. Clin J Oncol Nurs 2006; 10: 795–802. 3. Franco T, Ford RC. Models of care delivery for hematopoietic stem cell transplant patients. In: Ezzone S, Schmit-Pokorny K, editors. Blood and Marrow Stem Cell Transplantation: Principles, Practice, and Nursing Insights, 3rd edn. Boston: Jones & Bartlett; 2007. pp. 423–9. 4. Kelley CH, Randolph S. The role of the homecare nurse throughout the continuum of blood cell transplantation. J Intraven Nurs 1998; 21: 361–6. 5. Holmes W, Kapustay PM, Walker F, Williams L, Ezzone S, editors. Peripheral Blood Stem Cell Transplantation: Recommendations for Nursing Education and Practice. Pittsburgh, PA: Oncology Nursing Press; 1997. pp. 1–44. 6. Nelson JP. The blood cell transplant program. Semin Oncol Nurs 1997; 13: 208–15. 7. Grant M, Cooke L, Bhatia S, Forman SJ. Discharge and unscheduled readmissions of adult patients undergoing hematopoietic stem cell transplanta-

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tion: implications for developing nursing interventions. Oncol Nurs Forum (online) 2005; 32: E1–8. Schulmeister L, Quiett K, Mayer K. Quality of life, quality of care, and patient satisfaction: perceptions of patients undergoing outpatient autologous stem cell transplantation. Oncol Nurs Forum 2005; 32: 57–67. Hacker ED, Ferrans C, Verlen E et al. Fatigue and physical activity in patients undergoing hematopoietic stem cell transplant. Oncol Nurs Forum 2006; 33: 614–24. Byar KL, Eilers JE, Nuss SL. Quality of life 5 or more years post-autologous hematopoietic stem cell transplant. Cancer Nurs 2005; 28: 148–57. Rexilius S, Mundt CA, Megel ME et al. Therapeutic effects of massage therapy and healing tough on caregivers of patients undergoing autologous hematopoietic stem cell transplant. Oncol Nurs Forum (online) 2002; 29: E35–43. Buchsel PC, Leum E, Randolph SR. Nursing care of the blood cell transplant recipient. Semin Oncol Nurs 1997; 13: 172–83. Johns A. Overview of bone marrow and stem cell transplantation. J Intraven Nurs 1998; 21: 356– 60. Poliquin CM. Conditioning regimens in hematopoietic stem cell transplantation. In: Ezzone S, Schmit-Pokorny K, editors. Blood and Marrow

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Stem Cell Transplantation: Principles, Practice and Nursing Insights, 3rd edn. Sudbury, MA: Jones & Bartlett; 2007. pp. 109–46. Wagner ND, Quinones VW. Allogeneic peripheral blood stem cell transplantation: clinical overview and nursing implications. Oncol Nurs Forum 1998; 25: 1049–57. Kapustay PM, Buchsel PC. Process, complications, and management of peripheral stem cell transplantation. In: Buchsel PC, Kapustay PM, editors. Stem Cell Transplantation: A Clinical Textbook. Pittsburgh, PA: Oncology Nursing Press; 2000. pp. 5.1–28. Sauer-Heilborn A, Kadlidlo D, McCullough J. Patient care during infusion of hematopoietic progenitor cells. Transfusion 2004; 44: 907–16. West F, Mitchell SA. Evidence-based guidelines for the management of neutropenia following outpatient hematopoietic stem cell transplantation. Clin J Oncol Nurs 2004; 8: 601–13. Kapustay PM. Blood cell transplantation: concepts and concerns. Semin Oncol Nurs 1997; 13: 151– 63. Shapiro TW. Intensive care management of the BMT patient: administrative and clinical issues. In: Buchsel P, Whedon MP, editors. Bone Marrow Transplantation: Administrative Strategies and Clinical Concerns. Boston, MA: Jones & Bartlett; 1995. pp. 69–96.

Nursing Role in Hematopoietic Cell Transplantation 21. Crawford SW. Critical care and respiratory failure. In: Thomas ED, Blume KG, Forman SJ, editors. Hematopoietic Cell Transplantation, 2nd edn. Malden, MA: Blackwell Science; 1999. pp. 712– 22. 22. Horak DA, Forman SJ. Critical care of the hematopoietic stem cell patient. Crit Care Clin 2001; 17: 671–95. 23. Saria MG, Gosselin-Acomb TK. Hematopoietic stem cell transplantation: implications for critical care nurses. Clin J Oncol Nurs 2007; 11: 53–62. 24. Plunkett P. Ethical issues in transplantation. In: Whedon MB, Wujcik D, editors. Marrow and Blood Stem Cell Transplantation: Principles, Practice, and Nursing Insights, 2nd edn. Boston, MA: Jones & Bartlett; 1997. pp. 506–23. 25. Hagen SA, Craig DM, Martin PL et al. Mechanically ventilated pediatric stem cell transplant recipients: effect of cord blood transplant and organ dysfunction on outcome. Pediatr Crit Care Med 2003; 4: 206–13. 26. Afessa B, Tefferi A, Dunn WF, Litzow MR, Peters SG. Intensive care unit support and acute physiol-

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treatment. Best Pract Res Clin Haematol 2007; 20: 311–27. Flowers MED, McDonald GB, Boeckh M et al. Long-Term Follow-Up After Hematopoietic Stem Cell Transplant: General Guidelines for Referring Physicians. Seattle, WA: Fred Hutchinson Cancer Research Center/Seattle Cancer Care Alliance; 2002. Deeg HJ. Delayed complications after hematopoietic cell transplantation. In: Thomas ED, Blume KG, Forman SJ, editors. Hematopoietic Cell Transplantation, 2nd edn. Malden, MA: Blackwell Science; 1999. pp. 776–88. Laffan A, Biedrzycki B. Immune reconstitution: the foundation of safe living after an allogeneic hematopoietic stem cell transplantation. Clin J Oncol Nurs 2006; 10: 787–93. Lilleby K. A nursing history of bone marrow and peripheral stem cell transplantation: recollections. In: Buchsel PC, Kapustay PM, editors. Stem Cell Transplantation: A Clinical Textbook. Pittsburgh, PA: Oncology Nursing Press; 2000. pp. 15.1– 8.

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David S. Snyder

Ethical Issues in Hematopoietic Cell Transplantation

Introduction Many of the ethical issues raised in hematopoietic cell transplantation (HCT) are common to those involved in other forms of medical therapy utilizing advanced technologies. These issues are informed by the traditional principles of patient autonomy and the importance of informed consent and confidentiality; justice, that is, the fair allocation of limited resources; beneficence and nonmaleficence; and fidelity or nonabandonment when the goals of treatment shift from cure to supportive care. However, certain unique features of HCT compound the complexity of these ethical issues, including: the young age of most patients; the fact that most of the diseases are life-threatening and often fatal within a short time in the absence of an HCT, yet are potentially curable with this treatment; the fact that a living donor is required who is often a minor; the ability to select potential donors for an affected sibling by utilizing preimplantation genetic diagnosis; the great financial expense of the procedure and controversies about reimbursement; and the fact that survivors of HCT may be cured of their primary malignant disease but may face other serious complications, such as sterility, chronic graftversus-host disease (GVHD), psychologic distress, sexual dysfunction, cognitive impairments, and occupational disability. In this chapter, some of the ethical issues in HCT are reviewed under the following general headings: who is eligible for HCT, informed consent for the patient, informed consent for the donor, alternative sources of stem cells, HCT for nonmalignant diseases, and end-of-life issues.

Who is eligible for HCT? Case scenario 1 A 49-year-old man with stage II multiple myeloma has a human leukocyte antigen (HLA)-identical sibling. He is eligible for a protocol utilizing tandem autologous HCT followed by a reduced-intensity regimen allogeneic HCT. His physicians feel this approach represents his best chance for long-term, disease-free survival and possible cure of his myeloma. His insurance plan considers this treatment plan experimental, and will only authorize a single autologous HCT. Case scenario 2 A 32-year-old man with chronic myeloid leukemia (CML) has no sibling donors available for allogeneic HCT. Although his company’s insurance Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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plan covers the costs of procurement and the actual transplant for a matched unrelated donor HCT, it will not pay for the search costs, which the patient cannot afford. Although the company sponsors a donor drive in the name of this patient, he cannot access any of the donors who might be identified. These two scenarios, which are based on actual cases, help to highlight some of the difficult issues involved in determining who undergoes an HCT procedure. This process involves both medical decision making based on clinical and protocol-determined eligibility criteria and thirdparty payer decision making. Each of these processes is ethically complex. First, the medical decision making: in the interest of justice, it is imperative that transplant teams develop standardized sets of criteria by which patients are selected for HCT. Some of these criteria are straightforward, such as the patient’s diagnosis and stage of disease, age, and adequacy of major organ function. Other criteria are more subjective and often relate to psychosocial issues that may influence the patient’s ability either to tolerate the HCT procedure or to comply with the long-term follow-up regimen. Multidisciplinary teams are needed that include not only physicians, but also nurses, psychologists, and social workers. It is important to avoid arbitrary and inconsistent decision making in deciding when not to offer HCT to a patient. For example, an HCT center may decide that a young patient with acute leukemia who has Down’s syndrome is not an acceptable candidate because of expected psychosocial management problems that might compromise the outcome. In one survey of 58 pediatric HCT centers, 16 leukemic children with Down’s syndrome had been transplanted at 10 centers. This was only about 20–25% of the predicted number based on incidence data. In fact, the outcomes were no different than those expected from a similar cohort of patients with acute leukemia who did not have Down’s syndrome. The author cautioned against physician bias toward these patients [1]. In an accompanying editorial, physicians were advised to use a utilitarian approach, that is, to recommend against HCT for children with Down’s syndrome only if data show poorer outcomes for them. Many HCT teams are concerned that patients with a history of active substance abuse may be poor candidates because of expectations of higher complication rates and poor compliance in their long-term care. A retrospective study from one transplant center identified 17 of 468 HCT patients as lifetime substance abusers (alcohol in 71%, marijuana in 30%, and opiates in 30%). When these 17 patients were paired with matched control subjects, a significant difference in survival probability was seen, 60% versus 10% (p = 0.0022) [2]. More data may be needed to substantiate these findings, but they do support the current practice at many HCT centers of excluding such patients. A survey of 597 HCT professionals (physicians, social workers, and nurses) was conducted in which 17 case vignettes were posed that

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highlighted a range of psychosocial issues that could potentially impact the long-term outcome of the transplant procedure [3]. The issues included suicidal ideation, use of addictive illicit drugs, a history of noncompliance, and others. At least 10% of responders indicated they would not proceed in all 17 vignettes, but there was a lack of unanimity for most of the cases in terms of recommendations to proceed or not. The decision making seemed contingent on perceptions of the severity and currency of the psychosocial issue and the ability to manage the patient’s potential noncompliance. A challenging case was recently published in which the issue of whether an incarcerated minor with relapsed acute leukemia should be offered an HCT, and who should pay for it [4]. This type of case highlights the sharp conflicts between a physician’s obligation to provide optimal care versus social justice. Is it cruel and unusual punishment to deny a prisoner a potentially life-saving treatment when the crime was not judged to be severe enough to justify the death penalty? Is it right for the state to pay for such a procedure when other law-abiding citizens without insurance or other resources are denied this type of treatment? There is a need for Federal and statespecific guidelines to establish when it is appropriate medically and socially for prisoners to receive technologically advanced and expensive healthcare. Decision making regarding third-party payer reimbursement is seen as a conflict over fairness and justice. Physicians as patient advocates are often in conflict with insurers who argue that HCT for certain diagnoses and disease stages is experimental and unproven. Physicians often believe that it is unfair to have patients accepted or denied access based on geography, their position in society or their ability to pay when a therapy is investigational, high cost or both. In analyzing patterns of utilization in four states, Mitchell et al. found that black patients, those enrolled in health maintenance organizations, those covered by Medicaid, and self-pay patients were less likely to receive HCT when admitted for either leukemia or lymphoma [5]. Insurers believe that it is unfair, and uneconomical, to be pressured by physicians and patients to pay for care that is not standard therapy [6]. Uniform and fair methodologies are needed to bridge these two views, rather than relying on costly litigation [7]. One approach is taxonomic, with a treatment modality labeled as experimental or standard. Thirdparty payers would cover experimental therapy if it met certain criteria, such as a phase III trial approved by the appropriate government agency, for example the Food and Drug Administration, and if the treatment were likely to benefit the patient. The treatment should be medically necessary, safe, and effective. The therapy must be as beneficial as any established alternative, and the improvement must be attainable outside of the investigational setting [8]. It is necessary to balance the premature dissemination of poorly studied, toxic, and expensive therapies to desperate patients by accepting limitations to HCT on the one hand, and by accepting that the patients’ best interests may be served by an experimental therapy on the other. Legal authorities caution that, although insurers may deny autologous transplant for a patient with cancer because the treatment is experimental, the more relevant question may be, “Is it the best option available for that particular patient with that particular disease?” The controversy in the recent past related to autologous HCT for breast cancer exemplifies these dilemmas. Welch and Mogielnicki highlighted some of the lessons that could be gleaned from this experience, including: (1) it is premature to discuss the cost-effectiveness of an intervention when its clinical effectiveness is unknown; (2) the National Institutes of Health should have an important role in determining what is experimental therapy; (3) public officials should not mandate coverage in the absence of clear data; and (4) the news media watchdog role should be extended to health care [9].

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An alternative approach is to reach a consensus based on available data of efficacy, duration of expected benefit, and the quality of wellbeing after the treatment [10]. Panels of expert physicians and insurers could review available data on a regular basis and develop grids that designated which diagnoses and disease stages would be covered, based on outcome analyses. Different categories could be considered, such as (1) diseases for which HCT is curative and may be the only curative therapy available, for example CML; (2) diseases for which HCT may not be curative but definitely prolongs survival, for example autologous transplant for CML, and possibly breast cancer and multiple myeloma; and (3) diseases for which the benefits of HCT are not yet known, for example autologous transplant for autoimmune diseases such as multiple sclerosis. One model is the Oregon Medicaid system, which adopted such an approach and covered HCT for all major hematologic malignancies including chronic lymphocytic leukemia, but did not cover chemotherapy for metastatic renal cell carcinoma. Such an approach is followed in California for Medi-Cal patients and by Blue Cross/Blue Shield. Another component of this approach is to restrict payments to “centers of excellence” that have demonstrated track records in their field. One of the most difficult challenges that the medical profession and the public at large face is how to assure that progress in developing new therapies will continue in a managed care system designed to minimize health-care expenses and to reimburse only for medically necessary and proven treatments. Who is going to pay for ongoing research that will lead to the breakthroughs of the future? One could argue that third-party payers have an ethical obligation to participate in this process by helping to underwrite the expenses involved in clinical research, and thereby to help establish which new treatment modalities are in fact effective and superior to established methods. Such new therapies may in fact be cheaper in the long run if they lead to definitive cures for patients and obviate the need for years of expensive, supportive chronic care.

Informed consent for patients undergoing HCT Case scenario 3 An 18-year-old woman with relapsed acute myelogenous leukemia (AML) has an HLA-matched brother available for allogeneic HCT. She has expressed an interest and willingness to undergo a transplant. However, during the informed consent process, she refuses to listen to any discussion about possible risks and complications from HCT. Can her doctors assume that she has given her consent and proceed to HCT? Respect for patient autonomy is reflected most critically in the informed consent process, in which patients are informed of their diagnosis, prognosis, potential benefits and risks of the proposed therapy, and alternative treatments available. Although this statement is true for any type of medical intervention, in the setting of HCT there are extra complexities, including the facts that many patients are minors, that almost all patients being considered for HCT have diseases that will likely be fatal without a transplant and are potentially curable with the procedure, and that patients who survive their original disease and the early transplant mortality are at risk for development of a wide variety of chronic complications that may impair their quality of life in many ways. After a patient has been told that he or she has a fatal disease and that a potentially curative procedure is available, how much does the patient really hear and/or retain about the potential risks and complications? How accurate are the available data about details of mortality and morbidity – including risk of sinusoidal obstructive syndrome (SOS), acute and chronic GVHD, cytomegalovirus and other infections, chronic radiation effects, avascular necrosis, sterility, the risk of second malignancies, and quality of life after transplant – and how many of these data

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are revealed to prospective HCT patients? If patients choose not to hear all of these specifics and make choices solely on the options of life and death, is this really informed consent? Must we insist on inflicting them with these truths? Can patients exercise their autonomy by choosing not to be fully informed? One study compared the process of informed consent as viewed by physicians or nurses in comparison to the view of patients [11]. For the HCT nurses, it was most important that patients know about the sideeffects and complications of the procedure. The physicians wanted the patients to know about the diagnosis, therapeutic options, and outcomes of treatment. The physicians’ views matched the patients’ most closely, as they made decisions based mainly on outcome and life or death. In another study of adult and pediatric patients and their physicians, the three main reasons that patients and their parents chose HCT were: (1) trust in their physician; (2) fear that their illness would get worse without the transplant; and (3) belief that the HCT would be a cure. Most of the adult patients remembered that complications could occur, but they could recall fewer than half of those mentioned. Most of the patients believed they had been given adequate information and that it was not too technical, although the physicians thought the information conveyed was in fact too technical and excessive. Most of the patients believed that their physician wanted them to undergo the BMT, and the patients made their decision based on that advice. One HCT nursing director suggested that the role of the HCT nurse in this process may be to ensure that patients have been completely informed, although the nurse must realize that patients may not be able to comprehend the complex details of treatment, and that the intensity of the choice between life and death may overshadow the risks of potentially life-threatening complications. The nurse can function as a patient advocate; which may create conflicts with the physician at times, and the nurse must not take away hope from the patient [11]. Lee et al. analyzed the degree of concordance or discordance between 313 patients and their physicians in estimating the chances of success after HCT [12]. There was significant concordance between patients and physicians when the outcomes were likely to be favorable, but when patients had more advanced disease, they tended to fail to recognize the higher risks of relapse and death. The authors speculated that physicians may tend to minimize the risks in their discussions with patients who have advanced disease. Divulging more information about these risks may not change the patients’ decisions to move forward with the HCT, but it may promote better psychologic adjustment after HCT. Andrykowski et al. focused on the question of whether patients had “returned to normal” following HCT, and examined differences between their expectations and actual outcomes, and the impact these differences might have on their sense of wellbeing [13]. They studied 172 diseasefree survivors from five HCT centers and found that only a minority felt they were back to normal. Before HCT, only 19% of patients had not expected to return to normal, and 47% anticipated they would. Following HCT, 32% (possibly up to 52%) stated they had not returned to normal. The discordance between pre-HCT expectations and their current functional status was associated with greater current psychologic stress. Despite this discrepancy, the survivors’ evaluation of their decision to pursue HCT was generally quite positive. The investigators asked why there would be such differences between pre-HCT expectations and actual outcomes. Some patients may hear the word “cure” pre HCT, and therefore expect that they will be “as good as new” after the transplant. Patients may not have been adequately informed of potential sequelae at the time of the HCT. Some patients may underestimate the risk of bad things happening to them, the socalled optimistic bias. This may be an adaptive mechanism to deny threats and to be unrealistically optimistic in the face of stress or adversity. In the short term, this adaptive mechanism may be beneficial, but

in the long run, it may promote psychologic distress because of unrealistic expectations. These findings suggest that providing details of risks and benefits during the informed consent process may have little impact on the patients’ decision making. Only a minuscule number of patients actually turn down HCT if it is offered, mainly because they view HCT as their sole chance of disease cure and survival. These studies suggest, however, that, following HCT, periodic discussions of possible complications and outcomes should continue to take place between patients and their physicians to help optimize the patients’ psychologic wellbeing. The issue of involving children in medical decision making is important since many candidates for HCT are minors. Most investigators recognize that children’s capacity for decision making and autonomy develops over time. The parents or guardians must give consent for their minor child, but the wishes and concerns of the child should be taken into account [14]. The American Academy of Pediatricians states that one “should not exclude children and adolescents from decision-making without persuasive reasons” [15]. It is generally accepted that, starting at age 7 or 8, children are capable of giving assent to any proposed intervention, and that their assent should be required to proceed. The assumption is made that the parents will act as surrogate decision makers for their children and make choices based on the best interests of the child. The courts may need to become involved if parents do not seem to be following this approach, for example if a parent is a Jehovah’s Witness and refuses blood transfusion for the child.

Informed consent for donors of hematopoietic cells Case scenario 4 A mother leaves the country with her 16-year-old son who is HLA matched with his 22-year-old brother who has acute leukemia, because she fears that if her younger son donates stem cells then he will die or get leukemia too. She feels she is going to lose one son and does not want to lose the other. The older son dies from refractory leukemia without HCT. Case scenario 5 In performing HLA typing on members of a large family because a relative has leukemia, it becomes evident that one of the siblings has a different father from the rest of the children. The source of cells for an allogeneic transplant is usually a living donor who may be closely related to the patient, who may be a minor, or who may be an unrelated volunteer. In each of these situations, the autonomy of the donor must be respected, as reflected in an appropriate informed consent process. Donors must be informed of the specifics of the procedure that they may undergo, especially the potential risks involved. The process must be free of coercion and must respect the confidentiality of the donor and the family when it is a family donor in question. Cultural diversity issues may complicate the search for a potential donor if unusual or irrational beliefs are brought to bear. Special and important considerations are raised when the donor is a minor. It is generally accepted that the risks involved in being a marrow donor are very small. Estimates of the incidence of life-threatening or incapacitating complications are about 0.29% for adult donors at several centers, and 0.4% in a pediatric population from a single institution [16]. A potential benefit of the donation process is that pre-existing medical problems that require intervention may be detected in the donor. In this study from Seattle, 206 medical problems were observed in the 1549 donor evaluations, with hypertension, obesity, and cardiac problems the most common (see Chapter 42) [17].

Ethical Issues in Hematopoietic Cell Transplantation

Many collection centers are using granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood cells (PBCs) from matched siblings or unrelated donors as their preferred source of cells [18–20]. Many of the risks associated with marrow harvests are minimized or prevented, such as complications from general anesthesia, the pain from multiple iliac crest punctures, local hematomas, and/or infection at the aspiration sites. The use of G-CSF for this purpose is considered to be generally safe except for donors who have certain underlying medical conditions such as hemoglobinopathies, autoimmune disease, or coronary artery disease. In a randomized study comparing PBC to bone marrow donations, the donors found the PBC collections to be the less burdensome and preferred method [21]. In a study of sibling donors for patients enrolled in a randomized trial comparing PBCs with bone marrow cells, Rowley et al. found that the intensity and nature of pain was very similar for the two groups, although all the PBC donors returned to normal function by 14 days compared with 80% of the bone marrow cell donors [22]. It is still not settled whether there is any overall advantage to the recipient from one source of cells over the other. Donor preference may be the deciding factor if there is no clear advantage between the two cell sources. In reviewing the experiences and attitudes of the first 20 volunteer unrelated donors at the University of Minnesota, Stroncek et al. found no serious physical or emotional after effects [23]. Nine of the 20 donors reported that a friend or family member had discouraged them. Nineteen said they would donate again, and 17 would advise others to donate. One donor had orthostatic hypotension for 1 day, and all donors received one or two units of autologous blood. The benefits of being a marrow donor for a family member or an unrelated recipient are a matter of debate. At one extreme, it is argued that there are no benefits to the donor, and therefore even the small risk of complications becomes more significant. Others state strongly that there are remarkable benefits to the donor of a psychologic nature, especially when donating to a family member, such as the satisfaction of helping to save a loved one’s life [24]. On balance, the very low risks associated with marrow donation and the fact that marrow is a renewable resource like blood, and that significant benefits are derived by the donor, make it ethically appropriate to encourage PBC and marrow donation. Of course, it is imperative that any coercion of potential donors be avoided. To that end, the HCT center should keep results of HLA typing confidential (or not even initiate testing) until all family members have had the opportunity to discuss privately their desire to proceed [11]. Another argument for strict confidentiality related to HLA typing results is that previously hidden facts about paternity may be brought to light to an unsuspecting family. Revelation of such information could have devastating effects on the entire family unit. The psychologic state of the donor must be respected. If the outcome is poor, especially if the patient fails because of GVHD, there will be a natural tendency for donors to blame themselves for the patient’s death. Appropriate counseling before and after the HCT can help lessen this burden for the donor. In light of these concerns, it has become the legal standard that no court can force anyone to undergo medical testing or a procedure that is intended only to benefit another individual. Confidentiality and noncoercion are especially important issues in relation to unrelated donors. The National Marrow Donor Program has very explicit policies and procedures designed to respect potential donors’ autonomy by requiring informed consent at every step of the process, including the time at which a donor is first listed in the registry; before collection of blood for confirmatory typing, infectious disease markers, and research samples; prior to notification of the transplant

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center that a donor is willing to proceed to donation; and before general anesthesia [25]. The donor’s privacy is of utmost importance to avoid any extraneous influences or pressures as the donor decides whether or not to donate. The identity of the donor is known only to the individual donor center, and many precautions are put in place to ensure this. Generally, the recipient and donor are allowed to contact each other directly only by mutual consent 1 year after the HCT. The National Marrow Donor Program considers these measures to be critical in promoting increased participation in the program by altruistic individuals. The World Marrow Donor Association reviewed the ethical issues for unrelated donors, focusing on respect for the donors’ autonomy, wellbeing, and needs. They stressed the importance of noncoercion and adequate informed consent, especially when requests for donation of a second cellular product are being considered [26]. The process by which decisions are made to use minors as marrow donors is somewhat controversial [27–29]. Some authors have questioned the manner in which informed consent is obtained to collect marrow from minors and have asked whether parents can truly give informed consent. The need to involve the minor in the consent process by seeking his or her assent is a widely accepted concept, especially for children over the age of 7 or 8 years. Delany has questioned whether it is really legal to harvest marrow from a minor and has labeled the process “altruism by proxy” [29]. One legal concept permits a medical procedure only if it serves the best interests of the child who undergoes it. If there is disagreement between caregivers, it may be necessary to go to court. A second concept argues that parents can give valid consent to treatments that are “not against the interests of the child.” This approach was developed to allow blood drawing from children whose legitimacy or parentage was at issue. Some have argued that this principle is relevant to any medical intervention carried out on children purely for other people’s benefit. Delany argues that marrow donation is not in the best interest of the donor, and that it may be against the child’s interests, especially if the potential donor is too young to have established an emotional bond with the intended recipient. She also contends that the parents are not well suited to give informed consent for the donor due to conflict of interests related to the sick child, and that an informal tribunal or other forum independent of the parents and medical advisors should be responsible for approving each proposed donation. This procedure would be analogous to the strategy developed by the Law Commission in England to safeguard mentally incapacitated adults from being inappropriately volunteered for marrow extraction. Month disagreed strongly with this interpretation of the issues [29]. She believed that the positive aspects for the child donor are considerable and include the saving of a sibling’s life and the benefit of many years of a whole family not burdened by the psychologic trauma of the death of a child. Further, since HCT is an accepted therapeutic option that can be lifesaving, and often offers the best cure rate, is there really any reason a morally competent sibling would not want to donate? The risks of donation are minimal, especially when compared with the risks of not donating marrow, that is, the death of a sibling. Savulescu argued that although the bone marrow donation is not in the donor’s medical interests, it may be in his or her overall interest [29]. To save the life of a loved one is one of the most important things one can do. Even for donors who are too young to understand at the time, the potential for a future sense of achievement and the love and gratitude of the saved sibling is important. There are benefits to the family unit as well. Parents should be the ones to give informed consent after they are educated about the risks and benefits of the donation, just as parents often make decisions with conflicting interests of their children, but with a commitment to the overall good of the family.

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An unusually difficult case was recently published involving a 15-yearold boy with relapsed AML who was incarcerated for sexually assaulting his 9-year-old sister [4]. The conflicts involved in the decision making about whether or not to offer the boy HCT were discussed in a preceding section of this chapter. The 9-year-old sister was the only HLA-matched relative available. Should she be asked to donate stem cells to her brother? Is there a reasonable alternative source of stem cells available for the patient, for example a matched unrelated donor? Is the mother the right person to give informed consent on her behalf? Should there be a legal guardian appointed to protect the interests of the sister? Would the potential harm to her, psychologic and physical, outweigh the benefits? Although these are questions relevant to almost every case involving a minor donor, the conflicts were clearly more acute in this case. The formal process for approving the use of a minor for marrow donation varies between countries and between centers within the same country. A survey of 52 HCT centers in Europe and the United States found that some centers had an ethics committee decide the validity of the therapeutic proposal [28]. In France, there is a law that requires a committee of three experts to assess the psychologic and medical consequences of donation of the organ by a minor, and notes that “the refusal of the minor donor to accept the removal shall always be respected.” In the United States, a Washington State court ruling stated that the advantages to the donor are undoubtedly greater than the risks, and defers to the parents to give informed consent. Some institutions require an authority outside the family and hospital, for example a “tutelary judge” or commission of experts, to make the decision. A questionnaire was sent to 70 HCT centers in North America by pediatricians at the MD Anderson Cancer Center to poll the HCT teams on their approaches to marrow donations by minors [30]. The impetus for sending the questionnaire was a case brought to the MD Anderson pediatricians in which they were asked to collect marrow from a young child to be donated to the child’s parent. There was a significant difference in size between the child and parent, and three HLA mismatches. The parent had relapsed AML. Ethical concerns were raised by hospital staff members. As a result, the MD Anderson Center developed a process in which the minor donor and surrogate are interviewed by a social worker, pediatrician, and anesthesiologist, none of whom are involved in the care of the recipient. If any concerns are raised, the case is referred to the ethics committee for consultation. Fifty-six (80%) of the centers responded to the questionnaire. There was consensus in endorsing the validity of parental consent. Most centers would use donors as young as 6 months old and would use them more than once if needed. The centers were willing to use minor donors for patients on experimental protocols, and the projected outcome of the HCT did not affect the decision to use the minor as a donor. Issues raised included the nontherapeutic nature of the donation, the vulnerability of the minor, and the potential conflict of interest when a parent acts as a surrogate for both the donor and a sick family member. Even greater conflict was seen if a parent was the intended recipient of the marrow, and most agreed that the other parent or both should be involved in the informed consent procedure. The majority of the centers and physicians had policies or practices of using the parents as the surrogate; a minority preferred to use a parent plus a child advocate or child protection agency. If there were disagreements between the parents, eight centers would cancel the HCT, and the rest would either seek consultation with the ethics committee, use an unrelated donor, refer the parents for another opinion or refer the decision to a child protection agency. When asked if the use of a child intentionally conceived as a marrow donor were ethical, most of the physicians answered that they would be willing to use such a donor. They would consider using umbilical cord blood (UCB) in that case as being safer for the donor.

The participation of pediatric donors in clinical research raises additional issues beyond standard clinical practice [31]. When can Institutional Review Boards (IRB) approve such activities? The IRB must focus on whether the proposed research procedure is different for the donor compared with a standard stem cell collection. If the proposed research adds or replaces procedures to the standard clinical donation, and thereby increases the risks to the donor, such a protocol must be approved by the Secretary of Health and Human Services with the advice of a panel of experts (category 46.407 of the Federal Regulations). Some have argued that minors who are asked to donate G-CSFprimed PBC should be considered to be research subjects, since the data about long-term risks in this setting are not well known. The parents would be the best persons to decide for their child donors, but IRB approval would be required [16,32].

Alternative sources of hematopoietic cells Case scenario 6 A couple decides to reverse a vasectomy to create a new baby to be a possible donor for their teenage daughter with CML who has no family or unrelated donors. HLA-matched siblings are usually considered to be the primary source of cells for HCT. For the 70% of patients who lack such a donor, alternative sources of stem cells include matched related donors other than siblings, unrelated volunteer donors, UCB cells, and possibly autologous stem cells in the appropriate clinical setting. In this section, the ethical issues raised by three potential sources of stem cells are discussed, namely, UCB [14,33–35], “children conceived to give life” [36], and xenografts from baboons or other primates. Since the first UCB HCT was performed in 1988, about 6000 such transplants have been carried out worldwide [37]. There are over 170,000 UCB units available in 37 banks in 21 countries [38]. The advantages of cord blood over adult marrow include: the lower risk of carrying viruses; a reduction in procurement time from the 4–6 months needed for an unrelated marrow donor, to 1–2 weeks; and the fact that cord blood cells are potentially usable across greater HLA disparities. The major disadvantages are the limited number of cells available, the fact that one cannot go back to the donor for more cells if needed, and the risk of transmitting a genetic disease not previously diagnosed in the donor’s family. Marshall raised several ethical issues related to cord blood cell collections and cord blood banks, starting with consent and privacy [39]. Is informed consent required for a product that is otherwise discarded as waste? Is the mother (or parents) the appropriate one(s) to give consent? Is consent required to conduct a battery of tests to detect possible infectious or genetic diseases, or both? Should donors, that is, the parents, be informed of the results of such tests? Who owns the cord blood? Obviously the donor himself cannot give informed consent, yet an extensive medical profile about the donor may be generated, raising concerns about invasion of privacy. Should consent be obtained for follow-up tests that are not consented to directly? What should be done with abnormal results from such tests? Should banks maintain medical files and genetic data, but in such a way that names and identifiers are delinked from the donated sample [40]? What are the obligations to inform the family if evidence of human immune deficiency virus or an abnormal gene is found? Some authors argue that the infant donors should be the first to benefit from use of their own cord blood cells [41]. Records should be kept of diseases detected and parents informed. Therapy should be offered for treatable diseases. Commercialization of cord blood cells for therapy should include a financial benefit for infants; for example, royalties from

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each donation should be placed in a national trust fund for research and treatment of children with serious diseases. Kurtzberg et al. argued that cord blood that was previously discarded material should be a public resource with no financial gain to any party [42]. Regulation of donor banks by an agency such as the Food and Drug Administration is needed. For example, there are companies that try to persuade parents that they should store the cells as insurance against future needs, when in fact the chances are remote that the donor would ever need or want to use the cord blood cells. There is a pressing need to protect the confidentiality of the information collected, to notify parents and children of the test results, and to assure equitable access to cord blood samples. For the health of the mother and baby, the collection process should be restricted to full-term, uncomplicated pregnancies, and should not interfere with good obstetrical practice at delivery. The American Academy of Pediatrics has addressed the issue of cord blood banking and issued a set of guidelines to help physicians regarding types and quality of blood banks (for-profit versus not-for-profit), ethical and operational standards including informed consent policies, financial disclosures, and conflict-of-interest policies [43]. Their three main recommendations are: (1) the storage of cord blood in private banks for later personal or family use should be discouraged unless there is a full sibling in the family with a known malignant or genetic condition that might benefit from cord blood transplantation; (2) cord blood donations should be encouraged when stored in a bank available for public use; and (3) private storage of cord blood as “biologic insurance” should be discouraged. Considerable attention and debate were generated in response to the actual case outlined above as case scenario 6. The practice of “conceiving a child to give life” is not a rare event [44]. Some question whether a good outcome for the recipient lessens the ethical dilemmas posed. It was argued that such a practice does not impose harm on persons or relationships within the family, and does not show lack of respect for the child conceived [11]. Others raised their concerns about performing prenatal testing to diagnose the genetic disease affecting the intended recipient and for HLA typing, and the potential for aborting in the case of a mismatch. One hematologist’s view of the concept is that such a child is conceived not to replace the sick sibling, but to give life to the sibling who would not survive without a transplant [44]. The benefits of this approach include the fact that the cord blood is disposable material, and that no anesthesia or blood transfusions are needed for the donor. From the recipient’s perspective, the transplant can be performed earlier since a marrow transplant could require a delay of 6 months or more. Alby described three characteristics of pregnancies conceived for HCT: (1) families in that situation face the trauma of the impending death of the sick child; (2) the pregnancy outcome with respect to HLA matching and the HCT results are both uncertain; and (3) biology has intruded into the family dynamics [36]. A family may attempt a cure at any cost, especially in an emergent situation such as acute leukemia. A child may be viewed subconsciously as a replacement child, but should never be reduced to the role of a therapeutic tool. In terms of family dynamics, a child may be viewed as bad or good based on HLA matching. Many are concerned that such a child is being conceived only for transplantation. Yet a child is always born for something: to maintain the integrity of a couple, to gratify the parents’ need for a family, to repair their vulnerability. All parents must go beyond the idealized view of their children and accept that they have an identity of their own, and all children have some symbolic role in the family fantasy. In that light, is there really anything wrong with conceiving a child to donate cells for the purpose of saving the life of a sibling? There are risks for children born for HCT, of both a physical and a psychologic nature. The physical risks are less from cord blood collec-

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tion than from marrow harvesting. Such a child would be born into a psychologically stressed family dealing with the emotional and financial trauma of caring for a sick child. In case of the death of the recipient, the new child is exposed to parental depression, identification with the deceased sibling, loss of identity if the parents cannot accept the death, and shared guilt with the parents. When the procedure is successful, the donor is seen as the savior, and both the child and the family must find a normal way of life again. Advances in in vitro fertilization techniques have opened the door to preimplantation genetic diagnosis (PGD). Candidate embryos can be screened in vitro for HLA matching [45] and for various genetic traits [46,47] as the basis for selecting which embryos to implant. HLA typing of embryos to select a potential stem cell donor for an afflicted relative has been proposed as a legitimate means of securing a donor for a patient that is in need of a life-saving transplant. The ethics of this approach have been reviewed, and the conclusions were that using PGD to choose a stem cell donor is unlikely to cause harm to anyone, is likely to be beneficial to some, is a reasonable use of limited health resources, and should be permitted in countries where PGD is already allowed [48]. Morgan et al. also concluded that the use of assisted reproductive techniques to create a stem cell donor can be considered to be ethically acceptable [49]. As a recent case in point, in the United Kingdom, the Human Fertilisation and Embryology Authority gave permission for PGD to be utilized to select an unaffected embryo to serve as a future hematopoietic cell donor for a sibling with thalassemia. The technique has been used in the United States since 2000 [50]. Pennings et al. argued that PGD-guided selection of embryos for this purpose is morally defensible on the condition that the procedure to be performed on the future child is acceptable for an existing child, and that the “instrumentalization” of the donor child does not demonstrate disrespect for the child’s autonomy and intrinsic worth [51]. Devolder considered the risks and benefits of utilizing preimplantation HLA typing for selecting embryos to be used as donors of stem cells for sick siblings and concluded that it would be unethical not to allow this procedure, since there is no indication of harm to the donor child, and some lives would be saved [52]. She argued further that it should be up to the parents to decide whether their current or future children can act as a donor for a loved one, and that the pool of potential recipients should not be restricted to siblings or other family members, but should be expanded to include anyone whom the couple loves. The ability to create multipotential stem cells in vitro from pluripotential embryonic stem cells is a subject of wide public debate. Legal and ethical distinctions have been drawn between human reproductive cloning and nuclear transplantation for the production of stem cells for therapeutic applications. Only the latter will be discussed here as a potential source of hematopoietic cells for use in HCT in the future. Many scientists, ethicists, government bodies, and medical organizations in the United States, Canada, and Europe have advocated for the legalization of research for the purpose of therapeutic cloning [53–58]. The potential to utilize such cells to treat a variety of medical conditions appears to be great, although significant technical hurdles are yet to be overcome before there can be clinical applications. Many serious ethical issues must be considered including the protection of human dignity, the source of unfertilized eggs and/or embryos to be used for research, informed consent from potential donors, the privacy of donors, and financial incentives to donate eggs and/or embryos. The utilization of an unusual source of stem cells generated considerable media attention when investigators attempted to reconstitute the immune system of a patient with acquired immune deficiency syndrome (AIDS) using stem cells collected from a baboon [59]. A number of scientific and safety issues are raised, including the risks of the baboon cells attacking the human host, the unknown ability of the baboon cells

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to mount an immune response against pathogens to which the AIDS patient is exposed, the concern that the patient will be made sicker by the conditioning regimen, and the question of whether the patient will have to fight off baboon viruses. Ethically, there are concerns about how to protect the rights of the first patients treated this way. How does one select the first patients for procedures that are not likely to benefit them, that is, the so-called “patient-pioneer” or “human guinea pig”? It is critical that the patient understand how unlikely it is that he or she will benefit, and that the experiments be done in a rigorous way so that specialists in the field can learn from the experience. There are fears of transmission of animal pathogens to humans, perhaps necessitating germ-free colonies of donor animals. Pigs are being used for solid organ transplants to humans, perhaps as a bridge to tide the patient over until a human organ becomes available. Barker proposed caution in xenotransplantation and a postponement of solid organ xenotransplants until the issue of informed consent to the infectious disease risks is adequately addressed in an open public policy process [60].

Transplantation for nonmalignant diseases Most HCT procedures are performed to treat malignant diseases for which HCT often represents the only curative option. Many nonmalignant diseases are also treated by HCT, especially in the pediatric patient population, and include primary marrow failure such as severe aplastic anemia and a variety of congenital immune deficiency syndromes. The balance of risk and benefit for patients with these diseases is clearly in favor of HCT since the diseases are often rapidly fatal, and curative alternative options are generally not available. The balance may not be so clearly defined for a variety of diseases that affect marrow-derived cells, which may be a source of significant morbidity for affected patients and possibly shortened life span, but are not considered to be rapidly fatal. Further, the data that demonstrate the long-term benefit from HCT for these conditions may be inconclusive or even nonexistent. Examples of this category of diseases would include hemoglobinopathies such as sickle cell disease, certain metabolic storage diseases, autoimmune diseases such as multiple sclerosis or rheumatoid arthritis, and acquired immune deficiency diseases, specifically AIDS. How does one select the right patient to be treated by a procedure that carries significant risk of morbidity and possibly early mortality? How can one demonstrate the efficacy of HCT in diseases that usually progress over years rather than weeks or months? How does a parent decide to consent to an HCT for a child with such a disease? This discussion focuses on hemoglobinopathies as a paradigm for these issues since there has already been a broad experience in performing transplants for thalassemia [61], and a body of literature about the ethical dilemmas involved is available. These issues were discussed at an international meeting on HCT in children held in March 1994 [33]. In regard to hemoglobinopathies, the difficult questions are whom to treat and when, and whether to offer HCT as a curative procedure to all affected children as early as possible. Some participants favored HCT for all patients with sickle cell disease who have an HLA-matched sibling in early childhood, given the high risk of morbidity in adult life. Others argued that it should be reserved for those who show clinical indicators of poor prognosis, as adopted by the ongoing national trial to recruit 30–60 children over a 5-year period. For thalassemia, the clinical course is more variable. Some proposed that HCT should be offered before any morbidity has occurred, while others would reserve it for children whose iron overload was well controlled and who were without liver disease. Giardini discussed the ethical issues of HCT for thalassemia in an editorial in 1995 [62]. He argued in favor of HCT since the risk of mortality from disease-related complications remains high even with

desferrioxamine chelation. The success of standard therapy depends on compliance of the patient and a health-care system that is able to carry it out. The cost of this effort is about $32,000 per patient per year in developing countries, and about $60,000 per year for adults in the United States. These patients require the input of specialists, including cardiologists, hepatologists, psychologists, and endocrinologists. In comparison, an HCT for hemoglobinopathies in 1991 cost about $173,250 in the United States and about half that in Europe. Giardini argued that it is ethically and economically appropriate to recommend HCT for thalassemia, especially given the progress in dealing with GVHD and cytomegalovirus, the availability of newer antibiotics, and so forth. Estimated outcomes for patients with class of risk I are 3% probability of death, 4% of rejection, and 93% of disease-free survival. For patients in class of risk II and III with organ damage from iron overload, the mortality from HCT is higher, but their expected survival with standard therapy is much lower. Although other experimental approaches show promise, such as artificial hemoglobin, new oral iron chelators, gene therapy, and agents to stimulate fetal hemoglobin, the fact is that currently HCT offers the only chance of cure of the disease. Kodish and associates examined the issue of parents’ decision making concerning HCT for children who were affected with sickle cell disease [63]. A questionnaire was presented to parents with hypothetical chances of cure versus mortality to see how they balanced these two outcomes. In the study of 67 parents, 54% were willing to accept some risk of short-term mortality. Thirty-seven percent would accept at least a 15% mortality risk in the short term, and 12% were willing to accept a 50% or more risk. Sixteen (24%) of the 67 parents would not accept HCT even if there was a 0% mortality risk. Differences were found between the group of parents who would accept some risk and those who would not. Parents who were highschool graduates, who were employed outside of the home or who had more than one child with sickle cell disease were more willing to take the risks. Of note, parents were more willing to take risks for girls than for boys. The parents’ decisions were not related to the clinical severity of their children’s illness, unlike the way the doctors might make such a decision. For parents with more than one child, their willingness may reflect their knowledge of the disease, or the burden of caring for children with sickle cell disease. Clearly, the concerns and values of the parents must be considered along with weighing the medical issues of risks and benefits. The difficult issue is what criteria to use to select patients to undergo HCT for sickle cell disease. As one model, the IRB at the University of Chicago in 1988 allowed two groups of children to be treated with HCT: children who already had had a stroke and therefore were receiving monthly transfusions to prevent the next stroke, and those with recurrent painful crises who had required hospitalization for at least 60% of the preceding year. Recent advances in HCT, such as the use of peripheral blood hematopoietic cells from allogeneic donors and reduced-intensity conditioning regimens, have been utilized in the treatment of hemoglobinopathies [64,65]. These advances help reduce the risks of short- and long-term toxicities for either the donor or the recipient, and thus create a more favorable risk–benefit ratio. Parents of minors with hemoglobinopathies will need to be informed about these innovations to help them make an appropriate decision for their children who may be candidates for HCT.

End-of-life issues Case scenario 7 A 50-year-old physician with CML is dying from SOS and GVHD with liver and respiratory failure early after HCT. He has no written advanced

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directive. He made his second wife promise that she would not let him stay on life support for more than a week if he had no hope of recovery. She is devoutly Catholic, but feels she made a commitment to her husband to withdraw life support. His adult children from his first marriage, who are atheists, say their father would never agree to withdrawal of life support, that he is ardently opposed to euthanasia, and that to him withdrawal of life support is no different from euthanasia. Respect for patient autonomy requires caregivers to determine the wishes of their patient with respect to resuscitation and life support. Advanced directives, such as a Power of Attorney for Health Care, are extremely effective vehicles to facilitate discussions between patients and their doctors, for patients to convey their wishes, and to name a surrogate to speak for them when they become incapacitated. Yet very few patients execute such documents or discuss these matters with their doctor. Such discussions about end-of-life issues are difficult for most physicians, nurses, and patients. They are even more difficult in the context of HCT, where the patients are generally young and, although they have potentially fatal diseases, often have a significant chance for cure with this treatment. During the informed consent process that leads up to the HCT, physicians are obligated to discuss the risks and complications of the planned therapy, including the chance of early mortality. As discussed above in the section on informed consent for patients, when faced with life and death choices, patients often block out the details of possible complications in the decision-making process. Physicians have trouble raising end-of-life issues in the context of proposing a potentially lifesaving therapy, in part for fear that patients will take away a negative message about their prognosis [66]. The fact remains that up to 15–20% of patients may succumb to early allogeneic transplant-related mortality. HCT patients often decompensate rapidly and unpredictably, for example from severe SOS or GVHD. There may not be an opportunity to discuss end-of-life issues when the patient is in respiratory failure on the way to the intensive care unit. The quality of patient care would be much enhanced if such discussions and appropriate documentation of patients’ wishes were carried out well before this point, and were then acted upon as dictated by the patient or the surrogate. Many of these patients will be maintained by intensive life support for extended periods of time, despite generally poor chances of recovery. At some point in the course of the HCT procedure, the goals of therapy must shift from cure and aggressive interventions to palliation and supportive care for a dying patient. Rigorous attention to pain control is essential throughout the HCT process and certainly during the period of dying. The principles of fidelity and nonabandonment require caregivers to make it clear to patients and their families that they will not be abandoned as the goals of treatment shift from cure to caring for the dying patient. Although it may be appropriate to withhold interventions that are medically futile, how is this to be determined? How much of our limited health-care resources should be expended providing what is ultimately futile medical care? How great an emotional cost do families have to pay? These are difficult questions that need to be addressed at both the macrolevel of society and the microlevel of individual patients and families. It is the patient (or surrogate) who is best able to understand and decide on the issues of his or her ability to pay for a treatment and the possibility of financial destitution of a family by continuing futile care. The surrogate must utilize substituted judgment to relay the decisions that the patient would have made if he or she had the capacity. In cases in which no surrogate is legally designated, an ethics committee may need to be involved to consider quality of life issues and death with dignity. To explore the variables involved in instituting do-not-resuscitate (DNR) orders for HCT patients, a retrospective review of 40 patients who died on the Seattle Veterans Administration Medical Center HCT

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unit was carried out [67]. This center has a DNR policy that requires physicians to document the discussion held with the patient/surrogate, and for the patient/surrogate to sign a DNR consent. Patients with fatal diseases often suspend their feelings about terminal illness in hopes of a cure through HCT. The dying trajectory of these patients is often sudden and unpredictable. The health-care providers often focus on aggressive medical management when these situations arise, and, as a result, the topic of death is avoided. The process of executing DNR orders was believed to facilitate the transition from aggressive care to supportive care in critically ill HCT patients. Of 42 deaths that occurred on the HCT unit during a 5-year period, six patients had a resuscitation attempt and 36 had DNR orders (two records were not available for review). The DNR consent was signed 26% of the time by the patients themselves, 32% by spouses, 24% by mothers, and 18% by other family members. The two groups of patients did not vary in terms of age, diagnosis or type of HCT. The non-DNR group developed life-threatening complications earlier in their course, whereas multisystem organ failure was the common factor among the DNR patients. The death of non-DNR patients may not have been anticipated because the complications that developed were emergent and were considered reversible. These conditions may have precluded discussions regarding DNR status. For the DNR patients, this designation occurred close to the time of death: 32% on the same day as death, 82% less than 5 days before death, and 18% 5–14 days prior to death. In 11 studies of HCT patients who required mechanical ventilation, the rates of survival to discharge from the hospital ranged from 0% to 11.1%, with a mean of 4.7% [68]. It would be clinically useful to define a subgroup of patients whose chance of survival is so low that both patients/surrogates and physicians can agree that intensive care can no longer fulfill the original goals of the HCT. Criteria to identify patients who are destined to die after HCT are needed to help reduce emotional and financial expenditures of families and institutions. At the Fred Hutchinson Cancer Research Center from 1980 to 1992, 25% of all HCT patients were ventilated. Patients who were more than 20 years old, had disease in relapse, and had grafts that were not HLA identical had a 50% chance of needing ventilation. Only 6.1% survived. To identify predictors of death in mechanically ventilated HCT patients, Rubenfeld and Crawford conducted a nested case-control study comparing all 53 survivors of ventilatory support with 106 matched control subjects who did not survive [68]. These patients were selected from the 865 individuals who were mechanically ventilated for at least 24 hours during that 12year period. Survivors were defined as those who were alive for 30 days after extubation and who were discharged from the hospital. Survival was statistically associated with younger age, lower score on the APACHE (Acute Physiology, Age, Chronic Health Evaluation) III scale, and shorter time from HCT to intubation. Of the 106 control subjects who died, 82% died while on the ventilator, and the remaining 18% died in the hospital a median of 18 days after extubation. Of the 53 survivors, median survival after extubation was 634 days (range 54 days to 12 years). Eighteen (34%) survived for less than 6 months and five (9%) for less than 1 year; 30 (57%) lived for more than 1 year. No patient who was intubated for more than 149 days survived. The survival rates changed from 5% to 16% over the most recent 5 years, which was not explained by changes in the age of patients, the rate or timing of intubation or the percentage of allogeneic transplants that were not HLA identical. The improved survival may be due to better antimicrobials and the use of cytokines. There were no survivors among the 398 patients who had lung injury and either required more than 4 hours of vasopressors or had sustained hepatic and renal failure. Using these three criteria, an accurate prediction of death could be made in the first 4 days for more than half of the patients who did not survive. Of the 60% of patients who developed any

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two of these three risk factors, half had done so by day 2, and 90% by day 4. On average, these risk factors developed six ventilator days and nine hospital days before death. If life support had been withdrawn on the first day that two out of three risk factors were met, and if death had followed swiftly, more than 7300 hospital days and 4800 ventilator days could have been avoided for the 812 patients who died. A multi-institutional study confirmed these observations in adult HCT patients, for whom the combination of mechanical ventilation and hepatic and renal dysfunction was associated with a probability of death of 98–100% [69]. A scoring system for pediatric patients undergoing allogeneic HCT called the Oncologic Pediatric Risk of Mortality (OPRISM) may be useful in predicting fatal events, and thus help parents decide to establish a supportive care strategy for their children [70]. Although most physicians and ethicists may agree that futile, inappropriate or unreasonable medical care should not be provided even if requested, how are these terms to be defined, and by whom? In cases in which the patient/surrogate continues to request treatments that the caregivers consider futile, the physician is not ethically required to provide that care, but is obligated based on the principle of nonabandonment to

facilitate the transfer of the patient to another physician who will. Consultation with the institutional ethics committee may be helpful in these most difficult situations.

Closing remarks The indications for HCT have expanded over the years to encompass a wide range of diagnoses and disease stages, including patients with early stages of hematologic malignancies considered to be at high risk for relapse, solid tumors, congenital disorders, and autoimmune diseases. Potential sources of stem cells have also expanded to include autologous or allogeneic cells from bone marrow, peripheral blood or cord blood from related or unrelated HLA-matched donors. It is imperative that the rights of both patients and donors be respected in step with these technologic advances. The ethical principle of justice requires caregivers and insurers to provide potentially life-saving yet high-risk procedures to HCT candidates in an open and equitable manner. Patient autonomy, nonmaleficence, and nonabandonment are guiding principles in making decisions about end-of-life care.

References 1. Arenson EB Jr, Forbe MD. Bone marrow transplantation for acute leukemia and Down syndrome: report of a successful case and results of a national survey. J Pediatr 1989; 114: 69–72. 2. Chang G, Antin JH, Orav EJ, Randall U, McGarigle C, Behr HM. Substance abuse and bone marrow transplant. Am J Drug Alcohol Abuse 1997; 23: 301–8. 3. Foster LW, McLellan LJ, Rybicki LA, Dabney J, Welsh E, Bolwell BJ. Allogeneic BMT and patient eligibility based on psychosocial criteria: a survey of BMT professionals. Bone Marrow Transplant 2006; 37: 223–8. 4. Opel DJ, Diekema DS. The case of A.R.: the ethics of sibling donor bone marrow transplantation revisited. J Clin Ethics 2006; 17: 207–19. 5. Mitchell JM, Meehan KR, Kong J, Schulman K. Access to bone marrow transplantation for leukemia and lymphoma: the role of sociodemographic factors. J Clin Oncol 1997; 15: 2644–51. 6. Vaughan WP, Purtilo RB, Butler CD, Armitage JO. Symposium: Ethical and financial issues in autologous marrow transplantation: a symposium sponsored by the University of Nebraska Medical Center. Ann Intern Med 1986; 105: 134–5. 7. Faber-Langendoen K. Ethical issues in the allocation and reimbursement of bone marrow transplantation. Leukemia 1993; 7: 1117–21. 8. Beatty PG. Bone marrow transplantation for the treatment of hematologic disease: Status in 1994. Exp Hematol 1995; 23: 277–88. 9. Welch HG, Mogielnicki J. Presumed benefit: lessons from the American experience with marrow transplantation for breast cancer. Br Med J 2002; 324: 1088–92. 10. Tamura T, Matsuzaki M, Harada H, Ogawa K, Mohri H, Okubo T. Upregulation of interferon-a receptor expression in hydroxyurea treated leukemia cell lines. J Invest Med 1997; 45: 160–7. 11. Downs S. Ethical issues in bone marrow transplantation. Semin Oncol Nurs 1994; 10: 58–63. 12. Lee SJ, Fairclough D, Antin JH, Weeks JC. Discrepancies between patient and physician estimates for the success of stem cell transplantation. J Am Med Assoc 2001; 285: 1034–8.

13. Andrykowski MA, Brady MJ, Greiner CB et al. ‘Returning to normal’ following bone marrow transplantation: outcomes, expectations and informed consent. Bone Marrow Transplant 1995; 15: 573–81. 14. Massimo L. Ethical problems in bone marrow transplantation in children. Bone Marrow Transplant 1996; 18(Suppl 2): 8–12. 15. Harrison C, Kenny NP, Sidarous M, Rowell M. Bioethics for clinicians. 9. Involving children in medical decisions. Can Med Assoc J 1997; 156: 825–8. 16. Pulsipher MS, Nagler A, Iannone R, Nelson RM. Weighing the risks of G-CSF administration, leukopheresis, and standard marrow harvest: ethical and safety considerations for normal pediatric hematopoietic cell donors. Pediatr Blood Cancer 2006; 46: 422–33. 17. Buckner CD, Petersen FB, Bolonesi BA. Bone marrow donors. In: Forman SJ, Blume KG, Thomas ED, editors. Bone Marrow Transplantation. Boston: Blackwell Scientific Publications; 1994. pp. 259– 69. 18. Anderlini P, Przepiorka D, Lauppe J et al. Collection of peripheral blood stem cells from normal donors 60 years of age or older. Br J Haematol 1997; 97: 485–7. 19. Bishop MR, Tarantolo SR, Jackson JD et al. Allogeneic-blood stem-cell collection following mobilization with low-dose granulocyte colonystimulating factor. J Clin Oncol 1997; 15: 1601–7. 20. Pavletic ZS, Bishop MR, Tarantolo SR et al. Hematopoietic recovery after allogeneic blood stem-cell transplantation compared with bone marrow transplantation in patients with hematologic malignancies. J Clin Oncol 1997; 15: 1608–16. 21. Heldal D, Brinch L, Tjonnfjord G et al. Donation of stem cells from blood or bone marrow: results of a randomised study of safety and complaints. Bone Marrow Transplant 2002; 29: 479–86. 22. Rowley SD, Donaldson G, Lilleby K, Bensinger WI, Appelbaum FR. Experiences of donors enrolled in a randomized study of allogeneic bone marrow or peripheral blood stem cell transplantation. Blood 2001; 97: 2541–8.

23. Stroncek D, Strand R, Scott E et al. Attitudes and physical condition of unrelated bone marrow donors immediately after donation. Transfusion 1989; 29: 317–22. 24. Anonymous. Ethics of organ transplantation from living donors. Transplant Proc 1992; 24: 2236–7. 25. Anonymous. National Marrow Donor Program® Donor & Patient Confidentiality Guidelines. Minneapolis. 2003. pp. 1–4. NMDPnetwork.nmdp. org. 26. Bakken R, van Walraven AM, Egeland T. Donor commitment and patient needs. Bone Marrow Transplant 2003; 33: 225–30. 27. Curran WJ. Beyond the best interests of a child – bone marrow transplantation among half-siblings. N Engl J Med 1991; 324: 1818–19. 28. Burgio GR, Nespoli L, Varrasi G, Buzzi F. Bone marrow transplantation in children: between therapeutic and medico-legal problems. Bone Marrow Transplant 1989; 4(Suppl 4): 34–7. 29. Delany L, Month S, Savulescu J, Browert P, Palmer S. Altruism by proxy: volunteering children for bone marrow donation. Br Med J 1996; 312: 240– 3. 30. Chan K-W, Gajewski JL, Supkis D, Pentz R, Champlin R, Bleyer WA. Use of minors as bone marrow donors: current attitude and management. J Pediatr 1996; 128: 644–8. 31. Peerzada JM, Wendler D. Hematopoietic stem cell transplant research with pediatric donors: when can institutional review boards approve it? Transplantation 2006; 81: 1616–20. 32. Pentz RD. Healthy sibling donation of G-CSF primed stem cells: a call for research. Pediatr Blood Cancer 2006; 46: 407–8. 33. Roberts I. Bone marrow transplantation in children: current results and controversies. Bone Marrow Transplant 1994; 14: 197–9. 34. Gluckman E, Eurocord Network Organisation. Ethical and legal aspects of placental/cord blood banking and transplant. Hematol J 2000; 1: 67– 9. 35. Dame L, Sugarman J. Blood money: ethical and legal implications of treating cord blood as property. J Pediatr Hematol Oncol 2001; 23: 409–10.

Ethical Issues in Hematopoietic Cell Transplantation 36. Alby N. The child conceived to give life. Bone Marrow Transplant 1992; 9(Suppl 1): 95–6. 37. Samuel GN, Kerridge IH, Vowels M, Trickett A, Chapman J, Dobbins T. Ethnicity, equity and public benefit: a critical evaluation of public umbilical cord blood banking in Australia. Bone Marrow Transplant 2007; 40: 729–34. 38. Duarte RF, Pamphilon D, Cornish J et al. Topical issues in unrelated donor haematopoietic stem cell transplants: a report from a workshop convened by the Anthony Nolan Trust in London – 2005. Bone Marrow Transplant 2006; 37: 901–8. 39. Marshall E. Clinical promise, ethical quandary. Science 1996; 271: 586–8. 40. Rubinstein P, Stevens CE, Adamson JW, Migliaccio G. Umbilical cord blood cells: informed consent. Bone Marrow Transplant 1995; 15: 160. 41. Ammann AJ. Placental-blood transplantation. N Engl J Med 1997; 336: 68–70. 42. Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335: 157–66. 43. Section on Hematology/Oncology and Section on Allergy/Immunology. Cord blood banking for potential future transplantation. Pediatrics 2007; 119: 165–70. 44. Schaison GS. The child conceived to give life. The point of view of the hematologist. Bone Marrow Transplant 1992; 9(Suppl 1): 93–4. 45. Verlinsky Y, Rechitsky S, Sharapova T, Morris R, Taranissi M, Kuliev A. Preimplantation HLA testing. J Am Med Assoc 2004; 291: 2079–85. 46. Grewal SS, Kahn JP, MacMillan ML, Ramsay NKC, Wagner JE. Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype-identical sibling selected using preimplantation genetic diagnosis. Blood 2004; 103: 1147–51. 47. Van de Velde H, Georgiou I, De Rycke M et al. Novel universal approach for preimplantation genetic diagnosis of {beta}-thalassaemia in combi-

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nation with HLA matching of embryos. Hum Reprod 2004; 19: 700–8. Boyle RJ, Savulescu J. Ethics of using preimplantation genetic diagnosis to select a stem cell donor for an existing person. Br Med J 2001; 323: 1240– 3. Morgan ER, Girod J, Rinehart JS. Having a child to save a sibling: reassessing risks and benefits of creating stem cell donors. Pediatr Blood Cancer 2007; 48: 249–53. Dyer C. Watchdog approves embryo selection to treat 3 year old child. Br Med J 2002; 324: 503. Pennings G, Schots R, Liebaers I. Ethical considerations on preimplantation genetic diagnosis for HLA typing to match a future child as a donor of haematopoietic stem cells to a sibling. Hum Reprod 2002; 17: 534–8. Devolder K. Preimplantation HLA typing: having children to save our loved ones. J Med Ethics 2005; 31: 582–6. Weissman IL. Stem cells – scientific, medical, and political issues. N Engl J Med 2002; 346: 1576–9. Anonymous. The environments of stem cells – biology, ethics, and policy. Can Med Assoc J 2002; 166: 1005. American Academy of Pediatrics Committee on Pediatric Research, Committee on Bioethics. Human embryo research. Pediatrics 2001; 108: 813–16. McLaren A. Ethical and social considerations of stem cell research. Nature 2001; 414: 129–31. Evers K. European perspectives on therapeutic cloning. N Engl J Med 2002; 346: 1579–82. Bruce DM. Stem cells, embryos and cloning – unravelling the ethics of a knotty debate. J Mol Biol 2002; 319: 917–25. Nowak R. Xenotransplants set to resume. Science 1994; 266: 1148–51. Barker JH, Polcrack L. Respect for persons, informed consent and the assessment of infectious disease risks in xenotransplantation. Med Health Care Philos 2001; 4: 53–70.

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61. Lucarelli G, Andreani M, Angelucci E. The cure of thalassemia by bone marrow transplantation. Blood Rev 2002; 16: 81–5. 62. Giardini C. Ethical issues of bone marrow transplantation for thalassemia [Editorial]. Bone Marrow Transplant 1995; 15: 657–8. 63. Kodish E, Lantos J, Stocking C, Singer PA, Siegler M, Johnson FL. Bone marrow transplantation for sickle cell disease – a study of parents’ decisions. N Engl J Med 1991; 325: 1349–53. 64. Yesilipek MA, Hazar V, Küpesiz A, Kizilörs A, Uguz A, Yegin O. Peripheral blood stem cell transplantation in children with beta-thalassemia. Bone Marrow Transplant 2001; 28: 1037–40. 65. Schleuning M, Stoetzer O, Waterhouse C, Schlemmer M, Ledderose G, Kolb H-J. Hematopoietic stem cell transplantation after reduced-intensity conditioning as treatment of sickle cell disease. Exp Hematol 2002; 30: 7–10. 66. Crawford SW. Decision making in critically ill patients with hematologic malignancy. West J Med 1991; 155: 488–93. 67. Kern D, Kettner P, Albrizio M. An exploration of the variables involved when instituting a do-notresuscitate order for patients undergoing bone marrow transplantation. Oncol Nurs Forum 1992; 19: 635–40. 68. Rubenfeld GD, Crawford SW. Withdrawing life support from mechanically ventilated recipients of bone marrow transplants: a case for evidence-based guidelines. Ann Intern Med 1996; 125: 625–33. 69. Bach PB, Schrag D, Nierman DM et al. Identification of poor prognostic features among patients requiring mechanical ventilation after hematopoietic stem cell transplantation. Blood 2002; 98: 3234–40. 70. Schneider DT, Cho J, Laws HJ, Dilloo D, Göbel U, Nürnberger W. Serial evaluation of the oncological pediatric risk of mortality (O-PRISM) score following allogeneic bone marrow transplantation in children. Bone Marrow Transplant 2002; 29: 383– 9.

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Richard P. McQuellon & Michael Andrykowski

Psychosocial Issues in Hematopoietic Cell Transplantation

Introduction Hematopoietic cell transplantation (HCT) is an aggressive, dynamic, and evolving medical procedure. It is used in the treatment of a variety of life-threatening diseases, often as a high-risk, best choice for patients with few treatment alternatives. Consequently, HCT is associated with the potential for a wide variety of serious physical, psychological, and social complications. In this chapter, we discuss the origin, recognition, and management of the psychological and social complications associated with HCT. We use the term “psychosocial” to refer to issues that are psychological and/or social in nature. Similarly, we use the term “HCT” to refer to a class of medical procedures that involve the transplantation of hematopoietic stem cells (HSCs) derived from peripheral blood, cord blood or bone marrow. For the most part, the literature examining psychosocial issues in HCT does not distinguish among stem cells of different origin. A set of generic psychosocial issues is germane regardless of the origin of stem cells or the underlying disease. We direct the major part of our discussion to psychosocial issues pertinent to adult HCT recipients. We also provide some discussion of psychosocial issues pertinent to donors and caregivers. The recognition and study of psychosocial issues associated with HCT has a relatively long history [1–3]. As HCT has gradually evolved as a medical procedure, some of the psychosocial issues associated with it have evolved as well. There have been several recent trends in HCT that can affect how recipients and their families experience the transplant, and consequently what type of psychosocial issues emerge. First, peripheral blood rather than bone marrow is now the main source of stem cell support. This eliminates the need for the bone marrow harvest and its concomitant general anesthesia requirements. Stem cells can now be harvested in a series of one to three sessions in an apheresis unit lasting approximately 1–3 hours. Before stem cell collection, stem cell donors are likely to be primed with growth factors in order to increase the concentration of HSCs in the peripheral blood. Second, older individuals are now being transplanted with greater frequency. This increases the likelihood of physical morbidity for recipients. It also increases the possibility that older caregivers may be called upon to provide supportive care to their loved one at a time when they may need considerable health care themselves. Third, the use of reducedintensity conditioning regimens is becoming more common. These Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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reduced-intensity regimens produce less physical morbidity and thus may often be done on an outpatient basis, resulting in less isolation of the patient and less exposure to the hospital environment. Fourth, with the advent of new testing methods, better matching in the allogeneic transplant setting is possible. This has the potential to reduce the incidence of graft-versus-host disease (GVHD) and the distress and psychosocial problems that accompany this serious and often life-threatening complication. Finally, much more information on potential clinical outcomes associated with HCT is available to potential HCT recipients and their families. For example, a prospective HCT recipient with lymphoma can now go to the website for the Center for International Blood and Marrow Transplant Research (CIBMTR; http://www.cibmtr.org/) and view Kaplan–Meier survival curves relevant to their specific disease status. There they would see the following: “Among 2,292 patients receiving autotransplants for follicular lymphoma between 1996 and 2004, 3-year probabilities of survival were 73% ± 1.5% and 52% ± 7% for chemosensitive and chemo-resistant disease, respectively.” This greater access to information regarding clinical outcomes may be either frightening or comforting to potential recipients and their families depending on their understanding of their disease and overall outlook. Another recent trend in HCT is the increased emphasis on post-transplant survivorship monitoring [4]. Greater emphasis is now placed on periodic and systematic monitoring of a range of clinical outcomes in recipients. This is likely to identify psychosocial problems that may be present and consequently improve the chances they will be addressed. Several important studies of recovery following HCT conducted with relatively large cohorts of recipients and caregivers have identified key potential problem areas and pointed to potential areas for psychosocial intervention [5,6]. Also, an important survey of 600 HCT survivors was conducted in 2006 by the Bone Marrow Transplant Information Network (http://BMTinfonet.org). When asked to identify the most significant issues facing them following transplantation, 73% of respondents endorsed emotional/psychological health as a problem. The results of the survey reflect a reality for a significant number of recipients: HCT is highly stressful, and psychosocial issues remain a primary concern for the majority of recipients for a long period of time after transplantation.

Psychosocial issues in HCT: recipients Two major sources of distress can be identified for HCT recipients during the course of treatment and recovery. First, HCT is often used after other conventional treatment options have failed in diseases that

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are virtually always life-threatening. Furthermore, HCT itself is associated with significant risk of mortality: treatment may actually hasten a patient’s death relative to what might have been expected had conventional treatment, or no treatment, been employed. The context of HCT is one of uncertain and yet ever present life-threat that serves as a significant source of stress for recipients, family caregivers, donors, and medical staff. In its simplest form, the potential HCT recipient is faced with this decision: high-risk treatment with a chance of cure or some alternative course of action with no chance of cure. A second source of distress in the HCT setting is the aggressiveness of the procedure. High-dose or reduced-intensity chemotherapy regimens with or without localized and/or total body irradiation produce acute side-effects (e.g. nausea, mucositis, hair loss, and fatigue) and long-term effects (e.g. chronic GVHD, secondary malignancies, etc.). In the best of circumstances, most HCT recipients typically experience an array of taxing physical stressors within the context of the psychological and social stress associated with medical uncertainty and life-threatening circumstances. Even when the transplant is a success and recipients resume their life outside of a closely monitored medical environment, the need for ongoing surveillance for recurrent disease is a periodic reminder of an ever-present awareness: they have entered a high-stakes medical scene and will remain there for the rest of their lives. Stages of HCT Over the course of the HCT process, recipients are confronted with an often highly predictable sequence of treatment-related events. The word “stage” refers to a variable time period that is characterized by specific tasks and physical and psychosocial stressors that recipients and their caregivers must negotiate. In the broader cancer survivorship literature, the time period from diagnosis through the long term has been described as the “seasons of survival” [7]. Descriptions of the number and nature of stages of HCT have varied [2,8]. We will organize our discussion of psychosocial issues confronting the HCT recipient around five relatively distinct stages: (1) the decision to undergo HCT; (2) pre-HCT preparation; (3) post-HCT hospitalization; (4) hospital discharge and early recovery; and (5) long-term recovery (Table 33.1). These stages represent a general map of the process and not an invariant sequence of events and stressors facing all recipients. Some psychosocial issues that appear during one stage of HCT may surface again at a later time. For example, the prospect of a shortened life span due to a life-threatening illness suggests patients may struggle with an intense awareness of their mortality at the time of their decision to undergo HCT [9]. Such concerns may fade into the background at the time of hospital discharge and early recovery, only to re-emerge as significant concerns during long-term recovery. Many of the physical stressors can be directly related to anxiety, depressive symptoms, and distress that may appear periodically during each stage of treatment and recovery. Stage 1: the decision to undergo HCT The decision process leading to HCT is usually highly stressful. Few individuals realize that the very fact they are contemplating HCT places them in a category where a natural life span may be less than likely. At best, recipients can expect a host of toxic short- and long-term sideeffects that typically exceed those associated with conventional therapeutic options. At worst, the recipient may die during the course of HCT, experiencing an earlier death relative to what might have occurred had other conventional or palliative therapeutic options been selected. Despite the evolution of the medical context of HCT, there has been little interest in how these recent changes may affect the decision matrix or the decision process more generally. Most of the extant research on

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HCT-related decision making has focused upon recipients’ provision of informed consent. This is a rather limited perspective on HCT-related decision making, however. The formal consent process is only the final step in an often protracted decision-making process. Earlier steps in the decision sequence include the decision to consider HCT as a treatment option and to undergo formal medical evaluation for HCT (i.e. medical referral for HCT). Medical referral for treatment. The decision to undergo HCT begins with its consideration as a treatment option. In most cases, the possibility of HCT is raised initially by the medical oncologist managing the patient’s care. In other instances, patients may raise the prospect of HCT with their physicians on their own. Regardless of the source of referral, up-to-date knowledge regarding HCT is critical at this juncture. This is particularly true when HCT is viewed as the only viable hope for disease cure. In these instances, patients are likely to quickly embrace HCT as their only alternative, disregarding the alternative of no further curative treatment. In effect, a psychological commitment to HCT may be made at the time this option is first raised and considered [10], perhaps before the patient possesses any substantial knowledge of the risks and benefits associated with HCT. This commitment may be immutable by the time the patient arrives at the transplant center for initial evaluation. The referring physician therefore plays a critical role in ensuring that the decision to undergo HCT is truly an “informed” decision. Selection and screening of candidates. The period of time leading up to HCT poses many psychosocial stressors. We have identified the pattern of some of these stressors in Table 33.1. The isolation procedures and physical hazards (e.g. infections and severe toxicity) associated with HCT have been assumed to have the potential to create great distress in prospective patients. Consequently, the American Society of Blood and Marrow Transplantation included a psychiatric/psychosocial assessment as part of the guidelines for treatment established in 1995 [11]. The intent of this assessment is to obtain information about a number of areas of functioning, including current psychological distress, past or present psychiatric history, current social support, coping style, history of medical noncompliance, and past or current problems with alcohol or substance abuse. While psychosocial considerations, such as a history of alcohol abuse or medical noncompliance, may be used to screen out candidates for solid organ transplantation [12], rejection of an HCT candidate strictly on the basis of psychosocial considerations is rare. Psychosocial information obtained during the pre-HCT evaluation is most often used to anticipate problems in caring for the patient and to develop an individualized plan of care over the course of HCT. For example, to facilitate care planning, Molassiotis developed a brief scale to prospectively identify patients prone to emotional difficulties during post-HCT hospitalization [13]. Standardized approaches to the pre-HCT psychosocial evaluation have been described, including the Psychosocial Levels System [14], the Transplant Evaluation Rating Scale (TERS) [15], and the Psychosocial Assessment of Candidates for Transplantation Scale (PACT) [16]. All three approaches focus, for the most part, upon the content areas indicated above. The PACT and the TERS yield comparable information [17], while the TERS appears to possess somewhat better psychometric properties than its precursor, the Psychosocial Levels System [18]. There is a need for more research in this area of screening and selection of HCT candidates. For the most part, the clinical utility of standardized approaches to pre-HCT psychosocial evaluation for selecting appropriate candidates for HCT remains to be empirically established. While it might be anticipated that inadequate social support, a history of treatment nonadherence or substance abuse problems would be associated with poorer post-HCT outcomes, this linkage has not been firmly

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Table 33.1 Themes, tasks and psychosocial issues associated with five stages of hematopoietic cell transplantation (HCT) Stage

Issue

*1. The decision to undergo HCT

Theme: active decision making • Confronting mortality and the possibility of death • Managing the uncertainty of treatment outcome • Considering alternative treatments • Financial considerations/insurance limitations • Psychosocial evaluation • Informed consent process • Symptoms of anxiety, depression, and distress

2. Pre-HCT preparation

Theme: aggressive treatment • Managing acute treatment side-effects • Adapting to isolation and hospital routine • Adopting the patient role • Confronting unfamiliar procedures and treatment (e.g. total body irradiation) • Separation from family and friends • Adjusting to altered body image (e.g. hair loss, weight loss, and Hickman catheter placement)

3. Post-HCT hospitalization

Theme: watchful waiting • Waiting for engraftment • Heightened physical and emotional vulnerability • Contending with the boredom of isolation • Maintaining morale and hope • Dealing with life-threatening complications • Encountering acute psychologic distress • Managing discouragement

4. Hospital discharge and early recovery

Theme: transition from intense medical surveillance • Managing the loss of daily psychosocial support of the medical team and allied health-care professionals (e.g. pastoral care) • Contending with the stress of frequent medical appointments, readmissions, and setbacks • Reintegration into valued social roles (e.g. parent, spouse or social companion) • Managing unexpected sequelae (e.g. profound fatigue) • Adapting to potential frustration, depressive symptoms, and anger • Complying with self-care guidelines and daily medicine regimen

5. Long-term recovery

Theme: establishing the new normal life • Re-establishing primary identity and relinquishing the patient role • Recovery of valued roles in the community, at work, and at home • Adjust to losses associated with transplantation, e.g. fertility • Return to employment • Accepting the possibility and reality of long-term effects (e.g. cataracts or second malignancies)

* Many of these issues continue over the course of the five stages. For example, confronting mortality and the prospect of death may be revisited more or less intensely each time the patient returns to the transplant center for ongoing evaluation and follow-up over the course of many years. Also, anxiety, depressive symptoms, and distress may manifest themselves over the course of the entire HCT process. We place these symptoms in stage 1, but they might just as easily emerge in any stage.

established. However, several studies suggest an association between psychosocial status at the time of HCT and important post-HCT outcomes such as survival [19–22] and risk for medical complications [21,23]. While there has been no solid link between pre-HCT psychosocial variables and post-HCT survival [24–28], this remains a provocative area of research. Hoodin and Weber conducted a systematic review of the literature and identified 15 methodologically sound studies that highlight psychosocial factors affecting survival after HCT [29]. Based on their review, they cautiously suggest that survival after HCT is not affected substan-

tially by depressed mood (in contrast to Loberiza et al., noted above), psychopathology or social support. However, longer survival may be related to less anxious preoccupation, higher fighting spirit, and better quality of life (QOL) ratings before transplantation. The authors concluded that this is an important area of inquiry, and that the literature is not sufficiently developed to provide strong evidence for a relationship between psychological variables and survival following HCT. Whatever impact psychosocial factors have on survival, they may be weak relative to patient characteristics and clinical factors such as histologic subtypes [28].

Psychosocial Issues in Hematopoietic Cell Transplantation

Provision of informed consent. Informed consent occurs in a face-toface meeting between the transplant physician and the recipient and caregivers either before and/or at the time of pre-HCT hospitalization. At this meeting, the multipage consent forms are gone over in more or less detail, depending on the preference of the recipient. Some recipients want to go over every word; others prefer to gloss over the document. In any case, the end product is a witnessed, legal document signed by the patient and physician. In theory, the consent process allows patients to make an informed and voluntary decision regarding whether to proceed with HCT. In practice, however, this ideal is unlikely to be realized [1,10]. There are several reasons for this, including the nature and amount of information required to make an informed decision, the stressful context within which an informed decision is made, and the reluctance of potential recipients to actively consider the risks associated with HCT. It is not uncommon for potential HCT recipients to come into the initial consultation with limited knowledge of their disease and their treatment options. More importantly, they may have little understanding of the morbidity and mortality associated with transplantation as well as survival rates without a transplant. Stiff et al. studied recipients’ understanding of their disease and treatment plan at the time of the initial HCT consultation in 99 HCT candidates [30]. Participants were assessed both before and after a 3-hour multidisciplinary team consultation that was held before transplantation. Prior to the consultation, about three-fourths of these potential recipients reported adequate information about their disease, yet a large percentage of patients lacked knowledge about their 1-year prognosis with or without any therapy. After the consultation, two-thirds of the patients reported they had obtained enough information to make an informed decision about whether or not to undergo HCT. This was compared with only 23.2% prior to this consultation. While the consultation clearly increased the number of individuals who believed they knew enough to make an informed treatment decision, the fact that one-third of respondents did not believe they had enough information to make an informed decision indicates that a substantial number of HCT recipients may not be making informed decisions to undergo this treatment. Significant elevations in psychological distress have been observed in adult HCT recipients within the 48 hours after provision of informed consent [31]. Most consent forms contain exhaustive descriptions and listings of the medical risks associated with HCT, sufficient to frighten even the most psychologically stable adult or parent. Furthermore, research has documented that many recipients are considerably distressed in the days prior to HCT [32–37]. Distress can compromise the consent process by inhibiting communication with health-care providers, limiting rational consideration of the risks and benefits of HCT, and inhibiting comprehension or memory for information communicated during the important consent discussions. Indeed, research suggests that distressed individuals may be more likely to agree to undergo experimental therapies [38]. HCT is being used increasingly as a “first-line” therapy for some medical conditions, making the treatment decision even more complex and distressing for all concerned. While HCT may increase the likelihood of cure or prolonged remission relative to other therapeutic options, these other options (e.g. conventional oral or intravenous chemotherapy) may be associated with less morbidity and virtually no short-term mortality risk. An example of this dilemma is seen in the treatment of chronic myeloid leukemia (CML). Conventional therapy for CML, while fairly benign, is not curative, and without further treatment the disease will likely ultimately accelerate. Patients with a suitable donor may be advised to consider undergoing HCT before their disease worsens. Given the increased morbidity and mortality associated with HCT, an optimal strategy might be to postpone HCT as long as possible.

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However, if CML enters an accelerated phase, risks for HCT-related morbidity and mortality increase relative to what the risks would have been if HCT had been implemented during the chronic, stable phase of the disease. This decision is now even more complicated with the recent success of the oral chemotherapeutic agent imatinib mesylate (Gleevec) for the treatment of CML. This agent has produced prolonged remissions in many CML patients and may even produce cure. The decision of whether and when to undergo HCT fundamentally involves a trade-off between the increased toxicity associated with HCT and the increased potential for disease cure, or at least long-term, disease-free survival. QOL considerations must be balanced against quantity of life considerations. There is evidence that patients are willing to risk substantial toxicity and increased risk of mortality for increased survival even if the outcome of treatment is uncertain [39,40]. Based on the premise that the HCT decision is based upon patients’ knowledge of HCT and their preferences for certain mortality and morbidity (e.g. QOL) outcomes, Sebban et al. developed a bedside decision board to assist CML patients and their physicians in deciding between HCT and other more conservative management options for CML [41]. They tested the decision board with 42 healthy hospital personnel and not HCT recipients. Satisfaction with treatment preference was higher for those exposed to the decision board compared with those presented with an abbreviated version of the decision board. Such decision tools could be a welcome addition to the pre-HCT decision context but require testing and evaluation with actual prospective HCT recipients before they can be advocated for routine use. Stage 2: pre-HCT preparation The second stage of HCT begins with the start of the preparative regimen, consisting of either high-dose or reduced-intensity chemotherapy, often in combination with total body irradiation. Typically, the recipient is hospitalized during this period, although it has become more common for patients to receive their pre-HCT preparative regimen on an outpatient basis. While side-effects vary as a function of the type and intensity of the preparative regimen, they are often severe, even with aggressive supportive care, and may continue to be experienced after preparative therapy has been completed. Critical psychosocial issues during the pre-HCT preparation phase center on: (1) adaptation to hospitalization, including becoming accustomed to the hospital routine and being confined to a single hospital room and the immediate surroundings; (2) learning proper infection control procedures (I would suggest not being too specific as these vary from center to center); and (3) maintenance of the recipient’s psychological strength and coping capacity. Simple hospital routines such as frequent vital sign monitoring in the middle of the night can be annoying. Pain and nausea are typically controlled through liberal use of antiemetic and analgesic medications, all of which can alter the recipient’s psychological status. Cognitive-behavioral therapies can also be used as adjunctive therapy for symptom control, especially with patients who are averse to antiemetic and analgesic medications. In a pair of well-designed studies, Syrjala et al. examined the utility of several cognitive-behavioral interventions (e.g. hypnosis, relaxation and imagery, and cognitive-behavioral coping skills training) for controlling pain and nausea associated with pre-HCT preparative therapy [42,43]. While hypnosis and coping skills training appeared to have little impact upon nausea and vomiting [42], all three interventions reduced pain associated with mucositis. Results suggest that hypnosis can significantly add to the effects obtained with standard analgesic medications. Widespread incorporation of cognitive-behavioral therapies into the management of HCT recipients may be difficult since implementation of these therapies is typically time-consuming and costly. However, more abbreviated cognitive-behavioral interventions could be developed

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and could be effective. For example, some evidence suggests a singlesession psychoeducational intervention designed to enhance coping with treatment side-effects can be effective in reducing nausea, fatigue, and anxiety in autologous HCT recipients [44]. As administration of the pre-HCT preparative regimen occurs either in the hospital or in an outpatient clinic setting close to the transplant center, this period of time can be used to plan for the management of recipients’ psychosocial needs as they will likely emerge during postHCT recovery [45]. If not completed already, a detailed psychosocial evaluation can be performed at this time. Identification of potential risk factors for psychosocial difficulties post HCT is key to their prevention and management. A proactive approach can be helpful as it establishes a relationship between the HCT recipient and a mental health-care professional (e.g. social worker, counselor, psychologist or psychiatrist) who might later help manage any emerging psychosocial difficulties. Elements of this proactive approach include identification of coping resources and deficits, discussion of psychosocial difficulties that might be anticipated, and training in behavioral skills to improve symptom management and facilitate coping. Some research has focused upon distress experienced during this preHCT phase of the transplant process. Trask and colleagues assessed psychological distress in 50 HCT recipients (both allogeneic and autologous) prior to transplantation [36]. Fifty percent and 51% of recipients reported clinically significant levels of emotional stress and anxiety, respectively. Twenty percent reported clinically significant levels of depression. Andorsky et al. assessed pre-HCT physical and mental functioning using the Medical Outcomes Scale Short Form 36 (SF-36) in 320 HCT recipients (allogeneic and autologous) [46]. Results indicated that mean mental functioning scores prior to HCT were no different from those of the general population. Of course, mean group scores can obscure the fact that a significant proportion of respondents might be experiencing significant distress. Nevertheless, this recent study suggests many current HCT recipients, perhaps a majority, may approach their transplant with reasonably good mental functioning. Stage 3: post-HCT hospitalization Upon completion of cytotoxic preparative therapy, infusion of hematopoietic cells takes place. Most recipients experience this brief infusion of stem cells as anticlimactic. However, completion of this infusion of stem cells highlights a phase of the HCT process characterized by profound physical vulnerability. During the weeks after stem cell infusion, recipients are monitored daily for evidence of stem cell engraftment and immune system reconstitution. Many recipients request printed copies of their daily laboratory results and diligently focus on key milestones indicating engraftment. Indeed, the mood shifts of recipients and family members often fluctuate daily depending heavily on the up or down movement of their “counts.” Once the side-effects of preparative therapy have resolved, and if no serious infectious complications have arisen, the recipient enters a period of hopeful waiting. Weakness and fatigue are common at this time, although the recipient may actually feel relatively good. Should serious medical complications develop, however, this quiescent state quickly ends. Given the life-threatening nature of many medical complications during this early post-HCT period, recipients often react to medical setbacks with considerable distress. A lengthy hospitalization, often characterized by a series of vexing medical complications, can be profoundly demoralizing, especially if a tentative discharge date had been set, only later to be postponed. Some recipients respond to a prolonged hospitalization with lethargy and decreased motivation, others with anger and agitation. In extremely rare cases, a recipient may attempt to leave the hospital against medical advice. At these times, the psychosocial team can play a critical, if not lifesaving, role by helping the

recipient to better cope with the stresses, disappointments, and lack of control characteristic of this post-HCT stage of the transplant process. The primary psychosocial issues during this early period of post-HCT recovery center on the promotion of continued effective coping with the physical and psychological stresses associated with HCT. In addition to anxiety and depressive reactions [47], recipients may experience neurocognitive symptoms or neurologic complications [48–50]. Episodes of delirium may occur and may last several hours to several days. Symptoms are diverse and can include alterations in level of consciousness, altered sensory or perceptual function (e.g. hallucinations), impairments in memory, concentration, and higher cognitive processes, mood swings, impaired judgment, and sleep disturbance [51]. HCT recipients differ in the amount of time they are required to spend in a dedicated hospital “transplant unit” before and after HCT. The point along this inpatient–outpatient continuum that characterizes a recipient’s experience is critical to understanding the particular psychosocial difficulties they may encounter. The more HCT is experienced as a lengthy inpatient procedure, the more likely recipients are to evidence psychosocial difficulties associated with prolonged confinement in an institutional setting (e.g. boredom, loss of control, loss of identity, social and physical isolation, interrupted sleep, etc.). These are all mitigated when HCT is an outpatient procedure. Other difficulties may be substituted, however. For example, while hospitalization and strict infection control procedures can be stressful, the immediate availability of supportive care from the medical team can be enormously reassuring to the recipient. Assistance with an emergency medical crisis is immediately available for recipients while they are hospitalized; such assistance may not be as readily available in the outpatient setting. This small difference can have a profoundly disconcerting impact on some recipients. Some recipients might even prefer the psychological security afforded by hospitalization on a specialized unit in lieu of the freedoms associated with a more outpatient-based HCT procedure. The stressors associated with HCT can bring out both the best and the worst in patients and their families. The so-called “difficult” patient or family member (someone who is angry, demanding, and often uncooperative with the HCT team) can pose many challenges. Certain principles underlie the successful management of such individuals, including frequent communication among medical team members, the formulation of a reasonable treatment care plan, and the consistent implementation of this treatment plan by all members of the team. Multidisciplinary team meetings can be very helpful in planning and ensuring a consistent course of treatment for challenging patients. Ongoing, effective communication with the difficult patient or family member is critical, along with continued attempts to identify, convey understanding of, and address the fears and anxieties likely underlying their difficult behavior. Despite the presence of multiple and significant stressors during the post-HCT hospitalization and the difficulty in conducting psychosocial research during this stage, there is some relevant research on this time period. Prieto and colleagues studied both the physical and psychological status of 220 inpatients receiving HCT [52]. Patients were assessed at hospital admission, on the day of their transplant, and 7 and 14 days after HCT. Anxiety was highest at hospital admission and decreased thereafter. The proportion of recipients who qualified as a “case” of anxiety as measured by the Hospital Anxiety and Depression Scale declined from 22.7% at the initial assessment to 8% 14 days after HCT. In contrast, the percentage of recipients qualifying as a “case” of depression increased from a low of 11.4% at the initial assessment to 21% 7 days after transplant. The authors suggest their figures might underestimate the prevalence of anxiety and depression symptoms because of the use of psychopharmacologic treatments during hospitalization. Zittoun et al. assessed QOL as well as depression and anxiety symptoms during

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hospitalization in 179 HCT recipients [53]. Recipients were assessed 10, 20, and 30 days after completion of their pre-transplant preparative regimen. At each assessment, approximately one-fourth to one-third of respondents reported clinically significant levels of depression and anxiety. The results of these studies suggest that significant psychological distress during the post-HCT hospitalization period is fairly common. Consequently, periodic monitoring of distress during hospitalization is recommended with short-term counseling and/or psychopharmacologic intervention utilized if needed. However, there are significant challenges to accurate assessment of mood and psychosocial status during this stage of the HCT process. In particular, accurate recognition of depression at this time can be problematic. Recipients’ eating and sleep–wake cycles are almost always disrupted to some degree, and fatigue and lack of energy are common side-effects of intensive HCT preparative regimens. A lack of energy may also be the result of anemia, sleep disturbance, and/or nutritional deficits. Persistent fear and worry can also be a significant energy drain. Lethargic recipients might be mislabeled as depressed, or, even worse, truly depressed recipients might be mislabeled as simply being fatigued due to the variety of lethargy-inducing factors potentially present in the HCT setting. Health-care providers must be open to considering the variety of underlying explanations for the significantly depressed or fatigued HCT recipient, carefully weigh the evidence supporting different competing explanations, and ultimately prescribe the appropriate course of treatment. Stage 4: hospital discharge and early recovery When engraftment has occurred, blood counts are near normal, and recipients are medically stable, they are ready to leave the HCT unit. HCT recipients may also be moved to a less restrictive setting such as a “step-down” unit in the hospital or lodgings close to the HCT center for a period of time after discharge from the hospital transplant unit. This intermediate move allows for continued careful monitoring of those recipients whose homes are distant from the transplant center. Frequent medical follow-up on an outpatient basis is the norm during the first several weeks and months post HCT. The period after treatment ends brings new challenges [54]. Hospital discharge and return home mark the beginning of an often long process of convalescence, recovery, and life reintegration. Critical psychosocial issues associated with the early recovery period include coping with any anxiety generated by the recipient’s gradual weaning from the intensive medical care available in the transplant unit, coping with the often profound physical debilitation experienced by recipients at this time, and adjusting to the need to vigilantly practice a regimen of self-care behaviors designed to minimize the risk of infection. While hospital discharge and a return home is an eagerly anticipated milestone, it can be highly distressing. Many recipients report anxiety at the realization they will no longer be under the constant surveillance and care of medical staff. Fears that life-threatening difficulties might emerge when appropriate medical assistance is not easily available can temper any excitement surrounding a return home or a move to temporary lodging outside the hospital. Frequent clinic follow-up is needed during the early recovery stage. Such follow-up necessitates either remaining close to the transplant center or regularly commuting many hours from the recipient’s home, which may be quite geographically distant from the center. Coping with physical debilitation is a significant challenge for nearly all patients in the early recovery stage. It is not uncommon for recipients to report generalized weakness and fatigue during the 6–12 months after HCT [37,55]. These symptoms can drastically interfere with resumption of routine activities characteristic of the recipient’s premorbid lifestyle. How recipients react to these symptoms and their functional limitations

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can be influenced by expectations for post-HCT recovery. While most recipients experience some physical limitations, the anticipated timeline for post-HCT recovery varies considerably [56]. Recipients and family members are often overly optimistic in their expectations. Many hope for a quick return to a healthy, fully functioning, “normal” life. Frustration, depression, and anger can result when these expectations are not met [10]. Most HCT recipients experience significant physical deconditioning as a result of their lengthy hospitalization, yet they are rarely enrolled in any kind of systematic program of physical rehabilitation. This is likely due to many factors including geographic distance between the transplant center and the recipient’s home. The few studies that examine the benefits of exercise programs for HCT recipients show great promise [57,58]. Routine screening following HCT for psychosocial distress is uncommon in spite of the fact that the procedure is physically and emotionally demanding. One study evaluated 80 autologous (44%) and allogeneic (56%) transplant patients prior to admission, at the first clinic visit after hospital discharge, and at 100 days post transplant [59]. A total of 44% of patients had symptoms of either depression, anxiety or post-traumatic stress disorder after transplantation. The measurement of pretransplant distress was associated with detection of distress after transplantation, suggesting that pretransplant assessment is predictive of post-transplant functioning. An important finding of the study was that there was an association between self-reported distress and noncompliance with medication following transplantation. This suggests the obvious to the clinician: distressed patients may be less likely to comply with the rigorous regimen of medications following transplantation. Promotion of appropriate self-care behavior is also a significant issue during the early recovery stage. Good self-care behavior is intended to reduce the risk of infection and includes both behavioral restrictions (e.g. avoid crowds) and the prescription of specific activities (e.g. engage in some form of physical exercise). Patients may be required to take up to 30 pills each day at varying schedules, an assignment which is taxing for even the most diligent. Failure to adhere to this self-care regimen can result in very serious medical complications and even death. Despite the importance of appropriate self-care after HCT, little research has examined this issue. Our own experience suggests adherence to self-care guidelines can be a significant problem after HCT in both adult and pediatric settings. We have known of one male HCT recipient who acquired a mortal wound infection after shaving with a forbidden straight razor and one patient who could not stop smoking and contracted a fatal fungal infection. While these are notable examples of nonadherence, less flagrant violations of self-care guidelines are common and can result in equally disastrous consequences. In this regard, there is some indication in the literature that screening might identify those at risk for nonadherence of the strict medication regimens [59].

Stage 5: long-term recovery Successful recovery after HCT is characterized by waning of the physical side-effects of treatment, decreased symptoms of disease, a return to premorbid levels of psychosocial and physical functioning, and a resumption of valued life roles. This process usually begins 3–6 months after HCT and can continue for up to 2 years or more [56]. Syrjala and colleagues have illustrated the recovery trends in patients with treatment-related distress and depression versus no depression, respectively, in a sample of allogeneic and autologous transplant recipients (Figs 33.1 and 33.2). It is critical to recognize, however, the wide variability in the “the trajectory of recovery” following HCT [37]. Unfortunately, some recipients never fully recover, either physically and/or psychosocially, while for others recovery requires a lengthy journey, a journey which may not end in the same place it began.

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Fig. 33.1 Mean scores for treatment related distress comparing autologous versus allogeneic transplant recipients over time. HCT, hematopoietic cell transplantation.

Fig. 33.2 Respondents with physical limitations over time, according to the presence or absence of depressive symptoms at baseline.

Physical and psychosocial late effects of HCT. One of the benchmarks often used by HCT recipients to gauge the progress of their long-term recovery is the extent to which they perceive they have “returned to normal” [60]. Clinical experience and empirical research both suggest that most disease-free HCT recipients will experience the sense that they have “returned to normal” within the first several years following transplant [10,37], although the recovery process may take as long as 3–5 years [56]. What is “normal,” of course, is highly dependent upon each recipient’s unique perspective. While acknowledging that they

have “returned to normal,” it is not uncommon for even disease-free HCT recipients to say that their life will never be the same. Even with the return of what they might consider a normal lifestyle, few recipients are left unchanged by their HCT. Recipients may evidence a variety of late physical effects, such as pulmonary problems, cataracts, sterility, endocrine dysfunction, chronic GVHD, and weakness/fatigue (with possible sexual dysfunction). Even in the absence of such late physical effects, most recipients experience some interruption in normal developmental tasks, for example attending school and developing peer group relationships in the case of pediatric recipients, or maintaining a career and establishing satisfactory marital and family relationships in the case of adult recipients. Recipients may also evidence a variety of psychosocial late effects such as anxiety, depression, strained interpersonal relationships or even post-traumatic stress disorder [61]. These psychosocial late effects may persist for months or years after HCT or they may first emerge several years after HCT. In all instances, the presence of significant psychosocial late effects exact a significant toll on a recipient’s QOL [32]. GVHD in its acute or chronic form can precipitate many psychosocial stressors for patients [62]. These can include additional medical appointments and accompanying stressors for caregivers, as well as symptoms of depression and anxiety. The extent to which GVHD is a psychosocial stressor seems intuitively obvious, yet its impact on mental health has yet to be sufficiently documented. There are some conflicting data in the literature regarding to what extent either acute or chronic GVHD affects mental health. In one study, the patients’ experience of acute or chronic GVHD in the 12 months following transplantation was not reflected in worse scores on the Mental Component Subscale or Physical Component Subscale of the SF-12 [62]. Clinical observation suggests that both acute and chronic GVHD could precipitate intense anxiety and/or depressive symptoms, and/or discouragement in patients seeking to return to normal following HCT. Indeed, we have counseled patients contending with bouts of chronic GVHD several years post transplantation whose functional activities and work life have been severely impaired by this complication. Hjermstad and colleagues studied health-related QOL, fatigue, anxiety, and depression in HCT recipients 3–5 years after transplantation [63]. A total of 248 patients (n = 61 allogeneic HCT, n = 69 autologous HCT, n = 118 conventional chemotherapy) were included in the study. Importantly, both the transplant and conventional chemotherapy patients reported more fatigue than population norm values after 3 years. No differences were found between the groups in terms of anxiety and depression. When comparing across groups, anxiety and depression were less of a problem than fatigue. The authors concluded with a strong recommendation for regular follow-up care for patients even after 3 years post transplant, with an emphasis on assessment of functional status and fatigue. Despite the presence of physical and psychosocial late effects, the majority of long-term HCT survivors report relatively satisfactory global QOL. For example, Broers et al. described how 90% of their sample of HCT recipients reported good-to-excellent QOL 3 years post-HCT [64], despite the fact that about 25% continued to experience significant functional limitations even 3 years after transplantation. Thus, recipients often acknowledge having quite good QOL while at the same time experiencing the continued presence of significant physical or psychosocial difficulties. This apparent paradox can be at least partially explained by some evidence that suggests patients with a chronic disease may alter their perspective on QOL, establishing a lower ceiling of acceptability and redefining what constitutes “good” QOL. This apparent paradox might also be explained by consideration of the potential of the transplant experience to exert a positive impact upon recipients’ lives. It is very important to note that the long-term impact

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of HCT is not uniformly negative. Recipients can and often do experience positive psychosocial sequelae of their transplant experience [65]. These positive psychosocial “late effects” are often subsumed under the label of “post-traumatic growth” and can include improved self-esteem, perception of a new meaning in life, redirected and re-established life priorities, enhanced capacity for compassion, improved family and social relationships, and renewed spiritual faith. Interestingly, HCT survivors’ reports of such “benefits” of the transplant experience often coexist with reports of significant physical and psychosocial difficulties. Andrykowski et al. compared HCT survivors with matched healthy controls on a variety of indices of physical and psychosocial functioning [65]. Relative to healthy controls, HCT survivors reported poorer physical, psychological, and social functioning. Conversely, however, HCT survivors also reported significantly more psychological and interpersonal growth, with the size of these statistically significant group differences also suggesting clinical importance. Hence, long-term post-HCT recovery can involve more than simply adjustment to the potential negative physical and psychosocial late effects of HCT and the quest to re-establish the premorbid status quo. Rather, long-term recovery can involve attempts to derive meaning from the HCT experience and identify benefits that may have paradoxically come from it. In the end, successful long-term recovery may involve the establishment of a fundamentally altered life equilibrium, a new normal if you will, one that incorporates both the negative as well as the positive impacts of the HCT experience. Management of late effects of HCT. Given that many HCT survivors report good QOL, derive benefit from their transplant experience, and view themselves as having returned to normal, some degree of complacency with regard to the management of the late effects of HCT is understandable. However, it may be misleading to adopt an overly rosy view of the long-term recovery of HCT recipients. These reports are probably best viewed as a testimony to the resilience of the human spirit when confronted by difficulties, rather than being a reflection of a survivor’s objective physical and psychosocial status. The fact remains that research has repeatedly demonstrated that many survivors continue to report a variety of clinically important physical and psychosocial difficulties long after transplant. Recognition of psychosocial late effects when they occur is best achieved by routine and periodic monitoring in HCT survivors. The CIBMTR, the European Group for Blood and Marrow Transplantation, and the American Society for Blood and Marrow Transplantation have developed screening guidelines for long-term HCT survivors [66]. These guidelines recommend a clinical psychosocial assessment conducted by a mental health professional at 6 months and 1 year following transplant and annually thereafter. An assessment of depressive symptoms, psychological distress, and sexual functioning is specifically recommended, as well as inquiry into family functioning and the psychosocial status and adjustment of the spouse or other primary caregiver. Guidelines also recommend inquiry into dietary and exercise habits, tobacco and alcohol use, and substance abuse. In addition to their impact upon general physical health, these health behaviors may be linked to the experience of distress and hence may be useful indicators of psychosocial difficulties. Management of the psychosocial late effects of HCT may involve some appropriate combination of pharmacotherapy, traditional counseling and psychotherapy, cognitive-behavioral intervention, and complementary and alternative medicine. Unfortunately, there have been very few efforts to carefully evaluate the impact of these interventions during the long-term recovery phase. Consequently, the specific evidence base for these interventions in the HCT setting is virtually nonexistent. However, there is a much stronger and growing evidence base regarding

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the impact of psychosocial or psychoeducational interventions on psychosocial adjustment in cancer survivors in general [67]. While the HCT survivor may evidence some unique needs or concerns relative to the larger population of cancer survivors, all share this common experience: they are survivors of a life-threatening and QOL-threatening illness and treatment process. As a result, interventions shown to be effective in minimizing distress and enhancing psychosocial adjustment in cancer survivors in general might be expected to evidence similar positive effects with HCT survivors. There is a critical need at the present time to build an evidence base to support efforts at management of the psychosocial late effects of HCT and the fostering of optimal psychosocial adjustment in HCT. There is a small but growing body of research examining the impact of physical activity and exercise interventions on both physical and psychosocial outcomes. In general, participation by HCT recipients in structured programs of exercise has been associated with better QOL, less distress, and less fatigue [57,58]. Finally, in approaching the management of psychosocial difficulties in HCT survivors, we suggest a perspective that views long-term recovery from HCT as a process and not a discrete event. Research has suggested recovery may be characterized by gradual passage through a series of psychosocial transitions [68]. These transitions are characterized by the changing physical and psychosocial concerns of primary importance to the HCT recipient [37,69]. Early in the course of postHCT recovery, recipients are likely to be most concerned about surviving the transplant and achieving a measure of medical stability. Psychosocial concerns are few at this time. As months and years pass, however, recipients begin to focus more upon psychosocial issues revolving around sexuality, occupational adjustment, social relationships, fears of recurrence, and coping with chronic physical limitations. Ultimately, most recipients achieve a level of psychosocial equilibrium and move on with their life. This view of recovery as a process has clear clinical implications. Specifically, staff should be aware that psychosocial concerns are likely to change over time as recipients gradually adapt to one set of concerns only to have others emerge and become salient. Early in the course of recovery, recipients might show no interest in addressing issues of sexual or occupational functioning. Later in the course of recovery, however, they may have great concern regarding deficits in these areas. As a result, clinical efforts to assist recipients’ recovery must focus on appropriate areas of concern at the appropriate time in the course of recovery [69].

Psychosocial issues in HCT: hematopoietic cell donors While the HCT donor is “matched” to the recipient with regard to human leukocyte antigen typing, the donor may be either a related family member or an unrelated volunteer donor. Until the mid 1980s, HCT donors were almost exclusively drawn from the ranks of human leukocyte antigen-matched relatives, and donation of bone marrow was the norm. In the last two decades, however, the number of unrelated volunteer donors has increased, as has the harvesting of hematopoietic cells from peripheral blood. In the United States, most unrelated volunteer donors are identified from a registry maintained by the National Marrow Donor Program [70]. Several important psychosocial issues arise in the context of hematopoietic cell donation. These issues revolve around identification of the short- and long-term physical and psychological outcomes associated with donation. While some parallels can be drawn between hematopoietic cell donation and both blood and living solid organ donation, these comparisons are imperfect. Significant differences exist among these three types of donation with respect to the risks associated with donation,

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the prior relationship between the donor and recipient, and the donor’s knowledge and direct experience of the medical outcomes experienced by the recipient of their donation. These differences are so great as to preclude simple translation of what is known about blood and solid organ donation to the donation of hematopoietic cells. For example, hematopoietic cell donation requires a much higher level of commitment and health risk than that associated with blood donation, but far less commitment and health risk than that associated with living solid organ donation. Thus, only minimal comparison can be made among these types of donation, and the uniqueness of hematopoietic cell donation must always be kept in mind. A number of studies have examined physical outcomes, primarily pain and physical disability, following the donation of hematopoietic cells from either bone marrow or peripheral blood [71–73]. Serious physical complications after either type of stem cell donation are rare. However, some localized pain and discomfort is typically associated with the harvesting of bone marrow [73,74]. Some pain and discomfort are also typically reported in conjunction with the administration of granulocyte colony-stimulating factor preparatory to harvesting of hematopoietic cells from peripheral blood [72,73]. Despite the pain and discomfort that accompanies harvesting procedures, most donors return to full normal activity within a few days [71,72]. Studies which have compared the two hematopoietic cell harvesting procedures have generally found that bone marrow donation is associated with somewhat greater pain and discomfort and/or a more gradual return to full normal functioning [71,72,74–77]. How patients experience the differences between the two harvesting procedures from a psychosocial perspective, that is, anxiety before, depressive symptoms after, etc., has not been studied. It is unlikely that one would be recommended over the other based on psychosocial criteria alone. In addition to the acute physical impact of hematopoietic cell donation, research has examined the long-term psychological and social outcomes associated with hematopoietic cell donation. Because of the obvious difference in the donor–recipient relationship between related and unrelated hematopoietic cell donors, the research has typically examined outcomes separately for these two groups. Several studies have examined post-donation outcomes in related hematopoietic cell donors [78,79]. In general, related donors tend to report little emotional distress associated with marrow donation and little change in their relationship with the recipient. Typically, the experience of related marrow donation is generally positive, and few if any regrets regarding donation are expressed. Reports of increased self-esteem, happiness, and life satisfaction are common among donors. However, a minority of donors do report negative experiences (e.g. estrangement from the recipient). A recent review of pediatric sibling hematopoietic cell donation suggests that distress following donation might be more prevalent in pediatric, as opposed to adult, hematopoietic cell donors [80]. More research in this area is merited, particularly research in the pediatric donor setting and research focusing upon identification of risk factors for poor long-term donor adjustment in both children and adults. Most of what is known about long-term psychosocial outcomes experienced by unrelated hematopoietic cell donors comes from a comprehensive study of several hundred adult, unrelated hematopoietic cell donors recruited through the National Marrow Donor Program [81,82]. Donors were assessed at several time points, including after provision of informed consent for donation as well as 1–2 weeks and 1 year post donation. Results suggested that a sizable minority of hematopoietic cell donors experienced donation as stressful and inconvenient when queried 1–2 weeks following donation, with 12% admitting to a fair degree of worry about their health [83]. When queried 1 year following donation, donors were generally quite positive about their donation. One year post donation, 87% of donors stated they believed their donation experience

was “very worthwhile,” while 91% indicated they would be willing to donate again. Some evidence suggested donors with longer marrow collection times (perhaps experiencing more acute pain during the harvesting procedure) and those experiencing lower back pain or difficulty walking following donation viewed their experience as more stressful and experienced less positive psychosocial outcomes. Psychosocial outcomes in related and unrelated HSC donors have been directly compared in a single study [84]. A total of 77 donors (41 unrelated and 36 related) completed a series of pre- and post-donation assessments. Related donors experienced more depressive symptoms both prior to and following donation, and they also reported more moderate and severe pain than unrelated donors following donation. Demographic differences and differences in intraoperative events during stem cell harvest were thought to be an unlikely explanation for these results. The authors suggested that differences in the motivations and pressures that underlie the act of donation might contribute to the greater morbidity in the related donor group. To what extent do the donor’s long-term psychosocial outcomes vary as a function of the actual transplant outcome? In the large study of unrelated donors described above, Butterworth et al. found that the death of the recipient rarely produced guilty feelings or feelings of personal responsibility [83]. Rather, grief was an almost universal response to the recipient’s death and was often surprisingly intense given the recipient was an unrelated stranger. Of course, these data describe the experience of unrelated donors. Given the existence of a prior donor–recipient relationship and the likelihood the donor may be attendant to the recipient’s death, more negative reactions, perhaps involving guilt and anger in addition to profound grief, might be expected among related donors. However, only two studies have examined related donor reactions following the death of the hematopoietic cell recipient. In a small study of 23 related donors, bereaved donors reported more depressive symptoms than nonbereaved donors 6 months after marrow donation [78]. However, an earlier and somewhat larger study found that bereaved and nonbereaved, related donors did not evidence any major differences in adjustment and attitudes as a function of the medical outcome of the transplant [84]. All in all, the available data suggest the negative impact of a recipient’s death on the long-term adjustment of both related and unrelated donors might be less than expected, with donors of both types experiencing few, if any, long term-negative outcomes. In summary, the available research suggests that serious physical and psychosocial complications are uncommon following hematopoietic cell donation. This is true for both related and unrelated donors, and for harvesting of stem cells from both bone marrow and peripheral blood. However, this does not imply that hematopoietic cell donation is a completely benign experience. A degree of acute pain and reduced functional status as well as negative psychological and social outcomes have been reported by some donors after their donation. Consequently, at least some monitoring of both the short- and long-term physical and psychosocial reactions of hematopoietic cell donors is warranted. Much of the research on donor reactions is at least a decade old at this time. As the HCT setting is continually evolving, it is important that research evolves as well to capture any changing trends in donor reactions to their donation experience. Current research priorities include early identification of donors at greatest risk for negative responses to donation, as well as the development of brief, supportive psychosocial interventions to minimize any negative post-donation reactions. This section has focused, for the most part, upon the adult hematopoietic cell donor. It is not uncommon for children to be called upon to serve as hematopoietic cell donors for a seriously ill sibling or even parent. In rare cases, children are conceived with the intention that they might serve as a hematopoietic cell donor for an ill sibling [85]. Pediatric hematopoietic cell donation raises important ethical and legal issues

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[86,87], and the psychosocial consequences of such remain to be adequately identified [88]. Clearly, this is an area in need of more research and informed ethical and legal deliberation.

Psychosocial issues in HCT: informal caregivers Caregivers are often the unsung heroes in the transplant process. Without them, the recipient would face a lonely, harrowing inpatient stay, and the healthcare providers would have less help in coaching, guiding, and motivating the recipient throughout the long treatment and recovery process. By informal caregivers we mean family members, friends, and acquaintances of the HCT recipient, who often play a crucial role in the transplantation process. They are typically expected to provide daily companionship, furnish emotional support, assist in the recipient’s performance of daily tasks and responsibilities, and even help with the provision of medical care. At the same time, they must make provision to either suspend their daily roles or continue them, a great challenge and burden for some. The need to perform in this caregiver role may extend for many months and even years beyond the actual time of transplantation. With recent trends for an earlier hospital discharge of HCT recipients, informal caregivers are asked to carry an increasingly heavy and prolonged caregiving burden. Almost without exception, caregivers perform their demanding role with healing care and grace. Caregivers that stay with their loved one during hospitalization for HCT are exposed to many of the same psychosocial stressors as recipients themselves. While informal caregivers do not experience the acute physical demands of transplantation, they are susceptible to the understandable worries and fears associated with their loved one’s treatment. Caregivers can also experience geographic dislocation, a radical change in normal daily activities, poor sleep, isolation from other family members and friends, financial stress, feelings of helplessness and inadequacy in the midst of the recipient’s suffering, and considerable outof-pocket expenses [89]. Despite their own considerable needs, HCT caregivers typically receive little care and support as the focus of care and concern is typically centered on the HCT recipient. Unlike the HCT recipient, however, caregivers are not permitted to assume the “sick role.” Assumption of the sick role gives the HCT recipient implicit permission to depend on caregivers for help with even the most basic of functional activities and to suspend their regular responsibilities. Caregivers, however, are subjected to increased expectations associated with the caregiver role. These expectations typically involve serving as a primary source of physical care and emotional support for the HCT recipient. Both the recipient and medical staff can hold these expectations. In addition, caregivers can also place heavy expectations upon themselves, asserting the need to “be strong” for the recipient. Being strong in this context often involves ignoring their own physical and psychological needs in lieu of a total focus upon those of the HCT recipient. In short, it is not easy being a caregiver in the HCT setting! Several general issues have been the focus of caregiver research in the HCT setting in a small but growing body of research. First, researchers have tried to identify the “needs” of HCT caregivers, broadly defined. Second, attempts have been made to document the nature, extent, trajectory, and consequences of the distress and burden experienced by HCT caregivers. Finally, attempts have been made to compare caregiver burden and distress associated with HCT treated in an inpatient setting versus HCT treated in a more outpatient setting. Only a few studies have attempted to directly assess the needs of HCT caregivers. This is surprising given that a careful explication of the nature and timing of needs experienced by caregivers is a necessary precursor to any effective clinical attempt to address these needs. In a study of 58 mostly spouse caregivers, the most prominent needs were related to information, particularly regarding home care after discharge

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and diagnosis, fear, and leisure activity deficits [90]. While the additional needs of HCT caregivers can be inferred from other research on caregiver stress and burden (described below), there is a clear need for additional research to directly document the spectrum of informational, psychological support, and practical needs of HCT caregivers. Such need assessments should focus on the entire trajectory of HCT treatment and recovery as it is likely that the specific needs of caregivers evolve and change over time. One strategy for identifying the nature and trajectory of the HCT caregiving experience is to contrast it to the status and experience of the HCT recipient. Comparison of the HCT caregiver with healthy controls or normative data also places the status of the HCT caregiver in an appropriate context. In a well-designed prospective and longitudinal study, Langer et al. studied 131 spouse caregiver–recipient dyads in the HCT setting from pre transplant through 24 months post HCT [91]. Comparison data were also collected from a sample of nonmedical healthy controls. While negative affect declined over time among both caregivers and recipients, caregivers reported more depression and anxiety relative to recipients and controls. Caregivers and recipients reported similar levels of marital satisfaction prior to transplant. However, caregivers reported lower levels of marital satisfaction relative to the recipient at 6 and 12 months post transplant. Bishop et al. compared current health-related QOL and post-traumatic growth in 177 spouse/partner caregivers and HCT recipient dyads and 133 recipient-matched, healthy controls [6]. Recipients were roughly 2–19 (mean 6.7) years post transplant. Caregivers and healthy controls reported better physical health than HCT recipients. However, both partners and recipients reported more depressive symptoms and sleep and sexual problems than controls. Caregivers reported less social support, dyadic satisfaction, and spiritual well-being and more loneliness than both recipients and healthy controls. Furthermore, caregivers reported less post-traumatic growth than HCT recipients. In short, while reporting better physical health status, HCT caregivers reported equal or significantly worse psychological and social status relative to HCT recipients. Recent trends toward shorter transplant-related hospital stays have resulted in a greater portion of HCT care furnished in the outpatient, ambulatory setting. While significant cost savings have been achieved without compromising clinical outcomes, identification of how this trend may affect the HCT caregiver is important [92]. It is assumed that caregiver burden increases with shorter inpatient hospital stays and a consequent shifting of greater responsibility for care to the outpatient setting. Thus, a third important research issue has been the extent of caregiver burden and distress associated with HCT in the inpatient setting relative to that associated with HCT in the outpatient setting. Grimm et al. compared the emotional responses and needs of 26 HCT caregivers from an inpatient setting with those of 17 caregivers from an inpatient/outpatient setting [93]. Caregivers were monitored from prior to transplant through 12 months post transplant. In general, few differences were found in caregiver distress between the inpatient and inpatient/outpatient settings. Surprisingly, some evidence suggested the inpatient/outpatient setting might be associated with less caregiver mood disturbance. Given the small sample size and the lack of random assignment to inpatient or inpatient/outpatient settings, these results are not definitive. However, this research does reinforce the importance of considering caregiver outcomes, in addition to cost and recipient outcomes, when evaluating the relative merits of different models of HCT care delivery. This is particularly true as the presence of an appropriate caregiver is a requisite for ambulatory, outpatient-oriented modes of HCT care delivery [92]. In summary, the presence of a committed caregiver during any inpatient hospitalization and throughout the long process of recovery is

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common and is an invaluable asset to the HCT recipient and professional staff on the unit. In fact, some preliminary data suggest the presence of a caregiver during HCT hospitalization is linked to better survival during the first year post HCT [94]. However, despite the clear value of the caregiver to the HCT recipient, the available research suggests the caregiving experience in the HCT setting can be physically, financially, emotionally, and socially stressful. Importantly, the negative impact of the HCT caregiving experience may linger for years and may equal or even exceed that of the HCT recipient experience, with fewer compensatory reports of psychological growth in caregivers relative to recipients. Given this, it is very surprising that we were unable to locate any research focused upon the provision of care to the HCT caregiver. This is an important area for future research.

Psychosocial issues in HCT: professional caregivers Provision of comprehensive, high-quality care to HCT recipients requires well-trained nursing, medical, and allied health-care professionals to work together under highly stressful conditions. All HCT inpatient units need to have the capacity for intensive care of patients; they are designed to provide for a wide range of patient needs. Due to the fragile nature of the immunocompromised patient, the medical status of HCT recipients is often highly volatile. This potential for rapid and profound shifts in the HCT recipient’s medical status requires that the health-care staff remain ever vigilant while managing an increasingly complex array of machines and medications. Alongside these acute life-threats are the mundane tasks such as daily walking and mouth hygiene which are required to avoid serious medical complications such as infection and pneumonia. Often, the bedside nurse must be the motivating force, encouraging compliance to the daily tasks of living, which can be overwhelming to a patient who is profoundly fatigued, nauseous, vomiting, and diarrhetic. This role can be especially burdensome with a patient who is not motivated to follow the daily treatment regimen. Furthermore, the health-care staff must often do their work while under close scrutiny from worried and vigilant family members who are present at the recipient’s bedside for extended periods of time. Some family members record all medication given and every entry to the hospital room of any health-care provider. Finally, despite the stresses posed by the difficult nature of their work and the difficult nature of the HCT setting, medical staff must respond appropriately and compassionately to the expressed and often unexpressed needs and concerns of the recipient and their family. This is clearly a challenging task. A very small body of research has examined professional caregivers in the HCT setting and attempted to document the stressful nature of their work. Molassiotis et al. surveyed 129 nurses and 26 doctors from 16 HCT centers [95]. One-half of their respondents reported emotional exhaustion, and 80% reported feelings of low personal accomplishment. While overt depression appeared to be less common than might be observed in the general population, signs of clinically significant anxiety were seen in more than 10% of respondents. Specific sources of distress that were reported by these professional caregivers included having to work with dying patients on a regular basis, coping with the overwhelming responsibilities of their role in the HCT setting, adjusting to rapid advances in HCT technology, and coping with the often excessive demands of patients and family members. The picture was not quite as bleak in a study of burnout and job satisfaction in HCT nursing staff [96]. Encouragingly, these investigators found burnout was low and job satisfaction was high among the nurses they studied. However, while most study respondents indicated they derived a sense of personal accomplishment from their work, the investigators reported that about 25% of study respondents appeared to evidence some worrisome symptoms of anxiety.

The psychological impact of the transplant outcome on medical staff should not be understated. The stakes are very high in the HCT setting. These high stakes, coupled with the need for medical staff to engage in frequent, lengthy, and often emotionally intense interactions with recipients and their family members, create a fertile environment for distress. It is not uncommon for members of the HCT team to become deeply attached to patients and family members. The consequences of this deep attachment range across the spectrum of human emotional experience, from deep feelings of sadness and distress when a patient expires, to joy and celebration when a patient “beats the odds” and is rewarded with extended life. More effort needs to be devoted to examining and understanding the emotional labor involved in the provision of care in the HCT setting and the development, implementation, and maintenance of support programs for the professional caregivers in the HCT setting. In sum, the work of professional caregivers in the HCT unit is highly demanding and stressful. The HCT unit requires highly flexible personnel who can respond quickly and appropriately to a variety of medical circumstances and emergencies. Their response needs to be both technically sound and sensitive to the emotional, social, and spiritual needs of HCT recipients and their family members. The difficulty in responding in this way increases when patients are critically ill and staffing is short. Given the extraordinary day-to-day pressures under which HCT units typically function, it is truly remarkable how often these units function at a very high level and indeed often serve as models of workplace cohesion.

Conclusion HCT is not a monolithic procedure, and broad and sweeping generalizations about the experience of recipients, donors, caregivers, and medical staff are necessarily limited. In fact, the description of the inpatient experience prior to HSC infusion runs from “It was like rolling off a log” to “It was the hardest thing I have ever done.” However, the core of the HCT experience is similar for nearly all recipients. Fundamentally, it is a life-threatening treatment for a life-threatening disease that can produce a range of physical and psychosocial morbidities. Based on the relevant literature and clinical experience, we have attempted to highlight and summarize some of the important psychosocial issues that arise over the course of transplantation and that are germane to recipients, donors, and caregivers in the HCT. In preparing this chapter, we noted a continued growth in the literature addressing psychosocial issues associated with HCT. However, this growth has been uneven. Most of the extant research has focused upon the adult HCT recipient. There is a clear need for more psychosocial research germane to the pediatric HCT setting, as well as more research examining the psychosocial needs of informal caregivers. In addition, there is a need for studies that identify risk factors associated with the difficulties encountered by recipients over the trajectory of diagnosis, treatment, and long-term recovery, as well as research that evaluates interventions to prevent or minimize psychosocial problems. While the focus of these intervention studies should be on the HCT recipient, attention should also be directed toward the development and evaluation of interventions to minimize distress and enhance psychosocial adjustment in the caregivers of these HCT recipients. The psychosocial care of recipients, donors, and caregivers remains a challenging task for all health providers from the early stages of decision making through long-term survivorship. This task will be enhanced by the recommended screening and preventive practices guidelines for long-term survivors established in 2006 [4]. These guidelines will draw attention to the biological, psychological, social, and emotional needs of recipients throughout their seasons of survivorship [7].

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Karen L. Syrjala & Samantha Burns Artherholt

Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients

Introduction Knowledge of health-related quality of life (QOL) during and after hematopoietic cell transplantation (HCT) has expanded rapidly in the past decade, although it always lags behind major changes in transplantation eligibility and methods. Cross-sectional and longitudinal surveys have described QOL in patients from pretransplant through at least 10 years post-transplant [1–13]. As a result, much is known about QOL outcomes over time for autologous and allogeneic myeloablative transplant recipients within and across diagnoses. The central consistent findings of these studies are that: (1) physical function returns to pretransplant level for approximately 75% of survivors by 1 year; (2) by 3 years, 80–90% of survivors are back to full-time work; and (3) the majority of patients navigate the challenges of HCT with good psychologic health. Notwithstanding these generally positive outcomes, a majority of patients will have residual problems in specific domains of QOL such as energy, sexual function, fertility, and musculoskeletal symptoms. Excellent measures and methods for evaluating quality of life have been validated with HCT patients. Population-normed instruments are available for comparison with HCT-related outcomes. Computer-adapted measures are being tested to facilitate routine, rapid assessment and immediately accessible results, enhancing the potential usability of QOL assessments in clinical care. Progress has been made in previously underconsidered groups impacted by HCT. Research has described pediatric outcomes, caregiver impacts, and cognitive deficits during and following treatment. Studies have begun to examine QOL in transplant recipients receiving reducedintensity conditioning regimens. However, a dearth of research remains in two areas: clinical trials to improve QOL outcomes, and QOL outcomes research following relapse post-HCT. In addition, research is lacking in documenting outcomes for families of patients who die. This chapter reviews what is known about QOL during and after HCT, and considers measurement issues necessary to understand when interpreting results or doing research that includes QOL outcomes. Other chapters discuss the application of QOL within outcomes research, in the psychosocial domain, and with sexual problems (see Chapters 29, 33, and 35).

Definition of health-related QOL Most QOL researchers would concur that “health-related QOL refers to the extent to which one’s usual or expected physical, emotional and Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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social wellbeing are affected by a medical condition or its treatment” [14]. Elements essential to any definition of QOL are multidimensionality and the patient’s perspective. With time, QOL has grown to encompass not only subjective evaluation, but also those behaviors that may be reported by patients but go beyond subjective experience to include function and activity reports. While medical outcome studies often define outcomes in terms of peak toxicity or time to an event such as relapse or death, QOL is usually described in relation to nontransplant controls or as change from pretransplant status. A central tenet is that QOL is relative to the current situation and the expectations of patients within the circumstances in which they find themselves. As an example, patients with major toxicities during transplant can report good psychosocial QOL and satisfaction with QOL because they believe they are doing as well as possible in a difficult situation, and they have the support they need from people important to them [6]. Despite this subjectivity, QOL correlates with medical, functional, and observational outcomes [15–17]. An important recent transition in outcomes research is the concept of patient-reported outcome (PRO). This concept has been instituted by the Food and Drug Administration for medical product development and labeling [18]. PRO measures include reports of disease symptoms, treatment adverse effects, functional status, or overall wellbeing. A useful guideline for determining appropriateness of a PRO is offered by Lipscomb et al.: “the concept measured by the PRO instrument should be relevant and specific to the population, condition, and treatment to yield clinically valid and useful information” [19]. This definition is somewhat narrower than that of QOL and linked more explicitly to medical outcomes. However, the term “PRO” is being broadly accepted as covering aspects of health status evaluation that come directly from those individuals being evaluated, without modification or interpretation by another observer. It seems likely that, over time, PRO will replace QOL as a term and focus of clinical trial endpoints, given their importance to medical treatment and potentially reimbursement decisions. However, components of QOL not directly linked to medical decision making, such as spirituality or social function outcomes, will continue to be of interest to patients and families preparing for treatment, recovery, and long-term survival needs.

Dimensions Concepts of QOL continue to adhere broadly to a three-level “pyramid” first described by Spilker [20]. He tops the pyramid with global assessment of wellbeing. The middle level includes multidimensional assessment of the physical, psychologic, economic, spiritual, and social domains. The base details subcomponents of each domain; for example, work and symptoms are aspects of the physical domain. Other

Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients

subcomponents include social and recreational activities, sexual function, relationships with the medical team, religion, perspective on life, personal control, and components of health and treatment that contribute to stress or worry for the patient. Ferrell and colleagues [21] interviewed 119 adult bone marrow transplant survivors at least 100 days post-transplant to determine what QOL meant to these patients. They categorized the many positives and negatives noted by survivors into themes and then into four domains matching those of Spilker: physical, psychologic, social and spiritual/existential wellbeing. Consistent with these definitions, the multidimensional QOL measures used in most HCT studies include physical, mental, and social function domains, in some cases with the addition of items specifically relevant to HCT questions of interest (Fig. 34.1). Table 34.1 lists measures commonly used to assess QOL components in HCT. Smith and colleagues [22] found that patients perceived health status and QOL as distinct from each other. Physical function weighed more heavily in assessments of health status, while emotional health weighed more heavily in judgments of QOL. Social function was not prominently considered in either global rating. Consistent with this emphasis on health outcomes, medical studies incorporating a QOL assessment have successfully used the shortened version of the Functional Assessment of Cancer Therapy – BMT Module (FACT-BMT) with only the physical wellbeing, functional wellbeing, and transplant-specific items. This abbreviated measure is called the Trial Outcome Index [23,24]. Researchers are attending to understanding resilience as they document that most patients with cancer and receiving HCT do very well in the psychologic domain. Four concepts being examined in detail are spirituality, optimism, benefit finding and “post-traumatic growth” [5,25–28]. In essence, the ability to find positive meaning beyond the diagnosis and to perceive gains as well as losses in the disease and transplant process predict better long-term adaptation. Most often gains are reported as greater appreciation for life, closer interpersonal relationships, reprioritizing “what really matters,” feeling closer to God, inner strength, and a sense of peace and thankfulness [5,21,29,30]. While these concepts do not appear in most health-related QOL measurement, they are usually noted as important outcomes by patients, clinicians, and researchers examining qualitative outcomes of HCT [28,31]. As measured by most scales, spirituality includes finding connection and meaning in life as well as practicing organized religion [29,32]. A challenge for QOL experts has been to determine a definition of spiri-

tuality distinct from religion and still specific enough to be measured rather than being all-inclusive of peaceful, positive feelings. Although research has reported relationships between spirituality and mortality, as well as other health outcomes, reviews conclude that most studies finding relationships between spirituality and health outcomes are methodologically flawed [29,30]. A survey of 1422 individuals from the general population in the United States reports that those who describe themselves as both spiritual and religious have less psychologic distress than those who are religious and not spiritual. However, associations to health status have not been adequately tested because significant sociodemographic differences cannot be controlled between these groups [32]. Benefit finding, optimism, and growth have been associated with improved outcomes in studies that have solid methodology [25–27], although long-term benefits to survival and QOL remain unclear [33]. When designing an assessment plan, the relevance of specific QOL dimensions to the cohorts and outcomes being examined merits careful consideration. For example, a study assessing African-American elderly and the health-related QOL relevancy of items found that spirituality was more relevant for these males and females than were many of the usual QOL content items [34]. As another example, if investigators are examining the QOL impacts of an antiviral medication, they will likely want to assess symptoms, and possibly physical and cognitive function, but evaluation of emotional or social function may not be warranted. In summary, QOL is always evaluated from the patient’s perspective. It is rated in part by internal comparisons with how much worse things could be. Thus, patients with poor physical health may report good psychologic or overall QOL. Increasingly, studies are measuring optimism and benefit finding as outcomes that may predict both mental health and medical outcomes. While many dimensions of QOL can be considered for evaluation, physical and mental components are usually included. In some cases, study hypotheses may focus on the PRO of symptoms and function, in which case mental health may not be salient.

Phases of HCT Transplantation is a dynamic and individual experience [31]. While fairly consistent patterns can be described, a patient will have his or her own trajectory of illness and recovery depending on both medical and

Physical Physical abilities or deficits Fatigue/stamina Musculoskeletal problems/mobility Other physical symptoms Sexual function Fertility Work capacity

Fig. 34.1 The principal dimensions of quality of life, and elements that have been evaluated within each of these dimensions.

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Social

Mental

Social support Economic and health resources Access to information Relationships Recreational activity Caregiver effects

Depression Distress/worry Post-traumatic stress Cognitive function Coping style/optimism Self-efficacy/perceived control Positive growth

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Table 34.1 Selected quality of life measures used in hematopoietic cell transplantation (HCT)

HCT specific

Minutes to complete

Number of items

Scores

Yes

12

30

9 Subscales, no total score

Yes

10

27 + 23 37

No No

4 5 5

23 27 36

5 Subscales plus BMT module (TOI includes 3 subscales) overall score 4 Subscales, 8 subscales, total score

Symptoms MDASI (MD Anderson Symptom Inventory) MSAS (Memorial Symptom Assessment Scale – Short Form)

Adaptable No

3 5

13 32

Total score 4 Subscales, total score

Mental health, emotional wellbeing BSI (Brief Symptom Inventory-Short Form) CESD (Center for Epidemiologic Studies – Depression) CTXD (Cancer and Treatment Distress) HADS (Hospital Anxiety and Depression Scale)

No No Yes Yes

4 5 3 4

18 20 18 14

3 Dimensions, global severity Total score 5 Subscales, mean total 2 Scores

Coping style Brief COPE

No

5–10

28

14 Subscales in 3 groups; can select from subscales

Social function MOS Social Support Survey (Medical Outcomes Study)

No

4

19

4 Subscales, overall index

No

3

12

Total score

Yes

10

40

9 Subscales, overall score, medical impact score

No

10

35

4 Subscales, total score

Relevant measures tested in HCT* Multidimensional QOL EORTC-QLQ-C30 (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire – C30) FACT-BMT (Functional Assessment of Cancer Therapy – BMT) or abbreviated Trial Outcome Index (TOI) PedsQL (Pediatric Quality of Life Inventory) plus Cancer Module SF-36 (Short Form 36 Health Survey)

Spiritual, existential wellbeing FACIT-SP (Functional Assessment of Chronic Illness Therapy – Spirituality and Wellbeing Scale) Sexuality SFQ (Sexual Function Questionnaire)

8 Subscales plus physical and mental components, no total score

Caregiver and family impact Caregiver QOL – Cancer

* Updated measure information is available from websites for many of the measures listed. Those noted below can be located with a web search of the name or with the web address listed. If not listed, no website was found and the author needs to be contacted. • EORTC-QLQ-C30 (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire – C30) http://www.eortc.be/home/qol/ExplQLQ-C30.htm • FACT-BMT (Functional Assessment of Cancer Therapy – BMT Module) http://www.facit.org/about/welcome.aspx • PedsQL (Pediatric Quality of Life Inventory and Cancer Module) http://www.pedsql.org/ • SF-36 (Health Survey Short Form 36) http://www.sf-36.org/ • MDASI (MD Anderson Symptom Inventory) http://www.mdanderson.org/departments/prg/dindex.cfm?pn=0ee78204-6646-11d5-812400508b603a14 • MSAS (Memorial Symptom Assessment Scale – Short Form), contact Victor T. Chang [email protected] • BSI (Brief Symptom Inventory) http://www.pearsonassessments.com/tests/bsi.htm • CESD (Center for Epidemiologic Studies – Depression), no restricted access, numerous websites provide measure and scoring • CTXD (Cancer and Treatment Distress, HCT-specific measure) http://www.fhcrc.org/science/clinical/biobehavioral/projects/ • HADS (Hospital Anxiety and Depression Scale) http://www.gl-assessment.co.uk/health_and_psychology/resources/hospital_anxiety_scale/hospital_anxiety_scale.asp • Brief COPE http://www.psy.miami.edu/faculty/ccarver/CCscales.html • MOS Social Support Survey http://rand.org/health/surveys_tools/mos/mos_socialsupport.html • FACIT-SP (Functional Assessment of Chronic Illness Therapy) http://www.facit.org/about/welcome.aspx • SFQ (Sexual Function Questionnaire) http://www.fhcrc.org/science/clinical/biobehavioral/projects/

Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients

psychosocial factors [35]. There are two methodologic concerns in past research that contribute some instability to results across studies. One is that much of the research is cross-sectional, clustering patients with widely varying times since transplant and diverse diseases or treatments. Another is the range of assessment instruments used to evaluate QOL outcomes, which prevents ready comparison across studies. Nonetheless, outcomes are more similar than different, lending confidence to the robustness of QOL results. The psychosocial components of the phases of HCT are only summarized here; a detailed review can be found in Chapter 33. Pretransplant Patients arrive at transplant with differing diseases, histories of treatment, levels of physical function, and psychologic, social, informational, and financial resources. Psychosocial predictors of outcome have received the most attention, although data indicate that pretreatment physical QOL also predicts transplant outcomes [1,6,12,36]. Distress, in particular, predicts an acute transplant symptom course, especially for pain and distress [7,36–38]. Psychosocial needs are greatest just prior to transplant if judged by the percentage of patients with clinically significant anxiety or depression at different phases of treatment and recovery [1,6,36]. The conclusion from the review of numerous studies is that pretransplant physical condition and psychologic status predict later physical and psychologic adaptation in addition to survival [1,6,9,10,39– 42]. These replicated findings support guidelines calling for routine assessments of psychosocial function [43,44]. Acute treatment During the immediate post-transplant weeks, assessments find that survival is the axis around which other aspects of QOL are evaluated. In consequence, patients attribute their experiences to physical rather than psychologic causes during this phase [6,36]. Patient focus is dominated by mouth pain, nausea, fatigue, hair loss, infection risks, adhering to treatment requirements, and efforts to maintain self-care. As would be expected, patients with acute graft-versus-host disease (GVHD) have measurable declines in QOL compared with those not experiencing acute GVHD [23,45]. As hospitalization has shortened and treatments for acute toxicities have improved, investigations have increasingly moved from a focus on acute QOL to recovery and long-term outcomes. Recovery Initial studies focused recovery from HCT on the first year, by which time physical function is known to return to pre-HCT levels for most survivors. Recent research has documented that, for allogeneic transplant recipients, recovery of QOL is a longer process, requiring 3 and even sometimes 5 years [1]. During recovery, QOL issues arise that may have received minimal attention during the acute transplant phase, such as work, family relations, infertility, and sexual function [1,35,46]. Multiple studies report that the following risk factors predict a slower or poorer return of function: physical health pretransplant, depression, lack of social support, lower education level, female sex, and medical complications during recovery, in particular chronic GVHD [1,6,7,9,10]. Older adults have more difficulties with medical recovery from highdose treatment and are less likely to return to work or social activities [3,7,47–49]. Risk of chronic GVHD or other medical sequelae increases with age, and active treatment for complications is associated with poorer QOL during recovery. On the other hand, younger women have been reported to have poorer performance status and more psychosocial concerns following HCT [35]. In all, while medical risks vary signifi-

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cantly with age, QOL is more noteworthy for a lack of differences based on age than for any remarkable distinctions based on age. While survivors have slower recovery in the years following transplantation if they struggle with chronic GVHD, or have pulmonary disease, infection or other medical complications [7,13,47,50,51], once these medical complications resolve, QOL improves to levels equivalent to those of patients who never had them [3,23,45,51]. Similarly, patients receiving allogeneic transplants may have more physical and emotional difficulties in the first year, but by 12 months, physical and psychologic difficulties do not differ between types of transplant [1,3]. Return to work justifiably receives a good deal of attention in recovery and long-term function reports. While 30–60% of survivors begin to return to work by the end of their first year post-transplant, return to full-time work continues to occur for patients into the third year [1,12,52,53]. Fortunately, by 3 years post-transplant, studies agree that 80–90% of survivors have returned to full-time work or school [1,7,12,54–57]. Interestingly, although the pace is slower, overall rates of return to work for HCT recipients are similar to those in patients with other cancers [57]. It appears that rates of return to work may differ to some extent by the country in which a patient lives, perhaps as a result of the different economic incentives or rehabilitation programs available [40,58–60]. Long-term function Longitudinal and cross-sectional studies find that, by 5 years, a large majority of adult survivors of HCT are functioning well in their return to “normal” life in the domains of physical, psychologic, social, existential, and overall subjective QOL [2,4,5,12,55,61,62]. However, specific deficits remain at rates higher than those of the general population. Chronic GVHD continues for some patients, and is an important predictor of poorer overall health and QOL [11]. Long-term survivors report more accumulated health problems, musculoskeletal dysfunction, and more limitations in sexuality and social function than case-matched controls [2,5,63]. Results are inconsistent in terms of whether HCT survivors have higher rates of psychologic dysfunction, although studies agree that a majority do well [2,5]. Survivors also report more difficulty obtaining life and health insurance coverage (although actual rates of coverage are comparable to rates in control groups) [2,55]. Sexual dysfunction is one of the most consistent long-term deficits found across studies and time points after treatment. Both men and women have lower rates of sexual activity and satisfaction after HCT than the general population, with women’s sexual dysfunction one of the most frequent and persistent long-term QOL problems [4,40,64–71]. Chapter 35 details these sexual function issues following HCT. Fatigue Fatigue is the most persistent symptom beyond the first year of recovery [45,48,56,72,73]. While fatigue during acute treatment usually refers to tiredness, fatigue in survivors is often described as lack of stamina and weakness. Many biologic mechanisms have been postulated to explain persistent fatigue following HCT or other cancer treatments. Considered among the potential causes are the effects of interleukins and interferons, anemia, hypothyroidism, metabolic abnormalities, infection, treatmentrelated hormone and immune suppression, total body irradiation (TBI), sleep disruption, lack of physical activity, depression, and the need for many medications [74]. Knobel et al. [75] examined fatigue in autologous survivors 3 years or longer post-transplant. While men did not differ from the general population, women reported significant problems with fatigue. Neither disease factors nor time since treatment predicted level of fatigue. For women, those treated in first remission reported more fatigue than

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women transplanted at more advanced stages of disease. Since biologic explanations were not detectable, this finding may reflect the relativity of subjective report. Women with less prior treatment may have noticed a greater contrast in their energy than women who came to transplant fatigued from prior treatment. Gonadal dysfunction also was not associated with post-transplant fatigue. Follicle-stimulating hormone and luteinizing hormone levels did not predict fatigue level, nor did estradiol or testosterone levels in male or female survivors. Likewise, hemoglobin and thyroid levels were unrelated to reported fatigue. Immune markers tested, including interleukin-6 and soluble tumor necrosis factor receptors p55 and p75, did not predict fatigue either. On the other hand, interleukins, interferons, depression, and numerous medications have been associated with fatigue in other non-HCT cancer studies [14,76]. Although common pathways continue to be investigated, successful treatments for fatigue have included psychostimulants, antidepressants, erythropoietin, and exercise [14,77,78]. Regardless of etiology, more than one researcher has demonstrated an improvement in fatigue with exercise interventions [78–80]. Cognitive function Cognitive performance is an area of great individual variability pretransplant. Studies of adults indicate that 20–56% enter transplant with cognitive deficits that could interfere with function [81–84]. To date, there is no indication that adult cognitive abilities decline more rapidly after HCT than with nontransplanted adults [85]. We completed a prospective, longitudinal study of neuropsychologic function in 142 adult allogeneic HCT survivors. Declines in functioning were evident from pretransplant to 80 days post-transplant across all cognitive and motor skills tested. By 1 year, function returned to pretransplant capabilities in information-processing speed, verbal fluency, and verbal memory. However, problems remained with motor strength and dexterity. Previous chemotherapy was associated with increased risk of impairment in verbal fluency and memory [83]. Similar findings have been reported in other studies, with deficits mainly in the areas of psychomotor speed, executive function, and information-processing speed [86–88]. Interestingly, cognitive complaints may be largely unreflective of cognitive performance, and QOL is more related to perceived deficits than actual impairment [86]. Further research is needed on risk factors for specific impairments, as no consensus has been reached regarding specific treatment-related risks [88,89]. Several investigators have described cognitive disorders in patients taking calcineurin inhibitors or glucocorticoids for GVHD. For the most part, these have been case reports. A number of mechanisms have been postulated as the cause, and unless stroke or other permanent brain events occur, reports have indicated that these effects resolve with discontinuation of the drug [90–92]. Longer-term neuropsychologic effects have been described [93]. Abnormalities in neurologic and magnetic resonance imaging exams from 8 months to 5 years after transplant were greater for patients with chronic GVHD, corticosteroid or cyclosporine use. Meanwhile, long-term cyclosporine use and age increased the risk for neuropsychologic impairment. Disease, donor status, and conditioning regimen have been unrelated to long-term neuropsychologic results [83,93,94]. Patients who experience acute delirium during the treatment phase of HCT may be at elevated risk for depression, anxiety, and fatigue at 30 days, and worse mental health, more anxiety, fatigue, and distress at 80 days post-HCT [95]. These patients also had worse executive and frontal lobe functioning, attention, and processing speed at 80 days than HCT recipients who did not have a delirium episode. In children, prospective studies to 3 years after HCT have found no decline in cognitive performance for those over the age of 5 at transplant [96,97]. However, patients under the age of 3 are at increased risk of

intellectual decline over time post-transplant [96,98]. Long-term outcomes do not differ based on TBI versus chemotherapy-only conditioning regimens [96,99,100]. Disparate findings, with researchers reporting cognitive deficits in one-third to two-thirds of survivors, may be attributable to inclusion requirements of the samples (autologous versus allogeneic, age range, or history of prior treatment). Alternatively, some disparity may result from differences in tests selected, from time points at which testing was done or from differences in HCT treatment at the sites where patients were examined. In considering neuropsychologic function before and after transplant, it is important to recognize that performance is susceptible to disruption from virtually any source. Similar to fatigue, cognitive dysfunction is a common endpoint of many physiologic and mental strains. As etiologies are further examined, causes will likely differ between patients, treatments, and time points. Late effects As late effects are more closely studied, it is clear that medical risks continue at a higher rate for long-term survivors than for nontransplanted adults and children [11,40,47,53,63,101]. It is also certain that infertility concerns impact a substantial minority of both male and female survivors [102]. However, it is not similarly clear whether there are other QOL late-onset complications among survivors. Medical late effects such as cardiac failure or second cancers are distinct events that can occur long after treatment has been completed. In contrast, physical and psychologic problems can wax and wane following treatment, and the prevalence of problems ranges widely across studies. Thus, it is challenging to discover whether QOL deficits accumulate after transplant more rapidly than would be expected in an aging population. We would speculate, however, that survivors remain vulnerable to late QOL complications as a response to increased medical risks. Chapter 105 presents a review of late medical risks. Relapse Little progress has been made in understanding the QOL of patients who relapse. Issues of family, social network, and spiritual needs become vital to patient outcomes as their physical health is jeopardized. The symptoms and course of QOL, whether proceeding to death or returning to remission, remain largely unexplored, in part because patients are often censored from studies when they relapse. This is changing as more patients live with active disease or move between relapse, second transplant, and remission status following HCT. In summary, the physiologic, and psychologic phases and impacts of HCT have been well defined. Common problems post-transplant include fatigue, cognitive difficulties, sexual dysfunction, social limitations, and efforts to manage continuing uncertainty and fear. Late medical effects can be expected to have related QOL impacts.

Risk factors and diversity in HCT outcomes Risk factors for some QOL outcomes have been identified. Differences in medical course and short-term outcomes have been documented for autologous and allogeneic stem cell recipients, for patients with different diseases or pretransplant histories, and for patients who receive TBI versus those who receive chemotherapy-only regimens. Another area of diversity is the wide range of patients and others affected by transplant who respond within their own developmental phase of life or within their roles: adult or child patients, caregivers who are parents of children or spouses of adults, and males or females whether as patients or as caregivers.

Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients

Conditioning regimen Few sustained differences in QOL have been confirmed relative to the use of TBI or chemotherapy only as a conditioning regimen. Research indicates that TBI is the best predictor of neuropsychologic deficits at 80 days post-transplant, but differences are no longer measurable after 1 year [47,103]. Two medical differences that could influence QOL are a greater incidence of cataracts in patients receiving TBI, and a greater likelihood of alopecia in patients receiving busulfan with cyclophosphamide [53]. Few studies document other differences predicted by conditioning regimen within the domains of QOL. Clinical reports and early toxicity data suggest that nonmyeloablative or reduced-intensity conditioning recipients will differ from myeloablative recipients in their course of morbidity and function [104,105]. Since populations receiving less-intensive conditioning have generally not been eligible for high-dose conditioning, it is difficult to compare QOL and function across the two groups. Nonetheless, comparisons have been somewhat informative. One prospective longitudinal study compared the QOL of patients receiving reduced-intensity conditioning and myeloablative HCT, and found that both groups returned to baseline QOL by 2 years post-transplant [106]. Compared with autologous transplant recipients, patients receiving reduced-intensity conditioning transplants report more anxiety but overall better QOL in the first year post-transplant [45]. A continued study of the long-term outcomes of patients receiving reduced-intensity conditioning transplants is underway. Chapter 71 discusses the differences in eligibility as well as treatment for reduced-intensity conditioning HCT recipients. Autologous, allogeneic HCT Type of donor has been compared directly in numerous studies examining risk factors for QOL outcomes. Few studies have found measurable differences in psychologic QOL over time between autologous, allogeneic, and unrelated transplant recipients, despite their medical and treatment differences [1,49]. Some studies have reported that autologous HCT recipients have better QOL in all domains than allogeneic transplant recipients [73,107], while others have found that allogeneic HCT recipients report less impairment [56,108]. Still others have found that any discrepancy in QOL based on transplant type disappears by 2 years post-transplant [3]. Despite the discrepant findings, it appears that transplant recoveries for autologous versus allogeneic HCT recipients are more similar than different, particularly in the psychologic domains. Male or female Little attention has focused explicitly on gender effects in QOL outcomes. Nonetheless, nearly all research documents some gender effects within QOL surveys or interventions. In reviewing these outcomes, it becomes rapidly apparent that female recovery is more complex than male recovery, at least based on self-reports [1,7,10,47–49,109]. Norms for males and females on psychologic tests are always separated because of the well-established fact that women endorse more symptoms than men given the same levels of health. Women report more fatigue and generally report higher levels of distress, even when using standardized scores within gender normed measures [1,110]. Two additional consistent findings across studies are that women have more sexual problems and are slower to return to work [1,47,64]. In summary, risk factors tend to be relatively stronger during the acute phase of transplantation, with few aspects of treatment, disease or individual characteristics distinguishing long-term QOL in survivors. The major treatment factors influencing QOL are the use of reduced-intensity conditioning regimens and immunosuppression post-HCT, particularly

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for calcineurin inhibitors. Females seem to have greater difficulty during and following HCT across numerous studies and outcomes. On the other hand, age and type of donor have had less consistent influence on outcomes. Allogeneic transplant and older age seem to be risk factors primarily for acute differences in QOL.

Pediatric QOL Both parents and children have documented elevated distress during acute treatment, with declining distress in the 6 months following HCT [111,112]. Risk factors for high levels of distress and low QOL include receiving a transplant from an unrelated donor, increasing age, and lower socioeconomic status [113,114]. By 1 year post-transplant, both mothers and children report good QOL [112,114], and most long-term survivors lead independent, productive lives in adulthood [115,116]. Nonetheless survivors of pediatric HCT are noted to have higher anxiety as well as feelings of sensitivity and vulnerability when compared with survivors of pediatric bone cancers [8]. Other researchers have reported that pediatric survivors do better than their peers in psychosocial domains [117]. The rate of successful return to school (85–95%) looks similar to rates of return to work in adult survivors [118]. In sum, results testify to the resilience of children not unlike the resilience of adults. The restriction to these overall positive outcomes is that it has not been possible yet to track many survivors past their twenties. Cohorts of mature adult survivors of pediatric HCT are not yet large enough to evaluate their QOL.

Caregivers and parents of pediatric transplant recipients Medical staff and patients could not manage HCT and recovery without the presence of a family caregiver or multiple caregivers. As central a role as families play, research on family caregivers is only recently making headway in evaluating QOL needs. QOL in caregivers both before and after HCT is low compared with norms and that of their patient counterparts [119–122]. Spouses are the principal source of support for most adults undergoing transplant, and patients relying on spouses for caregiving report better QOL than those relying on others (such as friends or children) [119]. Caregivers report more distress than patients do during the acute transplant period [121,123,124]. However, research also indicates that the stressful impact of supporting a loved one through HCT can last years beyond the active caregiving phase [125]. Parents of pediatric HCT patients report significant distress in the course of their child’s treatment. Although some studies report that mothers of pediatric HCT patients suffer the greatest trauma in the course of their child’s treatment [126], other researchers find that mothers and fathers both report significant distress symptoms [127,128]. Younger mothers and those experiencing more depression or anxiety at the time of HCT are more likely to continue to be distressed 18 months later [126]. Other caregivers manage remarkably well from early evidence, despite their levels of acute distress.

Interventions to improve QOL outcomes Clinical trials to improve QOL have been surprisingly infrequent in HCT populations. During acute treatment, several studies have demonstrated that pain and distress are improved with hypnosis or other psychosocial care [38,129–131]. Several clinical trials have demonstrated that fatigue is reduced with exercise programs [78–80,132,133]. In reviewing the limited randomized controlled trial research, it seems that interventions have had modest effects, while the cost-effectiveness can be questioned

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[58,134]. As next steps, researchers need to define which QOL deficits benefit from intervention, in which high-risk patients, and at what points in time.

Measuring QOL What to measure and how to measure it is challenging when QOL is an HCT outcome. While PRO data can be rich and informative, inadequate appreciation of data collection requirements necessary for high-quality data and the importance of minimizing nonrandom missing data have limited the clinical value of many early QOL studies. In selecting measures of QOL in HCT recipients, the ideal is to use measures with established norms, reliability, and validity in HCT cohorts. In addition, an availability of broader population norms is valuable to permit a comparison of HCT results with those of other groups. However, the use of generic measures tested across broad populations has a downside as well. There is an inherent trade-off in benefits of generic versus specific QOL measures. HCT-specific measures may be more precise and informative but are then only relevant to an HCT-specific problem. Another challenge is the changing nature of what is relevant to measure based on the dramatic changes in QOL over the phases of HCT. Finally, there is the added value and potential reduction of missing data when using online or multiple electronic methods for data collection. In QOL measurement, a “response shift” is expected as patient conditions change medically. Patients naturally reprioritize concerns, and self-report is, by human nature, relative to expectation rather than being tied to medical status. In this process, health may decline, but many patients still report good psychologic function [6,135]. Another factor that can shift response is the changing relevance of assessment dimensions. While physical symptoms may be the central focus during acute treatment, focus shifts in long-term follow-up to functional abilities and emotional adaptation [35,46]. This is a major reason why excellent measures in one circumstance may be of less value at a different time. If no patients work in the 3 months after HCT, there is no gain from asking about work, and to do so would seem out of touch with patient experiences. However, after the first year, not asking about work would be a major oversight. Generic measures There are three QOL measures routinely selected when providing a “generic” assessment of HCT outcomes: the FACT-BMT, the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire – C30 (EORTC QLQ-C30), and the Short Form 36 Health Survey (SF-36), which is not a cancer-specific measure. One or another of these is used in most cancer QOL studies. Each of these well-standardized, reliable, and valid measures has some questions in common with the others and asks some questions that are not represented in the others. Yet each of them has been used effectively in HCT research. The FACT-BMT asks questions specific to the treatment of cancer and the HCT process, and focuses its emotional wellbeing content on concerns specific to the disease and treatment. It does not evaluate physical abilities per se. As such, it is quite disease- and treatment-oriented and may be less relevant during recovery and follow-up. The “BMT module” of added questions contains items that elaborate the more common HCT symptoms and concerns [136]. The EORTC QLQ-C30, in contrast, has a more explicit focus on functional abilities, what a patient is able to do physically, and specific symptoms, and a rather brief psychologic review. The HCT module, the EORTC QLQ-HDC29, measures side-effects and HCT-specific issues. Psychometric studies of this new module are ongoing [137]. The SF-36, meanwhile, is a health-related QOL measure not specific to disease

[138]. Its evaluation of physical ability corresponds most closely to that of the EORTC QLQ-C30 [139]. It provides a brief assessment of psychologic function, and it has minimal emphasis on physical symptoms. Given its general health orientation across populations and lack of symptom evaluation, the SF-36 is appropriate for use in the years posttransplant as issues become less HCT symptom-specific. Research has documented that these QOL measures are correlated but do not share enough variance to be considered equivalent [139]. Investigators find that the instruments have analogous global health items (correlation r = 0.82; 67% of the variance is shared). But the physical and psychologic dimensions of the EORTC QLQ-C30 and FACT share only limited variance. Virtually no variance is shared between the EORTC QLQ-C30 and FACT on scales of social wellbeing or cognitive function [139]. With its focus on treatment-related issues, the FACT covers the areas of greatest influence on patients during acute treatment, whereas the EORTC QLQ-C30 provides assessments that may be relevant through recovery. Each of these measures has normative information on use with HCT. Each has translations in multiple languages, and all are appropriate to capture issues faced by HCT patients. These scales have particular value for their abilities to provide a normative reference for a sample or an individual patient. For comparison, Table 34.2 lists the subscales for these three QOL measures. Figure 34.2 shows the capacity of the SF-36 to distinguish the function of 10-year HCT survivors and matched controls. Numerous other measures exist that are relevant to HCT but are not as widely used. Hence comparative data are not as readily available. The Sickness Impact Profile (SIP) [140], with 12 subscales, effectively tracks decline and recovery in health-related QOL before and after HCT [12], but is not cancer specific and is no longer widely used. Other scales have relevance in specific circumstances but do not have the extent of testing in HCT to provide normative information. Still, these multidimensional measures of QOL might be of relevance depending on the goals of assessment. The Cancer Rehabilitation Evaluation System – Short Form (CARES-SF), the Functional Living Index-Cancer (FLIC), and the Memorial Symptom Assessment Scale – Short Form (MSAS-SF) are some notable examples [108,141,142]. For a detailed consideration of measures, the reader is referred to books on this topic or articles comparing measures [143,144].

Mental composite t score Role emotional Social function Mental health Vitality -Physical composite t score Physical function Role physical Bodily pain General health 40

Controls Survivors

P = .01

P = .007 P = .002 P = .06 P = .02 P = .02 P = .03 P = .004 P = .10 P = .001

50

60 70 80 Mean score

90

100

Fig. 34.2 Health-related quality of life standardized mean scores and standard errors of the means on eight domains and two composite scales (physical and mental) on the Short Form 36 Health Survey (SF-36) for 10year survivors compared with case-matched controls. (Reproduced from the American Society of Clinical Oncology and Syrjala et al. [2] with permission.)

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Assessment of Quality of Life in Hematopoietic Cell Transplantation Recipients Table 34.2 Subscales of the three most widely used global health-related quality of life measures

Subscales

Summary scores Number of items

EORTC-QLQ-C30

FACT-BMT

SF-36

PedsQL plus Cancer

Physical functioning Emotional functioning Social functioning Role functioning Cognitive functioning Global quality of life Pain Nausea/vomiting Fatigue EORTC QLQ-HDC29 No – only subscales

Physical wellbeing Functional wellbeing Emotional wellbeing Social/family wellbeing BMT scale

General health Physical function Role – physical Body pain Vitality (fatigue) Mental health Role – emotional Social function

Physical function Emotional function Social function School function Cancer module

Total score

Total scale score

30 in core 29 added in EORTC QLQ-HDC29

27 in core 23 added in BMT scale

Physical composite t-score Mental health composite t-score 36

23 in core 27 in cancer module

See text for abbreviations.

Specific measures

0.6

Since generic measures are unlikely to have the sensitivity to capture the details of main interest in a clinical trial, measures specific to anticipated outcomes may need to be added to a generic tool. Considering symptom and function outcomes helps to clarify this point. Patients can be assessed for general physical function with a measure such as the EORTC QLQ-C30 or SF-36, but if an intervention is targeted explicitly to reducing fatigue, these scales may not have the sensitivity to capture outcome changes [75]. A specific fatigue measure will provide greater sensitivity to differences in outcomes. These focused forms will also provide investigators with more detail on where changes are occurring: is sleep or stamina improved; or does the patient feel more alert? A construct central to cancer QOL and HCT specifically is the concept of disease- and treatment-specific distress: what are the worries, stresses or fears specific to a patient having a transplant that contribute to how he or she is doing? General cancer distress measures are advocated for routine clinical and research use by the National Comprehensive Cancer Network guidelines [145]. Studies find that HCT-specific distress is a better predictor of pain, nausea, and stress responses during acute treatment than is depression or other general mood indicators [37,38], while depression is a better predictor of long-term outcomes [1]. Figure 34.3 shows the mean level of transplant-specific distress in patients over time in comparison to their general depression scores. The difference in these two measures visually demonstrates why a generic measure may not always be specific or sensitive enough to capture selective information.

0.4

Mean z scores

-0.4 -0.6 -0.8 -1.0 -1.2

Depression Distress Physical Limits

-1.4 5 s ar

ye

When a patient is unable to provide a self-report of QOL, a proxy responder may be the only option. This proxy is often a nurse or caregiver, or the parent of a young child. Research on the validity of proxy responses

-0.2

s ar ye

Proxy or parent

0.0

3

The gold standard for QOL assessment is a patient completing written or computer assessment with a staff person who is available to answer questions and to assure that the patient is competent and responding as instructed, as well as to review responses and correct skipped or double responses while the patient is present. However, often the choice is between no information or mailed, phone or proxy responses with someone answering for the patient.

0.2

ar ye 1 ys da 90 -tx e Pr

Alternative QOL measurement strategies

P < .001, each time course differed from the others

Fig. 34.3 Trajectories of physical limits, depression, and distress from before hematopoietic cell transplantation (HCT) to 5 years after HCT. Scores are transformed to z-scores to be on the same scale. Physical function and depressive symptoms recovered by 1 year and on average were stable thereafter. Distress related to disease and treatment differed from depression over time and declined gradually over the 5 years following treatment. Patients were 319 adult, male and female recipients of allogeneic or autologous HCT who had been treated with myeloablative therapy, with nonrelapsing survivors followed to 5 years. (Reproduced from JAMA and Syrjala and Zaza [164] with permission.)

indicates that, on average, parents and other proxies differ equally in responses when compared with patient reports [146,147]. Proxies, including parents, tend to underestimate patients’ symptoms. In contrast, they tend to rate global QOL and mental health lower than patient self-reports [146]. Fortunately, proxies are more accurate when they are rating concrete, observable events. Another finding from this research is that clinicians and parents underestimate the QOL information a child or patient with limited communication ability can still transmit. Of interest, children’s self-reports of their own health status significantly match physician ratings, while parents’ ratings do not [15]. When evaluating pediatric QOL, there are several assessments to consider [15]. The Pediatric Quality of Life Inventory (PedsQL) includes

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both a patient report and a parent report evaluating QOL for pediatric cancer patients on dimensions parallel to the FACT or EORTC QLQC30 [147–150]. Multiple versions suited to the developmental ages of children are comparable. A companion Cancer Module provides treatment-specific assessment of symptoms [151]. Both of these measures have well-established reliability and validity, although it is not clear that they have been used with HCT patients. In contrast, a relatively new measure is the Child Health Ratings Inventory (CHRIs), with its DiseaseSpecific Impairment Inventory-HSCT (DSII-HSCT; a transplantspecific module). Preliminary studies in HCT cohorts indicate good reliability, and moderate correlations between child and parent reports of QOL [152]. Given the large number of measures translated for age and language, it should be possible for the majority of patients to complete a general QOL measure appropriate to their needs if they can read. Alternatively, most measures can be administered orally, including by telephone if necessary. Administration by computer, mail or telephone interview The shortening of QOL measures, the increased attention to symptoms, and the necessary brevity of physician time have led researchers to explore the use of automated assessment systems, with some success. Testing of QOL assessment across modalities with HCT or other cancer patients substantiates the transferability of written measures to a touchscreen or other bedside computer [153–156]. Measures confirmed to be equally reliable and valid with written, mailed, and computer formats include the EORTC QLQ-C30, the SF-36, the FACT, and Memorial Symptom Assessment Scale [153–156]. Completeness and quality of data are improved with the computer administration of measures [154]. Software can immediately catch skipped items, eliminate double responses, and reduce burden by intentional skip patterns using item response rules. In contrast, at least one error occurred in 44% of paper response forms. As important, patients generally prefer the electronic response version, and it takes somewhat less time to complete. When onsite assessment is not an option, web and telephone methods are also effective. Bush et al. [4] demonstrated the feasibility, high compliance and satisfaction of a web-based system for collecting QOL data at home in the year following transplant. An automated telephone symptom monitoring for HCT recipients has been successfully tested and compared with written reporting [157]. A computerized system (Interactive Voice Response) can be set to contact patients at home at any designated day or time selected by the patient. If the patient does not respond, the system will call after set periods as often as programmed. The technology can be programmed to download information to a physician or nurse if critical set points are reached. In a comparison of Interactive Voice Response versus paper and pen, HCT patients responded at the same rate as on paper and reported the same symptom severity. In addition, 98% found the system easy to use [157].

Clinical utility of QOL data After a major review of oncology studies, a National Cancer Institute working group concluded that QOL assessment in cancer trials is fea-

sible, has scientific quality, can yield interpretable findings, and brings added value to cancer care decision making, especially when there is a substantial QOL difference but no survival difference between competing therapies [158]. Although the collection of QOL data is frequent in research settings, the frequency of its use in clinical settings is much lower [159,160]. Lee et al. [160] found that most HCT physicians consider providing QOL data to be part of their responsibility when informing patients of the risks of treatment. However, a majority of physicians do not believe that this QOL information influences patients’ decisions on whether to undergo HCT. Only 53% of physicians surveyed report using published QOL data to inform their practice. More effort is needed to translate QOL findings into usable information for clinical practice. One important component of usability is consensus on what constitutes a “clinically meaningful difference,” also called a “minimum important difference.” Increasingly, accepted strategies consider a meaningful difference to be a change in scores or a difference between groups of onethird to one-half a standard deviation of the observed scores [161,162]. Monitoring and discussion of QOL with HCT recipients has the potential to increase patient involvement in care, reassure patients, and improve patient satisfaction. Use of QOL assessments can improve physician–patient communication and enhance patient QOL outcomes [163].

Conclusion QOL before, during, and after HCT has been well described. Studies agree that most patients regain high levels of physical and psychologic QOL by 3 years following transplantation. At the same time, a subset of 5–20% of patients will have medical problems, such as chronic GVHD, that impinge long-term on QOL. Even for patients who return to full-time work and normal social activities, and those who have good psychologic and physical function, specific areas of difficulty have been identified. Recognized long-term problems include fatigue, cognitive deficits, musculoskeletal symptoms, and sexual dysfunction for both males and females. The impact on QOL of medical late effects is receiving more attention but remains an area deserving of further investigation. To date, few clinical trials have been published that improve QOL during either acute treatment or long-term recovery. By nature, QOL shifts with the situation a patient is in, reflecting not only observable abilities, but also expectations for the immediate circumstance. As a result, very ill patients can report good QOL in some areas. In assessing QOL, many options have been tested with HCT patients and work well. A generic QOL measure provides valuable normative information that permits a comparison of scores with patients of comparable age, gender, and/or disease. At the same time, specific, focused measures such as those for fatigue or sexual function may be needed to provide adequate sensitivity when defining the nature of a target problem or to detect clinical trial differences. Fortunately, there are many effective examples of QOL evaluation with HCT patients, at all ages and phases of treatment and survivorship. Finally, a remaining challenge is to determine a method that facilitates use of QOL data in clinical decision making.

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D. Kathryn Tierney

Sexuality Following Hematopoietic Cell Transplantation: An Important Health-related Quality of Life Issue

Introduction For more than two decades, investigators have explored the healthrelated quality of life (HRQOL) of hematopoietic cell transplant (HCT) recipients. Findings from these studies indicate that the majority of HCT recipients report good-to-excellent HRQOL [1–7], and have identified both positive and negative sequelae that influence HRQOL. Positive sequelae noted following HCT include a heightened appreciation for life, improved interpersonal relationships, personal growth, changes in priorities, and enriched spirituality [8–11]. Sequelae that negatively influence the HRQOL of HCT recipients are well documented and include fatigue or decreased physical strength [12–14], lack of or perceived lack of social support [3,12], cognitive changes [3], inability to resume previous role responsibilities [13,15], chronic graft-versus-host disease (GVHD) [12,13], recurrent infections [13], emotional distress [3,13], insomnia [3,6,13,14], and alterations in sexual health [3,4,12,13]. Investigators have an opportunity to improve the HRQOL of HCT recipients by exploring strategies to minimize the negative sequelae and enhance the positive ones. Alterations in the sexual health of HCT recipients are an important HRQOL issue and the focus of this chapter. First, the concept of sexuality will be explored, followed by what is known about sexual health in HCT recipients, including the types of alteration, their etiologies, and the impact of these alterations. Assessment of sexual health and possible interventions to improve sexual functioning and decrease sexual dissatisfaction will then be described. Finally, areas for future research will be explored.

The concept of sexuality In order to fully appreciate the impact of the diagnosis and treatment of cancer on an individual’s sexuality, one first has to consider a broad view of sexuality. Sexuality encompasses much more than sexual activity. Sexuality includes the view a person holds of him- or herself as a sexual being. Gender roles and what it means to be a man or a woman are components of sexuality [16]. Body image, self-confidence, and self-esteem all influence one’s sexuality and its expression. Sexuality is shaped by age, developmental stage, culture, sexual beliefs, expectations, sexual preferences, past intimate relationships and experiences,

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

and the sexual partner. Sexuality is expressed not only in sexual activity, but also in appearance, attitudes, values, roles, and relationships [16,17]. Life-threatening illness does not eliminate the need of individuals for intimacy, affection, and emotional connection to others. The World Health Organization views sexuality as an integral component of the human experience, which can enrich and enhance personality, communication, and love [18]. The American Society of Clinical Oncology and the Institute of Medicine are working to raise awareness of the needs of cancer survivors, and calling for the development of survivorship care plans that address psychosocial needs, including sexuality [19]. Standards for the scope of nursing practice published by the Oncology Nursing Society and the American Nurses Association mandate that nurses assess and intervene to treat sexual dysfunction [20]. Cancer survivors have identified altered sexual health as a distressing consequence of the diagnosis and treatment of cancer that negatively influences HRQOL. One study found that two variables, altered sexuality and family distress, exerted the most negative effect on measures of social wellbeing in long-term cancer survivors [21]. Cancer patients ranked loss of sexual feelings as the sixth most severe side-effect of chemotherapy treatment, preceded by effects on loved ones, loss of hair, fatigue, altered role responsibilities, and effects on social activities, respectively [22]. Altered sexual health following HCT has been reported by 22–70% of recipients [23,24], and for many these changes persist for years following treatment [3,25,26]. HCT recipients report that alterations in sexual health negatively affected their HRQOL [3,4,12,13]. Changes in sexual health following treatment for a life-threatening malignancy are complex. Sexual health may be altered as a direct result of physiologic changes due to the cancer or treatment. In addition to physiologic changes, alterations in sexual health may result from psychosocial variables such as anxiety, depression, altered relationships, and the uncertainty that accompanies the diagnosis and treatment of cancer. An individual’s sexuality is often expressed within the context of a relationship with another individual. Therefore, altered sexual health in one member of the couple will affect the sexual health of the other. The sexual partner may also experience depression, anxiety, and uncertainty, further contributing to alterations in sexual health. The American Psychiatric Association, in the Diagnostic and Statistical Manual of Mental Disorders, collectively describes sexual dysfunctions as a group of disorders that may affect one or more phases of the sexual response cycle [27]. These disorders are characterized by changes, physiologic or psychologic, that adversely influence sexual functioning, leading to psychologic distress or stress within relationships. Diagnoses for disorders of the sexual response cycle are shown in Table 35.1.

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Table 35.1. Diagnoses of disorders of the sexual response cycle. Hypoactive sexual desire disorder Female sexual arousal disorder Male erectile disorder Orgasmic disorders Sexual pain disorders, including dyspareunia and vaginismus Data from [27].

These diagnoses are based on the four phases of the sexual response cycle [28,29]. The first phase, desire, is the interest in sexual activity and includes sexual fantasy and thoughts. The concept of desire has biologic, behavioral, and cognitive components and is poorly understood [30,31]. Desire is strongly linked to the second phase of the sexual response cycle, arousal [31]. The subjective experience of excitement and pleasure, and the associated physiologic changes, characterize the arousal phase. In men, arousal leads to an erection due to a complex sequence of neurovascular and cellular physiologic changes [32,33]. Arousal in women is associated with vasocongestion leading to vaginal lubrication and swelling [31]. Orgasm, the third phase, is a period of intense sexual pleasure and is accompanied by the release of sexual tension and rhythmic muscular contractions of the perineal tissues and reproductive organs. Emission and ejaculation are the two phases of orgasm in men. Relaxation and an evaluation of the sexual experience are noted in the final phase of the sexual response cycle, resolution. The phases are not necessarily linear but are strongly interconnected. It must be emphasized that sexuality is a much broader concept than the sexual response cycle; however, the sexual response cycle offers a standardized approach to assessing and diagnosing alterations in sexual health.

Altered sexual health in HCT recipients There has been some work specifically investigating the alterations of sexual health experienced by HCT recipients. One noteworthy study evaluated the sexual health of HCT recipients longitudinally, reporting that, in both genders, the incidence and types of alteration in sexual health increased from before HCT to the last assessment 3 years post HCT [26]. However, the majority of information on the altered sexual health of HCT recipients is derived from studies focusing on the multidimensional construct of HRQOL and not specifically designed to evaluate sexual health. These HRQOL investigations have highlighted the incidence and the types of altered sexual health faced by HCT recipients. Numerous HRQOL studies have reported diminished sexual satisfaction following HCT [4,5,12,34–36], and other studies have reported sexual dysfunction [13,24,26,37]. Altered sexual health is often cited as negatively impacting HRQOL of HCT recipients [3,4,12,13]. A summary of findings from investigations specifically designed to explore the sexual health of HCT recipients is shown in Table 35.2.

Etiology of altered sexual health in HCT recipients Alterations in sexual health may be due to a number of biologic, physiologic, psychologic, and social variables, and the complex interactions among these variables. Consequently, the etiology of altered sexual health will rarely be limited to a single cause. In HCT recipients, alterations in sexual health may be due to the malignancy, the diagnosis, therapy antecedent to HCT, the preparative regimen utilized for transplant, and the complications and treatment of complications following HCT. In male HCT recipients, reported alterations in sexual health include hypoactive sexual desire disorder, erectile dysfunction (ED),

ejaculatory dysfunction, hormonal changes, and infertility. Alterations in the sexual health of female HCT recipients include hypoactive sexual desire disorder, premature ovarian failure (POF) and menopause, arousal disorders, dyspareunia, and infertility. Biologic and physiologic variables in men The effects of the high-dose preparatory regimen on the hypothalamicpituitary-gonadal axis in men are well documented. Damage to the gonads from the preparative regimen leads to a loss of the secretion of the sex steroids, testosterone and inhibin, leading to an absence of negative feedback to the hypothalamus and pituitary, resulting in elevated levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The hypothalamus secretes gonadotropin-releasing hormone (GnRH) into the circulation, stimulating the release of the gonadotropins, FSH and LH, from the pituitary. FSH and LH control gonadal function, resulting in the production of gametes and the synthesis of sex steroids. The Leydig cells in the testicles, under the influence of LH, start androgen synthesis and secretion. FSH initiates spermatogenesis, which is then maintained by testosterone [38,39]. Negative feedback to the hypothalamus and pituitary occurs when testosterone and inhibin levels rise, inhibiting the secretion of GnRH and the gonadotropins [40]. Both radiation and chemotherapy damage gonadal tissue, leading to decreased secretion of sex steroids and thus a loss of the negative feedback inhibition at the level of the hypothalamus and pituitary. The result is the characteristic findings of elevated levels of FSH and LH indicative of infertility [38]. The degree of gonadal damage will depend on age, the dose of radiation and chemotherapy, and the type of chemotherapy administered [38,41]. Irradiation directly to the testes in doses of 12–15 cGy produces irreversible azoospermia [39]. Radiation and chemotherapy, particularly alkylating agents, result in a dose-dependent depletion of the germinal epithelium lining the seminiferous tubules of the testes, and this depletion results in azoospermia, testicular atrophy, and infertility [41,42]. Features of germinal cell depletion consist of small testicular size and reduced testicular volume [41]. Endocrine values in men with germinal cell depletion include elevated FSH (25–90 mIU/mL), elevated LH (8–25 mIU/mL), low-to-low-normal testosterone (200–700 ng/100 mL), decreased testosterone production rate (3.5 mg/day), and decreased free testosterone (8.6 ng/100 mL) [41]. Testosterone is strongly correlated with sexual desire and arousal [43]. Levels of testosterone in male HCT recipients often remain within normal limits as the Leydig cells are relatively resistant to the effects of chemotherapy and radiation [39,44]. However, subtle damage to the Leydig cells, evidenced by a reduction in the amount of testosterone produced and a decrease in free testosterone level, may be found on more extensive hormonal evaluation [45]. Men over the age of 45 may be at increased risk of testosterone insufficiency [46]. Elevated prolactin levels have been found in some men following HCT and may indicate damage to the hypothalamus [42,47]. Hyperprolactinemia has been associated with infertility, ED and decreased libido [43,47–49]. Radiation damage to the thyroid may result in hypothyroidism or subclinical hypothyroidism [42,50], and levels of free testosterone and bioavailable testosterone are reduced in men with hypothyroidism [51]. ED is defined as the inability to achieve or maintain an erection sufficient for satisfactory sexual intercourse. The etiology of ED may be hormonal, vascular, neurologic, secondary to medications, psychologic or a combination of these factors [49]. Cavernosal arterial insufficiency has been reported as a cause of ED in men following HCT, with the risk being higher in those men receiving total body irradiation compared with those receiving chemotherapy only [52]. Pelvic irradiation can cause penile arterial insufficiency contributing to ED in some men [42,49,52,53].

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Table 35.2. Investigations of altered sexual health following hematopoietic cell transplantation (HCT). Study

Measurement(s)

Findings

n = 51 men Mean age 33 (range 19–49) years Mean time from HCT 58 (range 6–154) months [47]

Investigator-designed questionnaire on sexual function Hospital Anxiety and Depression Survey Hormonal assessment

15% diminished desire 24% ED 13% delayed ejaculation 9% dry ejaculation 20% premature ejaculation

n = 31 Median age 38 (range 16–56) years Assessed pre and 3 months post HCT [94]

Derogatis Interview for Sexual Functioning

Pre/3 months post-HCT: 36%/48% sexual dysfunction 65%/52% sexual dissatisfaction 36%/40% decreased desire 14%/11% arousal problems 43%/43% orgasm problems 25%/25% ED

n = 29 men Mean age 35 (range 18–61) years Mean 36 (range 7–97) months post HCT [48]

Psychosexual Functioning Questionnaire – male version Psychosocial Adjustment to Illness Scale Hormonal assessment

38% 21% 38% 48%

n = 16 men Mean age 36 (range 20–49) years Median of 3 years (range 7–96 months) post HCT [95]

Investigator-modified Psychosocial Adjustment to Illness Scale Hormonal assessment

25% diminished desire 12% intermittent ED

n = 102 Mean age 37 (SD ± 9.9) years Assessed pre HCT and 1 and 3 years post HCT [26]

Investigator-modified Derogatis Sexual Functioning Inventory Symptom Checklist-90

In women pre/3 years post HCT: 40%/52% arousal disorder 14%/46% orgasm disorder 30%/52% lubrication problems 14%/33% pain In men pre/3 years post HCT: 15%/20% arousal disorder 4%/6% orgasmic disorder 13%/22% getting an erection 13%/16% keeping an erection

n = 168 Median age 33 years Median time from HCT 14 months [68]

Questionnaire on sexual functioning and fertility EORTC QLQ-C30 Patient ranking of perceived changes in sexual relationships

Autologous/allogeneic HCT recipients: 44%/51% decreased desire 48%/57% decreased sexual activity 27%/42% decreased pleasure 29%/45% decreased sexual ability

n = 126 Mean age 27 (range 9–50) years Mean 47 (range 6–149) months post HCT [24]

“Faces” Scale of Life Satisfaction Hormonal assessment when possible

24% ED 13% ejaculation difficulties 22% of all subjects reported diminished sexual satisfaction

n = 64 Median age 39 (range 20–65) years Mean 52 (range 19–89) months post HCT [96]

EORTC QLQ-C30 Leukemia-BMT specific module Questionnaire on sexual functioning and fertility Questionnaire on patient perception of changes in HRQOL

Autologous/allogeneic HCT recipients: 21%/60% diminished desire 30%/68% decreased sexual activity 18%/47% decreased pleasure 29%/53% diminished ability to engage in sexual activity

diminished desire altered body image ED sexual dissatisfaction

ED, erectile dysfunction; EORTC QLQ-C30, European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30; HRQOL, health-related quality of life.

Hypertension is a major contributor to ED in healthy men [49,51] and a common side-effect of calcineurin inhibitors utilized for GVHD prophylaxis and treatment. ED and diminished intensity of orgasm may be caused by chemotherapy agents associated with peripheral neuropathies

administered before HCT or with the preparative regimen [54]. Other lifestyle factors that may contribute to ED include cigarette smoking, alcohol use, and high cholesterol levels [49,55]. The immunosuppressant sirolimus (Rapamune) commonly causes hyperlipidemia.

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Findings of fatigue and decreased physical stamina are often reported following HCT [12–14]. All phases of the sexual response cycle can be disrupted by fatigue and weakness [17,43]. Biologic and physiologic variables in women In women, the effects of the high-dose preparatory regimen on the hypothalamic-pituitary-gonadal axis are understood. Loss of the secretion of estradiol from the ovaries damaged by the preparative regimen leads to an absence of negative feedback to the hypothalamus and pituitary, resulting in elevated levels of FSH and LH. As in men, the hypothalamus secretes GnRH into circulation, stimulating the release of the gonadotropins FSH and LH from the pituitary. FSH initiates growth and maturation of the ovarian follicles, and LH controls ovulation and corpus luteum formation [38]. Negative feedback to the hypothalamus and pituitary occurs when estradiol levels rise, inhibiting the secretion of GnRH, FSH, and LH [40]. Both radiation and chemotherapy damage gonadal tissue, leading to decreased secretion of estradiol and thus a loss of the negative feedback inhibition at the level of the hypothalamus and pituitary. In women, the findings of elevated levels of FSH and LH are indicative of infertility and menopause [38]. The degree of gonadal damage will depend on age, the dose of radiation and chemotherapy, and the type of chemotherapy administered [38,41]. A single high dose of total body irradiation produces ovarian failure in all women [38]. Alkylating agents and radiation therapy have cytotoxic effects on both dividing and resting ovarian cells. Ovarian failure is more likely in women over the age of 25 [41,44]. Serial ultrasound examinations have demonstrated that, almost immediately following the preparative regimen, the ovaries show evidence of structural damage, shrinkage, and loss of follicles [46]. In addition to decreased levels of estradiol and elevated levels of FSH and LH, a small number of women tested had evidence of abnormal androgen function [42]. Radiation damage to the thyroid may result in hypothyroidism or subclinical hypothyroidism [42,50], which can contribute to POF [56]. Estrogen deficiency is associated with a multitude of symptoms, including hot flashes, night sweats, insomnia, mood swings, irritability, depression, vaginal dryness, atrophy and fibrosis, pruritus, urogenital symptoms, changes in cognitive function, changes in appearance, reduced bone density, and cardiovascular disease [44,57,58]. POF results in symptoms of estrogen deficiency that are more pronounced and severe due to the sudden loss of estrogen [23,58]. Table 35.3 summarizes results from investigations of POF in female HCT recipients. Findings from an ongoing longitudinal investigation evaluating the effects of POF on sexuality indicate that many women report symptoms of estrogen deficiency and alterations in sexual health on the first assessment completed prior to HCT [59]. In 48 women evaluated pre HCT, 46% reported hot flashes, with 27% reporting the hot flashes to be moderate-to-severe in intensity. Thirty-five percent reported vaginal dryness during sexual intercourse, and 60% reported diminished libido. Of the 28 women in this sample who reported sexual activity in the preceding month, 36% reported decreased arousal, 29% described decreased lubrication, 25% experienced pain during vaginal penetration, and 47% reported problems with orgasm. These findings indicate that therapy antecedent to HCT contributes to symptoms of POF and alterations in sexual health. In female HCT recipients, the etiology of vaginal atrophy is multifactorial and includes the effects of POF, the preparative regimen, in particular radiation therapy, and chronic GVHD of the vagina. Estrogen deprivation results in the vaginal mucosa becoming thinner with less lubrication, leading to symptoms of itching, dryness, and burning, increased risk of infections, and dyspareunia [58,60]. Additionally, estrogen deficiency affects urogenital tissues, leading to dysuria, urinary frequency, incontinence, and an increased risk of urinary tract infections

[60,61]. Radiation may contribute to vaginal narrowing and stenosis [42]. One investigation reports the incidence of vaginal chronic GVHD as 36% 1 year post HCT, increasing to 49% by 2 years [62]. Symptoms of vaginal chronic GVHD include cystitis, excoriation, ulceration, adhesions, and stenosis [62–64]. Decreased physical stamina and fatigue are commonly reported following HCT [12–14]. Fatigue and weakness can cause disruptions in all phases of the sexual response cycle [17,45]. Psychosocial variables Investigations of HRQOL in HCT recipients report a number of longlasting psychologic issues, which may alter sexual health. Long-term survivors of HCT report depression [8,65], anxiety [8,13,65], decreased self-confidence and self-esteem [12,66], altered body image [46,48,66,67], and distress secondary to infertility [66,68]. A sense of wellbeing, mood, and energy all significantly influence a woman’s sexuality [43]. Body image scores for both male and female HCT recipients were significantly lower compared with a healthy sample [67]. Higher levels of psychologic distress correlated with poor body image, disruption in sexual relationships, and reduced sexual satisfaction in one study [24]. In men, higher levels of psychologic distress pre HCT significantly predicted for sexual dissatisfaction 3 years post HCT [26]. In both genders, the desire, arousal and orgasm phase of the sexual response cycle can be altered by psychologic distress, especially depression and anxiety [30,69]. Psychologic stress, especially anxiety, depression, and relationship stress, can contribute to ED [49]. POF is more than an early transition into menopause as it is associated with a number of psychologic stresses including feelings of lost youth and femininity, and altered body image due to changes in appearance [39,58]. A woman’s sense of femininity and what is means to be a woman may be negatively altered by infertility [16,70]. Similarly, in men, infertility may alter their sense of virility and masculinity [16,70]. Infertility and alterations in gender roles may result in a loss of self-confidence [46,66,67,70]. If a couple has not completed childbearing, infertility may lead to distress within the relationship. Additionally, single individuals may be hesitant to establish new intimate relationships for fear of rejection secondary to their infertility [30,71,72]. Not surprisingly, infertility was more distressing for younger individuals or those who had not completed their families [66,68]. The time from the diagnosis of cancer or life-threatening illness through treatment and recovery following HCT is lengthy, psychologically difficult, and filled with uncertainty. During HCT and the early recovery phase, a caregiver is required who assumes significant responsibility for the care of the HCT recipient. Often this caregiver is the sexual partner. The effects of role changes on intimate relationships are not yet understood. Several recent publications have cited the burdens of caregiving and the significant problems faced by caregivers following HCT. In a longitudinal investigation evaluating marital satisfaction, findings indicate that the spousal caregivers experienced higher levels of depression and anxiety at 6 months and 1 year post HCT than the HCT recipient and a healthy normative population [73]. Additionally, marital satisfaction scores decreased for the caregiver from pre HCT to the 1-year assessment, whereas marital satisfaction scores for the HCT recipient remained stable. The decreased marital satisfaction scores were significantly more pronounced if the caregiver was female. The sexual partner may experience psychologic distress including anxiety, depression, and uncertainty [69,71,74], which can lead to alterations in sexual health. Findings from an ongoing longitudinal investigation indicate that nearly half of the 28 spouses/partners of women preparing for HCT experienced alterations in sexual health [59]. Specifically, seven (25%) indicated slight problems performing sexual

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Table 35.3. Investigations of premature ovarian failure in women post hematopoietic cell transplantation (HCT). Study

Measurement(s)

Findings

n = 15 Mean age 22 (range 17–30) years Assess pre HCT and 3–4 months post HCT [63]

Hormonal assessment Pelvic ultrasound Interview

53% 25% 25% 33% 73% 40% 53%

n = 37 Mean age 30 (range 17–46) years Time from HCT 13–84 months [23]

Gynecologic examination Hormonal assessment

83% vasomotor symptoms (hot flashes, sweating, irritability) 76% vaginal dryness and dysuria 76% dyspareunia 70% sexual problems (decreased desire, altered self-image, anxiety, decreased self-confidence)

n = 36 Mean age 26 (range 14–43) years Mean 4 years (range 8 months to 9 years) post HCT [66]

Questionnaire regarding menopausal symptoms, sense of femininity, sexual difficulties, social activities and coping Interview for effects of hormone therapy on symptoms

61% hot flashes 36% night sweats 81% vaginal dryness Problems in 22 sexually active women: 73% decreased desire 64% orgasm problems 82% problems with sexual intercourse

n = 30 Mean age 37 (range 25–49) years Mean time with ovarian failure 16.6 months (range 6–36 months with an additional subject 4 years and another 9 years) [80]

General and gynecologic examination Complete blood count, liver function tests, lipid panel, haemostasis parameters, hormone levels Medical history Pelvic ultrasound, mammography, and computerized bone mineralometry Investigator-designed questionnaire for menopausal symptoms

90% vasomotor symptoms, hot flashes, night sweats, and palpitations 61% musculoskeletal pain 54% vulvovaginal atrophy with dyspareunia, itching, and burning 54% mood changes with anxiety, depression, irritability, and headache 45% atrophic skin changes 42% urinary tract symptoms 26% weight gain 16% insomnia 16% memory changes Gynecologic examination revealed cervical and uterine atrophy, loss of pubic hair, pale mucous membranes, vaginal dryness, stenosis, and decreased vaginal elasticity

n = 44 Median age at evaluation 30 (range 18–43) years Median 357 (range 261–4628) days post HCT [97]

Physical examination Laboratory studies Gynecologic examination Interview for menopausal symptoms and sexual functioning

Received radiation and chemotherapy: 67% hot flashes, night sweats, insomnia, and mood changes Received chemotherapy only: 38% hot flashes, night sweats, insomnia, and mood changes In 30 sexually active women: 77% vaginal dryness 53% diminished libido and arousal 60% dyspareunia 70% diminished pleasure Gynecologic findings: tissue atrophy, loss of pubic hair, small uterus, introital stenosis, and atrophic vulvovaginitis

n = 74 postpubertal allogeneic HCT recipients Median age 30 years Mean 49 (range 4–118) months) post HCT [61]

Gynecologic examination Hormonal assessment Interview regarding HRT, menopausal symptoms, and sexual activity

78% hot flashes 61% genitourinary symptoms 94% of 52 sexually active women reported difficulties with intercourse Gynecologic findings: vulvovaginal atrophy, loss of pubic hair, and decreased thickness of genital mucosa

HRT, hormone replacement therapy.

hot flashes cystitis symptoms irritability mood changes depression diminished libido dyspareunia

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activities, three (11%) indicated constant problems, and two (7%) indicated being totally unable to perform sexual activities. The couple may find that resuming sexual activity after HCT is difficult due to the long period of forced abstinence. The HCT recipient may be struggling with body image changes and concerns about their sexual functioning. Both the HCT recipient and the spouse/partner may be suffering from depression and anxiety that is interfering with the desire phase of the sexual response cycle. As a recent cancer survivor stated (personal communication), it can take time for couples to find one another again. The psychosocial adjustment of HCT recipients is influenced by the presence or absence of social support [3,7,12]. Social support has been found to facilitate healthy adjustment following HCT, with strong family relationships predictive of better psychosocial adjustment [75]. Decreased social support negatively influenced the psychosocial coping of HCT recipients even years following treatment [3]. If a major source of social support for the HCT recipient is the sexual partner and relationship stress arises due to alterations in sexual health, the recovery process may be jeopardized.

Assessment and interventions for altered sexual health Barriers to addressing issues of sexuality and sexual health include personal discomfort, lack of education and limited time on the part of the healthcare professional [17,53,76], and reluctance of the part of patients to disclose concerns. Strategies to overcome professional barriers include increasing awareness of the types of sexual problem experienced in the population served, gaining experience in assessing sexual health, acquiring knowledge of interventions, and identifying available resources [77]. One approach to beginning an assessment of sexual health is to simply ask patients if they have resumed sexual activity. One report demonstrated that by asking two specific questions during an office visit – “Are you sexually active?” and “Are you having any sexual difficulties or problems at this time?” – the number of sexual concerns revealed increased from 3% who spontaneously disclosed concerns to 16% [78]. Interventions for addressing sexuality begin before HCT, when disclosure of possible alterations in sexual health should be discussed. In addition to stating the risks of infertility, POF, and menopause, describing possible alterations in sexual health is an essential element of informed consent. Information regarding possible alterations in sexual health should be included in the consent form and the educational materials provided by transplant centers. These education materials can serve as a valuable resource to HCT recipients during the recovery process. The educational materials should include information on when it is safe to resume sexual activity, what precautions are needed, when to consult a healthcare professional with concerns, and a list of resources. HCT recipients experience higher levels of psychologic distress when there is a discrepancy between their expectations and the outcomes following HCT [65]. This finding suggests that one intervention for improving post-HCT sexual health and HRQOL is education aimed at minimizing the discrepancy between expectations and possible outcomes. It has been shown that individuals have a remarkable capacity to adapt to changes and re-establish norms [10]. Educating HCT recipients and their spouses/partners regarding possible changes in sexual health facilitates their ability to adapt. Simply informing couples that decreased libido is common for many months following HCT is one place to start. By initiating a discussion of sexual health before HCT, the healthcare provider has accomplished two goals in addition to providing informed

consent. First, the healthcare provider has identified him- or herself as a resource for sexual health concerns. Second, they have validated that sexuality and altered sexual health are legitimate concerns. Ideally, discussions of sexual health will include the sexual partner. Including the sexual partner may facilitate a discussion between the couple, identify the partner’s anxieties, and enlist the partner’s support for coping with changes. The transplant team should identify a group of specialists for referrals to help address alterations in the sexual health of HCT recipients. Several referrals may be needed to address physiologic, biologic, and psychosocial variables that could be contributing to altered sexual health. These resources should include a dedicated and interested gynecologist and urologist, reproductive specialists, and mental health professionals for counseling couples [53]. Grieving lost fertility may occur following HCT, and assessment of the emotional response to infertility should be evaluated. A number of accepted and investigational approaches for fertility preservation include sperm or oocyte cryopreservation, cryopreservation of testicular or ovarian tissue, hormonal suppression of ovarian function, and embryo cryopreservation [39]. Other reproductive techniques that may be attempted in the post-HCT setting include in vitro fertilization and in vitro fertilization by intracytoplasmic sperm injection [39]. A detailed discussion of fertility preservation procedures and success rates is beyond the scope of this chapter, but the reader is referred to reference [39] for a more detailed discussion. Much of the responsibility for fertility preservation before HCT rests with the referring oncologist/hematologist as most patients have already received therapy that may have affected fertility prior to arriving at the transplant center. However, keeping informed of advances in assisted reproductive technologies that may be utilized by HCT recipients and making appropriate referrals are an important responsibility of the transplant team. Alterations in sexual health are nearly always related to a combination of biologic, physiologic, and psychosocial variables. To comprehensively assess alterations in sexual health, the healthcare provider must assess multiple biologic, physiologic, and psychosocial variables and the interactions among these variables. In making the diagnosis of a sexual disorder, considerations include the onset of the symptoms, the situational context, the frequency of occurrence, and the psychologic and physiologic conditions that may be contributory. There has been an increased focus on the sexuality of cancer patients and alterations that arise during and following treatment; however, there is a paucity of studies investigating treatment strategies [17]. Key factors for the successful treatment of any alteration in sexual health will be open communication between the couple and a desire on the part of both for sexual activity. Hypoactive sexual desire disorder Sexual desire is the least well understood and most difficult aspect of altered sexual health to treat. Desire is strongly linked to the second phase of the sexual response cycle, arousal, so individuals with hypoactive sexual desire disorder are likely to experience problems with arousal. Many HRQOL investigations of HCT recipients have demonstrated that hypoactive sexual desire disorder and arousal problems are common. The dynamic relationships between the phases of the sexual response cycle cannot be overemphasized. Women who experience dyspareunia may quickly lose interest in sexual activity. Similarly, in men, loss of desire for sexual activity may be secondary to ED. In these situations, interventions need to be targeted at treating the dyspareunia or ED, and not specifically focused on decreased desire. In many cases, the discussion of sexual difficulties with the HCT recipient and partner can be therapeutic. The information, reassurance

Sexuality Following Hematopoietic Cell Transplantation: An Important Health-related Quality of Life Issue

that treatment options are available, and opportunity to explore alterations in sexual health with a healthcare provider can often effectively address many areas of concern [17,79]. In fact, the most effective intervention may be for the healthcare provider to begin a dialog on issues of sexuality [53]. Fatigue, altered body image, decreased self-confidence, depression, anxiety, and uncertainty are key variables to assess when evaluating hypoactive sexual desire disorder. Depression and anxiety in both the HCT recipient and the sexual partner should be evaluated. Assessment of the relationship with the intimate partner needs to be evaluated for stress, discordance of expectations, and adequacy of communication. When couples were asked if there were any arguments or problems related to alterations in their usual sexual activities before HCT, 24 (86%) of the spouses/partners and 20 (71%) of the women reported no arguments, yet the majority reported alterations in sexual activities [59]. These data raise the question as to whether the alterations in sexual health were not a concern of the couple before HCT or whether they were simply not discussing the sensitive issues surrounding altered sexual health. If depression is identified as a contributing factor for hypoactive sexual desire, antidepressant therapy can be helpful. However, the selection of the antidepressant is critical. Delayed or absent orgasm may occur with selective serotonin reuptake inhibitors, which may necessitate a change in dose or alternative antidepressant [53]. Resources for the recovering HCT recipient and his or her partner include educational materials from the transplant center, support groups, and Internet discussion groups. Additionally, the American Cancer Society publishes two books, one for men and one for women, titled “Sexuality and Cancer,” authored by Leslie R. Schover. Testosterone replacement in men may restore sexual desire and arousal [43]. In male HCT recipients with low-to-low-normal testosterone levels, testosterone replacement therapy was effective in improving sexual desire [52]. The role of testosterone in female sexual desire and arousal is not clearly understood, and the use of testosterone therapy in women is controversial [43]. However, some women may find improvement in sexual desire and arousal with testosterone replacement therapy [43,53,80].

POF, vaginal atrophy and dyspareunia Assessment of women with POF includes medical history, physical examination, gynecologic examination, laboratory analysis, hormonal evaluation, medication profile, and a review of coexisting health problems and degree of fatigue. The hormonal testing should include estradiol, FSH, LH, androgen level, and thyroid function tests. Many symptoms of estrogen deficiency can be ameliorated or minimized with hormone replacement therapy (HRT). HRT is effective in relieving hot flashes, improving sleep, maintaining vaginal elasticity and lubrication, and decreasing changes in the appearance of the skin and changes in breast size. One study found that symptoms of estrogen deficiency were eliminated in 22 of 27 women [66], and two other investigations reported almost immediate relief of symptoms in the majority of women treated [23,61]. In a more recent investigation, HRT effectively relieved hot flashes in 66% of female HCT recipients within 10 days (range of 7–15 days) [80]. The HRT also relieved other symptoms; specifically, insomnia was reduced in 66%, improved psychologic and emotional states were noted in 66%, and 53% had a decrease in vulvovaginal atrophy after 5 (range 4–12) weeks of therapy. On gynecologic examination, the appearance of the genitalia returned to normal following HRT. The authors conclude that HRT should be offered to all women with no medical contraindications soon after HCT.

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There are many gaps in knowledge relating to the use of HRT for the treatment of POF in HCT recipients. There are several studies that suggest that currently prescribed HRT may not be adequate for HCT recipients [24,42,66]. The evidence for questioning the adequacy of currently prescribed HRT is the findings of low estradiol levels despite HRT, the large number of women receiving HRT not having menstrual cycles, and the degree of vaginal dryness found despite HRT. Data from one study revealed that, at 3 years post HCT, 76% of the women were taking HRT yet 52% cited lubrication and arousal problems, 33% experienced dyspareunia, and 46% experienced difficulties with orgasm [26]. Findings from this investigation indicate that when HRT is initiated may be important to maximize benefit. Those women who had not started HRT by 1 year post HCT experienced distressing sexual dissatisfaction at 3 years even though they were all taking HRT by this time. The benefits of HRT in relieving symptoms of estrogen deficiency are clear, but the selection of the HRT for female HCT recipients is challenging and requires additional study. One author recommends low-dose estrogen replacement be administered for the first month and increased to full dose in the second month if the woman has only recently become estrogen deficient [56]. However, if she has been estrogen deficient for 12 or more months, low-dose estrogen replacement should be initiated with a gradual increase to maintenance doses by 6 months to avoid sideeffects. A recommended estrogen dose in young women with POF is the equivalent of 1.25 mg of conjugated equine estrogens [56]. In women with an intact uterus, progestins should be added [56]. Careful selection of the type of HRT is critical as some oral contraceptives may reduce circulating free testosterone by increasing sex hormone-binding globulin levels, resulting in diminished sexual desire [43]. In women with adequate estrogen replacement and complaints of diminished desire, fatigue, and a poor sense of wellbeing, testosterone replacement may be beneficial [56,80]. The use of HRT following physiologic menopause has been an area of debate and controversy for some time. Recently, however, the results of the Women’s Health Initiative Study have reported that the risks of HRT – breast cancer, heart attack, stroke, and blood clots – outweigh the benefits in women following physiologic menopause [81]. The risks of HRT in female HCT recipients may not be the same as the risks of HRT in women following physiologic menopause. For females with POF, HRT is administered to compensate for the premature loss of endogenous hormones [39,56]. Selection of the type of HRT should be guided by the woman’s symptoms and preferences, and designed to prevent future complications such as osteoporosis [80]. In general, women with POF should continue HRT until the age of physiologic menopause [56]. Due to the complexities of HRT and the gaps in knowledge regarding HRT for female HCT recipients, a prudent course of action is to refer women to a gynecologist for a thorough evaluation and individualized approach to HRT. This individualized approach must encompass not only the woman’s symptoms and preferences, but also a broad assessment of factors that may increase the risks associated with HRT. These factors include a family history of cancer and cardiovascular disease, genetic markers, hypertension, and hyperlipidemia, as well as lifestyle issues such as exercise patterns, obesity, diet, and smoking history. Nonhormonal strategies proposed for relieving symptoms of estrogen deficiency include the use of herbs, vitamins, yoga, acupressure, acupuncture, exercise, and diet modifications. Few of these interventions have been rigorously tested. In a recent systematic evidence review, Remifemin, a formulation of the herb black cohosh, has evidence to support its use in the relief of hot flashes [82]. Antidepressants are another nonhormonal strategy that can ameliorate some symptoms of menopause. Hot flashes were reduced by 60% with the use of venlafaxine (Effexor) [83]. Other antidepressants that may be

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helpful include paroxetine (Paxil), fluoxetine (Prozac), and citalopram (Celexa) [84]. Gabapentin (Neurontin), clonidine hydrochloride (Catapres), and bellergal (Bellergal-S) are other nonhormonal agents that may decrease hot flashes [84]. Resources are available to women who want to explore nonhormonal strategies for the relief of menopausal symptoms. Several that have been well received by female HCT recipients include: “The Estrogen Decision,” by Susan Lark, published by Celestial Arts in 1999, and “Natural Menopause,” by Susan Perry and Kate O’Hanlan, published by Perseus Books in 1997. These books provide information on the role of HRT and alternative strategies in alleviating symptoms of menopause. Nonhormonal strategies for managing menopausal symptoms will require a consistent commitment by the woman to maximize potential benefits. Female HCT recipients with vaginal atrophy, narrowing, and dyspareunia may benefit from the consistent use of vaginal lubricants and dilators. Vaginal lubricants should be water soluble to avoid the risk of infections. Vaseline is not water-soluble and therefore should not be used as a lubricant. K-Y Jelly® (Johnson & Johnson, New Brunswick, NJ), vitamin E, Replens® (LDS Consumer Products, Cedar Rapids, IA), and Astroglide (Biofilm Inc., Vista, CA) can all be used as a lubricant prior to sexual intercourse. Replens can be used as a vaginal moisturizer as well as a lubricant. As a vaginal moisturizer, one applicator of Replens can be inserted into the vagina three to four times per week at bedtime. Astroglide has been reported by some users to last longer during sexual activity. These products can be found at the local drug store or via the Internet. Dilators can be obtained from a gynecologist or often through the radiation therapy department. Women should lubricate the dilator and then insert the dilator into the vagina until it is slightly uncomfortable and lay with the dilator in place for 10–15 minutes three or four times per week. As the vaginal tissues begin to stretch, the woman can increase the depth of penetration and gradually increase the size of the dilator. The level of commitment for pursuing these measures in a consistent manner will optimize results. A woman experiencing dyspareunia should assume a position during sexual intercourse that allows her to control both the rate and depth of penetration, enabling her to stop penetration if she becomes uncomfortable. Women worried about lubrication and their response to sexual stimulation can be encouraged to self-stimulate, masturbate, as a way to explore their response without simultaneously being concerned about their partner’s needs [85]. A number of medications may affect sexual functioning in women, including antiandrogens, sedatives, antidepressants, and stimulants [86]. Evaluation of the medication profile and adjustment of medications may improve sexual functioning. Chronic GVHD of the vagina can also contribute to vaginal atrophy, stenosis, and dyspareunia. Epithelial cell damage, mononuclear cell inflammatory infiltration, fibrosis, and atrophy are histologic features of chronic GVHD [87]. Additional features of chronic vaginal GVHD include inflammation, stricture formation, narrowing, and obliterated introitus [64,88]. A comprehensive program for the prevention of vaginal chronic GVHD incorporating patient education, topical estrogen, early initiation of HRT, vaginal dilatation in the absence of sexual activity, and regular gynecologic examination minimized the development of severe GVHD of the vagina [62]. These investigators also outlined treatment for established chronic GVHD of the vagina, which included topical steroids, topical cyclosporine, and vaginal dilatation in women who had evidence of vaginal narrowing. Fifteen of the 28 women treated for chronic GVHD of the vagina had complete resolution of symptoms, eight noted improvement, and in five women the symptoms remained stable. Unlike other reports of chronic GVHD of the vagina [64], no patients in this report required surgical intervention.

Erectile dysfunction Assessment of ED includes a medical history and physical examination, a review of the medication profile, evaluation of coexisting health problems such as hypertension, anemia, hyperlipidemia, and fatigue, and an endocrine evaluation. Hormonal testing should include total testosterone, free (bioavailable) testosterone, sex hormone-binding globulin, FSH, LH, prolactin, and thyroid function [49,52]. Additionally, the transplant team can make a referral to a urologist for a comprehensive evaluation of the functional and structural capacity of the penis [89]. In men, testosterone is strongly linked to both sexual desire and arousal. Testosterone replacement therapy often restores sexual desire and arousability in the subset of men with low testosterone levels, and testosterone replacement therapy may also improve erectile function [43,52]. Testosterone replacement therapy is available as an intramuscular injection, transdermal patches, gel and oral forms. Testosterone enanthate (Delatestryl) and testosterone cypionate (Depo-Testosterone) are two testosterone replacement products available [49,89]. The recommended dose is 100–200 mg intramuscularly every 2–4 weeks [89]. Testosterone replacement is contraindicated in men with a history of prostate cancer, and routine monitoring of prostate-specific antigen levels should be performed at 6-month intervals [90]. Testosterone replacement therapy can contribute to coronary artery disease by its effect of lowering high-density lipoprotein levels [89]. Hyperprolactinemia observed in some male HCT recipients is associated with decreased desire and ED. The use of bromocriptine mesylate (Parlodel) can be effective in lowering prolactin levels [91]. Performance anxiety can lead to ED and subsequently decreased libido. Suggesting self-stimulation, masturbation, to men may improve their confidence in their erectile function in a setting where they do not need to worry about their partner’s needs [85]. Cognitive interference can lead to ED, so encouraging the man to stay very focused on sensations during sexual activity will help minimize negative thoughts and performance concerns. Suggesting that the couple prolong foreplay will help to ensure that the male is aroused and may also improve erectile function. Antihypertensives, anticholinergics, antihistamines, cardiac medications, antidepressants, alcohol, and other recreational drugs can all contribute to ED. A thorough review of the medication profile is an important component of the assessment for the etiology of ED. Reducing doses or trying alternative medications may result in improved sexual functioning. Sildenafil (Viagra) and other phosphodiesterase inhibitors such as vardenafil (Levitra) and tadalafil (Cialis) have made an important contribution to the effective treatment of men with ED. Sildenafil does not improve sexual desire but increases the penile response to sexual stimulation [32,49]. Sildenafil should be taken approximately 1 hour prior to sexual activity in a dose of 20–100 mg orally. The side-effects of sildenafil are generally mild and well tolerated, and include disturbed color vision, headache, flushing, and dyspepsia [32]. Sildenafil is contraindicated in men taking nitrates for angina or hypertension. A number of other strategies for the treatment of ED, such as cavernosal injections, vacuum devises, and surgical interventions, are available, but in general these strategies are not well accepted by men [53]. One group of investigators hypothesized that a combination of testosterone replacement therapy combined with sildenafil would improve erectile functioning in men following HCT [92]. The sample consisted of eight men aged 22–58 years who were 2–24 months post HCT with evidence of hypogonadism, ED, decreased libido, ejaculatory disorders, and documented cavernosal arterial insufficiency. The men were treated with a combination of testosterone replacement therapy given monthly for 6 months and sildenafil taken one to two times per week. Results

Sexuality Following Hematopoietic Cell Transplantation: An Important Health-related Quality of Life Issue

indicated that all eight men had an improvement in erectile function allowing for satisfactory sexual intercourse, seven men had resolution of ejaculatory dysfunction, and all had an improvement in both total and free testosterone levels. The authors concluded that this combination therapy is a safe and effective therapy for ED in HCT recipients and should be evaluated in a larger clinical trial.

Areas for future research Over the past two decades, investigators have explored the HRQOL of HCT recipients and have identified a number of positive and negative sequelae that influence HRQOL. The next step is for investigators interested in improving the HRQOL of HCT recipients to begin to design and test interventions to minimize the negative sequelae, particularly strategies to minimize alterations in sexual health. Throughout this chapter, areas for future study have been raised. Designing and testing an education program to help HCT recipients and their spouses/partners develop realistic expectations of possible changes in health, including alterations in sexual health, may bring expectations and possible outcomes closer together and thereby decrease psychologic distress. Many research questions can be generated regarding the HCT recipient’s caregiver. One question concerns what the issues and concerns of caregivers are during and after HCT. In addition, what strategies can be designed and tested to address caregivers’ concerns? Additional investigation is needed to develop insights into the effects on intimate relationships when the spouse/partner is the caregiver. Investigations of the biologic, physiologic, and psychosocial variables that underlie alterations in sexual health need to continue. The identification of variables that can predict which HCT recipients are at risk for alterations in sexual health is needed. The effects of nonmyeloablative transplantation on fertility and sexual health have not been fully explored. Anxiety and depression are known to be long-term psychologic issues for some HCT recipients. What intervention strategies reduce these negative emotional consequences? Questions regarding HRT therapy for female HCT recipients include: (1) what is the most efficacious combination of hormone replacement?; (2) does testosterone therapy benefit women by improving libido and arousal?; (3) when is the best time to begin HRT?; (4) how well does HRT protect women from the long-term sequelae of POF, such as osteoporosis and cardiovascular disease?; (5) what strategies can improve

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adherence to the use of HRT?; and (6) what nonhormonal strategies are effective in alleviating symptoms of estrogen deficiency? The monitoring and prophylactic measures described earlier for vaginal chronic GVHD were effective in preventing the development of severe GVHD, avoiding the need for surgical intervention [62]. What additional preventative strategies can be employed? Since most HCT recipients during the time of risk for chronic GVHD have returned to their homes and referring physicians, what can the HCT team do to see this program implemented by a woman’s local gynecologist? The understanding that cavernosal arterial insufficiency is a significant cause of ED in HCT recipients led to the development of a treatment strategy that included the use of testosterone replacement therapy plus sildenafil, which was very effective in a small number of male HCT recipients [92]. This intervention strategy needs to be investigated in a larger sample of men suffering from ED post HCT.

Conclusion Sexuality is a broad concept encompassing much more than sexual activity, and remains an important aspect of being human regardless of the diagnosis and treatment of life-threatening illness. The understanding of alterations in sexual health following HCT is not complete. It is clear that the etiology of alterations in sexual health involves biologic, physiologic and psychosocial components, as well as the complex interactions among these variables. While understanding the etiology of alterations in sexual health is not complete, enough knowledge exists to allow healthcare professionals to begin to address this critical aspect of HRQOL. A beginning step is to include an assessment of sexual health as part of routine care following HCT. It has been shown that an open discussion of sexuality with a healthcare professional can be therapeutic and address many areas of concern. Developing a referral network of other healthcare professionals, including gynecologists, urologists, reproductive specialists, and mental health professionals, enables the HCT team to further assist HCT recipients with alterations in sexual health. While the focus of this chapter has been alterations in sexual health following HCT, it must be stated that many HCT recipients report satisfying sexual relationships after transplant [6,24,93]. The challenge for the HCT team is to identify those HCT recipients who are experiencing alterations in sexual health and intervene to improve sexual functioning, decrease sexual dissatisfaction, and thereby improve their HRQOL.

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Susan K. Stewart

Hematopoietic Cell Transplantation: The Patient’s Perspective I still find it almost bizarre that I would be the one to get so sick. I was very physically active. I rode my bike 2,000 miles a year, swam about 100 miles and was active in my children’s lives. Leukemia happens to people you read about in the newspaper during their appeals for donors or money for transplants. It doesn’t strike enormously healthy, happy and vital 39-year-old family men. HCT survivor Mike Eckhardt, 1995

Preparing for transplant Like Mike, most people have never contemplated the notion that they may some day have an illness that requires treatment with hematopoietic cell transplantation (HCT). Regardless of whether a patient is newly diagnosed or previously learned of the illness and has exhausted other therapies, the prospect of HCT is traumatic for both the patient and his or her loved ones. Having lived through this experience myself in 1989, after being diagnosed with acute myelogenous leukemia, I know first hand how difficult it can be to process information under duress, make life or death decisions, and cope emotionally with the HCT experience. In 1990, I created Blood & Marrow Transplant Information Network (BMT InfoNet; www.bmtinfonet.org) to help other families navigate the HCT experience. Through lay-language books about HCT, survivorship symposia, and peer counseling, BMT InfoNet services more than 10,000 patients and their loved ones each year. Their experiences help inform this chapter. Patients are overwhelmed with fear: fear that the transplant will not cure; fear that the transplant might kill; fear of pain; and fear that the side-effects of treatment will be intolerable. Many are also angry and/or depressed over the loss of control over their wellbeing. A disease now controls their body, and the patient must rely on a team of strangers to save his or her life. Preparing a patient for the rigors of HCT against this backdrop of fear, anger, and/or depression is challenging. Some patients are so overwhelmed emotionally that they hear and process little of what a physician tells them. They can process only discrete packets of information, are overwhelmed by details, and prefer to leave much of the decision making in the hands of the physician. At the other end of the spectrum are the patients who attempt to regain control over their body by accumulating as much information as possible about their disease and treatment options. They want complete, detailed information about the risks and benefits of treatment. They will search external sources, such as the Internet, for information. They want to be fully engaged as partners in their care. Since patients’ styles of coping with troubling medical information vary greatly, it is important to ascertain in advance how much information a patient wants, and how best to deliver it. Level of education, cultural biases about discussing illness and death, the degree of trust a

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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patient has in the medical establishment, as well as individual coping styles will define the best method of discussing treatment options with a patient. Asking how much detail the patient wants when discussing medical treatments, and whether he or she prefers to hear it all at once or in small segments, enables a physician to tailor the delivery of information to best suit each individual patient. Despite these differences, there are certain universal elements of communication that are helpful to bear in mind when preparing a patient for transplant.

Put the risks into perspective When a patient meets with the transplant team, a long list of potential treatment-related complications is described. For many, this is the first time they have heard a detailed discussion of the risks associated with HCT. The prospect of developing these complications is frightening for patients, and many leave the consult with the transplant team more depressed than when they arrived. It was like a whirlwind, a dream. One day our child was a normal 15-year-old boy who would live to be 80. The next day we were staring at blackboard diagrams about transplants, and hearing doctors tell us our son might die. It wasn’t real. We didn’t understand. All we could do was hug each other and cry. (Lorraine Boldt, mother of transplant survivor)

Patients need help putting the risks associated with HCT into perspective. Grouping the complications into (a) those that always occur, (b) those that often occur, and (c) those that seldom occur provides patients with a manageable framework for understanding relative risks and coping emotionally with the prospect of complications. Knowing that the risk of developing severe liver damage is small, while the risk of developing mucositis is high, for example, conveys a far more accurate picture of what a patient should expect. Without such a framework, patients may imagine the risk of developing very serious complications to be greater than it is. It is also important to distinguish between those side-effects that are temporary and those that may persist long term. Most people believe they can deal with a problem for a discrete period of time. Knowing in advance what the normal range of duration is for each complication enables patients to better cope with the difficulty when it arises. Without such information, patients can become distressed if a complication persists, and may become concerned that it signals relapse, disease progression or another serious problem.

Hematopoietic Cell Transplantation: The Patient’s Perspective

It is equally important to discuss how complications will be managed. Although a detailed discussion of each therapy is not necessary, patients should know that treatment for each complication exists. This is particularly important when discussing painful complications, since some patients fear pain more than death. Many patients do not automatically assume that pain medication will be available to relieve discomfort. This must be explicitly stated. Leave out this discussion and a patient can assume that he or she will suffer tremendous pain, or that the complication is not treatable, and this can increase the emotional load.

Communication in lay language Although many patients are well educated, most are unfamiliar with the medical terms used routinely by transplant personnel. Terms like “CBC”, “aspirate,” “Hickman,” “bilirubin,” “TPN,” and even “stem cells” are often foreign to people without medical training. Even more commonly used words such as “prognosis” and “remission” may be meaningless to patients who have never used these terms. Most patient advocacy groups, as well as the general print media, strive to use language that can be understood by a person with a fifth- or sixth-grade education. Given that HCT patients are under a great deal of emotional stress and may not be able to process information as easily as the average person, medical procedures and terms should be described as simply as possible. In the absence of a clear explanation, patients may misinterpret the message being conveyed by the physician. Using precise language also improves a patient’s understanding of what he or she is about to experience. For example, when describing a painful procedure, using words such as “hurt” or “pain” does not adequately prepare a patient for the sensation he or she will feel. Terms like “pressure”, “stinging sensation” or “dull ache” convey a more precise idea of the sensation. Precise language will help reassure patients that these sensations are normal when they occur.

Repetition and reinforcement is important The volume of information that HCT patients receive is overwhelming, and few completely digest the information during the first presentation. Patients who are emotionally overwhelmed may shut out complicated or frightening information, and later recall only part or none of what was said. It’s important to repeat information at several points during treatment. A patient who was unable to absorb critical information when it was initially presented may be better prepared to absorb it later after the initial shock about the diagnosis and the difficult treatment he or she is about to undergo wanes. Some patients have found video- or audiotaping the initial consultation with the transplant physician to be helpful. Such a record enables them to double-check their understanding of what was said later when reflecting upon the information. It also helps them catch important details they may have missed during the initial presentation. Suggesting this option to patients can provide them with a valuable tool, and can reassure them that the physician wants them to be as informed as possible about the impending treatment. Suggesting that patients bring a support person to the consultation whose sole purpose is to take notes can also be helpful. Proactively suggesting this as well as taping the conversation will help those who might be too distraught to think of these tools on their own. Encouraging patients to ask questions can also enhance understanding. Some patients come from a cultural background where questioning a physician is considered rude or disrespectful. Inviting them to ask questions to confirm their understanding can give them “permission” to do so. Inviting questions means more than simply saying, “Do you have any questions?” Inviting questions in a way that elicits more than a

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yes/no answer may allow patients to better formulate and state their concerns. For example, a physician might say, “Some patients don’t understand what I mean when I say they will become neutropenic. Do you understand what I mean by neutropenic, or should I explain it more clearly?” Such a question normalizes a confusion the patient may have and affirms the physician’s responsibility to make the explanation as clear as possible. Patients who have questions during treatment sometimes do not feel that they have an adequate opportunity to present them to the physician. Creating an atmosphere that encourages questions from patients is essential. A physician who appears busy or distracted can discourage patient questions without meaning to do so, leaving the patient feeling anxious and uninformed. Sitting down when entering a patient’s hospital room, for example, signals to the patient that the physician has time to focus on the patient’s concerns. Consciously making an effort to engage a patient in conversation, rather than simply delivering news, can create an atmosphere that elicits patient concerns. Suggesting that the patient maintain a notebook of questions that arise when the physician is not present can also help patients organize their thoughts.

Different learning styles It is well accepted by educators that individuals have different styles of processing information and learning. A person who is primarily a visual learner does best when charts or pictures accompany the explanation. Seeing the person with whom he or she is talking, rather than just hearing an explanation, enhances understanding. An untidy environment can interfere with this patient’s ability to absorb information. Auditory learners, on the other hand, do best with verbal explanations. They are more likely to use telephone support systems, such as helplines, than pore over charts or diagrams that explain a problem. They are easily distracted by sounds. Kinesthetic learners learn by doing. They like hands-on explanations and can be distracted by movement. When educating transplant patients, accommodating the variety of learning styles is important. A person who is primarily a visual learner can miss key information if the education plan relies solely on verbal explanations. Patients who are primarily auditory learners can have difficulty absorbing print information. Kinesthetic learners may benefit from a tour of the facilities where procedures will be performed, with an opportunity to handle equipment that will be used during transplant. Although it may be difficult to ascertain each individual’s learning style, providing the patient with a menu of learning tools will maximize the likelihood that he or she will absorb important information. Augmenting verbal explanations of procedures with written publications or audiovisual aids produced by organizations like BMT InfoNet or the National Marrow Donor Program (NMDP; www.marrow.org) enables a patient to choose the information vehicle that best suits his or her needs.

Discussing pain control is important Many patients fear pain even more than death. Yet while all transplant teams inform patients about potential complications, some neglect to discuss the steps that will be taken to manage pain. They assume that patients know that pain medication will be administered as needed. Many patients, however, have had little experience with hospitalization and painful medical procedures, and do not automatically assume that pain relief will be available. Others have undergone painful medical procedures without adequate pain control and have little reason to assume that pain will be managed more effectively by the transplant team.

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Explicitly telling patients that pain control medications will be used to prevent and control pain, and educating them about when to ask for pain relief, can ease their anxiety. Many patients try to be stoic, asking for pain medication only after pain becomes intense. Explaining that pain is easier to manage before it becomes intolerable, normalizing the use of pain medications in the transplant setting, and encouraging patients to request relief before pain becomes unbearable can minimize the discomfort and anxiety a patient experiences. If a patient-controlled analgesia device will be available, letting the patient know that he or she will have some control over the administration of pain medication can make the prospect of pain more manageable. It is equally important to warn patients and caregivers in advance about possible side-effects of pain medications. Most are unprepared for the change in mental status that accompanies the use of some medication, and both patients and caregivers can become alarmed when this occurs. Patients can see or hear things that are not real and talk nonsensically. Caregivers who observe this without understanding that this is a side-effect of the medication, and patients who later realize they have been hallucinating, can become emotionally distraught. Assuring both patient and caregiver that these drug reactions are within the range of normal and are temporary can alleviate unnecessary anxiety. Fear of addiction discourages some patients from asking for pain relief. Patients, as well as many physicians, do not realize that drug addiction is a psychological phenomenon, whereas drug dependence is a physical phenomenon. Although a patient may develop a physical dependence on a drug, he or she can be weaned from the drug by tapering the dosage. An addiction, however, requires psychological interventions to address the underlying cause of the addiction, as well as tapering or discontinuation of the drug. Patients who have never before had a drug addiction are unlikely to develop an addiction to pain medications administered as part of HCT [1].

Discuss psychological difficulties One important area of patient education that is often overlooked is a discussion of the psychological difficulties that patients and family members encounter during transplant. Patients may be told that the transplant will be a “difficult” or “stressful” experience, but these words fail to describe or normalize the type and depth of emotions the patient and his or her family are likely to experience. If not given this information, a patient becoming angry or depressed during treatment and recovery may assume that there is something wrong – that he or she is a weakling or is not coping as well with the stress as most patients. After all, the patient reasons, if these emotional difficulties were common among HCT patients, the medical team would have said so in advance, just as they warned of the other medical complications. Some become angry with themselves for not coping well, which exacerbates their emotional distress. As difficult as the transplant experience is for patients, it can be even more difficult for the family member/caregiver who is keenly aware of the patient’s progress or lack thereof. The person upon whom the caregiver normally relies for support – the partner – may be the patient, who is ill equipped to deal with anyone’s emotional load besides his or her own. Usual support networks, like family members or friends, may fail to comprehend the severity of the situation and be unprepared to address the caregiver’s needs. It is important that the health-care team be explicit with both the patient and caregiver about the range of emotions they may experience during and after transplantation. Anger about the disease, resentment over the loss of control of the patient’s wellbeing, fear about medical procedures or possible treatment failure, and depression about the slow pace of progress are typical emotions patients and caregivers face. The

patient may express distress by lashing out at the medical team or a loved one. Assuring both patient and caregiver that these emotions are normal, and explicitly telling them what types of assistance are available to help them cope, maximizes the likelihood that they will seek and accept help, should they need it. Patients need to understand that HCT is an extraordinary experience and that they can expect to need more than their everyday coping tools to manage the stress. Normalizing the use of antianxiety or antidepressant medications in this circumstance, and assuring the patient that, in the absence of a history of substance abuse, drug dependency is not likely, can alleviate patients’ concerns about using these tools. For some patients, the acute care period is the time of greatest stress. For others, the post-transplant period poses the greatest psychological challenge. The support system provided by the hospital and medical personnel has been curtailed, the busy daily schedule of tests, doctor visits and other hospital routines is now replaced by long days of isolation and inactivity, and the challenge of resuming a normal lifestyle can be daunting. When I left the hospital, I thought the tough part was over. I’ve never been so wrong. The inpatient part was the easiest part of the bone marrow transplant process. I was focused and fired up. . . All the worldly things, such as my role as a husband and father, were secondary to winning the inpatient battle. Other routine things such as house payments, medical bill, career and church weren’t even on my mind. Nurses and doctors took care of me. It wasn’t easy, but I felt I was making significant progress toward beating my disease. The clarity of purpose and sense of progress were lost when I came home. Instead of feeling like a successful patient, I felt like a failed person. All those worldly things, such as my roles as husband and father that I had ignored in the hospital, came roaring back. They were once again important and I felt woefully inadequate in those roles. The steroids and cyclosporine made me extremely emotional and irrational. I would cry because I had too much milk on my cereal. I couldn’t sleep (steroids), couldn’t shower (Hickman), couldn’t read (no concentration), couldn’t drink coffee in the morning (nausea), couldn’t exercise (no strength) and couldn’t get close to my children (might get an infection). Even taking my medicine was confusing and overwhelming. I spent time worrying about things I couldn’t control. I was convinced I was going to run out of money, lose my house, my dog, etc. There was no measured progress anymore. I felt I was regressing. I was in an emotional rut. [2]

Emotional difficulties can surface weeks or months after transplant. Unexpected rehospitalization for a complication can cause distress. Some experience symptoms similar to post-traumatic stress syndrome: a sight or smell may trigger an unpleasant memory of the transplant experience, leaving the patient shaken. Returning to work, school or a social world that has moved on during transplantation can make a patient feel isolated. Fear of relapse causes many patients to avoid long-term planning. It can take many months before a patient is able to get through a single day without thinking about the transplant. A recent study has confirmed what many caregivers report: the intensity and duration of transplant-related distress is in some ways greater for caregivers than for patients long term [3]. The caregiver must deal not only with his or her own fears about the patient’s health, but with those of other family members as well. The caregiver may be thrust into new roles, assuming responsibilities once handled by the recovering spouse. These tasks, coupled with his or her own emotional turmoil, can create significant distress. Some who handled the acute phase of transplant well find themselves emotionally drained and unable to fully function many months later:

Hematopoietic Cell Transplantation: The Patient’s Perspective In some ways, it was like we were no longer married. We were never alone. Our whole relationship – everything – was based on illness, medical procedures, hospital and family. Trying to resume a normal relationship was not possible. Family was continually around – always. I understood, but some days I didn’t want to share my husband. I wanted him all to myself. I had to take over all of his responsibilities – paying bills, yard work, etc. Instead of being an equal partner, it was like having another child to worry about. I felt like I’d lost the man I married and just wanted him back.

Psychosocial support networks for patients and their families A number of support networks are available for families facing the emotional challenges associated with transplant. BMT InfoNet has a Caring Connections Program that links HCT patients and family members with survivors who can provide emotional support. Patients and survivors are paired according to diagnosis, type of transplant, and other factors the patient may deem important such as gender, transplant center, religion, etc. Survivors are screened to ensure they provide emotional support, rather than medical information, and the pairs communicate by phone or email. This service is particularly helpful for patients who do not have access to, or prefer not to communicate by email. Patients can access this service at www.bmtinfonet.org/patient.html. Many patients and family members find list-servs on the internet helpful. A list-serv is an electronic mailing list that enables a person to discuss issues with a large number of peers online, and to offer and get support. The list-serv is managed by an individual who typically screens postings before emailing them to the entire list, although the screening is generally limited to weeding out commercial solicitations and hostile emails. Members can opt to receive individual emails from other members after they have been posted, or a daily digest of emails that enables them to determine, by the subject heading, whether any of the issues discussed in the posts are of interest to them. Patients can respond to a posting on the list-serv by emailing the entire list or by privately emailing the individual who posted it. Discussions on HCT-related list-servs are lively, and many patients find the list-serv an invaluable place to vent their feelings and concerns with others who understand and will empathize. A great strength of listservs is that they can validate a person’s feelings, and normalize his or her experience. A downside is the potential for sharing medically inaccurate or inappropriate information, although patients can easily verify the information with their own physician. More often, valuable information about medical interventions that have worked with other patients is shared which the patient can discuss with his or her physician. The Association of Online Cancer Resources (ACOR; www.acor.org) maintains a list-serv for HCT patients (BMT-Talk, 1443 subscribers), one for patients with graft-versus-host disease (GVHD, 348 subscribers), and list-servs organized by diagnosis. Anyone can subscribe via the ACOR website by following the links under Mailing Lists. A real-time online chat room also exists for HCT patients and caregivers. BMT-Support (www.bmtsupport.org) meets several times weekly. There are separate chat rooms for patients and for caregivers. Participants log in at the specified time, and the live online chat is moderated by a survivor with nursing training. Disease-specific organizations, such as the Leukemia & Lymphoma Society or International Myeloma Foundation, offer local or online support programs for patients. While some transplant patients find these helpful, others find discussions with people who have not undergone a transplant unsatisfying. A list of organizations that provide disease-spe-

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cific information and support can be found on BMT InfoNet’s website at www.bmtinfonet.org/resource. General support groups for caregivers are offered by the Well Spouse Foundation (www.wellspouse.org) and by general cancer support organizations such as Gilda’s Club (www.gildasclub.org), the Wellness Community (www.thewellnesscommunity.org), and local cancer support centers. Young adult patients often find resources targeted to their age group helpful. The Ulman Cancer Fund for Young Adults (www.ulmanfund. org) provides a peer support program similar to BMT InfoNet’s Caring Connections Program. Young adults can be linked with others whose life has been affected by transplant and read their stories online. The fund also provides educational scholarships to young adults diagnosed with cancer. Planet Cancer (www.planetcancer.org) also offers online support programs. Some patients or their family members require individual counseling to help deal with their emotional distress. Although transplant centers may provide psychiatric help and counseling while the patient is undergoing transplant, that support system is often unavailable when patients return home. It can be difficult to find a local mental health professional who is experienced in dealing with cancer or transplant related issues. The American Psychosocial Oncology Society (www.apos-society.org) offers a help-line for patients and families seeking a local specialist who is trained in working with cancer patients and can address their needs.

Quality of life post transplant From the patient’s perspective, successful HCT does not simply mean that the patient survives. It also means that he or she resumes a reasonably normal life after transplant. Significant survivorship challenges are often not discussed during the acute phase of treatment, and even at discharge, patients may not be adequately prepared for the psychological stress that ensues. They may also not be equipped with coping strategies for medical difficulties such as depression, infertility, cognitive problems, chronic fatigue, and sexual difficulties that can occur after HCT. In 2006, BMT InfoNet invited HCT survivors and their family members to complete an online survey to determine issues of concern to survivors post transplant. Invitations were issued by email to persons on the Blood and Marrow Transplant Newsletter mailing list, and via the home page of BMT InfoNet’s website. Five hundred and eighty persons completed the survey. Respondents identified themselves as: • an HCT survivor – 68%; • the parent of a survivor – 12%; • the spouse/significant other of survivor – 11%; • the child of a survivor – 1%; • the sibling of a survivor – 1%; • other (caregivers or transplant center staff) – 8%. Forty percent had an autologous transplant, 34% had an allogeneic transplant with a related donor, and 26% had an allogeneic transplant with an unrelated donor. Respondents reported their last date of transplant as follows: • 2001–06 – 55%; • 1996–2000 – 25%; • 1990–95 – 17%; • pre-1990 – 3%. When asked which issues, based on their experience, were most important to transplant survivors, respondents checked the following options (more than one answer being allowed): • Emotional/psychological health of transplant survivor – 72%. • Detecting and preventing long-term complications – 71%. • Fatigue – 63%. • Emotional/psychological health of the survivor’s family – 50%.

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• Chronic GVHD – 49%. • Learning and memory problem – 46%. • Health insurance – 44%. • Educating local physicians about transplant survivors’ needs – 43%. • Secondary cancers – 41%. • Sexuality after transplant – 37%. In response to an open-ended question, survivors expressed frustration over the lack of resources to deal with the psychological burden after transplant, including significant depression. They also expressed concern with problems that interfered with their ability to resume work or school and function effectively, such as cognitive deficits or fatigue. Input from this survey was used to develop a 1-day survivorship symposium for HCT survivors in Oakbrook, Illinois, April 2007. Presentations by experts on fatigue, psychosocial problems post transplant, cognitive issues, chronic GVHD, sexuality, infertility, insurance, and employment issues were featured. Three hundred and fifty survivors and caregivers from throughout the United States attended this educational event, which also featured psychosocial support groups where survivors could network with each other. For most, this was the first opportunity post transplant to hear presentations on issues that were significantly impacting their daily life, such as psychosocial difficulties, cognitive issues, and insurance issues, and to discuss them with other survivors. Not only were their experiences validated, but also they learned tools for managing some of the problems. Evaluations were overwhelmingly positive, and more forums of this type were requested. Many who attended the session on infertility remarked that they had been poorly equipped by their transplant program for this side-effect of treatment. Not only had prophylactic measures, such as sperm banking, been ignored in some cases, but few resources were offered them when it was confirmed they were infertile, either because of prior treatment or because of the transplant. One attendee reported that she had not been advised that infertility was a potential complication of transplant. For some patients, the prospect of infertility post transplant is as upsetting as the diagnosis itself. Disappointment over the prospect of infertility is not limited to patients who have never had children but extends to some individuals who already have children and would like to expand their family. Patients’ emotional distress can be lessened if they are advised that it is sometimes possible to conceive a child post transplant. Men may be able to bank a sufficient quantity of viable sperm before treatment to enable attempts at pregnancy post transplant, provided that prior therapy has not led to aspermia. Some women may be able to conceive a child with assisted reproduction techniques and a donor oocyte. While these steps do not guarantee a successful pregnancy, the technology is improving each year, and it comforts many patients to know that there is at least a chance of conceiving or carrying a child to term. Even if a woman does not become infertile immediately after transplantation, the chemotherapy or radiation administered as part of the HCT can reduce the number of viable oocytes and cause her to become prematurely menopausal later on. Thus, women who wish to conceive a child post transplant should be counseled that their window of opportunity may have shortened, and plans for pregnancy should be pursued as early as practical [4]. Many transplant survivors experience significant, long-term changes in sexuality following treatment. Problems including lack of libido, inability to perform, and pain are reported by persons with both normal and abnormal hormone levels post transplant. As one man stated, “It seems like my brain has been cut off from the rest of my body. I can conjure up all sorts of juicy sexual images in my brain, but can’t get the rest of my body to perform.”

Changes in sexuality can seriously stress a relationship, particularly if the survivor’s partner believes that the problem is purely psychological rather than physical. While fatigue and psychological trauma do, indeed, affect sexuality, it is important that patients and their partners know that physical changes occur following transplant that may affect the survivor’s sexuality as well. Patients should be advised that they may need to develop new approaches to intimacy after transplant to enable both partners to have a satisfying sexual relationship. Patients should be encouraged to report sexual difficulties and be provided with referrals to experts who may be able to help resolve some of the problems. A significant issue that is often overlooked in discussions with patients is changes in cognitive abilities they may experience, both short term and long term. Much has been written about learning difficulties observed in children following HCT, but little note has been taken of the cognitive changes experienced by some adult HCT survivors. Problems reported by survivors include poor concentration, memory problems, stuttering, difficulty in spelling, inability to perform jobs that were previously mastered, and difficulty in learning new tasks. Many survivors have been able to overcome the problem by changing the way that they manage information, such as making lists, writing down all appointments, keeping notes, and so forth. For others, the solution is not so simple, and the problem may interfere with their ability to perform a job or learn a new skill. One survivor who was a sculptor, said, “Before my HCT, conceptualizing the design and drawing it was the most difficult part of my work. When it came to the actual sculpting, I could do it without even thinking. However, after my HCT, I could barely concentrate on the sculpting. Conceptualizing and drawing the design became impossible. No one has been able to explain why this happened, or suggest what I can do to relieve the problem.” One woman who underwent high-dose chemotherapy and autologous HCT was delighted that her company held her computer operator job for her while she underwent treatment. However, when she returned to work 4 months later, she found that the nature of the job had changed. She needed extensive training to perform the new tasks. After 2 weeks of training, she feared she would be fired. As she explained, “When I’m at work and in training, all the explanations make perfect sense. But then I come home, fix dinner, and clean up. When I sit down to review the day’s lesson I’m totally confused. By the following morning, I’ve completely forgotten everything I learned the previous day.” Although most patients do not develop cognitive problems as severe as those experienced by the two persons described above, patients should be advised that changes in cognitive abilities sometimes occur. If cognitive problems do develop, survivors will be less frustrated if they know that their problem is not unique. Advance warning about this potential problem can also relieve tension in the home. Tired and distraught family members will understand that the survivor’s new forgetfulness is not simply laziness, but a consequence of the treatment.

Chronic GVHD Survivors with chronic GVHD face a unique set of challenges post transplant that can severely impact their quality of life. Preliminary results of a 2007 survey conducted by BMT InfoNet of more than 300 transplant survivors who have or had chronic GVHD found that chronic GHVD interfered with the daily activities of nearly two-thirds of patients and nearly 40% of other family members. Forty-one percent reported financial difficulties as a result of chronic GVHD, and nearly a third reported marital difficulties. Fifty-seven percent of survivors with chronic GVHD reported sadness or depression, and 41% said the mental health of other family members had been negatively impacted as well.

Hematopoietic Cell Transplantation: The Patient’s Perspective

Survivors with significant chronic GVHD often feel quite isolated. There are few HCT support groups available for patients after transplant, and disease-specific support groups seldom include a significant number of survivors who have been through transplant, let alone those who have experienced chronic GVHD. Improvements in treatment are rare, and opportunities to network and learn from other survivors are quite limited. For those with internet access, a list-serv on ACOR provides a forum for patients with chronic GVHD. List members discuss their symptoms, therapies they have tried to manage them, and clinical trials they have participated in. Often, a newly diagnosed person or a list member who has become overwhelmed with the difficulties presented by chronic GVHD shares his distress with list-mates, and the outpouring of emotional support is quite impressive. When the patients with chronic GVHD surveyed by BMT InfoNet were asked what resources would be helpful, 98% said a central website with chronic GVHD information, 93% said a print newsletter about chronic GVHD, and more than 8% said a DVD with information about chronic GVHD, a local meeting with transplant doctors to discuss chronic GVHD or a local support group would help. BMT InfoNet is exploring the feasibility of hosting a chronic GVHD website as a centralized resource for patients.

Other resources for patients and survivors As described earlier, a number of resources are currently available for families dealing with the prospect of HCT as well as survivors. BMT InfoNet BMT InfoNet provides online resources for patients during all phases of treatment: • Transplant center directory (www.bmtinfonet.org/centers): an online directory of transplant programs in the United States and Canada providing detailed information on each program, including the medical director and key medical personnel; whether or not the center is accredited by the Foundation for Accreditation of Cellular Therapy and/or is an affiliate of the NMDP; the number of transplants performed, by type, in each of the three previous years; patient age and donor match criteria; and contact information. Patients can search the database by name of center, state, diagnosis, and age of patient (pediatric or adult). Data posted are provided directly to BMT InfoNet from each transplant center and are updated annually. • Blood and Marrow Transplant Newsletter (www.bmtinfonet.org/ newsletters/index.html): an archive of back newsletter issues, written in lay language, featuring advances in research, patient vignettes, and resources of interest to HCT patients and survivors. • BMT InfoNet books (www.bmtinfonet.org/books.html): access to patient handbooks about HCT and being a caregiver, and a calendar featuring transplant survivors, including one patient guide about HCT in Spanish. • Survivorship forum DVD: videotaped presentations and slides from BMT InfoNet’s Celebrating a Second Chance at Life Survivorship Conference. • Caring Connections Program (www.bmtinfonet.org/patient.html): a moderated peer support program linking patients with survivors who can provide emotional support. • Resource directory (www.bmtinfonet.org/resource): a list of organizations that provide disease-specific information and financial support to HCT patients. • E-newsletter: an electronic bulletin with information of interest to patients.

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• Help-line (www.bmtinfonet.org/contact.html): a service enabling patients to pose questions to a panel of medical experts. • Survivorship forums: regional conferences to educate patients and survivors. • Attorney referral service (www.bmtinfonet.org/attorney.html): for patients having difficulty securing insurance approval for their transplant or related treatment. • Directory of drugs used during transplant (www.bmtinfonet.org/ drug): describes the purpose of the drug in the transplant setting, and potential side-effects grouped by likelihood of occurrence. In addition, patients can phone 888-5897-7674 for personalized help with any of the above services. National Marrow Donor Program The NMDP provides resources for patients undergoing a HCT with an unrelated donor, some of which are also appropriate for patients transplanted with a related donor or undergoing an autologous transplant: • Information about the donor search and HCT process. • Transplant center directory: this details the cost of HCT with an unrelated donor at transplant programs in the United States as well as medical personnel at each center; the number of transplants performed for specific diseases; raw and risk-adjusted survival rates; and contact information. • DVDs: for special interest groups including young adults, pediatric patients, and older patients. • List of cord blood banks: including contact information and criteria for donating cord blood. • Financial guide: a workbook to help patients organize their finances. • Survivorship newsletter: discussing some of the issues survivors face 3 months to 2 years after transplant. Some of the online information is available in Spanish and other languages. The NMDP’s Office of Patient Advocacy (OPA) provides phone counseling for patients undergoing HCT with an unrelated donor, including help with tracking donor searches and research on disease- and treatment-related topics. • National BMT-Link (www.nbmtlink.org) offers a guide for HCT caregivers, a DVD entitled The New Normal, and a peer support program for HCT patients. Periodic teleconferences and an in-person support group for Michigan residents are also offered. Center for International Blood & Marrow Transplant Research The Center (http://cibmtr.org/PUBLICATIONS/guidelines.html) provides consensus guidelines on long-term follow-up care for patients after HCT. Recommendations are outlined for 6-month, 1-year, and annual check-ups. A separate version is available for patients and physicians. The information can be downloaded, or hard copies can be ordered. The Children’s Oncology Group The Children’s Oncology Group (www.survivorshipguidelines.org) provides long-term follow-up guidelines for pediatric cancer survivors. National Institutes of Health Sponsored Clinical Trials (http://clinicaltrials.gov) lists federally supported clinical trials with separate descriptions available for health-care professionals and the lay public. Included are enrollment criteria, contact information for principal investigators, and a brief description of the trial’s objectives.

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The Clinical Center (http://clinicalcenter.nih.gov) in Bethesda, MD, provides free clinical care to patients who qualify for one of their clinical trials. Protocols for some HCT patients are available. Other resources • The Children’s Hospital Oakland Research Institute (www.chori.org) offers free cord blood banking for families who have a child with a disease that is treatable by HCT. • Private HLA typing (www.bonemarrowtest.com) to determine whether a person is a potential donor for a family member is sometimes requested by a physician without providing guidance to the potential donor on how to be tested. Some DNA labs provide this service only through a contract with a medical facility. Tepnel Diagnostics will arrange the service directly with the potential donor and provides necessary testing supplies and results via mail. • Disease-specific information and fundraising or financial assistance for HCT patients is provided by a number of organizations. A list of such groups is maintained by BMT InfoNet (www.bmtinfonet.org/ resource). • The ACOR (www.acor.org) provides information about various diagnoses and treatment options, as well as list-servs where patients and survivors can connect. The HCT group (BMT-Talk) and GVHD group are both very active and supportive lists. • BMT-Support (www.bmtsupport.org) offers a real-time online chat room each week for HCT patients and caregivers. The chat is moderated by an HCT survivor with a nursing background.

Although HCT creates enormous challenges for patients and their loved ones, many survivors find personal growth in the experience. When asked what he would advise patients about to go through transplant, Steve Dugan, a 4-year survivor of an allogeneic HCT, offered this advice: Expect to be amazed by how much strength you find in yourself. Expect the shock of what’s happening to you to continue for some time. Stolid acceptance of your situation is just not realistic. Expect to feel very alone and, at times, even detached from your surroundings. Expect to be shown, by the person closest to you, what love really means – maybe for the first time in your life. For me, that was, and always will be, my wife and best friend, Joyce. In Greg Allman’s words: “When life’s game gets so hard, I hold the highest card – the Queen of Hearts.” That’s Joyce! Expect there to be days when you say to yourself, “I just can’t do this anymore.” Expect to make outrageous promises to God in exchange for clemency. Expect to find a whole new group of heroes – nurses. Expect for your real friends to show up. Be prepared for surprises – both good and bad. Expect to be truly shaken – and, at times, depressed, by your physical appearance. Just remember: The mirror reflects what you look like – not who you are. Expect to catch yourself sometimes staring at your children, not wanting to miss a single breath they take.

Conclusion Undergoing HCT is a very difficult experience both physically and emotionally. Working with a compassionate medical team that is attuned not only to the patient’s physical needs, but to his or her emotional needs as well, is a blessing that all patients will deeply appreciate. Being fully armed with information about long-term quality of life issues will help make the HCT patient’s transition from being a successful patient to a successful survivor much easier. Transplant physicians need not be the sole source of information for patients and survivors. Directing patients to resources outside the transplant program’s area of expertise such as experts in fertility, sexuality, cognitive retraining, fatigue management, and psychosocial support will expand a patient’s support network and enhance the likelihood that the survivor and his or her family will live well, even in spite of ongoing medical concerns.

But most importantly: Expect and believe that someday soon you will look at this whole episode of your life – in the rearview mirror.

Kathleen Jones, who underwent two HCTs after being diagnosed with chronic myeloid leukemia, summarizes a perspective shared by many survivors: The bone marrow transplants affected my life in every way. I am thankful to wake up every morning. I have learned compassion and empathy for those unable to be normal. I have learned not to spend time worrying about little unimportant things. I have learned to appreciate sunrises and sunsets. I have learned what it means when others are kind. I have learned the value of my family in my life, and just how precious life is, and how much I want to live it.

References 1. Syrjala K. Relieving pain. BMT Newsletter 1993; 16. 2. BMT Newsletter 1996; 33. 3. Bishop M, Hahn E, Brady M et al. The gift of life comes with a price: the impact of hematopoietic cell

transplant on the long-term quality of life of survivors and their spouses. Clinical Abstract, American Society for Blood and Marrow Transplantation/ Center for International Blood & Marrow Transplant Research Tandem Meeting, 2004.

4. Rinehart J. Director, Division of Reproductive Endocrinology and Infertility at Evanston Northwestern Healthcare, Evanston, IL. Presentation at BMT InfoNet Celebrating a Second Chance at Life Survivorship Symposium, April 2007.

Section 4 Sources of Hematopoietic Cells for Hematopoietic Cell Transplantation

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Phyllis I. Warkentin & Elizabeth J. Shpall

Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation

Introduction Hematopoietic cell transplantation (HCT) is now established therapy for many serious congenital and acquired diseases of hematopoietic origin, as well as many high-risk malignancies. Hematopoietic cells (HCs) obtained from bone marrow, peripheral blood cells, and placental and umbilical cord blood cells are common graft sources derived from autologous, related or unrelated donors. HCs are also being used for functions other than as progenitor cells, such as post-transplant donor lymphocyte infusion [1]. Cellular therapies have also expanded beyond hematopoietic and immunologic reconstitution to new therapies using mesenchymal stem cells, dendritic cells, and gene-modified and other cells, and to efforts at regenerative cellular interventions [2]. Rapid evolution of such therapies has heightened concerns about the safety, purity, potency, and efficacy of these cellular products.

Governmental regulation Federal regulation of human cells, tissues, and cellular and tissue-based products (HCT/Ps) is based upon the authority of the Public Health Services (PHS) Act and the Federal Food, Drug, and Cosmetic Act. Under these Acts, the Food and Drug Administration (FDA) is empowered to enact binding legislation in the form of rules, which are intended to implement, interpret or prescribe law or policy. In 1997, the FDA published its approach to the regulation of HCT/Ps [3]. The goal of the regulatory framework is a unified and tiered approach to the regulation of traditional and new products, including HCs, that would provide more uniformity with only the amount of regulation necessary to protect the public health [3]. Products that are expected to be at a higher risk for disease transmission or at risk for contamination during collection or processing, such as allogeneic cells or cells highly manipulated ex vivo, are subject to more regulation than those products with less risk, such as autologous or minimally manipulated products. The FDA regulations are based on five public health and regulatory concerns: (1) prevention of the transmission of communicable disease; (2) assurance that necessary processing controls exist to prevent contamination of cells and tissues and to preserve their integrity and function; (3) assurance of clinical safety and effectiveness; (4) assurance of necessary product labeling, including permissible promotion for proper

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

product use; and (5) establishment of a mechanism for the FDA to communicate with the cell and tissue industry. A Tissue Reference Group, comprised of representatives of the Center for Biologics Evaluation and Research and the Center for Devices and Radiological Health, was also proposed and established to provide a single reference point within the FDA for all regulatory questions about HCT/Ps. The regulatory structure for the wide range of cellular therapies described above has been defined in Part 1271 of Chapter 21 of the Code of Federal Regulations (21 CFR 1271) [4]. This regulation was created to establish a uniform system for registration of manufacturers of HCT/ Ps, to delineate donor eligibility criteria, and to describe current good tissue practices and other procedures to prevent the introduction, transmission, and spread of communicable diseases. This regulation covers the spectrum of hematopoietic cellular products, from those not manipulated ex vivo, to stem cell-based therapies involving highly processed cells, cultured, differentiated or otherwise manipulated ex vivo, that may be used for other than their normal function, may be used for metabolic purposes, and/or may be combined with nontissue components [5,6]. This regulation includes HCT/Ps regulated solely under the PHS Act, Section 361, those which meet the following criteria: 1 The product is minimally manipulated. 2 The product is intended for homologous use only, as reflected by the labeling, advertising or other indications of the manufacturer’s objective intent. 3 The product does not involve combination of the cells or tissue with another article (excepting water, crystalloid, or sterilizing, preserving or storage agent). 4 The product does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function (unless autologous, first- or second-degree relative donor or for reproductive use) [4]. The regulations in 21 CFR 1271 also apply to those products regulated as “biologic products” under the PHS Act Section 351. These are cells or tissues that are highly processed, used for other than their normal function, combined with nontissue components, and/or are used for metabolic purposes [5]. Other regulations also apply to these “biologic products,” including current Good Manufacturing Practices and other portions of Chapter 21. Part 1271 of 21 CFR includes the Registration Final Rule, the Donor Eligibility Final Rule, and the Current Good Tissue Practices (cGTP) Final Rule. Subpart A of 21 CFR 1271 describes the scope of the regulation and its relevant definitions. Subpart B describes the requirements for establishment registration and listing of products. Any establishment that participates in the manufacture of HCT/Ps must register with the

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FDA initially and annually, and maintain an updated list of HCT/Ps at least every 6 months. Subpart C defines the requirements for donor eligibility, including donor screening and testing, which is considered to be part of manufacturing, and therefore subject to regulation. Donor screening is the review of relevant medical records for indications of past or present infection and for risk factors for a relevant communicable disease. Donor testing is performing laboratory tests on a specimen, generally a blood specimen, collected from the donor to determine if she or he has been exposed to or infected with a relevant communicable disease or its agent. Infectious disease screening and testing requirements are based on risk. Screening and testing are not required for autologous donors. For allogeneic donors of HCs (that include viable leukocyte rich products), relevant communicable diseases and agents include at least the following: human immunodeficiency virus type 1, human immunodeficiency virus type 2, human T-lymphotrophic virus type I, human lymphotrophic virus type II, hepatitis B virus, hepatitis C virus, Treponema pallidum, and human transmissible spongiform encephalopathy, including Creutzfeldt–Jakob disease. Subparts D, E, and F of 21 CFR 1271 constitute the cGTP rule, including provisions for inspection and enforcement. The cGTP rule is a set of regulations intended to prevent the introduction, transmission, and spread of communicable disease by helping to ensure that products do not contain relevant communicable disease agents, that products are not contaminated during manufacturing, and that the function and integrity of products are not impaired through improper processing [4]. GTP regulations include requirements for the establishment and maintenance of a comprehensive Quality Management Program; adequate organizational structure; sufficient personnel; adequate facilities; environmental control and monitoring; adequate equipment, supplies, and reagents for the processes carried out in the facility; proper processing, including process change and process validation; proper labeling, claims, and labeling controls; storage, receipt, distribution, and other records; maintenance of complaint files; reporting adverse reactions and product deviations; and procedures for tracking the product from recipient to donor and from donor to recipient or ultimate disposition. In addition to such rules, the FDA issues guidance documents under the provisions of Good Guidance Practices, delineated in the Code of Federal Regulations [7]. Guidance documents describe the Agency’s interpretation of a regulatory issue related to design, production, labeling, promotion, manufacturing, and testing of regulated products; the processing, content, and evaluation or approval of submissions; or inspection and enforcement policies [8]. Guidance documents may be also related to specific issues within a regulation where there may have been confusion among persons or establishments subject to the regulation. Several of these documents are of particular relevance, including the following: Quality Systems Approach to Pharmaceutical CGMP Regulations [9]; Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) [10]; Regulation of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) Small Entity Compliance Guide [11]; and Certain Human Cells, Tissues, and Cellular and Tissue-Based Product (HCT/Ps) Recovered From Donors Who Were Tested For Communicable Diseases Using Pooled Specimens or Diagnostic Tests [12]. Additional guidances are published as Draft Guidance, but still represent the current Agency thinking related to issues in the manufacture of cellular therapy products [13]. Governmental regulation of HC therapy at the state level is fragmented. Many US states have little specific regulation. Some states have adopted mechanisms of qualifying HCT programs and facilities, such as the certificate of need process. Other states have developed a licensure process, often heavily dependent on the standards established by profes-

sional societies. Some states, such as New York, have identified the public health concerns in cellular transplantation, and have adopted direct and specific regulations for processing of and storage facilities for HCT/Ps [14,15]. A number of states have adopted, or are considering, a mechanism of approval for transplantation that requires accreditation by a professional organization. At the present time, both Massachusetts and Maryland require accreditation by the Foundation for the Accreditation of Cellular Therapy (FACT) to perform HCT within those states.

Voluntary professional accreditation Foundation for the Accreditation of Cellular Therapy Historical background FACT was founded in 1995 to promote quality patient care and laboratory practice in HCT through its program of professional standards and voluntary accreditation for the procurement, processing, and transplantation of HC products [16,17]. FACT was initially founded as the Foundation for the Accreditation of Hematopoietic Cell Therapy (FAHCT). The name was changed in December 2001 to encompass, in addition to HC products and therapies, the new and exciting therapies using mesenchymal stem cells, dendritic cells, targeted lymphocytes, genetically modified cells, pancreatic islets, and others. This change followed the lead of the parent organization, the International Society for Hematotherapy and Graft Engineering, which changed its name in 2001 to the International Society for Cellular Therapy (ISCT). FACT is the accreditation arm of two professional societies dedicated to improvement and progress in cellular therapy. The ISCT was formed in 1992 as a professional society of scientists and physicians working in HC manipulation. Its membership includes most of the major HCT programs worldwide. The Regulatory Affairs Committee of ISCT developed the first draft of Standards for Hematopoietic Cell Collection and Processing in 1994. The other parent society of FACT is the American Society of Blood and Marrow Transplantation (ASBMT), formed in 1993 as a professional society of physicians and investigators involved in the clinical conduct of HCT. The ASBMT Clinical Affairs Committee developed the first Clinical Standards for Hematopoietic Cell Transplantation. Believing that quality care can only be achieved if both clinical and laboratory issues are addressed, the International Society for Hematotherapy and Graft Engineering laboratory standards and the ASBMT clinical standards were merged into a single document in December 1994, forming the foundation for the first edition of FAHCT’s Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation, published in 1996 [18]. A companion accreditation manual was published subsequently to provide guidance to applicant facilities and personnel and to FAHCT inspectors [19]. The accreditation manual included each standard, the checklist items related to that standard to be evaluated during the accreditation process, and additional guidance information, including rationale for the standard, explanations, definitions, and examples of alternative methods, approaches, and organizations that would be considered to be in compliance with the standards. The manual also addressed questions and concerns that had been submitted during the period of public comment to the draft first edition of the Standards. The first edition of FAHCT’s Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation was unique in the breadth and depth of activities covered [18]. Standards applied to hematopoietic progenitor cells (HPCs), defined as: self-renewing and/or multipotent stem cells capable of maturation into any of the hematopoietic lineages; lineage-restricted pluripotent progenitor cells; and committed progenitor cells, regardless of tissue source (bone marrow, umbilical cord blood, peripheral blood or other tissue source). These Standards

Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation

also included “therapeutic cells,” defined as nucleated cells from any tissue source (marrow, peripheral blood, and umbilical cord blood) collected for therapeutic use other than as HPCs. FAHCT Standards applied to all phases of the collection, processing, storage, and administration of these cells, including various manipulations such as removal or enrichment of various cell populations, expansion of HC populations, and cryopreservation. These Standards do not address the collection, processing or administration of erythrocytes, mature granulocytes, platelets, plasma or plasma-derived components, or products intended for transfusion support. The FACT Standards define an infrastructure required for the safe and efficacious collection, processing, storage, and use of HCs. They define the minimum education and experience necessary for staff participating in these activities, and require an ongoing assessment of activities. There is a minimum requirement that patient outcome be monitored by at least the tracking of neutrophil and platelet engraftment. FACT Standards do not define a required structure or form for the transplant program, nor do they prescribe the clinical use of HCs. In 1997, it became apparent that the field of placental and umbilical cord blood banking was more complex than addressed in the first edition of the FAHCT HPC Standards, and that additional standards were required to address these complexities. Representatives of FAHCT, ISCT, and ASBMT collaborated with members of NetCord, an international organization of independent cord blood banks, to draft additional standards for cord blood banking and to establish a parallel accreditation program. The first edition of NetCord–FACT International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release was developed by consensus of international experts in the field, initially published in June 2000, and revised in 2002 [20]. These NetCord–FAHCT Standards superseded all relevant sections relating to cord blood in the first edition of FAHCT’s Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation, excepting those clinical standards related to the transplantation of cord blood cells. Now in the third edition, these international standards require all cord blood banks to maintain a comprehensive quality management program, to document the training of all collection and processing staff, to utilize validated methods, supplies, reagents, and equipment, to maintain product tracking, and to maintain details of clinical outcome [21]. These Standards form the basis for the voluntary accreditation of cord blood banks worldwide. Fifteen cord blood banks from the United States, Europe, and the United Kingdom have achieved FACT–NetCord accreditation. FACT representatives have also worked with colleagues from the European Group for Blood and Marrow Transplantation (EBMT) and ISCT-Europe, to establish the Joint Accreditation Committee of ISCTEurope and EBMT (JACIE) [22]. The primary aim of JACIE is to improve the quality of HCT in Europe through its accreditation and education programs, and to work toward international harmonization of standards and regulations. JACIE adopted the first edition of the FAHCT Standards in 1999 [23]. The second edition of the Standards was jointly reviewed by FACT and JACIE [24]. Most recently, the third edition of the Standards, published in 2006, was jointly developed and entitled FACT–JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration [25]. FACT and JACIE collaborated in three training workshops in Barcelona, Spain (January 2000, March 2001, and May 2002) to share accreditation tools and experience, and to initiate the European accreditation program. Following a pilot project in Spain between 2000 and 2003, during which FACT inspectors performed the first on-site survey, the JACIE accreditation program was fully implemented in January 2004 with support from the European Union under the Public Health Programme (2003–08). The JACIE accreditation process is similar but not

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identical to the FACT process described below. Since 2004, 41 centers in 13 countries in Europe have been accredited by JACIE. JACIE has developed various documents based on the FACT model, and made use of on-line publications, registration, and inspection and accreditation processes. During this process, JACIE inspectors and staff found that almost all centers were functioning at a high level of excellence, with the majority having only minor deficiencies noted at the on-site inspection. When formally surveyed, these centers reported that implementation of JACIE accreditation required a significant investment of time and resources, but all believed that the result was a demonstrable improvement in the accredited program [26]. Additional information and documents are available on the JACIE website (www.jacie.org). FACT is now an established nonprofit organization with a central office and staff in Omaha, Nebraska. The core of FACT is its active Board of Directors, comprising an equal number of representatives from ISCT and ASBMT, the Presidents-elect of these two parent organizations, the FACT Medical Director, and the Chairperson of the Standards Committee, who represents ASBMT, ISCT or both. Standing Committees of the Board oversee the activities of the Foundation. The FACT Board of Directors approves all publications and sets the agenda for the Foundation. The infrastructure of the organization includes Chief Executive and Operations Officers, Medical Director, Director of Standards and Training, Quality Assurance Director, Information Technology Director, and Accreditation Coordinators. Standards All FACT Standards are developed by consensus of experts active in the field. Wherever possible, standards are based on established evidence from the literature. Standards are also reviewed by legal counsel and internally for technical accuracy, consistency, and regulatory compliance. Every effort is made to incorporate sound recommendations that foster quality medical and laboratory practice; however, no Standards can guarantee the successful outcome of cellular therapies. Draft standards are published for comment by members of ASBMT, EBMT, ISCT, NetCord, other practitioners in cellular therapy, and the general public. Each comment is discussed and carefully considered by the Standards Committee, and incorporated as appropriate. All Standards require compliance with applicable law, but, as appropriate, requirements of the Standards may exceed the minimum regulatory requirements. In addition, these Standards are not intended to be the only means of complying with the standards of care in a community or industry. Standards are developed by the Standards Subcommittees and Oversight Committees. A Standards Committee Chairperson is appointed by the FACT Board of Directors for a term of 3 years to encompass the development and publication of one edition of each set of standards, cellular therapy and cord blood banking. FACT–JACIE Standards are developed by three subcommittees: Clinical, Collection, and Laboratory Processing. Each subcommittee has a FACT representative and a JACIE representative as a co-chair, as well as additional members representing both organizations. An Oversight Committee, including all co-chairpersons, ensures consistency among the sections of the edition. NetCord– FACT Standards for Cord Blood Banking are developed by separate subcommittees for Collection, Laboratory, and Quality Management and Banking. NetCord and FACT representatives co-chair the subcommittees, which also include additional experts in cord blood banking. FACT–JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration These Cellular Therapy Standards are designed to provide minimum guidelines for facilities and individuals performing HCT and related cellular therapies. FACT Standards require that all clinical, collection,

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and processing facilities develop and maintain a comprehensive Quality Management Plan that includes at least the following components: defined organizational structure; personnel requirements; process development; agreements; outcome analysis; audits; management of errors, accidents, and adverse events; document control; product tracking; and, where appropriate, validation and qualification [27]. The current edition also includes many of the regulatory requirements from the United States FDA and the Directives of the European Union, including donor eligibility and product labeling [4,28–30]. The cellular therapy product proper names as defined in Standards are consistent with the names and definitions proposed for inclusion in the official terminology of ISBT 128 [31,32]. Standards for each of the services or facilities participating in the cellular therapy program describe facility requirements, standard operating procedures, and personnel requirements for the area, including minimum education, training, experience, and competencies for each position. In addition, all services participating in a cellular therapy program are expected to maintain active and clear communications with each other. Histocompatibility testing (human leukocyte antigen-A, B, and DR) must be performed by a laboratory accredited by the American Society for Histocompatibility and Immunogenetics or the European Federation for Immunogenetics. All other laboratory testing must be performed by laboratories appropriately licensed and/or accredited for the specific assays. Clinical standards define a blood and marrow transplant program as an integrated medical team housed in geographically contiguous or proximate space with a single Clinical Program Director and common staff training programs, protocols, and quality management systems. The Clinical Program must use HC collection and processing facilities that meet FACT–JACIE Standards with respect to their interactions with that clinical program. Clinical standards also enumerate support staff; cover donor evaluation, selection, eligibility, and consents; provide minimal guidelines for administration of cellular product therapy, including the preparative regimen of high-dose therapy; describe the appropriate management of clinical research and Institutional Review Board-approved protocols; and require the maintenance of complete and accurate records. Standards for cell collection define elements common to both bone marrow- and apheresis-derived peripheral blood HCs, as well as detail those requirements unique to each cell source, such as administration of mobilizing growth factor, potential need for a central venous catheter, and general anesthesia for marrow harvest. The intent is to provide the framework for donor evaluation and cell collection that will foster safe care for both patient and donor. Comprehensive laboratory standards detail requirements for personnel; process controls; inventory management; validation and qualification of facilities, supplies, reagents and equipment; labels and labeling; storage; transport; and records. The intent is to establish that the laboratory is operated in a responsible and responsive manner, that deviations in processes or products are noted, and that the recipient’s physician is aware of any adverse event that could compromise the cellular therapy product. Laboratory and collection personnel are expected to follow clinical outcome as one measure of product safety and efficacy. There are several notable changes in the third edition of the HPC Standards. The document has been restructured so that the three sections are aligned and consistent throughout. International language, understood uniformly, is utilized whenever possible, and the regulatory requirements of the European Directive have been included with those of the FDA where applicable. Specifically, many of the requirements detailed in the GTP regulations have been incorporated. The Quality Management requirements have been expanded to include specifically detailed elements. Pediatric competencies have been added for the Collection Services as well as the Clinical Program. The minimum number

requirements have been changed to allow smaller programs performing autologous transplant only to be eligible for accreditation; however, programs with more than one clinical site must still treat at least five new patients per year in each site. There are now minimum number or duration requirements for both the collection service and processing laboratory. FACT–JACIE Standards and the accompanying accreditation guidance manual are available in print and online at the FACT website (www.factwebsite.org). NetCord–FACT International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release These Standards are intended for the field of cord blood banking, in which a cord blood bank is defined as an integrated team responsible for the collection, processing, testing, banking, selection, and release of cord blood units [21]. It is important to note that the Standards begin with the processes of maternal donor recruitment, consent, and screening, and the process of collection, rather than only covering those processes occurring in the laboratory. The Standards apply to both the banks responsible for cord blood units collected, stored, and reserved for use by a designated individual or family (“private” banking), as well as those banks responsible for units collected, stored, and donated for use by unrelated recipients. There are some differences between Standards for family units and those for unrelated donor units. Most Standards are similar; however, the methodologies employed to meet these Standards may be somewhat different in the two situations. The nature of the collection sites and the relationships among the bank, the cord blood unit collector, the donor, and the collection facility are among the prominent differences in the Standards for related and unrelated units. Similar to the Cellular Therapy Product Standards, these standards require that each cord blood bank establish and maintain a comprehensive quality management program that covers all aspects of the operation and includes at least the following: organizational structure; personnel requirements, qualifications, training, and competency; systems for document creation, review, control, and maintenance; quality assessments and audits; detection, investigation, reporting, corrective action, and follow-up of errors, accidents, biological product deviations, adverse events, and complaints; validation, qualification, calibration, and maintenance of equipment, supplies, reagents, and materials; inventory control for reagents and products; process controls; systems for product identification, labeling, and tracking; outcome analysis; facilities and safety management; donor suitability determination; vendor qualification; and agreements with third parties. The bank staff is required to follow clinical outcomes from each unit released for transplant in sufficient detail to ensure that the procedures in use continuously provide a safe and effective product. There are standards for unique issues that may face a bank, such as inventory transfer or interruption of operations at established collection or laboratory sites. Comprehensive processing, storage, and labeling standards are consistent with ISBT 128 terminology and labeling requirements [31,32]. To accompany the third edition of the Cord Blood Banking Standards, there is, for the first time, a guidance manual that provides explanations, examples, and clarifications, similar to that accompanying the HPC Standards [33]. Both the third edition Standards and guidance manual are available in print from the FACT office and online at the FACT website (www.factwebsite.org). Accreditation process FACT accreditation is a voluntary process based on documented compliance with the published standards as assessed by an evaluation of submitted written information and an on-site inspection of the applicant program or facility. The evaluation of submitted materials is completed

Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation Table 37.1 Foundation for the Accreditation of Cellular Therapy (FACT) inspector qualifications FACT hematopoietic progenitor cell inspector • Meet all educational and experience requirements for the position • Individual member of ISCT, ASBMT, American Society for Apheresis, or NetCord • Affiliated with FACT-accredited or applicant facility or cord blood bank • Has attended a FACT or FACT–NetCord training course, passed a written examination, and completed successfully a relevant inspection as a trainee • Has submitted formal application, confidentiality and other required agreements Clinical program inspector • Is a licensed physician • Has a minimum of 2 years’ experience in hematopoietic progenitor cell transplantation Apheresis inspector • Has a relevant doctoral, nursing or biological science degree • Has completed formal training in apheresis or has at least 1 year’s experience in peripheral blood progenitor cell collection by apheresis as a director, physician, or supervisor or associate supervisor Cell processing facility inspector • Has a relevant doctoral or biological science degree • Has at least 2 years’ experience as director, medical director or supervisor of a cellular therapy processing facility Cord blood bank inspector • Individual member of organizations above, plus ISCT-Europe, EBMT or JACIE Cord blood bank collection inspector • Has a relevant doctoral, nursing or biological science degree • Has at least 1 year’s experience as a collection supervisor in a cord blood bank, or is an active FACT or JACIE clinical or collection inspector Cord blood bank laboratory inspector • Has a relevant doctoral or biological science degree • Has at least 1 year’s experience as director, medical director or supervisor of a cord blood bank laboratory or hematopoietic progenitor cell processing laboratory See text for abbreviations.

by the Accreditation Program Chairman, designee, or appropriate member of the inspection team. One of the strengths of the program is that all inspectors are active in the field of HC therapy, and meet the minimum qualifications for all inspectors for the FACT Accreditation Program. All inspectors are unpaid volunteers who meet the minimum FACT inspector qualifications as listed in Table 37.1. To promote uniformity and consistency in the process and fairness to applicant facilities, it is important that inspectors have a common, up-to-date understanding of the principles of the FACT Standards, and of the approach that the organization takes to the inspection and accreditation. In addition, all inspectors are annually required to submit documentation of potential conflicts of interest, and to verify agreement with the confidentiality and other FACT policies. Although the FACT–JACIE HPC Standards are common, the accreditation processes for FACT and JACIE are separate. Currently, FACT accreditation is open to programs in North America, Australia, and China. JACIE accreditation is applicable in Europe. In addition, the international NetCord–FACT process for cord

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blood bank accreditation, managed by the FACT staff, is a parallel but separate process. The goal of the accreditation process is to raise the bar of performance for all HCT programs and the support services that contribute to their activities, in the expectation that these improvements will lead to better patient outcomes. It is not the goal to be punitive, but rather to be educational and helpful to enable capable and committed personnel to achieve accreditation. Assessments by peers and experts in the field contribute to the ability of FACT to accomplish this goal. The process for initial FACT accreditation of HCT, illustrated in Fig. 37.1, is as follows: 1 Registration. A Program or Facility Director will register interest in accreditation for one or more of the transplant-related services using the demographic registration form available from the FACT office or website. The FACT Accreditation Office staff will review the registration information and determine if the program or facility is eligible for accreditation. 2 Application. An inspection packet is sent to eligible applicants, detailing accreditation requirements and process, including: (a) instructions for completion of the formal application process; (b) a list of documents that must accompany the formal application (licenses, evidence of Board certification, Quality Management documents, etc.); and (c) the Inspection Checklist that lists each of the items to be inspected during the on-site inspection. The applicant personnel will answer each item on the checklist as it pertains to their program, and submit the completed checklist to the FACT office. The inspector will verify each of these items at the on-site inspection, using the checklist as part of the formal inspection report. Use of this checklist ensures that the program is measured against the Standards, and that the inspection will be complete. Completion and submission of the checklist by the applicant is expected within 12 months of its receipt. 3 On-site inspection. The submitted materials are reviewed by a designated FACT Accreditation Coordinator. The results of this review are shared with the applicant, giving the applicant the opportunity to complete missing items or make any corrections to potential problems detected by the Coordinator. The applicant team submits potential dates for the on-site inspection when all key personnel will be available to participate in the process. The inspection team is selected from the available inspectorate based upon the size and complexity of the applicant program, assuring that members of the team have the training and experience necessary to assess all HCT activities. Potential inspectors are asked to recuse themselves if they perceive a potential duplicity of interest that could interfere with the objectivity of the inspection. The proposed inspection team is identified to the applicant facility for approval before the assignment is complete. Inspectors may also be replaced if the applicant perceives a potential conflict of interest. Approximately 3–4 weeks prior to the scheduled on-site inspection, a copy of the application materials is sent to each inspector for review prior to departure for the program. Any missing documents should be requested during this time, and questions clarified. The on-site inspection should be completed in 1 day, starting with introductions and ending with a summation of major observations, but not a final determination of accreditation status. A written report of the observations by the inspection team is submitted to the FACT Accreditation Coordinator as soon as possible after the on-site inspection, including any documents collected on-site, the completed checklist, and a report of all citations observed. 4 Accreditation Committee Review. The FACT Accreditation Coordinator will review the inspectors’ report and all submitted documents, prepare a summary report, and present this report to the Accreditation Committee. The Accreditation Committee is chaired by the FACT

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Application and fees submitted to FACT office

Program or facility eligible?

Inspection scheduled, team assigned

Reapply

No

On-site inspection conducted

Applicant fees returned

Inspection reports and documents returned to FACT office

Yes Inspection instructions and checklist to applicant

FACT staff document review and summary

Inspection materials returned, reviewed by FACT staff

Applicant materials complete?

Inspection results presented to FACT Accreditation Committee No

Applicant contacted for omissions Outcome and next steps determined

Yes

Applicant notified

Deficiencies corrected

Yes

Reinspection required? No Deficiencies corrected

Accreditation awarded

Medical Director, and has a membership of active inspectors representing all three of the major areas (clinical, collection, and laboratory), the chair of the Standards Committee, at least some persons with several years’ experience on the committee, and at least some members of the Board of Directors. Individuals serve 2-year terms on this committee, and are eligible for election to repeat terms. The Accreditation Committee meets at least monthly by conference call, and reviews in detail every inspection report. The possible outcomes are listed in Table 37.2. The Board of Directors retains ultimate responsibility for these outcomes, handles any complaints or appeals, hears and decides contentious issues, and makes any precedent-setting decisions. The Program Director is notified of the outcome, with any instructions necessary to complete the process and achieve accreditation. 5 Accreditation. Accreditation is awarded based on documented compliance with Standards. Any deficiencies identified must be corrected and these corrections documented. The Accreditation Committee may

Fig. 37.1 Foundation for the Accreditation of Cellular Therapy (FACT) Accreditation Program: accreditation process.

review this documentation if needed. Accreditation is valid for 3 years. Accredited Programs are announced in the newsletters of the ISCT and ASBMT, and on the FACT website. 6 Annual report. All accredited programs and facilities report annually on the number of autologous and allogeneic HCT patients treated in the previous 12 months, and on any significant changes in personnel, location or complexity of service. In addition, programs may be asked to document continued compliance with Standards by supplying evidence of implementation of corrections required to achieve accreditation. 7 Accreditation renewal. Programs are expected to have completed the renewal process prior to the expiration of the prior accreditation. At least 9 months prior to expiration of accreditation, each accredited program will receive information and documents from the FACT Accreditation Office to begin the application for renewal. The renewal process is essentially identical to initial accreditation. The applicable standards will be the edition that is current on the day of the on-site inspection.

Hematopoietic Cell Procurement, Processing, and Transplantation: Standards, Accreditation, and Regulation

The first FACT on-site inspection occurred in September 1998; and the first accreditation was awarded in February 1999. Since that time, over 200 programs have submitted an initial application for accreditation. Currently, there are 155 accredited programs in North America, representing an estimated 90% of eligible programs. There are over 160 active inspectors, trained at one or more of 22 North American Training Workshops. Additionally, cord blood bank inspectors have been trained at six International Training Workshops, and 15 cord blood banks have been accredited. In the FACT accreditation process, observations made at the on-site inspection are recorded on the checklist and are determined to be in compliance with the Standards or not in compliance. A deficiency is the failure to comply with a mandatory requirement, stated in the Standards as “shall.” A variance from recommendation is the failure to follow a recommended practice, stated in the Standards as “should.” The inspection summaries of the 145 programs or facilities inspected under the first edition of the FACT Standards (1996) were reviewed by the FACT Accreditation Office to determine the most frequently cited deficiencies and variances from recommendation. This review included only the initial inspection results for these programs, although several Table 37.2 Foundation for the Accreditation of Cellular Therapy Accreditation Program: potential inspection outcomes Accreditation No deficiencies or variances from recommendation observed at the on-site inspection or documented on submitted materials. Full accreditation for 3 years awarded, effective on the date of the Accreditation Committee review Minor deficiencies Minor deficiencies noted at the on-site inspection and/or documented on the submitted materials. Full accreditation can be awarded upon written documentation of satisfactory correction of all deficiencies. The medical director or designee may determine the adequacy of the facility’s response, or the Accreditation Committee may decide to review the responses. Incomplete or unsatisfactory responses may result in a required repeat of the on-site inspection for all or part of the program Significant deficiencies Significant deficiencies, either serious in nature and/or numerous. Full accreditation requires correction of all deficiencies, written documentation of these corrections, and satisfactory completion of a focused reinspection of the areas (clinical, collection, and/or laboratory) where excessive deficiencies have been noted. The results of the focused reinspection will be reviewed by the Accreditation Committee, who will determine the accreditation status of the program Non-accreditation Failure to meet eligibility criteria or to respond to requirement to document corrected deficiencies

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accreditation renewal inspections were also performed under this first edition of Standards. The results from the first 76 programs have been published [34]. Similar to the results observed by JACIE in its accreditation process, the results of recent on-site FACT inspections demonstrate that most programs are functioning at a high level of quality and have addressed at least most of the Standards. Deficiencies observed generally represent failure to completely address a Standard. The most common deficiencies cited across all areas of the HCT programs were deficiencies related to standard operating procedures, both in format and in content. In many cases, the standard operating procedures for entire processes were absent. In addition, clinical programs frequently had deficiencies in data management, in defining and performing quality management activities, and in documentation of personnel qualifications. Cell collection facilities often had not validated processes in use, and often had elements missing in both the informed consent process and on the product collection label. Cell processing laboratories also frequently omitted required label information. Standard operating procedures most often missing or incomplete were those for assessment of staff training and competency, microbial monitoring of products, resolution of ABO/Rh discrepancies, and processes for the return and reissue of previously released HC products. The most commonly observed deficiencies based on inspections related to the third edition of FACT Standards are listed in Table 37.3. Quality Management and Policy and Procedure deficiencies were the most commonly cited in all three areas: Clinical Program, Collection Facility, and Processing Facility. Specifically, the deficiencies that were observed in all areas included missing policies and procedures for customer-reported product failures, concerns or complaints, lack of procedures and/or approval for planned or unplanned deviations, and absence of documentation of corrective action and/or evaluation of its effectiveness. Management of products with positive microbial cultures was frequently cited; however, most programs addressed some but not all of the specific points required by the Standards. Audits were also commonly cited in Collection and Processing Facilities. Frequently, audits were not used effectively as a means for identifying problems and improving operations, were not reviewed to identify trends or opportunities for improvement, or were conducted by either unqualified personnel or personnel directly responsible for the work being audited. Also across all sections, policies and procedures were commonly cited for lack of specific documents or specific processes, such as annual review or documentation of training prior to implementation. Donor consents were commonly cited for failure to document each of the required elements. Significance of FACT accreditation FACT accreditation helps a cancer program attain its ranking among America’s Best Hospitals, published by US News & World Report [35]. As of April 2007, FACT accreditation for allogeneic HCT was awarded one point toward best hospital status. FACT accreditation for autologous HCT only was awarded one-half point. In addition, US News & World Report improved its usefulness and relevance of ranking of pediatric

Table 37.3 Most commonly cited deficiencies related to the third edition of the Foundation for the Accreditation of Cellular Therapy Standards Clinical program

Collection facility

Processing facility

Quality Management (B4) Policies and Procedures (B5) Donor Selection, Evaluation, and Management (B6) Therapy Administration (B7) Personnel (B3)/Data Management (B8)

Quality Management (C4) Policies and Procedures (C5) Labels (C7) Donor Selection, Evaluation, and Management (C6) Cellular Therapy Product Collection Procedure (C8)

Quality Management (D4) Policies and Procedures (D5) Labels (D7) Storage (D9) Process Controls (D6)

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hospitals by including FACT accreditation as a factor in the selection of the Top Ten America’s Best Children’s Hospitals [35]. FACT–JACIE Standards have achieved international acceptance, as best demonstrated by the joint authorship and committee membership in Standards development. In Australia, the Therapeutics Good Administration has accepted the collection and laboratory standards as the regulation for the field. In the United States, cooperative groups require institutions entering patients on HCT trials to have FACT accreditation. Some states, including Massachusetts, Minnesota, and others require or recommend FACT accreditation to operate an HCT program in the state. Most insurance companies require HCT programs to disclose accreditation status as part of the application for Center of Excellence designation. AABB The AABB (formerly the American Association of Blood Banks) is the professional society of 8500 individuals involved in blood banking and transfusion medicine [36]. It also represents over 2200 institutional members, including community and Red Cross blood collection centers, hospital-based blood banks, transfusion services, and cell processing laboratories that collect, process, distribute, and transfuse blood components and HCs. The AABB has a long history of standard-setting activity, having published its first edition of Standards for a Transfusion Service in 1958, the same year it began its program of on-site inspections and accreditation. AABB also publishes Standards for Immunohematology Reference Laboratories, Perioperative Autologous Blood Collection and Administration, and Relationship Testing Laboratories. Approximately 160 programs involved in HC collection or processing worldwide have been accredited by AABB. Originally published as separate volumes, AABB now has current standards for cellular therapy products and services that encompass HC and cord blood services [37–39]. Standards documents of the AABB are based upon a quality management framework. All standards within the documents are of equal importance. Each standard is stated once; then it applies throughout. The quality management standards, combined with the technical requirements, form the total requirements of AABB. Although very different in structure, the content of the AABB Standards for Hematopoietic Cell Services parallels the FACT Standards as published in the first edition. Accreditation by AABB is based upon submission of demographic information and an on-site inspection, coordinated by a paid, full-time quality assessor. Accreditation is valid for 2 years. National Marrow Donor Program The National Marrow Donor Program (NMDP) was founded in 1987 as a cooperative effort of the AABB, the American Red Cross, the Council of Community Blood Centers, and the United States Navy to facilitate volunteer unrelated donor marrow transplantation. It comprises a network of cooperating facilities, including transplant centers, donor centers, marrow collection centers, apheresis centers, cord blood banks, recruitment groups, cooperative donor registries, contract laboratories, and cell and serum repositories. The Health Resources and Services Administration has regulatory authority over NMDP. Since its inception, NMDP has established standards for membership participation. A Standards Committee composed of experts in various aspects of HCT is in place to provide continuous review and revision of these standards. NMDP Standards cover cells obtained from marrow, peripheral blood, and umbilical cord blood. The Standards include standards for participating centers and groups, personnel qualifications, required support services, policy and procedure requirements, confidentiality, recruitment of the unrelated donor, donor medical and laboratory screening and testing, informed consent, donation and transplant process, a few collection, packaging, labeling, and processing standards, and

records requirements; the informed consent process; progenitor cell packaging, labeling, and transportation; and quality control, patient rights, and records. Standards for laboratory processing are less detailed than those of FACT–JACIE, NetCord–FACT or AABB, since most of the processing laboratories and the procedures performed there are an integral part of the recipient’s transplant program, and participating cord blood banks are required to be otherwise accredited. Oversight of investigational procedures is from the Institutional Review Board, the FDA if applicable, and FACT or AABB if the program participates. NMDP Standards are intended to serve the discrete function of qualifying groups, centers, and banks for participation in the registry. NMDP Standards include standards for quality assessment and improvement, and are generously supplemented by operational policies and procedures of the registry. NMDP regularly audits the data submitted by participating programs, collects annual reports, and may perform on-site inspections as deemed necessary.

Future directions In an era when regulation and voluntary professional standards coexist, it is important that the professional organizations continue to lead the field by setting those quality standards demonstrated to be important in patient outcome, to provide the educational materials necessary to programs to meet and exceed regulations, and to continue communication with regulatory bodies to ensure clear understandings and enable appropriate regulation. Recently, several initiatives have been jointly sponsored by professional organizations to serve the needs of the HCT centers and patients. An interorganizational task force developed a Circular of Information for Cellular Therapy Products that can be used in association with cellular therapy products in any center. Another group developed a donor history questionnaire for allogeneic cellular therapy donors to assist staff in meeting requirements of donor eligibility determination. This questionnaire is periodically updated, and is available on the FACT website. In addition, FDA and professional organizations have several mechanisms to communicate concerns as regulations are promulgated and Standards revised. Some of these mechanisms include the presence of an FDA liaison on various Standards and Advisory Committees, a Uniform Donor History Questionnaire Task Force, and participation in education programs, public workshops, and public advisory meetings. In addition, biannual liaison meetings between representatives of the Center for Biologics Evaluation and Research, the Office of Cellular, Tissue and Gene Therapies, and the professional societies representing active professionals in the cellular therapy community continue as a venue for the communication by industry of issues of concern. In December 2005, the C.W. Bill Young Cell Transplantation Program was enacted as a new structure to support unrelated donor HCT in the United States. This legislation includes provisions to increase the potential use of cord blood units in HCT by increasing the national inventory of such units and managing the distribution of cord blood units. In addition, it creates the Stem Cell Therapeutic Outcomes Database, under which outcomes data must be collected on all persons who are allogeneic donors or recipients of HCT, regardless of the tissue source of the product. This will permit the publication of center-specific survival rates for all allogeneic transplants, and the collection of data on uses of HCs for new therapeutic applications such as regenerative medicine. The impact of this legislation remains to be seen. In concept, the development of meaningful and appropriate outcomes measures is supported by experts in HCT, who believe that scientifically based outcomes reporting will lead to improved center quality and best serve both patients and centers [40]. Participation of members of the HCT community will be essential to optimize the data collection and analysis process.

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References 1. Appelbaum FR. Hematopoietic-cell transplantation at 50. N Engl J Med 2007; 357: 1472–5. 2. Rosenzweig A. Cardiac cell therapy – mixed results from mixed cells. N Engl J Med 2006; 355: 1274– 7. 3. Food and Drug Administration, Department of Health and Human Services. A proposed approach to the regulation of cellular and tissue-based products. Fed Regist 1997; 62: 9721–2. 4. Code of Federal Regulations. Washington DC: Office of the Federal Register National Archives and Records Administration, US Government Printing Office; 2006. Chapter 21, Part 1271. 5. Kessler DA, Siegel JP, Noguchi PD et al. Regulation of somatic-cell therapy and gene therapy by the Food and Drug Administration. N Engl J Med 1993; 329: 1169–73. 6. Halme DG, Kessler DA. FDA Regulation of stem cell-based therapies. N Engl J Med 2006; 355: 1730–5. 7. Code of Federal Regulations. Washington DC: Office of the Federal Register National Archives and Records Administration, US Government Printing Office; 2006. 10.115(a). 8. Witten C. FDA and Tissue Regulation. Presented to the World Health Organization, Geneva, Switzerland, June 7–9, 2006. Available at http://www. fda.gov. 9. Guidance for Industry: Quality Systems Approach to Pharmaceutical CGMP Regulations (2006). Available at http://www.fda.gov/cber/gdlns/qualsystem.htm. 10. Guidance for Industry: Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) (2007). Available at http://www.fda.gov/cber/gdlns/ tissdonor.htm. 11. Guidance for Industry: Regulation of Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) Small Entity Compliance Guide. Available at http://www.fda.gov/cber/gdlns/hctpcompl. htm. 12. Guidance for Industry: Certain Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps) Recovered From Donors Who Were Tested For Communicable Diseases Using Pooled Specimens or Diagnostic Tests. Available at http:// www.fda.gov/cber/gdlns/hctppool.htm. 13. CBER Guidances/Guidelines/Points to consider. Available at http://www.fda.gov/cber/guidelines. htm.

14. Ciavarella D, Linden JV. The regulation of hematopoietic stem cell collection and storage: the New York State approach. J Hematother 1992; 1: 201–14. 15. Linden JV, Preti RA, Dracker R. New York State Guidelines for cord blood banking. J Hematother 1997; 6: 535–41. 16. Warkentin PI. Voluntary accreditation of cellular therapies: Foundation for the Accreditation of Cellular Therapy. Cytotherapy 2003; 5: 299–305. 17. Warkentin PI. Professional standards for cellular therapies: Foundation for the Accreditation of Cellular Therapy (FACT). In: Gee AP, editor. The Cellular Therapy Book of the Production Assistance in Cellular Therapy Group. New York: Springer (in press). 18. Foundation for the Accreditation of Hematopoietic Cell Therapy. Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation. Omaha, NE: FACT; 1996. 19. Foundation for the Accreditation of Hematopoietic Cell Therapy. Accreditation Manual. Omaha, NE: FACT; 1996. 20. NetCord/Foundation for the Accreditation of Cellular Therapy. NETCORD-FACT International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release, 2nd edn. Omaha, NE: NetCord/FACT; 2002. 21. NetCord/Foundation for the Accreditation of Cellular Therapy. International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release, 3rd edn. Omaha, NE: NetCord/FACT; 2007. 22. Kvalheim G, Urbano-Ispizua A, Gratwohl A. FAHCT–JACIE Workshop on Accreditation for blood and marrow progenitor cell processing, collection and transplantation. Barcelona, Spain. Cytotherapy 2000; 2: 223–4. 23. Joint Accreditation Committee of ISHAGE-Europe and EBMT. Standards for blood and marrow progenitor cell processing, collection and transplantation. Cytotherapy 2000; 2: 225–46. 24. Foundation for the Accreditation of Cellular Therapy. Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation, 2nd edn. Omaha, NE: FACT; 2002. 25. Foundation for the Accreditation of Cellular Therapy and Joint Accreditation Committee of EBMT and Euro-ISCT. FACT-JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration, 3rd edn. Omaha, NE: FACT-JACIE; 2006.

26. Samson D, Slaper-Cortenbach I, Pamphilon D et al. Current status of JACIE accreditation in Europe: a special report from the Joint Accreditation Committee of the ISCT and the EBMT (JACIE). Bone Marrow Transplant 2007; 39: 133–41. 27. LeMaistre CF, Loberiza FR Jr. What is quality in a transplant program? Biol Blood Marrow Transplant 2005; 11: 241–6. 28. Directive 2004/23/EC of the European Parliament and of the Council (7 April 2004). Available at http://eur-lex.europa.eu/en/index.htm. 29. Commission Directive 2006/17/EC (8 February 2006). Available at http://eur-lex.europa.eu/en/ index.htm. 30. Commission Directive 2006/86/EC (24 October 2006). Available at http://eur-lex.europa.eu/en/ index.htm. 31. Ashford P, Distler P, Gee A et al. Standards for the terminology and labeling of cellular therapy products. Transfusion 2007; 47: 1319–27. 32. Ashford P, Distler P, Gee A et al. ISBT 128 implementation plan for cellular therapy products. Transfusion 2007; 47: 1312–18. 33. NetCord/Foundation for the Accreditation of Hematopoietic Cell Therapy. Accreditation Guidance Manual. Omaha, NE: FACT; 2008. 34. Warkentin PI, Nick L, Shpall EJ. FAHCT accreditation: common deficiencies during on-site inspections. Cytotherapy 2000; 2: 213–20. 35. Comarow A. What it takes to be the best. US News & World Report, July 23–30, 2007, pp. 90–4. 36. Warkentin PI. Regulations and standards for hematopoietic progenitor cell facilities. In: Snyder EL, Haley NR, editors. Hematopoietic Progenitor Cells: A Primer for Medical Professionals. Bethesda, MD: AABB Press; 2000. pp. 201– 19. 37. American Association of Blood Banks. Standards for Hematopoietic Progenitor Cell and Cellular Product Services, 3rd edn. Bethesda, MD: AABB; 2002. 38. American Association of Blood Banks. Standards for Cord Blood Services. Bethesda, MD: AABB; 2001. 39. AABB. Standards for Cellular Therapy Products and Services, 2nd edn. Bethesda, MD: AABB, 2007. 40. Committee on Hematopoietic Cell Therapy Quality Outcomes. ASBMT Committee Report White Paper on Measurement of Quality Outcomes. Biol Blood Marrow Transplant 2006; 12: 594–7.

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Dennis L. Confer, John P. Miller & Jeffrey W. Chell

Bone Marrow and Peripheral Blood Cell Donors and Donor Registries

Introduction Hematopoietic cell (HC) donors provide bone marrow or peripheral blood progenitor (hematopoietic) cells (PBPCs) for more than 20,000 allogeneic HC transplant (HCT) recipients annually. Donations from related allogeneic HC donors continue to outnumber those from unrelated donors, even though at least 70% of HCT candidates lack human leukocyte antigen (HLA)-matched sibling donors. This suggests that only a fraction of the potential patient population is currently being served. As unrelated donor registries grow and international collaboration increases, more patients will have the opportunity for transplantation. Even under the most optimistic scenarios, however, alternative approaches will also be necessary. Umbilical cord blood transplantation (see Chapter 39) and haploidentical transplantation (see Chapter 46) are strategies that will play increasingly important roles in meeting the needs of patients. This chapter discusses the evaluation of HC donors, the risks and side effects of donation, and the logistics of HC collections.

Products from HC donors Bone marrow was until recently the most common graft source for allogeneic HCT [1–4], but since 1995, the use of PBPCs has increased dramatically. Data from the Center for International Blood and Marrow Transplant Research (CIBMTR) show that, in the period 2001–04, about 70% of adult allogeneic transplant recipients received PBPC grafts. National Marrow Donor Program (NMDP) statistics confirm the dramatic growth in the use of PBPCs for unrelated donor transplantation (Fig. 38.1). PBPCs are now the most common choice for both autologous and allogeneic transplantation worldwide. Under normal physiologic conditions, a small number of CD34+ HCs circulate in the peripheral blood, but their concentration is too low to enable efficient collection of an adequate HC dose for transplantation. However, in the mid-1980s, it was discovered that HCs could be mobilized from the bone marrow into the blood following chemotherapy and/or the administration of hematopoietic growth factors such as human recombinant granulocyte colony-stimulating factor (rhG-CSF; filgrastim, Neupogen) and human recombinant granulocyte–macrophage colony-stimulating factor (rhGM-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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CSF; sargramostim, Leukine). Around the same time, advances in apheresis instrumentation and collection techniques occurred such that an adequate number of mobilized HCs (1.0–5.0 × 106 CD34+ cells/kg of patient weight) could be collected from the patient (in the case of autologous transplantation) or an allogeneic donor in one to three daily large-volume apheresis procedures. PBPCs are used almost exclusively for autologous transplantation as durable engraftment occurs more rapidly than with bone marrow. In the allogeneic setting, perhaps because of a larger total dose of T cells and alteration of T-cell function, PBPCs are associated with increased risk of chronic graft-versus-host disease (GVHD). In the matched-sibling setting, both retrospective and prospective studies have demonstrated faster engraftment and higher rates of chronic GVHD with PBPCs compared with marrow, but the impact of PBPC transplantation upon survival and disease-free survival has varied. A recent meta-analysis of nine randomized trials failed to demonstrate a difference in overall survival for PBPC recipients compared with bone marrow recipients [5]. Eapen et al. [6] published CIBMTR data for children receiving HLA-identical sibling grafts showing that PBPCs were associated with higher transplant-related mortality and lower overall survival. The picture in the unrelated donor setting is also unclear. A multicenter prospective randomized trial is currently underway in the Blood and Marrow Transplant Clinical Trials Network to compare survival between recipients of unrelated donor PBPCs and bone marrow. This trial will also assess the impact of PBPCs and bone marrow on both donor and recipient quality of life measures.

Donors of allogeneic bone marrow and PBPC Donors of bone marrow and PBPCs for allogeneic transplantation are either blood relatives of the transplant recipients or unrelated, anonymous volunteers. Related donors Related allogeneic HC donors are usually first-degree relatives of their recipients. Most are full siblings because of the much higher likelihood of complete HLA identity. The family of genes responsible for encoding the proteins of HLA-A, B, C, and DR are colocated in a short region on chromosome 6, and as such are usually inherited en bloc as a single haplotype. Thus, there are only four combinations possible with the two haplotypes of each parent. Between two siblings, the likelihood of two identical HLA haplotypes, a single identical haplotype, and no identical haplotypes is, therefore, 25%, 50%, and 25%, respectively. A parent and

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Number of transplants

3300 3000

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PBHC

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2700 2400 2100 1800 1500 1200 900 600 300 2005

2003

2001

1999

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1993

1991

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Fig. 38.1 Annual transplants from unrelated donors facilitated by the National Marrow Donor Program since its inception. Annual figures include products collected outside the United States and imported, as well as those collected within the United States and exported. Prior to July 1999, only bone marrow was used for initial transplantation, but peripheral blood progenitor cells (PBPCs) were available in the setting of retransplantation. Stippled bars, bone marrow; solid bars, PBPCs; open bars, cord blood units.

their offspring are usually only half-matched (haploidentical), except in the uncommon situation where both parents happen to each possess an identical or nearly identical haplotype. Given that the average size of modern families is just over two offspring, when one child needs an allogeneic transplant, the likelihood of an HLA-identical sibling match is 25–30%. The likelihood of a haploidentical match (full-sibling, halfsibling, parent or child) is much higher, but transplantation using such donors is not yet routine. Unrelated donors and unrelated donor registries Most potential transplant candidates do not have a fully HLA-matched family donor, a limitation that became apparent early in the history of allogeneic transplantation. By the early 1970s, however, case reports began to appear documenting the feasibility of HCT from HLA-matched unrelated donors [7–9]. Such donors were usually gleaned from the files of HLA-typed platelet donors at community blood centers or large hospitals. In 1974 in the UK, the Anthony Nolan Registry was established and dedicated to recruiting and maintaining a file of HLA-typed volunteer bone marrow donors. Within a few years, similar efforts were established in countries throughout Europe, the Americas and Asia. Today, most of the world’s unrelated donor registries collaborate under the auspices of the World Marrow Donor Association (WMDA). The WMDA was organized in 1988 to promote international cooperation and the exchange of HC products [10–12]. The association has established standards and provides guidance on the organization and operation of unrelated donor registries [13,14]. Working groups of the WMDA address issues in quality assurance, registry operations, ethics, finances, and transplant program services [13]. More recently, the WMDA has created an accreditation program for international registries, a Serious Events and Adverse Effects Registry (SEAR) to collect data on serious donor incidents, and a similar registry (SPEAR) to collect productrelated problems and product-related adverse events. The WMDA annually surveys unrelated donor registries around the world [15]. Sixty-seven registries responded to the 2006 survey. Together, these registries listed more than 11,800,000 potential unrelated

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HC donors. Approximately 70% of these donors have been tissue typed for HLA-A, B, and DR, while the remainder has been typed only for HLA-A and B. Most of the registries serve one or more transplant centers in their country; together, the registries reported 1189 transplant centers with potential access to unrelated donors. The Japan Marrow Donor Program reported the largest number of transplant centers, with 192. The WMDA respondents reported collecting 8418 unrelated donor marrow and PBPC products in 2006. In 3269 instances (39%), the collected products were exported to another nation. Germany performed 3029 collections in 2006, which was the most of any nation and represented 36% of the world total. Germany exported 1877 (62%) of the products collected, mostly within the European Union and to North America. The United States was the largest consumer of HC products, with 2293 (27% of the world total), of which 776 (34%) were imported. Worldwide, PBPC and bone marrow collections numbered 5416 (64%) and 3002 (36%), respectively. Registries differ on their policies concerning contact between donors and their transplant recipients. In a recent WMDA survey, 88% of respondents indicated that they allow anonymous contact, for example letter exchanges, between donors and recipients, but only 35% will allow exchange of identifying information. After the first anniversary of transplantation, the NMDP will allow United States donors and recipients to meet provided each consents to release their private information to the other. Much of the world’s available inventory of unrelated adult donors and cord blood units can be accessed through Bone Marrow Donors Worldwide (BMDW). HLA data from 59 registries and 37 cord blood banks, representing more than 11.2 million adult donors and 265,000 cord blood units, were summarized by BMDW in mid-2007. BMDW provides a highly important resource, but is limited by incomplete participation and lack of a comprehensive search function. Given the potential for extreme growth in unrelated donor transplantation, with more than 28,000 transplants annually,1 it is important that a complete worldwide listing of adult donors and cord blood units is made available, together with comprehensive search and matching services. The largest registry in the world is the NMDP, with more than 4.4 million United States registrants, followed by the German National Bone Marrow Donor Registry (ZKRD), with more than 2.8 million. The history of the NMDP registry has been recently reviewed [16]. The NMDP recruits about 350,000 new donors annually. The vast majority of these are recruited at community donor drives, which are held at numerous locations including shopping malls, blood centers, college campuses, community centers, and churches. More than 1000 drives are held each month. New recruits are screened to confirm good health and the absence of high-risk behaviors. Informed consent is administered, after which DNA samples are collected for HLA typing. Blood samples for DNA-based typing have been largely replaced by swabs obtained from the buccal mucosa. HLA typing is performed at an intermediate level of resolution by high-throughput contracted laboratories. Once completed, HLA information is combined with an assigned donor ID and donor demographic information for listing in the NMDP donor database. Private donor information necessary for making direct donor contact is held in separate highly secured computer systems.

1

A total of 12,000 HLA-matched sibling transplants representing 30% of potential allogeneic recipients means that there are 28,000 potential alternative donor recipients (12,000/0.3 − 12,000 = 28,000).

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Donor eligibility and qualification Prior to HC donation, donors must be evaluated to ensure that: (1) they can safely donate, (2) their HCs will be safe for the recipient, and (3) they understand fully what they are being asked to do [17–19].

Regulatory requirements In 1997, the United States Food and Drug Administration (FDA) proposed an approach to the regulation of cells and tissues designed to minimize the risk of contamination and maintain the integrity and function of cell- and tissue-therapy products, including PBPCs and umbilical cord blood HC (bone marrow for transplantation remaining exempt from these regulations because of Congressional legislation) [20]. Subsequently, in 2004, the FDA published three final rules in the US Code of Federal Regulations that describe, among other things, the requirements for registration of establishments that “manufacture” HCs, for determination of donor eligibility, and for compliance with Good Tissue Practices [21]. The FDA determined that minimally manipulated PBPCs and umbilical cord blood HCs, collected from related donors, would be regulated under section 361 of the Public Health Service Act. These regulations are designed to reduce the risk of transmission of relevant communicable diseases, either acquired from the donor or introduced into the products during manufacturing. For HC products from unrelated donors, however, the FDA felt there was a need for additional manufacturing controls, and chose, therefore, to regulate these products under Section 351 of the Public Health Service Act. Section 351 sets additional requirements for Good Manufacturing Practices, which must be followed to ensure the safety, potency, and efficacy of HC products from unrelated donors. The FDA had chosen to phase in its regulatory framework, and this process was incomplete as of mid-2007. Once it is fully implemented, unrelated donor PBPCs must be collected under either an Investigational New Drug application or a Biologics License Application approved or accepted by the FDA. Both related and unrelated donors of PBPCs must be determined to be eligible or ineligible by the screening criteria outlined in the Donor Eligibility Final Rule and the companion FDA guidance document, both of which also provide instructions for correct product labeling [22,23]. The FDA regulatory framework is not unique; similar regulatory frameworks have arisen in the European Union, Canada, Australia and elsewhere. A uniform feature of every regulatory system is establishment of controls to prevent transmission of infectious agents by HC products. There are numerous infectious diseases that, if present in the HC donor, would pose a definite or theoretical risk to the transplant recipient. It is clear that HCs can transmit many of the same infections that are transmissible by blood transfusion including hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV). In addition, some congenital or acquired conditions in the donor, such as genetic defects and immune system abnormalities, may be transmissible to the recipient. Minimizing the risk of disease transmission requires combining the information obtained from a targeted behavioral/medical history, a search for the physical signs of disease, and the results of laboratory testing for specific pathogens [22,23]. HC donors, similar to blood donors, should complete a written questionnaire that has been specifically designed to elicit medical history and identify behaviors or activities that may increase the risk of infectious disease transmission [17–19,22,23]. Questions regarding high-risk behaviors, nonprescription drug use, and skin-breaching procedures, such as tattooing and piercing, are included, as well as questions to assess residence in regions where exposure to malaria or the agent of bovine spongiform encephalopathy may occur. The donor screening

questionnaire is intended to detect information that may place the transplant recipient at increased risk for transplantation-transmissible diseases. Testing of the donor’s blood cannot substitute for direct questioning because the donor might have a “window-period” infection, the blood tests may be falsely negative or, equally important, the testing does not include all potentially transmissible diseases. Indeed, suitable screening blood tests for some disorders, such as Creutzfeldt–Jakob disease and variant Creutzfeldt–Jakob disease, do not currently exist. Positive responses on a proper donor-screening questionnaire may lead to donor disqualification or the donor may be determined to be ineligible by FDA criteria. Donors may also become ineligible as a result of physical examination findings or testing for relevant transmissible infectious diseases, as described below. In the United States, FDA regulations provide a mechanism for collecting HC products from ineligible donors [22,23]. In this case, the transplant physician may determine that there exists a state of “urgent medical need,” which means that a suitable alternative donor or graft source is not readily available, and the risks of alternative therapies are greater than the risk of proceeding with the ineligible donor. If the transplant physician elects to proceed with an ineligible donor, the declaration of urgent medical need must be documented, and appropriate paperwork and labeling must accompany the PBPC product. It should be emphasized that donor eligibility from a regulatory standpoint is distinct from donor suitability. A donor may be eligible, but have medical conditions, as described below, that make him or her unsuitable for donation. The donor physical examination, discussed in greater detail below, should detect behavioral and experiential stigmata, such as recent tattoos, piercings, illicit drug use, and so on, as well as signs of significant conditions, including specifically sepsis and vaccinia [22]. In addition, a sample of the donor’s blood must be tested within 30 days prior to donation for at least the following infectious diseases: HIV 1/2, HBV, HCV, Treponema pallidum, human T-cell lymphotrophic virus I and II, West Nile virus, and cytomegalovirus (CMV). For infusion in the United States, testing must be performed in a certified laboratory, which uses test kits specifically licensed or approved for donor screening and which follows the test kit manufacturer’s instructions. Testing that is performed with an unlicensed test kit or a test kit that is licensed for a different purpose (e.g. diagnosis or monitoring) is a violation of FDA regulations. Testing of HC donors for Chagas’ disease is not currently required, although FDA-licensed kits are available for this purpose. Testing for prior infections with varicella-zoster virus, Epstein–Barr virus, and toxoplasmosis, while not required, may also be desirable. The results of regulated and nonregulated infectious disease testing must be available and reviewed prior to the initiation of preparative conditioning therapy for the recipient [19,22,23]. Donors with a confirmed positive test for HIV must not donate. As already described, donors with prior exposure to HBV and HCV may be used when there are no suitable alternatives and the potential gain outweighs the risk. Strategies for managing hepatitis exposure in donors and recipients have been reviewed [24]. In one instance, interferon-alpha pretreatment of a marrow donor apparently prevented transmission of HCV to the recipient [25]. Donors who have recovered from HBV infection can safely donate marrow [26]. HCT recipients with active HBV infections may, in fact, benefit from transplantation with a donor who shows evidence of HBV immunity [27–30]. Further, it may be that natural immunity, reflecting recovery from HBV infection, is more likely to benefit the transplant recipient than vaccination-related immunity [30]. Transplant recipients whose donors are positive for hepatitis B surface antigen, however, are at high risk for post-transplant hepatic complications and

Bone Marrow and Peripheral Blood Cell Donors and Donor Registries

transplant-related mortality [31,32]. These issues are fully discussed in Chapter 95. CMV-seronegative recipients may benefit from having CMVseronegative donors, but this is a controversial issue. Two analyses of data from the NMDP have shown that, although recipients who are CMV seropositive have poorer outcomes, donor CMV status has no impact on unrelated donor HCT outcome [33,34]. Similarly, it has been suggested that CMV-seropositive patients may benefit from CMV-seropositive donors [35]. Ljungman et al. [36], analyzing data from the European Blood and Marrow Transplant Group, reported that CMV-seropositive recipients of unrelated donor transplantation experienced improved survival and reduced transplant-related mortality when their donors were also seropositive, compared with recipients whose donors were seronegative. This effect was not seen among recipients of HLA-identical sibling transplants. A study of chronic myelogenous leukemia recipients showed those with seropositive donors experienced a more rapid rise of high avidity anti-CMV antibodies following transplant [37]. CMV seropositive recipients with seronegative donors had a slower rise of antiCMV antibodies, which were initially of low avidity. CMV-related issues are further discussed in Chapter 90. Medical eligibility HC donation is not without risk. As described below, adverse events are uniformly encountered, but these are generally minor and short lived. Serious adverse events, including death, may, however, occur. It is essential that, prior to donation, each donor is assessed to ensure the absence of conditions that might increase donor risk to unacceptable levels. To ensure objectivity and preservation of the donor’s best interests, this assessment should be performed by a physician who is not directly involved in the care of the proposed recipient. History and physical The donor undergoes a medical history and physical examination appropriate for the anticipated donation procedure. The history, which addresses risk to the donor, as opposed to the screening history described above, should focus on matters relevant for the anticipated donation, including the psychologic issues discussed below. For all donors, these would include a review of known health problems, a listing of medication and allergies, and a review of the family history. Marrow donors should be questioned about prior surgical procedures and types of anesthesia received. Marrow donors should also have a careful review of systems directed toward neurologic, respiratory, cardiovascular, and musculoskeletal problems. PBPC donors should be questioned about prior whole blood or apheresis donations. The review of systems for PBPC donors should include a careful cardiovascular and neurologic review as well as specific questions about a history of venous access problems, autoimmune diseases, and splenic disorders. The donor’s physical examination should focus upon the neurologic, respiratory, and cardiovascular systems. In addition, marrow donors need an assessment of the oral airways and an evaluation of access to the iliac crests. Donors with a history of musculoskeletal symptoms need a careful examination of the spine and lower extremities. The examination of PBPC donors should additionally include evaluation of venous access, and an abdominal examination for splenomegaly.

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donors should also have determinations of alkaline phosphatase (AP) and lactate dehydrogenase (LDH). Some physicians will also recommend evaluations of immunoglobulin levels and a screen for monoclonal proteins in the blood [38]. Additional evaluations of donors may include urinalysis, chest X-ray, and electrocardiogram. HC donors whose intended recipients suffer from inherited conditions, such as hemoglobinopathies or inborn errors of metabolism, may require specific testing to rule out carrier states that could affect transplant outcome. Donors with sickle cell trait and thalassemia minor can serve as donors in successful bone marrow transplants [39]. As discussed below, however, donors with S-beta thalassemia, SC or other complex sickle hemoglobinopathies should not receive rhG-CSF. Successful transplantation from a donor with hemoglobin H disease has been reported [40]. Barquinero et al. [41] suggested that marrow donors with trisomy 21 and Fanconi syndrome heterozygotes may be less suitable because their recipients experienced a high frequency of poor graft function. A pregnancy assessment must be performed for female donors with childbearing potential [18,19]. Pregnancy is considered a contraindication to marrow donation, but successful marrow collection from pregnant donors has been reported [42,43]. Pregnant women cannot be PBPC donors as hematopoietic growth factor administration is contraindicated. One PBPC donor has been reported, however, who received filgrastim while 2–3 weeks’ pregnant without any adverse impact on the pregnancy or delivery [44].

Ethical and psychosocial issues Psychologic aspects of marrow donation The psychologic condition of the donor should be assessed. In particular, what are the donor’s motivations for considering the HC donation? Is the donor acting out of a genuine desire to help, or are there other motives involved – perhaps an unrealistic expectation of reward or personal gain? Switzer et al. [45] interviewed 343 unrelated marrow donors predonation, and then immediately and 1 year post donation. They determined that donor motives could be classified into six categories, and that an individual donor might express motives in more than one category. Donor motives varied between men and women, and were associated with reported levels of predonation ambivalence and with both psychologic and physical difficulties post donation [45]. Compared with unrelated donors, related donors may have different motivations, and may be subject to increased emotional and physical stress [46,47]. Discussions of the psychologic issues affecting donors post donation are presented in Chapter 33. Occasionally, donors may be subjected to coercion [48,49]. In one instance, a potential donor’s unwillingness to donate for her sister attracted media attention, forcing the donor to reconsider her decision [48]. Switzer et al. [45] have reported that unrelated donors who feel they were pressured, either encouraged or discouraged about donation, are less likely to have a positive donation experience. In an early report of unrelated donors, nine of 20 reported being discouraged from donation by a relative or a friend [50]. Donor consent for HC donation

Laboratory and procedural evaluations of donors The laboratory evaluation of all HC donors should include the following: complete blood count with white blood cell differential, ABO/rhesus grouping, serum electrolytes, glucose, alanine aminotransferase, bilirubin, creatinine or blood urea nitrogen, total serum protein and albumin, and prothrombin time and partial thromboplastin time [19]. PBPC

HC donors must provide written consent prior to donation [18,19]. The model of informed consent for research, as detailed in multiple documents [51,52], is appropriate for the HC donor even in the absence of planned research. Like the research subject, the HC donor is a volunteer who must be provided with full and complete information about the planned procedures. This approach is also consistent with recommenda-

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tions developed by the WMDA. This means that donors must receive a clear description of the proposed donation, its risks, and the alternatives to the procedure. They must also have the opportunity to raise questions and to have these satisfactorily addressed. Materials and programs have been developed to assist with providing donor education and information [53]. HC donors as research subjects It is important to determine circumstances that make the HC donor a bona fide research subject. This can be complicated because much of the research involving HC donors also involves the HCT recipient. Several tests may be applied to help determine whether a particular activity constitutes research involving the HC donor. These include the following: (1) Is there collection of individually identifiable information or product for the purpose of research?; (2) Is there collection of information or product in support of an FDA Investigational New Drug application or an FDA Investigational Device Exemption?; and (3) Is there an interaction with a donor that would not be occurring in the absence of the research? Affirmative responses to any of these questions serve to define research. Examples of research wherein the HC donor becomes a research subject include the following: ex vivo culture or “expansion” of donor cells, insertion of new genes into donor cells, establishment of immortal donor cell lines, and nonstandard genetic analysis of donor DNA. Standard genetic analysis includes, for example, DNA-based HLA typing, hemoglobinopathy testing, and assessment of donor/recipient chimerism. An example of genetic research would be the identification of single nucleotide polymorphisms within particular donor genes for the purpose of assessing impact on transplant outcomes. The complex risks and consent issues created by genetic analysis of DNA have been reviewed [54]. It is worth emphasizing that donors are research subjects when they are asked to provide HCs for patients on experimental transplant protocols where the patient could not otherwise receive a transplant were it not for the protocol (question 3 above). This would include protocols investigating allogeneic transplantation for totally new indications, for example lung cancer or breast cancer, and allogeneic transplantation with a new preparative regimen wherein patients are only eligible for the new regimen if they are ineligible for standard regimens. In both of these situations, the HC donation, even if it is a standard donation, is occurring because of the research protocol.

Donation procedures Bone marrow Marrow is typically collected from the posterior iliac crest of the donor under general or regional anesthesia [55]. The posterior superior iliac spine is the primary landmark for initiating the marrow collection. Marrow is aspirated through large-bore needles into glass or plastic syringes that have been rinsed with a heparin-containing solution. Typically, the iliac crest is entered through a skin puncture or a small skin incision. Once the cortical bone has been penetrated, a small amount of marrow (5–10 mL) is aspirated by applying vigorous suction. The syringe is removed, and its contents are immediately transferred to an anticoagulant solution. Commonly, the aspiration needle is then advanced several millimeters and the process repeated. Several aspirations may be obtained from a single bone puncture as the needle is repeatedly advanced. Through a single skin entry site, the bone may be entered multiple times. A typical collection may involve 200–300 marrow aspirations obtained through a few or several skin punctures or incisions. Occasionally, marrow is also collected from the anterior iliac crests. In

a report from Seattle, prior to 1983, the anterior crests were aspirated in 69% of collections [56]. In current practice, aspiration from the anterior crests is uncommon, but may occur if the posterior marrow space has been compromised by trauma, radiation or some other insult. The aspiration of marrow produces an admixture of marrow cells from the bone cavity and capillary blood. The ratio of marrow to blood appears to depend upon the technique employed. Short, vigorous aspirations are generally thought to produce a higher concentration of marrow cells relative to peripheral blood. Even though the dilution of marrow cells with peripheral blood is variable, the adequacy of marrow collections is determined by the nucleated cell count of the mixture. For many years, the target cell dose for allogeneic marrow collection was over 2.0 × 108 nucleated cells/kg recipient body weight. Sierra et al. [57] evaluated the effect of marrow cell dose in 174 unrelated donor bone marrow transplant patients with high-risk acute leukemia. Their analysis showed a beneficial effect of marrow cell doses above the study group median (3.65 × 108 nucleated cells/kg). As a result of this analysis and similar studies, many centers now routinely seek marrow cell doses of 4 × 108 nucleated cells/kg. Overnight hospitalization of donors was once routine but is now uncommon at most centers. When the marrow collection is completed in the morning, most donors will be ready for discharge by late afternoon. Donors are given instructions about care for their dressings, and precautions about fever or signs of infection. Some centers will routinely provide narcotic-containing analgesics upon discharge, but nonnarcotics will suffice for most donors. Women are often treated with several weeks of oral iron, but this is not necessary for male donors. Decisions regarding transfusions of blood vary between collection physicians. Allogeneic (homologous) blood should not be used without a clear indication and, if indicated, must be irradiated and CMV-safe. When autologous blood units are available, some physicians will routinely transfuse these either intraoperatively or following completion of the collection. Other physicians will not transfuse even autologous blood unless there is an indication upon completion of the collection. The need for autologous blood has been questioned [58,59].

Peripheral blood HCs Requirements for establishment of a PBPC collection program have been reviewed [60]. The PBPC collection site is usually a blood center or the apheresis unit of a hospital. Donors typically remain as outpatients throughout the mobilization and collection process. Filgrastim is currently the most commonly used mobilizing agent, although lenograstim (not available in the United States) yields similar numbers of CD34+ cells [61,62]. Although also not yet licensed in the United States, the chemokine (CXC) receptor-4 (CXCR4) antagonist AMD3100 effectively mobilizes PBPCs in autologous and allogeneic donors [63,64]. The rate of engraftment with cells from related donors appears similar for the filgrastim- and AMD3100-mobilized PBPCs [64]. Combination of AMD3100 with filgrastim may further result in higher numbers of CD34+ cells [63]. Filgrastim is usually administered at a daily dose of 10–16 μg/kg donor weight, which may be given as a single subcutaneous injection or divided into twice-daily injections. PBPCs are collected using a continuous-flow cell separation device. Collections begin after the fourth or fifth dose of filgrastim. Success with shorter courses of rhG-CSF has been described [65]. Usually, cannulae are placed in the antecubital veins. From one line, blood is withdrawn into the apheresis machine at a rate of 30–70 mL/min. The continuous-flow centrifuge is set to separate a fraction composed predominantly of mononuclear cells and platelets from the other blood elements. The mononuclear cell/platelet

Bone Marrow and Peripheral Blood Cell Donors and Donor Registries

fraction, which is rich in HCs, is retained, while the remainder is reinfused to the donor via the return line. During a single 3-hour procedure, it is possible to process between 9 and 12 L of whole blood. The resulting yield of CD34+ cells is correlated with the precollection concentration of CD34+ cells in the donor’s blood. In an effort to reduce donor inconvenience, expedite PBPC collection, and reduce overall cost, there has been increasing interest in largevolume (16–25 L) leukapheresis. Following large-volume leukapheresis, the total yield of CD34+ cells may exceed the calculated maximum based upon the donor’s predonation peripheral blood CD34+ content [66]. This observation suggests that, during the leukapheresis procedure, CD34+ cells are continually released into the blood stream. Several effective apheresis devices are available. Most apheresis instruments have similar CD34+ cell collection efficiencies, but they may vary in the number of platelets collected, which may be of importance in donors with low predonation platelet counts [67,68]. There is wide variability between donors in the number of CD34+ cells that will be mobilized and collected following stimulation with filgrastim [69]. The exact dose of CD34+ cells appropriate for allogeneic transplantation has also not been established, but the usual target is 4–6 × 106 CD34+ cells/kg recipient weight [66,70–74]. For the majority of adult recipients, it appears that one or two 12 L aphereses, or a single large-volume procedure, will provide sufficient HCs for successful engraftment [69,75].

Adverse events associated with HC donation An adverse event is any untoward medical occurrence. Serious adverse events are those adverse events that pose a threat to the individual’s life or functioning. Serious adverse events include at a minimum those adverse events that: (1) are fatal, (2) are immediately life-threatening, (3) require or prolong hospitalization, (4) result in a significant or persistent disability, or (5) represent a congenital anomaly. Both marrow and PBPC donations are associated with adverse events. Fortunately, serious adverse events are not common. In the NMDP experience, hospitalization is the most common reason for an adverse event to be rated Serious. Unexpected adverse events, which may be either Serious or not, are those that are not described in a protocol, consent form, Investigator’s Brochure or the medical literature. Adverse events associated with marrow donation Marrow donors routinely experience minor adverse events [55,56,76,77]. In a review of 1270 related-donor marrow collections in Seattle, it was reported that all donors experienced some component of pain [56]. Stroncek et al. [77] reviewed the experiences of the first 493 unrelated marrow donors in the NMDP. When surveyed 2 days after donation, 75% reported fatigue, 68% had pain at the collection site, and 52% experienced low back pain. Data from 9601 NMDP marrow collections, reported by donor sex, are shown in Table 38.1. Women report more adverse events in general. Only bandage pain is reported more often in men. Older donors report significantly fewer adverse events related to pain, nausea, lightheadedness, and vomiting. Fever is, however, more likely to be reported by older donors. Not surprisingly, in the NMDP experience, some adverse events were dependent upon how the collection procedure is performed. For example, regarding the type of anesthesia, general anesthesia was administered in 78% of cases, epidural anesthesia in 15%, and spinal anesthesia in 7%. Nausea, vomiting, and sore throat are all significantly more common following general anesthesia than following regional anesthesia; fever and fainting are less likely to occur. Similar results were seen in a small controlled study [78].

549

Table 38.1 Symptoms reported by National Marrow Donor Program bone marrow donors (n = 9601) Symptom

Women (n = 4106)

Men (n = 5495)

Tiredness* Collection site pain* Back pain Nausea* Sore throat* Pain sitting* Lightheadedness* Headache* Vomiting* Intravenous site pain* Fever Bandage pain* Bleeding at site* Fainting*

85% 78% 67% 63% 62% 62% 53% 40% 39% 37% 22% 19% 10% 7%

76% 75% 68% 40% 57% 57% 42% 32% 17% 23% 22% 26% 8% 5%

* p < 0.05 between men and women.

Observations among unrelated donors may not apply to related marrow donors. In a Seattle study of post-donation pain, male donors tended to report more pain and used more codeine-containing analgesics than did female donors [76]. This study also concluded that pain relief was incomplete with acetaminophen plus codeine, and that donors might benefit from more potent narcotic analgesics. Stroncek et al. [77] examined how side-effects and recovery varied with respect to marrow volume collected, duration of the marrow collection, and duration of anesthesia. The total marrow volume removed, measured as mL/kg of donor weight, was only modestly correlated with most symptoms. The duration of the marrow collection procedure, which reflects not only the volume collected, but also the ease of collection, was the single factor most strongly correlated with increasing frequencies of immediate and delayed symptoms, and with longer time to complete recovery. Longer collections increase the frequency of symptoms and prolong recovery. Sixty-three percent of the 493 NMDP donors reported complete recovery, as defined by the donor, within 14 days. An additional 24% recovered between 2 weeks and 1 month, but 13% of donors took more than a month until they felt fully recovered. Minor complications occur in 6–20% of marrow donations [56,77,79]. These include such events as hypotension, syncope, severe postspinal headache, excess pain, and minor infections. By definition, these complications resolve within a few to several days of onset. Laboratory and radiologic abnormalities also accompany marrow donation. Anemia is the most significant laboratory finding. Marrow harvesting also causes significant, but transient, elevations in serum alkaline phosphatase and ostoecalcin [80]. X-rays, computed tomography scans, and radionuclide scans disclose abnormalities in the pelvis and sacrum that may persist for weeks or months [81]. Abnormalities disclosed by magnetic resonance imaging may correlate with symptoms post donation [82]. Serious adverse events following marrow donation The frequency of serious adverse events following marrow donation is estimated at 0.1–0.3%. A review of data collected between 1969 and 1983 on 2248 allogeneic marrow donations reported to the International Bone Marrow Transplant Registry and 1160 additional donations occur-

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Table 38.2 Major bone marrow donor complications reported to the International Bone Marrow Transplant Registry, 1980–89 (n = 8296) Complication

Number of reports

Myocardial infarction Severe anemia Anaphylaxis during anesthesia Prolonged paralysis after anesthesia Pulmonary embolism Severe back pain Acute renal failure from incompatible blood transfusion Anaphylaxis from incompatible blood transfusion Hepatitis B virus infection Intervertebral disk prolapse Malignant hyperthermia Paroxysmal tachycardia Pulmonary edema Retroperitoneal hematoma Severe hypotension during anesthesia Severe vasovagal reaction

3 3 2 2 2 2 1 1 1 1 1 1 1 1 1 1

Data courtesy of M. M. Horowitz, Center for International Blood and Marrow Transplant Research.

ring in Seattle revealed nine total incidents (0.27%) [83]. These included one case of aspiration pneumonia, two cases of deep vein thrombosis (including one occurrence of pulmonary embolism), three serious bacterial infections (including two with bacteremia), two instances of serious cardiac arrhythmia, and one case of cerebral infarction. The International Bone Marrow Transplant Registry data were updated in 1991 in an examination of 8296 allogeneic donations occurring between 1980 and 1989 (Horowitz, personal communication). Twentyfour instances of life-threatening or incapacitating complications were identified for an incidence of 0.29% (Table 38.2). In the review of the first 493 NMDP marrow donors, only one case, which was potentially a life-threatening complication-apnea and bradycardia, was identified (0.2%) (77). The NMDP has classified major and/or life-threatening complications into five risk categories: anesthesia, infection, mechanical injury, transfusion, and others. Anesthesia risks These are related to the procedures employed and the agents administered. Rarely do significant complications accompany procedures such as intravenous line insertion, endotracheal intubation, and lumbar puncture. Several NMDP donors have experienced profound bradycardia during anesthesia, including regional anesthesia (spinal or epidural), that required emergency treatment. In each case, recovery was prompt and complete. Five serious cases of potential anesthetic complications, including hypotension, hypoxia or cardiac arrest, were identified among 1549 marrow donors at Seattle [79], who were not included in earlier reports [56,83]. A marrow donor who developed life-threatening malignant hyperthermia has also been reported [84]. Infection risks Infections may occur at the sites of marrow collection or line insertions. Infections at distant sites, for example pneumonia, have also been reported [83]. Serious infections have been rare in the NMDP experience. One donor developed bacterial sepsis shortly after marrow dona-

tion. The medical literature also contains additional reports of serious infections following marrow collection [56,83]. Infectious osteomyelitis appears to be a relatively rare complication following marrow collection [79,85]. Mechanical injury risks The procedure of marrow collection may lead to local tissue injuries. These may include bone damage, nerve damage or entry of a collection needle into a blood vessel, an organ or the spinal canal. Hemorrhage, which may be delayed, can create severe pain from compression of soft tissues [86]. Sciatic pain lasting up to 18 months has been reported in a related marrow donor [56]. Prolonged and significant pain may occur following marrow collection, leading to permanent disabilities. Fractures of the iliac crests have also been reported [56,87]. Visceral injuries are apparently very rare, although a case of retroperitoneal hematoma has been reported to the CIBMTR (Table 38.2). Transfusion risks Neither autologous nor allogeneic blood transfusion is free of serious risks. Allogeneic transfusions may cause transfusion reactions, transmit viral infections, and (rarely) transmit bacterial infections. The CIBMTR analysis (Table 38.2) included a case of HBV transmission and two cases of incompatible blood transfusion. Autologous blood has largely replaced allogeneic blood for the transfusion of both related and unrelated marrow donors. Buckner et al. [56], describing the Seattle experience through 1983, reported allogeneic blood transfusions in 21% of 1135 related marrow donors. Allogeneic blood was more often used in women (35%) and in children under the age of 10 years (38%). Among 23 infants under the age of 2 whose marrow was harvested between 1975 and 1986, 22 received allogeneic blood transfusions [88]. In an update of the Seattle experience, only 10% of 1126 adult donors with autologous blood stored required allogeneic blood transfusions [79]. Most NMDP donors receive autologous blood transfusions [77]; NMDP Standards discourage the administration of allogeneic blood [19]. Among 15,400 NMDP marrow collections through 2005, allogeneic blood was administered in only 51 instances (0.3%). In most of these, transfusion of allogeneic blood could have been avoided. Another strategy for avoiding allogeneic transfusion is to recover autologous red cells from the collected bone marrow. This process has been successfully applied in children donors without compromising the quality of the marrow product [89]. Administration of epoetin-alpha (recombinant human erythropoietin, rhEPO) or other erythropoiesis-stimulating agents may also diminish the likelihood of allogeneic blood transfusion following marrow donation [90,91]. Erythropoiesis-stimulating agents marketed in the United States are not approved for administration after bone marrow donation. The FDA issued an alert in November 2006 concerning the safety of erythropoiesis-stimulating agents that included new requirements for label warnings. Other risks Fat embolism has been documented following marrow donation [92]. An unusual case of small bowel mechanical obstruction, which necessitated an exploratory laparotomy, has been reported [93]. The donor had a distant history of prior intestinal obstruction following appendectomy. It was postulated that perhaps the prone positioning of the donor during the marrow collection had allowed an intestinal volvulus to develop. In another report, a donor with unsuspected Addison’s disease experienced grand mal seizures and acute adrenal insufficiency following marrow collection [94]. A flare of systemic lupus erythematosus following marrow donation has also been reported [95].

Bone Marrow and Peripheral Blood Cell Donors and Donor Registries

551

Table 38.3 Death occurring in association with hematopoietic cell donation Age

Sex

Hematopoietic stem cell source

Proximity to donation

Cause of death

Reference

40 46 57 35 35 35 47 57 62

M M F M F M F F F

Marrow Marrow Marrow Marrow Marrow Marrow PBPCs PBPCs PBPCs

Prior Prior Immediate Immediate Immediate After Immediate After After

Cardiac arrest Cardiac arrest Ventricular fibrillation Respiratory arrest Myocardial infarction Pulmonary embolism Sickle crisis Stroke Cardiac arrest

[81] * † ‡ [82] § [83] [84], ¶ **

PBPCs, peripheral blood progenitor cells. * Confer, personal communication, 1994. † Scheafer, personal communication, 1997. ‡ Onozawa, personal communication, 1997. § Boogaerts, personal communication, 1997. ¶ Gajewski, personal communication, 1997. ** Aul and Heyll, personal communication, 1997.

Marrow donation and risk of death Death has occurred among normal marrow donors. A review of 7857 marrow collections reported to the CIBMTR revealed two deaths [96]. Table 38.3 lists nine documented deaths occurring within a few days of HC donation [97–100]. The aforementioned cases from the CIBMTR are not included in Table 38.3 because it cannot be determined whether they may represent duplicate reports. As is evident in Table 38.3, two deaths actually occurred prior to scheduled marrow donations. Whether the stress of impending donation somehow contributed to these events is unknown. Similarly, it is difficult to know whether death in the days immediately following HC donation is causally related. Overall, it appears the risk of death in proximity to the procedure is approximately 1 in 10,000.

Table 38.4 Symptoms reported by National Marrow Donor Program peripheral blood progenitor cell donors, excluding reports of bone pain Symptom

All donors (n = 1080)

Adverse events associated with PBPC donation

Myalgia Headache Malaise Insomnia Nausea Sweats Other flu-like symptoms Anorexia Fever Chills Vomiting

54% 52% 49% 28% 15% 14% 12% 11% 6% 6% 2%

Like marrow donors, most PBPC donors will experience adverse events. Most of these are related to the administration of hematopoietic growth factors, most commonly filgrastim (rhG-CSF), lenograstim (a glycosylated rhG-CSF formulation, not marketed in the United States) or sargramostim (rhGM-CSF), that are intended to increase the concentration of HCs in the donor’s blood. Rowley et al. [101] reported the experiences of 69 sibling donors who participated in a randomized comparison of bone marrow and PBPC transplantation. The frequency and intensity of symptoms between the two groups of donors was no different. Bone pain is the most prominent adverse event among PBPC donors, occurring in 25–86% of donors receiving filgrastim [75,102–109]. Bone pain likely results from altered bone metabolism, which is reflected by increased bone-derived alkaline phosphatase and decreased serum osteocalcin [110]. The pain is typically diffuse but most prominent in the spine, hips or pelvis, and ribs [109]. Headache is also common, occurring in 38–70% [109,111]. The discomfort from bone pain improves with mild analgesic therapy, such as acetaminophen or a nonsteroidal antiinflammatory drug. Other symptoms seen with filgrastim include nausea and vomiting (approximately 10%), myalgia (approximately 20%), fatigue (around 15%), insomnia (approximately 10%), and injection site reactions (rare). Symptoms are probably less frequent at filgrastim doses below 10 μg/kg daily. Pain symptoms resolve promptly with discon-

tinuation of filgrastim, and are rarely sufficient to cause premature cessation, or even dose reduction [75,102,107,109,112,113]. The NMDP has evaluated data on more than 1000 adult unrelated PBPC donors, all receiving filgrastim at 10 μg/kg/day for 4 or 5 days. Bone pain is reported by 85% of these donors and is the single most common adverse event. The pain begins after a single filgrastim injection and plateaus after two or three injections. Bone pain resolves promptly after discontinuation of filgrastim. Additional side-effects among NMDP donors are summarized in Table 38.4. HC mobilization with filgrastim also causes numerous alterations in serum chemistries and blood cell counts. LDH, AP, and alanine aminotransferase increase two- to four-fold after five daily doses of filgrastim [75,102,107,109]. Anderlini et al. [75] reported that isoenzymes LDH-4 and LDH-5 comprise most of the LDH increase. Gamma-glutamyl transferase levels are unaffected by filgrastim, suggesting that increased AP is not hepatic in origin. The serum levels of potassium, blood urea nitrogen, and magnesium may show minimal declines during filgrastim treatment [109]. The white blood cell (WBC) count, and in particular the absolute neutrophil count, increase dramatically during filgrastim therapy

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[75,102,107,109]. These increases are dose dependent. At daily filgrastim doses of 10 μg/kg or greater, the total white count may reach 70–80 × 109/L by day 5. Eighty to 90% of the white cells will be neutrophils or band forms. It is generally recommended that the filgrastim dose be reduced if the WBC count exceeds 70–75 × 109/L [60,98]. In the NMDP experience, only five of 1084 donors (0.5%) receiving filgrastim at 10 μg/kg/day demonstrated a WBC count over 75 × 109/L in the preapheresis blood sample on day 5. The median WBC figure among NMDP donors pre apheresis was 36.8 × 109/L, with the 25th and 75th percentiles at 29.9 and 45.2 × 109/L, respectively. A decline in platelet counts accompanies daily filgrastim administration [109,114]. Among NMDP donors, the median decline from baseline to day 5 pre-apheresis is modest, only 16 × 109/L, but the range of platelet count change is large, from a decline of 156 × 109/L to an increase of 103 × 109/L. Frank thrombocytopenia (platelet counts less than 140 × 109/L) was present in 17 of 1084 (1.6%) of NMDP donors by day 5 pre apheresis. The platelet decline appears to be dose independent and occurs even at filgrastim doses of 2 μg/kg daily [109]. The apheresis procedure used in PBPC collection is also a source of adverse events. Securing peripheral venous access at the antecubital veins may produce bruising, hematoma or minor bleeding. Anticoagulation with acid–citrate–dextrose solution may elicit symptoms of hypocalcemia: perioral numbness, paresthesias, and carpopedal spasms [60,66]. These symptoms may be ameliorated with oral calcium supplementation, but are more effectively managed with an intravenous infusion of calcium during apheresis. Reducing the blood flow rate may also improve symptoms of hypocalcemia, but at the expense of prolonging the collection procedure. Another approach that has been used is to supplement the anticoagulation with heparin to diminish the amount of acid–citrate–dextrose required [115]. Peripheral WBC counts will fall after the collection of mobilized PBPCs. Mild neutropenia is usual for a few weeks [111]. Decreases in hemoglobin and hematocrit values are minimal, although in one report hemoglobin levels decreased by an average of 1.2 (range 0.3–2.2) g/dL with each apheresis procedure [74]. Thrombocytopenia is the most consistent and significant finding with PBPC apheresis [66,74,75,106,109,112,114,116–118]. The preapheresis platelet count reproducibly declines between 20% and 30% with each collection, and does not begin to recover until 3–4 days after the last collection. Following two procedures, it is common to encounter total platelet counts below 100 × 109/L [66,74,75,106,112,114,116,117]. In the NMDP experience, the incidence of thrombocytopenia less than 75 × 109/L is 1% (11 of 1084) after a single apheresis procedure (median blood volume processed of 12 L), but rises to 11% (90 of 787) after two procedures. No instances of life-threatening hemorrhage secondary to thrombocytopenia have been reported. Nevertheless, avoidance of aspirin during mobilization and collection, and nonsteroidal antiinflammatory drug therapy during collection is prudent. Some authors have recommended discontinuing PBPC leukapheresis if the platelet count falls below 70–80 × 109/L [60,98]. An alternative to discontinuation of PBPC collection is the recovery and reinfusion of platelet-rich plasma from the leukapheresis product. Each product obtained by standard techniques may contain 3–5 × 1011 total platelets, which is approximately equal to a platelet apheresis product. Mild lymphopenia is routine following PBPC donation and persists for 8–10 weeks [117,119]. The cause of this lymphocytopenia has not been determined, but clinical sequelae have not been reported. The most significant immediate problem of PBPC apheresis is inadequate venous access. Central lines to perform apheresis are required in 0–8% of collections [74,98,108,109,117]. The NMDP has tried to minimize the use of central venous lines. In spite of this, 41 of the first 395 NMDP PBPC donors required a central line. Central lines were 10 times

more common among women than men (37 of 179 women [21%] versus 4 of 216 men [2%]). Central line complications are uncommon, but include pneumothorax, hemorrhage, and infection. To eliminate the risk of pneumothorax, some authors have recommended the femoral approach to central venous access for PBPC collection [60]. A disadvantage of femoral catheterization is the requirement for immobility, which, if more than one apheresis procedure is necessary, will force overnight hospitalization of the donor. Large-volume leukapheresis, which involves the processing of three or more total donor blood volumes, allows the collection of large numbers of HCs in a single procedure and may be appropriate when femoral catheterization is required [66]. Serious adverse events following PBPC donation Filgrastim administration may precipitate severe sickle crisis in persons with sickle cell anemia or complex sickle cell hemoglobinopathies [97,120]. Indeed, a 47-year-old woman with hemoglobin SC disease, who had never had any symptoms from her condition, suffered a fatal sickle crisis during filgrastim mobilization of PBPCs intended for her sister with chronic myelogenous leukemia [97]. It remains to be clarified whether persons with sickle trait (hemoglobin AS) are at any increased risk from filgrastim. Kang et al. [121] safely mobilized and collected PBPCs from nine donors with sickle trait. These donors did experience higher symptom scores during mobilization than did eight simultaneous control donors, but there were no symptoms suggestive of painful sickle crisis. There have been five reports of spontaneous splenic rupture apparently related in some way to extramedullary hematopoiesis [122–126]. In three cases, the spleen was surgically removed, but conservative management was effective in two cases. Platzbecker et al. [127] performed ultrasound evaluations of spleen size before and after rhG-CSF mobilization in 91 healthy PBPC donors. Although no adverse splenic events occurred in this group, significant increases in spleen length and width were routinely documented. No donor factors or clinical findings (change in WBC, increase in AP, etc.) correlated with the degree of splenic enlargement. Similar findings were reported in independent studies by Stroncek et al. [128,129], which also demonstrated that splenic enlargement may persist for up to 10 days in some donors. Based on these studies, ultrasound does not appear to be useful for predicting the risk of hemorrhage into the spleen. Growth factor is probably contraindicated in donors with a history of autoimmune disorders [130–134]. Flares of rheumatoid arthritis and ankylosing spondylitis have been reported in non-PBPC donors following therapy with filgrastim or sargramostim [130,134]. In patients with normal thyroid function, but pre-existing antithyroid antibodies, therapy with sargramostim caused thyroid dysfunction and one case of goiter [131]. A variety of eye inflammatory responses have been reported among allogeneic PBPC donors. These have included marginal keratitis, episcleritis, and iritis occurring during therapy with filgrastim [132,133,135]. Chest pain in association with PBPC donation is occasionally encountered and is noncardiac in origin [109,116]. However, an instance of myocardial infarction following PBPC leukapheresis has been reported [112]. The donor in this instance had a history of coronary artery disease and had previously suffered myocardial infarction. The donor recovered uneventfully, but a planned second leukapheresis procedure had to be canceled. PBPC donation and the risk of death As with bone marrow donation, death at the time of, or in close proximity to, PBPC donation has been reported [97,98]. Table 38.3 lists three

Bone Marrow and Peripheral Blood Cell Donors and Donor Registries

deaths among PBPC donors. Only one of these, the 47-year-old woman with hemoglobin SC disease, was clearly related to the PBPC donation [97]. It is unclear whether the risk of death associated with PBPC donation differs from that of bone marrow donation. Long-term donor follow-up The long-term safety profile of growth factor therapy in normal individuals has not been established. Three cases of leukemia have been reported occurring months to years after PBPC donation [136,137]. In each case, the donor who developed leukemia was the HLA-matched sibling of the transplant recipient, who also had leukemia. It is known that siblings of persons with leukemia have a two- to fivefold increased annual incidence of leukemia [138–140]. Several investigators have performed serial evaluations on the peripheral blood of normal PBPC donors using a variety of techniques to evaluate chromosomal content, DNA stability, replication timing, and gene expression profiling [141–144]. In every instance except one, filgrastim-induced alterations during HC mobilization have been transient; Nagler et al. [143] reported persistent aneuploidy of chromosome 17 in the lymphocytes of PBPC donors 6–9 months after mobilization. There have been several reports providing 1–5 years of follow-up on limited numbers of PBPC donors [70,74,106,145,146]. In no instance have serious adverse events attributable to filgrastim been identified. Cavallaro et al. [44] evaluated 101 PBPC or granulocyte donors who had received filgrastim between 3 and 6 years previously. Ninety-four donors were able to be interviewed; one of the remaining ones had died 15 months after donation from a drug overdose, and the six others could not be interviewed. Among the 94 donors, 11 had experienced adverse medical events during the follow-up period, but none of these related to the hematopoietic system. Two donors had developed cancer during the follow-up period, one breast cancer and the other prostate cancer. Anderlini et al. [147] recently reported on the follow-up of 343 PBPC donors, of whom 281 (82%) were able to be interviewed. With a median follow-up of 39 (range 7–80) months, there were no instances of leukemia or other hematopoietic adverse events. The NMDP conducts annual follow-up of PBPC donors. More than 4000 donors have been followed for between 1 and 9 years, providing nearly 10,000 donor–years of observation. Twenty cases of cancer at various sites have been reported, but there have been no instances of leukemia or lymphoma [148]. PBPCs versus marrow: which is easier for donors? Switzer et al. [149] evaluated 70 unrelated bone marrow donors who subsequently provided a second HC product (25 PBPC and 45 bone marrow). Donors whose second product was PBPCs reported that, in comparison to their prior bone marrow donation, PBPC donation was less difficult physically, required less time, and was more convenient. In randomized trials of PBPC versus bone marrow among sibling donors, the distinction between the two procedures is less clear. Similar levels of anxiety and pain are reported, and time to complete recovery may also be similar [150,151]. When differences are observed, however, in acute symptoms, fatigue, time to recovery or late complications, these tend to favor PBPC donation [151–153].

Special considerations Infants and children as HC donors Safety Children may safely donate marrow [56,88,154–156]. They are probably more likely than are adults to receive allogeneic blood transfusion, but

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serious complications among children donors have been rare [56,88,156]. The use of children as HC donors must consider the ethical and legal issues addressed below. Sanders et al. [88] reported on 23 marrow donors under the age of 2 years. Harvested volumes were between 11.5 and 19.3 mL/kg donor weight. One 14-month-old donor was discovered during predonation evaluation to have a stage 1 neuroblastoma. The tumor was resected at the time of marrow donation. Aside from this case, no serious complications were encountered, but 22 of the 23 infants required allogeneic blood [88]. Successful marrow collection from a 3.95 kg donor has also been reported [156]. The collection volume represented two-thirds of the donor’s total blood volume, essentially necessitating exchange transfusion. Another case reported collecting 335 mL of marrow from a 9.4 kg infant (total blood volume 750 mL) [155]. In this instance, recovery of autologous red cells from the collected marrow enabled avoidance of allogeneic blood transfusion. In a survey of pediatric marrow transplant physicians, only seven of 56 responders were unwilling to collect marrow from infant donors 0–6 months of age [154]. There was little agreement, however, on the management of large-volume collections. Of 52 respondents, six would limit collections to 25% or less of the donor’s blood volume, whereas 24 placed the limit at 50% or higher. Twenty-two respondents preferred to manage large-volume collections in two stages [154]. Children appear similar to adults with respect to their response to filgrastim and PBPC collection [157]. They tolerate filgrastim similarly and provide high yields of CD34+ cells. Central venous access may be required more often for successful collection from children [157]. A report described PBPC collections in five children aged 4–13 years [115]. The children each received filgrastim at a dose of 6 μg/kg every 12 hours for 3 or 4 days. One or two leukapheresis procedures were performed. Three children were donating for siblings, and two were donating for a parent. No child required a central venous line, and no immediate complications were encountered. Pulsipher et al. [158] recently reviewed the clinical, ethical, and research issues surrounding the administration of filgrastim to children. Information, consent, and assent Children who are being evaluated as potential HC donors deserve special attention. They may have very limited understanding of their family member’s illness and of the donation process. Their fears and concerns are often complex and accompanied by significant ambivalence [159– 163]. With hapoloidentical transplantation protocols, children may also be considered as donors for parents, which raises added concerns about understanding, stress, and informed consent [164]. Consent for children presents special concerns. In general, children are only considered for related-donor HC donation. In most instances, parents are expected to consent for their children, which may create conflict of interest situations. When they are later asked, children have reported that they were forced to donate by families or physicians or, short of being forced, that they felt they had no real choice in the matter [162,163]. Consent issues may become very complex as evidenced in one case report concerning a pediatric donor who had been sexually abused by her sibling recipient [165–167]. A survey of pediatric transplant physicians in the United States confirmed that most felt the role of consent appropriately rested with the parents [154]. Outside the United States, “altruism by proxy” has stirred debate in the medical literature [168–170]. In some locales, it remains the standard practice to appoint legal guardians to determine whether HC donation is in the child’s best interest. When governing law allows parents to render consent, it is incumbent on the physician, at a minimum, to recognize the potential for conflict

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of interest, to seek expert ethical guidance when needed, and to ensure to the extent possible that the child donor is a willing and informed participant. Children who are able must provide assent for donation [171]. Whether children can serve as unrelated HC donors is a matter of debate. In 2000, the Washington state legislature passed a law intended to pave the way for minors to become unrelated HC donors [172]. The law states simply, “A person’s status as a minor may not disqualify him or her from bone marrow donation.” This legislation was the direct result of a campaign by a 15-year-old male who wanted to be tested as a potential donor for a young Hawaiian man with leukemia [173]. The NMDP has continued its policy that prospective donors must have reached the legal age of majority (18 years throughout most of the United States) prior to registration. Subsequent donations following an initial HC donation A person can donate HCs repeatedly. Several unrelated donor registries have accumulated experience with donors who donate repeatedly for different recipients. The Japan Marrow Donor Program reported on 137 marrow donors who donated for a second recipient over a 12-year timespan and 5305 total donations [174]. Through 2005, with a total of more than 20,000 donations, the NMDP had had 495 donors provide two donations for separate recipients, and 18 provide products for three different recipients. Such donors may be preferentially selected by transplant physicians because they usually have high-quality HLA-typing data and are known to be highly motivated. Most second donations of marrow or PBPCs are intended for the original transplant recipient and used for treatment of graft failure, poor graft function or disease recurrence. Buckner et al. [56], reporting on 1160 marrow donors, identified 99 who donated marrow twice, usually within a 2-month period. There was a modest decline in the average total number of nucleated cells collected with the second donation. Stroncek et al. [175] reported on 16 second-donation donors at the University of Minnesota. There was a trend toward lower total cells and lower cell concentrations in the second collections. When fewer than 90 days separated the collections, the decrement in cell concentration was statistically significant [175]. Donors whose second donation occurred within 60 days of the first were frequently transfused with allogeneic blood (five of eight within 60 days versus one of eight later than 60 days). Consecutive PBPC collections are also feasible. Stroncek et al. [146] collected PBPC products 1 year apart in 19 healthy volunteers. There were no differences in premobilization blood counts, the response to filgrastim stimulation or the CD34+ cell yield between the first and second collections. Similar results were reported by Anderlini et al. [70], who described 13 allogeneic PBPC donors providing a second donation at a median of 5 (range 1–13) months. No differences were identified in pre-apheresis WBC counts of the donors or in the yield of CD34+ HCs. In more recent studies, de la Rubia et al. [176] noted a decline in the total CD34+ cell yield following a second PBPC mobilization and collection. Donor symptoms were no different between the two donations. Similar results were noted by Platzbecker et al. [177], who also found that female sex and a low peripheral blood CD34+ cell count during the first mobilization predicted for a low CD34+ yield with the second donation. Through 2005, with more than 20,000 transplants facilitated, 684 NMDP donors had provided additional HC support for their original marrow recipients. In 476 instances (70%), the second product was

PBPCs, compared with 208 providing bone marrow. Currently, about one donor in 25 will provide additional HC support for their transplant recipient. Donor experiences during second marrow or PBPC collections have not differed from the first donations. Switzer et al. [149] compared the reactions of second-time NMDP donors to PBPCs and to marrow. All of these donors had initially provided marrow as their HC product. Although the donors asked to provide PBPCs as a second-time product expressed more initial reluctance, they ultimately reported fewer donation-related side-effects than did second marrow donors. The donors who had provided bone marrow followed by PBPCs expressed a strong preference for the latter procedure [149]. In the NMDP experience, the most frequent subsequent donation request today is for peripheral blood lymphocytes collected by apheresis. Indications for donor lymphocytes include accelerated immune reconstitution, treatment of viral infection, immunomodulation (particularly in the setting of reduced-intensity conditioning), and graft-versus-tumor therapy [178]. Approximately 5% of NMDP donors currently provide apheresis lymphocyte products for their recipients. Growth factor-mobilized bone marrow In efforts to accelerate the engraftment rate for bone marrow transplantation, several groups have explored filgrastim administration prior to bone marrow collection [179–182]. These reports, a combination of single-arm trials and comparison studies, suggest indeed that rhG-CSF pretreatment of the marrow donor may accelerate the recovery of neutrophils and platelets. When rhG-CSF was administered in one report at relatively low dose (2 μg/kg/day), accelerated engraftment was not observed [183]. Morton et al. [184] reported a randomized comparison between PBPC- and filgrastim-primed bone marrow. Engraftment of neutrophils and platelets was no different between the two groups, but patients receiving filgrastim-primed bone marrow had significantly less steroid-refractory acute GVHD, less chronic GVHD, and fewer days of immunosuppressive therapy. Taken together, these preliminary observations suggest that it may be possible to achieve the rapid engraftment of PBPCs without the added risk for increased acute and chronic GVHD [182,184,185]. These studies, however, have important implications for HC donors because many of the most significant and nonoverlapping adverse events of PBPC and marrow donation are combined in a single procedure. While reduced volumes of marrow collection may be possible [107,179], this may not fully offset the added risks of filgrastim administration. This would seem to be an area where carefully designed, prospective clinical trials are needed.

Summary Allogeneic HC donation is safe. Serious adverse events are uncommon, and death is exceedingly rare. Nevertheless, all donors must be carefully evaluated and fully informed prior to HC donation. Emerging regulatory requirements must be anticipated and met. The two common procedures for obtaining an HC graft – bone marrow and PBPC collection – differ in their acute and longer-term impacts upon the donors. The long-term safety data for both procedures with respect to preservation of a functional, benign hematopoietic system is incomplete. It is highly likely that annual numbers of allogeneic HCT will continue to grow. Based on estimates of current need, the numbers of related and unrelated allogeneic HCTs will easily double within the next several years.

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marrow harvest – a retrospective study in 50 unrelated marrow donors. Bone Marrow Transplant 2005; 35: 667–73. Bortin MM, Buckner CD. Major complications of marrow harvesting for transplantation. Exp Hematol 1983; 11: 916–21. Hosoya N, Miyagawa K, Mimura T et al. Malignant hyperthermia induced by general anesthesia for bone marrow harvesting. Bone Marrow Transplant 1996; 19: 509–11. Riley D, Evans TG. Osteomyelitis complicating bone marrow harvest [Letter]. Clin Infect Dis 1992; 14: 980–1. Drake P. Hemorrhage after bone marrow harvest: a case presentation. Clin J Oncol Nurs 2000; 4: 29–31. Klumpp TR, Mangan KF, MacDonald JS, Mesgarzadeh M. Fracture of the ilium: an unusual complication of bone marrow harvesting. Bone Marrow Transplant 1992; 9: 503–4. Sanders J, Buckner CD, Bensinger WI et al. Experience with marrow harvesting from donors less than two years of age. Bone Marrow Transplant 1987; 2: 45–50. Kletzel M, Olezewski M, Danner-Koptik K, Coyne K, Haut PR. Red cell salvage and reinfusion in pediatric bone marrow donors. Bone Marrow Transplant 1999; 24: 385–8. Akiyama H, Tanikawa S, Takamoto S et al. Recombinant human erythropoietin (rhEPO) administration to marrow donors. Bone Marrow Transplantation (BMT) Team. Int J Hematol 1995; 62: 145–9. York A, Clift RA, Sanders JE, Buckner CD. Recombinant human erythropoietin (rh-Epo) administration to normal marrow donors. Bone Marrow Transplant 1992; 10: 415–17. Baselga J, Reich L, Doherty M, Gulati S. Fat embolism syndrome following bone marrow harvesting. Bone Marrow Transplant 1991; 7: 485– 6. Wolf HH, Heyll A, Hesterberg R et al. Mechanical ileus following bone marrow harvesting: an unusual complication [Letter]. Bone Marrow Transplant 1994; 14: 179–80. Agura ED. Seizure in a normal marrow donor: Addison’s disease unmasked. Bone Marrow Transplant 1994; 13: 215–16. Sturfelt G, Lenhoff S, Sallerfors B et al. Transplantation with allogeneic bone marrow from a donor with systemic lupus erythematosus (SLE): successful outcome in the recipient and induction of an SLE flare in the donor. Ann Rheum Dis 1996; 55: 638–41. Anderlini P, Rizzo JD, Nugent ML et al. Peripheral blood stem cell donation: an analysis from the international bone marrow transplant registry (IBMTR) and European Group for Blood and Marrow Transplant (EBMT) databases. Bone Marrow Transplant 2001; 27: 689–92. Adler BK, Salzman DE, Carabasi MH et al. Fatal sickle cell crisis after granulocyte colony-stimulating factor administration. Blood 2001; 97: 3313–4. Anderlini P, Körbling M, Dale D et al. Allogeneic blood stem cell transplantation: considerations for donors [Editorial]. Blood 1997; 90: 903–8. Blazar BR, Lasky LC, Perentesis JP et al. Successful donor cell engraftment in a recipient of bone marrow from a cadaveric donor. Blood 1986; 67: 1655–60.

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114. Murata M, Kanie T, Hamaguchi M et al. Unrelated bone marrow transplantation from the National Marrow Donor Program. Int J Hematol 1997; 66: 239–43. 115. Körbling M, Chan KW, Anderlini P et al. Allogeneic peripheral blood stem cell transplantation using normal patient-related pediatric donors. Bone Marrow Transplant 1996; 18: 885–90. 116. Majolino I, Buscemi F, Scime R et al. Treatment of normal donors with rhG-CSF 16 micrograms/ kg for mobilization of peripheral blood stem cells and their apheretic collection for allogeneic transplantation. Haematologica 1995; 80: 219–26. 117. Martinez C, Urbano-Ispizua A, Rozman C et al. Effects of G-CSF administration and peripheral blood progenitor cell collection in 20 healthy donors. Ann Hematol 1996; 72: 269–72. 118. Stroncek DF, Clay ME, Smith J et al. Comparison of two blood cell separators in collecting peripheral blood stem cell components. Transfus Med 1997; 7: 95–9. 119. Körbling M, Anderlini P, Durett A et al. Delayed effects of rhG-CSF mobilization treatment and apheresis on circulating CD34+ and CD34+Thy1dim CD38− progenitor cells and lymphoid subsets in normal stem cell donors for allogeneic transplantation. Bone Marrow Transplant 1996; 18: 1073–9. 120. Wei A, Grigg A. Granulocyte colony-stimulating factor-induced sickle cell crisis and multiorgan dysfunction in a patient with compound heterozygous sickle cell/B+ thalassemia. Blood 2001; 97: 3998–9. 121. Kang E, Areman E, David-Ocampo V et al. Mobilization, collection, and processing of peripheral blood stem cells in individuals with sickle cell trait. Blood 2002; 99: 850–5. 122. Becker PS, Wagle M, Matous S et al. Spontaneous splenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): occurrence in an allogeneic donor of peripheral blood stem cells. Biol Blood Marrow Transplant 1997; 3: 45–9. 123. Falzetti F, Aversa F, Minelli O, Tabilio A. Spontaneous rupture of spleen during peripheral blood stem-cell mobilisation in a healthy donor. Lancet 1999; 353: 555. 124. Balaguer H, Galmes A, Ventayol G, Bargay J, Besalduch J. Splenic rupture after granulocytecolony-stimulating factor mobilization in a peripheral blood progenitor cell donor. Transfusion 2004; 44: 1260–1. 125. Dincer AP, Gottschall J, Margolis DA. Splenic rupture in a parental donor undergoing peripheral blood progenitor cell mobilization. J Pediatr Hematol Oncol 2004; 26: 761–3. 126. Kroger N, Renges H, Sonnenberg S et al. Stem cell mobilisation with 16 microg/kg vs 10 microg/ kg of G-CSF for allogeneic transplantation in healthy donors. Bone Marrow Transplant 2002; 29: 727–30. 127. Platzbecker U, Prange-Krex G, Bornhäuser M et al. Spleen enlargement in healthy donors during G-CSF mobilization of PBPCs. Transfusion 2001; 41: 184–9. 128. Stroncek D, Shawker T, Follmann D, Leitman S. G CSF induced spleen size changes in peripheral blood progenitor cell donors. Transfusion 2003; 43: 609–13. 129. Stroncek DF, Dittmar K, Shawker T, Heatherman A, Leitman SF. Transient spleen enlargement in

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145. Sakamaki S, Matsunaga T, Hirayama Y, Kuya T, Niitsu Y. Haematological study of healthy volunteers 5 years after G-CSF. Lancet 1995; 346: 1432–3. 146. Stroncek D, Clay M, Herr G et al. Blood counts in healthy donors 1 year after the collection of granulocyte-colony-stimulating factor-mobilized progenitor cells and the results of a second mobilization and collection. Transfusion 1997; 37: 304–8. 147. Anderlini P, Chan FA, Champlin RE, Korbling M, Strom SS. Long-term follow-up of normal peripheral blood progenitor cell donors treated with filgrastim: no evidence of increased risk of leukemia development. Bone Marrow Transplant 2002; 30: 661–3. 148. Confer DL, Miller JP. Long-term safety of filgrastim (rhG-CSF) administration. Br J Haematol 2007; 137: 77–8. 149. Switzer GE, Goycoolea JM, Dew MA, Graeff EC, Hegland J. Donating stimulated peripheral blood stem cells vs bone marrow: do donors experience the procedures differently? Bone Marrow Transplant 2001; 27: 917–23. 150. Fortanier C, Kuentz M, Sutton L et al. Healthy sibling donor anxiety and pain during bone marrow or peripheral blood stem cell harvesting for allogeneic transplantation: results of a randomised study. Bone Marrow Transplant 2002; 29: 145–9. 151. Kennedy GA, Morton J, Western R et al. Impact of stem cell donation modality on normal donor quality of life: a prospective randomized study. Bone Marrow Transplant 2003; 31: 1033–5. 152. Bredeson C, Leger C, Couban S et al. An evaluation of the donor experience in the Canadian multicenter randomized trial of bone marrow versus peripheral blood allografting. Biol Blood Marrow Transplant 2004; 10: 405–14. 153. Heldal D, Brinch L, Tjonnfjord G et al. Donation of stem cells from blood or bone marrow: results of a randomised study of safety and complaints. Bone Marrow Transplant 2002; 29: 479–86. 154. Chan KW, Gajewski JL, Supkis D, Jr. et al. Use of minors as bone marrow donors: current attitude and management. A survey of 56 pediatric transplantation centers. J Pediatr 1996; 128: 644–8. 155. Chan KW, Stanley CE, Wadsworth LD. Bone marrow collection from a 9.4-kg donor avoiding allogeneic blood transfusion [Letter]. Transfusion 1987; 27: 441–2. 156. Urban C, Weber G, Slavc I, Kerbl R. Anesthetic management of marrow harvesting from a 7week-old premature baby. Bone Marrow Transplant 1990; 6: 443–4. 157. de la Rubia J, Diaz M, Verdeguer A et al. Donor age-related differences in PBPC mobilization with rHuG-CSF. Transfusion 2001; 41: 201–5. 158. Pulsipher MA, Levine JE, Hayashi RJ et al. Safety and efficacy of allogeneic PBSC collection

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Hal E. Broxmeyer & Franklin O. Smith

Cord Blood Hematopoietic Cell Transplantation

Introduction Hematopoietic cell transplantation (HCT) from human leukocyte antigen (HLA)-matched related donors has been successfully used for the treatment of children and adults with a high risk of recurrent hematologic malignancies, genetic immunodeficiencies, metabolic disorders, hemoglobinopathies, and marrow failure syndromes. Unfortunately, the majority of patients who could potentially benefit from allogeneic HCT do not have suitably matched related donors. To address this problem, the National Marrow Donor Program (NMDP) was established in 1986 [1,2]. Despite the success of this program, suitable HLA-compatible bone marrow (BM) and peripheral blood (PB) donors cannot be identified for all patients in need of allogeneic transplantation [3]. Therefore, clinical investigators have, over the past two decades, explored the suitability of umbilical cord blood (UCB) hematopoietic cells as an alternative source of hematopoietic stem cells (HSCs). The world’s experience suggests that UCB is an acceptable alternative to BM.

History of UCB transplantation The potential use of umbilical UCB as a source of HSCs was proposed in 1982 in a private discussion held by Edward A. Boyse, Hal E. Broxmeyer, and Judith Bard [4,5]. Dr Boyse felt it was a waste of precious tissue to not use the UCB that was being discarded, and thought that the mature cells in UCB could be used or generated for transfusion purposes. Dr Broxmeyer alerted Dr Boyse to the possibility that UCB likely contains HSCs, and that if there were enough HSCs in UCB, the UCB could be used for transplantation purposes. Subsequent to these discussions, with the stem cell and hematopoiesis expertise of Dr Broxmeyer, and the immunology and genetics background of Dr Boyse, a number of in vitro studies with human UCB [6,7] and in vivo studies with mouse blood [7] were performed to document the feasibility of this proposal. Initial studies used hematopoietic progenitor cell (HPC) assays, which served as surrogate assays for HSCs, to compare numbers of primitive cells in UCB versus BM [6–8]. Early studies assessed whether previously untrained obstetrical healthcare professionals were able to collect UCB that was free of bacterial and fungal contamination, determine the range and average volume of

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

UCB collected, and assay samples for the total number of nucleated cells and HPCs using in vitro culture methods following overnight shipment of UCB units. During the course of these experiments, a number of UCB units were cryopreserved and stored in liquid nitrogen freezers at the Indiana University School of Medicine for potential clinical use as a proof of principle cord blood bank. These studies were followed by experiments in which lethally irradiated mice received transplantations of blood from near-term or term donors, which contained sufficient numbers of HSCs to reconstitute hematopoiesis [7]. The first UCB HCT was performed for a 5-year-old child with Fanconi’s anemia by Dr Gluckman and colleagues in October 1988 [9]. UCB from the patient’s HLA-identical sibling was collected in Durham, NC, by Dr Gordon Douglas, New York University Medical Center; it was shipped to Indiana University where it was cryopreserved by Dr Broxmeyer’s laboratory and hand-delivered to Paris, where the patient underwent his preparative regimen and UCB HCT [4–6,9,10]. The patient had durable engraftment of donor hematopoiesis and survives without hematological manifestations of the treated disease. The next four UCB transplantations, for three children with Fanconi’s anemia and one with juvenile myelomonocytic leukemia, were performed using UCB also collected at a distant obstetrical unit by Dr Douglas and banked at Indiana University [4–7,9–12]. Four of the first five UCB recipients had engraftment of donor cells, with one patient with Fanconi’s anemia having graft failure. In 1991, two children with leukemia received high-dose preparative regimens for UCB HCT from related donors [11,13]. Following these first, largely successful transplant procedures, UCB as an alternative source of HSCs was utilized by others, and these transplants were initially reported by an International Cord Blood Transplant Registry [14]. It is now estimated that at least 350,000–400,000 UCB units have been banked, with at least 6000 UCB transplantations performed worldwide [15]. UCB has now been utilized as a hematopoietic cell source for numerous malignant and nonmalignant diseases in children and adults.

Collection of UCB UCB can be collected for several purposes. Public UCB banks collect UCB for the allogeneic transplantation of any recipient for whom the UCB unit is a suitable HLA match and of sufficient cell dose. The vast majority of the world’s experience with UCB transplantation has been facilitated by publicly banked UCB. UCB can also be collected and stored by expectant parents for potential use by the newborn infant (e.g. autologous transplantation). To date, three autologous transplants

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using previously cryopreserved UCB have been reported for recurrent acute lymphoblastic leukemia [16], neuroblastoma [17], and severe aplastic leukemia [18]. UCB can also be stored for a family member in need of a HCT procedure (e.g. directed allogeneic donor transplantation). While autologous and directed allogeneic collections and storage have typically been performed by private (for-profit) banks, the National Heart, Lung and Blood Institute (NHLBI) of the National Institutes of Health supported a public UCB bank at the Children’s Hospital Oakland in California for this purpose [19]. A similar directed allogeneic UCB bank was established by the National Blood Service of England [20]. These directed allogeneic UCB banks stored UCB from children with hemoglobinopathies and genetic diseases, and from the normal siblings of children with malignant and nonmalignant disease who were in need of a transplant procedure. The number of directed allogeneic transplants is small, with only 17 (3.4% of collected units) allogeneic transplants performed using units from the NHLBI-supported UCB bank at the Children’s Hospital of Oakland [19] and only 13 allogeneic transplants (5.6% of collected units) from the National Blood Service in England [20]. The majority of directed allogeneic transplants facilitated by these UCB banks were for nonmalignant diseases. UCB can be collected from anonymous donors, on local Institutional Review Board-approved studies, for laboratory-based research. Due to the complexities inherent in UCB banking at both public and private programs, the American Academy of Pediatrics issued a policy statement to provide information to physicians so they could provide guidance to parents considering donation of their newborn’s UCB to one of these banks [21]. Recommendations were provided regarding ethical and operational standards, inclusion of policies on informed consent, financial disclosures, and physician, institutional, and organization conflict of interest for those that operate or have a relationship with a UCB banking program. A number of techniques have been proposed for the optimal collection of UCB, considered to be a UCB unit with sufficient volume of blood, total nucleated cells, and CD34+ cells. In addition, the unit would contain a low number of maternal T cells [22], and would be free of transmissible infectious agents. As engraftment is closely correlated with the number of infused cells, numerous variables in the UCB collection process have been examined in attempts to maximize the cell dose. Early studies suggested that factors that may increase these numbers include volume of UCB collected, number of nucleated cells or number of CD34+ cells, larger birth weight, fewer prior live births, and birth order, with a larger number of cells in first-born children [23]. Other factors that appear to increase cell dose include prolonged stress during delivery, placing the infant on the mother’s abdomen after delivery [24], collection prior to delivery of the placenta, cesarean section [25–27], early clamping of the umbilical cord [24,28], and normal saline flush of the umbilical vessels [27,29]. In addition, there is a direct correlation of gestational age with nucleated cell count, but an inverse relation to CD34+ cell count [23,30]. More recent analyses from several large public UCB banks have further identified factors predictive of cell dose [31–33]. In the NHLBIsupported Cord Blood Transplantation (COBLT) study banking program, a total of 11,077 units were cryopreserved [33]. Higher cell doses were obtained from cesarean section deliveries as opposed to vaginal deliveries, and in higher birth weight newborns. Lower cell doses were obtained from African-Americans. There was a significant correlation between CD34+ cell dose and colony-forming unit (CFU)-granulocyte– macrophage (GM), CFU-granulocyte–erythrocyte-macrophagemegakaryocyte (GEMM), burst forming unit-erythroid (BFU-E), and total CFUs [32]. Despite this information, there remains considerable debate about the optimal collection method. The American College of

Obstetricians and Gynecology [34] and the American Academy of Pediatrics [35] has recommended that standard obstetric procedures not be altered to facilitate UCB collections. Cord blood for public allogeneic use is generally collected at a limited number of sites in a single geographic location, with dedicated, trained personnel performing the collections according to standard operating procedures established by the UCB bank. For directed allogeneic or autologous use, UCB is generally collected at the birth location by local obstetrical care providers. The American Academy of Pediatrics has provided guidance regarding recommended procedures for physicians engaged in UCB collection for related and unrelated banking [21]. Recommendations include the collection of UCB in bags containing citrate– phosphate–dextrose anticoagulant, processing and cyropreservation within 48 hours of collection, use of standardized freezing and storage conditions, use of attached segments that can be used for testing and confirmation of identity, storage of extra cells and plasma, infectious disease testing according to United States Food and Drug Administration regulations, use of only UCB banks that are accredited by the Foundation for the Accreditation of Cellular Therapy (FACT) and follow FACT banking standards, and the storage of UCB units under liquid nitrogen or equivalent temperatures. Despite these variable methodologies, the cellular characteristics of UCB units are reasonably constant and distinct from BM and mobilized PB. Single UCB units generally contain a 10fold smaller dose of nucleated cells and CD34+ cells than that typically transplanted with BM or mobilized PB [23]. However, these units are enriched in HPCs [6,8]. Despite efforts to bank as many UCB units as possible, it has been shown that a very large percentage of potential UCB donors are ineligible for donations to public UCB banks [36–38]. In the COBLT study, 34,799 potential donors were screened, with 20,710 giving consent to participate in the study [33]. A total of 17,207 ethnically diverse units were collected between 1998 and 2001, with only 11,077 (64%) of units cryopreserved. Of these quarantined units, only 79% met eligibility criteria with subsequent HLA typing and entry into the search registry. The chief reasons for not using potential UCB donors are the presence of sexually transmitted diseases in the mother, maternal fever during delivery, medications administered to the mother, maternal diseases, complications of delivery, presence of infections, and complications and problems with the placenta or umbilical cord. Overall, experience suggests that UCB donation is a safe procedure for both mother and newborn.

UCB banking The first UCB bank was created at the Indiana University School of Medicine, with the first UCB HCT performed using units cryopreserved and stored in this bank [4–7]. As a result of the interest in UCB transplantation generated from this preliminary transplant experience, other UCB banks were established [39–45]. It is estimated that 350,000– 400,000 UCB units have been collected, tested, and cryopreserved by these worldwide banks [15]. Establishment of a quality UCB bank requires attention to a number of specific issues including donor recruitment, donor consent, donor evaluation, labeling, UCB collection, UCB processing, cryopreservation, histocompatibility testing, infectious disease testing, genetic disease testing, confirmation of recipient histocompatibility testing, tracking and allocation methods, transportation of UCB from the collection site to bank, and from UCB bank to the transplant center, thawing methods, and protection of confidentiality of donors and recipients [46,47]. Therefore, a number of organizations have created standards to ensure the quality of UCB banks and UCB units, including the American Association of Blood Banks (AABB), the American Red Cross, the NHLBI

Cord Blood Hematopoietic Cell Transplantation

COBLT Study, the European Group for Blood and Marrow Transplantation (EBMT), FACT/NETCORD, the Group for the Collection and Expansion of Hematopoietic Cells, the International Society for Cellular Therapy (ISCT), the Joint Accreditation Committee of ISCT-Europe and EBMT, and the NMDP. While minor differences exist in these standards, they are generally consistent. Because of concerns about the loss of nucleated cells, UCB that was initially collected for transplantation was cryopreserved without removal of red blood cells or further separation [6]. With this method of cryopreservation, large volumes of unseparated UCB required a large freezer for storage. Subsequently, numerous investigators explored different methods of UCB separation including the use of Ficoll, Percoll, methylcellulose, gelatin, starch, and lysis to remove red blood cells and recover nucleated cells [28,48–51]. These methods have allowed for more efficient storage of UCB units, although it is not yet clear what the best separation procedure is, or if the collections should undergo any separation at all, in terms of efficient recovery of hematopoietic stem and progenitor cells, and subsequent engraftment of recipients. UCB units that are banked for allogeneic transplantation undergo histocompatibility typing using conventional serological and molecular, DNA-based techniques for class I antigens, and molecular HLA typing for class II alleles. Because the amount of UCB that is available for HLA typing and infectious disease testing is limited, molecular methods, including the polymerase chain reaction and sequence-specific oligonucleotide probe methods, are used to better define class I and class II alleles while using very small amounts of UCB cells [52,53]. While these molecular techniques better define specific alleles, the optimal level of HLA typing for UCB and the clinical impact of higher degrees of resolution are not currently known [54]. Due to the rapidly evolving field of immunogenetics and the need for a standard worldwide nomenclature, the World Marrow Donor Association has provided guidelines for HLA nomenclature, with a recent validation of this system [55]. In addition to ABO, rhesus and HLA typing, UCB banked for transplantation is tested for infectious agents in accordance with the requirements and recommendations of regulatory bodies as listed above. Specifically, UCB units and the mother’s blood are routinely tested for hepatitis B and C, human immunodeficiency virus, human T-cell lymphotropic virus, cytomegalovirus, and syphilis [39,40]. Since some infectious agents can be transferred in liquid nitrogen, newly collected UCB units are typically kept in quarantine until infectious disease testing is complete. If newly collected UCB units are found to be free of potentially transmissible infectious agents, the units are placed into long-term liquid nitrogen storage or equivalent temperatures. In addition to this testing, UCB banks also elicit a history of genetic diseases in the family, travel of donors to places that have a high frequency of transmissible infections, and other high-risk behaviors, including intravenous drug use and high-risk sexual behavior, upon which UCB units may be excluded from the bank. These screening questions are similar to those used for blood donor screening by the AABB. Upon thawing, units of UCB that were cryopreserved for up to 15 years demonstrated 83 ± 12% recovery of nucleated cells, with 95 ± 16%, 84 ± 25% and 85 ± 25% recovery of CFU-GM, BFU-E, and CFUGEMM [56], values essentially equal to those same samples defrosted at 10 years after cryopreservation [57]. Similar results have been noted after 21 years of storage (Broxmeyer, unpublished observations). (Plate 39.1(a) shows colonies derived from UCB multipotential and granulocyte–macrophage progenitor cells of UCB units defrosted after being cryopreserved for 21 years.) The proliferative capacity of these early progenitor cells was intact for colonies formed from the cryopreserved cells, self-renewal potential of CFU-GEMM was high, as assessed by

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replating of colonies into secondary dishes in vitro, and extensive ex vivo expansion was possible. CD34+ cells separated from the 15-year defrosts were able to multilineage-engraft sublethally irradiated nonobese diabetic with severe combined immunodeficiency syndrome (NOD/SCID) mice, suggesting high-quality recovery of HSCs [56]. Laboratory studies demonstrated that UCB cells frozen for several years can be thawed, gene transduced, and ex vivo expanded [56,58,59]. The longest a UCB collection has been stored prior to use for successful cord blood transplantation is in the range of 10–12 years (Rubinstein, personal communication.) UCB banking has raised a number of ethical, regulatory, and legal issues, including questions about recruitment, confidentiality, ownership, informed consent, and fairness in the allocation of this valuable resource. Considerable efforts have been made to define these issues to ensure the appropriate operation of UCB banks [46,47]. It was recognized that there needed to be a national system within the United States to oversee UCB collection, banking, distribution, and usage [60]. Recent federal legislation (discussed below) focused on coordinating these activities and ensuring the safety of this valuable national resource.

Searching for a UCB donor Several potential advantages of unrelated donor UCB transplantation over unrelated donor BM or mobilized PB are the ready availability of banked UCB units, a shorter time to acquisition of UCB with a more rapid time to transplantation, and the ability to tolerate greater degrees of HLA disparity. It was suggested that if four out of six and three out of six antigen matches are clinically acceptable, donors would be identified 99% of the time for patients of all races [61]. Based upon traditional search methods and strategies, several groups have demonstrated that the time required for identification of a suitable hematopoietic cell source and the time to transplantation is more rapid for unrelated donor UCB than for unrelated donor BM [62,63]. A number of questions were raised in a report from the United States Institute of Medicine of the National Academies of Science, including adequacy of cord blood inventories, standardization of the collection of cord bloods, and their processing, storage, documentation, and quality control [64]. In the context of these concerns, the United States Congress provided money under the Stem Cell Therapeutics and Research Act of 2005 to establish a US National Cord Blood Bank Program. This effort fell to the Health Resources and Service Administration (HRSA). Information on the Blood Stem Cells Program of HRSA, kindly provided by Robert L. Baitty, MPP, of HRSA follows. More information on this can be found on the following websites: http://bloodcell.transplant.hrsa.gov and http://marrow.org. This program deals with implementation of the C.W. Bill Young Cell Transplant Program and focuses in part on: the national cord blood inventory; the related cord blood donor demonstration project; program infrastructure including a cord blood coordinating center; and an advisory council. This stem cell therapeutic and research act (public law 109–129) was signed on December 20, 2005 with the aims to increase the number of unrelated donor transplants, to provide a public inventory of high-quality UCB units from diverse populations, and to increase the number of UCB units available for research. HRSA’s implementation was guided by a single point of access for patients and physicians to all sources of blood stem cells, the collection of high-quality diverse UCB units, and complete data on clinical outcomes of transplants. The NMDP was chosen as a Cord Blood Coordinating Center, and six National Cord Blood Inventory Banks were initially chosen. An HRSA Advisory Council on Blood Stem Cell Transplantation has recently been instituted to evaluate amongst other issues, the quality of UCB blood banks and UCB units.

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Clinical results While there is now extensive clinical experience for the transplantation of related and unrelated donor UCB, including a large, prospective national trial (COBLT), to date there are no prospective, randomized clinical trials comparing UCB with BM or mobilized PB. However, there are an increasing number of studies comparing outcomes of UCB transplantation to related and unrelated donor marrow transplantation. Related donor UCB transplantation Two large case series of related donor UCB transplants have been reported by the International Bone Marrow Transplant Registry (IBMTR) [65] and Eurocord [66]. These retrospective series have shown survival rates of approximately 60% at 1 year. However, of greater importance are results of the IBMTR and Eurocord report in a retrospective cohort controlled analysis of children under 15 years of age transplanted for malignant and nonmalignant diseases with HLA-identical UCB or BM [66]. In this study, 2052 unmanipulated BM recipients were compared with 113 UCB recipients. Transplants were performed between 1990 and 1997 at 207 transplant centers worldwide. The median period of followup was 27 (range 3–85) months. Significant differences in these two patient groups were identified, with UCB recipients being younger (5 years versus 8 years; p < 0.001), of lower weight (median weight 17 kg versus 26 kg; p < 0.001), and less likely to receive methotrexate (MTX) for graft-versus-host disease (GVHD) prophylaxis (28% versus 65%; p < 0.001). The median UCB cell dose was 4.7 × 107 nucleated cells/kg (range <1.0 × 108 to 36 × 107/kg). The cumulative incidence of neutrophil engraftment by day 60 was 0.89 (95% confidence interval [CI] 0.82–0.94) for UCB versus 0.98 (95% CI 0.97–0.99) for BM ( p < 0.001). The cumulative incidence of platelet recovery to 20,000/mm3 was 44 days after UCB and 24 days after BM, with a cumulative incidence of platelet engraftment by day 180 of 0.86 (95% CI 0.78–0.92) for UCB versus 0.96 (95% CI 0.94–0.97) after BM ( p < 0.001). The cumulative incidence of grade II–IV acute GVHD was 0.14 (95% CI 0.08–0.22) for UCB versus 0.24 (95% CI 0.22–0.26) for BM ( p = 0.02). The 3-year cumulative incidence of chronic GVHD was 0.06 (95% CI 0.02–0.13) for UCB versus 0.15 (95% CI 0.13–0.17) for BM ( p = 0.02). However, there was no difference in the probability of overall survival at 3 years (0.64 (95% CI 0.53–0.74) for UCB versus 0.66 (95% CI 0.64–0.68) for BM), with no difference in relapse-related deaths. This study demonstrated a lower risk of acute and chronic GVHD with UCB, a slower rate of neutrophil and platelet recovery in the first month post transplant with UCB, but similar survival in both UCB and BM recipients. These findings support the use of HLA-matched related donor UCB as an acceptable alternate to BM for children with HLA-identical siblings. Unrelated donor UCB transplantation As with related donor UCB transplantation, the vast experience with unrelated donor UCB transplantation is the result of retrospective case reports of patients [66,67], rather than the result of prospective clinical trials [68]. This experience is complemented by emerging results from the prospective NHLBI COBLT study with reports of unrelated donor UCB transplantation in adults [69], children with lysosomal and peroxisomal storage diseases [70], and infants and young children with leukemia [71]. Among the largest of these series is a study by the New York Cord Blood Program that reports the outcome of unrelated donor UCB transplantation in 861 patients [72]. The majority of UCB recipients in this

series were children (age 0–11 years) (67%) and patients with hematologic malignancies (67%). The majority of patients received UCB grafts that were HLA mismatched at HLA-A, B or DRB1, with 87% of grafts mismatched for one or two antigens. The Kaplan–Meier estimate for neutrophil engraftment (absolute neutrophil count ≥500/mm3) was 0.93. As in other reports [66,68,73], the most powerful factor predictive of neutrophil engraftment was the cell dose of the UCB unit. However, the analysis also found that the time to neutrophil recovery was associated with HLA disparity, with a median neutrophil engraftment on day 23 for recipients of HLA-matched UCB versus day 28 for recipients of HLA-mismatched UCB ( p = 0.0027). Acute GVHD was also associated with increasing HLA disparity. The incidence of grade III–IV acute GVHD was 0.08 for recipients of HLA-A, B, DRB1-matched UCB and 0.28 in mismatched UCB recipients ( p = 0.006). Multivariate analysis demonstrated GVHD and stage of disease as the best predictors of disease relapse. In this series, the Kaplan–Meier estimate at 3 years for overall survival was 0.27 (95% CI 0.40–0.55) for patients with hematologic malignancies, and 0.48 (95% CI 0.40–0.55) for patients with genetic diseases. A number of reports have compared the results of unrelated donor UCB transplantation with those of unrelated donor BMT [74–77]. In the first of these case-controlled retrospective reports, recipients of between zero and three HLA antigen-mismatched UCB were compared with recipients of HLA-A, B, DRB1-matched unrelated donor BM [74]. Analyses were based upon the type of GVHD prophylaxis administered. All UCB recipients received cyclosporine (CSP) and methylprednisone. BM recipients received either CSP and MTX (26 pairs), or CSP and methylprednisone and T-cell depletion (31 pairs). This analysis demonstrated a probability of neutrophil engraftment at day 45 of 0.88 (95% CI 0.75–1.00) for UCB versus 0.96 (95% CI 0.89–1.00) for BM (CSP plus MTX) ( p = 0.41), and 0.85 (95% CI 0.72–0.98) for UCB versus 0.90 (95% CI 0.80–1.0) for BM (T-cell depletion) ( p = 0.32). The probability of platelet engraftment by day 180 was 0.72 (95% CI 0.50–0.94) for UCB versus 0.76 (95% CI 0.54–0.98) for BM (CSP plus MTX), and 0.84 (95% CI 0.64–1.00) for UCB compared with 0.84 (95% CI 0.64– 1.00) for T-cell-depleted BM. Analyses were performed using matchedpair analysis based on the type of GVHD prophylaxis administered. The incidences of acute and chronic GVHD were similar for both stem cell sources. The Kaplan–Meier estimate of overall survival at 2 years was 0.53 (95% CI 0.31–0.75) for UCB recipients versus 0.41 (95% CI 0.22–0.60) for BM (CSP plus MTX) ( p = 0.40), and 0.52 (95% CI 0.30–0.73) for UCB versus 0.56 (95% CI 0.30–0.70) for BM (T-cell depletion) ( p > 0.80). The single institution, case-controlled analysis suggested that, despite significantly greater HLA disparity among UCB recipients, the probabilities of neutrophil and platelet engraftment, acute and chronic GVHD, and survival were similar between recipients of zero to three antigen-mismatched unrelated donor UCB and HLA-matched unrelated donor BM. Similar results were demonstrated in an analysis performed by Eurocord [63]. In this analysis, 541 children with acute leukemia who received zero to four HLA antigen-mismatched UCB (n = 99) were compared with recipients of zero to three HLA-antigen mismatched T-cell-depleted unrelated donor BM (n = 180) or recipients of zero to two HLA antigen-mismatched T-replete BM (n = 262). While comparisons were made after adjustments to these three groups, distinct differences were noted among the three groups. Most notably, UCB recipients received a higher number of HLA-mismatched grafts (92%) versus recipients of T-cell-depleted BM (43%) and versus T-replete BM recipients (18%) ( p < 0.001). The probability of neutrophil engraftment by day 60 was 0.80 (95% CI 0.70–0.90) for UCB, 0.90 (95% CI 0.84–0.96) for T-cell-depleted BM, and 0.96 (95% CI 0.95–0.97) for

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T-replete BM at a median of 32 (range 11–56), 16 (range 9–40), and 18 (range 10–40) days ( p < 0.001), respectively. Platelet recovery occurred at a median of 81 (range 16–159) days for UCB, 29 (range 8–165) days for T-cell-depleted BM, and 29 (range 8–141) days for T-cell-replete BM ( p < 0.001). The probability of grade III–IV acute GVHD was less in recipients of UCB (p = 0.22) and T-cell-depleted BM (p = 0.08) versus T-cell-replete BM ( p = 0.30). Further, the probability of chronic GVHD was lower in UCB and T-cell-depleted marrow recipients compared with T-cell-replete BM ( p = 0.02). While early TRM was highest among UCB recipients ( p < 0.01), nonadjusted estimates of survival (for patient, disease, and transplant differences) at 2 years were similar: 0.35 (95% CI 0.25–0.45) for UCB, 0.41 (95% CI 0.33– 0.49) for T-cell-depleted BM, and 0.49 (95% CI 0.43–0.55) for T-cellreplete BM. Overall mortality was greatest in recipients of T-cell-depleted BM ( p < 0.07). A report from the Center for International Blood and Marrow Transplant Research compared the outcome of matched related sibling donor (n = 101), unrelated donor BM (n = 85), and UCB (n = 81) in children less than 18 months with acute leukemia [76]. Treatment-related mortality was 6%, 15%, and 31% after matched sibling, unrelated donor marrow and UCB, respectively, with no difference in risk of relapse, and overall and leukemia-free survival among the different stem cell sources. A recent meta-analysis compared the outcome of unrelated donor UCB versus unrelated donor BM in children and adults [77]. A total of 161 children and 316 adults received unrelated UCB transplants, with 316 children and 996 adults receiving unrelated donor BM transplants. In children, the incidence of chronic GVHD was lower in unrelated donor UCB grafts (relative risk 0.26), while the incidence of acute GVHD (grade III–IV) did not differ. In adults, TRM and disease-free survival were not different based upon hematopoietic cell source. Taken together, these retrospective and meta-analyses suggest that unrelated donor UCB and T-cell-replete unrelated donor BM have different risks and potential advantages, with similar overall survival. The data support the overall findings suggesting that unrelated donor UCB is a reasonable hematopoietic cell source for children and adults without a matched related donor, giving comparable survival to unrelated donor BM. UCB transplantation in adults As a result of the limited number of cells available in the finite volume of collected UCB, there was initial concern that UCB might not contain a sufficient number of cells to reliably engraft larger children, adolescents, and adults. There is now an emerging experience for UCB transplantation in adults [69,78–86]. The first large series of UCB transplantation in adults reported 68 adults (median age 31.4 years, range 17.6–58.1) with hematologic malignancies, marrow failure syndromes, and a single patient with an inborn error of metabolism [78]. The median weight of patients was 69.2 (range 40.9–115.5) kg. Seventy-one percent of patients received UCB grafts that were mismatched for two or more HLA antigens, with patients receiving a median cell dose of 1.6 × 107 (range 0.6 × 107–4 × 107) nucleated cells/kg and a median of 1.2 × 105 (range 0.2 × 105–16.7 × 10 5) CD34+ cells/kg. The probability of neutrophil engraftment was 0.90 (95% CI 0.85–1.0) at a median of 27 (range 13–59) days. Similar to other reports, this study also demonstrated an association between a higher number of cells in the cryopreserved UCB unit and the rate of neutrophil recovery. Platelet recovery (>50,000/mm3) occurred at a median of 99 (range 42–228) days. The probability of acute GVHD of grade III–IV by day 100 was 0.20 (95% CI 0.11–0.29) without a significant association between the grade

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of acute GVHD and the degree of HLA mismatching ( p = 0.70). The probability of chronic GVHD was 0.38 (95% CI 0.23–0.52). At a median follow-up of 22 (range 11–51) months, 19 of 68 patients were alive (28%) with 18 of them disease free. Thirty-two of 68 (47%) patients died of transplant-related complications. Patients receiving UCB grafts containing more than 1.2 × 105 CD34+ cells/kg had longer survival than patients receiving less than this number of CD34+ cells ( p = 0.05). However, event-free survival was not associated with HLA disparity ( p = 0.07), patient age ( p = 0.42) or the type of malignancy. The results are similar to those reported by Eurocord for 107 adults [75]. The COBLT study prospectively studied the use of UCB in adults [69]. Thirty-four adults with a median age of 34.5 (range 18.2–55) years received four of six (n = 23), five of six (n = 10) and six of six (n = 1) antigen-matched UCB grafts for various malignant hematologic diseases. The cumulative incidence of neutrophil and platelet engraftment by day 42 was 0.66 (median 31 days) and 0.35 by day 180 (median 117 days). The cumulative Kaplan–Meier estimate for survival at day 180 was 0.30. Causes of death included recurrent disease (25%), acute GVHD (25%), chronic GVHD (11%), infection (26%), graft failure (11%), and graft rejection (4%). Recent efforts in adults have focused on the use of reduced-intensity preparative regimens attempting to reduce the high incidence of nonrelapse mortality (NRM). A study of 21 adults with advanced Hodgkin’s lymphoma reported the results of unrelated UCB (n = 9) or matched sibling donation after a reduced-intensity preparative regimen of busulfan, fludarabine, and total body irradiation [82]. All patients had sustained engraftment by day 60, although patients receiving a matched sibling donor graft had a more rapid rate of engraftment. In this small study, the cumulative incidence of acute GVHD, chronic GVHD, and day 100 TRM was no different among hematopoietic cell sources. Progression-free survival at 2 years was also no different, suggesting, with limited patient sampling, comparable outcomes between hematopoietic cell sources in adults with Hodgkin’s lymphoma receiving a reduced-intensity preparative regimen. Figure 39.1 shows the cumulative incidence of neutrophil and platelet recovery and adjusted probability of leukemia-free and overall survival after matched and unmatched BM versus mismatched UCB transplantation in adults with acute leukemia [83]. The issue of low UCB cell doses for large patients has prompted several groups to study the transplantation of 2 UCB units [84–86]. The first of these studies reported 23 adults (median age 24 years) receiving two partially HLA-matched UCB units after high-dose conditioning [84]. The median infused cell dose was 3.5 × 107 (range 1.1–6.3 × 107) nucleated cells/kg. All evaluable patients engrafted at a median of 23 days. Interestingly, by day 100, a single UCB unit predominated. Disease-free survival was 57% at 1 year. Two subsequent trials have studied the transplantation of 2 UCB units after reduced-intensity preparative regimens. In one of these studies [86], 21 adults (median age 49 years) received 2 UCB units after a preparative regimen of fludarabine, melphalan, and antithymocyte globulin (ATG). The median engraftment of neutrophils was at 20 days. NRM was 14%, with a disease-free survival at 1 year of 67%. Patients demonstrated several patterns of donor chimerism, with a single unit predominating in some patients, whereas both units were present in others. The largest study to date for double UCB unit transplantation has recently been reported [84]. One hundred and ten adults (median age 51 years) received 2 UCB units after a preparative regimen of fludarabine, cyclophosphamide, and a single fraction of total body irradiation. Neutrophil engraftment was demonstrated in 92% of patients. The incidence of acute (grade III–IV) GVHD was 22%, with 23% of patient

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(a)

(b)

(c)

(d)

Fig. 39.1 Transplantation outcomes with mismatched cord blood, and matched and mismatched bone marrow for treatment of adults with acute leukemia. (a) Recovery of neutrophils, (b) recovery of platelets, (c) adjusted probability of leukemia-free survival, and (d) adjusted probability of overall survival. It is seen that human leukocyte antigen (HLA)-mismatched cord blood should be considered an acceptable source of hematopoietic stem cell grafts for adults in the absence of an HLA-matched adult donor. (Reproduced from [83], with permission.)

developing chronic GVHD. TRM and survival at 3 years were 26% and 45%, respectively. Figure 39.2 shows neutrophil and platelet recovery, and 3 year event-free and overall survival for patients receiving 1 or 2 UCB units after reduced-intensity conditioning in adults with hematologic disease. While mixed donor chimerism was frequently demonstrated soon after transplantation, all patients demonstrated engraftment of a single UCB unit when assessed at later time points. No factors, including CD34+ cell content, total nucleated cell dose, HLA matching, ABO typing, gender match or order of unit infusion were predictive of the predominant unit. The experience in adults suggests that the probability of engraftment and incidence of acute and chronic GVHD are tolerable, but NRM is high with traditional high-dose preparative regimens. Recent preliminary reports of double UCB unit transplants performed after reducedintensity conditioning regimens are encouraging, suggesting that this approach may overcome critical issues of cell dose and high TRM. However, there are unanswered questions and some concerns with transplantation of multiple UCBs. First, only one UCB wins out, and it is not yet predictable which will win. Second, there may be enhanced GVHD with multiple UCBs, which could negate the advantage of UCB, that is, the lowered incidence of GVHD elicited by single UCB transplants.

Immune reconstitution after UCB transplantation As a result of the low number of transplanted cells and the “naïve” pattern of immune cells in UCB, it was initially thought that immune reconstitution following UCB transplantation was delayed in comparison to BM and mobilized PB. It was reported that UCB transplant recipients had a high incidence of infectious complications, although it was not apparent if these infections were the result of delayed immune recovery, delayed neutrophil engraftment or the general use of high-dose steroids for GVHD prophylaxis in UCB recipients. The pattern of immune recovery following UCB transplantation has now been well described for children and adults [68,73,87–93]. The reports demonstrate a remarkably similar pattern, with prompt recovery of natural killer (NK) cells by 2–3 months post transplant, of B cells by 6–9 months post transplant, and of CD8+ T cells by 8–9 months, but delayed recovery of CD4+ T cells with a return to normal numbers at approximately 1 year. In studies involving both children and adults, the recovery of CD4+ T cells was slower in adults, consistent with the pattern of recovery for BM and PB [93]. Mitogen stimulation responses are noted in the normal range by 6–9 months post transplant. Studies of T-cell receptor diversity and T-cell receptor excision circles [89–91] further suggest efficient thymic regeneration mechanisms following

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(a)

(b)

(c)

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Fig. 39.2 Cord blood hematopoietic cell transplantation (HCT) after reduced-intensity conditioning in adult patients with hematologic disease. (a) Cumulative neutrophil recovery by day 42, (b) unsupported platelet recovery greater than 50 × 109/L at 6 months, (c) cumulative proportion of 3-year event-free survival, and (d) cumulative proportion of 3-year overall survival. (c and d) Patients receiving either 1 (- -) or 2 (—) cord blood units. The findings support the use of umbilical cord blood after reduced-intensity conditioning as a strategy for extending the availability of HCT, particularly for older patients. (Reproduced from [84], with permission.)

UCB transplantation that are similar, if not superior, to that of BM. The prospective COBLT study included an assessment of immune recovery after unrelated donor UCB transplantation [92]. UCB recipients were studied for the presence of T lymphocytes with herpesvirus antigen-specificity. Overall, 66 of 153 recipients developed antigenspecific T lymphocytes to at least one of the herpesviruses. In a subsequent analysis, it was demonstrated that patients who developed antigen-specific T-lymphocyte responses had a significantly lower probability of relapse (p < 0.003) with improved relapse-free survival (p < 0.0001) [94]. Future directions for clinical UCB transplantation Studies to date demonstrate the importance of cell dose on the pace of engraftment and the ability to engraft larger patients. In addition, cell dose is predictive of NRM. It is also clear that lower cell dose increases the deleterious effects of HLA mismatch [69,95,96]. Several strategies have been explored to address the problems of cell dose and high NRM in adults. The transplantation of 2 UCB units following reducedintensity conditioning regimens are showing encouraging results. These approaches complement the expansion of UCB banks with an increasingly large number of UCB units with a larger cell dose, thereby making larger UCB units more available for transplantation. Hematopoietic growth factors (e.g. granulocyte colony-stimulating factor [G-CSF]) have been routinely used after UCB transplantation. More limited is the use of other growth factors (e.g. interleukin 11 [IL-11]). However, to date, there are no reliable data to suggest the benefit of growth factors following UCB transplantation. Another approach for facilitation of neutrophil and platelet engraftment is the ex vivo expansion of UCB prior to transplantation [97]. Twenty-five adults and 12 children with hematologic malignancies (n =

34) or breast cancer (n = 3) received an infusion of UCB expanded in G-CSF, megakaryocyte growth and development factor, and stem cell factor (SCF; also called steel factor). Patients received a median of 0.99 × 107 nucleated cells/kg (expanded plus unexpanded UCB). The median time to neutrophil and platelet engraftment was 28 (range 15–49) and 106 (range 38–345) days, respectively, with engraftment in all patients who survived 28 days. Grade III–IV acute GVHD occurred in 40% of patients, with extensive chronic GVHD in 63% of patients. This information notes a greatly increased rate of acute and chronic GVHD compared with noncultured cells. With a median follow-up of 30 months, 35% of patients survived. This study and others suggest the ability to expand ex vivo more committed progenitors, with limited ability to expand long-term repopulating cells. The inability of ex vivo-expanded cells to contribute to long-term hematopoiesis may be the result of increased apoptosis, initiation of the cell cycle, and disrupted homing of HSCs. Future studies will explore the safety and efficacy of ex vivo expansion, different expansion conditions, and manipulation of signaling pathways (including Notch, Wnt, bone morphogenetic protein 4, and Tie2/angiopoietin-1), as well as other pathways noted below and intracellular mediators (phosphatase and tensin homolog and glycogen synthetase kinase-3, etc.), to expand HSCs with less effect on differentiation [98].

Characteristics of primitive hematopoietic stem and progenitor cells in UCB The number of nucleated cells available for UCB transplantation is greatly limited compared with that available for BMT. However, the frequency of primitive cells in UCB, as assessed by progenitor cell numbers as determined by assays of colony-forming cells [6,8], is greater than that of BM. Numbers of CFU-GM, BFU-E, and CFUGEMM present in the first 65 UCB collections performed in the

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Broxmeyer laboratory have been published [57]. Most of these values fell within the limits of the corresponding numbers present in BM used for successful BM transplantation [8]. It was noted that the progenitor cell content of UCB units was more rigorously associated with major covariants of post-transplantation survival than was the nucleated cell count, and that progenitor cell content was a better indication of UCB grafts [99]. As important is the quality of these cells in UCB compared with BM. UCB includes cells with extensive proliferative capacity and replating ability in vitro (Fig. 39.3 (a–c)) [6,8,100–108]. UCB progenitors respond very rapidly to stimulation by combinations of cytokines. Moreover, primitive human UCB cells appear to have great capacity to engraft/repopulate the hematopoietic system of mice with severe combined deficiency syndrome (SCID) (Fig. 39.3 (d)). The proliferative characteristics of primitive UCB cells are extensive. However, why engraftment of neutrophils and, especially, platelets is slower with UCB than BM cells still remains a serious unanswered question. This difference may in part reflect the more immature nature of UCB versus BM primitive cells. The more immature status of UCB primitive cells as a total population is suggested by studies evaluating

the loss of telomeric DNA with age [109], and by a higher ratio of more immature to mature colony-forming cells in UCB than in BM [8]. Efforts to accelerate neutrophil recovery in recipients of UCB transplantation have not been very rewarding. Low numbers of megakaryocyte progenitors in UCB grafts have been implicated in the delay in platelet recovery after UCB transplantation [110], but the problem may also be in the maturation capacity of the megakaryocytes from UCB. Cord blood megakaryocytes derived from megakaryocyte progenitors do not complete maturation as well as cells from BM or PB [111]. More intricate knowledge of the cytokine regulation of blood cell production in the context of microenvironmental stromal cell interactions may be necessary for the development of better intervention methodologies to accelerate engraftment of UCB cells. There are a number of cell surface phenotypic markers of primitive cells that help to define these populations. For the most part, these markers, which include CD34, CD38, Thy1, c-kit, fms-related tyrosine kinase-3 (Flt3), and rhodamine-123, are the same for primitive cells found in UCB and BM [112]. These primitive cells express CD34 antigens and low or absent levels of CD38, Thy1, c-kit, and rhodamine-123,

Fig. 39.3 Proliferative Capacity of Human Cord Blood Hematopoietic Progenitor and Stem Cells. A) Colony formation by multipotential (CFU-GEMM) (lower-left)- and granulocyte macrophage (CFU-GM) (upper-right)- progenitor cells from cord blood cells thawed after 21 years in a frozen cryopreserved state, B) Colony formation from a high proliferative potential (Bi and Bii) and a smaller proliferative potential (Biii and Biv) progenitor cell arising from a single high CD34-expressing single cell sorted into a single microtiter plate (i and iii are at original magnification x 20; and ii and iv are at original magnification x 80). Reprinted from Figure 2 of [102] with permission from Blood; C) Secondary CB CFU-GEMM colony derived from replating of a single CFU-GEMM colony into a secondary plate, a measure of self-renewal of CFU-GEMM. See reference104 for more information, D) immunohistochemical staining of CD34+ cord blood cells that engrafted NOD/SCID mouse bone marrow using human Ki67, a marker of cell proliferation. See Orazi et al238 for more information. Conclusion: Cord blood progenitors and stem cells have extensive proliferative capacity.

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and are positive for Flt3. Human CD34+ cells contain HSCs and HPCs, and since CD34 is also found on other cells (e.g. endothelial cells), this marker alone is not a perfect indicator of the actual number of HSCs or the composition of HSCs and HPCs of a graft. CD133 has also been used to identify populations enriched for HSCs and HPCs. In the human system, CD34+ cells can be subtyped into a CD38− population, considered to contain the earliest cells including HSCs, and a CD38+ population in which HPCs are highly enriched and HSCs are few or absent. The complexity and heterogeneity of the CD34+CD38− population is highlighted by the demonstration that, in the human SCID repopulating cell (SRC) assay, fewer than 1 : 600 CD34+CD38− cells appear to be a human SRC [107], and the SCID assay is currently our best assay for evaluating human HSCs. Second generation NOD/SCID (IL-2 common γ chain receptor null) mice are now available. The use of these mice, which manifest enhanced repopulation by human cells and for longer periods of time, may allow adjustments as to the frequency of human SRCs from cord blood. At present, it is possible to more rigorously define murine than human HSCs phenotypically. Mouse HSC functional assays depend on repopulation of lethally irradiated mice in either a competitive or a noncompetitive situation. In the human SRC assay, only sublethal irradiation dosages are given to recipient mice, and human cells have not been proven to rescue lethally irradiated SCID mice. Murine HSCs [113,114] are phenotypically characterized into long-term (Sca-1hiThy1loLin−Mac1−c-kit+) and short-term (Sca-1hiThy1loLin−Mac-1loc-kit+) repopulating cells [115]. The signaling lymphocyte activation molecule (SLAM) family is a group of 10 or 11 cell surface receptors tandemly arranged at a single locus on chromosome 1, and includes CD150, CD244, and CD48. Murine BM HSCs were found to be highly purified in a phenotypically defined population of CD150+CD244−CD48− cells [116]. In contrast, nonself-renewing multipotential HPCs were defined as CD244+CD150−CD48−, and most restricted progenitors were designated as CD48+CD244+CD150−. A similar pattern of SLAM family receptor expression was found on murine fetal liver HSCs [117]. Fetal liver HSCs of mice were CD150+CD48−CD244− , with the vast majority of the fetal liver HPCs found in the CD48+CD244−CD150− or CD48+CD244+CD150− cells. More rigorous phenotyping of murine than human HPCs is also available [118,119]. A recent review documents myeloid lineage commitment from HSCs with the murine system [120]. More definitive phenotypic categorizing of human HSCs and HPCs is needed. In this regard, a clonogenic subpopulation of CD34+CD38− cells was identified that expressed high levels of CD7 and possessed only potential for lymphoid cell development [121]. These cells also expressed CD45RA and HLA-DP, but were low or absent in expression of c-kit and Thy1. Methodologies for assessment of the functional properties of HSCs and HPCs have been published [112]. Efforts are underway to profile HSC and HPC populations at a genomics [122–126] and proteomics [127,128] level, and will no doubt be accelerated for human cells by advances in technology, but may be hindered by lack of more precise phenotypic markers of human HSCs and HPCs that better recapitulate subset functional heterogeneity.

Endothelial progenitor cells and mesenchymal stem/stromal cells in cord blood In addition to HSCs and HPCs, other immature non-HSCs have been identified in UCB. An immature subset of high-proliferative endothelial progenitor cells (EPCs) has been characterized. This cell achieved at least 100 population doublings and had the capacity to form colonies in secondary and tertiary dishes with maintenance of high telomerase activity [129]. EPCs from UCB had greater proliferative potential than those

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from adult tissue sources. EPCs were redefined by clonal analysis and using HSC/HPC principles [130]. Mesenchymal stem/stromal cells (MSCs) produce bone, fat, and cartilage and have great proliferative potential, but whether or not they can be categorized as stem cells is still an open question [131]; hence the cells are sometimes designated mesenchymal stromal, rather than stem, cells (see Chapter 9). MSCs are found in cord blood and have demonstrated immune-modulatory activity [132–141]. Sometimes these cells are found in low frequency in UCB or are absent; thus, great variability is noted in the frequency of these cells in different cord blood collections. Whether or not this variability is technical or has significance for transplant outcome remains to be determined. EPCs and MSCs are still not well defined phenotypically, and enhanced characterization of phenotypes, that recapitulate functions, will increase the potential for use of these cells from cord blood. Efforts towards this for MSCs have been published [142].

Ex vivo expansion of UCB stem cells Since the numbers of HSCs and HPCs that can be obtained from single collections of UCB is limited [6,8,57,100], and these numbers appear to be dose limiting, especially in the context of higher-weight individuals and adults [9,78], a number of different options to deal with this problem have been evaluated. These include attempts at ex vivo expansion of HSCs and HPCs, the use of combined collections of UCB, mixture of UCB with other tissue sources of HSCs, enhancement in the homing capacity of HSCs and HPCs, and intramarrow injections of cells to bypass potential loss of cells through the circulation. Investigators have placed extensive laboratory efforts into expanding UCB HSCs ex vivo. It is clear that primitive UCB cells can be greatly expanded ex vivo. However, these studies demonstrated greater expansion of the more mature cells than the most immature cells in this primitive population. Also, a dissociation of SRC phenotype and function was noted during expansion of UCB CD34+CD38− cells [143], and the fate of functional SRCs has varied greatly among groups studying their expansion ex vivo. These studies leave open the question of whether the long-term marrow repopulating human HSCs are truly being expanded under these conditions and, if so, by how much? Clearly, loss of these most primitive cells is undesirable. While UCB cells that have been subjected to ex vivo expansion culture maneuvers have been transplanted into recipients [97], clinical benefit of these cells has not yet been demonstrated. The capacity to ex vivo expand HSCs is most likely limited by our lack of knowledge of the process of self-renewal. A number of cytokines have been used in attempts to “ex vivo” expand human HSCs/HPCs. The most basic starting combination of cytokines for this effort includes SCF, Flt3 ligand, and thrombopoietin (TPO). Other molecules, such as stromal cell-derived factor (SDF)-1/CXCL12, have also been implicated as useful agents for these efforts. Information on these and other cytokines involved in hematopoiesis, with potential effects on ex vivo expansion, have been described [144]. SDF-1/CXCL12 has recently been found to enhance the replating capacity of multipotential (CFU-GEMM) and macrophage (CFU-M) progenitor cell-derived colonies in vitro [145], a measure of self-renewal capacity of HPCs, and also to greatly enhance the ex vivo expansion of human cord blood HPCs (Broxmeyer, unpublished observations). It remains to be determined if SDF-1/ CXCL12, in combination with SCF, Flt3 ligand, and TPO or other cytokines, will ex vivo expand human HSCs. Information on how the intracellular signals triggered by the cytokines alone and in combination, and in the absence and presence of stromal cell support, are mechanistically mediated for proliferation, self-renewal, and differentiation could greatly enhance the possibilities of modulating HSCs for expansion ex vivo and in vivo. A number of intracellular signaling molecules have

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been identified for such functions; these include the cyclin-dependent kinase inhibitors p21cip1/waf1, p27kip1, members of the STAT transcription factor family and their associated JAK proteins, Notch ligands, Wnt family members, β-catenin, glycogen synthase kinase-3, sonic hedgehog, bone morphogenic proteins, members of the Hox family of transcription factors, and other transcription factors such as PU.1, GATA-1, growth factor independent-1 [144], and Forkhead 0 family members [146]. HSC self-renewal may involve symmetric and asymmetric division of the cells where at least one daughter cell maintains the pluripotentiality of the parent HSC. It may be that the best possibilities for ex vivo expansion will occur in synthetic three-dimensional models with specific types of scaffold [147]. In addition to ex vivo expansion of HSCs/HPCs, fully mature red blood cells have been generated ex vivo from populations of CD34+ UCB cells [148].

Gene therapy of UCB stem cells UCB HSCs are logical cellular vehicles for dissemination of the products of newly introduced genes. There is in vitro evidence that primitive UCB cells may be more efficiently transduced than equivalent cells in BM using retroviral vectors [149,150]. Primitive UCB cells are efficiently transduced by retroviral vectors [58,150,151]. A limited number of attempts at gene therapy were performed utilizing UCB in a clinical setting years ago [152]. Three children with adenosine deaminase (ADA)-deficient SCID received transplants of noncryopreserved autologous UCB CD34+ ADA gene-transduced cells in a reduced-intensity conditioning setting. Engraftment of the retrovirally ADA genetransduced cells was successful, but the level of engraftment was low [153,154]. Enhanced effectiveness of this procedure as a clinical effort awaits elucidation of better vectors and improved methodology to transduce the earliest noncycling HSCs, with maintenance of the self-renewal capacity of the transduced cells and high, but controlled, expression of the introduced genes.

Homing of UCB stem cells Enhanced homing of the small numbers of HSCs found in single collections of UCB is another potential option to increase the effectiveness of UCB transplantation. HSCs are nurtured and produced in the BM, where various cell types provide an appropriate niche for their development [155–162], although what this niche actually encompasses is not completely agreed upon [163]. While some subsets of mouse HSCs appear to home with absolute efficiency to the marrow, as a total population of HSCs they do not. It is likely that only a minority of HSCs that are infused into human recipients actually home to an appropriate microenvironment. Enhanced homing could result in a reduction in the number of cells necessary for engraftment and might shorten the time to engraftment. A clearer understanding of adhesion molecules and the agents that enhance expression and activation of these adhesion molecules will no doubt be of use. The cytokine family of chemokine molecules, along with other cytokines, may be intimately involved in the migration and homing of HSCs. Chemokines are chemoattractant molecules involved in chemotaxis (directed cell movement, as contrasted to chemokinesis, random cell movement) of leukocytes [164]. SDF-1/CXCL12 [165,166], CKβ-11/ CXCL19 [167], and SLC/CCL21 [168] have been implicated in the in vitro chemotactic migration of primitive cells from UCB and BM, and these chemokines act in concert with SCF [166], CKβ-11/CCL19, and SLC/CCL21, and are relatively specific for chemotaxis of macrophage progenitor cells [167,168], while SDF-1/CXCL12 is chemotactic for a wide range of progenitor cells (CFU-E, BFU-E, CFU-GEMM, etc.) [165,166]. SDF-1/CXCL12 and its receptor CXCR4 have been implicated in the homing of human stem cells into SCID mice [169–171],

and retroviral vector-induced overexpression of CXCR4 results in enhanced chemotaxis of cells to SDF-1/CXCL12 [172]. Some intracellular signals have been definitely linked mechanistically to the SDF-1/CXCL12 chemotaxis of primary HPCs. These include the phosphatases SHIP [173] and SHP-1 [174]. HPCs from mice functionally deleted in SHIP and SHP-1 were enhanced in sensitivity to SDF-1/CXCL12 chemotaxis, suggesting that SHIP and SHP-1 act to negatively regulate SDF-1/CXCL12-induced chemotaxis. Also implicated in this migration of HSCs/HPCs are transforming growth factor-β [175] and protein phosphatase 2A [176]. Since SDF-1/CXCL12 also acts as a survival factor for HPCs and HSCs [reviewed in 177–179], and blocks the myelosuppressive effects of inhibitory members of the chemokine family [180], this chemokine may play a dual role in homing to, as well as survival within, the marrow microenvironment. There are a number of different isoforms of SDF-1/CXCL12 [181]. While all demonstrated chemotactic properties, only the α, β, and ε forms enhanced the survival and replating capacity of HPCs. In addition, mice that are null for chemokine receptors, such as CXCR4 (the receptor for SDF-1/CXCL12) [182], CXCR2 (a receptor for IL-8) [183], and CCR1 (a receptor for macrophage inhibitory protein1 alpha) [184], have been used to suggest that these chemokine receptors may also be involved in the trafficking of murine HPCs. CXCR4−/− mice die perinatally with little or no hematopoiesis in the marrow. CXCR2−/− mice demonstrate a hyperplasia of hematopoietic cells, especially in the spleen, and CCR1−/− mice have abnormal tissue distribution of progenitor cells. Treating UCB HSCs or HPCs ex vivo with cytokines, or transducing these cells with genes for receptors or cytokines involved in homing, may permit the use of fewer HSCs for optimal engraftment [185–188]. Decreasing the amount or activity of CD26/DPPIV (dipeptidylpeptidase IV), a membrane-bound extracellular peptidase that cleaves dipeptides from the amino terminus of polypeptide chains, results in enhanced chemotaxis to SDF-1/CXCL12 [189]. CD26/DPPIV can cleave the chemokine CXCL12/SDF-1α at its position 2 proline and inactivate CXCL12/SDF-1. CD26/DPPIV is expressed by a subpopulation of CD34+ cells isolated from UCB and these cells have DPPIV activity. Amino-terminal truncated CXCL12/SDF-1α lacked the ability to induce migration of CD34+ UCB cells, but inhibited normal CXCL12/SDF-1αinduced migration. Inhibition of endogenous CD26/DPPIV activity on CD34+ UCB cells enhanced the migratory response of these cells to CXCL12/SDF-1α. Pretreatment of mouse BM cells with inhibitors of CD26 (e.g. Diprotin A, a tripeptide, or Val-Pyr, a dipeptide), or use of marrow cells from mice functionally deleted in CD26 (= CD26−/−), resulted in enhanced engraftment of self-renewing, long-term repopulating, and competitive HSCs [190]. Enhanced engraftment of human UCB CD34+ cells into NOD/SCID mice was observed after a 15-minute pretreatment of the CD34+ cells with Diprotin A to inhibit CD26 [191,192]. Treating recipient NOD/SCID mice with Diprotin A enhanced engraftment of G-CSF-mobilized human CD34+ cells, which expressed low or absent levels of CD26 [193], and pretreating lethally irradiated congenic mice with Diprotin A enhanced engraftment of untreated donor mouse BM long-term competitive repopulating and self-renewing HSCs [194]. It is possible that pretreating donor cells as well as recipients to inhibit CD26 may be more effective than treating only the donor cells or the recipients. Since CD26 can truncate certain growth factors [187], it is possible that inhibiting CD26 will have additional effects, perhaps enhancing the growth-promoting activities of these cytokines in vivo for enhanced recovery in a stem cell transplantation setting. Efforts are also ongoing to determine if direct injection of HSCs into the bone in a preclinical model will enhance the engrafting capability of these cells [195,196]. The concept here is to bypass loss of cells from

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the circulation that do not home well to the BM microenvironment. There is not enough information available yet in a clinical setting to know how effective this type of maneuver will be.

Immune cells An original concern with UCB transplantation was that UCB collections might contain maternal cell contamination, and thus elicit a severe and life-threatening GVHD reaction [7,9,10]. However, the original studies using relatively insensitive methods did not find any maternal T-cell contamination [7,9–11]. Further studies using much more sensitive technology found that maternal T cells do contaminate UCB collections in many cases [197–201], but the frequency of contaminating T cells was extremely low. This low frequency of contaminating T cells, in the absence of clinical data suggesting an enhanced incidence of GVHD in UCB transplantation [7,9,14,65–67,73], brings into question the clinical meaning of such a low frequency of T-cell contamination. Although the incidence of GVHD is apparently low in UCB transplant recipients, even in an unrelated HLA-disparate setting [65–67,73], the extent of GVHD has thus far not correlated well with the disparity of HLA between donor and recipient [65,73]. The apparently low incidence of GVHD noted in most UCB transplant recipients may reflect the biological activities of UCB T lymphocytes [202,203]. UCB T lymphocytes generate less cytotoxic T-cell activity than do adult T cells, even after repeated allogeneic stimulation in vitro [203–205]. Whereas UCB T lymphocytes respond as well as adult BM or blood T lymphocytes to the proliferation-inducing activity of a primary allogeneic stimulation, UCB T cells become unresponsive to secondary allogeneic stimulation, whereas adult T cells proliferate to an even greater extent in response to secondary allogeneic stimulation compared with their response to the primary allogeneic stimulation [206]. Investigation into the possible mechanisms mediating this UCB T lymphocyte unresponsiveness to secondary allogeneic stimulation has implicated the ras intracellular signal transduction pathway [207]. Stimulated lymphocytes from UCB have reduced the production of cytokines, such as interferon-gamma (IFN-γ) and tumor necrosis factoralpha (TNF-α), which may be involved in the pathophysiology of GVHD. As transcription factors of the nuclear factor of activated T cell (NFAT) family play a role in transcription of IL-4, IFÑ-γ, GM-CSF, IL-13, TNF-α, CD69, FasL, and CD40L genes [208], it is possible that NFAT-1, found to be low or not expressed in UCB T cells [209], might be involved in the lowered amplification of donor UCB T-cell alloresponsiveness against recipient antigens, and in the reduced GVHD noted in UCB transplantation. Cord blood T cells have also been shown to manifest distinct chemokine responsiveness by different chemokine receptor repertoires than T cells from adult blood [210], which may also be involved in differences noted in the extent of GVHD after UCB compared with BMT. Further analysis of UCB T cells focused on CD8+ T cells [211]. CD28 is a co-stimulatory molecule. It is the CD28−CD8+ T-cell subset that has been associated with cytotoxic T-lymphocyte (CTL) effector function. Cord blood is composed of CD8+ T cells with few or no CD28− CTLs. However, a combination of cytokine stimulation and activation of another co-stimulatory molecule in the TNF receptor family (41BB) leads to CTL effector function in the following sequence. IL-15 and IL-12 preferentially induced 41BB on CD28−CD8+ UCB T cells. Costimulation of these cells with anti-41BB restored expression of CD28 on these cells, as well as memory markers such as CD45RO and CCR6. The memory-type CD28+ T cells so generated acquired greatly enhanced CTL activity in association with increased content of granzyme B, a cytolytic mediator. Interestingly, the CTL activity could be almost completely abrogated by a soluble form of 41BB. In a follow-up study, it was noted that transforming growth factorbeta and 41BB modulated UCB CD8+ T-cell effector differentiation

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[212]. Transforming growth factor-beta is an immunosuppressive cytokine that inhibits CTL immune responses, and suppresses differentiation of naïve UCB CD8+ T cells. This effect was abrogated by 41BB costimulation, but not by CD28 or another member of the TNF receptor family, CD30. It was noted that 41BB co-stimulation suppressed TGFβ-induced phosphorylation of Smad2. Critical roles for IL-4 and IL-12 were reported for regulating 41BB effects on TGF-β-mediated suppression. It has been suggested that ex vivo expanded, matured, and activated UCB T cells with IL-2, IL-12, anti-CD3, and IL-7 may be of value for adoptive cellular immunotherapy post umbilical UCB transplantation [213]. It is possible that 41BB activation is also of importance in this type of adaptive immunotherapy. Forkhead Box P3+ (FOXP3+) T cells are unconventional as they suppress other immune cells and play critical roles in immune tolerance [214,215], factors that impinge on GVHD. FOXP3+ regulatory T cells belong to the T-cell receptor-alpha beta group. It was found that FOXP3+ T cells could be divided into CD45RA+ (naïve-type) and CD45RO+ (memory-type) cells. T cells undergo changes in trafficking receptors according to their stages of activation and differentiation [216]. In UCB, CD45RA+FOXP3+ T cells are in the majority, but CD45RO+FOXP3+ T cells become a majority in adult PB. A role for CD45RA+FOXP3+ UCB T cells and the switch to CD45RO+FOXP3+ adult T cells in GVHD is to be evaluated. It was originally thought that a lower GVHD incidence inherent in UCB transplantation might manifest in a lowered graft-versus-leukemia (GVL) effect with UCB cells. While increased relapse rates have not been obvious so far in UCB transplantation for malignant diseases, more experience in this area is needed before any firm conclusions can be drawn. NK cells have been implicated in mediating the GVL effect [217,218]. While UCB generally manifests low NK activity compared with that found in adult BM and blood, the NK cell activity of UCB and adult cells is readily activated in vitro by cytokines, such as IL-2 and IL-12 [217–219], suggesting that UCB NK cells should be as capable as adult BM or blood NK cells to mediate the GVL effect. Interestingly, a subset of NK cells with the surface phenotype CD16+CD56−, which has not yet been identified in the adult blood of normal individuals, has low lytic activity and might represent a precursor of mature NK cells. The lytic activity of CD16+CD56− cells can be enhanced by cytokine stimulation [220]. Functional quantitative activities of other UCB immune cells are also distinct from their counterparts found in the adult [202,217]. Selected cytokine production by T cells and dendritic cell (DC) antigen presentation are reported to be lower in UCB than adult blood cells. DCs are active antigen-presenting cells that initiate and maintain adaptive immune responses. Both lymphoid (HLA-DR+, CD123+, CD11c−, CD33−) and myeloid (HLA-DR+, CD123+, CD11c+, CD33+) DCs have been identified in UCB, although the majority of these cells had a lymphoid morphology [221]. The 41BB ligand was found to mediate maturation of CD11c+ myeloid DCs derived from CD34+ UCB [222]. In addition to enhanced expression of CD11c, other antigens, such as major histocompatibility class II, CD86, and 41BB ligand, were increased after stimulation of UCB CD34+ cells with granulocyte–macrophage colonystimulating factor (GM-CSF), TNF-α, Flt3 ligand, and SCF. Stimulation of the 41BB ligand on these DCs resulted in the capacity of these cells to produce IL-12, results that may be of use in the design of DC-based vaccines with enhanced activity. Freshly isolated UCB lymphoid DCs did not induce a potent allostimulation for naïve UCB T cells. CD34+ UCB cells can generate DCs. A CD7+CD45RA+ subpopulation of CD34+ UCB cells was found to have the dual capacity to generate NK cells and DCs [223]. Immature DCs induce tolerance, while mature DCs induce inflammatory immune responses. The question addressed was whether or not DCs

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could be generated that manifested anti-inflammatory properties in the immature and mature states [224]. Highly purified UCB monocytes were induced to differentiate into IL-10hiIL-12absent DCs in the presence of macrophage colony-stimulating factor (M-CSF) and IL-4, in a GM-CSFindependent manner. These M-CSF/IL-4-generated DCs, in contrast to GM-CSF/IL-4-generated DCs, induced decreased T helper type 1 differentiation and proliferation of naïve CD4+ T cells in both primary and secondary mixed leukocyte reaction, and showed tolerogenic potential, suggesting that these M-CSF/IL-4-generated DCs may be useful for suppressing unwanted immune responses. TGF-β added to the combination of M-CSF and IL-4 induced the generation of immune inhibitory UCB DCs that released cytokines that enhanced ex vivo expansion of human UCB HPCs induced with SCF, Flt3 ligand, and TPO [225]. TGFβ/M-CSF/IL-4-generated DCs secreted lower concentrations of progenitor cell inhibitory cytokines, demonstrated enhanced responses to bacterial lipopolysaccharide (LPS)-induced activation of ERK, JNK, and p38 MAP kinases, and were less potent in activating T cells than were DCs generated with TGF-β/GM-CSF/IL-4. The uniqueness of TGF-β /M-CSF/IL-4-generated DCs to silence immunity while promoting expansion of HPCs may be of potential therapeutic value. Functional activity of DCs in vivo likely relates to the migratory capacity of DCs. GM-CSF/IL-4/LPS DCs generated from UCB monocytes manifested decreased chemotaxis to respective CCR7 and CXCR4 ligands CCL19 and SDF-1/CXCL12 when compared with similarly generated DCs from adult blood monocytes [226]. Decreased chemotaxis of GM-CSF/IL-4/LPS-generated UCB DCs to CCL19 was associated with decreased expression of CCR7 on these cells compared with those generated from adult monocytes. However, UCB DCs expressed higher levels of CXCR4 than the adult DCs, and decreased chemotaxis of UCB DCs to SDF-1/CXCL12 was associated with a stronger and sustained activation of ERK.

Additional information and conclusion Experience with UCB HCT is encouraging. However, worldwide experience with UCB transplantation is still relatively limited. The use of UCB for HCT has advantages and disadvantages [227]. Among the advantages are rapid availability, ability to more rapidly schedule the transplant as the UCB units are stored and ready for use, the apparent reduced need for an exact HLA match, and induction of a less severe GVHD compared with BM. It offers the potential of targeting collections in minority populations, groups that are currently underrepresented in the NMDP registry. Compared with matched or mismatched BM, greater degrees of HLA disparity appear to be tolerated with UCB. As allogeneic UCB banks reach sufficient size, the time required to acquire a donor unit should decrease, allowing transplantation to occur at an earlier time. The establishment of the HRSA-funded UCB Coordinating Center at the NMDP, and enhanced collections of quality UCBs to be banked for future use, should increase the availability of UCB units for transplantation. Disadvantages include the limited numbers of cells collected and stored, a relatively limited inventory enabling at least four-of-six matches with an adequate cell dose for many patients (e.g. differing races and ethnicity), the lack of additional cell collections from the UCB donor, and the noted variability in terms of UCB unit quality after thawing of the stored cryopreserved cells. As with non-UCB sources of hematopoietic cells for transplantation, there are a number of factors associated with transplant outcome [228]. These include the donor, graft, patient, conditioning regimen and prophylactic therapy for GVHD and GVL, treatment-related mortality, relapse, and disease-free survival. While there is increasing information on this with UCB for HCT, there is clearly more information needed,

especially for adult recipients. Thus far, it appears that UCB can be used to treat all malignant and nonmalignant disorders currently treated with BM and mobilized PB cells. As the criteria for selecting the best UCB unit for adults are better defined, and more effective conditioning and prophylactic measures are used, it is likely that the results of UCB HCT will improve. It is known that HLA mismatches play an important role in engraftment, GVHD, and survival, but with UCB, the effect of HLA mismatches is partially abrogated by increased UCB cell dose [228,229]. Results of unrelated UCB HCT for patients with Fanconi’s anemia have demonstrated that factors that can be modified easily, including better UCB unit selection, and a fludarabine-containing regimen has a significantly enhanced therapeutic endpoint for patient survival [230]. In addition to a delay in neutrophil and platelet recovery when UCB is used for HCT, there is also some evidence of delayed immune cell reconstitution, which appears to reflect impaired thymopoiesis and skewing of late memory T cells [231]. There are also reports of an increased risk for opportunistic infections after UCB HCT, mainly of viral origin [232]. Infection-related mortality is a primary or secondary cause of death (in the presence or absence of another major problem such as GVHD) in 50% or more of deaths after UCB HCT. This infection-related mortality within the first 100 days after transplant was higher among recipients of mismatched UCB than HLA-matched or mismatched BM [233], although beyond 100 days there was no significant difference in the three groups. Interestingly, recipients of UCB HCT are reported to manifest similar risks of infection with cytomegalovirus, responses to antiviral therapy, and survival following this viral infection compared with BM or mobilized PB HCT [234]. Not only is UCB HCT associated with lower levels of acute GVHD, but chronic GVHD may be more responsive to therapy after UCB HCT [235]. Post-transplant donor-acquired leukemia is a rare event in HCT, and there is not any evidence that this occurs more frequently in UCB HCT compared with other tissue sources of HCT [236]. However, more information in this area is clearly warranted for all tissue sources of HCT. Since the volume of each UCB available for collection is limited, means to enhance the number or quality of HSCs in UCB is needed. Manipulation of UCB through ex vivo expansion is still a sought-after endpoint, but enhancing the capacity of the UCB HSCs to home to an appropriate microenvironment in the marrow may serve a similar purpose. In the meantime, the problem of limiting numbers of UCB cells is being addressed by use of double UCB for transplantation. In the unrelated UCB setting, it is not possible to reacquire UCB from the donor when the recipient experiences graft failure or relapse. Ex vivo expanded cells stored for future use or the use of another stored UCB unit may circumvent these possible problems. Despite proposals to require long-term tracking of unrelated UCB donors, there is currently little clinical information available on unrelated UCB donors, such that the possibility exists that genetic diseases may be inadvertently transferred to UCB recipients. Enhanced genetic testing in the future will be of use here. Neutrophil, and especially platelet, engraftment in UCB recipients is prolonged. In vivo or ex vivo use of cytokines or expanded mature cells may be of help. Newly identified cytokines and greater knowledge of the intracellular signaling events they trigger for proliferation and self-renewal of HSCs will be helpful. Future anticipated efforts in the field of UCB transplantation include: (1) greater success in efficiently engrafting adults and higher-weight children with a single UCB collection, thus decreasing the potential for enhanced GVHD possible with the use of multiple UCB units; (2) enhanced downmodulation of GVHD and upmodulation of the GVL effect, perhaps by using MSCs, DCs or other immune cell types; mechanistic insight into MSC-mediated immune suppression is increasing [237]; and (3) knowledge of whether UCB is or is not also efficacious for regenerative medicine in a nonhematopoietic situation.

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40

Alan W. Flake & Esmail D. Zanjani

In Utero Transplantation

Introduction For most of human history, the fetus has been shrouded in mystery, obscured by multiple layers of maternal tissue, and protected from the scrutiny and intervention of well-meaning physicians. In the past three decades, this private world of the fetus has been invaded by a vast array of imaging techniques and methods for fetal tissue sampling, allowing the early gestational diagnosis of selected congenital hematologic disorders. Over the past 10 years, progress has accelerated in a number of technologies that are destined to revolutionize the field of prenatal diagnosis. The convergence of knowledge from the human genome project, molecular genetic diagnosis, isolation of fetal cells or DNA from the maternal circulation, and techniques of high through-put gene analysis will, in all likelihood, allow population screening and diagnosis of every genetically based hematologic disorder, early in gestation. In many instances, the early prenatal diagnosis of a disease provides a rationale for prenatal treatment [1]. This rationale is particularly compelling for diseases that cause death or irreversible organ damage before birth. In this circumstance, by the time of birth, the damage has already been done. Examples of successful treatment of lethal fetal disease are now numerous and include fetal transfusion for erythroblastosis fetalis [2] and prenatal correction of selected anatomic malformations [3]. Another compelling rationale for fetal therapy exists when there are biological advantages for prenatal therapy relative to postnatal therapy. In this circumstance, unique aspects of fetal development may allow the treatment of a disease with reduced morbidity, mortality, and cost compared with treatment after birth. This is the rationale for in utero hematopoietic stem cell (HSC) transplantation. It is the purpose of this chapter to review the theoretical, experimental, and, at the present time, limited clinical support for this approach.

An experiment of nature Perhaps the most compelling argument for the potential of in utero hematopoietic cell transplantation (IU-HCT) comes from an experiment of nature. Owen [4], in 1945, observed that dizygotic cattle twins that shared a placental circulation were chimeric for their sibling’s blood cells after birth. Subsequent investigators confirmed that chimeric animals were specifically tolerant for skin grafts [5] and organ grafts [6]

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

from their sibling donor. Natural chimerism arising from shared placental circulation has also been observed in a number of other species, most notably primates [7] and humans [8]. The cotton-top tamarin, a New World primate species, has a high incidence of natural chimerism, with documentation of stable donor cell chimerism of greater than 80% in some animals. The incidence of human chimerism in monochorionic, dizygotic pregnancies is around 8% when sensitive detection methodology is utilized, and natural human chimeras with all levels of donor cell chimerism, including marked donor cell predominance [9], have been observed. These observations are proof in principle for IU-HCT. They confirm that circulating allogeneic stem cells can effectively compete and stably engraft an early gestational recipient, and that, at least in specific circumstances, normal host hematopoiesis is not prohibitive to the engraftment of donor cells. The primary question is, “What are the ideal circumstances to permit experimental and clinical recapitulation of this experiment of nature?”

Fetal immunologic tolerance Owen’s observations of natural chimerism were followed by Burnet’s [10] theory of immunity, which subsequently led to the experimental work of Billingham and Medawar [11] promoting the concept of “acquired” immunologic tolerance. These studies suggested that the early presentation of cellular antigen resulted in specific immunologic tolerance. Their studies documented that tolerance could only be achieved during a period of immunologic immaturity and was best accomplished by the transplantation of living cells. More recent studies have documented the role of the fetal thymus in self-recognition and tolerance induction [12,13]. The human thymus becomes populated by prethymocytes at 8–9 weeks’ gestation [14]. Thymocytes undergo a series of differentiation and selection events in the thymus before becoming mature functional lymphocytes. The details of thymic processing are still being investigated, but the critical events have now been defined [15]. Thymocytes are positively selected for recognition of self-class I or class II major histocompatibility complex (MHC) antigens, which are presented by thymic cortical epithelial cells of thymic stromal origin. Lack of MHC recognition results in programmed cell death. Thymocytes that recognize self-MHC are then negatively selected by high-affinity recognition of “self”-antigen in association with self-MHCs. Thymic deletion occurs in the medullary region of the thymus and is mediated by thymic epithelial cells and thymic dendritic cells that are derived from HSCs. The end result is a repertoire of single positive (CD4+ or

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CD8+) functionally competent lymphocytes that recognize foreign antigen in association with self-MHC. Single positive (post-thymic) lymphocytes are first seen in the peripheral blood circulation at around 12–14 weeks’ gestation [16]. Theoretically, introduction of foreign cells prior to completion of this process would result in thymic processing of foreign cells as “self” with secondary specific tolerance on the basis of clonal deletion. With improved understanding, it is clear that this early assumption in the field of IU-HCT is an oversimplification. In reality, during normal development, self-reactive T-cell clones routinely escape thymic deletion by a number of mechanisms [17–20]. It has been recently appreciated that these cells rarely cause autoimmune disease due to peripheral mechanisms of tolerance primarily involving regulatory T cells [21,22]. In the circumstance of clinical or experimental IU-HCT, donor cells are delivered after the process of thymic selection has begun, and donor antigen presentation in the host thymus may or may not be optimal. While partial deletion of donor reactive lymphocytes after IU-HCT has been clearly demonstrated [23,24], it is likely that a significant number of donor reactive cells escape thymic deletion after IU-HCT, and that the induction of peripheral regulatory mechanisms will be important for success in IU-HCT. We [25], and others [26,27], have documented evidence of an immune barrier after IU-HCT, which will be elaborated on below. In addition, while T cells may be implicated, there are other mechanisms of potential rejection after IU-HCT, including B-cell mediated responses and the innate immune system, that remain relatively poorly understood.

The fetal hematopoietic environment A biologically unique aspect of IU-HCT is the fetal receptive environment. Whereas postnatal bone marrow transplantation (BMT) generally requires myeloablative or at least minimal conditioning to achieve engraftment, such conditioning would be prohibitively toxic to the fetus. Thus, engraftment following in utero HSCs, by necessity, must depend upon competitive population of available receptive sites with a goal of

achieving an adequate level of mixed chimerism to have therapeutic effect for a given target disorder. The receptive sites in the early gestational environment are undoubtedly dynamic and are dependent upon the gestational age of the fetus. Fetal hematopoiesis is characterized by an orderly series of migrational events that proceed from the yolk sac or periaortic splanchnopleura, or both, to the fetal liver, and finally to the bone marrow [28,29]. Thus the migration of fetal hematopoiesis is probably best viewed as a sequential development of organ-specific microenvironmental niches of increasing HSC affinity [30]. At the developmental stage that corresponds to fetal immunologic immaturity in multiple species, the primary receptive site is the fetal liver. Studies in which tracking of transplanted cells have been performed in the sheep [31] and mouse [32,33] models suggest that the pattern of engraftment after IU-HCT recapitulates ontogeny; i.e. prior to bone marrow formation cells home to the fetal liver but once the bone marrow forms, even fetal liver-derived cells will preferentially home to the fetal bone marrow. The early dogma of this field predicted that the rapid expansion of the fetal hematopoietic compartment would favor the availability of open niches for engraftment of donor cells, and that there would be a “window of opportunity” prior to immune competence. While “space” is available prior to population of the bone marrow, this has proven to be only partially true (Fig. 40.1). While it is certainly true that the immunologic opportunity exists, the efficiency of engraftment of bone marrow cells after in utero transplantation is no better than that after postnatal myeloablative BMT, with only approximately 5% of cells homing to and engrafting receptive sites in the fetal liver [32,33]. This suggests that the fetal receptive environment, with a few disease-specific exceptions, is highly competitive and that strategies will need to be developed to improve the competitive capacity of donor cells to achieve significant engraftment.

Animal studies that support in utero transplantation The first experimental studies that supported engraftment of donor cells after in utero transplantation were the classic studies of Billingham and

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Fig. 40.1 A schematic of normal hematopoietic ontogeny depicting the concepts of an early immunologic “window of opportunity” and increasing host hematopoietic competition. The late limit of immunologic tolerance in humans has not been defined and may extend into later gestation in immunodeficiency disorders such as severe combined immunodeficiency disease.

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Fig. 40.2 Maximal donor cell engraftment occurs during the mid- to later stages of the preimmune period. This period is associated with the development of bone marrow in sheep which is populated by donor stem cells in competition with endogenous hematopoietic cells. The failure of donor cells to engraft in very young fetuses may be explained by the absence of suitable receptive sites.

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Fig. 40.3 Levels of engraftment plateau with transplantation of log-fold increases in donor cells. This is most consistent with a model of saturation kinetics with engraftment limited by available receptive sites. An identical curve is seen in the xenogeneic human–sheep model.

40

Medawar [11], who documented donor-specific tolerance for skin grafts after fetal or neonatal transplantation in mice. Although chimerism was not analyzed, it can now be assumed that tolerant animals were chimeric for donor cells. Large animal models In order to determine whether engraftment of allogeneic HSCs could be achieved in a large animal model, we developed an in utero HSC transplant approach in sheep that results in multilineage allogeneic engraftment without the need for myeloablation [34] and, under appropriate conditions, without graft-versus-host-disease (GVHD] [35]. Engraftment after IU-HCT has for unexplained reasons been easier to achieve in the sheep model than in other species and man, raising concern regarding the clinical applicability of findings in the sheep model. However, the sheep model has provided a number of basic observations that have translated well on a developmental stage-for-stage basis with other animal models. The relationship of gestational age at transplantation and engraftment has been examined carefully in the sheep model with confirmation of the concept of a presumed immunologic “window of opportunity.” Late gestational transplantation results in failure of engraftment, with loss of the ability to engraft roughly corresponding to the gestational age at which fetal lambs reject allogeneic skin grafts (75 days gestation) (Fig. 40.2). A similar window exists in the murine model where engraftment can be achieved across full MHC barriers by IU-HCT at embryonic days 14–15, but not in newborn mice. The relationship between dose of donor cells and engraftment has also been examined in the sheep model. Logfold increases in the dose of transplanted cells initially increased engraftment, but this rise rapidly plateaued, suggesting saturation of available receptive sites (Fig. 40.3) [36]. If this theory were true, then transplantation of cells in divided doses should allow the development of new receptive sites between transplants and should improve engraftment. Indeed, in the allogeneic sheep model, transplantation of the same total number of cells in three divided doses increases engraftment by a considerable margin (Fig. 40.4). The importance of route of administration of donor cells has also been examined in sheep. Higher levels of donor cell engraftment have been

Donor cells (%)

35 28.3 ± 6.9

30 25 20

n = 13

15

10.9 ± 3.6

10 5

n=8

0 3

1 Number of injections

Fig. 40.4 The effect of dividing cell dose on engraftment. Transplantation of the same total number of cells as a single injection versus a series of three injections given 1 week apart is shown. The engraftment with divided doses is significantly higher, supporting the concept that engraftment is limited at any particular time by the number of available receptive sites.

obtained when cells are administered by intraperitoneal injection than by intravenous injection. Although we are unable to provide an explanation for this finding, this difference has been observed on many occasions, and for that reason we have utilized the intraperitoneal approach for most studies. In more recent murine studies, we have utilized the vitelline vein for intravascular injection [25,33,37]. This has allowed injection of far greater doses of cells (20 million versus 5 million per fetus), resulting in a higher frequency of engraftment of congenic recipients (100% maintaining long-term chimerism) with similar levels of chimerism. Interestingly, it has not resulted in a significantly increased frequency or level of chimerism in allogeneic recipients. In the context of tracking studies demonstrating equivalent homing, expansion, and migration early after IU-HCT in congenic and allogeneic strains, this suggests that the frequency of chimerism is limited by immunologic barriers and that the level of chimerism is limited by competitive barriers. Other large animal models in which IU-HCT has been attempted include primates [38–43], pigs [44,45], goats [46,47], and dogs [48–51]. Of these, the primate and pig have been most successful. It has been shown in the rhesus model that fetal liver-derived hematopoietic cells

580

Chapter 40

can engraft and maintain long-term low-level multilineage chimerism [38], but that T-cell depleted adult bone marrow resulted in polymerase chain reaction-detectable chimerism at best [43]. In the baboon model, similar low levels of chimerism have been obtained with CD34-enriched adult-derived cell sources [40,41]. In the baboon model, the add-back of T cells to the CD34-enriched donor cells improved the level of chimerism but was limited by GVHD [42], results very similar to those in the sheep model [52]. The use of fetal immune suppression (steroids and antithymocyte globulin) resulted in a transient but not sustained improvement in peripheral blood donor chimerism levels [41]. Recently, interesting results in the swine leukocyte antigen-defined miniature swine model have been reported. Injection of a mixture of T-cell-depleted bone marrow with addition of unmanipulated whole bone marrow to a level of 1.5% T-cell content into the intrahepatic portion of the umbilical vein resulted in long-term, multilineage chimerism at easily measurable levels by flow cytometry in five of seven surviving pigs at 2 months and in two long-term survivors up to 70 weeks of age [45]. Tolerance for donor swine leukocyte antigen was demonstrated by transplantation of donor-matched kidney allografts without the need for immunosuppression [44]. These results need to be reproduced and expanded upon but have major implications for clinical application of IU-HCT as primary therapy or as a means achieving donor-specific tolerance to facilitate postnatal cellular or organ transplantation. The murine model With the exception of the sheep and recently the pig model, engraftment in other species has been difficult to achieve. The first studies in the mouse were the classic studies of the correction of genetic anemia by Fleischman and Mintz [53] in W/Wv mice by transplacental injection of normal HSCs. The W/Wv mouse strain is genetically deficient in the c-kit receptor, resulting in a selective advantage for normal stem cells [54]. In this model, a single normal stem cell can engraft and reconstitute normal hematopoiesis after IU-HCT [55]. Blazar et al. [56] demon-

100 Pep3b n=6 n=4

CD45+ cells (%)

80 70

B6

Congenic

50 40 n=4

20 10

n=6

n=7 n = 28

0 0 (a)

20

90

SJL

Liver Cord blood

B6

80

60

30

100

Liver Cord blood CD45+ cells (%)

90

strated in a murine severe combined immunodeficiency disease (SCID) model that in utero transplantation of congenic or fully allogeneic adult bone marrow could result in full T-cell and partial B-cell reconstitution and a functionally intact immune system. These studies demonstrated the important concept that in experimental circumstances of a selective donor stem cell or lineage advantage, IU-HCT can result in functional reconstitution and disease correction. However, most potential therapeutic applications of IU-HCT do not provide a competitive advantage to donor cells, and cumulative experimental and clinical experience suggests that the prenatal environment presents significant barriers to engraftment. The likely barriers to engraftment have been reviewed elsewhere [57] and can be categorized as a lack of available space, host cell competition, and immunologic barriers. In order to systematically examine the relative importance of these barriers and develop strategies to overcome them, the development of the normal allogeneic murine model was critical. The available inbred and transgenic strains, defined immunology, large litter size, minimal cost, and short gestation of the mouse allow studies to be performed that simply cannot be considered in other species. In addition, the mouse is, stage for stage, very similar to the human with respect to hematopoietic and immunologic ontogeny. In utero HSC transplants can be performed early in gestation (11–15 days) during a time when hematopoiesis is confined to the fetal liver, and prior to completion of thymic processing and the appearance of mature lymphocytes in the peripheral circulation. Finally, the normal mouse initially proved very difficult to engraft, making it a legitimate model for investigation of barriers to engraftment. We, and others, were only able to achieve microchimerism for several years in hematopoietically normal mouse strains [24,27,58–61]. To assess the issue of receptive space available in the fetal environment, we have assessed early events after IU-HCT in different studies with different modes of cell administration (intraperitoneal and intravascular) [32,33]. The results were remarkably consistent, demonstrating that engraftment efficiency was no better in the fetal recipient than in the adult BMT recipient and that only approximately 5% of donor bone marrow cells home to the fetal liver (Fig. 40.5). In response to the initial

40

n = 13

60

100

Allogeneic

40 30 n = 16 n=5

n=8 n=9

n=4

n=4

n = 16

n=8

0

120

Time (hours)

n = 11 n=7

50

10

n = 24 80

60

20

n = 10

n = 28

70

0

20

40

60

80

100

120

Time (hours)

(b)

Donor cell type

FL chimerism at 4 hours (%)

No. of CD45+ cells per recipient FL

No. of homed CD45+ cells

No. of CD45+ cells in donor innoculum

Homing efficiency (%)

Pep 3b BM SJL BM

19 ± 6.3 16.9 ± 7.7

2.6 ± 0.8 × 105 2.6 ± 0.8 × 105

4.8 ± 1.6 × 104 4.3 ± 1.9 × 104

9.9 × 105 9.9 × 105

4.9 ± 1.6 4.4 ± 2.0

(c)

Fig. 40.5 These graphs depict the triphasic kinetic profiles for the frequency of donor cells circulating within the recipient fetal peripheral blood (cord blood) or lodged within the fetal liver at various time points (1.5–96 hours) after in utero transplantation in the murine model. The nearly identical profiles for both the congenic (a) and the fully allogeneic (b) strain combinations are shown. The donor cell frequency is calculated as the percentage of cells expressing the donor CD45.1 isoform among all CD45.1 cells within the host cord blood or fetal liver. (c) Efficiency calculations for the homing of donor hematopoietic cells to fetal liver (FL) in this model. Only approximately 5% of transplanted cells home and engraft. (Adapted from [32], with permission.)

In Utero Transplantation

Donor cells* (%)

study, we improved our cell preparations and performed a dose escalation study to investigate the effect of increasing cell doses. With improved delivery of higher numbers of HSCs, we were able to achieve macrochimerism in multiple allogeneic strain combinations. The level of chimerism was dependent upon cell dose, on cell source, with fetal liver being far superior to adult bone marrow at equivalent doses, and on strain combination (Fig. 40.6). The second barrier of host cell competition may be the most important. It is clear from studies of in utero transplantation utilizing mouse strains that have a genetic deficiency in HSCs [53] or a specific lineage deficiency [56,62,63] that hematopoietic repopulation or lineage replacement can be achieved with very low levels of HSC engraftment and, in the case of c-kit-deficient mice, engraftment of a single HSC [55]. A strategy to improve engraftment would therefore be to selectively improve donor cell competitive capacity by conferring a homing or engraftment advantage at the time of transplantation. We have recently demonstrated that improving donor cell homing to the fetal liver by blockade of the dipeptidyl-peptidase CD26 (preventing inactivation of the chemokine stromal cell-derived factor-1) [64,65] can significantly increase short- and long-term engraftment in the murine model [66]. This is an important proof in principle that manipulation of a single component of the homing/engraftment sequence can have a major effect on engraftment. Similarly, as described below, the conferment of a competitive advantage after birth to donor

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

n = 16

cells after establishment of low-level chimerism by in utero transplantation results in conversion to high-level or complete chimerism [23,67,68]. Thus, assuming no immune rejection, after initial engraftment, the size of the donor-derived hematopoietic compartment will depend upon the ability of donor cells to compete with host hematopoiesis. Finally, the immunologic barriers to engraftment remain to be defined. The early gestational fetal environment immunologically differs from the postnatal environment and can be considered in the context of specific and innate immunity. There is little evidence that the fetus, prior to maturation of T cells in the thymus, can mount a specific immune response. Long-term persistence of allogeneic [34] or xenogeneic [69,70] cells and multilineage hematopoiesis in the sheep model suggest that donor-specific tolerance can be achieved by IU-HCT. Our more detailed analysis in the murine system clearly documents that, when engraftment is successful, tolerance is achieved by a predominant mechanism of bidirectional deletion of alloreactive lymphocytes augmented by peripheral mechanisms of tolerance after IU-HCT [23,67,71,72]. Tolerance is consistently present in animals with levels of peripheral blood chimerism of greater than 1% (macrochimerism) [23] but inconsistent in microchimeric animals [24]. We have documented a predominant mechanism of high-level clonal deletion in macrochimeric animals by analysis of relevant Vβ-T-cell receptor consistent with thymic processing of donor antigen (Fig. 40.7).

15

n=6

n = 11

70 60

n = 14

50

10

n = 18

40 30 5

20

n = 16 SJL

Pep

n=9

Donor strain (10 cells)

10

106

Balb/c 6

(a)

581

107 TCD

Donor cell dose

(b)

n = 12 105

(c)

106

5 × 106

Donor cell dose

Fig. 40.6 The effect of strain combination, dose, and cell source on donor cell engraftment after in utero hematopoietic cell transplantation. (a) Peripheral blood chimerism in B6 recipients of adult bone marrow from three different strain donors. (b) Peripheral blood chimerism of Balb/c recipients of B6 bone marrow at low and high doses. (c) Peripheral blood chimerism in B6 recipients of various doses of SJL fetal liver, demonstrating the dramatically higher levels of dose-dependent engraftment achieved with fetal liver-derived donor cells. (All levels measured at 6 months of age.) TCD, T-cell depleted; B6, C57BL6; Pep, C57BL/6; Ly5.1:Pep3B. *Note the difference in scale of the y-axis for the three figures.

B6 control (n = 6) BALB control (n = 6) BALB chimera (n = 12)

6

12

5

10

4 3

*† * *†

2

*

8 6

*

4

1

2

0 (a)

BALB/c control (n = 6) SWR → BALB/c chimera

14

Vβ6+ (%)

Donor CD3+ cells (%)

7

0 Vβ5.1/2

Vβ11

Vβ12

(b)

CD4+

CD8+

Fig. 40.7 Evidence of bidirectional clonal deletion as a mechanism of tolerance in macrochimeric animals created by in utero hematopoietic cell transplantation (IU-HCT). Donor and host strains are chosen based on the presence or absence of class II I–E, which is required for the presentation of mammary tumor virus (mtv) antigens. Mtv antigens are superantigens encoded by Mtv oncogenes in the host genome that are normally deleted in the host strain but not the donor strain. Thus, donor antigen presentation in the thymus can be limited to donor-derived antigen-presenting cells (APCs), i.e. direct antigen presentation, or to host-derived APCs, i.e. indirect antigen presentation. (a) Efficient deletion of donor host-specific Vβ T-cell receptor (VβTCR) in chimeric mice by direct antigen presentation after IU-HCT. *p < 0.001 chimeric mice versus B6 control; †p < 0.05 chimeric mice versus Balb/c control. (b) Partial deletion of host donor-specific VβTCR by indirect antigen presentation in SWR→Balb/c chimeras after IU-HCT *p < 0.001. (Adapted from [23], with permission.)

Chapter 40

These data, along with the observation by ourselves and others that there was no difference in engraftment between congenic and allogeneic cells after IU-HCT [56,58,73–75] formed the basis of our belief for many years that there was not a significant adaptive immune barrier to allogeneic engraftment. We have now re-examined this question in the murine model using much higher doses of cells (enriched HSC and bone marrow mononuclear cells [MNCs]), made possible by the use of the intravascular injection technique [37]. In this circumstance, where there is an excess of donor HSCs, there are major differences in the frequency of engraftment between congenic and allogeneic HSCs or bone marrow. In addition, using new methodology for tracking of injected cells, we have found that all animals are engrafted by allogeneic cells, and that there are no differences in early engraftment, expansion or migration between congenic or allogeneic cells up to 2 weeks after transplantation. However, by 5 weeks, only 30% of the allogeneic animals remain chimeric and go on to become long-term chimeras, whereas 100% of congenic animals maintain their engraftment [25]. This argues against a role for the innate immune system (which should act early after transplantation) and clearly implicates the adaptive immune system. The explanation for the variability in engraftment and inconsistent achievement of tolerance remains to be defined. A possibility, however, is that significant numbers of donor-reactive cells escape thymic deletion after IU-HCT, and that whether a graft is rejected or tolerance occurs depends on the balance of donor-reactive T cells and donor-specific T-regulatory cells. In summary, experimental results from animal models support the rationale and feasibility of IU-HCT. Although engraftment can be achieved clinically in circumstances of a donor cell selective advantage, the primary limitation to clinical application for most target disorders is the relatively low level of donor cell expression achieved. The future of IU-HCT depends upon developing successful strategies to overcome the barriers to engraftment, specifically the immune response, and host cell competition. There are a number of strategies being tested in animal models that show considerable promise and will be discussed below.

Strategies for successful in utero HSC transplantation The potential clinical applications of IU-HCT fall into two general categories. The first is reconstitution or replacement of a stem cell or lineage defect or deficit. The second is prenatal tolerance induction for facilitation of postnatal cellular transplantation. In order for either strategy to be successful, an adequate number of donor HSCs must initially engraft to provide ongoing hematopoiesis at a therapeutic level or to provide adequate thymic presentation of antigen for stable tolerance induction. One of the major problems clinically is the inability to achieve greater than polymerase chain reaction-detectable engraftment using enriched cell populations. The achievement of “microchimerism” is probably inadequate for either purpose as it appears to be associated with the engraftment of very few if any HSCs and does not reliably induce tolerance by a mechanism of clonal deletion. Therefore one of the major clinical challenges is to achieve higher initial levels of HSC engraftment. As conventional myeloablation is not an attractive option in the fetus, an alternative approach has been to attempt to increase available “space” by stromal co-transplantation [76,77]. Although the mechanism remains unclear, it has now been documented in both the allogeneic and xenogeneic sheep models that stromal co-transplantation leads to increased short- and long-term levels of donor cell engraftment in the bone marrow and donor cell expression in the peripheral blood. In the allogeneic model, the co-transplantation of adult stroma (plastic adherent bone marrow cells) resulted in greater enhancement of early donor peripheral

blood expression (60 days after transplantation) than fetally derived stroma, and the enhancement of expression was sustained for at least 30 months (Fig. 40.8). This observation, in combination with previous findings of delayed contribution of bone marrow-engrafted donor cells to peripheral expression in the sheep model [31], suggests that fetal stroma may be deficient or immature in its capacity to support definitive hematopoiesis, adding an additional rationale for co-transplantation of stroma, particularly in circumstances where high levels of donor cells are required early after in utero transplantation. There are a variety of other potential approaches to improve initial engraftment but these await experimental support. At the present time, attempts at in utero reconstitution or replacement have only been successful in circumstances where a competitive advantage exists for donor cells. It would therefore follow that, in order to achieve replacement of host cells in a competitive environment, one would need to either increase the competitive capacity of donor cells or reduce the competitive capacity of host cells, or both. A particularly promising approach is to increase the competitive capacity of donor cells postnatally after establishing low-level chimerism and tolerance by IU-HCT. This approach is based on the observation that engraftment can be achieved without cytoreduction in syngeneic models, arguing that, in the presence of tolerance or effective immunosuppression, cytoreduction may not be required [78,79]. This observation has been validated in allogeneic systems by the success of a variety of nonmyeloablative strategies for postnatal BMT in circumstances of host immune deficiency or tolerance [80,81]. We have applied three nonmyeloablative strategies in the mouse model to alter the competitive balance of the donor and host cell compartments in tolerant mixed chimeric animals after IU-HCT. In the first strategy, donor lymphocyte infusion was utilized to induce a graftversus-host hematopoietic effect. This resulted in a donor lymphocyte dose-dependent conversion of mixed chimerism to complete donor chimerism across a fully MHC-mismatched strain combination (Fig. 40.9). Surprisingly, only one of 56 animals receiving the high dose of donor lymphocytes with conversion to complete donor chimerism developed evidence of GVHD, suggesting that the mixed chimeric recipient created by IU-HCT may be a favorable candidate for donor lymphocyte infusion-induced enhancement of engraftment relative to the postnatal irra-

30 25 Donor hemoglobin (%)

582

20 HSC + stroma 15 10 HSC alone

5 0 5

15

25

35

Months post-transplant

Fig. 40.8 Co-transplantation of adult sheep stromal cells results in a sustained increase in donor-derived hematopoiesis. Sustained long-term engraftment that has persisted for more than 30 months after transplantation is shown, where the donor hemoglobin levels in peripheral blood in animals receiving adult bone marrow stroma (n = 4) are significantly higher (p < 0.01) than those receiving adult T-cell-depleted bone marrow mononuclear cells alone (n = 3). (Reproduced from [76], with permission.)

In Utero Transplantation

diation-induced chimera. Potential reasons for reduced GVHD in our study include the lack of irradiation with its accompanying proinflammatory cytokine milieu, which may lower the threshold for GVHD in nonhematopoietic tissues, or the presence of a highly active, nonirradiated thymus for the production of regulatory T-cell populations. These animals maintain donor-specific tolerance with bidirectional deletion of alloreactive lymphocytes, and normal immune response to third-party or novel T-cell-dependent antigens. This study is indicative of the potential for this approach as it is, to our knowledge, the first time that complete allogeneic chimerism has been achieved across full MHC barriers in the complete absence of cytoreductive or immunosuppressive therapy [23].

n = 17 n = 10

n=6 n = 14

The second minimally ablative approach that we have investigated was suggested by results of marked enhancement of engraftment in the syngeneic nonmyeloablative model when low-dose total body irradiation (TBI) was administered prior to transplantation [82]. When we administered graded doses of minimal TBI to our tolerant mixed chimeric animals following IU-HCT and then retransplanted the animals with 30 million T-cell-depleted donor bone marrow cells, we observed a TBI dose-dependent enhancement of engraftment to levels of greater than 90% donor peripheral blood expression with the highest TBI dose of 276 cGy [67] (Fig. 40.10). Similar results were obtained in a more recent study utilizing single-agent (busulfan) chemotherapy for conditioning the host [68]. These studies confirm the predominant importance of

n=6

n = 17 n=5

80

80

40 20

n=6

60 40 20

0

0 0 1 2 3 4 6 8 12 16 20 24 36 48

(a)

Weeks after DLI n=9 n = 10

0 1 2 3 4 6 8 12 16 20 24 36 48 (b)

n=9 n = 15

Weeks after DLI

n=7

n = 13 n=8

8 week old, low level chimeras ( <10%) 100

80

80

60 40

n=8 n = 15

n = 11

8 week old, high level chimeras ( >10%)

100

Donor cells (%)

Donor cells (%)

n=8 n = 14

4 week old, high level chimeras ( >10%) 100

Donor cells (%)

Donor cells (%)

4 week old, low level chimeras ( <10%) 100

60

583

20

60 40 20

0

0 0 1 2 3 4 6 8 12 16 20 24 36 48

(c)

Weeks after DLI n=6

0 1 2 3 4 6 8 12 16 20 24 36 48 (d)

n=7

Weeks after DLI

n=6

4 week old, SJL/J → B6 chimeric mice

Donor cells (%)

100 30 × 106 splenocytes 15 × 106 splenocytes 5 × 106 splenocytes PBS 30 × 106 splenocytes (naïve mice)

80 60 40 20 0 0

(e)

2

4

8

12

16

20

24

Weeks after DLI

Fig. 40.9 Engraftment profiles following postnatal donor lymphocyte infusion (DLI) in chimeric animals created by in utero hematopoietic cell transplantation (IU-HCT). DLI was performed in 4- (a,b) or 8- (c,d) week-old chimeras with either low-level (a,c) or high-level (b,d) chimerism using three doses of donor splenocytes. (e) Results of DLI performed at 4 weeks of age in a second allogeneic strain combination. (Reproduced from [23], with permission.)

Total donor chimerism (%)

584

Chapter 40 Table 40.1 Potential advantages of in utero hematopoietic stem cell transplantation

100 80

Immunologic tolerance

60 40 20 0 0

2

3

4

5

6

0 cGy (n = 12) 276 cGy (n = 14)

(a) Total donor chimerism (%)

1

7

8

10 12 14 16 20 24 28 32

82.5 cGy (n = 12) Control (n = 12)

Small fetal size and total cell no. Preempts clinical disease Stem cell biology: expansion, migration

→ No HLA restriction/immunosuppression → Transplantation massive donor cell doses/kg → Avoidance of morbidity → Expansion and distribution of engrafted donor cells*

138 cGy (n = 12) * Assumes equal or superior competitive capacity of donor cells. HLA, human leukocyte antigen.

100 80 *

60

*

40 20

#

0 0

1

2

3

4

(b)

5

6

7

8

10 12 14 16 20 24 28 32

Weeks post TBI/BMT 0 cGy (n = 12) 276 cGy (n = 14)

82.5 cGy (n = 12) Control (n = 12)

138 cGy (n = 12)

Fig. 40.10 In utero hematopoietic cell transplantation (IU-HCT) followed by a postnatal low-dose total body irradiation/bone marrow transplantation (TBI/BMT) regimen results in high levels of allochimerism. Chimeric mice after IU-HCT received one of four 4 doses of TBI followed by a postnatal Tcell-depleted BMT with cells congenic (B6Pep3b) to the allogeneic prenatal donor at 4 (a) or 8 (b) weeks of age. Control mice were 4-week-old naïve Balb/c males that received 276 cGy TBI followed by tail vein infusion of 30 × 106 T-cell-depleted B6 bone marrow cells. At all time points, there is no difference between chimerism levels in mice boosted at 8 and 4 weeks of age (p < 0.05) with the exception of the two marked points (*). Levels of chimerism were statistically different between each irradiation dose (p < 0.05) with the exception of the point marked #. (Reproduced from [67], with permission.)

competitive capacity for maintenance and expansion of the donor cell compartment in circumstances of mixed chimerism and suggest that similar strategies could be clinically successful once adequate chimerism is established by IU-HCT for the induction and maintenance of donor-specific tolerance.

Considerations for clinical application Consideration of the above supports the rationale for prenatal HSC transplantation in selected circumstances. The potential clinical advantages of the prenatal approach over postnatal BMT are summarized in Table 40.1. These potential advantages must be weighed against the associated maternal and fetal risk. It should be emphasized that prenatal transplantation does not preclude conventional postnatal therapy. From an ethical perspective, it would be inappropriate to withhold beneficial postnatal therapy if prenatal transplantation failed or was only partially effective [83]. Donor cell source Among the many potentially important variables for in utero transplantation is the source of donor cells. This, by necessity, is influenced by

the important clinical issues of susceptibility of the early gestational fetus to GVHD and the risk to the maternal–fetal unit of transmissible viral, bacterial, and fungal infection. It makes intuitive sense that HSC from preimmune fetal sources may offer immunologic and functional advantages over adult-derived HSCs with respect to engraftment and the risk for GVHD. In fact, in the sheep, baboon, and murine models, fetal liver-derived cells provide significantly better engraftment than adult bone marrow without the risk for GVHD [34–36,39,84,85]. However, the use of fetal liver for human in utero transplantation presents practical problems that may severely limit its use. We found that more than 85% of human fetal livers obtained from abortus specimens over a 3year period were contaminated with bacterial and fungal pathogens, and only three of 18 ectopic/spontaneous abortus tissues were genetically normal. Rice et al., using the United States Pharmacopoeia Assay, reported a 79% rate of bacterial and fungal contamination, making the specimens unsuitable for clinical use [86]. Even when the tissues were sterile, quality control evaluations required that the tissue be frozen until used. In contrast to fetal sources, newborns (cord blood) and adults are readily available sources of donor HSCs that do not present any significant quality control or ethical issues. However, the presence of mature T cells in both sources raises concern for GVHD, and this concern is justified by experimental data. When bone marrow MNCs from adult sheep were transplanted into unrelated normal fetuses of different gestational ages and pregnancy was allowed to go to term, significant donor cell engraftment occurred in a number of recipients, but all chimeric lambs developed severe GVHD and died. GVHD occurred at different times after transplantation and was not accompanied by significant graft loss [87]. GVHD is secondary to the inoculum of mature donor T cells given at the time of transplantation rather than to de novo generation of T cells from donor progenitors and is dose related, as would be expected [52]. The potential of cord blood to cause GVHD in the fetus has not been fully evaluated. We have observed GVHD after allogeneic but not after xenogeneic cord blood transplantation (Zanjani, unpublished data). As there are significant engraftment benefits of cord blood, the use of cord blood as a donor source needs further evaluation in animal models. One approach to decreasing the risk of GVHD is to simultaneously Tcell deplete and enrich for HSCs. In general, the use of highly enriched HSC populations for in utero transplantation has been disappointing. In the congenic murine model, low levels of chimerism can be achieved with very large doses of highly enriched HSCs, but we have observed no engraftment in allogeneic recipients [25], and clinical experience in optimal circumstances has likewise shown no engraftment [88]. The addition of T cells has been demonstrated to improve engraftment in several animal models but always at the risk of GVHD [42,52,89,90]. This suggests the need for an as yet undefined cell population to facilitate engraftment of HSCs in the prenatal recipient, without simultane-

In Utero Transplantation

ously inducing GVHD, analogous to the situation in postnatal allogeneic BMT. Maternal and fetal risk Maternal and fetal risk can be divided into procedural and nonprocedural risk. The risk of fetal loss with chorionic villous sampling has been well documented and is less than 1% [91]. The risk of procedure-related fetal loss with intraperitoneal or intravascular transplantation of HSCs in the first to second trimester is unknown, but extrapolation from the extensive experience with intraperitoneal transfusion for fetal rhesus disease is reasonable. There are differences, however, which need elaboration. A number of factors increase the risk of fetal loss in treatment of rhesus disease that are not present with IU-HCT. Intraperitoneal transfusion, when used for rhesus disease, involves placement of a catheter into the fetal peritoneal cavity via the lumen of a 16-gauge needle with infusion of a relatively large volume of cells. In contrast, IU-HCT is usually carried out using no larger than a 22-gauge needle and involves less than 1 mL injectate. The early gestational age and small size of the fetal recipient of IU-HCT, on the other hand, may increase the relative risk of in utero stem cell transplantation. In balance, the increased risk of fetal loss following a single in utero transplant procedure is probably on the lower end of the range of risk for intraperitoneal transfusion, which has been estimated to be between 0.8% and 3.5% in various series [92], and we currently estimate a risk of around 1% per transplant when counseling patients. It goes without saying that these risk estimates apply only to centers with extensive experience with these techniques. Nonprocedural risk to the mother and fetus includes transmissible infectious disease and GVHD. The risk of transmissible infectious disease is dependent upon the donor cell source and donor cell screening process. This represents the primary obstacle to the use of fetal tissue, as there currently exist no quality controls on procurement, processing, and donor screening. With adult-derived tissues, standard protocols and procedures used to screen donors for HCT can be utilized. Clinical bone marrow or stem cell laboratories can be utilized, and the high level of quality control established for cell processing for HCT can be maintained. The risk of fetal GVHD is also dependent on the donor tissue used. Postnatal experience in immunocompromised HCT recipients suggests that transplantation of more than 1 × 105 mature T cells per kilogram introduces the risk of GVHD [93]. Undoubtedly, fetuses with immune deficiency disorders are at greater risk than other disease groups for this complication. It is well documented that newborns with SCID have a significant incidence of maternal versus fetal GVHD related to the transplacental passage of maternal T cells [94,95]. The seven reported recipients of CD34-enriched paternal marrow with different types of SCID received doses of 1.1 × 105 T cells per kilogram fetal weight, or less, per injection and none had clinical evidence of GVHD [96–98]. Thus, based on available evidence, this appears to be a relatively safe minimal dose of T cells for clinical in utero transplantation. An important area in the future will be definition of facilitating populations required to achieve higher levels of engraftment after IU-HCT in hematopoietically competitive recipients. These populations will need to be carefully assessed in experimental models for their propensity to induce GVHD prior to clinical application.

Diseases potentially amenable to IU-HCT Theoretically, in the future, any disease that can be diagnosed early in gestation and can be cured by postnatal BMT could potentially be treated by IU-HCT. In reality, each disease is biologically unique, and with the limitations of current knowledge regarding the requirements for engraft-

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Table 40.2 Diseases potentially amenable to in utero hematopoietic stem cell transplantation Rationale = selective advantage for donor cells SCID: X-linked* ZAP70 Jak 3 ADA deficency Wiskott–Aldrich syndrome Chromosal breakage syndromes: Fanconi’s anemia Bloom’s syndrome Rationale = minimal engraftment requirement Hyper IgM syndrome Chronic granulomatous disease * Has been successfully treated by in utero hematopoietic stem cell transplantation. ADA, adenosine deaminase, IgM, immunoglobulin M; SCID, severe combined immunodeficiency syndrome.

ment, only a few are reasonable candidate diseases to consider (Table 40.2). Diseases with a selective advantage for normal cells By far the most favorable biology for IU-HCT is circumstances in which there is a survival or proliferative advantage for normal cells. This is clearly the case in the majority of SCID disorders. The most common type of SCID is X linked (SCID-X) and is due to a mutation in the gene encoding the common gamma chain (γc), a member of the cytokine receptor superfamily and an essential component for normal function of the interleukin-2 and multiple other cytokine receptors [99]. The resultant cytokine unresponsiveness causes a block in T-cell development and a severe deficiency of mature T cells. B cells, although present in normal or even increased numbers, are also dysfunctional. Other characterized mutations in cytokine receptor signaling pathways (i.e. Jak 3 or ZAP 70), or adenosine deaminase deficiency, resulting in SCID, should also be favorable candidate diseases for IU-HCT. Based on the available clinical and experimental evidence it is likely that any member of this group of disorders can be effectively treated by IU-HCT, using established protocols, with results comparable to the reported results for SCID-X. Ideally, clinical trials of IU-HCT for SCID would be established, and the results compared with early postnatal transplantation protocols, to determine whether there is a biologic advantage favoring IU-HCT. Unfortunately, such trials may not be possible due to the rarity of these diseases and the perception that postnatal therapy is adequate [100]. In addition, the recent impressive results of postnatal gene therapy for SCID-X may make prenatal treatment unnecessary [101], although concerns regarding insertional mutagenesis may require re-examination of the gene therapy approach [102]. Another immunodeficiency disorder where a selective advantage for normal cells exists is Wiskott–Aldrich Syndrome (WAS). WAS is a severe immunodeficiency and platelet deficiency disease arising from mutation(s) in the WASP gene, which in normal cells encodes an intracellular protein able to interact with other proteins relevant to the control of cytoskeletal organization [103]. Immunodeficiency is mainly due to progressive T-cell malfunction. Salient defects of WAS T cells are a CD3-restricted impairment in proliferative responses and cytoskeletal abnormalities, including the frequent appearance of T cells with

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atypical morphology [104]. Direct evidence of a selective advantage for normal cells is documentation of nonrandom inactivation of the X chromosome in multiple lineages of peripheral blood cells and early lineage hematopoietic cells in carriers of WAS, similar to that seen in the T-cell lineage in carriers of SCID-X [105,106]. The combination of a selective advantage for normal hematopoietic progenitors and a proliferative defect in host T cells should provide favorable biology for successful IU-HCT. This disease has been successfully treated with matched sibling BMT, but the results of mismatched marrow transplants have been poor [107,108], adding to the justification for attempting IUHCT. However, IU-HCT has not yet been appropriately tested in a fetus with WAS. Another group of diseases that could benefit from IU-HCT are those in which somatic mosaicism and in vivo selection have been documented to occur. In these diseases, there is presumably a survival advantage for the spontaneously corrected cells [109]. Such correction has been noted in adenosine deaminase SCID [110], Fanconi’s anemia [111], and Bloom’s syndrome [112], the latter two of which are chromosomal breakage syndromes. In both Fanconi’s anemia and Bloom’s syndrome, mitotic recombination was documented as the molecular mechanism of somatic reversion. This represents an experiment of nature documenting the improvement in a disease by clonal expansion of a single spontaneously corrected HSC and suggests that low level engraftment achieved in utero could eventually replace host hematopoiesis as progressive bone marrow failure occurred. True clinical cure of either disease is unlikely as they are associated with other pleiotropic manifestations, such as an increased rate of malignancy, that are unlikely to be reversed by hematopoietic reconstitution alone. Specific nonhematopoietic disorders of bone metabolism may also be attractive target disorders for IU-HCT. A recent report of rescue of osteopetrotic mice with the same mutation as approximately half of human patients with the autosomal recessive diseases by IU-HCT is intriguing [113]. In this study, complete phenotypic correction associated with osteoclast engraftment was achieved, despite the fact that there are an abundance of osteoclasts present in the disorder that are not functional. There is also interest in the treatment of osteogenesis imperfecta by prenatal replacement of “mesenchymal stem cells” (MSCs) or stromal progenitor cells, and a clinical case has been reported. The experimental basis for application of IU-HCT toward this disease, however, needs further development. Engraftment of MSCs after intraperitoneal transplantation in xenogeneic systems occurs but is very low in frequency [114]. In the murine system, we have been unable to achieve survival with intravascular injection of murine or human MSCs due to clogging of umbilical vessels. The premise that typical MSCs isolated by plastic adherence give rise to osteoblasts has been challenged [115] by the observation that hematopoietic cells and osteoblasts are derived from a common progenitor in the nonplastic adherent fraction of bone marrow. The question is then whether IU-HCT can engraft the osteoblast compartment. Although the target disorders remain limited, it is not unreasonable to consider further attempts at IU-HCT using current methodology in the context of the discussion and disease entities described above. Having said that, it is clear that this remains an experimental approach and should be limited to institutions with active research programs, IUHCT, appropriate clinical protocols in place, and the clinical expertise to optimally test the approach for this limited subset of disorders. It should be emphasized that at the present time, because of the extremely limited clinical experience at individual centers, the optimal approach to IU-HCT remains debatable.

Clinical experience with IU-HCT The clinical experience with IU-HCT thus far supports the existence of the barriers discussed above and the inadequacy of current approaches to overcome them. There have now been well over 40 attempts to transplant fetuses for a variety of congenital disorders. Most of these cases have been adequately presented in multiple reviews [116–118] and will not be revisited here. The majority of these efforts have been performed in a suboptimal fashion with respect to the gestational age of the fetus, the disease entity chosen for treatment or the donor cells utilized, and with current knowledge would be predicted to fail. However, more recently, there have been a number of optimally performed transplants with high doses of appropriate donor cells delivered at early gestational time points [88,98,119–121]. The only clear successes have been in B+ but not B− forms of SCID (predominantly SCID-X), with results reported thus far in several patients that are at least comparable to results with nonmyeloablated postnatal transplantation. Fetuses that have been successfully transplanted have stable split chimerism with the T-cell lineage of donor origin and all other lineages of host origin. At least some of the reported cases maintain a partial B-cell functional deficit, but a detailed comparison of B-cell function and T-cell–B-cell cooperation between preand postnatal nonmyeloablated recipients has not been performed [122]. A detailed analysis of T-cell reconstitution in these patients has been published [98] and supports thymic T-cell reconstitution with a diverse repertoire of T cells and full cellular immunity. In addition, one patient has been reported to have complete humoral immunity [123]. All other hematopoietic target diseases, i.e. other immune deficiency disorders, hemoglobinopathies, and inborn errors of metabolism have either failed to engraft or have had inadequate engraftment for therapeutic effect. There has been an intriguing case report published describing improvement in the anticipated phenotype after engraftment of a fetus with osteogenesis imperfecta by in utero transplantation of fetal liver-derived MSCs [124]. However, because of the variability of genotypic/phenotypic correlation in this disorder, and the somewhat minimal engraftment reported, further follow-up is required.

Conclusion IU-HCT is currently in its early stages of development, but holds considerable promise as a therapeutic approach for the treatment of a large number of congenital hematologic diseases. Despite only recent, limited evidence of clinical efficacy, interest in the field continues to gain momentum. Parallel advances in prenatal screening, molecular diagnosis, and the Human Genome Project make it highly likely that opportunities for application of this approach will increase. However, at this point in the evolution of IU-HCT, there are more questions than answers. Widespread clinical application is premature based on the extremely limited clinical success that has been achieved. The biology of each disease is unique, and expectations of success or failure can only be based on sound clinical investigation guided by experimental work in relevant animal models. The barriers to prenatal engraftment need to be investigated and understood prior to further clinical efforts in diseases where host cell competition is prohibitive. Clinical centers should be associated with an active research effort to solve the remaining problems with this potentially promising clinical approach. In the near future, advances in our understanding of stem cell biology in the context of the prenatal microenvironment may allow IU-HCT to achieve its full potential.

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malignant diseases. Crit Rev Oncol Hematol 2001; 39: 25–9. Sykes M, Preffer F, McAfee S et al. Mixed lymphohaemopoietic chimerism and graft-versuslymphoma effects after non-myeloablative therapy and HLA-mismatched bone-marrow transplantation [see comments]. Lancet 1999; 353: 1755–9. Stewart FM, Zhong S, Wuu J, Hsieh C-C, Nilsson SK, Quesenberry PJ. Lymphohematopoietic engraftment in minimally myeloablated hosts. Blood 1998; 91: 3681–7. Flake AW. Prenatal intervention: ethical considerations for life-threatening and non-life-threatening anomalies. Semin Pediatr Surg 2001; 10: 212–21. Zanjani ED, Ascensao JL, Harrison MR, Tavassoli M. Ex vivo incubation with growth factors enhances the engraftment of fetal hematopoietic cells transplanted in sheep fetuses. Blood 1992; 79: 3045–9. Shaaban AF, Milner R, Kim HB, Flake AW. Fetal liver is a superior donor source for the engraftment of prenatally transplanted allogeneic hematopoietic stem cells. Exp Hematol 1999; 27(Suppl 1): 121, Abstract 320. Rice HE, Hedrick MH, Flake AW, Donegan E, Harrison MR. Bacterial and fungal contamination of human fetal liver collected transvaginally for hematopoietic stem cell transplantation. Fetal Diagn Ther 1993; 8: 74–8. Zanjani ED, Mackintosh FR, Harrison MR. Hematopoietic chimerism in sheep and nonhuman primates by in utero transplantation of fetal hematopoietic stem cells. Blood Cells 1991; 17: 349– 63. Muench MO, Rae J, Barcena A et al. Transplantation of a fetus with paternal Thy-1(+)CD34(+)cells for chronic granulomatous disease. Bone Marrow Transplant 2001; 27: 355–64. Hayashi S, Hsieh M, Peranteau WH, Ashizuka S, Flake AW. Complete allogeneic hematopoietic chimerism achieved by in utero hematopoietic cell transplantation and cotransplantation of LLME-treated, MHC-sensitized donor lymphocytes. Exp Hematol 2004; 32: 290–9. Bhattacharyya S, Chawla A, Smith K et al. Multilineage engraftment with minimal graft-versushost disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated, sensitized T cells and adult T cell-depleted bone marrow in fetal mice. J Immunol 2002; 169: 6133–40. Rhoads G, Jackson L, Schlesselman S et al. The safety and efficacy of chorionic villus sampling for early prenatal diagnosis of cytogenetic abnormalities. N Engl J Med 1989; 320: 609–17. Bowman J. Hemolytic disease (erythroblastosis fetalis). In: Creasy RK, Resnik R, editors. Maternal-fetal medicine principles and practice, 3rd edn. Philadelphia: WB Saunders; 1994. pp. 730– 3. Mackinnon S, Papadopoulos EB, Carabasi MH et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease. Blood 1995; 86: 1261–8. Alain G, Carrier C, Beaumier L, Bernard J, Lemay M, Lavoie A. In utero acute graft-versus-host disease in a neonate with severe combined immu-

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41

Thomas C. Shea & John F. DiPersio

Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy

Introduction High-dose chemotherapy has become an important therapeutic strategy for many malignancies. These regimens are often severely myelosuppressive or myeloablative, and autologous peripheral blood progenitor cells (PBHCs) have been used to provide rapid and sustained hematopoietic recovery to support such treatment. In the past, autologous bone marrow (BM) transplantation was used in this setting but, because of the less invasive collection methods and more rapid resolution of neutropenia and thrombocytopenia, PBHC transplantation has largely replaced BM transplantation for the support of high-dose chemotherapy [1–3]. Both hematopoietic stem cells (HSCs) and progenitor cells populate the resting BM. Stem cells are single cells that are clonal precursors capable of giving rise to both identical stem cells (self-renewal) and a defined set of differentiated progenitors. Progenitor cells are multipotent precursor cells that lack the ability to self-renew but, like stem cells, maintain the capacity to differentiate and proliferate. Despite this important difference, these terms often are used interchangeably in the literature [4]. The ability of PBHCs to reconstitute hematopoiesis was first described in 1980 and followed by additional reports in 1985 and 1986 [5–9]. Under steady-state conditions, hematopoietic progenitor cells circulate in the peripheral blood (PB) compartment at a frequency of approximately 100 cells/mL of whole blood. This low frequency of PBHCs in the circulation made collecting adequate numbers of these cells a cumbersome process. As a result, methods to mobilize these cells from the marrow compartment and increase their numbers in the peripheral circulation have been developed to foster collection of sufficient cells for clinical use. The work by Abrams et al. [5] and Richman et al. [10] in animal models demonstrating an increase in PBHCs following the administration of subablative chemotherapy, and by Socinski et al. and Gianni et al.in patients following cytokine administration, have led to the widespread application of this approach for supporting high-dose therapy [5,10–12].

Identification and enumeration Human HSCs express CD34 and Thy1 at a high level, and c-kit at a low level and do not express Lin and CD38 [4]. The ability of a mobilized

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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apheresis product to engraft and permanently repopulate the BM depends on its content of both stem cells and progenitor cells. In the transplant setting, this can be evaluated using several assays. Long-term cultureinitiating cells have been used to assess the ability of mobilized cells to repopulate the BM, but this assay is time-consuming and subject to inconsistency between laboratories. Functional assays measuring the quantity of cells committed to a particular lineage include colony-forming unit (CFU) and burst-forming unit (BFU) assays. CFUgranulocyte–macrophage (CFU-GM), CFU-granulocyte–erythrocyte– macrophage–megakaryocyte (CFU-GEMM), and BFU-erythroid (BFU-E) have been correlated with time to engraftment [11–14]. Because these assays require significant laboratory manipulation, results often reflect technical variability and are difficult to compare. More recently, quantification of CD34-expressing cells has been used to assess the quality of the apheresis product and its adequacy for repopulating the BM. CD34+ cell content correlates closely with CFUGM, BFU-E, and CFU-GEMM, as well as time to engraftment of both neutrophils and platelets [14–17]. CD34+ cell quantification is also subject to intralaboratory variation, but standardization of procedures can reduce this variation [18,19]. Efforts have also been undertaken to identify subsets of the CD34 cell population that are critical to BM reconstitution, such as CD34+CD33−, CD34+CD33+, or CD34+CD38− cells [14,17,20–23]. One study found CD34+CD33− cells to be more reliably predictive of hematopoietic recovery rates than CD34+ cells [21], and one recently reported that CD34 cell number predicted early platelet recovery, while CD34+CD38− cells were more predictive of long-term platelet engraftment [24].

Mechanisms of progenitor cell mobilization HSCs in the BM exist in a highly organized three-dimensional microenvironment consisting of a diverse group of cell types including stromal cells and an associated extracellular matrix rich in fibronectin, collagens, and various proteoglycans. HSCs are physically situated in specific BM niches. Osteoblasts appear to play a pivotal role in HSC function via a physical interaction that exists between HSCs and BM osteoblasts, thus defining the osteoblastic niche [25]. Other subpopulations of HSCs exist in close proximity with sinusoidal endothelial cells, thus defining the “endothelial or vascular niche” [26]. It is assumed that each of these niches differentially regulates HSC trafficking (homing and mobilization), as well as basic functions such as survival, proliferation, selfrenewal, and differentiation. To enter the circulation, HSCs must transmigrate through the vascular barrier (termed the BM–blood barrier). This barrier or endothelial sinus is composed of endothelial cells, a basement membrane, and a layer of adventitial cells. Electron

Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy

microscopic studies demonstrate that there are numerous sites within the endothelial cells where luminal and abluminal membranes are fused. It is thought, although not definitely proven, that HSCs migrate through these “diaphragmed fenestra” when HSCs are mobilized from the BM to the PB. A number of adhesion molecules has been implicated in HSC trafficking. These include stromal cell-derived factor-1 (SDF-1/CXCL12), very late antigen-4 (VLA-4 or α4 integrin), c-kit, CD62 ligand (CD62L), P- and E-selectins, and CD44 [27–30] . In addition to these candidate adhesion molecules, a number of cell-associated proteases, including neutrophil elastase (NE), cathepsin G (CG), metalloproteinase-9 (MMP9 or gelatinase B), and CD26 (dipeptidylpeptidase IV), have been implicated in mediating the release of HSCs from the BM into the PB via cleavage of the candidate adhesion molecule tethers mentioned above [31–35] . The kinetics of HSC mobilization can vary dramatically depending upon the mobilizing agents used. Granulocyte colony-stimulating factor (G-CSF) induces maximum HSC mobilization in 4–5 days, while granulocyte–macrophage colony-stimulating factor (GM-CSF) induces maximum stem cell mobilization in 5–6 days. When chemotherapy and G-CSF or GM-CSF are used together, mobilization is optimal after 10–14 days. In contrast, mobilization by chemokines (interleukin-8 [IL-8] or GRO-β) or chemokine receptor antagonists (AMD3100) result in mobilization of HSCs in minutes to hours [31,36,37]. The phenotype of BM versus PB HSCs is different. Relative to BM HSCs, a higher percentage of mobilized PB HSCs are in G0 or G1 phase of the cell cycle. HSCs are selectively mobilized after the M phase of the cell cycle, providing a potential explanation for the preponderance of HSCs in G0 and G1 of the cell cycle in the blood after G-CSF treatment [38]. In addition, consistent with the downregulation of various adhesion molecules contributing to mobilization, circulating HSCs display reduced expression of VLA-4 [39,40], c-kit [40], and chemokine (C-X-C motif) receptor 4 (CXCR4) [41].

Evidence for the pivotal role of SDF-1/CXCR4 in HSC mobilization and trafficking There is convincing evidence that the interaction of SDF-1/CXCL12 with its cognate receptor, CXCR4, generates signals that regulate HSC trafficking in the BM. SDF-1 is a CXC chemokine constitutively expressed in the BM by stromal cells and BM osteoblasts [42]. SDF-1 is a potent chemoattractant agent for HSCs and has been shown to regulate HSC survival, adhesion, and cell-cycle status. Analyses of CXCR4-deficient mice have established that both SDF-1 and CXCR4 are necessary for normal migration of HSCs from the fetal liver to the BM, and for the efficient retention of these progenitors in the BM [43,44]. Elevation of SDF-1 levels in the blood by the administration of SDF-1 or by the injection of adenoviral vector expressing SDF-1 is associated with significant mobilization of HSCs into the blood. Likewise, injection of a competitive inhibitor of CXCR4 (AMD3100), induces rapid mobilization (9 hours in humans and 3 hours in mice) of both human and mouse HSCs [36,37,45]. Finally, inhibition of signaling via the chemokine receptor CXCR4 with pertussis toxin results in rapid mobilization of HSCs from the BM into the blood. Consistent with these data are studies demonstrating that neutrophil mobilization in response to G-CSF is impaired in mice deficient in GRK6 (G protein-coupled receptor kinase-6), which participates in the regulation of neutrophil chemotaxis by CXCR4 signaling [46]. Interruption of SDF-1/CXCR4 signaling/interaction represents a pivotal and key step in HSC mobilization by G-CSF. SDF-1 levels in the BM decline sharply during G-CSF mobilization [25,47–49]. The decrease in SDF-1 levels in the BM after treatment with G-CSF

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correlates well with the magnitude of HSC mobilization [50]. There is evidence to suggest that CXCR4 (expressed on HSCs) and SDF-1 (expressed on BM stromal cells and osteoblasts) may be proteolytically cleaved and inactivated during G-CSF treatment [49]. The mechanisms that regulate SDF-1 levels in the BM remain unclear. Cleavage of SDF-1 by NE and CG might regulate residual SDF-1 levels in the BM extracellular matrix via proteolytic cleavage [25,49]. The role of proteolytic cleavage of SDF-1 by these proteases as the mechanism of G-CSF-induced mobilization has been brought into question by elegant studies in which mice deficient in both NE and CG mobilized HSCs normally in response to G-CSF treatment [50]. Recent data provide compelling evidence that G-CSF induces a dramatic transcriptional downregulation of SDF-1 mRNA in BM stromal cells. The downregulation of SDF-1 mRNA in response to G-CSF occurs in a temporally consistent way with G-CSF-induced mobilization of HSCs (maximum downregulation of SDF-1 and HSC mobilization occurring in 4–5 days) [51]. These data provide prima facie evidence that G-CSF regulates SDF-1 levels and thus HSC mobilization via transcriptional downregulation, but they do not eliminate the possibility that G-CSF may also induce mobilization in part by activation of cell-associated proteases. Role of proteases Significant data suggest that a highly proteolytic environment is induced in the BM during HSC mobilization by G-CSF [52]. The neutrophil serine proteases, NE and CG, accumulate in the BM with kinetics that mirror those for HSC mobilization [52]. In addition, expression of SERPINA1 and SERPINA3, naturally occurring inhibitors of these proteases, are markedly decreased during G-CSF treatment [53]. NE and CG are capable of proteolytic cleavage of key BM-associated adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), SDF1, and c-kit [25,54]. The study by Levesque suggests that G-mobilization in NE- and CG-deficient mice is normal after G-CSF treatment, suggesting that proteases distinct from NE and CG may be essential [50]. MMP-9 also accumulates in the BM plasma following mobilization with G-CSF, IL-8, and chemotherapy [32,52]. Further, neutralizing antibodies to MMP-9 attenuate IL-8-induced mobilization in primates [32]. Again, however, mice deficient in MMP-9 mobilized HSCs normally in response to IL-8 and G-CSF, raising questions as to the role of this metalloproteinase in G-CSF-induced mobilization [33,55]. Finally, the membrane-bound extracellular serine protease CD26 has been implicated in chemokine- and cytokine-induced mobilization. This protease has been demonstrated to inactivate SDF-1 via proteolytic cleavage [34]. G-CSF-induced mobilization is only modestly defective in CD26deficient mice or in wild-type mice treated with a moderately specific inhibitor to CD26 [34,35], minimizing the key role of this protease in G-CSF-induced mobilization. In summary, although many proteases have been implicated in HSC mobilization, studies using mice deficient in specific proteases or combinations of proteases have not clearly implicated any single or combination of proteases mechanistically in cytokine-, chemokine-, or chemotherapy-induced HSC mobilization. Effect of other cell adhesion molecules on HSC mobilization VLA-4 is expressed on the majority of HSCs in a low-affinity state. In response to cytokines such as G-CSF, IL-3, stem cell factor, and GMCSF, VLA-4 can be rapidly activated to promote adhesion to fibronectin. Its primary ligand on the BM microenvironment is VCAM-1, which is constitutively expressed by both BM endothelial cells and BM stromal cells. Preclinical murine studies demonstrated that specific neutralizing

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Chapter 41

antibodies to VLA-4 or VCAM-1 can lead to HSC mobilization with intermediate kinetics [27]. In addition, like CXCR4, mice that are conditional “knockouts” of VLA-4 (and CXCR4) have high circulating levels of HSCs [29]. Of interest is that the effect of blocking VLA-4 on inducing HSC mobilization is significantly enhanced in CD18 (β2integrin)-deficient mice, suggesting that both VLA-4 and β2-integrins may participate along with CXCR4 in tethering HSCs to the BM microenvironment [55]. Although small-molecule VLA-4 antagonists have been developed and tested for the treatment of inflammatory disorders such as asthma, no preclinical or clinical trials have been reported using small-molecule antagonists of the VLA-4/VCAM-1 axis for HSC mobilization. CD44 is one of two receptors (the other being the hyaluronanmediated motility receptor [RHAMM]) for the glycosaminoglycan hyaluronic acid. Expression of both CD44 and RHAMM is reduced in mobilized HSCs compared with BM-bound HSCs, suggesting a role of hyaluronic acid receptors in HSC mobilization [56]. Further, mice deficient in CD44 demonstrate impaired HSC mobilization in response to G-CSF [57], and the treatment of mice with neutralizing anti-CD44 antibodies results in modest mobilization with intermediate kinetics [58]. Finally, blockade of human CD44 with human CD44-specific neutralizing antibodies results in decreased homing of human HSCs to the mouse BM using a NOD-SCID xenotransplant model [58]. Selectins (both P- and E-selectins) are constitutively expressed in BM endothelial cells and are induced in response to multiple inflammatory stimuli. Mice deficient in endothelial selectins exhibit marked leukocytosis and increased levels of circulating HSCs [30,59]. In addition, homing of HSCs is defective in P- and E-selectin-deficient mice [60,61]. Consistent with these observations, these same groups have demonstrated that fucoidan, a sulfated glycan and an in vitro inhibitor of both selectins, when given in vivo to mice, resulted in significant HSC mobilization [60,61]. Of interest is that these same studies demonstrated that fucoidan effectively mobilized HSCs even in L-, P-, and E-selectindeficient mice, suggesting that fucoidan induces HSC mobilization through nonselectin pathways.

Clinical applications PBHCs have become the preferred source of autologous stem cells to support the use of high-dose, myeloablative chemotherapy in a variety of hematologic malignancies, solid tumors, autoimmune diseases, and nonmalignant hematologic disorders such as sickle cell disease [1,2,62– 64], and are being utilized widely in allogeneic transplants as well [3,65–68]. Current mobilization regimens include single-agent and cytokine combinations as well as combinations of chemotherapy and cytokines. Following mobilization, PBHCs are collected using a variety of apheresis techniques.

Collection techniques Once PBHC mobilization is initiated, the timing and volume of apheresis are critical to maximizing yield. For steady-state mobilization with recombinant growth factors (in the absence of chemotherapy), the peak values of mobilized progenitor cells are observed after 4–6 days of treatment in most series [22,69–72]. Hematopoietic progenitor cells are usually collected with an apheresis machine using a technique that is similar to that used for the collection of platelets. Small comparison studies have not clearly demonstrated the superiority of one machine over another, but at least one report suggested that use of the COBE Spectra unit resulted in a more rapid collection and higher yield of CD34+ cells in patients undergoing apheresis in preparation for autologous transplantation for multiple myeloma [73].

Chemotherapy-based mobilization results in an increase in the time required to initiate collection and less predictability as to when this will occur, as maximal mobilization occurs following resolution of the hematologic nadirs produced with chemotherapy-induced myelosuppression. While a number of studies indicate that the combination of chemotherapy and cytokines leads to an increased yield of progenitor cells compared with cytokines alone, the variability inherent in blood count recovery following chemotherapy can present a challenge in trying to schedule apheresis procedures. Collections are usually initiated when the white blood cell count (WBC) recovers from more than 1 to over 3 WBC × 109/L [74,75], although monitoring daily CD34+ cell content in the PB has also been reported to be predictive of PBHC yield. In one study, a CD34+ cell count of 50 cells/mL or more PB predicted a CD34+ cell yield of 2.5 × 106 or more CD34+ cells/kg body weight in a single apheresis [76]. Other studies have found that a percentage of PB CD34+ cells of 0.5% or more predicted a high mobilization yield and early engraftment [77,78], and that baseline circulating CD34 cells in the blood predict for mobilization success [79]. Similar results have been demonstrated in children, where a circulating CD34 cell count greater than 40/μL was associated with a high probability of successful apheresis [80]. In harvesting PBHCs, the goal is to minimize the number of apheresis procedures required to achieve the target progenitor cell dose. To date, the optimal apheresis volume that achieves this goal has not been defined. Large volume apheresis (>15 L) has been investigated as a way to decrease the number of aphereses. Initially, there was concern that the large volumes would impair CD34+ cell quantity harvested over collection time. In one study evaluating large-volume apheresis in myeloma patients being mobilized for autologous transplant, the quantity of CD34+ cells harvested during the first hour of collection was similar to that collected during the last 2 hours, while the CD34+ cell content collected during subsequent days was not affected [81]. This demonstrates that large-volume apheresis can allow the collection of more CD34 cells per session than smaller volumes. Another study examining the kinetics of large-volume apheresis showed that CD34+ cell recruitment from the BM to the PB starts in the second half hour of the collection and remains steady during the next 4 hours [82]. However, other groups have not shown an advantage for using larger apheresis volumes to collect PBHCs. In one randomized trial comparing apheresis volumes of 7 L and 10 L, the use of a 10 L volume did not decrease the number of aphereses required to collect 2 × 106 CD34+ cells/kg or more [83]. Another group compared an 8 L with a 12 L volume after cyclophosphamide (CY) and etoposide mobilization followed by G-CSF. The median number of CD34+ cells collected per apheresis did not differ between the two volumes, but the larger volume increased the time on the apheresis machine by 1 hour [84]. Most reports have demonstrated a correlation between total CD34+ cell yield and the blood volume processed, resulting in varied approaches among institutions including individualized, patient-specific, collection techniques that are frequently based on practical issues such as scheduling and cost [85].

Identifying the optimal cell yield Several groups have attempted to determine a minimum threshold CD34+ cell dose necessary for rapid and sustained engraftment [76,77,86]. These groups have identified thresholds ranging from 1 × 106 to 3 × 106 CD34+ cells/kg body weight. Other investigators have suggested an optimal CD34+ cell dose of 5 or more to 8 or more × 106 CD34+ cells/kg, with which neutrophil and platelet recovery is accelerated, the demand for other supportive measures (antibiotics or transfusion) is decreased, and therapy schedule is more likely to be preserved

Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy

[15,70,87–89]. One report highlighted the fact that, although a higher dose of CD34+ cells is unlikely to shorten the median duration of neutropenia or thrombocytopenia in a broad patient population, it will reduce the number of “outlier” patients with prolonged times to engraftment requiring prolonged transfusion support (Fig. 41.1) [87]. While some investigators suggest that more primitive CD34+ cell subsets such as CD34+CD33− or CD34+CD38− cells are better predictors of durable engraftment, most centers continue to rely on total CD34+ cell numbers as the most reliable indicator of an adequate PBHC population [1,2,21,24].

Cytokine-induced mobilization

Probability

G-CSF and GM-CSF have been used alone to mobilize PBHCs from cancer patients at doses from 3 to 24 μg/kg/day by subcutaneous injection. Most studies evaluating single-agent cytokines report increases in circulating CD34+ cells or in peak levels of CD34+ cells or CFU-GMs compared with that found in the unmobilized, steady state [10,11,70,89– 93]. A number of studies have compared the use of G-CSF and GM-CSF

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

CD34+ cells × 106/kg 1.0 2.0 5.0 10.0

7

14

21

28

Days

Fig. 41.1 This figure demonstrates the achievement of platelet recovery to >50,000/mL post transplant on the y-axis and days post transplant on the xaxis as a function of the number of CD34+ cells infused. Note that the median number of days to platelet recovery does not change, but that the frequency of patients with prolonged time to recovery decreases markedly as doses exceed 5 × 106 CD34+ cells/kg infused. Adapted with permission from Glaspy et al. [92].

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for mobilization both alone and in conjunction with chemotherapy [22,94–96]. While most have found that G-CSF provides a higher yield of PBHCs, both growth factors reliably mobilize cells into the circulation with minimal toxicity (Table 41.1) [22,94,96]. A dose-response to G-CSF, with higher doses resulting in increased CD34+ peripheral cells, has been demonstrated. One group compared G-CSF doses of 5 and 10 μg/kg/day [72], while other groups compared G-CSF doses of 10 or 24 μg/kg/day [97]. In these studies, the higher dose resulted in superior collections of CD34+ cells (Table 41.2) [72,84,93,96–103]. Higher doses of GM-CSF have likewise been shown to improve the yield of CD34+ cells [93]. Higher doses of G-CSF have also been used successfully to mobilize and collect cells in patients who have failed to collect following lower cytokine doses [102]. The dosing schedule of G-CSF has also been evaluated, comparing once-a-day to twice-a-day doses of G-CSF in normal donors. Administering G-CSF 5 μg/kg twice daily rather than 10 μg/kg once daily resulted in a higher CD34+ cell yield and a lower number of apheresis procedures [103]. Trials in normal donors providing hematopoietic progenitor cells for allogeneic transplant recipients and others using a split versus single daily dose of G-CSF in conjunction with chemotherapy for mobilization of progenitor cells prior to autologous transplants for lymphoma or myeloma have not found a benefit for one schedule over the other [104,105]. Thus, to date, there is no compelling evidence that one schedule is preferable over the other, and patient/donor convenience and comfort should probably be the determining factor at this time. Other cytokines capable of PBHC mobilization as a single agent include IL-12, IL-3, and stem cell factor (SCF) [106–108], but none to date has been approved by the United States Food and Drug Administration for this indication, nor do they offer a compelling advantage over G-CSF or GM-CSF. The timing of apheresis initiation following cytokine administration has also been examined. One study of normal donors receiving G-CSF at 10 μg/kg/day showed that the CD34+ cell yield was threefold higher when apheresis was started on day 5 versus day 6 [22,72]. On the day of apheresis, one study found the peak time for cell mobilization was approximated 10 hours following G-CSF injection [109]. Chemokine receptor 4 (CXCR4) and stromal cell derived factor 1α (SDF-1α) are a receptor–ligand couplet that is important in the normal adhesion and release of CD34 cells from the marrow compartment. Both of these molecules are degraded by proteases released from neutrophil degradation, thus contributing to CD34 mobilization following the neu-

Table 41.1 Comparison of mobilization with granulocyte colony-stimulating factor (G-CSF), pegylated G-CSF, and granulocyte–macrophage colonystimulating factor (GM-CSF) Number of patients

Comparison

Observation

262

G-CSF + chemo versus GM-CSF + chemo Pegylated G-CSF versus equivalent to G-CSF 10 μg/kg four times a day × 7 doses G-CSF at 6 μg/kg versus GM-CSF at 8 or 16 μg/kg

G-CSF group: 1.7-fold increase in CD34+ cell yield G-CSF pegylated G-CSF 100 mg × 1 dose

36 29 44 156

Chemo, chemotherapy.

G-CSF + chemo versus GM-CSF + chemo

G-CSF group: 5.4-fold higher CD34+ cell yield No difference in G-CSF versus GM-CSF-induced CD34+ cell yield G-CSF group: 3.5-fold increase in CD34+ cell yield

Reference [93] [107] [25] [74] [95]

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Chapter 41

Table 41.2 Comparisons of cytokine doses and combinations for mobilization Number of patients

Comparison

Observation

Cytokine doses 95

G-CSF: 5 μg/kg/day versus 10 μg/kg/day

G-CSF 10 μg g/kg qd group: fourfold increase in CD34+ cell yield No statistically significant difference in CD34 cells collected in any one group over the others Equivalent CD34 yields with slightly faster WBC recovery with filgrastim 30 of 40 patients mobilized a mean of 9.8 × 106 CD34 cells, and 10 required additional G-CSF

82 45 40 24

50

Mobilization with chemotherapy + filgrastim, molgramostim or lenograstim Chemo + pegfilgrastim, 6 or 12 mg/day, or filgrastim 8 μg/kg/d Chemo + pegfilgrastim 6 mg f/b G-CSF if needed Chemo + G-CSF 8.5 μg/kg/d versus chemo + pegfilgrastim 12 mg/day 2-day earlier WBC recovery with pegfilgrastim, and statistically insignificant lower CD34 yield Chemo + G-CSF: 8 μg/kg versus 16 μg/kg

144

No growth factor versus GM-CSF: 125 μg/m2/day versus 250 μg/m2/day

128 72

Chemo + G-CSF: 5 μg/kg versus 10 μg/kg G-CSF G-CSF: 400 μg/m2 qd versus 200 μg/m2 bid in healthy donors G-CSF: 10 μg/kg qd versus 5 μg/kg bid to healthy volunteers in a crossover design with a washout period in between

4

Cytokine combinations 30 Chemo + G-CSF versus chemo + GM-CSF followed by G-CSF 35 Chemo + G-CSF versus chemo + GM-CSF versus GM-CSF followed by G-CSF 156 Chemo + G-CSF versus chemo + GM-CSF followed by G-CSF 40

G-CSF or GM-CSF versus GM-CSF followed by G-CSF

48

GM-CSF versus GM-CSF+G-CSF versus GM-CSF followed by G-CSF in allogeneic donors

174

G-CSF versus GM-CSF followed by G-CSF in allogeneic donors

Reference

[68] [123] [124] [122] [125]

G-CSF 16 μg/kg group: 1.7-fold increase in CD34+ cell yield GM-CSF 125 μg/m2/day and 250 μg/m2/day groups: fiveand 12-fold increase in PBHCs compared with no growth factor No benefit from higher-dose G-CSF No benefit from split dose Split dose led to an approximately twofold increase in CD34+cells/donor

[97] [73]

[98] [108] [105]

No difference in CD34+ cell yield

[99]

No difference in CD34+ cell yield

[100]

No difference in CD34+ cell yield (but both regimens yielded 3.5-fold more CD34+ cells than chemo + GM-CSF) GM-CSF f/b G-CSF group: 2.3-fold increased yield of CD34+ cells GM-CSF f/b G-CSF group: increased yield of CD34+ cells compared with GM-CSF alone or concurrent GMCSF+G-CSF GM-CSF f/b G-CSF group: 1.4-fold increase in CD34+ cell yield no difference in OS, DFS, relapse or GVHD

[95]

[101] [102]

[103]

bid, twice daily; chemo, chemotherapy; DFS, disease-free survival; f/b, followed by; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colonystimulating factor; OS, overall survival; PBHC, peripheral blood hematopoietic cell; qd, every day.

trophilia observed after administration of G-CSF [25,115]. It has been noted that inhibition of this binding occurs in the presence of AMD3100, a reversible bycyclam inhibitor of the binding between these molecules. Preclinical studies attest to the capacity of this molecule to facilitate the mobilization of CD34 cells that are capable of long-term marrow repopulation in hematopoietically ablated dogs [111] and primates [112]. This drug was initially tested in patients infected with human immune deficiency virus as an inhibitor of viral cell entry, but was subsequently noted to also mobilize both neutrophils and hematopoietic progenitor

cells in this population and patients with cancer [36,45,113–115]. Injection of AMD3100 in doses between 80 and 240 μg/kg subcutaneously was associated with only mild abdominal bloating, injection site erythema, and perioral paresthesias. Optimal doses, when given alone to myeloma patients, non-Hodgkin’s lymphoma (NHL) patients, and normal volunteers, was 240 μg/kg subcutaneously [36,45,115]. Unlike mobilization with G-CSF or GM-CSF, the activity of AMD3100 is much more rapid, and leads to an increase in circulating progenitor cells within 6–9 hours of administration.

Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy

Combination cytokine- and cytokine-plus chemotherapy-induced mobilization Cytokine combination regimens continue to be investigated as avenues through which the mobilization of PBHCs might be improved. These cytokine combinations have been used both with and without antecedent chemotherapy. The concurrent and sequential use of G-CSF and GMCSF has been investigated (Table 41.2). While some trials have found no significant difference in progenitor cell yields following G-CSF alone compared with sequential GM-CSF and G-CSF [96,116], others have found a significant increase in yields in both the autologous [99,100] and the allogeneic setting [101]. However, the additional yield of PBHCs resulting from such approaches does not seem warranted, given the added expense and modest improvement in PBHC yield compared with the yield from G-CSF alone. Recently, a pegylated form of G-CSF has been developed and approved for use to augment neutrophil recovery following chemotherapy. This compound, pegfilgrastim, has also been used to mobilize CD34 cells when administered following chemotherapy, and, in most phase II, trials appears roughly equivalent to daily doses of G-CSF [117–121]. There have not, however, been any randomized trials demonstrating comparable efficacy to daily G-CSF, so its use in this setting cannot be routinely recommended. SCF is a cytokine that acts on primitive multilineage hematopoietic progenitor cells. In a randomized controlled phase III trial of patients with high-risk breast cancer, steady-state mobilization with G-CSF alone was compared with G-CSF plus SCF. The administration of G-CSF plus SCF resulted in a significant reduction in the number of aphereses required to achieve the desired CD34+ cell yield [122]. In two phase II studies in heavily pretreated lymphoma patients, patients receiving GCSF plus SCF had a higher median CD34+ cell collection than patients receiving G-CSF alone [123,124]. In another trial, however, there was no significant improvement in CD34+ cell yield when it was combined with G-CSF and compared with G-CSF alone in patents who had failed prior mobilization prior to autologous transplantation [125]. Because the improvement in PBHC yield following the addition of SCF to G-CSF was relatively modest, and the use of SCF increased the risk of histamine related side-effects, this drug was not approved by the Food and Drug Administration for this indication, and it is unlikely that this drug will become widely available for use in the United States despite its potential utility in hard-to-mobilize patients. IL-3 has been used alone and in combination with G-CSF or GM-CSF with or without mobilization chemotherapy [126–128]. In these studies, both cytokines acted synergistically with IL-3 to mobilize PBHCs. When given sequentially after mobilization chemotherapy in heavily pretreated patients, the G-CSF–IL-3 combination resulted in an adequate CD34+ cell yield for autologous stem cell support [129], but the additional IL-3-associated toxicity, including headache, fever and malaise, has prevented its widespread availability for this purpose. A synthetic fusion molecule combining IL-3 and G-CSF has been developed but was found inferior when compared with G-CSF alone for mobilization [130]. IL-11, a cytokine used to stimulate platelet generation, has been studied as a mobilization agent in conjunction with chemotherapy and G-CSF. The apheresis products mobilized with this regimen contained adequate numbers of CD34+ cells and reconstituted the BM following myeloablative therapy [130], but the value of this combination over G-CSF alone has not been clearly demonstrated. Because IL-2 stimulates cellular immunity and T-cell progenitors, it has the potential to promote antitumor immunity within an autograft. These characteristics make IL-2 an attractive candidate for costimulation of immunologic effector cells in concert with other cytokines, such as G-CSF, that are more effective for progenitor cell mobilization. While IL-2 may enhance the number and function of

595

antitumor effector cells, it unfortunately also impairs the mobilization of the CD34+ cells [131–132], thereby limiting its application in the context of hematopoietic progenitor cell mobilization. Even so, as long as the CD34+ dose was adequate, IL-2-mobilized progenitor cells were able to repopulate the BM after myeloablative chemotherapy [133]. As noted, G-CSF induces HSC mobilization with prolonged kinetics (4–6 days). Chemokines such as IL-8 and GRO-β induce a rapid release of both proteases (NE, CG, and MMP-9) and HSCs from the BM into the blood. This effect by IL-8, GRO-β and the N-terminal truncated portion of GRO-β, SB-251353, currently in early clinical testing, occurs in 30 minutes to 9 hours in mouse and man [31,32,134,135]. AMD3100 was shown to be a modest mobilizing agent when given alone to patients with myeloma and NHL [115]. When given either alone or after 4 days of G-CSF, optimal mobilization (two- to three-fold above the level of CD34/ml induced by G-CSF alone) occurred approximately 6–9 hours after the dose of AMD3100. The combined use of AMD3100 (240 μg/kg subcutaneously the night before each apheresis) plus G-CSF (10 μg/kg/day subcutaneously × 4 days) was significantly more effective than G-CSF or AMD3100 alone for the mobilization of HSCs in patients with myeloma and NHL [114]. Data from the 25 patients in this phase II study demonstrated that none of the patients initially given AMD3100 plus G-CSF failed to reach the minimum target of more than 2 × 106 CD34/kg after four apheresis collections. In contrast, nine of 25 patients failed to reach this target when initially given G-CSF alone. All nine of the patients that failed to mobilize with G-CSF alone were successfully remobilized with G-CSF and AMD3100. After mobilization with AMD3100 plus G-CSF, 56% of the patients reached the minimum CD34 cell dose after one apheresis, and all patients reached the minimum target after 2 days of apheresis. Mobilization with G-CSF alone resulted in only 36% of the patients reaching this minimum target after 2 days of apheresis [114]. Thus, AMD3100 appears to be an active mobilizing agent in man, especially when combined with G-CSF. These data have provided the foundation for two large randomized trials in which myeloma and NHL patients were mobilized with either G-CSF or G-CSF plus AMD3100 in preparation for autologous stem cell transplantation. The primary endpoint of the NHL trial is a level greater than 5 × 106 CD34/kg in four or fewer apheresis collections, while the primary endpoint of the myeloma study is 6 × 106 CD34/kg (sufficient for tandem autologous stem cell transplants). The results of these trials are currently pending. Myelosuppressive therapy stimulates stem cell and progenitor cell proliferation. This results in a larger pool of progenitors than exists at baseline steady state. Some of these progenitors will egress into the PB. Early mouse studies demonstrated that combination therapy with CY plus G-CSF resulted in an increased egress of stem cells and progenitor cells from the BM compared with G-CSF alone or CY alone [136]. Human studies have shown that patients mobilized with CY plus G-CSF had more rapid platelet engraftment following their autograft than patients mobilized with CY alone, suggesting that the combination regimen resulted in increased numbers of mobilized PBHCs (Table 41.3) [95,137–140]. In a randomized trial comparing mobilization with G-CSF alone with CY plus G-CSF in patients with refractory lymphoma, the addition of CY resulted in a threefold increase in CD34+ cell yield, but this did not translate into a shorter time to engraftment [138]. A small randomized study compared 5 μg/kg daily G-CSF plus CY, 2 g/m2 with 10 μg/kg G-CSF and found no difference in regard to CD34+ yield or count recovery post transplant, but increased toxicity with the addition of CY [141]. Another study comparing CD34+ cell yield within the same patient found that 21 of 22 patients who did not mobilize an adequate CD34+ cell dose following G-CSF alone were successfully mobilized with high-dose CY plus G-CSF [142]. A randomized, crossover trial

596

Chapter 41

Table 41.3 Comparison of cytokine alone versus cytokine + chemotherapy Number of patients

Comparison

Observation

Reference

47

G-CSF alone versus chemo + G-CSF

[126]

96 21

GM-CSF followed by G-CSF versus chemo + G-CSF G-CSF alone versus chemo alone versus chemo + GM-CSF

Chemo + G-CSF group: 2.9-fold increase in CD34+ cell yield Chemo + G-CSF: 2.7-fold increase in CD34+ cell yield G-CSF alone and chemo + GM-CSF groups: fourfold increase in CD34+ cell yield compared with chemo alone group Standard chemo + G-CSF group: 2.1-fold increase in CD34+ cell yield Intensive chemo + G-CSF group: 5.5-fold increase in CD34+ cell yield Equivalent CD34 yields and post-transplant count recovery with fewer days of hospitalization and fever with G-CSF alone

152

79

G-CSF alone versus standard chemo + G-CSF versus intensive chemo (di-CEP) + G-CSF

Randomized trial of G-CSF 10 μg/kg/d versus cyclophosphamide 2 g/m2 + G-CSF 5 μg/kg/day

[127] [94]

[128]

[147]

Chemo, chemotherapy; di-CEP, cyclophosphamide, etoposide, and cisplatin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colony-stimulating factor.

compared CY plus G-CSF with GM-CSF plus G-CSF within the same patients. The CY-containing regimen resulted in a 2.7-fold increase in CD34+ PBHCs over that resulting from the cytokines alone [139]. Another randomized, phase III trial compared regimens of (1) G-CSF, (2) GM-CSF, and (3) sequential GM-CSF then G-CSF following myelosuppressive chemotherapy. Compared with patients receiving GM-CSF alone, patients receiving G-CSF alone yielded more CD34+ cells/kg, had fewer aphereses, and had a faster time to neutrophil recovery. There were no significant differences between groups receiving G-CSF alone and receiving sequential GM-CSF followed by G-CSF [96]. These studies illustrate the improved potency of regimens containing both myelosuppressive chemotherapy and hematopoietic growth factors. At the same time, these increased PBHC yields have been accompanied by greater toxicity as a result of the concurrent administration of chemotherapy, and a potential increase in the risk of myelodysplasia compared with the use of cytokines alone [143]. The optimal doses of G-CSF have been investigated in a number of studies. In a randomized controlled trial, patients received either 8 or 16 μg/kg/day G-CSF following standard mobilization chemotherapy. With the higher dose of G-CSF, the CD34+ PBHC yield was significantly increased more than threefold, and the time to WBC engraftment was significantly decreased [98]. A small dose-escalation study by Lefrere compared G-CSF doses of 50, 75, 100, 125, and 10 μg/m2 along with chemotherapy and found they achieved adequate CD34+ collections in 84 of 96 patients, suggesting that lower doses of G-CSF may be adequate for the great majority of patients [144]. Efforts have been made to determine an optimal CY mobilization dose as well. Intermediate-dose CY, 4 g/m2, has been used with G-CSF to collect PBHCs. Several studies have shown that a higher dose, 7 g/m2, results in higher PBHC yield, but also leads to increased toxicity without a significant reduction in time to engraftment [95] and no overall improvement in outcome [145]. In a more recent historical cohort study, patients with multiple myeloma received either 4 or 7 g/m2 CY; the higher dose did not result in a higher yield of CD34+ PBHCs but increased the patients’ risk of febrile neutropenia [146]. Even with higher doses, some patients will not mobilize an adequate CD34+ cell product with CY. Similar reports have described good progenitor cell mobilization but increased toxicity, and no improvement in CD34 yield

or clinical outcome, with the addition of etoposide to CY plus G-CSF [147]. Often these patients are quite heavily pretreated. In patients who failed mobilization with CY, etoposide [148,149] and high-dose cytarabine [150], or a combination of the two, have been used with good results [151]. Optimal timing of G-CSF administration after mobilization chemotherapy is also uncertain. In one study, G-CSF doses ranging from 250 μg/day as a fixed dose up to 10 μg/kg/day were administered four or more days following completion of chemotherapy and led to adequate (>2 × 106) or optimal (>5 × 106) CD34 cells/kg in the majority of patients [152]. It also appears that among the various preparations of GCSF that are available, there is no significant advantage for one preparation (filgrastim, lenograstim or molgramostim) over another [118]. Because no single mobilization chemotherapy regimen is clearly superior to the others, another mobilization approach is to incorporate PBHC mobilization into a cycle of disease-specific chemotherapy. In patients with hematologic malignancies, regimens containing ifosfamide, carboplatin, and etoposide (ICE), ifosfamide, epirubicin, and etoposide, and or etoposide, cytosine arabinoside, cisplatin, and prednisone (ESHAP) have been used successfully for concurrent tumor cytoreduction and mobilization chemotherapy [153–159]. Other standard regimens such as CY, doxorubicin, vincristine, and prednisone (CHOP) [160], cisplatin, cytarabine, and decadron (DHAP) [161], and vincristine, doxorubicin (adriamycin), and dexamethasone (VAD) [162], have also been used to mobilize and collect CD34+ cells. It is also important to note that the addition of the anti-CD20 monoclonal antibody rituximab prior to CD34 mobilization and collection does not decrease CD34 yield [163–167] but may reduce contaminating tumor cells in the CD34 product.

Factors affecting yield of PBHC mobilization It is important to note that most patients mobilize adequate numbers of CD34+ cells using a regimen of G-CSF alone. Although the addition of chemotherapy improves CD34+ yield, this comes at the expense of increased short-term toxicity and, possibly, the increased risk of secondary myelodysplastic syndrome [143]. Even with chemotherapy–growth factor combination regimens, it may be difficult to achieve an adequate

Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy

CD34+ cell yield in some patients. Often, cancer patients undergoing mobilization therapy for PBHC harvest to support high-dose chemotherapy have been treated extensively with chemotherapeutic agents and may have malignancy involving their BM. These factors impair the ability to mobilize PBHCs. Even in normal, previously untreated donors, mobilization with G-CSF results in a wide variability of CD34+ cell doses, with 5–15% of donors mobilizing a suboptimal yield of less than 2.5–5 × 106 CD34+ cells/kg [168]. This variability makes predicting how well any one patient will mobilize quite difficult. However, several studies have identified predictors of poor PBHC yield. The amount of myelosuppressive therapy (both chemotherapy and radiation therapy) received prior to mobilization is the most important factor affecting CD34+ yield [15,169–171]. In particular, stem cell toxic agents including nitrogen mustard, chlorambucil, procarbazine, melphalan, carmustine, and over 7.5 g CY have been strongly associated with poor mobilization [8,170,172–174], as has the prolonged use of fludarabine, a purine analogue commonly used for treatment of follicular lymphoma and chronic lymphocytic leukemia [175–177]. The number of chemotherapeutic regimens – more than six [178] and 11 or more [171] – and duration of exposure to chemotherapy (>12 months) [169] also predict poor mobilization, but shorter courses of alkylating agents do not necessarily preclude good CD34+ cell mobilization [179,180]. Timing and intensity of the most recent cytotoxic drug administration relative to mobilization and stem cell collection may also influence mobilization [181]. A short time interval since last chemotherapy, of less than 6 months [188] and less than 65 days [189], has been found to be predictive of poor mobilization. Finally, previous radiation, hypocellular marrow, and refractory disease have been associated with poor mobilization [171]. Strategies to manage patients who do not mobilize well include dose escalation of cytokines, BM harvesting, and remobilization with a more intensive or novel regimen (Table 41.4) [184]. One center has found that infusing a suboptimal PBHC product in combination with harvested BM can achieve short and long-term engraftment [185]. Others have used high-dose G-CSF at 16 and 32 μg/kg/day to successfully mobilize patients previously failing standard myelosuppressive chemotherapy plus G-CSF mobilization [186,187]. One study found G-CSF alone to be superior to chemotherapy plus G-CSF for second mobilization in patients failing their first mobilization regimen [188], and new approaches such as the use of chemokine-associated agents like AMD310 will likely experience increased utilization in the setting of primary mobilization failures. Techniques to prospectively identify poor mobilizers have been investigated. One group found the PB CD34+ and CFU-GM response to a single dose of 12 μg/kg G-CSF to be predictive of PBHC mobilization [189]. Other groups have found that steady-state PB CD34+ cells predicted response to G-CSF mobilization [190–191]. Identifying these

poor mobilizers prospectively would assist clinicians in choosing an optimal mobilization regimen and decrease the time and expense that results from failed mobilization and apheresis procedures.

Tumor contamination Although PBHC products are less frequently contaminated with tumor cells than BM, tumor cells have been detected in patients with breast cancer, small-cell lung cancer, lymphoma, multiple myeloma, and leukemia [138,192–195]. In fact, it has been shown that PBHC mobilization can also cause breast cancer and small-cell lung cancer cell recruitment into the PB [192]. In one study, all of the 16 multiple myeloma patients mobilized tumor cells with CD34+ cells at the time of PBHC harvest [142]. In a study that compared outcomes of patients with NHL receiving high-dose therapy followed by autologous transplantation, patients receiving tumor-free autografts had a significantly improved survival over those receiving minimally contaminated autografts, demonstrating the importance of decontaminating the PBHC product [194]. Another group attempted to predict tumor cell contamination in the mobilized product by examining pre-harvest PB and BM, but there was no association between the presence of tumor cells in the preharvest samples and the harvested PBHC product [196]. An important analysis of the International Bone Marrow Transplant Registry and European Bone Marrow Transplant registry datasets was undertaken by Bierman et al. [197], who reported that patients undergoing either purged or syngeneic donor transplants for NHL had a lower likelihood of relapse post transplant than did patients undergoing unpurged transplants for this disease. While there was no improvement in overall survival in the purged group, this analysis nonetheless attests to the potential importance of tumor cell contamination in the infusion product as one cause for treatment failure following high-dose therapy for these patients. Several reports from the group in Milan using in vivo purging with rituximab for mantle cell lymphoma patients indicated that tumor cell polymerase chain reaction-negative products could be obtained in the majority of patients, and that this was associated with a durable remission of more than 50% at 5 years [198,199]. This is felt to be the principal reason why recent reports from several centers have suggested that long-term transplant results are improved for patients who receive rituximab prior to their autologous transplant for B-cell lymphomas [167,200]. One strategy to reduce tumor contamination in mobilized PBHCs is to perform a positive CD34+ cell selection. This approach resulted in a 2–5 log decrease in malignant cells [192,201], but it can only be applied to CD34− malignancies. In patients with chronic myeloid leukemia receiving autologous PBHCs, positive selection of CD34+ human leukocyte antigen-DR− cells was associated with a superior cytogenetic

Table 41.4 Guidelines for the “hard to mobilize” patient 1 2 3 4 5

597

Increase chemotherapy dose: 4–7 g/m2 CY appears more effective than 2–4 g/m2 Increase growth factor dose (up to 10–24 μg/kg G-CSF) and consider use of cytokine combinations such as G-CSF + GM-CSF Consider bone marrow harvest Prolong duration between last chemotherapy and planned collection Use investigational agents such as SCF, IL-11, GRO-β, AMD3100

bid, twice daily; CY, cyclophosphamide; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; SCF, stem cell factor.

598

Chapter 41

Table 41.5 Preclinical mobilization studies Preclinical compound

System

Observation

Reference

GRO-β (SB-251353)

Mice/primates

[54], [141]

Flt3 ligand Antibodies to LFA-1/Mac integrins Antibodies to VLA-4 IL-8 IL-17 AMD3100; a CXCR4 antagonist

Mice/primates Mice Mice Mice/primates Mice Mice/primates/dogs

Fivefold increase in CFU and CD34+ cells relative to G-CSF alone More primitive Lin− cells than with G-CSF alone Marked increase in CFU or CD34+ cells and synergy with G-CSF 2.7-fold increase in cobblestone and CFU/mL 2–4-fold increase in CFU/mL Increase in CFU/mL Increase in CFU/mL Increase in circulating progenitor cells capable of long-term repopulation in transplant models

[45], [46] [31] [3], [181], [182] [42], [43] [183] [37], [115–117]

See text for abbreviations.

Table 41.6 Clinical mobilization studies with investigational compounds Compound

Number of patients

Observation

Reference

G-CSF versus G-CSF + IL-3 fusion protein (leridistim) IL-11 + G-CSF + ICE chemo G-CSF + SCF versus G-CSF alone

267

G-CSF + IL-3 fusion protein: increased toxicity but no increase in PBHC yield Good yield with combination but no comparison available G-CSF + SCF group: 63% achieved target of 5 × 106 CD34+ cells/kg G-CSF alone group: 47% achieved target of 5 × 106 CD34+ cells/kg No benefit with addition of SCF

[118]

G-CSF + chemo or G-CSF-alone mobilization failure patients were remobilized with G-CSF 10 μg/kg/day + SCF 20 μg/d G-CSF alone and G-CSF + AMD3100 were given to each patient who underwent apheresis after each G-CSF + chemo + 100 μg/kg/day human growth hormone

8 children 203 20

25

16

In each case, CD34 yields were higher with the combination than with G-CSF alone; 14 of 25 patients collected > 5 × 106 CD34 cells in 2 days with the combination as opposed to 4 of 25 with G-CSF alone Compared with prior failed mobilization with chemo + G-CSF without growth hormone, CD34 yields increased sixfold

[119] [112] [130]

[119]

[224]

Chemo, chemotherapy; G-CSF, granulocyte colony-stimulating factor; ICE; ifosfamide, carboplatin, and etoposide regimen; IL, interleukin; PBHC, peripheral blood hematopoietic cell; SCF, stem cell factor; qd, every day.

response, suggesting successful purging of BCR/ABL-expressing cells [202]. In multiple myeloma patients, both positively selected CD34+Thy1+Lin− progenitor cells and B-cell lineage-depleted PBHCs have been transplanted to support high-dose chemotherapy with elimination of detectable tumor contamination and good engraftment [195], but there has been no improvement in overall survival [203]. Although these techniques are promising, available results have not demonstrated a clear benefit as PBHCs are depleted in the selection process and overall survival has not been improved.

Future directions While the majority of patients receiving conventional mobilization regimens do produce adequate PBHC products, a significant, and not always predictable, proportion of the population does not. Patients at risk for poor mobilization yields represent a challenging clinical scenario requiring improved mobilization strategies. Higher cytokine and marrowsuppressive chemotherapy doses can produce improved CD34+ cell yields [72,98,138,204]; however, these higher yields do not always translate into a shorter time to engraftment. Cytokine combinations have also been used in heavily pretreated patients, resulting in modestly

increased CD34+ PBHC yield [123,124]. Clearly, innovative approaches are needed to address these patients. Novel means of mobilizing PBHCs continue to be investigated with a number of preclinical compounds under investigation (Tables 41.5 and 41.6) [1,2,31,45,49,114,115,122,135,202–208]. Manipulation of the adhesive interactions between hematopoietic progenitor cells and the BM stroma has much promise. The expression of adhesion molecules on mobilized CD34+ cells by different growth factors is being investigated [209–210]. Pharmacologic modification of the expression or functional state of adhesion receptors could improve PBHC collection. These interactions could be blocked using monoclonal antibodies or competitive inhibitors of receptor–ligand binding. Anti-integrin monoclonal antibodies already have been shown to increase circulating hematopoietic progenitor cells in nonhuman primates [27,208] and in mice [205]. Manipulation of the chemokine environment represents an exciting and potentially most readily available opportunity for improving mobilization. A truncated GRO-β chemokine agonist has been shown to induce a rapid mobilization of HSCs in mice and primates [31,135], as has IL-8 [211,212]. Preclinical and clinical investigations with compounds such as fms-related tyrosine kinase-3 (flt-3) ligand [206,207], defibrotide [213], human growth hormone [214], pegylated G-CSF, SCF, IL8, and IL-17

Mobilization of Autologous Peripheral Blood Hematopoietic Cells for Cellular Therapy

599

Table 41.7 Recommendations for “standard” mobilization regimens Regimen

Advantages

Disadvantages

G-CSF: 10–24 μg/kg/day by qd or bid schedule

Adequate yield in most patients Low toxicity Reliable scheduling Low risk of MDS Appropriate for use in allogeneic patients Higher CD34+ cell yield than with G-CSF alone Appropriate to consider for use in allogeneic patients

Lower yield than G-CSF + chemo

GM-CSF followed by G-CSF

Chemotherapy + G-CSF: CY 2–4 g/m2 CY ± VP-16 Standard chemotherapy regimen

Higher CD34+ cell yield than cytokine alone Enhanced cytoreduction pre transplant High CD34 yields Continued successful cytoreduction

More expensive than G-CSF alone No proven benefit over G-CSF alone in allogeneic or autologous patients Greater toxicity Increased risk of MDS/AML post transplant Uncertain scheduling; risk of MDS Inappropriate for allogeneic patients

AML, acute myeloid leukemia; bid, twice daily; chemo, chemotherapy; CY, cyclophosphamide; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; MDS, myelodysplastic syndrome; VP-16, etoposide.

also hold promise but require additional study prior to clinical use [122–124,130,206,207,215]. How to best integrate these newer cytokines and chemokines designed to manipulate the progenitor cell–BM stroma interactions and whether these can provide a synergistic approach to PBHC mobilization remains to be determined.

Summary The use of high-dose therapy and autologous PBHC-supported transplantation has become an essential component of contemporary therapy

for hematologic malignancies. The majority of patients will undergo successful mobilization and collection of sufficient PBHCs with standard approaches to permit safe and effective autologous transplant procedures (Table 41.7). Despite these advances in PBHC acquisition, a significant minority of patients will have inadequate yields of PBHCs, and will continue to be the focus of research efforts designed to understand better the mechanisms of PBHC mobilization and to identify improved methods for their collection and administration.

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179. de la Rubia J, Blade J, Lahuerta JJ et al. Effect of chemotherapy with alkylating agents on the yield of CD34+ cells in patients with multiple myeloma. Results of the Spanish Myeloma Group (GEM) Study. Haematologica 2006; 91: 621–7. 180. Gupta S, Zhou P, Hassoun H et al. Hematopoietic stem cell mobilization with intravenous melphalan and G-CSF in patients with chemoresponsive multiple myeloma: report of a phase II trial. Bone Marrow Transplant 2005; 35: 441–7. 181. Keane C, Gibbs S, Seymour JF et al. The HyperCVAD chemotherapy regimen has an adverse long-term impact on the ability to mobilize peripheral blood stem cells, which can be readily circumvented by using the early cycles for mobilization. Hematol Oncol 2006; 24: 159–63. 182. Perry AR, Watts MJ, Peniket AM, Goldstone AH, Linch DC. Progenitor cell yields are frequently poor in patients with histologically indolent lymphomas especially when mobilized within 6 months of previous chemotherapy. Bone Marrow Transplant 1998; 21: 1201–5. 183. Tarella C, Zallio F, Caracciolo D, Cherasco C, Bondesan P, Gavarotti P. Hemopoietic progenitor cell mobilization and harvest following an intensive chemotherapy debulking in indolent lymphoma patients. Stem Cells 1999; 17: 55–61. 184. Stiff PJ. Management strategies for the hard-tomobilize patient. Bone Marrow Transplant 1999; 23(Suppl 2): 29–33. 185. Bentley SA, Brecher ME, Powell E, Serody JS, Wiley JM, Shea TC. Long-term engraftment failure after marrow ablation and autologous hematopoietic reconstitution: differences between peripheral blood stem cell and bone marrow recipients. Bone Marrow Transplant 1997; 19: 557– 63. 186. Lie AKW, Hui CH, Rawling T et al. Granulocyte colony-stimulating factor (G-CSF) dosedependent efficacy in peripheral blood stem cell mobilization in patients who had failed initial mobilization with chemotherapy and G-CSF. Bone Marrow Transplant 1998; 22: 853– 7. 187. Gazitt Y, Freytes C, Callander N et al. Successful PBSC mobilization with high-dose G-CSF for patients failing a first round of mobilization. J Hematother 1999; 8: 173–83. 188. Fraipont V, Sautois B, Baudoux E et al. Successful mobilization of peripheral blood HPCs with G-CSF alone in patients failing to achieve sufficient numbers of CD34+ cells and/or CFU-GM with chemotherapy and G-CSF. Transfusion 2000; 40: 339–47. 189. Mijovic A, Pagliuca A, Mufti GJ. The ‘G-CSF test.’ The response to a single dose of granulocyte colony-stimulating factor predicts mobilization of hemopoietic progenitors in patients with hematologic malignancies. Exp Hematol 1999; 27: 1204– 9. 190. Brown RA, Adkins D, Goodnough LT et al. Factors that influence the collection and engraftment of allogeneic peripheral-blood stem cells in patients with hematologic malignancies. J Clin Oncol 1997; 15: 3067–74. 191. Fruehauf S, Schmitt K, Veldwijk MR et al. Peripheral blood progenitor cell (PBPC) counts during steady-state haemopoiesis enable the estimation of the yield of mobilized PBPC after granulocyte colony-stimulating factor supported

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203. Stewart K, Vescio R, Schiller G et al. Purging of autologous peripheral-blood stem cells using CD34 selection does not improve overall or progression-free survival after high-dose chemotherapy for multiple myeloma: results of a multicenter randomized controlled trial. J Clin Oncol 2001; 19: 3771–9. 204. Sheridan WP, Begley CG, To LB et al. Phase II study of autologous filgrastim (G-CSF)-mobilized peripheral blood progenitor cells to restore haemopoiesis after high-dose chemotherapy for lymphoid malignancies. Bone Marrow Transplant 1994; 14: 105–11. 205. Velders GA, Pruijt JF, Verzaal P et al. Enhancement of G-CSF-induced stem cell mobilization by antibodies against the b2 integrins LFA-1 and Mac-1. Blood 2002; 100: 327–33. 206. Molineux G, McCrea C, Yan XQ et al. Flt-3 ligand synergizes with granulocyte colonystimulating factor to increase neutrophil numbers and to mobilize peripheral blood stem cells with long-term repopulating potential. Blood 1997; 89: 3998–4004. 207. Brasel K, McKenna HJ, Charrier K, Morrissey PJ, Williams DE, Lyman SD. Flt3 ligand synergizes with granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor to mobilize hematopoietic progenitor cells into the peripheral blood of mice. Blood 1997; 90: 3781–8. 208. Craddock CF, Nakamoto B, Andrews RG et al. Antibodies of VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice. Blood 1997; 90: 4779–88. 209. Gazitt Y, Shaughnessy P, Liu Q. Expression of adhesion molecules on CD34+ cells in peripheral blood of non-Hodgkin’s lymphoma patients mobilized with different growth factors. Stem Cells 2001; 19: 134–43. 210. Ford CD, Greenwood J, Anderson J, Snow G, Petersen FB. CD34+ cell adhesion molecule profiles differ between patients mobilized with granulocyte-colony-stimulating factor alone and chemotherapy followed by granulocyte-colonystimulating factor. Transfusion 2006; 46: 193–8. 211. Laterveer L, Lindley IJ, Hamilton MS, Willemze R, Fibbe WE. Interleukin-8 induces rapid mobilization of hematopoietic stem cells with radioprotective capacity and long-term myelolymphoid repopulating ability. Blood 1995; 85: 2269–75. 212. Laterveer L, Lindley IJ, Heemskerk DP et al. Rapid mobilization of hematopoietic progenitor cells in rhesus monkeys by a single intravenous injection of interleukin-8. Blood 1996; 897: 781–8. 213. Carlo-Stella C, Di Nicola M, Longoni P et al. Mobilization of primitive and committed hematopoietic progenitors in nonhuman primates treated with defibrotide and recombinant human granulocyte colony-stimulating factor. Exp Hematol 2004; 32: 68–75. 214. Carlo-Stella C, Di Nicola M, Milani R et al. Use of recombinant human growth hormone (rhGH) plus recombinant human granulocyte colonystimulating factor (rhG-CSF) for the mobilization and collection of CD34+ cells in poor mobilizers. Blood 2004; 103: 3287–95. 215. Schwarzenberger P, Huang W, Oliver P et al. IL-17 mobilizes peripheral blood stem cells with short- and long-term repopulating ability in mice. J Immunol 2001; 167: 2081–6.

42

John G. Gribben

Removal of Tumor Cells from the Hematopoietic Graft

Introduction Despite the success of the use of combination chemotherapy for the treatment of advanced-stage malignancies, the majority of these patients die of their disease. In an attempt to overcome drug resistance, high-dose chemotherapy is used with curative intent both in patients with previously relapsed disease, and increasingly as consolidation therapy in high-risk patients in first complete remission (CR). The myeloablation induced by high-dose therapy can be reversed by autologous or allogeneic hematopoietic cell transplantation (HCT). Autologous cells have several potential advantages over allogeneic cells for HCT. Autologous HCT overcomes the need for a human leukocyte antigen-identical donor, eliminates the risk of graft-versus-host-disease and enables the use of chemotherapy dose escalation for a large number of patients with hematologic and solid tumors [1–4]. One of the major obstacles to the use of autologous HCT after highdose chemotherapy is that contaminating tumor cells might be infused back to the patient and would then contribute to subsequent relapse. To enable the use of autologous HCT, a variety of methods have been developed to “purge” malignant cells. The aim of purging is to eliminate any contaminating malignant cells and leave relatively intact the hematopoietic cells that are necessary for engraftment. The development of purging techniques has led subsequently to a number of studies of autologous HCT in patients with either a previous history of or even overt peripheral blood (PB) or bone marrow (BM) involvement at the time of stem cell collection [2,5–9]. These clinical studies have demonstrated that purging can result in a depletion of malignant cells in vitro without significant impairment of hematologic reconstitution. The rationale for removing tumor cells from hematopoietic cells might appear compelling, yet the issue of purging remains controversial. The finding that the majority of patients who relapse after autologous BM transplantation do so at sites of prior disease has led to the widespread view that purging of autologous marrow cannot contribute to outcome in those patients who are destined to relapse from their endogenous disease. Several clinical trials testing the efficacy of purging by comparison of infusion of purged versus unpurged autologous hematopoietic cells have not demonstrated a survival advantage for the use of purging. These issues, in addition to the lack of availability of effective purging strategies that are approved for widespread use, particularly

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

with the larger number of cells now collected using PB hematopoietic cells (PBHCs) rather than BM, have made it difficult to design and perform definitive studies to address the contribution of purging. In assessing the potential value of purging, three basic questions have to be addressed. First, what is the evidence that residual malignant cells are contained within autologous BM or PBHC collections? Second, can these tumor cells be purged using currently available techniques? Third, do reinfused tumor cells contribute to relapse and does removal of these cells lead to improved outcome after treatment?

Detection of residual disease in autologous hematopoietic cells The likelihood that autologous hematopoietic cells are contaminated with neoplastic cells is determined by a number of clinical variables. BM and PB involvement is extremely rare in some tumors such as testicular or ovarian cancers, more common in non-Hodgkin’s lymphoma (NHL) and in solid tumors including small cell lung cancer, neuroblastoma and breast cancer, and invariable in the leukemias. Generally, the higher the stage of the tumor, the more likely it is that the BM and PB are involved. The ability to detect malignant cells within the circulation is dependent upon the sensitivity of the assay used (Table 42.1). Since the limit of detection of BM infiltration by histologic examination is 5% and approximately 1011–1012 cells are collected, collection from a patient whose BM is judged to be normal by histologic examination may still contain as many as 5 × 109 malignant cells. More sensitive assays to detect the presence of malignant infiltration, such as immunocytochemistry, flow cytometric analysis, molecular biologic techniques, and clonogenic assays, have greatly increased the sensitivity of detection of malignant cells beyond that possible by histology. These techniques have all demonstrated the presence of minimal residual disease (MRD) in patients in whom there is no morphologic evidence of tumor infiltration in PBHC collections, as outlined in detail in Chapter 26. Immunocytochemistry Immunocytochemical techniques have been most widely used to detect MRD in solid tumors. Detection rates and levels of sensitivity vary widely depending upon the tumor type, the stage of disease in the patient population being studied, and also the methodology used to detect the tumor cells. The use of monoclonal antibodies (mAbs) that recognize cytokeratins is one of the most widely used methods, particularly in breast cancer [10]. Since there are no true tumor antigens that are

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Table 42.1 Sensitivity of tumor cell detection Method

Sensitivity

Light microscopy Southern blot analysis Fluorescence in situ hybridization Flow cytometry Immunocytochemistry (alkaline phosphatase–antialkaline phosphatase) Clonogenic assays Polymerase chain reaction

1 in 101–102 1 in 102 1 in 102 1 in 104 or greater 1 in 104–106 1 in 104–106 1 in 105–106

In a model in nonobese diabetic mice with severe combined immunodeficiency disease, in all subtypes of human acute myeloid leukemia (AML) analyzed except acute promyelocytic leukemia, cells capable of expanding in these mice were exclusively CD34+ CD38−, which are found at a low frequency with the leukemic blast population [14]. Unfortunately, the conditions for clonogenic tumor growth and the characteristics of the tumor stem cell have not been well characterized in the majority of tumors. Sensitive culture techniques have demonstrated clearly that clonogenic malignant cells can be grown from BM with no morphologic evidence of infiltration [15–20]. At least for breast cancer, there appears to be a good association between immunocytochemical and clonogenic assays for detection of MRD [19,20].

Residual tumor cells in PB mononuclear cell collections recognized using these techniques, great care must be taken in interpretation of data to ensure that normal cells that express these antigens are not also scored as tumor cells. It is one great advantage of immunocytochemical techniques that it allows morphologic examination of the positively stained cells, although it is not always possible to determine whether a stained cell is malignant. Additional markers that stain cycling cells, such as Ki-67, may improve the ability to discern malignant cells from background normal cells. Molecular biologic techniques The underlying principle for the application of molecular biologic techniques to the diagnosis of human malignancies lies in the detection of clonal proliferation of tumor-specific chromosomal translocations or gene rearrangements such as those occurring at the antigen receptors. The use of the polymerase chain reaction (PCR) has greatly increased the sensitivity of detection of MRD. Nonrandom chromosomal translocations are ideal candidates for PCR amplification if the DNA sequences at the chromosomal breakpoints are known. For example, cloning of the t(14;18) breakpoints involving the bcl-2 proto-oncogene on chromosome 18 and the immunoglobulin heavy chain locus on chromosome 14 has made it possible to use PCR amplification to detect lymphoma cells containing this translocation [11]. Using this technique, residual lymphoma cells were detected in the BM at the time of initial assessment and following induction or salvage therapy of all patients with advancedstage NHL containing the bcl-2 translocation [11–13]. Although a number of leukemias and lymphomas are characterized by nonrandom chromosomal translocations, the majority of malignancies, especially the solid tumors, do not demonstrate such nonrandom chromosomal translocations and are less suitable for detection by PCR amplification. PCR techniques can still be used to detect genes expressed within tumor cells that should not normally be detected within PB and can act as a surrogate marker for tumor detection, as described in more detail below. Cell culture techniques The biggest disadvantage of molecular and immunocytochemical techniques in detection of MRD is that these techniques do not differentiate between clonogenic tumor cells and cells that have lost the potential to proliferate. Clonogenic tumor assays have the capacity to detect the tumor cells that may be most relevant for subsequent relapse. The precise nature of the clonogenic tumor cell that can grow in the host and contribute to subsequent relapse has still to be defined. The cell capable of initiating human disease must have the potential for self-renewal and be capable of proliferating.

PBHC collections are now preferred over autologous BM harvests due to reduced engraftment time and ease of attainment; this issue is discussed in more detail in Chapter 41. Although the level of tumor contamination may be at a lower concentration in PBHC collections compared with BM, a number of studies have demonstrated that PBHCs may still be contaminated with tumor cells so that this source of hematopoietic cells may also require further processing to separate the hematopoietic cells from the tumor cells. In a multi-institutional study of PBHC transplantation in patients with advanced multiple myeloma receiving high-dose chemotherapy, tumor cells were detected in the leukapheresis products from eight of 14 unselected patients and ranged from 1.13 × 104 to 2.14 × 106 malignant cells/kg [21]. A 2.7–4.5 log reduction in contaminating myeloma cells was achieved using CD34 selection. In a retrospective analysis, cryopreserved BM aspirates from 83 patients with high-risk stage II, III, and IV breast cancer were obtained after induction chemotherapy but before stem cell harvest. All samples had no evidence of BM infiltration by morphologic assessment. PCR for cytokeratin 19 was performed, and results correlated with the probability of relapse following high-dose therapy and autologous HCT. The incidence of detection of cytokeratin 19 positivity assessed by PCR analysis in BM increased significantly with advancing stage: 52% for 19 stage II, 57% for 14 stage III, and 82% for 50 stage IV patients (p = 0.0075) [22]. Paired PBHC and BM samples from 48 patients with breast cancer were analyzed using immunocytochemical and clonogenic tumor colony techniques [19]. Immunocytochemistry detected tumor cells at a significantly higher rate in BM than in PBHCs (p < 0.005). Tumor cells were detected in 13 of 133 PBHC specimens (9.8%) from nine of 48 patients (18.7%), and in 38 of 61 BM specimens (62.3%) from 32 of these 48 patients (66.7%). Clonogenic tumor colonies grew in 21 of 26 specimens positive by immunocytochemistry. No tumor colony growth was detected in 30 of 32 specimens that were negative by immunocytochemistry. Immunocytochemical detection of tumor involvement in BM and PBHCs was correlated significantly with in vitro clonogenic growth (p < 0.0001). In the resting state, PB contains fewer tumor cells than BM in some malignancies, but mobilization with chemotherapy and growth factors may mobilize tumor cells as well as stem cells [23]. A greater number of PBHCs than BM cells must be collected so that the cell dose infused becomes an important determinant, in that the total number of tumor cells, rather than the concentration of such cells, is likely to be more relevant. This issue was addressed in a study in patients with multiple myeloma [24]. Quantitative PCR analysis of the immunoglobulin heavy chain variable region sequence of the patient’s myeloma cells was performed to assess tumor burden in samples from PBHC collections and BM harvests from 13 patients with multiple myeloma. As expected, the

Removal of Tumor Cells from the Hematopoietic Graft

percentage of tumor cells contaminating the BM harvest (median 0.74%) was higher than in the PBHC specimens (median 0.0024%). Because of the increased total number of cells used for PBHC transplantation, the increase in total number of contaminating cells in the BM versus PBHC autografts was less pronounced (BM versus PBHC tumor contamination ratios ranging from 0.9 to >4500; median 14) [24]. In patients with NHL, there was less than 1 log difference in the concentration of tumor cells in BM than in PBHCs. When allowance was made for the greater number of PBHCs used, there was no difference in the total number of tumor cells within the collected products [25]. Taken together, these data demonstrate that it is naïve to assume that PBHC collections are free from contaminating tumor cells. Clinical trials examining the question of tumor contamination and its clinical significance for subsequent outcome after HCT using PBHCs continue.

Strategies for purging of malignant cells At the same time that techniques were being developed to demonstrate the existence of MRD, attempts were being made to develop methodologies to deplete contaminating malignant cells without impairing hematopoietic progenitor cells, including negative purging with pharmacologic agents and mAbs, positive selection with immunoadsorption, immunomagnetic and flow cytometry sorting techniques, ex vivo culture, treatment with cytotoxic T cells, and virally mediated purging (Table 42.2). In the United States, ex vivo manipulation of PBHCs or BM is regulated by the Food and Drug Administration [26]. Most studies performed have utilized pharmacologic or immunologic maneuvers to remove malignant cells from the autologous marrow by a process of negative selection. An alternative strategy is to select positively the hematopoietic stem cells (HSCs) based upon expression of surface antigens such as CD34. These studies are described in detail in Chapter 5. The studies have been hampered largely by the relative inefficiency of CD34 selection techniques [27]. Preclinical studies have examined the potential role of positive selection of CD34 cells followed by negative depletion steps to remove residual contaminating tumor cells [28–31].

Table 42.2 Methodologies for purging tumor cells Physical separation

Pharmacologic Immunologic

T-cell mediated Viral

Size Density Osmotic lysis Lectin agglutination Hyperthermia Pulsed electric field 4-hydroperoxycyclophosphamide Asta-Z Uncoupled monoclonal antibodies Complement-mediated lysis Immunomagnetic beads Directly coupled Chemotherapeutic agent Toxins Magnetic beads Radionuclide

607

Pharmacologic purging A number of antitumor agents have been used for in vitro elimination of tumor cells, but most studies have used active analogs of cyclophosphamide or ifosphamide. The rationale for this approach was the finding that there was relative sparing of HSCs by cyclophosphamide in vivo, and by activated metabolites such as 4-hydroperoxycyclophosphamide (4-HC) [32], due to the relatively high levels of aldehyde dehydrogenase in HSCs that inactivates active metabolites of cyclophosphamide. This was rapidly adapted for clinical use [33–36], with 4-HC used more widely in the United States and mafosfamide used more extensively in Europe. Pharmacologic purging has been hampered by the availability of suitable agents. The Food and Drug Administration did not approve 4-HC for clinical use because of the lack of a prospective, randomized clinical trial, leading to unavailability of this drug for several years. Further development of 4-HC is under review, but studies have continued with mafosfamide. While 4-HC largely spares primitive HSCs, it produces dosedependent toxicity against tumor cells and reduces committed progenitors such as granulocyte–macrophage colony-forming units by up to 99% [37]. The major complication with the use of pharmacologic purging has been the delayed neutrophil engraftment times. Median time to neutrophil engraftment in patients with AML was 29 days using 4HC-purged BM [33] but 22 days using unpurged BM [38]. There is considerable interpatient variability in drug sensitivity, and some centers use assays to assess dose for an individual patient [39]. Amifostine is a prodrug converted by alkaline phosphatase to the active sulfhydryl compound WR-1065, which protects normal cells by scavenging free radicals and binding to active derivatives of antineoplastic agents. Amifostine shortens the engraftment period of 4-HC-purged BM [40]. Other agents that have been investigated include etoposide, which spares hematopoietic progenitor cells and stromal cells compared with leukemia or lymphoma cells [41], 6-hydroxydopamine [42], alkyl lysophospholipids [43], the photosensitizing agent merocyanine with light exposure [44], and guanine arabinoside [45].

Characteristics of ideal mAbs for purging Because of their specificity, mAbs make ideal agents to identify and target such malignant cells. A number of mouse anti-human mAbs have been generated with specificity for human cell surface antigens by immunizing mice with human malignant cells or malignant cell membranes. Despite the hope that unique tumor-specific cell surface proteins would be recognized, all of the cell surface antigens identified to date on neoplastic cells of the hematopoietic or solid tumor malignancies represent normal differentiation antigens, and true leukemia-, lymphoma- or cancer-specific antigens have not been identified. The most important factor to be determined is that the mAb targets the malignant cell as specifically as possible but has no effect on the HSCs necessary for marrow engraftment. The principle for the selective depletion of contaminating residual tumor cells from HSCs is illustrated in Fig. 42.1. Likely mechanisms of failure of immunologic purging include antigenic heterogeneity, whereby not all tumor cells express the targeted antigen, and the relative inefficiency of the purging methodology employed to “purge” the targeted cells. The ideal characteristics of mAbs for purging are shown in Table 42.3. The targeted antigen should be present at high density on the cell surface to increase the efficiency of subsequent cell killing or removal. To limit the effect of antigenic heterogeneity of expression on the target cell, multiple mAb cocktails are employed targeting multiple antigens. Since mAbs are not by themselves toxic, they must be used in combination with other agents to kill the targeted cell. The most widely studied

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methods of immunologic purging are complement-mediated lysis, immunomagnetic bead depletion, and immunotoxins. The potential advantages and disadvantages of each of these techniques are shown in Table 42.4. If mAbs are used with complement, complement-fixing isotypes must be used, the most efficient being immunoglobulin M. For immunologic purging using complement-mediated lysis or immunomagnetic bead separation, it is important that the antigen–antibody complex remains on the cell surface and is not internalized. If immuno-

Anti-CD20 mAb Lineage antigens CD19 NHL cells

CD20

Complement

Anti-CD34 mAb Stem cells

CD34

Stem cell collection

Selection/Depletion

Reinfusion of tumor-free stem cells

Fig. 42.1 Principles of immunologic purging. Targeted antigens are present on the surface of malignant cells but are not expressed on hematopoietic progenitors. Malignant cells escaping the purging procedure are likely not to express or express only weakly the targeted antigens. mAb, monoclonal antibody; NHL, non-Hodgkin’s lymphoma.

Table 42.3 Ideal target antigens for tumor cell purging • • • • • •

Not expressed on hematopoietic progenitors Expressed on clonogenic tumor cells High density of expression on malignant cells Limited heterogeneity of expression on tumor cells Lineage restriction Depending on strategy for purging – ability to modulate

toxins are used, the targeted antigen–antibody complex should be internalized to ensure intracellular delivery of the cellular toxin. Complement-mediated lysis The earliest preclinical studies utilized the ability of mAb to fix complement to the mAb-coated cells, which were then eliminated by complement-mediated cytotoxicity. Complement-mediated cytolysis was previously the most commonly employed method for immunologic purging, due in part to its efficiency, specificity, and relatively low cost. In most studies, rabbit complement has been used to circumvent the problem of homologous species restriction, the process whereby cells are generally resistant to lysis by complement from the same species. The ideal complement source must be toxic to cells coated with mAb but not toxic to cells that have not been coated with antibody. There are, however, major disadvantages of using complement. There is considerable variability among different lots of complement so that each new lot must be tested for nonspecific toxicity. There are nonspecific cell losses that occur because of the need for cell-washing steps. In addition, complement-mediated lysis is inefficient when the neoplastic cells only weakly express the targeted antigen. Regulatory issues related to the use of non-human sources of protein for human cell manipulation have markedly decreased the use of complement-mediated lysis for clinical purging. Among the factors that may influence the efficiency of complementmediated lysis are the density of surface antigen expression, antigen modulation, and resistance to complement lysis. In addition, failure of immunologic purging using complement-mediated lysis could be attributed to three possible mechanisms. First, the clonogenic tumor cells might not express, or only express weakly, the cell surface antigens expressed by the majority of tumor cells. Second, modulation of one or more of the surface antigens following attachment of the mAb to its ligand might limit complement-mediated lysis. Third, a subgroup of patients may have malignancies that are intrinsically more resistant to complement-mediated lysis. Tumor cell killing by antibody-mediated complement activation results from osmotic cell lysis following disruption of the semipermeable properties of the cell membrane. An actively metabolizing cell is capable of turning over its cell membrane. This phenomenon may result not only in antigen modulation, but also neutralization of the lytic effects of the complement. Previous studies have shown associations between biochemical events in the cell and sensitivity to complement-mediated lysis [46,47]. An anticomplement factor has been described in normal BM cells that limits complement activation, not only on the cells that

Table 42.4 Comparison of methods of immunological purging Method of tumor depletion

Nature of antibody–antigen interaction

Complement-mediated lysis

Antibody must fix complement High antigen density expression Antigen should not modulate Antibody–antigen complex must internalize High antigen density expression

Immunotoxins

Immunomagnetic bead depletion

All antibodies are candidate targets High antigen density expression

Technical issues

Resistance to tumor cell

Expense

Screen complement lots for toxicity Multiple antibodies additive Multiple cycles are required Nonspecific cell loss Longer incubation time More easy to standardize Difficult to assess efficacy of purge Nonspecific cell loss Easy to standardize Simple and rapid Fewer cycles required

Antigen heterogeneity Resistance to complement lysis

Low

Antigen heterogeneity Few antigen targets internalize Resistance to toxin mechanism

Intermediate

Antigen heterogeneity Antigen shedding

High

Removal of Tumor Cells from the Hematopoietic Graft

produce the factor, but also on antibody-coated cells within the BM [48]. These anticomplementary effects may be overcome by repeated treatments with complement, and previous studies have suggested that the use of repeated treatment cycles is more efficient in removing contaminating tumor cells than single treatment cycles. This approach is timeconsuming, increases the expense of the procedure in terms of both reagents and laboratory staff effort, and may increase the nonspecific loss of HSCs. The continuous infusion of fresh complement while removing media containing the used complement may increase the efficiency and time taken to perform the procedure [49]. Studies have been reported that address whether these mechanisms of complement resistance can be overcome by chemical engineering of mAbs [50] or by neutralization of CD59 activity [51]. Expression of membrane-bound regulators of complement activation including CD46, CD55, and CD59 protect nucleated cells from complement-mediated injury. Increased expression of these molecules may be a mechanism by which tumor cells protect themselves from inflammatory responses and complementmediated injury. Populations of cells that survive following mAb purging appear to be more resistant to subsequent treatments with the same mAb and complement, presumably because of the emergence of subpopulations of cells with a relative decrease in the surface expression of the targeted antigens [52]. Such changes in relative expression of antigen density have also been observed following treatment with chemotherapy. It is important to demonstrate that the tumor to be purged expresses the targeted antigen, not only at the time of diagnosis, but also at the time of hematopoietic stem harvest. This is increasingly the case now that humanized mAbs are in routine use in the treatment of patients with a variety of malignances. Although the emergence of target antigen-negative tumor cells is relatively rare, this has been observed [53–56]. The combination of immunologic and pharmacologic purging appears to be more efficient than either method alone in eliminating clonogenic Burkitt cell lines from human BM [57]. The effectiveness of purging small cell lung cancer cell lines was also significantly increased when mAb and complement-mediated lysis were used in combination with the cyclophosphamide derivative Asta-Z 7557, although there was significant reduction in myeloid colony growth [58]. In T-cell malignancies, 2′-deoxycoformycin has been used in combination with anti-T-cell mAbs to eliminate clonogenic malignant human T cells [59,60]. A combined approach was taken to attempt to eliminate multidrug-resistant leukemic cell lines from BM using a mAb directed against the cell surface product of the multidrug-resistance gene [61]. This treatment did not affect normal committed precursors. Following antibody purging, the addition of etoposide enhanced the purging efficacy and resulted in a 4.6 log reduction of malignant cells. Furthermore, this antibody was effective when used against the patients’ leukemic blasts, suggesting that this approach was effective and selective for removal of multidrug resistance-expressing cells from the graft. Magnetic bead depletion The use of immunomagnetic beads has the advantage that there is no biologic variability between lots as has been observed with complement. A number of particles have been developed that are directly attached to the primary mAbs used for purging. These reagents have the advantage of allowing more rapid and simpler purging procedures. Instruments to remove the immunomagnetic beads are now available commercially, and immunomagnetic bead depletion is used increasingly as a method of eliminating malignant cells for HCT. The use of immunomagnetic beads was originally developed to facilitate depletion of neuroblastoma cells since the available mAb did not fix complement [62]. A number of studies have been performed for a variety of other malignancies,

609

including small cell lung cancer, breast cancer, acute lymphoblastic leukemia (ALL), myeloma, and lymphomas, as described below. The efficacy of mAbs and immunomagnetic beads in removing Burkitt’s lymphoma cells from normal BM has been demonstrated in a number of studies. Clonogenic lymphoma cell assays have demonstrated that different anti-B-cell mAbs differ in their efficiency of depleting lymphoma cells from 1.9 to 2.8 log in one cycle. When three mAbs were used in a cocktail, the efficiency of purging increased to 3.3 log following a single cycle of treatment, and 5 log after two cycles. Treatment of the BM with beads alone or with the mAb did not significantly reduce the number of HSCs as assessed by colony assays [63]. A cocktail of two mAbs was used to assess the relative efficiency of purging of two different immunomagnetic particles [64]. The efficacy of purging the cell line NALM-6 was assessed using immunofluorescence or colony assays. Log tumor cell kill was significantly better using BioMag particles (3.1 log) than Dynabeads (1.8 log) following a single cycle of treatment. These beads differ in size, and the smaller particles appeared to remove cells more efficiently. There was no significant difference in the efficiency of purging after two treatment cycles, and greater than 4.5 log of tumor cell depletion was obtained using either immunomagnetic bead. In preclinical experiments, BM samples from patients with follicular lymphoma that were contaminated with up to 20% lymphoma cells were purged with either a three- or a four-mAb cocktail followed by immunomagnetic bead depletion. This resulted in the loss of all PCRdetectable cells after three cycles of treatment in all 25 patients studied [65]. After two treatment cycles of immunomagnetic bead depletion, four mAbs were more efficient than three mAbs for purging, and immunomagnetic beads were more efficient than complement-mediated lysis. The results also demonstrate that multiple cycles of immunomagnetic bead depletion may still be required to remove PCR-detectable lymphoma cells, although immunomagnetic bead depletion had no significant effect on myeloid colony-forming assays, suggesting that repeated cycles of immunomagnetic bead depletion might be performed safely. Theoretically, attachment of a single magnetic particle may be sufficient to allow removal of the targeted cell in a magnetic field, and some lymphoma cells may have a sufficiently low density of expression of the targeted antigens to allow elimination with immunomagnetic beads but not to allow complement-mediated lysis. Immunotoxins Purging of autologous marrow in vitro using immunotoxins is a particularly promising approach. Several exquisitely effective candidate toxins have been identified that mediate their cytotoxic function by inhibiting cellular protein synthesis. Because the mechanism of killing by toxins is different from that of chemotherapeutic agents, they are capable of killing cells that are resistant to chemotherapy [66]. These toxins are cytotoxic to both normal and malignant cells, and must be targeted to the malignant cell to demonstrate specificity. The combination of these toxins with a mAb to target delivery specifically to the neoplastic cells is a theoretically attractive proposition [67]. If native toxins were to be conjugated to mAb, the resultant immunotoxin would still be capable of binding to nonspecific targets by binding to the toxinbinding site on normal cells. This nonspecific binding is overcome by modification of the toxin moiety to delete the binding site but leave the toxin domains intact. The most widely studied toxins have been ricin, Pseudomonas exotoxin, and diphtheria toxin. Most experience of in vitro marrow purging has been with ricin. Multiple anti-T-cell intact ricin immunotoxins have been evaluated as potential purging agents [68]. The cocktail containing all four immunotoxins in equimolar concentrations eliminated more than 4 log of clonogenic leukemic cells at a dose that spared more than 70% of the pluripotent HSCs.

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Other approaches Flowing hematopoietic cells through pulsed electric fields effectively purges myeloma cells without sacrificing functional stem cells [69]. A novel approach for the separation of malignant cells was reported for 16 patients with B-cell malignancies using floating immunobeads [70], in which low-density polypropylene beads precoated with rat antimouse mAb were added to the harvested autologous BM following incubation with anti-B-cell mAb. This approach resulted in 75% recovery of mononuclear cells with an 83% recovery of myeloid progenitors. Oncolytic viruses are being investigated as a novel purging strategy. DNA viruses such as genetically engineered adenoviral vectors have been used to deliver either a prodrug-activating enzyme or express wild-type p53 selectively in tumor cells in ex vivo purging protocols [71]. Assessment of the efficacy of purging The identical techniques used to assess whether a hematopoietic cell collection contains residual tumor cells can be used to assess whether tumor cells remain present after immunologic purging, including culture systems, clonogenic assays, and PCR analysis. It is more difficult to assess the efficacy of pharmacologic purging using molecular techniques, since the malignant cells are not physically removed. Studies have demonstrated that different anti-B-cell mAbs differ in their efficiency to deplete lymphoma cells [63]. Multiple rounds of treatments were more efficient than single treatments, and the combination of two or more antibodies was more efficient than a single mAb to eliminate tumor cells [72,73]. PCR has been used to assess the efficacy of immunologic purging in both models using cell lines [74] and patient samples [13,65]. The efficacy of mAbs and immunomagnetic beads has also been demonstrated in a number of studies [28,63,65]. Clonogenic lymphoma cell assays have demonstrated that the efficiency of purging increased significantly when three mAbs were used in a cocktail. A cocktail of two mAbs was used to assess the relative efficiency of purging of two different immunomagnetic particles [75]. Log tumor cell kill was significantly better using BioMag particles (3.1 log) versus Dynabeads (1.8 log) following a single cycle of treatment. There was no significant difference in the efficiency of purging after two treatment cycles, and greater than 4.5 log of tumor cell depletion was obtained using either immunomagnetic bead. Addition of more antibodies and the use of immunomagnetic beads compared with complement mediated

lysis results in more efficient depletion of lymphoma cells in all patient samples tested [65]. Using a single cycle of treatment with multiple mAbs and beads, approximately 2.5 log of small cell lung cancer lines could be depleted, although there was variability in the efficiency of purging different cell lines [76]. In parallel studies, there was no significant toxicity noted to myeloid progenitors. Using two small cell lung cancer lines, immunomagnetic bead depletion was shown to result in a 4–5 log reduction of cancer cells and did not adversely affect BM colony growth [77]. AntiCD15 mAb, expressed on a variety of human cancer cell lines, was capable of depleting up to 3 log of breast cancer cells from normal contaminated marrow using immunomagnetic bead depletion but minimally affected normal hematopoietic progenitors [78]. The combination of 4-hydroperoxycyclophosphamide and immunomagnetic bead depletion removed 4–5 log of clonogenic breast cancer cells [79]. Evaluation of the purging efficiency of an immunotoxin prepared by conjugating anti-CD7 with pokeweed antiviral protein revealed that approximately 3 log of clonogenic T cells could be eliminated. The addition of 2′-deoxycoformycin and deoxyadenosine to the immunotoxin resulted in the elimination of up to 6 log of the T-cell line but also resulted in decreased myeloid progenitor colony assay growth [60].

Contribution of infused tumor cells to relapse The finding that the majority of patients who relapse after autologous HCT do so at sites of prior disease has led to the widespread view that purging of autologous marrow could contribute little to subsequent outcome after autologous HCT. Although few direct studies have compared the infusion of purged versus unpurged HSCs, indirect approaches can be made to assess the clinical significance of immunologic purging. Methods to assess the clinical utility of purging are shown in Table 42.5. Clinical studies of purging Pharmacologic purging Pharmacologic purging has been most extensively studied in AML, where it has been shown that ex vivo purging of BM with 4-HC or mafosfamide is feasible and can result in long-term disease-free survival in patients with high-risk AML [33,34,80–82]. Studies have also been

Table 42.5 Impact of immunological purging on engraftment

Disease Complement AML AML ALL NHL Immunomagnetic beads Neuroblastoma ALL Immunotoxins ALL ALL

Number of patients

Antigen/monoclonal antibody

Days to neutrophils >500/μL

Days to platelets >20,000/μL

Reference

138 12 54 100

CD14, CD15 CD33 CD10, CD19, CD7 CD10, CD20, B5

27 (1st CR) 30 24 27

38 45 40 29

[3] [99] [97] [6]

91 8

UJ13A, Thy 1, UJ127.11, UJ181.4 CD10, CD9

28 15

42 27

[104] [102]

13 14

CD7 CD5, CD7

17 27

40 NS

[105] [106]

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CR, complete remission; NHL, non-Hodgkin’s lymphoma.

Removal of Tumor Cells from the Hematopoietic Graft

611

Table 42.6 Approaches to demonstrate clinical efficacy of purging Surrogate endpoints • Depletion of polymerase chain reaction-detectable cell • Depletion of clonogenic tumor cells Gene transfer of marker gene Randomized trial • Purged versus unpurged autologous hematopoietic cells

Fig. 42.2 Cumulative probability of relapse is significantly reduced in children with acute myeloid leukemia in whom in vitro purging has been performed. Reproduced with permission from [89].

performed in NHL [83] and myeloma [84]. An analysis of 1393 patients registered with the European Bone Marrow Transplant Registry (EBMT) demonstrated an advantage of purged BM versus unpurged sources of HSCs [85]. Similar results have been obtained in smaller single-center studies [86–88]. A multicenter analysis of the use of 4-HC for marrow purging in AML patients in first remission also demonstrated an advantage for purging, with a leukemia-free survival of 56% (range 47–64%) for purged versus 31% (range 18–45%) for unpurged marrow [9]. In a retrospective EBMT analysis of 387 children who underwent autologous HCT in first CR, probability of relapse was decreased after in vitro purging as shown in Fig. 42.2 [89]. In contrast to AML, few studies in ALL have addressed the use of pharmacologic purging. A study reporting the outcome in patients registered with the EBMT demonstrated no difference in outcome for patients receiving purged compared with unpurged sources of HSCs [90]. The role of purging in transplants for solid tumor has been reviewed and remains controversial [91]. Immunologic purging Immunologic purging was first performed in NHL and has been most widely studied in this disease [6,8,13,92,93]. Additional studies have been performed in multiple myeloma [27,94,95], ALL [96,97], AML [98], breast cancer [99], small cell lung cancer [58], and neuroblastoma [100,101] among others. These studies have confirmed that immunologic purging can be performed safely and that subsequent hematopoietic engraftment is not significantly delayed. No randomized prospective study has demonstrated whether the removal of occult or overt neoplastic cells resulted in improved disease-free survival [27,95]. Whether the failure to demonstrate an advantage of purging is due to the relative inefficiency of the purging technique or the intrinsic resistance of the tumor cells to the high-dose chemotherapy approach used is not clear. The results obtained in the larger reported trials using immunologic purging are shown in Table 42.6. Complement-mediated lysis In a clinical trial of 138 patients with AML, BM purging was performed using two mAbs and complement-mediated lysis [3]. One hundred and ten patients were in CR at the time of transplantation (23 in first CR, 87 in second or third CR). Engraftment was prompt in most patients, and

only one very heavily pretreated patient in third CR failed to engraft. Engraftment was faster in those patients infused with larger numbers of colony-forming units. This study did not compare results obtained using purged versus unpurged marrow, but the relapse-free survival of the patients in second and third remission appears to be comparable to that obtained following allogeneic transplantation in similar-risk patients. Anti-CD33 mAb and complement-mediated lysis was used to purge the BM of 12 patients with AML [98]. Patients had durable but delayed engraftment, and platelet engraftment was particularly delayed in some patients. Of note, granulocyte–macrophage colony-forming unit colony growth was markedly reduced following purging. In a multicenter study, autologous HCT was used in 54 patients with ALL [96] with purging using anti-CD10, anti-CD19, and anti-CD7 mAbs and rabbit complement. The transplant related mortality was 5% and engraftment was rapid. Of note, this study was not designed to demonstrate efficacy. One hundred patients with B-cell NHL were treated at the DanaFarber Cancer Institute with autologous BM purged when patients were in CR or minimal disease state [6]. Notably, 69 patients had a prior history of histologic marrow involvement, and 37 patients had overt marrow disease at the time of harvest. This study was associated with low treatment-related mortality. Engraftment was rapid in all cases. The results of the use of HCT in patients with follicular lymphoma in first complete or partial remission have also been reported [8,102]. These studies not only address the outcome for those patients receiving autologous HCT, but also enrolled patients on an “intent-to-treat basis” and suggested that virtually all patients with follicular lymphoma could achieve minimal disease state before proceeding to autologous HCT. Long-term follow-up of patients treated at the Dana-Farber Cancer Institute was reported on 153 patients with B-cell NHL with autologous BM purged when patients were in CR or minimal disease state [2]. Notably, 47% of patients had overt marrow disease at the time of collection. This study was associated with low treatment-related mortality, and engraftment was rapid in all cases. Immunomagnetic bead depletion The first clinical studies of purging using immunomagnetic beads were performed for children with neuroblastoma [101]. Immunomagnetic bead depletion was used to purge 123 BMs before autologous stem cell transplantation in 91 cases of neuroblastoma [103]. In this study, 59 patients received a single graft and 32 patients received two sequential procedures. Although the procedure resulted in a significant loss of mononuclear cells, there was little evidence of additional toxic effects on myeloid progenitors. Immunomagnetic beads were used to deplete leukemic cells from the marrows of patients with common ALL [101]. In this study, the BM of 18 patients was purged using a cocktail of three mAbs, although only eight of these patients were subsequently treated with high-dose therapy and autologous HCT. Engraftment was rapid in all cases although slower than that observed in patients with neuroblastoma.

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Immunotoxins Fewer clinical trials have been reported using immunotoxins for purging. Seven patients with high-risk T-cell ALL and six patients with T-cell NHL were treated by autologous BMT following purging with anti-CD7 ricin A immunotoxin [104]. Incubation of the marrow with up to 10−8 mole/L had no significant effect on HSC progenitors as assessed by colony assay growth or by subsequent delay of engraftment. Using a different approach, autologous marrow from 14 consecutive patients with T-cell ALL was purged with a combination of two immunotoxins, anti-CD5 and anti-CD7 linked to intact ricin, plus 4-HC [105]. The efficacy of purging was assessed using multiparameter flow analysis, cell sorting, and leukemic progenitor cell colony assay. Following purging, no blast colonies were observed in the marrows of 11 of 13 evaluable patients. Engraftment occurred in 13 of the 14 patients, and the median time to reach an absolute neutrophil count greater than 500/ μl was 27 days. Despite the apparent efficiency of purging, nine patients relapsed, the majority of them shortly after HCT, suggesting that in this study relapse was most likely due to failure of the high-dose therapy to ablate endogenous disease in these high-risk patients. Outcome after successful immunologic purging

Probability of event-free survival

In studies at the Dana-Farber Cancer Institute, PCR amplification of the t(14;18) was used to detect residual lymphoma cells in the BM before and after purging to assess whether efficient purging had any impact on disease-free survival [13]. In this study, 114 patients with B-cell NHL and the bcl-2 translocation were studied. Residual lymphoma cells were detected in all patients in the harvested autologous BM. Following three cycles of immunologic purging using the anti-B-cell mAb J5 (anti-CD10), B1 (anti-CD20), and B5, and complement-mediated lysis, PCR amplification detected residual lymphoma cells in 57 of these patients. The incidence of relapse was significantly increased in patients who had residual detectable lymphoma cells compared with those in whom no lymphoma cells were detectable. The long-term follow-up results of this patient cohort by the result obtained after purging is shown in Fig. 42.3. This study demonstrates that patients who were infused with a source of hematopoietic cells that was free of detectable lymphoma cells had improved outcome compared with those who had residual detectable

100

PCR neg (57 patients, 14 relapses)

80 60 p <0.0005

40 20

PCR pos (57 patients, 52 relapses)

0

2

4

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Fig. 42.3 The disease-free survival of lymphoma patients who were infused with autologous bone marrow with no polymerase chain reaction (PCR)detectable lymphoma cells (PCR neg) was significantly improved compared with those infused with BM containing residual PCR-detectable lymphoma (PCR pos). All patients had PCR-detectable lymphoma cells in their bone marrow before immunologic purging. (Unpublished update of data from a previously published study [13,65].)

lymphoma. This finding was independent of the histology of the lymphoma, the degree of BM infiltration at the time of BM harvest or remission status at the time of autologous HCT. These findings suggest that the detection of residual lymphoma cells is associated with or provides a surrogate marker for subsequent relapse. A major problem with this finding is that the majority of patients who relapse do so at sites of previous disease, suggesting that the major contribution to subsequent relapse came from endogenous disease. In 60 consecutive patients with a PCR detectable bcl-2 translocation who had undergone immunologic purging and autologous BMT, there was also an association between the presence of residual lymphoma cells after purging and the presence of circulating lymphoma cells that could be detected as little as 2 hours after infusion of autologous BM. It is possible that these circulating lymphoma cells are capable of homing back to the sites of previous disease and that these sites provide the microenvironmental conditions conducive for cell growth. Additional studies have provided further indirect evidence that the use of sources of hematopoietic cells that are contaminated with malignant cells is associated with poorer outcome after HCT. In NHL, two additional studies have demonstrated that the presence of residual lymphoma cells within the collected cells is associated with poorer outcome [18,106]. Studies at the University of Nebraska have demonstrated that those patients who are infused with morphologically normal BM containing clonogenic lymphoma cells have an increased incidence of relapse after HCT [17,18]. Even in metastatic breast cancer, in circumstances where it is likely that endogenous disease in the patient contributes highly to subsequent failure, the presence of contaminating breast cancer cells may be associated with poor outcome after high-dose therapy. In a retrospective analysis, cryopreserved BM aspirates from 83 patients with high-risk stage II, III, and IV breast cancer were obtained after induction chemotherapy but before BM harvest [22]. All samples had no evidence of BM infiltration by morphologic assessment. PCR for cytokeratin 19 was performed, and results correlated with the probability of relapse following high-dose therapy and autologous BMT. The incidence of detection of cytokeratin 19 positivity assessed by PCR analysis in BM increased significantly with advancing stage. Furthermore, in patients with advanced-stage breast cancer, detection of message for cytokeratin 19 in BM was associated with a significantly higher (p = 0.0002) incidence of subsequent relapse. The probability of relapse at 3 years after autologous BMT for PCR-positive patients was 32% for stage II/III and 94% for stage IV patients. Patients with no PCRdetectable disease had better outcome, with a probability of relapse of 10% for stage II/III and 14% for stage IV patients [22]. None of the studies listed above provides definitive proof that infusion of residual cells at the time of autologous HCT contributes to relapse since it is possible that the detection of residual cancer cells at the time of HCT is associated with an inherently worse prognosis in these patients. This could be due to the fact that patients who are not purged could have a higher tumor burden than patients who are purged successfully. Patients who purge successfully could have a higher level of expression of the target antigen on the cell surface of the clonogenic tumor cell or be more sensitive to complement-mediated lysis, and this could be associated with an inherently different outcome. Irrespective of the mechanism, the finding of residual malignant cells in autologous hematopoietic cells does appear to provide a powerful prognostic surrogate marker for relapse, independent of other clinical parameters. A retrospective analysis of the EBMT Lymphoma Registry compared the outcome of 270 patients whose collections had been purged with 270 case-matched control patients [107]. A variety of purging methodologies was used. In this study, there was no advantage in outcome if patients received purged products. Patients with low-grade NHL did not have a

Removal of Tumor Cells from the Hematopoietic Graft

significantly improved progression-free survival if the BM was purged (p = 0.1757), but they did have a significantly improved overall survival (p = 0.00184). In this study, time to hematologic engraftment, response to autologous BMT, and number of procedure-related deaths were similar in patients who received purged and unpurged grafts. Only one randomized trial, the Chemotherapy, Unpurged, Purged (CUP) trial, has attempted to evaluate the role of purging prospectively [108]. Although 140 patients were registered from 36 centers internationally, only 89 were randomly assigned. No benefit of purging was demonstrated, but there was no attempt to assess whether the purging strategy used was successful in eradicating tumor cells. Marker gene studies demonstrate that infused cells contribute to relapse Transfection of a marker gene into clonogenic malignant cells ex vivo provides a method to assess the fate of malignant cells within the autologous hematopoietic cells, as described in more detail in Chapter 10. If the majority of cells at the site of relapse expressed the marker gene, this would provide compelling evidence that infused malignant cells contribute to relapse. Since the efficiency of transfection is low using existing technology, a negative result would still not be definitive. Results published to date have demonstrated that when relapse occurs, there is evidence of malignant cells, with the marker gene suggesting strongly that the reinfused malignant cells contributed to relapse [109– 112]. In these studies, the neomycin resistance gene was retrovirally transferred to mark BM harvested from eight patients with childhood tumors, including leukemia and neuroblastoma. The marked marrow cells were subsequently reinfused as part of an autologous HCT. The marker gene could be identified in the malignant cell populations in the majority of patients at relapse. Analysis of tumor cell DNA for discrete marker gene integration sites suggested that at least 200 malignant cells, each capable of tumor formation, were introduced at HCT and contributed to relapse [112]. The authors concluded that autologous BM might contain a multiplicity of malignant cells that subsequently contribute to relapse. In a study in patients with myeloma, genetically marked autologous CD34+-enriched BM or PB were transplanted with a mixture of transduced cells and unmanipulated graft, and were followed with regular time points of analysis. Vector integration and transgene expression were analyzed by colony assays and PCR. The transgene could be detected for up to 5 years post-transplant in normal BM cells, even in remission following relapse, and no side-effects related to retroviral gene transfer were observed. There were no marked myeloma cells observed in the patients either in remission or in relapsing disease, with no indication that infused cells contribute to subsequent relapse [113]. In vivo purging The availability of humanized antibodies allows the use of mAbs to be given in vivo, unlike the murine mAbs that can only be used for ex vivo manipulations. Humanized mAbs have been given as additional systemic therapy to eliminate tumor cells from the PBHC collection as an in vivo purging approach [114–117]. This clearly has the advantages that it is easier to perform, does not require a cell manipulation laboratory, and allows treatment not only of the collected stem cells, but also of the whole patient. Moreover, this approach can be repeated following infusion of the autologous hematopoietic cells. A disadvantage at present is the limited availability of suitable antibodies such that only one antigen is being targeted. Most clinical experience has been with the anti-CD20 mAb rituximab. Successful in vivo purging with rituximab before autologous HCT was demonstrated in 15 patients with follicular and mantle cell NHL, treated

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with two cycles of intensive chemotherapy, each followed by growth factor infusion and two cycles of rituximab [116]. There was no PCRdetectable disease in the CD34+ harvested HSCs in 93% of those receiving rituximab compared with 40% in 10 patients who were similarly treated but without the addition of rituximab. Chemoimmunotherapy produced complete molecular and clinical remissions in all 14 evaluable patients receiving rituximab, including in six mantle cell lymphomas with significantly longer disease-free survival. The administration of rituximab with high-dose sequential chemotherapy with autologous HCT was assessed in 32 patients with advancedstage NHL [115]. Rituximab was delivered concurrently with high-dose chemotherapy to exploit the in vivo purging properties of the drug, as well as at the end of treatment to target MRD. PCR analysis was carried out in indolent lymphoma patients: in all nine follicular NHL and three of six CD5-positive NHL PCR-negative PB progenitor cell harvests were collected. An updated report now includes 92 patients with advanced-stage follicular NHL and demonstrates a projected long-term progression-free survival exceeding 80% for patients in whom a source of HSCs can be collected that are free of PCR-detectable lymphoma cells [117]. In 18 patients with NHL (15 with follicular and three with mantle cell NHL) treated with three cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), followed by stem cell mobilization using high-dose cytosine arabinoside and mitoxantrone with rituximab, the number of PCR-detectable lymphoma cells decreased progressively with treatment as measured in both the BM and PB [118]. In 17 of 18 patients, stem cells sufficient for transplantation were obtained in a single harvest. mAb therapy is also being studied after autologous HCT. In 35 patients with relapsed and refractory aggressive NHL who received autologous HCT, followed by 4-weekly infusions of rituximab among the first four patients, then repeat rituximab at 6 months in the remaining 31 patients, estimated 2-year disease-free survival was 86%, while overall survival was 85% in the 29 patients completing therapy [119]. In a phase II study, 34 patients with chronic lymphocytic leukemia received alemtuzumab consolidation in an effort to improve the quality of their response to fludarabine-based induction, and the feasibility and tolerability of subsequent PB collection and autologous HCT was assessed. PBHC collection was successful in 24 (92%) of 26 patients, and 18 patients underwent autologous PBHC transplantation [120]. Radioimmunotherapy using iodine-131-tositumomab with high-dose etoposide and cyclophosphamide has also been used in combination with autologous HCT. In 52 patients with relapsed B-cell NHL, followed by autologous HCT, the estimated overall and progression-free survival was 83% and 68% at 2 years. This compared favorably with a nonrandomized control group that received high-dose etoposide, cyclophosphamide, and total body irradiation during the same time period [121]. This is also feasible in older patients [122]. Iodine-131-tositumomab has also been assessed with BCNU (carmustine), etoposide, cytosine arabinoside, and melphalan (BEAM) chemotherapy in 23 patients with primary induction failure or relapsed NHL (four with follicular lymphoma, 14 with diffuse large cell lymphoma, and five with mantle cell lymphoma), followed by autologous HCT. Early results are encouraging in this highrisk population [123]. Yttrium-90-ibritumomab has also been combined with BEAM and autologous HCT, and results suggest that this approach is relatively safe and may improve outcome in patients with refractory lymphoma [124]. Design of randomized clinical trials A randomized trial using purged versus unpurged autologous marrow capable of providing a definitive answer would require a multicenter study of several hundred patients. One international collaborative trial

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Chapter 42

to determine the role of autologous HCT and the role of purging in follicular NHL has been conducted, and although the study demonstrated an advantage of high-dose therapy over conventional therapy, there was no benefit for purging. However, despite the international collaborative nature of this study, only 89 patients were enrolled [108]. In multiple myeloma, a randomized phase III trial using purged versus unpurged autologous PBHCs was performed using CD34 selection [27]. After CD34 selection, tumor burden was reduced by a median of 3.1 log, with 54% of CD34-selected products having no detectable tumor. In this study, there was no improvement in disease-free or overall survival. Short-term and long-term engraftment were equivalent in the two arms of the study. Immune resconstitution including lymphocyte subset recovery and immunoglobulin levels were identical in both arms at 1 year. There was no difference in infection rates at all time points. Although purging appears to have no significant toxicity, it is expensive and there are no definitive data that unpurged marrow contributes to relapse. The expense of purging would increase the cost of autologous HCT considerably for those patients who may not require purging. Although data do not prove that purging is essential, they are consistent with the interpretation that MRD in the marrow may contribute to relapse. Lastly, data suggest that even if purging is performed, it is likely to have benefit only if it is successful in eradicating residual tumor cells. Many studies performed to date have not analyzed whether the purging procedure performed has successfully eradicated detectable tumor. While purging procedures remain suboptimal and require specialized

skills, it has not been possible to perform adequate studies to resolve fully whether purging has any benefit. The availability of in vivo purging greatly decreases the complexity of assessing the impact of immunologic purging, and ongoing studies are assessing the impact of in vivo administration of mAbs before, at the time of, and following autologous HCT.

Summary In vitro and in vivo methods exist that are capable of eradicating minimal and overt disease from autologous hematopoietic cells. The evidence that such eradication of tumor cells results in improved disease-free or overall survival is circumspect at best. Techniques are now available to assess whether purging techniques have successfully eradicated tumor cells from the autologous hematopoietic cells from a variety of tumor types. It is important to continue to assess whether successful eradication of detectable tumor from the source of autologous HSC results in improved outcome. Ex vivo manipulation of human cells is now federally regulated in the United States, limiting the applicability of methodologies and devices used for purging clinical samples. Such procedures should be continued only in the clinical trial setting since it is in these circumstances that it will eventually be possible to determine the clinical impact of purging. The availability of mAbs for in vivo purging strategies has greatly increased interest in immunologic purging and the impact of eradication of detectable disease in autologous HCT.

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82. Smith BD, Jones RJ, Lee SM et al. Autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide purging for acute myeloid leukaemia beyond first remission: a 10-year experience. Br J Haematol 2002; 117: 907–13. 83. Morel P, Laporte JP, Noel MP et al. Autologous bone marrow transplantation as consolidation therapy may prolong remission in newly diagnosed high-risk follicular lymphoma: a pilot study of 34 cases. Leukemia 1995; 9: 576–82. 84. Fouillard L, Laporte JP, Labopin M et al. Autologous stem-cell transplantation for non-Hodgkin’s lymphomas: the role of graft purging and radiotherapy posttransplantation – results of a retrospective analysis on 120 patients autografted in a single institution. J Clin Oncol 1998; 16: 2803– 16. 85. Reiffers J, Labopin M, Sanz M et al. Autologous blood cell vs marrow transplantation for acute myeloid leukemia in complete remission: an EBMT retrospective analysis. Bone Marrow Transplant 2000; 25: 1115–19. 86. Chao NJ, Stein AS, Long GD et al. Busulfan/etoposide – initial experience with a new preparatory regimen for autologous bone marrow transplantation in patients with acute nonlymphoblastic leukemia. Blood 1993; 81: 319–23. 87. Linker CA, Ries CA, Damon LE, Rugo HS, Wolf JL. Autologous bone marrow transplantation for acute myeloid leukemia using busulfan plus etoposide as a preparative regimen. Blood 1993; 81: 311–18. 88. Linker CA, Ries CA, Damon LE, Rugo HS, Wolf JL. Autologous bone marrow transplantation for acute myeloid leukemia using 4-hydroperoxycyclophosphamide-purged bone marrow and the busulfan/etoposide preparative regimen: a followup report. Bone Marrow Transplant 1998; 22: 865–72. 89. Locatelli F, Labopin M, Ortega J et al. Factors influencing outcome and incidence of long-term complications in children who underwent autologous stem cell transplantation for acute myeloid leukemia in first complete remission. Blood 2003; 101: 1611–19. 90. Gorin NC, Aegerter P, Auvert B. Autologous bone marrow transplantation for acute leukemia in remission: an analysis of 1322 cases. Haematol Blood Transfus 1990; 33: 660–6. 91. Nieto Y, Jones RB, Shpall EJ. Stem-cell transplantation for the treatment of advanced solid tumors. Springer Semin Immunopathol 2004; 26: 31–56. 92. Freedman AS, Takvorian T, Neuberg D et al. Autologous bone marrow transplantation in poorprognosis intermediate-grade and high-grade Bcell non-Hodgkin’s lymphoma in first remission: a pilot study. J Clin Oncol 1993; 11: 931–6. 93. Freedman AS, Neuberg D, Gribben JG et al. High-dose chemoradiotherapy and anti-B-cell monoclonal antibody-purged autologous bone marrow transplantation in mantle-cell lymphoma: no evidence for long-term remission [see comments]. J Clin Oncol 1998; 16: 13–18. 94. Anderson KC, Andersen J, Soiffer R et al. Monoclonal antibody-purged bone marrow transplantation therapy for multiple myeloma. Blood 1993; 82: 2568–76. 95. Vescio R, Schiller G, Stewart AK et al. Multicenter phase III trial to evaluate CD34(+) selected versus unselected autologous peripheral blood

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43

Norbert Schmitz

Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation

Introduction The existence of hematopoietic cells (HCs) in the peripheral blood (PB) was postulated in 1909 by Alexander Maximow [1]. In 1962, Goodman and Hodgson were first to prove that circulating HCs were capable of restoring irradiation-induced marrow aplasia in mice [2]. The first clinical transplantation was published in 1980 by Abrams et al., who transfused large numbers of syngeneic blood leukocytes to a patient with Ewing’s sarcoma [3]. In 1985 and 1986, four different teams reported successful hematopoietic reconstitution by autologous blood-derived HCs in cancer patients [4–7]. Although these latter case reports provided the long-sought proof of principle, the low concentration of HCs in unmanipulated PB remained a major obstacle to the wider use of clinical transplants from PB, especially in the allogeneic setting where the exposure of healthy individuals to mobilizing cytotoxic drugs was not possible. Only with the advent of granulocyte-colony stimulating factor (GCSF) and the discovery that this and other cytokines were able to substantially increase the concentration of CD34+ cells in PB [8,9] did autologous and, 5–6 years later, allogeneic PBHC transplantation (PBHCT) begin to enter the clinic. With regard to PBHCT, in 1989 Kessinger et al. showed that T-cell-depleted blood HCs were able to restore hematopoiesis after myeloablative therapy [10]. Because of fears that the high numbers of T lymphocytes contained in PBHC harvests would induce severe graft-versus-host disease (GVHD) in most recipients, allogeneic PBHCT replete of T cells was first used in emergency situations such as the failure of a previous marrow graft [11] or the inability of the donor to undergo general anesthesia for marrow collection [12]. After pilot studies using unmanipulated PB from human leukocyte antigen (HLA)-identical sibling donors were published in 1995 [13–15], a surge of allogeneic PBHCTs ensued. Currently, about 70% of all allogeneic transplants in Europe and worldwide are performed with PB instead of marrow [16].

The donors When allogeneic PBHCT entered the clinical arena, the experience with healthy individuals donating HCs mobilized into the blood was also

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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limited. The major issues which had to be addressed were the following: 1. How could HCs be harvested most effectively? 2. Did harvest products from the PB contain stem cells in quantities and quality which would allow for timely, complete, and durable engraftment of allogeneic lymphohematopoiesis? 3. What were the acute side-effects and long-term sequelae of the mobilization and collection procedure? For further information regarding donors for HCT see also Chapter 38. Mobilization and collection of HCs from PB The first attempts to “mobilize” HCs into the PB of normal donors followed the experience gained with granulocyte transfusions [17], and with the mobilization of autologous HCs in patients with lymphoma and other diseases where the stem cell pool was not compromised by marrow infiltration or extensive cytotoxic therapy [18,19]. G-CSF at doses between 5 and 16 μg/kg/day was administered to the potential donor for 5–7 days. Such treatment allowed the collection of CD34+ cell numbers ranging from around 2 to more than 20 × 106/kg recipient body weight in most instances. Infusion of the harvest products into recipients with leukemia or lymphoma after myeloablative therapy resulted in reliable and surprisingly fast engraftment [13–15]. Tens of thousands of healthy individuals have now undergone mobilization and collection of PBHCs using a variety of harvest protocols [20–24]. A joint analysis from the International Bone Marrow Transplant Registry (IBMTR) and the European Group for Blood and Marrow Transplantation (EBMT) [25] summarized the experience with 1488 donations of PBHCs from HLA-identical siblings (n = 1322), other relatives (n = 149) or unrelated donors (n = 15). Nearly all donors had received G-CSF (filgrastim, lenograstim) for mobilization. Approximately 40% of the donors had undergone a single apheresis, 45% underwent two, 11% underwent three, and 5% had to undergo four or more leukaphereses to collect the number of HCs deemed necessary for engraftment by the investigator. The Spanish National Donor Registry reported that G-CSF was administered to 466 donors at a median dose of 10 (range 4–20) μg/kg/day for a median of 5 (range 4–8) days. The mean CD34+ cell dose collected was 6.9 (range 1.3–36) × 106/kg, with only 14 donors (2.9%) not achieving the minimum target number of 2 × 106 CD34+ cells/kg [26]. A more recent single-center study reported on 400 related and unrelated donors who had received 10 μg/kg/day subcutaneously of glycosylated or nonglycosylated G-CSF once daily for 4 consecutive days,

Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation

with apheresis commenced on day 5 [27]. The target cell number of 4 × 106 CD34+ cells/kg was reached after one (63%) or two aphereses in 81% of donors. Most other donors collected between 2 and 4 × 106 CD34+ cells/kg, while only 11 donors (<3%) collected less than 2 × 106 CD34+ cells/kg with a maximum of two aphereses. Based on these and similar data, G-CSF at a dose of 10 μg/kg given for 4 or 5 consecutive days is considered the standard regimen for mobilization of allogeneic HCs [28,29], although other G-CSF doses as low as 2 μg/kg or as high as 40 μg/kg (in the autologous setting [30]) given once or twice daily have been used for mobilization, with a clear trend towards higher CD34+ cell numbers being collected after administration of higher doses of growth factor. Commencing leukapheresis on day 4 or 5 with any commercially available continuous flow blood cell separator will allow the collection of adequate numbers of HCs in nearly all healthy individuals with one or two daily collections if two to four times the donor’s blood volume is processed. CD34+ concentrations in PB decrease when G-CSF is continued for 7 days and beyond. Therefore, adequate timing of aphereses and effective collection procedures are key to achieve optimum harvest products. Other variables such as age, sex, body weight or baseline CD34+ cell concentration in the blood of the donor have also been described to correlate with the stem cell yield [27], but these findings remain controversial [31,32], and none of these parameters will allow precise prediction of the stem cell yield. Recently, pegfilgrastim, a covalently bound conjugate of filgrastim and monomethoxypolyethylene glycol with a significantly longer elimination half-life than unconjugated G-CSF, at a single dose of 12 mg has been used to mobilize allogeneic PBHCs in healthy family and unrelated donors [33]. Although harvests in all 25 donors were successful, further experience with long-term follow-up of donors and recipients will be necessary before pegfilgrastim can generally be recommended. Growth factors other than G-CSF or its variants, mostly granulocyte– macrophage colony-stimulating factor in conjunction with G-CSF, have occasionally been used for mobilization in healthy individuals, without obvious advantages over the administration of G-CSF alone [34,35]. Interleukin-3 (IL-3) [36], stem cell factor [37], and other experimental cytokines like fms-related tyrosine kinase-3 (Flt3) ligand [38] have been shown to increase the CD34+ cell concentration in the PB of rodents, primates or humans, but have never been used clinically because of their side-effects, which would put healthy volunteers at unnecessary and potentially severe risk. AMD3100 is an inhibitor of chemokine (CXC) receptor-4/stromal cell-derived factor-1α (CXCR4/SDF-1α) binding that mobilizes CD34+ cells into PB. In normal volunteers, the administration of AMD3100 after 4–5 days of G-CSF resulted in a 3–3.5-fold increase of circulating CD34+ cells. This agent has been successfully used in patients with myeloma and lymphoma [39] but awaits further study in healthy donors. Specific clinical situations, such as T-cell depletion of the graft, nonmyeloablative preparatory regimens, or unrelated, mismatched and haploidentical transplants [40], will need high CD34+ cell numbers to safeguard against the increased risk of graft failure associated with any of these procedures. It is for these reasons that the search for the optimum mobilization and collection procedure is ongoing. Characteristics and dose of HCs from PB or marrow As the human hematopoietic stem cell still is elusive, the precise answer to the question of how many true hematopoietic stem cells are contained in a typical PB collection product awaits further study. It is, however, possible to describe the quality and quantity of committed and more immature HCs as they appear in mobilized PB, and to compare the findings with marrow-derived HCs. This comparison would indicate that

619

primitive and committed HCs are present in mobilized PB at least as frequently as in a typical bone marrow (BM) harvest. Using the CD34+ antigen, which can easily be detected by flow cytometry, as a surrogate marker for the human stem cell, pilot studies found that mobilized PB would contain two to five times more HCs than BM. This has been confirmed by the results of the large randomized trials [41–44]. What is the minimum CD34+ cell number to guarantee quick and durable engraftment? And should a certain number of CD34+ cells not be exceeded? A definite answer to both questions is not yet possible. However, it seems reasonable to recommend the collection of at least 2-4 × 106 CD34+ cells/kg recipient weight, because clinical experience indicates that the risk of graft failure is increased if less than this threshold number is transplanted. It is also difficult to tell if there should be an upper limit for CD34+ cell numbers and if higher numbers would be harmful if transferred to the recipient. Animal experiments [45] and clinical data indicate that the desirable number of HCs needed to ensure engraftment will depend on a number of interdependent parameters, the most important of which are the immunosuppressive potential of the conditioning regimen, the HLA disparity between donor and recipient, the number of T cells infused, and the regimen used for GVHD prophylaxis. For a “standard” transplant from an HLA-identical sibling donor after myeloablative conditioning, doses between 4 and 10 × 106 CD34+ cells/ kg may be optimal, while other situations, for example haploidentical transplantation, will need much higher numbers of CD34+ cells to secure engraftment. High CD34+ cell numbers have generally been recommended because a significant correlation with improved survival has been demonstrated [46,47]. It probably is less important if high CD34+ cell numbers come from BM or PB, although one study reported that rich marrow (containing >2.7 × 108/kg nucleated cells) might result in superior outcome compared with mobilized PB [48]. On the other hand, because PBHC collection products containing high CD34+ cell numbers also contain more T cells, it has been postulated not to use transplants from PB with very high CD34+ cell numbers. Przepiorka et al. evaluated risk factors for acute GVHD after allogeneic PBHCT and found a sharp increase of GVHD if more than 6.3–10.0 × 106 CD34+ cells/kg were transplanted [49]. It will be difficult, however, to confirm this finding with prospective data because such high numbers of marrow or PBHCs are harvested from a small fraction of donors only. A number of investigators have looked at immunophenotypically, genotypically or functionally characterized subtypes of HCs in more detail. Körbling et al. reported that G-CSF at a dose of 12 μg/kg/day increased the concentrations of PB CD34+ cells and more primitive subsets such as CD34+Thy1dim and CD34+Thy1dimCD38− cells by 16.3-, 24.2-, and 23.2-fold, respectively. The mean apheresis yield of CD34+Thy1dim and CD34+Thy1dimCD38− cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 × 104 and 27.2 × 104, respectively, indicating an additional “peripheralization” effect of G-CSF on primitive CD34+ cell subsets [50,51]. Other reports [52] confirmed these findings but also showed that the immunophenotypic profiles of HCs from blood or marrow differed in several respects (Table 43.1) (reviewed in [53]). The low expression of CD71 and the decreased retention of rhodamine-123 in CD34+ cells from mobilized PB suggest that these cells are not actively proliferating or metabolically active [54]. Kinetic data corroborate the phenotypic findings, further demonstrating that relatively more CD34+ cells from PB exhibit characteristics of quiescent cells [55]. Steidl et al. [56] reported on the molecular phenotype of BM-derived and circulating CD34+ cells from mobilized PB. Gene expression profiling confirmed that CD34+ cells from BM cycle more rapidly, whereas CD34+ cells from PB include more quiescent stem and progenitor cells.

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Chapter 43

Table 43.1 Characteristics of CD34+ hematopoietic cells (HCs) from bone marrow (BM) or mobilized peripheral blood (PB) Characteristics

BM

Mobilized PB

Phenotype CD13+, CD33+ CD38− HLA-DR− CD10+ CD19+ CD7+ c-kit VLA-4

Standard Standard Standard Standard Standard Standard Standard

Higher Higher Higher Less Less Less Less

Metabolic activity CD71+ Rhodamine

High High

Low Low retention

Normal Normal Active Standard Standard

Increased Increased Nonactive Less Less Increased Increased Increased

Clonogenicity Colony formation LTC-IC Cycling status Genotype cell cycle progression DNA synthesis GATA 2 N-myc Apoptotic activity LTC-IC, long-term culture-initiating cells.

Prosper et al. [57] investigated the number of long-term cultureinitiating cells (LTC-ICs) in the PB of normal donors treated with GCSF (7.5–10 μg/kg/day) and found that CD34+CD38+ cells and CD34+HLA-DR+ cells sorted from mobilized PB contained 0.5–5% of cells capable of sustaining hematopoiesis in long-term culture for up to 5 weeks. This is at least five times more than the number of LTC-ICs found in steady-state BM or nonmobilized PBHCs. However, 90–95% of the LTC-ICs present in CD34+CD38+ or CD34+HLA-DR+ fractions of mobilized PB were not able to sustain hematopoiesis for 8 weeks, as is the case with close to one-third of CD34+CD38+ and CD34+HLA-DR+ cells from nonmobilized PB. Taken together, phenotypic, genotypic, and functional analyses of HCs from BM or PB demonstrate that stem and progenitor cells are contained in both compartments. HCs from PB may be enriched in LTCICs and other clonogenic cells, they are metabolically less active, cycle less frequently, and show increased apoptotic activity. Stem cell biology including the differences of CD34+ cells from mobilized PB or BM remains an area of active research because a better understanding of basic phenotypes and function is paramount to optimize the peripheralization, harvesting, and clinical use of HCs.

T lymphocytes and other immune cells in mobilized blood There are many differences in the composition of PBHC harvests and marrow grafts if cells other than HCs are considered. Most importantly, PB harvests contain approximately 10 times more T cells than BM; the ratio of CD4+:CD8+ lymphocytes, however, is identical. This had already been noticed with autologous PBHC harvests [58], and was the most

important reason why allogeneic transplants with PB were accepted as an alternative to BM only after studies had demonstrated that the infusion of such high T-cell numbers was not regularly followed by the occurrence of severe GVHD. To some extent, the numbers of T cells contained in PBHC harvests relative to the number of CD34+ cells seem to depend on the mobilization protocol and the day of apheresis. Leukapheresis on day 5 after administration of G-CSF should give the highest stem cell yield with relatively low numbers of T cells contained in the final product [58]. The observation that the high number of T cells in PB harvests did not lead to a dramatic increase in the incidence and severity of GVHD in patients allografted with mobilized PB prompted researchers to investigate the effects of G-CSF administration on the number and function of immune cells in more detail. With regard to T cells, Pan et al. [59] were first to show that pretreatment of healthy individuals with G-CSF polarizes T cells towards the production of type 2 cytokines (IL-4 and IL-10), while type 1 cytokine production (interferon-gamma and IL-2) is reduced. Other effects capable of explaining the surprisingly low incidence of severe GVHD after transplantation of allogeneic PBHCs are downregulation of allogeneic immune responses by posttranscriptional inhibition of tumor necrosis factor-alpha production [60], and the comobilization of CD4+ and CD 8+ T cells. Kusnierz-Glaz et al. [61] demonstrated that CD4+CD8+ αβ T cells are markedly enriched in the leukapheresis products of G-CSF-treated normal donors, and that these cells, further enriched in low-density fractions of mobilized PB, significantly suppress the mixed lymphocyte reaction. Mielcarek et al. [62,63] calculated that the number of CD14+ cells in mobilized PB was 50-fold greater than in marrow. These large numbers of monocytes suppress T-cell proliferative responses to alloantigen by increased IL-10 production and decreased secretion of IL-12 and tumor necrosis factor-alpha. Arpinati et al. demonstrated that the number of T helper type 2 (Th2) cell-inducing dendritic cells (pre-DC2s) was increased in mobilized PB. As pre-DC2s lead to polarization of naïve CD4+ T cells toward a T helper type 2 phenotype, this finding also may explain why transplantation of allogeneic PBHC does not lead to more severe GVHD in a larger fraction of patients [64]. Recently, Franzke et al. [65] showed that T cells do express G-CSF receptors after G-CSF treatment, and G-CSF can thus directly modulate T-cell immune responses. Among other possible mechanisms, G-CSF seems to diminish T-cell receptor-mediated activation and proliferation, downregulate leukocyte function associated antigen-1 alpha (LFA-1α) thus affecting the homing process of the graft and abrogating donor Tcell activation to host antigens), and upregulate GATA-binding protein3 (GATA-3), the master regulatory factor for induction of a T helper immune response. Natural killer cells are five to 10 fold more frequent in mobilized PB than in BM. Joshi and colleagues analyzed peripheral blood mononuclear cells from G-CSF-mobilized blood cell harvests for their immunologic function and compared them with peripheral blood mononuclear cells from steady-state nonmobilized donors [66]. They found a significant decrease in NK- and lymphokine-activated killer cell-mediated cytotoxicity for G-CSF-mobilized effector cells. They also reported a significant decrease in both T-cell and B-cell mitogen responses with G-CSF-mobilized versus nonmobilized cells. Neutrophils not only increase in number after administration of GCSF, but their function also is enhanced immediately after G-CSF administration, as shown by upregulation of Fcγ RIII receptors, cell surface CD11b and CD66 molecules, and elevated levels of plasma elastase [67]. It is not clear which of the mechanisms described contributes to which extent to the striking observation that the infusion of 1 to 2 log more T cells into the recipient does not result in the development of severe GVHD in more patients grafted with PB.

Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation

Side-effects and long-term sequelae of mobilization and collection of HCs The overall short- and long-term effects of G-CSF have repeatedly been reviewed [68,69]. The acute toxicities of G-CSF administration have been reasonably well described, and comprise bone pain, headache, and fatigue as the most frequent side-effects. However, myalgias, chest pain, anxiety, insomnia, night sweats, fever, anorexia, weight gain, nausea and/or vomiting, and local reactions at the injection site have also been described [26,68,70]. Side-effects have been mild to moderate in the vast majority of donors and usually resolve within 48 hours of discontinuation of G-CSF. They can be treated successfully by the administration of minor analgesics (acetaminophen or ibuprofen) in most instances, and these side-effects have not been reported to require cessation of G-CSF administration or cancellation of the PBHC harvest. There is some indication that the frequency and severity of side-effects of G-CSF is dose dependent [71,72]. Therefore, doses as low as possible should be used. A number of side-effects of G-CSF administration and the harvest procedure on blood chemistry, the coagulation system, and baseline hematologic parameters already described in patients with hematologic malignancies also occur in normal individuals. Laboratory abnormalities caused by G-CSF include transient increases of lactate dehydrogenase and alkaline phosphatase, uric acid, alanine aminotransferase, and/or glutamyl transpeptidase, and a decrease in serum potassium and/or magnesium [21,29,73]. Several authors have shown that G-CSF can induce a mild hypercoagulable state, as indicated by in vitro testing. Increases in fibrinogen and factor VIII levels, a reduction in protein C and S levels [74], and increases in prothrombin fragments, thrombin–antithrombin complexes, D-dimers, and platelet aggregation have all been reported [75–77]. Mildto-moderate decrements in platelet, lymphocyte, and granulocyte levels have been described following the collection of PBHCs [17,78]. Thrombocytopenia may partly be due to the administration of G-CSF itself [79] or the leukapheresis procedure. Cytopenias are usually mild and selflimited, although more profound thrombocytopenia following repeated large-volume leukaphereses has been reported [80]. A number of studies have been presented which compare the experience of donors who participated in one of the randomized trials comparing BM transplantation (BMT) with PBHCT [81–84]. It was reassuring that none of these reports described life-threatening complications or long-lasting discomfort in any of the donors. The study reports generally confirm that the side-effects of PBHC harvesting are related mainly to G-CSF administration, while the side-effects of BM harvesting are caused by the harvest procedure itself. The incidence of adverse events occurring in 5% or more of donors of PB or BM who participated in the large European trial are presented in Table 43.2. Some of these studies also measured the emotional status and pain of donors prior to the harvest procedure and at defined time points after the collection of BM or PB. In general, the symptom burden, the levels of maximal and average pain, and anxiety were similar, although symptom peaks occurred at different time points during G-CSF administration (in PB donors) or shortly after BM harvesting. A quicker resolution of symptoms in PBHC donors was also described [82,83]. Only the Scandinavian trial reported a significant difference in the total burden of complaints in the two groups of donors, favoring the PB donors. This study also found that donors would prefer to donate PB rather then BM if asked to donate again [84]. This observation is in line with a report from unrelated donors [85], and a previous report in cancer patients who also preferred to donate autologous PB rather than BM [86]. Serious or lethal complications of G-CSF administration and leukapheresis are rare. At least five cases of splenic rupture associated with the harvest procedure have been reported [87–91]. Therefore, special

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Table 43.2 Subject incidence of adverse events occurring in >5% of donors. Date from the EBMT study comparing bone marrow transplantation (BMT) with peripheral blood progenitor cell transplantation (PBPCT) [44]

Any adverse event

BM donors (n = 166)

PBHC donors (n = 164)

95 (57%)

107 (65%)

Harvesting procedure-related adverse events Any harvest-related 91 (55%) Access pain 39 (23%) Anemia 17 (10%) Back pain 16 (10%) Nausea 10 (6%) Arthralgia 8 (5%) Vomiting 8 (5%) Skeletal pain 3 (2%)

61 2 0 4 1 1 0 10

(37%) (1%) (0%) (2%) (1%) (1%) (0%) (6%)

Filgrastim-related adverse events Any filgrastim-related Musculoskeletal Headache LDH increased Alkaline phosphatase increased

96 71 19 14 8

(59%) (43%) (12%) (9%) (5%)

BM, bone marrow; LDH, lactate dehydrogenase; PBHC, peripheral blood hematopoietic cell.

attention should be paid to donors presenting with an enlarged spleen or a history of splenic disorders. Routine abdominal ultrasound has been recommended to reduce splenic complications. Also, cases of severe cardiovascular events [28,92], acute lung injury [93], acute iritis [94], pyogenic infections [95], gouty arthritis [96], and anaphylactoid reactions [97] have been reported. Such case reports, as well as concerns that G-CSF might perturbate normal hematopoiesis and increase the risk for developing malignant disorders of the blood, including myelodysplasia and leukemia [98], caused investigators to propose an international donor registry more than 10 years ago [28]. The first report on severe donor events in a very large cohort of donors was compiled only recently, emphasizing that a global approach to document and evaluate the acute and long-term effects of stem cell collection in healthy donors is mandatory. The report by Halter et al. [91] summarizes all deaths and severe complications of PB or marrow donations for 51,024 recipients receiving a first HCT. Data were generated retrospectively from questionnaires sent out to the principal investigators of 338 transplant centers who participated in the annual EBMT activity surveys. Any serious event occurring within 30 days after donation and the occurrence of any hematologic malignancy in a donor had to be registered. Overall, five donor deaths occurred (one after BM and four after PB donation; incidence 0.98 per 10,000 donations) and 37 severe complications (12 in BM and 25 in PB donors; p < 0.02) were reported. Nineteen hematologic malignancies (eight after BM and 11 after PB donation; p = 0.4) were reported. Three deaths (pulmonary embolism, subarachnoidal hematoma, and cardiac arrest) were clearly related to the donation, whereas two cardiac arrests on days 15 and 17 after PB donation may or may not have been related to the harvest procedure. The severe, potentially lifethreatening complications mostly were cardiovascular events, hemorrhages, and splenic rupture. The hematologic malignancies reported were acute myeloid leukemia (three), acute lymphoblastic leukemia (three), lymphoma (seven), nasopharyngeal plasmacytoma, chronic

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lymphoblastic leukemia, and myeloproliferative syndrome (one case each), and other nonspecified disorders. In Chapter 38, Confer et al. summarize data available on nine deaths which occurred worldwide after donation of BM or PB. It is unclear how much overlap might exist between their cases and those reported by Halter et al. Also, it remains uncertain what number of donations formed the basis to Confer’s report. In any case, these data, together with unpublished reports from other registries, suggest that life-threatening and fatal accidents are rare but do occur in donors of PB or marrow stem cells. Therefore, the World Marrow Donor Association recently created a Serious Events and Adverse Effects Registry to collect and evaluate information on serious donor events. The leukapheresis itself is generally well tolerated, but side-effects caused by the anticoagulant and placement of central venous access have been reported. Although any effort should be made to avoid a central venous access, 2–7% of donors ultimately will need it because peripheral veins cannot be used or the necessary blood flow cannot be established [13–15]. Rarely, donors have experienced tetany or severe symptoms of hypocalcemia caused by the anticoagulant used throughout the leukapheresis procedure [81].

The recipients In 1995, pilot studies from the United States and Germany [13–15] indicated that transplantation of allogeneic PBHCs resulted in fast and stable engraftment; GVHD did not seem to occur more frequently than after marrow grafting. Larger series from other transplant centers or cooperative groups [99,100], as well as retrospective comparisons with BMT [101,102], confirmed that transplantation of allogeneic mobilized PB seemed a viable alternative to marrow transplantation. The largest retrospective analysis presented by IBMTR and EBMT compared results of 288 PBHCTs with 536 BMT procedures from HLAidentical sibling donors [103]. The paper confirmed that recipients of PBHCs attained more rapid recovery of neutrophils and platelets; significant differences in relapse rates and the incidence of grade II–IV acute GVHD between marrow and PBHCT were not observed. The incidence of chronic GVHD at 1 year, however, was significantly higher after PBHCT (65%) than after BMT (53%). Treatment-related mortality was lower, and leukemia-free survival rates were higher with PBHCT in patients with advanced leukemias, but were comparable in standardrisk leukemias. The randomized trials Between 1998 and 2002, the results of eight prospective randomized trials comparing the transplantation of allogeneic PBHCs with that of BM were published [41–44,104–107]. These studies were almost exclusively done in patients with leukemia, patients with other hematologic malignancies were included only rarely, and nonmalignant disorders were not represented at all. The donors were HLA-identical siblings, and BM or PBHCs were infused without any manipulation. The trials differed substantially with respect to the number of patients included, the status of the diseases, the conditioning regimes used, the doses of HCs and T cells infused, the GVHD prophylaxis administered, and a number of other variables that may have influenced outcome. Still, the results of these trials allow a number of important conclusions, which are summarized below. Engraftment Transplantation of allogeneic PBHCs results in faster engraftment of HCs of all lineages compared with BMT. Neutrophils exceed a threshold of 0.5 × 109cells/L between 2 and 6 days earlier with PBHC than after

BMT. In the United States trial, the neutrophils surpassed 0.5 × 109/L on day 16 (range 11–29 days) with PB, and on day 21 (range 13–36 days) with BM (p < 0.001) [41]. The Canadian study reported neutrophil recovery on day 19 (range 12–35 days) with PB, and on day 23 (range 13–68 days) with BM [43]. Patients in the EBMT study had the shortest time interval from transplantation to an absolute neutrophil count of greater than 0.5 × 109/L: 12 days for PB and 15 days for BM [44]. However, this trial was the only one of the large randomized studies that used G-CSF (5 μg/kg/day) also after transplantation, and day 11 methotrexate was not included. Two other randomized trials proved that the administration of G-CSF at a dose of 10 μg/kg/day after PBHCT can accelerate neutrophil recovery by 3–4 days [108,109]. Platelet recovery after allogeneic PBHCT is also faster by approximately 6 days: a platelet count of 20 × 109/L was reached on day 13 (range 5–41 days) with PB, and on day 19 (range 7–74 days) with BM in the United States study (p < 0.001). The Canadian study reported platelet recovery after 16 (range 0–100) days for patients grafted with PB, and 22 (range 0–100) days for patients grafted with BM (p < 0.0001). The EBMT study had a median time to platelet recovery greater than 20 × 109/L of 15 days for patients transplanted with PB, and 20 days for patients transplanted with BM (p < 0.0001). This difference translated into a median of 8 (range 1–68) days platelet transfusions in the PBHCT group, and a median of 10 (range 2–71) days in the BMT group (p = 0.0029). It is well recognized that factors other than the source of HCs influence the pace of hematologic recovery. While G-CSF accelerates neutrophil recovery, methotrexate given for prophylaxis of GVHD retards hematopoietic reconstitution [110]. The occurrence of GVHD, infection, the grade of genetic disparity between recipient and donor, and splenomegaly have all been reported to affect the speed of hematopoietic recovery, and may explain the slight variations in times to neutrophil and platelet recovery seen in the randomized trials. Graft failures after PBHCT are not more frequent than after BMT. Comparative studies of hematopoietic chimerism after PBHCT and BMT support the notion that transplantation of PB results in fast, complete, and durable complete chimerism [111]. Immune recovery Recovery of immunity after unmanipulated allogeneic PBHCT is at least as quick as after BMT, but may be faster with respect to some subgroups of cells with immune functions. Circulating naïve (CD4+CD45RA+) and memory (CD4+CD45RO+) helper T cells, B cells, and monocytes are found in higher numbers after PBHCT compared with BMT between 1 and 11 months post transplant [112]. Natural killer cell numbers did not significantly differ between patients grafted with PB or marrow over the same time period. Proliferative responses to T- and B-cell mitogens (phytohemagglutinin, pokeweed mitogen, tetanus toxoid, and Candida) were significantly increased in recipients of PB from HLA-identical donors [112]. Storek et al. [113] studied 115 patients who had been randomly assigned to receive allogeneic marrow or mobilized PB from HLAidentical donors. Between 1 month and one year after transplantation, the counts of CD4+CD45RAhigh and CD4+CD45RAlow/− T cells were significantly higher after PBHCT. T cells appeared equally functional when challenged by phytohemagglutinin or herpesvirus antigen. Median serum immunoglobulin G levels were similar in both groups of patients. Of note, the authors also reported that particularly severe infections were significantly reduced after PBHCT. The reduced risk was most prominent for fungal infections, intermediate for bacterial infections, but low for viral infections. This latter phenomenon may be a consequence of the marked loss of memory T cells which occurs after allogeneic PBHCT [114].

Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation

Another interesting observation published by Lapierre et al. [115] was that, 30 days after PBHCT, anti-A and/or anti-B blood group titers were significantly higher after PBHCT compared with BMT, suggesting that immunohematologic reconstitution after PBHCT is quicker than after BMT. This observation could explain the cases of acute hemolysis described after ABO-incompatible PBHCT [116,117]. On the other hand, transfusion requirements are no different in ABO-, rhesus- or Kell-different transplantation; however, ABO-mismatched patients were reported to show poorer survival than matched patients after PBHCT [118]. Acute GVHD The incidences of acute GVHD grade II–IV were similar for both groups of patients in all larger studies except for the largest one [44], which noted a 13% increase of grade II–IV acute GVHD (p = 0.013) and a 12% increase of grade III–IV acute GVHD with PB (p = 0.0088) (Table 43.3). The EBMT study also was the only study where the maximum grade of acute GVHD was the primary endpoint, and randomization was stratified for some of the known risk factors of GVHD. Other differences in the design of the EBMT study and the other trials were the inclusion of good-risk patients only, the omission of methotrexate on day 11, and the use of G-CSF post transplant. Omission of day 11 methotrexate has been reported to predispose recipients to develop GVHD [110], although this finding was not supported by data from the international metaanalysis [119]. It also remains in question if the high numbers of T cells transferred with a typical PBHC graft are responsible for the higher incidence of acute GVHD. Although such a correlation between GVHD and T-cell numbers was detected in the EBMT study, another study failed to demonstrate such a correlation [120].

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Chronic GVHD The large randomized studies all showed at least a trend to more chronic GVHD with PBHCs, and two studies reported statistically significantly more chronic GVHD after PBHCT [42,44] (Table 43.4). There was also more extensive chronic GVHD in patients having received PBHCs compared with BM cells. This difference was significant in the EBMT trial and in the studies chaired by Blaise et al. [42] and Vigorito et al. [107]. Follow-up reports from the United States trial [121], the French trial [122], and the EBMT trial [123] have been published with a median follow-up of 41, 45, and 36 months, respectively. Flowers et al. still did not observe more chronic GVHD after PBHCT, and the clinical characteristics of chronic GVHD were similar after PBHCT or BMT [121]. However, the updated study showed that chronic GVHD was more difficult to control when it occurred after PBHCT compared with BMT: the number of successive treatments needed to control chronic GVHD was higher after PBHCT, and the duration of glucocorticoid treatment was also longer (p = 0.03 for both endpoints). In contrast, Mohty et al. found a highly significant difference in the 3-year cumulative incidence of chronic GVHD in the PBHC group compared with the BM group (65% versus 36%; p = 0.004) [122]. They also observed significantly more extensive chronic GVHD. Chronic GVHD needed further immunosuppression and led to longer periods of hospitalization. No survival difference was observed thus far. The 3-year follow up of the prospective EBMT trial (Table 46.4) and the 5-year follow up of the retrospective IBMTR/EBMT study also showed more chronic GVHD in patients after PBHCT [124]. Overall, there is evidence that PBHCT causes more chronic GVHD or at least chronic GVHD which is more difficult to treat. The picture is not yet complete, however, and further updates from the large randomized trials are eagerly awaited. Relapse rates

Table 43.3 Acute graft-versus-host disease (GVHD) grades II–IV Reference

PB (%)

BM (%)

p-value

Bensinger et al. [41] Blaise et al. [42] Couban et al. [43] Heldal et al. [104] Powles et al. [106] Schmitz et al. [44] Vigorito et al. [107]

64 44 44 21 50 52 27

57 42 44 10 47 39 19

p = 0.35 Not given p > 0.9 Not given Not given p = 0.013 p = 0.53

All percentages represent cumulative incidences at day +100 post-transplant except for the studies by Blaise et al. [42], Heldal et al. [104] and Powles et al. [106] where the actual percentage of patients with acute GVHD at day +100 is given.

Knowing that the graft-versus-leukemia effect is exerted mainly by donor T cells and that around 10 times more T cells are transferred with a typical PB collection product, it was anticipated that transplantation of PB cells would result in a reduction of relapse rates after PBHCT. Animal studies [125] and results of nonrandomized studies [126] also pointed in that direction. The randomized studies have not consistently confirmed these expectations so far, although the United States study [41] and the small British study [106] indeed reported significantly fewer patients with relapse after PBHCT than after BMT. Transplant-related mortality Retrospective analyses had suggested a trend in favor of lower transplant-related mortality (TRM) after PBHCT. Specifically, the EBMT/ IBMTR study showed a significant advantage in TRM for patients trans-

Table 43.4 Chronic graft-versus host disease (GVHD) First report

Follow-up report at 3 years

References

PB

BM

p-value

Reference

PB

BM

p-value

Bensinger et al. [41] Blaise et al. [42] Couban et al. [43] Schmitz et al. [44]

(46%) (50%) 85% (40%) (67%)

(35%) (28%) 69% (30%) (54%)

0.54 <0.03 0.62 (0.37) 0.0066

Flowers et al. [121] Mohty et al. [122]

(63%) 65%

(52%) 36%

0.33 0.004

Schmitz et al. [123]

73% (36%)

55% (19%)

0.003

% overall chronic GVHD; (% in brackets), extensive chronic GVHD. BM, bone marrow; PB, peripheral blood.

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planted with PB for advanced leukemias [103]. The randomized United States study reported a cumulative incidence of transplantation-related deaths of 21% in the PB group and 30% in the BM group; this difference, however, was not significant [41]. The Canadian study also found a reduction in nonrelapse deaths after PBHCT as opposed to BMT. At day 100 after transplantation, the actuarial probabilities of death were 7.4% for the PB group and 16.1% for the BM group, respectively (p = 0.07) [43]. Until now, significant differences have not been reported from any of the randomized studies, and it remains to be determined if subgroups of patients with specific diseases or disease status benefit from the transplantation of PB because TRM is reduced. Survival Survival is the most relevant endpoint of studies comparing BMT with PBHCT. This is particularly true because different sources of HCs may have different effects on secondary endpoints such as TRM, relapse rate or GVHD, and the interplay of these may give different results for overall survival if different clinical situations are considered. The three largest randomized studies came to different conclusions with respect to survival. The EBMT study had overlapping survival curves for patients grafted with PB or BM [44]. The United States study reported a difference in overall survival of 12% at 2 years between the two groups of patients, favoring PBHCT; this difference was borderline significant (p = 0.06) [41]. The subgroup of patients with more advanced disease had a statistically significantly better survival (57%) with PB than with BM (33%) (p = 0.04). The Canadian study reported better overall survival for the whole group of patients transplanted with PB (68%) compared with the BMT group (60%) at 30 months (p = 0.04) [102]. As in the United States study, the survival benefit mainly rested with the advanced-disease patients, while patients with early disease did not show significant differences. In a subgroup analysis, survival was significantly better for patients with chronic myelogenous leukemia (CML), and a trend in this direction was observed for patients with myelodysplastic syndrome (MDS), while patients with AML had comparable survival with either source of HCs.

Marrow or mobilized blood: the meta-analysis An individual-patient data meta-analysis using data from nine randomized trials enrolling 1111 adult patients has recently been published [127]. The study confirmed that PBHCT results in faster neutrophil and platelet engraftment than BMT. PBHCT was shown to cause more grade III–IV acute GVHD, and more overall and extensive chronic GVHD (68% versus 52% at 3 years; p < 0.000001) compared with BMT (Fig. 43.1). PBHCT decreased relapse rates in both late-stage (33% versus 51% at 3 years; p = 0.02) and early-stage (16% versus 20% at 3 years; p = 0.04) disease. Nonrelapse mortality was no different between PBHCT and BMT. Overall survival and disease-free survival were only significantly improved in patients with late stage disease (overall survival 46% versus 31% at 3 years; p = 0.01). The major results of the meta-analysis are depicted in Fig. 43.2. The study also made clear that the vast majority of data (75%) comparing PBHCT with BMT were generated in patients with early disease (CML in chronic phase, AML, and acute lymphoblastic leukemia in first complete remission, and early MDS) and that CML – a diagnosis hardly treated by allogeneic transplantation in first chronic phase nowadays – was the most frequent diagnosis (approximately 40% of all patients treated). Mobilized BM for allogeneic transplantation Several investigators have used G-CSF-stimulated BM for hematopoietic reconstitution after myeloablative therapy, and compared engraftment and the development of GVHD with patients who were rescued with G-CSF-mobilized PB [128]. A small prospective study (57 patients being randomized) comparing G-CSF-primed BM with G-CSFmobilized PBHCs concluded that engraftment after transplantation of either source of HCs was comparable; however, more steroid-refractory acute GVHD, chronic GVHD, and prolonged requirement for immunosuppressive therapy was found after PBHCT [129]. Survival did not significantly differ. Larger randomized studies would be needed to clearly define the role of G-CSF-stimulated BM compared with G-CSFmobilized blood.

Fig. 43.1 (a) Time-to-event plots showing the absolute risk for development of extensive stage of chronic graft-versus-host disease (GVHD) in patients with hematologic malignancies. (b) Time-to-event plots showing the absolute risk for development of any stage of chronic GVHD in patients with hematologic malignancies. Abs diff, absolute difference; BMT, bone marrow transplantation; PBSCT, peripheral blood stem cell transplantation. (Reproduced from [127], with permission.)

Peripheral Blood Hematopoietic Cells for Allogeneic Transplantation

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Fig. 43.2 Summary forest plot showing the effects of allogeneic peripheral blood stem (hematopoietic) cell transplantation (PBSCT) versus bone marrow transplantation (BMT). If the square is to the left of the solid line, the odds ratio (OR) is better in the group receiving peripheral blood hematopoietic cells, but if the confidence interval (CI) crosses this line, this result is not statistically significant (p < 0.05). aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; O – E, observed minus expected; Redn., reduction; SD, standard deviation; Var., variance. (Reproduced from [127], with permission.)

Allogeneic PBHCT in diseases other than leukemia

Allogeneic PBHCT in children and adolescents

The prospective studies comparing BMT with PBHCT, as well as most retrospective analyses, included patients with leukemia and a few with lymphoma or myeloma. It is therefore largely unknown how patients with diseases other than leukemia would fare after either procedure. Some years ago, EBMT published their experience with BMT or PBHCT in patients with MDSs [130]. The results of this retrospective study were similar to the large retrospective analysis of patients with acute leukemias and CML published by the IBMTR and EBMT: earlier hematopoietic recovery, and similar incidences of acute, but more chronic GVHD with PB. The 2-year TRM was significantly reduced with PB except for patients who had refractory anemia or high-risk cytogenetics. Treatment failure was significantly decreased with PB. Estimates of the 2-year event-free survival were 50% with PBHC versus 39% with BM. Gahrton et al. compared PBHCT with BMT in patients with multiple myeloma [131]. They did not find significant differences between BMT and PBHCT for TRM, acute and chronic GVHD, progression-free survival or relapse rates; overall survival tended to be better in the BMT group (53% versus 44% at 3 years; p = 0.05). A recent EBMT study compared outcomes of 692 patients with severe aplastic anemia grafted with either mobilized blood or marrow from HLA-matched sibling donors. Hematopoietic recovery and grade II–IV acute GVHD were similar after PBHCT and BMT. In patients aged over 20 years, chronic GVHD and overall mortality rates were similar after PBHCT and BMT. In patients aged 20 or less, however, rates of chronic GVHD and overall mortality were higher after transplantation of PBPC than after BMT. In younger patients, the 5-year probabilities of overall survival were 73% and 85% after PBHCT and BMT, respectively. Corresponding probabilities for older patients were 52% and 64% [132]. These data indicate that BM grafts should be preferred to PBHCT in young patients with severe aplastic anemia, and that results of PBHCT versus BMT may vary according to the underlying disease. No comparisons of BMT with PBHCT are available so far for patients with lymphoma.

A study from the IBMTR cautioned against the use of PBHCT in children and adolescents [133]. One hundred forty-three PBHCT and 630 BMT procedures from HLA-identical sibling donors in patients aged 8–20 years with acute leukemia were compared. Risks of grade II–IV acute GVHD were similar; however, the incidence of chronic GVHD was higher after PBHCT. The cumulative incidences of chronic GVHD at 3 years were 33% and 19% after PBHCT and BMT (p = 0.001). In contrast to reports in adults, rates of TRM, treatment failure, and mortality were also higher after PBHCT. The 3-year probability of overall survival, adjusted for other significant factors, was 48% and 58% after PBHCT and BMT, respectively (p = 0.01). These data serve as a cautionary note before a major change in the source of HCs used for allogeneic transplantation of children comparable to the situation in adults will occur. Cord blood is an attractive alternative, particularly for children, and further studies comparing cord blood transplantation, BMT, and PBHCT would be helpful to better define the role of either source of HCs. T-cell-depleted mobilized blood Because of concerns that the large numbers of T cells contained in mobilized PB would cause more frequent and severe GVHD, the interest in T-cell-depletion strategies was high from the beginning. Soon after the first transplants with mobilized PB had been reported, a number of studies using T-cell-depleted mobilized PB were initiated [134,135]. Early results, however, were disappointing because – among other causes – depletion methods were far from optimal. With T cell numbers in excess of 1 × 105 T cells/kg recipient body weight remaining in the graft, it was not too surprising that acute and severe GVHD still occurred with incidences comparable to those observed after transplantation of unmanipulated PBHCs. With further experience, technical problems were solved, and it became possible to routinely purge PB collection products to a residual T-cell

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content of below 1 × 104 T cells/kg. Such minimal T-cell load is not sufficient to elicit clinical GVHD even in the haploidentical setting [40]. One of the major problems of T-cell depletion at the time when BM was exclusively used as the source of HCs was the increase in graft failures occurring after transplantation. Because CD34+ cell numbers are much higher in mobilized PB, and the eventual loss of HCs caused by the depletion process can be compensated for by further leukaphereses, graft failure is less of a problem after the transplantation of T-depleted mobilized PB. Urbano-Ispizua et al. reported that the graft failure rate was still 18% after T-cell-depleted PBHCT if fewer than 2 × 105 CD3+ T cells remained in the graft, while the incidence was very low (1%) if more than 2 × 105 T cells were infused [136]. It should be noted, however, that in this study relatively low numbers of CD 34+ cells (3.4 × 106/kg) had been infused into patients who subsequently suffered graft failure. Other studies suggest that the transplantation of very high numbers of CD34+ cells can overcome the graft failure risk in the HLA-identical [137] and the mismatched setting [40] regardless of the residual number of T-cells in the graft. Transplantation of CD34+-selected cells from mobilized PB to patients with early myeloid malignancies was reported to result in less GVHD, less TRM, and better disease-free survival if compared with unmanipulated PBHCT [138]. The randomized study published by Cornelissen et al. [139] made clear that the results of CD34 selection of PB or marrow are largely dependent on the efficacy of the T-cell-depletion strategy. When relatively high numbers of CD3+ T cells (median 3 × 105/kg) are transplanted after CD34+ selection of PB, the incidence of acute GVHD remains high (52% grade II–IV), and TRM and survival are negatively influenced. In summary, the availability of HCs from PB is important in the context of T-cell depletion strategies, because GVHD can virtually be eliminated by vigorous depletion strategies without a dramatic increase of graft failure rates when high to very high numbers of HCs are transferred. T cells, however, are mediators of the graft-versus-leukemia effect and contribute to quick immune reconstitution after transplantation. For these reasons, transplantation of T-depleted mobilized blood has largely been restricted to cases of haploidentical transplantation where high numbers of CD34+ cells are needed [140]. Unrelated donor transplants In contrast to the HLA-identical setting, no randomized trials comparing allogeneic PBHCT with BMT in unrelated donors have been published.

However, most donor registries have become more open to the idea of having their donors donate mobilized PB instead of BM, and activities in this direction continue (see Chapter 38). For the time being, the available information on the outcome of transplants with mobilized PB in unrelated donors is limited. The largest study so far was published in 1999 by Ringdén et al. [141]. In a retrospective comparison with BMT, they reported faster engraftment with PB; acute GVHD, 1-year TRM, and overall survival did not significantly differ after PBHCT or BMT. The study had only 45 patients in each arm, and 18 more patients had received CD34+-selected PB, so observations must be rated as preliminary, and no definite conclusions can yet be drawn.

Conclusion and future developments A clear-cut picture of the advantages and disadvantages of G-CSF administration and collection of HCs from PB has evolved over recent years. Long-term effects of PBHC and marrow harvesting do occur and make close follow-up of donors unavoidable. This is also necessary because the demand for HCs will steadily increase, and older individuals as well as donors with comorbidities will increasingly be involved. Many potential donors and medical community members currently prefer to work with HCs from PB. This is a consequence of the results of the clinical trials available so far, but is also dictated by recent developments in other areas of blood and marrow transplantation, including nonmyeloablative conditioning, exhaustive T-cell depletion, and transplantation across major HLA barriers, all of which require high to extremely high numbers of HCs that usually cannot be harvested from BM. In the near future, patients will likely receive cocktails of HCs plus an array of other cells with distinct immune functions tailored to each patient’s needs. Probably, all of these cells will be able to be harvested or grown from the PB. One of the most exciting questions today is whether, with our increased understanding of basic immunology, it will be possible to dissect GVHD from the graft-versus-leukemia effect, both exerted by donor T cells. Future work will also show if blood can act as a source of truly pluripotent stem cells that can be used in the treatment of nonhematopoietic diseases, including cardiac failure, Parkinson’s disease, and other degenerative disorders.

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its reactivation. Clin Immunol 2006; 119: 261– 71. Lapierre V, Oubouzar N, Aupéring A et al. Influence of the hematopoietic stem cell source on early immunohematologic reconstitution after allogeneic transplantation. Blood 2001; 97: 2580– 6. Oziel-Taieb S, Faucher-Barbey C, Chabannon C et al. Early and fatal immune haemolysis after so-called “minor” ABO-incompatible peripheral blood stem cell allotransplantation. Bone Marrow Transplant 1997; 19: 1155–6. Salmon JP, Michaux S, Hermanne JP et al. Delayed massive immune hemolysis mediated by minor ABO incompatibility after allogeneic peripheral blood progenitor cell transplantation. Transfusion 1999; 39: 824–7. Erker CG, Steins MB, Fischer RJ et al. The influence of blood group differences in allogeneic hematopoietic peripheral blood progenitor cell transplantation. Transfusion 2005; 45: 1382–90. Bensinger W; Stem Cell Trialists’ Collaborative Group. Individual patient data meta-analysis of allogeneic peripheral blood stem cell transplant vs bone marrow transplant in the management of hematological malignancies: indirect assessment of the effect of day 11 methotrexate administration. Bone Marrow Transplant 2006; 38: 539– 46. Cao TM, Wong RM, Sheehan K et al. CD34, CD4, and CD8 cell doses do not influence engraftment, graft-versus-host disease, or survival following myeloablative human leukocyte antigen-identical peripheral blood allografting for hematologic malignancies. Exp Hematol 2005; 33: 279–85. Flowers ME, Parker PM, Johnston LJ et al. Comparison of chronic graft-versus-host disease after transplantation of peripheral blood stem cells versus bone marrow in allogeneic recipients: long-term follow-up of a randomized trial. Blood 2002; 100: 415–19. Mohty M, Kuentz M, Michallet M et al. Chronic graft-versus-host disease after allogeneic blood stem cell transplantation: long-term results of a randomized study. Blood 2002; 100: 3128–34. Schmitz N, Beksac M, Bacigalupo A et al. Filgrastim-mobilized peripheral blood progenitor cells versus bone marrow transplantation for treating leukemia: 3-year results from the EBMT randomized trial. Haematologica 2005; 90: 643–8. Schmitz N, Eapen M, Horowitz MM et al. Longterm outcome of patients given transplants of mobilized blood or bone marrow: a report from the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation. Blood 2006; 108: 4288–90. Glass B, Uharek L, Zeis M et al. Allogeneic peripheral blood progenitor cell transplantation in a murine model: evidence for an improved graftversus-leukemia effect. Blood 1997; 90: 1694– 700. Elmaagacli AH, Beelen DW, Opalka B et al. The risk of residual molecular and cytogenetic disease in patients with Philadelphia-chromosome positive first chronic phase chronic myelogenous leukemia is reduced after transplantation of allogeneic peripheral blood stem cells compared with bone marrow. Blood 1999; 94: 384–9. Stem Cell Trialists’ Collaborative Group. Allogeneic peripheral blood stem-cell compared with

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from peripheral blood in patients with myeloid malignancies in early phase: a case control comparison with unmodified peripheral blood transplantation. Bone Marrow Transplant 2001; 28: 349–54. 139. Cornelissen JJ, van der Holt B, Petersen EJ et al. A randomized multicenter comparison of

CD34(+)-selected progenitor cells from blood vs from bone marrow in recipients of HLAidentical allogeneic transplants for hematological malignancies. Exp Hematol 2003; 31: 855– 64. 140. Aversa F, Terenzi A, Tabilio A et al. Full haplotype-mismatched hematopoietic stem-cell trans-

plantation: a phase II study in patients with acute leukemia at high risk of relapse. J Clin Oncol 2005; 23: 3447–54. 141. Ringdén O, Remberger M, Runde V et al. Peripheral blood stem cell transplantation from unrelated donors: a comparison with marrow transplantation. Blood 1999; 94: 455–64.

44

Scott D. Rowley

Cryopreservation of Hematopoietic Cells

Introduction Storage of hematopoietic cells (HCs) is required for transplantation, whether for the brief period required to deliver a freshly harvested bone marrow (BM) or peripheral blood HC (PBHC) product to an allogeneic recipient, or for the long-term (potentially decades) banking of cord blood HCs (CBHCs) intended for unrelated-donor transplantation. Room temperature or refrigerated (4º C) storage may be appropriate for products stored for a few hours to a few days. Cryopreservation allows longer-term storage and is the storage technique for virtually all products intended for autologous HC transplantation (HCT). Although cryopreservation of HCs is not a requirement for treatment with myeloablative, dose-intensive conditioning regimens, and patients have recovered marrow function after reinfusion of HCs stored for several days at 4º C, a progressive loss of HCs occurs during nonfrozen storage. The definition of ideal conditions (e.g. temperature of storage, initial processing of the cells, cell concentration, and additives) for the nonfrozen storage of cells may slow this loss. In contrast, although some unavoidable loss of HCs occurs with marrow or PBHC processing and cryopreservation, progressive loss over months to years of proper storage is not obvious and may not occur if optimal storage conditions are maintained. Cryopreservation allows the administration of multiple-day transplant conditioning regimens as well as the elective storage of cells for patients to be transplanted at a subsequent point in a course of treatment. That HCs can be successfully cryopreserved is evident from the successes of autologous and allogeneic HCT in regenerating marrow function after marrow-lethal conditioning regimens. Engraftment failure is generally not attributed to HC cryopreservation. Although some investigators have correlated poor HC cryopreservation with delayed engraftment after transplantation [1,2], this deleterious effect is most likely to be observed with products containing borderline quantities of HCs. Most cryopreservation laboratories use a variation of the technique outlined in Table 44.1, including differences in cryoprotectant concentration, product volume, and storage temperature. Cryopreservation research, however, has been a focus of few transplantation teams, and the biologic consequences of HC cryopreservation is one of the least understood aspects of HC processing. This lack of understanding of the effects of cell freezing on the cellular structure and the function of the cell popu-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

lations required for hematologic and immunologic reconstitution of the patient hinders the ability to adapt techniques to handle modified cell collections such as cells expanded in vitro, or to assess the consequences of accidental or intentional deviations from laboratory protocol. Although easily performed, HC cryopreservation and reinfusion are not without risk of toxicity to both the HC inoculum and the recipient of these cells. There are multiple aspects to successful cryopreservation of HCs. Each of these variables affects the survival of the cells and, as with any manufacturing process, must be rigidly controlled for reproducible results. Nonfrozen storage of HC products Cryopreservation is not required for short-term storage of HCs. Nonfrozen storage is a less-costly alternative, especially for the immediate transportation of products and for the transplantation of patients conditioned with brief courses of chemotherapeutic agents with short in vivo half-lives. Although most products are infused or processed within a few hours of collection, products collected for unrelated donor HCT may be shipped over long distances, requiring, sometimes, over 24 hours of transit time. Similarly, products intended for autologous HCT may be transported to regional processing centers, again requiring 24–48 hours of transit time before processing [3,4]. The major advantages of cryopreservation are the lack of progressive loss of HCs over time and the greater flexibility in the timing of HCT relative to the collection of HCs, including the ability to modify or postpone a transplant conditioning regimen already started. A progressive loss of HCs occurs during nonfrozen storage. One author reported a 61% loss of myeloid colony-forming progenitor cells (colony-forming units granulocyte–macrophage [CFU-GMs]) from marrow after 72 hours of storage at 4º C [5]. Yet another found only a 3% loss of CFU-GMs from BM stored for 96 hours, but a 95% loss if the source of the cells was peripheral blood [6]. As part of a clinical trial described below, Preti et al. compared the survival of mature hematopoietic progenitor cells isolated from marrow during frozen or nonfrozen storage, and reported an immediate loss of myeloid (CFU-GM) progenitors of 33% during cryopreservation and thawing [7]. In contrast, cells stored at 4º C showed a progressive, linear loss of total nucleated cells, cell viability, and HCs cloned in vitro. The quantity of erythroid colony-forming progenitor cells (burst-forming units erythroid [BFUEs]) in these nonfrozen samples became significantly less than that of cryopreserved samples only after 5 days of storage. The difference for myeloid progenitor cells (CFU-GMs) was not yet significant even after 9 days of storage.

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Table 44.1 Hematopoietic cell cryopreservation: basic technical considerations Item

Principle

Example

Prefreeze processing Cryoprotectant

Reduction of mature blood cells that are poorly cryopreserved Protection of the cells from ice formation and dehydration. May be “colligative” or “polymeric” or a combination of both Reduces cryopreservation injury Provides a suspension of cells and diluent for cryoprotectants Cooling rates are dependent on the cell being frozen and the cryoprotectant used Storage temperature must protect from ice recrystallization and progressive cell damage

Separation of buffy coat cells or light-density cells Colligative: dimethylsulfoxide, glycerol Polymeric: Hydroxyethyl starch Autologous plasma or plasma protein concentrates Saline solutions Hematopoietic cells in dimethylsulfoxide are cooled at 1–3º C/min

Plasma protein Solvent Cooling Storage

These differing reports demonstrate that storage conditions such as the concentration of cells, chemicals added, product volume, storage bag, and temperature of storage will affect the survival of cells kept in nonfrozen storage. Most published reports of noncryopreserved storage describe storage at 4º C. This condition provides a stable temperature compared with storage at ambient temperatures, which can be quite variable (and which must be considered during the shipping of products). What temperature is optimal is not known and will probably depend upon such concerns as the prestorage processing, the quantity and concentration of mature blood cells, the buffering capacity of the solution, and the gas-diffusion capacity of the storage container. Beaujean et al., for example, reported a much lower pH for PBHC products stored overnight at room temperature compared with storage at 4º C [4]. Although they did not find a difference in the recovery of progenitor cells in their experiments, this effect may be damaging to HCs in other circumstances. Antonenas et al. also reported a better survival of PBHCs stored at 2–8º C but no effect of storage temperature on BM cells [8]. Yet another report describes better survival of HCs stored at 37º C and at 25º C compared with cells stored at 4º C [9]. These warmer storage temperatures are comparable to techniques being developed for the in vitro expansion of HCs. None of these studies entailed marrow reconstitution as the experimental endpoint. The conflicting reports about optimal storage temperatures are therefore difficult to interpret. Storage conditions for nonfrozen storage have not been tested in an engraftment model, so proper storage conditions are not adequately defined. For laboratories intending to store HCs for prolonged periods without freezing, rigorous validation of the storage conditions must be pursued. There is limited experience with the autologous transplantation of noncryopreserved HCs. Used primarily in conjunction with high-dose melphalan or cyclophosphamide conditioning [5,7,10–12], refrigerated storage was supplanted by cryopreservation as most transplant centers developed multiday conditioning regimens. HCs appear to maintain viability for several days after collection if carefully stored. Preti et al. compared, in a nonrandomized retrospective analysis, the engraftment kinetics of 54 patients who received cryopreserved marrow cells with 45 patients who received refrigerated cells [7]. The refrigerated cells were stored for a median of 4 days (range 3–9 days). The cryopreserved cells were stored a median of 69 days (range 5–981 days) at −80º C using a cryoprotectant mixture of dimethylsulfoxide (DMSO) and hydroxyethyl starch (HES). Almost all patients were conditioned with a regimen consisting of carmustine (BCNU), etoposide, and cyclophosphamide. This group found no significant difference in engraftment kinetics for these two groups, although a small, few-days difference would not have been detected with the limited numbers of patients in this study.

Nitrogen liquid or vapor-phase. Addition of polymeric cryoprotectant may allow storage at warmer temperatures

Cryopreservation theory The chemistry and biology of mammalian cell cryopreservation The recovery of viable mammalian cells after cryopreservation and thawing became possible with the discovery that glycerol could be used for the freezing of bovine sperm [13]. Subsequent experiments demonstrated that marrow cells frozen with glycerol could be used to reconstitute the marrow function of irradiated mice [14]. Considerable exploration of the cryobiology of various mammalian and nonmammalian cells ensued. These studies defined the mechanism of cell damage incurred during cryopreservation and techniques to moderate that damage. The current understanding of cryobiology contends that ice crystal formation during cooling is the primary cause of cell damage [15]. This damage can be classified into two categories. At rapid rates of cooling, intracellular ice crystals may form, resulting in mechanical disruption of the cell and immediate cell death. At slower rates of cooling, ice crystal formation preferentially occurs in the extracellular space, resulting in increasing osmolality as free water is incorporated into the growing ice crystals. This loss of free water results in the concentration of extracellular solutes such as sodium that do not freely penetrate the cell membrane, extreme hyperosmolality, and dehydration injury. For example, the molality of sodium chloride (NaCl) in a saline solution at −10º C is about 5.3 m, and that at −20º C about 10.5 m. Intracellular ice formation may be limited by cooling cells slowly. At slow rates, it is probable that ice nucleation will occur first in the larger volume outside the cell. Progressive dehydration of the cell will result if the rate of cooling is slow enough to allow water to shift to the extracellular space and be incorporated into the growing ice crystals. The optimal cooling rate differs for different cell types and appears to be defined by the permeability of water through the cell membrane [16]. Progressive dehydration with concentration of intracellular solutes prevents the supercooling of intracellular water and protects the cells from the formation of intracellular ice crystals. Ice that happens by chance to nucleate first within a cell will result in the death of that cell, but will also serve to promote the freezing of extracellular water as the ice crystal penetrates the cell membrane. The corollary is not true in that extracellular ice does not penetrate into the cell and promote intracellular ice formation, presumably because of some feature of the cell membrane. Glycerol and DMSO are colligative cryoprotectants that prevent dehydration injury by moderating the increased concentration of nonpenetrating extracellular solutes during ice formation, and by decreasing the amount of water absorbed by (in equilibrium with) the ice crystals at a defined temperature. “Colligative” refers to properties dependent upon the number of particles (solute) and not the composition of the particles. Freezing is the crystallization of water, and the freezing point

Cryopreservation of Hematopoietic Cells

is the temperature at which ice crystals can be sustained in equilibrium with water. The freezing point of water is depressed by the addition of solute. For any particular mixture of solute(s) and water, there will be a defined temperature at which ice crystals can initially form. Unlike pure water, ice crystal formation and growth in aqueous solutions occurs over a temperature range. Growing ice crystals absorb free water and exclude solute particles. The incorporation of water into ice results in concentration of the solute and further depression of the freezing temperature of the remaining water, thereby preventing additional ice formation unless further cooling occurs. Thus, temperature (and pressure) defines the equilibrium between ice and the nonfrozen solution. With further cooling, a temperature is eventually reached at which the solute itself crystallizes (eutectic point). The molality of a solution in equilibrium with ice is therefore determined by the temperature of the solution, not the initial concentration of the solute, as depicted by the equation m≈

273 − T 1.9

in which m represents the osmolal concentration of the solute and T is the temperature in degrees Kelvin [17]. Similarly, the fraction of water not frozen (q) is defined by the equation q≈

1.9 Mo 273 − T

where Mo is the osmolal concentration of the solution before freezing. In our example above, a saline solution at −20º C has an NaCl molality of about 10.5 m. For a three-component system such as DMSO and NaCl in water, both solutes will contribute to the molality of the unfrozen solution in the same relative proportion found in the initial solution. The molality (before freezing) of 10% DMSO (volume in volume [v/v]) in normal saline (140 mM) solution is about 1.6 m, with DMSO contributing about 10 times the molality contributed by NaCl to the medium. At −20º C, this molal ratio between DMSO and NaCl will be maintained. The molality contributed by NaCl will be about 1.05 m, or about seven times the molality of NaCl in a solution without ice. DMSO freely penetrates the HC cell membrane so that the intracellular concentration of DMSO will equal the extracellular concentration throughout the temperature range, assuming adequate movement of water from the cell as the extracellular ice crystals form and grow. The addition of a penetrating cryoprotectant to an aqueous saline solution therefore reduces the osmotic stress across the cell membrane at −20º C from about 75 times to about seven times that of an ice-free solution. According to this theory, colligative cryoprotectants must be capable of penetrating the cell to avoid merely contributing to the molality of the extracellular medium, and must be nontoxic to the cells at the concentration required for this effect. The tolerance of cells to freezing at slow cooling rates depends on the ability of the cells to withstand osmotic stress, and initial studies of the osmotic behavior of HCs have been reported [18]. The osmotic tolerance of granulocytes is much less than that for lymphocytes or CFU-GMs, probably accounting at least in part for the difference in survival after cryopreservation [19]. With DMSO concentrations of less than 10%, the degree of dehydration caused by concentration of the nonpenetrating solutes during freezing will be greater because nonpenetrating solutes will contribute proportionately more to the molality of the nonfrozen solution in the extracellular medium. With higher concentrations of DMSO, the osmotic stress will be less. The optimal concentration for a colligative cryoprotectant depends upon the osmotic tolerance of the cell to be frozen, the toxicity to the cell of high concentrations of the cryoprotectant [20,21], and the presence of other cryoprotectants. The ability of a cryoprotectant to penetrate the cell and its subcellular organelles explains, at least in part, the relative efficacy of different colligative cryoprotectants for different cell species.

633

Colligative properties do not explain the cryoprotection achieved by freezing cells in solutions of macromolecules such as HES. Solutions of high molecular weight, polymeric cryoprotectants, contain relatively few particles and, moreover, do not freely penetrate the cell. These cryoprotectants may protect the cell by forming a viscous, noncrystalline, glassy shell that retards the movement of water, thereby preventing progressive dehydration as water is incorporated into the extracellular ice crystals [22]. Solutions of some compounds, when present in sufficiently high concentrations, will solidify to an amorphous glass without first forming ice crystals, a process termed vitrification [23]. The “glasstransition temperature” (Tg) depends upon both the structure and concentration of the solute. At very high concentrations of cryoprotectants (6.3 M for DMSO [23]), the Tg is higher (warmer) than the temperature at which ice crystals can form, thereby preventing crystallization during cooling and its resulting mechanical and osmotic stresses. The practical difficulty with achieving vitrification arises from the necessity to use these very high concentrations of cryoprotectants. Vitrification has been used for the cryopreservation of murine embryos, for example, with a solution of 20% DMSO, 15.5% acetamide, 10% propylene glycol, and 6% polyethylene glycol [24]. Prolonged exposure to this solution was toxic, however, with complete loss of viability after 30 minutes at 4º C. Kurata et al. plunged CBHCs protected by high concentrations of ethylene glycol directly into liquid nitrogen [25]. The recovery of HCs in samples preserved with the highest concentrations of ethylene glycol was similar to that for samples more conventionally cryopreserved with 10% DMSO (Table 44.2). The toxicity of the cryoprotectant solution was not addressed by these authors. Also, the rapid cooling and warming rates required are easier to achieve with the small cell aliquots (0.5 mL) tested than with the large volumes characteristic of HC products. With lower concentrations of cryoprotectants, including extracellular macromolecular compounds and many sugars, crystallization occurs at warmer temperatures than the vitrification temperature and ice crystals will form during cooling. However, glass transition may occur even in the presence of ice crystallization. With the formation of ice crystals and resulting loss of free water, the concentration of the cryoprotectant in the nonfrozen water will increase. With decreasing temperature causing increasing cryoprotectant concentration, a point will be reached at which the solution forms a glass. At this “limiting glass-transition temperature” (T′g), the solution suddenly becomes viscous, retarding if not stopping the flow of water through the extracellular matrix. This has been proposed as the mechanism for cryoprotection afforded by the extracellular cryoprotectants [22]. In one study of peripheral blood monocyte cryopreservation using extracellular cryoprotectants, a limiting glasstransition temperature of −20º C was optimal, which was achieved using a 20% solution of HES [22]. Substances forming glasses at higher or lower temperatures were less effective. Pure water forms a glass at about

Table 44.2 Recoveries of nucleated cells and hematopoietic cells after cryopreservation with ethylene glycol or dimethylsulfoxide (DMSO) Cryoprotectant solution

Cell recovery

CFU-GM recovery

8 M ethylene glycol 4 M ethylene glycol 2 M ethylene glycol 10% (v/v) DMSO

89.5 ± 8.5 83.6 ± 9.4 75.0 ± 17.4 88.5 ± 12.4

66.6 ± 20.8 36.4 ± 23.6 14.7 ± 24.6 69.1 ± 9.7

CFU-GM, colony-forming unit granulocyte–macrophage. Adapted from [25].

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−139º C [26]. The addition of cryoprotectants such as DMSO, glycerol or HES raises the T′g [27]. This model of cryopreservation requires adequate dehydration to occur to concentrate intracellular solutes and decrease the probability of intracellular ice formation, but glass formation at an appropriate temperature (please recall that temperature defines the osmolality of the unfrozen aqueous solution) to prevent excessive dehydration of the cell. Cells are not frozen in simple two- or three-component solutions but rather in complex solutions containing salts, sugars, penetrating cryoprotectants with or without extracellular cryoprotectants, and plasma proteins. Phase-transition temperatures (such as T′g) for these solutions have not been published. The improved survival observed with increasing protein concentration [28], and, possibly, by combined penetrating and extracellular cryoprotectants [29], may be explained at least in part by these theories on ice and glass formation and their effects on the cell. Moreover, the existence of glass formation may explain the relationship between storage temperature and survival of HCs over time. Below the T′g temperature, enlargement of previously formed ice crystals by recrystallization of unfrozen water cannot occur, and the cells are protected from progressive mechanical disruption. (The growth of an existing ice crystal through the process of recrystallization is thermodynamically more favorable than the nucleation of a new ice crystal.) The optimal storage temperature is below the T′g for the cryoprotectant solution used. Warmer temperatures may be used, but at risk of cell damage, a risk that is dependent upon the temperature of storage and the stability (viscosity) of the solution at that temperature. This simplified review of the effects of freezing and the cryoprotectant properties of penetrating and polymeric cryoprotectants does not completely explain the processes involved during freezing, and more detailed reviews of the freezing of aqueous solutions and mammalian cells have been published [30]. In addition to mechanical and dehydration injuries, cooling itself may be damaging to the cell [31]. Colligative effects alone are not sufficient to explain the cryoprotectant properties of DMSO or glycerol. Other freely penetrating chemicals such as urea and dimethylsulfone (DMSO2) do not function as cryoprotectants for mammalian cells [32,33], and some chemicals such as ethanol and guanidine may actually function as cryosensitizers [34]. Obviously, the chemical structure of the cryoprotectant is important in the survival of mammalian cells, and a molecular interaction between the cryoprotectant and protein or lipid molecules appears necessary for optimal cryopreservation [35– 38]. Examples of selective damage to cell membrane proteins or structure from freezing are illustrated by the loss of response to giant cell tumor-conditioned medium of myeloid progenitor cells after cryopreservation [39], the decrease in L-selectin expression by CD34+ cells after cryopreservation and thawing [40], and the lineage-specific effects on granulocyte recovery for patients receiving cells frozen with HES and DMSO in combination [41]. Induction of apoptosis during cell cooling The recently demonstrated relationship of apoptosis with hypothermia may help explain HC losses during cooling and cryopreservation. Separate from cell necrosis resulting from the mechanical and dehydrating effects of cryopreservation, the induction of apoptotic pathways during cooling or after thawing may lead to further losses of HCs. Stroh et al. demonstrated caspase activation during the freeze–thaw process and reported improved survival of cells with the addition of a caspase inhibitor to the cryopreservation solution [42]. Benefit could be achieved also with the treatment of cells immediately after thawing, and the benefit was particularly significant for cells stored at warmer temperatures (e.g. −70 °C). Baust et al. describe the development of a commercial family of cryopreservation solutions used for the freezing and post-thaw processing of HCs [43]. Preclinical studies report improved recovery of

CD34+ cells and CFU-GMs from cord blood units frozen with these solutions, but clinical studies demonstrating improved engraftment kinetics are not yet published [44]. The physics of cooling and warming of cell products The rationale for slow rates of cooling and rapid rates of warming is explained by the mechanical and dehydration injuries resulting from the formation and growth of ice crystals. Heat transfer is a physical process, not a biologic or chemical process. Cooling is a physical process dependent on properties of the object being cooled, such as the difference in temperature between the object and its environment, and the size and thermal conductivity of the object. The rate of heat transfer is described by the equation q(r,t) = −k∇T(r,t) in which q(r,t) represents the heat-flux vector at a particular point on the object and a particular time, −k represents the thermal conductivity of the material, and ∇T(r,t) represents the temperature gradient vector at that particular time and place. Objects with a greater thermal conductivity because of composition or smaller size will gain or lose heat more rapidly, as will objects placed in an environment with a much larger difference in temperature between the object and its environment. Most centers use electronic rate-controllers to achieve an optimal cooling rate, but satisfactory rates of cooling can be achieved by simply immersing an appropriate volume of marrow or PBHCs into a −80º C mechanical freezer [45–48]. Rowley cooled 50 mL aliquots in storage bags and found reproducible cooling rates of about 3º C/min when placed in a −80º C freezer, but rates between 10º C/min and 16º C/min when placed in a −135º C freezer [45]. Clark et al. studied the effects of marrow volume on the cooling rates before and after the transition phase, and the duration of the transition phase for cells placed either in freezing bags or vials [48]. As would be predicted by the equation describing heat transfer, bags containing larger volumes cooled much more slowly and required longer duration of the transition phase (Table 44.3). Cryopreservation using an immersion technique is not “uncontrolled,” unless the physical parameters used by the laboratory are not controlled, resulting in widely varying rates of cooling. Before immersion techniques are adopted by individual laboratories, the rate of cooling and reproducibility for the type of bag and volume of cells must be determined and documented. The advantages of immersion cooling techniques are the avoidance of a rate-controlled apparatus and the decrease in personnel time required. The disadvantages are the lack of a recording documenting the rate of cooling, the limited adaptability to different volumes and containers, and possible detrimental effects related to the duration of transition phase, which may be critical if borderline quantities of cells are harvested.

Cryoprotectant solutions DMSO DMSO, glycerol, and a variety of other chemicals may serve as colligative cryoprotectants that protect the cell from excessive dehydration as extracellular water is drawn into growing ice crystals. The cryoprotectant properties of glycerol were described in 1949 [13], and those of DMSO 10 years later [49]. Both have been used for the cryopreservation of BM. The rapid diffusion of DMSO through the cell membrane, and the difficulty in removing glycerol before reinfusion (infusion of DMSOcontaining products being generally tolerated), have made DMSO the favored agent for HC cryopreservation.

Cryopreservation of Hematopoietic Cells Table 44.3 Effect of product volume on cooling rate after immersion into a –70º C freezer: rates of cooling before and after the transition phase, and the duration of the transition phase for samples of the stated volumes immersed into a −70º C refrigerator

635

Cooling velocity (ºC/min) Volume (mL)

Pre transition

Post transition

Duration of transition phase (min)

30 60 90 120

6.1 3.1 1.6 1.8

2.0 1.6 1.0 1.0

0.5 3.0 8.6 12.8

Adapted from [48].

DMSO, a byproduct of paper manufacturing, is a hygroscopic polar compound developed originally as a solvent for chemicals such as insecticides, fungicides, and herbicides [50]. Pure DMSO is a colorless, virtually odorless liquid (specific gravity 1.108, molecular weight 78.13 g/mol), although industrial grades may have a strong sulfur odor [51]. The serum half-life of DMSO is about 20 hours, although that of DMSO2, a renally excreted metabolite, is 72 hours [51]. A small proportion of DMSO is reduced to dimethylsulfide, which is expired through the lungs for about 24 hours after administration, and which accounts for the characteristic odor resulting from DMSO infusion. The optimal concentration of either DMSO or glycerol for the cryoprotection of HCs appears to be about 10%, although concentrations as low as 5% have been used successfully for HCT [52]. In their original report, Lovelock and Bishop demonstrated a dose–response with improving red cell survival as the concentration of DMSO was increased to 15% [49]. The effect of yet higher concentrations was not reported. Subsequently, Ragab et al. studied the survival of HCs from human donors after freezing with various concentrations of DMSO [28]. They found a significant increase in progenitor cell recovery when the concentration of DMSO was increased from 7.5% to 10%. No further improvement was found with an increase to 12.5%. This improvement resulted from an increase in nucleated cell recovery from 17.2% to 35.6% and 32.1%, respectively, after washing. Cell recovery before the post-thaw wash did not differ, nor did the numbers of colonies per 105 cells plated. The loss of cells during the wash steps that was found in this study may or may not reflect events occurring during direct intravenous infusion commonly used clinically. Donaldson et al. studied the recovery of CD34+ cells cryopreserved in varying concentrations of DMSO and HES and reported an increase in CD34+ cell recovery from 12.2 ± 10.0% (mean ± standard deviation) to 85.4 ± 28.4% as the concentration of DMSO was increased from 2.5% to 5.0%, but no further improvement with further increase in concentration to 10% (HES concentration kept constant at 4% weight in volume [w/v]) [53]. Varying the concentration of HES in the presence of 5% (v/v) DMSO had no effect on CD34+ cell recovery. HES HES is a polymeric substance containing chains of different molecular weights. Initially explored as a cryoprotectant for red blood cells, it was found also to be an effective cryoprotectant for a variety of other cells [22,54]. Macromolecular cryoprotectants may be used as single agents, but the major focus in the study of HC cryopreservation using extracellular cryoprotectants has been their use in combination with penetrating cryoprotectants. In one early study, the addition of polyvinylpyrrolidone, another macromolecular cryoprotectant, to glycerol or DMSO improved the cryopreservation of murine cells compared with the use of a penetrating agent alone [55]. Stiff et al. froze human cells in a combination of 5% DMSO, 6% HES, and 4% human serum albumin, and reported

improved progenitor cell survival as determined using in vitro cultures [29]. They subsequently successfully used this mixture of cryoprotectants to cryopreserve the marrow of 60 patients [46]. No engraftment failure was attributed to this technique, or after the cryopreservation of peripheral blood-derived HCs [47]. Only two clinical studies comparing engraftment outcomes for patients receiving products stored in DMSO/HES or DMSO alone have been reported. The first trial, limited to 12 patients in each arm, did not detect any difference in the kinetics of engraftment, although the small number of patients treated limited the power of the study to detect the small differences in engraftment speed that might be expected from small differences in cryoprotectant efficacy [56]. The other trial, a single-blind phase III study of 294 patients, found 1-day differences in the times to achieve a white blood cell count of 1 × 109/L or more and an absolute neutrophil count (ANC) of 0.5 × 109/L or more, and time to discontinuation of antibiotics for recipients of cells frozen with the combination cryoprotectant solution [41]. No differences were found for times to achieve endpoints of platelet engraftment (either 20 × 109/L or 50 × 109/L), indicating that this effect was probably lineage specific. Furthermore, the effect was more pronounced for recipients who received more than the median number of CD34+ cells, suggesting a specific effect on a limited cell population (Table 44.4). No adverse effects from the use of the combination cryoprotectant were noted by these authors. Protein Plasma proteins exert cryoprotectant effects, possibly by modifying the viscosity or the glass-transition temperature of the cryoprotectant solution. Lymphocytes can be cryopreserved using serum alone [32,57]. The addition of serum proteins to the cryoprotectant solution appears to improve HC survival. In one study using human marrow cells, myeloid progenitor cell survival, which was 41.1 ± 8.0% in the absence of serum, increased significantly to 64.8 ± 14.2% with 15% serum and 75.4 ± 14.8% with 50% serum [28]. Similarly, recovery of murine spleen colony-forming cells (CFU-Ss) increased from 18.2% to 100.5% when frozen in 10% DMSO with 10% serum added [58]. In this second study, cryopreservation with 10% fetal bovine serum was less effective (25.4% recovery) than that with homologous serum, which may reflect the low protein content that is a characteristic of fetal sources of serum. The effect of protein content or source on engraftment speed has not been defined, but anecdotal experience would suggest that the presence of protein is important. Engraftment will occur for CD34+ cells frozen without the addition of protein [59]. The 33 days (median) to achieve an ANC of over 500/μL and 46 days to achieve a platelet count of over 20,000/μL in that study that did not use protein in the cryoprotectant solution, however, were much slower than the 23 days and 22 days for ANC and platelet engraftment reported by Shpall et al. using a similar marrow-processing technique but different cryoprotectant solution containing protein [60].

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Table 44.4 Engraftment kinetics of patients receiving peripheral blood hematopoietic cells frozen in dimethlysulfoxide (DMSO) or DMSO and hydroxyethyl starch (HES) Patients below the median*

Patients above the median*

Outcome

DMSO

DMSO/HES

p value

DMSO

DMSO/HES

p value

Granulocyte engraftment WBC ≥ 1.0 × 109/L ANC ≥ 0.1 × 109/L ANC ≥ 0.5 × 109/L ANC ≥ 1.0 × 109/L

11 10 11 13

11 10 11 12

0.71 0.86 0.87 0.87

11 10 11 12

10 9 10 11

0.02 0.03 0.01 0.02

Platelet engraftment Platelets ≥ 20 × 109/L Platelets ≥ 50 × 109/L Last transfusion

11 14 9

12 15 9

0.25 0.10 0.67

9 12 8

9 12 8

0.36 0.28 0.75

ANC, absolute neutrophil count; WBC, white blood cell count. * Median times to achieve the designated outcome for patients classified by cryoprotectant used and stratified by quartile of CD34+ cell dose/kg (first and second quartiles combined versus third and fourth quartiles combined). The median cell doses for patients receiving products frozen with DMSO alone was 6.4 × 106/kg, and for those receiving cells frozen with DMSO/HES was 6.5 × 106/kg. p values are based on weighted log rank test, which compares the two estimated recovery curves. Reproduced with permission from [41].

Virtually all cryopreservation solutions currently in use contain plasma proteins added as either part of the cryopreservation solution or during cell processing. The range in concentration and source of protein varies among the various transplant groups; the source of protein is similarly diverse, although fetal bovine serum (used in some early trials of marrow transplantation) is no longer used. The use of albumin solutions is appealing because of the ability to achieve high, uniform concentrations of protein while avoiding the marrow fat, cellular debris, anticoagulants, and risk of cryoglobulins associated with the use of autologous plasma collected during initial processing of the HC product. A unique group of proteins are the antifreeze glycoproteins found in some species adapted to survival in cold environments. Some of these proteins are extremely efficient at preventing the recrystallization of ice and have been shown to enhance the cryopreservation of pig and murine embryos [61]. In studies of red cell cryopreservation, this effect was limited to a narrow concentration range, with higher concentrations actually increasing the growth of extracellular ice, with concomitant cell damage [62]. It cannot be assumed that the addition of these proteins will improve HC cryopreservation survival. Salt and sugar content Metabolically inactive cryopreserved cells do not require the complex medium preparations used for ex vivo growth of HCs. Successful cryopreservation has been achieved using commercially available pharmacologic salt solutions. However, the solution in which the cells are suspended should not be viewed as inert. For example, many tissue culture media contain compounds that may increase the sensitivity of cells to freezing (cryosensitizers) [34]. Also, as noted in the second equation cited above, the fraction of unfrozen water is determined by the initial osmolality of the solution. At least for red cells, cryopreservation in hypotonic solutions increases post-thaw hemolysis [63]. Various sugars may function as cryoprotectants. Leibo et al. found 50% survival of CFU-S after cryopreservation of murine marrow cells in 0.35 M sucrose without protein or other cryoprotectants [64]. Optimal rates of cooling, as with other extracellular cryoprotectants, were

between 16 and 70º C/min. Cooling at slower rates (<10º C/min) was associated with much poorer cryosurvival. Using a cell line derived from human kidney, Vos and Kaalen showed cryoprotectant properties for a number of sugars, including glucose, mannitol, and sorbitol, at concentrations greater than 0.1 M [32]. The amount of glucose in tissue culture media is in the millimolar range, which is negligible compared with the concentration shown by Vos and Kaalen to be an effective cryoprotectant in their system. These sugars do not freely penetrate the cell membrane, and function as extracellular cryoprotectants. Sugars may serve to stabilize the cell membrane during freezing or dehydration [36,37]. It has also been suggested that glucose may protect against the cytotoxicity observed with high concentrations of DMSO [65].

Cryopreservation technique Evaluation of cryopreservation efficacy A major impediment to the study of HC cryobiology is the inability to culture primitive HCs in vitro. The only “assay” for the cryosurvival of these cells is engraftment after transplantation, the rate of which depends more on patient diagnosis and marrow function, quantity of cells harvested, and any ex vivo treatment performed than on the cryosurvival of HCs as reflected by in vitro cultures [2]. Experimental models include engraftment of human cells in immunodeficient NOD/SCID mice [66]. Initial studies of blood and HC cryobiology used either metabolic assays or dye exclusion assays [67]. These assays are irrelevant, if only because the rarity of HCs in the harvested cells precludes a correlation between cell metabolism or dye uptake and HC cryosurvival. Techniques that measure the viability of the whole population of cells are of no use in determining HC viability in that a complete kill of HCs would change the proportion of cells with vital dye uptake by less than 1%. HCs cannot be identified morphologically, and moreover, metabolic and dye exclusion tests do not measure the proliferative capacity of these cells (Table 44.5) [67]. Progenitor cell assays such as culture for CFU-GMs after thawing can be predictive of engraftment [1,2], but the validity of these assays must be determined for each group of patients and the assay used. DMSO

Cryopreservation of Hematopoietic Cells Table 44.5 Protective effect of various additives for murine marrow cryopreservation: viability of bone marrow cells cryopreserved with various additives as shown by vital dye stains and the ability of these cells to rescue an animal from irradiation

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Cryoprotectant

Viability by vital stain (%)

Animal survival (%)

Dimethylsulfoxide (15%) Glycerol (15%) Serum (30%) Bovine serum albumin (15%) Dextran (15%) Saline

91 75 90 100 62 3

70 70 0 19 24 0

Adapted with permission from [67].

must not be present in concentrations greater than 0.1% in in vitro cultures [68,69], so some technique of dilution must be undertaken before cell culture. Rapid dilution of DMSO after thawing exposes the cells to osmotic shock. Some authors have reported satisfactory recovery of HCs after rapid dilution of thawed cells [1,70]. Others, however, have demonstrated a distinct advantage to serial dilution [28,69,71]. Deoxyribonuclease (DNAse) can be added to lyse clumps that form. The addition of sorbitol or other macromolecular substances after thawing may be of value to help prevent osmotic shock [72,73]. These techniques, of course, differ from those of direct intravenous infusion. Some centers routinely freeze “test vials” containing small aliquots of cells alongside the greater quantity of cells contained in freezing bags. The different cooling properties of vials compared with bags diminishes the reliability of these small aliquots for determining cryosurvival [48,74]. If an attempt is made to use small aliquots for clinical decisions, a system of cooling, thawing, washing, and culture that correlates with engraftment kinetics must first be developed. Processing of HC products for cryopreservation One of the basic concepts of clinical HC cryopreservation is the heterogeneity of BM and blood cell populations. HC cryopreservation involves the cryobiology of not only HCs, but also the mature blood cells contained in the harvested product. HCs comprise a very small portion (generally <1%) of the BM, PBHC or cord blood stem cell product. Furthermore, HC products consist of HCs heterogeneous over a range of cell maturation; accessory cells are also important to engraftment. BM consists of HCs, mature blood cells, and noncellular material such as fat. Cryopreservation techniques that are optimal for HCs will not preserve mature blood cells. The standard technique for red cell cryopreservation uses glycerol, granulocytes cannot be cryopreserved successfully, and platelets are frozen with lower concentrations of DMSO which must be added at a controlled rate to avoid osmotic shock to the cell [75]. The presence of mature blood cells affects HC cryopreservation in at least three ways. First, the large proportion of mature blood cells collected may hinder the laboratory processing if clumping before freezing or after thawing is induced by damaged granulocytes or platelets. Second, damaged cells may cause infusion-related toxicity. The infusion of marrow that was frozen without depletion of mature blood cells was associated with acute renal failure in three of 33 patients in one study [76]. This problem presumably resulted from massive hemolysis of the poorly preserved red blood cells. Third, if the cells are frozen at a set concentration, the presence of large numbers of mature blood cells requires that the cells be frozen in large volumes. Patients receiving such products are at considerable risk for serious infusion-related toxicity from the large quantities of cryoprotectant used. In addition, the concentration of HCs before storage reduces the space required, a logistical

consideration for the management of large banks of cells. CBHCs intended for unrelated donor transplantation are routinely concentrated to minimize the space required for the large number of products necessary in the banks providing products for patients lacking other sources of HCs. Therefore, cryopreservation of HCs can be facilitated by the prefreeze depletion of mature blood elements. A number of apheresis or cellwashing devices are capable of processing the large quantities of cells harvested. Some apheresis devices provide enrichment of mononuclear cells relative to granulocytes, providing a “cleaner” preparation for processing and cryopreservation. Density-gradient separation of lightdensity cells further enriches for HCs, although at the expense of additional cell losses and cost of processing. At the extreme of prefreeze processing is the extensive purification of HCs by isolation of CD34+ cells, which reduces the quantity of mature blood cells and the amount of cryoprotectant infused, leading to a reduction in infusion-related toxicity. Collection of buffy coat cells with depletion of red blood cells is the minimum processing required for cryopreservation of marrow. PBHCs collected by apheresis contain small proportions of red cells and generally do not require further separation, although volume reduction will decrease the amount of cryoprotectant used. Cord blood cells are processed to reduce the storage volume required for the large banks of cells intended for allogeneic transplantation [73]. Cell concentration The cell concentrations used in most protocols are often driven by such practical considerations as the desire to freeze more than one bag, or to minimize the total volume and number of bags of product stored. PBHCs differ from BM products because of their much larger nucleated cell quantity that must be processed by the laboratory. Although many protocols set limits for the concentration of nucleated cells, few also define the maximum or minimum concentrations or quantities of red cells, granulocytes or platelets. The effect of these cells on HC cryopreservation is not defined, but a potential effect is illustrated by a report of poor post-thaw recovery of red blood cells frozen at high cell concentrations, which may relate to the limited space in channels of nonfrozen water between the growing ice crystals during cooling and increased packing of cells [77]. Most studies involving cryopreservation of HCs at high cell concentrations were not designed to detect subtle damage from this practice. The cryopreservation of PBHCs at an average 3.7 × 108 nucleated cells/ mL (range 0.4–8.0 × 108/mL) with no obvious correlation between cell concentration and engraftment kinetics has been reported [78]. Although there also was no correlation between cell concentration during freezing and the post-thaw recovery of CD34+ cells, the recovery of CFU-GMs appeared to decrease at higher cell concentrations. This observation has been confirmed by others [79,80]. Although cell

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cryopreservation at high concentrations of nucleated cells is feasible, Rowley et al. suggested that infusion of PBHCs frozen at high concentrations resulted in increased risk for neurologic events during or shortly after cell infusion [81]. The development of techniques that enrich for CD34+ cells presents a different challenge to the cryopreservation laboratory. These techniques typically recover about 1% of the nucleated cells initially present. Cryopreservation of these cells in the same volume used for unselected cells would result in very low cell concentrations. Dicke et al. cite one study of murine marrow cryopreservation that showed stable CFU-S cryosurvival at cell concentrations between 5 × 106 and 2 × 108 cells/mL [71]. At cell concentrations of 1 × 106 cells/mL and below, however, CFU-S cryosurvival dropped precipitously. This decline may be related to nonspecific losses such as adhesion to surfaces and post-thaw wash steps. Similar studies of the effect of cell concentration have not been reported for human HCs. CD34+ cell-enriched products may be frozen in small vials or bags with adjustment of the cooling technique for the smaller volume. Rates of cooling and warming Different cells have different optimal cooling rates, and the optimal cooling rate is also dependent on the type and concentration of cryoprotectant used. In general, the higher the concentration of a colligative cryoprotectant, the slower the optimal cooling rate (Table 44.6). The optimal cooling rate when using a colligative cryoprotectant also falls within a narrow range. In contrast, optimal rates for cooling when using extracellular cryoprotectants are generally more rapid, and the cells tolerate a broader range (Table 44.6). Ma et al. used in vitro cultures of human bone marrow to show optimal cooling at 1º C/min for human hematopoietic progenitor cells suspended in 10% DMSO [82]. The recovery of colony-forming cells fell at rates slower than 1º C/min or faster than 3º C/min. Lewis et al. studied the impact of cooling rates before and after the plateau phase (immediately after the “heat of fusion”), and of the duration of the plateau phase on the recovery of murine CFU-Ss frozen in 12% glycerol [83]. No difference was found for preplateau cooling rates ranging from 0.8º C/min to 4º C/min. Increasing the duration of the plateau phase from 0 to 16 minutes resulted in a drop in spleen colonies from 16.7 to 12.0. Increasing the rate of the postplateau cooling similarly decreased CFU-S recovery. Clinically, Gorin et al. associated a rapid cooling rate with delayed engraftment for eight recipients of autologous marrow [84]. Virtually all the reports regarding the optimal rate of cooling for HCs do not discuss the temperature at which time rate becomes irrelevant. This author has routinely increased the rate to 10º C/min when the product reached −40º C and transferred the bags to the storage refrigerators when a temperature of −80º C has been reached [41]. At −40º C, water will spontaneously nucleate so supercooled water cannot exist below this temperature [85]. Leibo et al. reported that most cell damage

Table 44.6 Effect of cryoprotectant on optimal cooling rate for murine colony-forming units-spleen (CFU-S) Cryoprotectant

Optimal cooling rate

CFU-S survival

0.4 M glycerol 0.8 M glycerol 1.25 M glycerol 0.35 M sucrose

100º C/min 18º C/min 1.8º C/min 16–70º C/min

18% 40% 65% 54%

Adapted with permission from [64].

in their murine model occurred at temperatures between −10º C and − 45º C [64]. Therefore, HCs may be tolerant of higher cooling rates after initial freezing of water. The warming rate is more critical when rapid rates of cooling are used because of the intracellular ice nucleation that occurs with rapid cooling. If warming is slow during the thawing process, growth of these ice crystals by recrystallization can occur. Cooling at slow rates limits intracellular ice nucleation, and mechanical disruption of the cell from ice recrystallization during warming is less likely. Leibo et al. found no difference in CFU-S recovery for cells in 1.25 M glycerol cooled at 1.7º C/min and warmed at either 1.8º C/min or 910º C/min [64]. Therefore, rapid warming is appropriate. Storage of cryopreserved cells Most laboratories store HCs below −120º C in mechanical (electrical) refrigerators or in either the vapor or liquid phase of nitrogen. The rationale for this method is the possible progressive growth of ice crystals at warmer temperatures as water migrates from smaller to larger crystals (recrystallization), a process that does not occur in pure water at temperatures below about −130º to −139º C [26,27], or in cryopreservation solutions cooled below the phase-transition temperatures of the mixture [26]. Critical phase-transition temperatures for HC cryoprotectant solutions containing DMSO or DMSO/HES have not been fully explored. Nor has the effect of storage or intermittent warming above these phase-transition temperatures on HC viability been determined. Initial attempts of autologous transplantation infused cells stored at −80º C, although the mild, probably marrow-sparing, pretransplant conditioning regimens used in those initial trials hinder interpretation of the results [86]. In other, preclinical studies, however, progressive loss of HCs was demonstrated for cryopreserved cells stored at relatively warm temperatures. BM cells stored for 25 weeks in 10% with 80% survival after 22 weeks’ storage at −70º C and 72% survival after storage for 26 weeks in liquid nitrogen [87]. Illustrative of the need to maintain stable storage temperatures for long-term HC cryopreservation is the report by Appelbaum et al., using a dog model of autologous transplantation, of the failure of engraftment in 30%, 66%, and 100% of recipients of 0.5 × 108 marrow cells/kg stored in the vapor phase of nitrogen for 9, 12, and 18 months respectively [88]. Storage at ultralow temperatures is not a requirement for successful engraftment if adequate numbers of HCs are harvested and if the duration of storage is limited. Under such conditions, moderate losses of HCs may occur without obvious delay in engraftment kinetics. The engraftment failure observed by Appelbaum et al. can be explained by their intentional cryopreservation and transplantation of borderline quantities of marrow cells and the probable instability of the storage temperature of their cells. Nitrogen vapor-phase refrigerators are noted for the temperature gradient formed. Temperatures as warm as −100º C may exist at the top of the refrigerator. Cells stored at this location will be exposed to additional warming upon opening of the refrigerator or lifting of the rack to gain access to the storage cassettes. Over a prolonged storage time, cells may be repetitively exposed to warming during normal operation of the refrigerator, and this warming may be progressively damaging to HCs. This temperature gradient in the vapor phase can be minimized by constructing frames from metals with better heat conductivity, such as aluminum. Rowley and Byrne found a gradient of only 5.9º C at 22 inches (56 cm) above the liquid when using an aluminum racking system compared with a gradient of 86º C for a similar refrigerator containing a racking system constructed of steel [89]. This design achieved stable low-temperature storage throughout the refrigerator.

Cryopreservation of Hematopoietic Cells

In addition to the issue of temperature gradients in vapor-phase refrigerators, many laboratories perceive storage in the liquid phase to be safer because of the larger quantity of nitrogen present. However, liquid nitrogen can serve as a reservoir for viruses, which may be of clinical importance for these high-potency pathogens that require only a few organisms contaminating the bag port for transmission of disease [90]. This problem was dramatically illustrated by the transmission of hepatitis B infection to at least three patients whose cells were immersed in the same liquid nitrogen refrigerator as those of the index case [91]. Molecular typing of the virus and its isolation from the detritus of the refrigerator on subsequent investigation confirmed this mode of transmission. It is possible that different cryoprotectant solutions may permit storage at warmer temperatures, although this has not been prospectively tested using appropriate models of engraftment. Many centers have adopted storage at −80º C because of its simplicity for short-term storage for patients expected to undergo transplantation within a few weeks or months of cell harvest. Mostly, this storage is used in conjunction with DMSO/HES cryoprotection. Stiff reported successful transplantation of products stored for up to 22 months at this temperature [92]. Whether this accomplishment results from the addition of HES to the cryoprotectant solution, thereby affecting the stability of the solution at this relatively warm temperature, or because enough HCs were harvested to allow progressive loss was not analyzed. Galmes et al. reported a progressive loss of CFU-GMs and BFU-Es from PBHC products stored at −80º C in either 5% or 10% DMSO (without HES) and recommended that duration of storage at this temperature be limited [93]. The duration of storage may be indefinite if adequate temperatures are maintained and appropriate cryopreservation techniques are used. Evidence supporting this premise is found in both laboratory and clinical experience. Parker et al. found no loss of colony-forming units culture (CFU-Cs) after storage of human marrow in vapor-phase nitrogen for a median of 42 months [94]. Cord blood stem cells maintain proliferative capacity and ability to engraft in a NOD/SCID murine model for at least 15 years [95]. Furthermore, limited numbers of patients have received HC products stored for prolonged periods of time. One report of experience from multiple centers reported transplantation of cells stored for up to 11 years [96]. In that study of 33 patients, however, the median duration of storage was only 2.8 years, the authors did not correlate duration of storage with engraftment kinetics or other markers of HC survival, and some products were treated ex vivo with mafosfamide, which is known to delay engraftment. Some patients experienced markedly prolonged post-transplant aplasia (up to 119 days), and two patients failed to engraft. The other study reported the outcome of transplantation for 36 patients who received marrow stored for 2–7.8 years, and found, in retrospective comparison to a control group who received cells stored for less than 2 years, no differences in success or speed of engraftment [97]. The products in that report were stored either in nitrogen vaporphase refrigerators or mechanical freezers at −135º C. The temperature of storage should be maintained during transportation of marrow. The availability of “dry shippers” in which the nitrogen is absorbed into the wall of the container simplifies the transportation of frozen cells. These “shippers” can maintain nitrogen vapor-phase temperatures of about −180º C for periods of 7–10 days (if stored upright). The amount of residual nitrogen at any time after filling is determined by weighing the container. Cells may be stored either in bags or vials constructed of plastics tolerant to cryogenic temperatures. The advantage of vials relates to the ease with which samples may be thawed for analysis. The large numbers of vials required for storage, the risk of explosion on warming from nitrogen seepage into poorly sealed vials, and the higher risk of microbial contamination during handling are reasons that favor bags.

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Post-thaw manipulation DMSO and HES need not be removed before infusion if consideration is given to the potential toxicities of these agents. Most products may be frozen at sufficiently high cell concentrations that the total cryoprotectant dose is held within tolerable limits. Clumping of damaged cells may occur after thawing, especially if the cells are manipulated. For these reasons, most centers infuse cells within a few minutes after thawing and without any post-thaw processing other than filtration. Most DMSO-associated toxicities are related to the quantity of DMSO infused, which can be reduced by washing the cells after thawing. The techniques described generally involve serial dilution to avoid osmotic shock, and resuspension in a protein-containing medium [98,99]. Beaujean et al. reported an average 73.9 ± 6.4% CFU-GM recovery (± standard error; range 20.5–158.2%) for 50 density-gradient separated marrows after thawing and washing [98]. The recovery for 12 peripheral blood products was 93.9 ± 7.0% (range 67.4–135.6%). Twelve of the marrow bags were stored at room temperature for 4 hours after washing, with an additional 20–25% loss of cells and CFU-GMs. These authors did not describe the speed of engraftment, compare engraftment kinetics to similar patients receiving unmanipulated cells, or correlate engraftment speed with either total cell or CFU-GM recoveries. However, there is no unequivocal evidence published to date that dilution of DMSO from HC products significantly affects engraftment kinetics. The risk of post-thaw clumping can be diminished by removing granulocytes and platelets from the cell product before freezing, or by the addition of acid–citrate–dextrose (ACD) or DNAse. Symptomatic hypocalcemia occurs with the infusion of citrate anticoagulants, which may predispose the patient to adverse neurologic events during the infusion [81]. DNAse is effective at lysing clumps but poses the risk of allergic or febrile reactions if a pharmaceutical-grade reagent is not used. Unlike DNAse, ACD will not lyse clumps that have already formed. Thawed cells should be infused through a standard blood administration set with an in-line filter of 170 μm pore size. It is possible to process HC products extensively after thawing, including the separation of CD34+ cells from cryopreserved PBHC products [100]. Cell clumping during the processing is, as stated, a complication that appears related to the quantity of mature blood cells contained in the product.

Cryoprotectant toxicity Toxicity to HCs High concentrations of cryoprotectants may incur direct toxicity to the cells being cryopreserved (similar to the effects of vitrification solutions on murine embryos previously discussed). In general, cells are more tolerant to cryoprotectants at reduced temperatures. For example, DMSO is also concentrated during the formation of ice. Optimal cryopreservation requires a balance between protection from freeze damage and the occurrence of cryoprotectant-induced toxicity. The toxicity of DMSO to the HCs has been described, and most laboratories minimize the time of exposure to DMSO of cells before and after cryopreservation. Using in vitro cultures, Douay et al. described a 23.5% recovery of CFU-Cs after 60 minutes’ exposure (at 4º C, without cryopreservation) and a 15% recovery after 120 minutes [101]. However, Rowley and Anderson were unable to confirm their findings of DMSO-induced progenitor cell loss in similar studies, and surmised that the difference between these reports might relate to the purity of the DMSO used [68]. DMSO is a potent solvent, easily contaminated. In the latter study, which used pharmaceutical-grade DMSO, the recoveries of nucleated cells, CFU-GMs, and BFU-Es were virtually 100% after exposure to 10% DMSO at either 4º C or 37º C. Only at 20% concentration of DMSO did cell clumping cause a decrease in cell recovery to 21.7%

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(and total CFU-GM quantity to 27.1%). The numbers of myeloid and erythroid progenitors per 5 × 104 cells plated dropped only after exposure to 40% DMSO (3.3% of control after 60 minutes’ incubation). Similarly, they found no loss of progenitor cells after thawing if the removal of DMSO was delayed even up to 60 minutes. Others have also reported no toxicity to HCs with short exposure to DMSO [82], although the direct addition of DMSO into the culture medium at 1% or greater concentration is toxic to cell culture [58,69]. Toxicity to the HCT recipient In contrast, a high incidence of generally mild, infusion-related morbidity with the reinfusion of either marrow or PBHCs has been reported by several centers [102–104]. DMSO itself has a variety of pharmacologic effects [50], which may be compounded by the presence of lysed blood cells, foreign proteins from tumor-cell purging procedures or contaminants from nonpharmaceutical grades of reagents used in the processing. The LD50 values (amount of DMSO required to kill 50% of test animals) reported for intravenous infusion of DMSO are 3.1–9.2 g/kg for mice and 2.5 g/kg for dogs [51]. The acute toxic dose of DMSO for humans has not been determined. If a large amount of cryopreserved material is to be infused, the infusion can be separated over 2 days to avoid complications from the infusion of excessive amounts of DMSO. The most dramatic toxicity is the rare anaphylactic reaction occurring during the initial administration of thawed cells. This appears to be an allergic reaction to DMSO, contaminants of DMSO (DMSO is a potent solvent, easily contaminated) or products of tissue culture medium once used for cryopreservation. Treatment of this complication is the same as for anaphylactic reactions to other medications and, after resuscitation of the recipient, the remainder of the cells may be administered cautiously. Nonallergic, profound hypotension may result from the intravenous infusion of DMSO, presumably from histamine-induced vasodilatation [50]. Skin flushing, dyspnea, abdominal cramping, nausea, and diarrhea, reported to varying degrees after HC infusion, can also all be attributed to DMSO-induced histamine release. These complaints resolve over a few hours and are treated symptomatically. DMSO has a variety of cardiovascular effects. In a series of 82 patients who were premedicated with diphenhydramine, Davis et al. observed increased blood pressure and decreased heart rate that were maximal about 1 hour after the completion of the marrow infusion [102]. A number of authors have noted cardiac arrest or high-degree heart block occurring during or immediately after the infusion of cryopreserved marrow or PBHCs [105–108]. In two series, the incidence of bradycardia (heart rate <60 beats/min) was 48.8% and 65%, that of second-degree heart block was 9.7% and 24%, and of complete (thirddegree) heart block was 4.8% and 5.9% [107,108]. In both reports, the median time of onset was about 3 hours after the completion of the infusion. In one series, the authors noted that the heart block was often episodic, occurring with episodes of emesis [107]. In both series, the cardiac rhythm abnormalities resolved spontaneously within 24 hours of infusion. In contrast, Lopez-Jimenez et al. found no cardiac rhythm changes in a prospective series of 29 patients [109]. A break of 20 minutes was allowed between bags of thawed cells in this last series, and it may be the slower overall infusion rate that accounted for the lack of rhythm changes. Alessandrino et al. noted bradycardia for recipients of BM but not PBHCs, describing a higher risk of this complication for recipients of products containing higher quantities of red blood cells in the product [110]. Although headache in up to 70% of recipients of cryopreserved cells has been reported [104], other central nervous system complications are rare and generally related to the amount of DMSO infused. Two recipients who received HC products containing a total of 225 mL and 120 mL, respectively, of DMSO developed reversible encephalopathy [111]. The

first patient underwent plasmapheresis, with a prompt improvement in mental status; the second patient recovered over 5 days without specific treatment. The weights of the patients were not cited in this report, but both patients probably received over 2 g DMSO/kg body weight. In an attempt to reduce the volume of DMSO used during the cryopreservation of PBHCs, Rowley et al. concentrated cells to very high concentrations, averaging over 3.7 × 108 nucleated cells/mL [78]. They subsequently reported several patients who experienced seizures during the administration of these cells [81]. The average cell concentration for these patients was 6.9 × 108 nucleated cells/mL (range 0.8–12.9 × 108 cells/mL), but the maximum amount of DMSO administered was only 0.6 g/kg body weight. Administration of similar quantities of DMSO during the infusion of BM frozen at much lower cell concentrations has not been associated with seizures, so it is likely that these events are related to the cell concentration, cell quantity or types of cell cryopreserved in a PBHC product. This laboratory previously added ACD (formula A) after thawing to prevent clumping. This practice was stopped after a high incidence of citrate toxicity was observed. The incidence of seizures has also decreased with the removal of ACD from the infusate. Recently, Calmeis et al. reported a relationship between the quantity of non-HCs in the product and infusional adverse events for recipients of products from which DMSO had been removed before infusion [112]. Similarly, Milone et al. noted a correlation of infusion rate with cardiovascular changes, but a relationship between the nonmononuclear cells in the products and noncardiovascular adverse events [113]. Marrows frozen in 10% DMSO (which itself is 1.4 m) have an average osmolality of 1794 mOsm/kg H2O after thawing, and infusion through a central venous catheter is preferred. Painful irritation may occur if thawed cells are infused through a peripheral vein. Despite the hyperosmolality of the products, serum osmolality is not greatly affected for patients receiving less than about 1 g DMSO/kg body weight. Davis et al. did not detect significant hemolysis in their clinical study, as reflected by a lack of major change in hematocrit [102]. Hemoglobinuria occurs frequently [104], presumably resulting from cryopreservation-induced hemolysis of red cells within the HC inoculum. In anticipation of the infusion of hemolyzed red blood cells, many centers manage patients with urinary alkylinization and mannitol diuresis using a strategy recommended for the treatment of acute hemolytic reaction to red cell infusion. This author discontinued this practice (but maintained prophylactic mediation with an antihistaminic agent and a corticosteroid) without an increase in postinfusion renal failure [41]. The infusion of large quantities of red blood cells in marrow products that were not red cell depleted before cryopreservation was associated with the development of acute renal failure in three of 33 patients at one transplant center [76]. In contrast to the extensive studies of DMSO-induced toxicity, little attention has been given to the toxicity of nonpenetrating cryoprotectants. HES toxicity to HCs has not been reported. HES is widely used as a blood volume expander during surgery and to enhance the collection of granulocytes from healthy donors by apheresis methodology, and significant systemic toxicity from the infusion of HES is rare.

Special considerations Cryopreservation of HCs intended for allogeneic transplantation Cryopreserved HC products are not commonly used in allogeneic transplantation. The availability of a volunteer donor at time of transplantation obviates the cost and risks inherent with the cryopreservation of cells. However, when concerns about the availability of the donor arise (e.g. foreign nationals, prisoners, substance abusers or overly apprehensive donors), the collection and cryopreservation of cells before initia-

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Table 44.7 Engraftment and outcome for transplantation of cryopreserved marrow from allogeneic donors Study 1

Number of patients Days to absolute neutrophil count >500/μL Days to platelet count >50,000/μL Acute GVHD ≥II Chronic GVHD Day 100 mortality

Study 2

Study 3

Fresh

Frozen

Fresh

Frozen

Frozen

33 16 28 57.5% ND 39.4%

10 19 23 20%* ND 30%

19 17 ND 64% 38% 32%

18 18 ND 78% 55% 26%

10 21 ND 6/8 1/5 4/9

GVHD, graft-versus-host disease; ND, no data. * Difference between fresh and frozen samples significant at p = 0.037. Adapted with permission from [114] (study 1), [115] (study 2), and [117] (study 3).

tion of a myeloablative conditioning regimen may be justified. There is growing interest in the use of CBHCs for use in related- or unrelateddonor transplantation. Obviously, the only practical method of storage of CBHCs involves cryopreservation, potentially for decades. Limited experience in the allogeneic transplantation of cryopreserved marrow from related or unrelated donors has been published [114–117]. The probability and rate of engraftment were similar for recipients of cryopreserved cells and retrospective control groups of patients in the two studies that performed this analysis (Table 44.7). Long-term survival of these patients also appeared similar. Eckardt et al. however, described a significantly lower incidence of acute graft-versus-host disease (GVHD) for the recipients of cryopreserved cells [114]. Stockschlader et al. reported a similar study that found no difference in the incidence of either acute or chronic GVHD [115]. Both studies used total body irradiation or busulfan-based conditioning regimens, and cyclosporine and methotrexate for prophylaxis against GVHD for most patients. Stockschlader et al. subsequently published their experience with cryopreserved cells from unrelated donors (Table 44.7). The incidence of severe acute GVHD for recipients of cryopreserved marrow was 75% [117], which is similar to the reported experience of transplantation of noncryopreserved cells from unrelated donors transplanted at a number of centers. Recipients of CBHCs experience slower engraftment compared with recipients of either marrow or PBHCs. It is likely that these observations result from the limited cell dose available in this source of HCs, and not from the cryopreservation process. The observation that granulocyte engraftment kinetics could be hastened by the post-thaw removal of DMSO for recipients of CBHC products could also be attributed to changes in the post-transplant immunosuppressive regimen (discontinuation of post-transplant methotrexate) or other modifications of the transplant regimen for the small number of patients reported [118]. These data indicate that HCs intended for allogeneic transplantation may be cryopreserved, but with the increased costs of the additional processing and the increased risks inherent with the use of cryopreserved cells. A potential benefit from a reduced risk of GVHD proposed by one center has not been confirmed by another. For these reasons, it is likely that the cryopreservation of cells from allogeneic donors will be limited to specific situations in which the practicality of having cryopreserved cells outweighs these risks. Tumor cell purging Some investigators have suggested that cryopreservation provides a purging mechanism that will reduce the risk of relapse after autologous

HCT [119,120]. In one study, which used a rat model of acute myeloid leukemia (AML), Hagenbeek and Martens demonstrated a 30% recovery of normal CFU-Ss but only a 1.4% survival of splenic colonies derived from the leukemic cell line. Allieri et al. cloned leukemia progenitor cells (AML-CFUs) from the peripheral blood of five patients with AML before and after cryopreservation and compared the recovery of these cells with the recovery of CFU-GMs and BFU-Es from marrow specimens from healthy donors. The percentage recovery of AML-CFUs was always less than 50% of the percentage recovery of the normal cells in a series of cryopreservation experiments. Questions about the relevance of the models studied by these investigators can be raised, and whether cryopreservation of cells reduces the risk of relapse after autologous transplantation is not answered by these two studies. The demonstration using genetically marked cells that cryopreserved marrow products can be a source of relapse in patients treated for AML, neuroblastoma, and chronic myeloid leukemia suggest that cryopreservation is not a highly efficient purging technique [121–123].

Summary The cryopreservation of HCs was explored from the earliest attempts to understand HCT. It should be obvious from this discussion that current cryopreservation techniques are satisfactory for the treatment of many patients, but no study has accurately quantified the HC losses resulting from cell freezing and the impact of these losses on engraftment kinetics after HCT. Virtually all centers freeze cells with DMSO alone or in combination with HES. The similarities between the techniques used by different transplant centers are much greater than the differences. Current cryopreservation techniques may reduce the HC content of the product, risking a potential delay in engraftment if inadequate quantities of HCs are harvested and stored. Moreover, there is considerable but generally minor toxicity from the currently used cryoprotectants, and the equipment and processing techniques are expensive. The ideal cryopreservation solution is one that will achieve reproducible high cell recoveries, allow rapid cooling to minimize laboratory processing times, and be free of the toxicities associated with DMSO. More recent studies are focused on developing an understanding of apoptosis as a result of cell cooling, as opposed to cell necrosis from ice crystal formation, which may lead to modifications of cryopreservation solutions. These modifications, possibly the simple addition of inhibitors of apoptosis, may enhance engraftment kinetics with considerable savings in patient-care costs.

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98. Beaujean F, Hartmann O, Kuentz M et al. A simple, efficient washing procedure for cryopreserved human hematopoietic stem cells prior to reinfusion. Bone Marrow Transpl 1991; 8: 291– 4. 99. Rubinstein P, Dobrila L, Rosenfield RE et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Nat Acad Sci U S A 1995; 92: 10119–22. 100. Bohbot A, Lioure B, Faradji A et al. Positive selection of CD34+ cells from cryopreserved peripheral blood stem cells after thawing: technical aspects and clinical use. Bone Marrow Transpl 1996; 17: 259–64. 101. Douay L, Gorin NC, David R et al. Study of granulocyte-macrophage progenitor (CFUc) preservation after slow freezing of bone marrow in the gas phase of liquid nitrogen. Exp Hematol 1982; 10: 360–6. 102. Davis JM, Rowley SD, Braine HG, Piantadosi S, Santos GW. Clinical toxicity of cryopreserved bone marrow graft infusion. Blood 1990; 75: 781–6. 103. Stroncek DF, Fautsch SK, Lasky LC, Hurd DD, Ramsay NKC, McCullough J. Adverse reactions in patients transfused with cryopreserved marrow. Transfusion 1991; 31: 521–6. 104. Okamoto Y, Takaue Y, Saito S et al. Toxicities associated with cryopreserved and thawed peripheral blood stem cell autografts in children with active cancer. Transfusion 1993; 33: 578– 81. 105. Vriesendorp R, Aalders JG, Sleijfer DT et al. Effective high-dose chemotherapy with autologous bone marrow infusion in resistant ovarian cancer. Gynecol Oncol 1984; 17: 271–6. 106. Rapoport AP, Rowe JM, Packman CH, Ginsberg SJ. Cardiac arrest after autologous marrow infusion. Bone Marrow Transpl 1991; 7: 401–3.

107. Styler MJ, Topolsky DL, Crilley PA et al. Transient high grade heart block following autologous bone marrow infusion. Bone Marrow Transpl 1992; 10: 435–8. 108. Keung Y-K, Lau S, Elkayam U, Chen S-C, Douer D. Cardiac arrhythmia after infusion of cryopreserved stem cells. Bone Marrow Transpl 1994; 14: 363–7. 109. Lopez-Jimenez J, Cervero C, Munoz A et al. Cardiovascular toxicities related to the infusion of cryopreserved grafts: results of a controlled study. Bone Marrow Transpl 1994; 13: 789–93. 110. Alessandrino P, Bernasconi P, Calder A et al. Adverse events occurring during bone marrow or peripheral blood progenitor cell infusion: analysis of 126 cases. Bone Marrow Transpl 1999; 23: 533–7. 111. Dhodapkar M, Goldberg SL, Tefferi A, Gertz MA. Reversible encephalopathy after cryopreserved peripheral blood stem cell infusion. Ar J Hematol 1994; 45: 187–8. 112. Calmeis B, Lemarie C, Esterni B et al. Occurrence and severity of adverse events after autologous hematopoietic progenitor cell infusion are related to the amount of granulocytes in the apheresis product. Transfusion 2007; 47: 1268–75. 113. Milone G, Mercukrio S, Strano A et al. Adverse events after infusions of cryopreserved hematopoietic stem cell depend on non-mononuclear cells in the infused suspension and patient age. Cytotherapy 2007; 9: 348–55. 114. Eckardt JR, Roodman GD, Boldt DH et al. Comparison of engraftment and acute GVHD in patients undergoing cryopreserved or fresh allogeneic BMT. Bone Marrow Transpl 1993; 11: 125–31. 115. Stockschlader M, Kruger W, Kroschke G et al. Use of cryopreserved bone marrow in allogeneic bone marrow transplantation. Bone Marrow Transpl 1995; 15: 569–72.

116. Stockschlader M, Hassan HT, Krog C et al. Long-term follow-up of leukaemia patients after related cryopreserved allogeneic bone marrow transplantation. Br J Haematol 1997; 96: 382–6. 117. Stockschlader M, Kruger W, tom Dieck A et al. Use of cryopreserved bone marrow in unrelated allogeneic transplantation. Bone Marrow Transpl 1996; 17: 197–9. 118. Kurtzberg J, Laughlin M, Graham ML et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996; 335: 157–66. 119. Hagenbeek A, Martens ACM. Cryopreservation of autologous marrow grafts in acute leukemia: survival of in vivo clonogenic leukemic cells and normal hemopoietic stem cells. Leukemia 1989; 3: 535–7. 120. Allieri MA, Lopez M, Douay L, Mary JY, NGuyen L, Gorin NC. Clonogenic leukemic progenitor cells in acute myelocytic leukemia are highly sensitive to cryopreservation: possible purging effect for autologous bone marrow transplantation. Bone Marrow Transpl 1991; 7: 101– 5. 121. Brenner MK, Rill DR, Holladay MS et al. Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients. Lancet 1993; 342: 1134–7. 122. Rill DR, Santana VM, Roberts WM et al. Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 1994; 84: 380–3. 123. Deisseroth AB, Zu Z, Claxton D et al. Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML. Blood 1994; 83: 3068– 76.

45

Jürgen Finke & Roland Mertelsmann

Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation

Introduction

Erythropoietin

The field of autologous and allogeneic hematopoietic cell transplantation (HCT) has changed considerably during the last decade due to the availability of cytokine-mobilized peripheral blood hematopoietic cells (PBHCs) as a graft source. Despite the more rapid hematopoietic reconstitution compared with the use of marrow-derived grafts, high-dose chemotherapy preceding HCT still induces a period of severe marrow aplasia with the risk of infections and bleeding, requiring prophylactic and therapeutic interventions. Sepsis during the time of neutropenia represents a major problem and may result in transplant-related death. Hematopoietic growth factors and other cytokines contribute to the regulation of cellular growth and differentiation of the lymphohematopoietic system. Early-acting growth factors contribute to the differentiation of pluripotent stem cells, capable of self-renewal, into multipotent and committed progenitor cells. Late-acting growth factors lead to further differentiation, eventually giving rise to mature cell elements with distinct function. However, almost all growth factors have multiple biologic activities, partly overlapping or synergistic with other hematopoietic growth factors, and can act on more than one cell type (Table 45.1). It has been demonstrated in murine models that in addition to very primitive long-term hematopoietic cells, other progenitor cells with a lower proliferative potential and restricted differentiation capabilities contribute to the hematopoietic reconstitution after HCT [1–3]. Xenotransplantation of human hematopoietic cells into severely immunodeficient mice revealed heterogeneity in the human transplantable stem cell compartment [4,5]. Kinetic as well as cell purification studies suggest that hematopoietic reconstitution in humans is dominated by different types of short-term repopulating cells during the first months after HCT [6,7]. Myeloid-restricted short-term repopulating cells play a major role during the first month after transplantation and are followed by a second type of short-term repopulating cells able to regenerate myeloid as well as lymphoid lineages for a still undefined period of time [4]. Engraftment kinetics after HCT can be influenced by the use of recombinant hematopoietic growth factors, and numerous studies, including randomized trials, have demonstrated the effects and established the role of specific growth factors after HCT.

Erythropoietin (EPO) was the first growth factor to be identified experimentally and to be made available as a recombinant protein. EPO is widely used for the successful treatment of anemia associated with renal failure. The anemia encountered in HCT recipients is partly due to relative EPO deficiency. Frequently, patients are already anemic prior to transplantation and present with elevated serum EPO levels [8]. Inappropriately low EPO levels in relation to the severity of anemia during the period following HCT are attributed to several causes such as the toxic effects of high-dose therapy and drugs like cyclosporine on EPOproducing cells of the kidney, as well as inhibitory effects of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) on hypoxia-induced EPO production.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Recombinant human EPO after autologous HCT Several trials have addressed the value of recombinant human (rHu) EPO after transplantation to accelerate red cell regeneration and decrease the need for red blood cell (RBC) transfusions. After autologous bone marrow transplantation (BMT), EPO has been used in phase II as well as randomized phase III trials alone or in combination with granulocyte colony-stimulating factor (G-CSF) or granulocyte–macrophage colonystimulating factor (GM-CSF) [9–13]. Although EPO was tolerated without side-effects and an increase in reticulocyte counts was attained, no obvious benefit regarding transfusional requirements, engraftment or treatment outcome was observed. In a small case series of 28 patients with religious objection to receiving blood products, a combination of EPO, G-CSF, intravenous iron, and epsilon-aminocaproic acid was given after autologous peripheral blood HCT (PBHCT). With two treatment-related deaths (8%) and four bleeding complications, the median fall of hemoglobin level was 4.7 g/% and the median total number of days with a platelet count of less than 10,000/μml was 4 days. This approach was judged to be safe without the use of any blood products post autologous PBHCT [14]. rHuEPO after allogeneic HCT After allogeneic marrow transplantation, EPO enhanced reticulocytosis, increased hemoglobin levels, and reduced time to transfusion independence, resulting in the need for fewer RBC transfusions. In a large multicenter, placebo-controlled, randomized trial, 106 patients were treated with EPO after allogeneic BMT, and 109 patients with placebo [11]. Patients received either 150 IU/kg/day EPO or placebo as a

645

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Chapter 45

Table 45.1 Growth factors available for clinical use Growth factor

Gene localization

Naturally induced by

Main effector lineage

Generic name

Erythropoietin

7q21

Hypoxia

Erythroid

Epoetin-alpha, -beta, darbepoetin-alpha

Granulocyte colonystimulating factor

17q11.2–q12

IL-1, TNF-α, endotoxin

Myeloid

Filgrastim, pegfilgrastim, lenograstim

Granulocyte–macrophage colony-stimulating factor

5q31.1

TNF-α, lipopolysaccharide

Myeloid/macrophage

Molgramostin, sargramostim

IL-3

5q31.1

Activation

Progenitor/myeloid/lymphoid

–

Stem cell factor

12q22–q24

Constitutive

Myeloid/lymphoid

Ancestim

Megakaryocyte growth and differentiation factor/ thrombopoietin

3q2–q28

Constitutive (thrombocytopenia)

Megakaryocytic/progenitor

–

IL-11

19q13.3–q13.4

IL-1

Lymphoid/myeloid/megakaryocytic

Oprelvekin

IL-2

4q26–q28

Activated T lymphocytes

T/NK/B lymphocytes

Aldesleukin

Keratinocyte growth factor

15q15–q21.1

Tissue damage, platelet-derived growth factor, transforming growth factor

Epithelial cells

Palifermin

IL, interleukin; NK, natural killer; TNF-α, tumor necrosis factor-alpha.

continuous intravenous infusion. Therapy started after BMT and was continued until independence from RBC transfusions for 7 consecutive days with stable hemoglobin levels of 90 g/L or above, or until day 41 after BMT. The reticulocyte counts were significantly higher with EPO from day 21 to day 42 after BMT. The median time to RBC transfusion independence was 19 (range 16.3–21.6) days with EPO and 27 (range 22.3 to over 42) days with placebo (p < 0.003). The mean (± standard deviation) numbers of RBC transfusions until day 20 after BMT were 6.6 ± 4.8 with EPO and 6.0 ± 3.8 with placebo. From day 21 to day 41, the EPO-treated patients received 1.4 ± 2.5 (median 0) transfusions, and the control group received 2.7 ± 4.0 (median 2) transfusions (p = 0.004). In the follow-up period from day 42 to day 100, 2.4 ± 5.6 transfusions were required with EPO and 4.5 ± 9.6 were required with placebo (p = 0.075). A multivariate analysis showed that acute graft-versus-host disease (GVHD), major ABO blood group incompatibility, age above 35 years, and hemorrhage significantly increased the need for transfusions. After day 20, EPO significantly reduced the number of RBC transfusions in these patient groups. For the whole study period, EPO significantly reduced the transfusion requirements in patients with GVHD grade III or IV from 18.4 ± 8.6 to 8.5 ± 6.8 U [11]. In another phase III trial in allogeneic BMT, 50 patients were randomized to treatment with EPO (n = 25) or placebo (n = 25) [8]. EPO was given at 200 U/kg daily for 4 weeks and 200 U/kg twice weekly for a further 4 weeks. There were no differences between the two groups regarding time to engraftment, fever, hospitalization, GVHD, infections, hemorrhages, transplant-related mortality (TRM), relapse, and survival. However, more patients in the control group had an elevated serum creatinine (43% versus 14%; p = 0.04). RBC transfusion requirements for the first 2 months after BMT were significantly lower in the EPO group compared with the control group (5 U versus 10 U; p = 0.04). Time to an unsupported hemoglobin level of over 70 g/L was shorter in patients treated with EPO (14 days versus 24; p = 0.03). No effect was seen on platelet engraftment or the number of transfused platelet units. According to the protocol, the study drug was reduced (hemoglobin >

100 g/L) or discontinued (hemoglobin > 120 g/L) for a mean of 3.6 weeks among 11 EPO patients compared with 1.9 weeks among seven controls (p = 0.02) [8]. Ninety-one patients between the ages of 17 and 58 years undergoing allogeneic transplants from sibling donors were entered into a doubleblind randomized trial to evaluate the effect of EPO at a dose of 300 U/ kg/day given three times a week by intravenous injection. Treatment ended when the hemoglobin exceeded 120 g/L and recommenced if hemoglobin fell below 120 g/L, at 150 U/kg/day. If hemoglobin exceeded 120 g/L on a further occasion, the dose of EPO was not given. Patients received 2 U of erythrocytes when hemoglobin dropped below 85 g/L. Univariate analysis revealed a significantly higher reticulocyte count, hemoglobin concentration, and bone marrow erythropoiesis after day 14 in the group receiving EPO, but this was not reflected in decreased RBC transfusions (7 ± 5 in controls versus 6 ± 5 in the EPO group). However, in a multivariate analysis, the administration of EPO was associated with an 18% reduction in RBC transfusion requirement when other variables were taken into account [15]. Allogeneic HCT with major ABO blood group incompatibility may be associated with a markedly prolonged time to RBC engraftment, immune hemolysis or pure red cell aplasia. Several reports suggested a curative effect of EPO for these patients [16,17]. In a patient with normal neutrophil and platelet engraftment and pure red cell aplasia despite documentation of an elevated endogenous EPO level (360 mU/mL; normal value < 19 mU/mL) during the 230-day period of absent erythropoiesis, erythroid engraftment was observed soon after the initiation of EPO at a dose of 50 U/kg daily [16]. A 32-year-old patient with acute myeloid leukemia developed pure red cell aplasia after major ABOincompatible BMT. After receiving rHuEPO and methylprednisolone, she developed reticulocytosis and hemolysis. She promptly recovered from hemolysis and pure red cell aplasia after plasmapheresis [17]. In the era of reduced-intensity conditioning using fludarabine-based conditioning regimens, a higher incidence of pure red cell aplasia may be expected in patients with high antidonor isoagglutinins in comparison

Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation

to myeloablative regimens [18], but this does not seem to be a frequently observed problem [19]. The role of EPO was examined in the context of reduced-intensity conditioning. A phase II trial addressed the role of EPO given either from day 0 (19 patients) or starting on day 28 (27 patients) after reducedintensity conditioning and allogeneic HCT. EPO was administered subcutaneously once weekly at a dose of 500 U/kg/week, and 14 patients receiving no EPO served as the control group. Up to 6 months post transplant, hemoglobin values were significantly higher in patients receiving EPO compared with those receiving no EPO, but transfusion requirements were decreased only in the first month in patients receiving EPO from day 0. Interestingly, a reduced T-cell chimerism below 60% negatively influenced hemoglobin levels post transplant [20]. Summary: clinical use of EPO after HCT In summary, EPO stimulates erythroid engraftment after BMT, resulting in a small but significant reduction of RBC transfusion requirements. Whereas trials after autologous transplantation showed negative results, the beneficial effect after allogeneic HCT was more prominent. Late after allogeneic HCT, individual patients with delayed or inadequate erythropoiesis with inappropriate reticulocyte counts and relatively low serum EPO levels, for example patients with renal impairment, chronic inflammation or chronic GVHD, benefit from the application of EPO.

Granulocyte colony-stimulating factor G-CSF is produced by many cell types and is rapidly induced by inflammatory stimuli. In vivo studies demonstrated that G-CSF is a potent myeloid growth and differentiation factor. The dose-dependent increase of peripheral blood neutrophils is caused by expansion of the myeloid compartment in the marrow, an accelerated production of mature neutrophils, and a rapid shift of mature neutrophils from the marrow sinusoids into the peripheral blood [21]. Side-effects of rHu G-CSF administration are usually mild and include bone pain, occasionally low-grade fever, and weight gain. After highdose therapy and HCT, transient pulmonary infiltrates that cause dyspnea, especially during the early phase of hematopoietic engraftment, can occasionally be observed radiologically and clinically. High-dose corticosteroids and diuretics are the treatment of choice for this complication. The entity of the clinical findings of fever, capillary leak, and pulmonary

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infiltrates, frequently also including a skin rash, observed during engraftment after HCT has been termed “engraftment syndrome,” and posttransplant G-CSF increases the incidence of this syndrome [22]. Two recombinant preparations of G-CSF are commonly in use: filgrastim produced in Escherichia coli and the glycosylated form, lenograstim, produced in a Chinese hamster ovary cell line. Filgrastim is also available in a pegylated formula, pegfilgrastim, for single-dose use. In the following, the term “G-CSF” is generally used for the various recombinant products.

G-CSF after autologous HCT G-CSF after autologous BMT Early data suggested that neutrophil recovery after autologous BMT was accelerated in G-CSF-treated patients compared with historical controls, exceeding 0.5 × 109/L at a mean of 11 days after marrow infusion compared with 20 days for controls, a significant difference. This reduction led to significantly fewer days of parenteral antibiotic therapy, 11 versus 18 days in controls, and less protective isolation in reverse-barrier nursing, 10 versus 18 days [23]. Similar data were obtained after autologous BMT in lymphoma patients [24]. Several large randomized trials have confirmed the significantly accelerated neutrophil engraftment after autologous BMT compared with placebo (Table 45.2). In the first, G-CSF was given to 315 patients after autologous or allogeneic BMT in a prospective, randomized, placebo-controlled multicenter trial [25]. One day after bone marrow infusion, 163 patients received lenograstim 5 μg/kg/day by 30-minute infusion, and 152 patients received placebo daily for 28 days or until neutrophil recovery. Neutrophil recovery to above 109/L for 3 consecutive days was seen earlier in G-CSF-treated patients (16 versus 27 days; p < 0.001). Time to neutrophil recovery above 0.5 × 109/L was reduced (14 versus 20 days; p < 0.001). The difference was significant both in autograft (20 versus 14 days; p < 0.001) and in allograft (20 versus 14 days; p < 0.01) patients, in children (20 versus 13 days; p < 0.001), and in adults. G-CSF-treated patients had fewer days of infection and of antibiotic administration, and also spent less time in hospital. However, the incidence of clinical and microbiologic sepsis was similar in both groups. There was no significant toxicity ascribed to G-CSF. Survival was similar between groups at both days 100 and 365 after transplantation [25].

Table 45.2 Randomized, placebo-controlled trials of granulocyte colony-stimulating factor (G-CSF) following hematopoietic cell transplantation Absolute neutrophil count >500/μL (day after transplantation)

Number of patients enrolled

Type of transplant

G-CSF given from day

G-CSF

Placebo

p-value

Reference

315 43 41 54 38 62 62 54 42

Auto/allo BMT Auto BMT Auto PBHCT ± BMT Auto BMT Auto PBHCT Auto PBHCT Auto PBHCT Allo PBHCT Allo PBHCT

+1 +1 +1 +1 +1 +1 +5 ±0 +1

14 10 10 12 (14) 10 9 10 11 12

20 18 18 20 14 12.5 12 15 15

0.001 0.0001 0.0001 0.0004 0.0001 0.0001 0.0008 0.0082 0.002

[25] [26] [27] [28] [30] [31] [33] [44] [45]

Allo, allogeneic; Auto, autologous; BMT, bone marrow transplantation; PBHCT, peripheral blood hematopoietic cell transplantation.

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Chapter 45

Patients with lymphoma were treated in a randomized, open-label trial to study the use of G-CSF as an adjunct to high-dose chemotherapy and autologous BMT [26]. Of 43 patients, 19 were randomized to receive filgrastim by continuous subcutaneous infusion at a dose of 10 μg/kg/ day, 10 to filgrastim 20 μg/kg/day, and 14 to a parallel control group that received no filgrastim. The median time to neutrophil recovery of 0.5 × 109/L or more after the day of autologous BMT was significantly accelerated to 10 days in the combined G-CSF groups compared with 18 days in control patients. The median number of platelet transfusions was identical in both groups. Clinical parameters, including the median number of days with fever (1 versus 4) and neutropenic fever (5 versus 13.5), were significantly improved in the G-CSF group compared with the control group. For patients treated with the two different dose levels of G-CSF, the neutrophil recovery and clinical results were similar [26]. Forty-one patients undergoing high-dose therapy followed by infusion of autologous PBHCT with or without bone marrow were randomized to receive G-CSF 5 μg/kg/day beginning on day +1, or no G-CSF [27]. The median time to a neutrophil count of 500/μL or more was significantly shorter – 10.5 days in the G-CSF group compared with 16 days in the control group. G-CSF was associated with statistically significant reductions in the time to neutrophil engraftment among patients who received PBHCs alone (11 versus 17 days) and in patients who received PBHCs in conjunction with bone marrow (10 versus 14 days) [27]. In 54 patients with malignant lymphoma, hematopoietic recovery after high-dose chemotherapy and autologous BMT was compared between patients randomized to receive 10 or 30 μg/kg/day of filgrastim or no growth factor [28]. After high-dose chemotherapy with a cyclophosphamide (CY), VP-16, BCNU (CVB) or a BCNU, etoposide, cytosine arabinoside, melphalan (BEAM) regimen followed by autologous BMT, filgrastim was administered by continuous intravenous infusion from the first day after autologous BMT until neutrophil recovery. When the filgrastim groups were compared with the control group, the major findings were: the median time to reach an absolute neutrophil count (ANC) of 0.5 × 109/L or more was 20 days in the control group and 12 and 14 days, respectively, in the filgrastim groups (p = 0.0004). The duration of neutropenia (ANC < 0.5 × 109/L) was reduced from 27 days in the control group to 11 and 13 days in the G-CSF groups (p = 0.0001). In addition, fewer days of febrile neutropenia were observed in the GCSF groups (5 and 6 days) than in the control group (10 days; p = 0.036). No significant effect on the total number of days with fever was observed [28]. An accelerated neutrophil engraftment was observed in 41 consecutive children undergoing autologous BMT for hematologic malignancies in comparison with a similar historical control group of 38 children who did not receive G-CSF after autologous BMT. Their ages ranged from 2 to 16 (mean 7.2) years. G-CSF was given at a dose of 10 μg/kg/day intravenously in a 2-hour infusion from day +1 until +28 or until the ANC was above 1 × 109/L [29]. G-CSF after autologous PBHCT The results obtained after autologous BMT were essentially confirmed in several phase II and III trials (Table 45.2) addressing the role of GCSF after myeloablative chemotherapy and autologous cytokine mobilized PBHCT [30–33]. Of note, since engraftment after PBHCT is generally accelerated compared with unstimulated BMT, the potential benefit of G-CSF given post PBHCT is likely to be smaller. Thirty-eight patients with lymphoproliferative disorders were randomized to receive low-dose filgrastim (19 patients) or placebo (19 patients) beginning on the first day after HCT [30]. All patients received more than 2.5 × 106 CD34+ cells/kg, which had been mobilized with

chemotherapy and filgrastim 300 μg from day 5. Neutrophil engraftment was significantly more rapid in patients who received G-CSF, with a median number of days until ANC was greater than 0.5 × 109/L of 10 (range 9–13) versus 14 (range 9–19). The time to reach an ANC greater than 1 × 109/L was 12 (range 9–14) versus 16 (range 10–25) days. The total number of patients who required intravenous antibiotic therapy was lower in the G-CSF-treated group (68%) compared with the placebo group (89%); also, the median number of days with fever and the duration of antibiotic therapy were shorter, although these differences did not reach statistical significance. However, although only three of 19 (16%) patients who received G-CSF required amphotericin, 11 of 19 (58%) who received placebo did require it, and amphotericin usage was significantly less in the G-CSF group (p = 0.029). Finally, inpatient stay was significantly shortened in those who received G-CSF, from 16 (range 13–23) to 13 (range 11–18) days [30]. In another prospective study, 34 patients had been randomized to receive lenograstim and 28 to receive no growth factor [31]. The median time to an ANC of over 0.5 × 109/L was 9 days in the G-CSF arm versus 12.5 days in the no-G-CSF arm (p = 0.0001). The median number of days to platelet independence, platelet transfusions, incidence of infection, and RBC transfusion was the same in both arms [31]. Factors influencing hematopoietic engraftment were analyzed within the French multicenter LNH93-3 trial treating patients with aggressive high-grade non-Hodgkin’s lymphoma with intensive chemotherapy including BEAM and autologous PBHCT [32]. In the patient population treated with the same first-line regimen, bone marrow involvement and infusion of fewer CD34+ cells delayed platelet recovery. Administration of G-CSF after PBHCT significantly reduced neutropenia [32]. The effect on accelerated neutrophil engraftment by G-CSF appears to be preserved when the start of application is delayed to day 5 after HCT. Sixty-two adult patients were randomized to receive filgrastim after PBSC infusion (n = 30), and 32 patients, the control group, received no cytokines [33]. G-CSF was administered subcutaneously from day +5 in the treated group at a dose of 5 μg/kg body weight per day. The numbers of CD34+ and mononuclear cells infused were similar in each group. Faster granulocyte engraftment was evident in the treated group (mean of 10 versus 12 days to achieve more than 0.5 × 109/L granulocytes; p = 0.0008), without differences in the incidence and severity of infections, number of days of fever or duration of antibiotic treatment between groups. Considering the economic costs, the median expenditure per inpatient stay was c5961 (range c4386–17,186) in the G-CSF group compared with c5751 (range c3676–15,640) in the control group (p = 0.47) [33]. Several randomized trials showed no delayed neutrophil engraftment when G-CSF was not given before days 3, 5, 6, or 7 after PBHCT [34–36]. Early (day +1) or delayed (day +7) G-CSF after immunoselected CD34+-autologous PBHCT significantly accelerated ANC recovery but did not reduce the amount of supportive treatment or the duration of hospitalization in 21 consecutive patients with hematological malignancies [37]. A randomized trial compared pegfilgrastim with filgrastim after highdose melphalan and autologous PBHCT in 37 patients with multiple myeloma receiving a single 6 mg dose of pegfilgrastim on day 1 post transplant versus daily subcutaneous injections of filgrastim 5 μg/kg starting on day 5 post transplant. The median duration of grade 4 neutropenia in the pegfilgrastim and filgrastim groups was 5 and 6 days, respectively (p = not significant). The results for the two groups were also not significantly different for time to neutrophil and platelet recovery, but the incidence of febrile neutropenia (61.1% versus 100%; p = 0.003) and the duration of febrile neutropenia (1.5 versus 4 days; p = 0.005) were lower in the pegfilgrastim arm, demonstrating the safety and efficacy of this approach [38].

Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation

G-CSF after allogeneic HCT Initial concerns over potential aggravation of GVHD or an increase of relapse in patients with myeloid leukemias tempered the use of growth factors after allogeneic BMT. Essentially, after allogeneic BMT, the same effects can be observed as after autologous transplantation. Schriber et al. reported the experience with 50 patients treated at a single institution using G-CSF after allogeneic sibling (n = 30) and matched unrelated (n = 20) BMT [39]. The time to an ANC or 500/μL of greater was significantly less in patients who received G-CSF and cyclosporine and prednisone for GVHD prophylaxis when compared with historical control patients receiving the same GVHD prophylaxis without G-CSF (10 versus 13 days; p < 0.01). A similar accelerated myeloid engraftment was observed for patients who received additional methotrexate for GVHD prophylaxis when compared with historical control patients receiving the same GVHD prophylaxis regimen (16 versus 19 days; p < 0.05) [39]. Similar results were obtained in children [40]. Effects of G-CSF in patients undergoing allogeneic BMT from volunteer unrelated donors were analyzed retrospectively [41]. Cohorts of patients received G-CSF (n = 22) or no G-CSF (n = 25) in a nonrandomized manner. Median time to ANC of over 500/μl was 14 days with G-CSF versus 16 days without G-CSF (p = 0.048). G-CSF did not influence platelet recovery and incidence of infectious complications [41]. Delayed treatment with G-CSF resulted in a reduction of G-CSF treatment from 19 days to 14 days (p = 0.0017) in 38 patients randomly assigned to receive G-CSF starting on day 1 or day 6 after allogeneic BMT [42]. Reducing the length of treatment by 5 days lowered G-CSF treatment costs by 26.3%, and postponing treatment with G-CSF had no influence on the hematologic recovery after allogeneic BMT [42]. Similar results were obtained in 69 patients after unrelated donor BMT randomly assigned to G-CSF starting on day 0, +5 or +10, resulting in equivalent rapid neutrophil engraftment [43]. A randomized, double-blind, placebo-controlled study was performed by Bishop et al. to determine the effects of G-CSF on hematopoietic recovery after allogeneic PBHCT [44]. Fifty-four patients with hematologic malignancies undergoing related, HLA-matched allogeneic HCT were randomly assigned to receive daily G-CSF at 10 μg/kg or placebo starting on the day of transplantation. The median time to achieve an ANC of greater than 0.5 × 109/L was 11 (range 9–20) days for patients who received filgrastim compared with 15 (range 10–22) days for patients who received placebo (p = 0.0082). The median time to achieve a platelet count exceeding 20 × 109/L was 13 (range 8–35) days for patients who received G-CSF compared with 15.5 (range 8–42) days for patients who received placebo (p = 0.79). There were no significant differences for independence from RBC transfusion, the incidence of acute GVHD or 100-day mortality between the groups. The authors concluded that the administration of G-CSF appeared to be a safe and effective supportive care measure following allogeneic peripheral blood HCT [44]. Forty-two adult recipients of allogeneic PBHCT from HLA-matched related donors were randomized to receive G-CSF 10 μg/kg per day subcutaneously from day 1 through neutrophil recovery or no growth factor support after transplantation [45]. There was no significant difference between the two groups in the number of CD34+ cells infused (median 4.8 versus 4.3 × 106/kg). GVHD prophylaxis consisted of tacrolimus and steroids for nine patients and tacrolimus and mini-methotrexate for 33 patients. The group receiving G-CSF had a shorter time to neutrophil levels above 0.5 × 109/L (day 12 versus day 15; = 0.002) and to neutrophil levels over 1.0 × 109/L (day 12 versus day 16; p = 0.01) [45]. After full haplotype-mismatched HCT in humans, the application of GCSF after transplantation markedly delayed T lymphocyte reconstitution, and omitting G-CSF led to a more rapid increase of T cells and improved anti-infectious defense in this specific situation [46].

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A retrospective analysis of the European Group for Blood and Marrow Transplantation addressed the use of G-CSF after allogeneic HCT in 1789 patients with acute leukemia receiving BMT and 434 patients receiving PBHCT from HLA-identical siblings, from 1992 to 2002 [47]. Among the BMT and PBHCT patients, 501 (28%) and 175 (40%), respectively, were treated with G-CSF during the first 14 days after transplantation. BMT and PBHCT patients treated with G-CSF had a faster engraftment of absolute neutrophils, but platelet engraftment was slower. In the BMT patients, GVHD grade II–IV was 50% in the G-CSF group versus 39% in the controls (relative risk [RR] 1.33; p = 0.007, in the multivariate analysis). The incidence of chronic GVHD was also increased (RR 1.29; p = 0.03). G-CSF was associated with an increase in TRM (RR 1.73; p = 0.00016) and had no effect on relapse, but it reduced survival (RR 0.59; p < 0.0001) and leukemia-free survival (RR 0.64; p = 0.0003) rates. No such effects of G-CSF were seen in patients receiving PBHCs [47]. A meta-analysis based on 34 randomized controlled trials of prophylactic G-CSF and GM-CSF after autologous and allogeneic HCT demonstrated a reduced risk of documented infections and duration of parenteral antibiotics, but not in infection-related mortality [48]. The absolute decrease in the risk of documented infection was 8%, and 13 patients would need to be treated with colony-stimulating factors in order to prevent one infection. Subgroup analyses regarding autologous and allogeneic HCT, the use of G-CSF and GM-CSF, or PBHCT and BMT showed similar results. Specifically, in all allogeneic subgroups, the use of CSFs was associated with less TRM and improved survival, although statistically not significant. Furthermore, colony-stimulating factors did not increase the risk of acute or chronic GVHD and TRM after allogeneic HCT [48]. This analysis demonstrates the importance of randomized trials and highlights the potential pitfalls of premature conclusions from retrospective analyses. G-CSF in murine allogeneic transplant models Apart from these clinical findings, data obtained in murine models of allogeneic HCT point towards possible clinically relevant effects of GCSF on the immune system. G-CSF polarized donor T cells towards a T helper 2 (Th2) cell type, resulting in decreased alloreactivity [49], and furthermore decreased the allostimulatory capacity of antigenpresenting cells [50]. Administration of recombinant G-CSF to C57BL/Ka mice markedly increased the capacity of peripheral blood mononuclear cells (PBMCs) to reconstitute lethally irradiated syngeneic hosts. T- and B-lineage lymphocytes were depleted about 10-fold in the bone marrow of the treated mice, and the T-cell yield in the blood was increased about fourfold. The ability of PBMCs or purified CD4+ and CD8+ T cells to induce acute lethal GVHD in irradiated BALB/c mice was reduced after the administration of G-CSF. This effect was associated with decreased secretion of interferon gamma and interleukin-2 (IL-2), and an increased secretion of IL-4. The donor cell inoculum, which was most successful in the rescue of irradiated allogeneic hosts, was the low-density fraction of PBMCs from G-CSF-treated mice. These low-density cells were enriched for CD4−CD8−NK1.1+ T cells and secreted about 10-fold more IL-4 than the unfractionated cells from the G-CSF-treated donors [49]. Another study investigated whether G-CSF administration to PBHCT recipients, both with and without donor G-CSF pretreatment, further modulated acute GVHD in a murine model of PBHCT [50]. Recipients of G-CSF-mobilized splenocytes showed a significantly improved survival and a reduction in GVHD score and serum lipopolysaccharide levels compared with control recipients. G-CSF treatment of donors, rather than recipients, had the most significant effect on reducing levels of TNF-α 7 days after transplantation. As a potential mechanism of the

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reduction in TNF-α, G-CSF was shown to decrease dendritic cell TNFα and IL-12 production to lipopolysaccharide. G-CSF modulated GVHD predominantly by its effects on donor cells, reducing the production of TNF-α. The authors concluded that G-CSF treatment of BMT recipients, without pretreatment of the donor, did not have an impact on acute GVHD [50]. Summary: clinical use of G-CSF after HCT In summary, G-CSF application after allogeneic or autologous bone marrow or PBHCT significantly accelerates the time to neutrophil engraftment without negatively influencing other parameters such as platelet engraftment or GVHD. This effect is more prominent after BMT and is preserved when the start of G-CSF application is delayed from day +1 to +5 or +7 after HCT. Apart from this, present randomized trials fail to show a clear beneficial effect on other factors such as the outcome of patients after HCT. In contrast to autologous HCT, the clinical situation is more complex after allogeneic HCT, and the present data do not allow definite conclusions regarding specific patient situations. The positive effect of G-CSF on acute GVHD in murine allogenic HCT models is not clearly seen in man. This is likely to be due to a more complex situation in the clinical transplantation of patients with different malignant diseases, to state of remission, and to other influencing factors. Despite the retrospective character of the above-mentioned European Group for Blood and Marrow Transplantation study, its results showing a negative influence of G-CSF after allogeneic HCT remains a caveat. The potential immune-modulating effects of G-CSF on mobilized stem cell grafts, as well as G-CSF application to the recipient and a possible aggravation of an “engraftment syndrome,” should be kept in mind when treating patients with G-CSF after allogeneic HCT. Granulocyte–macrophage colony-stimulating factor GM-CSF can be produced by many different cell types, particularly activated T cells. In vitro, GM-CSF promotes the growth and expansion of granulocytic and monocytic, and, in combination with EPO, also multilineage colony-forming units. Multiple functions of mature neutrophils and macrophages, such as tumoricidal activity, superoxide production, antibody-dependent cell-mediated cytotoxicity, phagocytic activity, microbial killing, and secretion of other cytokines are stimulated [51,52].

Furthermore, GM-CSF, when used in vitro, induces the differentiation of malignant antigen-presenting cells with potent T-cell stimulatory capacity [53]. In humans, GM-CSF causes a dose-dependent increase in mainly blood neutrophils and eosinophils, but also macrophages and lymphocytes. There is no effect on the erythroid or megakaryocytic lineage. After application, patients may experience low-grade fever, fatigue, myalgias, and dyspnea. After higher doses, significant fluid retention, pericarditis, pleuritis, and a capillary leak syndrome may develop [54,55]. Commercially available recombinant preparations of GM-CSF (molgramostin and sargramostim) differ in their state of glycosylation. However, this difference does not appear to be clinically relevant. GM-CSF after autologous HCT In a randomized, double-blind, placebo-controlled trial, 128 patients undergoing autologous BMT for lymphoid malignancies were enrolled [56]. Fifty-six patients received GM-CSF in a 2-hour intravenous infusion daily for 21 days, starting within 4 hours of marrow infusion, and 63 patients received placebo. The patients given GM-CSF had a recovery of the neutrophil count to 500 × 106/L 7 days earlier than the patients who received placebo (19 versus 26 days; p < 0.001), had fewer infections, required 3 fewer days of antibiotic administration (24 versus 27 days; p = 0.009), and required 6 fewer days of initial hospitalization (median, 27 versus 33 days; p = 0.01). There was no difference in the survival rate at day 100 after HCT [56]. The data were essentially confirmed by other large randomized trials in autologous BMT [57] and PBHCT (Table 45.3) [58], and in a trial where GM-CSF and G-CSF were used sequentially after autologous transplantation [59]. In contrast, no statistical difference with regard to hematopoietic engraftment of any lineage or clinical parameters was seen in 50 patients of a randomized trial comparing GM-CSF with placebo after autologous PBHCT with GM-CSF-mobilized grafts [60]. GM-CSF was compared with G-CSF post autologous transplantation in a randomized trial with 42 patients with breast cancer randomized to receive filgrastim versus molgramostim subcutaneously at a dose of 5 μg/kg starting on day 6 after PBHCT. PBHCs were collected in all patients after stimulation with filgrastim and infused following highdose chemotherapy. The median days to reach more than 0.5 × 109/L

Table 45.3 Randomized, placebo-controlled trials of granulocyte–macrophage colony-stimulating factor (GM-CSF) following stem cell transplantation

Number of patients enrolled 128 81 343 44 50 40 57 109

Absolute neutrophil count >500/μL (day after transplantation)

Type of transplant

GM-CSF given from day

GM-CSF

Placebo

p-value

Reference

Auto BMT Auto BMT Auto BMT Auto PBHCT Auto PBHCT Allo BMT (sib) Allo BMT (sib, T-deplet.) Allo BMT (sib; CyA, Pred)

±0 ±0 +1 +1 +1 +1 ±0 ±0

19 15 19 14 12 13 16 13

26 28 27 21 14 16 20 17

0.001 0.001 0.001 0.001 0.22 (n.s.) n.s. n.s. 0.0001

[56] [57] [58] [58] [60] [64] [65] [66]

Allo, allogeneic; Auto, autologous; BMT, bone marrow transplantation; CyA, Pred, prophylaxis for graft-versus-host disease with cyclosporine A and prednisone; n.s., not significant; PBHCT, peripheral blood hematopoietic cell transplantation; sib, matched sibling donor; T-deplet., T-cell-depleted graft.

Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation

granulocytes was similar for patients receiving G-CSF (10.5 ± 0.8 days) and GM-CSF (10.2 ± 0.9 days). No significant differences were observed in platelet engraftment or in the number of platelets transfused. Time of discharge was 2 days earlier in the G-CSF arm (15 ± 4.2 versus 17.4 ± 4.7 days; p = 0.04). Finally, the incidence of adverse side-effects attributable to the cytokines (G-CSF or GM-CSF) was equivalent and only present in 19% of the patients [61]. Another study compared the use of G-CSF (5 μg/kg/day) with sargramostim (GM-CSF) 500 μg/day from day 0 until neutrophil recovery (ANC > 1500/μl) in patients with breast cancer or myeloma who had PBSCs mobilized with the combination of CY, etoposide, and G-CSF [62]. Twenty patients with breast cancer or myeloma received GM-CSF, and 26 similar patients received G-CSF. The patients were comparable for age and stage of disease, and received grafts that were not significantly different. Platelet recovery, transfusion requirements, fever days, and use of antibiotics were similar in both groups. The recovery of neutrophils, however, was faster using G-CSF. Achievement of an ANC of more than 500/μl and more than 1000/μl was reached in the GM-CSF group at 10.5 ± 1.5 and 11.0 ± 1.7 days, respectively, whereas with GCSF only 8.8 ± 1.2 and 8.9 ± 2.2 days were required (p < 0.001). This study suggests that neutrophil recovery occurs more quickly when using G-CSF in comparison to GM-CSF at the specific doses used following autologous PBHCT [62]. A randomized trial addressed the in vivo effect of GM-CSF and GCSF administration on the immune recovery of 24 patients who underwent autologous BMT [63]. G-CSF contributed to a significantly faster recovery of CD8+ cells (p = 0.03). The CD8+ cell regeneration consisted mainly of activated cells (CD38+/HLA-DR+) which lacked the CD11b antigen. In contrast, GM-CSF favored the regeneration of CD4+ cells (through both the CD45RO+ and CD45RA+ subset), leading to a higher CD4+ : CD8+ ratio (p = 0.007). Furthermore, the use of hematopoietic growth factors did not seem to exert a significant influence on the recovery of natural killer cells and B lymphocytes [63].

GM-CSF after allogeneic HCT GM-CSF was also tested in randomized trials after allogeneic transplantation. In a randomized, double-blind trial in patients undergoing HCT for leukemia, 20 received GM-CSF and 20 received placebo for 14 days after allogeneic, matched-sibling BMT [64]. The neutrophil count recovered to 0.5 × 109/L 3 days earlier in the GM-CSF group than in the placebo group (not significant). No difference in GVHD or relapse was reported. In a prospective randomized, placebo-controlled trial involving 57 patients receiving T-cell-depleted marrow transplants, GM-CSF led to higher neutrophil and monocyte counts for 6–10 days during the time period of 2–3 weeks after transplantation, resulting in fewer pneumonias [65]. In a prospective, multicenter, randomized, double-blind, placebocontrolled trial, GM-CSF or placebo was administered by 4-hour intravenous infusion starting on the day of marrow infusion (day 0) to day 20 [66]. All patients received marrow grafts from HLA-identical siblings and cyclosporine and prednisone for GVHD prophylaxis. Fifty-three patients received GM-CSF and 56 received placebo. The time to achieve an ANC of over 0.5 × 109/ L was significantly shortened in GM-CSFtreated patients (day 13 versus 17). The incidence of grade III–IV mucositis and infection were significantly reduced and the duration of hospitalization was modestly shortened by 1 day (p = 0.02) in GM-CSFtreated patients. No differences in platelet recovery, erythrocyte recovery, incidence of veno-occlusive disease, GVHD severity, relapse or survival were observed [66]. Of note, in no trial was methotrexate used as GVHD prophylaxis, and when considering GM-CSF post HCT, the

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concomitant use of a growth factor and an antimetabolite may be counterproductive. A long delay in hematologic recovery after BMT, especially graft failure, can extend and amplify the risks of infection and hemorrhage, compromise patients’ survival, and increase the duration and cost of hospitalization. GM-CSF was evaluated in 37 patients with marrow graft failure after allogeneic (n = 15), autologous (n = 21) or syngeneic (n = 1) BMT [67]. GM-CSF was administered by 2-hour infusion at doses between 60 and 1000 μg/m2/day for 14 or 21 days. At doses of less than 500 μg/m2, GM-CSF was well tolerated and did not exacerbate GVHD in allogeneic transplant recipients. No patient with myelogenous leukemia relapsed while receiving GM-CSF. Twenty-one patients reached an ANC of 0.5 × 109/L or above within 2 weeks of starting therapy, while 16 did not. None of seven patients who received chemically purged autologous marrow grafts responded to GM-CSF. The survival rates of GM-CSF-treated patients were significantly better than those of a historical control group [67]. A prospective, randomized trial compared GM-CSF (250 μg/m2/day for 14 days) versus sequential GM-CSF for 7 days followed by G-CSF (5 μg/m2/day for 7 days) as treatment for primary or secondary graft failure after BMT [68]. Eligibility criteria included failure to achieve a white blood cell count of 100/μL or more by day +21 or 300/μL or more by day +28, no ANC of 200/μL or above by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, eight related allogeneic, and five autologous), and 24 received GM-CSF followed by G-CSF (12 unrelated, seven related allogeneic, and five autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC of 500/μL or more) occurred between 2 and 61 (median 8) days after therapy, while those receiving GM-CSF + G-CSF recovered at a similar rate of 1–36 days (median 6 days; p = 0.39). Recovery to independence from RBC transfusion was slow, occurring 6–250 (median 35) days after enrollment with no significant difference between the two treatment groups (GMCSF median 30 days; GM-CSF + G-CSF median 42 days; p = 0.24). Similarly, independence from platelet transfusion was delayed until 4– 249 (median 32) days after enrollment, with no difference between the two treatment groups (GM-CSF median 28 days; GM-CSF + G-CSF median 42 days; p = 0.38). Recovery times were no different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone, yielding 100-day survival estimates of 96 ± 8% for GM-CSF versus 71 ± 18% for GM-CSF + G-CSF (p = 0.026). Sequential growth factor therapy with GM-CSF followed by G-CSF offered no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT [68]. Summary: clinical use of GM-CSF after HCT In summary, GM-CSF accelerates neutrophil engraftment after autologous and allogeneic transplantation. However, due to a lack of advantage over G-CSF, GM-CSF is not routinely used for this purpose. GM-CSF has been used effectively in bone marrow failure states. The immunostimulating effects of GM-CSF, with its capacity to differentiate antigenpresenting cells, are employed with or without concomitant donor lymphocyte infusions for relapse of leukemia after allogeneic transplantation, and a beneficial role of the combination of alpha-interferon and GM-CSF is suggested [69]. Furthermore, GM-CSF may have a place as an adjunct in post-transplant vaccination trials and in anti-infectious strategies, for example in treating patients with invasive fungal infections by augmenting the phagocytic capacities of monocytes and macrophages.

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Interleukin-3 IL-3, also known as multi-CSF, is a multilineage hematopoietic growth factor produced by T lymphocytes and mast cells that acts on early and committed cell populations of all lineages. In vivo, IL-3 administration is often accompanied by severe side-effects with fever, malaise, chills, headache, arthralgias, and urticaria [70]. Because of these side-effects, a dose-escalating study using IL-3 alone had to be stopped at 2 μg/kg/ day [70]. Used alone, IL-3 did not enhance neutrophil engraftment compared with the use of GM-CSF [71]. IL-3 was used in combination with G-CSF in 54 consecutive patients with refractory or relapsed lymphoma [72]. Four of 31 patients (12.9%) who received IL-3 subcutaneously experienced one severe adverse event, defined as World Health Organization (WHO) grade 3–4 toxicity (fever, n = 2; pulmonary toxicity, n = 2) and were withdrawn from the study. The addition of IL-3 resulted in a significant improvement of multilineage hematopoietic recovery, lower transfusion requirements, fewer documented infections, and a shorter stay in the hospital [72]. In summary, due to the side-effects and only modest positive impact on hematopoiesis, IL-3 or its modifications have not been licensed for clinical use after HCT.

Stem cell factor Stem cell factor (SCF), also known as steel factor, kit-ligand or mast cell growth factor, promotes the proliferation and differentiation of the most primitive hematopoietic progenitor cells into committed progenitor cells. Recombinant human SCF (ancestim) has been developed for clinical use in combination with G-CSF to optimize stem cell mobilization from the marrow into the peripheral blood for apheresis and to provide a sustained increase in the number of hematopoietic stem cells capable of engraftment [73]. When used for hematopoietic cell mobilization, a considerable number of patients reacted with delayed-type hypersensitivity such as skin rash, dyspnea, angioedema, and cardiovascular symptoms. No trials with the use of SCF after HCT have been published. In a single case report, an unexpectedly rapid rise in platelet count was observed with complete hematological recovery after the BEAM regimen in a patient who could not be rescued by autologous transplant but who received G-CSF, epoetin-alpha, and SCF [74]. The authors attributed these findings to this specific growth factor combination, including SCF [74]. Presently, there is no proven indication for SCF after transplantation, and there is no licensed product available for clinical use.

Thrombopoietin/megakaryocyte growth factor Thrombopoietin (TPO) stimulates the growth and differentiation of megakaryocytes and proplatelets via the receptor c-mpl. It is predominantly produced by hepatocytes, and serum levels are inversely correlated with platelet counts. Two ligands for the mpl receptor with broad in vitro megakaryocytic proliferative, maturational, and differentiation capacity have been developed and tested in clinical trials. One is a modified (PEGylated) form of a recombinant truncated mpl ligand, called megakaryocyte growth and development factor (MGDF) [75]. The other is the recombinant glycosylated version of the naturally occurring TPO molecule [76]. In a randomized trial, 47 patients with stage II, III or IV breast cancer undergoing autologous HCT were allocated to placebo (n = 13) or to one of five sequential dose cohorts of PEG-rHuMGDF (n = 34), resulting in a median time to platelet recovery of 11 and 12 days for the placebo and combined PEG-rHuMGDF groups, respectively [77]. In an open-label phase I study, TPO was administered intravenously by bolus

after autologous BMT without the development of neutralizing antibodies [76]. A case of pancytopenia associated with the development of neutralizing antibodies to TPO occurred in a patient who had undergone chemotherapy repeatedly with multiple cycles of subcutaneous administration of PEGylated rHuMGDF [78]. Samples of the patient’s bone marrow showed trilineage hypoplasia with absence of myeloid, erythroid, and megakaryocyte progenitor cells, but with elevated endogenous levels of EPO, G-CSF, and SCF. The subcutaneous route of application of TPO in this trial may have been of importance for the development of anti-TPO antibodies. The role of recombinant megakaryocyte growth factors after HCT has not been thoroughly tested and remains unclear. Furthermore, due to reports on the development of neutralizing antibodies and thrombophilia in vivo, the above-mentioned recombinant products have not been licensed for clinical use. After the experience of these first-generation thrombopoietic growth factors, several second-generation thrombopoietic factors are under development, as summarized in a review by Kuter [79]. AMG 531 is a TPO-mimetic peptide linked to human immunoglobulin G fragments, and weekly subcutaneous applications in healthy volunteers and patients with immune thrombocytopenia resulted in elevated platelet counts without major side-effects apart from mild headache at the day of injection. Phase III trials are presently underway [79]. Further developments include the orally available small molecules eltrombopag and AKR-501 with TPO-agonistic properties [79]. These developments will likely result in clinically available platelet stimulating drugs.

Interleukin-11 IL-11 is a naturally occurring cytokine produced by fibroblasts and bone marrow stromal cells and is a growth factor with pleiotropic effects overlapping those of other growth factors. In vivo, IL-11 administration stimulates megakaryopoiesis and increases peripheral platelet and neutrophil counts [80], an effect that can be enhanced by combination with IL-3. A recombinant preparation of IL-11, oprelvekin, produced in E. coli, is licensed for clinical use for the prevention of severe thrombocytopenia and reduction of the need for platelet transfusions following myelosuppressive chemotherapy. Major side-effects include fluid retention and edema. A placebo-controlled, randomized trial with 80 breast cancer patients tested the effects of recombinant IL-11 on platelet recovery after highdose chemotherapy and autologous transplantation with PBHC support. The results did not demonstrate that treatment with rHuIL-11 significantly decreased platelet transfusion [81]. In a murine allogeneic BMT model of GVHD directed against major histocompatibility antigens and minor antigens, an immunomodulating effect of IL-11 with prevention of lethal GVHD while preserving the graft-versus-leukemia effect was demonstrated [82]. IL-11 decreases cytokine release and increases survival in murine BMT models. In these systems, it reduces gut permeability, partially polarizes T cells to a Th2 phenotype, downregulates IL-12, prevents mucositis, and accelerates recovery of oral and bowel mucosa. In a randomized, double-blind pilot study, IL-11 was administered with cyclosporine/MTX prophylaxis after conditioning with CY/total body irradiation (TBI) and allogeneic stem cell transplantation for hematologic malignancies [83]. Patients received IL-11 50 μg/kg/day subcutaneously or placebo. Only 13 patients (10 IL-11, three placebo) were enrolled because the early stopping rule for mortality was enforced. Of 10 evaluable patients who received IL-11, four died by day 40 and one on day 85. Deaths were attributable to transplant-related toxicity. One of three placebo recipients died of a suicide; the other two are alive. Patients receiving IL-11 had severe fluid retention and early mortality,

Use of Recombinant Growth Factors After Hematopoietic Cell Transplantation

making it impossible to determine whether IL-11 given in this schedule could reduce the rate of GVHD. Grade II–IV acute GVHD occurred in two of eight evaluable patients on IL-11 and one of three patients on placebo. The primary adverse events of the study were severe fluid retention resistant to diuresis (average weight gain 9 ± 4%) and multiorgan failure in five of 10 evaluable patients. The authors concluded that IL-11 as administered in this trial for GVHD prophylaxis in allogeneic transplantation could not be recommended [83].

Interleukin-2 IL-2 is produced by activated T lymphocytes and is an important regulator of the immune system, acting on T, B, and natural killer cells. IL-2 knockout mice develop T cells normally, but they suffer from generalized fatal immunoproliferative disorders and loss of self-tolerance. Recombinant IL-2, aldesleukin, is licensed for the treatment of metastatic renal cell carcinoma. Several phase II trials used IL-2 after autologous HCT for patients with lymphoma or acute myeloid leukemia as an immunomodulator to reduce relapse rates. Fifty-six patients with advanced lymphoma received autologous peripheral blood-derived hematopoietic grafts incubated with IL-2 followed by IL-2 4 mIU/m2 for the first 4 weeks post transplant and IL-2 maintenance. The 3-year progression-free survival of 31% was similar to that of a previous study cohort receiving no IL-2 and was not dependent on the actual IL-2 dose received; IL-2-related toxicity was considerable but reversible [84]. A similar approach was chosen in 59 patients with stage II–IV breast cancer, testing IL-2 in a randomized way [85]. Three-year progressionfree survival was 53% and 48% for patients receiving or not receiving IL-2, without any statistical difference. Of note, some of the patients developed the clinical syndrome of acute GVHD [85]. High-dose IL-2 (9 mIU/m2/day × 4) was given to 39 patients with acute myeloid leukemia in first complete remission, about one-third with good-risk core binding factor leukemias, after engraftment from autologous HCT, resulting in a 2-year progression-free survival of 74% and a TRM rate of 4% [86]. Without large randomized trials, the role of IL-2 in the context of autologous HCT remains unclear. In a pilot trial, the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and nine patients with chronic myeloid leukemia after allogeneic HCT were examined [87]. Overall, IL-2 treatment resulted in a median 1.9-fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a median 9.7-fold increase in FOXP3 expression in CD3+ T cells [87]. Successful treatment with IL-2 0.5 mIU/ m2/day by continuous intravenous infusion of a patient with progressive multifocal leukencephalopathy developing after allogeneic HCT was reported [88], highlighting the role of IL-2 as a potent immunomodulator after allogeneic HCT. Furthermore, IL-2 has an effect by enhancing the antileukemic action of donor lymphocyte infusions for relapsing leukemia after allogeneic HCT [89], a topic covered in a different section of this book.

Keratinocyte growth factor Keratinocyte growth factor (KGF), also called fibroblast growth factor7, is a mediator of epithelial cell proliferation and a growth factor for hepatocytes and pneumocytes. KGF is naturally produced in mesenchymal cells and acts on a wide variety of epithelial cells. It has been shown to be protective in radiation- or chemotherapy-induced organ damage. In murine models of allogeneic transplantation, it protected the gut from lethal GVHD [90]. Interestingly, in murine transplantation models, KGF protected thymic epithelial cells from cytotoxic damage when given prior to conditioning [91]. Furthermore, pharmacologic doses of KGF allow regeneration of GVHD-induced thymic damage, and therefore

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KGF appears to exert a potent effect on thymic epithelial cell function, which in turn allows for normal T lymphopoiesis to occur during acute GVHD [92]. Palifermin, the N-truncated form of rHuKGF-1, is available for the prevention of mucosal damage after intensive chemotherapy or radiation therapy [93]. KGF in autologous HCT A phase I multicenter, dose-escalating trial was performed with rHuKGF for mucositis prevention in lymphoma patients conditioned with the BEAM protocol prior to autologous HCT [94]. A dose of 60 μg/kg/day for 3 days prior to high-dose BEAM chemotherapy was chosen based on safety and preliminary efficacy. With this dose, a reduction in duration of severe ulcerative mucositis from 4.6 (placebo) to 0.8 days was observed [94]. In a case series of patients treated prophylactically with KGF during high-dose chemotherapy and autologous HCT, reduction of severe oral mucositis and related symptoms, but not infections, dietary intake, time to engraftment or cumulative dose and duration of narcotic administration, was observed compared with patients without KGF treatment [95]. KGF improved thymus-dependent T-cell reconstitution with elevated T-cell receptor excision circles after autologous CD34(+) PBHC transplantation in rhesus macaques conditioned with myeloablative TBI [96]. KGF has been proved to significantly reduce the duration and severity of oral mucositis after high-dose TBI-containing therapy in randomized trials [97,98]. In one trial, KGF 60 μg/kg/day was given for 3 days prior to TBI and on days 0, 1, and 2 after transplantation. With this, the incidence of oral mucositis of WHO grade 3 or 4 was 63% in the KGF group and 98% in the placebo group (p < 0.001). The median duration of oral mucositis of WHO grade 3 or 4 was 3 (range 0–22) days in the KGF group and 9 (range 0–27) days in the placebo group (p < 0.001), and patients reported less soreness and use of opioid analgesics in the KGF group [98]. Side-effects were skin rash, pruritus, erythema, and taste alteration of mild-to-moderate severity, and were transient. KGF in allogeneic HCT KGF was tested in a randomized, double-blind, placebo-controlled, dose-escalation study regarding safety, engraftment, and influence on GVHD (n = 69 patients) compared with placebo (n = 31) in patients conditioned with CY/TBI or busulfan/CY and given methotrexate along with a calcineurin inhibitor (cyclosporine or tacrolimus) for GVHD prophylaxis. All patients received three doses before conditioning and either three (cohort 1), six (cohort 2) or nine (cohort 3) doses after HCT. Palifermin doses were 40 μg/kg/day (cohort 1 only) or 60 μg/kg per day (all cohorts). Six patients (two on placebo, four on palifermin) experienced a total of 11 dose-limiting toxicities (most often skin, respiratory or oral mucositis). The most common adverse events included edema, infection, skin pain or rash. Palifermin was associated with a reduced incidence and severity of mucositis in patients conditioned with CY/TBI but not busulfan/CY. KGF was regarded as safe in allogeneic HCT but had no significant effect on engraftment, acute GVHD or survival in this trial [99].

Conclusion In several situations, hematopoietic growth factors have a defined role after HCT. In particular, randomized trials have documented faster erythroid engraftment with EPO after allogeneic BMT, and G-CSF resulted in accelerated neutrophil engraftment after allogeneic and autologous marrow as well as PBHCT. Regarding other endpoints, the results are

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equivocal. The value of these growth factors given after HCT has to be seen in the light of pros and cons of the individual patient and specific transplant situation. Faster engraftment is only one aspect, and other factors, such as number of days in hospital or in isolation, transfusion requirements, possible cytokine-induced engraftment syndromes, or

influence on the immune system, especially after allogeneic HCT, as well as costs, have to be taken into consideration. In the future, growth factors preventing or repairing tissue damage, modulating the immune system post transplantation, and optimizing platelet engraftment will be of special interest.

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Claudio Anasetti, Franco Aversa & Andrea Velardi

Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors

Introduction Hematopoietic cell transplantation (HCT) from human leukocyte antigen (HLA)-matched siblings has become the treatment of choice for many hematologic diseases, but fewer than 40% of patients have an HLAmatched sibling [1]. Registries of HLA-typed volunteers have been established worldwide to provide HLA-matched unrelated donors for HCT (see Chapter 47). The chance of finding an unrelated donor matched for HLA-A, B, C, and DR depends on the HLA diversity of the population and varies with race, ranging from 75% in Caucasians to less than 50% for United States ethnic minorities (http://www.marrow.org). A limitation in the use of unrelated donors derives from the long duration of the search, which may allow disease progression in patients who urgently need transplantation, such as those with acute leukemia. Umbilical cord blood offers the advantages of easy procurement, no risk to the donors, low risk of transmissible infections, and immediate availability of the cryopreserved graft (see Chapter 39). However, engraftment is a major concern when the harvested cord blood contains a low number of mononuclear cells. Older patient age and HLA disparity are poor risk factors for engraftment and survival after transplantation of unrelated cord blood hematopoietic cells [2,3]. An alternative source of hematopoietic cells is from relatives who are partially matched for HLA antigens. Almost all patients have at least one HLA partially matched family member, parent, sibling or child, who is immediately available as donor. Transplants of T-replete marrow or growth factor-mobilized peripheral blood progenitor cells (PBPCs) from relatives mismatched for a single HLA-A, B or DR antigen have met with acceptable success rates using standard protocols commonly employed for transplants from HLA-identical siblings [4,5]. Conversely, transplants of T-replete marrow from donors mismatched for two or three HLA-A, B, and DR antigens have resulted in an extremely high incidence of severe acute graft-versus-host disease (GVHD) [4,6]. Marrow T-cell-depletion has been associated with increased risk of graft rejection, but the use of T-depleted PBPCs has enhanced the probability of engraftment despite donor mismatch for two or three HLA antigens, as well as carrying a low incidence of acute and chronic GVHD [7,8]. In this chapter, we first present principles for the selection of related donors with the least degree of HLA disparity for use in conventional

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

transplants, and later present more recent developments in the field of T-depleted PBPC transplants from donors mismatched for two or three HLA-A, B, and DR antigens.

Donor selection Function and polymorphism of HLA The HLA system (described in detail in Chapter 12) includes at least 12 genetic sites, named HLA loci, located on the short arm of human chromosome 6. Each HLA locus is highly polymorphic because it is occupied by multiple alternative forms of an HLA gene, designated HLA alleles, any of which may be carried by a given individual. HLA alleles encode class I HLA-A, B, and C antigens, and class II HLA-DR, DQ, and DP antigens. Class I HLA antigens are expressed on the surface of all nucleated cells in the body, while class II HLA antigens are expressed on the surface of antigen-presenting cells (APCs) such as dendritic cells, monocytes, B cells, and activated T cells. It is the function of HLA molecules to bind antigenic peptides and activate the immune response in a specific manner. In order to accommodate the need for the binding of ever-changing environmental and microbial antigens, HLA molecules have evolved through gene duplication, gene conversion, recombination, and point mutation to acquire an enormous degree of polymorphism, most evident across different ethnic groups. Class I HLA molecules present antigenic peptides and activate cytotoxic CD8+ T cells, while class II HLA molecules present antigenic peptides and activate helper CD4+ T cells. Both classes of HLA molecule lead to T-cell activation through the binding of specific T-cell antigen receptors. In the case of HLA-incompatible HCT, donor and recipient differ not only for one or more types of HLA molecule, but also for the thousands of antigenic peptides that each mismatched HLA molecule can bind and present to foreign T cells [9]. By this process, HCT leads to the activation of an enormous number of both donor and host T cells that mount a response, its strength depending on the degree of HLA mismatch. T-cell recognition of antigenic peptides presented by mismatched HLA molecules can result both in graft rejection and GVHD. HLA class I molecules also bind and activate specific receptors on natural killer (NK) cells. Recent developments on the role of NK cells in transplantation are presented later in this chapter, in the section on Tdepleted grafts, where the effects of NK cell function have become apparent. HLA class I and II molecules also function as antigens by eliciting antibody responses by B cells. It is thought that anti-HLA antibodies can mediate a hyperacute rejection of allogeneic HCTs [10].

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HLA haplotypes and segregation in families The HLA antigens inherited together from one chromosome of each parent are referred to as an HLA haplotype (Fig. 46.1). When a family study is performed in search of an HLA-identical sibling donor, parents should also be typed for HLA-A, B, and DR, and each of the four parental HLA haplotypes identified. Parental haplotypes can segregate among the offspring in four different combinations, and the chance that any one sibling is HLA identical with another is 25%. If an HLA-identical sibling is not found, it is possible to find family members who are partially matched for HLA, even though they have not inherited the same two haplotypes, because certain HLA antigens and haplotypes are frequent. The parental haplotypes should be reviewed for homozygosity or sharing of antigens. The parents, siblings and children, and other relatives who share a haplotype with the patient may have a second haplotype that is partially matched with the patient. Figure 46.1(a) illustrates a family in which the parents have matching haplotypes, “b” and “c.” The patient (sib 1, “a/c”) has a unique HLA type, and none of the siblings is HLA identical with the patient. However, the patient and the father (“a/b”) share the paternal “a” haplotype and are matched for the HLA-A, B and DR antigens of their unshared haplotypes (“b” and “c”). Figure 46.1(b) illustrates a family in which the

Parental sharing

Homozygous parent

A B DR

A B DR a b

c

d

A B DR

a b

c d

a c sib#1

a d sib#2

A B DR a c sib#1

a d sib#2

b c sib#3

(a)

b d sib#4 (b)

Fig. 46.1 Segregation of human leukocyte antigen (HLA) haplotypes and partial sharing of HLA antigens within a family. (a) The two parental haplotypes “b” and “c” are identical, so that sibling 1 (haplotypes “a” and “c”) is compatible with one of these parents (haplotypes “a” and “b”). (b) One parent is HLA homozygous (haplotype “c” and “d” being identical), so that sibling 1 (haplotypes “a” and “c”) and sibling 2 (haplotypes “a” and “d”) are compatible.

mother (“c/d”) is HLA homozygous. The patient (sib 1, “a/c”) and sib 2 (“a/d”) share the paternal “a” haplotype and each has inherited one of the two maternal haplotypes (“c” and “d”). Because of the serendipitous similarity of the maternal “c” and “d” haplotypes, sib 1 and sib 2 are HLA matched. Donor and recipient matching Potential donors are those relatives who share by inheritance one HLA haplotype with the patient and are variably mismatched for 0, 1, 2 or 3 HLA-A, B, and DR loci of the unshared haplotype. A more precise classification of matching, useful in predicting the risk of rejection and GVHD after HLA-incompatible T-replete transplants, takes into consideration the vector of incompatibility in situations where donor or recipients are homozygous at one of the mismatched loci (Table 46.1) [6]. If recipient and donor are overall incompatible for one HLA locus but the recipient is homozygous at the mismatched locus, the mismatch does not contribute a risk for GVHD. Conversely, if recipient and donor are overall incompatible for one HLA locus but the donor is homozygous at the mismatched locus, the mismatch does not contribute a risk for graft rejection. Originally, some of the polymorphisms of HLA class I and II antigens were defined serologically by typing with alloantisera obtained from women with a history of pregnancy. The development of genetic typing has demonstrated that few HLA antigens represent unique alleles [11]. For example, HLA-A2 is defined as an antigen by serology, but DNA sequencing has identified as many as 89 distinct alleles, designated from HLA-A*0201 to A*0299, all of which are expressed with a unique amino acid sequence but are recognized by the same anti-HLA-A2 antibody (http://www.anthonynolan.org.uk/HIG). Some other unique HLA-A*02 alleles are not expressed as proteins (for example, A*0215N), they are instead defined as “null” and are immunologically silent. Once a null allele is identified, it should be ignored for donor selection. Distinction between the variants of polymorphic HLA antigens appears to be functionally relevant because, for example, each of the seven B27 antigen variants can be distinguished by specific cytotoxic T-cell clones [12]. The clinical importance of HLA-A, B, C, and DRB1 allelic differences that cannot be distinguished by serologic typing was demonstrated in studies of unrelated donor HCT where mismatching was associated with increased GVHD and worse survival [13–19]. Increasing evidence has related DPB1 mismatching with graft failure, acute GVHD, and protection from leukemia relapse [20–25]. However, the relevance of donor disparity for DQB1 or DPB1 for the post-transplant survival of patients with malignancy remains disputed [21–26]. In the process of

Table 46.1 Mismatching according to vector of genetic disparity for human leukocyte antigen (HLA)-A, B, and DR Vector† Example

Haplotype

Recipient

Donor*

Overall

Rejection

Graft-versus-host disease

Heterozygous recipient and donor

Mismatched Shared* Mismatched Shared Mismatched Shared

A2, B44, DR7 A1, B8, DR3 A1, B35, DR1 A1, B8, DR3 A2, B44, DR4 A1, B8, DR3

A3, B7, DR2 A1, B8, DR3 A3, B35, DR1 A1, B8, DR3 A1, B8, DR4 A1, B8, DR3

3

3

3

1

1

0

2

0

2

Homozygous recipient Homozygous donor

Incompatible antigens are represented in bold. * Recipient and donor are related and share HLA haplotype. † Number of mismatches at HLA-A, B or DR loci.

Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors

donor selection, we must consider that recipients and donor pairs, who have not inherited the same HLA haplotypes but are assumed HLA identical on the basis of serologic typing alone, might be reclassified as mismatched for one or more HLA alleles using high-resolution DNA typing technology. Crossmatching Patients may be alloimmunized by pregnancy or blood transfusions, and sensitization to donor alloantigens increases the risk of graft failure [10,27,28]. Crossmatch testing can determine whether the recipient has been sensitized to HLA antigens of the donor, and is indicated before the selection of an HLA-mismatched donor for HCT. Standard assays for crossmatching the patient’s serum with donor lymphocytes include complement-dependent microcytotoxicity and multicolor flow microfluorimetry [29,30]. HLA-specific antibodies can be detected by enzymelinked immunosorbent assay, flow cytometry or Luminex technologies that are likely to replace the more cumbersome direct crossmatch [31–33]. Probability of identifying an HLA partially compatible family donor One report from Germany found that the probability of identifying an HLA-matched sibling was 40.7%, a matched relative 4.0%, a one HLAA, B or DR locus-mismatched sibling 3.1%, and a one-locus mismatched relative 10.4% [1]. The best chance of finding a donor for patients without an HLA-matched or a one-locus-mismatched sibling is an unrelated donor search, because this is currently successful in up to 75% of cases. If the initial search identifies one or more HLA-A, B, and DR matched unrelated donors, there is no obvious justification to undertake an extended family search beyond siblings, because not only would the probability of finding a suitable donor be lower, but also the donor typing effort and the cost of finding donors would be greater [1]. Computer programs have recently been developed that can calculate the probability of finding a suitable related or unrelated donor based on patient typing [1,34–36], and the results of such programs can be utilized to define the best search strategy for an individual patient. Candidates for allogeneic HCT with no suitably matched donor and a short life expectancy might be better served by immediate transplantation from a relative mismatched for one entire HLA haplotype, or by a partially matched unrelated umbilical cord blood transplant, than by a lengthy search for a completely matched donor.

659

necessary to assess the presence of donor cells. Functional studies in patients with graft failure have demonstrated that residual host T lymphocytes are cytotoxic against donor alloantigens, and the patient’s serum may be active in the antibody-dependent cell-mediated cytotoxicity test against donor cells [37,38]. These findings have indicated that alloimmune-mediated rejection is a mechanism for graft failure after HCT from HLA-incompatible donors. If a high-dose conditioning regimen is administered, either primary or secondary graft failure is usually associated with persistent aplasia. In certain patients, recovery of autologous myeloid cells can occur, especially if the patient is pretreated with reduced-intensity conditioning regimens and administered granulocyte–macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) after graft failure [39]. If there is no recovery of myeloid function despite growth factor therapy, a second transplant from the same or a different donor can be attempted, but the success of second transplants has been limited [10]. Risk factors. HLA mismatch. The relevance of HLA compatibility to sustained engraftment was first analyzed in 269 patients with hematologic neoplasms who underwent T-replete marrow transplantation from a family member who shared one HLA haplotype with the patient, but differed to a variable degree for the HLA-A, B, and DR antigens of the unshared haplotype [10]. These 269 patients were compared with 930 patients who received marrow from siblings with an identical HLA genotype. All patients were treated with cyclophosphamide (CY) and total body irradiation (TBI) followed by infusion of unmodified donor marrow cells. The incidence of graft failure was 12.3% among recipients of marrow from an HLA partially matched donor compared with 2.0% among recipients of marrow from an HLA-matched sibling (p < 0.0001) (Table 46.2). The incidence of graft failure correlated with the degree of HLA incompatibility in the host-versus-graft direction. Graft failure occurred in three of 43 transplants (7%) from donors who were matched with their recipient for HLA-A, B, and DR, in 11 of 121 donors (9%) incompatible for one HLA locus, in 18 of 86 (21%) incompatible for two loci, and in one of 19 (5%) incompatible for three loci (p = 0.03). Therefore, donor HLA incompatibility is a significant risk factor for graft failure. Results of unrelated donor transplants point to HLA-C and DPB1 mismatch as risk factors for graft failure that might account for the graft failure observed after transplantation from family donors matched for HLA-A, B, and DR [13,20]. The reasons for the apparently lower incidence of graft failure with a transplant incompatible for three loci compared with two loci reported in that study are not obvious [10]. Transplants with the highest degree of mismatch were conducted predominantly in children who received a higher marrow cell dose. It is

Transplantation of T-replete marrow grafts Transplant outcome The relevance of HLA incompatibility between donor and recipient has been analyzed in patients with hematologic disorders who received HCTs of T-replete marrow grafts. The patient and related donor shared one HLA haplotype but differed to a variable degree for the HLA-A, B, and DR antigens of the unshared haplotype. The results of these transplants have shown the effect of HLA incompatibility on graft failure, GVHD, and survival, formally demonstrating that HLA antigens constitute the major histocompatibility complex (MHC) in humans. Graft failure Clinical presentation. Primary graft failure is likely to occur if severe granulocytopenia persists for 21 days after transplantation of marrow or PBPCs, but secondary graft failure may also occur after initial engraftment (see Chapter 80) [10]. Studies of chimerism (see Chapter 25) are

Table 46.2 Effect of HLA incompatibility on marrow graft failure

Failure of engraftment Late graft failure All failures

HLA-nonidentical donor (n = 269) number of patients (%)

HLA genotype-identical donor (n = 930) number of patients (%)

p value

23 (8.5%) 10 (4.1%) 33 (12.3%)

15 (1.6%) 4 (0.4%) 19 (2.0%)

<0.0001 <0.0001 <0.0001

HLA, human leukocyte antigen Modified with permission from Anasetti et al. [10].

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Chapter 46

also possible that the higher degree of mismatch led to a more rapid and intense graft-versus-host reaction that blunted host immune responses that mediate graft rejection. There is an additional effect of HLA disparity in particular donor and recipient combinations [40]. When the recipient is homozygous at a mismatched HLA locus, the degree of HLA disparity is greater in the direction of rejection than in the direction of GVHD (Table 46.1). Transplants in recipients homozygous at one or more mismatched loci had a significantly higher incidence of graft failure than transplants in heterozygous recipients. Graft failure after a transplant mismatched for one or more HLA loci occurred in 11 of 76 (15%) homozygous recipients compared with 22 of 350 (6%) heterozygous recipients. The effect of the vector of incompatibility on graft failure was significant in a multivariable analysis (p = 0.005). The association between recipient homozygosity for the mismatched locus and increased graft failure has been confirmed in a subsequent study of HLA-mismatched unrelated donor transplants [41]. These results are consistent with the concept that the graft-versus-host reaction protects from graft failure. Presumably, the graft-versus-host reaction is directed against residual immune cells of the recipient that are responsible for graft failure. In cases where the degree of disparity in the donor exceeds the degree of disparity in the host, the graft-versus-host reaction is less and the risk of graft failure is increased.

provided as treatment for the underlying disease. Most patients with severe combined immunodeficiency lacking both T and NK cells have been engrafted with HLA-incompatible HCT despite receiving neither pretransplant conditioning nor post-transplant immunosuppression [42]. Patients with leukocyte adhesion deficiency (see Chapter 78), a congenital defect resulting in failure to express the leukointegrin β2 chain on white cells including T and NK cells, demonstrate low resistance to engraftment with T-depleted transplants from HLA-incompatible donors [43]. Thus, β2 leukointegrins have a critical role in the rejection of HLA-incompatible HCTs. Patients with chronic myeloid leukemia (CML) have a higher risk of graft failure than patients with acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). The incidence of graft failure was examined in non-T-depleted marrow grafts from HLA partially matched relatives in patients treated with the same conditioning regimen of CY (120 mg/kg) plus hyperfractionated TBI (1320–1440 cGy) followed by post-transplant immunosuppression with cyclosporine (CSP) and a short course of methotrexate (MTX). Graft failure occurred in 11 of 84 (13%) patients with CML compared with five of 133 (4%) patients with other diagnoses (odds ratio = 3.5, 95% confidence interval [CI], range 1.1–10.5, multivariate p = 0.03) [44]. Patients with acute leukemia may be at lower risk for graft failure because of the immunodeficiency induced by repeated treatment with chemotherapy agents [45].

Recipient sensitization. Prior patient immunization to donor HLA antigens has a profound effect on engraftment. In a study of marrow transplants from HLA-incompatible relatives, graft failure occurred in 13 of 21 (62%) patients with a positive pretransplant crossmatch of patient serum reactive against donor T or B lymphocytes, compared with 31 of 501 (7%) patients with a negative crossmatch (p = 7.8, E-10) [10,40]. Among all the microcytotoxicity assays, the antiglobulin crossmatch and the B-cell crossmatch at 22°C or 37°C correlated best with graft failure [26]. Autoreactive antibodies were observed in four patients who were all successfully engrafted. Alloimmunized patients who tested positive for anti-HLA antibodies by screening against a random cell panel but were crossmatched negative with the donor did not demonstrate an increased risk of graft failure [30]. Ottinger et al. [27] also found that positive serum crossmatch is a predictor for graft failure and poor survival after peripheral blood HCT from an HLA-mismatched donor. In unrelated donor transplantation, flow cytometry and Luminex technology can detect patient antibodies specific against the donor mismatched HLA-A, B, and DPB1 alleles and predict for graft failure [28]. If complete patient and donor HLA genotyping is available, assays that detect antibodies to individual HLA alleles of the donor are available. In the presence of a positive antidonor crossmatch, graft failure is likely the result of alloimmune rejection mediated both by sensitized radioresistant host T cells and by antibody-dependent cell-mediated killing. Removal of antidonor antibody through a plasma exchange followed by CY (120 mg/kg) and TBI (1200–1575 cGy), the addition of total lymphoid irradiation (600 cGy in four fractions over 2 days) or antithymocyte globulin (ATG) (10 mg/kg/day, day 2 before transplantation [day −2] to day 3 afterwards [day +3]) to the standard regimen of CY and TBI has not resulted in consistent marrow engraftment [10,40]. If a patient has antibodies against an HLA-incompatible family donor, there is no effective measure to offset the risk of graft rejection, and the best option for engraftment is to identify an HLA-matched unrelated donor, or a related or unrelated donor that is mismatched for an HLA antigen against which the patient is not sensitized.

Conditioning regimen. Pretransplant conditioning with CY (120 mg/kg over 2 days) and TBI (920–1575 cGy) allowed engraftment in 88% of 269 patients transplanted from HLA partially matched relatives [10]. Thus, a high dose conditioning that includes TBI is not uniformly effective in facilitating engraftment of HLA-incompatible marrow or PBPC grafts. A more effective high-dose regimen containing TBI was developed for T-depleted transplants: CY was replaced by ATG, fludarabine and thiotepa, agents that contributed immunosuppressive activity without additional toxicity (see below, under T-cell-depleted transplants) [46]. Pretransplant immunosuppression with CY alone (200 mg/kg for 4 days) allowed sustained engraftment in only four of 11 patients with severe aplastic anemia transplanted from donors incompatible for one HLA-A, B or DR locus, and none of three patients transplanted from a two-locus incompatible donor [47]. Increasing the immune-suppressive potency of the pretransplant conditioning regimen can minimize the incidence of immune-mediated rejection. The addition of thiotepa (15 mg/kg) and rabbit ATG (15 mg/kg) to CY (150 mg/kg) allowed sustained engraftment of marrow or PBPCs in all 17 patients with hematologic malignancies transplanted from a onelocus mismatched donor, and six of seven patients transplanted from a two-locus mismatched donor [48]. Busulfan (BU) (13–16 mg/kg over 4 days) in addition to CY (120–200 mg/kg over 2–4 days) was used for 11 patients with myelodysplastic syndrome (MDS), lymphoma or myeloma. Nine patients transplanted from one-HLA-locus incompatible donors and one of two transplanted from two-HLA-locus incompatible donors engrafted, suggesting that BU adds to the immunosuppression produced by CY alone [49]. However, BU/CY was less effective in allowing engraftment of marrow from HLA-mismatched donors in patients with thalassemia, where sustained grafts were achieved in only eight of 15 patients mismatched at one HLA locus, two of five patients mismatched at two loci, and none of three mismatched at three loci [50]. Thalassemia patients are not only more immunocompetent than leukemia patients but are also sensitized to histocompatibility antigens by multiple blood cell transfusions. The addition of thiotepa to BU/CY decreased significantly the risk of graft failure in thalassemia patients transplanted from HLA partially compatible unrelated donors [51]. Humanized anti-CD52 antibody alemtuzumab (100 mg over 5 days) was added to fludarabine

Patient diagnosis and immune competence. The risk of graft failure is low in patients who are immunodeficient by virtue of a congenital disorder or as the result of prior cytotoxic or immunosuppressive therapy

Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors

Post-transplant immunosuppression. Post-transplant immunosuppression decreases the risk of graft failure. Patients transplanted from HLA partially matched donors had a graft failure rate of 5% when treated with CSP plus short-course MTX, but 9% when treated with MTX alone (p = 0.03) [40]. This observation is consistent with the report from the Center for International Blood and Marrow Transplant Research (CIBMTR) showing that, in T-depleted marrow transplants from HLAidentical sibling donors, the use of post-transplant immunosuppression with CSP plus MTX was associated with a lower risk of graft failure than CSP alone [53]. In a study of 89 T-replete marrow transplants from HLA haplotype partially matched relatives, graft failure occurred in 18% of patients treated with a reduced-intensity regimen of fludarabine 150 mg/m2, CY 29 mg/kg, and TBI 200 cGy before transplant, and CY 50–100 mg/kg after transplant (Fuchs, unpublished data) [54]. These data strongly imply that postgrafting CY decreases the risk of graft failure.

1.0

Acute GVHD grade III–IV

(120 mg/m2 over 4 days) and CY (2000 mg/m2 over 4 days) to develop an immune-suppressive regimen that does not produce myeloablation. With T-replete PBPC transplantation from HLA partially matched related donors, primary engraftment was achieved in 46 of 49 (94%) patients, and secondary graft failure occurred in four (8%) patients, so overall sustained engraftment was 86% [52]. The benefits of a reducedintensity conditioning regimen were the relatively low (31%) nonrelapse mortality and low (16%) incidence of grades II–IV acute GVHD.

661

p = 0.03

0.8

0.6 Both incompatible (n = 175) Recipient incompatible (n = 22)

0.4

Neither incompatible (n = 48) 0.2

Donor incompatible (n = 25)

0.0 0

20

40

60

80

100

Days after transplantation

Fig. 46.2 Effect of the vector of human leukocyte antigen (HLA) mismatch on acute graft-versus-host disease (GVHD). Probability of grade III–IV acute GVHD in patients who received a marrow transplant from a haploidentical relative. Transplants incompatible for one HLA-A, B or D/DRB1 locus are classified as “donor incompatible” in the case of homozygous recipients where mismatch is only in the direction of graft-versus-host, as “recipient incompatible” in the case of homozygous donors where mismatch is only in the direction of host-versus-graft, and as “both incompatible” in the case where donor and recipient are both heterozygous. Transplants from a haploidentical relative whose unshared haplotype is matched to the patient’s are classified as “neither incompatible.”

Graft-versus-host disease Clinical presentation of acute GVHD. Acute GVHD occurs early after HLA-incompatible transplants. The median onset of acute GVHD was 14 days after HLA-incompatible transplants, compared with 22 days after HLA-identical sibling transplants [4]. Acute GVHD after HLAincompatible transplants can be associated with a hyperacute syndrome characterized by fever 7–10 days after transplantation, fluid retention, low central venous pressure, low serum albumin, pulmonary edema, and renal failure (see Chapter 86). If the syndrome cannot be reversed promptly by the use of immunosuppressive agents, the patient may die from respiratory failure and other complications [55]. Risk factors. HLA mismatch. After HCT from partially matched relatives, the incidence and severity of acute GVHD correlates with the degree of HLA incompatibility for HLA-A, B, and DR. More recent data from unrelated donor transplants indicate that mismatch at HLA-C, DQ, and DP also contributes to the risk of GVHD [13,24–26]. When patients received MTX as single agent for GVHD prophylaxis, the incidence of grades II–IV GVHD was 34% for recipients of HLA-identical sibling marrow and increased progressively up to 84% for recipients of HLA-A, B, and DR locus-incompatible marrow [4]. In patients transplanted from HLA nonidentical donors, acute GVHD is more frequent not only in the skin, but also in the gastrointestinal tract and liver. Among patients transplanted for CML, the incidence of gastrointestinal involvement was 10% in HLA-identical sibling transplants and 22% in a one-HLA locusincompatible transplant. The incidence of liver involvement was 13% in HLA-identical sibling transplants compared with 36% in a one-HLA locus-incompatible transplant [44]. The vector of HLA incompatibility affects the risk of acute GVHD. Because HLA-homozygous recipients are matched with their donors in the direction of GVHD, transplants incompatible for one HLA locus in homozygous recipients are associated with lower incidence of acute GVHD than in heterozygous recipients (p = 0.03) (Fig. 46.2) [6]. The incidence of acute GVHD for homozygous recipients of onelocus-incompatible transplants is similar to that seen in HLA-matched

transplants. Conversely, HLA homozygosity in the donor does not affect the incidence of GVHD. Pretransplant anti-T-cell antibody therapy. With alemtuzumab (100 mg over 5 days) administered to 49 patients before transplantation of T-replete PBPCs from HLA partially matched related donors, the incidence of acute GVHD of grades II–IV was 16%, suggesting that a large fraction of donor T cells infused in the graft were depleted by alemtuzumab in vivo [52]. Investigators from Peking University (Beijing, China) utilized rabbit ATG (Thymoglobulin) 10 mg/kg over 4 days, or porcine ATG 80 mg/kg over 4 days before the transplant of T-replete marrow cells plus PBPCs from HLA partially matched related donors, and post-transplant immune suppression with MTX/CSP plus mycophenolate mofetil [56]. All 171 patients achieved full donor chimerism and hematopoietic reconstitution. Acute GVHD grade II–IV and III–IV was observed in 55% and 23% of patients, respectively, with no suggestion for an association between degree of HLA disparity and risk of acute GVHD. A separate report from the same institution compared the outcome of HLA partially matched transplants in patients pretreated with Thymoglobulin with the outcome of HLA-matched sibling transplants, without Thymoglobulin. All patients received the same postgrafting immune suppression with MTX/CSP plus mycophenolate mofetil. The cumulative incidences of acute GVHD grade II–IV were 32% (95% CI 25–39%) versus 40% (95% CI 32–48%) in the matched and mismatched cohorts, respectively (p = 0.13). The risk of acute GVHD grade II–IV was significantly higher, and GVHD had a more rapid onset after HLAmismatched transplants. Clinical manifestation of grade III–IV GVHD after mismatched transplants included severe (44%) and bloody diarrhea (23%), hepatic dysfunction (41%), skin rash (31%), and noncardiac edema (18%) [57]. While the relative contribution of pretransplant ATG and post-transplant mycophenolate mofetil to GVHD prevention cannot be discerned

662

Chapter 46

Acute GVHD grade II–IV

1.0

p <0.0001

HLA mismatch: 3 loci

0.8

2 loci 1 locus

0.6

0 locus

0.4

HLA-identical sibling

Patient age. Patient age does not appreciably affect the risk of grade II–IV acute GVHD after HLA-incompatible transplants, but younger age is associated with a decreased risk of acute GVHD grade III–IV (RR 1.23; 95% CI 1.05–1.44 per decade of patient age; p = 0.04) [6]. Posttransplant immunosuppression with CSP and MTX compared with MTX alone has not resulted in improved outcome for patients of any age after transplants incompatible at two or three HLA loci [6]. Chronic GVHD

0.2 0.0 0

20

40

60

80

100

Days after transplantation

Fig. 46.3 Effect of donor and recipient human leukocyte antigen (HLA) mismatch on acute graft-versus-host disease (GVHD). Probability of grade II–IV acute GVHD according to the degree of matching for HLA-A, B, and D/DRB1 in patients transplanted from a haploidentical relative or an HLA genotype-identical sibling. Patients received unmodified marrow and methotrexate plus cyclosporine for GVHD prophylaxis.

from these studies, the data indicate that the administration of ATG just before HCT can deplete T cells in vivo and diminish the risk of GVHD. Whether ATG can offset the GVHD risk from multiple HLA disparities between Caucasian donors and recipients remains to be established. Post-transplant immunosuppression. Initial transplants from HLAincompatible related donors were performed in patients receiving MTX as the only immunosuppressive agent. When it became apparent that the combination of CSP and MTX was superior to either agent alone in HLA sibling transplants, the same regimen was adopted for HLA partially matched transplants. The risk of severe acute GVHD of grade III–IV was significantly less and the time of onset was delayed by the use of CSP and MTX (relative risk [RR] 0.35; p < 0.0001). The incidence of severe grade III–IV acute GVHD was decreased from 53% to 28% in one-locus-incompatible recipients and from 63% to 47% in two-locusincompatible recipients. Recipients incompatible for one HLA-A or B locus had a significantly lower incidence of acute GVHD when receiving CSP and MTX rather than MTX alone, but there was no apparent benefit from CSP and MTX in patients incompatible for one HLA-DR locus. Thus, the addition of CSP to a regimen of MTX was most effective in transplants incompatible for HLA-A or B. A major remaining task is to overcome HLA disparity for HLA-DR, either alone or in combination with HLA-A or B. Therefore, despite post-transplant immunosuppression with CSP and MTX, HLA incompatibility has remained an important risk factor for GVHD (Fig. 46.3) [6]. In a multivariate analysis of 474 patients who received CSP and MTX or MTX alone and achieved sustained engraftment, HLA incompatibility was a significant risk factor for acute GVHD (RR 1.95 per HLA locus; p < 0.0001) [6,40]. Substituting CSP with tacrolimus was not adequate to prevent GVHD after transplantation from HLA-DRincompatible relatives [58]. Post-transplant CY (50 or 100 mg/kg) was administered in addition to tacrolimus and mycophenolate mofetil to prevent GVHD in 89 patients who received T-cell-replete marrow transplant from partially HLA-mismatched related donors [54]. The incidence of acute GVHD of grades II–IV was 35%, acute grade III–IV GVHD 10%, and chronic GVHD 22%. These data indicate that posttransplant CY can decrease the incidence and severity of acute and chronic GVHD after T-cell-replete grafts from HLA-incompatible donors.

Marrow transplants from HLA partially matched family members have been associated with a higher probability for developing clinical extensive chronic GVHD (49% versus 33%) and an earlier median day of onset (159 versus 201 days) compared with transplants from HLAidentical siblings [59]. Consistent results have been found by the CIBMTR [60]. The duration of immunosuppressive treatment for chronic GVHD is prolonged after HLA disparate-donor transplants, with a hazard ratio of 0.8 (95% CI 0.7–0.9) per mismatched HLA locus (p = 0.007) [61]. Intriguing results were reported by Lu and collaborators using ATG treatment before T-cell-replete marrow or PBPC transplantation from HLA partially matched relatives. The 2-year incidence of chronic GVHD was 55% (95% CI 46–64%) after mismatched HCT with ATG compared with 56% (95% CI 47–64%) after HLA-identical sibling HCT with no ATG (p = 0.9) [57]. Graft-versus-leukemia effect Relapse of leukemia after HLA-identical sibling transplants is less frequent in patients who develop clinically significant GVHD compared with syngeneic or allogeneic transplant recipients without GVHD (see Chapter 18). Patients with ALL after transplantation from HLA partially matched donors in whom GVHD developed also had a lower risk of relapse than patients without GVHD [6]. However, the probability of leukemic relapse in patients without clinically significant GVHD was the same whether the marrow donor was an HLA-identical sibling or an HLA partially matched relative. These data suggested that HLA disparity in absence of acute GVHD has no obvious antileukemic effect. In one analysis, chronic but not acute GVHD was associated with a lower relapse rate in patients with AML or CML [6]. Because recipients of one-HLA locus-incompatible transplants have a higher incidence of acute GVHD than recipients of HLA-identical sibling transplants, one would expect to see a lower incidence of leukemic relapse. A study from the CIBMTR comparing the outcome of 1222 HLA-identical sibling transplants to that of 238 transplants from one-HLA locus-incompatible donors failed to detect different rates of relapse [7]. One study found an association of donor mismatch for one HLA antigen with a lower incidence of leukemic relapse in patients transplanted from unrelated volunteers [62]. The use of T-cell-replete grafts of G-CSF-mobilized PBPCs from one-HLA antigen-mismatched related or unrelated donors has been associated with a lower risk of post-transplant relapse in patients with CML [63]. The apparent increase in antitumor effect with PBPCs compared with marrow is likely related to the number of donor T cells transplanted [64]. Immune reconstitution After transplantation, new T cells are derived from donor hematopoietic stem cells (HSCs) following positive and negative selection within the host thymus. T lymphocytes recognize immunogenic peptides presented by self-HLA but not by mismatched allogeneic HLA molecules, a phenomenon defined as HLA restriction. Positive T-cell selection is facilitated by HLA molecules expressed on thymic epithelial cells, whereas negative selection is controlled predominantly by HLA molecules expressed on marrow-derived APC [65]. Thus, new T cells that are positively selected on thymic epithelium can optimally recognize antigen

Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors 1.0

Survival

0.8

HLA-identical sibling (n = 472) 0.4

0 locus mismatch (n = 6)

1 locus mismatch (n = 50)

2 or 3 locus mismatch (n = 27)

0.0 0

3

(a)

6

9

12

15

Years after transplantation 1.0

0.8

Survival Effect of HLA mismatch. Studies from Seattle in patients receiving HCT for AML, ALL, CML, MDS or lymphoma have evaluated the role of HLA-A, B, and DR incompatibility on survival after T-cell-replete marrow transplants. HLA-DR subtypes, termed HLA-D, were defined by functional testing with homozygous typing cells and utilized in the definition of a match. Survival in patients receiving post-transplant immunosuppression with MTX alone was similar after transplantation from an HLA-identical sibling or a one-HLA antigen-incompatible family donor. However, survival was lower in patients transplanted from a family donor incompatible for two or three HLA loci [4]. Subsequent studies of patients whose post-transplant immunosuppression consisted of CSP and MTX also showed that the degree of overall HLA incompatibility was inversely correlated with the probability of survival (Fig. 46.4(a)). Survival was predicted more precisely by matching classified according to the degree of overall incompatibility than to the incompatibility for each vector alone, because the degree of overall incompatibility best reflects the risks of both graft rejection and GVHD. In a multivariate proportional hazard regression analysis, the factors associated with lower survival were leukemia in marrow relapse at the time of transplant, and the degree of overall HLA incompatibility between donor and recipient. Post-transplant immunosuppression with CSP and MTX compared with MTX alone did not have a beneficial effect on survival. A large study from the CIBMTR compared the outcomes of marrow transplants from HLA-identical siblings, HLA partially matched related donors, and unrelated donors for treatment of leukemia [60]. The definition of donor and recipient histocompatibility differed from the Seattle criteria because matching for the DR locus was based exclusively on serologic data and not on cellular or DNA typing. The probability of leukemia-free survival for patients transplanted for early-stage leukemia, CML in chronic phase, AML or ALL in first remission is presented in Fig. 46.4(b). A multivariable analysis of transplant-related mortality showed an increase in risk with the use of a one-HLA locus-mismatched relative compared with an HLA-identical sibling, and for the use of a

0.6

0.2

Leukemia-free survival

in the context of marrow-derived APCs in peripheral tissues only if there is sharing of HLA antigens between donor and recipient [66]. Mature T cells transplanted into an HLA-disparate recipient may not recognize antigens presented by host APCs, thereby failing to help immune reconstitution. These premises justify why donor and recipient matching for at least one HLA haplotype is the minimal requisite for donor selection. Extrathymic pathways of immune reconstitution after transplantation are predominant in adults as thymus function begins to decline relatively early in life, usually before the age of 20 years. Therefore, T cells that repopulate adult transplant recipients are derived predominantly from the relatively small number of mature donor T cells infused with the hematopoietic cell inoculum [67]. Immunosuppressive regimens should be developed that are effective in preventing GVHD while sparing donor T cells that do not recognize host alloantigen but contribute to long-term immune reconstitution. Both acute and chronic GVHD also contribute to immunodeficiency. Because adult patients have very poor thymic function, T-celldepletion has been associated with severe and prolonged postgrafting immunodeficiency [68]. Recent data on T-cell receptor excision circles as a marker for recent thymus emigrants has demonstrated that the human thymus continues to function at low levels until late in life, providing hope that effective treatment can be developed to restore immunity quickly in T-cell-deficient adults [69]. Preclinical data on interleukin-7 (IL-7) and keratinocyte growth factor are promising for this application [70,71].

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HLA-identical sibling (n = 805) 0.6

0.4

1 locus mismatch (n = 104)

0.2

2 locus mismatch (n = 24)

0.0 0 (b)

1

2

3

4

5

Years after transplantation

Fig. 46.4 Effect of donor and recipient human leukocyte antigen (HLA) mismatch on survival after hematopoietic cell transplantation. Probability of survival in patients with chronic myeloid leukemia in chronic phase, acute myeloid leukemia in first remission or acute lymphoblastic leukemia in first or second remission, according to the degree of donor and recipient HLA incompatibility. (a) Results from a single center study. (Reprinted from [6], with permission.) (b) Results from a study of the International Bone Marrow Transplant Registry. (Reprinted from [60], with permission.)

two-HLA locus-incompatible relative compared with a one-HLA locusincompatible relative. Results of matched unrelated donor transplants were similar to results of one-antigen-mismatched related donor transplants, while results for one-antigen-mismatched unrelated donor transplants were similar to results for two-antigen-mismatched related donor transplants [60]. A large proportion of patient and donor pairs serologically compatible at DR are mismatched for one or two DRB1 subtypes [18], and mismatching for DRB1 correlates with allele mismatching at HLA-B and DQB1 (see Chapter 47). Therefore, transplants assessed as mismatched for only one HLA locus in the CIBMTR report are expected to be more genetically disparate than transplants assessed as mismatched for one HLA locus in the Seattle report. Taken together, these data demonstrate that a limited degree of HLA disparity between donor and recipient can be tolerated with T-cell-replete marrow in transplants for hematologic malignancies, but a higher degree of donor HLA disparity reduces survival. Clinical data on allogeneic HCT for leukemia or MDS were analyzed from the database of the Japan Society for Hematopoietic Cell

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Transplantation. Data from T-cell-replete transplants with a single class I HLA mismatch (n = 70), single class II mismatch (n = 42) or two or three locus mismatch (n = 30) were compared with 2805 transplants from an HLA-identical sibling. Serologic HLA mismatch, higher age, and high-risk disease were identified as independent risk factors for both shorter survival and the development of grade III–IV acute GVHD. The degree of HLA disparity also appeared to correlate with survival. In standard-risk patients, survival after one-locus-mismatched HCT was significantly shorter than that after HLA-matched HCT. However, in high-risk patients, survival after a one-locus-mismatched or a matched HCT was identical [72]. A single-center study from Milwaukee compared transplantation outcomes in patients with hematologic malignancies who received marrow grafts from either HLA-matched unrelated, one-antigen-mismatched unrelated or highly mismatched family donors [73]. All patients received a standardized conditioning regimen and a uniform GVHD prophylaxis schedule – with the exception of mismatched related recipients, who received ATG as additional postgrafting immunosuppression. There was a higher probability of survival for matched unrelated transplants (58%) than either mismatched unrelated (34%; p = 0.01) or mismatched related transplants (21%; p = 0.002). This study supports the fact that patients lacking an HLA-matched family donor can be offered a matched unrelated donor, if available. With the limitations imposed by the low degree of HLA typing resolution and the small sample sizes, the CIBMTR and Milwaukee studies both found no detectable advantage to using a one HLA-A, B or DR antigen-mismatched unrelated versus a more HLAdisparate family donor. Novel approaches at GVHD prevention: effects on outcome. Pretransplant ATG plus post-transplant mycophenolate mofetil. The outcomes of HLA-mismatched related HCT (n = 135) for leukemia were compared with those of HLA-identical sibling HCT (n = 158) performed during the same time period in Beijing, China [57]. The donor was mismatched for one (n = 21), two (n = 62) or three (n = 52) HLA-A, B or DR loci. All patients received conditioning with BU/CY. In addition, ATG was administered before mismatched HCT only. Unmanipulated marrow and/or PBPCs were used for transplantation. All patients received postgrafting immune suppression with MTX/CSP plus mycophenolate mofetil. All patients achieved full engraftment. The cumulative incidences of grade II–IV acute GVHD in the matched and mismatched cohorts were 32% versus 40%, respectively (multivariate p = 0.02), while chronic GVHD did not differ between the cohorts (p = 0.97). Two-year incidences of treatment-related mortality and relapse for matched versus mismatched were 14% (95% CI 9–20%) versus 22% (95% CI 15–29%) (p = 0.10), and 13% (range 8–19%) versus 18% (range 10–27%) (p = 0.40), respectively. Two-year adjusted overall survival and leukemia-free survival were 72% (95% CI 64–79%) versus 71% (95% CI 62–77%) (p = 0.72), and 71% (95% CI 63–8%) versus 64% (95% CI 54–73%) (p = 0.27), respectively. Multivariate analyses showed that advanced disease stage and a diagnosis of acute leukemia had an increased risk of relapse, treatment failure, and overall mortality, whereas donor HLA mismatch did not affect these outcomes. These data suggest that the addition of pretransplant ATG and post-transplant mycophenolate mofetil to standard regimens may offset the risks associated with donor HLA-mismatching. Post-transplantation, high-dose CY. Two independent clinical trials from Johns Hopkins and Seattle evaluated the safety and efficacy of highdose, post-transplantation CY to prevent graft rejection and GVHD after outpatient reduced-intensity conditioning and T-cell-replete bone marrow transplantation from partially HLA-mismatched related donors (Fuchs, personal communication) [54]. Patients with advanced hematologic

malignancies (n = 87) or paroxysmal nocturnal hemoglobinuria (n = 1) were treated with post-transplantation CY (50 or 100 mg/kg), tacrolimus, and mycophenolate mofetil (15 mg/kg orally two or three times daily). Graft failure occurred in 15 (18%) of the 84 evaluable patients, and was fatal in two. The cumulative incidences of grade II–IV and grade III–IV acute GVHD by day 200 were 35% and 10%, respectively, and that of chronic GVHD at 1 year was 22%. The cumulative incidences of nonrelapse mortality and relapse at 1 year were 19% and 50%, respectively. Overall and event-free survivals at 2 years after transplantation were 35% and 24%, respectively. These data strongly suggest that post-transplantation CY can decrease the risk of graft rejection, GVHD, and mortality associated with donor HLA disparity. A prospective, multicenter trial has been planned by the US Blood and Marrow Transplantation-Clinical Trial Network to confirm and extend this observation.

Transplantation of T-cell-depleted hematopoietic cell grafts Risk of graft failure with T-cell-depleted marrow Numerous clinical trials have demonstrated that extensive ex vivo depletion of T cells from the marrow graft without post-transplant immunosuppression prevents acute and chronic GVHD and associated morbidity (see Chapter 85). Unfortunately, marrow T-cell depletion is associated with an increased risk of graft failure that is proportional to the degree of donor HLA incompatibility. A study from the CIBMTR on transplantation from donors incompatible for two or three HLA-A, B, and DR antigens found an incidence of graft failure of 42% with T-cell-depleted marrow compared with 28% in non-T-cell-depleted marrow ( p < 0.03) [7]. In a small series of patients with leukemia transplanted from a twoor three-HLA antigen-mismatched donor at Memorial Sloan Kettering Cancer Center, New York, the use of T-cell depletion was also associated with an incidence of graft failure approaching 50%. In contrast, T-cell-depleted marrow from parents incompatible for a full HLA haplotype engrafted almost all patients with severe combined immune deficiency [74]. In immune competent hosts, resistance to engraftment is mediated primarily by host T lymphocytes [37]. We presume that with T-celldepleted marrow grafts, the balance between competing host and donor T cells shifts in favor of the host, resulting in unopposed host-versusgraft reaction. In rodents, resistance to MHC-incompatible HCT can be overcome by adding BU, thiotepa or anti-T-cell antibodies to the hostconditioning, or by increasing the number of donor T cells or hematopoietic cells in the donor graft [75]. In leukemia patients transplanted from HLA-mismatched donors, a regimen of TBI, CY, thiotepa, and ATG was inadequate for engraftment of T-cell-depleted marrow, but was adequate for engraftment of T-depleted PBPCs [8]. Partial marrow T-cell depletion Since even a small number of T cells in the marrow inoculum can overcome allogeneic resistance, investigators have developed strategies for partial T-cell depletion. Removal of less than 2 logs T cells from the donor marrow requires, however, the administration of post-transplant immunosuppression for the prevention of GVHD. In 201 patients, conditioning comprised high doses of TBI, CY, cytarabine, and etoposide, while GVHD prophylaxis consisted of partial marrow T-cell depletion with either anti-CD3 murine monoclonal antibody OKT3 (n = 143) or T10B9 (n = 58), plus post-transplant immunosuppression with ATG, CSP, and glucocorticoids [76]. Engraftment occurred in 98% of patients, grade II–IV acute GVHD in 13%, and chronic GVHD in 15%. The 5year transplant-related mortality, however, was 51% and was caused

Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors

predominantly by opportunistic infections. The incidence of relapse was 31%, and the 5-year disease-free survival was 18%. Patient age greater than 15 years, active disease at transplant, donor age greater than 25 years, and a donor mismatched for three HLA-A, B, and DR antigens adversely affected outcome. This study demonstrated that partial T-cell depletion plus postgrafting immunosuppression can prevent GVHD after marrow transplantation from donors mismatched for one HLA haplotype, that the risk of graft failure is low, but that mortality from opportunistic infections is unacceptably high. Hematopoietic cell dose and use of PBPCs Preclinical studies Animal models showed that high doses of T-cell-depleted marrow cells can overcome the MHC barrier without causing GVHD [75]. When purified HSCs were transplanted in irradiated mice, stable hematopoietic chimeras were generated in all cases, but 10–60 times the number of HSCs were required for the survival of mice transplanted across MHC disparities compared with no MHC disparities. Cell-dose escalation allowed full donor cell engraftment even in mice that had been presensitized by donor lymphocytes, in mice whose immune system had been partially reconstituted with the infusion of small numbers of host T cells before the allogeneic transplant, and in mice pretreated with sublethal doses of TBI low enough to spare recipient T lymphocytes. Human CD34+ cells exhibited “veto” activity in vitro, that is, they neutralized allogeneic cytotoxic T-lymphocyte precursor cells directed against their antigens but not against a third party [77,78]. Early myeloid CD33+ cells were also endowed with marked veto activity, which is not found in late myeloid cells expressing CD14 or CD11b. Therefore, soon after transplantation, infused CD34+ cells and their CD33+ progeny might inhibit residual anti-donor cytotoxic T-lymphocyte precursors from the recipients through deletion mediated by tumor necrosis factor-alpha [79]. Transplant studies in Perugia: the benchmark protocol The hypothesis that escalation of the hematopoietic cell dose can facilitate engraftment of T-cell-depleted, HLA-mismatched transplants was first tested in patients with advanced leukemia. In 1993, the Perugia

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Bone Marrow Transplant Center developed a transplant protocol using the combination of G-CSF-mobilized T-depleted PBPCs and T-celldepleted marrow cells for transplantation from HLA-mismatched relatives [8]. Donor and recipients were incompatible for two or three HLA-A, B, and DR antigens of the unshared haplotype. Greater than 3 log T-cell depletion was achieved by lectin agglutination followed by the E-rosetting technique. T cell levels in the graft were a median of 1–2 × 105/kg recipient body weight, and CD34+ cells showed a median of 10 × 10 6/kg recipient body weight. The average dose of GM-CFU was seven- to 10-fold greater in the combined product than in marrow alone. Conditioning included TBI (8 Gy at an instantaneous dose rate of 16 cGy per minute with the lungs shielded to receive 7 Gy), thiotepa (10 mg/kg over 2 days), CY (120 mg/kg over 2 days) and rabbit ATG (25 mg/kg over 5 days). All patients received G-CSF after transplantation until engraftment and no planned post-transplant immunosuppression. Sixteen of 17 patients with advanced leukemia achieved primary engraftment. Acute GVHD grade II–IV occurred in one patient, and there were no cases of chronic GVHD. Two patients relapsed, and nine died of transplant-related complications, which were mainly infections. Six of 17 patients survive [8]. After the first pilot study of T-cell-depleted PBPCs plus marrow, the attention of the Perugia team focused on optimizing the graft-processing protocol, because the original lectin and E-rosetting techniques were technically demanding, time-consuming, labor intensive, and needed expert staff. In 1995, the protocol was modified by replacing the lectin agglutination with positive immunoselection of CD34+ cells, using the Ceprate system (CellPro Inc., Washington, DC, USA), after one round of E-rosetting (Table 46.3). With the Ceprate protocol, the median T-cell dose was 2 × 104/kg, 1 log less than with the lectin protocol [46]. To minimize the extrahematologic toxicity of the conditioning regimen, fludarabine (40 mg/m2/day for 5 days) replaced CY, based on data from a murine model where fludarabine/TBI provided similar immunosuppression to TBI/CY [80]. In addition, the dose of total lung irradiation was decreased from 7 to 4 Gy. The modified protocol was tested in 43 adult patients with high-risk acute leukemia. Primary sustained engraftment was achieved in 41 of 43 (95%) patients. Grade II–IV GVHD did not occur. The 2-year disease-free survival was 36% for 20 patients with

Table 46.3 Evolution of the Perugia protocol for T-cell-depleted hematopoietic cell transplantation (HCT) from human leukocyte antigen (HLA) haplotypeincompatible related donors 1993–1995

1995–1998

1999–2006

HSC source

BM + PBPCs (n = 36)

PBPCs (n = 175)

Graft processing

SBA E-rosetting TBI Thiotepa CY ATG Yes 10.8 2.2 [8]

BM+PBPC (n = 29) PBPCs (n = 15) E-rosetting CD34-selection by Ceprate SC TBI Thiotepa Fludarabine ATG No 10.5 0.2 [46]

Conditioning regimen

G-CSF post-transplant Graft content (median) References

CD34 cells (×106/kg) CD3 cells (×105/kg)

CD34-selection by CliniMACS TBI Thiotepa Fludarabine ATG No 12.8 0.1 [86]

Conditioning regimen used for transplantation of T-cell-depleted peripheral blood HCT in Perugia since 1995. TBI is administered at the dose of 800 cGy in a single fraction at 16 cGy/min. Thiotepa is administered in a single dose of 10 mg/kg. Rabbit antihuman thymocyte globulin (ATG; Fresenius, Bad Homburg, Germany) is administered at 5 mg/kg/day for 4 days. Fludarabine is administered at 40 mg/m2/day for 4 days. SBA, soy bean agglutinin. See text for other abbreviations.

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AML, and 17% for 23 patients with ALL ( p = 0.05), and all survivors recovered a normal performance status [46]. Since 1999, CD34+ cells were positively selected from peripheral blood using the CliniMACS device (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany), which is a one-step, fully automated instrument [81]. Besides providing a good yield of a highly purified CD34+ cell population, the Miltenyi protocol ensures a median of 4.5 log T-cell depletion and a 3.2 log B-cell depletion, which helps prevent Epstein– Barr virus-associated lymphoproliferative disorders in patients who receive ATG and a T-cell-depleted HCT [82]. Post-transplant G-CSF administration was omitted because preliminary data suggested it induced immunosuppression in HLA haplotype-incompatible transplant recipients [83]. Primary engraftment was achieved in 94 of 101 patients, and six more engrafted after a second transplantation. Acute GVHD developed in eight patients, and chronic GVHD in five. Thirty-eight patients died of causes other than leukemia. Relapse occurred in nine of 66 patients receiving transplantation in remission, and in 17 of 38 receiving transplantation in relapse. Event-free survival at 1 year was 48% and 46%, respectively, for the 42 AML and 24 ALL patients receiving transplantation in remission, and 4% for the 38 patients with AML or ALL receiving transplantation in relapse [84]. These clinical trials demonstrated that a highly immune-suppressive conditioning regimen of TBI, fludarabine, thiotepa, and ATG combined with positively CD34-selected PBPC cells can overcome the engraftment barriers across HLA disparities in leukemia patients. In addition, CD3+ cell doses up to 2 × 104/kg do not cause severe GVHD if ATG is administered as part of the conditioning regimen. Effective GVHD prevention hinders the antileukemia effects mediated by T cells against histocompatibility antigens on recipient leukemia [53,85], but donor NK-cell alloreactivity against the recipient contributed to control posttransplant relapse in AML patients (see the section on NK alloreactivity below) [85–90]. Studies in Tübingen and Memphis: T-cell negative selection from PBPCs and reduced-intensity conditioning Handgretinger and collaborators modified the Perugia protocol by substituting OKT3 for ATG, and shifting from positive CD34+ cell selection to negative selection through the use of CD3- and CD19-coated CliniMACS microbeads, so as to infuse both CD34+ and CD34− cells, including CD8+ T cells, NK cells, and other accessory cells [91]. In children with leukemia or lymphoma, Lang et al. [92] compared positive HSC selection with CD34- (n = 39) or CD133-coated magnetic microbeads (n = 14) to T- and B-cell depletion using CD3- and CD19-coated microbeads (n = 11). Primary engraftment was 85% after CD34-positive selection, 72% after CD133-positive selection, and 91% after CD3/ CD19-negative selection. The incidence of grade II–IV GVHD was similar after CD34- or CD133-positive selection (3% and 7%, respectively), but increased to 27% after CD3/CD19-depletion. Despite the rise in GVHD, the investigators reported a lower than 10% incidence of fatal viral infections, suggesting that rapid CD3+ cell recovery had occurred. It remains unclear whether CD3/CD19-negative selection provides any advantage over positive CD34-selection, or whether OKT3 is as effective as ATG. To reduce toxicity, Lang et al. designed a conditioning regimen consisting solely of chemotherapy: fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), and OKT3 (5 mg/day, from day −5 to day +14) and no post-transplant immunosuppression [93]. Engraftment was prompt, with full donor chimerism after 2–4 weeks in all patients. The incidence of grade II–IV GVHD was 48%. In the first 100 days, six of 29 (20%) patients died of treatment-related complications. This chemotherapy-only regimen is promising, and a prospective phase I–II study is ongoing.

Studies in Japan using conventional high-dose conditioning regimens In Japan, a transplant trial of CD34+ cells selected with the Isolex device (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) enrolled 135 children, 64 of whom received two-HLA locus-mismatched grafts, and 43 three-locus-mismatched grafts [94]. Median CD34+ cell doses were 3.2 × 106/kg for patients receiving marrow only, 5.5 × 106/kg for those receiving PBPCs only, and 4.9 × 106/kg for those receiving marrow and PBPCs, and median T-cell doses were 6.0, 9.4, and 12.1 × 104/kg, respectively. The conditioning regimens before HCT included TBI in 77% of the cases and ATG in 52%; therefore they were in general less intense and immunosuppressive than those developed in Perugia (Table 46.3). Patients needed post-transplant immunosuppression to prevent GVHD. Graft failure occurred in 13% of patients with hematologic malignancies, and 40% of patients with nonmalignant disease. The incidence of GVHD was 10% when ATG was included in the conditioning and 27% when it was not included. Disease-free survival at 5 years was 39% in standard-risk patients and 5% in high-risk patients. These results confirm that CD34+ PBPCs do not ensure consistent engraftment of HLA-mismatched transplants unless an optimized conditioning regimen is administered, and that GVHD prevention requires postgrafting immune suppression if T-cell depletion is suboptimal. Infection prophylaxis The majority of nonrelapse deaths after HLA-mismatched transplants are related to cytomegalovirus (CMV) and Aspergillus infection [84,85]. Therefore, patients transplanted in Perugia with a T-cell-depleted, HLAmismatched graft receive prophylaxis for both CMV and Aspergillus. For all CMV-positive recipients, prophylaxis consists of ganciclovir (10 mg/kg/day) from −9 to day −2, and foscarnet (90 mg/kg/day) from day +4 after to day +20. Antifungal prophylaxis is liposomal amphotericin B (1 mg/kg/day) from day −5 to day +20, followed by oral itraconazole solution (400 mg/day) from day +21 to +120. Despite this prophylactic regimen, patients remain susceptible to opportunistic infections for several months. The risk of life-threatening infectious episodes plateaus after about 1 year, supporting laboratory data indicating that immune reconstitution is complete in transplant recipients who do not receive immunosuppression and do not suffer from chronic GVHD. Consequently, careful monitoring on an outpatient basis is recommended for the first 8–10 months after transplantation to detect and promptly treat infections at an early stage. Immune reconstitution Several mechanisms are responsible for patient immunodeficiency after transplantation of T-cell-depleted hematopoietic cells from HLAincompatible donors. In adults, early immune recovery stems from the expansion of mature T cells in the graft. However, in HLA-mismatched transplants with T-cell-depleted grafts, the number of T cells in the graft must be sufficiently low to prevent GVHD, and anti-T-cell antibodies administered in the conditioning regimen further deplete the few transplanted T cells. Intense conditioning regimens induce tissue damage that prevents T-cell homing to peripheral lymphoid tissues, where the generation and maintenance of T-cell memory take place [95,96]. Administration of G-CSF after transplantation is immunosuppressive as it blocks IL-12 production by APCs and decreases T-cell responses to pathogens in vitro and in vivo after transplantation [83]. Since the administration of G-CSF after transplantation was omitted in the Perugia trials, engraftment rates have remained unchanged, and recovery rates of immune parameters, including APC production of IL-12, have improved [83]. Several strategies are under investigation to hasten post-transplant immune recovery and decrease mortality from opportunistic infections

Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors

without causing GVHD. Genetic manipulation of donor lymphocytes with a suicide gene is a promising strategy to effectively eliminate pathogenic T cells [97]. Should GVHD develop after in vivo transfer of donor lymphocytes genetically engineered with the herpes simplex virus thymidine kinase suicide gene, transduced cells can be eliminated by ganciclovir treatment. In 17 patients, cells positive for thymidine kinase provided protection against CMV reactivation and disease. Overall, the cumulative infectious mortality at 6 months post transplant was 12%, with only 6% CMV-related mortality. Reinfusion of nonalloreactive T cells into patients previously transplanted with T-cell-depleted grafts from HLA-mismatched donors may lead to immune reconstitution without GVHD. Such approaches involve the co-incubation of donor T cells with recipient APCs in the presence of agents that can selectively eliminate or inactivate host-reactive T cells. In two studies, donor T cells were exposed ex vivo to recipient alloantigen and treated with an immunotoxin specific for the IL-2 receptor alpha chain [98,99]. This approach was effective in selectively eliminating alloreactive T cells without eliminating T cells reactive to third-party antigens. Based on preclinical data that antigen presentation in the absence of CD28 co-stimulation induces a state of T-cell unresponsiveness, one clinical trial employed host APCs and soluble cytotoxic T lymphocyte antigen-4 immunoglobulin (Ig) to present host alloantigen to donor T cells while blocking the CD28 co-stimulatory pathways. This approach resulted in ex vivo donor T-cell unresponsiveness to the HLA-mismatched cells of the recipient [100]. Transplantation of marrow replete with alloantigen-unresponsive T cells led to primary engraftment in 11 of the 12 children, and three cases of acute GVHD despite post-transplant immunosuppressive therapy. An update of this study after treatment of an additional 12 patients (five receiving grafts tolerized in vitro by host antigen and B7-specific antibodies) reported acute GVHD grade II–III in 35% of patients [101]. This approach appears to have the potential to facilitate engraftment and perhaps immune reconstitution, but does not appear to eliminate GVHD. Another strategy is to adoptively transfer pathogen-specific donor immune cells. Encouraging results were achieved using ex vivo-expanded EBV-specific allogeneic cytotoxic T lymphocyte clones to prevent or manage EBV-associated diseases, including post-transplant lymphoproliferative disorders [102]. Donor CD4+ T-cell clones were raised against Aspergillus fumigatus and CMV antigens, and screened for cross-reactivity to host alloantigens [103]. Nonhost-reactive CD4+ T-cell clones, presumably devoid of GVHD potential, were pooled and infused into recipients soon after a T-cell-depleted HLA-mismatched transplant. The maximum safe dose (1 million CD3+ cells/kg recipient body weight) appeared clinically effective and did not cause GVHD. CMV reactivation and Aspergillus galactomannan antigenemia disappeared over time, while the frequencies of anti-Aspergillus- and anti-CMV-specific T-cell clones increased. These diverse approaches show that adoptive cellular therapy is feasible and can be effective in fighting infections after HLA-incompatible HCT.

activate SHP-1 and SHP-2 phosphatases for inhibitory signal transduction. KIR2DL1 recognizes HLA-C alleles characterized by a Lys80 residue (HLA-Cw4 and related; “Group 2” alleles). KIR2DL2 and KIR2DL3 (which are allele variants) recognize HLA-C with an Asn80 residue (HLA-Cw3 and related; “Group 1” alleles). KIR3DL1 is the receptor for HLA-B alleles sharing the Bw4 supertypic specificity (Tables 46.4 and 46.5). Another type of human NK-cell inhibitory receptor involved in HLA recognition is CD94-NKG2A. It binds to the nonconventional class I molecule HLA-E [107]. Several HLA class I alleles provide signal sequence peptides that bind HLA-E and allow its expression at the cell surface. Consequently, it is expressed in every individual. Inhibitory KIRs, CD94/NKG2A, and HLA-class I genes determine individual NK-cell repertoires during development [108,109]. As they are located on different chromosomes, receptors and ligands segregate independently in human pedigrees. The HLA class I genotype selects a self-tolerant repertoire by dictating which KIR and/or NKG2A receptor combinations are to be used as inhibitory receptors for self-HLA class I. Consequently, every functional NK cell in the mature repertoire expresses at least one inhibitory receptor for self-HLA: coexpression of two or more receptors is less frequent. Since inhibitory KIRs recognize specific groups of HLA class I molecule, that is, HLA-C group 1, HLA-C group 2 or HLA-Bw4 alleles, NK cells with the potential to exert allogeneic reactions use KIRs as inhibitory receptors for self-HLA class I [86–90,110–112]. NK cells that express, as their only inhibitory receptor for self-HLA, a KIR for the class I group that is absent on allogeneic targets sense the missing expression of the self-class I KIR ligands and mediate alloreactions (“missing self” recognition) (Fig 46.5). Most individuals can exert allogeneic reactions as they possess a full complement of inhibitory KIR genes. The KIR2DL2 and/or KIR2DL3 receptors for HLA-C group 1 are present in all; the KIR2DL1 receptor for HLA-C group 2 is found in 97% and the KIR3DL1 receptor for HLA-Bw4 alleles is found in approximately 90% of people [90,113,114]. Individuals expressing group 1 HLA-C alleles possess the KIR specific for group 1 HLA-C alleles (KIR2DL2 and/or KIR2DL3) and are alloreactive against cells from individuals who do not express group 1

Table 46.4 Human leukocyte antigen (HLA) class I specificity of the main inhibitory killer immunoglobulin-like receptors (KIRs) expressed by human natural killer (NK) cells KIR genes*

Encoded protein

HLA specificity†

KIR2DL1

P58.1 receptor

HLA-C group 2 i.e. Cw2, Cw4, Cw5, Cw6 Sequence: Asn77, Lys80

KIR2DL2/3

P58.2 receptor

HLA-C group 1 i.e. Cw1, Cw3, Cw7, Cw8 Sequence: Ser77, Asn80

KIR3DL1

P70/NKB1 receptor

HLA-Bw4-associated i.e. B27

Role of NK cell alloreactivity in HLA-incompatible HCT NK-cell activation is regulated by a balance between inhibitory and activating receptors (see Chapter 13). In humans, currently 16 inhibitory killer Ig-like receptors (KIR) genes and pseudogenes are known to codify for inhibitory and activating KIRs. Inhibitory KIRs recognize amino acids in the COOH-terminal portion of the MHC class I α1 helix [104–106]. They possess two (KIR2D) or three (KIR3D) extracellular C2-type Ig-like domains and a long cytoplasmic tail (L) containing immunoreceptor tyrosine-based inhibition motifs, which recruit and

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* KIR2D refers to receptor molecules with two immunoglobulin-like domains, whereas KIR3D refers to those displaying three immunoglobulin-like domains. Receptors having a long inhibitory cytoplasmic tail are designated as L (long), whereas those having a short activating tail are termed S (short). † Two groups of HLA-C alleles can be distinguished on the basis of alternative amino acid sequence motif at position 77 and 80 of the α1 helix.

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Table 46.5 Human leukocyte antigen (HLA)-C group 1, HLA-C group 2 and HLA-Bw4 group alleles Group 1 HLA-C alleles (Ser77, Asn80) Cw1 Cw3 (except C*0307, C*0310,† and C*0315)

Cw7 (except C*0707 and C*0709) Cw8 Cw12‡ (except C*1204 and C*1205) Cw13 Cw14 (except C*1404§) C*1507 Cw16 (except C*1602)

Group 2 HLA-C alleles (Asn77, Lys80)

HLA-Bw4 alleles

Cw2 C*0307 and C*0215

B5 B13 B17 B27

Cw4 Cw5 Cw6 C*0707 and C*0709

B37 B38 B44 B47

C*1204 and C*1205

B49 B51

Cw15 (except C*1507) C*1602 Cw17 Cw18

B52 B53 B57 B58 B59 B63 B77 B*1513 B*1516 B*1517 B*1523 B*1524

Each serologically defined group includes all alleles except where noted. † C*0310 (Ser77, Lys80) belongs to both HLA-C groups 1 and 2 [142]. C*0310 blocks natural killer (NK) cells expressing all HLA-C-specific receptors, but does not block clones expressing the Bw4 receptor. ‡ C*1207 (Gly77, Asn80) cannot be assigned to either group based on its amino acid sequence, and still needs to be tested functionally. § C*1404 (Asn77, Asn80) does not belong to either HLA-C group 1 or 2 and does not block NK cells expressing any HLA-C-specific receptor [142]. Expression of C*1404 in a patient behaves with respect to NK-cell recognition as if the patient does not express that HLA-C allele.

HLA-C alleles. Individuals who express group 2 HLA-C alleles and possess the KIR specific for group 2 HLA-C alleles (KIR2DL1) exert NK alloreactions against cells from individuals who do not express group 2 HLA-C alleles. HLA-Bw4+ individuals who also express the Bw4-specific KIR3DL1 receptor may have NK cells that are alloreactive against Bw4− cells. These KIR ligand mismatches occur often in donor– recipient pairs that are disparate for one HLA haplotype (Table 46.6). When exerted in the donor-versus-recipient direction, NK cell alloreactivity emerged as a crucial factor in improving outcomes of haploidentical transplantation. The opportunity for NK cell alloreactivity reduced the risk of leukemia relapse, did not cause GVHD, and markedly improved event-free survival in a series of haploidentical transplants (57 patients with AML, 20 of whom were transplanted from NK-alloreactive donors) [87]. In an updated analysis [90], 112 patients with AML received HLA-incompatible HCT from NK-alloreactive (n = 51) or non-NKalloreactive donors (n = 61). For patients in remission at the time of HCT, the cumulative incidence of relapse was significantly lower after transplantation from NK-alloreactive donors (3% versus 47%; p < 0.003) (Fig. 46.6(b)), and this translated into an improved event-free survival

Recipient

Donor NK cell

HLA HLA-C group 2 Cw2

KIR2DL1

HLA-C group 1 Cw1

KIR2DL1 2/3

HLA-Bw4 B51

KIR3DL1

HLA-C group 2 Cw2/Cw4

missing HLA-C group 1

HLA-Bw4 B27

Fig. 46.5 Donor-versus-recipient natural killer (NK) cell alloreactivity in human leukocyte antigen (HLA)-mismatched transplants. NK-cell alloreactions are generated between donors and recipients who are killer immunoglobulin-like receptor (KIR) ligand-mismatched in the graft-versushost direction. In these circumstances, donor NK cells expressing, as their only inhibitory receptor for self-HLA, a KIR for the class I group that is absent in the recipient sense the missing expression of the self class I ligand on allogeneic targets and mediate alloreactions. This figure illustrates the example of a donor–recipient pair with a combination of mismatched HLA-C antigens, resulting in donor NK-cell alloreactivity against recipient targets. The top donor NK cell expresses KIR2DL2/3 and is blocked by HLA-C group 1 antigens Cw1 on donor cells and Cw8 on recipient cells. The bottom donor NK cell expresses KIR3DLI that is blocked by Bw4 on both recipient and donor cells. The middle donor NK cell expresses KIR2DL1 that is blocked by donor (self)-HLA-Cw2 (an HLA-C group 2 antigen), but not by the recipient HLA-Cw3 (an HLA-C group 1 antigen). Consequently, this NK cell is alloreactive and lyses the recipient’s cells (lightning bolt).

Table 46.6 Donor–recipient human leukocyte antigen (HLA) class I combinations associated with natural killer (NK)-cell alloreactivity in the graft-versus-host direction Recipient HLA type

HLA type of NK alloreactive donor*

Group 1 HLA-C, HLA-Bw4 Group 1 HLA-C, Group 1 HLA-C, Group 2 HLA-C, Group 1 HLA-C Group 2 HLA-C

Group 2 HLA-C,

No NK alloreactive donor

Group 2 HLA-C HLA-Bw4 HLA-Bw4

HLA-Bw4 Group 2 HLA-C Group 1 HLA-C Group 2 HLA-C and/or HLA-Bw4 Group 1 HLA-C and/or HLA-Bw4

* Recipients who express class I alleles belonging to the three major class I groups (HLA-C group 1, HLA-C group 2, and HLA-Bw4 alleles) will block all NK cells from every donor. Donors may exert donor-versus-recipient NK cell alloreactivity when HLA-C and HLA-B typing shows KIR-ligand mismatches in the graft-versus-host direction, that is, the recipient does not possess one HLA-C allele group (C1 or C2) and/or the HLA-Bw4 group that are present in the donor. In the donor’s HLA typing, the alleles listed are associated with the potential to exert NK-cell alloreactions against the specific HLA type of the recipient, whatever other alleles may be present. The HLA-C group 1 receptor genes (KIR2DL2 and/or KIR2DL3) are present in 100% of individuals. High-frequency alloreactive NK clones are detected in these individuals. The HLA-C group 2 receptor gene (KIR2DL1) is present in approximately 97% of donors. When the gene is present, high-frequency alloreactive NK clones are detected in donors. The KIR3DL1 HLA-Bw4 receptor gene is present in only around 90% of individuals. Even when the gene is present, alloreactive NK clones occur in highly variable frequencies and are detected in two-thirds of donors. Thus, functional assessment of the donor NK repertoire appears necessary.

Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors

Fig. 46.6 Transplantation from haploidentical natural killer (NK)alloreactive donors controls relapse in acute myeloid leukemia and improves event-free survival. (a) Relapse in patients transplanted in chemoresistant relapse from NK-alloreactive versus non-NK-alloreactive donors. (b) Relapse in patients transplanted in any remission from NK-alloreactive versus nonNK-alloreactive donors. (c) Event-free survival in patients transplanted in relapse from NK-alloreactive versus non-NK-alloreactive donors. (d) Eventfree survival in patients transplanted in complete remission from NKalloreactive versus non-NK-alloreactive donors. (Data from [90].)

(67% versus 18%, P = 0.02) (Fig. 46.6(d)). For patients in relapse at the time of HCT, donor NK cell alloreactivity was associated with no difference in post-transplant relapse (32% versus 37%, p = NS) (Fig. 46.6(a)), although it was associated with improved event-free survival (0.48%, 95% CI 0.29–0.78; p < 0.001) (Fig. 46.6(c)). Based on these data, donor NK-cell alloreactivity against recipient cells has become a selection criterion for HLA-disparate donors when a T-cell-depleted protocol is employed. The 67% probability of surviving event-free for AML patients transplanted in any remission from NK-alloreactive donors is in the range of best survival rates after transplantation from unrelated donors and cord blood units. The 34% event-free survival of patients transplanted in chemoresistant relapse from NK-alloreactive donors is also remarkable. Transplantation from non-NK-alloreactive, HLA haplotype-incompatible donors appears justified only for AML patients in remission, where it leads to an 18% event-free survival. Lack of an NK-alloreactive donor is a contraindication to transplantation for patients in chemoresistant relapse as very few survive. Several observations suggest alloreactive NK cells are responsible for favorable transplantation outcomes. Transfer of human alloreactive NK cells into NOD-SCID mice eradicated previously transplanted human AML cells [87]. KIR ligand mismatches correlated with the ability of

669

donor NK-cell clones to kill cryopreserved hematopoietic recipient cells, including leukemic cells [86,87,90]. Most importantly, engrafted HSCs give rise to an NK-cell repertoire that includes donor-versus-recipient alloreactive NK clones that kill cryopreserved hematopoietic recipient cells, including leukemic cells [86]. Donor-versus-recipient alloreactive NK clones are detected in vivo in recipients for up to 1 year after transplant [90]. One immediate consequence of these clinical findings is exploitation of NK-cell alloreactivity in haploidentical donor selection. The search for NK-alloreactive donors may require extension beyond the immediate family to aunts, uncles, cousins, etc. In the clinical studies above, NKalloreactive donors were found for around 50% of patients, which approaches the maximum because one-third of the population express class I alleles belonging to all three class I groups recognized by KIRs and block NK cells from every donor. How is an NK alloreactive donor selected? Recipients who express alleles belonging to one or two of the three class I allele groups recognized by KIRs may find NK-alloreactive donors. Donors who are HLAC group mismatched with their recipients possess high-frequency NK clones that are alloreactive against recipients’ target cells [86–90]. Thus, high-resolution HLA-C typing is a good predictor of NK-cell alloreactivity (Tables 45.5 and 45.6). Since 3% of individuals do not possess the KIR2DL1 gene [90,105,114], the combination of a KIR2DL1− donor and a recipient without HLA-C group 2 alleles could result in a 1.5% incidence of false positivity. KIR2DL1 gene typing of the donor may be necessary to assess the NK-alloreactive potential of this combination. In HLA-Bw4 mismatches, even when the KIR3DL1 gene is present in the donor (~90% of individuals) [90,105,114], NK repertoire studies show that alloreactive NK clones are nondetectable in around one-third of individuals [90]. In some, allelic variants in the HLA-Bw4 inhibitory NK receptor gene KIR3DL1 may not allow full receptor expression at the cell membrane and affect NK cell inhibition by the HLA-Bw4 ligand [115,116]; others apparently express alloreactive NK clones in very low frequencies. Thus, for HLA-Bw4 mismatches, functional assessment of the donor NK repertoire appears necessary. As approximately half of unrelated donor transplants are mismatched for one or more HLA class I alleles, donor versus recipient NK cell alloreactivity may also occur in this setting. However, some retrospective studies show no advantage in transplantation from KIR ligandmismatched donors [117–121]. Unrelated donor transplant protocols are heterogeneous in conditioning regimens, patient populations, and underlying diseases. They most commonly use T-cell-replete bone marrow harvests or, nowadays more frequently, peripheral blood progenitors that contain approximately 4 log more T cells than haploidentical grafts. Relatively few transplanted HSCs, combined with the high T-cell graft content and post-transplant immune suppression, have been associated with poor reconstitution of potentially alloreactive, KIR-bearing NK cells [122,123]. Other studies have observed an increased graft-versusleukemia effect of KIR ligand-mismatched transplants [124–129]. A marked survival advantage was reported in patients who received ATGs pre transplant to produce in vivo T-cell depletion, and a graft containing two- to three-fold more nucleated cells than usual in unrelated donor transplantation for a population containing a high proportion of children [124]. Prospective studies are needed to determine whether strategies such as high doses of HSCs, T-cell depletion, and no post-transplant immune suppression, which harness donor-versus-recipient NK-cell alloreactivity in haploidentical transplantation, can be implemented to improve outcome in more closely HLA-matched unrelated donor transplants. Since the original report that NK-cell alloreactivity in haploidentical transplantation rests upon KIR ligand mismatching and donor NK-cell

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recognition of “missing self” on recipient targets [87], the “missing ligand” model has been proposed as a powerful algorithm for predicting favorable transplant outcomes not only in haploidentical transplants [130,131], but also in matched sibling [132] and in unrelated donor transplants [133]. Under the perturbed conditions that exist after an HCT, it is hypothesized that NK allogeneic reactions occur when KIR ligand-matched donors possess an “extra” KIR for which neither donor nor recipient has an HLA ligand. These donors may carry KIR-bearing NK cells in an anergic/regulated state that, upon transfer into the recipient, are hypothesized to become activated and exert a graft-versus-leukemia effect. However, even though self-tolerant NK cells that do not express inhibitory receptors for self-MHC have been described in mice [134– 137] and humans [138], no studies have as yet determined whether tolerant NK cells acquire/resume cytotoxic effector function after transplant. When an adult series of AML patients transplanted from HLA incompatible donors was analyzed according to the “missing ligand” algorithm, the “missing ligand” transplant recipients disappointingly had a worse prognosis than patients transplanted from NK-alloreactive (KIR ligand-mismatched) donors [90]. While differences in diseases, age of patients, and transplantation protocols, such as ATG [90] versus no ATG in the conditioning regimen [130–138], or peripheral blood CD34+ cells [90] versus bone marrow [137,138] as a source of hematopoietic cells, may account for these conflicting results, the analysis shows that donor NK-cell recognition of “missing self” on recipient targets is essential for triggering powerful NK-cell allogeneic reactions that are beneficial for transplant outcomes. Effects of donor activating KIR genetics on donor-versusrecipient NK cell alloreactivity Activating KIRs, which regulate NK- and T-cell functions, are molecular homologs of the inhibitory KIRs with shorter cytoplasmic tails (S) [104–106,119,139–141] and a charged residue in their transmembrane domain that allows association with immunoreceptor tyrosine-based activation motif-containing signaling polypeptides. Knowledge of their ligand specificity is limited [142–145]. These studies have reported a weak interaction between KIR2DS1 and Lys80 HLA-C molecules, despite its homology to KIR2DL1, and an even weaker interaction between KIR2DS2 and Asn80 HLA-C molecules, despite its homology to KIR2DL2 and KIR2DL3. Unlike inhibitory KIRs, activating KIRs exhibit extensive variation in gene number and content, and this leads to heterogeneity within the general population and diverse ethnic groups [114]. Indeed, activating KIRs may not even be present in approximately 25% of Caucasians who are homozygous for the so-called “group A” KIR gene haplotypes that contain inhibitory KIR genes, and the KIR2DS4-activating KIR gene that in two-thirds of individuals encodes for a nonfunctional protein. On the other hand, 75% of Caucasians are either heterozygous or homozygous for “group B” haplotypes, which carry not only inhibitory KIR genes, but also various combinations of activating KIR genes KIR2DS1-2-3-5 and KIR3DS1. Several studies have evaluated the role of KIR genetics in allogeneic HCT, with diverse results. Transplantation from donors carrying activating KIR genes was associated with improved control of leukemia relapse after related HLA-identical transplantation [146], improved survival after unrelated donor transplantation [147], and less CMV reactivation after related HLA-identical transplantation [148]. After matched sibling, unrelated, and partially T-cell-depleted HLA haplotype-incompatible transplants, donor activating KIR genetics were reported to adversely affect outcomes, mainly through an increased incidence of acute GVHD [149–154]. Thus, with T cells in the graft, donor activating KIR genes may result in excessive T-cell alloreactivity and GVHD.

One recent analysis hypothesized that this may not occur after extensively T-cell-depleted HLA haplotype-incompatible transplants [155]. In a series of 84 haploidentical transplants for AML, the impact of donor KIR genetics (group A versus group B KIR gene haplotypes) was assessed separately in NK-alloreactive and non-NK-alloreactive transplants. Forty-seven recipients were transplanted from NK-alloreactive donors (12 with group A KIR gene haplotypes versus 35 with B haplotypes), and 37 recipients from non-NK-alloreactive donors (8 with group A KIR gene haplotypes versus 29 with B haplotypes). KIR gene haplotypes had no impact in non-NK-alloreactive transplants. In transplants from NK-alloreactive donors, the presence of group B haplotype KIR genes in the donors was associated with reduced transplant-related mortality, largely infection-related (B versus A haplotypes: 20% versus 67% transplant-related mortality; p < 0.005). In multivariate analyses taking into account disease status at transplant, age, patient and donor sex, conditioning regimens, and the number of CD34+ and CD3+ cells in the graft, activating KIR genotype was the only significant variable predicting protection from transplant-related mortality (RR 0.24, 95% CI 0.14– 0.42; p < 0.01), and resulted in a trend towards better event-free survival (60% versus 33%, p < 0.1). When the number of activating KIR genes in the donor was taken into account, donors carrying three or more activating KIR genes provided significant protection from transplant-related mortality (12% versus 67%; p < 0.003) and significantly better event-free survival compared with A haplotype donors that had fewer than three activating KIR genes (71% versus 33%; p = 0.02). In multivariate analysis, transplantation from alloreactive donors carrying three or more group B haplotype activating KIR genes was the only variable predicting protection from transplantrelated mortality (RR 0.40, 95% CI 0.27–0.58; p < 0.02) and significantly improved event-free survival (RR 0.56, 95% CI 0.32–0.98; p < 0.05). Thus, while NK-alloreactive donors protect against leukemia relapse, those who also carry activating KIRs protect against infectious mortality and help improve survival. The chance of finding an NK alloreactive donor is around 50% of HLA haplotype-incompatible transplants, and the odds of finding an NK-alloreactive donor carrying activating KIRs are about 30% of HLA haplotype-incompatible transplants. Protection against infection may be mediated directly by NK cells or indirectly through other mechanisms. Activating KIRs could enhance NK-cell cytokine secretion and cytotoxicity against pathogen-infected cells in the context of missing self. A notable example of direct recognition of a pathogen by an activating NK receptor is provided by murine CMV protein m157 and the murine Ly49H NK receptor [156]. In humans, progression to acquired immune deficiency syndrome is slower in patients who have both KIR3DS1 and the HLA-Bw4 allotype, the putative ligand of KIR3DS1 [157]. More NK cells expressing NKG2C are present in CMV-exposed individuals, suggesting this activating NKcell receptor plays a role in the immune response to this infection [158]. Therefore, associations between activating NK receptors and enhanced immunity against infections are well documented. Activating KIRs could also help control infections indirectly through the interaction between NK cells and dendritic cells [159–165]. NK cells regulate dendritic cell homeostasis and maturation. Mature dendritic cells can, in turn, activate NK cells. In vivo, NK– dendritic cell interactions in lymphoid organs or nonlymphoid tissues can lead to T helper type 1 cell polarization. NK cells in lymph nodes provide the early interferongamma production, which is essential for T helper type 1 polarization [166]. Consequently, the interaction between NK and dendritic cells influences the quality and the strength of the adaptive immune response. The clinical data suggest that either or both these mechanisms could operate in HLA haplotype-incompatible transplants from NK-alloreactive donors who possess activating KIRs. These studies have improved criteria for donor selection.

Hematopoietic Cell Transplantation from Human Leukocyte Antigen Partially Matched Related Donors

Conclusion Transplant results have demonstrated that an increasing degree of donor HLA incompatibility is associated with a proportionally increased risk of graft failure, GVHD, and transplant-related mortality. Initial studies utilizing T-cell-replete marrow transplants found that donor incompatibility for two or three HLA antigens represented a formidable barrier to success, did not allow sufficient control of GVHD, and led to unacceptably low patient survival. As post-transplant immunosuppression protocols have failed to control GVHD from highly mismatched donors in the majority of studies, great effort has been invested to test the use of T-cell-depleted marrow grafts. Such studies showed a decrease in acute and chronic GVHD, but at the expense of increased graft failure and leukemia relapse, and impaired immune reconstitution, leading to no advantage in survival. The most recent advance in HLA-mismatched HCT has been the use of hematopoietic growth factors to mobilize PBPCs. Transplantation from a full HLA haplotype-mismatched family

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member is currently a viable option for patients with acute leukemia at high risk of relapse who urgently need a transplant and do not have a matched unrelated donor. The mismatched transplant relies for its success on the combined action of: 1 high-dose conditioning regimens to ensure the lowest possible residual leukemia burden and maximal degree of immunosuppression; 2 very high doses of hematopoietic cells to ensure engraftment across the HLA barrier; 3 extensive T-cell depletion of the graft to prevent GVHD; 4 a lack of post-transplant immunosuppression to ensure undisturbed immune reconstitution; 5 donor-versus-recipient NK-cell alloreactivity to facilitate engraftment, prevent GVHD, and exert antileukemia effects; 6 NK-alloreactive donors who also carry activating KIRs to protect against infectious mortality and help improve survival. Exploiting NK cell alloreactivity may enhance the efficacy and safety of HLA-mismatched transplants.

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Effie W. Petersdorf

Hematopoietic Cell Transplantation from Unrelated Donors

Introduction The transplantation of hematopoietic stem cells from unrelated volunteer donors has increased exponentially over the last two decades and now accounts for almost 15% of all allogeneic transplants performed worldwide (http://www.marrow.org) [1]. The increased safety and efficacy of the procedure have contributed to the increasing numbers of patients referred for unrelated donor hematopoietic cell transplantation (HCT) at an earlier stage in their disease. Major advances that have contributed to the improved outcomes after unrelated HCT include the development of less toxic conditioning regimens and improved graft-versus-host disease (GVHD) prevention and treatment strategies, recognition of the importance of comprehensive and precise donor human leukocyte antigen (HLA) matching, the availability of molecular technologies for the early detection of recurrent malignancy, the development of immunotherapeutic strategies for the treatment of recurrent disease, and improved supportive care of patients. As the unrelated HCT clinical experience matures, there has been a greater appreciation for the many factors that influence the success of unrelated donor HCT. Some factors that impact transplant outcome can be modified and/or choices are available, and thus provide the patient and physician with a means to plan the transplant at a time when these factors are optimal. Perhaps the best example of a modifiable factor is the HLA-match status of the donor. When a suitably matched donor is not available, a search can be continued until a better matched donor is identified, provided the patient’s clinical status remains stable and the disease is well controlled. Other factors such as patient age cannot be modified, and yet other variables, such as comorbid medical conditions, may be difficult to modify. It has long been recognized that the success of allogeneic transplantation from related or unrelated donors is strongly influenced by the underlying malignancy and the burden of disease at the time of transplantation. For the individual patient, when to time the transplant, how the transplant should be performed, and from which donor, can be a complex decision that requires careful assessment of the risks and benefits. Central to the success of an unrelated donor transplant is the ability to surmount the “transplantation barrier.” The concept that GVHD is a complex disease of polygeneic etiology shaped by “environmental” factors, such as the conditioning and immunosuppressive regimens, provides a framework for understanding the immunogenetics of the transplantation barrier. The known genetic determinants involved in GVHD

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

and the graft-versus-tumor (GVT) effect are genes of the classical (and possibly nonclassical) HLA and the natural killer (NK) families, cytokine, and immune response genes. Identification of single genes of importance in transplant outcome is but the first step in understanding the basis of the transplantation barrier. The role of gene–gene interactions is increasingly recognized, and the inflammatory environment in which these interactions occur provides a basis for understanding individual patient susceptibility to GVHD and the strength of GVT. In turn, the impact of genetic effects varies with different conditioning regimens (high dose and reduced intensity) and immunosuppressive therapy (including manipulation of the graft, T-replete versus T-deplete grafts, and different methods for depletion), and with different cell sources (bone marrow [BM], peripheral blood progenitor cells [PBPCs] or cord blood). Indeed, elucidation of the many genetic and environmental factors that influence outcome will require methodical analysis of very large well-characterized transplant populations with complete clinical data. The recent availability of a sequence of the human genome (http:// www.hapmap.org) provides the much-needed map to identify the genes that constitute a major component of the transplantation barrier. With the technical ability to detect single-nucleotide differences in gene sequences between recipients and donors, however, our understanding of the extent and nature of human diversity is far ahead of what is known about the functional consequences of that genetic variation. Ultimately, how best to integrate genetic information into day-to-day clinical practice to benefit the individual patient will require continued clinical research on both the genetics front as well as in the development of novel prevention and treatment strategies. Currently, HLA typing for donor selection and risk assessment provides an immense tool when counseling patients and planning transplant treatment strategies. Inclusion of NK genetic information into the assessment of potential donors and for planning the future transplant approach is not far behind, as is the potential clinical use of cytokine polymorphism data for planning immunosuppressive preventive and treatment strategies. In this chapter, the major advances that have contributed to the increased safety and efficacy of unrelated donor transplantation are described. The reader is directed to Chapters 12, 13, 38, 48, and 84 for topics related to unrelated donor HCT.

Role of unrelated donor HCT in the treatment of hematologic malignancies The chief determinant of transplant outcome is the delicate balance between the benefit of GVT and the risk of transplant-related morbidity and mortality (TRM). One of the most challenging aspects of planning transplantation for patients who lack a related donor is determining who

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Years after Transplantation

Fig. 47.1 Impact of disease stage and human leukocyte antigen allele matching at the time of unrelated donor transplantation. (a) Low-risk chronic myeloid leukemia (chronic phase [CML-CP] and transplantation within 2 years of diagnosis). (b) Intermediate-risk hematologic malignancies (CML-CP and transplantation more than 2 years from diagnosis; CML acute phase and CML blast phase/remission [CML-BP/rem], and transplantation within 3 years of diagnosis; acute leukemia in remission; myelodysplastic syndrome-refractory anemia). (c) High-risk hematologic malignancies (CML-BP; CML acute phase and CML-BP/rem, and transplantation more than 3 years from diagnosis; acute leukemia in relapse; myelodysplastic syndrome beyond refractory anemia). (Reproduced from [2].)

can benefit from an unrelated HCT, when the transplant should be performed, how the transplant should be carried out, and which donor should be chosen. In general, patients derive the greatest benefit from unrelated HCT when transplantation can be performed while carrying the lowest leukemic burden, using HLA-matched donors, and timed when the patient can best tolerate the transplant procedure. The dramatic impact of disease stage is best seen among recipients of HLA-A, B, C, DRB1, and DQB1 allele-matched unrelated donor HCT. In a single-center study, transplantation for good risk (CML-chronic phase [CML-CP] and transplantation within 2 years of diagnosis), intermediate risk (CML-CP and transplantation more than 2 years from diagnosis; CML-accelerated phase [CML-AP] and blast-phase/remission [CML-BP/rem] and transplantation within 3 years of diagnosis; acute leukemia in remission; myelodysplastic syndrome-refractory anemia) and high-risk (CML-BP, CML-AP, and CML-BP/rem and transplantation more than 3 years from diagnosis; acute leukemia in relapse; myelodysplastic syndrome beyond refractory anemia) disease was 78%, 45%, and 35%, respectively, at 10 years (Fig. 47.1) [2]. The strong influence of disease diagnosis and stage at time of transplantation among allelematched transplant recipients has recently been validated in a National Marrow Donor Program (NMDP) and Center for International Blood and Marrow Transplant Research (CIBMTR) study wherein 5-year survival for good-, intermediate-, and high-risk leukemia was 63%, 48%, and 31%, respectively [3]. Patient survival was not affected by donor age, donor parity, donor cytomegalovirus status or donor gender. Given the pronounced effect of disease stage on transplant outcome, disease-specific strategies for timing HCT rely on prognostic indicators for each disease, outlined below. One of the most important aspects of planning an unrelated donor transplant is to avoid, whenever possible, the “urgent” donor search situation. As detailed in Chapter 12, the availability of high-throughput HLA typing laboratory support has decreased the time required to identify well-matched donors. Depending on the patient’s genotype, it is currently feasible to offer transplantation for high-risk patients within 3 months. For patients with high-risk features at diagnosis, prompt initiation of an unrelated donor search provides sufficient time for a suitable donor to be identified and for the transplant procedure to be performed when the disease is in remission. For all patients, but particularly high-risk patients, the identification of back-up donors provides options and avoids delays when the primary donor is unable to donate, or when the patient’s condition changes (http://www. asbmt.org/policystat/policy.html) [4,5]. A study from the Netherlands demonstrates that 46 of 502 donor work-ups ended up with the donor

being deferred, 78% due to medical reasons and 22% due to nonmedical reasons. For those cases for which a back-up donor was already identified (50%), the transplant was scheduled within 2 weeks. When back-up donors were not available, the median delay to transplant was 18 weeks [5]. The reader is referred to Chapters 38 and 48 for specific aspects of the evaluation of unrelated donors. Optimizing unrelated HCT requires strategies to reduce TRM. The development of safer high-dose and reduced-intensity regimens has significantly lowered organ toxicity [6]. High-dose and reduced-intensity [7,8] regimens have greatly expanded options for older patients and patients whose underlying medical conditions place them at higher risk for toxicity with the use of myeloablative regimens. The development of high-dose and reduced-intensity conditioning (RIC) regimens provide a means to treat early MDS, and clinical experience demonstrates that the GVT effect is operational [7,9]. Further, trends for lower relapse after unrelated compared with matched sibling HCT suggest a more intense GVT effect that arises from undetected genetic disparity among unrelated individuals [8]. The source of hematopoietic cells may influence the transplant outcome [10]. Differences between BM and PBPCs on early and late events mirror those observed after HLA-identical sibling transplantation. In a single-center study, no significant differences in the risks of acute and chronic GVHD between PBPC and BM recipients were found compared with BM [11]. In a CIBMTR analysis, there was a 56% incidence of grade II–IV acute GVHD and a 54% incidence of chronic GVHD at 3 years with PBPCs, compared with 45% and 39%, respectively, for BM [12]. No differences in relapse or TRM in the first 9 months post transplant were observed; however, of patients surviving 9 months, late TRM was higher with PBPCs than with BM and led to lower overall survival (OS), particularly for patients transplanted for good-risk CML. These retrospective results should soon be clarified by the results of a large national Blood and Marrow Transplant Clinical Trials Network study that is currently in progress. Acute myeloid leukemia As reviewed in Chapters 53 and 54, cytogenetics at the time of diagnosis of acute myeloid leukemia (AML) is a robust indicator of response to induction chemotherapy and to HCT [13–18]. Low-risk de novo AML patients include those with the t(8;21) and inv(16) karyotypes, and the t(15;17)(q22;q12–21)+ acute promyelocytic leukemia subtype. These patients enjoy a high response rate to conventional chemotherapy and a

Hematopoietic Cell Transplantation from Unrelated Donors

low (35%) probability of disease recurrence after first complete remission (CR1) is attained. For low-risk patients who lack an HLA-identical sibling donor, unrelated HCT in CR1 is associated with a high TRM that outweighs the benefits of the GVT effect compared with chemotherapy [19]. When a patient with good-risk features relapses, however, HCT in second complete remission (CR2) is indicated. The high-risk AML group comprises patients with −5, −7, inv3 or complex karyotypes, defined as containing at least three chromosomal aberrations. The probability of disease recurrence among high-risk patients is 80% after CR1, and the probability of achieving CR2 is very low. For these patients, allogeneic HCT in CR1 remains the best option to prevent relapse. Survival, disease-free survival (DFS), and relapse after unrelated HCT for high-risk AML in remission may yield survival rates at least comparable to those obtained after related donor HCT, especially when the transplant procedure can be performed in remission (http://www.cibmtr.org) [15,20]. A second group of very high-risk patients includes those who fail primary induction chemotherapy. Application of unrelated HCT for salvage in these patients requires individualized risk assessment [14]. The most challenging (and largest) group of patients with de novo AML are within the intermediate-risk group. Recent data suggest that this group can be further divided into a better-risk and a poorer-risk group based on nucleophosmin (NPMI) and fms-related tyrosine kinase3 (FLT3) status (see Chapter 53) [21]. Intermediate-risk patients have a 50% probability of disease recurrence after CR1, and the chances that CR2 can be attained are low. For these patients, balancing the risks of TRM with the benefits of the GVT effect is critical. In a retrospective CIBMTR analysis of unrelated HCT for AML in CR1 and CR2, the 5-year DFS for CR1 patients with favorable, intermediate, and poor-risk cytogenetics was 29%, 27%, and 30% respectively; OS at 5 years for the same three groups was 30%, 29%, and 30%, respectively [14]. When unrelated HCT was performed in CR2, 5-year DFS for patients with favorable, intermediate, and high-risk cytogenetics was 42%, 38%, and 37%, respectively; OS at 5 years was 35%, 45%, and 36%, respectively. Patients with high-risk cytogenetics had a high risk of relapse after HCT, but the overall outcome after transplantation was superior to that with chemotherapy. Relapse after HCT was higher for patients with unfavorable cytogenetics whether the transplant was performed in CR1 or CR2, and there was evidence that there was less of a GVT effect with transplantation for high-risk AML in CR1 or CR2. These data suggest that, for patients with unfavorable cytogenetics, an unrelated HCT in CR1 provides the optimal approach. DFS for patients with intermediate-risk cytogenetics is comparable to that achieved with conventional chemotherapy, and therefore unrelated HCT for these patients is best deferred until after CR1. This final conclusion may change as more data concerning the impact of NPMI and FLT3 on outcome emerge. Acute lymphoid leukemia The results of unrelated HCT for acute lymphoid leukemia (ALL) are approaching those after matched related HCT [22], with data from the CIBMTR demonstrating 42% survival after unrelated and 48% after sibling HCT (http://www.cibmtr.org). The prognostic indicators for high-risk adult ALL can be used to plan allogeneic transplantation [23– 25]. Factors associated with a high risk of disease recurrence include older patient age at diagnosis, total white blood cell count greater than 30,000/μL at diagnosis, and presence of the t(4;11)+ pro-B cell karyotype, t(9;22) translocation, hypodiploidy, and the rare t(1;19) karyotype (see Chapters 55 and 56). When an HLA-matched sibling is available to serve as a donor, transplantation for ALL is indicated regardless of risk group [26]. Use of an

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unrelated donor for CR1 remains untested. The findings of a recent evidence-based review support unrelated donor transplantation for highrisk ALL in CR1, but not for standard-risk ALL [27]. For patients in CR2, transplantation from related or unrelated donors yields comparable survival, both superior to chemotherapy alone and to autologous HCT. Some very high-risk patients who relapse after chemotherapy may be salvaged with unrelated donor HCT [28]. Myelodysplastic syndrome Allogeneic transplantation is the only known cure for MDS (see Chapter 57). Since this disease is generally a disorder of older patients, a careful assessment of individual risk factors that may increase TRM is critical. The disease morphology, blast count, neutrophil count, disease duration, patient age, and cytomegalovirus serostatus all influence transplant outcome for MDS [29]. The International Prognostic Scoring System stage at the time of transplantation strongly correlates with the risk of post-transplant disease recurrence, and can aid in the identification of patients who might benefit from HCT early after diagnosis. Data on transplantation for early MDS are limited, but available evidence shows a low probability of disease recurrence, which may be offset in some patients by higher TRM [30]. When the disease has advanced with excess blasts, and when cytogenetics indicates higher-risk disease (−5 or −7, or complex cytogenetic abnormalities), initiation of the unrelated donor search and careful monitoring of marrow function are critical. When MDS has advanced to AML, high relapse rates are seen after transplantation [31]. Chronic myeloid leukemia The introduction of up-front bcr–abl tyrosine kinase (TK) targeted therapy has significantly changed the indications for unrelated HCT for CML [32]. As reviewed in Chapter 51, frequent monitoring of hematologic, cytogenetic, and molecular response to TK inhibitors is essential, and may be required every 3–6 months. When there is evidence for loss of response by quantitative polymerase chain reaction (PCR) for bcr– abl, transplantation should be considered [33]. The major challenge in planning unrelated HCT is the rapidity with which suitable unrelated donors can be identified when clinical response is lost and a search of unrelated donor registries suggests that potential HLA-matched unrelated donors are not available. In these circumstances, data from patients transplanted in the pre-TK era demonstrate that the use of donors with selected HLA mismatches should be considered [2,3]. When there is no initial response to TK inhibitor therapy, initiation of an search for unrelated donors is warranted. In the pre-TK inhibitor era, increased length of time from diagnosis to transplantation was identified as a marker of poorer transplant outcome [34,35]. Today, the majority of patients defer transplantation until there is evidence of loss of TK inhibitor response. The implications of delayed transplantation in this clinical setting remains to be evaluated. A second area of research is focused on the potential impact of pretransplant therapy with TK inhibitor agents on transplant outcome [36,37]. Although use of imatinib prior to transplantation has not been shown to increase organ toxicity, engraftment, DFS or survival, patients with a suboptimal response to imatinib have a higher hazard of mortality than patients who achieve a complete cytogenetic response [37]. Most transplant outcomes data for CML are derived from the pre-TK inhibitor era and demonstrated superior DFS when transplantation could be performed early in the course of the disease [11,32,35,38,39]. After myeloablative transplantation, earlier transplantation from an HLA-A, B, C, DRB1, DQB1 allele-matched unrelated donor is associated with superior survival compared with transplantation later in the disease and

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Chapter 47

from HLA-matched or mismatched donors (Fig. 47.1). Given the negative effect of the longer time interval from diagnosis to transplantation on OS, delays due to an exhaustive search for suitable unrelated donors require careful assessment of the risks and benefits for the individual patient. When an initial search does not yield HLA-matched donors, continuation of the donor search may or may not yield a better matched donor. During the search, the disease may or may not remain stable. Retrospective analysis of the effect of disease stage and donor HLA matching in a single-center study suggests that when an unrelated donor search is unlikely to yield matched donors, there is increased mortality when transplantation is performed later in the course of the disease even when an HLA-matched donor is eventually identified (Plate 47.1) [2]. Hence, exhaustive efforts to find the best match may theoretically increase the success of transplantation; however, a prolonged donor search may only delay transplantation without necessarily increasing the benefit to the patient if a matched donor cannot be identified. Patients receiving a transplant for the treatment of CML since 1997 have a 10–15% overall better outcome compared with historical patients. These improved outcomes are the result of many advances, including improved prevention and treatment of GVHD, donor HLA matching, and supportive care [11,35,38–44]. Good-risk patients have achieved the greatest improvement in transplant outcome, with OS rates of 80% and a TRM of 17%. The development of RIC and nonmyeloablative HCT regimens has greatly expanded treatment options for CML patients with advanced age or medical contraindications to standard high-dose conditioning. The largest series of reduced-intensity transplants shows a 43% 3-year DFS and 69% OS when transplantation can be performed in chronic phase 1 (CP1) [45]. Patients with CML require careful longitudinal monitoring for early detection of disease relapse [46]. For patients with recurrent disease after transplantation, donor lymphocyte infusion can provoke a robust and effective GVT response [47].

Optimizing unrelated HCT: genetic factors that influence outcome The main goal of the immunogenetic clinical research effort is to reduce TRM by understanding the risks associated with genetic disparity and donor–recipient genetic variation on transplant outcome. The classical HLA genes, HLA-A, B, C, DR, DQ, and DP, have been extensively studied and provide the basis for current standards of practice (http:// www.nmdp.org). As described in Chapter 12, the discovery of novel alleles over the past three decades and the development of molecular techniques to define the sequence diversity of HLA genes have had an immense impact on the prospective evaluation and selection of unrelated donors. More recently, the study of NK alloreactivity in GVHD and the GVT effect has led to sentinel discoveries on the role of the innate immune system in allogeneic HCT. Elucidation of the genetic diversity of cytokine and immune response genes has opened new research initiatives in the integrated pathways that bridge the adaptive and innate immune systems. GVHD is commonly observed after unrelated HCT, and is a complex phenotype shaped by both genetic and “environmental” factors. As a complex phenotype, GVHD has a polygenic etiology. The genes known to be involved in the pathogenesis of GVHD are the HLA, killer immunoglobulin-like receptor (KIR), and cytokine genes. Although the recipient’s genotypes are not modifiable, the potential to select a donor with the “optimal” genetic make-up to reduce GVHD and TRM is feasible. The environmental factors that influence a given patient’s risk of GVHD (as well as the effects that can be measured from HLA, NK, and cytokine genetic polymorphisms) are contributed by the chemotherapy- and radiation-containing regimens used to prepare the patient, the immunosuppressive agents used to prevent and treat GVHD, and the source of donor

stem cells, to name but three important factors. To a great extent, these conditions can be modified to fit each patient’s need, and there has been tremendous progress in the development of novel approaches such as RIC to increase the safety of unrelated HCT. Recognition that a complex phenotype such as GVHD arises from the effects of more than one gene is an important platform for future research in immunogenetics. Gene–gene interactions may vary under widely different transplant procedures and with different approaches for GVHD prevention and treatment, and might explain the heterogeneity of results reported from different studies. As more information becomes available on the extent of HLA, NK, and immune response gene diversity in human populations, it is appreciated that the race of the recipient and donor plays a major role in defining the genetics of a given transplant. A full understanding of the genetic basis of host-versus-graft (HVG) and graft-versus-host (GVH) alloreactivity will come from the continued evaluation of racially and ethnically diverse populations. This section reviews the sentinel advances in the identification of the immunogenetic risk factors in unrelated donor HCT: major histocompatibility (MHC) region genes and haplotypes, HLA–NK interactions, and cytokine and immune response genetics. HLA system Major advances have shaped the selection of unrelated donors for transplantation in the 40 years since the discovery of the HLA system (see Chapter 12). With each newly defined locus, the sophistication with which the laboratory methods could discriminate unique antigenic epitopes increased, and paved the way to understanding the importance of HLA matching of unrelated donors. The breakthrough in technology came in the mid-1980s with the discovery of the PCR. Discrimination of single-nucleotide differences between serologically related antigens was now feasible, and the application of PCR-based methods led to an exponential increase in the number of newly discovered alleles. The advent of PCR technology marked the start of the modern DNA era with respect to high-resolution typing and matching of unrelated donors. Molecular methods were first developed for the HLA-DR gene because sequences were available for selected antigens and permitted the design of PCR primers and oligonucleotide probes for HLA-DRB1 exon 2. During this time, typing for class I HLA-A and B was still performed using alloantisera and serologic methods, and typing for HLA-C was not routinely performed because sera were not available for all HLA-C determinants. The earliest studies on HLA matching in unrelated donor HCT defined the impact of allelic differences at HLA-DRB1 or DQB1 among HLA-A, B serologically matched transplants [48–52]. Later, when DNA-based methods were developed for class I loci, undetected allelic mismatches among class I serologically matched pairs were uncovered. Re-evaluation of locus-specific risks became feasible, and permitted redefinition of the relative importance of matching at the five classical loci, HLA-A, B, C, DRB1, and DQB1 [2,3,53–60]. The inclusion of legacy data has required substantial efforts to retrospectively characterize donor and recipient samples and define the precise numbers and kinds of allele mismatches. This effort has included the retyping of donor–recipient pairs for loci that were not originally defined at the time of transplantation; it is not surprising, therefore, that not only has a substantial degree of incompatibility been uncovered in the retyping effort, but also the patterns of mismatching for the classical loci are highly complex (see Chapter 12). Retrospective analysis has been the chief study design used in HLA outcomes research. The history of HLA typing and the development of HLA nomenclature and ontogeny presented in Chapter 12 provide the basis for many of the challenges faced when interpreting the results of retrospective studies of HLA matching. Interpretation of single-center

Hematopoietic Cell Transplantation from Unrelated Donors

or registry data must consider which loci were tested, at what resolution, how the loci are modeled (which depends on the study hypotheses), and how non-HLA factors that influence outcome are included in multivariable regression models. The incidence of mismatching at the locus/loci of interest and the number of transplant pairs available for analysis will influence the statistical power that one has to address various hypotheses of interest, and therefore the level of complexity that these hypotheses can accommodate. When the sample size is limited, simple models that require the most assumptions are often necessary. For example, suppose one is interested in testing the relatively simple hypothesis that single-locus HLA mismatches are associated with an increased risk of post-transplant complications. If one combines all single-locus mismatches into a single group and compares the risk associated with mismatching against that associated with matching, one assumes that all mismatches contribute in a similar manner to the clinical endpoint. These assumptions may not be true, however, and if the sample size is not sufficiently large, this assumption cannot be confirmed with sufficient confidence. When interpreting the results of such an analysis, it is important that one understands that the results apply only in the context of these assumptions. Larger sample sets may allow certain assumptions to be relaxed, thereby permitting more complex questions to be addressed. Using the above example, locus-specific risks associated with a single HLA-A, B, C, DRB1 or DQB1 mismatch may be measured if there are sufficient donor–recipient pairs in each of the mismatch groups to allow reasonable statistical power; this approach could then evaluate whether a mismatch at a particular locus conferred similar or different risks to an alternate locus. Note that such an analysis makes the assumption that all mismatches at a particular locus confer the same risk. Since many variables unrelated to HLA have an impact on the same clinical endpoints that are also affected by HLA disparity, multivariable models should adjust for all clinical variables that are known to affect outcome to account for the possibility that imbalances in these factors may exist between groups that are being compared. When interpreting the HLA effect that is being modeled, one needs to understand the assumptions that are associated with the way in which the HLA and non-HLA factors are modeled. For example, suppose one is interested in modeling the impact of a mismatch at HLA-A, but pairs that are mismatched at other loci are included in the analysis [51,55,56]. One can adjust for the mismatches that occur at other loci, in addition to non-HLA factors such as severity of disease and age. When interpreting the effect of a mismatch at HLA-A, however, one must consider how the factors other than HLA-A are modeled, and if the HLA-A effect is assumed to be the same across all categories of the non-HLA factors. Such an assumption is made unless there are appropriate interaction terms included in the regression model. The lack of a term that models the interaction between mismatching at HLA-A and some other factor then implies that one assumes that the effect of a mismatch at HLA-A is the same across all levels of this other factor. For example, if this other factor models the degree of mismatching at class II DRB1 and DQB1 loci, the assumption would be that the impact of a mismatch at HLA-A among patients matched for all four class II alleles is the same as that among patients mismatched for a single class II allele, among patients mismatched for two class II alleles, among patients mismatched for three class II alleles, and among patients mismatched for all four class II alleles. Similarly, if one includes in the regression model a term for severity of disease without an accompanying term for interaction, interpretation of the impact of a mismatch at HLA-A is, in the context, the assumption that the impact is the same across all levels of disease severity. Instead of adjusting for HLA, an alternate approach is to define mutually exclusive groups of donor–recipient pairs defined by the match/mis-

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match status at each HLA locus [2,3]. The advantage of this approach is that it eliminates any potential effect of other loci to the locus under examination by not requiring the assumption that the impact of a mismatch at a particular locus is the same across all categories of the other loci that are being adjusted. Because each group is mutually exclusive, however, the chief limitation of this approach is that each comparison group may be limited by small numbers of pairs. One could also use a combination of these approaches, where mutually exclusive groups are defined but that do not necessarily exhaust all possibilities, with adjustment for loci or combinations not considered in the group definitions. Whichever approach one chooses, it is critical to the interpretation of results that one understands the assumptions that are made in the modeling. Seemingly different results are not necessarily contradictory, as the results must be interpreted in the context of the assumptions that underlie the modeling. The following section focuses on recently published large, well-controlled studies in which donor and recipient samples were each characterized at the currently highest resolution possible for HLA-A, B, C, DRB, DQB1, and DPB1 alleles (Table 47.1). The matched donor A “matched” donor may be defined on the basis of high-resolution allele definition at four loci – HLA-A, B, C, DRB1 (“8/8”) – or five loci – HLA-A, B, C, DRB1, DQB1 (“10/10”). Several historic studies have met the criteria outlined above (high-resolution typing at all loci, sufficient power to detect statistically significant differences when they exist, and multivariate analysis to control for non-HLA variables that affect clinical outcome). These studies used the 10/10 definition and demonstrate that patients have superior DFS after HLA-matched unrelated HCT compared with any mismatching [2,3,54,55]. The impact of more complete and precise donor HLA matching is especially dramatic for a subset of good-risk patients undergoing HCT for AML, MDS, ALL, and CML; transplantation using a high-resolution matched unrelated donor can yield survival rates that are very similar to those seen after HLA-identical sibling transplantation (http://www.marrow.org) [61–66]. Most recently, a new concept that a minimal level of matching can be defined among HLA-A, B, C, DRB1, and DQB1 genes has been evaluated [3]. In this analysis of 3860 patients transplanted from unrelated donors following myeloablative conditioning, high-resolution matching at HLA-A, B, C, and DRB1 was associated with highest survival rates. Relative risks associated with single-locus and multilocus mismatching are discussed below, and suggest that one isolated HLADQB1 mismatch does not adversely affect outcome. Comparison of 10/10 was comparable to 9/10 when the single mismatch was at HLADQB1, indicating that the 8/8 high-resolution matched unrelated donor can be used as a predictor for patient survival. Multilocus mismatching (8/10) was associated with adverse outcome (see below), and therefore prospective HLA-DQB1 typing is required to define the total number of mismatches when a donor is already known to have one HLA-A, B, C or DRB1 mismatch. Although donor HLA match status is a strong risk factor for posttransplant complications, the recipient’s underlying diagnosis and stage of disease at the time of transplantation remain the most important prognostic features that affect DFS. Two studies demonstrate the importance of early transplantation and good control of disease on clinical outcome [2,3]. As noted earlier, among 10/10 high-resolution matched recipients from a single-center study, OS at 10 years post-transplant for good-, intermediate-, and high-risk leukemia was 78%, 45%, and 35%, respectively (Fig. 47.1) [2]. One-year survival from HLA-matched transplants analyzed by the CIBMTR for patients in similar risk categories

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Chapter 47

Table 47.1. Role of human leukocyte antigen matching in unrelated donor transplantation

Mismatch Status

Locus

Graft failure

Graft-versus-host disease

Single

A

[53,54,58]

B

C

Survival

Comments

[3,54–56]

[2,3,54–56]

[53,54,58]

[3,56]

[2,55,56]

[53,58,67]

[54,56]

Antigen mismatches are associated with higher risks than allele mismatches [55] TRM is increased with any mismatch [3] Antigen mismatches are associated with higher risk than allele mismatches [55] TRM is increased with any mismatch [3] Single allele or antigen mismatches are detrimental, notably HLA-C in low-risk patients [2] Antigen mismatches are associated with higher risk than allele mismatches [3,55] TRM is increased with any mismatch [3] Antigen mismatches are associated with higher risk than allele mismatches [55] There is a trend for lower survival [3]

DRB1

Two or more

Epitopes

DQB1 DPB1 Class

Graft-versus-leukemia effect

[54]

[2,48,53,56]

[69] [53,58]

[2,49,50] [57,74] [53,77]

[2,55,68]

[2,3,55]

[2] [57] [18,53,56,58,62,77]

Class II Classes I and II DPB1 DQB1

[53,54,56,77] [53,77]

Class I

[72]

[72]

[75]

[69,75]

[73]

[73]

HLA-DP

[57,74] [2]

[69]

Classes I and II

Multiple class I allele mismatches are as detrimental as a single antigen mismatch [58]

[53,54,56,77] [53,56,77] [57] [2]

HLA-DQ mismatch in combination with others is detrimental [2] Residue 116 substitutions in class I are detrimental [72] HVG vector mismatches at HLA-DPB1 increase graft failure after HCT for thalassemia [69] 15 high-risk allele mismatches are identified 6 class I residues are identified as nonpermissive [73]

Summary of studies that meet the criteria for an interpretable study: large populations typed at high-resolution for a minimum of five loci (HLA-A, B, C, DRB1, and DQB1) and use of multivariable biostatistical methodology. HVG, host-versus-graft; TRM, transplant-related mortality.

was 63%, 48%, and 31%, respectively [3]. These data demonstrate that when matched donors can be identified, transplantation outcome is optimal when the transplant can be performed in early remission.

The mismatched donor The likelihood of identifying 8/8 or 10/10 matched donors is dependent on the recipient’s genotypes and haplotypes, and the size and composition of the donor registries. Currently, over 11.3 million volunteer donors are registered worldwide, and the probability of a search yielding a matched donor ranges from 20% to 80% depending on recipient HLA (http://www.worldmarrow.org; http://www.nmdp.org). When a recipient encodes two common HLA haplotypes, for example HLA-A1, B8, DR3, and HLA-A3, B7, DR2, the strong linkage disequilibrium (LD) generally favors a high degree of matching not only at each HLA locus

(A*0101 for A1), but also for the HLA alleles linked on the haplotype (A*0101, B*0801, DRB1*0301, and A*0301, B*0702, DRB1*1501) (see Chapter 12). When a recipient has a less common genotype, or less common combination of alleles on a haplotype, the probability of identifying a 10/10 or 8/8 matched donor is dependent on the donor pool. When a recipient has inherited a recombination event that generates an unusual linkage of HLA alleles, it may be exceedingly difficult to identify a fully matched donor; in this situation, limiting a mismatch to a single locus may be the only option. When a 10/10 matched donor cannot be identified, current data provide the following algorithm: 1 A single HLA-DQB1 mismatch may be forgiving (i.e. an 8/8 match at HLA-A, B, C, and DRB1); however, combinations of mismatches including DQB1 may increase mortality [2,3]. 2 “Permissible” HLA mismatches defined by polymorphism for selected HLA class I residues that participate in the peptide repertoire or direct

Hematopoietic Cell Transplantation from Unrelated Donors

contact with the T-cell receptor (TCR) may aid in the selection of the best mismatched donor. 3 Multilocus mismatches are poorly tolerated and should be limited or avoided. Heterogeneity in the literature with regard to the effects of mismatching for specific HLA loci must be interpreted in light of the typing technology that was available at the time of the study. In general, a comparison of results from studies of large numbers of transplant pairs, and which robust biostatistical methods were used to measure independent effects, indicates that the degree to which single-locus mismatches increase post-transplant risk is defined by qualitative differences in the locus and the specific combination of donor–recipient allele disparity.

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contribute [55]. Recently, extension of the NMDP study to a larger study population shows that each HLA-A, B, C or DRB1 mismatch is associated with a 9–10% lower OS compared with 8/8 allele-matched patients [3]. Among all single-locus mismatches, disparity for HLA-A or DRB1 was each associated with higher mortality than disparity for HLA-B or C. Mismatching at HLA-A, B, and C were each associated with an increased risk of GVHD. In an update from the JMDP, GVHD risk was associated with HLA-A, B, C, and DPB1 allele mismatching [70]. These studies may have come to different conclusions regarding the relative contributions of class I and class II mismatching because of different allele and antigen mismatches between patients and donors. GVT effects

Graft failure Graft failure and increased mortality in patients following the use of HLA-C mismatched unrelated donors provided the earliest clinical data supporting HLA-C as a classical transplantation antigen [60,67,68]. The role of HLA-C in graft failure was most pronounced among patients with good-risk CML [58]. Large-scale analysis of the importance of donor HLA matching and engraftment [44,54–56] provided the impetus for the inclusion of HLA-C into the prospective evaluation of unrelated donors in the late 1990s. Most recently, HLA-DP mismatching has been associated with an increased risk of graft failure after unrelated HCT for thalassemia [69]. Acute GVHD Information on the importance of HLA-DRB1 and DQB1 in GVHD became available in the 1990s as a direct result of the availability of DNA-based methods that permitted retrospective retyping of legacy samples for these loci [48–52]. Many of these early studies of class II allele matching were performed in HLA-A, B serologically matched study pairs because high-resolution typing methods for HLA-A, B, and C were not available. As such, the potential additive risks associated with undetected HLA-A, B, C allele mismatching among DRB1/DQB1 mismatched cases was not yet defined [48–52]. In one early study of HLA-A, B, DR serologically matched unrelated donor transplants, the presence of DRB1 allele mismatching was associated with a statistically significantly increased risk of clinically severe acute GVHD compared with DRB1 allele matching [48]. This study did not examine allele disparities at HLA-DQB1. An expanded dataset later permitted evaluation of both HLA-DRB1 and DQB1 genes, and uncovered a synergistic effect of two-locus mismatching [52]. Several singlecenter and registry studies of unrelated transplants confirmed and extended the findings of HLA-DR and DQ disparity and the risk of clinically significant acute GVHD [49–51]. Of these, an NMDP analysis of 831 CML transplants confirmed a role for HLA-DRB1 allele mismatching in risk for grade III–IV acute GVHD [51]. It was not until the late 1990s, when high-resolution class I typing methods became available, that more detailed locus-specific risk assessment was feasible. The NMDP reported the findings of 1874 transplant patients in whom HLADRB1 mismatching was associated with increased risk of acute GVHD [55], and confirmed the role of prospective donor evaluation and matching for this locus. The first large study describing the effect of class I on GVHD risk was published by the Japan Marrow Donor Program (JMDP) [54]. HLAA and C allele disparity were each independent risk factors for severe acute GVHD; interestingly, no contribution from class II was found. In a recent update of the JMDP experience, HLA-A, C, B, and DRB1 were each found to be independent risk factors for grade III–IV acute GVHD [56]. HLA-A allele mismatching was also found to be a risk factor for severe GVHD in the NMDP analysis (relative risk 1.33; range 1.0–1.7; p = 0.04); however, HLA-B and C allele mismatches did not appear to

HLA incompatibility is associated with an increased risk of clinically severe acute GVHD and lowered post-transplant relapse indicative of GVT effects. Elimination of recipient leukemia cells by GVT mechanisms can provide a therapeutic advantage, particularly for patients with a high risk of relapse after transplantation. The most comprehensive analysis of donor matching and post-transplant relapse was conducted by the JMDP in five-locus allele-typed donor–recipient pairs [54,56]. Mismatching at the HLA-C locus in combination with mismatching at HLA-A/B and/or DR/DQ was associated with a lower 3year relapse rate compared with complete matching or mismatching at HLA-A/B alone [54]. Most recently, HLA-DPB1 has been shown to participate in GVT effects in two large studies of unrelated donor HCT [57,70]. Alleles and antigens Nomenclature has been developed to describe HLA determinants that have been defined by their sequence (allele) or their phenotype (antigen); high-resolution sequence (allele-level) data can furthermore be used to infer the parent antigen when serology is not available (see Chapter 12). When an allele-matched donor is not available, selection among mismatched donors frequently involves the consideration of donors with allele or antigen mismatches. In a single-center study of graft failure after high-dose unrelated HCT, donor–recipient mismatching for HLAA, B or C antigens conferred greater risk for graft failure than did single-allele mismatches at these loci [58]. However, the risk of graft failure increased dramatically when multiple-allele mismatches at class I were present, providing evidence for additive effects of multilocus mismatches. These observations suggested that the number and nature of nucleotide substitutions that participate in the peptide binding groove and TCR contact residues could in part explain the immunogenicity of alleles and antigens. Three studies have compared risks associated with allele and antigen mismatches in large populations. In the 2004 NMDP study, risks associated with antigen-level mismatches were higher than those seen with allele-level mismatches at the same locus [55], suggesting that the constellation of residue differences between donor and recipient HLA antigens could provoke the strongest alloimmune responses. This study found poorer outcome in recipients with antigen mismatches at HLA-A and B compared with recipients with allele mismatches at the same loci [55]. In both a single-center analysis and the 2006 CIBMTR study, allele mismatches were as detrimental as antigen mismatches when stage of disease was taken into account; the potential exception was HLA-C in the CIBMTR cohort [2,3]. These data are consistent with the graft failure study [58] where there was a predominance of HLA-C mismatches, and allele disparities did not contribute to the increased risk. Taken together, these studies demonstrate that, when measuring the effects of HLA mismatching, large well-characterized populations are needed, and studies are best carried out when the variables that also affect clinical outcome can be accounted.

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Models for evaluating permissible HLA mismatches DNA-based methods provide an unprecedented level of accuracy to define HLA genes. Although a high frequency of allele disparity can be defined among serologically identical donor–recipient pairs, the favorable clinical outcome of many of these patients strongly suggests that not all mismatches are detrimental. Identification of mismatches that do not lead to increased risk of GVHD and mortality will provide a means to broaden the application of mismatched unrelated HCT when matched donors are not available. Several approaches have been used to better understand the sequence variation that governs permissivity. The functional assay of cytotoxic T-lymphocyte precursor (CTLp) cells measures CD8+ T-cell-mediated alloreactivity that arises from donor recognition of recipient sequence polymorphisms encoded in exons 2 and 3 (α1 and α2 domains) of class I HLA genes (see Chapter 12), for example HLAC residues 97, 99, 113, 114, and 116. The CTLp assay has been used to identify mismatch combinations associated with GVHD; the lack of a CTLp response may indicate permissible mismatches [71]. Identification of amino acid substitutions at residues involved in peptide binding and/or TCR contact may provide important information on the rules that govern permissible HLA mismatches. The first study to identify substitutions in the class I heavy chain that correlate with GVHD risk examined a population of HLA-B-mismatched donor–recipient pairs [72]. Substitutions at residue 116 were associated with a significantly increased risk of GVHD and TRM compared with transplants without substitutions at residue 116, and predicted the involvement of residue P9 in peptide binding as a possible mechanism. A new model that integrates information on the structural differences induced by the amino acid substitutions of class I and II molecules was recently tested in a large retrospective study of 4866 unrelated donor transplants facilitated by the JMDP [73]. The Tyr9–Phe9 amino acid mismatch of HLA-A and the Tyr9–Ser9, Asn77–Ser77, Lys80–Asn80, Tyr99–Phe99, Leu116–Ser116, and Arg156–Leu156 mismatches of HLA-C were each associated with a significantly increased risk of severe acute GVHD. Interestingly, positions 77 and 80 of HLA-C are “linked” in that they comprise the ligand recognized by NK KIR receptors (see below). Accordingly, when the analysis was further restricted to donor– recipient pairs matched for the KIR ligands, HLA-C disparities at residues 95, 156, and 163 correlated strongly with GVHD risk. This is the first study to provide evidence that functional HLA mismatches can be defined by discrete amino acid substitutions, and provides a platform for the analysis of similar combinations of allele and antigen mismatches. Most recently, HLA-DP has served as an important model for tolerable mismatches because over 80% of otherwise HLA-matched donors and recipients are HLA-DP mismatched due to weak LD (see Chapter 12). Risks of graft failure and GVHD are associated with HLA-DPB1 mismatching [57,69,70,74,75]. Higher rates of GVHD are accompanied by lower rates of relapse, and suggest that HLA-DP can participate in the GVT effect [57]. Coding region polymorphisms have been evaluated with the aid of functional assays, and permissible groups of HLA-DP epitopes have been identified that participate in GVHD and graft failure [69,75]. Multilocus mismatches The effects of HLA disparity on risk of graft failure, GVHD, and mortality can be measured by the total number of detectable mismatches. As the number of HLA disparities increases, the risks of graft failure [53,56,58], GVHD [3,49,52–56,76], and mortality [2,18,53,54,56,58,62,77] increase. Studies differ with respect to which combinations of locus mismatching produce the most significant risks. Early studies in class I serologically matched pairs identified combined DR and DQ mismatching to be a risk factor for GVHD [49–52]. When

DNA typing for class I became available, retrospective analysis of donor–recipient pairs typed for 10 alleles uncovered multilocus class I and II mismatch effects on GVHD [3,53–56]. Analysis of multilocus mismatches in the newest CIBMTR study confirms the tally effect of increasing number of mismatches associated with significantly worse survival [3]. OS after 8/8, 7/8, and 6/8 transplantation were 52%, 43%, and 33%, respectively, demonstrating a 9– 10% survival decrement with each additional HLA mismatch. Although the presence of a single HLA-DQB1 mismatch was the most tolerable in terms of the five classical loci (see above), the presence of an HLADQB1 disparity in addition to mismatches at HLA-A, B, C or DRB1 was associated with worse outcome, indicating that prospective HLADQ typing is indicated when the choice must be made among donors with known HLA-A, B, C or DR mismatches. A new paradigm: MHC haplotype Comprehensive and precise donor matching for the classical HLA alleles can lower but does not eliminate risks after HCT from unrelated donors. As described in Chapter 12, the MHC is the most gene-dense region of the human genome, encoding over 300 genes, an estimated 30% of which are believed to have immune-related function. These observations form the basis for the hypotheses that two unrelated individuals who share the same HLA alleles may differ for undetected MHC resident genetic variation, that this variation may contribute to increased risk of GVHD after transplantation, and further that the HLA haplotype can be used to measure functional variation in HCT. Several recent lines of evidence support the role of other MHC-resident genes in transplant outcome. Recently, data have emerged implicating HLA-E in transplant outcome in which increased risk for bacterial infections and corresponding TRM at day 180 post transplant were found in recipients transplanted from HLA-E*0101,0101 homozygous unrelated donors [78]. Two recent studies have used microsatellite (Msat) markers to map new MHC resident variation of clinical importance in transplantation. Donor–recipient variation for tumor necrosis factor (TNF) was associated with lower survival among Japanese patients who developed GVHD [79]. A subsequent study of North American and Western European Caucasian 10/10-matched recipients and donors by the International Histocompatibility Working Group uncovered signals from five MHC markers associated with increased or decreased risk of GVHD and mortality [80]. The Msat data point to the existence of non-HLA MHC-linked variation that is clinically relevant, and the need for new approaches to map novel genes. To address the unmet needs of disease-association mapping within the MHC requires information on donor haplotypes. The current standard of practice is to perform gene-by-gene matching between recipients and unrelated donors as a surrogate for the haplotype matching that is feasible between genotypically identical sibling pairs. Because HLA allele-matched unrelated donors and recipients are not related to one another, it is conceivable that the physical linkage of donor HLA alleles to one another is different than the recipient’s haplotypes (Fig. 47.2). Given the substantial information supporting haplotype-linked variation, one may hypothesize that 10/10-matched unrelated donors and recipients who have the same haplotype linkage of HLA alleles are more similar to each other with respect to undetected MHC variation than are 10/10 allele-matched pairs whose HLA haplotypes differ. In order to test this hypothesis, new tools for physically linking HLA alleles are required. The lack of availability of the unrelated donor’s parents precludes a definitive pedigree definition of the donor’s haplotypes. As an alternative, several laboratory methods have been developed to permit “phasing” of markers as to whether they are encoded on the same (cis) or the

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Fig. 47.2 Human leukocyte antigen matched unrelated donors and recipients may be haplotype matched or haplotype mismatched [82].

alternative (trans) haplotype. Current phasing methods include the use of single-molecule PCR, sperm typing, allele-specific PCR, pyrosequencing, bacterial artificial chromosome cloning, polymerase colony, construction of somatic cell hybrids, atomic force microscopy, rolling circle amplification, and mass spectrometry [81]. Although these methods can define single-nucleotide polymorphism (SNP) haplotypes, not all are suitable for haplotyping the multilocus MHC region due to the extensive HLA polymorphism and uneven distribution of coding HLA variation, as well as the physical distance between each HLA locus. To meet the challenge of phasing the highly polymorphic HLA-A, B, DR genes that span over 4 megabases in distance, a novel long-range phasing technique has recently been developed [81]. The phasing method uses oligonucleotide arrays specific for the two known HLA-B alleles encoded in a sample, to separate 2 megabaselong DNA fragments containing the HLA-A-B and HLA-B-DRB1 haplotypes. Once each fragment is isolated, the HLA-A and DRB1 alleles are determined by hybridization to oligonucleotide arrays specific for the two known HLA-A and DRB1 alleles encoded in the sample. This method permits the definition of long-distance haplotypes and is useful for delineating the cis/trans relationships of HLA genes. HLA allele identity between a transplant recipient and unrelated donor does not guarantee haplotype identity. In a study of 10/10 allele-matched unrelated donor–recipient pairs, application of the MHC phasing technique uncovered a 20% frequency of haplotype mismatching [82]. Haplotype mismatching was associated with a significantly increased risk of grade III–IV acute GVHD. The increased risk of GVHD was offset by lower relapse, leading to similar OS (Fig. 47.3). This study demonstrates that variation linked to the haplotype is functional and that the HLA haplotype can be used as a surrogate marker for GVHD risk. Prospective donor matching for HLA haplotypes in addition to HLA genes may lower risks of acute GVHD. By physically linking HLA-A with B with DR on the same strand of DNA, this technique can be used to identify novel MHC polymorphisms that confer post-transplant risks. KIR genes in unrelated HCT The role of NK cells in the immunobiology of transplantation provides new avenues for optimizing allogeneic transplantation [83]. The majority of the data for NK in clinical transplantation are derived from the haploidentical related transplant experience. More recently, clinical observations on the role of HLA ligands and inhibitory KIRs in HLA genotypically identical related donor transplantation have become available, and data on the role of HLA ligand–KIR interactions in unrelated donor HCT are emerging. The demonstration of lower post-transplant disease recurrence in selected populations of mismatched transplants is particularly important for patients with high-risk disease, and opens the

potential for integrating recipient ligand and donor KIR information into donor selection to fully maximize NK-mediated antitumor effects [83,84]. That the HLA and KIR genetic systems segregate independently by classical Mendelian genetics has implications for HLA-matched and HLA-mismatched, related and unrelated donor HCT. The reader is referred to a comprehensive review of the basic immunobiology of NK cells in Chapter 13, and of the role of NK cell alloreactivity in related donor transplantation in Chapter 46. The following section focuses on the recent clinical observations of HLA ligand–KIR interactions in unrelated HCT. Inhibitory KIR receptors NK cell receptors have inhibitory or activating potential, and it is the balance of these effector functions that results in the NK-cell response to altered or loss of self MHC antigens [85]. There are substantially more data available on the importance of the inhibitory KIRs in allogeneic transplantation compared with activating receptors. Inhibitory NK cell receptors include the KIRs and the immunoglobulin-like transcripts encoded on chromosome 19q13.4. Genetic KIR variation is generated through allelic as well as haplotype diversity [85,86]. The ligands for the inhibitory KIRs are encoded by HLA class I molecules, and, in this way, the MHC bridges the innate and adaptive immune systems. The inhibitory receptors KIR2DL3, KIR2DL2, and KIR2DL1 recognize epitopes presented by HLA-C antigens, while the KIR3DL1 receptor recognizes the HLA-Bw4 expressed by some HLA-B antigens, discussed below. The immunoglobulin-like transcript-1 receptor recognizes HLA-A, B, C, G, E, and F as its ligands. A second structurally distinct group of inhibitory receptors consists of CD94 covalently bound to NKG2A, known as the C-type lectin receptors; these genes are encoded on chromosome 12p12–p13 and serve as the receptor for HLA-E. The inhibitory KIR receptors and their HLA-C and B ligands are the best studied. The ligand specificity of inhibitory KIR receptors is governed by residues 77 and 80 of HLA-C and by the HLA-Bw4 epitope present on some HLA-B and A molecules [85]. High-resolution typing of the transplant recipient and donor for HLA-A, B, and C genes provides all the information required to determine the presence/absence and heterozygosity/homozygosity of ligands, donor–recipient mismatching for ligands, and specificity for inhibitory KIRs. Based on HLA-C residue 77/80 polymorphism, two groups of HLA-C ligands are distinguished. The Ser77/Asn80 polymorphism is encoded by HLA-C group “C1” molecules (HLA-Cw1; Cw3 except Cw*0307, *0310, *0315; Cw7 except Cw*0707, *0709; Cw8; Cw12 except Cw*1205, *12041/2; Cw13; Cw14 except Cw*1404; Cw*1507; and Cw16 except Cw*1602), and is the ligand for the inhibitory KIR2DL2 and 2DL3 receptors. The Asn77/Lys80 polymorphism is encoded by the HLA-C group “C2”

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Fig. 47.3 Impact of haplotype matching after human leukocyte antigen (HLA)-A, B, C, DRB1, DQB1 allele-matched unrelated donor hematopoietic cell transplantation. (a) Probability of grade III–IV acute graft-versus-host disease. (b) Probability of relapse. (c) Probability of transplant-related mortality. (d) Probability of survival. (Reproduced from [82].)

molecules (HLA-Cw2; Cw*0307; Cw*0315; Cw4; Cw5; Cw6; Cw*0707, *0709; Cw*1205, *12041/2; Cw15 except Cw*1507; Cw*1602; Cw17; and Cw18) and is the ligand for the KIR2DL1 receptor. The third ligand group is specified by the presence of the HLA-Bw4 epitope present in B5, B13, B17, B27, B37, B38, B44, B47, B49, B51, B52, B53, B58, B59, B63, B77, B*1513, B*1516, B*1517, B*1523, and B*1524 sequences; the Bw4 ligand is recognized by the inhibitory KIR3DL1 receptor. Models for donor inhibitory KIR-mediated killing Donor NK cell alloreactivity against host cells can arise in the HLAmismatched as well as the HLA-matched setting. As described in Chapter 46, the sentinel discovery by the Perugia team that recipient target cells are susceptible to lysis via donor NK-cell recognition of HLA ligands has provided a model for understanding the HLA–NK genetic systems [84,87,88]. In the original observations, GVH vector KIR ligand mismatching occurs when HLA class I ligands are present in the donor but absent in the recipient [87]. In the setting of HLA-mismatched transplantation (haploidentical mismatched related donors and recipients and HLA-mismatched unrelated donors and recipients), recipients may lack the appropriate ligand that is present in the donor due to the HLA mismatch (Table 47.2). The recipient’s target cells are

susceptible to alloreactive donor NK cell killing (GVH) because the donor NK cells are not inhibited. The recipient target cells include residual host leukemia or tumor cells. As an example, in the mismatched setting between a donor who has both a C1 (HLA-Cw8) and a C2 (HLACw6) epitope and a recipient who has only C1 (HLA-Cw8, Cw8 homozygous), donor inhibitory KIRs are not engaged and recipient targets are lysed. With regard to the Bw4 epitope, donor inhibitory KIRs are not inhibited with a Bw4+ donor and a Bw4− recipient. For example, this situation is present in a B44,18+ donor and a B18,18+ recipient (donor Bw4 [B44], Bw6 [B18]; patient Bw6 [B18], Bw6 [B18]), and also in a B44,52+ donor and a B18,35+ recipient (donor Bw4 [44], Bw4 [B52]; patient Bw6 [B18], Bw6 [B35]). The haploidentical HLA-mismatched, T-cell-depleted transplants in the Perugia studies received a high CD34+ cell dose and no postgrafting immunosuppression, conditions that promote rapid NK-cell recovery [88]. The NK-cell-mediated reduction of relapse has been observed in myeloid but not lymphoid malignancies, presumably because NK cells do not express adhesion receptors to LFA-1-deficient ALL cells. The patients with AML who received transplants from KIR ligand-mismatched donors had significantly improved 5-year survival rates compared with patients who received KIR ligand-matched transplants (60% versus 5%; p = 0.0005). Donor KIR ligand mismatching was associated

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Table 47.2 Clinical importance of human leukocyte antigen killer immunoglobulin-like receptors (HLA-KIRs) in unrelated donor transplantation Ligand Model†

Mechanism

Recipient

Donor

Vector

Examples‡

Mismatched ligand [70,84,87, 91–97,99]

Due to the donor–recipient HLA class I mismatch, the recipient’s target cells lack the class I allotype present in the donor

C1, C1 and Bw4, Bw4

C1, C2 and Bw4, Bw6

No C2 in recipient; GVH

C1, C2 and Bw4, Bw6

C1, C2 and Bw6, Bw6

No Bw4 in donor; HVG

C1, C1; Bw4, Bw4 or C1, C1, Bw4, Bw6

No C2 ligand for donor KIR2DL1 No C2 ligand for donor KIR2DL1 and no Bw4 for 3DL1

HLA-B15 versus B13 mismatched haploidentical donor and recipient: Donor B*2705 (Bw4), Cw*0101 (C1); B*1501 (Bw6), Cw*0401 (C2) Recipient B*2705 (Bw4), Cw*0101 (C1); B*1301 (Bw4), Cw*0302 (C1) HLA-B18 versus B52 mismatched haploidentical donor and recipient: Donor B*1501 (Bw6), Cw*0401 (C2); B*1801 (Bw6), Cw*0701 (C1) Recipient B*1501 (Bw6), Cw*0401 (C2); B*5201 (Bw4), Cw*1202 (C1) Recipient B*1301 (Bw4), Cw*0302 (C1); B*2705 (Bw4), Cw*0101 (C1) or B*1301 (Bw4), Cw*0302 (C1); B*0801 (Bw6), Cw*0701 (C1)

Missing ligand [77,97– 100]

Despite HLA identity, host cells are missing the class I ligand to provide the inhibitory signal

C1,C1; Bw6,Bw6 C2, C2; Bw4, Bw4 or C2, C2; Bw4, Bw6 C2,C2; Bw6, Bw6

No C1 for donor KIR2DL2

No C1 for donor KIR2DL2 and no Bw4 for donor 3DL1

Recipient B*3901 (Bw6), Cw*1203 (C1); B*0702 (Bw6), Cw*0702 (C1) Recipient B*4405 (Bw4), Cw*0202 (C2); B*1302 (Bw4), Cw*0602 (C2) or B*4405 (Bw4), Cw*0202 (C2); B*1501 (Bw6), Cw*0401 (C2) Recipient B*1801 (Bw6), Cw*0501 (C2); B*1302 (Bw6), Cw*0602 (C2)

C1, HLA-C group 1 allotypes: the KIR2DL2 and 2DL3 receptors recognize Ser77 and Asn80. C2, HLA-C group 2 allotypes: the KIR2DL1 receptor recognizes HLA-C allotypes encoding Asn77 and Lys80. Bw4: The inhibitory receptor KIR3DL1 recognizes the HLA-Bw4 epitope expressed on certain HLA-B and HLA-A molecules. GVH, graft-versus-host; HVG, host-versus-graft. † Mechanism/hypothesis tested in the references listed. ‡ Donor and recipient HLA-B-C haplotypes.

with no graft failure, no acute GVHD, and a 0% 5-year probability of relapse in patients with AML. In contrast, KIR ligand matching was an independent risk factor for poor transplant outcome (15.5% incidence of graft failure, 13.7% incidence of acute GVHD, and 75% 5-year probability of relapse). Alloreactive NK clones were isolated from patients transplanted from HLA haploidentical related donors wherein the recipient did not have the epitope present in the donor [87]. The alloreactive NK-mediated lysis of recipient targets was blocked using target cells that expressed the missing epitope. Most recently, the Perugia experience has demonstrated that risks of relapse and mortality correlate most strongly with the NK alloreactivity of the haploidentical donor [89]. In unrelated donor HCT, the impact of ligand mismatching depends on disease diagnosis (lymphoid versus myeloid), the conditioning regimen, the use of pretransplant antithymocyte globulin or ex vivo Tcell depletion methods [70,90–100]. Genotyping of HLA and KIR genes was performed in a recent study to measure risks after T-cell-replete HCT for ALL, AML, MDS, and CML in three groups of donor–recipient pairs: HLA class I antigen matched (KIR ligand matched); class I antigen mismatched but KIR ligand matched; and HLA class I and inhibitory KIR ligand mismatched [99]. The 1-year OS for these three groups was 59%, 49%, and 30%, respectively. The mismatched groups had lower OS and event-free survival compared with the matched group; further, the group with both HLA and KIR mismatching had higher relapse and TRM than the other groups. A detrimental effect was

observed for patients who lacked inhibitory KIR receptors; among HLAmatched cases, those who lacked the C1 or C2 ligand had lower OS compared with patients with either present. Patients who lacked C1 or C2 had higher TRM compared with patients who had all ligands present. The data from this study suggest that inhibitory KIR ligand mismatching and missing inhibitory KIR ligands confer higher risk after T-replete unrelated donor HCT. KIR-ligand mismatching had adverse effects on acute GVHD and rejection and no survival benefit for patients undergoing T-replete unrelated HCT in a recent analysis of 1790 transplants by the JMDP [70]. This study demonstrates an important role for HLA-C, DPB1, KIR ligand mismatching in the GVHD vector on post-transplant relapse. Namely, donor–recipient HLA-C disparity was associated with reduced relapse among patients with ALL. HLA-DPB1 mismatches were associated with lower relapse among CML transplants. Mismatching for the KIR2DL ligand in the GVH vector was associated with an increased risk of relapse for ALL. An increased risk of rejection was observed for KIR2DL ligand mismatches in the host-versus-graft vector. The risk of acute GVHD was increased with disparity for HLA-A, B, C, DPB1, and KIR ligand mismatching in the GVHD vector; mismatching for HLA-A, B, DQB1, and KIR ligand in the GVH vector increased mortality. The ligand mismatch mechanism of NK-cell alloreactivity is operational when there is an HLA class I disparity between the transplant recipient and donor. When a recipient and donor are HLA matched, the

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recipient may lack the appropriate ligand for donor KIR receptor. This “missing recipient ligand” mechanism is also present between HLAmismatched individuals, and therefore the lack of ligand is independent of donor–recipient HLA match status. The lack of recipient ligand may involve the HLA-C group C1 and C2 epitopes and the Bw4 epitope of HLA-B (Table 47.2). As an example, when the recipient encodes C1, C2, and Bw4, all the ligands for donor inhibitory KIRs are present, and there is no donor NK-cell alloreactivity. When the recipient is C1, C1 homozygous, the C2 group ligand is missing and may lead to donor NK alloreactivity because the donor KIR2DL1 is not engaged. The same situation arises in a C2, C2 homozygous patient; however in this situation, the donor KIR2DL3 (receptor for C1 group) is not engaged. When the recipient is Bw4−, the 3DL1 is not engaged and this leads to lysis of the target cell. Missing ligand in the recipient has been associated with improved outcome after unrelated donor HCT [97,100]. The International Histocompatibility Working Group retrospectively measured risks associated with missing recipient ligand and donor–recipient mismatched ligand in patients who received T-replete BM or PBPCs after high-dose conditioning from either HLA-matched or HLA-mismatched unrelated donors [97]. Among HLA-mismatched recipients, the presence of homozygosity for HLA-Bw6 and C1 or C2 (missing ligand) was associated with a decreased hazard of relapse. Most of the protective effect was contributed by the subset of patients who were missing C2 or Bw4. The impact of missing ligand was most pronounced in patients with AML, CML, and ALL, and the effect was observed among HLA-mismatched and not HLA-matched transplants. Among HLA-B- and/or HLA-C-mismatched pairs, there was no difference between ligand-mismatched and ligandmatched cases. There was a trend for lower relapse among patients who were missing ligand compared with patients with ligands present. Taken together, these data indicate that missing ligand in the recipient is a useful indicator for lowered risk of post-transplant relapse. The missing ligand model was recently tested in a large dataset of unrelated donor transplants for CML, AML, MDS, and ALL facilitated by the NMDP [100]. When compared with patients with all ligands present, patients who were missing one or more ligands had lower posttransplant disease relapse. Among the subset of good-risk CML patients, missing recipient ligand was an independent risk factor for clinically severe grade III–IV acute GVHD, indicating that NK-cell alloreactivity may play an indirect role in the pathogenesis of GVHD. Interestingly, among patients transplanted for high-risk leukemia and haploidentical donors, the Perugia experience demonstrates that donor NK alloreactivity is a better predictor of transplant outcome compared with missing ligand [89].

KIR haplotypes and activating receptors High-resolution typing of class I provides the necessary data to test hypotheses related to the clinical importance of ligand mismatching and ligand absence. Direct information on KIR haplotype content and allelic diversity provides the necessary data needed to test hypotheses on the predictive power of donor KIR genotype and haplotype on transplant outcome [98,101]. Two broad groups of KIR haplotype are recognized and are referred to as “group A” and “group B” [85,86]. Group A haplotypes encode primarily inhibitory receptors and the activating KIR2DS4 gene. Group B haplotypes exhibit extensive diversity by encoding more activating genes, including KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, and KIR3DS1. Inhibitory KIR receptors have greater affinity for class I ligands than do activating KIR receptors. HLA serves a dominant role in the selection of the peripheral repertoire of NK-cell inhibitory KIRs through its function as a ligand. During normal development, NK cells

that express inhibitory KIRs with specificity for self-class I are selectively enriched. Although both inhibitory and activating KIRs are expressed on NK and some T cells, inhibitory KIRs are dominant. The known ligands for the activating receptors are MHC class I molecules. KIR2DS1 and KIR2DS4 have specificity for HLA-C, CD94/ NKG2C for HLA-E, and NKG2D for MHC class I polypeptide-related sequences A and B, and retinoic acid early transcript-1/UL16 binding protein (RAET1/ULBP). The activating receptors display diversity [86]. The number of activating KIR genes, the presence of KIR ligand mismatching, and the presence of KIR A and B haplotypes are each predictive of outcome after high-dose conditioning and T-cell-depleted unrelated donor transplantation [102]. Specifically, transplantation from donors with group A KIR haplotypes or donors with lower numbers of activating KIR genes was associated with reduced relapse and improved DFS among AML/MDS recipients and to lesser extent among CML patients. There were no haplotype effects observed in patients with ALL. These data suggest that not only is the HLA ligand an important factor, but also the composition of donor KIR haplotypes may influence the risk of relapse and DFS. These observations point to the potential usefulness for prospective integration of recipient and donor KIR genotype information into donor selection to fully maximize NK-cell-driven antileukemic effects.

Impact of cytokine and immune response gene variation on outcome of HCT from unrelated donors GVHD arises as a consequence of donor T-cell-mediated recognition of recipient HLAs not shared by the donor, and/or recognition of minor histocompatibility determinants. Additionally, the lack of donor inhibition of NK-cell KIRs may lead to lower GVHD risk, either because the donor and recipient differ for class I or because the recipient is missing the HLA ligand for which the donor has an inhibitory receptor. The activation of T-cell and NK-cell pathways that lead to GVHD is mediated by cytokines [103]. The activation of NK cells by immature dendritic cells in the periphery is facilitated by interleukin (IL)-2, IL-12, IL-15, and IL-18, and NK-cell cytotoxicity can be induced by TNF produced by dendritic cells. In lymphoid tissues, the IL-2- and IL-12induced activation of NK cells leads to granulocyte–macrophage colonystimulating factor and interferon-gamma (IFN-γ) production; these events induce T helper (Th) cell differentiation, notably polarization of Th1 cells. The intensity of tissue injury and inflammation of GVHD may be modulated through the effects of allelic diversity within the coding and promoter regions of cytokine and immune response genes [104]. These polymorphisms located in the 5′ and 3′ regulatory sequences influence transcription and cytokine serum levels, and may also play a role in the release of cytokines during the three classical phases of the GVHD pathway. Recipient genetic variation for cytokine genes is hypothesized to exert its effect during the peritransplant period, when direct tissue injury from chemotherapy and radiation therapy initiates the activation of the cytokine storm; both recipient and donor genetic variation is hypothesized to modulate the severity of inflammation in the GVHD process. The events that lead to clinical GVHD are classically described in three stages [103]. The inciting injury begins as early as the conditioning regimen when irradiation and chemotherapy directly damage host tissues and cause the release of the proinflammatory cytokine TNF-α. TNF-α serum levels peak during the conditioning regimen and correlate with increased risk of death (see below). The inflammatory environment promotes increased expression of HLA and minor histocompatibility antigens, recognition of which by donor T cells leads to the expansion of donor T-cell clones. This response is amplified by the production of cytokines by CD4+ and CD8+ T cells. Further augmentation of injury of

Hematopoietic Cell Transplantation from Unrelated Donors

host tissues from donor T cells ensues. The cytokines implicated in this phase include IL-1, IL-2, IL-8, and IL-10 [103]. The severity of GVHD may be further increased or diminished through the effects of INF-γ, and Th1 and Th2 cytokines, in particular IL-1, IL-2, IL-8, and IL-10 [105– 107]. The final phase is characterized by cytotoxic T-cell-mediated tissue destruction. Sequence variation in cytokine and immune response genes provides information on the pathways involved in GVHD and practical strategies for assessing the potential risk of individual patients. Most of the published literature on cytokine and immune response genes has examined the effects of polymorphism of single genes after HLA-matched sibling donor HCT, with TNF-α and IL-10 being the two best studied genes. Data in unrelated donor transplantation are limited but growing [108– 110]. Side-by-side comparison of clinical results necessitates complete information on the transplant conditioning regimen (high-dose versus reduced-intensity), immunosuppressive agents (T-replete versus T-cell depletion, and methods for the latter), and source (related donor, unrelated donor, cord blood unit or granulocyte colony-stimulating factormobilized PBSCs versus BM). Since the distribution of cytokine gene and immune response gene variation differs worldwide, the racial and ethnic background of the study population is another important feature. In this regard, most of the early observations in this field were made in predominantly Caucasian transplant recipients and donors. Recently, extension of these analyses to more diverse populations demonstrates differences in the frequencies of SNPs, variable number of tandem repeat sequences, Msat markers, and alleles and haplotypes, particularly for the TNF locus, which displays strong positive LD within the class III region of the MHC [111–115]. Comparison of results from different studies is optimal when similar methodologies are used (e.g. genotyping of Msat versus individual SNPs versus SNP haplotypes, and assessment of risk of chronic GVHD among cases of acute GVHD), when the same polymorphisms are tested (e.g. TNF-α Msat alleles versus SNPs), and when a common gene nomenclature is employed. Side-by-side comparison may be otherwise difficult. This section will review the current literature that supports the hypothesis that genetic variation of TNF-α, IL-1, IL-2, IL-10, and IL-18 modulates risks of GVHD and mortality after unrelated HCT. Although other cytokines, such as IL-6, IL-7, IL-15, IFN-γ, and transforming growth factor-beta, have been implicated in the pathogenesis and severity of GVHD after HLA-identical sibling transplantation, as yet there are few or no data for these genes in the setting of unrelated donor HCT, and therefore they are not discussed. The literature on cytokine gene and immune response gene polymorphisms in related donor transplantation is vast, and the reader is referred to Chapter 16 and to outstanding comprehensive reviews on this topic [104]. Phase I: injury of host tissues and the initiation of the cytokine storm In this phase, the skin, liver, and gastrointestinal tract of the recipient are the target organs of injury. The proinflammatory cytokines TNF-α and IL-1, together with the anti-inflammatory cytokine IL-10, are among the first to participate in the cytokine storm. TNF-a and TNF receptor. TNF-α plays a multifaceted role in the pathogenesis of GVHD. TNF-α is produced by macrophages, monocytes, NK cells, and T cells, and functions as a Th1 cytokine by inducing apoptosis in target tissues and increased expression of HLA alloantigens, as well as by promoting further production of the IL-1, IL-6, and IL-10 cytokines [103]. The TNF-α and lymphotoxin-alpha genes are encoded within the HLA class III region, and are in strong positive LD with certain HLA-B and DRB1 genes. Polymorphism of TNF-α has been described using Msat and sequencing methods [104].

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The TNF promoter has extensive SNP diversity at positions −1031, −863, −857, −376, −308, −238, the most extensively studied polymorphisms of which are −308G/A and −863C/A. Rigorous comparison of studies of TNF-α variation and transplant outcome is difficult because the studies differ with respect to the polymorphisms tested and the clinical demographic features of the transplant populations. Confounding the analysis of TNF is the strong LD within the MHC, which has precluded definitive risk assessment of this gene independent of other tightly linked MHC genetic variation. In this setting, the data on TNF-α variation in unrelated HCT are heterogeneous. TNF-α-associated GVHD risk has been observed with TNF −308 and −857 in some but not other studies [108–110,116]. Data suggest that the risk of grade III–IV acute GVHD after unrelated HCT correlates with the level of TNF-α production [117–119], and that TNF-α polymorphisms are associated with higher TRM and lower OS [108,110,120]. Variation at residue 196 arginine (R) and methionine (M) in the TNFRII receptor affect outcome after unrelated HCT [109]. IL-1A and IL-1B. The IL-1 gene family is encoded on chromosome 2 and consists of IL-1A, IL-1B, and the IL-1 receptor agonist. IL-1A is an intracellular regulator, and the IL-1A promoter has a C/T SNP at −899 which alters gene transcription and expression. Presence of the IL-1A −889T SNP in unrelated donors was associated with improved 1-year survival, the effect of which was additive when both donor and recipient encoded −889T alleles [121]. Recently, an analysis conducted in a large homogeneous population of patients transplanted for CMLCP1 did not find significant differences in outcome according to the presence or absence of the IL-1A −889T SNP [122]. The IL-1B gene encodes an extracellular protein. The IL-1B promoter has a T/C SNP at −511, and the T allele is associated with increased binding of transcription initiation factors. In a single-center study of unrelated transplant recipients, the presence of the −511T allele in the recipient, the donor, or both recipient and donor was associated with a significantly reduced risk of mortality and TRM that was independent of GVHD. The −511T donor genotype correlated more strongly with outcome than did recipient genotype at this position [121]. IL-10. IL-10 maps to chromosome 1q31–q32 and is a member of the IFN receptor superfamily. IL10 is anti-inflammatory and functions as a Th2 cytokine. IL-10 reduces HLA expression, lowers CTL recognition of HLA and strongly inhibits TNF-α, IL-1α, IL-1β, IL-6, IL-12, and IFN-γ [123]. The IL-10 promoter has several polymorphic positions (−3575, −2763, −1082, −1064, −819, and −592), of which the −1082A allele is associated with lower in vitro IL-10 production [124]. As with TNF-α, there is substantially more information on the functional significance of IL-10 in related donor compared with unrelated HCT. Central to the hypotheses is that IL-10 sequence diversity may correlate with IL-10 production, and in this way influence risk of acute and chronic GVHD. Most studies of IL-10 variation in allogeneic HCT have defined an inverse relationship between IL-10 levels and risk of acute GVHD; that is, high IL-10 levels, or the presence of genetic variants associated with high production of IL-10, correlate with a low incidence of GVHD and mortality, supporting the observation that IL-10 has anti-inflammatory protective effects. Recipient homozygosity for the −592AA promoter genotype was associated with decreased risk of severe acute GVHD and death after unrelated HCT [125–128], and may be a result of increased IL-10 production associated with the −592A/C and A/A genotypes. These data suggest that the antigen-presenting cells of recipients homozygous for AA are high IL-10 producers capable of inducing tolerance in donor T cells [125]. In unrelated HCT studies of IL-10 polymorphisms other than −592, GVHD and mortality risk have been reported with −1082 and −1064 SNPs [116,120].

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Phase II: Activation of donor T cells In this phase, donor T-cell recognition of host MHCs and minor antigens leads to T-cell proliferation and differentiation. Several pathways contribute to the activation of T cells in this phase. Th1 reponses amplify the effects of the cytokine storm and involve INF-γ, IL-2, IL-12, and IL-18. Th2 responses are mediated by IL-10, IL-4, and IL-18. Data are currently available on the impact of genetic variation of IL-2 and IL-18 on GVHD risk after unrelated HCT. IL-2. IL-2 is a proinflammatory cytokine. The promoter −330T/G SNP is associated with early and sustained production. The hypothesis that genotypes associated with high production of the cytokine may increase risk of GVHD has been tested in unrelated donor transplant recipients [129]. Recipients encoding the IL-2 −330G allele had an increased risk of acute GVHD, and the presence of at least one G allele was associated with a twofold increase in risk of acute GVHD. IL-18. Interest in IL-18 has been motivated by its ability to regulate both Th1 and Th2 responses, and induce the secretion of growth factors in murine models [103]. As a Th1-inducing cytokine, it can reduce the severity of acute GVHD when given after high-dose conditioning. When administered to the donor, IL-18 reduces the severity of acute GVHD as a Th2-inducing cytokine. IL-18 can preserve GVT effects after HCT [130]. IL-18 is produced by macrophages, T cells, dendritic cells, and keratinocytes. The promoter haplotype comprising −137G/C, −607C/A, and −656G/T has been analyzed in 157 unrelated transplants [131]. Although there was no association between patient/donor haplotypes and risk of GVHD, the recipient GCG haplotype was associated with lower TRM

at day 100 and 1 year post-transplant, and with improved survival. Patients with acute GVHD after unrelated HCT have high levels of IL18 [132], and serum levels are reduced after successful GVHD therapy. Phase III: inflammatory effectors In this phase, cellular and inflammatory effectors produce the end-organ target damage that comprises the clinical GVHD phenotype. The two major cytokines that participate in this phase are TNF and IL-1. Injury of the gastrointestinal tract and skin caused by direct toxicity from the conditioning regimen and from GVHD causes leakage of bacterial toxins and byproducts. Mononuclear phagocytes are stimulated and secrete TNF and IL-1.

Conclusion The success of unrelated HCT as a curative therapy has come through innovative research in transplantation genetics and biology, the development of safer conditioning regimens, more effective GVHD preventive and therapeutic strategies, advances in the monitoring and detection of early disease recurrence, and improved supportive care of patients. The ultimate goal of individualizing treatment strategies will require a more complete understanding of the correlation between genotype and phenotype, and the impact of different transplant procedures on transplantrelated toxicities and GVT effects. In the future, the optimal transplant approach for a given patient may include combined modalities of immunogenetic measures for the prevention of GVH and promotion of GVT effects, together with the use of post-transplant immunotherapy to lower risk of disease recurrence.

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61. Drobyski WR, Klein J, Flomenberg N et al. Superior survival associated with transplantation of matched unrelated versus one-antigen-mismatched unrelated or highly human leukocyte antigen-disparate haploidentical family donor marrow grafts for the treatment of hematologic malignancies: establishing a treatment algorithm for recipients of alternative donor grafts. Blood 2002; 99: 806–14. 62. Chalandon Y, Tiercy JM, Schanz U et al. Impact of high-resolution matching in allogeneic unrelated donor stem cell transplantation in Switzerland. Bone Marrow Transplant 2006; 37: 909–16. 63. Saarinen-Pihkala UM, Gustafsson G, Ringden O et al. No disadvantage in outcome of using matched unrelated donors as compared with matched sibling donors for bone marrow transplantation in children with acute lymphoblastic leukemia in second remission. J Clin Oncol 2001; 19: 3406–14. 64. Yakoub-Agha I, Mesnil F, Kuentz M et al. Allogeneic marrow stem-cell transplantation from human leukocyte antigen-identical siblings versus human leukocyte antigen-allelic-matched unrelated donors (10/10) in patients with standard-risk hematologic malignancy: a prospective study from the French Society of Bone Marrow Transplantation and Cell Therapy. J Clin Oncol 2006; 24: 5695–702. 65. Hows JM, Passweg JR, Tichelli A et al. Comparison of long-term outcomes after allogeneic hematopoietic stem cell transplantation from matched sibling and unrelated donors. Bone Marrow Transplant 2006; 38: 799–805. 66. Kennedy-Nasser AA, Leung KS, Mahajan A et al. Comparable outcomes of matched-related and alternative donor stem cell transplantation for pediatric severe aplastic anemia. Biol Blood Marrow Transplant 2006; 12: 1277–84. 67. Petersdorf EW, Longton GM, Anasetti C et al. Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors. Blood 1997; 89: 1818–23. 68. Tiercy JM, Passweg J, van Biezen A et al. Isolated HLA-C mismatches in unrelated donor transplantation for CML. Bone Marrow Transplant 2004; 34: 249–55. 69. Fleischhauer K, Locatelli F, Zecca M et al. Graft rejection after unrelated donor hematopoietic stem cell transplantation for thalassemia is associated with nonpermissive HLA-DPB1 disparity in host-versus-graft direction. Blood 2006; 107: 2984–92. 70. Morishima Y, Yabe T, Matsuo K et al. Effects of HLA allele and killer immunoglobulin-like receptor ligand matching on clinical outcome in leukemia patients undergoing transplantation with T-cell-replete marrow from an unrelated donor. Biol Blood Marrow Transplant 2007; 13: 315– 28. 71. Heemskerk MB, Roelen DL, Dankers MK et al. Allogeneic MHC class I molecules with numerous sequence differences do not elicit a CTL response. Hum Immunol 2005; 66: 969–76. 72. Ferrara GB, Bacigalupo A, Lamparelli T et al. Bone marrow transplantation from unrelated donors: the impact of mismatches with substitutions at position 116 of the human leukocyte antigen class I heavy chain. Blood 2001; 98: 3150–5.

73. Kawase T, Morishima Y, Matsuo K et al. Highrisk HLA allele mismatch combinations responsible for severe acute graft versus host disease and implication for its molecular mechanism. Blood Blood-2007; 110: 2235–41. 74. Petersdorf EW, Gooley T, Malkki M et al. The biological significance of HLA-DP gene variation in haematopoietic cell transplantation. Br J Haematol 2001; 112: 988–94. 75. Zino E, Frumento G, Marktel S et al. A T-cell epitope encoded by a subset of HLA-DPB1 alleles determines nonpermissive mismatches for hematologic stem cell transplantation. Blood 2004; 103: 1417–24. 76. Keever-Taylor CA, Bredeson C, Loberiza FR et al. Analysis of risk factors for the development of GVHD after T cell-depleted allogeneic BMT: effect of HLA disparity, ABO incompatibility, and method of T-cell depletion. Biol Blood Marrow Transplant 2001; 7: 620–30. 77. Loiseau P, Busson M, Balere M-L et al. HLA association with hematopoietic stem cell transplantation outcome: the number of mismatches at HLA-A, -B, -C, -DRBI or -DQBI is strongly associated with overall survival. Biol Blood Marrow Transplant 2007; 13: 965–74. 78. Tamouza R, Rocha V, Busson M et al. Association of HLA-E polymorphism with severe bacterial infection and early transplant-related mortality in matched unrelated bone marrow transplantation. Transplantation 2005; 80: 140– 4. 79. Li S, Kawata H, Katsuyama Y et al. Association of polymorphic MHC microsatellites with GVHD, survival, and leukemia relapse in unrelated hematopoietic stem cell transplant donor/recipient pairs matched at five HLA loci. Tissue Antigens 2004; 63: 362–8. 80. Malkki M, Gooley TA, Horowitz MM et al. Mapping MHC-resident transplantation determinants. Biol Blood Marrow Transplant 2007; 13: 986–95. 81. Guo Z, Hood L, Malkki M, Petersdorf EW. Longrange multilocus haplotype phasing of the MHC. Proc Natl Acad Sci USA 2006; 103: 6964–9. Erratum in Proc Natl Acad Sci USA 2006; 103: 9374. 82. Petersdorf EW, Malkki M, Gooley TA et al. MHC haplotype matching for unrelated hematopoietic cell transplantation. PLoS Medicine 2007; 4: e8. 83. Passweg JR, Stern M, Koehl U et al. Use of natural killer cells in hematopoetic stem cell transplantation [Review]. Bone Marrow Transplant 2005; 35: 637–43. 84. Aversa F, Tabilio A, Velardi A et al. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med 1998; 339: 1186–93. 85. Parham P. MHC class I molecules and KIRs in human history, health and survival [Review]. Nat Rev Immunol 2005; 5: 201–14. 86. Marsh SG, Parham P, Dupont B et al. Killer-cell immunoglobulin-like receptor (KIR) nomenclature report, 2002. Immunogenetics 2003; 55: 220–6. 87. Ruggeri L, Capanni M, Casucci M et al. Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation. Blood 1999; 94: 333–9.

88. Ruggeri L, Capanni M, Urbani E et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: 2097–100. 89. Ruggeri L, Mancusi A, Capanni M et al. Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood 2007; 110: 433–40. 90. Sun JY, Gaidulis L, Dagis A et al. Killer Ig-like receptor (KIR) compatibility plays a role in the prevalence of acute GVHD in unrelated hematopoietic cell transplants for AML. Bone Marrow Transplant 2005; 36: 525–30. 91. Giebel S, Locatelli F, Lamparelli T et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 2003; 102: 814– 19. 92. Davies SM, Ruggieri L, DeFor T et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor. Blood 2002; 100: 3825–7. 93. Bornhauser M, Schwerdtfeger R, Martin H et al. Role of KIR ligand incompatibility in hematopoietic stem cell transplantation using unrelated donors. Blood 2004; 103: 2860–1. 94. Schaffer M, Malmberg KJ, Ringden O et al. Increased infection-related mortality in KIRligand-mismatched unrelated allogeneic hematopoietic stem-cell transplantation. Transplantation 2004; 78: 1081–5. 95. Beelen DW, Ottinger HD, Ferencik S et al. Genotypic inhibitory killer immunoglobulin-like receptor ligand incompatibility enhances the long-term antileukemic effect of unmodified allogeneic hematopoietic stem cell transplantation in patients with myeloid leukemias. Blood 2005; 105: 2594– 600. 96. Farag SS, Bacigalupo A, Eapen M et al. The effect of KIR ligand incompatibility on the outcome of unrelated donor transplantation: a report from the Center for International Blood and Marrow Transplant Research, the European Blood and Marrow Transplant Registry, and the Dutch Registry. Biol Blood Marrow Transplant 2006; 12: 876–84. 97. Hsu KC, Gooley T, Malkki M et al. KIR ligands and prediction of relapse after unrelated donor hematopoietic cell transplantation for hematologic malignancy. Biol Blood Marrow Transplant 2006; 12: 828–36. 98. Leung W, Iyengar R, Turner V et al. Determinants of antileukemia effects of allogeneic NK cells. J Immunol 2004; 172: 644–50. 99. Sun JY, Dagis A, Gaidulis L et al. Detrimental effect of natural killer cell alloreactivity in Treplete hematopoietic cell transplantation (HCT) for leukemia patients. Biol Blood Marrow Transplant 2007; 13: 197–205. 100. Miller JS, Cooley S, Parham P et al. Missing KIR-ligands is associated with less relapse and increased graft versus host disease (GVHD) following unrelated donor allogeneic HCT. Blood 2007; 109: 5058–61. 101. Cook M, Briggs D, Craddock C et al. Donor KIR genotype has a major influence on the rate of cytomegalovirus reactivation following T-cell replete stem cell transplantation. Blood 2006; 107: 1230–2.

Hematopoietic Cell Transplantation from Unrelated Donors 102. Kroger N, Binder T, Zabelina T et al. Low number of donor activating killer immunoglobulin-like receptors (KIR) genes but not KIR-ligand mismatch prevents relapse and improves disease-free survival in leukemia patients after in vivo T-cell depleted unrelated stem cell transplantation. Transplantation 2006; 82: 1024–30. 103. Reddy P, Ferrara JL. Immunobiology of acute graft-versus-host disease [Review]. Blood Rev 2003; 17: 187–94. 104. Dickinson AM, Middleton PG. Beyond the HLA typing age: genetic polymorphisms predicting transplant outcome [Review]. Blood Rev 2005; 19: 333–40. 105. Liu J, Anderson BE, Robert ME et al. Selective T-cell subset ablation demonstrates a role for T1 and T2 cells in ongoing acute graft-versus-host disease: a model system for the reversal of disease. Blood 2001; 98: 3367–75. 106. Nikolic B, Lee S, Bronson RT et al. Th1 and Th2 mediate acute graft-versus-host disease, each with distinct end-organ targets. J Clin Invest 2000; 105: 1289–98. 107. Daneshpouy M, Socie G, Lemann M et al. Activated eosinophils in upper gastrointestinal tract of patients with graft-versus-host disease. Blood 2002; 99: 3033–40. 108. Takahashi H, Furukawa T, Hashimoto S et al. Contribution of TNF-alpha and IL-10 gene polymorphisms to graft-versus-host disease following allo-hematopoietic stem cell transplantation. Bone Marrow Transplant 2000; 26: 1317–23. 109. Ishikawa Y, Kashiwase K, Akaza T et al. Polymorphisms in TNFA and TNFR2 affect outcome of unrelated bone marrow transplantation. Bone Marrow Transplant 2002; 29: 569–75. 110. Keen LJ, Defor TE , Bidwell JL et al. Interleukin10 and tumor necrosis factor alpha region haplotypes predict transplant-related mortality after unrelated donor stem cell transplantation. Blood 2004; 103: 3599–602. 111. Delaney NL, Esquenazi V, Lucas DP et al. TNFalpha, TGF-beta, IL-10, IL-6, and INF-gamma alleles among African Americans and Cuban Americans. Report of the ASHI Minority Workshops: Part IV. Hum Immunol 2004; 65: 1413– 19. 112. Leffell MS, Vogelsang GB, Lucas DP et al. Association between TGF-beta expression and severe GVHD in allogeneic bone marrow transplantation. Transplant Proc 2001; 33: 485–6.

113. Cox ED, Hoffmann SC, DiMercurio BS et al. Cytokine polymorphic analyses indicate ethnic differences in the allelic distribution of interleukin-2 and interleukin-6. Transplantation 2001; 72: 720–6. 114. Hassan MI, Aschner Y, Manning CH et al. Racial differences in selected cytokine allelic and genotypic frequencies among healthy, pregnant women in North Carolina. Cytokine 2003; 21: 10–16. 115. Zachary AA, Bias WB, Johnson A et al. Antigen, allele, and haplotype frequencies report of the ASHI minority antigens workshops. Part 1. African-Americans. Hum Immunol 2001; 62: 1127–36. Erratum in Hum Immunol 2002; 63: 337. 116. Nordlander A, Uzunel M, Mattsson J, Remberger M. The TNFd4 allele is correlated to moderateto-severe acute graft-versus-host disease after allogeneic stem cell transplantation. Br J Haematol 2002; 119: 1133–6. 117. Schots R, Kaufman L, Van Reit, I et al. Proinflammatory cytokines and their role in the development of major transplant-related complications in the early phase after allogeneic bone marrow transplantation. Leukemia 2003; 17: 1150–6. 118. Remberger M, Jaksch M, Uzunel M, Mattsson J. Serum levels of cytokines correlate to donor chimerism and acute graft-vs.-host disease after haematopoietic stem cell transplantation. Eur J Haematol 2003; 70: 384–91. 119. Visentainer JE, Lieber SR, Persoli LB et al. Serum cytokine levels and acute graft-versus-host disease after HLA-identical hematopoietic stem cell transplantation. Exp Hematol 2003; 31: 1044– 50. 120. Bettens F, Passweg J, Gratwohl A et al. Association of TNFd and IL-10 polymorphisms with mortality in unrelated hematopoietic stem cell transplantation. Transplantation 2006; 81: 1261– 7. 121. MacMillan ML, Radloff GA, Defor TE et al. Interleukin-1 genotype and outcome of unrelated donor bone marrow transplantation. Br J Haematol 2003; 121: 597–604. 122. Mehta PA, Eapen M, Klein JP et al. Interleukin-1 alpha genotype and outcome of unrelated donor haematopoietic stem cell transplantation for chronic myeloid leukaemia. Br J Haematol 2007; 137: 152–7. 123. Korholz D, Kunst D, Hempel L et al. Decreased interleukin 10 and increased interferon-gamma

124.

125.

126.

127.

128.

129.

130.

131.

132.

691

production in patients with chronic graft-versushost disease after allogeneic bone marrow transplantation. Bone Marrow Transplant 1997; 19: 691–5. Turner DM, Williams DM, Sankaran D et al. An investigation of polymorphism in the interleukin10 gene promoter. Eur J Immunogenet 1997; 24: 1–8. Hempel L, Korholz D, Nussbaum P et al. High interleukin-10 serum levels are associated with fatal outcome in patients after bone marrow transplantation. Bone Marrow Transplant 1997; 20: 365–8. Takatsuka H, Takemoto Y, Okamoto T et al. Predicting the severity of graft-versus-host disease from interleukin-10 levels after bone marrow transplantation. Bone Marrow Transplant 1999; 24: 1005–7. Holler E, Roncarolo MG, Hintermeier-Knabe R et al. Prognostic significance of increased IL-10 production in patients prior to allogeneic bone marrow transplantation. Bone Marrow Transplant 2000; 25: 237–41. Baker KS, Roncarolo MG, Peters C et al. High spontaneous IL-10 production in unrelated bone marrow transplant recipients is associated with fewer transplant-related complications and early deaths. Bone Marrow Transplant 1999; 23: 1123– 9. MacMillan ML, Radloff GA, Kiffmeyer WR et al. High-producer interleukin-2 genotype increases risk for acute graft-versus-host disease after unrelated donor bone marrow transplantation. Transplantation 2003; 76: 1758– 62. Reddy P, Teshima T, Hildebrandt G et al. Interleukin 18 preserves a perforin-dependent graftversus-leukemia effect after allogeneic bone marrow transplantation. Blood 2002; 100: 3429– 31. Cardoso SM, Defor TE, Tilley LA et al. Patient interleukin-18 GCG haplotype associates with improved survival and decreased transplantrelated mortality after unrelated-donor bone marrow transplantation. Br J Haematol 2004; 126: 704–10. Fujimori Y, Takatsuka H, Takemoto Y et al. Elevated interleukin (IL)-18 levels during acute graft-versus-host disease after allogeneic bone marrow transplantation. Br J Haematol 2000; 109: 652–7.

48

Ann E. Woolfrey

Donor Selection for Hematopoietic Cell Transplantation

Introduction Most patients referred for allogeneic hematopoietic cell transplantation (HCT) lack a human leukocyte antigen (HLA)-matched sibling; thus, an alternative donor must be identified. Possible sources for an alternative donor include unrelated (volunteer) donors (URDs), unrelated umbilical cord blood (UCB) units, and extended family members. The suitability of each donor source depends upon the disease being treated, the urgency of the transplant procedure, and the available protocols. To date, there have been no randomized studies comparing outcome of the various donor sources that could guide the selection of an alternative donor. The optimal alternative donor will be matched for HLAs with the patient, but a less well-matched donor may be appropriate for patients with aggressive malignancies in the interest of shortening the time to HCT. This chapter will discuss factors relevant to selection of alternative donors for HCT.

Selection of URDs Transplants of HLA-matched URD marrow for treatment of hematologic malignancies have been reported to result in outcomes similar to those using HLA-matched sibling donors (see Chapter 47 for a complete description of HCT from URD) [1–3]. Recipients of URD grafts have a higher risk of nonrelapse mortality (NRM), balanced by a lower risk for relapse. In contrast, patients with nonmalignant disorders, including severe aplastic anemia, Fanconi’s anemia, and inherited disorders, have a higher risk for mortality following HCT from URD compared with HCT from HLA-matched sibling donors, because there is no graft-versus-leukemia benefit to outweigh the higher incidence of graft-versus-host disease (GVHD). Several factors should be considered in selection of the optimal URD in order to reduce NRM, the most important of which is the degree of HLA match. Within the past decade, high-resolution typing techniques have been developed to allow identification of polymorphic alleles among class I and class II HLA antigens. Petersdorf et al. studied 300 patient–donor pairs matched for HLA-A and B by serologic typing, and matched for the DRB1 alleles, and demonstrated that only half of the pairs were matched at the allele level match for all five loci (HLA-A, B, C, DRB1, and DQB1), and that one-quarter were mismatched for

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

692

multiple alleles [4]. The ability to distinguish allele-level mismatches has allowed investigation of the relevancy of patient–donor mismatching. Subsequent studies show that the impact of patient–donor mismatching depends on the disease being treated, and within disease, risk groups depend upon the degree of HLA mismatch and the locus of HLA mismatch. Initial studies of patients with chronic myeloid leukemia (CML) found an increased risk for graft failure when donors had multiple mismatches that involved at least one class I allele [4–7]. The Seattle group found that the incidence of graft failure was 29% when the mismatch involved more than one class I allele mismatch, and 12% for mismatched pairs involving both class I and class II alleles (p = 0.003 and 0.01, respectively), compared with 2% or less when either no mismatch or a mismatch confined to a single HLA-A, B, C, DRB1, and DQB1 allele was present. The risk of developing acute GVHD grades III–IV also was influenced by the number and class of mismatched alleles. The highest risk for severe acute GVHD was observed for multiple mismatches involving both class I and class II alleles (hazard ratio [HR] 2.0, p = 0.02). Pairs with a single class I mismatch did not have a significant increase in acute GVHD compared with matched recipients, but a single class II mismatch or multiple class I mismatches both appeared to confer a higher (although not significant) hazard of severe GVHD. An important limitation of these studies was that patients were mainly Caucasian; therefore results may not be transferable to other ethnic populations. For example, studies from Japan found that mismatching of HLA-A and B, but not class II HLA, decreased survival [6]. The number of tolerated mismatches appears to differ according to the disease being treated. The Seattle group studied the effect of a single HLA allele mismatch on survival in 242 patients classified as having low-risk disease (patients with CML in chronic phase receiving HCT within 2 years of diagnosis), 183 as having high-risk disease (patients with acute leukemia beyond first remission), and 304 as having intermediate-risk disease (all other patients) [8]. Among patients with lowrisk disease, mismatch of a single antigen or allele was associated with a significant decrease in survival and increase in NRM, while there was no difference in risk for relapse (Fig. 48.1(a)). Mismatch at the HLA-C locus conferred the highest risk for mortality (HR 3.18, confidence interval [CI] 1.74–5.82), which did not appear to be due to natural killer–killer immunoglobulin-like receptor (NK–KIR) ligand matching. In contrast, among patients with high-risk disease, a donor with a single HLA mismatch had no effect on risk for mortality. In the Seattle dataset, the relevance of HLA mismatching among intermediate-risk or high-risk patients could be appreciated only when

Donor Selection for Hematopoietic Cell Transplantation 2.5

Relative risk of death

* 2 1.5

* *

*

*

NMDP 2007

Seattle 2004 IR/HR

Match 1 Allele mismatch 2 Allele mismatch

1 0.5 0 NMDP 2004

(a)

Seattle 2004 LR

3

Relative risk of death

2.5

* *

2 5 or 6 of 6 HLA match 4 of 6 HLA match

1.5 1 0.5 0

(b)

NYBC 1998

Minnesota 2002

Eurocord 2004

Fig. 48.1 Increasing the number of human leukocyte antigen (HLA) mismatches of either unrelated marrow or unrelated cord blood correlates with an increase in risk of death. The relative risk of death for recipients of unrelated donor marrow grafts (a) matched for 10 of 10 HLA alleles (black bars) is set at 1.00 for comparison with grafts matched for nine of 10 (dark gray bars) and eight of 10 (light gray bars) HLA alleles. Shown are results from the 2004 National Marrow Donor Program (NMDP) analysis [9] of 1874 pairs, the 2007 NMDP analysis of 3857 pairs [10], and the 2004 Seattle [8] analysis of 661 pairs categorized as intermediate- or high-risk (IR/HR) leukemia, and 207 pairs categorized as low-risk (LR) disease. Among cord blood recipients (b), the relative risk of death for recipients of grafts matched for five or six of six HLA antigens (black bars) is set at 1.00 for comparison with grafts matched for four of six HLAs (gray bars). Shown are results from the 1998 New York Blood Center (NYBC) analysis [32] of 562 pairs, the 2002 Minnesota analysis [34] of 102 pairs, and the 2004 Eurocord analysis [35] of 550 pairs. *Statistically significant difference in relative risk compared with the control group (black bar).

Table 48.1 Effect of a single human leukocyte antigen (HLA) mismatch on survival, non-relapse mortality, and acute graft-versus-host disease (GVHD)

693

two or more disparities existed. Among 164 such patient–donor pairs, increasing from one to two or more HLA mismatches significantly decreased overall survival (HR 1.40, CI 1.08–1.82). These results have been substantiated by studies conducted through the National Marrow Donor Program (NMDP) (Fig. 48.1(a)). To investigate the relationship between HLA mismatch and outcome, the NMDP retrospectively typed 1874 patient–donor pairs to analyze the impact of HLA-allele level matching [9]. Outcome endpoints of engraftment, acute GVHD, chronic GVHD, and mortality were examined according to locus of donor–recipient HLA disparity. Donor–recipient disparity of class I HLA loci HLA-A, B, C, and DRB1 were independently associated with a statistically significant increase in the risk for mortality. In addition, mismatching at HLA-A was associated with higher risk for both acute and chronic GVHD. Among class I HLA mismatches, those that only could be detected by high-resolution typing (allele-level mismatches) did not appear to increase the risk for poor outcome, whereas a significant effect on outcome endpoints was associated with mismatches detected by low-resolution typing (antigen-level mismatches). Specifically, mortality was significantly higher for antigenlevel mismatches at HLA-A (relative risk [RR] 1.44, CI 1.20–1.74; p < 0.0001), B (RR 1.46, CI 1.19–1.80; p < 0.0003), C (RR 1.21, CI 1.06– 1.40; p < 0.007), and DRB1 (RR 1.23, CI 1.04–1.45; p = 0.01). The 2008 NMDP analysis of 3857 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia, CML or myelodysplastic syndrome confirmed the lack of association of HLA-DQ mismatch independently with survival [10]. The earlier NMDP study employed multivariate modeling, whereas the most recent NMDP study used subset analysis, which showed that a single mismatch for HLA-A, B, C or DRB1 was associated with a higher risk for transplant-related mortality (TRM) and acute GVHD, and that mismatch for HLA-A, C, and DRB1, but not HLA-B, was associated with statistically worse survival compared with the eight of eight HLA allele-matched pairs (Table 48.1). In contrast to the previous NMDP report, the effect of a mismatch at the allele level was equivalent to a serologic or antigen mismatch. A donor with multiple HLA mismatches increased the risk for mortality, in a degree-dependent fashion, HR being 1.25 and 1.65 (p < 0.0001) for a seven and six of eight loci mismatch, respectively, compared with eight of eight matched pairs. The Japanese Marrow Donor Program experience supports the idea that disparities involving HLA-class I alleles are independent risk factors for acute GVHD, TRM, and overall survival. In the Japanese analysis, the addition of HLA-C allele disparity with other HLA allele mismatches increased the risk of acute GVHD in a synergistic fashion [6,11]. HLA class I allele mismatches also were associated with a significantly higher incidence of graft failure when compared with patients with allele matched donors.

Survival

Nonrelapse mortality

Acute GVHD

Single mismatched locus

Relative risk

p-value

Relative risk

p-value

Relative risk

p-value

None HLA-A HLA-B HLA-C HLA-DRB1 HLA-DQB1

1.00 1.35 1.19 1.16 1.36 0.97

0.0003 0.19 0.03 0.06 0.77

1.00 1.52 1.41 1.32 1.40 1.08

<0.0001 0.02 0.0005 0.07 0.50

1.00 1.44 1.62 1.47 1.21 1.03

0.002 0.003 <0.0001 0.42 0.86

(Lee, unpublished data.)

694

Chapter 48

Taken together, these results support donor identification strategies that limit HLA mismatch. Mismatch at the allele level must be identified as it contributes equally to antigen mismatch with respect to outcome. If mismatch is unavoidable, a tolerable mismatch will depend upon the ethnicity of the recipient. Among Caucasian recipients, mismatch at HLA-DQB1 appears most tolerated, followed by mismatch at HLA-B. In contrast, in Japanese recipients, HLA-A or B mismatches fare the worst. These data do not define tolerable mismatches for other ethnic groups, since adequate power could not be attained due to the wide range of diverse haplotypes and insufficient numbers of non-Caucasian patients. Further, the results of these large retrospective studies must be viewed in the context of high-dose conditioning predominately for marrow grafts as treatment for hematologic malignancy. It remains uncertain whether these results can be extrapolated to reduced-intensity regimens, or to peripheral blood progenitor cell (PBPC) grafts, wherein the number of tolerable HLA mismatches remain to be defined. Further, just as the number of tolerable mismatches differs for patients with low-risk compared with higher-risk malignancies, so too will nonmalignant disorders require definition of the number of tolerable, if any, HLA mismatch. As studies further define risks of HLA mismatches, particularly in nonCaucasian populations, delineation of “low risk” from “high risk” mismatches may be possible [12]. Until then, the donor selection process should endeavor to identify the least mismatched donor within the time allowed by the clinical situation. Major advances have been made in understanding the role of NK-cell activity after marrow grafting, but the value of selecting an NK alloreactive donor has not been established. NK biology and NK-cell KIRs are described in detail in Chapter 13. Several studies have evaluated the impact of inhibitory KIR ligand mismatching, defined as the absence of one donor KIR ligand class I allele in the recipient. A study of 130 patients treated at three European centers involved patients given high-dose conditioning, unmanipulated marrow or granulocyte colonystimulating factor-mobilized peripheral blood stem cells (PBSCs), pretransplant thymoglobulin, and GVHD prophylaxis with cyclosporine and short-course methotrexate [13]. Inhibitory KIR epitope mismatch between donor and recipient was associated with significant improvement in disease-free (87% versus 39%; p = 0.007), NRM (6% versus 40%), and overall survival at 4.5 years (87% versus 48%; p = 0.006) compared with recipients of KIR epitope-matched grafts. In contrast, retrospective analyses from Japan and Minnesota evaluated 1449 and 175 URD transplants, respectively, and found no benefit for inhibitory KIR ligand incompatibility [14,15]. While most of these latter patients did not receive T-cell-depleted grafts, thought necessary for promoting NK alloreactivity, another study of 190 patients most of whom received antithymocyte globulin also showed no benefit; in fact, survival was lower and NRM higher among recipients of KIR ligandmismatched grafts [16]. Selection of the optimal URD also must consider whether the donor expresses HLA antigens to which the recipient has been sensitized. A study of 522 patients found a ninefold greater incidence of graft rejection among those with antibodies to donor HLAs, detected by crossmatch testing, compared with those with negative crossmatch tests [17]. These results suggested that antibody-dependent cytotoxicity reactions could lead to graft rejection. To determine whether the recipient has been sensitized to HLA, recipient serum is screened against a panel with known HLA antigen phenotypes. Historically, this test used a panel of fresh or frozen lymphocytes; subsequently, techniques have been developed to create solid surfaces coated with purified HLA molecules, resulting in enhanced sensitivity and specificity compared with cell-based assays. The percentage of phenotypes in the panel that are seropositive is referred to as the percent

Table 48.2 Indications for a crossmatch study PRA result

HLA match*

Prefinal crossmatch†

Final crossmatch‡§

Positive Negative Negative

Match or mismatch Match Mismatch

Yes No Yes

Yes No¶ Yes

HLA, human leukocyte antigen; PRA, panel reactive antibody. * HLA match is defined as phenotypically matched for HLA-A, B, C, DQB1 (by DNA typing methods at least to an intermediate resolution level) and allele matched for DRB1. † Prefinal crossmatch: serologic crossmatches with a serum sample drawn within the previous 6 months. ‡ Final crossmatch: all serologic and flow cytometry crossmatch tests. The serum sample must be drawn within 30 days of transplantation. § If the prefinal crossmatch was performed with serum drawn within 30 days of transplantation, no final crossmatch is necessary. ¶ Special attention must be paid to PRA-negative patients at the time of admission, since alloimmune status may change between prefinal (confirmatory) testing and admission.

panel reactive antibody (PRA), and a positive result typically is defined as over 10% PRA. If the recipient PRA screen is positive, additional testing should be done to identity the HLA antibody specificity and to determine if the antibody is donor directed. Crossmatch studies, which detect antibodies in recipient serum directed against antigens expressed on donor cells, confirm that the antibody is donor directed and clinically relevant. The indications for crossmatch testing, performed according to the patient’s alloimmune risk status and the degree of HLA matching with the donor, are shown in the Table 48.2. Antibodies may be directed against class I HLA antigens (HLA-A, B, and C) and/or HLA Class II HLA antigens (HLA-DR, DQ, and DP). When HLA-DP antibodies are identified, typing of donors and recipients for HLA-DP may aid in the interpretation of crossmatch results. Sometimes non-HLA-specific antibodies or other proteins result in falsepositive crossmatch results. Since HLA expression on solid-phase assays is variable and very sensitive, false-positive results may occur when recipient antibodies react with plastic or other reagents. The detection of a donor-directed antibody by the solid-phase methods does not always predict a positive crossmatch. The limitations of each assay require that the HLA antibody specificity and crossmatch results be evaluated in conjunction with each other. Consideration of other donor-related factors is justified when more than one donor of equivalent HLA-match has been identified. Selection of a cytomegalovirus (CMV)-seronegative donor reduces the risk of CMV disease from 10% to 3% among CMV-seronegative recipients [18]. A large megafile analysis from the European Group for Blood and Marrow Transplantation (EBMT) showed that CMV serology was an independent factor predicting survival for CMV-seropositive patients, favoring those who received grafts from seropositive compared with seronegative donors [19]. The contribution of donor age or gender to NRM may be important for certain diseases. The 2001 NMDP analysis, which included 6978 patients, found both male gender and younger age to be independently associated with lower risk for GVHD, and younger age with improved survival [20]. Importantly, the analysis suggested that these factors were more important in the situation wherein the donor was HLA-mismatched. Age and gender were not found to be important in a recent NMDP analysis [10].

Donor Selection for Hematopoietic Cell Transplantation

Selection of UCB units UCB characteristically differs from marrow in a number of ways, described in detail in Chapter 39. Two important characteristics influence unit selection: the relative reduction in both number of cells and immunologic reactivity. The median dose of total nucleated cells (TNCs), CD34+ cells, and CD3+ cells in a UCB unit is approximately 10 times lower than that of a bone marrow graft [21,22]. Reduced cell numbers may be offset by a higher capacity for replication, as indicated by higher cell-cycle rates and longer telomeres in UCB progenitor cells [23,24]. Immune mediator cells in UCB have been characterized as relatively immature compared with marrow cells. UCB lymphocytes have less mature T- and B-cell phenotypes, reduced response to alloantigen, and lower capacity to generate inflammatory cytokines [25–28]. These biologic differences have a significant effect on outcome following UCB transplant, and therefore must be considered in the selection of an UCB unit. Relative immune immaturity has allowed transplantation of HLAmismatched UCB units. Conventionally, determination of the degree of HLA matching between recipient and UCB graft has been made according to serologic typing at HLA-A and B, along with high-resolution typing to distinguish DRB1 alleles. Hence, a UCB unit matched by serologic typing at HLA-A and B and matched for the DRB1 allele has been considered a full (or six of six locus) match. This definition of matching ignores mismatches at HLA-C and DQB1, as well as allelelevel mismatches at the HLA-A and B loci. Not surprisingly, around one-third of conventionally typed units will have at least one additional undetected HLA mismatch at HLA-A, B, C, DRB1, and DQB1, when retyped by high-resolution methods [29]. Using high-resolution methods to retrospectively type 122 recipient–UCB pairs, Kogler et al. showed that approximately 60% of conventionally typed four-to-six loci-matched units actually were mismatched at three or more alleles [30]. The relevance of occult HLA mismatching will not be known until analysis of a large registry set is undertaken. Accordingly, further discussion of the effect of HLA matching will consider only conventional typing. All large studies of UCB transplants have shown an association between the degree of HLA mismatch and the risk for graft failure, NRM, and GVHD, and some studies have found an association with disease-free survival (DFS). Initial observations in 65 UCB transplants by the Eurocord registry in 1997 and 562 transplants by the New York Blood Center (NYBC) in 1998 indicated that HLA mismatch was associated with a lower probability of neutrophil and platelet recovery, and, in the latter study, a higher probability of acute GVHD and lower probability of survival [31,32]. A subsequent NYBC analysis of 607 UCB transplants found that the degree of HLA mismatch correlated directly with the probability of NRM [33]. Particularly among patients who did not develop acute GVHD, HLA mismatch was associated with high risk of death from infection, implying a potential negative affect on immune reconstitution. The Minnesota group study of 152 UCB transplants showed that survival after transplant of an UCB unit matched at four of six loci was significantly worse compared with those matched at five or six loci. Together with the NYBC study, the results indicated that the step down from five or six of six loci-matched to four of six loci-matched UCB results in about a 2–2.4-fold increase in mortality (Fig. 48.1(b)) [34]. In direct contrast, the subsequent Eurocord report, which analyzed 550 patients, confirmed the association of HLA mismatch with a probability of neutrophil engraftment and acute GVHD grades III–IV, but also showed an association with lower probability of relapse and thus no apparent affect on survival [35]. While the high replication activity of cells within UCB allows the transplant of a hematopoietic stem cell dose that otherwise would be considered unacceptable in a marrow graft, the cell dose of a UCB unit

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is a critical factor in determining success. The importance of cell dose was observed in the first large studies, and correlated with probability of neutrophil engraftment and platelet recovery [31,32]. Based on the Eurocord results, the lower limit of an acceptable unit generally has been considered to be around 2 × 107 TNCs/kg recipient weight, as determined by TNCs in the unit before cryopreservation [36]. In acknowledgment of the importance of cell dose, UCB banks subsequently made efforts to improve the collection volumes. Until recently, difficulty in the identification of sufficiently sized units has restricted UCB transplants to smaller patients; hence, most subsequent studies have been performed in pediatric patients. These studies have confirmed the association of cell dose and engraftment, and in the most recent Eurocord analysis that included 550 UCB transplants, TNC count was found to be associated with risk for acute GVHD [35]. Together, these studies support a minimum TNC dose of approximately 2 × 107/kg recipient weight. Further, stepwise increases in TNC dose appear to be correlated with reduction in NRM, and there does not appear to be an upper limit over which TNC dose seems detrimental for survival [37]. The best method to measure UCB progenitor cell dose for clinical use has not been established. Several studies show superior predictive value using CD34+ cell dose [34,38]. In the Minnesota studies, the number of CD34+ cells/kg recipient weight was associated not only with graft recovery, but also with NRM and survival. Some studies have found that the doses of TNC and CD34+ measured after thawing may have superior predictive value compared with values obtained before unit cryopreservation [33]. Post-thaw assays have no practical value for unit selection, but this may be relevant to optimal methodology as some studies suggest that, when comparing cell dose before and after cryopreservation, there is a considerable fall-off in TNCs, whereas CD34+ cell numbers remain relatively stable. Hence, the dose of CD34+ obtained before cryopreservation may be a more accurate depiction of the post-thaw progenitor cell content. Unfortunately, however, the CD34+ cell count appears to be less consistent between laboratories, compared with TNC count; thus, with respect to unit selection, the TNC count may improve ability to compare the cell dose of units stored in separate banks. The lower limit of CD34+ cell dose has not been established firmly, but generally a unit with fewer than 1.7 × 105 CD34+ cells/kg recipient weight has been considered inadequate [34,38]. Other investigators have suggested that graft progenitor cell count, measured by colony-forming cell assay, is a better predictor of engraftment, although no association with survival has been suggested [36]. Current data support the utilization of either TNC or CD34+ cell dose as measure of unit suitability. The selection of an optimum UCB unit must take into account both cell dose and HLA match, and there continues to be debate about which factor, if either, should be considered more important. After the recognition of the importance of cell dose, most UCB banks have improved collection methods, consequently in the future HLA match may become the predominant selection criteria. Until then, most centers have established an algorithm for cord blood selection that prioritizes the best HLA match among units that have a cell dose above the acceptable minimum level, either TNCs of over 2.0–2.5 × 107 or CD34+ cells over 1.7 × 105/kg recipient weight. This approach may be too simplistic, since the largest studies have shown an interaction between cell dose and HLA mismatch, such that the negative effects of HLA mismatch may be overcome to a certain extent by higher cell dose [33]. Hence a more refined algorithm considers the importance of both HLA match and cell dose, such that the minimum TNC limit of 2.0–2.5 × 107/kg recipient weight is acceptable for UCB units matched for five or six of six loci, while a dose of 4.0–5.0 × 107/kg recipient weight is preferred for four of six HLA-matched units [33]. Consideration of cell dose in addition to HLA match has the most profound implications for adults. While it is now possible to identify an

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Table 48.3 Scheme for selection of double cord units Each unit must contain a minimum of 1.5 × 107 TNCs/kg Each unit must be matched with the recipient for at least 4 of 6 HLA loci First unit: select the unit with the least HLA disparity with the recipient Determine need for second unit based upon the algorithm: Match grade TNC dose TNC dose <3.0 × 107/kg 6/6 ≥3.0 × 107/kg <4.0 × 107/kg 5/6 ≥4.0 × 107/kg 4/6 None Any 5. Match second unit to the first unit for 4 of 6 HLA loci

1. 2. 3. 4.

HLA, human leukocyte antigen; TNC, total nucleated cell.

UCB unit matched for four of six loci for almost all patients, the number of usable units decreases markedly when cell dose is considered [39]. Indeed, the multicenter Cord Blood Transplantation (COBLT) study found lower than 10% survival in the adult arm of the protocol, representative of the fact that most received UCB units with multiple HLA mismatches and low cell dose, given that the median TNC count was 2.3 × 107/kg recipient weight [29]. These difficulties have prompted exploration of methods to enhance cell dose, such as transplantation of more than one UCB unit or expansion of UCB progenitor cells. Single-center studies of double UCB units in adult recipients suggest improved DFS (discussed in detail in Chapter 39). One such study of 21 adult patients found that a second UCB unit could increase the median TNC count to 4.0 × 106/kg recipient weight, higher than that reported for single units in the adult patient arm of the COBLT study, consistent with the hypothesis that higher cell dose improved outcome [40,41]. The observation that only one of the two UCB units engrafts long term suggests a supportive role for the additional unit. Algorithms to aid in multiple unit selection have been devised, based upon HLA match and cell dose of each unit. One reasonable algorithm for the selection of two UCB units, based on the Minnesota experience, is shown in Table 48.3. Without large numbers of patients to analyze, ambiguity remains regarding the allowable minimum cell dose, HLA mismatch with recipient, and unit-to-unit HLA matching. Reports of supporting single UCB transplant with third-party granulocyte-colony-stimulating factor-mobilized PBPCs suggest that HLA matching between cell products may not be relevant [42]. Much more important appears to be the degree of HLA match of the recipient to each UCB unit, since either may become the engrafting unit [41]. Recipient sensitization to donor alloantigen is more difficult to assess prior to UCB transplant, since donor cells are not available for crossmatch assays. A reasonable approach is to screen recipients for HLA antibodies by PRA assay, discussed in the previous section. Recipients with a positive PRA can be tested further for HLA antibody specificity. If it is determined that the patient has an antibody to a specific HLA antigen, this information may provide guidance for unit identification. If a recipient antibody to a specific HLA-A, B or DR antigen is identified, it is prudent to avoid units with this specific HLA antigen whenever possible. If, however, the recipient antibody is directed to a specific HLA-C, DQ or DP antigen, it will require additional HLA typing of the unit, which may not be possible for units without attached segments. While it is reasonable to avoid, whenever possible, units which express HLA antigens for which the recipient is sensitive, there have not been sufficient studies to determine the true relevance of recipient sensitization to outcome of UCB transplants.

Recipient A

B

A*0201 *2401 Rejection B*4402 *4402 DRB1*0401 *0103

1 HLA mismatched donor GVHD

A*0201 *2401 Rejection B*4402 *4402 DRB1*0401 *0103

C

A*0201 *2401 B*4402 *4402 DRB1*0401 *0103

A*0201 *2401 B*4402 *4402 DRB1*1201 *0103 A*0201 *2401 B*4001 *4402 DRB1*0401 *0103

GVHD

A*0201 *0201 B*4402 *4402 DRB1*0401 *0103

Fig. 48.2 The vector for graft-versus-host disease (GVHD) or graft rejection depends upon homozygosity of the mismatched human leukocyte antigen (HLA) locus. Shown on the left is the HLA typing of a potential recipient homozygous for HLA-B*4402. On the right are shown the HLA typing of three potential donors mismatched for a single HLA antigen. In panel A, both recipient and donor are heterozygous for the mismatched HLA antigen (DRB1*0401 versus DRB1*1201); therefore, the mismatch will generate alloreactivity in both the GVHD and the rejection vectors. In panel B, the recipient is homozygous at the mismatched HLA locus (B*4402 versus B*4001); therefore, the mismatch will generate alloreactivity only in the direction of rejection. In panel C, the donor is homozygous at the mismatched HLA locus (A*0201 versus A*2401); therefore, the mismatch will generate alloreactivity only in the direction of GVHD. (Data from [17].)

Selection of partially matched related donors A haploidentical or partially matched related donor (PMRD) is defined as sharing one distinct inherited haplotype (genetically identical) with the patient; the unshared haplotype may be HLA matched (phenotypically identical) or mismatched at one or more HLA loci. Most patients have a PMRD available, typically a parent or sibling. An in-depth discussion of HCT from PMRDs can be found in Chapter 46. With respect to donor selection, the limit of tolerable HLA mismatches has not been defined for transplantation of unmanipulated PMRD grafts, because studies that analyze large numbers of pairs have not employed highresolution HLA typing. Published studies have examined outcome risks associated with HLA mismatch defined by serologic typing for HLA-A and B plus identity for DRB1 alleles. The contribution of mismatching at HLA-C or DQB1, or of allele-level mismatching at HLA-A or B, thus is unknown. Nonetheless, these studies provide some guidance for selection of PMRDs. An analysis of 1199 patients given marrow grafts donated from related donors found a sixfold increase in graft failure among recipients of grafts from PMRDs mismatched for zero to three HLA-A, B, and DRB1 antigens of the nonshared haplotype compared with those who received HLA-identical sibling (genetically HLA-identical) grafts [17]. Further, the number of HLA disparities in the PMRD grafts related significantly to the incidence of graft failure, which occurred in 7% of zero-locus-incompatible, 9% of one-locus-incompatible, and 21% of two-locus-incompatible transplants. The relative disparity between donor and host histocompatibility antigens, determined by the vectors of HLA incompatibility, also affects engraftment (Fig. 48.2). Recipients homozygous at one or more mismatched loci had a threefold increase in graft failure compared with heterozygous recipients. Thus the situation in which host-versus-graft vectors are not counterbalanced by equivalent graft-versus-host vectors should be considered less desirable [43].

Donor Selection for Hematopoietic Cell Transplantation

Donor HLA disparity also correlates significantly with the risk for acute GVHD, such that disparity of multiple HLA loci results in a prohibitive incidence of GVHD if the graft is not depleted of T cells [44,45]. Several studies report significantly lower DFS among recipients of grafts mismatched for two or three HLA loci compared with those mismatched for zero or one HLA locus. An EBMT analysis of 121 PMRD transplants compared outcome for recipients of grafts mismatched at zero or one HLA to those mismatched for two or three HLAs, and found that recipients of zero or one HLA mismatch had significantly improved leukemia-free survival (p = 0.03). When disease phase was considered, however, there was no difference between patients with early-stage disease receiving two- or three-HLA-mismatched grafts compared with patients with late-stage disease and better HLA match [46]. These studies, together with knowledge gained from analyses of URD transplants, indicate that high-resolution typing will help discriminate PMRD potentially mismatched for zero or one HLA-A, B, C, DRB1 or DQB1 locus. Until further information is gained, risk assessment of HLA matching among PMRDs should be similar to that discussed above for URD selection, particularly if the graft will not be depleted of T cells. PMRDs with more than one HLA mismatch may be the only available donor for some patients. The immunologic capacity of the donor graft for overcoming residual host immunity is of primary significance in determining engraftment in the PMRD setting. When T cells are depleted from the donor graft in an effort to prevent severe GVHD, rates of graft failure increase significantly, at least fivefold for recipients of genotypically HLA-identical grafts, and approximately twofold for recipients of histoincompatible marrow grafts [44]. Considerable effort investigating the mechanisms of hematopoietic cell engraftment resulted in the current understanding of the important role of hematopoietic stem cell dose. Early murine models demonstrated that engraftment of allogeneic marrow required a 10-fold higher number of cells compared with syngeneic marrow [47]. Similar models showed that host alloreactivity conferred by the presence of residual (or experimentally added-back) host T cells could be overcome by increasing the number of donor cells, theoretically by manipulating competition for marrow space in favor of the donor stem cells [48]. In histoincompatible models, engraftment of T-cell-depleted marrow was shown to require greater numbers of donor marrow cells compared with non-T-celldepleted grafts [49,50]. Stem cell dose also appeared to be the critical factor in determining engraftment of T-cell-depleted histoincompatible marrow when intensity of immunosuppression was held constant [51]. In this experiment, mice irradiated at sublethal doses and transplanted with PMRD marrow were highly resistant to engraftment as a consequence of residual host immunity and challenge from strong alloantigenic stimuli. High cell doses of T-cell-depleted marrow resulted in stable donor chimerism and appeared to induce tolerance to donor alloantigen. These studies provide support for the hypothesis that CD34+ cells have veto activity, capable of neutralizing donor cytotoxic T cells directed against alloantigen [52]. The use of mobilized PBSCs to attain high cell dose was reported by Aversa and colleagues [53,54]. After two to four apheresis procedures followed by CD34+ selection or T-cell depletions, the graft contained a median CD34+ cell dose of 13.9–16 × 106/kg recipient weight and a median CD3+ cell dose of 0.27–1.43 × 105/kg recipient weight. The subsequent study confirmed the ability to transplant high CD34+ cell doses resulting in 95% engraftment rate following a high-intensity regimen. Correlation of CD34+ cell dose with risk for graft failure has been confirmed by other groups after different conditioning regimens [55]. The requirement for maximal CD34+ cell dose guides donor selection toward a preference for adult donors able to tolerate multiple apheresis procedures. In contrast, if a marrow graft is planned instead of PBPC graft, studies suggest that younger donor age may be preferable [56].

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Studies by the Perugia group suggest that, in the absence of T cells, NK-cell alloactivity plays an important role in outcome of PMRD grafts. NK cells are essential effector cells of the innate immune system that, without prior activation, recognize and lyse target cells. The biology of NK cells is reviewed comprehensively in Chapter 13. In brief, NK-cell cytolytic activity is regulated by inhibitory and activating signals generated by the binding of NK-cell surface receptors, the KIRs. Negative regulation occurs when inhibitory KIRs bind to specific HLA class I molecules; hence, target cells expressing the appropriate HLA class I molecules are protected from NK-cell cytolysis, a mechanism termed “missing self” [57], reviewed in [58] and in Chapter 46. In the setting of allogeneic HCT, NK-cell alloreactivity arises in the situation where the recipient lacks the inhibitory ligand for donor KIRs. A list of KIRs and their associated ligands can be found in Chapter 46. Class I HLA epitopes involved in NK-cell allorecognition include the Bw4 epitope, present on approximately 40% of HLA-B alleles, and the allelic HLA-C1 and C2 epitopes, one of which is present on all HLA haplotypes and which have approximately equal frequencies [59,60]. The existence of potential donor alloreactive NK cells can be deduced based upon a comparison of donor and recipient HLA class I type. The “missing self” mechanism for NK alloreactivity rests on the direct identification of NK clones in the donor capable of activation provided the recipient lacks the inhibitory ligand. Such a description of NK-cell alloreactivity is an oversimplification of NK-cell receptor–ligand biology, as some individuals do not have the inhibitory KIR gene anticipated based on the HLA typing. KIR genes also include activating receptors, and NK cells express a variety of other classes of surface activating and inhibitory receptors. Velardi and colleagues screened the KIR genotype of 162 patients and found that 97% bore KIR2DL1 (receptor for HLA-C group 2 alleles), 100% expressed KIR2DL2/3 (receptors for HLA-C group 1 alleles), and 94% bore KIR3DL1 (receptor for HLA-Bw4 alleles). Thus, prediction of NK alloreactivity based solely on KIR ligand incompatibility would be invalid for 3% of donors who do not possess the KIR2DL1 receptor for group 2 HLA-C alleles, and 6% of donors who lack the gene for the HLA-Bw4 inhibitory receptor KIR3DL1 [61–64]. This group also showed that direct identification of NK alloreactive clones in the donor was useful for optimum donor selection. Taken together, these results indicate that selection of the optimal NK alloactive PRMDs should be based upon combined information gained from the class I HLA typing of donor and recipient, the genetics of inhibitory KIRs of the donor, and, in some instances, the functional assessment by analysis for donor NK alloreactive clones. Testing of extended family members may uncover a suitable NK alloreactive donor when none has been identified within the immediate family. Selection of an NK alloreactive donor is discussed fully in Chapter 46. Potentially relevant to the selection of PMRDs is the “missing ligand” mechanism for donor NK activity, which takes into consideration that alloreactive donor NK clones may develop after HCT provided the recipient lacks at least one KIR ligand. The mechanism for “missing ligand”-induced NK alloreactivity is discussed fully in Chapter 46. In contrast to the mechanism of the “missing self,” there is no requirement for a mismatch of the class I HLA ligand between donor and recipient. Hence, the “missing ligand” mechanism potentially would encompass two-thirds of recipients, who will lack one or more of the class I HLA ligands for KIRs. In this model, KIR genotyping is vital to identify a donor with potential to express an alloreactive KIR. Potential NK alloreactive donors are listed in Table 48.4, according to both mechanisms for “missing self” and for “missing ligand.” The Perugia group demonstrated the role for NK alloreactivity in protecting against relapse after T-cell-depleted PMRD grafts [61,65]. Among 92 PMRD graft recipients analyzed, significantly lower rates of

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Table 48.4 Selection of an “NK alloreactive” donor Mechanism

Missing self

Missing ligand

Recipient HLA typing allele group: HLA-C group 1, HLA-C group 2, and HLA-Bw4 HLA-C group 1 and HLA-C group 2 HLA-C group 1 and HLA-Bw4 HLA-C group 2 and HLA-Bw4 HLA-C group 1 HLA-C group 2 HLA-Bw4

NK alloreactive donor must express:* No alloreactive donor possible Bw4-related alleles HLA-C group 2-related alleles HLA-C group 1-related alleles HLA-C group 2 or HLA-Bw4-related alleles HLA-C group 2 or HLA-Bw4-related alleles HLA-C group 1 or HLA-C group 2-related alleles

Genotype of KIR inhibitory receptor: No alloreactive donor possible KIR2DL1 KIR2DL2/3 KIR2DL1 or KIR3DL1 KIR2DL2/3 or KIR3DL1 KIR2DL1 or KIR2DL2/3

HLA, human leukocyte antigen; KIR, killer immunoglobulin-like receptor; NK, natural killer cell. * Since 3% of individuals do not possess the KIR2DL1 gene, the combination of a KIR2DL1-negative donor and a recipient without HLA-C group 2 alleles could result in a 1.5% incidence of false positivity. KIR2DL1 gene typing of the donor may be necessary to assess the NK alloreactive potential of this combination. In HLA-Bw4 mismatches, even when the KIR3DL1 gene is present in the donor (around 90% of individuals), NK repertoire studies show that alloreactive NK clones are not detectable in about one-third of individuals. Thus, for HLA-Bw4 mismatches, functional assessment of the donor NK repertoire is necessary. Data from [64].

rejection and GVHD were found among the KIR epitope-mismatched graft, and among AML patients there was a sevenfold increase in DFS (p < 0.009). Subsequently, the Perugia group analyzed the effect of NK alloreactivity in 112 patients with AML [65]. Before transplant, NK alloreactive clones were detected in all KIR epitope-mismatched donors whose recipients did not express HLA-C group ligands, and two-thirds whose recipients did not express HLA-Bw4 alleles, whereas none were detected in donors whose recipients expressed the class I HLA groups present in the donor. Multivariate analysis confirmed that donor versus host KIR “ligand mismatch” was an independent factor for survival, associated with a twofold reduction in death or relapse among all patients (p < 0.001), including those with relapsed disease at time of HCT (DFS 30% versus 6%; p = 0.04). In contrast, when the analysis took into consideration patients who lacked expression of at least one KIR ligand, in which there was potential for NK alloreactivity according to the mechanism for “missing ligand,” no survival advantage was discerned. While these results have not been confirmed by others [66], they suggest that PMRD selection should consider extension of the search for a donor beyond immediate family members, guided by KIR genotyping [65]. These results also have implications for URD transplants, discussed previously, and suggest that KIR genotyping will be necessary to donor selection. Investigators have speculated that exposure to noninherited alloantigen during pregnancy might induce a level of tolerance that could be exploited in the selection of PMRDs. Indeed, the long-term presence of very small numbers of fetal cells has been detected in about 80% of mothers, and maternal cells detected in about 65% of offspring, consistent with transference of maternofetal tolerance. Recent studies have sought to determine the permissibility of PMRD grafts when the mismatched haplotype comprises the noninherited maternal antigens (NIMAs), exposed to the recipient in utero (Fig. 48.3) [67]. Ichinohe reported 35 patients with hematologic malignancy given PMRD T-cellreplete grafts mismatched for two or three HLA A, B, and DR antigens. Donors were a mother, a child or in some cases a sibling in whom maternal microchimerism was detected. Overall, the incidence of GVHD grades III–IV was 24%, lower than expected, and HCT from siblings or offspring donors was associated with the lowest risk of GVHD (p = 0.04). The concept of NIMA mismatching was supported further by a retrospective International Bone Marrow Transplant Registry (IBMTR) study that included 269 patients given T-cell-replete grafts and 50 given T-celldepleted grafts from PMRDs mismatched for one or two HLA-A, B, and

Fig. 48.3 A scheme of three different types of noninherited maternal antigen (NIMA)-complementary human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplantation (HCT). (A) HCT from mother to offspring: a graft-versus-host reaction is directed against the inherited paternal HLA antigens (IPAs), while a host-versus-graft reaction is directed against the NIMAs of offspring 1 (Mb). (B) HCT from offspring to mother: a graft-versus-host reaction is directed against the NIMAs of offspring 2 (Ma), and a host-versus-graft reaction is directed against IPAs. (C) Stem cell transplantation between NIMAmismatched siblings who shared the inherited paternal HLA haplotype. These siblings are bidirectionally mismatched for NIMAs in both graft-versus-host and host-versus-graft directions. (This research was originally published in Blood. Ichinohe et al. Feasibility of HLA-haploidentical hematopoietic stem cell transplantation between noninherited maternal antigen (NIMA)-mismatched family members linked with long-term fetomaternal microchimerism. Blood 2004; 104: 3821–8 [67]. © the American Society of Hematology.).

Donor Selection for Hematopoietic Cell Transplantation

DRB1 antigens [68]. The multivariate analysis showed that, compared with sibling donors mismatched for NIMAs, both parental donors and sibling donors mismatched for noninherited paternal antigens had significantly higher risk for acute GVHD grades II–IV, and that parental donors had significantly higher risk for chronic GVHD and NRM. These results are preliminary, and should not yet be used to guide donor selection when planning for T-cell-replete grafting. However, they support the hypothesis that exposure to NIMAs may confer long-term tolerance among haploidentical sibling donors.

Selection among donor types Criteria used to select the optimal URD, UCB or PMRD have been derived from multivariate analyses of large numbers of transplants. In contrast, the question of whether one donor source should be preferred over another can only be answered by randomized studies, of which there are currently none. Studies that seek to compare outcome between any donor type, whether HLA-matched sibling, HLA-matched URD, UCB or PMRD, have been of a retrospective nature. Consideration of the results from retrospective studies must take into account the problem of selection bias, wherein poor-risk patients die before HCT can be performed. In general, the time lapse between the decision to undergo HCT and donor identification is greater for recipients of alternative donor grafts, increasing the probability that poor-risk patients will not be included in these groups. In counterbalance, the perception of an increased risk associated with alternative donor HCT may compel physicians to withhold referral of patients until the disease has progressed to an advanced stage. Finally, except for HLA-matched siblings, retrospective studies have defined HLA matching based upon serologic typing for HLA-A, B, and DRB1 allele typing. Thus, when analyzing comparative studies of URD transplants, it should be recognized that approximately 50% of URDs defined as “six of six” HLA matched actually will be mismatched for one or more HLA alleles, which would be detected using the current standards for HLA typing. The best understanding of the effect of donor source comes from large retrospective analyses of registry data. The strength of registry studies is the capability of including large numbers of transplants, providing greater statistical power to detect differences among recipient groups. Several studies have compared the outcome of alternative donor grafts with that of HLA-matched sibling donor grafts. An IBMTR study examined the outcome of 2055 transplants for treatment of hematologic malignancies, which included grafts from 1224 HLA-identical sibling donors, 340 PMRDs, and 491 URDs [69]. Compared with the recipients of HLA-identical sibling grafts, the recipients of alternative donors had significantly worse performance scores and more advanced disease, among other differences. The multivariate analysis found that NRM was significantly higher for recipients of alternative donors compared with HLA-matched sibling donors, and for HLA-mismatched donors compared with HLA-matched URDs. In contrast, the incidence of relapse was significantly lower for recipients of alternative donors compared with HLA-matched sibling donors. There was no difference in DFS with any donor type for patients with advanced leukemia, whereas DFS was higher for patients with early- or intermediate-phase disease given HLAmatched sibling grafts compared with alternative donor grafts, and for those given HLA-matched URD grafts or one-HLA-mismatched related donor compared with other HLA-mismatched grafts. Other variables significantly associated with improved survival included younger age, lower white blood cell count at diagnosis, more than 2 years’ duration between the diagnosis of CML and HCT, less-intense conditioning regimens, and T-cell depletion. Weisdorf and colleagues compared outcome for patients with CML who received either HLA-matched sibling donor (n = 450), six of six

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HLA-matched URD (n = 2062) or partially matched URD (n = 402) grafts using data from the NMDP database [70]. Significant differences between groups included, among others, longer time from diagnosis to transplant, younger age, and a higher proportion with advanced disease phase for the URD group. Multivariate analysis found the risk of death or relapse to be approximately 1.8-fold greater for recipients of HLAmatched URD compared with HLA-matched sibling donor grafts, independent of disease phase, also significant. An important caveat to the application of these findings is the inability to assess the relative contribution of HLA allele matching, which has become standard using current HLA-typing technologies, particularly for the URD groups. Registry studies also provide comparative information about outcome among different alternative donor groups. The Eurocord group reported separately outcome for pediatric and adult patients with acute leukemia given unrelated UCB compared with URD marrow transplants. The pediatric study analyzed 99 unrelated UCB and 262 URD transplants reported consecutively from 1994 through 1998 [71]. Again, significant differences were found between groups, including younger age, more advanced disease, and shorter first remission duration among the UCB group, as well as differences in transplant regimens. These differences, among others, were used to adjust comparisons between groups, which detected lower DFS and a twofold increase in NRM among the UCB compared with URD recipients (p < 0.01). Predominantly, the differences in adjusted outcomes appeared within the first 100 days after HCT. In contrast, no difference in survival was reported for adult patients in the Eurocord comparison of 98 single UCB unit recipients to 584 recipients of six of six HLA-matched URD marrow grafts, reported to the registry between 1998 and 2002 [72]. Different results were found in an analysis of adults given single-unit UCB (n = 150) compared with those given six of six HLA-matched URD (n = 367) or one-antigenmismatched URD (n = 83) T-cell-replete marrow grafts reported to the IBMTR. The UCB group was younger and smaller, and included a higher percentage of non-Caucasian ethnicity, a lower percentage of patients with CML, and a greater percentage of advanced disease phase, among other significant differences. Compared with recipients of HLAmatched URD marrow, recipients of UCB or mismatched URD marrow had a higher risk for death from any cause (HR 1.66, 1.53, respectively; p < 0.001) [73]. Retrospective analyses generated from single-center experiences also contribute to our understanding of outcome differences. The advantage of a single-center study is the tendency toward less variability in patient selection, transplant regimen or supportive care between the comparative groups. Most single-center studies lack sufficient numbers of patients, such that true differences in outcome endpoints may not be detected. The Milwaukee group compared outcome for the recipients of six of six HLA-matched URD (n = 81), five of six HLA-matched URD (n = 58), and PMRD (n = 48) grafts given the same preparative regimen [74]. Survival was significantly better for recipients of HLA-matched URD (58%) compared with mismatched URD (34%) or PMRD (21%) transplants (p = 0.002) The Essen group also compared outcome for recipients of HLA-matched sibling donor (n 004 138) versus HLAmismatched related (n = 86) and 10 of 10 HLA-matched unrelated (n = 101) grafts following a standard regimen for treatment of hematologic malignancies [75]. A significantly higher proportion of patients with alternative donors had more advanced disease compared with those with HLA-matched sibling donor grafts (p = 0.001). In contrast to the previous study, while survival was correlated with advanced disease and older recipient age, there was no association of donor type with survival. Recipients of alternative donor grafts had approximately twofold higher risk for GVHD (p = 0.03) and twofold lower risk for relapse (p = 0.01). The Seattle group compared the outcome of consecutively treated pediatric patients with acute lymphoblastic leukemia given URD (n = 78) or

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HLA-identical sibling donor (n = 74) grafts, and found no difference in DFS (unpublished data) (Fig. 48.4). Single-center studies that compared UCB recipients generally could not control for differences in the transplant regimen, although other dif-

ferences between recipients have been limited. The Tokyo group compared consecutively treated adult patients given unrelated UCB (n = 100) with those given grafts from related donors (n = 71), of whom 17 were mismatched for one or two HLA-A, B or DRB1 antigens. No significant difference was found between UCB and related donor HCT groups with respect to DFS [76]. The Minnesota group reported a matched-pair analysis of pediatric patients given UCB or URD grafts. Survival and NRM for 26 UCB recipients were no different than for recipients of unmanipulated URD grafts [77]. A larger study of 60 UCB recipients comparing outcome against 52 URD marrow recipients, matched for five or six of six HLA-A, B, and DRB1 antigens, also did not show significant differences in survival or NRM [78]. In all studies of alternative donor HCT, phase of disease has been found to be the most important factor predictive of outcome [1,35,46,79,80]. Thus, the urgency for transplant should be considered an independent variable that must be weighed against optimization of donor selection. As an illustration of the counterbalance between time to transplant and donor optimization, patients with chronic-phase CML given a 10 of 10 HLA-matched URD graft more than 1 year after diagnosis had no better chance of survival than those given a nine of 10 HLA matched URD graft within 1 year of diagnosis [8]. Disease phase appears to be the overwhelming determinant of outcome for patients with acute leukemia [1,2]. In general, DFS according to phase of acute leukemia at time of transplant ranges approximately around 40–60% for first remission, 25–50% for second remission, and less than 10% for relapse. Patients with the highest leukemia burden, reflected by having circulating blasts or a high percentage of blasts in the marrow, have very little

Percent leukemia free

100

80

CR1 (p = 0.93)

60 CR2 (p = 0.80) 40

20 REL (p = 0.20) 0 0

2

6

4

8

10

Years fom transplant

Fig. 48.4 Disease-free survival after hematopoietic cell transplantation (HCT) for treatment of acute lymphoblastic leukemia in children according to phase of disease at time of transplant. Outcome is shown for recipients of human leukocyte antigen-matched sibling donor (n = 74, dashed line) and URD (n = 78, solid line) grafts in first complete remission (CR1), second complete remission (CR2) or relapse (REL) at time of HCT. The p-value compares the outcome between donor types at each phase of disease.

HLA type patient and family

Initiation of search to donor selection process

1–2 weeks

Consent and financial clearance 1–2 weeks

3–5 weeks

Initial donor selection 1 week

Donor selection process to HCT

URD 8–10 HLA allele typing reported 4–6 weeks

PRMD immediate

typing reported 1–2 weeks DRB1 allele type selected UCB

PRMD 1–3 weeks UCB 2–6 weeks

UCB DRB1 allele

KIR genotype Donor NK assays 1–2 weeks

2–4 weeks HLA allele type selected URD 4–8 weeks

URD 4–14 weeks

Fig. 48.5 Schema of the search process for alternative donors (see text for abbreviations). The left-hand panel summarizes the typical search times required to get a patient to HCT with an alternative donor. The right-hand illustration shows the specific steps involved in the alternative donor search. Regardless of donor type, the first stage in the search process includes all HLA typing, documentation of consent for search, and financial clearance. The time required for the first stage varies according to the different HLA typing laboratories and transplant centers, and generally takes about 3–4 weeks. Subsequently, the time required for donor selection depends on the type of donor and the extent of HLA typing information available from the registry. In general, a PRMD will be identified immediately from the initial HLA typing report of the patient and his or her family. However, a delay of several weeks will be necessary if optimization for NK alloreactivity is considered. Suitable UCB also may be identified quickly from the initial registry search, but often the unit of interest lacks DRB1 allele typing. In this case, a delay will result while DRB1 allele typing is obtained, depending upon the individual cord blood banks. Finally, a suitable URD may be identified quickly from the initial registry search. However, even in the best case where allele-level HLA typing is available through the registry, there will be an obligate time before HCT in order for the donor to be evaluated and determined to be medically fit for donation. In many cases, however, complete allelelevel HLA-typing will need to be requested, which may add several more weeks to the process of donor selection.

Donor Selection for Hematopoietic Cell Transplantation Table 48.5 Algorithm for donor selection Standard timeline 1. Initial search parameter

䊏 HLA-A, B, C, DRB1, DQB1 10 of 10 allele match 䊏 HLA-A, B, C 9 of 10 allele match 2. Review patient status and search results every 8 weeks; upgrade to urgent timeline if indicated Urgent timeline (transplant goal ≤3–4 months) 1. Initial search parameter 䊏 HLA-A, B, C, DRB1, DQB1 10 of 10 or 9 of 10 allele match 䊏 Isolated HLA-DQB1 mismatch (allele or antigen) 䊏 Isolated HLA-A, B, C antigen mismatch 䊏 HLA-DRB1 allele mismatch with or without HLA-DQB1 mismatch 2. Review patient status and search results at 4 weeks; upgrade to critical timeline if no donor is identified Critical timeline (transplant goal ≤1 month) 1. Prioritize first available: 䊏 HLA-A, B, C, DRB1, DQB1 10 of 10 or 9 of 10 allele match 䊏 Isolated HLA-DQB1 mismatch (allele or antigen) 䊏 Isolated HLA-A, B, C antigen mismatch 䊏 HLA-DRB1 allele mismatch with or without HLA-DQB1 mismatch 䊏 Umbilical cord blood HLA-A, B, and DRB1 4–6 of 6 matches with ≥2.0 × 107 nucleated cells/kg recipient weight 䊏 Haploidentical donors: HLA-allele type parents, siblings, children or extended family members 䊏 Unrelated donor: HLA-A, B, C mismatch involving two separate loci 2. Review within 3–4 weeks or as soon as a donor is identified. HLA, human leukocyte antigen.

chance for long-term survival [1]. Consensus as to donor selection, which balances disease risk with donor optimization, has been put forth by a number of authors [81–83]. Accordingly, selection of the optimal donor is most accurately defined as selection of the best donor available within the timeframe specified by the risk of disease progression. Pragmatically, the first step in donor selection should be an assessment of the risk for disease progression or relapse. Clearly, disease risk depends upon the current available therapies, so over time the risk for progression may change. The best illustration is the impact of recently developed tyrosine kinase inhibitor therapy on the risk for progression of CML: whereas previous analyses showed that HCT within 1 year of diagnosis correlated with improved outcome

701

[3], this conclusion likely cannot be applied to patients who achieve cytogenetic remission with a tyrosine kinase inhibitor therapy. Whenever possible, risk assessment should include information from assays of biologic markers of disease; for example, detection of minimal residual disease in a patient with acute leukemia should prompt a decision in favor of an alternative or less “optimal” donor rather than further delay [84,85]. As soon as the risk of progression is estimated, a time limit for donor optimization should be determined. The Seattle approach classifies patients into three risk groups: critical (risk of disease relapse or progression within 1–2 months), urgent (risk of disease relapse or progression within 3–4 months), and standard. The strategy for donor identification will be shaped by the speed at which the search can be conducted at a particular center, the number of potential URDs in the donor registries, and the level of HLA typing in the registry report. Figure 48.5 illustrates a typical donor search timeline. The initial phase, which includes HLA allele-level typing of the recipient, patient consent for donor search, and financial clearance, often takes 1–4 weeks away from the functional process of donor selection. The time from screening the donor registry typing to donor identification depends heavily on the level of HLA typing reported by the registry: DRB1-typed UCBs or HLA allele-level-typed URDs will be quickly identified, whereas incompletely typed donors will slow down the process to the extent that donor sample must be obtained for additional HLA typing. Finally, when few potential allele-matched URDs exist in the registries, the search should be broadened early in the process to include potential UCB or PRMD grafts. A reasonable system for donor selection, which considers both disease risk and donor optimization, is outlined in Table 48.5. Periodic review of the status of both the patient and the donor search will ensure that the timeline remains consistent with the risk for disease progression.

Conclusion and future directions Many patients given allogeneic HCT will receive a graft from an alternative donor. Survival rates after URD grafts have improved steadily, in part due to improved donor selection criteria based on current understanding of the role of HLA allele-level matching. The rapid progress in improving outcomes of UCB and PRMD grafts has rested as well on the ability to optimize donor characteristics. Studies currently in progress worldwide address important questions about the roles of HLA haplotype or KIR matching in URD HCT, and the roles of crossmatch studies and extended HLA typing for UCB grafts. Most importantly, advances in alternative donor HCT procedures currently assure the option of HCT for most patients. The urgency of the transplant procedure should dictate donor selection as the best HLA match available within the specified timeframe. Randomized studies comparing the outcome of UCB with URD HCT have been proposed, and hopefully in time comparing PRMD with URD HCT. While the complexities of study design and the difficulties of conducting clinical trials that randomize donor source are significant, these studies are the only means to determine the relevance of donor source, particularly important for patients with multiple options. For patients with limited donor options, future progress must entail improvements in alternative donor HCT procedures, including reduced intensity regimens, enhanced techniques for immune reconstitution, and tumor control.

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74. Drobyski WR, Klein J, Flomenberg N et al. Superior survival associated with transplantation of matched unrelated versus one-antigen-mismatched unrelated or highly human leukocyte antigendisparate haploidentical family donor marrow grafts for the treatment of hematologic malignancies: establishing a treatment algorithm for recipients of alternative donor grafts. Blood 2002; 99: 806–14. 75. Ottinger HD, Ferencik S, Beelen DW et al. Hematopoietic stem cell transplantation: contrasting the outcome of transplantations from HLA-identical siblings, partially HLA-mismatched related donors, and HLA-matched unrelated donors. Blood 2003; 102: 1131–7. 76. Takahashi S, Ooi J, Tomonari A et al. Comparative single-institute analysis of cord blood transplantation from unrelated donors with bone marrow or peripheral blood stem-cell transplants from related donors in adult patients with hematologic malignancies after myeloablative conditioning regimen. Blood 2007; 109: 1322–30. 77. Barker JN, Davies SM, DeFor T et al. Survival after transplantation of unrelated donor umbilical cord blood is comparable to that of human leukocyte antigen-matched unrelated donor bone marrow: results of a matched-pair analysis. Blood 2001; 97: 2957–61. 78. Barker JN, Hough RE, van Burik JA et al. Serious infections after unrelated donor transplantation in 136 children: impact of stem cell source. Biol Blood Marrow Transplant 2005; 11: 362–70. 79. Aversa F, Terenzi A, Tabilio A et al. Full haplotypemismatched hematopoietic stem-cell transplantation: a phase II study in patients with acute leukemia at high risk of relapse. J Clin Oncol 2005; 23: 3447–54. 80. Lu DP, Dong L, Wu T et al. Conditioning including antithymocyte globulin followed by unmanipulated HLA-mismatched/haploidentical blood and marrow transplantation can achieve comparable outcomes with HLA-identical sibling transplantation. Blood 2006; 107: 3065–73. 81. Grewal SS, Barker JN, Davies SM, Wagner JE. Unrelated donor hematopoietic cell transplantation: marrow or umbilical cord blood? [Review]. Blood 2003; 101: 4233–44. 82. Zuckerman T, Rowe JM. Alternative donor transplantation in acute myeloid leukemia: which source and when? [Review]. Curr Opin Hematol 2007; 14: 152–61. 83. Marks DI, Khattry N, Cummins M et al. Haploidentical stem cell transplantation for children with acute leukaemia. Br J Haematol 2006; 134: 196–201. 84. Bruggemann M, Raff T, Flohr T et al. Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia. Blood 2006; 107: 1116–23. 85. Zhou J, Goldwasser MA, Li A et al. Quantitative analysis of minimal residual disease predicts relapse in children with B-lineage acute lymphoblastic leukemia in DFCI ALL Consortium protocol 95-01. Blood Epub 2007 May 7. doi: 10.1182/blood-2006-09-045369.

Section 5 Hematopoietic Cell Transplantation for Acquired Diseases

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

49

George E. Georges & Rainer Storb

Hematopoietic Cell Transplantation for Aplastic Anemia

Epidemiology Aplastic anemia (AA) is a rare disease with an incidence of three cases per million per year in the United States and western Europe and about 15 cases per million per year in East Asia [1,2]. When treated with supportive care including blood transfusions and antibiotics, only about 28% of patients are alive 2 years after diagnosis [3]. More than 50% of patients with severe AA (SAA) may die within 6 months of diagnosis without definitive therapy [4]. Effective therapies for AA consist of either immunosuppressive therapy (IST) with agents such as antithymocyte globulin (ATG), or allogeneic hematopoietic cell transplantation (HCT).

Clinical description The disease is characterized by pancytopenia and marrow parenchyma that is lacking hematopoietic elements. Typically, the marrow cavities are filled with fat, and the aspirates show mainly lymphocytes, plasma cells, and fibroblasts. The differential diagnosis of a marrow aspirate with this appearance includes myelodysplastic syndrome (MDS), clonal T-cell disorders, and AA associated with paroxysmal nocturnal hemoglobinuria (PNH). One of the most difficult entities to distinguish from AA is hypocellular myelodysplasia (see Chapter 57), but the difference is important as the two disease entities are treated differently. The conditioning regimen for HCT for MDS often contains busulfan or total body irradiation (TBI) as for acute leukemias, whereas conditioning regimens without radiation are preferred for patients with AA (see below). Cytogenetic analysis of marrow is sometimes useful in distinguishing between hypocellular MDS and AA. Marrow cytogenetics are normal in AA in contrast to MDS, and, in contrast to hypocellular myelodysplasia, developing blood cells are usually normal in appearance. Occasionally, there may be dysplastic erythroid cells in AA [5]. However, the presence of dyshematopoiesis in nonerythroid cell lines is more consistent with MDS [6]. SAA is defined as marrow cellularity less than 25% with at least two of the following: 1 absolute neutrophil count less than 0.5 × 109/L; 2 platelet count less than 20 × 109/L; 3 absolute reticulocyte count less than 40 × 109/L [7].

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Very severe AA is defined by a neutrophil count less than 0.2 × 109/L and at least one other peripheral blood criterion of SAA and marrow features consistent with SAA [8].

Etiology AA may be the result of a variety of causes, including ionizing radiation, benzene, and chemotherapeutic agents [6,9,10]. Drugs causing idiosyncratic marrow injury include chloramphenicol, phenylbutazone, sulfonamides, gold, and anticonvulsants such as felbamate (reviewed in [5,6]). Rarely, AA is associated with viral diseases such as non-A, non-B, non-C hepatitis, parvovirus (pure red cell aplasia) or Epstein–Barr virus, and autoimmune disorders such as eosinophilic fasciitis [5,6]. Some cases of AA result from congenital or hereditary disorders, including Diamond–Blackfan anemia and Fanconi’s anemia. A diagnosis of dyskeratosis congenita may be apparent when typical clinical features of nail dystrophy, reticulate skin pigmentation and leukoplakia are present. Mutations in telomerase complex component (TERC and TERT) can be found in about 5% of adult patients with apparent acquired AA who lack the above clinical abnormalities and who typically show a lack of or poor response to IST (“cryptic dyskeratosis congenita”) [6,11,12]. In most patients, the disease etiology is unknown (idiopathic or primary AA).

Molecular and clinical biology Possible pathophysiologic mechanisms of AA include quantitative and/ or qualitative deficiencies of hematopoietic stem cells (HSCs), a defective marrow microenvironment, impairment of cellular interactions needed to sustain hematopoiesis, and immunologic suppression of marrow function. The result is a reduction in morphologically recognizable precursor cells in the marrow aspirate [13,14]. The variable response of SAA to therapy and the diverse evolution of the disease with time are perhaps a reflection of the multiple pathophysiologic mechanisms. The observation that the infusion of syngeneic marrow without prior immunosuppressive conditioning results in cure of the disease in approximately 50% of patients suggests that a defect in HSCs is a likely cause in these patients [15–17]. While most patients with AA have normally functioning stromal cells [15,16], there are a few in whom defects in the marrow microenvironment have been thought to have a role in the pathogenesis of their disease [18]. Recent evidence suggests that many cases of AA may be due to immune-mediated destruction of marrow. The immune attack leads to bone marrow failure. The responsiveness of AA to IST is evidence for the immune pathophysiology [13].

707

708

Chapter 49

Hematopoietic cell transplantation This chapter summarizes the results of marrow HCT for SAA over the past 30 years. Early reports of marrow HCT for SAA identified three major transplant-related problems: 1 graft rejection; 2 acute graft-versus-host disease (GVHD); 3 chronic GVHD [19–21]. The incidence of graft rejection has decreased in part because of the use of more effective immunosuppressive conditioning regimens, and in part because of changes in transfusion practices (see below). The incidence of acute GVHD has also decreased owing to improved GVHD prevention protocols. The incidence and mortality of chronic GVHD may also have declined slightly. These changes, in turn, have resulted in significant improvement in survival. With longer follow-up, late sequelae including impaired growth and development among children [22] and secondary malignancies [23] have become better understood. Syngeneic grafts Patients with syngeneic donors were among the first to receive marrow HCTs. Six of 12 patients in Seattle showed complete and sustained marrow recovery after HCT without preceding immunosuppression. In the other six patients, second marrow infusions were performed after conditioning with 200 mg/kg cyclophosphamide (CY). Ten of 12 patients were alive with an overall survival of 83% and follow-up ranging from 1.5 to 30 years [24]. The International Bone Marrow Transplant Registry (IBMTR) reviewed data on 40 syngeneic transplants worldwide. Twenty-three patients did not receive an initial conditioning regimen, and eight of the 23 had full hematologic recovery, while 15 required second transplants that were preceded by conditioning regimens. Twelve of 17 patients whose initial HCT was preceded by conditioning had full hematologic recovery. Four of these 17 patients died within 20 days of transplantation: one from fungal pneumonia, one from acute respiratory distress syndrome, and two from diffuse alveolar hemorrhage. One patient required a second transplant with conditioning for a full hematologic recovery. While initial sustained engraftment was higher in patients who had transplants with conditioning, their 10-year survival was lower (70%) than that of patients who did not receive conditioning initially (87%) [25]. These observations suggest that syngeneic HCT can be attempted without conditioning, and, if the graft fails, a second marrow infusion with a conditioning regimen is usually successful without compromising overall survival. The results of syngeneic HCT are consistent with the concept that some cases of AA are caused by a defect in HSCs that can be corrected by simple intravenous infusion of syngeneic marrow. However, there are other cases in which the infused twin marrow HCT either fails to engraft or is rejected following a period of transient recovery of hematopoietic function. The etiology of the aplasia in these cases may be T-cell mediated or be the result of unknown factors whereby the dysfunction of marrow can be overcome by conditioning with CY and a second marrow infusion [13,26].

Human leukocyte antigen-identical related bone marrow transplantation The largest number of transplants for SAA performed to date have been from human leukocyte antigen (HLA)-matched sibling donors. In preparation for allogeneic HLA-identical HCT, recipients have been treated by intensive immunosuppression to prevent rejection of the grafts. Immunosuppressive agents have included CY given either alone at a

dosage of 50 mg/kg/day intravenously on each of 4 successive days, or combined with ATG. In other patients that received earlier, less effective regimens, CY at a dosage of 60 mg/kg/day for 2 days was combined with TBI or limited field radiation, such as total lymphoid irradiation or thoracoabdominal irradiation. Table 49.1 summarizes recent reports on transplant outcome. The median age of patients reported in studies was approximately 25 years or less, with a range of 1–63 years. The conditioning regimens and agents used for GVHD prophylaxis varied widely, even within studies. Most transplanted patients have been previously transfused, a variable that has been shown to adversely affect transplant outcome by increasing the risk of graft rejection. The survival rates have improved with time in part because of decreased incidences of graft rejection, the result of changes in transfusion policies, and improved conditioning programs [27], and in part because of a decreased incidence of acute GVHD owing to the introduction of cyclosporine (CSP) [28] and the combination of methotrexate (MTX) and CSP [29]. Graft rejection Graft rejection occurs because of genetic disparity between donor and recipient. Graft rejection may also be due to persistent host T cells that mediated the bone marrow failure in the first place. Two forms of graft rejection may be seen. Primary rejection is defined by the absence of any sign of hematologic function of the graft, and late rejection is defined as graft loss after initial graft function. Patients with primary or late graft rejection can often be rescued with a second HCT [30]. Graft rejection was formerly a frequent problem among patients who received CY alone for conditioning. Graft rejection at the Fred Hutchinson Cancer Research Center, Seattle, was seen in over 35% of patients transplanted in the early 1970s (Fig. 49.1) [27]. A report from the European Group for Blood and Bone Marrow Transplantation (EBMT) showed a rejection incidence of 32% among patients transplanted before 1980 [31]. However, as described in the following section, significant progress has been made with respect to decreasing the risk of graft rejection. With modern conditioning regimens, graft rejection has become the exception, but it is important to review the interventions necessary to prevent graft rejection. A major factor associated with graft rejection has been sensitization to histocompatibility antigens through previous blood product transfusions, as shown by extensive animal studies and confirmed in clinical trials [32–36]. Figure 49.2 shows the historical data that previously transfused patients had a lower overall survival compared with untransfused patients [37], mainly because of complications of graft rejection. Data from animal studies showed that dendritic cells in the transfusion product are important in sensitizing recipients to disparate minor histocompatibility antigens of the transfusion (and marrow) donor [38]. Treating blood products with 20 Gy of gamma irradiation in vitro before transfusion almost eliminated sensitization to minor histocompatibility antigens and prevented rejection of dog leukocyte antigen-identical marrow grafts (Table 49.2) [39,40]. Other methods to reduce graft rejection in the canine model included the use of platelet and red blood cell transfusions that were leukocyte depleted (Table 49.2) [34]. These data strongly suggested that all human patients with AA who are potential candidates for HCT should be given blood products that are both irradiated and leukocyte poor [41]. In historical studies, patients with AA who proceeded to bone marrow transplantation (BMT) without prior blood transfusion had a reduced incidence of graft rejection. However, less than 15% of patients were untransfused prior to transplant [37]. Other methods were therefore developed to overcome marrow graft rejection. In the 1970s, an inverse relationship was observed between the number of donor marrow cells

13

31

1994–2001

1991–1998

1991–2002

1990–2003 1995–2003

1978–2001

1990–2001 – 1989–2003 1999–2004

1997–2005

1998–2001 2000–2005

2004–2006

2007

2000

2007

2005

2007

2004

2002

2003

2004

2006

2006

2003

2006

2007

IBMTR, Champlin et al. [56]

GITMO, Locatelli et al. [54]

EBMT, Locasciulli et al. [103]

EBMT, SAA Working Party*

EBMT, Schrezenmeier et al. [47]

Paris, Ades et al. [105]

Hamburg, Kroger et al. [106]

Seoul, Kim et al. [86]

Toronto, Gupta et al. [87]

NHLBI, Srinivasan et al. [57]

Jerusalem, Resnick et al. [59]

Tunis, Abdelkefi et al. [104]

Singapore, Koh et al. [142]

Tamil Nadu, India, George et al. [58]

–

CY + ATG, CY ± TBI

5–43 (20)

25–48 (43)

4–39 (19)

9–55 (20)

11–65 (27)

4–46 (17)

16–50 (28)

7–43 (25)

–

n = 343

age >20:

BM BM BM PBPC

CY + ATG CY + PCB + ATG CY + Cam CY (120) + FLU

PBPC

PBPC (80%)

FLU (90) + 2 Gy TBI

CY (120) + FLU (180) ± ATG

BM

PBPC (70%)

(180) + ATG CY + ATG

BM (30%)

CY (120) + FLU

(125) + ATG

BM

CY + TAI (n = 98) CY + ATG (n = 31)

PBPC: n = 134

BM: n = 558

various

CY + ATG (54%),

age ≤20: n = 349;

–

CY + ATG

± varied

BM

BM

CY (n = 60) CY + ATG (n = 70) CY

BM

CY + ATG

–

1–67 (18.7)

4–46 (19)

1–51 (24)

2–63 (25)

source

Conditioning program

CSP + MTX

+ MMF

CSP + MTX

CSP + MTX

CSP

CSP + varied

CSP ± Cam

CSP + MTX

CSP + MTX

Varied CSP + MTX

or other

CSP + MTX

CSP + MTX

CSP or −

CSP + MTX

5

3

0

16

0

0

24

15

–

11 11

CSP + MTX

CSP versus CSP + MTX

4

CSP + MTX

GVHD

29

50

11

8

69

14

11

15

42 0

PBPC: 14

BM: 10

–

–

38 30

18 11

24

Acute

GVHD (%)

32

50

4

13

46

4

12

17

82

75

86

84

77

81

89

86

58 90

age >20: 52

age >20: 30 64 42

age >20: 64 age ≤20: 73

age ≤20: 85

76

(1991–96) 74 (1997–02) 80

78 94

74 80

88

Survival (%)

age >20: 31 age ≤20: 27

age ≤20: 12

–

–

44 30

21 32

26

Chronic

(1.8)

0.3–4.3 (2.0)

0.4–3.5 (1.5)

0.2–8.0 (3.9)

0.5–5.4 (1.75)

0.5–12 (4.9)

0.1–6.6 (2.5)

0.2–11.6 (5.8)

(13.6) (4.4)

0.4–11.0 (5)

5-year survival

2.0–13.2 (3.6)

0.6–7.8 (4.0)

0.4–10.2 (6.3)

0.5–16.4 (9.2)

years (median)

follow-up in

Range of

In all series, the majority of patients were previously transfused. ATG, antithymocyte globulin; BM, bone marrow; Cam, alemtuzumab; CSP, cyclosporine; CY, cyclophosphamide (200 mg/kg, unless otherwise indicated); EBMT, European Group for Blood and Marrow Transplantation; FLU, fludarabine (total dose in mg/m2); GITMO, Gruppo Italiano Trapianti di Midoleo Osseo; GVHD, graft-versus-host disease; IBMTR, International Bone Marrow Transplant Registry; MTX, methotrexate; NHLBI, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD; PBPC, granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells; PCB, procarbazine (37.5 mg/kg); TAI, thoracoabdominal irradiation; TBI, total body irradiation; –, data not reported. * Additional interactive Cox survival model analysis for transplant outcome based on EBMTR data of neutrophil count, age, and year of transplant (up to 1990) is available at: http://www.ebmt.org/4Registry/registry5.html.

35

8

13

33

113

21

129

692

352

1275

71

130

81

1988–2004

2005

Seattle, Kahl et al. [53]

years (median)

patients

(%)

rejection

plant

Graft Prevention of

of

Hematopoietic

Age range in

Number

report

Year of trans-

Transplant team, reference

Year of

Table 49.1 Selected recent reports of human leukocyte antigen (HLA)-identical hematopoietic cell transplantation for severe aplastic anemia (SAA)

Hematopoietic Cell Transplantation for Aplastic Anemia

709

710

Chapter 49 100

50

No preceding blood transfusion (n = 53) 75

22/63 Survival (%)

% Graft rejection

40

30

20

Previously transfused (n = 115) 50

25

15/102

p = 0.04

13/110 10

0 0

3/81

3

6

9

12

15

18

Years following marrow transplantation

0 £1975

1976–80

1981–88

>1988

Year of marrow graft

Fig. 49.1 The incidence of graft rejection versus year of transplantation for patients in Seattle receiving marrow from human leukocyte antigen-identical siblings. All patients were conditioned with cyclophosphamide-containing regimens. The numbers above the bars indicate the number of rejections per number of patients transplanted. (Data from [24,53].)

Fig. 49.2 The effect of transfusion status on actuarial survival for patients treated between 1978 and 1991. Tick marks denote censoring times of surviving patients. The p-values were calculated using the log-rank test and are two-sided. (Reproduced from [37], with permission.)

Table 49.2 Transfusion sensitization prior to transplantation with hematopoietic grafts from dog leukocyte antigen-identical littermates after 9.2 Gy total body irradiation

Group

Preceding blood product transfusion on days −24, −17, and −10*

A B

None From marrow donor

C

From two different unrelated dogs on each of the 3 successive days Leukocyte-poor red blood cell transfusions from the donor

D

E

Leukocyte-poor platelet transfusions from the donor

2000 cGy in vitro gamma irradiation

Total number of dogs studied

Number of dogs with sustained engraftment (%)†

Not applicable Yes No Yes No No

62 20 27 16 27 14

61 (95%) 17 (85%) 0 (0%) 15 (94%) 10 (37%) 9 (64%)

No

15

8 (53%)

Statistical significance

} p value < 0.001 } p value = 0.001 p value = 0.001 (compared with group A) p value <0.001 (compared with group B with no irradiation) p value <0.001 (compared with group A and group B with no irradiation)

* All received donor buffy coat infusions in addition to marrow, except for 21 dogs which did not receive any preceding transfusions and 12 dogs who received unirradiated blood from their marrow donors or unrelated dogs. Data from [32,34,39,179,180]. Approximately 50 mL of heparinized blood per transfusion. † The p values were calculated using Fisher’s exact text.

infused and the risk of graft rejection in patients conditioned with CY [42]. A low incidence of graft rejection was seen with a high (>3 × 108 cells/kg) marrow cell dose, and vice versa. On the basis of that observation, an attempt was made to increase the number of hematopoietic cells transplanted by infusing unirradiated donor buffy coat cells in addition to the marrow [43]. While effective in reducing the incidence of graft rejection and improving survival compared with patients given marrow alone, the infusion of donor buffy coat cells was subsequently abandoned because of the increased risk of de novo chronic GVHD [44,45].

Historically, another approach to overcome graft rejection that was subsequently abandoned included intensifying the conditioning regimen, for example using CY combined with TBI or limited field radiation. While effective in reducing the incidence of rejection, radiation-based regimens were associated with worse long-term survival due to a higher rate of transplant-related mortality (TRM) and an increased risk of secondary cancer, as well as problems with growth, development, and fertility (see below and Chapters 104, 105, and 106). Recently, there was an interest in the use of granulocyte colonystimulating factor (G-CSF) mobilized peripheral blood hematopoietic

Hematopoietic Cell Transplantation for Aplastic Anemia

cells (G-PBHCs) as the source of HSCs for allogeneic transplantation. G-PBHCs contain an increased number of CD34+ hematopoietic progenitor cells and an approximately 10-fold increased numbers of T cells compared with marrow [46]. With the infusion of a larger number of donor cells, G-PBHCs could potentially decrease the risk of graft rejection in patients with SAA. EBMT registry data indicated that approximately 20% of HLA-identical sibling transplants for SAA in the years 1995–2003 used G-PBHCs as the HSC source [47]. The use of G-PBHCs resulted in a significantly increased risk of chronic GVHD and decreased overall survival. For patients with AA, there is no compensatory benefit of a graft-versus-leukemia effect provided by PBHCs; therefore, G-PBHCs should be avoided. Animal studies showed synergistic immunosuppressive effects between ATG and alkylating agents such as procarbazine and CY, as assessed by the criteria of skin graft prolongation and overcoming marrow graft rejection [48,49]. On the basis of these experimental animal data, the regimen of CY and ATG was developed and was initially used to rescue patients who had rejected their first graft. Fifteen of 19 previously transplanted patients who were reconditioned with CY and ATG had successful second marrow grafts, and 50% became longterm survivors [50]. More recently, survival of patients after second HCT increased to 83% at the Fred Hutchinson Cancer Research Center, a result not significantly different from survival after the first transplant [30]. Because of the success of CY and ATG in second transplants, the combination was chosen as a conditioning regimen for first transplants from HLA-identical family members, beginning in 1988. This led to the initial report of significantly improved outcome of HCT compared with the historical regimen of CY alone with buffy coat infusions [50]. Changes in transfusion policies (consistent use of irradiated, leukodepleted blood products), and improved conditioning regimens (combined CY + ATG) before transplant have led to significant decreases in graft rejection [31]. In addition, postgrafting immunosuppression aimed at controlling GVHD may also have a role in controlling host-versus-graft reactions. A retrospective analysis by the EBMT suggested that the use of CSP as GVHD prophylaxis has decreased the rejection rate compared with MTX [31], but a randomized prospective study from Seattle failed to show differences in rejection [27,29] when long-term MTX was compared with a short course of MTX combined with CSP. Studies in animals showed MTX and CSP to be superior to CSP alone, not only in controlling GVHD, but also in suppressing host-versus-graft reactions, thereby enhancing engraftment [51]. More recent data demonstrated that the combination of mycophenolate mofetil (MMF) and CSP provided even better control of host-versus-graft reactions than MTX and CSP [51,52]. Thus, postgrafting immunosuppression used for GVHD prevention also has to be considered when evaluating new conditioning regimens for their efficacy in decreasing the risk of graft rejection. Updated results from the Seattle study of CY and ATG for patients with SAA confirmed that graft rejection had become a minor problem (Fig. 49.3a). Of the 81 consecutive patients enrolled, 76 had received multiple transfusions, and 44 had failed IST. The ages of the patients ranged from 2 to 63 years. After transplantation, 81 patients received MTX and CSP for GVHD prophylaxis. There was a 3.7% incidence of graft rejection between 1 and 7 months after transplantation (Fig. 49.3a). Of the three patients with graft rejection, two were alive following successful second HSC transplantation. With a median follow-up of 9.2 (range 0.5–16.4) years, the overall survival rate was 88% (Fig. 49.3e) [53]. Some transplant centers have continued to use CY without ATG as the conditioning regimen for younger patients receiving marrow HCT from HLA-identical siblings. Results from a prospective randomized multicenter trial sponsored by the Gruppo Italiano Trapianti di Midolio Osseo (GITMO) and the EBMT that compared CSP alone versus MTX

711

and CSP for GVHD prophylaxis in 71 patients with SAA showed that the overall incidence of graft rejection was 8% [54]. Several factors may explain why the GITMO/EBMT results with CY conditioning alone were superior to the historic data from the Seattle group in the 1980s. Since the initial Seattle report, there has been increased awareness in the medical community of the benefit of leukodepleted and irradiated blood products for patients with SAA. Seventy of 71 patients in the GITMO/EBMT study had been transfused with a median of 6 units of blood products prior to transplantation. Although not specifically addressed in the GITMO/EBMT study, the widespread use of irradiated and leukodepleted blood products for transfusion support prior to transplantation for patients with AA may have contributed to the lower incidence of graft rejection seen since the 1980s. The GITMO/EBMT study included predominantly younger patients, many of whom were not treated with a trial of immunosuppression prior to proceeding to allogeneic HSC transplant. In contrast, the updated multicenter trial of CY and ATG included older, heavily transfused patients whose prior treatment with IST had failed [53,55]. In a multicenter prospective trial conducted over 7 years, 134 patients were randomly assigned to receive CY alone or in combination with ATG. The majority of patients had not received prior IST. Patients received T-cell-replete bone marrow from an HLA-identical sibling, and GVHD prophylaxis consisted of CSP and MTX. The 5-year probabilities of survival were 74% and 80% in the CY and the CY + ATG groups, respectively. Graft failure rates were similar, at 18% and 16%, respectively. Fifteen of 22 patients with graft failure underwent second HCT from their initial donor, but three patients in the CY alone group required more than two transplantations. At last follow up, 12 of the 22 patients with graft failure were alive. Unfortunately, this randomized study was underpowered to detect a statistically significant difference in survival, so the data are inconclusive as to whether the combination of CY and ATG leads to superior outcome after BMT [56]. Perhaps for younger patients (under age 20 years) who have not received prior IST for AA and who have received only a very limited number of blood product transfusions, all of which have been irradiated and leukodepleted, the risk of graft rejection following HLA-identical sibling BMT is low, and the addition of ATG to CY conditioning regimen may not be necessary. However, recent data indicate that transplantation of a high cell dose of donor marrow (corrected for donor peripheral blood cell content) results in significantly increased risk for chronic GVHD [53]. Thus, to avoid possible graft rejection when the marrow cell dose is restricted, ATG should be administered with CY. The addition of fludarabine (FLU) to a reduced dose of CY with ATG has been recently evaluated as an alternative approach to overcoming graft rejection. However, studies of FLU + CY + ATG were confounded by the use of G-PBHCs instead of bone marrow to help overcome the risk of graft rejection. The incidence of graft rejection was low, but the incidence of severe acute GVHD was high, and the overall outcome was not superior to the accepted standard CY + ATG regimen [57–59]. In addition to CY and ATG for conditioning following first graft rejection, there have been anecdotal reports for treating graft failure. One regimen included Campath-1G, a monoclonal antibody that reportedly eliminates radiation-resistant and CY-resistant host T cells [60]. One case report described the use of G-PBHCs without reconditioning [61], and another transplant center has employed the use of rabbit ATG [62] to overcome graft failure. In summary, results from clinical trials to date indicate that combined CY and ATG conditioning followed by MTX and CSP as postgrafting immunosuppression is the most effective and reliable regimen to prevent graft rejection following HLA-identical BMT for SAA. Similar survival might be obtained without ATG in younger, relatively untransfused patients. The use of the least toxic transplant regimen associated with

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(a)

(d) Probability of cancer

Probability of rejction

0.25 0.20 0.15 0.10 0.05 0.00 0

4 8 Months after HCT

1.00 0.75 0.50 0.25 0.00

12

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3

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0

5

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(e) 1.00 Probability of cancer

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(b)

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20

60 40 Days after HCT

80

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(c) Prevalence of chronic GVHD

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the lowest incidence of graft rejection avoids the need for having to deal with this potentially life-threatening complication after transplant. For first transplants, the use of G-PBHCs is not appropriate given the increased risk of chronic GVHD. Treatment guidelines for SAA include uniform adherence to the following: 1 the use of irradiated and leukodepleted blood products before transplant; 2 for patients with an HLA-identical sibling donor, proceed to BMT as first-line therapy; 3 the well-established and well-tolerated conditioning regimen of CY and ATG; 4 infusion of 2.5 × 108 donor marrow cells (corrected for donor peripheral blood cell count)/kg; 5 avoid use of G-PBHCs; 6 postgrafting immunosuppression with combined MTX and CSP.

Mixed donor–host hematopoietic chimerism Transient mixed chimerism is common in patients after marrow allografts for AA. In one study, almost 60% of patients had mixed chimerism in peripheral blood or bone marrow after HCT; two-thirds of these eventually converted to complete donor-type hematopoiesis, while the remain-

20

Fig. 49.3 Cumulative incidences of (a) graft rejection and (b) developing acute grade II– IV graft-versus-host disease (GVHD) in 81 consecutive patients with aplastic anemia given human leukocyte antigen-identical marrow grafts following cyclophosphamide and antithymocyte globulin conditioning, and GVHD prophylaxis with methotrexate and cyclosporine. (c) Prevalence of chronic GVHD. (d) Probability of malignancy after hematopoietic cell transplantation (HCT). (e) Overall survival. (Reproduced from [53], with permission. Copyright 2005 Blackwell Publishing Ltd.)

der rejected their grafts [63]. One other study showed that T-cell mixed chimerism in two patients conditioned with CY predated late graft failure [64]. In a larger study of 116 patients with AA transplanted from sex-mismatched HLA-identical siblings, 54% were mixed chimeras detected in either blood or marrow. While patients with mixed chimerism appeared to have a higher incidence of graft rejection (14%) than those who were complete chimeras (9%), this difference was not statistically significant [65]. A study of 45 patients with AA who received HLA-identical sibling BMT and serial monitoring with short tandem repeat polymerase chain reaction chimerism analysis reported that 72% developed complete donor chimerism, 11% had stable mixed chimerism, and 17% had an increase in host chimerism after transplantation. Patients with increasing host chimerism after transplantation had a 50% incidence of graft failure and an overall survival of 38%. Patients with complete donor or stable mixed chimerism had superior survival. None of the patients with stable mixed chimerism developed chronic GVHD [66]. These results suggest that monitoring chimerism after allogeneic HCT is useful to predict the subsequent transplant outcome. T-cell chimerism can now be routinely determined after HCT. Chimerism analysis is an indispensable tool to monitor the kinetics of engraftment and the ultimate fate of the graft; it also appears to be useful to predict impending graft rejection.

Hematopoietic Cell Transplantation for Aplastic Anemia

Chronic GVHD (see Chapter 87) While significant progress has been made in reducing the incidences of graft rejection and acute GVHD, chronic GVHD continues to be a major

(a) Probability of survival

Once engraftment has been accomplished, acute GVHD may develop, usually within the first 6 weeks after HCT. Grade II–IV acute GVHD has an adverse effect on survival in patients transplanted for SAA. Early Seattle data showed an actuarial probability of survival at 11 years of 45% for SAA patients with preceding grade II–IV acute GVHD compared with 80% survival among patients with grades 0 and I acute GVHD [67]. Similarly, subsequent IBMTR data showed a 31% 5-year actuarial probability of survival for SAA patients with grade II–IV acute GVHD compared with 80% among patients with no or mild acute GVHD [68]. During the 1970s, MTX was the most commonly used agent given after BMT to prevent acute GVHD, and 35% of MTX-treated SAA patients developed grade II–IV acute GVHD [67]. Because of the high mortality associated with acute GVHD, a number of studies were carried out in the 1980s to investigate whether the incidence of this complication could be reduced and survival improved. CSP was initially studied as a single agent to prevent acute GVHD. Retrospective analyses showed that CSP favorably influenced the survival of more recently transplanted SAA patients compared with historical control patients given MTX alone [69–72]. A number of transplant centers combined prednisone (PSE) and CSP in hopes of improving control of acute GVHD. A randomized prospective study of patients with high-risk lymphohematopoietic malignancies showed that the combination of CSP and PSE was only marginally better than CSP alone in preventing acute GVHD, but this came at the expense of a significant increase in chronic GVHD [73,74]. Another study of patients with leukemia or lymphoma found a combination of MTX, CSP, and PSE more effective in preventing acute GVHD than CSP and PSE [75]. Encouraging data in experimental animals led, in 1981, to the introduction in the clinic of a combination of MTX (15 mg/m2 given at 24 hours after completion of bone marrow infusion and 10 mg/m2 given on days 3, 6, and 11 after BMT) combined with CSP. One randomized trial from Seattle showed a significant reduction in incidence and severity of acute GVHD in 22 SAA patients given the combination versus 24 patients receiving MTX alone [29], and overall survival was higher in patients who received the combination of drugs (Fig. 49.4) [29,76]. Similar findings were made in patients with leukemia where the combination of MTX and CSP was found to be superior to CSP alone in preventing acute GVHD [77]. In a later nonrandomized retrospective analysis of IBMTR data from 595 patients with SAA who had received BMT from matched siblings, patients who received either CSP alone or CSP with MTX compared with MTX alone had a statistically significant improved survival rate of 69% versus 56%, respectively [68]. Importantly, no grade IV acute GVHD was seen in patients given MTX and CSP. The reduction in acute GVHD resulted in improved survival, a finding that was especially impressive among pediatric patients with SAA [69]. One retrospective analysis suggested that optimal doses of both MTX and CSP were important in controlling acute GVHD [78]. Because of the success of the combination of MTX and CSP, a randomized study was carried out to compare MTX + CSP with CSP + MTX + PSE for patients with hematologic malignancies and SAA. Among patients with HLA-identical donors, the addition of PSE increased the risk of acute and chronic GVHD unless the PSE was administered after the completion of a short course of MTX [79]. A subsequent randomized trial of MTX + CSP versus MTX + CSP + “late PSE” was completed in patients with leukemia; this suggested improved survival in the MTX and CSP arm (p = 0.1) [80]. The prospective randomized study by GITMO/EBMT confirmed the initial Seattle report and showed that the combination of MTX and CSP

resulted in superior 5-year estimated survival compared with the use of CSP alone for SAA patients transplanted with an HLA-identical sibling marrow graft. The incidence of acute GVHD in the MTX + CSP group versus CSP alone was 30% and 38%, respectively [54]. Updated results of the study with the Seattle regimen consisting of CY and ATG, with HLA-identical marrow followed by combined MTX and CSP immunosuppression in 81 consecutive patients showed that the cumulative incidence of grade II–IV acute GVHD was 24% (Fig. 49.3b) [53]. The severity of GVHD observed was grade II (21.6%), grade III (0%), and grade IV (2.4%). Thus, despite the increased age of patients in the Seattle regimen study compared with the GITMO/EBMT study, the overall incidence of acute GVHD was less in the Seattle study. Similarly, the randomized CY versus CY + ATG multicenter study with GVHD prophylaxis of MTX + CSP reported an acute GVHD incidence of 18% and 11%, respectively [56]. These findings suggest that the incorporation of ATG in the conditioning regimen may be effective in preventing acute GVHD, particularly in the older patient age groups. ATG may have an effect on depletion of the infused donor T cells.

1.00 MTX + CSP (n = 22)

0.75

MTX (n = 24)

0.50

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Acute GVHD (see Chapter 86)

713

0.60

0.40

0.20 MTX (n = 24) MTX + CSP (n = 22)

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10 15 Years after marrow grafts

20

80

Fig. 49.4 Survival of patients with aplastic anemia given marrow grafts from human leukocyte antigen (HLA)-identical siblings after conditioning with cyclophosphamide (without antithymocyte globulin). Shown are (a) Kaplan– Meier estimates for overall survival and (b) prevalence of chronic graftversus-host disease (GVHD) among patients in a randomized prospective trial comparing methotrexate (MTX) alone with MTX and cyclosporine (CSP) for GVHD prophylaxis. Most of the patients enrolled in the trial were adults because of Food and Drug Administration restrictions. (Reproduced from [76], with permission.)

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Chapter 49

complication of marrow grafting for AA. Unlike the graft-versusleukemia effect associated with chronic GVHD, which can improve survival of patients with leukemia, there is no benefit of chronic GVHD in patients transplanted with AA. Historically, the incidence of chronic GVHD varied depending on the patient groups studied. A few small series of children transplanted from HLA-identical siblings reported incidences of 0–25% [81–83], whereas the incidence was as high as 40–60% among other series that included adult patients and those who received supplemental buffy coat infusions [84,85]. In most current series of matched sibling transplants that included adults, the incidence of chronic GVHD was 10–30% [47,53,54,56,59,86,87] (Table 49.1). Early studies identified a history of acute GVHD as the most important risk factor for chronic GVHD [44,88]. Older patients also experienced more chronic GVHD. De novo chronic GVHD occurs without preceding acute GVHD. Besides donor buffy coat infusions, other risk factors, including increased patient age and preceding corticosteroid therapy, have been associated with de novo chronic GVHD development [44,89]. Most recently, infusion of a high corrected marrow cell dose (greater than 3.4 × 108 cells/kg) was associated with a significantly increased incidence of chronic extensive GVHD (hazard ratio 7.7) and lower overall survival [53]. G-PBHCs increase the risk of chronic GVHD, which is more protracted and less responsive to treatment compared with that associated with bone marrow [90]. Because of the excellent overall survival of SAA patients after HLA-identical BMT given combined CY and ATG conditioning and CSP and MTX for GVHD prophylaxis, it will be very unlikely for future studies to demonstrate any significant benefit of utilizing T-cell-depleted or CD34-enriched PBHCs over HLA-identical bone marrow as the stem cell source. Therefore, for patients with SAA, HLA-identical bone marrow with a target corrected cell dose of 2.5 × 108 nucleated cells/kg body weight is the optimal source of HSCs. Chronic GVHD is associated with significant morbidity and requires prolonged IST, although ultimately immunosuppression can be discontinued in most patients as chronic GVHD resolves [53,91]. In the past, up to one-third of affected patients died, often from infections. PSE, azathioprine, CY, procarbazine, CSP, thalidomide, MMF, tacrolimus (FK506), and extracorporeal photopheresis, given either alone or in combination, have all been used for treatment of chronic GVHD (see Chapter 87) [91–96]. Recent evidence suggests that persistent chronic GVHD is a risk factor for the development of solid tumors late after transplantation. In addition, in the Seattle survey of long-term survivors after allogeneic transplantation for AA, chronic GVHD was a risk factor for development of cataracts, lung disease, bone and joint disease, and depression [97]. In the Seattle experience, since the introduction of the CY and ATG conditioning regimen and the discontinuation of added buffy coat cell infusions, the prevalence of chronic GVHD has significantly decreased. Chronic GVHD was not only less frequent but also appeared to be more responsive to therapy in patients conditioned with CY and ATG and given marrow alone compared with historical control patients treated with CY and marrow plus buffy coat. In the updated Seattle transplant regimen report with HLA-identical sibling marrow grafts consisting of CY and ATG followed with MTX and CSP, the cumulative incidence of chronic GVHD observed was 26% (Fig. 49.3c). In all surviving patients, chronic GVHD responded to therapy with complete responses. Of the patients with chronic GVHD, 83% survived long term. The peak incidence of chronic GVHD was at 1 year after transplantation. By 7 years, no patients required IST for chronic GVHD [53]. The incidences of chronic GVHD in the GITMO/EBMT trial with CY conditioning and postgrafting immunosuppression with either CSP alone or MTX + CSP were 30% and 44%, respectively. The majority of

patients with chronic GVHD had limited disease of the skin; the overall incidence of extensive chronic GVHD was 9% [54]. Most of the patients in the GITMO/EBMT study were children, who had a lower incidence of chronic GVHD compared with adults. Prompt diagnosis of chronic GVHD combined with aggressive initiation of IST with the combination of CSP and PSE has resulted in an apparent decrease in the duration of time needed for inducing remission of chronic GVHD, and the overall response to treatment has improved over time. However, this improvement has not been associated with a significant increase in survival for patients with SAA who develop chronic GVHD [93,98]. Thus, mortality associated with chronic GVHD remains a significant problem for patients following allogeneic HCT. These observations suggest that improvements in the prevention of chronic GVHD must be sought to further improve patient survival. Perhaps prompt diagnosis and treatment of chronic GVHD may help improve response to treatment and overall survival. However, in a randomized prospective study comparing CSP continued at “therapeutic levels” for 24 months versus 6 months after HCT for patients at highest risk of developing chronic GVHD, for example those with preceding acute GVHD or corticosteroid therapy, and older patients, there was no difference in overall survival between the two groups [94]. In the setting of HLA-identical sibling HCT, CY + ATG conditioning and GVHD prophylaxis with MTX + CSP, the most effective means to prevent chronic GVHD, may be achieved by limiting the infused donor marrow to a target cell dose of 2.5 × 108 total nucleated cells/kg (corrected for donor peripheral blood cell count). Taken together, current evidence indicates that the Seattle regimen of CY and ATG conditioning followed by HLA-identical marrow transplantation and combined MTX and CSP postgrafting immunosuppression appears to provide the lowest risks of graft rejection and acute and chronic GVHD compared with other transplantation regimens. Interstitial pneumonia The incidence of interstitial pneumonia occurring after transplantation in patients with SAA has decreased over time. In 1989, Weiner et al. [99] reviewed IBMTR data from 547 patients with AA receiving HLA-identical marrow grafts for the risk of interstitial pneumonia. They found an overall incidence of interstitial pneumonia of 17%. Cytomegalovirus (CMV) pneumonia affected 37% of patients, 22% had pneumonia from other organisms, and in 41% of cases no organisms were identified. The case fatality rate was 64%. At that time, four factors predicted the development of interstitial pneumonia: 1 the use of MTX rather than CSP after HCT; 2 moderate-to-severe acute GVHD; 3 TBI compared with CY in the preparative regimen; 4 increased patient age. Results from 329 patients treated for SAA in Seattle from 1974 to 1990 showed the overall incidence of interstitial pneumonia was 16%. Forty-four percent of affected patients had CMV pneumonia, 37% had pneumonia resulting from a variety of other infectious organisms, and 19% had pneumonia of unknown etiology. The overall mortality was 7%. TBI and acute GVHD were significant risk factors for the development of interstitial pneumonia, but there was no adverse influence of patient age or of MTX prophylaxis on the development of interstitial pneumonia [27]. More recent studies have confirmed that grade II–IV acute GVHD is the major risk factor for the development of interstitial pneumonitis [100]. A study of idiopathic lung disease after transplant found that patients with AA conditioned with CY had a significantly lower incidence (less than 5%) than those transplanted for malignant diseases who were conditioned with TBI or busulfan-containing regimens [101]. The

Hematopoietic Cell Transplantation for Aplastic Anemia

case mortality rate for these studies and more recent Seattle data, all of which contained large numbers of patients with malignant diseases, was 71–75% [100–102]. Over time, avoidance of TBI, improved prophylaxis for acute GVHD with MTX and CSP, and improved supportive care, including the prompt diagnosis and treatment of infectious agents such as CMV (by DNA detection methods), have all contributed to the decreased incidence of idiopathic interstitial pneumonitis. In the recent update of the patients with SAA treated with the Seattle regimen, only one of 81 patients died of idiopathic interstitial pneumonia [53]. Survival Overall survival following HCT for SAA has increased over the last 30 years. During the 1970s, survival rates of 40–60% were commonly seen, whereas the survival rates now range from 74% to 100% [47,53,54,56, 58,59,86,87,103–106] (Table 49.1). Figure 49.5 shows improved survival among Seattle patients transplanted after July 1988 (88%) compared with patients transplanted before 1977 (41%) and with patients transplanted between 1977 and 1988 (61%). All patients transplanted since 1985 received MTX and CSP for GVHD prophylaxis, and since 1988 all were conditioned with CY and ATG rather than CY alone. Updated results employing the Seattle transplant regimen (CY and ATG conditioning, HLA-identical marrow HCT, and combined MTX and CSP postgrafting immunosuppression) confirmed that, with a median follow-up of 9.2 (range 0.5–16.4) years, the survival rate was 88% [53]. In this study of 81 consecutive SAA patients, the median age was 25 and the age range 2–63 years. The randomized GITMO/EBMT trial comparing CSP alone with combined MTX + CSP for patients conditioned with CY alone showed an estimated 5-year survival of 94% in the MTX and CSP group, and 78% for those in the CSP alone group [54]. The difference in survival between the two groups was statistically significant. This study confirmed the initial Seattle results demonstrating the superiority of postgrafting immunosuppression with combined MTX and CSP. The multicenter randomized study of CY versus CY + ATG showed that the 5-year survival was 74% for the CY alone group and 80% for the CY + ATG group [56].

100 7/1988–2004 (n = 81) 75 % Survival

1977–6/1988 (n = 193) 50

1970–1976 (n = 82)

25

0 0

10 20 Years following transplantation

30

Fig. 49.5 Hematopoietic cell transplantation from human leukocyte antigen (HLA)-identical family members after cyclophosphamide (CY). Overall survival indicated by transplant year cohort. The combination of methotrexate and cyclosporine was used for graft-versus-host disease (GVHD) prophylaxis in some patients since 1981 and in all patients since 1985. All 81 patients transplanted since July 1988 were conditioned with CY and antithymocyte globulin without supplemental buffy coat cell infusions.

715

Analysis of transplant registry data has been helpful to identify important trends influencing survival over time. The reported results are in general less favorable than the optimal conditioning regimens reported by some transplant centers because multiple conditioning and GVHD prophylaxis regimens are used by registry members, some of which have been suboptimal. A retrospective multicenter multiregimen IBMTR study reported that 5-year survival after HLA-identical sibling transplants had increased from 48% in 1976–80 to 66% in 1988–92 [85]. Because of the introduction of CSP for GVHD prophylaxis, the risk of GVHD decreased. Improved long-term survival was because of decreased mortality in the first 3 months post transplant. The incidence of reported graft failure did not significantly change, ranging from 20% in 1976–80 to 16% in 1988–92. However, only 9% of reported transplants in 1988–92 included CY and ATG conditioning. IBMTR data for 1699 patients receiving HLA-identical sibling transplants for SAA between 1991 and 1997 showed a 5-year probability of survival of 75% (95% confidence interval [CI] 92–98%) for 874 patients less than 20 years of age, 68% (CI 64–72%) for 696 who were aged 21–39 years, and 35% (CI 17–53%) for 129 who were 40 years or older [107]. Survival was highest in untransfused patients transplanted early in the course of their disease and who were without active infection at the time of transplant. Only a minority of patients reported to the registry between 1991 and 1997 received CY and ATG conditioning with combined MTX and CSP post-grafting immunosuppression. More recently, since children with HLA-identical siblings have been increasingly referred for transplantation as first-line therapy, and because the majority of transplant centers now administer CY + ATG conditioning with MTX + CSP for GVHD prophylaxis, the survival trends have continued to improve. Between 1995 and 2003, 305 patients age 20 years and under that received bone marrow as the HSC source had a 5-year probability of survival of 85%. The 253 patients greater than age 20 years had a 5-year survival of 64%. Survival was significantly worse for the 134 patients that received G-PBHCs as the graft source due to the increased incidence and severity of chronic GVHD [47]. EBMT data for 1275 patients receiving HLA-identical sibling transplantation between 1991 and 2002 confirmed and extended the IBMTR results. The year of the transplant, the age of the patient, and the interval from diagnosis to transplant predicted outcome after HCT. There was an improvement in 5-year survival rates when comparing patients transplanted in 1991–96 and 1997–2002: 74% versus 80%, respectively. In the years 1997–2002, actuarial survival was 91% for children (<16 years of age) and 74% for adults. ATG was added to the conditioning regimen in 20% of patients, and their overall survival was 81% versus 73% among patients not receiving ATG [103]. Several other studies found a survival of more than 85% for patients conditioned with the regimen of CY and ATG [55,82,104–106]. In contrast, reports of patients conditioned with CY and irradiation had survival rates ranging from 57% to 78% [105,108,109], although follow-up on some of these reported studies was short. Pediatric patients (younger than 19 years) generally fared better than adult patients in four studies [27,69,81,82], with survival rates of 94–100%. A recent EBMT report showed 85% survival among children receiving CY + ATG conditioning and BMT [47]. Recent EBMT data with CY + ATG conditioning and HLA-identical BMT for different patient age groups are summarized in Fig. 49.6. A striking difference in transplant outcome for AA between radiation and nonradiation-based conditioning regimens was reported from a single center by Ades et al. From 1978 to 1992, 100 patients received a CY + thoracoabdominal irradiation conditioning regimen, with an overall survival at 15 years of 58%. From 1995 onwards, 33 patients (26 under age 30 years) received CY + ATG conditioning with MTX + CSP for GVHD prophylaxis, and had a 94% overall survival [105].

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100

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0–10 yr. 40

10–20 yr. 20–30 yr. 30–40 yr.

20

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Fig. 49.6 Data from the European Group for Blood and Marrow Transplantation (EBMT) Aplastic Anemia Registry for 352 patients conditioned with cyclophosphamide and antithymocyte globulin and receiving matched sibling hematopoietic cell grafts between 1990 and 2003. Shown is 5-year overall survival stratified by patient age. (Courtesy of Dr Jakob Passweg, on behalf of the Working Party on Aplastic Anemia of the EBMT.)

In summary, radiation-containing regimens were widely used in the late 1970s and 1980s because of their better immunosuppressive properties, resulting in a decreased incidence of graft rejection. However, the use of radiation has been associated with the risks of interstitial pneumonia, growth impairment, development of secondary solid tumors (see below), and lower overall survival compared with CY and ATG. Radiation is no longer recommended as an initial preparative component for patients with HLA-matched sibling donors. Transplantation for older patients An analysis of all 20 patients with SAA who were above the age of 40 (range 40–63) years and underwent BMT from HLA-identical siblings in Seattle with CY + ATG conditioning and MTX + CSP GVHD prophylaxis between July 1988 and January 2006 showed an overall survival of 70% with a median follow-up of 7 years. Three patients died prior to engraftment from pre-existing infections (n = 2) or heart failure (n = 1). Three patients died from infections at 3, 6, and 7 months after BMT, related to acute or chronic GVHD. The overall incidence of acute and chronic GVHD was 47% and 37%, respectively. The median time to discontinuation of immune suppression was 6 (range 6–46) months [110]. These data support the use of BMT for patients with AA well above the age of 40 years and for otherwise healthy patients up to 65 years.

For hepatitis-associated AA, there was concern that liver damage caused by hepatitis might lead to increased liver damage from CY, or that the damaged liver might not be able to enzymatically activate CY. Neither of these concerns proved valid [111,112]. Marrow grafts were successful, even when CY was administered at times of highly abnormal liver function tests. Also, there were no long-term sequelae related to the HCT procedure in these patients. Survival was in excess of 80% [111]. Fanconi’s anemia is covered in detail in Chapter 79, but it is often included in reports describing patients with SAA. Fanconi’s anemia is an autosomal recessive disease with progressive pancytopenia, chromosomal fragility, and hypersensitivity to DNA crosslinking agents that can progress to acute leukemia. Currently, the only successful treatment for marrow failure in patients with Fanconi’s anemia is HCT. Conditioning regimens used in the past to prepare patients with Fanconi’s anemia for transplantation have been associated with considerable toxicity and TRM [113,114]. In addition, irradiation and high-dose CY are associated with a very high risk of secondary malignancies [115]. It has recently been recognized that, unlike patients with idiopathic AA, those with Fanconi’s anemia can be conditioned with lower doses of CY than conventionally used because of their unusual sensitivity to toxicity from alkylating agents [116]. Results from Curitiba, Brazil and Seattle show that patients with Fanconi’s anemia can be transplanted successfully without incorporation of irradiation into the conditioning regimen with substantially lower doses of CY than used in the past. Nineteen patients transplanted with 100 mg/kg CY and HLA-identical marrow with MTX and CSP postgrafting immunosuppression had 79% survival at 10 years and sustained donor engraftment [117]. More recently, further dosage reduction of CY to 15 mg/kg/day given for 4 consecutive days (total dose 60 mg/kg) has been successful in achieving durable donor engraftment accompanied with decreased toxicity and an excellent long-term survival of 93% with follow-up to 7.9 years [118]. Similar results with CY 60 mg/kg combined with ATG have been reported [119]. Thus, the optimal dosage of CY that will allow sustained engraftment with minimal toxicity appears to be 60 mg/kg. Dyskeratosis congenita is a rare inherited syndrome of ectodermal dysplasia that is associated with AA in 50% of cases [120]. The initial trials using conventional conditioning regimens were associated with significant morbidity and mortality [121,122], particularly an increased incidence of late chronic pulmonary and vascular complications. Recent reports have shown successful outcomes in patients given a reducedintensity conditioning regimen such as the Seattle nonmyeloablative regimen of FLU 90 mg/m2 + 2 Gy TBI [123] or, alternatively, CY (60 mg/kg) + ATG [124]. HCT has also been used to treat SAA resulting from PNH and Diamond–Blackfan anemia [125–127]. Among nine patients transplanted for PNH, six were alive without evidence of disease 2–20 years after HCT [126]. Recently, the reduced-intensity conditioning regimen of FLU 90 mg/m2 + 2 Gy TBI has been successful for patients with PNH [128]. Of 61 patients with Diamond–Blackfan anemia who were refractory to PSE and other therapies reported to the IBMTR, 41 received HLA-identical sibling transplantation, with 76% survival at 4 years [129]. Late effects Long-term survival

Influence of etiology of AA on the outcome of the marrow grafts It is now well established that HCT can cure AA resulting from causes other than an idiopathic etiology. This finding has included patients with hepatitis-associated AA, Fanconi’s anemia, and dyskeratosis congenita.

The long-term outcome among 212 patients transplanted for SAA has been reported [97]. Ninety-three percent of patients were conditioned with CY with or without ATG, and 89% were given transplants from HLA identical siblings (8% had mismatched related donors, 2% had unrelated donors, and 1% had syngeneic donors). The 20-year survival among patients without chronic GVHD (n = 125) was 89%, compared

Hematopoietic Cell Transplantation for Aplastic Anemia

with 69% among those with chronic GVHD (n = 86). Of the 17 patients who died between 2.5 and 20.4 years after transplant, 13 had chronic GVHD, while four did not. Among the 13 patients with chronic GVHD, two died of human immunodeficiency virus (HIV) disease, five died of pulmonary failure (one from pulmonary complications of dyskeratosis congenita), three died of septicemia, and three died of squamous cell carcinoma. Among the four patients who died without chronic GVHD, one died of suicide, one in an automobile accident, one of HIV disease, and one of pulmonary complications from dyskeratosis congenita. All of the cases of HIV disease occurred before blood transfusion testing for HIV antibodies had become standard. Nearly all of the patients who survived beyond 2 years returned to a fully functional life [97]. Gonadal function and fertility (see Chapters 97 and 98) Gonadal function in patients conditioned only with CY + ATG often returns to normal. Among 65 women between the ages of 13 and 25 years who received CY, all had evidence of recovery of ovarian function, whereas among women aged 26–38 years, 37% developed primary ovarian failure [22,130–133]. These data suggested that older age was a risk factor for ovarian failure. Testicular function had returned to normal in most men aged 14–41 years who received CY + ATG only for conditioning [22]. Many successful pregnancies had been reported after conditioning regimens containing CY + ATG only. At 20 years post transplant, the probability that a female patient would become pregnant after transplantation for AA was 47%, and the probability that a male patient would father a child was 50% [97]. In contrast, pregnancy and fathering a child after conditioning with TBI were rare events [134]. There is insufficient information regarding fertility after FLU-based regimens. Growth and development (see Chapter 104) A study of hormonal function and growth after HCT found that children conditioned with CY only had normal height, normal thyroid, and adrenal function, and no growth hormone deficiency. However, in children given TBI as part of the conditioning, while prepubertal growth in the first 3 years after transplant was normal, the final height was lower than target height [135]. Therefore, TBI-containing regimens should be avoided in children if possible. Both children and adults treated with TBI-containing regimens were also more likely to develop thyroid dysfunction after transplantation [22], and these patients should be monitored with yearly thyroid function tests. Secondary malignancies (see Chapter 106) Since the first patients have been transplanted successfully for AA over 33 years ago, data have been accumulated on the incidences and types of secondary malignancies resulting from HCT. Generally, secondary malignancies following HCT are uncommon, but they may be fatal. Deeg et al. [115] described results in 700 patients with AA who had an estimated 14% incidence of secondary malignancies at 20 years. The cancer risk among transplanted patients was ninefold higher than that among the general population. Overall, 18 of 621 patients (excluding those with Fanconi’s anemia) developed secondary malignancies a median of 91 months post transplant. Of the tumors, five were lymphoid malignancies seen a median of 3 months after transplant, and 13 were solid tumors occurring a median of 99 months after transplant. Nine of the solid tumors were squamous cell carcinomas of the head and neck, and most of these patients were cured following surgical resection. All of the patients with lymphoid malignancies have died, whereas only 46% of those with solid tumors have died. Significant risk factors for the development of secondary malignancies in this series included the use of azathioprine for chronic GVHD treatment, increased age, and use of irradiation in the conditioning regimen [115]. With regimens not

717

containing irradiation, there was a cumulative incidence of secondary cancers at 10 years of 1.4% [136] compared with a 22% (standard error 11%) incidence at 8 years [137] with radiation-containing regimens. Other studies have also implicated irradiation-based conditioning regimens as risk factors for late malignancies [23,105], so these regimens should be avoided in HLA-identical HCT candidates. The incidence of secondary malignancy for 81 patients receiving CY + ATG conditioning is shown in Fig. 49.3(d). Chronic GVHD is also a risk factor for the development of solid tumors after transplant for AA, with an estimated 17–30% probability at 20 years for patients with both acute and chronic GVHD [53,97]. Carcinomas of the oropharyngeal mucosa developed in patients with a history of chronic GVHD. These findings were also confirmed in a larger study of 19,229 patients who had undergone transplantation between 1964 and 1992 that showed chronic GVHD was associated with a significantly increased risk of squamous cell carcinoma of the buccal cavity and skin [138]. The severity of chronic GVHD and duration of IST for chronic GVHD appear to be risk factors for secondary malignancy. These findings emphasize the importance of avoiding infusion of buffy coat or G-PBHCs to prevent chronic GVHD in patients with AA.

Newer reduced-intensity conditioning regimens Considerable interest has been generated recently with the development of reduced-intensity conditioning regimens which can decrease TRM and permit successful allogeneic HCT of older or medically infirm patients with hematologic malignancies, renal cell cancer or inborn errors of metabolism (see Chapter 71) [52,139–141]. The current standard CY + ATG regimen for SAA is nonmyeloablative, although it is a more intensive conditioning regimen compared with the least intensive nonmyeloablative regimen of FLU 90 mg/m2 + 2 Gy TBI. Whether or not an alternative conditioning regimen such as FLU or reduced-dose CY could be less toxic or better tolerated and yet as effective as CY 200 mg/kg + ATG for SAA patients remains to be determined. Recent results with regimens containing FLU 180 mg/m2, CY 120 mg/kg, and ATG with patients receiving primarily G-PBHCs as the HSC source showed a low incidence of graft failure, but a markedly increased rate of acute GVHD and no improvement in overall survival [57]. Another report of 13 primarily young patients receiving the same regimen described less acute GVHD but similar overall survival [59]. To date, there has been no evidence of improvement in survival or reduction in TRM with the intensified FLU with reduced CY-based regimens. A recent report of six patients with SAA who received FLU 90 mg/m2 and 2 Gy TBI, a G-PBHC graft, and the triple-drug GVHD prophylaxis regimen of CSP + MMF + MTX showed that all six patients survived with donor engraftment, although follow-up was brief [142]. These results suggest that alternatives to the standard CY + ATG regimen are worthwhile considering only in the context of clinical trials for patients at high risk for medical complications from the CY 200 mg/kg and ATG regimen. Given the historical experience with high rates of graft rejection for transfusion-sensitized SAA patients, the potential benefit of a slight reduction in conditioning regimen toxicity must be very carefully balanced against the increased risk of complications associated with graft rejection or acute and chronic GVHD. Grafts from alternative marrow donors Transplantation of marrow obtained from unrelated or HLA-mismatched related donors has been less successful than grafts from HLA-identical siblings. There are probably two major reasons for this discrepancy: 1 There is an increased risk of transplant-related complications because of the greater genetic disparity between donors and recipients.

Chapter 49

2 Most patients are not considered candidates for HCT from alternative donors until they have failed attempts at IST. By the time these patients come to transplantation, they are often refractory to platelet transfusions, have received multiple antibiotics because of life-threatening infections, and are often severely infected at the time of transplantation. In order to improve patient survival, transplantation from alternative donors should be considered at earlier time points before patients become severely ill. HLA-nonidentical related donors (see Chapter 46) Data from Seattle and the EBMT group indicate that patients with HLA phenotypically matched related donors have better overall survival, ranging from 64% to 100%, than patients with HLA-mismatched related donors, whose survival is generally 50% or less [143,144]. For patients with phenotypically HLA-matched donors, irradiation proved not to be a necessary component of the conditioning regimen, and survival of CY-conditioned patients was similar to that of patients with HLA-identical sibling donors [143]. For patients with HLA-mismatched related donors, conditioning with CY with or without ATG did not provide sufficient immunosuppression to prevent graft rejection in most patients, and none of the patients achieved long-term survival. High rates of acute and chronic GVHD occurred among those few patients who did engraft [143]. For subsequently transplanted patients with HLA-mismatched related donors, the use of CY combined with TBI decreased the incidence of graft failure and increased survival, but pre- and peritransplant infections and posttransplant GVHD have remained major problems [143]. The optimal dose of TBI for these patients to prevent rejection with a minimal amount of regimen-related toxicity is not known. The general trend is that increasing HLA disparity adversely affects transplant outcome [143,144], consistent with findings in patients transplanted for leukemia [145]. A recent IBMTR survey showed 49% projected survival for one HLA-locus-mismatched HCT, similar to Seattle data that showed 50% survival for these patients [146]. The IBMTR series reported 35% survival for two or more HLA-loci-mismatched recipients, with other studies reporting similarly poor outcomes [143].

to define the minimum effective dose of TBI sufficient to achieve engraftment for patients with AA transplanted with unrelated donor marrow [150]. The starting dose of TBI was 6 Gy (three fractions of 2 Gy) given after three doses of ATG 30 mg/kg/day, combined with four consecutive infusions of CY 50 mg/kg/day. The TBI dose was to be escalated in increments of 2 Gy if graft failure occurred in the absence of prohibitive toxicity, and de-escalated for toxicity in the absence of graft failure. A total of 87 patients were enrolled between 1994 and 2004: 62 patients received HLA-A, B, C, DR, and DQ allele-matched marrow transplants based on molecular typing, and 25 patients received marrow from donors that differed by one or two alleles, or one HLA antigen. The ages of the patients ranged from 1.3 to 53.5 years, with a median age of 18.6 years. The time interval from diagnosis to transplantation was 2.8–328 months, with a median of 14.6 months. All patients had received multiple transfusions and a median of three courses of IST. All 20 patients treated with 6 or 4 Gy TBI, CY, ATG, and HLA-matched marrow engrafted, and survival was 50%. Of the 35 patients receiving 2 Gy TBI, CY, ATG, and HLA-matched marrow, one rejected the graft, and 23 (66%) were alive (Fig. 49.7). Grade 3 or greater pulmonary toxicity occurred in eight of 30 patients conditioned with 6 or 4 Gy TBI, and in one of 35 patients conditioned with 2 Gy TBI. The incidence of acute GVHD was 69%, with eight deaths attributable to acute or chronic

(a)

80 200 cGy 60 400/600 cGy

40

20

Unrelated donors (see Chapter 47)

0 0 (b)

1

2

3

4

5

6

7

8

9

10

7

8

9

10

100 HLA-nonidentical 80

Percent survival

Most patients with AA who do not have suitably HLA-matched related donors receive IST as the first-line treatment of choice. They proceed to unrelated donor BMT only if they fail to respond to IST. Historically, rates of TRM have been high. In a National Marrow Donor Program retrospective analysis of 141 patients transplanted between 1988 and 1995, the overall survival at a median of 36 months after transplant was 36% [147]. Eighty-six percent of these patients received a radiationcontaining conditioning regimen, 74% received HLA-matched marrow, while 26% received marrow mismatched for at least one HLA-A, B or DR antigen; 32% received T-cell-depleted marrow, and all but 13% received a CSP-containing regimen to prevent GVHD. Eighty-nine percent achieved sustained engraftment, and 52% of patients developed grade II–IV acute GVHD. In a pilot study, five patients received unrelated donor transplants following CY and ATG conditioning as used for recipients of HLAidentical donor marrow. Three patients experienced graft failure, and only one patient survived long-term [148]. Other groups reported that high-dose irradiation regimens, although effective in achieving engraftment, resulted in an increased incidence of fatal organ toxicity without increasing the probability of survival. In the past decade, high-resolution HLA typing and identification of an optimized conditioning regimen has resulted in improved survival after unrelated donor HCT [149]. A collaborative multicenter prospective National Marrow Donor Program-sponsored study was undertaken

100 HLA-identical

Percent survival

718

60 400/600 cGy 40 200 cGy 20

0 0

1

2

3 4 5 6 Years after transplant

Fig. 49.7 Outcome of unrelated donor hematopoietic cell transplantation for patients with aplastic anemia. Survival by total body irradiation (TBI) dose in patients that received antithymocyte globulin as part of their conditioning regimen. Shown separately are patients with human leukocyte antigen (HLA) allele-matched (a) and HLA-nonidentical unrelated donors (b). Results with TBI doses of 400 and 600 cGy were pooled. (Reproduced from [150], with permission. Copyright 2006, American Society of Hematology.)

Hematopoietic Cell Transplantation for Aplastic Anemia

GVHD and infection. Chronic GVHD requiring therapy developed in 52% of patients. Nine patients did not tolerate ATG and instead received CY 120 mg/kg and fractionated 12 Gy TBI; five of the nine survived [150]. Age was an important risk factor for survival. Patients who were under the age of 20 years at the time of HLA-identical unrelated BMT conditioned with 2 Gy TBI + CY + ATG had a 78% 5-year survival, compared with 50% for patients older than 20 years. Recipients of HLA-nonidentical unrelated donor grafts had an overall survival of 40% [150]. The IBMTR data for unrelated donor HCT show that, among 181 patients transplanted with low- or intermediate-resolution HLA-matched unrelated donor marrow between 1988 and 1998, the 5-year survival probability was 39% [146]. As multiple suboptimal preparative regimens were used, and high-resolution HLA typing was not applied, the results are inferior to the new regimen identified by Deeg et al. for unrelated HCT. Most unrelated transplants were performed late in the course of disease after failure to respond to one or more courses of immunosuppression. Further, it is now accepted that the widespread adoption of improved molecular techniques for high-resolution HLA typing will result in better outcomes for recipients of HLA-matched unrelated donor HCT [151]. Table 49.3 summarizes the marrow transplant results of patients who received marrow grafts from HLA-matched unrelated donors. In these studies, patients who were younger than 20 years had better survival. As with transplants using HLA-mismatched related donors, nonradiationcontaining conditioning regimens provided insufficient immunosuppression to prevent rejection among patients receiving unrelated marrow grafts [148]. More recent studies have endeavored to achieve engraftment with low-intensity TBI doses [150,152–154]. A recent EBMT study used FLU, low-dose CY (1200 mg/m2), and ATG, and reported a high rate of graft rejection (18%) but a lower incidence of acute and chronic GVHD [155]. Alternative approaches have been to use alemtuzumab instead of ATG in an otherwise similar regimen to the EBMT study [156], or the use of CD34+-selected G-PBHCs [157]. However, transplant outcomes with these approaches have been inferior to those reported from Deeg et al. [150]. In summary, the National Marrow Donor Program-sponsored study reported by Deeg et al. showed improved outcome of patients, and identified a conditioning regimen of 2 Gy TBI, CY, and ATG that achieved reliable engraftment with significantly decreased organ toxicity [150]. These results, combined with the recent advances in molecular HLA typing, now strongly suggest that patients who have unrelated donors fully HLA matched for both class I and II, including HLA-C, by high-resolution DNA-based typing may have superior long-term outcome with HCT over IST. If a high-resolution 10 of 10 HLA-allele matched donor is identified, unrelated donor HCT should be considered early in the course of treatment of patients with SAA, even as first-line therapy, particularly for patients 20 years old or younger. Because of the time interval involved in the donor search, unrelated donor HCT might not be feasible as first-line therapy for acquired SAA. However, patients could proceed to unrelated donor HCT within 4 months if response to IST was unsatisfactory. As with the HLA-identical sibling setting, transfusion with leukodepleted irradiated blood products prior to transplantation is critical to decrease the risk of graft rejection. Although confirmatory data are lacking, younger patients (less than 35 years of age) with one HLA-allele-mismatched donors could also proceed to BMT if treatment with one or possibly two optimal IST regimens had failed [150]. Umbilical cord blood A potential advantage of using umbilical cord blood (UCB) as a source of HSCs for unrelated donor transplantation in severe AA is that HLA

719

mismatching is better tolerated, and therefore may be considered when a fully HLA allele-matched marrow donor is not available. Published data for UCB transplantation in acquired AA are very limited. Further, the low cell dose obtained from a UCB donation poses particular problems in AA, where an increased HSC dose is important to help maximize engraftment. Initial reports of cord blood transplantation for SAA were very discouraging because of a very high incidence of graft rejection and transplant-related death [158]. The recent use of double UCB transplants as a means of increasing the HSC dose for adult recipients has been successful in achieving a high engraftment rate in high-risk MDS/leukemia. For individual patients, only 1 of the 2 units engrafted long term [159]. A recent study from Guangzhou, China demonstrated engraftment in seven of nine adult patients with acquired AA, with sustained mixed chimerism [160]. In five of the patients, 2 units of UCB were infused, one of which engrafted. Follow-up was very limited; further studies of UCB in acquired adult SAA are indicated. Nontransplant approaches The value of immunosuppressive agents for the treatment of AA was first described by Mathé et al. in Paris in 1970 [161]. They observed recovery of autologous hematopoiesis in patients conditioned with antilymphocyte globulin (ALG) administered in preparation for transplants of marrow from HLA-mismatched donors. Later prospective studies showed that the mismatched marrow graft did not contribute to the responsiveness, and subsequently ALG or ATG was used alone to treat patients with AA. More recently, CSP with or without corticosteroids has been added to ATG or ALG [162]. For patients who are not candidates for first-line BMT, the combination of ATG and CSP is the currently recommended immunosuppressive regimen for AA. The response rate to IST (as defined by transfusion independence) for patients with severe AA was 60–70% at 3–4 months [163,164]. Long-term follow-up from a German prospective, multicenter randomized trial comparing ATG alone with ATG + CSP reported an overall survival of 54% and 58%, respectively, at 11 years (Fig. 49.8a). The addition of CSP significantly improved early response to ATG treatment, but because of the efficacy of salvage CSP treatment, there was no overall survival benefit. Failure-free survival, defined as survival in the absence of relapse, no response, PNH, MDS, acute myeloid leukemia (AML) or solid tumors, favored the CSP regimen (39% versus 24%; Fig. 49.8b). Normalization of blood counts occurred in one-third of patients. Risk of relapse was 38%. Remissions were CSP dependent in 26% of patients treated with CSP, necessitating long-term maintenance of the drug. Clonal or malignant diseases developed in 25% of the patients. The actuarial probability of developing hemolytic PNH, MDS/AML or a solid tumor after IST was 10%, 8%, and 11% at 11 years, respectively [164]. The addition of G-CSF to ATG and CSP when administered daily for 3–4 months resulted in improved neutrophil recovery but conferred no benefit in terms of trilineage hematologic recovery or improved survival [165]. The use of high-dose G-CSF for prolonged duration with IST is a risk factor for development of MDS/AML with monosomy 7, particularly in children [166]. A recent European survey included 840 patients who received a first-line IST with (43%) or without (57%) G-CSF. The incidences of MDS/AML in patients who did or did not receive G-CSF were 10.9% and 5.8%, respectively. A significantly higher hazard (1.9) of MDS/AML was associated with use of G-CSF. Relapse of AA was not associated with a worse outcome in patients who did not receive G-CSF as first therapy, whereas relapse was associated with a significantly worse outcome in those patients who received G-CSF [167]. To address this issue definitively, a multicenter prospective EBMT study

1994–2004

1988–1998

1993–2000 1998–2004 1993–2000 2000–2005 1999–2004

2006

2006

2002 2005 2001 2006 2005

NMDP, Deeg et al. [150]

EBMT, Passweg et al. [146]

JMDP, Kojima et al. [154]

EBMT, Bacigalupo et al. [155]

Nagoya, Kojima et al. [152]

Houston, Kennedy-Nasser et al [181]

Seoul, Lee et al. [182]

13

13

15

33

154‡

232

87

Number of patients

15–34 (23)

5–18 (12)

3–19 (11)

3–37 (14)

1–46 (17)

1–55 (16)

1–53 (18.6)

Age range in years (median) GVHD prophylaxis CSP + MTX

CSP + MTX, Varied

MTX + CSP/FK506 CSP + MTX MTX + CSP/FK506 CSP/FK506 MTX + CSP

Conditioning regimen CY + ATG + 2 Gy TBI, n = 46; CY + 4 or 6 Gy TBI + ATG*, n = 41 CY + TBI + others, varied

TBI/LFI + CY ± ATG CY + ATG + FLU 5 Gy TBI + CY + ATG CY + 2 Gy TBI + Campath CY + ATG or CY + FLU or CY + FLU + ATG

Acute grade II–IV

GVHD (%)

Chronic

Survival (%)

1.2–10.2 (7)

Range of follow-up (years) (median)

0

8

0

18

11

23

15

33

11

29

31

9

7

27

30

74

85

100

73

56

0.3–4.2 (3.1)

0.5–4.0 (2.5)

0.2–7.2 (4.25)

0.5–6.0 (1.7)

0.25–6.8 (2.4)

0.2–11.6 (5.1) HLA-matched (low resolution, n = 181): 17 48 29 39 HLA-mismatched (low resolution, n = 51): 24 37 24 36

HLA-allele matched (n = 62): 1 70 52 61† HLA-allele/antigen mismatched (n = 25): 12 75 57 44

Graft failure (%)

ATG, antithymocyte globulin; Campath, alemtuzumab; CSP, cyclosporine; CY, cyclophosphamide; EBMT, European Group for Blood and Marrow Transplantation; FK506, tacrolimus; FLU, fludarabine; GVHD, graft-versus-host disease; HLA, human leukocyte antigen; JMDP; Japan Marrow Donor Program; LFI, limited field irradiation; MTX, methotrexate; NMDP, National Marrow Donor Program; TBI, total body irradiation. * Includes nine patients that did not tolerate ATG, and received CY (120 mg/kg) + 12 Gy TBI. † 5-year survival among 23 patients age ≤20 years conditioned with 2 Gy TBI and disease duration less than 1 year was 85%. ‡ 79 donor pairs matched at HLA-A, B, and DRB1 loci by DNA –typing; 75 mismatched (DNA typing showed 51 mismatched at one HLA locus, and 12 at two or more loci).

Year of HCT

Year of report

Transplant team, reference

Table 49.3 Selected recent reports of hematopoietic cell transplantation (HCT) from unrelated donors

720 Chapter 49

Hematopoietic Cell Transplantation for Aplastic Anemia

study, CY induced prolonged pancytopenia, particularly in patients with pretreatment absolute neutrophil counts greater than 0.5 × 109/L, which contributed to the increased risk of infectious deaths. The authors concluded that high-dose CY without allogeneic HCT was a dangerous choice for treatment of AA. In a subsequent report of late complications, relapse of disease and clonal evolution of cytogenetic abnormalities occurred in both the ATG and the CY treatment groups [171]. Given these results, high-dose CY alone without subsequent HCT is not recommended as an alternative IST regimen for the treatment of SAA.

Cumulative proportion surviving

(a) 1.0 0.8 ATG + CSP

0.6

ATG

0.4 P = 0.6

0.2

Comparison of IST and HCT

0.0 0

3

6

9

12

15

12

15

Years after start of treatment (b) 1.0 Proportion surviving failure-free

721

0.8 0.6 ATG + CSP

0.4

ATG 0.2 P = 0.04 0.0 0

3

6

9

Time to treatment failure (years)

Fig. 49.8 Nontransplant therapy for 84 patients with aplastic anemia. Patients were randomized to receive antithymocyte globulin (ATG) alone or ATG + cyclosporine (CSP). (a) Overall survival. Patients treated with (ATG + CSP) or without (ATG) CSP had similar overall survival times. (b) Failurefree survival. Patients treated with ATG + CSP had longer failure-free survival times than patients treated without CSP (ATG). In contrast to the outcome with hematopoietic cell transplantation, the majority of patients are not cured with immune-suppression therapy (Reproduced from [164], with permission. Copyright 2003, American Society of Hematology.)

comparing IST with or without G-CSF is currently in progress, but the routine use of G-CSF with ATG and CSP outside of randomized clinical trials is not currently recommended. Alternative treatment to ATG or ALG and CSP has also been explored. In a study that extended over 9 years, 10 patients were treated with a high dose of CY (180 mg/kg) without marrow rescue, and seven patients responded with increases in peripheral blood counts so that they no longer met the criteria for SAA. Six patients were alive and without relapse or clonal disorders with a median follow-up of 10.8 years [168]. A more recent study of 19 patients treated with 50 mg/kg/day CY for 4 consecutive days without CSP but with G-CSF support reported an 84% probability of 2-year survival and a 65% probability of complete remission at 4 years [169]. Recovery of neutrophil counts was delayed, and the median time to independence from transfusion of red blood cells and platelets was 11 months. However, a randomized trial conducted at the National Institutes of Health that compared high-dose CY with ATG (both treatment groups including CSP) was terminated early because of excessive infectious mortality in the CY group [170]. In the National Institutes of Health

Several reports have compared the effectiveness of conventional IST to HCT. There have been no randomized trials comparing IST with HCT. Recent studies have consistently shown that there has been a clear survival benefit for patients who underwent HLA-matched HCT [8,37,103,110,172,173]. An earlier large Seattle trial compared the outcome of 168 patients treated with BMT and 227 patients receiving IST between 1978 and 1991. In this study, many of the patients treated with BMT already had failed initial IST. Overall survival in this study at 15 years was 69% for the BMT patients and 38% for patients receiving IST (p < 0.001) [37]. The higher overall survival with BMT compared with IST was seen in all groups of patients under 40 years of age. In a long-term study of 100 consecutive children under the age of 17 years with SAA, the Nagoya transplant team confirmed that HCT was superior to IST [172]. Similar to the results from Seattle, an updated retrospective survival analysis was completed based on the initial treatment offered to 2479 consecutive patients with SAA reported to the EBMT registry from 1991 to 2002 [103]. A total of 1567 patients received BMT and 912 patients received IST. The actuarial 10-year survival was 73% and 68% for patients treated with BMT or IST, respectively (p = 0.002). Comparing the two time periods 1991–1996 to 1997–2002, BMT outcome for all patients improved significantly with time, with overall survival of 69% and 77%, respectively (p = 001). Survival improved over time both for recipients of matched sibling donor transplants (74% and 80%; p = 0.003) and for alternative donor recipients (38% and 65%; p = 0.0001) transplants. After BMT, survival in children was 79%, compared with 68% in adults (p < 0.0001). In multivariate analysis, favorable predictors of BMT outcome were younger age, transplant after 1996, an HLA-identical sibling donor, a brief time interval from diagnosis to transplantation, and no irradiation in the conditioning regimen (p < 0.001). In contrast to the outcome with BMT, there was no significant improvement over time for patients receiving IST (69% survival for patients treated from 1991 to 1996, and 73% survival for patients treated from 1997 to 2002; p = 0.29). Survival after IST was significantly better in children compared with adults (81% versus 70%; p = 0.001). Overall survival after IST for patients with severe AA was better in children (83%) than adults (62%) (p = 0.0002) [103]. One important difference between IST and HCT is that immunosuppression alone may not correct the underlying marrow abnormality. About 30% of IST patients may relapse, that is, have a return of their marrow aplasia, and a substantial proportion may develop clonal abnormalities including MDS and AML [26,163,167,174,175]. In a report of 50 children treated with a combination of G-CSF and CSP, 22% developed MDS or AML [176]. In another study of immunosuppression with ATG and CSP, survival was 72% at 2 years, but 10% of patients had developed PNH [177]. One study compared the incidence of secondary malignancies among patients with HCT versus IST. Forty-two malignancies were noted in 860 patients receiving IST, while nine were reported in 748 patients who received HCT. Overall, the 10-year cumulative incidence of cancer was 18.8% after IST, while the rate was 3.1% after HCT. In this study,

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MDS and acute leukemia were seen exclusively in patients treated with IST, while the incidence of solid tumors after IST versus HCT was similar [175]. The relatively high incidence of malignancies after IST was in agreement with earlier work by Tichelli et al. [178], who reported an incidence of PNH, MDS, and acute leukemia as high as 57% at 8 years after treatment. Thus, long-term survival curves after IST have as yet not become stable in distinction to those after HCT. For some patients, the decision to proceed with BMT versus IST as first-line therapy depends on failure-free survival and quality of life issue analyses. One retrospective study compared quality of life outcomes between 52 transplanted patients and 155 patients receiving IST during the period 1976–1999. Overall and event-free survival were similar between the two groups. However, quality-adjusted time without symptoms and toxicity analysis showed that, compared with BMT, IST-treated patients had longer periods of time with (1) symptoms from drug toxicity, (2) transfusion dependency, (3) partial remission, and (4) secondary clonal disorders. Transplanted patients spent more time in complete remission without drugs, and had longer periods free from symptoms [173]. In most registry studies of IST versus HCT, patients received IST because they lacked an HLA-identical family member donor and were generally not offered unrelated donor transplants as salvage if they failed IST because, at the time of these studies, very little had been published about successful unrelated donor HCT for AA. With the recent increased success of unrelated donor HCT, the salvage rate for patients first given a trial of IST may be considerably greater than seen in the past. This result could change the relative benefits of early transplantation from an unrelated donor versus an initial trial of IST. In summary, over the past three decades, overall survival has improved among patients with AA undergoing allogeneic HCT. There has also been improvement in survival among patients treated with IST, although the improvement was not as significant as with allogeneic HCT. Based on a review of results to date, HCT with CY + ATG conditioning and MTX + CSP GVHD prophylaxis provides superior overall survival and should be recommended as first-line therapy over IST for patients up to age 65 with an HLA-identical sibling. Patients under age 20 years with a 10 of 10 HLA allele-matched unrelated donor should proceed directly to HCT with the CY + ATG + 2 Gy TBI conditioning regimen followed by MTX + CSP GVHD prophylaxis. Older patients or patients who do not have an HLA allele-matched unrelated donor should receive first-line IST with CSP + ATG. If there is failure of IST after 4 months, patients should proceed to an unrelated donor HCT.

Conclusion There are at least two reasons for the improved survival of patients with AA who were treated by HLA-identical HCT. One is the decreased incidence of graft rejection. The decline in rejection has resulted from the more judicious use of transfusions before HCT, the removal of sensitizing white blood cells from transfusion products, and improvements in the immunosuppressive qualities of the conditioning programs used to prepare patients for transplantation. Irradiation-based programs have been effective, but at the price of more transplant-related complications, and the CY + ATG combination is just as successful in preventing rejection with better long-term survival. With regard to transfusions before HCT, in vitro irradiation of all blood products may further reduce the risk of sensitization to minor histocompatibility antigens in the future [32,34]. The second reason for improved survival has been a decrease in the incidence and severity of acute GVHD through the introduction of better GVHD prevention regimens, for example combined MTX and CSP. The incidence of chronic GVHD may be decreasing, but mortality from it has not changed much despite prompt therapy. Therefore, better ways of preventing chronic GVHD are necessary, such as targeting the total corrected donor marrow cell dose to 2.0–2.5 × 108 cells/kg [53]. As more patients become long-term survivors, the problem of long-term sequelae from the initial conditioning programs and from certain postgrafting immunosuppressive agents, such as azathioprine, must be considered, in particular secondary cancer. In future studies, perhaps less toxic conditioning programs can be developed. Radiation-based regimens should not be used in HLAidentical recipients because of the higher likelihood of inducing secondary cancer, the deleterious effect on fertility, and the potential detrimental effects on growth and development for pediatric patients. For patients without HLA-matched sibling donors, transplantation from HLA-mismatched related donors or unrelated donors should be considered. Prompt initiation of an unrelated donor search is needed after diagnosis of SAA in case there is failure to respond to IST within 4 months. Radiation appears to be an essential part of the currently used conditioning regimen for patients with alternative donors to prevent rejection. Recent results with unrelated donor marrow transplantation indicate that 2 Gy TBI, CY, and ATG conditioning, and combined MTX and CSP postgrafting immunosuppression, has been sufficient to prevent graft rejection and avoid TBI-induced organ toxicity [150]. The use of molecular-based methods for identifying optimally HLA allele-matched unrelated donors is critical to further improve survival after transplant.

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157. Benesch M, Urban C, Sykora KW et al. Transplantation of highly purified CD34+ progenitor cells from alternative donors in children with refractory severe aplastic anaemia. Br J Haematol 2004; 125: 58–63. 158. Rubinstein P, Carrier C, Scaradavou A et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998; 339: 1565–77. 159. Barker JN, Weisdorf DJ, Defor TE et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005; 105: 1343–7. 160. Mao P, Zhu Z, Wang H et al. Sustained and stable hematopoietic donor–recipient mixed chimerism after unrelated cord blood transplantation for adult patients with severe aplastic anemia. Eur J Haematol 2005; 75: 430–5. 161. Mathé G, Amiel JL, Schwarzenberg L et al. Bone marrow graft in man after conditioning by antilymphocytic serum. Br Med J 1970; 2: 131–6. 162. Frickhofen N, Kaltwasser JP, Schrezenmeier H et al. Treatment of aplastic anemia with antilymphocyte globulin and methylprednisolone with or without cyclosporine. N Engl J Med 1991; 324: 1297–304. 163. Rosenfeld S, Follmann D, Nunez O, Young NS. Antithymocyte globulin and cyclosporine for severe aplastic anemia: association between hematologic response and long-term outcome. J Am Med Assoc 2003; 289: 1130–5. 164. Frickhofen N, Heimpel H, Kaltwasser JP, Schrezenmeier H. Antithymocyte globulin with or without cyclosporine A: 11-year follow-up of a randomized trial comparing treatments of aplastic anemia. Blood 2003; 101: 1236–42. 165. Gluckman E, Rokicka-Milewska R, Hann I et al. Results and follow-up of a phase III randomized study of recombinant human-granulocyte stimulating factor as support for immunosuppressive therapy in patients with severe aplastic anaemia. Br J Haematol 2002; 119: 1075–82. 166. Kojima S, Ohara A, Tsuchida M et al. Risk factors for evolution of acquired aplastic anemia into myelodysplastic syndrome and acute myeloid leukemia after immunosuppressive therapy in children. Blood 2002; 100: 786–90. 167. Socie G, Mary JY, Schrezenmeier H et al. Granulocyte-stimulating factor and severe aplastic anemia: a survey by the European Group for Blood and Marrow Transplantation (EBMT). Blood 2007; 109: 2794–6. 168. Brodsky RA, Sensenbrenner LL, Jones RJ. Complete remission in severe aplastic anemia after high-dose cyclophosphamide without bone marrow transplantation. Blood 1996; 87: 491–4. 169. Brodsky RA, Sensenbrenner LL, Smith BD et al. Durable treatment-free remission after high-dose cyclophosphamide therapy for previously untreated severe aplastic anemia. Ann Intern Med 2001; 135: 477–83. 170. Tisdale JF, Dunn DE, Geller N et al. High-dose cyclophosphamide in severe aplastic anaemia: a

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50

Robert P. Witherspoon

Hematopoietic Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder that has a varied clinical spectrum [1–3]. Patients may present with intermittent hemolysis, thrombotic events, or pancytopenia initially indistinguishable from idiopathic aplastic anemia. Low-frequency PNH clones may actually be present in a good proportion of patients with aplastic anemia and may appear in association with other hematopoietic diseases, such as myelodysplasia, acute myelogenous leukemia or T-cell leukemia [4]. PNH may also develop following immunosuppressive therapy for aplastic anemia [5,6]. Some patients present with only mild symptoms, while others have life-threatening complications of thrombosis, hemolysis or marrow failure with infection from neutropenia or bleeding from thrombocytopenia. The disease often has a waxing and waning course, sometimes over 15 or more years, and it is difficult to predict when a life-threatening complication will occur [7].

of expression of CD59, one of the PIG-anchored antigens on the cell surface, and a return of the normal apoptosis pattern [12]. Another view is that the mutational frequency of the PIG-A gene on the X chromosome is innately high, and that polyclonal mutated cells exist in normal individuals in small numbers. They have no intrinsic growth advantage, but they happen to be resistant to marrow damage. They are not under pressure to take over normal hematopoiesis unless marrow damage occurs. Clinical PNH develops when immune-mediated bone marrow injury damages normal bone marrow, but the mutated PIG-A cells resist injury. These cells expand under the stress of restoring hematopoiesis. These two hypotheses are fundamentally different. The first one suggests an independent clonal process resulting from a mutation that could potentially evolve to a malignancy and may explain the association, though rare, of PNH with myelodysplasia and leukemia. The second one suggests a process that allows for benign clonal expansion that persists for years and is rarely followed by a hematopoietic malignancy [10].

Epidemiology and etiology Numerous studies have elucidated the molecular basis for PNH. The abnormal clone of cells has a defect in the formation of the phosphatidyl inositol glycans that are part of the anchor proteins on the cell surface that protect red blood cells and other cells from lysis by complement. The defect is due to an acquired somatic mutation in the phosphatidyl inositol glycan-A (PIG-A) gene on the X chromosome of the hematopoietic stem cell [8,9]. The PIG-A gene is responsible for directing the protein synthesis of enzymes that enable the biosynthesis of the PIG anchor proteins as shown in the two left-hand panels of Fig. 50.1. The evolution to clinical expression of the disease is not completely understood. One view is that an environmental toxin or some other unknown mechanism damages the bone marrow. The damage results in somatic mutations in the PIG-A pathway in the hematopoietic stem cell (reviewed in [10]). Numerous mutations have been identified in PNH, some of which completely inactivate the pathway or result in partial inactivation (reviewed in [11]). Some of the PNH clones expand through a selective advantage of resisting apoptosis. This notion is supported by the finding that cell lines from PNH patients show reduced sensitivity to apoptosis induced by irradiation [12]. Restoration of gene activity at the PIG-A mutation site in cell lines has been associated with restoration

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Molecular and clinical biology Although studies of PNH have primarily focused on the red blood cell, the clone affects other cell lines as well [13,14]. The normal anchor proteins are identified on the surface of red blood cells and blood mononuclear cells by CD14, CD16, CD24, CD55, CD59, and CD66b and can be detected by flow cytometry. The acquired mutations result in loss of expression of the anchor proteins and a failure to detect the anchor proteins by flow cytometry [13,15]. The extent to which cells bearing the defect circulate in the blood of affected individuals is dependent on several factors, including (1) the stage of differentiation of the progenitor cell containing the mutation, (2) the type of PIG-A mutation that may cause either a deletion or alteration in the quantity of the enzyme needed for the biosynthesis of the anchor proteins, and (3) the often simultaneous presence of normal unaffected cells in the circulation [14]. Thrombosis in PNH patients is one of the life-threatening complications, and means to predict which patients are at high risk for thrombosis are needed. Patients with clinical symptoms of abdominal pain, hemoglobinuria, esophageal spasm, and impotence are more likely to have thrombotic events [16]. Geographic differences in the incidence of thrombosis have been observed. Latin Americans and AfricanAmericans are at higher risk compared with Europeans and other groups. Ethnicity, therefore, is a risk factor for thrombosis in PNH [17]. The pathogenesis of thrombotic events associated with PNH is not well understood. Complement-mediated hemolysis, impairment of the fibrinolytic system, platelet activation, and leukocyte-derived tissue

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Chapter 50

factors are thought to be responsible [18–20]. These tissue factors may act as procoagulants in the blood of PNH patients and predispose them to thromboses. These circulating particles can be detected in patients with PNH and patients with aplastic anemia with a PNH clone, but not in patients with aplastic anemia without a PNH clone [19]. These findings indicate that platelet activation results in procoagulant phospholipid particles in the blood of PNH patients. The propensity for thrombosis is related to the size of the PNH clone. Among 49 patients with a PNH clone measured in granulocytes, thrombosis occurred in 12 of 22 patients who had more than 61% PNH granulocytes, whereas none of the remaining 27 patients with less than 61% PNH granulocytes had thromboses [16]. Such markers of increased risk of thrombosis in the blood can be of value clinically in identifying which patients are at risk and who could be candidates for anticoagulation and possibly early transplantation.

Clinical description of PNH A diagnosis of PNH should be considered when a patient presents with nonautoimmune hemolytic anemia or aplastic anemia. It should also be included in the differential diagnosis of individuals presenting with a life-threatening thrombosis or with recurrent thromboses (Table 50.1). PNH takes its name from the notion that a drop in blood pH during sleep

Normal X Chromosome

Terminal complement activation

PIG-A mutation

CD59

cell

cell

C5

C5

cell

C5

Eculizumab block

C5a

C5a CD55

Attack on cell surface

PNH with eculizumab

PIG-A mutation

PIG-A gene

Formation of CD55 anchor proteins

PNH

CD59

cell

cell

cell

No lysis

Lysis

No lysis

leads to activation of complement components with subsequent nocturnal hemolysis [11]. In fact, patients may recognize a low level of continuous hemolysis only with urination in the morning after free hemoglobin has accumulated overnight in the urine. However, PNH is somewhat a misnomer because the hemolysis is not necessarily nocturnal or paroxysmal. As the abnormal PNH clone expands due to the selective resistance to apoptosis, and perhaps due to selective pressure to restore hematopoiesis after marrow damage, the hemolytic events can be expected to occur more frequently. The time-honored tests to diagnose patients in whom PNH is suspected have been the sugar water hemolysis and Ham’s tests. However, these tests are positive only when abnormal cells remain in circulation after a major hemolytic episode. The recognition of the loss of the anchor proteins associated with CD59 and other antigens has revolutionized the diagnosis by making it possible to quantitatively determine the percentage of cells affected. The recognition of the abnormalities of expression of other anchor proteins including CD14 and CD55 on affected monocytes, the expression of anchor proteins CD16, CD24, CD59, and CD66b on neutrophils, and that of CD24 and CD59 on lymphocytes makes it possible to establish the diagnosis by flow cytometry of cells that remain in circulation [13,14]. Flow cytometry has become the standard screening test to diagnose patients earlier in the course of PNH. PNH and idiopathic aplastic anemia are closely related disorders [21]. Patients who appear to have idiopathic aplastic anemia are found by flow cytometry to have small percentages of cells in circulation that do not express CD14, CD16 or CD59 [22]. More sensitive tests have been developed that raise questions about the relationship between the two different diseases [23]. Aerolysin is a toxin that binds to cells via the anchor proteins encoded by the PIG-A gene. Since PNH red cells cannot bind the toxin, they survive in an aerolysin-based assay. Using this assay, residual PNH cells can be detected in samples from PNH patients but not in cells from patients with myelodysplasia or in normal control samples. However, as many as 60% of the cells obtained from patients who have idiopathic aplastic anemia and not PNH by flow cytometry before treatment with the aerolysin toxin show characteristics of PNH after treatment [23] (Table 50.1). This sensitive test indicates that previously undetectable PNH cells of different lineages circulate in patients with untreated aplastic anemia, and points to the possibility of clonality in the early stages of aplastic anemia.

Nontransplant treatment of PNH

Fig. 50.1 Pathway showing formation of anchor proteins that protect cells from lysis by activated complement in normal individuals, subjects with paroxysmal nocturnal hemoglobinuria (PNH), and the effect of eculizumab to prevent activation of C5 to C5a. C5, fifth component of complement; C5a, activated fifth component of complement; CD55, CD59, anchor proteins; PIG-A, phosphatidyl inositol glycan-A gene.

Immunosuppressive therapy has been used for the treatment of PNH, most often when the presentation is that of aplastic anemia. However, the use of corticosteroids, cyclosporine, granulocyte colony-stimulating factor, and antithymocyte globulin (ATG) does not eradicate the abnormal clone, and patients may continue to be symptomatic from the disease

Table 50.1 Clinical and laboratory characteristics of paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia and myelodysplasia

Clinical features Aplastic/cytopenia Nonautoimmune hemolytic anemia Thrombosis Laboratory findings Ham’s test on red blood cells Flow cytometry detection of CD55 and CD59 on red blood cells and leukocytes Aerolysin detection of PNH type II and III cells

Normal control

PNH

Aplastic anemia

Myelodysplasia

− − −

+ + +

+ − −

+/− − −

− +

+ −

+/− +/−

− +

−

+

+/−

−

Hematopoietic Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria

[24–27]. In a small number of patients, high-dose cyclophosphamide (CY) without marrow infusion has resulted in autologous marrow recovery without persistence of the PNH clone [28]. By far the most significant advance in nontransplant therapy for PNH is the recent development of eculizumab, a humanized monoclonal antibody against the complement protein C5, thereby blocking terminal complement activation as shown in the far right side panel of Fig. 50.1. Eculizumab prevents hemolysis in patients with PNH. In a randomized placebo-controlled, multicenter phase III trial, 21 of 43 patients assigned to eculizumab achieved stabilization of hemoglobin levels and transfusion independence, whereas none of 44 patients assigned to placebo achieved those outcomes. Eculizumab reduced intravascular hemolysis by 86% and was associated with improved quality of life measures [29]. Thrombosis was not a primary outcome in the study, so it is not known whether thrombosis is abrogated. Further studies will be needed to learn whether eculizumab will have any effect on the frequency of thrombosis, how long patients can remain under treatment, and whether long-term therapy is feasible [30]. For patients with the aplastic manifestations of PNH, it is not expected that eculizumab will have an impact.

Transplantation for PNH Hematopoietic cell transplantation (HCT) from a human leukocyte antigen (HLA)-identical sibling for a patient with PNH was first reported in 1973 [31]. This patient was found to have PNH at the time of presen-

729

tation of severe aplastic anemia and had a positive sucrose hemolysis or Ham’s test. In subsequent reports, most patients had life-threatening disease from marrow aplasia, and hemolysis played a less important role at the time of HCT [32–35]. Subsequently, patients with hemolysis or thrombosis were evaluated for transplantation. Overall, the number of individuals who have been transplanted is small (Table 50.2). The experience in Seattle with transplantation for nine patients was summarized in 1992 [36]. Subsequently, 19 additional patients have been transplanted, and the results are updated in Table 50.3. Among the total of 28 patients, 12 presented with the severe aplastic anemia phase of PNH and had severely hypocellular marrows. The remaining 16 patients presented with hemolysis or thrombosis and had moderately cellular to hypercellular marrows. One of these (patient 28) also had documented thromboses associated with the presence of the factor V Leiden abnormality. Data from Table 50.3 are summarized in Table 50.4. Conditioning for grafting of the patients with aplastic anemia who had HLA-identical sibling donors consisted of 200 mg/kg CY with or without ATG in five patients, and 16 mg/kg busulfan (BU) with 60 mg/ kg CY in two patients. One of these patients with aplastic anemia received two grafts from the same donor. The patient with aplastic anemia and the haploidentical parental graft was conditioned with 120 mg/kg CY and 1200 cGy total body irradiation (TBI). The syngeneic recipient with aplastic anemia received an infusion of marrow without conditioning. One patient with aplastic anemia received an unrelated umbilical cord blood HCT after myeloablative conditioning

Table 50.2 Published reports of transplantation for paroxysmal nocturnal hemoglobinuria (PNH) Author

Number of patients

Storb [31] Fefer [32] Kolb [33]

1 1 2

Antin [34] Szer [35]

4 4

Kawahara* [36]

9

Saso [37]

57

Bemba [54] Raiola [55] Graham [46] Hershko [47] Endo [45] Woodard [41] Suenaga [44] Markiewicz [42] Van den Heuvel-Eibrink [38] Lee [40] Ditschkowski [39] Lee [49] Hegenbart [48] Flotho [43] Takahashi [50] Grosskreutz [51]

16 7 1 1 1 3 1 2 5 1 1 5 7 2 5 1

Type of donor and number of each

Conditioning regimen

PNH after transplant

Allogeneic 1 Syngeneic 1 Allogeneic 1 Syngeneic 1 Allogeneic 4 Allogeneic 3 Syngeneic 1 Allogeneic 7 Syngeneic 2 Allogeneic 55 Syngeneic 2 Allogeneic 16 Allogeneic 7 Syngeneic 1 Syngeneic 1 Syngeneic 1 Allogeneic 3 Allogeneic Allogeneic Allogeneic Allogeneic Allogeneic Allogeneic Allogeneic Allogeneic Allogeneic Allogeneic

Yes No Yes No Yes Yes No Yes No Yes No/Yes Yes Yes Yes No No Yes Yes – RI Yes Yes Yes – RI Yes Yes – RI Yes – RI Yes Yes – RI Yes – RI

No Yes No Yes No No Yes No† Yes No Yes No No No Yes Yes No No No No No No No No No No No

RI, reduced-intensity conditioning regimen. * Reference [36] contains cases previously reported in [31,32,35]. † One allogeneic recipient (patient 8; Table 50.3) had a return of the PNH clone 2.7 years after transplantation and is asymptomatic.

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Table 50.3 Patients in the Seattle Transplant Program

Age

HCT donor

Paroxysmal nocturnal hemoglobinuria presentation

1 2 3

23 19 14

HLA ID SIB Syngeneic HLA ID SIB

A A A

4 5 6

16 29 38

HLA ID SIB Syngeneic HLA ID SIB

7

20

8 9 10 11 12 13 14 15 16 17 18 19 20

37 22 25 32 29 33 21 42 39 41 19 21 35

Haplo parent/Haplo SIB HLA ID SIB HLA ID SIB HLA ID SIB HLA ID SIB Matched Unrel 1Ag MM parent HLA ID SIB HLA ID SIB HLA ID SIB 1Ag MM child MM SIB HLA ID SIB Unrel cord blood

21 22 23 24 25 26 27 28

36 33 18 32 14 67 41 31

Matched Unrel HLA ID SIB HLA ID SIB Matched Unrel Unrel cord blood Matched Unrel PBSC HLA ID SIB PBSC Matched Unrel PBSC

Patient number

Conditioning regimen

GVHD acute grade/chronic

Status

Survival post transplant at last contact

Cause of death

Hepatitis C Suicide

0/No 0/No II/Limited

A D D

>35.0 years 25.2 years 28.5 years

A H A

CY None PA PA PA BC CY BU CY CY None BC CY

0/No 0/0 II/Clin Ext

A A D

>27.7 years >25.2 years 9.3 years

A

CY TBI ATG BC CY

No/NA

D

39 days

T H H H H T, H A T H H H H T

BU CY BU CY BU CY ATG BU CY ATG BU CY ATG BU CY ATG BU CY ATG BU CY ATG BU CY BU CY ATG BU CY ATG BU CY ATG CY TBI 1350

0/Clin Ext II/No II/NA IV/Clin Ext IV/NA III/NA II/No 0/NA 0/Subclin II/Clin Ext III/Clin Ext 0/Sub Clin III/Clin Ext

A D D D D D A D A A D A D

>16.6 years 14.6 years 105 days 83 days 58 days 81 days >10.9 years 11 days >5.0 years >8.1 years 4.1 years >7.0 years 169 days

T A A H A A H, A, ICMF A

ATG BU CY ATG CY ATG BU CY BU CY ATG CY TBI 1350 FLU TBI 200 BU FLU ATG FLU TBI 200

II/Clin Ext II/Limited III/Clin Ext 0/0 II/Clin Ext II/Clin Ext II/0 0/0

A A A A D A A D

>6.5 years >6.1 years >6.1 years >1.0 years 1.7 years >1.0 yrs >1.6 yrs 176 days

Human immunodeficiency virus Pulmonary hemorrhage

Hepatitis C Fungal pneumonia GVHD Infection GVHD Infection Fungal pneumonia Bleeding varices

Respiratory failure Post-transplant lymphoproliferative disorder

Myocardial infarction

Possible cerebral hemorrhage

GVHD, graft-versus-host disease. HCT donor: 1Ag, 1 antigen; haplo, haploidentical; HLA ID SIB, human leukocyte antigen-identical sibling; MM, mismatched; PBSC, peripheral blood stem cells; Unrel, unrelated. Paroxysmal nocturnal hemoglobinuria presentation: A, aplastic anemia; H, hemolysis; ICMF, idiopathic chronic myelofibrosis; T, thrombosis. Conditioning regimen: ATG, antithymocyte globulin; BC, donor buffy coat; BU, busulfan; CY, cyclophosphamide; FLU, fludarabine; PA, procarbazine + ATG; TBI 1350, 1350 cGy total body irradiation; TBI 200, 200 cGy total body irradiation. GVHD acute grade/chronic: Clin ext, clinical extensive; NA, not applicable; Subclin, subclinical. Status: A, alive; D, dead.

with ATG, BU, and 1350 cGy TBI. Two patients received matched unrelated peripheral blood stem cells following conditioning with fludarabine 90 mg/m2 body surface area and 200 cGy TBI. The conditioning regimens for the HLA-identical siblings with thrombotic or hemolytic presentations of PNH consisted of BU CY plus ATG in four patients, and BU CY in three patients. One patient with a history of idiopathic chronic myelofibrosis, hemolysis, and pancytopenia received an HLA-identical sibling peripheral blood stem cells graft after BU, fludarabine 120 mg/m2 body surface area, and ATG. The recipients of the parental donor graft and the child donor graft received BU CY plus ATG, or BU CY respectively. The mismatched sibling received BU CY and ATG. Among three recipients of unrelated donor marrow, two were conditioned with BU CY and ATG, and one with BU CY. One

patient received CY, ATG, and 1350 cGy TBI for an unrelated umbilical cord blood graft. The syngeneic marrow recipient did not receive conditioning. Fifteen of the 28 patients developed acute graft-versus-host disease (GVHD) (10 grade II, three grade III, and two grade IV), and 10 subsequently developed clinically extensive chronic GVHD. PNH recurred 9–17 years after transplant in both syngeneic recipients, and 2.7 years after transplantation in one allogeneic recipient [36]. Fourteen patients died. The causes of death included GVHD complicated by infection in two patients, fungal pneumonia in two patients, and a gastrointestinal bleed in one patient. The latter patient had thromboses of the portal and splenic veins from PNH, which led to bleeding varices. One patient rejected the haploidentical parental graft and died of pulmonary

Hematopoietic Cell Transplantation for Paroxysmal Nocturnal Hemoglobinuria

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Table 50.4 Summary of patients in the Seattle Transplant Program (n = 28): donor type, conditioning regimen, and survival Aplastic anemia presentation HLA identical sibling CY ± ATG BU CY Haploidentical family TBI 1200 CY Unrelated umbilical cord TBI 1350 BU ATG Unrelated donor reduced intensity TBI 200 FLU Syngeneic None Survival 100 days post transplant Survival at last contact Alive and well Chronic GVHD

12

Hemolytic thrombotic presentation HLA identical sibling BU CY ± ATG BU FLU ATG Haploidentical family BU CY ± ATG Mismatched sibling BU CY ATG Unrelated umbilical cord TBI 1350 CY ATG Unrelated donor BU CY ± ATG Syngeneic None Survival 100 days post transplant Survival at last contact Alive and well Chronic GVHD

16

5 2 1 1 2 1 11 6 3 3

7 1 2 1 1 3 1 9 8 3 5

ATG, antithymocyte globulin; BU, busulfan; CY, cyclophosphamide; FLU, fludarabine; GVHD, graft-versus-host disease; TBI 1200, 1200 cGy total body irradiation; TBI 1350, 1350 cGy total body irradiation.

hemorrhage shortly after a second transplant from another haploidentical family member. One patient died of a post-transplant lymphoproliferative disorder 169 days after an unrelated umbilical cord blood transplant. One died from human immunodeficiency virus (HIV) infection 9.3 years after transplantation. One died of hepatitis C 14.6 years after transplant, and one syngeneic recipient died 25.2 years after transplantation as a result of complications of a liver transplant for hepatitis C. One developed myelodysplasia in the donor cells 26.9 years after transplant and had a second successful myeloablative transplant from a different HLAidentical sibling following BU CY, but committed suicide about a year later, 28.5 years after the first transplant. One each died of probable cerebral hemorrhage, myocardial infarction, and respiratory failure of unknown cause 176 days, 1.7 years, and 4.1 years, respectively after HCT. Fourteen patients are surviving more than 1–35 (median 7.6) years after transplantation. Six of these individuals are well, and eight have clinically extensive chronic GVHD. These results show that patients with the aplastic anemia presentation of PNH do well; none of the eight aplastic anemia patients who received HLA-identical sibling or syngeneic grafts died from the complications

Fig. 50.2 Survival 4 months after transplant. The solid line (-) represents eight patients with the aplastic anemia presentation of paroxysmal nocturnal hemoglobinuria (PNH) who had human leukocyte antigen-identical sibling or syngeneic donors compared with 16 patients with the hemolytic or thrombotic presentations with any type of donor (---).

of transplantation (Fig. 50.2). One of these died later from HIV infection before the era of blood screening for HIV. In contrast, eight of the 16 patients who received a transplant from any type of donor for the hemolytic or thrombotic presentations of PNH died. Five of these patients died within 120 days after transplantation of complications from GVHD with nonfungal infection, fungal infection or variceal bleeding. Three died later, one each of hepatitis C, post-transplant lymphoproliferative disorder, and respiratory failure presumably related to chronic GVHD. The International Bone Marrow Transplant Registry has reported the results of 57 patients with PNH who were transplanted at 31 different centers [37]. Thirty-nine of these patients presented with PNH as the only diagnosis. Sixteen evolved from PNH into an aplastic anemia presentation by the time of HCT, and two developed PNH syndrome after initially presenting with aplastic anemia. The donors were HLAidentical siblings in 48 cases, syngeneic donors in two cases, a haploidentical family member in one case, and HLA phenotypically identical, unrelated donors in six cases. These patients received conditioning consisting of BU CY for 30 patients, CY/TBI for 12, limited field irradiation and CY for 11, and CY alone for three patients. One identical twin recipient did not receive any pretransplant conditioning. GVHD prophylaxis consisted of methotrexate or cyclosporine plus methotrexate in 39 patients, and cyclosporine plus corticosteroids in 11 patients. Six patients received T-cell-depleted grafts, and one identical twin recipient did not receive prophylaxis. Graft failure occurred in 11 of 48 patients evaluable for engraftment. Of those with engraftment, 34% developed grade II or greater acute GVHD, and 33% of patients surviving beyond 90 days developed chronic GVHD. Overall, 30 of the 57 patients survived. Among those 48 patients receiving HLA-identical sibling grafts, 27 (56%) survived compared with one survivor among seven patients (14%) receiving HLA-nonidentical family member or unrelated donor grafts. Six patients in three reports had matched sibling donors for hemolytic or aplastic presentation, and five survived transplantation with myeloablative conditioning regimens without PNH recurrence [38–40]. One report describes three patients with the aplastic anemia presentation of PNH given unrelated donor bone marrow grafts following T-cell depletion of marrow. The post-transplant course was complicated by an

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Epstein–Barr virus-positive lymphoproliferative disorder that responded to cytotoxic T-cell therapy specific for this virus. Another patient had veno-occlusive disease and another severe hemolytic uremic syndrome and hemorrhagic cystitis. However, they all recovered and are alive without PNH 2.5–5.1 years after transplantation [41]. Three reports describe seven unrelated donor transplant recipients with myeloablative conditioning regimens for aplastic or hemolytic presentations of PNH, five of whom survived without subsequent recurrence of their disease [38,42,43]. These data suggest that survival after HCT for the aplastic phase of PNH using HLA-identical sibling donors is quite good. However, transplantation is successful in only about half of the cases with nonaplastic presentations of PNH using HLA-identical sibling donors. Results using partially matched family member donors are poor. Finally, although the results using matched unrelated donors are encouraging, the reports are few.

Transplants following reduced-intensity conditioning Because of reduced toxicity, nonmyeloablative or reduced-intensity conditioning regimens are appealing. One report describes a patient with long-standing hemolysis who had acute renal failure after an episode of hemolysis and infection. He received HCT from an HLA-identical sibling after conditioning with 8 mg/kg BU, 0.66 mg/kg cladribine, and 5 mg/kg ATG, with cyclosporine for GVHD prophylaxis. The patient subsequently had sustained engraftment and no detectable PNH-positive cells [44]. The dosage of BU in this case was close to the dosage used in myeloablative regimens, and one could argue that this case was not a typical reduced-intensity transplant. The reduced-intensity conditioning regimens often used may be insufficient for sustained donor engraftment in patients with nonaplastic presentations because even those syngeneic recipients of marrow for the aplastic phase of PNH required conditioning regimens to achieve sustained donor engraftment (Table 50.2) [32,33,36,37,45–47]. Furthermore, sufficient conditioning therapy perhaps in combination with GVHD may be necessary to eliminate the PNH clone because syngeneic recipients who did not receive pretransplant conditioning eventually had a return of PNH disease years later [35,36]. Except for one patient, recipients of allogeneic stem cells had sustained elimination of the disease (Table 50.3) [36]. These results suggest that a graft-versus-PNH type of effect in either a myeloablative or reduced-intensity allogeneic transplant procedure makes an important contribution toward eliminating the PNH clone. In this regard, seven patients with PNH – two with aplastic anemia and five with the nonaplastic presentation – received reduced-intensity transplants from matched related (n = 2) or unrelated (n = 5) donors after conditioning with 90 mg/m2 body surface area fludarabine and 200 cGy TBI. These patients achieved complete donor chimerism by day 56 and lost all signs of the PNH clone by flow cytometry. Unfortunately, three

patients died of treatment-related complications, one from acute pancreatitis, one from infection during treatment for chronic GVHD, and one from bleeding after liver biopsy during assessment for chronic GVHD. Four patients are alive with follow-up ranging from 1 to more than 3 years after HCT [48]. There have been reports of individual cases treated by reduced-intensity HCT approaches with successful elimination of PNH that support the idea that immunologically based eradication of PNH cells is possible following reduced-intensity conditioning regimens (Table 50.2) [40,49–51]. Further studies of the reduced-intensity approach are warranted, especially for patients who cannot undergo conventional HCT because of an increased risk of transplant-related mortality due to pretransplant organ dysfunction.

Autologous transplantation for PNH An interesting hypothesis has been put forward by Musto et al. [52]. They demonstrated that flow cytometry could select CD34+/CD59+ cells from PNH patients who had a mixed population of circulating normal and abnormal cells. If these cells do not bear the mutation in the PIG-A gene, a rationale to mobilize and select autologous normal hematopoietic stem cells for autologous transplantation exists, although no autologous transplant cases have been reported [27].

Conclusion The diagnosis of PNH alone should not be the sole indication for HCT therapy. Defining the risk of a fatal complication in an individual PNH patient is still the most important determination to make before recommending HCT. Eculizumab may modify the risk of hemolysis, although it does not seem likely it will affect the aplastic phase. Whether it will modify the risk of thrombosis is unclear, and the feasibility of long-term administration is uncertain at this time. Risk factors for a poor outcome in the natural history of PNH are thrombosis at initial presentation, evolution to pancytopenia and aplastic anemia, thrombocytopenia at diagnosis, evolution to myelodysplasia or leukemia, and age over 55 years at diagnosis. Patients with infection at diagnosis are at greater risk for thrombosis [7]. When the patient has a life-threatening presentation with aplastic anemia and an HLA-identical sibling or identical twin donor is available, HCT is recommended. Unrelated donors have been used to achieve successful grafts in patients with idiopathic aplastic anemia, and HCT with a matched, unrelated donor for the life-threatening aplastic phase of PNH is warranted, although the experience is small [38,41–43,53]. Reducedintensity conditioning for HCT should be explored further, especially for patients with severe PNH who cannot tolerate a full myeloablative transplant due to comorbid medical conditions. In all other phases of disease, the care should be supportive, and transplantation should be done when the disease becomes life-threatening, or reliable predictors are developed to identify which patients with the nonaplastic phases of PNH are at particularly high risk of death from PNH.

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Jerald P. Radich & Ravi Bhatia

Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia

Introduction Chronic myeloid leukemia (CML) is a malignant disorder of hematopoiesis resulting from the clonal expansion of a primitive hematopoietic cell. At the outset of disease, this malignant stem cell retains the capacity to differentiate, leading to marked marrow hyperplasia and increased numbers of myeloid cells and platelets in the peripheral blood. The natural history of untreated CML is a relatively benign chronic phase (CP) lasting on average approximately 3 years followed by an accelerated phase (AP) lasting several months, eventually terminating in a rapidly fatal blast crisis (BC). CML was the first malignant disease found to be consistently associated with a specific cytogenetic abnormality, the Philadelphia (Ph) chromosome, and was one of the first diseases clearly curable by transplantation. The advent of the tyrosine kinase inhibitor (TKI) imatinib mesylate (Gleevec) has changed the landscape of CML therapy, and has moved transplantation from the up-front therapy of choice to an option for salvage therapy.

Incidence and epidemiology CML represents 14% of all new leukemia cases in the United States, with an annual incidence of 1.6 cases per 100,000 per year [1]. The median age at diagnosis is 67 years, and the incidence sharply rises with age [2]. Exposure to ionizing radiation (e.g. survivors of the atomic bomb) is the only known risk factor [3].

Molecular biology Nowell and Hungerford in 1960 [4] described the presence of a small chromosome in metaphase preparations of marrow from patients with CML. This abnormal chromosome, termed the Philadelphia chromosome after the city where it was discovered, was later shown by Rowley to be the result of a translocation between chromosomes 9 and 22 [t(9; 22)(q34;q11)] [5]. The result of this translocation is the fusion of the BCR (breakpoint cluster region) gene on chromosome 22 to the ABL (Abelson leukemia virus) gene on chromosome 9. The translocation results in the production of an abnormal BCR fusion protein, which is a constitutively active cytoplasmic tyrosine kinase [6–9]. The Ph chromosome is found in cells of myeloid, erythroid, megakaryocytic, and B-lymphoid origin, demonstrating the stem cell nature of the disease. As the disease accelerates and enters BC, additional cytogenetic

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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abnormalities develop, including duplication of the Ph chromosome and trisomy 8 [10]. In murine models, BCR-ABL can cause myeloproliferative diseases similar to human CML, although the CP has been difficult to simulate [11–13]. Epidemiologic models of human CML incidence suggest a multihit disease [14]. In vitro studies suggest that the BCR-ABL expression allows cells to become cytokine independent [15], protects cells from apoptotic responses to DNA damage [16], and increases adhesion of hematopoietic cells to extracellular matrix proteins [17]. The normal ABL protein is a nonreceptor tyrosine kinase with important roles in signal transduction and the regulation of cell growth [18]. The BCRABL protein, unlike normal ABL, is constitutively active and has increased kinase activity, leading to continuous activation of a number of cytoplasmic and nuclear signal transduction pathways including STAT, RAS, JUN, MYC, and phosphatidylinositol-3 kinase [8].

Clinical description Most CML patients (>90%) present in CP, often diagnosed incidentally during routine examination. Symptoms, when present, usually include fatigue, weight loss, bony aches, and abdominal discomfort from splenomegaly. Patients generally present with leukocytosis, thrombocytosis, and anemia; the marrow is hypercellular. The cytogenetic examination shows the Ph chromosome in over 90% of patients, and in the remaining 10%, cryptic or complex translocations can be detected by fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) assays. Without therapy, CML evolves from CP to AP and eventually to BC. In approximately 25% of patients, there is no intervening AP between CP and BC. Indeed, molecular studies show that there is no clear distinction between AP and BC [19]. Various definitions of AP have been developed. Two of the more commonly employed are those of Sokal et al. [20] and the International Bone Marrow Transplant Registry (IBMTR) [20] (Table 51.1). In general, AP is characterized by symptoms of fever, night sweats, weight loss, and bone pain, difficulty in controlling blood counts using conventional therapy, increased numbers of blasts and early myeloid cells in marrow and peripheral blood, and evidence of karyotypic evolution. The most common cytogenetic changes associated with disease evolution are an additional Ph chromosome, trisomy 8, isochrome i(17q), and trisomy 19 [21]. BC has been defined as having more than 30% blasts and promyelocytes in the bone marrow or peripheral blood, or the development of extramedullary blastic infiltrates [20], although a recent reclassification by the World Health Organization has lowered the blast cut-off to 20%. In approximately two-thirds of patients, the blasts are predominately myeloid, while in the remaining third the blasts have a lymphoid phenotype [22].

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Table 51.1 Definition of accelerated phase chronic myeloid leukemia Sokal criteria [20]

IBMTR criteria [82]

Peripheral blood or marrow blasts ≥5% Basophils >20% Platelet count ≥1000 × 109/L despite adequate therapy Karyotype evolution Frequent Pelger–Huet-like neutrophils; nucleated crythrocytes, megakaryocytic nuclear fragments Marrow collagen fibrosis Anemia or thrombocytopenia unrelated to therapy Progressive splenomegaly Leukocyte doubling time <5 days Fever not otherwise explained

Leukocyte count difficult to control with conventional therapy Rapid doubling time of leukocytes (<5 days) Peripheral blood or marrow blasts ≥10% Peripheral blood or marrow blasts plus promyelocytes ≥20% Peripheral blood basophils and eosinophils ≥20% Anemia or thrombocytopenia unresponsive to therapy Persistent thrombocytosis Karyotypic evolution Progressive splenomegaly Development of chloromas or myelofibrosis

IBMTR, International Bone Marrow Transplant Registry.

The natural history of CML has a median survival from diagnosis of approximately 3 years [22]. A number of prognostic scoring systems have been developed with the goal of predicting the length of CP in individual patients, the best known and widely used being by Sokal et al. [23]. In an algorithm, spleen size, percentage of circulating blasts, platelet count, and age were identified as prognostic factors for patients in CP. However, the Sokal scale was based on therapies considered archaic today (busulfan [BU] and splenectomy), and newer systems for patients treated with interferon (IFN) have resulted in newer prognostic scoring systems [24,25].

Non-transplant therapies The pre-imatinib era The earliest chemotherapy for CML, circa the 1950s and 60s, was with BU [26] and hydroxyurea [27]. In randomized trials, hydroxyurea was shown to prolong survival of patients with CP CML compared with BU [28,29], but both therapies were considered palliative. IFN-α was first reported to have activity in CP CML in 1986 [30]. The mechanisms by which IFN works in CML are not understood, but it has been hypothesized that IFN controls proliferation or adhesion of the malignant progenitor in CML, or that it functions by stimulating an immune response to CML. IFN can induce hematologic remissions in 70–80% of CP cases. However, partial or complete cytogenetic responses (defined as having less than 34% Ph+ cells in the marrow) can occur in 20–30% of patients. The cost of the increased activity is toxicity, as side-effects are common with IFN and include fatigue, myalgia, arthralgia, weight loss, depression, memory changes, and autoimmune disorders. In fact, 14–25% of patients on the randomized trials discontinued IFN as a result of side-effects. In an effort to improve response rates and duration, several investigators have combined low-dose cytarabine with IFN. While a benefit was suggested in one study, the results looked less promising in a second [31,32]. In both trials, the addition of cytarabine substantially increased gastrointestinal and hematologic toxicity. Tyrosine kinase inhibitors The advent of TKI therapy has fundamentally changed the approach of treating CML. The icon of TKI therapy is imatinib mesylate (STI 571, Gleevec). Imatinib is a small-molecule inhibitor of several protein tyrosine kinases, including the ABL tyrosine kinase, c-KIT, and plateletderived growth factor. Druker et al. [33] found that imatinib specifically

inhibited or killed proliferating myeloid cell lines containing BCR-ABL but had no effect on normal cells. A phase I trial was initiated in 1998 testing imatinib in patients with CP CML who had failed therapy with IFN [34]. Of 54 patients who received oral doses of imatinib of 300 mg/ day or more, 53 had a complete hematologic response (CHR), usually within 4 weeks of starting the drug. In addition, cytogenetic responses were seen in 54% of patients. In a companion study, 58 patients with CML in myeloid or lymphoid BC or Ph+ acute lymphoblastic leukemia (ALL) in relapse were studied [35]. Partial or complete responses were seen in 60% of patients with CML in myeloid BC, and 70% of patients with CML in lymphoid BC or recurrent Ph+ ALL had partial or complete responses. Unfortunately, most of the responses in patients with CML and all of the responses in patients with ALL were brief, usually less than 4 months. The toxicity profile of imatinib in these phase I studies suggests that it is more toxic than hydroxyurea but far easier to take than IFN. Nausea, edema, and muscle cramps were seen in approximately 50% of patients, with diarrhea, vomiting, rash, and headache seen in one-third [36]. The initial phase I results were confirmed in broader phase II studies of imatinib, and, based on these results, the drug was approved by the United States Food and Drug Administration in May 2001 for treatment of CP CML refractory to IFN, AP, and BC. Subsequently, 1106 patients were entered onto a phase III study comparing imatinib with IFN plus cytarabine for the treatment of newly diagnosed CP CML [37]. At 12 months, complete cytogenetic remission (CCyR) was seen in nearly 70% of the imatinib treated patients versus 7% with IFN and cytarabine therapy. Disease progression was seen in 1.4% of the imatinib group versus 10.3% of the patients treated with IFN. Crossovers as a result of intolerance occurred in 1% of imatinib- and 19% of IFN-treated patients. Based primarily on a higher rate of disease progression with IFN plus cytarabine, the study was closed by the study’s independent monitoring board, with the conclusion that imatinib is the initial nontransplant treatment of choice for patients with newly diagnosed CP CML. A follow-up on the study confirmed the dramatic response of CML to imatinib. At a median follow-up of 60 months, the overall survival of patients who received imatinib as initial therapy was 89%, and progression to advanced phase occurred in only 7% [38]. Eighty-seven percent of patients achieved a CCyR at some point of their imatinib therapy. Nearly 70% of patients remained in CCyR at the 5-year mark. Despite the impressive clinical efficacy of imatinib, development of resistance and intolerance are a concern. Resistance to imatinib can be either primary (intrinsic), where patients fail to respond to treatment, or secondary (acquired), where resistance develops after an initial response

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[39]. In the International Randomized IFN vs. ST1571 (IRIS) trial, an estimated 16% of patients with newly diagnosed CP CML failed to achieve a major cytogenetic response (MCyR) after 12 months of imatinib, and approximately 24% failed to achieve a CCyR after 18 months of imatinib therapy [40]. Acquired resistance was also observed in 17% of cases, with 7% of patients progressing to AP or BC CML [38]. Like all other treatments for CML, the rates of efficacy, resistance, and relapse greatly favor treatment in CP rather than the advanced phases (AP or BC) of the disease [41,42]. Resistance to imatinib is described as either primary, where patients fail to achieve treatment milestones, or secondary, when patients relapse once they had had a good initial response to therapy. Primary resistance is likely through BCR-ABL-independent mechanisms, such as alterations in drug transporter influx (octamer-binding transcription factor-1 [Oct-1]) and efflux (ATP-binding casette [ABC] proteins) [43–45]. In addition, signaling pathways besides BCR-ABL may contribute to disease evolution and imatinib resistance, such as the SRC-family kinases, which are substrates and downstream mediators of BCR-ABL signaling [46–48]. Several mechanisms underlie “acquired” (secondary) imatinib resistance, including amplification of the Ph, and the acquisition of point mutations in the ABL kinase domain of the BCR-ABL fusion protein [39,49,50]. These KD kinase domains are the most common cause of imatinib resistance (40–90%), and inhibit imatinib binding and efficacy by either inducing a structural change towards the active conformation of the protein, causing a shift in the equilibrium of BCR-ABL from inactive (which imatinib binds) to active (which imatinib is unable to bind), or by mutating critical binding sites in the molecule. Approximately 90 different ABL mutations have been found [51]. The likelihood of acquiring an ABL mutation increases with the phase of disease and length of time from diagnosis to treatment. Thus, the acquisition of ABL mutations increases with advanced-phase disease and in CP patients with a longer (>1 year) time from diagnosis to initiating imatinib therapy [52]. Imatinib resistance is often associated with a rapid progression to advanced-phase disease, especially with mutations in the ATP phosphate-binding loop [53–55]. The 3-year survival rate for imatinibresistant patients with CP CML was 72%, dropping to 30% for patients with AP CML, and 7% for BC CML [54]. This underlines the need for new agents to treat imatinib-resistant CML as well as monitoring strategies to detect cases failing imatinib therapy.

Monitoring disease response on imatinib There are several treatment milestones for patients with newly diagnosed CML placed on imatinib therapy. Several layers of monitoring can be used to guide physicians as to when to continue imatinib or when other therapies, such as “second-generation” TKIs, or transplantation, should be considered.

prognostic factor for survival, and should therefore be considered a goal of therapy [57,58]. Failure to achieve any reduction in the number of Ph+ cells after 6 months of imatinib therapy and failure to achieve a MCyR response after 12 months of imatinib therapy predicts for a less than 20% chance of ever achieving a CCyR [56]. Additionally, studies have shown that achieving a MCyR at 3 months is associated with prolonged time to disease progression in patients with late CP and AP CML [59,60]. Molecular response FISH (see Chapter 26) has a somewhat limited role in BCR-ABL monitoring. At diagnosis, it should not replace conventional cytogenetics, since it cannot detect other chromosomal changes indicative of AP disease. It can, however, be used in the diagnostic setting if the patient has an unaspirable bone marrow. FISH is not adequately sensitive for following low levels of residual disease once a patient has achieved a CCyR. The preferred method of sensitive molecular monitoring is quantitative reverse transcriptase (RT)-PCR (see Chapter 26). The measurement of BCR-ABL transcript level by RT-PCR is the most sensitive method to monitor disease burden, and can be combined with cytogenetics to predict outcome. Results from the IRIS trial showed that patients who had achieved a CCyR after 12 months of imatinib had a significantly lower risk of disease progression than patients without a CCyR. Patients who had both a CCyR and a “major molecular response” (MMR; a >3-log reduction of BCR-ABL transcript level from an aggregate baseline of 30 newly diagnosed patients) at 12 months of imatinib therapy had a 100% probability of remaining progression free at 24 months. In comparison, patients with a CCyR and a less than 3-log reduction of BCR-ABL had 95% progression-free survival (PFS), while patients who did not achieve a CCyR by 12 months had an 86% PFS [57]. Five-year follow-up data from this study revealed that no patient who achieved a MMR by 12 months had progressed to advanced disease [38]. Earlier responses (<6 months of imatinib therapy) may also be a predictor of long-term outcome. Several studies have shown that both overall survival and PFS are improved in patients who achieve a cytogenetic response at 3 or 6 months [60–63]. In addition, early molecular responses have also been shown to be associated with better outcome, as those patients who fail to achieve a 1–2-log reduction after 3 months of therapy are less likely to ever achieve an MMR [64–66]. In a large study of cases in CP, it was shown that the achievement of an MMR at 3, 6, and 12 months was highly correlated with the duration of CCyR [64]. For example, for those patients who achieved a CCyR after 3 months of therapy, the subset that also achieved a MMR at that time had a 0% risk of relapse in the next 2 years, compared with a 20% risk for those without an MMR. This trend held true for those in CCyR at 6 and 12 months as well. Thus, early molecular response by peripheral blood testing can give an early indication of which patients will fail to achieve cytogenetic and molecular milestones.

Hematologic response Achieving CHR by 3 months is a clear treatment goal. Although imatinib induces a CHR in most (>95%) patients with CP CML, failure to achieve such a response is considered an indicator of a treatment failure and warrants a change in therapy [56]. Cytogenetic response Cytogenetic monitoring of the level of Ph+ metaphases is the strongest prognostic factor for predicting long-term response to imatinib once patients have achieved CHR. Achieving a CCyR is an independent

Mutational analysis of the ABL kinase domain While there is a relatively low incidence of imatinib-resistant BCR-ABL mutations arising in patients with “early” CP CML, mutations are more frequent in patients with late CP or advanced-phase disease [53]. Thus, regular mutation screening is appropriate for patients with advanced disease. Additionally, patients who respond suboptimally to imatinib or show an increase in the level of BCR-ABL transcripts should also be screened [51]. Such regular mutational monitoring could facilitate early identification of potential mutant clones and allow treatment to be switched prior to further disease progression.

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Table 51.2 Treatment response to imatinib therapy Response

3 months

6 months

12 months

18 months

Failure Suboptimal Optimal

No HR No CHR CHR and 1–2-log decrease in BCR-ABL

>95% Ph 35–95% Ph <35% Ph

>35% Ph 1–35% Ph 0% Ph (CCR) and >3-log decrease in BCR-ABL

>0% Ph 0% Ph and <3-log decrease in BCR-ABL transcripts 0% Ph and >3-log decrease in BCR-ABL transcripts

CCyR, complete cytogenetic response; CHR, complete hematologic response; HR, hematological response; Ph, Philadelphia chromosome.

Recommendations for response assessment

High-dose imatinib

Time-based landmark responses have been established in order to identify patients who are unlikely to respond to imatinib (Table 51.2) [56,58,67]. Patients can be sorted into groups according to treatment response – that is, failure, suboptimal or optimal. Those in the failure category are considered to be unresponsive to imatinib and should be switched to another treatment (a second-generation TKI or transplantation). Patients who respond suboptimally may still benefit from imatinib, but are less likely to have a favorable long-term outcome, and alternative therapies should be considered. The current recommendations for monitoring response to imatinib treatment in CML are outlined in the recently updated National Comprehensive Cancer Network guidelines [58]. Cytogenetic testing is recommended at 6 months and 12 months after the start of therapy. At 18 months, another cytogenetic evaluation should be conducted if a CCyR has not been achieved by 12 months. Alternative therapies should be considered for patients without: (1) a CHR by 3 months; (2) any sign of a cytogenetic response by 6 months; (3) a partial cytologic response by 12 months; or (4) a CCyR by 18 months of therapy. If the patient appears to be responding to therapy (especially once a CCyR is obtained), quantitative PCR measurement of BCR-ABL in the peripheral blood is recommended every 3 months. If a rise in BCR-ABL is detected and confirmed by two measurements taken approximately 1 month apart, the frequency of measurements can be increased to once a month. Rising levels of BCR-ABL transcripts should also prompt mutation screening. For patients in CP, screening for ABL kinase domain mutations is recommended if there is an inadequate initial response. Additionally, mutation analysis should also be performed on patients with any indication of a loss of response, including hematologic or cytogenetic relapse and a rise in BCR-ABL transcript level. Given the high frequency of BCR-ABL mutations associated with advanced-stage disease, routine mutational screening should be performed in these patients every 3 months regardless of treatment response.

High-dose imatinib (600–800 mg/day) is an option in patients with CP CML who respond suboptimally to treatment with 400 mg/day imatinib. The basis for this therapeutic approach stems from studies demonstrating that some BCR-ABL mutations have only intermediate resistance to imatinib [68], and that BCR-ABL amplification and overexpression sometimes confer resistance. Thus, perhaps increased imatinib may overpower BCR-ABL in certain resistant cases. While studies have shown that high-dose imatinib can produce responses in patients who have relapsed on or are refractory to standard-dose imatinib [69,70], these responses are generally not dramatic or durable. Thus, with the advent of new agents, this strategy is not generally recommended for patients with advanced-phase disease and is not appropriate for patients harboring highly resistant BCR-ABL mutations or BCR-ABLindependent resistance.

Treatment options after first-line imatinib failure There are several treatment options that are now available for patients with imatinib intolerance, who have a poor initial response to therapy or who relapse later. “Second-generation” TKIs are now available and are recommended for the treatment of patients who are resistant to imatinib. For those patients who have a suitable matched related or unrelated donor, hematopoietic stem cell transplantation is an option that has the longest track record of potential cure. Lastly, clinical trials are available for imatinib-intolerant or imatinib-resistant cases, and this option should be considered with high priority.

Secondary TKI therapy Dasatinib is an oral, multitargeted kinase inhibitor, including BCR-ABL, SRC, c-KIT, and platelet-derived growth factor receptor-beta. Dasatinib has greater than 300-fold greater activity against native BCR-ABL in vitro as compared with imatinib, and binds both the active and inactive BCR-ABL conformation. Dasatinib has activity against all imatinibresistant BCR-ABL mutations with the exception of T315I [71,72], and was recently approved by the United States Food and Drug Administration and European Medicines Evaluation Agency for the treatment of imatinib-resistant or imatinib-intolerant CML in any phase and Ph+ ALL [73–76]. Indeed, in CP patients resistant to or intolerant of imatinib, a CCyR was achieved in approximately 40% of cases overall after 8 months of therapy [75]. Moreover, a recent randomized study compared dasatinib with high-dose imatinib in patients with imatinib-resistant CP CML (at doses of imatinib from 400 to 600 mg/day). Patients on dasatinib had CCyR rates of 40%, compared with 16% on high-dose imatinib. The rate of MMR was also greater with dasatinib compared with highdose imatinib, at 16% versus 4%, respectively [54]. Dasatinib may also play a role in the treatment of advanced-phase CML. Several studies have demonstrated efficacy in advanced-phase disease. For example, 27% and 43% of myeloid and lymphoid BC, respectively, achieved a CCyR on dasatinib. Unfortunately, the response was rather short lived, as the median PFS was 5 and 3 months for myeloid and lymphoid BC, respectively [73,74]. Nilotinib has also shown activity in patients with imatinib-resistant or imatinib-intolerant CP and AP CML [77,78]. Nilotinib has yielded CCyR in approximately 40% of CP and 20% of AP cases, respectively. The estimated overall survival for CP patients was 95%, and 79% for AP patients. It should be noted that it is, however, impossible to directly

Chapter 51

compare the results for dasatinib and nilotinib in these trials, since the definitions of imatinib resistance and intolerance differ between them.

Management of the newly diagnosed CML patient in the imatinib era Given the success of imatinib, and the promise of newer TKIs, one must ponder if a chapter devoted to transplantation of CML is destined to the medical history shelf. Is transplantation for CML dead? Here is what we know: 1 Imatinib is remarkably effective for patients treated in CP, as greater than 75% of patients obtain a CCyR. In the IRIS trial, approximately 70% of cases remain in a CCyR at 5 years of follow-up. Treatment of advanced-phase (accelerated or blast disease) is associated with much poorer outcomes. 2 Allogeneic transplantation is generally associated with 10-year survivals of 75% or better for patients in CP, but survival likewise falls in accelerated or blast-phase disease. 3 Relapse occurs for patients in CP treated with imatinib, but outcome can be effectively monitored by sensitive RT-PCR assays. Current recommendations from advisory panels, such as the European Leukemia Net and the National Cancer Care Alliance, state that all CP patients start on imatinib therapy, but allow for consideration of transplantation based on the patient’s age, preference, and response to initial imatinib [58,67]. The tacit assumption is that, given the excellent results of both imatinib and transplantation, a contemporary randomized trial comparing the methods would be very unlikely. There are also questions that make the decision on how early to transplant problematic. For example, given that TKI do not appear to kill the CML stem cell, is relapse inevitable? Given that many relapses stemming from ABL point mutations act aggressively, will these patients be especially hard to cure with transplantation? And, while short-term TKI therapy does not seem to impact negatively on transplant results, what will happen with long-term exposure to these agents? There have been a few attempts to compare up-front transplantation with nontransplant therapy [79]. A recent trial enrolled 621 patients with CP CML; of these, 354 were considered eligible for transplantation and “biologically randomized” based on the availability of a related donor. Of the 123 patients who received a transplant, the 10-year estimate of survival was 53%. Those 219 patients without a related donor were treated with IFN until imatinib became available later in the trial. Imatinib was then offered to patients whose disease did not respond to IFN. The 10-year estimate of survival in this group was 52%. The survival curves of these two groups show that the transplant group suffered a higher early mortality, with a relative flattening of the survival curves, whereas the nontransplant group had a better early outcome, with the survival curves continuing to drop. The crossover of the curves came at 8 years. It should be noted that the “nontransplant” group contained patients who later underwent an unrelated transplant (their survival being 69%). It is unfortunately hard to translate this study to contemporary practice. Given the remarkable success of imatinib as first-line therapy, it would be expected that the survival curves for patients treated with imatinib up front would be superior to that of the IFN–imatinib group in this study. In addition, most transplant centers would be disappointed with the survival statistics in the transplant arm of this study of long-term outcomes, which were approximately 50%. All things considered, imatinib is a reasonable initial treatment of choice for patients with CML, and in most treatment guidelines it is recommended as the initial therapy for CP patients. For patients with CP disease, imatinib can be initiated, with a concurrent work-up of family donors, and if needed, unrelated donors. Criteria for imatinib failure or suboptimal response are in evolution (Table 51.3). Certainly

failure to achieve a CHR at 3 months of treatment is an indication to switch therapy. At 6 months of therapy, patients should have some cytogenetic response, and by 12 months, patients should achieve an MCyR, with the elimination of two-thirds of their Ph chromosomes on cytogenetic examination. By a conservative approach, patients should achieve a CCyR by 18 months of therapy. Patients who relapse after a CCyR, especially those with ABL point mutations, should consider alternative therapy, including transplantation. For patients diagnosed in AP or BC, initial treatment with imatinib or dasatinib is recommended, but since responses are generally short, all such patients should be evaluated for transplantation and, if found to be appropriate candidates with suitable donors, should proceed to transplantation as soon as possible.

Allogeneic hematopoietic cell transplantation The outcome of allogeneic hematopoietic cell transplantation (HCT) in CML is influenced by many factors, most importantly the phase of disease, but also the type of donor used, the nature of the stem cell product, and the age of the patient. Phase of disease As with all treatments of CML, outcomes are far superior for HCT for patients in CP compared with advanced-phase disease. The Seattle team began studying HLA-matched sibling transplants for CP CML in 1979, and in 1982 reported initial results from 10 patients [80]. Shortly thereafter they published the first large study on 167 patients transplanted through 1983 from matched siblings [81]. These results have been updated through November 2002, as shown in Figs 51.1 and 51.2, and show that 40% of patients transplanted in CP survive; results deteriorate with transplantation done in advanced phases. Data from the International Bone Marrow Transplant Registry (IBMTR) between 1994 and 1999 show a probability of survival of 69% (standard deviation 2%) for 2876 patients transplanted within the first year from diagnosis, and 57% (standard deviation 3%) for 1391 patients transplanted more than 1 year from diagnosis (Fig. 51.3) [82]. Contemporary results from selected single institutions continue to demonstrate the excellent outcomes with HCT. For example, the Seattle group has recently reported on their most recent trial using a preparative regimen of targeted BU plus cyclophosphamide (CY) in 131 consecutive CP CML patients. Survival 3 years

Annals paper (Tx through 1983) Updates as of November 2002

1

0.8

Survival

738

0.6

Blast phase in remission (n = 12)

0.4

Chronic phase (n = 67)

0.2

Accelerated phase (n = 46) Blast phase (n = 41)

0 0

5

10

15

20

25

Years

Fig. 51.1 Kaplan–Meier probabilities of survival by phase at the time of transplantation for 166 patients with chronic myeloid leukemia who received transplants through 1983 from human leukocyte antigen-identical siblings, first published in 1986 [81]. The results are updated as of November 2002. Tx, treatment.

Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia Annals paper (Tx through 1983) Updates as of November 2002

1

1.0 Overall survival

0.8

0.8 Disease-free survival

0.6

0.4

Probability

Relapse

739

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0.2

Accelerated phase (n = 46)

0.6

0.4

0.2

0 5

0

10

15

20

Relapse

25 0.0

Years

0

Fig. 51.2 Cumulative evidence of cytogenetic relapse by phase at the time of transplantation for 166 patients with chronic myeloid leukemia who received transplants through 1983 from human leukocyte antigen-identical siblings, first published in 1986 [81]. The results are updated as of November 2002. Tx, treatment.

1

2

3

4

5

6

Years after transplant

Fig. 51.4 Kaplan–Meier probabilities of survival and disease-free survival and the cumulative incidence of relapse among 131 patients with chronic myeloid leukemia in chronic phase transplanted from matched siblings following a preparative regimen of targeted busulfan and cyclophosphamide [83].

100

Probability (%)

80

HLA-identical sibling, <1 year (n = 2876)

the small but definite percentage of patients cured still provides a justification for transplantation because there is no other curative therapy available for such patients.

60 HLA-identical sibling, ≥1 year (n = 1391) 40

Preparative regimen 20 p = 0.0001 0 0

1

2

3

4

5

6

Years

Fig. 51.3 Probability of survival after allogeneic transplants for patients with chronic myeloid leukemia in chronic phase transplanted between 1994 and 1999 and reported to the International Bone Marrow Transplant Registry, by disease duration [82]. HLA, human leukocyte antigen.

post transplant was 86%, and 87% of surviving patients were molecularly negative for BCR-ABL messenger RNA by RT-PCR analysis (Fig. 51.4) [83]. Outcomes in advanced-phase disease (AP and BC) are inferior to those for CP. As noted in Fig. 51.1, and other studies, the outcomes of matched sibling transplants for AP CML are worse than those seen in CP [84,85]. In a Seattle analysis of 58 patients with AP CML transplanted from human leukocyte antigen (HLA)-identical siblings, the 4year probabilities of survival and event-free survival were 49% and 43%, respectively, and the actual probability of relapse, censoring for other causes of death, was 12% [86]. Of particular interest was the observation that the probability of survival was 66% for those who were declared to be in AP solely because of cytogenetic clonal evolution, but only 34% if factors other than or in addition to cytogenetic progression (such as blast count) were the reasons for declaring disease progression. In virtually all studies, the outcome of transplantation for patients in BC is very poor, because of both a high risk of disease recurrence and a high incidence of transplant-related deaths [81,87,88]. The Seattle team had performed transplants for 100 patients in blast phase before 1993, with event-free survivals of 43%, 18%, and 11% at 100 days, 1 year, and 3 years, respectively. Although these results are disappointing,

The majority of patients treated in the early 1980s received a preparative regimen of 120 mg/kg CY, followed by total body irradiation (TBI). In the initial series reported by Thomas et al. [81], the TBI dose was 2.0 Gy on each of 6 successive days. A randomized trial comparing the TBI dose of 12 Gy in six exposures to 15.75 Gy in seven exposures found that the relapse rate was decreased with the increased TBI dose, but at the expense of an increased nonrelapse mortality (NRM); thus, neither survival nor disease-free survival was improved with the higher TBI dose [89]. In 1987, Tutschka et al. [90] described the use of a preparative regimen consisting of 16 mg/kg BU administered over 4 days combined with 60 mg/kg CY on each of 2 successive days, reporting excellent results in a limited number of patients with myeloid malignancies. Similar results were noted by others [87], and in 1988 a randomized trial compared the BU–CY regimen with CY plus 12 Gy TBI. No differences were found between the CY–TBI and BU–CY treatment groups in survival at 3 years (80% for both), relapse (13% for both) or eventfree survival (68% for CY–TBI and 71% for BU–CY) [91]. An update of this study was published in 1999 [92], and a further update in 2002 shows overall survival of 78% at 10 years with BU–CY versus 64% with CY–TBI (Fig. 51.5). The absorption and subsequent metabolism of BU varies considerably from patient to patient. When the concentration of BU in the plasma of patients transplanted for CML using a standard BU–CY regimen was measured, it was shown that patients with a steady-state BU concentration less than the median value (<917 ng/mL) of the cohort had a significantly higher risk of disease recurrence and worse overall survival than those with levels over 917 ng/mL [93]. Measurement of BU metabolism on the first day of therapy allowed the adjustment on subsequent dosing to maintain a steady-state BU concentration at 900–1200 ng/mL. A subsequent report of 131 consecutive CP CML patients transplanted with HLA-identical relatives enjoyed a 3-year survival of 86%, a relapse rate of only 8%, and NRM rate of 14% [83]. Remarkably, there were

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Chapter 51 Protocol 428

1

1.0

BU – CY (n = 73) 0.8 Probability of survival

0.8

Estimates

Survival CY – TBI (n = 69)

0.6

0.4 CY – TBI (n = 69) 0.2

Relapse

0.6 <40 (n = 48) 40 –50 (n = 54) >50 (n = 29) Censor

0.4

0.2 BU – CY (n = 73)

0 0

2

4

6

8

10

12

14

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0.0 0

1

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3

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Years after transplant

Fig. 51.5 Kaplan–Meier probabilities of survival and the cumulative incidences of relapse among 142 patients with chronic myeloid leukemia in chronic phase transplanted from matched siblings following a preparative regimen of either busulfan plus cyclophosphamide or cyclophosphamide plus total body irradiation. Results of this randomized trial were originally published in 1994 and are here updated as of November 2002.

no significant differences in outcome related to patient age (Fig. 51.6). Other preparative regimens have also been reported to provide excellent disease control with acceptable toxicities in the treatment of CML in CP, including the regimen of TBI plus etoposide, reported by the groups from City of Hope National Medical Center and Stanford University [94], and the use of CY plus 500 cGy TBI given as a single dose at high-dose rate [95]. Donor type In 1979, Fefer et al. [96] published results of identical twin (syngeneic) transplants in four patients with CP CML treated with dimethyl BU, CY, and a single 920 cGy exposure TBI. All four recovered with Ph− normal hematopoiesis. In 1982, the same group reported on 22 CML recipients of twin transplants, including 12 treated in CP [97]. As at August 2002, seven of the 12 CP patients were alive at 20.8–26.3 years after transplant, of whom five were in their initial complete remission, one was alive in remission after a second transplant, and one was alive in relapse. These data demonstrate that a high-dose preparative regimen can, in some cases, cure CML even without the benefit of an allogeneic graftversus-leukemia (GVL) effect. Only approximately one-third of patients have HLA-matched family members to serve as donors. Thus, considerable research into the use of alternative donors for transplantation in CML has been conducted. In Chapter 47, details concerning the use of unrelated donors for transplantation in CML are presented. As discussed in that chapter, while early results with matched unrelated donor transplantation in CML suggested results somewhat poorer than those seen with matched siblings [98], advances in donor selection, prophylaxis for graft-versus-host disease (GVHD), and supportive care have resulted in continued improvements in outcome, so that now selected single institutions are reporting results almost equivalent to those seen with matched siblings, and registry data are showing 65% survival at 5 years among younger patients transplanted within a year of diagnosis [99–101]. In the Seattle experience, survival in CP, AP, and BC is similar between matched-related and unrelated donors (with a caveat that unrelated donor transplants have a lower exclusion for patient age). Details about the use of mismatched

Fig. 51.6 Kaplan–Meier probabilities of survival according to age among 131 patients with chronic myeloid leukemia in chronic phase transplanted from matched siblings following a preparative regimen of targeted busulfan and cyclophosphamide [83].

related donors are presented in Chapter 46, and the use of cord blood transplants is included in Chapter 39. The experience with autologous transplantation in CML is detailed below. Source of hematopoietic stem cells Bone marrow has served as the source of stem cells for the large majority of patients transplanted for CP CML from matched siblings. Two separate large randomized trials involving patients with a variety of hematologic malignancies receiving transplants from matched siblings have been published demonstrating that the use of filgrastim (granulocyte-colony stimulating factor)-mobilized peripheral blood hematopoietic cells (PBHCs), when compared with bone marrow, leads to more rapid myeloid and platelet recovery, no significant difference in acute or chronic GVHD, and an overall survival advantage. These studies were not prospectively designed to address the role of peripheral blood versus bone marrow for individual disease states. In the studies by Bensinger et al. [102] and Couban et al. [103], there was a trend towards improved survival in CML patients with the use of peripheral blood. In the study by Couban et al. among 109 CML patients, survival at 3 years was 80% with peripheral blood versus 65% with bone marrow. Both of these studies used conventional GVHD prophylaxis. Lastly, the results of a randomized study of CP CML showed no statistically significant differences in outcome between the bone marrow and peripheral blood groups. Relapse rates were lower in the peripheral blood group, but chronic GVHD (and subsequent complications thereof) higher, balancing the score [104]. Patient age Initial studies of transplantation for CML in CP from HLA-matched siblings suggested that results might deteriorate with increasing patient age. Given the median age of patients with CML at diagnosis, these observations significantly limited the proportion of patients who were candidates for transplantation. As methods for GVHD prophylaxis and supportive care were improved, the impact of age on outcome appeared to diminish. In 1993, the Seattle group published results of 33 patients with CP CML, aged 50–60 years, and reported an 85% survival at 5

Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia

years [105]. The relative lack of effect of age up to age 65 on outcome of matched sibling transplantation for CML in CP has been reconfirmed in the most recent Seattle experience utilizing the targeted BU–CY preparative regimen (Fig. 51.6). However, age seems to be more clearly related to transplant outcome in the unrelated donor setting [106].

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preventing GVHD without loss of a GVL effect by combining T-cell depletion with an intensified conditioning regimen and delayed reinfusion of viable donor lymphocytes [116].

Relapse following transplantation Time from diagnosis to transplant Early studies showed that an increased interval from diagnosis to transplant was associated with a worse transplant outcome, even for patients in CP at the time of transplant. This finding was confirmed by others and is evident in the most recent IBMTR data (Fig. 51.3) [107,108]. Reasons for the effect of delay on outcome are not readily apparent. The effect of delay includes a slightly higher relapse rate and a slight increase in NRM. Nonetheless, the aggregate effect of delay on outcome is readily apparent and, as discussed below, has been a major consideration in the management of patients with CP CML. One hypothesis for the effect of delay on outcome focused on the effects of prior therapy. An early report from the IBMTR strongly suggested that exposure to low-dose BU led to a worse outcome with subsequent transplantation [107]. Some reports suggested that exposure to IFN might worsen the outcome of unrelated donor transplant, but data on the effect of IFN on matched sibling transplantation were less clear. In a recent German report of 856 patients randomized to hydroxyurea, BU or IFN, 197 went on to transplant. Although there was no overall difference in transplant outcome according to the initial treatment, the 5-year survival from transplant was only 46% for the 50 patients who received IFN within the last 90 days before transplant, but was 71% for the 36 who did not (p = 0.0057) [109]. These observations suggest that IFN should be avoided, if possible, in the months immediately preceding allogeneic HCT. There have been several studies on the effect of prior imatinib and transplant outcomes. Early reports warned of an increase in regimenrelated toxicity and mortality, especially from hepatic causes [110]. Larger studies have failed to show a deleterious effect of pretransplant imatinib [111,112]. A study of 140 patients with CML showed no difference in regimen-related mortality, survival or relapse in patients who received imatinib versus 200 historical controls [113]. GVHD prophylaxis The form of GVHD prophylaxis used in the treatment regimens also influences the outcome of transplantation for CML, especially in CP. Prior to 1984, most patients were given either single-agent methotrexate (MTX) or single-agent cyclosporine (CSP) as GVHD prophylaxis, and randomized trials demonstrated the relative equivalence of the two approaches [114]. A subsequent randomized trial showed that a regimen combining four doses of MTX with CSP was superior to single-agent CSP prophylaxis in reducing the incidence of GVHD and in improving overall survival for CP CML patients [115]. A number of variations from the standard MTX–CSP regimen have been explored, including adding prednisone or substituting tacrolimus (FK506) for CSP, but none has shown an overall survival advantage. Prevention of GVHD by removing T cells from the donor marrow was explored in a number of transplant studies in the 1980s. Although successful in reducing the incidence of GVHD, T-cell depletion in CML was associated with high rates of graft failure and relapse, leading to poorer disease-free and overall survival [88]. These findings illustrated the critical role of the GVL effect in eradicating CML following allogeneic transplantation. Because of these observations, T-cell depletion was largely abandoned as a method to control GVHD in CML transplants. However, there has been renewed interest in the possibility of

GVL effect in CML While evidence for a GVL effect can be found in many settings, nowhere is it as strong as in the setting of allogeneic transplantation for CML. Evidence in support of such an effect includes, first, the higher rates of relapse following syngeneic and T-cell-depleted transplants compared with nonmodified allogeneic transplants [88,117]. Second, following non-T-cell-depleted transplants, there is a strong inverse association between acute and chronic GVHD and relapse [118,119]. Third, response rates to donor lymphocyte infusions (DLIs) to treat post-transplant relapse range from 50% to 100% in various reports, higher than in any other malignancy [120,121]. The markedly increased relapse rates seen with T-cell depletion argue that it is largely a T-cell-mediated process. The targets of T cells might include minor histocompatibility antigens shared by most cells in the body, thus accounting for the association of GVL with GVHD. Alternatively, there may be polymorphic minor histocompatibility antigens with expression limited to hematopoietic tissue. A number of such antigens have been identified [122,123], and their role as targets for GVL could explain the observation that relapse rates are less following nonT-cell-depleted transplants than with T-cell-depleted transplants, even in patients who develop no signs of GVHD. A third possible category of targets for the GVL effect in CML is the overexpression of protein targets in CML cells. For example, PR3 is a neutral serine proteinase with expression largely restricted to very immature myeloid cells. Molldrem et al. [124] detected CD8+ cytotoxic T cells specific for PR3 in the blood of CML patients who had been treated successfully with allogeneic transplantation or with IFN. When disease recurred, these responses were lost. T-cell responses could be directed at the tumor-specific peptides spanning the BCR-ABL fusion region, but there is little evidence for the presence of high numbers of T cells with specificity against BCR-ABL in transplant patients. Understanding the cells and their targets responsible for the potent GVL effect seen in CML will be critical to the development of more effective, less toxic transplant-based therapies in the future.

Reduced-intensity conditioning in CML Given the power of the GVL effect following standard transplantation for CML, investigators have begun to study reduced-intensity or nonablative transplant approaches in an attempt to avoid the toxicities of high-dose preparative regimens while retaining the potentially powerful GVL effects. These approaches (discussed in more detail in Chapter 71) are of particular relevance for patients with CML given that their median age at diagnosis is 67 years. In contrast to myelodysplastic syndrome and acute myeloid leukemia, few papers evaluating reduced-intensity conditioning (RIC) regimens in CML have been published. The Seattle Consortium published results with a nonmyeloablative regimen of fludarabine and TBI or 2 Gy TBI in 24 patients with CML in first CP, second CP or AP. All patients received peripheral blood progenitor cells (PBPCs) from matched related donors [125]. Five of 25 patients died from NRM, and the 100-day NRM rate was 4%. The 2-year estimates of OS were 70% for patients in first CP, and 56% for patients beyond first CP. However, results using unrelated donors were not as initially encouraging because of high rates of graft rejection (45%).

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Chapter 51

Investigators at Hadassah-Hebrew University Hospital published results in a younger cohort of patients (median age 35 years) using an RIC regimen of fludarabine 180 mg/m2, oral BU 8 mg/kg, and antithymocyte globulin 20–40 mg/kg in 24 patients with CML in CP. Nineteen patients received matched related donor and five received unrelated donor PBPCs [126]. Six patients had mixed chimerism, and three required additional therapy with DLI. The 100-day NRM was 0%, and there were only three mortalities in the series, all secondary to GVHD. The estimated 5-year overall and relapse-free survival was 85%. At a median of 42 months from HCT, all patients had a complete molecular response. The European Group for Blood and Marrow Transplantation group has reported results on 186 patients with CML (median age 50 years) who received RIC [127]. Patients with all stages of CML were included, with various RIC regimens. In addition, there was a mix of stem cell product source (PBHCs versus bone marrow), and donors (HLA matched and mismatched related and unrelated donors). The 100-day NRM was 6.1%, and the 2-year NRM was 23.3%. The 3-year overall and relapsefree survival were 58% and 37%, respectively. At least 40% of patients attained a complete molecular response, and at least 62% a CCyR. All of these studies indicate that RIC is a viable strategy in patients with CML; however, longer follow-up is necessary to determine the ultimate utility of RIC for CML, particularly in the era of TKIs. The combination of debulking patients with imatinib, then following with RIC and HCT seems particularly attractive.

Monitoring minimal residual disease The fact that CML is always associated with the BCR-ABL translocation, coupled with the development of PCR techniques capable of detecting and quantifying extraordinarily small numbers of abnormal cells among a large population of normal ones, makes CML an ideal setting to test the capabilities of minimal residual disease (MRD) monitoring to provide insight into the disease and direct therapies. Details about MRD detection are provided in Chapter 26. With nonquantitative PCR techniques, it is possible to detect a single CML cell in a population of 105–106 cells. Such a qualitative assay was found predictive of relapse in 346 patients tested post transplantation [128]. In over 40% of patients, PCR assays were positive for residual disease at 3 months post transplant, but this finding was not predictive of outcome, suggesting that eradication of the CML clone post transplant takes an extended period of time. In contrast, at 6 or 12 months post transplant, the risk of being PCR positive dropped to approximately 25%, and here the assay was a powerful predictor of outcome, as 42% of PCR-positive patients subsequently relapsed, compared with 3% of PCR-negative patients (p < 0.0001). The predictive power of PCR among long-term survivors is somewhat weaker. When studied at 18 months or more post transplant, only 1% of 289 BCR-ABL negative patients subsequently relapsed, compared with 14% of 90 BCR-ABL positive patients [129]. The phenomenon of long-lived PCR positivity without relapse has been seen by several groups, and is discussed under “dormancy” in Chapter 26. The advent of quantitative PCR assays allowed fine-tuning of the association of MRD and relapse. Accordingly, attempts have been made to develop quantitative PCR assays and apply them to the post-transplant CML setting. Olavarria et al. [130] studied 138 patients who received allografts, using a quantitative RT-PCR assay performed at 3–5 months post transplant, and were able to define patients as having a low risk (16%), an intermediate risk (43%) or a high risk (86%) of relapse based on the quantification of the PCR signal [131]. A more complete discussion of MRD in CML is found in Chapter 26.

Treatment of post-transplant relapse The pace of disease progression after post-transplant relapse is variable. Indeed, if relapse is defined at a molecular (PCR) level, some patients may never progress, or at least may not progress for a very long time. Similarly, patients whose only evidence of relapse is detection of low levels of Ph+ metaphases may remain stable for many years, and even patients with clinical relapse may not progress rapidly. Two retrospective EBMT studies have examined CML patients who relapsed after transplant [132,133]. Stage at transplant and relapse, time from diagnosis to transplantation and from transplantation to relapse, and type of donor (matched sibling versus volunteer) all were shown to affect survival. Specifically, recipients of matched sibling grafts for CP disease with a short interval from diagnosis to transplant but a long interval from transplant to relapse did particularly well, with a likelihood of being alive at 10 years after post-transplant relapse of 42%. An appreciation of the likely tempo of progression is necessary when considering treatment interventions. An increasing number of potential interventions are available to the patient who has relapsed after allogeneic transplantation for CML. Treatment with IFN can produce both clinical and cytogenetic remission in patients who have relapsed after transplantation [134,135]. Results with IFN appear better if treatment is initiated at the time of cytogenetic relapse instead of waiting until hematologic relapse, and in some cases IFN induced molecular remissions. In addition, intervention with IFN for patients who were PCR positive for BCR-ABL at 6–12 months post transplant demonstrated a low relapse rate of approximately 10%, and molecular remissions in most patients [83]. Nonetheless, early treatment of relapse in CML has been supplanted by imatinib. Given its dramatic activity as initial therapy for CML, it is hardly surprising that imatinib mesylate should also be active as post-transplant therapy [136,137]. In these studies. patients have been in various types of relapse (frank hematologic, cytogenetic or molecular relapse), and most were either “imatinib naïve” or not resistant to the drug prior to transplant. The results of the studies provide a uniform theme. As expected, response mirrors stage of disease, and CHR was seen in 90– 100% of CP cases, 50–80% of AP, and 20–40% in BC. A CCyR was accomplished in over 40%, again substantially higher in CP than advanced-phase cases. As expected, however, survival for blast cases is poor, with PFS of around 10% at 2 years. Lastly, imatinib appears to be tolerated given early after transplantation to prevent relapse in high-risk Ph+ cases [138]. Twenty-two patients (15 with Ph+ ALL and seven with CML) were given imatinib at a median of 28 days post engraftment. Seventeen of 19 adults and all three children tolerated imatinib at the targeted dose (400 mg/day for adults and 260 mg/m2/day for children), and 19 completed the planned course of 1 year. At a median follow-up of around 1.4 years, five of seven of the CML patients and 12 of 15 of the Ph+ ALL cases were in a molecular remission. The approach of early TKI therapy, coupled with sensitive molecular monitoring, may allow for prevention or early intervention of disease reoccurrence in CML, and may give new hope to patients necessitating transplant in advancedphase disease. Since the initial description of DLI as treatment of post-transplant relapse, experience with this approach has grown considerably and is detailed in Chapter 72. There are now a large number of studies demonstrating CCyR rates of 50–100% in patients treated for clinically relapsed CP CML (reviewed in [121]). Response rates tend to be higher for patients treated earlier at the time of cytogenetic relapse, and lower for patients in AP. There is a trend towards improved response rates in patients treated within 2 years after transplantation [139]. The two major complications of DLI are transient marrow failure and the development of GVHD. Marrow failure only occurs in patients

Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia

treated in hematologic relapse and likely reflects clearance of host hematopoiesis before donor hematopoiesis recovers; it is thus of particular concern for patients in full-blown hematologic relapse with no evidence of residual donor hematopoiesis [140]. Treatment earlier in the course of relapse can avoid this complication. The overall incidence of GVHD following DLI ranges around 50% in most series. Although complete remissions can be achieved in the absence of GVHD, with currently used approaches there is a close correlation between development of GVHD and achievement of complete responses to DLI [139]. Most early studies of DLI involved a single infusion of a relatively large number of donor T cells. In a doseescalation study conducted in patients who had recurrent CML after T-cell-depleted transplants, Mackinnon et al. [141] found that 5 × 107/kg T cells were necessary to control CML, but that the incidence of GVHD also increased with higher T-cell doses. Dazzi et al. [142] have since reported that large numbers of T cells are tolerated with less GVHD if administered in a fractionated fashion rather than as a single bulk dose. A recent report from the EBMT provides further support for starting at lower doses of lymphocytes and escalating dosage as required. In their retrospective study of 298 patients, they found that beginning with a lower initial cell dose was associated with less GVHD, less myelosuppression, equal response rates, and better survival [143]. They recommend an initial cell dose no higher than 0.2 × 108/kg. Second transplants have been used to treat patients who have developed recurrent CML following a first transplant. In general, preparative regimens based on chemotherapy only have been used for patients previously exposed to TBI, and TBI-based regimens have been used when the first regimen relied only on chemotherapy. The numbers of second transplants reported in CML are small, yet the data are consistent, reporting regimen-related mortality in the region of 40%, with long-term disease-free survival of around 25% [144]. As expected, survival in BC is quite rare. With the availability of IFN, DLI, and now imatinib, the use of second transplants has diminished substantially.

Autologous transplantation The rationale for autologous transplantation in CML has been experimental and clinical evidence for persistence of polyclonal Ph− progenitors capable of reconstituting hematopoiesis in CML patients [145–151], which is most dramatically demonstrated by the high rate of CCyR seen in CML patients treated with imatinib [40]. Transplantation of autologous Ph− cells may allow restoration of Ph− hematopoiesis. Another rationale for autologous transplantation has been that co-transplantation of Ph− and Ph+ cells appears to favor engraftment of the Ph− clone [151–153]. Reduction of numbers of Ph+ stem cells could reduce the chance of acquisition of additional mutagenic events responsible for transformation from CP to AP and BC.

Historical experience with autologous transplantation for CML Initial studies carried out using unmanipulated autologous marrow or blood cells collected from patients in CP indicated that autologous transplantation could re-establish CP in patients with more advanced disease and induce cytogenetic responses in a small proportion of CML patients [152,154–158]. Although these responses were usually transient, these studies suggested that autologous transplantation might extend survival for CML patients beyond that expected with conventional chemotherapy. Subsequently, attempts were made to improve the results of autologous transplantation by purging malignant progenitors from infused cells prior to transplantation.

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Several approaches to selectively deplete malignant CML progenitors or to select benign progenitors from CML autografts ex vivo have been evaluated. These include long-term bone marrow culture [159,160], exposure to IFN-α [161], and treatment with 4-hydroxyperoxy-CY [162]. More recently, treatment of grafts with antisense oligodeoxynucleotides directed against the BCR/ABL breakpoint junction and against c-Myb, exposure to granulocyte–macrophage colony-stimulating factor, and photodynamic therapy using a dibromo-rhodamine derivative (TH9402) have been reported. These studies indicate that depletion of Ph+ progenitors by ex vivo graft manipulation is possible and may be associated with cytogenetic remission post transplant. However, remissions were of relatively short duration, indicating that purging was either ineffective or insufficient to prevent relapse post transplantation secondary to residual leukemic cells in the patient. Delayed or partial engraftment was a significant problem, possibly reflecting damage to benign stem cells as a result of the purging process. Another approach to depleting malignant cells from the graft is to treat patients prior to harvesting marrow or peripheral blood for autografting (in vivo purging). Enrichment of benign progenitors may be related in part to differential kinetics of regeneration of Ph− and Ph+ progenitors in CML patients treated with high-dose chemotherapy. Autografting of a selected group of CML patients with good cytogenetic responses to intensive treatment was associated with a high incidence of cytogenetic responses following transplantation in some studies [163– 167], but not as compelling in other reports [168–178]. Ph− PBHC collections were obtained in 15–80% of patients. Moreover, high-dose chemotherapy was often associated with significant toxicity and delayed recovery. Insufficient progenitor cell yields to allow transplantation were seen in up to 15% of patients. Several groups have reported a higher frequency of Ph− collections from patients treated earlier in the disease course and in patients in a goodprognosis group [168,173,177–179]. Less intensive chemotherapy regimens with reduced toxicity appear to be as effective as more intensive regimens in mobilizing Ph− PBPC products [176,177,180]. A second course of intensive chemotherapy did not improve Ph− PBPC collection [175]. Mobilized PBHC collections may yield larger numbers of benign progenitors than steady-state or primed bone marrow [177]. Although the level of Ph− cells in the autograft appeared to predict for cytogenetic response post transplant [177,181], it is possible that response to conventional chemotherapy identifies a group of good-risk patients. An alternative approach to in vivo purging involves collection of PBHCs by growth factor mobilization following induction of cytogenetic responses by treatment with IFN-α [182–185]. This approach appears to be feasible and well tolerated, and usually results in the collection of adequate numbers of cells, but is applicable to only a selected group of patients. The role of autologous HCT has been the subject of a number of studies covering a period of more than 20 years. McGlave et al. compiled results of 200 consecutive autologous transplants at eight different transplant centers in Europe and North America between June 1984 and January 1992 [186,187]. The median survival time of 142 patients with CP CML was not reached, and the survival probability was 58%. However, the majority of surviving recipients of autologous transplant had evidence of residual disease. In the absence of controlled clinical trials, it was not possible to make any definite conclusions regarding effects of autologous transplantation on survival of CML patients. Subsequently, several randomized studies were initiated but none was completed, since accrual to these trials was severely affected by the advent of imatinib as a very effective treatment for CML. A metaanalysis of six such trials in which patients were randomly allocated to receive autologous HCT or an IFN-based regimen did not show an advantage for HCT. There was no evidence of a difference in survival (odds ratio 0.99, 95% confidence intervals 0.67–1.46) or in best hematologic or cytogenetic response achieved in the first year.

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Chapter 51

Table 51.3 Results of peripheral blood hematopoietic cell (PBHC) collection from patients in complete cytogenetic remission on imatinib

Author

Patients

Target numbers of CD34 cells collected

PBHC products Ph− or BCR/ABL− by fluorescence in situ hybridization

Drummond [191] Hui [192] Kreuzer [193] Perseghin [194] Bhatia [195]

58 32 18 25 39

23/58 19/32 13/18 20/25 34/39

26/31 NA NA NA 31/35

+

As discussed above, imatinib has proven remarkably effective in treatment of CML [41,59,188–190]. Historically, autologous transplant was considered as a treatment option for CML patients without a suitable matched donor. However, trials of autologous HCT now need to be considered in the context of the excellent results of imatinib treatment, and the availability of second-generation kinase inhibitors with significant activity in imatinib-resistant CML patients. Since patients in CCyR on imatinib treatment continue to demonstrate evidence of residual leukemia cells, and some patients do relapse [57], pilot studies have been initiated to collect PBHCs from patients who have received imatinib and achieved CCyR, for use for autologous HCT in case of later progression. Given that these patients are in CCyR at the time of collection, this approach may allow reliable collection of adequate numbers of Ph− PBPCs in a larger proportion of patients. The results of five such studies are summarized in Table 51.3 [191–195]. These results support the feasibility of collection of Ph− PBPCs from patients responsive to imatinib treatment. The PBPC collection process is well tolerated, and acquiring Ph collections is more consistent than with chemotherapy and IFN-based strategies. Although the target number of CD34+ cells is usually attained, a subset of patients fail to reach this target, and many patients mobilize relatively suboptimally and require multiple collections, possibly as a result of effects of imatinib on normal hematopoiesis and the mobilization process [188]. Collection of PBPCs after a brief discontinuation of imatinib appears to result in greater success. Other factors, such as damage to normal stem cells from disease and prior therapeutic exposure and microenvironmental abnormalities, could contribute to this defect [196]. Collections are usually Ph− by routine cytogenetics and FISH. Although PBPC products without molecular evidence of disease are collected in a few patients, in most patients residual BCR-ABL+ cells can be detected using sensitive PCR assays for BCR-ABL transcripts. Collection of PBHCs from patients responsive to imatinib may form the framework of future attempts to perform autologous transplantation in CML; however, additional strategies may be required to further deplete BCR-ABL+ cells from PBHC collections. Achievement of durable disease-free survival after autologous HCT will require development of improved methods for graft purging as well as improved post-transplant therapy to treat residual disease.

Approaches to eliminate residual disease and prevent disease recurrence PBPC collections from patients treated with imatinib are usually Ph− by routine cytogenetics and FISH. However, small numbers of residual BCRABL+ cells are detectable by PCR in most patients. Studies using retroviral transduction of the Neo resistance gene to mark CD34+ marrow cells from CML patients prior to autologous transplantation indicate that infusion of leukemia cells in the autograft may contribute to relapse post transplant [197]. Therefore methods to eliminate residual disease and obtain BCRABL− collections in a larger proportion of patients are required.

PBHC products BCR-ABL− by polymerase chain reaction 1/9 1/11 5/13 1/25 30/32

Purging techniques previously employed in clinical trials have been limited by incomplete depletion of malignant stem cells and damage to normal stem cells, leading to problems related to delayed or failed engraftment. In vivo purging with chemotherapy may be effective in further reducing the leukemia contamination in autografts obtained from imatinib-sensitive patients. However, imatinib is suppressive towards normal hematopoiesis, and increased myelotoxicity and delayed recovery may be concerns. Other purging approaches may be applicable. Short interfering RNA technology has been described as a potent method for specific targeting of BCR-ABL gene expression [198,199]. Advances in gene targeting and gene transfer technology may allow improved suppression of BCR-ABL gene expression in the future. Another possible approach is in vitro exposure to high concentrations of imatinib or other agents with more potent activity against the p210BCR-ABL kinase [200]. These inhibitors may be combined with other antileukemic agents or with inhibitors of downstream signaling pathways to enhance killing of BCR-ABL+ cells during short-term exposure [201,202]. CML progenitors demonstrate enhanced proliferation and differentiation, and reduced self-renewal, in response to growth factor stimulation [203–205], and pilot studies have demonstrated the feasibility of autologous transplantation using marrow cells purged by ex vivo long-term culture or ex vivo granulocyte–macrophage colony-stimulating factor treatment of the autograft [206]. Yang and colleagues reported that a strategy that combines brief exposure to imatinib and mafosfamide followed by 2 weeks culture with cytokines eliminates BCR-ABL(+) cells from aphereses from CP CML patients, while preserving normal progenitors [207]. Recently, Krause et al. have shown that BCR-ABLexpressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML [208]. Studies of allogeneic transplant for CML clearly indicate the importance of a GVL effect to prevent and treat relapse in CML after transplant. This suggests that achievement of durable remissions after autologous transplantation requires improved antileukemic therapy to treat residual disease post transplantation. One possible approach is immune targeting of malignant CML progenitors. Approaches that have been considered include interleukin-2 administration, natural killer cell administration, and infusions of ex vivo co-stimulated autologous T cells. An alternative strategy is to generate cytotoxic T-lymphocytes directed towards other antigenic targets expressed at abnormally high levels or aberrantly expressed in leukemic cells, including the Wilms’ tumor (WT1) gene product [209] and PR1, an HLA-A2.1-restricted peptide from proteinase-3, expressed at increased levels in CML blasts [210–214]. There has also been interest in dendritic cell vaccine development for use in CML. CML dendritic cells constitutively express BCR-ABL and are expected to express other leukemia-associated antigens. The feasibility of generation of dendritic cells from CML mononuclear cells and CD34+ cells has been shown [215–218], clinically applicable methods to generate autologous BCR/ABL+ dendritic cells

Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia

from monocyte precursors in CML patients have been developed, and several small pilot studies have been reported. The clinical benefits of dendritic cell vaccination in CML remain to be determined.

Conclusion CML serves as an excellent model for how insights into the molecular basis of a malignant disease and the body’s response to that disease can be translated into powerful diagnostic tools and highly effective therapies. While the simultaneous development of imatinib and improvements in transplant techniques have made it harder to advise patients on

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which initial therapeutic path to take, both paths are superior to anything that could previously be offered to these patients. In the near future, the shape of CML therapy may be altered by several forces: the prospect that more powerful TKIs, treating CML initially, may have more of an impact, changing the natural history of disease, progression, and resistance; the possibility that diagnostic tests, using molecular or protein signals, may allow us to predict which patients will respond best to which therapy; and the use of molecular monitoring to change therapy before relapse occurs. CML is not a public health menace, but it has taught us more than most, if not all, malignancies about the intersection of biology and clinical practice. CML will continue to teach us in the future.

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139. Collins RHJ, Shpilberg O, Drobyski WR et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997; 15: 433– 44. 140. Keil F, Haas OA, Fritsch G et al. Donor leukocyte infusion for leukemic relapse after allogeneic marrow transplantation: lack of residual donor hematopoiesis predicts aplasia. Blood 1997; 89: 3113–17. 141. Mackinnon S, Papadopoulos EB, Carabasi MH et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versusleukemia responses from graft-versus-host disease. Blood 1995; 86: 1261–8. 142. Dazzi F, Szydlo RM, Craddock C et al. Comparison of single-dose and escalating-dose regimens of donor lymphocyte infusion for relapse after allografting for chronic myeloid leukemia. Blood 2000; 95: 67–71. 143. Guglielmi C, Arcese W, Dazzi F et al. Donor lymphocyte infusion for relapsed chronic myelogenous leukemia: prognostic relevance of the initial cell dose. Blood 2002; 100: 397– 405. 144. Cullis JO, Schwarer AP, Hughes TP et al. Second transplants for patients with chronic myeloid leukaemia in relapse after original transplant with T-depleted marrow: feasibility of using busulphan alone for re-conditioning. Br J Haematol 1992; 80: 33–9. 145. Coulombel L, Kalousek DK, Eaves CJ, Gupta CM, Eaves AC. Long-term marrow culture reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosomepositive chronic myelogenous leukemia. N Engl J Med 1983; 308: 1493–8. 146. Verfaillie CM, Miller WJ, Boylan K, McGlave PB. Selection of benign primitive hematopoietic progenitors in chronic myelogenous leukemia on the basis of HLA-DR expression. Blood 1992; 79: 1003–10. 147. Leemhuis T, Leibowitz D, Cox G et al. Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow. Blood 1993; 81: 801–7. 148. Delforge M, Boogaerts MA, McGlave PB, Verfaillie CM. BCR/ABL- CD34(+)HLA-DR- progenitor cells in early chronic phase, but not in more advanced phases, of chronic myelogenous leukemia are polyclonal [In process citation]. Blood 1999; 93: 284–92. 149. Goto T, Nishikori M, Arlin Z et al. Growth characteristics of leukemic and normal hematopoietic cells in Ph+ chronic myelogenous leukemia and effects of intensive treatment. Blood 1982; 59: 793–808. 150. Carella AM, Dejana A, Lerma E et al. In vivo mobilization of karyotypically normal peripheral blood progenitor cells in high-risk MDS, secondary or therapy-related acute myelogenous leukaemia. Br J Haematol 1996; 95: 127–30. 151. Wang JC, Lapidot T, Cashman JD et al. High level engraftment of NOD/SCID mice by primitive normal and leukemic hematopoietic cells from patients with chronic myeloid leukemia in chronic phase. Blood 1998; 91: 2406–14. 152. Haines ME, Goldman JM, Worsley AM et al. Chemotherapy and autografting for chronic

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Allogeneic and Autologous Transplantation for Chronic Myeloid Leukemia

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180. Carella AM. Autografting with non-clonal mobilized hematopoietic progenitor cells in CML. Leukemia 2000; 14: 954–5. 181. Talpaz M, Kantarjian H, Liang J et al. Percentage of Philadelphia chromosome (Ph)-negative and Ph-positive cells found after autologous transplantation for chronic myelogenous leukemia depends on percentage of diploid cells induced by conventional-dose chemotherapy before collection of autologous cells. Blood 1995; 85: 3257– 63. 182. Archimbaud E, Michallet M, Philip I et al. Granulocyte colony-stimulating factor given in addition to interferon-alpha to mobilize peripheral blood stem cells for autologous transplantation in chronic myeloid leukaemia. Br J Haematol 1997; 99: 678–84. 183. Reiffers J, Taylor K, Gluckman E et al. Collection of Ph-negative progenitor cells with granulocytecolony stimulating factor in patients with chronic myeloid leukaemia who respond to recombinant alpha-interferon. Br J Haematol 1998; 102: 639– 46. 184. Meloni G, Russo D, Baccarani M et al. A prospective study of alpha-interferon and autologous bone marrow transplantation in chronic myeloid leukemia. The Italian Co-operative Study Group on Chronic Myeloid Leukemia. Haematologica 1999; 84: 707–15. 185. Hernandez-Boluda JC, Carreras E, Cervantes F et al. Collection of Philadelphia-negative stem cells using recombinant human granulocyte colony-stimulating factor in chronic myeloid leukemia patients treated with alpha-interferon. Haematologica 2002; 87: 17–22. 186. McGlave PB, De Fabritiis P, Deisseroth A et al. Autologous transplants for chronic myelogenous leukaemia: results from eight transplant groups. Lancet 1994; 343: 1486–8. 187. Bhatia R, McGlave PB. Autologous stem cell transplantation for the treatment of chronic myelogenous leukemia. Cancer Treat Res 1997; 77: 357–74. 188. Druker B, Tamura S, Buchdunger E et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996; 2: 561–6. 189. Druker BJ, Talpaz M, Resta DJ et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344: 1031–7. 190. Talpaz M, Silver RT, Druker B et al. A phase II study of STI571 in adult patients with Philadelphia chromosome positive chronic myelogenous leukemia in accelerated phase. Blood 2000; 96: 469A. 191. Drummond M, Lennard A, Brummendorf T, Holyoake T. Telomere shortening correlates with prognostic score at diagnosis and proceeds rapidly during progression of chronic myeloid leukemia. Leuk Lymphoma 2004; 45: 1775–81. 192. Hui CH, Goh KY, White D et al. Successful peripheral blood stem cell mobilisation with filgrastim in patients with chronic myeloid leukaemia achieving complete cytogenetic response with imatinib, without increasing disease burden as measured by quantitative real-time PCR. Leukemia 2003; 17: 821–8. 193. Kreuzer KA, Kluhs C, Baskaynak G, Movassaghi K, Dorken B, le Coutre P. Filgrastim-induced stem cell mobilization in chronic myeloid leukae-

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52

Charlotte M. Niemeyer & Franco Locatelli

Hematopoietic Cell Transplantation for Juvenile Myelomonocytic Leukemia

Introduction Heterogeneity in presentation along with a clinical and morphologic picture mimicking a variety of infectious agents and metabolic disorders has long precluded a rational approach to the clinical management of children with juvenile myelomonocytic leukemia (JMML). The recent discovery that mutations in PTPN11 (protein-tyrosine phosphatase nonreceptor-type 11, the gene encoding protein SHP-2) are the most common molecular lesions in JMML has greatly improved our understanding of the molecular pathogenesis of this disorder. Further, it has enormously facilitated diagnosis by allowing mutational analyses. With clinical diagnosis confirmed by molecular markers, allogeneic hematopoietic cell transplantation (HCT) can be performed early in the course of the disease. In addition, monitoring of minimal residual disease (MRD) following transplantation has become feasible. This chapter summarizes the clinical and molecular features of JMML and reviews the role of HCT.

Classification and epidemiology JMML is a clonal haematopoietic disorder of early childhood characterized by excessive proliferation of monocytic and granulocytic cells. Following a first case description in 1924 [1], Jean Bernard and his coworkers in Paris carefully described the clinical picture in a group of 20 children in 1962 [2]. They referred to the disorder as subacute and chronic myelomonocytic leukemia and identified prognostic factors for survival [3]. Following the demonstration that the Philadelphia chromosome was absent in infants with chronic myelomonocytic leukemia (CMML), Hardisty coined the term “juvenile chronic myelogenous leukemia” (jCML) [4], and it became common practice to contrast jCML with Philadelphia chromosome-positive CML [5]. At the same time, British colleagues proposed the term “monosomy 7 syndrome” as a separate entity in children with myeloproliferative disorders and monosomy 7. This infantile monosomy 7 syndrome had clinical features similar to jCML, but lower hemoglobin F (HbF) levels [6]. Meanwhile, the French–American–British group had rationalized the morphologic classification of myelodysplastic syndrome (MDS) in adults [7]. Some investigators argued for the application of modified French–American– British criteria in children and used the term “chronic myelomonocytic leukemia” instead of jCML [8,9].

To avoid further confusion, in 1996, an international working group proposed the term JMML and established criteria for its diagnosis [10]. JMML incorporates those disorders previously referred to as jCML or CMML of infancy, as well as some cases of the infantile monosomy 7 syndrome [11]. To account for both the myelodysplastic and proliferative features noted in JMML, the recent World Health Organization classification placed the entity in the group of myelodysplastic/myeloproliferative disorders [12]. JMML accounts for about 2–3% of all childhood hematologic malignancies [13]. Incidence studies from Denmark and British Columbia show a JMML incidence of 1.2/million children per year [13], while a much lower incidence of 0.6/million has been reported from the United Kingdom [14]. It is unknown whether these differences are due to different geographic incidence rates or to variations in reporting.

Clinical presentation JMML is seen predominately in infants, with a median age at diagnosis of 2 years [3,8]. About 9% of patients are diagnosed by the age of 4 months, whereas only 8% are 6 years or older. There is a male predominance with a male-to-female ratio of 2 : 1. Pallor, fever, infection, skin bleeding, and cough are the most commonly presenting symptoms [8]. Marked splenomegaly and hepatomegaly are generally present and may give rise to abdominal discomfort. Occasionally, spleen size is normal at diagnosis, but it rapidly increases thereafter. Gut infiltrates may predispose to diarrhea and gastrointestinal infections. About half the patients have lymphadenopathy [8,15]. In addition, leukemic infiltrates may give rise to markedly enlarged tonsils. Dry cough, tachypnea, and interstitial infiltrates on chest X-ray are signs of peribronchial and interstitial pulmonary infiltrates. JMML rarely involves the central nervous system, although a small number of patients with central nervous system chloroma [8] and with ocular infiltrates have been described. Leukemic skin infiltrates are common. Most often they present as eczematous eruptions or indurated raised lesions with a central clearing. In addition to these often nonspecific lesions, juvenile xanthogranulomas may be seen. In some children, xanthogranulomas are associated with multiple café-au-lait spots and the clinical diagnosis of neurofibromatosis type 1 (NF1) [16]. JMML in NF1

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

The diagnosis of NF1 with multiple café-au-lait spots can be established in up to 11% of children with JMML [3,6]. As estimated from these

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data, the risk of developing JMML for patients with NF1 is 200–350fold higher than in patients without NF1 [17]. A positive family history for NF1 is known in about half of the cases of NF1 and JMML. While clinical parameters generally do not differ between patients with or without NF1, NF1 is more common among patients who are diagnosed after the age of 5 years. In addition, children with NF1 have a higher platelet count and a higher percentage of blasts in the bone marrow than patients without NF1 [8].

JMML-like disorder in Noonan’s syndrome Various clinical abnormalities and dysmorphisms, including Noonan’s syndrome, have been observed with a frequency of about 7% in patients with JMML [17]. Noonan’s syndrome is an autosomal dominant disorder characterized by short stature, distinct facial anomalies, developmental delay, and a typical spectrum of congenital heart defects, including pulmonic stenosis, hypertrophic cardiomyopathy, and septal defects. Within the first few months of birth, some infants with Noonan’s syndrome develop a disorder that is clinically and hematologically indistinguishable from JMML [18]. In these children, specific germline mutations in PTPN11 or KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) are identified (see the section on “Molecular pathogenesis” below). Most of these cases show spontaneous resolution of myeloproliferation without treatment [18,19], but a more aggressive behavior has been noted in others. To avoid unnecessary treatment, a careful watchand-wait strategy is recommended for JMML-like disorder in association with Noonan’s syndrome.

Table 52.1 Diagnostic criteria modified from [10] I.

Clinical and hematologic features (all three features mandatory) • Peripheral blood monocyte count >1 × 109/L • Blast percentage in peripheral blood and bone marrow <20% • Splenomegaly

II. Oncogenetic studies (one parameter sufficient) • Somatic mutation in PTPN11* or RAS* • NF1 mutation or clinical diagnosis of NF1 • Monosomy 7 III. In the absence of one parameter listed under II, the following criteria have to be fulfilled: • Absence of Philadelphia chromosome (BCR–ABL rearrangement) (mandatory) • And at least two of the following criteria: • Spontaneous growth or granulocyte–macrophage colony-stimulating factor hypersensitivity in colony assay • Hemoglobin F increased for age • Myeloid precursors on peripheral blood smear • White blood count >10 × 109/L • Clonal abnormality apart from monosomy 7 * In patients diagnosed within the first year of life, germline mutations (Noonan’s syndrome) have to be excluded.

100 Normal

60 40 20 0 0

2

4

6

8

10

12

14

12

14

100 80 Hemoglobin F (%)

Leukocytosis, thrombocytopenia, and anemia are common findings in JMML patients. The median white blood cell (WBC) count is 33 × 109/L [3,8,15]. In contrast to Philadelphia-positive CML, the WBC count rarely exceeds 100 × 109/L. A presenting WBC count under 10 × 109/L is occasionally noted at presentation, particularly in children with monosomy 7 [6,8]. Microscopy of the peripheral blood smear is the most important step in establishing the diagnosis. Immature monocytes, along with myelocytes, metamyelocytes, and nucleated red cells, are usually evident. Almost all cases show a striking monocytosis, often with dysplastic cell forms, and an absolute monocyte count exceeding 1 × 109/L is required for the diagnosis of JMML (Table 52.1) [10]. Often, a few blasts can be noted on peripheral blood smears, but the blast percentage rarely exceeds 20% [8]. Thrombocytopenia is a common finding, with about 14% of children having a platelet count at diagnosis of below 20 × 109/L. Most patients have a hemoglobin concentration between 7 and 11 g/100 mL. Red cells are generally normocytic, while macrocytosis is noted in some patients with monosomy 7 [8]. BM findings in JMML are not by themselves diagnostic, but rather are consistent with the diagnosis. The aspirate shows a high cell number with predominance of granulocytic cells at all stages of maturation, except for the few cases in which the erythroid series predominates. Monocytosis in bone marrow is generally less impressive than that in blood [3,8]. The marrow blast percentage is moderately elevated, but never reaches the level seen in acute leukemia. Megakaryocytes are reduced in number or absent in about two-thirds of cases. A remarkable feature of many JMML cases with a normal karyotype is a markedly increased synthesis of HbF (Fig. 52.1) resulting from a high number of circulating F cells [20]. In addition, other fetal red cell characteristics, such as increased expression of the “i” antigen and decreased carbonic anhydrase levels, are present [21].

Hemoglobin F (%)

80

Hematologic features

Monosomy 7 60 40 20 0 0

2

4

6

8

10

Age (years)

Fig. 52.1 Concentration of hemoglobin F for 273 children with juvenile myelomonocytic leukemia and either normal karyotype or monosomy 7.

Chromosomal abnormalities Chromosomal studies of leukemic cells show monosomy 7 in approximately 25% of JMML patients, with other abnormalities in 10%, but the majority (65%) of children have a normal karyotype [6,8,15]. While

Hematopoietic Cell Transplantation for Juvenile Myelomonocytic Leukemia

clinical characteristics of patients with monosomy 7 do not differ from those of patients with a normal karyotype, patients with monosomy 7 display some characteristic hematologic features [8]. Besides a lower median WBC count, red blood cells are often macrocytic, and erythropoiesis in bone marrow is more pronounced than in cases with a normal karyotype. In addition, patients with monosomy 7 present with a normal or only moderately elevated HbF, which is often elevated in patients with normal karyotype (Fig. 52.1). Among the chromosomal abnormalities other than monosomy 7, loss of material on the long arm of chromosome 7 is frequent [8]. As mentioned before, JMML lacks the Philadelphia chromosome and the BCR–ABL fusion gene.

Molecular pathogenesis Two important characteristics of JMML cells can be demonstrated in vitro. First, JMML cells from peripheral blood or bone marrow give rise to an excess number of monocyte–macrophage colonies when cultured in semisolid media in the absence of added growth factors. This so-called spontaneous proliferation of JMML myeloid progenitors depends on endogenous production of cytokines such as interleukin–1, granulocyte–macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha by monocytes. It can be completely abolished by prior depletion of adherent monocytes [22]. Second, following vigorous adherence depletion, JMML mononuclear cells exhibit a striking left shift of the GM-CSF dose–response curves compared with normal controls [23]. Although not absolutely specific, this GM-CSF hypersensitivity of myeloid progenitors has become a hallmark of JMML and an important diagnostic tool. Studies on human leukemic cells and mouse models provide evidence that GM-CSF hypersensitivity is due to a selective inability to downregulate RAS-dependent signaling pathways (Fig. 52.2). In approximately 25% of cases of JMML, the pathological activation of the RAS signaling cascade results from oncogenic mutations of NRAS (neuroblastoma RAS viral (v-ras) oncogene homolog) or KRAS [24,25]. Members of the RAS family of signaling proteins regulate cellular proliferation by cycling between an active guanosine triphosphate (GTP)bound state (RAS-GTP) and an inactive guanosine diphosphate-bound

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state (RAS-GDP). Mutant RAS alleles encode proteins that accumulate in the GTP-bound conformation. It has recently been demonstrated that oncogenic RAS is sufficient to initiate a rapidly fatal myeloproliferative disorder in mice associated with hyperproliferation, tissue infiltration, and aberrant responses to growth factors [26]. The conversion from active RAS-GTP to the inactive RAS-GDP state is facilitated by GTPase activating proteins. Neurofibromin, the protein encoded by the gene for NF1, functions as a GTPase activating protein and negatively regulates RAS. As described above, 11% of children with JMML have constitutional NF1 [3]. These patients carry one intact and one deficient allele of the NF1 tumor suppressor gene in the germline. Loss of the normal NF1 allele is common in JMML cells from children with NF1 [27], resulting in severely deregulated RAS signaling with aberrant growth of haematopoietic progenitor colonies in vitro [28]. Two recent studies indicate that a common mechanism of NF1 inactivation is uniparental disomy replacing the wild-type NF1 allele with the second copy bearing the NF1 mutation [29,30]. Shortly after the discovery of PTPN11 mutations in about 50% of patients with Noonan’s syndrome, specific germline PTPN11 alterations were identified in the young children with Noonan’s syndrome who developed a JMML-like disorder within the first few weeks of life [31,32]. Subsequently, somatic PTPN11 mutations were uncovered in JMML cells from 35% of children with nonsyndromic JMML [31,32]. The PTPN11 gene encodes for SHP-2, a protein tyrosine phosphatase that relays growth signals from activated growth factor receptors to other signaling molecules, including Ras. Most PTPN11 mutations are predicted to disrupt the autoinhibition of the catalytic phosphatase domain by the N-terminal src-homology 2 domain, thereby promoting the active conformation of the protein [33]. Somatic mutations observed in patients with JMML differ from those mutations found in patients with Noonan’s syndrome and JMML-like disorder, and from those detected in patients with Noonan’s syndrome alone [34]. In elegant functional experiments, several groups have shown that somatic PTPN11 mutations associated with sporadic JMML exhibit stronger biochemical and biologic effects than germline PTPN11 mutations, leading to the concept that only milder SHP-2 activation may be tolerated during embryonic development [35–37]. Murine model systems suggest that the phenotype exhibited by PTPN11 mutations is mediated through hyperactive RAS signaling. Recently, a patient with Noonan’s syndrome and JMML-like disorder has been found to harbor a de novo germline KRAS mutation [38]. Rare de novo KRAS mutations were subsequently identified in several additional cases of Noonan’s syndrome, indicating that the functional consequences of alterations in the PTPN11 or RAS genes overlap. Taken together, mutations of NF1, KRAS, NRAS or PTPN11 are present in mutually exclusive subsets in 75% of children with JMML. It is likely that the remaining approximately 25% of cases harbor lesions in yet undefined genes of the same pathway. Somatic point mutations greatly facilitate the diagnosis of JMML. They will also allow for the detection of allele-specific MRD following HCT, and be useful for the response evaluation of future window trials with targeted therapies.

Natural course and prognostic factors

Fig. 52.2 Simplified Ras signaling pathway. Proteins involved in the pathogenesis of juvenile myelomonocytic leukemia (JMML) (due to biallelic inactivation of NF1 or somatic mutations of PTPN11, KRAS or NRAS) are indicated in black.

JMML is a rapidly fatal disorder for most children if left untreated. The median survival time without HCT is about 1 year, and the probability of 10-year survival without HCT is 6%. Low platelet count, age above 2 years at diagnosis, and high HbF levels at diagnosis are the main predictors of short survival (Fig. 52.3) [3,6,8]. In a retrospective series of 110 cases, all children presenting with a platelet count of 33 × 109/L or less had died within a year from diagnosis, while those with higher

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Chapter 52

1.0 0.9 0.8 0.7 0.6 P

0.5 0.4 0.3 0.2

Log-Rank p = 0.0001

0.1 years 0.0 0

1

2 Plt ≥

3

4

33¥109/L,

5

6

7

8

9

10

11

12

age < 2 yr : 0.08, SE=0.08 (N = 27, 7 alive)

Plt ≥ 33¥109/L, age > 2 yr : 0.05, SE=0.05 (N = 19, 1 alive) Plt < 33¥109/L,

: 0.00, SE=0.00 (N = 24, 0 alive)

Fig. 52.3 Survival of patients without BMT according to platelet (Plt) count and age at diagnosis. (Reproduced from [105], with permission.)

[47]. There are, however, no data indicating that therapy with mercaptopurine influences the duration of survival. A great variety of other antineoplastic drugs and combination-type therapies have been applied, but are generally associated with poor responses [47,48]. As in other malignant disorders, steroids can be helpful in controlling pleural or pericardial effusion. Most approaches to intensive chemotherapy are derived from treatment protocols for acute myeloid leukemia (AML). Clinical remissions and long-term survival after AML-type combination therapy have been reported in small series [43,49]. Study CCG 2891 of the Children’s Cancer Study Group for treatment of childhood AML, MDS, and JMML included 13 children with JMML [44]. Seven of the 12 patients with JMML who received intensive induction chemotherapy achieved hematologic remission. Other investigators have pointed out that intensive chemotherapy is notably unsuccessful in patients with aggressive disease, and durable remissions may not be achievable [47,48,50–52]. The current JMML study of the Children’s Oncology Group includes cytoreductive therapy consisting of fludarabine and high-dose cytarabine concomitantly with 13-cis-retinoic acid prior to HCT.

Interferon-alpha and retinoic acid counts and age less than 2 years at diagnosis had a median survival of 3 years [8]. British investigators devised a scoring system where HbF of 10% or higher and a platelet count of 33 × 109/L or less negatively affect prognosis [6]. Blastic transformation is infrequent in JMML, and most untreated patients succumb to respiratory failure due to pulmonary infiltration of mature leukemic cells. Some young children with JMML (i.e. those diagnosed before 1 year of age) may experience a longer clinical course characterized by temporary clinical improvement in the absence of therapy. Although these children generally rapidly progress at a later age, some exceptional patients (without Noonan’s syndrome) spontaneously recover without any intervention. Japanese investigators recently claimed that the specific RAS mutations NRAS/KRASG12S might identify such patients [39]. Data from the European Working Groups of MDS in Childhood (EWOGMDS) do not support this hypothesis [40]. In the absence of reliable markers that prospectively identify these rare cases with spontaneous resolution, and in view of the clear superiority of HCT over other treatment modalities, prompt HCT for every patient with JMML, except children with Noonan’s syndrome, should be recommended.

Nontransplant approaches To ameliorate disease activity prior to HCT, various chemotherapeutic regimens have been administered [41–44]. More recently, novel agents have been evaluated in investigational phase II window trials in previously untreated JMML patients [45,46]. Although none of these regimens has been shown to affect overall survival, some studies have demonstrated prolonged responses. Comparative evaluation of the efficacy of different therapies is hampered by the variability of response with respect to WBC and platelet count, and liver or spleen size. Complete hematologic remission is difficult to define in a disease characterized by excessive myeloproliferation but low blast count. Hence, no generally accepted response criteria have been established. Low-dose and intensive chemotherapy Clinical and hematologic responses in JMML have most consistently been obtained with mercaptopurine, administered either as a single agent [3,47] or in combination with low-dose cytarabine [41,47] or etoposide

The apparently increased sensitivity of JMML cells to interferon-alpha [53] prompted some investigators to use this cytokine in treatment of the disease. Besides some clinical improvements [47], cases without objective response [54,55] have been reported. A prospective study of the Pediatric Oncology Group was stopped for excessive toxicity [56]. None of the evaluable patients had a partial or complete response. By contrast, in a JMML patient relapsing after allogeneic HCT, interferonalpha induced a sustained and complete remission [57]. It is unknown whether a direct antileukemic effect or an induced graft-versus-leukemia (GVL) reaction was responsible for this response. Spontaneous growth of JMML myeloid progenitors in vitro can be inhibited by 13-cis-retinoic acid (isotretinoin) [58]. Based on these laboratory observations, 10 children with JMML were treated with isotretinoin 100 mg/m2 daily [45]. Two children achieved complete remission, four had a partial or a minimal response, and four had disease progression. It is noteworthy that a large proportion of these children were below the age of 2 years, a factor known to characterize patients with a less aggressive course. In a subsequent phase II trial of the Pediatric Oncology Group, 22 evaluable patients were accrued; five complete and four partial responses were observed [59]. Other investigators did not observe significant clinical responses with retinoic acid [47,60], and its value in JMML remains questionable.

Therapy targeting Ras-dependent pathways The understanding of the important role of GM-CSF and the downstream Ras signaling transduction pathway has led to the design of therapeutic strategies targeting individual components of the pathway. The GM-CSF receptor has been targeted by the GM-CSF analog E21R. In a child with JMML relapsing after HCT, the administration of two cycles of E21R resulted in a transient clinical and hematologic response lasting for about 60 days [61]. Ras proteins are synthesized as precursor molecules in the cytoplasm, becoming activated in part by the addition of farnesly moiety catalyzed by the enzyme farnesyl-protein transferase. One farneslytransferase inhibitor, R115777 (Zarnestra) has been studied by the Children’s Cancer Group in an up-front phase II window study in newly diagnosed patients with JMML [62]. Overall response rate was based on changes

Hematopoietic Cell Transplantation for Juvenile Myelomonocytic Leukemia

in WBC count and organomegaly. Among the 38 patients receiving more than two courses, 22 had a complete or partial response. There was no relationship between farnesyl-protein transferase inhibition or response and the presence of RAS/PTPN11 mutation or inhibition of prenylation in vitro [62]. Other agents specifically designed to target components of the RAS–RAF–MAP and phosphoinositide-3 kinase/ protein kinase B [63] are still to be studied in the future. Although these strategies may not be sufficient to abolish the malignant clone by themselves, they could have an important role in future regimens of multimodality therapy.

Hematopoietic cell transplantation Early experiences The first HCT in JMML was reported from the group in Seattle in 1979 [64]. Subsequently, in 1988, the same investigators published a series of 14 children including the first case. Six of the 14 children transplanted from a human leukocyte antigen (HLA)-identical sibling or partially matched family donor remained in remission for up to 11 years after HCT [65]. In subsequent case reports and small series, a total of 48 children receiving HCT were reported by 1996 [65–76]. These early experiences indicated that HCT was capable of curing approximately a third of the children with JMML. Relapse was the major course of treatment failure and occurred early in the post-transplant course. In contrast, nonrelapse mortality (NRM) in this young patient population was low. Because patients with JMML are young and often do not have other siblings, partially matched family member donors and unrelated donors (URDs) were introduced in JMML transplantation in the early 1980s, although HCT experiences with alternative donor sources were limited at that time [47,65,68,77–83]. Most of these grafts were partially Tlymphocyte depleted in vitro. Event-free survival (EFS) after alternative donor transplantation was inferior to that of HLA-identical sibling HCT recipients, the most important problems encountered being represented by the risks of increased incidence of graft rejection, graft-versus-host disease (GVHD), and leukemia relapse. In a retrospective multicenter review of 43 children treated with HCT between 1987 and 1995, and registered to the EWOG-MDS data base, the probability of EFS for 18 patients transplanted from a matched URD or a two- to three-antigendisparate relative was 22% at 5 years, compared with 38% for 25 patients transplanted from an HLA-identical sibling donor (p < 0.5) [79]. There was a significant difference in the incidence of chronic GVHD between children transplanted from compatible/one-antigen-mismatched relatives and from alternative donors (23% and 87%, respectively; p < 0.005). Also, probabilities of NRM for children given HCT from an HLA-identical/one-antigen-disparate relative or from a matched URDs/ mismatched relative were 9% and 46%, respectively [79]. Similarly, children with JMML undergoing transplantation from URDs facilitated by the US National Marrow Donor Program (NMDP) between 1990 and 1997 had a probability of EFS of 24% at a median follow-up of 2 years [83]. In this study, patients developing grade III or IV GVHD had a significantly poorer survival. In contrast, chronic GVHD was associated with a decreased risk of death [83]. A more favorable outcome for alternative donor HCT was reported in a single-center experience from Toronto: eight of 12 consecutive patients remained in remission with a median follow-up of 31 months [81]. However, median age at HCT for these 12 children was only 1.9 years, compared with 2.8 years and 3.4 years in the EWOG-MDS and NMDP cohorts, respectively. Since age remains the most important prognostic factor for survival and relapse following transplantation, this difference may explain at least in part the better outcome of the Toronto patients.

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Preparative regimen During the early experiences, most children with JMML undergoing HCT received total body irradiation (TBI) as part of the preparative regimen. In fact, TBI has been recommended by the North American JMML Working Party until recently. There have been a number of reports indicating more deleterious long-term morbidity in young children after TBI compared with busulfan (BU)-based regimens [84]. To avoid severe radiation-induced growth retardation, endocrine, and neuropsychological sequelae, and second malignancies, several groups introduced non-TBI preparative regimens for HCT in JMML [72,74,75,78,85–87]. Most investigators chose BU and cyclophosphamide (CY) combined with etoposide (VP-16) [72,87] or melphalan [78,79,88], whereas others employed BU, cytarabine (Ara-C) and melphalan [47]. There are no reports indicating that TBI offers an advantage over a BU-containing preparative regimen. In a retrospective study on 27 children with JMML transplanted between 1990 and 1997 in Japan, the probability of EFS at 4 years was 54%, with no difference in outcome between TBI and non-TBI regimens. In contrast, the retrospective EWOG-MDS study demonstrated for the group of children given HCT from an HLA-identical sibling or a one-antigen-disparate relative, that the use of BU was associated with a lower probability of relapse (38% versus 78%, respectively), which resulted in a significantly better EFS in comparison to the use of TBI (62% versus 11%, respectively; p < 0.01) [79]. A significantly higher relapse rate in patients prepared with TBI was also demonstrated in a literature review on 65 children with JMML transplanted prior to 2000 [82]. EWOG-MDS chose a preparative regimen consisting of the three alkylating agents BU, CY, and melphalan [78]. Melphalan had been shown to increase the antileukemic efficacy of a BU/CY regimen in children given an unpurged autologous HCT [89]. In addition, a preparative regimen consisting of three alkylating drugs that have noncellcycle specific action appears potentially capable of eradicating stem cell disorders, such as JMML, in which at least a portion of clonogenic cells are dormant out of cycle. In the currently largest prospective study, 100 consecutive JMML patients registered in the EWOG-MDS were given unmanipulated grafts after a preparative regimen with BU 16 mg/kg, CY 120 mg/kg, and melphalan 140 mg/m2. All but three children experienced prompt engraftment. The 5-year probability of EFS for 48 patients given a graft from an HLA-identical sibling and 52 children transplanted from an HLA-matched or one-antigen/allele-disparate URD was 55% (95% confidence interval [CI] 41–70%) and 49% (95% CI 35–63%), respectively (p = NS) [88]. With a cumulative incidence of NRM of 13%, and no significant difference between sibling and URD transplants, this result indicates that the preparative regimen was safe. In addition, it confirmed that the use of URDs offers minimal or possibly no significant disadvantage compared with employing an HLA-identical sibling [86]. Figure 52.4 gives an update for EFS, NRM, and relapse incidence for the EWOG-MDS study [88] after a median observation time of patients alive of 5.8 (range 2.3–12) years.

Nonrelapse mortality With current supportive care, recent multicenter trials indicated that the probability of NRM in patients transplanted for JMML is not influenced by the use of either an HLA-identical sibling donor or a URD, ranging in the order of 11% [86], 13% [88], and 18% [83]. This surprisingly low mortality in a generally much compromised patient population transplanted with an intensive preparative regimen might be explained by the young patient age and the experience of centers dedicated to HCT in infants. Infections and hepatic toxicity account for most of the deaths.

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Relapse Relapse is the major cause of treatment failure of HCT in patients with JMML undergoing HCT [79,80,82,83,86,88]. It generally occurs within the first year after transplantation, the peak being observed after a median time of 2–6 months [83,88]. Older age [79,86,88], increased percentage of HbF [83,88], abnormal karyotype [86], and blast percentage above 20% [88] have been identified as patient-specific risk factors for disease recurrence. Age above 2 years at diagnosis and a high HbF have been found to predict short survival in studies analyzing the natural history of JMML in patients not treated with HCT [3,6,8].

1.0

GVHD and GVL effect

0.8

% (95% CI)

In the multivariate analysis of the prospective EWOG-MDS study, age remained the major significant factor for prediction of disease recurrence. The cumulative incidence of relapse for children less than 2 years of age at diagnosis, 2–4 years, and older than 4 years was 18% (95% CI 10–32%), 46% (95% CI 30–70%), and 73% (95% CI 53–98%), respectively, resulting in probability of EFS of 64% (95% CI 51–76%), 50% (95% CI 31–70%), and 16% (95% CI 0–34%) [88]. An update on these data with a median observation time of patients alive of 5.8 (range 2.3–12) years is shown in Fig. 52.5. The poor outcome of older children with JMML demands new strategies such as a better preservation of a GVL effect. It also points to the fact that HCT shortly after diagnosis is indicated even in patients less than 1 year of age.

Survival = 58% (46–70) n = 100; E = 40

0.6

EFS = 52% (42–62) n = 100; E = 48

0.4

RI = 35% (27–46) n = 100; E = 35

0.2

TRM = 13% (8–22) n = 100; E = 13

0.0 0

2

4 6 8 Years after transplantation

10

12

Fig. 52.4 Overall survival, event-free survival (EFS), relapse incidence (RI), and transplant-related mortality (TRM) of 100 children with juvenile myelomonocytic leukemia transplanted after a conditioning regimen including busulfan, cyclophosphamide, and melphalan from a matched related (n = 48) or a matched unrelated (n = 52) donor. Update with a median observation time of patients alive of 5.8 (range 2.3–12) years of data published by Locatelli et al. [88]. CI, confidence interval; E, events.

The most frequently used regimens for GVHD prophylaxis consist of cyclosporine for patients transplanted from an HLA-identical sibling donor, and of cyclosporine with methotrexate and anti-thymocyte globulin for URD transplantation [82,88]. The prospective EWOG-MDS study reported a cumulative incidence at day 100 of grade II–IV GVHD of 40% (95% CI 31–51%), whereas that of grades III–IV acute GVHD was 17% (95% CI 11–26%) [88]. There was no difference in the incidence of grade II–IV GVHD between matched sibling donor (46%) and URD donor (35%) transplants (p = NS). No patient- or transplantationrelated variable was significantly associated with the development of acute GVHD in multivariate analysis [88]. Acute GVHD grade III–IV has been associated with poor overall survival in JMML patients [83,86]. Due to the young age of JMML patients, incidence rates for acute and chronic GVHD tend to be lower than in other leukemic diseases. Chronic GVHD in children transplanted for JMML has been reported in 17% of children at risk in the prospective EWOG-MDS study [88], in 25% of patients in the NMDP cohort [83], and in 10 of 24 cases in the Japanese review [86]. Interestingly, in contrast to EWOG-MDS reporting the lowest cumulative incidence of chronic GVHD, investigators from the United States and Japan noted a better overall survival for children

1.0

1.0

0.8

≥ 4 years = 68% (49–94)

0.6 2–4 years = 46% (30–70) 0.4

Probability (95% CI)

Cumulative incidence (95% CI)

0.8

< 2 years = 63% (50–75) 0.6

2–4 years = 50% (31–69) 0.4 ≥ 4 years = 21% (1–40)

< 2 years = 20% (12–33) 0.2

0.2 < 2 years: n = 56; E = 11 ≥ 4 years: n = 17; E = 12

0.0 0

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< 2 years: n = 56; E = 21 2–4 years: n = 27; E = 13

2–4 years: n = 27; E = 12

p = 0.0001

2

3

Years after transplantation

4

5

p = 0.008

>4 years: n = 17; E = 14

0.0 0

1

2

3

4

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Years after transplantation

Fig. 52.5 Relapse incidence (left panel) and event-free survival (right panel) by age at diagnosis of 100 children with juvenile myelomonocytic leukemia transplanted after a conditioning regimen including busulfan, cyclophosphamide, and melphalan from a matched related (n = 48) or a matched unrelated (n = 52) donor. Update with a median observation time of patients alive of 5.8 (range 2.3–12) years of data published by Locatelli et al. [88]. CI, confidence interval; E, events.

Hematopoietic Cell Transplantation for Juvenile Myelomonocytic Leukemia

suffering from chronic GVHD compared with those without [83,86]. These data suggest a possible GVL effect directed against JMML cells. A number of reports describe the efficacy of withdrawal of immunosuppressive therapy in patients with relapsed JMML [47,57,73,80,82, 90,91]. In addition, less-intensive GVHD prophylaxis is generally employed during second HCT, a procedure resulting in similar longterm survival to that of first HCT despite the usage of the same donor [92]. Taken together, there is sufficient evidence that GVL plays a major part in eradicating JMML cells following allogeneic transplantation. Not surprisingly, HCT after in vitro depletion of donor T lymphocytes is hampered by a high relapse rate (EWOG-MDS registry, unpublished observation). Stem cell source and umbilical cord blood transplantation Most HCT in JMML has been performed with unmanipulated bone marrow cells. HCT with peripheral blood hematopoietic cells does not result in a better overall survival or lower relapse rate [88]. Cord blood has been used as an alternative source of hematopoietic stem cells to treat patients with a variety of haematologic malignancies, including JMML [88,93,94]. In a recent review on behalf of Eurocord-European Group for Blood and Marrow Transplantation and EWOG-MDS on 42 children with JMML given unrelated cord blood transplantation, the 2year probability of EFS was 45%, patients younger than 2.6 years having a better outcome [95]. The cumulative incidence of NRM at 2 years was 16%, similar to what has been reported in studies using bone marrow grafts. Despite a greater HLA disparity, the probabilities of grade II–IV acute GVHD (31%) and chronic GVHD (16%) were similar to those reported from studies employing conventional grafts. Eleven patients relapsed, the 2-year cumulative incidence of relapse being 22%. In conclusion, unrelated cord blood transplantation is a suitable option for children with JMML lacking an HLA-identical sibling donor. A search for unrelated cord blood can be initiated simultaneously with that for unrelated bone marrow donors. Chimerism studies and MRD As in other hematologic malignancies, serial quantitative chimerism studies using short number of tandem repeat markers have been shown to identify patients with increasing mixed chimerism who are at high risk for relapse following transplantation for JMML [96]. Because it can be demonstrated that rapidly increasing mixed chimerism is invariably followed by relapse of JMML [96], immediate withdrawal of immunosuppressive therapy is indicated after the first detection of mixed chimerism. Since severe GVHD is uncommon after early cessation of immunosuppression in this young patient population, this maneuver appears safe and effective [96]. However, in some patients, rapid progression of disease occurs in the absence of a previously documented mixed chimerism status. More sensitive methods, such as the application of disease-specific markers, may increase sensitivity, thus allowing the detection of a smaller percentage of residual leukemic cells. For this purpose, a fluorescence-based, allele-specific polymerase chain reaction assay to detect the most common RAS or PTPN11 mutations has recently been developed [97]. Prospective studies will be required to demonstrate whether this method will allow modification of post-transplant immunosuppression early enough to prevent hematologic relapse. Donor lymphocyte infusion Treatment options for patients relapsing with leukemia after HCT are limited. Withdrawal of immunosuppressive drugs is usually the first

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measure, which by itself can control leukemia in a limited number of patients [47,57,73,80,82,90,91,98], suggesting a GVL effect in JMML. In case of nonresponse and for patients suffering disease recurrence after discontinuation of immunosuppressive agents, donor lymphocyte infusion (DLI) or second HCT may be considered. There are several reports of successful therapy with DLI of relapsed JMML [99–101]. Two of three patients had a normal karyotype, one monosomy 7. Following chemotherapy (n = 2) or splenectomy (n = 1), DLI from the matched (n = 2) or one HLA locus-mismatched (n = 1) URD had been administered. GVHD was observed in two patients, bone marrow failure in one, and all patients were alive at the time of reporting. These case reports suggest that at least some relapsed JMML patients can benefit from DLI. However, reporting bias is likely to exaggerate the benefit of this form of therapy in single successful cases. In a retrospective study from the EWOG-MDS, the efficacy of DLI was studied in 21 JMML patients receiving DLI for mixed chimerism (n = 7) or relapse (n = 14) [98]. Response rates were significantly higher in patients receiving a higher total T-cell dose (≥1 × 107/kg) and in patients with an abnormal karyotype. None of the six patients receiving DLI from a HLA-identical sibling donor responded. In total, six of the 21 patients responded to DLI, but only one of the responders remained alive in remission, two relapsed, and three died of complications. Although JMML can be sensitive to DLI, the overall outcome of patients responding to DLI is poor due to complications and relapse. Second transplantation Several reports on the results of second HCT for relapse after first HCT in patients with various types of leukemia have been published. These analyses generally show a poor outcome with a high rate of relapse and NRM. Because relapse remains the major cause of transplant failure, second HCT had been attempted in a number of patients. Chang et al. reviewed the reports on 13 patients undergoing second transplantation, seven of whom were alive and disease free with a median follow-up of 53 months [102]. A recent retrospective review of EWOG-MDS confirmed that second HCT can be an effective salvage therapy for children with JMML relapsing after a first grafting procedure [92]. Twenty-four patients were given a second allograft for relapse diagnosed at a median of 160 days after first HCT. Median time between first and second HCT was 260 days. The same donor was employed for both first and second HCT in 19 of the 24 patients. Ten patients (43%) were alive in remission after a median observation time of 3.3 years. The probability for leukemia-free survival at 3 and 5 years was 50% (95% CI 30–70%) and 32% (95% CI 8–56%). While none of the six patients who developed chronic GVHD after second HCT experienced leukemia recurrence, eight of 14 patients without chronic GVHD relapsed after second HCT (p = 0.04). It is reasonable to speculate that reduced immunosuppressive therapy employed during the second grafting procedure resulted in a stronger GVL effect and favorable outcome. Eighteen of the 24 patients had received a BU-containing regimen for first HCT and a TBI-based regimen for second HCT, and nine of 18 were alive in remission. A report on HCT in JMML from the Seattle group included six patients who underwent second HCT using TBI/CY ± BU and BU/CY regimens for first and second HCT, respectively [103]. Only one of the six patients was alive in remission after second HCT. In view of these data and of the results obtained in the EWOG-MDS study, it seems reasonable to employ a non-TBI regimen to avoid late sequelae for first HCT, and to reserve TBI for those patients with JMML who may need a second HCT. In conclusion, current results on second HCT in JMML are superior to those obtained with DLI and, surprisingly, similar to those reported for first HCT.

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Splenectomy and other treatment prior to transplantation Although some investigators suggest that aggressive chemotherapy can ameliorate the disease in some children with JMML [43,44], there is no evidence that it reduces relapse incidence or improves survival following transplantation [88]. Therefore, intensive chemotherapy prior to HCT for JMML is not recommended by most investigators. Likewise, none of the other therapy approaches has been shown to have an impact on post-transplant survival. Whether epigenetic drugs or agents targeting the Ras pathway prior to HCT may be beneficial is currently unknown. Splenectomy remains a controversial intervention for children with JMML. In Philadelphia chromosome-positive CML, it has not been shown to improve survival after allogeneic HCT. However, gross enlargement of the spleen may give rise to abdominal discomfort and excessive transfusion requirements. Therefore, early splenectomy has been recommended for amelioration of the disease [47,104], and in the last study of the Children’s Oncology Group, all clinically stable patients were scheduled for splenectomy. In a retrospective study of 72 children with JMML who received an allogeneic hematopoietic cell graft, splenectomy prolonged survival independently of other risk factors (authors’ own unpublished observation). Although it has been common practice to remove large spleens prior to HCT [79,81], the benefit of splenectomy for prevention of posttransplant relapse is unknown [47,85]. In the prospective EWOG-MDS

study, splenectomy before HCT, as well as spleen size at time of the allograft, did not have an impact on post-transplantation survival, relapse rate or time to engraftment [88]. The results of this study are not in favor of the indiscriminate use of splenectomy before transplantation. Splenectomy at a young age is associated with an increased risk of life-threatening infections, and in the patient series by Bunin et al., one JMML patient developed Streptococcus pneumoniae sepsis following transplantation despite antibiotic prophylaxis [81]. The indication of performing splenectomy has to be carefully evaluated for each single child. The presence of massive splenomegaly with evidence of hypersplenism and/or refractoriness to platelet transfusion can be an argument for considering this procedure in order to promote engraftment, to hasten hematologic recovery, and to lower the risk of hemorrhagic complications.

Outlook Currently about 60% of children with JMML can be cured following first or second HCT. Reduction of relapse incidence for older children will be one of the major challenges for future clinical trials. Lessimmunuosuppressive therapy for GVHD prophylaxis and careful monitoring of MRD following transplantation will be required. Preparative regimens with better antileukemic efficacy can be designed, but major advances in transplant biology in children with JMML may depend on new insights into the etiology and molecular genesis of the disease.

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45. Castleberry RP, Emanuel PD, Zuckerman KS et al. A pilot study of isotretinoin in the treatment of juvenile chronic myelogenous leukemia. N Engl J Med 1994; 331: 1680–4. 46. Castleberry RP, Loh ML, Jayaprakash N et al. Phase II window study of the farnesyltransferase inhibitor R115777 (Zarnestra®) in untreated juvenile myelomonocytic leukemia (JMML): a Children’s Oncology Group study. Blood 2005; 106: 727a. 47. Lutz P, Zix-Kieffer I, Souillet G et al. Juvenile myelomonocytic leukemia: analyses of treatment results in the EORTC Children’s Leukemia Cooperative Group (CLCG). Bone Marrow Transplant 1996; 18: 1111–16. 48. Laver J, Kushner BH, Steinherz PG. Juvenile chronic myeloid leukemia: therapeutic insights. Leukemia 1987; 1: 730–3. 49. DeHeredia CD, Ortega JJ, Coll MT, Bastida P, Olivé T. Results of intensive chemotherapy in children with juvenile chronic myelomonocytic leukemia: a pilot study. Med Pediatr Oncol 1998; 31: 516–20. 50. Festa RS, Shende A, Lanzkowsky P. Juvenile chronic myelocytic leukemia: experience with intensive combination chemotherapy. Med Pediatr Oncol 1990; 18: 311–16. 51. Hasle H, Kerndrup G, Yssing M et al. Intensive chemotherapy in childhood myelodysplastic syndrome. A comparison with results in acute myeloid leukemia. Leukemia 1996; 10: 1269– 73. 52. Bergstrasser E, Hasle H, Rogge T et al. Treatment of juvenile myelomonocytic leukemia other than hematopoietic stem cell transplantation: a retrospective analysis and definition of response criteria. Pediatr Blood Cancer 2007; 49: 629–33. 53. Estrov Z, Lau AS, Williams BR, Freedman MH. Recombinant human interferon alpha-2 and juvenile chronic myelogenous leukemia: cell receptor binding, enzymatic induction, and growth suppression in vitro. Exp Hematol 1987; 15: 127– 32. 54. Mirro J, Dow LW, Kalwinsky DK et al. Phase I–II study of continuous-infusion high-dose human lymphoblastoid interferon and the in vitro sensitivity of leukemic progenitors in nonlymphocytic leukemia. Cancer Treat Rep 1986; 70: 363–7. 55. Hazani A, Barak Y, Berant M, Bar-Maor A. Congenital juvenile chronic myelogenous leukemia: therapeutical trial with interferon alpha-2. Med Pediatr Oncol 1993; 21: 73–6. 56. Maybee D, Dubowy R, Krischer J, Buchanan G, Bowman W, Dicker J. Unusual toxicity of high dose alpha interferon (aIFN) in the treatment of juvenile chronic myelogenous leukemia (JCML). Proc Am Soc Clin Oncol 1992; 1: 950a. 57. Ohta H, Kawai M, Sawada A et al. Juvenile myelomonocytic leukemia relapsing after allogeneic bone marrow transplantation successfully treated with interferon-alpha. Bone Marrow Transplant 2000; 26: 681–3. 58. Emanuel PD, Zuckerman KS, Wimmer R, Cohn S, Chaffee S, Castleberry RP. In vivo 13-cis retinoic acid therapy decreases the in vitro GM-CSF hypersensitivity in juvenile chronic myelogenous leukemia (JCML). Blood 1991; 78(10 Suppl 1): 170a. 59. Castelberry RP, Chang M, Maybee D, Emanuel P. A phase II study of 13-cis retinoic acid in

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75. Rubie H, Attal M, Demur C et al. Intensified conditioning regimen with busulfan followed by allogeneic BMT in children with myelodysplastic syndromes. Bone Marrow Transplant 1994; 13: 759–62. 76. Donadieu J, Stephan JL, Blanche S et al. Treatment of juvenile chronic myelomonocytic leukemia by allogeneic bone marrow transplantation. Bone Marrow Transplant 1994; 13: 777–82. 77. Chown SR, Potter MN, Cornish J et al. Matched and mismatched unrelated donor bone marrow transplantation for juvenile chronic myeloid leukaemia. Br J Haematol 1996; 93: 674–6. 78. Locatelli F, Pession A, Comoli P et al. Role of allogeneic bone marrow transplantation from an HLA-identical sibling or a matched unrelated donor in the treatment of children with juvenile chronic myeloid leukaemia. Br J Haematol 1996; 92: 49–54. 79. Locatelli F, Niemeyer C, Angelucci E et al. Allogeneic bone marrow transplantation for chronic myelomonocytic leukemia in childhood: a report from the European Working Group on Myelodysplastic Syndrome in Childhood. J Clin Oncol 1997; 15: 566–73. 80. MacMillan ML, Davies SM, Orchard PJ, Ramsay NK, Wagner JE. Haemopoietic cell transplantation in children with juvenile myelomonocytic leukaemia. Br J Haematol 1998; 103: 552–8. 81. Bunin N, Saunders F, Leahey A, Doyle J, Calderwood S, Freedman MH. Alternative donor bone marrow transplantation for children with juvenile myelomonocytic leukemia. J Pediatr Hematol Oncol 1999; 21: 479–85. 82. Matthes-Martin S, Mann G, Peters C et al. Allogeneic bone marrow transplantation for juvenile myelomonocytic leukaemia: a single centre experience and review of the literature. Bone Marrow Transplant 2000; 26: 377–82. 83. Smith FO, King R, Nelson G et al. Unrelated donor bone marrow transplantation for children with juvenile myelomonocytic leukaemia. Br J Haematol 2002; 116: 716–24. 84. Michel G, Socie G, Gebhard F et al. Late effects of allogeneic bone marrow transplantation for children with acute myeloblastic leukemia in first complete remission: the impact of conditioning regimen without total-body irradiation – a report from the Socíété Française de Greffe de Moelle. J Clin Oncol 1997; 15: 2238–46. 85. Maguire AM, Vowels MR, Russell S et al. Allogeneic bone marrow transplant improves outcome

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96. Yoshimi A, Niemeyer CM, Bohmer V et al. Chimaerism analyses and subsequent immunological intervention after stem cell transplantation in patients with juvenile myelomonocytic leukaemia. Br J Haematol 2005; 129: 542–9. 97. Archambeault S, Yoshimi A, Kratz C et al. Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia-moving beyond clinical assessment. Blood (submitted for publication). 98. Yoshimi A, Bader P, Matthes-Martin S et al. Donor leukocyte infusion after hematopoietic stem cell transplantation in patients with juvenile myelomonocytic leukemia. Leukemia 2005; 19: 971–7. 99. Worth A, Rao K, Webb D, Chessells J, Passmore J, Veys P. Successful treatment of juvenile myelomonocytic leukemia relapsing post stem cell transplant using donor lymphocyte infusion. Blood 2003; 101: 1713–14. 100. Pulsipher MA, Adams RH, Asch J, Petersen FB. Successful treatment of JMML relapsed after unrelated allogeneic transplant with cytoreduction followed by DLI and interferon-alpha: evidence for a graft-versus-leukemia effect in nonmonosomy-7 JMML. Bone Marrow Transplant 2004; 33: 113–15. 101. Neudorf S, Nourani A, Kempert P, Shen V, Sender L, Kirov I. Chemotherapy and donor leukocyte infusions for relapsed juvenile myelomonocytic leukemia (JMML). Bone Marrow Transplant 2004; 33: 1069. 102. Chang YH, Jou ST, Lin DT, Lu MY, Lin KH. Second allogeneic hematopoietic stem cell transplantation for juvenile myelomonocytic leukemia: case report and literature review. J Pediatr Hematol Oncol 2004; 26: 190–3. 103. Yusuf U, Frangoul HA, Gooley TA et al. Allogeneic bone marrow transplantation in children with myelodysplastic syndrome or juvenile myelomonocytic leukemia: the Seattle experience. Bone Marrow Transplant 2004; 33: 805–14. 104. Steinherz PG, Exelby PR, Young J, Watson RC. Splenectomy after angiographic embolization of the splenic artery in patients with massive splenomegaly and severe thrombocytopenia, in juvenile subacute myelomonocytic leukemia. Med Pediatr Oncol 1984; 12: 28–32. 105. Niemeyer CM, Arico M, Basso G et al. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. European Working Group on Myelodysplastic Syndromes in Childhood (EWOG-MDS). Blood 1997; 89: 3534–43.

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Frederick R. Appelbaum

Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia

Introduction

Molecular and cellular biology

Acute myeloid leukemia (AML) defines a spectrum of myeloid malignancies with many common features but also considerable variability in underlying biology and clinical outcomes. Because the majority of patients will not be cured with conventional chemotherapy, hematopoietic cell transplantation (HCT) plays an important role in the management of this disease. However, given the multiple forms of transplantation available (allogeneic related, allogeneic unrelated, autologous, reduced intensity, etc.) together with the variability in AML, careful evaluation and planning are required to optimally integrate HCT into the care of each patient.

Cell of origin

Epidemiology and etiology In 2005, approximately 12,000 Americans developed AML. The incidence of AML is roughly constant over the first three decades of life and then increases almost exponentially [1]. In the large majority of cases, no clear etiology can be found, although exposure to benzene, ionizing radiation, and cigarette smoke is associated with an increased incidence of AML. With increasing use of radiation and chemotherapy to treat malignancy, treatment-related AML has become more frequent and now accounts for 6–8% of AML cases [2]. AML following alkylating agent exposure typically has a latency period of 5–7 years and first appears as a myelodysplastic syndrome (MDS) with abnormalities of chromosomes 5 and 7. AML following topoisomerase II exposure has a shorter latency without an MDS prodrome, and often has chromosomal abnormalities involving 11q23. The incidence of AML is increased in several genetic syndromes. Syndromes with impaired DNA repair, including Bloom’s syndrome, ataxia telangiectasia, and Fanconi’s anemia, are associated with an increased incidence of AML and relative intolerance to high-dose transplant preparative regimens. The incidence of AML is also increased in several congenital cytopenia syndromes, including Blackfan-Diamond and Kostmann’s syndromes. A familial syndrome due to germline mutations of RUNX-1 results in thrombocytopenia and a high probability of eventual AML development.

AML is a clonal disorder, with all leukemia cells in an individual patient descending from a common leukemia stem cell. Efforts to identify the nature of the AML stem cell have been based on the ability of a cell to establish leukemia when transferred to an immunodeficient mouse. Such studies indicate that leukemia stem cells make up a relatively rare subpopulation of the leukemia, accounting for only 0.2–100/106 leukemic cells, and have a primitive CD34++CD38− phenotype, no matter the degree of differentiation of the rest of the leukemia cells [3]. Pathology Although the leukemia stem cell may have a primitive phenotype, the bulk of leukemia cells in any individual will exhibit morphologic evidence of some differentiation. For more than three decades, the French– American–British system has been used to classify AML, dividing cases among eight categories (M0–M7) according to the degree of differentiation and lineage (Table 53.1) [4]. While of historic interest, AML morphology is of little significance once the underlying molecular abnormality is taken into consideration. Immunophenotype AML blasts can also be defined according to the combination of myeloidassociated antigens expressed on the cell surface. The most immature cases express CD34, CD117, and CD33. With further maturation, cases express CD13 and lose expression of CD34. Acute promyelocytic leukemias (APLs) express CD15s but do not express human leukocyte antigen (HLA)-DR. AML cases with monocytic differentiation express CD14, while those with erythroid differentiation exhibit CD36 and CD71. Acute megakaryocytic leukemias react with antibodies to CD61. AML cases sometimes express a B- or T-cell-associated antigen (CD19 or CD2), but this is of no apparent clinical significance. Molecular pathology and cytogenetics

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Animal models of AML suggest that most cases require multiple mutations for full expression of the disease, including mutations that block differentiation and others that result in inappropriate proliferation [5]. These mutations can be divided into those that are recognizable by

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Table 53.1 World Health Organization classification of acute myeloid leukemia Acute myeloid leukemia with recurrent genetic abnormalities Acute myeloid leukemia with t(8;21)(q22;q22), (AML1/ETO) Acute myeloid leukemia with abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(q13;q22), (CBFb/MYH11) Acute promyelocytic leukemia with t(15;17)(q22q12), (PML/RARα) and variants Acute myeloid leukemia with 11q23 (MLL) abnormalities Acute myeloid leukemia with multilineage dysplasia Following MDS or MDS/MPD Without antecedent MDS or MDS/MPD, but with dysplasia in at least 50% of cells in 2 or more myeloid lineages Acute myeloid leukemia and myelodysplastic syndromes, therapy related Alkylating agent/irradiation-related type Topoisomerase II inhibitor-related type (some may be lymphoid) Others Acute myeloid leukemia, not otherwise categorized Classify as: Acute myeloid leukemia, without maturation (FAB M0) Acute myeloid leukemia minimally differentiated (FAB M1) Acute myeloid leukemia with maturation (FAB M2) Acute myelomonocytic leukemia (FAB M4) Acute monoblastic/acute monocytic leukemia (FAB M5) Acute erythroid leukemia (erythroid/myeloid and pure erythroleukemia) (FAB M6) Acute megakaryocytic leukemia (FAB M7) Acute basophilic leukemia Acute panmyelosis with myelofibrosis Myeloid sarcoma FAB, French–American–British system; MPD, myelodysplastic syndrome. See text for other abbreviations.

particular chromosomal abnormalities and those that require other molecular studies for their recognition. Recurrent cytogenetic abnormalities Core binding factor translocations. Core binding factor-alpha (CBFα) and CBF-β normally combine to form a heterodimeric transcription factor required for hematopoietic differentiation. The t(8;21) abnormality seen in 8% of adult AML cases results in fusion of the gene encoding CBF-α on chromosome 8 with a gene (MTG8) on chromosome 21. Inv(16), seen in approximately 7% of AMLs, results in the fusion of the gene encoding CBF-β to the smooth muscle myosin heavy chain gene. In both cases, the fusion protein acts as a dominant negative regulator of transcription. As will be discussed in more detail, CBF AMLs tend to occur in younger individuals and have a relatively favorable prognosis [6]. Retinoic acid receptor-alpha translocations. APL represents about 8% of adult AML cases and is essentially always associated with t(15;17), a translocation that fuses the promyelocytic gene (PML) on chromosome 15 with the retinoic acid receptor-alpha gene (RARα) on chromosome 17. The resultant fusion protein acts as a dominant negative, in part by recruiting nuclear co-repressor and the molecules sin3 and histone deacetylase [7]. All-trans-retinoic acid (ATRA) appears to

have unique activity in this disease by virtue of its ability to bind to the fusion protein, resulting in a change in its configuration and release of the attached nuclear co-repressors. Other rare translocations involving RARα, including t(11;17) and t(5;17), give rise to leukemia that clinically resembles APL but is much less responsive to ATRA [8]. Mixed-lineage leukemia mutations. Translocations involving 11q23 are seen in approximately 7% of adult AML cases. While t(9;11) is the most common, t(6;11), t(10;11), t(11;17), and t(11;19) are also seen. Murine studies suggest that the MLL gene product positively regulates homeodomain (HOX) genes, which are required for normal development. Thus, a current hypothesis is that MLL fusion proteins result in increased expression of HOX genes resulting in increased self-renewal [9]. Mutations involving 5q, 7q, and 20q. Abnormalities in 5q, 7q or 20q are particularly frequent in AML in the elderly, AML secondary to prior exposure to alkylating agents, and AML following MDS [10]. These leukemias have a relatively poor prognosis. Although it has been hypothesized that the recurrent appearance of these abnormalities might be explained by the existence of classic tumor suppressor genes at these locations, no such genes have been identified. Mutations not detectable on routine cytogenetics Tyrosine kinase receptor mutations. Receptor tyrosine kinases are a family of proteins with extracellular, transmembrane, and intracellular domains with tyrosine kinase activity. Ligand binding of these proteins leads to receptor dimerization and subsequent phosphorylation of adaptive proteins, which in turn activate RAS and other downstream proteins. Activating mutations in the gene for fms-related tyrosine kinase-3, FLT3, a prototypic receptor tyrosine kinase, are found in 30–35% of patients with AML. The majority of mutations are internal tandem duplications (ITDs), while approximately 25% of mutations are point mutations. The incidence of FLT3 mutations is higher in older individuals and those with higher white cell counts at diagnosis. The presence of FLT3 mutation implies a poorer outcome with conventional chemotherapy, and this trend is increased with increasing size of the ITD and with higher allelic ratios of mutated to wild-type genes in marrow specimens [11,12]. Mutations in other receptor tyrosine kinases are sometimes seen in AML, including mutations in FMS and KIT. RAS mutations. RAS is a guanosine diphosphate-binding protein activated by various tyrosine kinases. Activation of RAS has multiple effects depending on the cell and its particular state, but can result in cellular proliferation and transformation. Mutations in RAS are found in 15–20% of AML cases. Such mutations prevent hydrolysis of RASguanosine triphosphate, thereby keeping RAS in the “on” position. For normal function, RAS must have a farnesyl or geranylgeranyl lipid attached, so compounds that inhibit farnesylation have been studied as possible therapies in AML. Nucleophosmin mutations. The nucleophosmin gene (NPM1) encodes a nucleolar phosphoprotein with multiple hypothesized functions. NPM1 is mutated in 30% of cases of AML and is associated with monocytic differentiation, normal cytogenetics, and an improved overall prognosis [13]. C/EBPa mutations. C/EBPα stands for CCAAT/enhancer binding protein-α, which is a transcription factor required for normal granulocyte differentiation. C/EBPα is mutated in 6–10% of cases of AML, and such cases tend to have M1 or M2 morphology, intermediate-risk cytogenetics, and a favorable clinical outcome [14].

Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia

Classification The World Health Organization has proposed a classification schema for AML that includes as subgroups AML with the most common recurrent cytogenetic abnormalities, AMLs that evolve from MDS, AMLs that are therapy related, and for those that do not fit into the first three categories, AML organized according to morphology (Table 53.1) [15].

Clinical and laboratory presentation The usual presenting symptoms of AML relate to decreased production of normal blood cells. Most patients are anemic at presentation and often complain of decreased exercise tolerance, fatigue, and headache. Thrombocytopenia is usually present, and approximately one-third of patients note easy bruising, bleeding gums or epistaxis. A third of patients will present with significant infections, usually of bacterial origin. In addition to symptoms related to various cytopenias, diffuse bone pain and/or tenderness is noted by approximately 25% of patients. Leukemic infiltrates can give rise to chloromas, gingival hyperplasia, and leukemia cutis. On presentation, most patients have a normochromic, normocytic anemia and are thrombocytopenic, with 50% having platelet counts less than 50,000/mm3. Most patients are granulocytopenic, but the total white count is more variable, with 25% having a white blood cell count above 50,000/mm3, 25% having a count below 5,000/mm3, and the remainder in between. The bone marrow is usually hypercellular and, by definition, must have greater than 20% blasts to be considered AML as opposed to MDS. Patients with APL usually have prolonged partial thromboplastin and prothrombin times, reduced fibrinogen, and other evidence of disseminated intravascular coagulation. Blood chemistries in patients with newly diagnosed AML are usually normal, but some patients with very aggressive disease may have evidence of tumor lysis syndrome. Lumbar puncture will show unsuspected central nervous system involvement with AML in 2% of cases. However, a randomized trial showed no advantage for central nervous system surveillance or prophylaxis in adult AML [16].

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Studies performed in the 1980s suggested that for patients over age 60, a reduced dose of daunorubicin (30 mg/m2/day for 3 days) is preferred. However, more recent studies have found that older patients with a reasonable performance status and lack of major comorbidities can tolerate the same aggressive regimens used in younger patients [19]. However, if patients are both elderly and have a poor performance status, the incidence of early death with intensive induction chemotherapy is in excess of 50%, and alternative forms of therapy should be considered for such patients [10]. Postremission chemotherapy If patients are given only induction chemotherapy, AML will inevitably recur, usually within 4–6 months [20]. Thus, some form of postremission therapy is required. Most contemporary protocols include repetitive cycles of high-dose cytarabine-containing consolidation chemotherapy for patients under age 60 who are not transplanted in first remission. Among the more widely used regimens are those reported by the Cancer and Leukemia Group B [21], which consists of four cycles of cytarabine 3 g/m2 twice daily on days 1, 3, and 5, and the regimen of the British Medical Research Council consisting of one cycle of standard “3 + 7” followed by a cycle of “MACE” chemotherapy that includes amsacrine, cytarabine, and etoposide, and a final cycle of high-dose cytarabine plus mitoxantrone [22]. No large randomized trial has directly compared the various commonly used consolidation regimens, but with such regimens, approximately 35–40% of patients can be expected to be alive in continuous first remission at 5 years from original diagnosis. Patients over age 60 are less able to tolerate such therapy, and are generally treated with several cycles of moderate-dose combination chemotherapy as consolidation, such as 2 days of daunorubicin and 5 days of conventional-dose cytarabine. With such therapy, only 10–15% of patients over age 60 can be expected to remain in remission 5 years after diagnosis. The poorer outcome in older patients is likely due, in part, to their diminished tolerance of intensive therapy, but an even greater factor is the changing biology of AML as patients age. AML in older patients is more often preceded by MDS, more often has an unfavorable cytogenetic profile, and more often expresses multidrug resistance, all negative risk features for achievement of CR and CR duration [23].

Nontransplant approach to treatment Remission induction

Chemotherapy for recurrent AML

If untreated, AML is usually a rapidly fatal disease with death from bleeding or infection occurring within several months. Occasional older patients may have a more smoldering form of AML that can be managed for many months with antibiotic and transfusion support. For the majority of patients, the initial approach to therapy is administration of combination chemotherapy in order to achieve a complete remission (CR). According to the International Working Group recommendations, morphologic CR is defined as less than 5% marrow blasts with recovery of peripheral counts to an absolute neutrophil count of over 1000/mm3 and a platelet count of over 100,000/mm3 [17]. For more than two decades, standard induction therapy for patients less than age 60 has consisted of an anthracycline such as daunorubicin, 45–60 mg/m2/day for 3 days, or idarubicin, 12 mg/m2/day for 3 days, combined with cytarabine given at a dose of 100–200 mg/m2/day by bolus or continuous infusion for 7 days [18]. Numerous variations on this standard “3 + 7” theme have been studied, including other doses and schedules of the anthracycline and cytarabine, the addition of other chemotherapeutic agents to the standard two-drug regimen, or the use of hematopoietic growth factors simultaneous with or following chemotherapy. None of these variations has so far consistently resulted in a significant improvement in CR rates or overall survival.

Patients who relapse after an initial remission achieve a second complete response 30–50% of the time [24]. Unfortunately, the median duration of second remission is generally short, averaging 6 months, and very few patients will remain in remission if treated only with chemotherapy. Mortality rates associated with reinduction have been 15–25%. Prognostic factors that have been consistently associated with an improved chance of achieving a second remission include younger age, favorable cytogenetic risk group, and longer duration of first remission [25,26]. The choice of reinduction chemotherapy depends on the prior treatment received by the patient as well as patient age and health. Younger patients are typically treated with a regimen containing an anthracycline plus high-dose cytarabine. The addition of cyclosporine to reverse multidrug resistance was shown to be efficacious in one randomized trial [27]. For patients thought not to be candidates for intensive chemotherapy, gemtuzumab ozogamicin, a humanized anti-CD33 antibody conjugated to the potent antitumor agent calicheamicin, is effective in inducing CR in 30% of patients, with less toxicity than seen with aggressive combination chemotherapy [28]. A considerable number of agents are being studied for recurrent AML, including FLT3 inhibitors, farnesyl transferase inhibitors, histone deacetylase inhibitors, and antiangiogenic agents, inducers of apoptosis and deoxyadenosine analogues, among others.

Chapter 53

The special case of APL Because APL is uniquely sensitive to ATRA, arsenic trioxide, and anthracyclines, its treatment differs from that of other AML subtypes. Initial induction therapy generally includes ATRA plus an anthracycline, with or without the addition of other chemotherapy. Complete response rates of 85–90% can be expected with such therapy. Induction failures are usually due to early bleeding events or less commonly drug resistance. Patients typically receive several cycles of consolidation therapy including ATRA and an anthracycline. A recently completed prospective randomized trial showed that the further inclusion of two cycles of arsenic trioxide as consolidation therapy significantly improved eventfree and overall survival. In previous studies, maintenance with ATRA was also been found to be beneficial. With modern chemotherapy, at least 75% of patients with APL can be expected to be alive and disease free 3 or more years after diagnosis [29]. Both age and white count at diagnosis are of prognostic importance, with best results seen in patients less than age 50 presenting with a white count of less than 10,000/mm3. The choice of treatment for recurrent APL depends on the specific clinical situation. Arsenic trioxide is the agent of choice for those patients who have not previously been exposed to the drug; in that setting, second CR (CR2) can be expected in 85% of cases [30]. Patients resistant to ATRA and arsenic may respond to gemtuzumab ozogamicin.

Hematopoietic cell transplantation Indications for HCT in AML among patients age less than 60 Primary induction failure Patients who fail primary induction chemotherapy have essentially no chance for cure with alternative chemotherapies, but several studies have shown that long-term survival is possible if such patients undergo allogeneic transplantation from an HLA-matched sibling. For example, the European Group for Blood and Marrow Transplantation (EBMT) published 21% 3-year disease-free survival (DFS) in a group of 88 patients with AML who were transplanted after failing at least two cycles of induction chemotherapy [31]. Similar results have been published by others [32]. Because of the time required to perform a search, there is less experience with the use of unrelated donors for primary induction failure, but the limited data available demonstrate that cure is possible in approximately 20% of cases [33]. These results emphasize the importance of HLA typing patients and family members shortly after the diagnosis of AML so that valuable time is not lost in identifying suitable donors should remission attempts fail. How quickly patients should be taken to transplant is often determined by logistical issues. However, most experts would suggest that there is little to be gained by persisting with standard chemotherapy if a complete response is not achieved after two cycles of conventional-dose induction, or one cycle containing high-dose cytarabine [34]. Refractory relapse Various definitions of the term “refractory relapse” exist, making simple statements about treatment outcome for such patients based on published studies difficult. A strict and relatively unambiguous definition of patients in refractory relapse includes only those who have failed their last reinduction attempt. While a second reinduction using an alternative regimen is sometimes tried, such efforts are rarely beneficial. For example, based on data from a retrospective study conducted by investigators at the MD Anderson Cancer Center, the likelihood of achieving a CR was less than 1% following administration of chemotherapy to patients whose AML failed to respond to the last initial reinduction

attempt [35]. Results with transplantation in this strictly defined group of patients are also relatively poor. With matched sibling transplants, DFS at 3 or more years was seen in only approximately 10% of such patients [36]. Similar results have been published using unrelated donors, with 7% 5-year DFS seen in 81 patients transplanted for refractory relapse [33]. Among those transplanted in refractory relapse, results are marginally better if patients have a lower leukemia burden without circulating blasts [33,36–38]. This has led to the approach of administering intensive chemotherapy in an attempt to clear circulating leukemic blasts prior to initiating a transplant procedure [39]. Whether these attempts at reducing tumor burden change the ultimate outcome of transplantation remains unknown. Many reports use a less stringent definition of refractory relapse. Hiddemann et al., for example, defined refractory relapse to include patients who relapsed within 12 months of first remission and those in second or subsequent relapse, irrespective of whether they had received any attempts at reinduction [25]. Transplant studies using this or other similar definitions of refractory relapse report modestly better outcomes than seen in studies restricted to patients who have failed their last reinduction attempt [40]. Second or subsequent remission Although occasional patients with AML in second remission treated with chemotherapy alone may have long second remissions, these patients are generally limited to those with favorable cytogenetics and a long first remission. A retrospective study focused on patients treated 15–20 years ago compared results of allogeneic HCT for 257 patients with AML in second remission with those of 244 patients treated with chemotherapy alone [41]. The study demonstrated a 26% 3-year DFS with transplantation versus 17% for those treated with chemotherapy, suggesting that, except for patients with favorable cytogenetics and long first remissions, allogeneic transplantation is indicated for patients with AML in CR2 with a matched sibling. Over the last two decades, there have been significant advances in the outcome of allogeneic transplantation from matched siblings, and recent data from the Center for International Blood and Marrow Transplantation Research (CIBMTR) report a 5-year survival of 50% (SD 2%) for patients with AML transplanted in second or subsequent remission (59% [SD 4%] for pediatric patients and 47% [SD 2%] for patients over age 20) (Fig. 53.1) [42]. Unfortunately, there have been no similar improvements in chemotherapy for recurrent disease, suggesting that the advantage of matched sibling transplantation over chemotherapy for AML in second remission persists.

100

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20 Advanced (N=1,113) P < 0.001

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Fig. 53.1 Survival following human leukocyte antigen-identical sibling transplantation with high-dose conditioning for acute myeloid leukemia in adults, 1998–2004, by disease status. (Reproduced from [42], with permission.)

6

Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia

Untreated first relapse

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Fig. 53.2 Survival following unrelated donor transplantation with high-dose conditioning for acute myeloid leukemia, 1998–2004, by disease status. (Reproduced from [42], with permission.)

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40 Intermediate (N=424)

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Fig. 53.3 Survival following autologous hematopoietic cell transplantation for adults with acute myeloid leukemia, 1998–2004, by disease status. (Reproduced from [42], with permission.)

Patients with AML in CR2 lacking matched siblings are candidates for either matched unrelated or autologous transplantation. The CIBMTR database reports 5-year survivals of 42% (SD 2%) for unrelated donor transplantation for AML in CR2 (Fig. 53.2) and 40% (SD 3%) with autologous transplantation (Fig. 53.3) [42]. The decision to pursue an unrelated versus an autologous transplant for the patients represented in the registry was undoubtedly affected by multiple considerations that could introduce considerable bias into any comparison between groups. No prospective randomized trial comparing autografting with unrelated donor transplants for AML in CR2 has been published. In an effort to diminish possible bias, single-institution and registry comparisons have been performed using either matched-pair analyses or proportional hazards regression adjusted for differences in prognostic variables [43– 45]. These studies consistently show decreased relapse rates but increased nonrelapse mortality with unrelated transplantation. In none of the studies was the difference in leukemia-free survival statistically significantly different. None of these studies was designed to address whether particular subsets of patients might benefit from one approach or the other, but the improved control of leukemia with unrelated transplantation but increased nonrelapse mortality suggests that unrelated transplantation might be favored in situations where there is a higher risk of relapse and a lower risk of toxicity (younger patients with shorter first remissions), while autologous transplantation might be favored in opposite settings (older patients with longer first remissions).

Whether patients with AML in untreated first relapse who are transplant candidates should go directly to transplant or should first undergo chemotherapy in an attempt to induce a second remission prior to transplant is an unanswered question. Frequently, the issue is moot since nonmedical issues including insurance clearance and transfer to a transplant center often require time and do not permit rapid initiation of the transplant procedure. However, with improving technologies applied to the detection of minimal residual disease, it is likely that in the near future an increasing number of patients will be diagnosed with incipient relapse, allowing greater lead time and thus making transplantation in untreated first relapse a more feasible approach. To date, there have been only a limited number of published studies describing the outcome of transplantation in untreated first relapse. An initial report from Seattle published in 1983 described 17 patients who received allogeneic matched sibling HCT in untreated first relapse with a 3-year DFS of 29% [36]. A follow-up report from the same group in 1992 described 126 similar patients and reported a 3-year DFS of 28% [46]. A very similar result was published by Brown et al. from St Louis, who reported a 28–30% survival rate at 5+ years for matched related donor transplants for AML in untreated first relapse [47]. Only limited data exist about the outcome of HCT for AML in untreated first relapse using other sources of marrow. A single study of autologous transplantation described 38 patients whose marrow had been stored in first remission who underwent autologous HCT at first relapse using a conditioning regimen of busulfan (BU) plus cyclophosphamide (CY) and post transplant received interleukin-2 with or without lymphokine-activated killer cells [48]. Eight of the 38 patients were alive in remission at the time of the report. Because of the time required to identify and schedule an unrelated donor, there are almost no reports of unrelated donor transplants for AML in untreated first relapse, but the few reported cases suggest an outcome not markedly different from that seen with matched related donors [49]. While the outcomes for transplantation in untreated first relapse are apparently not as favorable as outcomes in second remission, it should be remembered that reinduction attempts are successful in, at best, 50% of cases, and those who fail reinduction either never are transplanted or are transplanted in refractory relapse. A thought experiment conducted using available data would suggest that among 100 patients subjected to reinduction, 50 would achieve CR2, and if every one of those patients were transplanted, approximately 20 would be cured (50 × 0.40). Among the other 50, 15–20% would die or suffer complications during reinduction that would preclude subsequent transplant, and of the remaining 30, perhaps three (10%) would be cured. Thus, this approach would yield 23 cured patients, which is less than the 28–30% cure rate reported for transplantation in untreated first relapse. Of course, considerable selection bias may have influenced those patients transplanted in untreated first relapse. In limited allogeneic experience of HCT in untreated first relapse, there was a trend towards better outcome in those who developed graft-versus-host disease (GVHD) and in those with less disease burden at the time of transplant, but no effect on the duration of first remission, while in the autologous experience, both increasing tumor burden and shorter first remission negatively impacted outcome [42–44]. Available data are very limited, but, based on what is currently known, patients with matched siblings or previously stored autologous stem cells might be considered for transplant in untreated first relapse if disease recurrence is detected at an early stage and logistical issues do not present a roadblock. First remission In 1979, Thomas et al. reported the results of 19 patients with AML in first remission treated with allogeneic HCT from HLA-identical siblings

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Chapter 53

Fig. 53.4 Kaplan–Meier estimates of survival and relapse among 19 patients with acute myeloid leukemia in first complete remission first reported by Thomas et al. in 1979. (Reproduced from [51], with permission.)

[50]. A recent follow-up of this experience reported that seven of these patients (37%) remain alive in remission 26–27.3 years following transplantation (Fig. 53.4) [51]. Thomas’s original findings were quickly duplicated by others [52,53]. In the early 1980s, several reports of small numbers of patients with AML in first remission treated with autologous transplantation were published [54,55]. Since that time, large numbers of patients with AML in first remission have been treated with matched related transplants, unrelated donor transplants or autologous transplants. The CIBMTR has data for 4240 patients receiving HLA-identical sibling (n = 3174) or unrelated donor (n = 1066) HCT for AML using high-dose conditioning in 1998–2004 [42]. The 3-year probabilities of survival were 60% (95% confidence interval 59–61%) with matched sibling transplants and 41% (95% confidence interval 39–43%) for unrelated donor transplants (Figs 53.1 and 53.2). The 3-year probability of survival following autologous transplantation for AML in first remission among 1934 adults was 44 (95% confidence interval 42–46% (Fig. 53.3). A question of great practical importance is how these results compare with those achievable using nontransplant approaches. Accordingly, a large number of prospective trials have been conducted attempting to answer this question. The general structure of most of these studies has been to enter newly diagnosed patients, treat with induction chemotherapy, and, once CR has been achieved, allocate those with HLAmatched donors to allogeneic transplantation in first remission, while those without donors are treated either with chemotherapy or autologous transplantation or are randomized between the two. A recent metaanalysis focusing on the comparative outcome of allogeneic transplant versus chemotherapy included all such studies conducted between 1995 and 2003, provided that the studies were written in English, included an intent-to-treat analysis, and presented survival data [56]. Five such studies were identified and included 3100 subjects, 1151 treated with allogeneic transplantation and 1949 receiving alternative forms of therapy [57–61]. For the entire cohort, there was a statistically significant advantage in overall survival associated with the use of allogeneic transplantation, with a hazard ratio of 1.15 (95% confidence interval 1.01–1.32; p = 0.037). The benefit was greatest for patients with unfavorable-risk cytogenetics with a hazard ratio of 1.24, and lowest for those with favorable-risk cytogenetics, with a hazard ratio of 0.90. For intermediate-risk patients, there was a nonsignificant trend in favor of allogeneic transplantation (hazard ratio 1.09). These results, in general,

mirror the results of most individual studies, but have the advantage of larger numbers. While cytogenetics is a powerful tool for assigning risk categorization, investigators have attempted to subdivide these groupings in an effort to reach clearer conclusions about the appropriateness of transplantation in first remission. Patients with favorable risk cytogenetics in general do well enough with initial chemotherapy that meta-analyses find no advantage for allogeneic HCT in first remission. However, a minority of patients with CBF gene mutations may also have mutations in c-KIT, and this predicts for a significantly worse outcome with chemotherapy [62]. Whether allogeneic transplantation is of advantage for this subgroup of patients is untested. Approximately 40% of patients with AML have no chromosomal abnormalities detected by routine karyotyping and are categorized as having intermediate-risk disease. When studied by molecular means, AML cells of these patients will have mutations of various genes, including NPM1 in 53%, FLT3-ITD in 31%, FLT-TKD in 11%, CEBPA in 14%, and NRAS in 13% [63]. Among AML cases with normal cytogenetics treated with chemotherapy, mutations in these genes considered individually appear to have only weak, if any, prognostic significance. However, certain combinations of mutations appear to have significant prognostic power. In particular, patients with AML treated with chemotherapy that are NPM1+ and FLT3-ITD− have an outcome almost as favorable as that seen in patients with favorable-risk cytogenetics, while all other combinations of NPM1 and FLT3-ITD do significantly worse [13,64]. In a prospective study reported by Schlenk et al. in which patients with matched siblings were treated with allogeneic transplant while those without received chemotherapy, transplantation was associated with a significant improvement in survival among all patients with normal cytogenetics except those whose leukemia was characterized as NPM1+/FLT3-ITD−, where the advantage of transplantation was not statistically significant [63]. Thus, current data would argue that, for younger patients with matched siblings, allogeneic transplantation while in first remission is indicated, except for those with favorable-risk cytogenetics and those with normal cytogenetics and NPM1+/FLT3ITD− AML. A question of practical importance is whether patients who will undergo matched sibling transplantation during first remission using a high-dose preparative regimen benefit from postremission chemotherapy prior to transplantation. No prospective randomized trial has directly addressed this question, but data from the International Bone Marrow Transplant Registry failed to find any obvious benefit [65]. Among 431 patients, treatment-related mortality, relapse rates, and the probability of 5-year survival were similar among those receiving no consolidation chemotherapy, standard-dose cytarabine consolidation, and high-dose cytarabine consolidation. Patients with AML in first CR (CR1) without matched siblings are potential candidates for either allogeneic transplantation using alternative donors or autologous transplantation. The sole prospective controlled study so far reported exploring the use of alternative donor transplantation for high-risk patients with AML in CR1 is the German AML 01/99 trial [66]. In this study, patients were defined as high risk if they had either unfavorable risk cytogenetics or more than 5% blasts remaining in the marrow on day 15 following the initiation of induction chemotherapy. A total of 234 high-risk patients were entered, with 137 achieving CR. Following consolidation, patients were treated with matched sibling transplantation, matched unrelated donor transplant or autologous transplant. The decision for unrelated versus autologous transplant was based on the availability of a matched unrelated donor and the date of trial entry. At 4 years, survival among patients treated with matched sibling transplantation was 68%, versus 56% among

Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia

recipients of unrelated donor transplants, and 23% after autografting (p = 0.01). The comparative utility of autologous HCT versus chemotherapy for patients with AML in CR1 has been the subject of numerous controlled randomized trials. A recent meta-analysis considered all such studies and focused on those that involved adult patients, were analyzed on an intent-to-treat basis, and had greater than 75 patients in each treatment arm [67]. Six trials were included that involved 4410 patients [22,57,68– 71]. Of these, 1044 achieved CR and were eventually randomized to continued treatment with either autologous HCT or further chemotherapy. Of the patients randomized, 835 received the intended treatment, including 360 patients in the transplant group (69%) and 475 in the control arm (91%). The combined results of these six trials indicated that autologous transplantation modestly improved event-free survival, but did not improve overall survival. The six trials that were included in this analysis employed different preparative regimens, different stem cell sources, and somewhat different treatment strategies. The failure of this meta-analysis to find an overall survival benefit with autologous transplantation does not necessarily mean that one or another of the approaches taken might not be of some benefit. For example, among the six studies, the two that showed the least evidence of benefit randomized patients early after remission induction to autologous transplantation versus intensive consolidation, while those studies that treated all patients with intensive consolidation chemotherapy and then randomized patients to subsequent transplant or not tended to show a greater advantage. Nonetheless, when considered in the aggregate, there is little compelling evidence from prospective randomized trials that autologous transplantation in first remission improves overall survival. Although the comparative outcome of autologous transplantation versus chemotherapy among patients in the various cytogenetic risk groups has not been extensively examined, two of the comparative studies noted above did include such an analysis. A report by Slovak et al. noted that patients with favorable cytogenetics did significantly better following autologous transplantation than with chemotherapy, while no such advantage was seen in the intermediate- and poor-risk groups [72]. In contrast, in the report from Burnett et al., the trend towards improved DFS with autologous transplantation was similar in all three cytogenetic risk groups [22].

HCT-specific issues in patients age <60 Preparative regimens The general topic of transplant preparative regimens is reviewed in Chapter 22. A limited number of trials have explored the question of optimal preparative regimens specifically for patients with AML undergoing matched sibling HCT [73]. Two prospective randomized trials have studied the issue of total body irradiation (TBI) dose when combined with a set dose of CY in patients with AML in CR1. One study showed that 12 Gy of fractionated TBI was superior to 10 Gy of TBI delivered as a single dose [74]. A second trial found that relapse rates were lower, but nonrelapse mortality higher, when the dose of fractionated irradiation was increased from 12 Gy to 15.75 Gy [75]. A single randomized trial has explored the form of chemotherapy in combination with TBI and found equivalence of melphalan and CY [76]. A final randomized trial compared CY plus TBI versus BU plus CY and found a significantly lower relapse rate with the TBI-containing regimen, resulting in improved DFS [77]. Subsequent retrospective registry data comparing 223 patients with AML in CR1 transplanted using a CY plus TBI regimen with a similar number of patients treated with BU plus CY found relative equivalence of the two regimens [77]. Reasons for the

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discrepancy between the randomized trial outcome and registry data are not obvious. Only a single small randomized trial has compared preparative regimens prior to autologous transplantation and found relative equivalence between CY plus TBI and BU plus CY [78]. Registry data since confirmed the relative equivalence of CY plus TBI and BU plus CY for patients with AML undergoing autologous HCT in CR1 [79]. While a number of alternative and novel approaches to the development of preparative regimens have been reported, no randomized trials other than those noted above have been published, and insufficient numbers of patients have been treated with any one alternative to allow for retrospective comparisons sufficiently meaningful to alter standard therapy. Source of hematopoietic stem cells The source of hematopoietic stem cells for transplantation can be defined both according to the relationship of the donor and recipient (autologous, matched sibling donor or matched unrelated donor) and according to their anatomic site (bone marrow, peripheral blood or umbilical cord blood). The broad topics of stem cell source are dealt with in greater detail in Chapters 37–48. The following discussion will be limited to specific issues of stem cell source selection in the treatment of AML. Sibling versus autologous versus unrelated donors The choice of matched sibling versus autologous versus unrelated donor transplant is, of course, driven by the clinical state of the patient and the availability of hematopoietic stem cells. The only clinical situation in which there are prospective data to guide the choice is in CR1. As noted above, two large meta-analyses have been performed addressing this question, and are in general agreement that allogeneic transplantation offers a survival advantage in this setting, particularly in patients with cytogenetically determined intermediate- and high-risk disease, whereas with autologous transplantation, although there may be some gain in DFS, no overall survival benefit is evident [56,67]. Also as noted above, the single German study that addressed the use of matched unrelated transplantation for patients with high-risk AML in CR1 found benefit with this approach compared with autologous transplantation [66]. Lack of prospective randomized trials makes the choice of donor for patients with more advanced disease less clear. General practice is to use a matched sibling whenever one is available. Retrospective comparisons of autologous transplantation versus unrelated donor transplants for patients with AML in second remission have demonstrated similar outcomes [43–45], but such comparisons are inherently limited by possible treatment selection bias, and most experts would favor matched unrelated transplants, particularly for younger patients, reserving autologous transplantation for those who are older, have significant comorbidities or have incompletely matched donors. Stem cell source for autologous transplantation Purging. Initial studies of autologous transplantation for AML utilized unmanipulated bone marrow. An obvious concern with autologous transplantation is that clonogenic leukemia cells might be reinfused with the remission marrow, a possibility formally demonstrated by Brenner et al. using gene marking of remission marrow [80]. Accordingly, various techniques were developed that in animal models are capable of eliminating clonogenic tumor cells from marrow mixtures without causing obvious harm to the marrow itself (see Chapter 42 for a thorough review). Subsequently, numerous phase I–II trials exploring these techniques were conducted. The most widely used approach was treatment of marrow in vitro with the potent alkylating agent 4-hydroperoxycyclophosphamide (4-HC). A retrospective analysis from the Autologous Blood and Marrow Transplant Registry reviewed 294 patients with

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AML who underwent autologous HCT in first remission. In a multivariate analysis, use of 4-HC was associated with improved DFS at 3 years (56% with purging versus 31% without) [81]. Despite encouraging retrospective data, no prospective randomized results have been published, and thus this approach is not approved by the United States Food and Drug Administration, and the drug is not currently commercially available. Peripheral blood versus bone marrow. Autologous peripheral blood hematopoietic cells (PBHCs) collected following chemotherapy and growth factor administration provide an alternative to autologous bone marrow as a source of stem cells for autografting. Use of PBHCs is clearly associated with faster engraftment and reduced early morbidity after autografting, but effects on overall survival are less clear because of the lack of an adequately sized prospective randomized trial. The EBMT conducted a large retrospective review of 1393 AML patients who underwent transplant using PBHCs, or purged or unpurged bone marrow [82]. The 2-year overall survival among the three groups was similar. A question of interest with the use of unpurged PBHCs is whether more intensive therapy prior to collection of hematopoietic stem cells might improve the purity of the stem cell yield, reduce tumor burden in the patient, and thereby decrease subsequent relapse rates. Tallman et al. conducted a retrospective analysis of registry data and reported that the risk of relapse and treatment failure was significantly less among patients receiving consolidation chemotherapy prior to hematopoietic stem cell collection than in patients receiving no consolidation [83]. They did not find a difference between groups receiving high-dose cytarabine consolidation regimens and those receiving standard dose regimens. Because of the retrospective nature of the analysis, it is difficult to know if the observed effect was at least in part due to drop-out of high-risk patients during the consolidation phase. Nonetheless, most current trials of autologous transplantation include at least one, and often several, cycles of consolidation prior to hematopoietic stem cell harvest and transplant. Hematopoietic stem cell source for allografting Bone marrow cell dose. Numerous studies have now demonstrated that bone marrow cell dose is closely linked with clinical outcome after both matched sibling and unrelated donor transplant for AML. This association was initially shown following unrelated donor transplantation for AML, where a marrow cell dose of greater than 3.0 × 108 nucleated cells/kg was found to be associated with a superior outcome [84]. Subsequently, a similar association has been found following matched sibling transplantation. For example, Rocha et al. showed that, among 572 patients undergoing allogeneic bone marrow transplantation for AML in CR1, both relapse and nonrelapse mortality were reduced in those patients receiving a nucleated cell dose of over 2.6 × 108, resulting in superior leukemia-free survival for this group [85]. Similar results have been published by others using the number of CD34+ marrow cells as the variable [86]. The explanation for the association of higher marrow cell dose with improved clinical outcome is not clear, but the association is maintained after accounting in multivariable analysis for patient age, sex, and other obvious potentially confounding factors. Bone marrow versus peripheral blood. The rapid engraftment seen with the use of autologous growth factor-mobilized PBHCs encouraged pilot studies of the use of allogeneic PBHCs. These pilot studies demonstrated rapid granulocyte and platelet engraftment, and, despite a far higher mature T-cell content than was present in marrow, no obvious increase in acute GVHD with the use of allogeneic PBHCs. Based on these encouraging pilot studies, a number of prospective randomized

trials were conducted comparing marrow versus growth factor mobilized PBHCs as the source of stem cells for matched sibling transplantation following standard high-dose preparative regimens. Recently, a meta-analysis of nine randomized trials enrolling 1111 adult patients was conducted and reported [87]. Compared with marrow, use of PBHCs was associated with more rapid neutrophil (p < 0.00001) and platelet (p < 0.00001) engraftment. While there was no difference in grade II–IV acute GVHD (54% with PBHCs versus 53% with BM), there was an increase in extensive chronic GVHD (47% versus 31%) and overall chronic GVHD (68% versus 52%) with the use of PBHCs. Use of PBHCs was also associated with a decrease in relapse at 3 years, both in late-stage and early-stage patients (21% versus 27% overall). Nonrelapse mortality was no different between the groups. When considering AML specifically, DFS and overall survival in late-stage patients was significantly improved with the use of PBHCs (odds ratio 0.45), while results were less definitive for early-stage patients. A large retrospective registry analysis from the EBMT yielded similar findings [88]. Thus, for patients with AML beyond first remission undergoing matched sibling transplantation following a high-dose preparative regimen, PBHCs are preferred, while for patients in first remission, the choice is less clear. More recently, pilot studies of the use of PBHCs for matched unrelated transplantation have been conducted [89,90]. These studies were encouraged by the results seen with the use of PBHCs for matched sibling transplantation coupled with the observation that survival appears to be improved with higher CD34+ cell doses following unrelated donor marrow transplantation. Results again showed rapid engraftment without intolerable GVHD, but more definitive conclusions cannot be drawn until the completion of prospective randomized trials, like the one being conducted by the Bone Marrow Transplant Clinical Trials Network. Unfortunately, these trials are unlikely to be sufficiently sized to address the use of unrelated PBHCs versus bone marrow for specific disease subsets. The general topic of peripheral blood versus bone marrow as a source of stem cells for HCT is considered in more detail in Chapter 43. Umbilical cord blood. Umbilical cord blood as a source of hematopoietic stem cells for HCT is the topic of Chapter 39. The amount of information about the utility of cord blood transplants specifically as treatment for adults with AML is limited. There are several relatively small series that have reported encouraging results. For example, Ooi et al. reported 18 adults with AML in CR1 transplanted using unrelated cord blood, with a 2-year DFS of 76.6% [91]. A recent review from Brunstein et al. summarizes other similarly small studies [92]. An International Bone Marrow Transplant Registry (IBMTR) report compared the outcome of cord blood transplants in 140 adults with leukemia with that seen in recipients of unrelated HLA-matched (n = 367) or singleantigen-mismatched (n = 83) marrow [93]. Outcomes with cord blood and one-antigen-mismatched marrow were very similar, but both were inferior to that seen with unrelated matched transplants. Results were not provided for specific leukemia subtypes or stages. The EBMT similarly compared outcomes for 98 adult recipients of cord blood transplants with acute leukemia with those seen in 584 recipients of unrelated bone marrow [94]. Multivariate analysis showed lower risks of GVHD with cord blood, but delayed engraftment. No significant differences in transplant-related mortality, relapse rates or leukemia-free survival were seen between the groups. When the analysis focused on subsets of patients, a trend towards improved 2-year leukemia-free survival was found for patients with AML treated with matched unrelated marrow compared with cord blood (42% versus 32%), while other subgroups appeared to benefit equally from cord blood versus marrow. The registry nature of the IBMTR and EBMT reports makes it impossible to account for biases in patient selection, and no attempts were

Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia

made to control for treatment regimen effects or differences in supportive care or other ancillary measures. Nonetheless, the reports suggest that the outcomes of unrelated bone marrow and cord blood transplants for adults with AML are sufficiently similar that, while one might prefer a matched unrelated donor, if none were available a partially matched cord blood with sufficient cell dose is a reasonable substitute. The majority of studies of adult cord blood transplants so far published utilized single cords as the source of stem cells and had a threshold for an acceptable cell dose (usually in the range of 2 × 107/kg), meaning that many possible candidates were not able to be transplanted because a cord of sufficient size could not be identified. The reason for the cell dose threshold is because of experience showing an increased incidence of graft rejection and poorer survival with lower cell doses. Recent experience combining multiple cords for a single transplant suggests that it might be possible to overcome this limitation, although there are as yet no randomized trials that confirm the suggested benefit [95]. GVHD prophylaxis The general principles of GVHD prophylaxis are discussed in Chapters 84 and 85. As outlined in Chapter 84, the results of prospective randomized trials suggest that combinations of methotrexate and either cyclosporine or tacrolimus are the preferred pharmacologic approaches for prevention of GVHD in patients with AML undergoing transplants from matched siblings or matched unrelated donors following high-dose preparative regimens [96]. As discussed in Chapter 84, many alternatives to these regimens are currently being explored, including, for example, the substitution of sirolimus or mycophenolate mofetil for methotrexate, or the addition of antithymocyte globulin during the peritransplant period. As yet, however, no prospective randomized trial has been reported showing superiority in terms of survival or DFS of any alternative to the standard combination of methotrexate and a calcineurin inhibitor for GVHD prevention in the setting of high-dose preparative regimens. As discussed in Chapter 85, removal of T cells from the donor stem cell inoculum as an alternative approach to GVHD prevention has been intensively studied. A variety of methods for T-cell removal have been developed, and preparative regimens with increased immunosuppressive effects have been constructed that appear capable of preventing the increased incidence of graft rejection otherwise associated with the use of T-cell depletion. To date, only a single prospective randomized trial examining the effects of T-cell depletion on transplant outcome has been reported [97]. In that trial, 405 patients with a variety of hematologic malignancies undergoing matched unrelated marrow transplantation following high-dose preparative regimens were randomized to GVHD prevention with either T-cell depletion (using counterflow centrifugal elutriation or antibody T10B9 plus complement) plus cyclosporine versus pharmacologic immunosuppression with methotrexate and cyclosporine. DFS at 3 years was 27% with T-cell depletion versus 34% without (p = 0.16). Among the 101 patients with AML, DFS at 3 years was 29% with T-cell depletion versus 33% without. Since the design and conduct of this trial, there have been numerous advances in unrelated donor selection, and both pharmacologic and T-cell depletion approaches to GVHD continue to evolve. But based on our current body of knowledge, there is insufficient evidence to argue for or against T-cell depletion in the treatment of AML. HCT for AML in patients age over 60 Compared with the extensive study of the use of HCT for treatment of AML in younger patients, transplantation for older AML patients is less well studied, and clinical indications are, accordingly, less well defined. Major reasons for this lack of information include the perception of

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many clinicians that older patients are less able to tolerate the toxicities of intensive conditioning regimens and that, because the incidence of GVHD rises with patient age, GVHD-associated complications in patients over age 60 might increase to intolerable levels. Although both these concerns have some merit, recent results show that age per se does not represent an absolute contraindication to transplantation. Clinical studies have shown that autologous HCT is possible in older patients, and although data are limited, in these studies nonrelapse mortality associated with regimens such as BU plus CY, or CY plus TBI, appear to be no higher in patients age 60–70 than has been seen in younger patients [98]. Admittedly, patients included in these studies were likely highly selected. No prospective trials of autologous transplantation in older AML patients have yet been conducted, so very little is really known about the broader applicability of this treatment strategy for this age group. In particular, we do not know how performance status in older patients affects outcome, and assuming some minimal status is required, what proportion of older patients would meet that criterion is unknown. Further, we have little information about difficulties in harvesting adequate numbers of hematopoietic stem cells for transplant from older AML patients. In addition, there are almost no data about outcome in broad representative patient populations. Thus, the role of autologous transplantation in older AML patients is unsettled. Similarly, very little is known about the use of high-dose preparative regimens followed by allogeneic transplantation for older adults with AML. Despite concerns for increased regimen-related toxicity and GVHD, there has been some experience with this approach in patients with myeloid malignancies; in a study of 52 patients with AML, chronic myeloid leukemia or MDS over age 60 treated with intensive preparative regimens and matched sibling transplants, nonrelapse mortality at 100 days and 3 years was 27% and 43%, respectively, and survival at a median of 4.6 years was 35% [99]. Because of the substantial morbidity of allogeneic transplantation in older individuals, the use of reduced-intensity conditioning (RIC) regimens has been increasingly explored in this patient population. This approach, which is reviewed in much greater detail in Chapter 71, is based on the observations that sustained allogeneic engraftment can be achieved with appropriate dose pre- and post-transplant immunosuppression, and that allogeneic engraftment is associated with a substantial graft-versus-tumor effect. While a certain amount of antileukemia effect is lost by omitting the high-dose therapy of the usual preparative regimen, it is hypothesized that the decreased toxicity will more than compensate for this loss. A number of studies of RIC followed by matched sibling and matched unrelated transplants for AML have now been published. Among the largest studies is that of Hegenbart et al., who reported on 122 patients with AML treated with a regimen of fludarabine (30 mg/m2/day for 3 days) and low-dose TBI [100]. The cumulative nonrelapse mortalities were 10% for related and 22% for unrelated recipients. Patients receiving transplants in CR1 had 2-year overall survivals of 44% after related and 63% after unrelated HCT. Results for patients in second remission at the time of transplant were likewise encouraging. In contrast, patients transplanted for active AML had a high likelihood of early leukemic relapse. Similar results have been published by others using a variety of reduced-intensity preparative regimens and forms of GVHD prophylaxis [101,102]. The encouraging results so far published concerning RIC in patients with AML raise a number of important questions. Two of the most compelling are the importance of dose intensity for transplantation of AML in remission (regardless of age) and the role of RIC with allogeneic transplantation in the treatment of older patients with AML. As yet, there have been no reports of prospective randomized trials comparing RIC with high-dose conditioning for the treatment of AML. There have

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been several retrospective looks at this issue [103,104]. The problems of selection bias in such studies are obvious and readily acknowledged by the authors, but the outcomes appear consistent. RIC transplants are generally associated with a higher leukemia relapse rate, but this is counterbalanced by substantially reduced transplant-related morbidity and mortality. The advantages for RIC appear more pronounced in older patients and those with greater comorbidities, while increased intensity appears to be of advantage in younger, healthier patients [105]. Whether RIC should be broadly used as standard consolidation therapy in older patients with AML in first remission is the question central to a number of current trials. Mohty et al. reported the first prospective study directly addressing this question [106]. In their study, 95 adults, median age 52 years, were allocated to RIC followed by allogeneic HCT if they had a donor, or to consolidation chemotherapy if they did not. Leukemia-free survival at 4 years was superior in the donor group compared with the no-donor group (54% versus 30% at 4 years; p = 0.01). Additional studies are needed to confirm these results, but given the generally poor prognosis associated with chemotherapy for AML in older patients, allogeneic HCT with RIC appears as an attractive alternative. Relapse following HCT Disease recurrence is a common cause of treatment failure following HCT and is the topic of Chapter 72. In AML treated either with chemotherapy or transplantation, the vast majority of relapses will occur within 3 years of treatment [107]. Chemotherapy is sometimes successful in inducing a remission in patients who have relapsed after HCT. In one study of 95 patients with AML who relapsed after allogeneic HCT, the CR rate with subsequent chemotherapy was 34% [108]. Not surprisingly, CR rates were higher for those transplanted in remission (47% versus 18%) and for those who relapsed at longer intervals from transplant. While subsequent remissions were possible, most of these were of limited duration, and, without other therapies, long-term survival was seen in only 5% of patients. By inducing an immunologic graft-versus-tumor effect, donor lymphocyte infusions (DLIs) can sometimes cause CRs in patients with a variety of malignancies who have relapsed after allogeneic HCT [109]. Recently, the EBMT has retrospectively analyzed the data of 399 patients with AML in first hematologic relapse after HCT whose treatment did (n = 171) or did not (n = 228) include DLI [110]. Estimated survival at 2 years was 21% (95% confidence interval 18–24%) for patients receiving DLI and 9% (95% confidence interval 7–11%) for patients not receiving DLI. After adjusting for differences between the groups, better outcome was associated with younger age, relapse occurring more than 5 months after HCT, and use of DLI. Among DLI recipients, a lower tumor burden at the time of DLI (<35% marrow blasts), female sex, favorable cytogenetics, and remission at the time of DLI all were predictive of a better outcome. Survival at 2 years was 56% (95% confidence interval 46–66%) for those given DLI while in chemotherapy-induced CR2. Second allogeneic transplants have been explored as possible therapy for patients who have recurred after an initial autologous transplant. Radich et al. reported on 59 patients, including 24 with AML, who were treated in this manner [111]. The 2-year DFS was surprisingly good for the AML patients, at 46%. However, the majority of these patients were in the pediatric age group, received an autograft with BU/CY conditioning, and were able to be treated with a TBI-containing regimen with their allograft. Results in adults and those treated with TBI during their initial autograft were not as encouraging. Second allogeneic transplantation after initial failure of allografting has also been studied. A survey conducted by the EBMT identified 170

patients with acute leukemia, including 85 with AML who underwent second allogeneic transplants after high-dose conditioning regimens for recurrent disease [112]. Both treatment-related mortality and the actuarial risk of relapse were high, at 46% and 59% respectively. Nonetheless, 25% leukemia-free survival at 5 years was reported. In multivariate analysis, a longer interval to relapse after first HCT, CR status at time of second HCT, use of TBI at second HCT, and development of acute GVHD at second HCT were associated with better outcomes. The leukemia-free survival was 52% at 3 years for those who relapsed more than 292 days after their first HCT and were in remission before receiving their second transplant. Because of the high treatment-related mortality associated with second allogeneic transplants using high-dose regimens (46% overall, and 53% among those transplanted within a year of their first transplant), investigators have more recently been studying the use of reduced-intensity allogeneic transplants for patients who have recurred after a first transplant. Baron et al. recently reported on 147 patients with hematologic malignancies, including 35 with AML or advanced MDS, who were treated with RIC and allogeneic transplantation after failing a conventional autograft or allograft [113]. The treatment-related mortality was 30%, and 44% of patients were alive at 3 years, including 29% of patients with AML or advanced MDS. Being in CR at the time of second transplant and developing chronic GVHD were both associated with improved survival. HCT for APL Because of the remarkable effectiveness of the inclusion of ATRA, and more recently arsenic trioxide, in the primary treatment of APL, there is no role of transplantation during first remission. Unfortunately, disease recurrence still occurs in approximately 10% of good-risk patients (those presenting with a white blood cell count <10,000/mm3) and 25% of poor-risk patients. Second remissions can commonly be achieved with the use or arsenic trioxide, especially if patients have not previously seen the drug, with ATRA-containing regimens if the patients have relapsed off ATRA maintenance, or with the use of gemtuzumab ozogamicin. HCT is generally recommended as appropriate therapy for patients with APL who suffer an initial relapse. De Botton et al. recently reported on the outcome of therapy in 122 patients with APL whose disease recurred after initial treatment with an ATRA-containing regimen [114]. The 7-year event-free survival was 60.6% for those treated with an autograft, 52.2% for those treated with an allograft, and 30.4% for those treated without transplantation (Fig. 53.5). Most of the patients treated with an autograft had marrow that was polymerase chain reaction (PCR) negative for the t(15;17) translocation at the time of hematopoietic stem cell harvest and transplantation, a selection strategy based on previous reports that patients who are PCR positive at the time of autografting for APL have a very high chance of recurrence [115]. The report by de Botton et al., together with those of others, would argue that autologous transplantation is appropriate therapy for patients with APL who achieve a PCR-negative second remission, while allogeneic transplantation should be considered as appropriate salvage therapy for all others [116].

Conclusion Since the last publication of this textbook 5 years ago, the biggest change in our understanding of AML has been the identification of mutations not recognized by routine cytogenetics and our appreciation of their prognostic importance. With this added information, a proposed algorithm for patients with newly diagnosed AML is presented in Fig. 53.6. With continued investigation into AML using gene expression arrays,

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messenger RNA patterning and proteomics, this algorithm will likely change in another 5 years, even if there are no advances in treatment modalities. A second major change relevant to transplantation of AML over the last 5 years has been the continued development of reduced-intensity preparative regimens. These studies show that, with appropriate preand post-transplant immunosuppression, engraftment can be reliably achieved, and that treatment-related mortality can be substantially reduced. This is at a price of increased disease recurrence, but the development of reduced-intensity regimens offers new hope for older individuals who would otherwise not be candidates for transplantation and, importantly, provides a foundation on which to add additional therapies such as radioimmunoconjugates specifically targeted at the tumor cell. Continued developments in the use of alternative donor transplants further expand the potential application of allogeneic HCT for the treatment of AML. Perhaps the most interesting advance in this area over the past 5 years has been the emergence of cord blood transplants as a possible alternative for adults with AML. Outcomes of matched unrelated donor transplantation for AML are approaching those seen with matched sibling donors [117]. If this approach continues to show promise, it would address a major problem faced by the patient with high-risk AML who has achieved CR1, does not have a suitably matched family member, and is at considerable risk for early relapse if a lengthy search for an unrelated donor is required.

Fig. 53.5 Survival of 122 patients with acute promyelocytic leukemia in second remission according to postremission therapy (autologous transplantation, allogeneic transplantation or other). Allo, allogeneic; Auto, autologous; SCT, stem cell transplantation. (Reproduced from [114], with permission.)

Diagnosis

Good risk (t8;21), inv(16) Normal with NPM1+FLT3 –

Induction

CR

No CR

Consolidation chemotherapy

Allo HCT if possible

Observe

Intermediate risk Normal (NPM1– or FLT3+) +8,+6,–y

Poor risk Del 5q, del 7q, –7 complex

Induction

Induction

CR

Matched sibling available

Matched sibling unavailable

Allo HCT

Consolidation chemotherapy

No CR

CR

Allo HCT if possible

Allo HCT with matched sibling, unrelated donor or cord blood

Observe

Fig. 53.6 Proposed algorithm for treatment of adults age less than 60 years with newly diagnosed acute myeloid leukemia. Allo, allogeneic; CR, complete remission; HCT, hematopoietic cell transplantation.

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While there have been important advances in HCT for AML over the last 5 years, there are also areas where the amount of progress has been disappointing. One area has been our inability to systematically apply existing technologies for measurement of minimal residual disease to the decision-making process of who should be transplanted in first

remission. It is also disappointing that we have been unable to develop broad clinically applicable methods to segregate the impressive power of the graft-versus-tumor effect from the toxicities of GVHD. These topics remain the focus of a great deal of research that, hopefully, will lead to real therapeutic advances in the near future.

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(TAD 9) in 150 patients with relapse after standardized first line therapy. Leukemia 1990; 4: 184–8. Davis CL, Rohatiner AZ, Lim J et al. The management of recurrent acute myelogenous leukaemia at a single centre over a fifteen-year period. Br J Haematol 1993; 83: 404–11. List AF, Kopecky KJ, Willman CL et al. Benefit of cyclosporine modulation of drug resistance in patients with poor-risk acute myeloid leukemia: a Southwest Oncology Group study. Blood 2001; 98: 3212–20. Sievers EL, Larson RA, Stadmauer EA et al. Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse. J Clin Oncol 2001; 19: 3244– 54. Tallman MS, Andersen JW, Schiffer CA et al. All-trans retinoic acid in acute promyelocytic leukemia: long-term outcome and prognostic factor analysis from the North American Intergroup protocol. Blood 2002; 100: 4298–302. Soignet SL, Frankel SR, Douer D et al. United States multicenter study of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol 2001; 19: 3852–60. Biggs JC, Horowitz MM, Gale RP et al. Bone marrow transplants may cure patients with acute leukemia never achieving remission with chemotherapy. Blood 1992; 80: 1090–3. Fung HC, Stein A, Slovak M et al. A long-term follow-up report on allogeneic stem cell transplantation for patients with primary refractory acute myelogenous leukemia: impact of cytogenetic characteristics on transplantation outcome. Biol Blood Marrow Transplant 2003; 9: 766–71. Sierra J, Storer B, Hansen JA et al. Unrelated donor marrow transplantation for acute myeloid leukemia: an update of the Seattle experience. Bone Marrow Transplant 2000; 26: 397–404. British Committee for Standards in Haematology, Milligan DW, Grimwade D et al. Guidelines on the management of acute myeloid leukaemia in adults. Br J Haematol 2006; 135: 450–74. Estey E. Treatment of refractory AML [Review]. Leukemia 1996; 10: 932–6. Appelbaum FR, Clift RA, Buckner CD et al. Allogeneic marrow transplantation for acute nonlymphoblastic leukemia after first relapse. Blood 1983; 61: 949–53. Wong R, Shahjahan M, Wang X et al. Prognostic factors for outcomes of patients with refractory or relapsed acute myelogenous leukemia or myelodysplastic syndromes undergoing allogeneic progenitor cell transplantation. Biol Blood Marrow Transplant 2005; 11: 108–14. Kebriaei P, Kline J, Stock W et al. Impact of disease burden at time of allogeneic stem cell transplantation in adults with acute myeloid leukemia and myelodysplastic syndromes. Bone Marrow Transplant 2005; 35: 965–70.

Hematopoietic Cell Transplantation for Adult Acute Myeloid Leukemia 39. Schmid C, Schleuning M, Schwerdtfeger R et al. Long-term survival in refractory acute myeloid leukemia after sequential treatment with chemotherapy and reduced-intensity conditioning for allogeneic stem cell transplantation. Blood 2006; 108: 1092–9. 40. Kurtzberg J, Waldmann TA, Davey MP et al. CD7+, CD4−, CD8− acute leukemia: a syndrome of malignant pluripotent lymphohematopoietic cells. Blood 1989; 73: 381–90. 41. Gale RP, Horowitz MM, Rees JK et al. Chemotherapy versus transplants for acute myelogenous leukemia in second remission. Leukemia 1996; 10: 13–19. 42. Center for International Blood and Marrow Transplant Research. http://www.cibmtr.org (Accessed September 20, 2007). 43. Busca A, Anasetti C, Anderson G et al. Unrelated donor or autologous marrow transplantation for treatment of acute leukemia. Blood 1994; 83: 3077–84. 44. Ringden O, Labopin M, Gluckman E et al. Donor search or autografting in patients with acute leukaemia who lack an HLA-identical sibling? A matched-pair analysis. Bone Marrow Transplant 1997; 19: 963–8. 45. Lazarus HM, Perez WS, Klein JP et al. Autotransplantation versus HLA-matched unrelated donor transplantation for acute myeloid leukaemia: a retrospective analysis from the Center for International Blood and Marrow Transplant Research. Br J Haematol 2006; 132: 755–69. 46. Clift RA, Buckner CD, Appelbaum FR et al. Allogeneic marrow transplantation during untreated first relapse of acute myeloid leukemia. J Clin Oncol 1992; 10: 1723–9. 47. Brown RA, Wolff SN, Fay JW et al. High-dose etoposide, cyclophosphamide, and total body irradiation with allogeneic bone marrow transplantation for patients with acute myeloid leukemia in untreated first relapse: a study by the North American Marrow Transplant Group. Blood 1995; 85: 1391–5. 48. Schiffman K, Clift R, Appelbaum FR et al. Consequences of cryopreserving first remission autologous marrow for use after relapse in patients with acute myeloid leukemia. Bone Marrow Transplant 1993; 11: 227–32. 49. Appelbaum FR, Pearce SF. Hematopoietic cell transplantation in first complete remission versus early relapse. Best Pract Res Clin Haematol 2006; 19: 333–9. 50. Thomas ED, Buckner CD, Clift RA et al. Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 1979; 301: 597–9. 51. Clift RA, Thomas ED. Follow-up 26 years after treatment for acute myelogenous leukemia [Letter]. N Engl J Med 2004; 351: 2456–7. 52. Blume KG, Beutler E, Bross KJ et al. Bonemarrow ablation and allogeneic marrow transplantation in acute leukemia. N Engl J Med 1980; 302: 1041–6. 53. Powles RL, Morgenstern G, Clink HM et al. The place of bone-marrow transplantation in acute myelogenous leukaemia. Lancet 1980; 1: 1047– 50. 54. Herve P, Rozenbaum A, Plouvier E et al. Autologous bone marrow transplantation in acute myeloid leukemia in relapse or in complete remission. Cancer Treat Rev 1982; 66: 1983–5.

55. Stewart P, Buckner CD, Bensinger W et al. Autologous marrow transplantation in patients with acute nonlymphocytic leukemia in first remission. Exp Hematol 1985; 13: 267–72. 56. Yanada M, Matsuo K, Emi N, Naoe T. Efficacy of allogeneic hematopoietic stem cell transplantation depends on cytogenetic risk for acute myeloid leukemia in first disease remission: a metaanalysis. Cancer 2005; 103: 1652–8. 57. Reiffers J, Stoppa AM, Attal M et al. Allogeneic vs autologous stem cell transplantation vs chemotherapy in patients with acute myeloid leukemia in first remission: the BGMT 87 study. Leukemia 1996; 10: 1874–82. 58. Keating S, de Witte T, Suciu S et al. The influence of HLA-matched sibling donor availability on treatment outcome for patients with AML: an analysis of the AML 8A study of the EORTC Leukaemia Cooperative Group and GIMEMA. European Organization for Research and Treatment of Cancer. Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto. Br J Haematol 1998; 102: 1344–53. 59. Slovak ML, Kopecky KJ, Cassileth PA et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/ Eastern Cooperative Oncology Group study. Blood 2000; 96: 4075–83. 60. Burnett AK, Wheatley K, Goldstone AH et al. The value of allogeneic bone marrow transplant in patients with acute myeloid leukaemia at differing risk of relapse: results of the UK MRC AML 10 trial. Br J Haematol 2002; 118: 385– 400. 61. Suciu S, Mandelli F, de Witte T et al. Allogeneic compared with autologous stem cell transplantation in the treatment of patients younger than 46 years with acute myeloid leukemia (AML) in first complete remission (CR1): an intention-to-treat analysis of the EORTC/GIMEMAAML-10 trial. Blood 2003; 102: 1232–40. 62. Cairoli R, Beghini A, Grillo G et al. Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study. Blood 2006; 107: 3463–8. 63. Schlenk RF, Corbacioglu A, Krauter J et al. Gene mutations as predictive markers for postremission therapy in younger adults with normal karyotype AML. Blood 2006; 108: 6a. 64. Döhner K, Schlenk RF, Habdank M et al. Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations. Blood 2005; 106: 3740–6. 65. Tallman MS, Rowlings PA, Milone G et al. Effect of postremission chemotherapy before human leukocyte antigen-identical sibling transplantation for acute myelogenous leukemia in first complete remission. Blood 2000; 96: 1254–8. 66. Krauter J, Heil G, Hoelzer D et al. Role of consolidation therapy in the treatment of patients up to 60 years with high risk AML. ASH Annual Meeting Abstracts 2005; 106: 172a. 67. Levi I, Grotto I, Yerushalmi R, Ben-Bassat I, Shpilberg O. Meta-analysis of autologous bone marrow transplantation versus chemotherapy in adult patients with acute myeloid leukemia in first remission. Leuk Res 2004; 28: 605–12. 68. Zittoun RA, Mandelli F, Willemze R et al. Autologous or allogeneic bone marrow transplantation

69.

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74.

75.

76.

77.

78.

79.

80.

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compared with intensive chemotherapy in acute myelogenous leukemia. N Engl J Med 1995; 332: 217–23. Harousseau J-L, Cahn J-Y, Pignon B et al. Comparison of autologous bone marrow transplantation and intensive chemotherapy as postremission therapy in adult acute myeloid leukemia. Blood 1997; 90: 2978–86. Cassileth PA, Harrington DP, Appelbaum FR et al. Chemotherapy compared with autologous or allogeneic bone marrow transplantation in the management of acute myeloid leukemia in first remission. N Engl J Med 1998; 339: 1649– 56. Reiffers J, Gaspard MH, Maraninchi D et al. Comparison of allogeneic or autologous bone marrow transplantation and chemotherapy in patients with acute myeloid leukaemia in first remission: a prospective controlled trial. Br J Haematol 1989; 72: 57–63. Slovak ML, Kopecky KJ, Cassileth PA et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/ Eastern Cooperative Oncology Group study. Blood 2000; 96: 4075–83. Appelbaum FR. Is there a best transplant conditioning regimen for acute myeloid leukemia? Leukemia 2000; 14: 497–501. Thomas ED, Clift RA, Hersman J et al. Marrow transplantation for acute nonlymphoblastic leukemia in first remission using fractionated or singledose irradiation. Int J Radiat Oncol Biol Phys 1982; 8: 817–21. Clift RA, Buckner CD, Appelbaum FR et al. Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: a randomized trial of two irradiation regimens. Blood 1990; 76: 1867–71. Helenglass G, Powles RL, McElwain TJ et al. Melphalan and total body irradiation (TBI) versus cyclophosphamide and TBI as conditioning for allogeneic matched sibling bone marrow transplants for acute myeloblastic leukaemia in first remission. Bone Marrow Transplant 1988; 3: 21–9. Blaise D, Maraninchi D, Archimbaud E et al. Allogeneic bone marrow transplantation for acute myeloid leukemia in first remission: a randomized trial of a busulfan-cytoxan versus cytoxan-total body irradiation as preparative regimen. A report from the Groupe d’Etudes de la Greffe de Moelle Osseuse. Blood 1992; 79: 2578–82. Ringden O, Labopin M, Tura S et al. A comparison of busulphan versus total body irradiation combined with cyclophosphamide as conditioning for autograft or allograft bone marrow transplantation in patients with acute leukaemia. Acute Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Br J Haematol 1996; 93: 637–45. Dusenbery KE, Daniels KA, McClure JS et al. Randomized comparison of cyclophosphamidetotal body irradiation versus busulfan-cyclophosphamide conditioning in autologous bone marrow transplantation for acute myeloid leukemia. Int J Radiat Oncol Biol Phys 1995; 31: 119–28. Brenner MK, Rill DR, Moen RC et al. Genemarking to trace origin of relapse after autologous bone-marrow transplantation. Lancet 1993; 341: 85–6.

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81. Gorin NC, Aegerter P, Auvert B et al. Autologous bone marrow transplantation for acute myelocytic leukemia in first remission: a European survey of the role of marrow purging. Blood 1990; 75: 1606–14. 82. Reiffers J, Labopin M, Sanz M et al. Autologous blood cell vs marrow transplantation for acute myeloid leukemia in complete remission: an EBMT retrospective analysis. Bone Marrow Transplant 2000; 25: 1115–19. 83. Tallman MS, Pérez WS, Lazarus HM et al. Pretransplantation consolidation chemotherapy decreases leukemia relapse after autologous blood and bone marrow transplants for acute myelogenous leukemia in first remission. Biol Blood Marrow Transplant 2006; 12: 204–16. 84. Sierra J, Storer B, Hansen JA et al. Transplantation of marrow cells from unrelated donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor HLA-matching, and marrow cell dose. Blood 1997; 89: 4226–35. 85. Rocha V, Labopin M, Gluckman E et al. Relevance of bone marrow cell dose on allogeneic transplantation outcomes for patients with acute myeloid leukemia in first complete remission: results of a European survey. J Clin Oncol 2002; 20: 4324–30. 86. Lee SH, Lee MH, Lee JH et al. Infused CD34+ cell dose predicts long-term survival in acute myelogenous leukemia patients who received allogeneic bone marrow transplantation from matched sibling donors in first complete remission. Biol Blood Marrow Transplant 2005; 11: 122–8. 87. Stem Cell Trialists’ Collaborative Group. Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: an individual patient data meta-analysis of nine randomized trials. J Clin Oncol 2005; 23: 5074–87. 88. Ringden O, Labopin M, Bacigalupo A et al. Transplantation of peripheral blood stem cells as compared with bone marrow from HLA-identical siblings in adult patients with acute myeloid leukemia and acute lymphoblastic leukemia. J Clin Oncol 2002; 20: 4655–64. 89. Ringden O, Remberger M, Runde V et al. Peripheral blood stem cell transplantation from unrelated donors: a comparison with marrow transplantation. Blood 1999; 94: 455–64. 90. Remberger M, Ringdén O, Blau I-W et al. No difference in graft-versus-host disease, relapse, and survival comparing peripheral stem cells to bone marrow using unrelated donors. Blood 2001; 98: 1739–45. 91. Ooi J, Iseki T, Takahashi S et al. Unrelated cord blood transplantation for adult patients with de novo acute myeloid leukemia. Blood 2004; 103: 489–91. 92. Brunstein CG, Baker KS, Wagner JE. Umbilical cord blood transplantation for myeloid malignancies. Curr Opin Hematol 2007; 14: 162–9. 93. Laughlin MJ, Eapen M, Rubinstein P et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med 2004; 351: 2265–75.

94. Rocha V, Labopin M, Sanz G et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004; 351: 2276–85. 95. Kim DW, Chung YJ, Kim TG et al. Cotransplantation of third-party mesenchymal stromal cells can alleviate single-donor predominance and increase engraftment from double cord transplantation. Blood 2004; 103: 1941–8. 96. Storb R, Deeg HJ, Fisher LD et al. Cyclosporine v methotrexate for graft-v-host disease prevention in patients given marrow grafts for leukemia: long-term follow-up of three controlled trials. Blood 1988; 71: 293–8. 97. Wagner JE, Thompson JS, Carter SL, Kernan NA. Effect of graft-versus-host disease prophylaxis on 3-year disease-free survival in recipients of unrelated donor bone marrow (T-cell Depletion Trial): a multi-centre, randomised phase II–III trial. Lancet 2005; 366: 733–41. 98. Gopal AK, Gooley TA, Golden JB et al. Efficacy of high-dose therapy and autologous hematopoietic stem cell transplantation for non-Hodgkin’s lymphoma in adults 60 years of age and older. Bone Marrow Transplant 2001; 27: 593–9. 99. Wallen H, Gooley TA, Deeg HJ et al. Ablative allogeneic hematopoietic cell transplantation in adults 60 years of age and older. J Clin Oncol 2005; 23: 3439–46. 100. Hegenbart U, Niederwieser D, Sandmaier BM et al. Treatment for acute myelogenous leukemia by low-dose, total-body, irradiation-based conditioning and hematopoietic cell transplantation from related and unrelated donors. J Clin Oncol 2006; 24: 444–53. 101. Tauro S, Craddock C, Peggs K et al. Allogeneic stem-cell transplantation using a reducedintensity conditioning regimen has the capacity to produce durable remissions and long-term diseasefree survival in patients with high-risk acute myeloid leukemia and myelodysplasia. J Clin Oncol 2005; 23: 9387–93. 102. Lowsky R, Takahashi T, Liu YP et al. Protective conditioning for acute graft-versus-host disease. N Engl J Med 2005; 353: 1321–31. 103. Alyea EP, Kim HT, Ho V et al. Impact of conditioning regimen intensity on outcome of allogeneic hematopoietic cell transplantation for advanced acute myelogenous leukemia and myelodysplastic syndrome. Biol Blood Marrow Transplant 2006; 12: 1047–55. 104. Shimoni A, Yeshurun M, Hardan I et al. Allogeneic stem-cell transplantation in patients with AML and MDS using myeloablative versus reduced-intensity doses of intravenous busulfan: the role of dose intensity. Blood 2004; 104 (Pt 1): 635a. 105. Sorror ML, Sandmaier BM, Storer BE et al. Comorbidity and disease status-based risk stratification of outcomes among patients with acute myeloid leukemia or myelodysplasia receiving allogeneic hematopoietic cell transplantation. J Clin Oncol 2007; 25: 4246–54. 106. Mohty M, de Lavallade H, Ladaique P et al. The role of reduced intensity conditioning allogeneic stem cell transplantation in patients with acute

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108.

109.

110.

111.

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113.

114.

115.

116.

117.

myeloid leukemia: a donor vs no donor comparison. Leukemia 2005; 19: 916–20. Appelbaum FR, Kopecky KJ. Long-term survival after chemotherapy for acute myeloid leukemia: the experience of the Southwest Oncology Group. Cancer 1997; 80: 2199–204. Mortimer J, Blinder MA, Schulman S et al. Relapse of acute leukemia after marrow transplantation: natural history and results of subsequent therapy. J Clin Oncol 1989; 7: 50– 7. Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia. Blood 1995; 86: 2041–50. Schmid C, Labopin M, Nagler A et al. EBMT Acute Leukemia Working Party. Donor lymphocyte infusion in the treatment of first hematological relapse after allogeneic stem-cell transplantation in adults with acute myeloid leukemia: a retrospective risk factors analysis and comparison with other strategies by the EBMT Acute Leukemia Working Party. J Clin Oncol 2007; 25: 4938– 45. Radich JP, Gooley T, Sanders JE et al. Second allogeneic transplantation after failure of first autologous transplantation. Biol Blood Marrow Transplant 2000; 6: 272–9. Bosi A, Laszlo D, Labopin M et al. Second allogeneic bone marrow transplantation in acute leukemia: results of a survey by the European Cooperative Group for Blood and Marrow Transplantation. J Clin Oncol 2001; 19: 3675– 84. Baron F, Storb R, Storer BE et al. Factors associated with outcomes in allogeneic hematopoietic cell transplantation with nonmyeloablative conditioning after failed myeloablative hematopoietic cell transplantation. J Clin Oncol 2006; 24: 4150– 7. de Botton S, Fawaz A, Chevret S et al. Autologous and allogeneic stem-cell transplantation as salvage treatment of acute promyelocytic leukemia initially treated with all-trans-retinoic acid: a retrospective analysis of the European acute promyelocytic leukemia group. J Clin Oncol 2005; 23: 120–6. Meloni G, Diverio D, Vignetti M et al. Autologous bone marrow transplantation for acute promyelocytic leukemia in second remission: prognostic relevance of pretransplant minimal residual disease assessment by reverse-transcription polymerase chain reaction of the PML/RAR alpha fusion gene. Blood 1997; 90: 1321–5. Kharfan-Dabaja MA, Abou Mourad YR, Fernandez HF, Pasquini MC, Santos ES. Hematopoietic cell transplantation in acute promyelocytic leukemia: a comprehensive review. Biol Blood Marrow Transplant 2007; 13: 997–1004. Moore J, Nivison-Smith I, Goh K et al. Equivalent survival for sibling and unrelated donor allogeneic stem cell transplantation for acute myelogenous leukemia. Biol Blood Marrow Transplant 2007; 13: 601–7.

54

Julie-An M. Talano, James T. Casper & David A. Margolis

Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia

Epidemiology and etiology Acute myeloid leukemia (AML) is the most common type of myeloid malignancy of childhood. The annual incidence of AML in the United States is approximately 7.5 per million, and it accounts for 15–20% of leukemias in individuals under the age of 20 years [1]. The incidence of childhood AML varies with age, with the highest rates in the first 2 years of life followed by a slow decline to a nadir at the age of 9 years and slowly increasing rates during the adolescent years [2]. In the United States, the incidence of AML is similar in Caucasian and AfricanAmerican children for all age groups. AML is a clonal disorder due to an acquired somatic mutation in a hematopoietic progenitor cell. It is a heterogeneous disease showing variability in the degree of commitment and differentiation of the cell lineage involved [3]. While the exact cause of AML has not been elucidated, a number of risk factors may predispose a child to the development of AML. Known risk factors include genetic predispositions as well as environmental factors. Down’s syndrome, Fanconi’s anemia, Shwachman’s syndrome, Kostmann’s granulocytopenia, Bloom’s syndrome, and neurofibromatosis are examples of genetic conditions associated with a known increased risk of AML [2]. More recently, a familial AML syndrome with monosomy of chromosome 7 has been reported [4]. Also, patients with severe aplastic anemia treated with granulocyte colony-stimulating factor (G-CSF) for a prolonged period of time may be at risk for development of AML [5]. Exposure to specific chemotherapy agents, such as alkylating agents or epidodophyllotoxins, has been associated with an increased risk of childhood AML [2]. Parental exposure to benzene and to pesticides and maternal alcohol consumption are factors for which causal evidence is suggestive but not conclusive [2].

Molecular and cellular biology The French–American–British Cooperative Group has developed the most comprehensive morphologic–histochemical classification system for AML. This classification system categorizes AML into subtypes called M0–M7 [6]. M0 does not show localized differentiation, M1 and M2 are more myeloid in nature, M3 acute promyelocytic leukemia (APML) is the hypergranular type, M4 and M5 are myelomonocytic–

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

monocytic, M6 is erythroid, and M7 is megakaryocytic. Between 50% and 60% of children with AML can be classified as having M1, M2, M3, M6 or M7 subtypes; approximately 40% have M4 or M5 subtypes [6]. The distribution between the groups is similar between adults and children except for M5, which may be increased in infants, and M6, which rarely occurs in children [6]. Cell surface phenotype analysis is also employed, and at least one of the myeloid markers (CD33, CD13, CD15, CD11b, CD14, CD36 or CD117) is seen in the vast majority of cases [6]. Type M7 (megakaryocytic) is classified based on its expression of the platelet glycoprotein antigens IIb/IIIa or Ib [6]. Lineage-associated B-lymphocytic antigens CD10, CD19, CD20, CD22, and CD24 may be present in 10–20% of cases of AML, but monoclonal surface immunoglobulin and cytoplasmic immunoglobulin heavy chains are usually absent; likewise, CD2, CD3, CD5, and CD7 lineage-associated T-lymphocytic antigens are present in 20–40% of all AML. The aberrant expression of lymphoidassociated antigens by AML cells is fairly common but in general has no prognostic significance [6]. More recently, and with increasingly recognized prognostic significance, the cytogenetic systems have been utilized based on the clonal chromosomal abnormality observed [3]. Conventional chromosome studies performed by skilled personnel can identify nonrandom clonal aberrations in at least 75% of patients with AML [6]. Multiple studies and groups of investigators have verified the link between cytogenetic classification and prognosis for childhood AML [7–10]. In summary, these studies find that t(15;17) patients have an excellent prognosis while patients with t(8;21) and inversion of chromosome 16 (inv16) are notable in that they have a better than average prognosis. On the other hand, AML with deletions of chromosome 5 or 7 is associated with a poor prognosis. Also, AML with mixed lineage leukemia gene rearrangements (t11q23) includes most cases of AML secondary to epipodophyllotoxins. These are associated with monocytic differentiation (M4 and M5), which has an unfavorable prognosis as well [11,12]. Further molecular characterization has shown that those children whose AML blasts have an internal tandem repeat of a tyrosine kinase gene (FLT3/ITD) have a significantly worse prognosis, and high ratios between mutant and WT-FLT3 further compromise prognosis [13]. Within FLT3-mutant cases, expression of the genes RUNX3 and ATRX can define high-, intermediate-, and low-risk prognostic groups [14]. Mutations in c-KIT occur in less than 5% of cases of AML but up to 10–40% of AML with core-binding factor abnormalities [15,16]. The presence of the activating c-KIT mutations in this subgroup of AML appears to be associated with a poor prognosis [16,17].

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The World Health Organization has proposed a new classification system linking the previous morphologic determinants with the biologic and genetic information to define a nomenclature with clinical relevance [18]. Gene expression profiling is rapidly becoming another method to subclassify AML [19]. Clearly, the classification of AML and its prognostic characteristics continues to evolve as more relevant molecular information is discovered.

The clinical presentation of the child with AML varies. Patients can present with very few symptoms, or unfortunately with life-threatening sepsis or hemorrhage. At diagnosis, the clinical signs reflect the lack of production of red blood cells, granulocytes, and platelets, which leads to anemia, infection, and bleeding. Infiltration of the skin is often seen in neonates with monocytic (M5) leukemia. Anemia can present as fatigue, pallor, headache, dyspnea, and congestive heart failure. Thrombocytopenia can cause petechiae, bruising, epistaxis, and bleeding. Disseminated intravascular coagulation can occur in any subtype of AML, but severe hemorrhagic problems can be seen in patients with the promyelocytic (M3) type of disease. The white blood cell count is variable and can be over 100,000/mL in about a quarter of the patients, which is a negative prognostic factor. Hepatomegaly or splenomegaly occurs in about 50% of patients with newly diagnosed AML. Other areas of extramedullary disease include enlargement of the lymph nodes, and chloromas (discrete tumors usually near the epidural areas or the orbits). Central nervous system involvement occurs in 5– 15% of newly diagnosed patients and can manifest as a central nervous system chloroma or meningeal infiltration with or without cranial nerve palsy. Symptoms can include headache, nausea, vomiting, photophobia, and papilledema [20].

Historical background The goal of treating the child who presents with AML is to attain a complete remission (CR) and cure while maintaining a reasonable quality of life. Over the past 25 years, survival rates for children with AML have remained lower than those for children with acute lymphoblastic leukemia (ALL), but these rates continue to improve. Because of the poor results obtained with chemotherapy in the 1970s, other treatment options were pursued. It was during this time that E.D. Thomas and his colleagues in Seattle demonstrated the curative effect of bone marrow transplantation (BMT) in a small number of patients with refractory leukemia [21]. As the remission-induction chemotherapy regimens for childhood AML improved, investigators applied BMT to children and young adults with AML who achieved a remission and had a matched sibling donor. In the early 1980s, BMT groups in Seattle, Minneapolis, and Baltimore reported disease-free survival (DFS) of approximately 65% for children with AML in first CR (CR1) [22–24]. Figure 54.1 illustrates the DFS and incidence of relapse for 38 children with AML in CR1 who received a BMT from human leukocyte antigen (HLA)-matched sibling donors in Seattle [22]. The conditioning regimen consisted of fractionated total body irradiation (TBI) to a dose of 1200 cGy and cyclophosphamide (CY) 60 mg/kg/day for 2 days. Methotrexate (MTX) was used for graft-versus-host disease (GVHD) prophylaxis. This regimen, with the addition of cyclosporine (CSP) and the use of only four doses of MTX, remains a standard treatment regimen more than 20 years later. The success of BMT (DFS = 65%) contrasted with DFS rates of less than 50% for patients treated with conventional chemotherapy during this same time period [25]. For this reason, many groups incorporated BMT into their AML treatment strategies if an HLA-matched sibling was available. Similar results were reported

0.8

Probability

Clinical description

1.0

Survival

Disease free survival

0.6

0.4

Relapse

0.2

0 0

1

2

3

4

5

6

7

8

9

Years

Fig. 54.1 Kaplan–Meier product limit estimates for probability of survival (---), disease-free survival (—) and relapse (-·-·-) of children who received transplants for acute myeloid leukemia in first remission. The tick marks indicate living patients. (Reproduced from [22], with permission.)

during the 1980s [22,25]. Relapse rates post BMT ranged from 0% to 30%. Only one study reported a DFS of less than 50%. The conditioning regimen used most often was similar to the Seattle TBI/CY regimen described above. GVHD prophylaxis usually consisted of MTX and CSP, alone or in combination. In all these studies, BMT appeared to be superior to chemotherapy for the treatment of childhood AML. However, one must be cautious when interpreting these results. Most of these studies were not prospectively designed and, as a result, allowed for selection bias related to patients. For example, some patients with a matched sibling donor may have been excluded from transplant because of therapy-related toxicity or early relapse. More recent data are based on large and, in most cases, prospective multi-institutional trials. These recent trials have demonstrated a DFS of more than 60% for matched sibling hematopoietic cell transplantation (HCT) in most studies (Tables 54.1 [95] and 54.2 [125]). Figure 54.2 shows the latest data compiled from the Center for International Bone Marrow Transplant Registry (CIBMTR) for matched sibling HCT. Because of advances in standard chemotherapy treatment regimens, hematopoietic cell source (cord blood and peripheral blood), and risk factor-based treatment, the questions at this time are: Who should receive an HLA-matched sibling HCT during CR1, and what is the role and timing of alternative donor transplants? Determining the appropriateness of any medical treatment rests in answering the pertinent question: What are the risks and benefits of the treatment proposed compared with other treatments? With that in mind, the purpose of this chapter is to evaluate the current role for HCT in caring for the child with AML. The following questions will be addressed: (1) What is the role and timing of matched sibling HCT? (2) What is the role for alternative donor HCT? (3) How does one treat the child who has relapsed after a HCT? (4) What are the long-term consequences related to HCT? By evaluating the data and answering these questions, an algorithm to help in the decision process can be constructed. Figure 54.3 is a proposed algorithm outlining our approach to the treatment of childhood AML. This chapter presents the data addressing the above questions and concludes with the rationale behind the algorithm illustrated.

777

Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia Table 54.1 Prospective comparisons of allogeneic BMT versus chemotherapy and/or autologous BMT in children with newly diagnosed AML N* Allo

DFS or EFS Auto

chemo

Allo

242/252

OS chemo

F/u‡

P§

Allo

45

33

5D

0.05

43

31

6D

0.33

297

46 ± 10

38 ± 6

5D

0.06

15

86 ± 13

58 ± 9

1.3 B

81

57

3B

33/37

51 ± 13

21 ± 8

27 ± 8

5D

0.03

113/117

52 ± 8

38 ± 6

36 ± 6

3E

0.01,†† 0.06

~62

→69

68

46

7D

0.02‡‡

70

55 ± 9

42 ± 8

47 ± 8

8D

0.001††0.01 60 ± 9

Study

Design† Age (years)

CCG 251 (Nesbit et al, 1994) AML 80 (Dahl et al, 1990) CCG 213 (Wells et al, 1994) ANZC CSG (Vowels et al, 1992) RAHC ANZ CCSG (Shaw et al, 1994) AIEOP LAM 87 (Amadori et al, 1993) POG 8821 (Ravindranath et al, 1996) MRC 10 (Stevens et al, 1998) CCG 2891 (Woods et al, 2001a)

P/I

0–21

79/89

P/I

0–19

15/19

42

P/I

0–21

83/113

P/C

0–15

15

42

P**/C

0–15

11/13

23/24

PR/I

0–15

22/24

23/35

PR/I

0–20

89

71/115

PR/I

0–15

61/85

44/50

PR/I

0–20

164/181

137/177

50 171/179

Auto

Auto

chemo

F/u¶

P§

50

36

5

0.04

52 ± 10

46 ± 10

5

0.13

40 ± 6

44 ± 6

3

0.007,†† 0.15

70

59

7

0.1,§§0.2¶¶

48 ± 8

53 ± 8

8

0.002,††0.05

*Numerator is the number treated as allocated and denominator is the number of patients allocated to the arm. †P, prospectively assigned to BMT if matched sibling available; R, randomized consolidation arms; I, intent-to-treat analysis; C, analysis restricted to patients treated correctly as assigned; A, analysis as treated. ‡Years follow-up. D, DFS from end of induction; E, EFS from diagnosis; B, EFS from BMT. §Allo versus chemo, except as noted. ¶Years of follow-up from end of induction. **Retrospectively defined subset for analysis from prospectively designed study. ††Allo versus auto. ‡‡Auto BMT versus chemo. §§HLA-matched donor available versus not available. ¶¶Auto BMT versus chemo. ANZ CCSG, Australia and New Zealand Childhood Cancer Study Group; RAHC, Royal Alexandra Hospital for Children; AIEOP, Associazione Italiana Ematologia ed Oncologia Pediatrica Cooperative Group. Reproduced from [95]

Treatment options for newly diagnosed patients The treatment of choice for the child with AML after attaining a first remission has been the central question in a number of multi- and singleinstitution studies as well as analyses by the CIBMTR and the National Marrow Donor Program (NMDP) over the past 20 years [26–28, 30–35,95,126]. Treatment options include intensive chemotherapy with or without a maintenance phase, allogeneic HCT using an HLA-matched sibling, or autologous HCT with or without purging of the marrow inoculum. Because only about 30% of children have an HLA-matched sibling, studies that have compared allogeneic matched sibling HCT with other treatments often have used the presence or absence of an HLA-matched sibling as a basis for “biologic randomization.” Thus, if an HLA-matched sibling were available, the patient would not be randomized but would undergo HCT. The remaining patients would be treated using a different plan, with survival and relapse rates compared between the HCT group and other treatment modalities. Tables 54.1 and 54.2 summarize the European, North American, and Australian studies that have been conducted using the concept of “biologic randomization” over the past 15 years. Since various induction and conditioning regimens were used, direct comparisons of the outcomes between studies are difficult. In an attempt to clarify this issue the authors of the journal Leukemia in 2005 invited cooperative groups from around the world to describe their experiences with pediatric AML according to specific guidelines [36]. They published 11

original manuscripts from this endeavor [37–47]. There were strict inclusion and exclusion criteria for the core group of AML patients studied to allow intergroup comparison. The inclusion criteria consisted of age 0–15 years, diagnosis of de novo AML, French– American–British Group subtypes M0–M7, and unclassifiable AML. The exclusion criteria were age over 15 years, AML as a secondary malignancy, myleosarcoma without significant bone marrow involvement, Down’s syndrome, preceding myelodysplastic syndrome (MDS), and pretreatment with cytostatic drugs (e.g. steroids) for more than 14 days. To summarize the data from the major cooperative groups as detailed in Tables 54.1, 54.2, and 54.3, patients were enrolled into various treatment arms, including allogeneic HCT, autologous HCT, and intensive chemotherapy [27–29,37–47,95,125]. Despite the use of different treatments and randomization strategies, examining the outcomes in these studies provides clues as to how to approach the child with AML in CR1. In general, the major groups reported an improved outcome over time with a 5-year overall survival (OS) ranging from 41% to 66%. Rates of early death and death in CR declined to 2–20% and 2–16%, respectively, and CR rates have increased to 70–93%. Improvements can be explained in part due to the importance of dose-intensification early in therapy and the need for intensive consolidation therapy, increased experience with intensive treatment, better facilities for supportive care, and better risk group stratification with subsequent risk group-adapted treatment. Relapse is the most frequent event and occurred after HCT approximately 30–40% of the time. Central nervous system relapses occurred

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Chapter 54

Table 54.2 Reproduced from Creutzig and Reinhardt. (125) Results according to post-remission treatment Relapse

TRM

Study group

Treatment given

n

% of total CR group %

n

%

n

%

pDFS (SE) %

AIEOP/LAM87 Amadori et al. (1993) n = 161 AML-BFM 93 Creutzig et al. (2001a,c) n = 471 EORTC Behar et al. (1996) n = 108 MRC AML 10 Stevens et al. (1998) n = 341

allo MRD SCT auto SCT Chth non-randomized allo MRD SCT Other SCT Chth

24 35 37 31 31 17 339

19 29 27 24 8 4 88

9 25 22 11 8 6 112

38 71 59 35 26

0 1 3 5 3 4 13

0 3 8 16 10

51 ± 13 21 ± 8 27 ± 8 34 ± 10 65 ± 9

4

61 ± 3

allo MRD SCT auto SCT Chth

13 2 69

15 2 82

allo MRD SCT

61

19

8

13

19 16

2 1 4

LAME‡ Michel et al. (1996) n = 171 NOPHO 93 Lie et al. (2001) n = 127¶ Spain Ortega & Olive (1998) n = 51

33

70 (donor)

69 ± 12 (7 years)

2

68 46

73 ± 11 66 ± 8 from d 155 66 ± 8 from d 155

72 ± 15 (71 ± 15)‡ 48 ± 10 (55 ± 10)‡

22 78

1 8

3 8

allo MRD SCT allo MUD SCT auto SCT Chth allo MRD SCT

19 3 12 57 31

4

14

1 2

60 ± 13 47 ± 7 81 ± 13

69

0

80 ± 12

auto SCT

46

31

P-value

n.g. n.g. (52% total group)

allo MUD SCT 4 auto SCT 60 stop Chth 50 (randomized) no SCT 144 (non-randomized)† allo MRD SCT 33 Chth 116

24 4 15 73 16 (incl. 2 PR)

Survival from SCT %

0.2 0.2

0.02 (0.10)§

79 ± 8

* Other SCT: MUD (n = 7), haploidentical SCT (n = 4), auto-SCT (n = 6). † One hundred and forty-four not eligible for randomization because of TRM (n = 19); early relapse, elected by parents/clinicians (n = 127). ‡ For update, see additional reference (Perel et al. 2002). § Age > 1 year. ¶ Down’s syndrome excluded. TRM, treatment-related mortality; pDFS, probability of disease-free survival; SE, standard error; CR, complete remission; SCT, stem cell transplantation; allo, allogeneic; auto, autologous; MRD, matched related donor; MUD, matched unrelated donor; Chth, chemotherapy.

100

Probability (%)

80 Early (n = 804)

60

Intermediate (n = 174) 40 Advanced (n = 165)

20 p < 0.001 0

0

1

2

3 Years

4

5

6

Fig. 54.2 Probability of survival after human leukocyte antigen (HLA)identical sibling transplants with high-dose conditioning for acute myeloid leukemia (AML), age under 20 years, over the period 1998–2004 by disease status Center for International Bone Marrow Transplant Registry (CIBMTR). This figure shows survival probabilities in patients less than 20 years of age among whom 3-year probabilities of survival were 63% ± 2%, 59% ± 4%, and 35% ± 4% after HCT for early, intermediate, and advanced disease, respectively. Early disease is defined as complete remission (CR) 1; intermediate disease is defined as CR2 or subsequent CR; advanced disease is defined as primary induction failure or relapse. (Data kindly provided by the CIBMTR.)

Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia

779

Induction chemotherapy

Complete remission

Matched sibling

Down’s syndrome APML Inv (16), t(8,21) (CLINICAL TRIAL)

HCT

Chemotherapy

Relapse

Relapse

HCT

Induction failure

No matched sibling

Monosomy 7 monosomy 5/5q-

Consider alternative donor HCT (CLINICAL TRIAL)

Chemotherapy

Chemotherapy

Cure

Relapse

Relapse

Alternative donor HCT

Alternative donor HCT

Relapse

Matched sibling

No matched sibling

HCT

Alternative donor HCT

Relapse

Cure

Relapse

Adoptive immunotherapy or second HCT

Fig. 54.3 An algorithm for the management of the child with acute myeloid leukemia. APML, acute promyelocytic leukemia; HCT, hematopoietic cell transplantation.

in 2–9% overall. There is no proven benefit to maintenance therapy except for APML, and in fact, the groups of patients randomized to maintenance therapy (CCG213 and LAME 91) showed inferior survival [26,47]. A major goal of the project was subgroup analyses. Subgroups such as Down’s syndrome OS 67–77%, OS in APML 75–88%, and OS with inv16 66–100% do relatively well across the groups with chemotherapy alone. Translocation (8;21) is a bit more controversial, with the majority of the groups reporting an event-free survival (EFS) of 50– 76%, except for Children’s Cancer Group Study 2891, which reported only a 41% EFS for this subgroup. In none of these trials did autologous BMT show significantly improved survival over intensive post-remission chemotherapy alone [36–47]. Figure 54.4 [48] illustrates the Children’s Cancer Group Study 2891 data, which led the authors to conclude that matched sibling allogeneic BMT in CR1 resulted in superior survival compared with chemotherapy or autologous transplant. Their conclusion has fueled debate over this issue in the past [49–51]. However, as the data have matured over time, OS on the allogeneic arm continues to drop due to long-term side-effects related to HCT, especially GVHD. As outcomes with intensive chemotherapy improve, one can ask whether children should be exposed to the potential morbidity associated with allogeneic HCT, especially for the low-risk cytogenetic subgroups. As seen in Fig. 54.5, improving outcome for the nontransplant modality diminishes the survival advantage for children who have a matched sibling donor available [29]. With the enhanced ability to predict outcome by incorporating cytogenetic data, current ongoing clinical trials are approaching transplantation in CR1 for patients with mediumand/or high-risk biologic features [49]. There is also the argument that patients who fail chemotherapy could be successfully transplanted in relapse or in second CR (CR2). Using this “salvage” approach, a group

of patients would not need to be exposed to transplant-related mortality (TRM) and harmful late effects. However, at the present time, the American Society for Blood-Marrow Transplantation position statement recommends matched sibling allogeneic HCT in CR1 (if one is available) over chemotherapy alone when performed outside the context of a clinical trial based on an evidence-based review of the literature. However, the Society does state that additional prospective data regarding risk subgroups may alter this recommendation [52,53]. Clearly, this issue will continue to be reassessed as more data are forthcoming.

Role of the preparatory regimen The preparatory regimen is but one aspect of the overall “transplant package,” which also includes the type of hematopoietic cell graft (related, unrelated, matched or unmatched) and the nature of GVHD prophylaxis. The choice of a preparatory regimen must take into account antileukemic efficacy as well as the risks of rejection, end-organ damage, and late effects. The effect that a preparatory regimen has on the outcome of an allogeneic transplant performed during CR1 may be significant. Most high-dose conditioning regimens used for AML have consisted either of chemotherapy-based busulfan (BU) and CY regimens or TBI with one or more chemotherapeutic agents [54,55]. Historically, the use of BU in children has been considered problematic because of the difficulty in having young children reliably ingest and retain BU pills, as well as the variable metabolism of BU in young children [56]. The emergence of BU pharmacokinetic data and intravenous BU has overcome inconsistent oral BU dosing, improved rates of engraftment, and decreased rates of sinusoidal obstructive syndrome (SOS). By combining test doses of BU (0.5 mg/kg) with follow-up pharmacokinetic sampling, two groups of investigators have reported

780

Chapter 54

Table 54.3 Outcome data of the most recently completed and matured studies from major groups concerning the well-defined core-group of de novo AML patients below 15 years of age. Reproduced with permission from Kaspers et al. [36] Study, years of enrolment and reference

Patient number

% of nonresponders

Early death rate (%)

CR rate (%)

5-year pEFS (%, with s.e.)

5-year pOS (%, with s.e.)

% death rate in CR

AIEOP92 (1992–2001)10 AML-BFM93 (1993–1998)3

160

5

6

89

54 (4)

60 (4)

7

427

10

7

83

51 (3)

58 (2)

4

CCG2891 (1989–1995)14

750

18

4

78

34 (3)

47 (4)

15

DCOG-ANLL 92/94 (1992–1998)7 EORTC-CLG 58921 (1993–2000)5 GATLA-AML90 (1990–1997)2

78

8

10

82

42 (6)

42 (6)

16

166

13

2

84

48 (4)

62 (4)

6

179

11

20

70

31 (4)

41 (4)

7

LAME91 (1991–1998)9

247

5

4

91

48 (4)

62 (4)

6

NOPHO-AML93 (1993–2001)8

223

5

2

92

50 (3)

66 (3)

2

PINDA-92 (1992–1998)11

151

5

26

68

36

36

4

POG8821 (1988–1993)12

511

19

4

77

32 (2)

42 (2)

8

PPLLSG98 (1998–2002)4

104

13

8

80

47 (5)

50 (5)

10

St. Jude-AML91 (1991–1997)13

62

16

3

79

44 (15)

57 (11)

?

303

3

4

93

49

58

10

UK-MRC AML10 (1988–1995)6

Cumulative doses of ara-C, VP16 and anthracyclines

% of total number of patients who underwent (allo-) SCT

No strict protocol guidelines 41.1 g/m2 950 mg/m2 300–400 mg/m2 14.6 g/m2 1100 mg/m2 180 mg/m2 33.2 g/m2 950 mg/m2 400 mg/m2 23.32–29.32 g/m2 1350 mg/m2 380 mg/m2 41.1 g/m2 1450 mg/m2 300 mg/m2 9.8–13.4 g/m2 400 mg/m2 460 mg/m2 49.6–61.3 g/m2 1600 mg/m2 300–375 mg/m2 7.64 g/m2 450 mg/m2 350 mg/m2 55.7 g/m2 2250 mg/m2 360 mg/m2 7.0–15.1 g/m2; 450–950 mg/m2; 420–600 mg/m2 3.8 g/m2; 1200 g/m2; 270 mg/m2 10.6 g/m2 500–1500 mg/m2 550 mg/m2

29 7

25

27

20

3

30

25

–

13

Not given, but a minority Not given

20

Please note that still the data are not completely comparable for various reasons. CR = complete remission; EFS = event-free survival; OS = overall survival; ara-C = cytosine arabinoside; VP16 = etoposide; SCT = stem cell transplantation. Cumulative dose of anthrayclines has been calculated applying the following arbitrary conversion factors to daunorubicin-equivalents; idarubicin 5×, mitoxantrone 5×, doxorubicin 1×. Some groups also applied amsacrine (LAME, UK), which has not been included in calculations of total athracyclines exposure.

dose adjustment of oral BU leading to individualized BU dosing, with improved engraftment rates and a decreased risk of SOS [57]. Michel et al. [30] tried to address the issue of the preparatory regimen in an analysis of children reported to the French registry who received either BU/CY 120, BU/CY 200, or a TBI-containing regimen. They retrospectively analyzed the outcome of 74 pediatric patients with AML treated with HLA-identical related allo-BMT in CR1. Except for the BU/CY 200 group, which had a mean white blood cell count that was higher than that of the TBI group, patient characteristics, including age,

were not statistically different. There was a lower EFS in the BU + CY 120 group (46 ± 24%, p = 0.07) than in the BU + CY 200 (82 ± 18%) and TBI (80 ± 14%) groups. These data suggest that patients treated with a more intense conditioning regimen had better EFS with only a slight increase in TRM. However, one must be careful in evaluating this study in view of the small number of patients in each treatment arm, its retrospective nature, and the lack of BU kinetic data. TBI has proven to be an excellent treatment modality for AML. Higher doses are associated with less relapse, but these benefits are

Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia

intravenously × eight doses (target area under the curve 900–1100 μM/ min), fludarabine 30 mg/m2/day × 6 days and antithymocyte globulin 2.5 mg/m2/day for one dose in related donors, and four doses for unrelated donors and umbilical cord blood recipients. For the entire cohort of patients, two patients rejected their grafts, and the overall TRM was 16%. Acute GVHD grade I–II occurred in 3 of 25 patients, and no grade III–IV GVHD was reported. Chronic GVHD occurred in 4 of 18 evaluable (22%) patients. For the entire cohort, 2-year EFS and OS are 49% (standard error 9.8) and 49% (standard error 11%) respectively. Of the eight patients surviving more than 1 year post transplant, four had AML. Although small numbers are reported, this is encouraging data for patients who have comorbidities that would prohibit a high-dose conditioning transplant [62].

1

0.75

Allo n = 114

Probability

Chemo n = 108 0.5

Auto n = 115

0.25 p = 0.0543 0 0

2

4

6

8

10

12

14

Years

Fig. 54.4 Overall survival from achieving remission for patients enrolled on the intensive timing arm of Children’s Cancer Group Study 2891 stratified by the post-induction therapy assigned (allogeneic BMT [Allo], autologous BMT [Auto], and chemotherapy [Chemo]). (Reproduced from [48], with permission.)

100

Donor No donor

Still alive (%)

75

70% 60%

50

25

Donor No donor

No. patients No. obs. Events exp. 85 25 31.9 230 91 84.1

p = 0.1

0 0 At risk: 85 Donor No donor 230

1

2

3

4

5

6

7

31 82

22 53

15 38

Years from CR 69 187

62 153

50 131

41 103

781

Fig. 54.5 Survival from complete remission (CR) by tissue typing status in the Medical Research Council 10 trial. Exp., expected number (from logrank analysis) obs., observed number of deaths in each arm. (Reproduced from [29], with permission.)

counterbalanced by an increase in regimen-related toxicity [58]. Several groups are using radioisotopes conjugated to monoclonal antibodies (CD33, CD45, and CD66) present on hematopoietic tissue and AML blasts to deliver the radiation dose in a more targeted and, hopefully, less toxic manner [59,60]. Reduced- intensity regimens have been developed primarily for use in older adults who cannot tolerate the regimen-related toxicity associated with standard high-dose conditioning [61]. While this strategy is showing promise in certain adult patient groups, preliminary data in abstract form are now available for pediatric AML. Previously published case series using reduced-intensity conditioning for pediatric malignant diseases only had one or two patients with AML. Pulsipher reported the Pediatric Blood and Marrow Transplant Consortium Study at the CIBMTR meetings in February 2007. Thirty-two pediatric patients (ages 2–20 years) have been enrolled with a variety of hematopoietic cell sources. Ten patients had AML (six CR2, three CR3, and one secondary AML). Patients were conditioned with BU 0.8 mg/kg

GVHD and graft-versus-leukemia effect The fine line between the benefit of an allogeneic immune response against the leukemia (the graft-versus-leukemia [GVL] effect) and the risk of an alloresponse against other tissues (GVHD) is aptly illustrated by studies comparing OS and the incidence of leukemic relapse in patients with acute GVHD (see Chapter 18) [63]. The role of GVHD/ GVL reactions to the cure of a particular disease can be assessed, in part, by examining how modulation of the GVHD prophylaxis affects outcome. For example, in a multicenter, prospective, randomized trial, the effect of altering the dose of CSP on outcomes for children with leukemia who receive matched sibling HCT was examined [64]. The patient cohort included a total of 59 children with either ALL (n = 47) or AML (n = 12). There was no difference in the incidence of chronic GVHD, and OS was not statistically different between the two CSP treatment groups. However Neudorf et al. recently reported data from the Children’s Cancer Group Study 2891. In this study, children with AML were randomly assigned to standard- or intensive-timing induction chemotherapy. Patients in CR1 with an HLA-identical related donor (or a single class I or class II mismatch) were nonrandomly assigned to receive a BMT conditioned with oral BU (16 mg/kg) and CY (200 mg/ kg). GVHD prophylaxis was MTX alone. One hundred and fifty patients received transplantation. This study demonstrated that there is a GVL effect. Univariate analysis showed that patients with grade I or II acute GVHD had better DFS 79% (95% confidence interval [CI] 60–87%) compared with patients who never developed GVHD, with a DFS of 63% (95% CI 47–75%) (p = 0.009). In the multivariate analysis, acute GVHD was the most significant factor associated with improved relapsefree survival [65]. Jernberg et al. found that chronic GVHD had a significant impact on relapse and survival. They evaluated 169 consecutive children who had undergone HCT for ALL and AML at their center. Median follow-up was 7 years. The 5-year probability of chronic GVHD was 34%. Median time to relapse was 24 months in children with chronic GVHD and 6 months in those without. The corresponding 5-year probabilities of relapse were 30% and 45%, respectively (p = 0.01). The 5-year probability of survival was 54%. Patients with chronic GVHD had a significantly better survival, 77% versus 51% (p = 0.01). In a Cox regression model, chronic GVHD independently decreased the risk of relapse (RR 0.44) and further predicted an increased chance of relapse-free survival (RR 1.7) and survival (RR 2.6). Acute GVHD was not an independent predictor for relapse or death in this study. This study is in support of a GVL effect in childhood leukemia related to chronic GVHD, reducing the risk of relapse and improving survival [66]. Recently, Parkman et al. [67] reported that successful immune reconstitution decreases leukemic relapse and improves survival in recipients of unrelated cord blood transplantation. One hundred and seventeen patients with ALL (n = 78) or AML (n = 39) were evaluated for their

782

Chapter 54

antigen-specific immune function after unrelated umbilical cord blood transplantation. They were specifically tested sequentially for their development of antigen-specific T-lymphocyte immunity to herpes viruses. The presence of an antigen-specific response resulted in a relapse-free survival advantage (p = 0.0001), which was primarily due to a decrease in the risk of relapse (p = 0.003). Notably in this study, neither acute nor chronic GVHD had any effect on the incidence of leukemic relapse. However, GVHD post umbilical cord transplant is typically less severe compared with bone marrow transplants or peripheral blood progenitor cell transplants. Further clinical research needs to be directed to this area. Whether a GVL effect can be separated from GVHD remains a point of intense study and beyond the scope of this chapter (see Chapters 14 and 18). Strategies to separate GVL and GVHD include the use of antigen-specific T-cell immunotherapy for leukemia [68] as well as utilizing delayed and/or modified donor leukocyte infusions (DLIs) [69,70]. Clinical GVHD may not be necessary for a GVL effect. For AML patients who receive donor marrow from an identical twin, there is a significantly higher incidence of relapse compared with patients who receive marrow from a matched sibling, but who do not develop clinical GVHD [71]. These data are consistent with the CIBMTR reports evaluating the incidence of acute leukemia relapse in patients receiving either an autograft, an identical-twin graft or an HLA-matched sibling graft without developing clinical GVHD [76]. The implication is that there is a GVL effect in patients receiving HLA-matched sibling grafts without clinical signs of GVHD.

Patients who may benefit from chemotherapy alone Because of the excellent outcome of children with Down’s syndrome using conventional chemotherapy, with 5-year EFS rates in excess of 80% [26], matched sibling HCT in CR1 is not recommended for this group of children. Similarly, because of the significant response seen with the targeted therapy of APML using all-trans-retinoic-acid and arsenic trioxide, patients with this subtype of AML are not offered matched sibling HCT in CR1 [73]. As previously discussed, the improved outcomes seen in children with AML who receive intensive chemotherapy alone have fueled the debate as to when to recommend a matched sibling allogeneic HCT in CR1 for the child with AML [27,49–51]. Controversy exists in determining whether children with favorable cytogenetics, defined as t(8;21) and inv16, should receive an HCT in CR1 [48]. Various international collaborative groups have shown that patients with inv16 and t(8;21) have a similar outcome with BMT compared with chemotherapy alone: a 5year OS for BMT of 68%, compared with 60–76% for chemotherapy [43,46]. Therefore, in current and future collaborative protocols for AML in the United States and in Europe, chemotherapy will be considered first-line therapy for patients with inv16 or t(8;21) in first remission, in an effort to spare the long-term side-effects of BMT. However, longterm follow-up of these patients will be important, especially for those patients who relapse and proceed to HCT in CR2.

Treatment of patients with AML beyond CR1 Management of the patient who fails conventional chemotherapy is complex after current first-line therapies for childhood AML. Approximately 40–50% of patients will relapse. If relapse occurs, especially for patients on therapy, the chance of cure with alternate chemotherapy remains poor. An HCT procedure with a matched sibling or an alternative donor offers the best chance for survival. The Children’s Cancer Group Study 2951 reported on using mitoxantrone and cytarabine induction for patients with relapsed or refractory

AML [74]. Patients with relapsed/refractory AML (n = 101) and secondary AML (n = 13) were entered. This induction regimen achieved a remission rate of 76% for relapsed/refractory patients and 77% for patients with secondary AML, with a 3% induction mortality rate. They concluded that this induction regimen was effective with reasonable toxicity in this cohort of patients. The French group in the LAME 89/91 trial has shown that, with reinduction therapy, CR2 was obtained in 71% (68 of 96) patients. The median duration of CR1 was 10 months. HCT was performed in 53 (78%) of these 68 patients. The DFS for children in CR2 was 45%. Multivariate analysis of re-treated children showed that the 5-year OS was higher if the CR1 had been longer than 12 months (54% versus 24%, respectively; p = 0.001). For patients attaining CR2, the 5-year DFS was not significantly different for matched sibling donor HCT (60%), autograft (47%) or alternative donor HCT (44%). They concluded that, after aggressive first-line therapy, one-third of children relapsing with AML could be cured [75]. Abrahansson et al. [76] reported on the outcome after relapse in children with AML for the Nordic society. Of the 122 patients who received reinduction therapy, 77% entered remission with 40 ± 5% survival. In early relapse (<1 year in CR1), survival was 21 ± 5%, compared with 48% ± 6% in late relapse. For children receiving reinduction therapy, survival in early relapse was 29 ± 6% and that in late relapse 51 ± 6%. Survival was 62 ± 6% in 64 children given HCT as part of their relapse therapy. The authors therefore concluded that children who received reinduction therapy, entered remission, and proceeded to HCT could achieve a DFS rate of 60%. Although there is no standard for assessing minimal residual disease (MRD) for childhood AML, improvements in MRD technology could help in making the decision to transplant early, i.e. before the standard definition of relapse (<5% blasts in the marrow) is met. Flow cytometry and DNA-based techniques such as fluorescence in situ hybridization and the polymerase chain reaction have been used to identify MRD in AML (see Chapter 26) [77–79]. For those patients who fail reinduction using standard chemotherapy, novel agents are being tested in children. Gemtuzumab, a humanized anti-CD33 monoclonal antibody conjugated to a modified cytotoxic agent, calicheamicin, has been effective in decreasing the leukemic cell burden and, in some cases, inducing a remission in patients with AML whose leukemic cells express CD33 [80]. Zwaan et al. [81] reported on 15 children with relapsed/refractory CD33+ AML who were treated with gemtuzumab ozogamicin (GO) monotherapy on a compassionate use basis. Eight children showed a reduction in bone marrow blasts to 5% or less. Arceci et al. reported on an open-label dose escalation study that evaluated the safety and efficacy of GO in children. Twenty-nine children ages 1–16 years of age received two doses ranging from 6 to 9 mg/m2. Toxicities included grade 3–4 hyperbilirubinemia (7%), elevated hepatic transaminases (21%), sepsis (24%), and grade 3–4 mucositis (3%). One patient treated at 9 mg/m2 developed SOS and defined the dose-limiting toxicity. Thirteen patients underwent HCT less than 3.5 months after the last dose of GO, and six patients (40%) developed SOS. Eight of 29 (28%) patients achieved overall remission, which was comparable in both patients with refractory (30%) and relapsed (26%) disease [82]. Current Children’s Oncology Group studies are using GO in combination with other chemotherapy agents in patients with de novo AML. The use of gemtuzumab in the post-transplant setting is associated with increased toxicity, specifically with SOS [83]. This agent may increase regimen-related toxicity in the pretransplant setting, but studies are still ongoing to collect these data. SOS has also been seen in the absence of HCT in these heavily treated patients [84].

Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia

Another agent that shows promise in relapsed/refractory disease is clofarabine, a novel nucleoside analog. Jeha et al. [85] reported on a phase I study of clofarabine in pediatrics with six dose levels. The study enrolled 17 patients with ALL and eight with AML. Among the eight with AML, one achieved CR and two had a partial remission, for an overall response rate of 37.5%. Multicenter phase II trials are ongoing for testing this agent in refractory disease. Approximately 15–20% of patients with AML have refractory disease and do not attain a remission after GO or other therapeutic options. HCT has been used successfully to cure approximately 10–20% of these refractory AML patients [86]. Factors predictive of outcome include blast count in the marrow, karyotype, the number of prior regimens, age, performance status, and availability of a donor. These prognostic factors should be considered prior to offering allogeneic transplant for primary refractory AML.

Alternative donor HCT for AML Our goal for this section is to make the reader aware of the treatment options available for the child who needs an alternative donor HCT for AML. The concepts of alternative donor transplants are reviewed in more detail in Chapters 46 and 47. Expanding the donor options if an HLA-matched sibling is unavailable has resulted in a significant advancement for the field of HCT. Alternative donor transplants for AML include (1) unrelated donor HCT utilizing bone marrow, peripheral blood or cord blood grafts, and (2) the use of partially matched or haploidentical related hematopoietic cell donors [54,87–92]. There have been no collaborative group prospective studies evaluating the efficacy of alternative donor transplants for AML in children. However, there are data from individual centers and the showing that alternative donor transplants for pediatric AML are successful (Fig. 54.6). Alternative donor transplants generally are associated with higher complication rates than matched sibling donor transplants due to the

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Fig. 54.6 Survival graph illustrating the outcomes of unrelated donor hematopoietic cell transplants (blood or marrow transplants) coordinated by the National Marrow Donor Program. Five-year survival for pediatric patients (<18 years of age) transplanted for acute myeloid leukemia (AML) is significantly improved for those patients transplanted in second complete remission compared with those transplanted in first complete remission or with advanced disease. However, a greater percent of patients transplanted in first complete remission had poor-risk cytogenetics compared with those patients who were transplanted in second complete remission (log-rank p-value < 0.0001). Unrelated transplant for AML in first complete remission is indicated if the patient has poor-risk cytogenetics at diagnosis or induction failure. (Data kindly provided by the NMDP.)

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greater risks of graft rejection and GVHD [93]. Recipients of alternative donor transplants have delayed immune reconstitution and are at increased long-term risk for infections because of the increased and prolonged immune suppression needed to prevent rejection and prevent and/or treat GVHD [94]. Consequently, alternative donor transplants are often reserved for patients who are considered to be at higher risk for, or have already failed, conventional therapy. For children with AML, this includes patients who (1) never enter a remission, (1) relapse after conventional therapy or autologous HCT, (3) develop AML after myelodysplasia, or (4) develop therapy-related AML. Additional factors may also be used to identify children with AML in CR1 whose outcome with intensive chemotherapy is likely to be inferior to the published outcomes for alternative donor transplants for children with AML [95]. For example, in the United Kingdom Medical Research Council 10 (MRC10) trial, children classified as poor risk (i.e. not entering CR or partial remission after one course of chemotherapy or with adverse cytogenetic abnormalities, such as monosomy of chromosomes 5 or 7, del(5q) or abnormalities of 3q, or a complex karyotype defined as more than four abnormalities) had a significantly inferior DFS of 32% at 7 years [29]. No child in this high-risk group who relapsed after having attained a remission and then relapsed survived [29]. The hypothesis that these children would fare better with an alternative donor transplant in CR1 deserves further study.

Bone marrow The role of T-cell depletion (TCD) to prevent GVHD in unrelated donor transplants has not been established [99]. There is no question that the incidence of severe (grade III–IV) GVHD is lower in the T-cell-depleted setting compared with the T-cell-replete group (5–10% versus 30–40%). However, whether this lower rate of GVHD will translate into a DFS advantage is still not clear. Data on the use of T-cell-replete HCT for pediatric AML comes from the Fred Hutchinson Cancer Research Center [96]. Of the 135 patients with AML in one report, 26 were under the age of 18 years. The primary conditioning regimen was CY/TBI. MTX/CSP was used for GVHD prevention. An important observation from the entire cohort is the relationships between OS and leukemic relapse based on the disease status of the patient (Fig. 54.7). Patients transplanted with higher-risk disease had an increased risk of failure due to relapse. The cohort of 16 patients receiving an alternative donor graft in CR1 suggests data to consider when evaluating the use of unrelated donor transplants for high-risk AML patients. All of these individuals had adverse features, such as delayed achievement of remission or high-risk cytogenetics. Their DFS was about 50%, which compares very favorably to that of high-risk patients who are treated with chemotherapy only (DFS approximately 30%) [29]. Data for T-cell-depleted HCT for AML come from the transplant group in Bristol, United Kingdom [92]. Of 39 AML patients treated, 33 were under the age of 19 years. Twenty-five of 39 patients were transplanted in CR2, with a median time of 3 months from relapse to transplantation. Fourteen individuals were transplanted in CR1 with major adverse prognostic features, such as failed remission induction (n = 5), antecedent MDS (n = 2), infantile leukemia with 11q23 abnormality (n = 22), and for other reasons. The Bristol approach primarily involved CY/TBI conditioning combined with Campath-1G in vivo and ex vivo for TCD together with CSP for GVHD prevention. The survival rate for this cohort is shown in Fig. 54.8. No patient in this study developed grade III or IV GVHD; however, there were five cases of chronic GVHD (four extensive, one limited). The total incidence of relapse was 13%. Infection-related complications were frequent and severe. There were 20 viral infections documented, with 12 of the 20 infections considered

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and, of these, 279 had AML. TCD, using various methods, was performed in 113 patients, while 166 patients received a T-replete transplant with MTX and CSP or FK506 (tacrolimus) for GVHD prophylaxis. While the survival at 5 years was better for the TCD group, it was not statistically different from that of the T-cell-replete group. Similarly, a prospective, randomized study comparing unrelated donor BMT using TCD plus CSP with T-replete grafts with MTX/CSP has shown equivalent DFS between the T-replete and T-deplete arms [100]. Of note, TCD was associated with less GVHD but no increase in relapse for patients with AML.

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Fig. 54.8 Probability of overall survival in 39 patients who received unrelated donor bone marrow transplantations for acute myeloid leukemia in remission using T-cell-depleted marrow. (Reproduced from [92], with permission.)

to be serious or life threatening. In the surviving cohort, the performance status is excellent. Similar to the Seattle report [97], the results for the group with high-risk features transplanted in CR1 seem to suggest that TCD may also be feasible for these individuals. Bunin et al. [98] reported on using partial T-cell depletion with unrelated donor grafts for children with leukemias from 1990 to 2001. The study reported on 76 unrelated donors and 28 partially matched related donors, 35 patients having AML. EFS for the entire cohort was 38.3%, and OS was 45.1% at 3 years. Relapse occurred in 22.8% of patients with AML/MDS. Grade III–IV GVHD was observed in only 6.7% of patients. The authors concluded that partial TCD allows the use of matched or mismatched unrelated donors or partially matched related donors with little mortality from GVHD, durable engraftment, and no increase in relapse risk. Analysis of data from the NMDP does not clearly define whether TCD results in superior outcomes compared with non-TCD strategies. Wagner et al. reported on 4938 transplants facilitated through the NMDP from 1987 to 1998 [99]. Children accounted for 1318 of the patients

An alternative source of hematopoietic cells for transplantation of children with AML is cryopreserved unrelated cord blood. Several studies have demonstrated that unrelated cord blood is a reasonable option for children lacking a suitably matched unrelated marrow donor (see Chapter 39) [101,102]. The Minnesota group has demonstrated that key variables in determining outcome for patients receiving unrelated cord blood transplants are CD34+ cell dose and HLA disparity [103]. Based on their data, an unrelated cord blood graft should have a CD34+ cell dose of at least 1.7 × 105 CD34+ cells/kg. There appears to be interplay between the cell dose and HLA disparity. For each degree of HLA disparity, there appears to be a critical infused cell dose below which survival is significantly impaired, particularly in recipients of unrelated cord blood with two or more HLA disparities. Hence, they argue, based on their data, that the choice of unrelated cord blood graft should be based primarily on CD34 cell dose and secondarily on degree of HLA disparity. In this dataset, there were 26 individuals with AML (one in CR1, 14 in CR2, one in > CR2, and 10 in relapse). The survival rate was two of four patients in the standard-risk group and 33% (95% CI 12–54%) for high-risk patients. A significant obstacle to survival in the AML cohort was relapse. Recently, Michel et al. reported the experience for EUROCORD and the use of unrelated cord blood for children with AML [104]. Ninety-five children were analyzed. The median age was 6 years, and the median weight was 21 kg. Twenty children were in CR1, 47 in CR2, five in CR3, and 23 not in remission. Forty-one had two or more HLA disparities. TRM was significantly lower for children who received a total nucleated cell dose of over 5.2 × 107/kg. The 2-year probability of

Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia

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leukemia-free survival was 42 ± 5% (59% ± 11% in CR1, 50% ± 8% in CR2, and 21% ± 9% for children not in CR). Notably, the results were particularly promising, even in CR2 patients who experienced an early relapse and for children with poor-risk cytogenetics. The authors concluded that unrelated cord blood transplantation is a therapeutic option with children who lack an HLA-matched sibling.

transplant approach potentially could take advantage of the unique role that alloreactive natural killer cells play in mediating the GVL effect. The data in adults with AML receiving megadose CD34+/low CD3+ haploidentical transplants are provocative, with a very low incidence of AML relapse for those patients transplanted in remission, perhaps because of alloreactive natural killer cells [108].

Haploidentical family donors

Relapse after HCT

Another potentially exciting modality for the treatment of children with AML is the use of a graft from a related haploidentical donor. Haploidentical transplants are attractive because most patients have a donor readily available and can proceed to transplant quickly. Godder and colleagues have reported data on 67 children treated for acute leukemia utilizing a partially matched family member [90]. The treatment plan included a partially TCD product with post-transplant immune suppression including antithymocyte globulin, CSP, and glucocorticoids. In this cohort, 24 of the 67 patients had AML; 75% of the AML patients had peripheral blasts. Donor–recipient pairs were mismatched in the GVHD direction for either three antigens (n = 10), two antigens (n = 8) or one antigen (n = 6). The estimated probability of survival at 3 years was 26%, and was similar for patients with ALL and AML. In the AML cohort, 39% of the transplants failed because of relapse. In particular, patients with circulating blasts had a very high risk of relapse. Neither GVHD nor rejection was a common cause of failure. In the multivariate statistical analysis, use of a younger donor (age <30 years) as well as absence of blasts at the time of HCT were found to be associated with better outcomes. This study demonstrated that HCT from a partially matched family member is feasible for children with AML. In contrast to the transplant protocol utilized by Godder et al., other centers are exploring the use of megadose CD34+/low CD3+ haploidentical transplantation as originally described by the group in Perugia, Italy [105]. Hematopoietic cells are collected by apheresis from donors who have been stimulated with G-CSF. The apheresis cells are positively selected on an immunomagnetic column, resulting in a relatively pure population of CD34+ cells. The number of T cells remaining is usually less than 50,000/kg. The theory underlying this strategy is that the megadose CD34+ infusion with low CD3+ cells permits engraftment across haplotype barriers without the development of GVHD, even though no post-transplant immune suppression is used. The Tübingen transplant group in Germany has published the largest pediatric series to date using this approach [91]. They reported on 63 pediatric patients (13 patients had AML, CML or juvenile myelomonocytic leukemia). The entire cohort had a 7% incidence of grade II acute GVHD with no grade III–IV seen. There was a 13% incidence of chronic GVHD. The 3-year DFS for AML/chronic myeloid leukemia (CML) was 18%. Marks et al. [106] reported on 34 pediatric patients with acute leukemia who underwent haploidentical transplantation. Seventeen patients had AML (13 being considered high risk). The actuarial survival at 2 years was 26% (95% CI 13–41%) for the entire cohort. All of the patients with AML who were not in remission at the time of transplant died. The overall low risk of GVHD observed to date is encouraging. However, the major barriers to survival have been relapse and infection. Clearly further advances in this field are needed to improve the GVL effect and more rapid immune reconstitution. Recently, the Tübingen group has published preliminary work on using CD3/CD19 depletion, which may improve engraftment and immune reconstitution after haploidentical transplantation. Their work is promising in high-risk patients lacking a suitable donor, and prospective phase I/II studies are ongoing [107]. For patients with AML, this

Despite the intensity of conditioning and the GVL effect, relapse of AML after either matched sibling or alternative donor HCT is not uncommon and often leads to death. The propensity for AML to recur despite intense conditioning points to the need for innovative therapies for the child who relapses after HCT. Second high-dose transplant procedure One option is a second high-dose HCT procedure. If the original donor was a matched sibling, the data suggest that a second transplant may result in salvage of about one-third of patients [109]. A study from the Société Francaise de Greffe de Moelle evaluated the use of second transplants in 150 individuals with leukemia (61 with AML), combining both adult and pediatric patients [110]. Although the recipients of grafts from unrelated donors were included in this study, the vast majority of the patients received grafts from matched sibling donors. For the pediatric cohort, univariate statistical analysis showed the 2-year post-second transplant OS to be 50 ± 18%, with a DFS of 47 ± 16% and TRM at 20 ± 18%. In the multivariate analysis, the factors that were associated with better prognosis were age less than 16 years and disease recurrence more than 1 year after the original transplant. Therefore, children whose disease recurs more than a year after the initial transplant should be considered for a second transplant procedure, especially if the original donor is available. Meshinchi et al. [111] reported outcomes for 25 pediatric patients who underwent a second allo-HCT (12 HLA-matched related, nine HLA mismatched related, and four HLA minor mismatched unrelated donor) for recurrent AML after a prior auto-HCT (n = 11) or allo-HCT (n = 14). Median time from first HCT to relapse was 6.2 months, and median time between first and second HCT 9.6 months. OS at 10 years after the second transplant was 48%, DFS was 44%, and TRM at day 100 was 12%. DFS for patients who underwent a second transplant in remission was higher than that for those who underwent transplantation while in relapse (70% versus 27%, p = 0.05). These data suggest that second HCT after a failed initial transplant results in long-term DFS in one-half of children with relapsed AML. A higher tumor burden at the time of second transplant was associated with a higher risk of subsequent relapse, and patients may benefit from reinduction therapy before second HCT. Adoptive immunotherapy In addition to second high-dose transplant regimens, adoptive immunotherapy, with or without chemotherapy, has been used to treat or prevent post-transplant relapse (see Chapter 72) [112–114]. Following the success seen with DLI for CML, there are case reports and retrospective reviews describing DLI for AML in adults and children [115,116]. These series vary as to whether chemotherapy and/or biologic response modifiers, such as interferon and/or interleukin-2, were used as an adjunct to DLI therapy. Occasionally, patients attain durable responses, while others either relapse with their diseases and/or develop GVHD. Kolb and the European Group for Blood and Marrow Transplantation (EBMT)

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have reported the largest number of AML patients treated with DLI at multiple institutions [115]. Five of 21 evaluable AML patients had achieved prolonged leukemia-free survival. Following the success of MRD-directed DLI for CML [117], it was hypothesized that improved identification of MRD also would lead to better responses after DLI for AML. Bader et al. [114] have utilized increasing levels of host chimerism as an indication for withdrawing CSP and/or using low-dose DLI for children with acute leukemia or MDS. Although the number of observations is small (12 children; three with AML and four with MDS), this pediatric study suggests that prophylactic immunotherapy may play a role in children with acute leukemia who exhibit increasing host chimerism. One prospective study evaluated the role of chemotherapy cytoreduction coupled with DLI to treat myeloid malignancies in patients who relapsed after HCT [112]. This multi-institutional study had 50 AML patients within a total cohort of 65 individuals. The age range was 2–59 years. The study utilized chemotherapy followed by G-CSF-mobilized DLI with a goal of delivering 1 × 108 CD3+ cells/kg along with CD34+ cells. The study objective was to transfuse DLI to confer a GVL effect as well as CD34+ cells to minimize aplasia after chemotherapy. For the entire cohort, the 2-year survival was 19% (95% CI 1–33%). Patients whose disease relapsed beyond 6 months from the initial transplant had a statistically higher probability of survival compared with those whose relapse occurred within 6 months of BMT (Fig. 54.9 [112]). The major barriers to survival in this study were early failures due to the ineffective induction of remission with the debulking doses of chemotherapy as well as a 23% incidence of TRM. This study provides evidence that reducedintensity conditioning with DLI can be efficacious for patients who relapse beyond 6 months post HCT. Porter et al. [118] investigated ex vivo activation of the DLI in an effort to augment the GVL effect. They hypothesized that failure of DLI to mediate an antitumor effect in most diseases was caused by inappropriate activation of the donor T cells in vivo to induce an antitumor response. They activated donor T cells by co-stimulation and expansion after exposure to magnetic beads coated with CD3 and anti-CD28. Eighteen patients participated in a phase I dose-escalation trial. Patients who had aggressive malignancies received induction chemotherapy, and all patients received conventional DLI followed 12 days later by activated DLI. Eight patients achieved CR, including two of four patients who had AML. Overall, 10 of 18 patients remain alive 11–53 months after receiving activated DLI. The authors concluded that adoptive trans-

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Fig. 54.9 Overall survival rates. —— indicates relapse after bone marrow transplantation (BMT) within 6 months; --- indicates relapse after BMT after 6 months. DLI, donor leukocyte infusion. (Reproduced from [112], with permission.)

fer with co-stimulated activated allogeneic T cells is feasible, does not result in excessive GVHD, and may contribute to durable remissions in diseases where conventional DLI has been disappointing. A recently described phenomenon that raises concerns about the efficacy of adoptive immunotherapy for AML is the occurrence of extramedullary relapse after HCT and DLI [119]. Choi et al. [120] tested the effects of cytoreductive chemotherapy, followed immediately by GCSF-primed DLI for 16 AML patients who relapsed after allogeneic HCT. Ten of these patients achieved CR, four of whom remain alive at a median follow-up of 1488 days after DLI. The 2-year OS was 31%. All five patients who relapsed after achieving CR with chemotherapy and DLI relapsed at extramedullary sites in the presence of continuous bone marrow remission. The pathophysiology of these observations remains unclear, but relapses in extramedullary sites, while the bone marrow is initially unaffected, suggest that the GVL effect may be compartmentalized.

Late effects associated with HCT When assessing the risks and benefits of different treatment plans, the long-term risks of each treatment option are of obvious relevance. Increasing numbers of children are surviving AML, and the long-term effects of treatment are becoming better appreciated. The late complications associated with HCT are reviewed in more detail specifically in Chapters 104, 105, and 106. The choice between transplant and chemotherapy for the child with AML who has a matched sibling is controversial, in part because of concerns that transplantation is associated with more profound late sequelae. Investigators at the Children’s Hospital of Philadelphia (CHOP) have examined the question of late effects for 52 survivors treated for AML, comparing chemotherapy regimens (n = 26) with transplants (n = 26). Most of the HCT recipients (17 of 26) had not received TBI [121]. The researchers found that the two treatment modalities had similar effects on growth and renal and cardiac function. Their data suggest an increased risk of infertility in the HCT group. Liesner and colleagues likewise compared chemotherapy and transplant cohorts and found that the risk of infertility was higher in the transplant group [122]. In contrast to the study from CHOP, Liesner et al. observed more growth retardation in the transplant cohort as compared with the chemotherapy group. Because of the similar antileukemia efficacy in AML of BU/CY 200 with TBI-based regimens [30], many centers utilize a non-TBI-based regimen. In a cohort of 45 children with AML comparing late effects using BU/CY versus CY/TBI, Michel et al. [123] have reported that the TBI group had more frequent decreases in height standard deviation scores, hypothyroidism, and cataracts. A concern for pediatricians related to TBI has been the worry that TBI will have a negative effect on cognitive functioning. This concern has been the focus of research done at the Medical College of Wisconsin [124]. Kupst and colleagues have prospectively evaluated the cognitive and psychosocial functioning of 153 children and adolescents [124]. Patients were evaluated pretransplant as well as at 1 and 2 years post HCT. One hundred and forty-two patients had received TBI. The data demonstrated that IQ tests, as a measure of cognitive function, remained stable over time and were not adversely affected by TBI. The strongest predictor of cognitive outcome was pre-HCT functioning. The range of the late effects reported in the literature points to the need for comprehensive follow-up for the survivors of AML, regardless of the treatment received. Children need to be monitored consistently for growth and sexual development, thyroid dysfunction, cataracts, second malignancies, and neuropsychological late effects. Close monitoring will then facilitate appropriate treatment.

Hematopoietic Cell Transplantation for Childhood Acute Myeloid Leukemia

Conclusion According to the Surveillance, Epidemiology and End Results (SEER) database, survival for children with AML has improved from 18.8%, from 1975 to 1977, to 53.1%, from 1996 to 2002 [2]. We now have even more successful chemotherapy protocols, several different donor sources for HCT, and adoptive cellular therapies, as well as targeted monoclonal antibody and molecular therapies. As more children survive their primary disease, we need to pay more attention to long-term sideeffects and second malignancies. Nevertheless, the current 53.1% survival rate clearly indicates that the cure of childhood AML is a work in progress. The data reviewed in this chapter provide a basis for decision making when the physician evaluates a child with AML. The algorithm in Fig. 54.3 should be used as a framework and not a rigid plan. The results with chemotherapy have improved, especially for patients with low-risk features. However, there remains an ongoing controversy regarding the interpretation of the data concerning HCT for AML in CR1. Due to the excellent results in children with AML employing chemotherapy alone, current experimental trials advocate HCT with a matched sibling in CR1 only for those patients with intermediate- to high-risk features of AML [125]. However in the absence of a clinical trial, the ASBMT consensus statement recommends a matched sibling HCT in CR1 when one is available. It is crucial that we continue to stratify patients based on the same risk parameters worldwide so that comparisons between treatment strategies are not biased. The advent of microarray technology along with the escalating information with regard to genetic markers should allow investigators to more precisely define AML based on biologic parameters. This information should at least lead to standardization and agreement as to risk assignment. If results with chemotherapy improve, or if TRM decreases, the algorithm in Fig. 54.3 could change. The argument has been made that patients who fail on the current chemotherapy protocols could proceed to HCT in early relapse or CR2. However, as conventional chemotherapy protocols for AML may have reached their toxicity limit, patients who fail chemotherapy may not be responsive to high-dose conditioning and HCT [126]. Strategies designed to employ molecular techniques to identify patients who continue to have leukemic cells early (i.e. before they develop a clinical relapse) are well underway. This concept could potentially allow for transplantation before the patient is refractory and in poor condition secondary to intense but ineffective treatment. Unfortunately, the molecular profile of AML blasts at diagnosis may not be the same at relapse [127]. The assumption is that patients whose AML blasts express FLT3/ITD at diagnosis will do so at relapse. However, recent reports demonstrate that this marker is not always reliable, as a subset of patients did not express FLT3/ITD at relapse [127]. Nevertheless, for those patients who can accurately be diagnosed with a molecular

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relapse or residual disease, moving quickly to HCT might be a potential strategy. It is important to ascertain at the time of AML diagnosis whether the patient has an HLA-matched sibling. If a matched sibling is available, and the patient has intermediate-risk or high-risk AML, as discussed earlier, we would recommend proceeding to transplant in CR1. Conversely, children with Down’s syndrome or APML should not be transplanted in CR1, as the current treatment results for those patients are excellent. Ideally, patients with an HLA-matched sibling and low-risk (inv16; t8;21) AML on current clinical trials would receive chemotherapy alone. In the absence of a randomized clinical trial, discussions with the patient and family regarding relative risks and benefits (as framed by the expertise of the physician) will need to guide the decision. If a child does not have a matched sibling, then the usual treatment plan includes primary intensive conventional chemotherapy. Alternative donor HCT may be considered in CR1 for high-risk cases or when the patient relapses during or soon after conventional therapy. The source of the alternative donor hematopoietic cells will depend on a number of factors, which include the underlying diagnosis and stage of disease, the degree of mismatch for the various potential hematopoietic cell sources, and the cell dose available based on the size of the patient.

Future directions It is clear that, especially in the pediatric population, hematopoietic cells from an unrelated source can be lifesaving. In fact, many institutions have reported no significant difference between outcomes for a wellmatched unrelated donor and an HLA-matched sibling [54,87,128]. The challenge remains how best to manipulate the hematopoietic cell inoculum, conditioning regimens, and GVHD strategies to ensure a successful outcome. For the proponents of unrelated cord blood HCT, the major challenges are securing an adequate cell dose in order to ensure engraftment and shorten the period of pancytopenia. Investigators are currently evaluating the ex vivo expansion of cells [129], the addition of mesenchymal cells [130], and the transplantation of multiple cord blood units [131]. The major challenges facing unrelated donor HCT are GVHD in the setting of a T-cell-replete graft, and infection and relapse in a T-celldepleted strategy. When the donor and recipient are HLA mismatched, these problems are magnified. Studies on the generation of cytotoxic T lymphocytes targeted to specific viruses [132] or to minor histocompatibility antigens [133] and leukemia antigens [134] are well underway. In the future, HCT is likely to advance toward a selective component therapy in order to become a less toxic and more effective treatment for a broader range of patients [135]. Because tumor cells use multiple mechanisms of immune evasion, a combination of several approaches, rather than a single treatment, will be necessary.

References 1. Linet MS, Ries LA, Smith MA, Tarone RE, Devesa SS. Cancer surveillance series: recent trends in childhood cancer incidence and mortality in the United States. J Natl Cancer Inst 1999; 91: 1051–8. 2. Ries LA, Melbert D, Krapcho M. SEER Cancer Statistics Review, 1975–2004. National Cancer Institute: Bethesda; 2007. 3. Bernasconi P, Boni M, Cavigliano PM et al. Molecular genetics of acute myeloid leukemia. Ann N Y Acad Sci 2002; 963: 297–305.

4. Kwong YL, Ng MH, Ma SK. Familial acute myeloid leukemia with monosomy 7: late onset and involvement of a multipotential progenitor cell. Cancer Genet Cytogenet 2000; 116: 170–3. 5. Kojima S, Ohara A, Tsuchida A et al. Risk factors for evolution of acquired aplastic anemia into myelodysplastic syndrome and acute myeloid leukemia after immunosuppressive therapy in children. Blood 2002; 100: 786–90. 6. National Cancer Institute. Childhood Acute Myeloid Leukemia/Other Malignancies PDQ

Treatment. Health Professionals Version. Bethesda, MD: National Cancer Institute; 2002. 7. Wells RJ, Arthur DC, Srivastava A et al. Prognostic variables in newly diagnosed children and adolescents with acute myeloid leukemia: Children’s Cancer Group Study 213. Leukemia 2002; 16: 601–7. 8. Chang M, Raimondi SC, Ravindranath Y et al. Prognostic factors in children and adolescents with acute myeloid leukemia (excluding children with Down’s syndrome and acute promylocytic

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55

Stephen J. Forman

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults

Introduction Acute lymphoblastic leukemia (ALL) is a hematologic malignancy of the bone marrow characterized by the rapid proliferation and subsequent accumulation of immature lymphocytes. ALL accounts for 20% of all acute leukemias that are seen in adults over the age of 20 years, and affects approximately two persons per 100,000 in the United States annually. Over the past two decades, there has been substantial improvement in the understanding of the molecular biology of the disease and in the management of adult patients who have this disorder. The success of therapy in children with ALL has continued to fuel the quest for similar success in adults utilizing the same principles that guide therapy in children, namely intensive induction and consolidation, maintenance therapy, and prevention of disease in extramedullary sites such as the central nervous system (CNS). Laboratory and clinical studies have helped refine the decisions concerning the indication and timing of allogeneic hematopoietic cell transplantation (HCT). This chapter reviews the biology of adult ALL, the relationship of specific disease characteristics to the natural history of the disease, and the role of allogeneic and autologous HCT in the management of adult patients with this disease.

predominantly AML, ALL can also occur after exposure to alkylating agents, epipodophyllotoxins, and other topoisomerase-2 inhibitors [4].

Signs and symptoms of disease Patients with ALL generally present with signs and symptoms of uncontrolled growth of leukemic cells in the bone marrow, lymphoid organs, and other sites of extramedullary disease. The resulting bone marrow involvement results in varying degrees of anemia, thrombocytopenia, and granulocytopenia with manifestations of pallor, fatigue, petechiae, bleeding, and fever. Liver, spleen, and lymph node enlargement is common. ALL frequently involves organs other than the bone marrow, and this spread is an important consideration in the treatment strategy. Many patients have extramedullary disease at diagnosis, and extramedullary relapse is a known complication. The most common sites of extramedullary disease include the CNS, testes, lymph nodes, liver, spleen, and kidney, with the CNS and testes having the most clinical significance. The occurrence of extramedullary relapse after achievement of remission frequently heralds the development of a bone marrow relapse and requires systemic chemotherapy in addition to local treatment such as radiation (testes) or intrathecal treatment (CNS).

Etiology

Morphology

The cause of ALL in adults remains unknown, and the associations are less distinct than they are with acute myeloid leukemia (AML) and are more closely identified with patients with pediatric leukemia. For instance, in children with Down’s syndrome, there is an 18-fold higher incidence of childhood ALL than in the general population [1]. In addition, there is an increased risk of ALL associated with inherited disorders such as Klinefelter’s syndrome, Fanconi’s anemia, Bloom’s syndrome, ataxia telangiectasia, and neurofibromatosis [2]. The incidence of acute leukemia, mainly AML but also ALL, was increased almost 20-fold in survivors of atomic bomb explosions who were exposed to more than 1 Gy of radiation. Although there was concern whether radiation exposure at Chernobyl led to an increased incidence of leukemia, this has not yet been shown to be the case [3]. The risk of development of ALL may also be increased after exposure to chemical agents such as benzene and secondary disease; although

Similar to the molecular and morphologic diversity of AML, ALL comprises a heterogeneous group of disorders based on morphologic, immunologic, and cytogenetic characteristics. Many of these have emerged as important prognostic indicators, and thus are relevant to decision making about the timing of allogeneic HCT. Classically, the French–American–British morphologic classification has defined three types of leukemic blasts: L1, L2, and L3. This classification is based on the spectrum of microscopic appearance of the cells. In contrast to childhood ALL, where 85% of the patients have L1 morphology, adult patients have more commonly L2 features. With the exception of distinguishing the L3 morphology and its associated cytogenetic and clinical behavior, as well as treatment differences, morphology has not been useful in guiding the treatment of adult patients with ALL.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Clonal origin of leukemic lymphoid cells Human ALL arises from a single progenitor cell that has undergone genetic damage leading to the disregulated growth and arrested differentiation. There is considerable evidence that each leukemic cell has descended from a single transformed progenitor, as cytogenetic studies

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show common numeric and structural chromosome abnormalities within discrete leukemic cell populations. Uniform rearrangements of immunoglobulin or T-cell-receptor (TCR) genes, as compared with a more diverse pattern of rearrangements observed in populations of normal T and B lymphocytes, is further evidence of the clonal development [5,6]. Additional evidence of clonality comes from studies of X chromosomelinked genes that are inactivated during embryogenesis and can be used to demonstrate the unicellular development of a leukemic cell population by detecting a single type of glucose-6-phosphate dehydrogenase in female patients [7]. In addition, the methylation patterns of restriction fragment length polymorphisms in X-linked genes detected by Southern blot analysis can also be used to show the clonal evolution from a single transformed progenitor [8].

defined by the expression of CD44 and CD25. After this time, the TCR gene becomes rearranged and proceeds through a phenotype that is double negative to one that is double positive. This process is followed by the gain of CD3 expression and then the separation into mature CD4 and CD8 single positive cells. There have been numerous studies that have confirmed that the antibodies utilized to define T-cell surface glycoproteins demonstrated a close relationship between the recognizable patterns of surface antigen expression on leukemic T cells and the normal stages of thymocyte development. In contrast to pre-B ALL, the clinical features of T-cell ALL include high blood leukocyte counts, a predominance in males, a higher incidence of CNS involvement, and radiographic evidence of a thymic mass in approximately half the patients.

Lineage-specific features of leukemic lymphoblasts

Mixed lineage leukemia

As noted above, malignant lymphoblasts share many features of normal lymphoid progenitor development with rearrangements of the immunoglobulin or TCR genes. In many cases, the leukemic cells appear to represent a clonal expansion of a lymphoid progenitor cell that is blocked at an early stage of either B- or T-cell differentiation [9]. However, studies suggest that leukemic lymphoblasts show an asynchronous gene expression with subtle variations in phenotype leading to aberrant regulation of gene expression and an altered phenotype compared with normal lymphocyte progenitors [9]. Nevertheless, it is an accepted concept that the leukemic cells can be classified according to their normal developmental stage.

Acute mixed lineage leukemia is defined by blast cells that coexpress markers of both lymphoid and myeloid origin [11]. Two distinct forms of these leukemias are recognized. The first is that of lymphoid morphology with coexpression of myeloid-associated antigens. The other has myeloid morphology and immunocytochemistry evidence of myeloperoxidase staining, with coexpression of cell surface antigens that are normally restricted to lymphoid cells. The origin of this type of leukemia is unknown, and it is not clear whether this represents aberrant gene expression due to specific gene alterations. Alternatively, it may be related to transformation at a time when the pluripotent cell retained the ability to differentiate into both myeloid and lymphoid cells, or to the immortalization of a rare cell that normally coexpresses both of these features. The literature is unclear about whether cases of lymphoid leukemia that express one or more myeloid surface antigens have a poor prognosis. Studies focused mostly on children suggest that myeloid expression in ALL was not an adverse prognostic feature [12]. However, the expression of T-lymphoid markers such as cytoplasmic CD3 or cell surface CD2 and CD7 in cells that are predominantly myeloid do identify patients who are less likely to achieve a complete remission (CR) with standard AML therapy than the induction therapy that would be used for ALL [13].

B-cell ALL The diagnosis of B-cell leukemia depends on the detection of surface immunoglobulin on leukemic blasts. This rare phenotype, however, accounts for only 2–3% of ALL cells and has the distinct morphology as noted above (L3). It is important to accurately diagnose this form of ALL because most investigators believe that acute B-cell leukemia is a disseminated form of Burkitt’s lymphoma, sharing cytogenetic, molecular genetic, immunologic, cytologic, and clinical features. For this type of ALL, responses and outcomes are better when treatment regimens are based on the treatment of Burkitt’s lymphoma and include rituximab, cyclophosphamide (CY), and rapid rotation of antimetabolite drugs in high doses [10]. Pre-B and early pre-B ALL Approximately 80% of ALL patients have a lymphoblastic phenotype corresponding to that of B-cell progenitors. These cases are identified on the basis of cell surface expression of CD19 and at least one other recognized B-lineage-associated antigen, which includes CD20, CD24, CD22, CD21, and CD79. Most of these leukemias, in addition, express the B-lineage ALL antigen CD10, that is, the common ALL antigen, as well as nuclear terminal deoxynucleotidyl transferase or CD34. Approximately a quarter of patients express cytoplasmic immunoglobulin μ heavy chain proteins, and are designated pre-B-cell ALL. T-cell ALL Leukemias of T-cell precursors are identified according to the sequence of expression of T-cell-associated surface antigens during normal thymic ontogeny. The earliest T-cell precursors are characterized by the lack of expression of CD4 and CD8 surface markers, but these double-negative thymocytes express CD7, terminal deoxynucleotidyl transferase, and cytoplasmic CD3, and proceed through a well-defined development,

Cytogenetic and molecular genetic analyses In ALL, the multiple acquired genetic abnormalities are likely related to the malignant transformation and distorted cell growth and differentiation seen in ALL. These can include structural abnormalities detected by standard chromosomal analysis and molecular analysis of the DNA. Chromosomal translocations are found in many cases of ALL and are broadly classified as being recurring lineage-restricted abnormalities, accounting for two-thirds of the translocations, or random translocations or abnormalities of chromosome number. Figure 55.1 shows the frequency and distribution of major chromosomal abnormalities in patients with adult ALL [14]. In addition, the genes affected by chromosomal translocations are shown in the bold type. Cytogenetic abnormalities represent the strongest independent prognostic variables for predicting the outcome of treatment. In several multicenter studies, clonal chromosomal abnormalities could be detected in approximately 62–85% of patients, with the major abnormalities being clonal translocations (9;22, 4;11, 8;14, 1;19, and 10;14), as well as other structural abnormalities, including 9p, 6q, and 12p [15–17]. When there are no structural abnormalities present, the abnormalities can be classified according to the modal number of chromosomes. Molecular analyses are used to detect gene rearrangements in ALL by polymerase chain reaction (PCR), Southern blot analysis or fluorescence in situ hybridization utilizing chromosome-specific probes. The most

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults

frequent molecular markers in ALL are BCR–ABL and ALL1–AF4 [18]. The Philadelphia chromosome (Ph) results from a translocation involving the breakpoint cluster region of the BCR gene on chromosome 22 and the ABL gene on chromosome 9, and can be demonstrated by molecular techniques. It occurs in about 25% of patients with adult ALL and increases with age. Approximately one-third of adult ALL patients with Ph+ ALL showed a BCR rearrangement, resulting in a 210 kD protein, similar to patients with chronic myelogenous leukemia (CML). Two-thirds, however, have an arrangement resulting in a 190 kD protein. The most frequent form of 11q23 abnormality in ALL is the t4;11 translocation. One partner gene is located on chromosome 11 and is named

BCR-ABL t(9;22)

Hyperdiploid >50 chromosomes 6% 25%

TCR translocations 7q35/TCRb 14q11/TCRad

2%

41%

6% Hypodiploid <45 chromosomes

4%

4% MYC 3% IG translocations t(8;14), t(2;8), t(8;22) E2A-PBX1 t(1;19)

No recurrent chromosomal abnormalities

7%

MLL fusions t(4;11) t(1;11) t(11;19)

2% TEL-AMLI t(12;21)

Fig. 55.1 Frequency and distribution of cytogenetic abnormalities in adult patients with acute lymphoblastic leukemia. The genes affected by these translocations are shown in bold type. (Reproduced from [14], with permission.)

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MLL for mixed lineage leukemia. This gene is fused to a gene located on chromosome 4 that is named AF-4 and is frequently detected in infant leukemia and in patients with early pre-B subtype (CD10−) cells. The overall incidence of this chromosomal translocation in adults is 5%. Table 55.1 summarizes molecular aberrations in ALL with associated immunophenotypic, cytogenetic translocations, and clinical features seen in adults.

Treatment of ALL in the adult The treatment of ALL begins with a proper diagnosis and immunologic and cytogenetic characterization of the disease. Bone marrow aspiration and biopsy, as well as lumbar puncture, are required for all patients. These are used for evaluating prognostic features, disease monitoring, and, in some circumstances, determining specifics of therapy, including the timing and indication for HCT. For patients with B-cell ALL, combination chemotherapy with rituximab has resulted in a high rate of remission and cure of the disease [10]. For patients with Ph− pre-B ALL and T-cell ALL, chemotherapy is usually divided into several phases, beginning with remission induction, consolidation therapy, CNS prophylaxis, and maintenance treatment [14,19]. Standard induction for therapy for pre-B ALL and T-cell ALL includes prednisone, vincristine, anthracyclines, mostly daunorubicin, but also L-asparaginase. Other drugs such as CY and cytarabine (ARA-C) are added in many protocols. Prednisone and prednisolone have been the most frequently administered, although dexamethasone has a higher anti-leukemic activity in vitro and possibly better penetration into the CNS. However, it may predispose patients to an increased risk of infection compared with prednisone. Although anthracycline dose intensity and schedule may play a role, many trials include a dose intensification with a 2–3-day schedule at the onset. CY is generally administered at the beginning of induction therapy, but a randomized trial by the Italian GIMEMA group comparing a three-drug induction with and without CY did not show a significant difference in overall survival [20]. Between 15% and 20% of adult patients do not achieve a CR after induction therapy, compared with less than 3% of patients with childhood ALL. Although less than 10% of adult patients die during

Table 55.1 Characteristics of immunologic subtypes of acute lymphoblastic leukemia (ALL) Subgroup (incidence) (synonyms)

Disease characteristics

Cytogenetic/molecular marker

Specific poor prognostic factors

Pro-B ALL (11%) (CD10− ALL)

High white cell count (>100,000/μL in 70%) CD13/CD33 coexpression (>50%) Incidence increasing with age (75% if >55 years) Partly CD20+ (45%) Large tumor mass (lactate dehydrogenase increased in >90%) Organ involvement (32%) CNS involvement (13%) CD20+ (>80%) Mediastinal tumor (60%) CNS involvement (8%) High white cell count (>50,000/μL) (46%) Subtypes: Early T (6%) Thymic T (12%) Mature T (6%)

70% t(4;11)/ALL1-AF4 (20% Flt3 in ALL1-AF4+) 4% t(1;19)/PBX-E2A (pre-B only)

High risk

Common-ALL (49%) Pre-B ALL (12%) Mature B ALL (4%) (L3-ALL, Burkitt’s leukemia)

T ALL (25%)

Adapted with permission from Gökbuget and Hoelzer [19].

White cell count >30,000–50,000/μL t(9;22)/BCR-ABL t(1;19)/PBX-E2A

t(8;14)/c-myc-IgH

20% t(10;14)/HOX11-TCR <20% t(11;14)/LMO/TCR 8%SIE-TAL1 4% NUP213-ABL1 33% HOX11 5% HOX11L2 50% Notch1

Early, mature T ALL White cell count >100,000/μL HOX11L2

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Chapter 55

induction, the mortality is related to age, with the major cause of death being infection. Patients who fail to achieve a remission, analogous to those patients who are slow to achieve a remission, have a very poor prognosis and are candidates for early HCT [19]. Several studies have demonstrated that allogeneic HCT performed after failure to achieve a CR can be curative in approximately 20% of patients [21,22]. Based on the intensity of the induction treatments now being utilized in adult treatment regimens, failure to achieve a remission is an indication for early HCT, rather than utilizing repetitive cycles of therapy that is not likely to be effective. Thus, for adult patients, human leukocyte antigen (HLA) typing of the patient and family members is an important test to perform early after diagnosis. CNS prophylaxis CNS leukemia occurs in about 6% of patients, with a higher incidence in T-cell ALL (8%) and mature B-cell ALL (13%). Treatment and prophylaxis of CNS leukemia usually consists of intrathecal methotrexate (MTX) alone or in combination with ARA-C or prednisone. Adult ALL patients who do not receive specific CNS treatment have a CNS relapse rate of 30%, similar to that observed in children. Thus, all patients should receive some form of CNS therapy. In most adult ALL trials, prophylaxis has included CNS radiation to 24 Gy, which reduces the CNS relapse rate to about 9%. It is not clear whether systemic high-dose treatment alone prevents CNS prophylaxis, but it may be possible to avoid radiation by combining early high-dose chemotherapy and intrathecal therapy. The risk of CNS relapse is associated with other prognostic factors, including T-cell ALL, B-cell ALL, extreme leukocytosis, elevated lactate dehydrogenase, and extramedullary organ involvement. Management of CNS relapse prior to HCT In general, the development of a CNS relapse in patients with ALL portends a poor prognosis, both for the effect it has on the CNS and because it is a harbinger of systemic relapse. Most patients with ALL have had some form of CNS prophylaxis with radiation or chemotherapy prior to their first systemic relapse. Although children with an isolated CNS relapse can still do well with the treatment of CNS and systemic reinduction therapy, adults with an isolated CNS relapse fare poorly, and such an event is an indication for HCT [23]. Patients who relapse in the CNS require additional therapy to control the disease prior to HCT. Previous prophylactic intrathecal therapy and CNS radiation does not necessarily preclude the use of total body irradiation (TBI) in the preparation for HCT. However, if a patient has had CNS irradiation and relapses, a second course of irradiation prior to a TBI-containing preparative regimen is not recommended as this may lead to more neurotoxicity following transplantation. In general, these patients can be managed with either intrathecal MTX or the combination of MTX, ARA-C, and hydrocortisone until there is clearing of leukemic cells from the spinal fluid. Following HCT, such patients should receive five intrathecal MTX injections during the first 100 days, followed by one monthly injection for 12–18 months or until toxicity precludes additional treatments. This approach, designed in Seattle, can result in control of the leukemia without substantially increasing the risk of leukoencephalopathy [24]. The risk of CNS damage caused by cranial radiation, intrathecal chemotherapy, and high-dose chemotherapy has been documented, and is related to the amount of intrathecal chemotherapy following the HCT procedure [24]. Currently, a common approach for patients with ALL who will be undergoing HCT, without any evidence of CNS disease, is to administer intrathecal MTX as prophylaxis for a total of five times prior to the HCT procedure. Cranial radiation is not necessary if the

patient then receives a preparatory regimen containing fractionated TBI. Maintenance therapy The optimal duration and form of maintenance therapy in adult ALL is unknown. The goal of maintenance treatment is to eliminate minimal residual disease (MRD) that may have persisted after induction and consolidation treatment. Standard maintenance is usually based on a combination of 6-mercaptopurine and MTX. Attempts in previous trials to omit maintenance after induction and consolidation therapy resulted in inferior results. In general, the cure rate with chemotherapy for adults with ALL is 35–40%. These results may be better in adolescents and younger adults utilizing more pediatric chemotherapy-based regimens, and are poorer in older adults, especially over the age of 60 [25,26]. Treatment of Ph+ ALL As noted above, translocation 9;22 is the most frequent genetic alteration in adult ALL, found in 20–30% of patients, and the incidence increases with age. The phenotype of this leukemia is almost exclusively CD10+ precursor B ALL, and there are only rare reports of its presence in Tlineage ALL. In this circumstance, the disease is likely related to the blast crisis of CML. Clinically, patients present with a variable white blood cell count, surface expression of CD19, CD10, and CD34, as well as the coexpression of myeloid markers, especially CD13 and CD33. The principles of initial diagnosis and treatment are similar to those of non-Ph+ ALL. Cytogenetic and molecular genetic analyses are required to establish the diagnosis of Ph+ ALL and can be obtained within a week of diagnosis. These studies are important because of the evolving treatment of the disease with targeted therapy. Historically, the prognosis of adult patients with Ph+ ALL treated only with chemotherapy has been poor, with less than a 10% probability of long-term disease-free survival (DFS). CR rates after induction therapy range from 60% to 90%; however, the median remission duration was inferior, between 9 and 16 months, with almost no long-term survivors. As discussed below, allogeneic HCT has been the treatment of choice for such patients, with many series showing better than 50% long-term survival in patients who undergo HCT in first remission. The disease has been even more difficult to treat in older patients with ALL where this cytogenetic abnormality actually is more common. The understanding of the leukemogenic role of the BCR–ABL oncogene and its dependency on the constitutive activation of the ABL tyrosine kinase led to the development of selective ABL inhibitors, with imatinib being the first to gain clinical approval. Its initial activity was demonstrated in phase I and phase II trials in CML, but also in ALL [27]. Although many patients with Ph+ ALL achieved a remission, there were very few long-term survivors. Only the subset of patients who then went to allogeneic HCT while in remission had a favorable outcome, with 50% of the patients disease free at 1 year [27]. Poor results of single-agent imatinib led to various investigations to explore the efficacy of imatinib as front-line therapy in combination with chemotherapy. Phase II trials combining imatinib (days 1–14 of each cycle), with hyperfractionated CVAD (CY, vincristine, adriamycin, and dexamethasone) chemotherapy had a very high remission rate after 21 days of treatment, suggesting synergy between imatinib and concurrent chemotherapy, with some patients achieving a molecular remission [28]. Similarly, encouraging results have been achieved in other centers in which imatinib was started after 1 week of induction therapy and then co-administered with chemotherapy during the remaining induction. In these trials, the 1-year event-free survival and overall survival was

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults

78–88%, with tolerability comparable to that seen with chemotherapy alone. Although the optimal schedule for combining imatinib with chemotherapy has not yet been established, both alternating and concurrent imatinib chemotherapy combinations have been studied, with a clear advantage of the simultaneous over the alternating schedule in terms of the achievement of a negative PCR for the BCR–ABL gene [29]. Those patients who undergo allogeneic HCT for ALL do not seem to have any detrimental effect of prior imatinib treatment. Given the increased incidence of Ph+ ALL in older patients with ALL and the difficulty of achieving a remission in these patients, imatinib has also been studied as front-line treatment in older patients with ALL, with an improved outcome compared with chemotherapy, and with better tolerability [30]. Several studies now suggest that imatinib-combined chemotherapy in newly diagnosed Ph+ ALL with subsequent treatment with allogeneic HCT has improved the results of treatment. With regard to the CNS, patients with Ph+ ALL are at significant risk for developing CNS leukemia [31]. Imatinib concentrations in the cerebrospinal fluid reach about 1–2% of serum levels and are not therapeutic. Thus, all patients with ALL, independent of their chromosome abnormality, require CNS prophylaxis [31]. Additional novel tyrosine kinase inhibitors with significantly more potent activity against the BCR–ABL gene have been developed and are in clinical trials. Currently, trials are being developed to compare combination chemotherapy and imatinib to allogeneic HCT in the era of tyrosine kinase inhibitors. As discussed below, the use of these drugs may have an impact on the pretransplant disease burden of the patient and reduce the risk of relapse following HCT. Prognostic factors in ALL The major prognostic factors for achieving a CR are advanced age and Ph+ ALL. These prognostic factors are of even greater importance in predicting the durability of remission and survival, and are utilized to assess the need for allogeneic HCT. Table 55.2 identifies some of the adverse prognostic factors for remission duration in adult ALL that have been identified in previous clinical trials and are utilized in determining a patient’s risk of relapse. Role of MRD In addition to age and cytogenetic analysis at the time of diagnosis, the most important prognostic factor, and a direct reflection of sensitivity to chemotherapy, is the rapid achievement of a CR. Thus, a slower time to achieving a remission is an indicator of relative chemoresistance, similar to what has been observed in pediatric patients. Those patients who take more than one cycle of induction chemotherapy have a poor long-term prognosis and a shorter remission duration [32–34]. A more quantitative approach to assess the response of an individual patient to chemotherapy is the measurement of MRD at various time

Table 55.2 Adverse prognostic factors for remission duration in adult acute lymphoblastic leukemia (ALL)

795

points after therapy. Quantitative assessment of tumor cell kill is emerging as an independent prognostic factor that reflects the resistance of the cells to chemotherapy and allows potential individualization of treatment [35,36]. The assessment allows the identification of potential patients at high risk for relapse despite achieving a morphologic remission and who may benefit from early HCT. Studies are being performed to determine the most predictive time point for measurement. It appears that, after consolidation, a high level of MRD at 10−4 is associated with a high risk of disease relapse, with a rising level of MRD on treatment also portending relapse [37,38]. In some studies, a high level of MRD after induction and consolidation has been identified as a high-risk feature despite the achievement of a morphologic remission and the absence of high-risk cytogenetics [37,38]. Conversely, the identification of patients who are sensitive to chemotherapy and achieve a low level of MRD (nondetectable) may identify a group of patients who do not need transplantation or can wait until there is clear evidence of relapse [37,38]. It also remains to be determined what the benefit of HCT may be in patients who are in first remission but have evidence of a new factor defining high-risk disease, that is, high MRD. At the present time, MRD measurement has certain limitations related to the technical procedure, which is time-consuming, expensive, and requires a specialized laboratory to conduct the studies. The testing also involves multiple evaluations with either immunophenotypic flow cytometry analysis or molecular analysis with patient-specific probes for gene rearrangements. Thus, the future of treatment of adult patients with ALL in first remission may be refined to determine those patients who are unlikely to benefit from further chemotherapy and should be considered for transplantation during first remission. Allogeneic HCT in first CR of ALL Allogeneic HCT in first CR (CR1) has generally been reserved for those patients who present with poor-risk features, such as those described earlier. In several phase II studies, patients with high-risk disease treated with allogeneic HCT had a DFS longer than would have been predicted, especially those with Ph+ ALL. Depending on the risk factors present at diagnosis in an individual patient, standard chemotherapy leads to continued remissions ranging from less than 10% to more than 50% [39,40]. Studies have been conducted indicating that HCT offers some groups of high-risk patients long-term disease survival rates of between 40% and 60% [41–47]. At the City of Hope and Stanford University, two series of patients with high-risk features who underwent allogeneic HCT in CR1 have been recently updated. Selection criteria included a white blood cell count of over 25,000/μL, chromosomal translocations t(9;22), t(4;11) or t(8;14), age older than 30 years, extramedullary disease at the time of diagnosis, and/or the need for more than 4 weeks to achieve a CR. Two-thirds of the patients had at least one risk factor, and the

Clinical characteristics

Immunophenotype

Cytogenetics/molecular genetics Treatment response

Higher age >35 years High white blood cell count >30,000/μL in B-lineage, 100,000/μL in T-cell ALL Pro B (B-lineage, CD10−) Early T (T-lineage, CD1a−, sCD3−) Mature T (T-lineage, CD1a−, sCD3+) t(9;22)/BCR–ABL or t(4;11)/ALL1-AF4, 1;19 Late achievement of complete remission >3–4 weeks Minimal residual disease positivity in remission

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Chapter 55

1.0 0.9 0.8

Probability

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

1

2

3

4

5

6

7 8 9 Time in years

10

1.0 Median DFS

DFS Probability

0.8

Sibling donor 25.6 months No sibling donor 13.8 months

3-year DFS 5-year DFS 47% 34%

45% 23%

11

12

13

14

15

Fig. 55.2 Probability of event-free survival (upper curve), overall survival (middle curve), and relapse (bottom curve) for 55 adult patients with high-risk acute lymphoblastic leukemia transplanted in first remission. (Reproduced and updated from [48], with permission.)

Table 55.3 European Cooperative Oncology Group–Medical Research Council UKALL XII/EC2993: outcome after allogeneic hematopoietic stem cell transplantation in Philadelphia chromosome-negative patients who had donors versus those who did not have donors

No.

5-year overall survival (%)

5-y relapse rate (%)

2-year nonrelapse mortality (%)

High risk* Donor No donor

401 171 230

40 36

39 62

39 12

Standard risk Donor No donor

512 218 294

63 51

27 50

20 7

0.6 45% 0.4 P = .007 0.2

0

18%

2

4 6 8 Time from CR achievement (years)

10

Fig. 55.3 Disease-free survival (DFS) according to genetic randomization. The group with a sibling donor comprised 100 patients, whereas that with no sibling donor included 159 patients. CR, complete remission. (Reproduced from [39], with permission.)

* High risk is defined as age 35 years or over, white cell count over 30,000/μL for patients with B-cell disease or over 100,000/μL for patients with T-cell disease, or time to attain complete remission of longer than 4 weeks.

undergoing allogeneic transplant in first remission. Table 55.4 shows a comparison of a number of other trials comparing chemotherapy to HCT. remaining patients had two or more high-risk features at presentation. The majority of these patients underwent allogeneic HCT within the first 4 months after achieving a CR. Allogeneic HCT during first remission led to prolonged DFS in this patient population, who would otherwise have been expected to fare poorly. At a median follow-up of greater than 5 years, the probability of event-free survival was 64%, with a relapse rate of 15% (Fig. 55.2) [48]. The French Group on Therapy for Adult ALL conducted a study comparing chemotherapy to autologous and allogeneic HCT [39]. Although the overall results of treatment did not show a treatment advantage for the group treated with allogeneic HCT, subgroup analysis revealed that those patients with high-risk disease, including patients with a t(9;22) and t(1;19) translocation, had a higher 5-year survival of 44% as opposed to 20% in the other two groups (Fig. 55.3). The recently completed Eastern Cooperative Oncology Group–Medical Research Council (ECOG–MRC) trial showed that allogeneic HCT resulted in improved disease control, with long-term benefit seen mostly in younger patients [49]. Table 55.3 shows the DFS and response rate for patients on this trial with the risk of relapse reduced following allogeneic HCT when compared with either chemotherapy or autologous HCT, showing the improved outcomes and better leukemia control for those patients

HCT for Ph+ ALL Historically, the dismal outcome with chemotherapy has led to trials focusing on the use of allogeneic transplantation for the treatment of adult Ph+ ALL. Most have been single-institution studies utilizing a variety of regimens, and the cure rate varies from 30% to 65%, dependent upon age and remission status [50,51]. Investigators from City of Hope and Stanford University have analyzed their experience in 79 patients with Ph+ ALL transplanted from HLA-identical siblings while in CR1 between 1984 and 1997 to determine long-term survival and disease control. All patients but one were conditioned with fractionated TBI (1320 cGy) and high-dose etoposide (60 mg/kg). The 3-year probability of DFS and relapse was 55% and 18%, respectively, with the latest relapse at 27 months. Beyond first remission, HCT is curative in a much smaller minority of patients but remains the treatment of choice (Fig. 55.4). The development of imatinib and other tyrosine kinase inhibitors for the treatment of BCR–ABL+ hematopoietic malignancy has changed the up-front treatment strategy, and also may affect the outcome after HCT. Recently, the feasibility of performing allogeneic HCT after first-line

797

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults Table 55.4 Comparison of several large trials for patient outcome in first complete remission in acute lymphoblastic leukemia (ALL) CR1

Group study, citation

Number of patients considered for HCT

Outcome measure

Chemo autologous therapy (%) HCT (%)

Allogeneic HCT (%)

p

ECOG–MRC JALSG-ALL93 LALA-87 LALA-87, high risk LALA-94 LALA-94, high risk GOELAMS 02, high risk PETHEMA 93, high risk

913 142 257 156 259 211 156 183

OS at 5 years OS at 6 years OS at 10 years OS at 10 years DFS at 3 years OS at 5 years OS at 6 years OS at 5 years

45 40– 31 11 34 21, 32 40 at 6 years 49

53 46 46 44 47 51 but median OS not reached 75 at 6 years 40

0.02 NS 0.04 0.009 0.007 Not stated 0.0027 0.56

HCT, hematopoietic cell transplantation; NS, not significant; OS, overall survival. (a) 1.0

0.9 p value = 0.0114

Survival probability

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21

Time (year) from date of transplant Disease status

1CR

>1CR

(b) 1.0 0.9

p value = 0.2780

Probability of relapse

0.8

Fig. 55.4 Probability of long-term disease-free survival and risk of relapse in 79 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia following allogeneic hematopoietic cell transplantation in first complete remission (CR1) and beyond first remission (>CR1). (a) Overall survival; (b) relapse rate. (Reproduced from [103], with permission.)

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

1

2

treatment with imatinib plus chemotherapy has been reported [52]. In this series, 29 adult patients who completed induction therapy were treated with allogeneic HCT, and the authors compared their results with those from 31 patients who had received transplantation in their unit

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21

Time (year) from date of transplant Disease status

1CR

>1CR

prior to the availability of imatinib treatment. The data suggest that the risk of relapse was significantly less in the imatinib group (3.5% versus 47.3%; p = 0.002), potentially reflecting a lower burden of residual disease at the time of HCT, and allowing a higher percentage of patients

Chapter 55 100

P < .001

Probability of DFS (%)

80 Imatinib group (78.1 ± 11.6%) 60 Historical group (38.7 ± 8.8%) 40

20

0 0

12

24 36 48 Months after SCT

60

72

P < .001

80 Imatinib group (78.1 ± 11.6%) 60 Historical group (38.7 ± 8.8%)

40

Fig. 55.5 Probabilities of disease-free survival (DFS) and overall survival in the imatinib group versus the historical group of patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. SCT, stem cell transplantation. (Reproduced from [52], with permission.)

20

0 0

12

24 36 48 Months after SCT

to come to transplantation in a “good” first remission. The results also indicated a superiority in DFS (76% versus 38%; p < 0.001) without much difference in transplant-related toxicities (Fig. 55.5). Thus, in the same way that imatinib may improve the up-front success of induction therapy and potential long-term outcome of patients with Ph+ ALL, entering transplantation with a lower burden of disease may improve the cure rate for such patients. Of additional interest is the follow-up of patients with ALL and the impact of the detection of MRD after allogeneic HCT. Radich et al. reviewed the results of 36 patients with Ph+ ALL who underwent allogeneic HCT and were monitored with sensitive assays of MRD [53]. Seventeen patients were transplanted in relapse, and 19 were transplanted in remission. Twenty-three patients had at least one positive BCR–ABL PCR assay after transplantation either before a relapse or without subsequent relapse. Ten of these 23 patients relapsed after a positive assay at a median time from first positive PCR assay of 94 (range 28–416) days. By comparison, only two relapses occurred in the 13 patients with no prior positive PCR assays. The unadjusted relative risk of relapse associated with a positive PCR assay compared with a negative assay was 5.7. Recent studies have also demonstrated the feasibility of administering imatinib following allogeneic HCT for BCR–ABL+ hematologic malignancy and can be used either preemptively or to treat any MRD detected after transplant prior to relapse instead of using donor lymphocyte infusions (DLIs) [54]. This strategy may improve the outcome of high-risk patients and facilitate improved long-term control of disease after HCT. The observation concerning the impact of pretransplant MRD suggests that it may be desirable to utilize second- and third-generation BCR–ABL inhibitors to reduce disease burden before transplant. Relapsed or primary refractory ALL ALL is refractory to primary chemotherapy in approximately 10–15% of patients, and allogeneic HCT can be successfully used to achieve both a remission and long-term control in approximately 20% of such patients. Of all those patients who do achieve a CR1 to primary therapy, approximately 50–70% will relapse. Relapsed ALL in an adult is not curable with standard chemotherapy, but remissions are sometimes achieved with reinduction using either a standard vincristine, prednisone, and anthracycline or an ARA-C-based regimen, particularly high-dose ARAC combined with an anthracycline or clofarabine [55–57], especially in patients with a long first remission. Recent studies confirm that, in the absence of HCT, adult patients with relapsed ALL have an extremely poor prognosis regardless of initial

60

72

1 P < 0.001 0.8 Probability of DFS

100

Probability of overall survival (%)

798

0.6 1st Remission (n = 41) 0.4 ≥2nd Remission (n = 46) 0.2

Relapse (n = 95)

0 0

2

4

6

8

10

12

Years after transplantation

Fig. 55.6 Long-term survival in patients with acute lymphoblastic leukemia demonstrating the impact of remission status on the outcome of transplant. DFS, disease-free survival. (Reproduced from [59], with permission.)

remission duration, and that transplantation, when feasible, is the only possible curative therapy [58]. Available data from the Center for International Blood and Marrow Transplant Research (CIBMTR) show that patients transplanted with an HLA-identical sibling donor for ALL in second CR (CR2) have an approximately 35–40% chance of longterm DFS, while those transplanted with disease not in remission have a DFS of only 10–20%. Figure 55.6 shows the overall DFS for patients with ALL, depending on their remission status, who underwent allogeneic HCT [59]. As in all series, these data do not account for those patients who suffer a relapse and do not undergo transplantation. Nevertheless, for an adult patient with ALL after a first relapse, allogeneic HCT is the best curative option. Unrelated HCT for ALL Historically, the outcome after transplantation from unrelated donors has been inferior to that observed after matched-sibling transplantation because of increased rates of graft rejection and graft-versus-host disease (GVHD) resulting from increased alloreactivity in this setting [60]. The National Marrow Donor Program (NMDP) reports a 5-year DFS of 35% in CR1 in adults and 46% in children, decreasing to 25% and 40%, respectively, in CR2. Over the past few years, improved results have been reported from several single-center studies, reflecting improvements in donor–recipient allele-level molecular matching in both class I and II histocompatibility genes, GVHD prophylaxis, and supportive

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults

799

care [61]. An NMDP study showed that younger donor and recipient age were associated with significantly improved outcomes. Recent reports suggest equivalent results for high-risk patients from either related or unrelated donors [62–64], making this a reasonable option for a patient who needs an allogeneic HCT for treatment of their disease.

relapsed ALL. It is for these reasons that HCT after reduced-intensity conditioning is of limited effectiveness in patients with ALL who are not in remission. Studies focused on developing antigen-specific T-cell immunotherapy for ALL may help augment the GVL activity of donor T cells (see Chapter 18) [71–76].

Role of graft-vs.-leukemia effect in patients with ALL

HCT after reduced-intensity conditioning for treatment of ALL

The low response rate in patients with ALL following DLI has led to questions about the significance of the graft-versus-leukemia (GVL) effect in preventing relapse in this disease. The GVL effect is derived from observations of a higher relapse rate after autologous or syngeneic HCT compared with allogeneic HCT, a lower incidence of relapse in patients who had GVHD, as well as increased relapse rates in recipients of T-cell-depleted marrow grafts. The most compelling argument for a strong GVL effect in ALL comes from both single-institution and registry data [65,66]. These studies show a consistent decrease in relapse rates in patients who develop GVHD compared with those patients who do not. Table 55.5 shows the rate of relapse after HCT for ALL in CR1 and the correlation with GVHD. The occurrence of acute, chronic or both forms of GVHD correlated with the best DFS. A study of 192 patients with ALL, most of whom were transplanted in second remission [67], evaluated the probability of relapse among patients without or with GVHD. Relapse was significantly higher in the group that had less than grade II GVHD. In patients without significant GVHD, the actuarial risk of relapse approached 80%, compared with 40% in those who developed grade II or higher GVHD. A subsequent study [59] confirmed this observation for both relapse and overall DFS. An evaluation of 1132 patients with T- or B-lineage ALL supports the observation that both acute and chronic GVHD are associated with a decreased risk of relapse in both of the major immunophenotypes of adult ALL [68]. Recent studies have also demonstrated the beneficial impact of chronic GVHD on reducing relapse in patients undergoing allogeneic HCT for ALL, including Ph+ ALL [69,70]. Although the data support the importance of a GVL effect in mediating a clinically useful antileukemic response in patients with ALL, the reasons for the limited beneficial effect for patients with relapsed ALL treated with DLI are not clear. The different outcomes may reflect differences in the ability of ALL cells to present antigen targets, the low frequency of T-cell precursors reactive with minor antigens presented by ALL cells, the susceptibility of ALL targets to lysis or kinetic differences in the way leukemic cells grow after HCT. Thus, cytoreduction with chemotherapy prior to DLI is a better strategy for patients with

Table 55.5 Relapse after transplantation for acute lymphoblastic leukemia in first complete remission Group

Relapse probability at 3 years (%)

Allogeneic, non-T depleted No GVHD Acute only Chronic only Both

44 ± 17 ± 20 ± 15 ±

Syngeneic

41 ± 32

Allogeneic, T-depleted

34 ± 13

GVHD, graft-versus-host disease. Reproduced with permission from Appelbaum [66].

17 9 19 10

Although there has been a large number of studies performed in evaluating the role of allogeneic reduced-intensity transplantation in patients with myeloid malignancies, multiple myeloma, and low-grade nonHodgkin’s lymphoma, there have been fewer studies conducted in patients with ALL. In general, the consensus has been that, for patients with ALL, high-dose chemoradiotherapy is required for an improved cure rate, but this approach is of limited use in patients over the age of 50. In addition, an evaluation of outcomes suggests that the graftversus-tumor effect is more effective against myeloid malignancies such as AML and CML, and B-cell malignancies of mature B cells such as low-grade non-Hodgkin’s lymphoma and multiple myeloma, but less so with a more undifferentiated B-cell disease such as pre-B ALL, especially if not in remission (see Chapter 71) [77–79]. Nevertheless, a few small studies have been conducted that suggest that there may be a role for reduced-intensity allogeneic transplantation even in this disease, particularly in older patients, with 34% achieving long-term remission in a report from the European Group for Blood and Marrow Transplantation (EBMT) [77]. A recent report of patients undergoing HCT utilizing either related, unrelated or cord blood donor and a fludarabine/ melphalan regimen showed an optimistic outcome in a group of patients who were either at high risk during first remission or transplanted after achieving a second or subsequent remission [80]. The results of the recent UKALL XII/ECOG E2993 study of adult ALL show high toxicity and limited improvement in DFS despite better disease control in older adult patients. Thus, there is increased interest in the development of clinical trials exploring these reduced-intensity approaches in older patients with ALL in remission who would otherwise be candidates for transplantation based on age, cytogenetics, MRD, and response to initial treatment. The poor outcome of older patients with ALL using standard chemotherapy makes this an important consideration for treatment in those patients. Regimen development for allogeneic HCT for ALL Historically, the most commonly used regimen for transplantation of patients with ALL is CY plus TBI. Several other different preparative regimens have been developed, each based on substituting a different chemotherapeutic agent for CY in combination with TBI for patients with ALL. High-dose fractionated TBI in combination with high-dose ARA-C has been employed by several centers, and, with the exception of a small series of pediatric patients at Case Western Reserve, there has been no significant improvement in DFS with this regimen in recipients of allogeneic HCT from sibling donors [81,82]. Investigators at Johns Hopkins University approached the problem by conducting trials substituting busulfan (BU) for TBI in order to decrease the long-term side-effects of TBI and to determine the efficacy of highdose combined alkylating therapy in eliminating leukemic cells [83,84]. These nonradiation-dependent regimens have shown activity in the treatment of advanced ALL, suggesting that TBI is not an absolute requirement for successful treatment of ALL by HCT. A retrospective analysis from the CIBMTR found that a conventional CY/TBI regimen was superior to a non-TBI-containing regimen of BU plus CY in children, with a 3-year survival of 55% versus 40% for BU/CY [85]. However,

Chapter 55

Radioimmunotherapy-based transplant regimens for ALL Studies in AML have shown lower relapse rates with higher doses of TBI, suggesting that methods that can selectively deliver radiation to sites of leukemia without increasing systemic toxicity might be of benefit to the patient. The use of tumor-reactive monoclonal antibodies conjugated with local acting radionucleotides such as iodine-131 (131I) or yttrium-90 (90Y) are being explored to accomplish the goal of decreased relapse (see Chapter 24). Initial studies conducted in Seattle in an animal model showed the feasibility of this novel approach, and subsequent phase I and II studies in patients have been initiated. These studies have demonstrated that initial targeting of marrow and other sites of leukemia could be accomplished utilizing 131I-conjugated monoclonal antibodies. The most recent studies have focused on a monoclonal antibody reactive with CD45, an antigen that is found on leukemic cells as well as normal hematopoietic tissue and, unlike CD33, does not internalize after antibody binding. A phase I trial of 131I anti-CD45 monoclonal antibody plus CY and TBI for advanced leukemia was completed [90]. This study focused on the biodistribution and toxicity of escalating doses of targeted radiation combined with 120 mg/kg CY and 12 Gy TBI followed by matched related or autologous HCT. Among 44 patients, five had ALL in relapse

(a)

Adjusted probability of LFS, %

despite these differences in survival, the risk of relapse was similar. A recent study of BU, fludarabine, and 400 cGy of TBI, which would be considered a high-dose regimen, showed a low transplant-related mortality (TRM) of 3% and a projected DFS of 65% [86]. The group at the City of Hope studied the substitution of etoposide (VP-16) for CY in combination with fractionated TBI (13.2 Gy) followed by allogeneic HCT [87]. A phase I–II trial indicated that a dose of etoposide of 60 mg/kg is the maximum tolerated dose when combined with a TBI dose of 1320 rads. In that study, 36 patients with ALL were treated, 20 of whom were in relapse. The actual DFS was 57%, with a 32% relapse rate, suggesting that the regimen had significant activity in patients with advanced ALL, a result confirmed in a subsequent trial from the Southwest Oncology Group [88]. A subsequent study from City of Hope/Stanford showed a 64% DFS for adult patients undergoing transplantation with this regimen in CR1 (see section on allogeneic transplant for ALL in first remission). The recently completed UKALL XII/ECOG E2993 trial, a comparative study of chemotherapy and autologous and allogeneic HCT, utilized this regimen for patients in CR1. A comparative analysis of TBI combined with either CY or etoposide chemotherapy was conducted to determine the relative efficacy of the chemotherapy in the transplant regimen [89]. The outcomes of 298 patients with ALL in CR1 or CR2 receiving HLA-matched sibling allografts after CY/TBI conditioning were compared with 204 patients receiving etoposide and TBI. In this analysis, four groups were compared based on the radiation dose: CY/TBI <13 Gy (n = 217), CY/TBI >13 Gy (n = 81), etoposide/TBI <13 Gy (n = 53), and etoposide/TBI >13 Gy (n = 151). Analyses of relapse, leukemia-free survival (LFS), and overall survival were performed separately for CR1 and CR2 patients. TRM did not differ by conditioning regimen. In CR1, there were also no significant differences in relapse, LFS or survival by conditioning regimen. In CR2, the outcomes differed among conditioning groups. In comparison with CY/TBI <13 Gy, the risks of relapse, treatment failure (inverse of LFS), and mortality tended to be lower with etoposide (regardless of TBI dose) or with TBI doses over 13 Gy. For both CR1 and CR2 patients, causes of death were similar among the groups; disease recurrence accounted for 47% of deaths. These data indicate that, for HLA-identical sibling allografts for patients with ALL in CR2, there is an advantage in substituting etoposide for CY or, when CY is used, in increasing the TBI dose to more than 13 Gy (Fig. 55.7).

100 80 60 Vp16-TBI (n = 129) Cy-TBI, TBI < 13 Gy (n = 118) Cy-TBI, TBI ≥ 13 Gy (n = 36)

40 20 0 0

1

2

3

4

5

4

5

Years (b)

Adjusted probability of LFS, %

800

100 80 60 40 Cy-TBI, TBI ≥ 13 Gy (n = 43) Vp16-TBI (n = 72) Cy-TBI, TBI < 13 Gy (n = 95)

20 0 0

1

2

3 Years

Fig. 55.7 Adjusted probability (derived from multivariate regression models) of leukemia-free survival (LFS) after human leukocyte antigen-identical sibling transplantations for acute lymphoblastic leukemia in first (CR1; a) or second (CR2; b) complete remission, according to the pretransplantation conditioning regimen (pointwise p value at 5 years for CR1 patients: etoposide (Vp16)-TBI versus Cy-TBI <13 Gy, p = 0.21; etoposide-TBI versus Cy-TBI ≥13 Gy, p = 0.17; Cy-TBI <13 Gy versus Cy-TBI ≥13 Gy, p = 0.59; pointwise p value at 5 years for CR2 patients: etoposide-TBI versus Cy-TBI <13 Gy, p = 0.002; etoposide-TBI versus Cy-TBI ≥13 Gy, p = 0.23; Cy-TBI <13 Gy versus Cy-TBI ≥13 Gy, p < .001). Cy, cyclophosphamide; TBI, total body irradiation. (Reproduced from [89], with permission.)

or refractory disease, and five were in second or third CR of ALL. Eighty-four percent of the patients had a favorable biodistribution of antibody, with a higher estimated absorbed dose of radiation to marrow and spleen than in normal tissues. Thirty-four patients received a therapeutic dose of 131I labeled with 76–612 mg 131I, designed to deliver an estimated radiation absorbed dose to liver of 3.5–12.25 Gy. In the group of nine patients treated for ALL, six of whom underwent allogeneic and three autologous HCT, two died of infection, four relapsed and three survived 10, 45, and 57 months after transplantation. This study demonstrated that 131I anti-CD45 antibody can deliver appreciable supplemental doses of radiation to the marrow (approximately 24 Gy) and spleen when combined with conventional fractionated TBI. Estimation of the ultimate benefit to DFS and improved safety of the regimen will await larger phase II studies in patients with ALL undergoing transplantation either in remission or in relapse. Given the efficacy of radiation-based regimens and a dose–response effect of radiation on leukemia, newer approaches to the delivery of radiation to the marrow are also being evaluated to increase the safety and the dose of radiation. Helical tomotherapy, used to focus and intensify local radiation treatment, can be utilized to treat the major marrowcontaining bones and offers a potential means to augment the dose of radiation to the marrow without increasing toxicity to other organs. This

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults

approach is now being combined with chemotherapy and evaluated in phase I–II trials [91]. Management of relapse after allogeneic transplant for ALL Once patients with ALL relapse after HCT, the prognosis is very poor. Similar to the approach in patients with AML and CML, manipulation of the antitumor effect mediated by the donor graft is often employed as a treatment strategy. Unfortunately, in patients with ALL, this therapy has not been as effective as it has been for patients with CML (see Chapter 72). A report from 25 North American BMT programs of 140 patients who received DLI showed that the CR rate was 60% in CML, with the responses being higher in patients with cytogenetic and chronicphase relapse compared with those with accelerated phase or blastic phase (75, 33, and 16%, respectively) [92]. The CR rates in relapsed AML and ALL are 15% and 18% respectively, similar to the blastic phase of CML. In that study, the development of acute and chronic GVHD following DLI was highly correlated with disease response. In a report from Europe, 40 patients with ALL received DLI as treatment for relapse, and of 29 evaluable patients, only one achieved CR [93]. Therefore, DLI as a sole therapy appears to have low potential for contributing to a remission and long-term control of disease in patients with relapsed ALL, and, if considered, should be a component of a chemotherapy-based treatment program. For those patients with Ph+ ALL, imatinib or dasatinib, either alone or with chemotherapy, is effective in helping some patients achieve another remission, although the duration of the remission is often quite short and DLI should be performed before another relapse occurs [94]. Autologous HCT for adult ALL There is much less experience with autologous transplantation for ALL, and studies have been focused primarily on those patients in either first or second remission who lacked a sibling or unrelated allogeneic donor. Some studies have utilized the same criteria for autologous transplantation as have been utilized for allogeneic transplantation, based on the idea that the preparative regimen does contribute to the cure of ALL since the allogeneic effect is less potent than in myeloid malignancies. Several groups have reported outcomes for large series of adults with ALL undergoing autologous HCT in first remission [95–98]. One study from France reported on 233 such patients with long-term DFS at 41% [97]. The most important prognostic factor was the interval between achieving a CR and proceeding to transplant, with those patients being transplanted later having the better DFS. This effect may represent the drop-out of high-risk patients who relapse before transplantation, or possibly the effect of consolidation therapy in reducing tumor burden administered prior to HCT. The ECOG–MRC report on over 1000 patients indicated an LFS of 36%, while the CIBMTR reported a similar plateau at 40% [95]. A long-term outcome analysis from NMDP–CIBMTR suggested equivalent results for autologous or unrelated donor transplants in first or second remission [99]. One randomized trial evaluated outcomes of adults with ALL in first remission treated with chemotherapy versus autologous transplantation. The French LALA-87 trial allocated patients aged under 40 with HLAmatched siblings to allogeneic transplantation, while the remaining patients received consolidation treatment with modest-dose chemotherapy or an autologous transplant [39]. There was a significant drop-out rate in the autologous arm due to early relapse, and the long-term followup showed no significant difference in overall survival between the two groups – 34% for autologous HCT and 29% for chemotherapy. This difference applied to both the standard- and high-risk groups. A large

801

trial involving collaboration between the ECOG Group and the MRC was recently reported comparing allogeneic transplant, autologous transplant or chemotherapy in all adult patients with ALL in first remission, and showed an inferior result for autologous transplantation versus continued chemotherapy [49]. Cell sources for HCT As is true for most patients undergoing allogeneic transplantation, peripheral blood has replaced bone marrow as the hematopoietic cell graft preference. A report from the EBMT presents a retrospective analysis in which the comparison outcome was made for 858 patients, 513 who were in first remission given marrow, versus 345 patients who were given blood [100]. Engraftment, as noted in many studies, was faster with peripheral blood but also resulted in an increased risk of chronic GVHD. LFS and overall survival did not differ. In a subsequent retrospective analysis, peripheral blood hematopoietic cells obtained from an unrelated donor were associated with an inferior outcome compared with bone marrow [101]. The incidence of 2-year TRM in 36 patients receiving blood was 61% compared with 47% in 66 recipients of bone marrow grafts. This resulted in a significantly different 2-year LFS of 32% versus 21%, and an overall survival of 34% versus 24% (p = 0.04). It appeared that younger patients had significantly lower TRM, but the relapse rates did not differ, indicating a probable similar antileukemic efficacy. Umbilical cord transplantation There is considerable interest in exploring the potential of umbilical cord transplant, but there are few data in patients with ALL. In a singleinstitution trial, the comparison was made between hematopoietic cells derived from umbilical cord transplant versus an unrelated donor following high-dose conditioning, which resulted in comparable outcomes [102]. In this study, there were only 16 adult patients with ALL, and the diagnosis of ALL predicted a high risk for relapse. At the present time, there are insufficient data to recommend use of this cell source outside of the context of a clinical trial, or unless an unrelated donor cannot be identified and there is an adequate CD34+ cell dose in the cord blood (see Chapter 39). Treatment strategy for ALL As discussed in this chapter, analysis of molecular genetic and clinical data treatment responses, as well as changes in the approach to treatment, has resulted in more refined recommendations as these relate to the timing of transplantation for the treatment of patients with ALL. Most studies indicate that allogeneic transplantation in first remission provides a better chance of DFS, especially in patients with high-risk disease. For patients who are younger than 50 without other high-risk features, better control of disease can be achieved by early allogeneic transplantation, as reported in the ECOG–MRC trial (Fig. 55.8). For patients who relapse, transplantation is the only treatment with the potential for cure, especially if a second remission can be achieved. Therefore, it is important to perform HLA typing at the time of diagnosis to determine what the transplant possibilities might be, either from related donors or through unrelated donor registries, especially for patients with high-risk features such as high white count, older age, slow rate of achieving remission or failure to achieve a remission. In addition, this information would be useful in planning for treatment in patients who suffer a relapse after achieving a remission. Clearly, identification of the Ph chromosome early after diagnosis gives patients an additional beneficial therapeutic option with the inclusion of tyrosine kinase inhibitors as part of the induction, consolidation, and maintenance regimen.

802

Chapter 55

100

Relapse rate Ph negative high risk

(b)

Relapse rate Ph negative standard risk

(a)

100

Donor Donor No donor n = 323

50

49% 24%

25 n = 239

No donor

75 Percent

Percent

75

50

63%

n = 261

37%

25 n = 204

P = < .00005

0

P = < .00005

0 0

1

2

3

4

5

6

7

8

9

10

0

1

2

3

4

Years

5

6

7

8

9

10

Years

Fig. 55.8 Comparison of relapse rate between allogeneic transplant and chemotherapy for (a) Philadelphia-negative patients at standard risk, and (b) Philadelphia-negative patients at high risk. See text for abbreviations. (Reproduced from [49], with permission.)

Diagnosis of ALL: cytogenetics, immunophenotyping, HLA typing

Ph + ALL

Induction/TKI treatment Induction therapy

No remission

Remission

Allogeneic transplant with related or unrelated donor

CR

Donor

No donor Consolidation and CNS phophylaxis

Allogeneic transplant

Continue chemotherapy/TKI treatment

High risk: Ph+, 4:11, high WBC slow response

Allogeneic HCT related or unrelated donor

Low risk

High risk due to age

Reduced intensity transplant on clinical trial If no transplant

Consider allogeneic transplant in first CR

Continue chemotherapy

Early relapse

Late relapse

Transplant

Reinduction

Transplant

Fig. 55.9 Suggested treatment and transplant algorithm for adult patients with acute lymphoblastic leukemia. TKI, thymidine kinase inhibitor; WBC, white blood cell. See text for other abbreviations.

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Adults

Trials are ongoing that will compare the outcome of this modern approach to ALL along with a more modern approach to ALL following transplantation. Figure 55.9 provides a suggested algorithm for the use of transplant in patients with ALL.

Future considerations For a number of years, there were few, if any, innovations in the treatment of ALL in the adult, especially in the development of new agents. However, over the last few years, a number of agents, including monoclonal antibodies to CD20, CD52, and CD22, and new drugs for T-cell ALL, such as nelarabine and imatinib, dasatinib, and nilotinib, for the treatment of Ph+ ALL may improve the treatment outcome for patients with ALL. The most important contribution may come from studies defining which patients are most likely to benefit from transplantation early in the course of their disease. Thus, the monitoring of MRD may have a predictive value for relapse, as a reflection of sensitivity to chemotherapy. This would be an important development for all patients. Those

803

patients, even those with higher-risk disease, who achieve a CR with lack of detection of MRD, may be spared the risk of transplantation, while other patients who have not achieved an adequate depth of remission would be better served by early transplantation, even if there were no overt high-risk features at the time of diagnosis. Thus, the introduction of MRD monitoring over the course of treatment might provide a new strategy for how to advise patients about the timing of transplantation. In addition, transplantation regimens will be developed to address patients with high-risk disease that could include radioimmunotherapy targeted to the CD45 antigen. Studies are also being conducted to determine the efficacy of utilizing antigen-specific T cells to augment a graftversus-tumor effect. These cells can be derived from normal donors and could be transduced to recognize the CD19 antigen present on nearly all patients with pre-B ALL. And, finally, given the results of chemotherapy treatment of older patients with ALL, studies of reduced-intensity conditioning should be pursued for such patients in order to explore the potential therapeutic role of the GVL effect in this high-risk patient population.

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59. Doney K, Hägglund H, Leisenring W, Chauncey T, Appelbaum FR, Storb R. Predictive factors for outcome of allogeneic hematopoietic cell transplantation for adult acute lymphoblastic leukemia. Biol Blood Marrow Transplant 2003; 9: 472–81. 60. Bachanova V, Weisdorf D. Unrelated donor allogeneic transplantation for adult acute lymphoblastic leukemia: a review. Bone Marrow Transplant 2008; 41: 455–64. 61. Cornelissen JJ, Carston M, Kollman C et al. Unrelated marrow transplantation for adult patients with poor-risk acute lymphoblastic leukemia: strong graft-versus-leukemia effect and risk factors determining outcome. Blood 2001; 97: 1572–7. 62. Chim CS, Lie AK, Liang R, Au WY, Kwong YL. Long-term results of allogeneic bone marrow transplantation for 108 adult patients with acute lymphoblastic leukemia: favorable outcome with BMT at first remission and HLA-matched unrelated donor. Bone Marrow Transplant 2007; 40: 339–47. 63. Dahlke J, Kroger N, Zabelina T et al. Comparable results in patients with acute lymphoblastic leukemia after related and unrelated stem cell transplantation. Bone Marrow Transplant 2006; 37: 155–63. 64. Kiehl MG, Kraut L, Schwerdtfeger R et al. Outcome of allogeneic hematopoietic stem-cell transplantation in adult patients with acute lymphoblastic leukemia: no difference in related compared with unrelated transplant in first complete remission. J Clin Oncol 2004; 22: 2816–25. 65. Horowitz MM, Gale RP, Sondel PM et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75: 555– 62. 66. Appelbaum FR. Graft versus leukemia (GVL) in the therapy of acute lymphoblastic leukemia (ALL). Leukemia 1997; 11: S15–17. 67. Cornelissen JJ, Carston M, Kollman C et al. Unrelated marrow transplantation for adult patients with poor-risk acute lymphoblastic leukemia: strong graft-versus-leukemia effect and risk factors determining outcome. Blood 2001; 97: 1572–7. 68. Passweg JR, Tiberghien P, Cahn JY et al. Graft-versus-leukemia effects in T lineage and B lineage acute lymphoblastic leukemia. Bone Marrow Transplant 1998; 21: 153–8. 69. Espérou H, Boiron JM, Cayuela JM et al. A potential graft-versus-leukemia effect after allogeneic hematopoietic stem cell transplantation for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia: results from the French Bone Marrow Transplantation Society. Bone Marrow Transplant 2003; 31: 909– 18. 70. Lee S, Cho BS, Kim SY et al. Allogeneic stem cell transplantation in first complete remission enhances graft-versus-leukemia effect in adults with acute lymphoblastic leukemia: antileukemic activity of chronic graft-versus-host disease. Biol Blood Marrow Transplant 2007; 13: 1083–4. 71. Appelbaum FR. Hematopoietic cell transplantation as immunotherapy. Nature 2001; 411: 385– 9. 72. Mutis T, Verdijk R, Schrama E, Esendam B, Brand A, Goulmy E. Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes specific for hematopoi-

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84. Tutschka PJ, Copelan EA, Klein JP. Bone marrow transplantation for leukemia following a new busulfan and cyclophosphamide regimen. Blood 1987; 70: 1382–8. 85. Davies SM, Ramsay NKC, Klein JP et al. Comparison of preparative regimens in transplants for children with acute lymphoblastic leukemia. J Clin Oncol 2000; 18: 340–7. 86. Russell JA, Savoie ML, Balogh A et al. Allogeneic transplantation for adult acute leukemia in first and second remission with a novel regimen incorporating daily intravenous busulfan, fludarabine, 400 CGY total-body irradiation and thymoglobulin. Biol Blood Marrow Transplant 2007; 13: 814–21. 87. Blume KG, Forman SJ, O’Donnell MR et al. Total body irradiation and high-dose etoposide: a new preparatory regimen for bone marrow transplantation in patients with advanced hematologic malignancies. Blood 1987; 69: 1015–20. 88. Blume KG, Kopecky KJ, Henslee-Downey JP et al. A prospective randomized comparison of total body irradiation-etoposide versus busulfan cyclophosphamide as preparatory regimens for bone marrow transplantation in patients with leukemia who were not in first remission: a SouthWest Oncology Group study. Blood 1993; 81: 2187–93. 89. Marks DI, Forman SJ, Blume KG et al. A comparison of cyclophosphamide and total body irradiation with etoposide and total body irradiation as conditioning regimens for patients undergoing sibling allografting for acute lymphoblastic leukemia in first or second complete remission. Biol Blood Marrow Transplant 2006; 12: 438–53. 90. Matthews DC, Appelbaum FR, Eary JF et al. Phase I study of 131I-anti-CD45 antibody plus cyclophosphamide and total body irradiation for advanced acute leukemia and myelodysplastic syndrome. Blood 1999; 94: 1237–47. 91. Wong JY, Liu A, Schultheiss T et al. Targeted total marrow irradiation using three-dimensional image-guided tomographic intensity-modulated radiation therapy: an alternative to standard total body irradiation. Biol Blood Marrow Transplant 2006; 12: 306–15. 92. Collins RH, Shpilberg O, Drobyski WR et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997; 15: 433–44. 93. Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia. Blood 1995; 86: 2041–50. 94. Wassman B, Pfeifer H, Scheuring U et al. Therapy with imatinib mesylate (Glivec) preceding

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56

Parinda A. Mehta & Stella M. Davies

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children

Introduction Acute lymphoblastic leukemia (ALL) of childhood includes a heterogeneous group of disorders, each with differing molecular genetic abnormalities and clinical behavior. Significant advances in chemotherapy regimens have been made over the last 50 years, such that more than 70% of children with ALL are now cured with chemotherapy [1]. The remarkable progress achieved in the treatment of ALL is the result of series of large-scale clinical studies conducted by cooperative clinical trials groups. In addition, biologic investigations have led to improvements in risk group identification and allowed administration of riskadapted therapy, improving the results of clinical trials. These advances in chemotherapy treatment have occurred in parallel with important changes in transplantation techniques, donor availability, and supportive care in hematopoietic cell transplantation (HCT). Together, these events have led to changes over time in indications for, and outcome of, HCT for childhood ALL. The indications for HCT in childhood ALL are likely to continue to change in future years as advances are made in chemotherapy, HCT, and understanding of biology. This chapter reviews the genetic characteristics of childhood ALL, which are central to accurate risk assessment, and discusses the biologic differences between childhood and adult ALL. Outcomes of sibling donor HCT are reviewed, followed by a discussion of the selection of conditioning regimens. For the majority of patients who have no available sibling hematopoietic cell donor, the use of alternative hematopoietic cell sources is an important therapeutic option, and outcomes of mismatched related marrow, unrelated marrow, and umbilical cord blood (UCB) donor transplants are reviewed. Two particular pediatric populations, infants with ALL and children with Down’s syndrome and ALL, require special consideration, and the appropriate use of HCT in these cases is discussed. Finally, data regarding the effectiveness of management of post-transplant relapse with a second transplant are presented.

Diagnosis and classification of ALL in childhood The last 20 years have seen an explosion in our knowledge of the phenotypic and genotypic characteristics of childhood ALL. The advent of monoclonal antibodies allowed the characterization of cell surface phenotypes of childhood ALL, and now categorization of leukemia

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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according to cell of origin is routine. Immunophenotyping demonstrates that most cases of childhood ALL are B lineage, while the remainder are T lineage (Table 56.1) [1]. B-lineage leukemias generally arise early in B-lymphocyte development before the maturation of surface immunoglobulin, and are termed “B precursor ALL.” Cases with cytoplasmic immunoglobulin are termed “pre-B ALL.” Cases with similar pre-B surface markers which do not express cytoplasmic immunoglobulin are termed “early pre-B ALL” [2]. Some mixed lineage phenotypes (expressing lymphoid and myeloid markers) have been observed. The association of B-cell and monocyte markers is especially important in childhood leukemia because it is seen frequently in the high-risk leukemias that arise in infants [3]. While immunophenotyping allows categorization of ALL by cell of origin, the development of molecular genetics has allowed for a finer classification of ALL into prognostically important subgroups on the basis of acquired genetic abnormalities. A large number of specific genetic alterations, commonly but not always associated with specific chromosomal translocations, have been described (summarized in Table 56.1), and are important predictors of outcome of chemotherapy. Some genetic abnormalities appear to be the consequence of mistakes in the normal process of DNA rearrangement used to generate immunologic diversity in lymphoid progenitor cells, as shown in Table 56.1. Examples include leukemias that involve immunoglobulin or T-cell receptor rearrangements. The functional result of such rearrangements is usually a fusion between the immunoglobulin or T-cell receptor gene and a “caretaker gene” or proto-oncogene, such as MYC, resulting in dysregulation of cellular proliferation. The most frequent genetic abnormalities in childhood ALL are translocations that result in fusion of two genes that are important in the regulation of gene transcription in hematopoietic cells, such as TEL, AML1, MLL or AF4. The fused genes produce a chimeric or fusion gene protein product that alters normal cellular function, frequently through regulation of signal transduction or transcriptional pathways [4,5]. It is important to note that the major genotypic forms of childhood ALL cannot be distinguished by morphology in most cases, and even by cytogenetics in some cases. An excellent example is the TEL-AML1 fusion that is generally cryptic in cytogenetic analysis and is important to identify by molecular analysis because of the excellent prognosis of these leukemias when treated with chemotherapy [6]. In contrast, BCR/ ABL rearrangements, and rearrangements of the MLL gene in infants, are associated with markedly inferior outcomes with chemotherapy [7–12]. Rapid identification of molecular abnormalities that can importantly affect the prognosis of ALL has allowed risk-adjusted therapy assignment to optimize outcomes. Despite advances in molecular diagnosis, cytogenetic studies in addition remain important for identifying leukemias with hyperdiploidy, confirming the results of molecular

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807

Table 56.1 Cellular genotype defines major forms of childhood acute lymphoblastic leukemia

Molecular genetic abnormality

Translocation

Biochemical defect

Associated features

TEL-AML1 (CBFα) fusion BCR-ABL fusion (p185)

t(12;21) cryptic t(9;22)(q34;q11)

B-cell lineage leukemia Transcription Signal transduction

Good prognosis with chemotherapy Poor prognosis with chemotherapy

E2A-PBX fusion

t(1;19)(q23;p13)

Transcription

MLL-AF4 fusion

t(4;11)(q21;q23)

Transcription

Pre-B phenotype, intermediate response to intensive chemotherapy Infants have poor prognosis Noninfants have intermediate prognosis Hyperleukocytosis FAB L3, extramedullary disease FAB L3, extramedullary disease FAB L3, extramedullary disease Good prognosis with chemotherapy Poor prognosis with chemotherapy

MLL-ENL fusion IGH-MYC fusion IGκ-MYC fusion IGλ-MYC fusion Hyperdiploidy Hypodiploidy (less than 45)

t(11;19)(q23;p13) t(8;14)(q24;q32) t(2;8)(p12;q24) t(8;22)(q24;q11) None None

Transcription Transcription Transcription Transcription Unknown Unknown

TAL1 (SCL) deletion

None

T-cell lineage leukemia Transcription

TCRδ-TAL1 (SCL) fusion

t(1;14)(p32;q11)

Transcription

TCRβ-TAL1 (SCL) fusion

t(1;7)(p32;q35)

Transcription

TCRα-MYC fusion TCRδ-RBTN1 fusion TCRδ-RBTN2 fusion TCRδ-HOX11 fusion TCRβ-LCK fusion

t(8;14)(q24;q11) t(11;14)(p15;q11) t(11;14)(p13;q11) t(10;14)(q24;q11) t(1;7)(p32;q35)

Transcription Transcription Transcription Transcription Signal transduction

analyses, and guiding the development of molecular assays to detect specific rearrangements.

Biologic differences between pediatric and adult ALL Chemotherapy outcomes in adults with ALL are notably inferior to results achieved in children. With intensive combination chemotherapy, more than 70% of children with ALL will be cured of their disease, while results in adult studies indicate survival rates of 30–40%, despite adoption of similar treatment strategies [1,13]. Biologic differences between adult and pediatric ALL likely influence response to chemotherapy and include age-dependent differences in immunophenotype, cytogenetic, and molecular genetic characteristics [14]. While the incidence of ALL increases in the elderly, there is also a marked peak in incidence in children between the ages of 2 and 5 years of age. The leukemias occurring within this peak are generally of Bprecursor origin, express CD10 surface antigen, and are hyperdiploid, all features associated with an excellent response to therapy [15]. Interestingly, occurrence of this peak appears to be related to improvements in socioeconomic status, as it did not occur in the early years of this century and does not currently occur in developing countries [16]. Worldwide, the lowest rates of childhood ALL are reported in black African children (4 per 106 annually), with rates approximately 10-fold higher in white Caucasian children [17–20]. These epidemiologic characteristics may indicate that this subset of leukemias specific to

Extramedullary disease, CD2+, CD10− Extramedullary disease, CD2+, CD10− Extramedullary disease, CD2+, CD10− Extramedullary disease Extramedullary disease Extramedullary disease Extramedullary disease Extramedullary disease

Recommended timing for blood or marrow stem cell transplantation

Following relapse Early in disease has been treatment of choice; clinical studies of targeted therapies may change this Following relapse

Contoversial; early if using research protocol Following relapse Following relapse Following relapse Following relapse Following relapse Following relapse Early in disease Following relapse Following relapse Following relapse Following relapse Following relapse Following relapse Following relapse Following relapse

childhood has a unique etiology. Experimental studies indicate that at least two molecular events are required for leukemogenesis, and the age of the children in this peak suggests that the first of these events take place in utero [21,22]. The nature of the later events which trigger overt leukemia have been the subject of speculation centering around possible infectious etiologies, perhaps related to delayed exposure to common viruses leading to an abnormal immune response [16–18,23,24]. Genetic studies of stored blood samples collected at birth have confirmed the presence of an abnormal clone at that time in at least a proportion of children with ALL, some of whom develop overt leukemia as late as their teenage years [25,26]. The majority of both adult and childhood ALL cases (70–75% of adult and 85% of pediatric cases) are derived from B-lineage cells. Approximately two-thirds of childhood ALL cases express early pre-B-cell markers, in contrast to 50% of adult cases [27–31]. This phenotype is associated with the best response to chemotherapy, regardless of patient age, perhaps because lymphocytes in this developmental stage are particularly susceptible to apoptosis, and so to killing by glucocorticoids and antimetabolites. Approximately 15% of pediatric and 25% of adult ALL cases express T-lineage markers [29,31]. Although some early studies indicated that T-lineage ALL was associated with poor outcome, treatment of these patients with intensive multidrug regimens has improved prognosis such that the T-cell phenotype is no longer an adverse risk factor in adults or children [32–36].

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Chapter 56

Table 56.2 Cooperative group studies of bone marrow transplantation for children with acute lymphoblastic leukemia in first complete remission (CR1)

Transplant group

Year of report

Number of patients

Remission status (n)

France [51] France [52]

1980–87 1982–92

32 21

Nordic [53] UK [54] USA [55] Italy [56] Spain [57]

1981–91 1985–90 1993–96 1995–2000 1993–2002

22 34 29 77 24

CR1 32 CR1 16 CR2 5 CR1 22 CR1 34 CR1 29 CR1 77 CR1 24

Preparative regimen

Follow-up (years)*

Disease-free survival (%)

Relapse (%)

TBI/CY BU + other chemotherapy

2.5 (0.6–6.8) 3.9 (1.8–6.4)

84.4 61.1

3.5 38.9

TBI/CY TBI/CY TBI/CY TBI/ETOP, BU/CY/ETOP TBI/CY BU/CY

>2 3.8 (1.1–5.9) 3.1 (0.1–4.6) 5 6.5 (1.3–12.4)

73 69 58.6 56.7 45

9 11 35 34 33

* Median and range. BU, busulfan; CY, cyclophosphamide; ETOP, etoposide; TBI, total body irradiation.

Approximately one-third of childhood early pre-B leukemia cases are hyperdiploid (>50 chromosomes), in contrast to less than 5% of adults. Hyperdiploidy is associated with a particularly favorable response to chemotherapy, perhaps because of the ability to accumulate high levels of methotrexate polyglutamates [37–39]. The most frequent structural rearrangement in childhood ALL, present in 20–30% of cases, is the translocation t(12;21)(p13;q22), which encodes a chimeric fusion product of the TEL and AML1 genes [40–44]. In addition to formation of the chimeric protein, the nontranslocated TEL allele is deleted in many cases. Loss of TEL activity is clearly a secondary event in some cases, occurring in a subclone of leukemia. This observation suggests that loss of TEL expression gives an additional proliferative advantage to the malignant cells, possibly because the normal TEL gene product can interact with the TEL-AML fusion protein in a dominant negative fashion [43]. The TEL-AML1 translocation seems to define a subgroup of childhood ALL characterized by age 1–10 years, a nonhyperdiploid karyotype, and an excellent prognosis [42]. The TEL-AML1 translocation occurs at a low frequency (<5%) in adult leukemia, which likely contributes to the poorer overall prognosis of adult ALL [45–48]. The Philadelphia chromosome t(9;22)(q34;q11) results in the production of a chimeric fusion protein derived from fusion of the BCR and ABL genes. In contrast to TEL-AML1, the Philadelphia chromosome is frequent in adult leukemias (25–30%), infrequent in childhood leukemias (3–5%), and associated with a reduced prognosis regardless of age. Philadelphia-positive leukemia often proves resistant to even the most intensive chemotherapy regimens [1,49,50]. The biologic characteristics described here indicate that the majority of children with leukemia have disease likely to be responsive to current chemotherapy regimens with acceptable toxicity, for example early preB disease with either hyperdiploidy or the cryptic TEL-AML1 fusion transcript. In contrast, adult patients commonly have biologic features associated with drug-resistant disease, such as the Philadelphia chromosome and less favorable immunophenotypes. The biologic differences between adult and pediatric ALL lead to important differences in the appropriate indications for transplantation for children with ALL compared with adults.

Outcome of sibling donor transplantation in children with ALL Transplantation in first remission Children with ALL with unfavorable biologic features, for example BCR-ABL or hypodiploidy, have poor outcomes with chemotherapy and

may benefit from HCT in first remission. Results of HCT for childhood ALL in first complete remission (CR1) are summarized in Table 56.2. Taken together, these studies generally show a reduction in relapse rate with transplantation in CR1, with increased treatment-related mortality (TRM) compared with chemotherapy. However, it is a continuing challenge for such analyses that study data no longer reflect current outcomes with chemotherapy by the time they are sufficiently mature for publication. It is important to note that many of the criteria used to define high risk in each of these studies (e.g. T-cell ALL and mediastinal mass, high white blood cell [WBC] count, and t(4;11)) are no longer associated with markedly inferior outcomes with modern chemotherapy and would not currently be considered an indication for HCT in first remission. Cooperative children’s cancer therapy groups worldwide have successfully investigated the role of chemotherapy in the treatment of childhood ALL for over 30 years [80–92]. Considerable effort has been directed towards identifying subsets of children with inferior prognoses so that additional or alternative therapies can be targeted to these children, and the use of risk-adapted therapies has been one of the major achievements in the management of childhood ALL. Over time, approaches to risk classification have become more complex and more effective in predicting prognosis [58]. The simplest level of ALL risk classification is the use of National Cancer Institute (NCI)/Rome standard criteria (age and WBC count at diagnosis) to broadly categorize leukemias, as high risk (WBC count ≥50,000/ μl or age ≥10 years) or standard risk (WBC count <50,000/ μl and age 1–9.99 years) [93]. These criteria are widely used to assign therapy for children with B-lineage ALL, but are less effective in categorization of children with T-lineage disease. The molecular genetics of the ALL blasts and the response of the disease to initial therapy (rapid or slow early response) are now routinely also used in risk classification, and intensified chemotherapy is given to children in poor prognostic groups [82,91,96]. Typically, only a minority of children with ALL is considered for HCT in first remission. In an effort to identify children with very-highrisk ALL who should be considered for HCT in first remission, Schultz et al. proposed a new classification system and treatment algorithm based on a combined analysis of clinical, biologic, and early response data predictive of event-free survival (EFS) from previous ALL studies conducted by the Children’s Cancer Group (CCG) and the Pediatric Oncology group (POG) [58]. A total of 11,779 children (age 1–21.99 years) with newly diagnosed B-precursor ALL were consecutively enrolled by the CCG (December 1988 to August 1995, n = 4986) and POG (January 1986 to November 1999, n = 6793). A total of 6238 patients had informative cytogenetic data and were included in the

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children

retrospective analysis. Very-high-risk group criteria defined in this analysis included extreme hypodiploidy (fewer than 44 chromosomes), t(9;22) and/or BCR/ABL fusion, and induction failure. These data are in agreement with reports from other cooperative study groups from the United States and Europe that report significantly inferior outcomes in childhood ALL with t(9:22), with hypodiploidy and in infants [80– 83,85–86,91–93]. These data support the early use of HCT in children with ALL falling into these categories, as outcomes with chemotherapy are clearly unsatisfactory. It will remain important to follow outcomes carefully, however, as new drugs such as imatinib, or other improvements in chemotherapy, may improve survival sufficiently to allow use of transplantation only in CR2, as TRM and late consequences of therapy are greater with transplantation than chemotherapy. The small number of high-risk ALL cases in specific leukemia subtypes has limited ability to determine the effectiveness of early HCT in increasing survival. In an effort to circumvent this limitation, data from a number of international study groups have been pooled to examine therapy outcomes for children with ALL carrying a Philadelphia chromosome [49]. Data on 326 children and young adults treated by 10 study groups were analyzed. The analysis showed that HCT from a matched sibling donor is superior to other types of transplantation, and to intensive chemotherapy alone in prolonging initial complete remission (Fig. 56.1). These data support the common clinical practice of offering HCT in first remission to children with Philadelphia chromosome-positive ALL and a matched sibling donor. However, for the majority of children without a matched sibling donor, there is less clear consensus on optimum management. The report from Aricò et al. included 21 children treated with unrelated donor HCT, and showed no improvement in survival in these cases compared with those treated with chemotherapy [49]. A report of 15 children with Philadelphia-positive ALL (nine in CR1 and six beyond CR1), treated with a T-cell-depleted unrelated donor HCT at a single center reported 2-year overall survival (OS) of 44% and disease-free survival (DFS) of 37% [94]. A report from Seattle, addressing children and adults with

Probability of leukemia-free survival

1.0

p < 0.001

0.8

0.6

0.4

Transplantation

0.2

Chemotherapy

0.0 0

12

24

36

48

60

72

Months after treatment Transplantation Chemotherapy

376 540

192 187

113 105

83 72

66 50

46 35

38 24

Fig. 56.1 Estimates of disease-free and overall survival (± SE) in 267 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with transplantation of bone marrow from human leukocyte antigen-matched related donors or chemotherapy alone. p = 0.002 for the comparison of the two treatments with respect to overall survival; p < 0.001 for the comparison with respect to disease-free survival.

809

Philadelphia-positive ALL, describes six of seven patients surviving after unrelated donor HCT in CR1 [95]. Investigators from the United Kingdom recently reported results of 42 children with Philadelphiapositive ALL treated on the Medical Research Council (MRC) ALL 97 trial [96]. Thirty-six (86%) achieved CR1 at the end of induction, and 28 underwent HCT. Eleven were transplanted using sibling donors, and 17 used unrelated donors (a donor source was not available for one patient). Outcomes were similar for both donor types. The EFS for sibling donor recipients was 45% (17–71%) and for unrelated donors 68% (39–85%) (p = 0.1 for both OS and EFS). Similarly, Talano et al. [97] reported encouraging results for the use of alternate donor transplantation for Philadelphia-positive ALL in CR1. Between 1987 and 2002, 29 patients were transplanted at the Children’s Hospital of Wisconsin, using either unrelated donors (n = 23) or a mismatched family donor (n = 6). EFS at 3, 5, and 10 years was 56%, 51%, and 46%, respectively. The TRM remains high at around 20%, and there is a 20% recurrence rate of disease post transplant. Continued investigation of the role of unrelated donor HCT in first remission for the children in this poorer-prognosis group is justified. More insight into different associated prognostic factors will also allow better stratification of patients to help improve the outcome. The multicenter report by Aricò et al. [49] was the first with sufficient cases for analysis of multiple prognostic factors within the Philadelphiapositive subgroup. There were important differences in outcomes in children with Philadelphia-positive ALL treated with chemotherapy, according to modified NCI/ Rome criteria, suggesting that chemotherapy may be an adequate treatment for those with the most favorable characteristics. Children 10 years old or younger with a WBC less than 50,000/ mm3 at diagnosis had a 5-year DFS of 49%. However, children of any age with a WBC at diagnosis of greater than 100,000/mm3 had a 5-year DFS of 20%. Additional data suggesting that a better-risk group of children with Philadelphia-positive ALL can be identified were reported by Roy et al. [96]. This UK MRC study (MRC ALL 97) showed improved survival for children with a good response to initial therapy (<25% blasts in the bone marrow within the first 2 weeks of treatment), with an EFS of 68% (43–84%) compared with 39% (18–59%) in the slow responders (>25% blast in the bone marrow after 2 weeks of treatment) (p = 0.03). Moreover, children presenting with a WBC lower than 50,000/mm3 had better OS than those with higher counts (p = 0.02). Recent studies have also explored the role of additional cytogenetic abnormalities in the outcome of Philadelphia chromosome-positive ALL. Over two-thirds of lymphoblasts with a Philadelphia chromosome contain secondary nonrandom chromosomal abnormalities [96,98]. An additional Philadelphia chromosome is found in about a third of cases of Philadelphia-positive ALL [99,100], and these patients have a better outcome [96,98]. In parallel with other children with ALL, the presence of hyperdiploidy [101] is also associated with a better outcome [96,98]. Those with a loss of chromosomal material (e.g. del(9) and del(7)) appear to have a significantly poorer outcome [96,98]. The tyrosine kinase inhibitor imatinib has been shown to have activity in childhood ALL, and current clinical trials are studying whether chemotherapy outcomes can be improved by including imatinib with intensive chemotherapy [102–108]. In addition, investigators have shown that imatinib is well tolerated when given prophylactically post transplantation, although current follow-up is too short to determine whether outcomes are improved [109]. Children with severe hypodiploidy (<45 chromosomes) have poor outcomes (<40% survival) with chemotherapy [110]. Raimondi et al. evaluated the cytogenetic records of 979 patients with ALL and reported that EFS was significantly lower for patients with less than 45 chromosomes compared with those with 45 or more (20% ± 10.3% versus 74.9% ± 1.6%; p < 0.001) [111]. Most recently, Nachman et al. [112]

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reported 139 patients with ALL and hypodiploidy (<45 chromosomes) collected from 10 different national ALL study groups and single institutions. In contrast to previous studies suggesting that near-haploid cases with 24–29 chromosomes have particularly poor outcome, this report found no difference in outcome between patients with 24–29, 33–39 or 40–43 chromosomes, with poor outcomes in all these cases. However, compared with patients with fewer than 44 chromosomes, patients with 44 chromosomes had a significantly better EFS (52.2% versus 30.1%; p = 0.01) and OS (69% versus 37.5%; p = 0.017). Twenty-nine children with hypodiploid ALL transplanted in first or second remission with a matched sibling donor between 1990 and 2001 have been reported to the International Bone Marrow Transplant Registry (IBMTR). Threeyear survival was 65% (95% confidence interval [CI] 45–80%), supporting the use of early HCT in this small subgroup (Eapen, personal communication, 2002). Cooperative group studies have contributed the important observation that initial response of childhood ALL to therapy is a strong indicator of long-term outcome, with better outcomes in rapid early responders [91–93]. Additionally, it has been shown that a proportion of slow responders (whether measured by clearance of peripheral blasts after 7 days of prednisone, or marrow examination after 7 or 14 days of induction chemotherapy) can be subsequently rescued by intensification of therapy [113,114]. Intensification of chemotherapy improves survival to around 70% in poor responders, so these children do not generally require transplantation in first remission. However, a small subset of slow responders fail to achieve remission after 28 days of chemotherapy, and for those children, if they do achieve remission, only 40% will survive, suggesting that early transplantation once remission is achieved might be of benefit [91]. Newer technologies are being developed to better assess response to therapy in childhood ALL, as early response is such a powerful prognostic indicator. The detection of minimal residual disease (MRD) using immunophenotyping or molecular detection of residual cells is increasingly being applied to clinical trials of chemotherapy for ALL (see Chapter 26) [115–120]. These studies have the potential to identify children who have an inadequate response to therapy or show early predictors of relapse, and might benefit from early intervention with HCT. Few studies have directly compared outcomes of children with ALL thought to be very high risk transplanted in first remission with matched chemotherapy-treated controls. Some of these studies have shown superior outcomes in transplanted cases [53,55,56]. In contrast, two consecutive MRC protocols identified cases considered to be very high risk for relapse (survival expected to be <40%), and offered sibling donor HCT to those with available donors [120]. The data showed a small benefit of 4.6% improvement in 10-year EFS in transplanted patients. However, when patients with a human leukocyte antigen (HLA)-matched donor were compared with those without, regardless of treatment received (intent-to-treat analysis), patients with a donor had an adjusted EFS 10.7% lower than those without a donor. Similarly, recently published results of the Programa para el Tratamiento de Hemopatías Malignas (PETHEMA) ALL-93 trial [57] failed to show any advantage of HCT over chemotherapy in CR1. These studies illustrate some of the challenges of transferring excellent single-center outcomes to cooperative group studies. Difficulties include heterogeneous populations, noncompliance with assigned therapy leading to selection bias, and transplantation at multiple smaller centers. Transplantation at centers with higher annual numbers of transplants and longer experience has been associated with improved outcomes [121,122]. Continuing investigation of the role of HCT in first remission will require careful attention to the optimization of statistical and clinical trial design, and the evaluation of newer transplantation

strategies, including newer hematopoietic cell sources and perhaps reduced-intensity conditioning regimens. Outcomes of transplantation in second or subsequent remission There are a number of single- and multiple-institution studies describing the outcome of HCT for patients with recurrent ALL. Many of the studies combine data for children and adults. In this chapter, where possible, the data from children are highlighted. A summary of results of sibling donor HCT for children with ALL in second or subsequent remission in single-institution and multi-institution studies is presented in Tables 56.3 and 56.4. Reported DFS ranges from 23% for patients with advanced disease, to 35–64% for cases transplanted in second remission [59–70]. The length of first remission is key in determining the outcome of relapsed ALL in children. Survival rates are typically poor with chemotherapy when relapse has occurred early (duration of first remission less than 3 years) [65,77,84]. However, a proportion of children with later relapses can be long-term survivors after re-treatment with chemotherapy [50,123–125]. The appropriate use of HCT or chemotherapy alone for children with relapsed ALL remains a controversial topic, and there have been no successful randomized studies to compare transplantation in second remission with further chemotherapy. A number of cooperative groups, including the Children’s Oncology Group, have attempted to compare outcomes of transplant and chemotherapy in randomized studies and have been unsuccessful, largely due to lack of acceptance of randomization by either or both of physicians and parents. In the absence of randomized data, the best available comparison comes from registry analyses. Eapen et al. compared 188 patients enrolled in POG chemotherapy trials and 186 children who received HLA-matched sibling transplants, treated between 1991 and 1997 (see above) [126]. For children with early first relapse (<36 months from diagnosis), risk of a second relapse was significantly lower after total body irradiation (TBI)-containing transplant regimens (relative risk [RR] 0.49, 95% CI 0.33–0.71%; p < 0.001) than after regimens based on chemotherapy alone. Patients receiving radiation-based regimens also had higher leukemia-free survival (LFS) and OS rates. In contrast, transplantation in CR2 did not offer advantage over chemotherapy for children with a late (≥36 months) relapse (p = 0.78). Transplant recipients who received a non-TBI-containing regimen fared poorly; recurrence rates were higher than those in children treated with chemotherapy alone, with correspondingly lower LFS and OS rates. In a smaller single-center study, investigators at the Memorial SloanKettering Cancer Center (MSKCC) compared HLA-matched sibling donor transplantation using hyperfractionated TBI and cyclophosphamide (CY) (n = 38) with chemotherapy (n = 37) for children with ALL in second remission [65]. DFS was improved in the transplantation group, and an advantage for HCT was seen in those with both short and long first remissions. However, these data likely do not reflect outcomes achievable with current chemotherapy protocols as children were treated between 1979 and 1992. The Italian Bone Marrow Transplant group reported on 57 children who received allogeneic transplantation for ALL in second remission and compared them with 230 patients who received chemotherapy following their relapse [84]. They demonstrated that patients who had an early (<30 months) first relapse had significantly longer DFS following allogeneic transplantation than treatment with chemotherapy. However, similar to the IBMTR analysis, this advantage was lost in patients with a later relapse (>30 months following diagnosis). A comparison of allogeneic BMT versus chemotherapy for children with ALL in second remission was reported from Spanish investigators first in 1989, and

1973–85 1979–85

1978–80 1979–91

1986–92 1988–93

1979–92 1990–97

Seattle [59] Memorial Sloan-Kettering [60]

Minnesota [61] Minnesota [62]

Boston [63]

Australia [64]

Memorial Sloan-Kettering [65] Australia [66]

37 20

26

17

15 123 (85 <18 years)

57 59

Number of patients CR2 57 CR2 31 CR3 12 CR4/relapse 16 ≥ CR2 15 CR1 19 CR2 68 CR3 36 CR2 14 CR3 3 CR1 2 CR2 22 ≥ CR3 2 CR2 37 CR2 17 CR3 1 Relapse 2

Remission status (n)

TBI/CY TBI/CY

CY/TBI CY/TBI (80) TBI/ARAC (15) TBI/CY (28) CY/ARAC/TBI CY/ETOP/TBI BU/CY/MEL

CY/TBI TBI/CY

Preparative regimen

NS 6.3 (4.5–8.5)

4.8 (2.5–6.2)

4.6 (3–7.3)

NS (1.4–10.4) CR2 5.1 (2.7–6.9) CR3 5.3 (3.9–6.5) CR4/relapse 6.2 (1.5– 7.0) 4 7.8 (1–12.7)

Follow-up (years)*

62 55

27

53

40 64 42 23 43 29

Disease-free survival (%)

* Median and range. ARAC, cytosine arabinoside; BU, busulfan; CR, complete remission; CY, cyclophosphamide; ETOP, etoposide; HCT, hematopoietic cell transplantation; Mel; melphalan; NS, not stated; TBI, total body irradiation.

Years of HCT

Transplant group

Single-institution studies

Table 56.3 Sibling donor bone marrow transplantation for children with acute lymphoblastic leukemia

19 NS

4

31

NS 13 25 64 NS 56

Relapse (%) Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children

811

NS

1981–89

1983–91 1983–93 1986–93 1980–88 1988–95

1995–99

1991–97

German [67]

Multiple in US [68]

IBMTR [69] France [70] Italy [71] Spain [72] IBMTR [73]

Spain [74]

COG & IBMTR [75]

186

67

255 42 46 21 627

213

51

Number of patients

PBSC CR1 CR2 ≥CR3 or relapse BMT CR1 CR2 ≥CR3 or relapse CR2

CR1 CR2 CR ≥ 3 relapse

CR1 CR2 CR3 CR2 CR2 CR2 CR2 CR1 CR2 CR ≥ 3 relapse

CR2

Remission status (n)

186

25 119 32 255 42 46 21 51 73 27 25 134 194 51 72 34 9 15 10 33 9 195

51

2.1

8.4 (1.3–13.3)

CY + TBI ± other TBI ± other BU + CY ± other

NS 3 (0.1–6) 2.8 (0.8–7.5) 14.5 (11.1–18.5)

NS

2.5 (0.1–5.6)

Follow-up (years)*

BU/CY CY/TBI

CY/TBI (451)

Multiple Multiple High-dose VCR/TBI/CY multiple BU/CY (176)

CY/TBI ETOP/TBI ARAC + TBI

Preparative regimen

43

27.1

54.9

41

28.7

35

45 17 30 40 41

NS

NS

Relapse (%)

53

50

40 53 58.2 42.8 35

38

5

Disease-free survival (%)

* Median and range. ARAC, cytosine arabinoside; BU, busulfan; CR, complete remission; CY, cyclophosphamide; ETOP, etoposide; HCT, hematopoietic cell transplantation; Mel; melphalan; NS, not stated; TBI, total body irradiation; VCR, vincristine.

Years of HCT

Transplant group

Multi-institutional studies

Table 56.4 Sibling donor bone marrow transplantation for children with acute lymphoblastic leukemia

812 Chapter 56

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children

updated in 1999 [72]. DFS in children undergoing transplantation was superior to that of the group of children receiving chemotherapy (43% versus 10%; p = 0.001), with notably long median follow-up of 14.5 (range 11.1–18.5) years. In 1991, the German pediatric cooperative groups (BFM and COALL) reported 51 children with second-remission ALL, all treated with similar initial chemotherapy, who then received a matched sibling donor transplantation after a relapse [67]. Comparison of HCT results with outcomes in children treated with chemotherapy for a bone marrow relapse showed that patients with an initial remission longer than 18 months had comparable survival whether treated with chemotherapy or bone marrow transplantation. Patients who had a very early relapse (first remission less than 18 months) or a relapse of T-cell ALL had a minimal chance of surviving following chemotherapy, and survival rates were significantly improved by allogeneic HCT. Outcomes of 56 children with relapsed ALL were reported in a singleinstitution study from Australia [66]. All patients with a matched family donor (n = 20) received transplantation, and the other patients received chemotherapy (n = 32) or alternative donor transplants (n = 2). EFS at 8 years was 55% in children with an available family donor compared with 9.2% in patients without a matched family donor (p = 0.002). All the above data, when taken together, indicate that HCT from a matched family donor is usually the best option for a child with relapsed ALL when relapse is early. Data are currently insufficient to determine whether similar benefit can be achieved with alternative donor HCT, although with an eightallele matched unrelated donor, results comparable to those seen with sibling donors can be achieved. The impact of graft-versus-host disease (GVHD) on quality of life, likely to be a larger problem with unrelated than related donors, remains to be established. The majority of studies indicate that children with later relapse can achieve similar survival with chemotherapy as with transplantation, so treatment options should be discussed carefully with the family. A careful reassessment of the morphologic, cytogenetic, and molecular genetic characteristics of the leukemia is essential at the time of relapse to confirm recurrence of the original disease and not development of a new secondary leukemia, either ALL or acute myeloid leukemia (AML), perhaps related to the use of chemotherapy [127,128]. Reinduction chemotherapy is successful in achieving remission in 70–90% of children with relapsed ALL. The problem of how to manage a patient when attempts at chemotherapy reinduction fail is not resolved. In some centers, such patients will be considered for allogeneic transplantation even in relapse. There are currently insufficient published data to predict outcomes accurately in these patients, although experience suggests extremely high TRM and a high post-transplant relapse rate because of resistant leukemic cells, limiting enthusiasm for such an approach. When considering the role of HCT in children with ALL, it is important to recognize that there has been continuous progressive improvement in chemotherapy outcomes, and that HCT can be associated with significant late morbidity [129–131]. Comparisons of HCT and chemotherapy outcomes must be contemporary and ongoing to allow appropriate therapeutic choices. Table 56.5 summarizes current approaches to indications for HCT for children with ALL.

Selection of conditioning regimens The conditioning regimen used to prepare a patient for a high-dose HCT for childhood ALL is expected to provide sufficient immunosuppression to facilitate donor cell engraftment, and cytotoxicity to assist in the eradication of residual leukemic cells. In the early years of transplantation, most regimens included TBI given in a single fraction. More

813

recently, TBI has typically been administered in multiple fractions to reduce toxicity. Hyperfractionated TBI combined with CY, now considered a standard regimen, was first introduced for the treatment of children with leukemia by investigators at MSKCC [60]. After the original reports of the use of CY and TBI, several investigators added other chemotherapeutic agents to the regimen, or substituted other drugs for CY in an attempt to improve outcomes. Although studies with some of these other combinations, such as TBI and Ara-C (cytosine arabinoside) showed initial promise [122], additional toxicity often occurred and the long-term outcome was often not appreciably changed. To specifically address the value of AraC in conditioning, data were combined from 14 centers that used a TBI and Ara-C regimen in a total of 213 allogeneic HCT recipients with ALL [68]. This study included children and adults, although 89% of cases were less than 25 years of age. The overall 3-year DFS was 38% (CI 31–45%), similar to that reported with other regimens [125]. Investigators at the University of Minnesota analyzed the results of four different regimens tested sequentially over 12 years (1979–1991) using histocompatible related donor marrow grafts [62]. One hundred twenty-three patients with ALL were treated, 92 of whom were less than 20 years of age, with a median follow-up of 7.8 years. The regimens studied included CY plus single-dose TBI (n = 35), CY plus fractionated TBI (n = 45), TBI plus high-dose Ara-C (n = 15), and hyperfractionated TBI plus CY (n = 28). Outcomes of all the regimens were similar, with 29% (CI 21–37%) DFS. The only notable difference among the regimens was that the regimen using TBI/Ara-C was associated with greater treatment-associated mortality, in agreement with other reports. In this comparison, the regimen originally reported from MSKCC using hyperfractionated TBI and CY was no more effective in prevention of leukemic recurrence and offered no substantial advantage over the prior regimens. Investigators at the Hôpital Saint-Louis in Paris, France, evaluated 42 children with ALL in second remission following allogeneic transplantation to address the value of multiple different conditioning regimens [70]. In this study, it was noted that there were no post-transplant relapses in children receiving the conditioning regimen of TBI, Ara-C, and melphalan (TAM). The authors suggested a possible advantage of using the melphalan-conditioning regimen in the eradication of ALL, although differences in outcome by conditioning regimen did not reach statistical significance in this small study. Investigators in Italy evaluated the feasibility of high-dose vincristine (4 mg/m2 over 4 days) with fractionated TBI and CY as a conditioning regimen for allogeneic bone marrow transplantation [71]. The 3-year EFS rate was 58.2% (40–76%), and the relapse rate was 30% (12–49%). Investigators from MSKCC have described the use of hyperfractionated TBI, thiotepa, and CY as a conditioning regimen for children with ALL receiving T-cell-depleted grafts from related or unrelated donors [132]. Preliminary reports suggest excellent outcomes, with 87% DFS at 3 years in 11 related marrow recipients and 73% DFS in 23 unrelated donor marrow recipients, all transplanted in first or second remission. More encouraging data come from investigators at the City of Hope National Medical Center and in Kiel, Germany, who have studied the substitution of etoposide (VP-16) for CY in the treatment of ALL patients receiving HCT [133,134]. Their studies suggested that etoposide and TBI are associated with a decreased relapse rate following transplantation for ALL, although data from a prospective randomized comparison are not available. Other investigators in Germany also used etoposide and fractionated TBI in 23 children with ALL in second remission [67]. Their data also suggested a reduced relapse rate in these patients compared with those receiving CY and TBI, with or without Ara-C.

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Table 56.5 Indications for transplantation in childhood acute lymphoblastic leukemia Donor type Stage of leukemia at time of BMT

Matched sibling or one-antigen-mismatched family member

Unrelated marrow or cord blood

CR1, Philadelphia chromosome positive

Indicated; poor results with chemotherapy [1,49], transplant studies suggest improved outcome [49,94,95,227–229]

Indicated for those with NCI high-risk characteristics [94–97]. Children with NCI good-risk characteristics may achieve equivalent results with chemotherapy [48]. Data should be contributed to registries to allow evaluation of outcomes

CR1, t(4:11) or other MLL rearrangement in infant <1 year old at diagnosis

Indicated; poor results with chemotherapy [11–15] Reported results suggesting improved outcome with BMT [230–233,191] involve small numbers and not all studies report benefit; careful prospective evaluation needed Note: some infants have biologic characteristics of betterrisk disease (above 6 months of age, germline MLL) and may have good outcome with chemotherapy

Poor results with chemotherapy, mixed results for outcome with unrelated donor stem cells [166,193–195]. Should be considered for patients with high-risk features. Data should be contributed to registries/group studies to allow evaluation of outcomes

CR1, >28 days to achieve CR

Transplant may be offered; limited data available comparing outcome with outcome of chemotherapy [70,86]

Transplant may be offered, particularly in cases with other highrisk features; data not available comparing outcome with outcome of chemotherapy

CR2, relapse with CR1 <36 months

Indicated. Outcome improved in transplant recipients in case-control comparison [66,70,77,125–126]

Indicated; good outcomes reported in significant series of cases [160–162,170,172]; however, careful case-control comparison is still needed to confirm improved outcome. Single-center and multicenter studies suggest outcomes can be equivalent to related donor BMT using URD matched at HLA-A, B, C, and DRB1 [163–164,171]

CR2, relapse with CR1 >36 months

Transplant typically offered; chemotherapy may offer an equivalent outcome [50,65,77,123,125–126]

Generally not offered to those with very late relapses; those with relapse just beyond 36 months may be considered. Singlecenter and multicenter studies suggest outcomes can be equivalent to related donor BMT using URD matched at HLA-A, B, C, and DRB1 [163–164,171]

CR3 and higher

Transplant indicated; cure with chemotherapy unlikely

Transplant indicated; cure with chemotherapy unlikely

Relapse

Results poor; transplant not generally offered unless in context of investigation of new BMT strategies

Results poor; transplant not generally offered unless in the context of investigation of new BMT strategies

Isolated extramedullary relapse

Controversial: transplant may be offered if CNS relapse occurs early (<18 months from diagnosis) and on therapy as outcomes with chemotherapy are poor in some reports [123]. Registry data suggest no benefit from transplant [234]. Not generally offered for testicular relapse or for CNS relapse occurring after end of therapy which can respond well to chemotherapy [123]

Transplant not generally offered unless associated with marrow relapse

BMT, bone marrow transplantation; CNS, central nervous system; CR1, first complete remission; NCI, National Cancer Institute; URD, unrelated donor.

An etoposide- and TBI-conditioning regimen has been tested in highrisk patients, primarily adults, undergoing bone marrow transplantation during a CR1, with good outcomes [135]. Jamieson et al. [136] reported 61% EFS and 62% OS for children transplanted in CR2 after a conditioning regimen of fractionated TBI (1320 cGY) and 60 mg/kg etoposide, with acceptable regimen related toxicity and TRM. Investigators from Toronto, Canada, compared CY and TBI (50 mg/kg CY for 4 days – a higher dose than most centers use – and 1200 cGy TBI in six fractions) with etoposide plus TBI (a single dose of 60 mg/kg of etoposide and 1200 cGy TBI in six fractions) in 107 children with ALL transplanted between 1990 and 2003 and reported no differences between the two conditioning regimens [137].

A recent large Center for International Blood and Marrow Transplant Research (CIBMTR) study compared 298 patients (adults and children) with ALL in CR1 or CR2 receiving HLA-matched sibling allografts after CY/TBI with 204 patients receiving etoposide/TBI [138]. Four groups were compared: CY/TBI <13 Gy (n = 217), CY/TBI of 13 Gy or more (n = 81), etoposide/TBI <13 Gy (n = 53), and etoposide/TBI of 13 Gy or more (n = 151). TRM did not differ by conditioning regimen, and in CR1 there were no significant differences in relapse, LFS or survival by conditioning regimen. In CR2, important differences in outcome among conditioning groups were identified. The risks of relapse, treatment failure, and mortality were lower in recipients of etoposide (regardless of TBI dose) or with TBI doses of 13 Gy or more

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children

in patients receiving CY. A striking reduction in relapse risk was observed in patients receiving 13 Gy or more TBI with CY when compared with CY/TBI using less than 13 Gy of radiation (p = 0.0016). These data are further evidence of the contribution of the conditioning regimen to disease control in transplantation for ALL. Concerns about the late effects of TBI in children have led to a number of studies of conditioning regimens using only chemotherapy. Early studies of busulfan and CY as a conditioning regimen focused on the treatment of myeloid malignancies, and data indicated that the regimen was adequate to achieve donor cell engraftment [139]. The use of a radiation-free conditioning regimen is an attractive option for young children if equivalent survival can be demonstrated as fewer late adverse effects on growth, endocrine, and cognitive function might be expected. A study from Australia evaluated a conditioning regimen combining busulfan and CY with single-dose melphalan in 25 patients with ALL, the majority in second remission [64]. Toxicity with this regimen was unacceptable, with 42% of the patients dying of regimen-related toxicity. There was a 50% incidence of interstitial pneumonitis, and 35% of the patients had severe hemorrhagic cystitis. Because of the severe toxicity, survival was not improved over that of a historical control group transplanted earlier using CY and TBI, despite a low relapse rate. An IBMTR analysis of children with ALL receiving HLA-identical sibling donor HCT compared outcomes with conditioning regimes of CY and TBI (n = 451) or busulfan and CY (n = 176) [73]. Patients from 144 institutions, transplanted between 1988 and 1995, were included, and patients were in first, second or subsequent remission or relapse at the time of transplantation. The 3-year probability of LFS was significantly higher in the group receiving CY and TBI compared with the group receiving busulfan and CY (Fig. 56.2). The risk of relapse was similar in the two groups, but TRM was higher in children receiving busulfan and CY. Transplantation not in remission, short duration of first remission (for transplants performed after first relapse), presence of a t(4;11) translocation in leukemic blasts, and use of T-cell depletion or combined methotrexate and cyclosporine, compared with cyclosporine or methotrexate alone, for GVHD prophylaxis were associated with lower survival rates. Bunin et al. [140] reported similar inferior outcomes of a busulfan and CY conditioning compared with TBI and CY in a small but prospective trial conducted by the Pediatric Blood and Marrow Transplant Consortium. Together, these reports indicate that, in the past, modifications of conditioning regimens for children with ALL have had limited success in improving outcomes. Inclusion of additional chemotherapeutic agents has, in some instances, led to increased morbidity and mortality without

Preparative regimen

1.0 0.8 TBI/CY (n = 451)

0.6 0.4

Bu/CY (n = 176) 0.2 p = 0.005 0.0 0

1

2

3

4

Years

Fig. 56.2 Leukemia-free survival after human leukocyte antigen-identical sibling donor transplant for childhood acute lymphoblastic leukemia according to conditioning regimen used.

5

815

significant improvement in long-term DFS. In addition, current data suggest reduced survival with radiation-free regimens. However, a number of recent studies indicate that the use of TBI and etoposide, instead of TBI and CY, or an increase in the TBI dose in the CYcontaining regimens, or the addition of thiotepa to TBI and CY might reduce post-transplant relapse and improve outcome [67,133,134,138]. These are important areas for future investigation.

Alternative donor HCT for children with ALL Mismatched family member transplantations in children with ALL Many of the problems of hematopoietic cell donor availability could be addressed using one-, two- or three-antigen-mismatched family member donors, who are available for almost every patient [141]. Early studies of highly mismatched related donor transplantations reported high rates of graft failure and GVHD, limiting enthusiasm for this approach [142,143]. Although newer transplantation approaches have shown improvement in outcomes, experience with transplantation using mismatched family member donors remains limited, and to a large extent focused in a small number of centers with particular interest and expertise in this strategy. There are few data regarding the use of these transplants for uniform patient populations, although there are several series summarizing overall experience at single institutions. In reports of heterogeneous patient groups, a number of authors have shown that results using a one-antigen-mismatched family member as a donor can be comparable to those achieved using a matched sibling donor, while risk of treatment failure increases incrementally with increasing HLA disparity [144– 147]. In keeping with this, a report of a series of 82 patients with leukemia (adults and children) treated with marrow grafts that had been T-cell depleted ex vivo with the monoclonal antibody T10B9 described a survival of 31% for the entire cohort [148]. It should be noted, however, that only one of 20 patients receiving a three-antigen-disparate graft was surviving at the time of reporting. Investigators in Perugia, Italy, have pioneered the use of a high degree of T-cell depletion together with infusion of a high dose of haploidentical CD34+ cells to overcome the problems of GVHD and graft failure associated with highly mismatched haploidentical grafts [149]. These authors demonstrated that donor-versus-recipient natural killer cell alloreactivity can eliminate leukemia relapse and graft rejection, and protect patients against GVHD in cases with AML. Unfortunately, activity against leukemia cells appeared to be specific for AML and not ALL blasts, and outcomes were not satisfactory in cases of ALL in this series. A pediatric study using a similar approach described outcomes in 27 children with ALL (seven in CR1, 10 in CR2, four in CR3, and six with refractory disease) [150]. Engraftment was rapid in 26 patients. Two patients who rejected the first graft and one patient with initial nonengraftment were successfully regrafted. Survival for the group overall is 33%, and 44% for children in remission at time of transplantation; none of the children transplanted without achieving remission survived. Relapse and infection were important causes of treatment failure. Investigators form Bristol, United Kingdom, reported their experience of using haploidentical family members as donors for 34 children with leukemia, of which 14 had ALL [151]. The conditioning regimen included CY and TBI (14.4 Gy in eight fractions). All patients received T-cell-depleted peripheral blood stem cell grafts with a median CD34 cell dose of 13.8 (range 4.2–35.1) × 106/kg, and 0.7 × 104 CD3+ cells/kg. The actuarial survival at 2 years was 26% (95% CI 13–41%). Eight patients survived disease-free with a median follow up of 62 months. Similar to results of other groups, relapse (14 patients) and adenoviral

Chapter 56

(six patients) or fungal (four patients) infections were the major causes of death. Outcomes for children with ALL treated with haploidentical transplantation are perhaps disappointing when compared with well-matched unrelated donor marrow or cord blood grafts, but may represent the only treatment option for children with infrequent tissue types. New directions in the use of adoptive cellular immunity, and improved pre-emptive monitoring and treatment of viral infections, appear to be promising in this respect and will continue to help investigators exploring this transplantation strategy. Unrelated donor HCT Donor selection The use of phenotypically matched unrelated donors has increased in parallel with the remarkable growth of registries of HLA-typed potential bone marrow donors, such as the National Marrow Donor Program (NMDP) in the United States, which facilitate the process of donor identification and marrow acquisition [152,153]. Additionally, refinements in HLA-typing technologies have allowed for more accurate matching at class I and class II loci [154,155]. Despite these improvements, donor identification can still be a logistical challenge, particularly for patients in whom remissions are likely to be short, such as those with ALL beyond first remission. An analysis of consecutive referrals to the University of Minnesota between September 1991 and August 1993 showed that an unrelated donor was identified in 37% of searches a median of 10 weeks after search initiation [156]. However, more recent data indicate a median time to donor identification of 49 days, suggesting that strategies by registries and transplant centers to reduce search times have been effective [157]. An improved search algorithm based on the conversion of HLA assignments of patients and donors into search determinants, and on allele and haplotype frequencies (HapLogic), is now routinely used by the NMDP registry, and greatly facilitates the identification of eight-allele-matched donors [158]. The use of less closely matched grafts can increase the pool of available donors, and data indicate that young patients can tolerate greater degrees of HLA mismatch than adult patients. A study of 211 consecutive unrelated donor marrow transplantations performed at the University of Minnesota showed that in younger (<18 years) recipients, survival was not significantly different after matched (HLA-A, B, and DRB1) or major mismatched (HLA-A or B locus) donor HCT (survival 53% versus 41% at 3 years; p = 0.4) [159]. In contrast, survival after matched unrelated donor HCT for adults was significantly better than that with mismatched donors (survival 30% versus 10% at 3 years; p < 0.01). These data were confirmed in a study, performed in Bristol, United Kingdom, of 50 children with ALL in second remission who received T-cell-depleted unrelated donor grafts [160]. In this study, 15 of 50 patients received marrow with a single locus mismatch at HLA-A, B or DRB1, and there was no significant difference in EFS between the matched and mismatched groups (51% versus 57%; p = 0.73) (Fig. 56.3). An update of this experience indicated an increased frequency of graft failure in recipients of HLA-mismatched T-cell-depleted grafts (11.8%, n = 52), but still no reduction in LFS compared with recipients of matched grafts (45% versus 40%; p = 0.65) [161]. Outcomes Initial reports of the results of unrelated donor transplantations necessarily included small, heterogeneous groups of patients. As donor availability has improved, larger single-center experiences and registry studies are now reported. Table 56.6 summarizes outcomes reported in published series including more than 25 children with ALL. Outcomes from different centers show some variability, which may reflect

heterogeneous patient populations or differing treatment strategies and provision of supportive care. Taken together, DFS for children transplanted in first and second remission is generally in the range of 40– 50%, and outcomes are not markedly different from those reported for related donor HCT (cf Tables 56.4 and 56.5). A number of single-center studies of pediatric HCT have demonstrated that, in children, equivalent results can be achieved using matched (HLA-A, B, and DRB1) unrelated donors and sibling donors, although the majority of these studies included heterogeneous diagnoses, not just children with ALL [161–164]. A multicenter study of 65 children with ALL in second remission showed a 5-year EFS of 39% in sibling donor HCT recipients and 54% in unrelated donor HCT recipients [164]. A single-center study from The Hospital for Sick Children, Toronto, including 62 children with ALL in first, second or third remission or relapse, also showed similar outcomes in sibling and unrelated donor HCT recipients [163]. Dahlke et al. [165] reported the outcome of 84 patients (35 children and 45 adults) with ALL undergoing sibling donor (n = 46) and unrelated donor (n = 38) transplantations between 1990 and 2001. There were no significant differences in survival, relapse, GVHD or TRM dependent on stem cell source. A recent CIBMTR report compared long-term survival after unrelated and sibling donor HCT for acute leukemia (ALL and AML) in children younger than 18 months [166]. Outcomes of unrelated donor HCT with bone marrow (n = 85) or cord blood (n = 81) grafts, and HLA-matched sibling donor HCT with bone marrow grafts (n = 101), for AML or ALL performed between 1990 and 2001 were compared. TRM rates were 31%, 15%, and 6% after unrelated donor bone marrow, cord blood, and matched-sibling HCT respectively. Disease status at transplantation was significantly associated with risk of relapse and likelihood of LFS. Although leukemia recurrence was lowest after unrelated donor HCT in first clinical remission, the OS and LFS rates were similar after matchedsibling and unrelated donor HCT, after adjustment for disease status. Relapse, OS, and LFS did not differ by graft type (bone marrow versus cord blood) or type of leukemia. Taken together, these data indicate that, in children, good outcomes can be achieved with unrelated donor HCT. Improvements in HLA typing have facilitated optimum donor selection, and currently an unrelated donor molecularly matched at HLA-A, B, C, and DRB1 is considered ideal, and likely equivalent to a genotypically matched sibling donor. Despite this, the translation of these single-center observations into improved survival for the overall population of children with

100 Event-free survival (%)

816

80

60

Mismatched Matched

40 0

12

24

36

48

60

72

84

Time from transplantation (months)

Fig. 56.3 Event-free survival after unrelated donor hematopoietic cell transplantation in children with acute lymphoblastic leukemia according to the degree of patient-donor matching at human leukocyte antigen-A, B, DR, and DQ. Donors were fully matched in 27 cases and mismatched in 23. No significant difference was seen in event-free survival between the two groups (51% versus 57%).

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children

817

Table 56.6 Outcome of unrelated donor bone marrow transplantation for children with acute lymphoblastic leukemia

Transplant group

Years of transplant

Number of patients

Remission status (n)

Single-institution series Bristol, UK [160]

1988–97

137

Seattle, USA [170]

1983–99

88

Minnesota, USA [162]

1985–94

35

Milwaukee, USA [172]

1986–91

25

Toronto, Canada [163]

1990–98

26

Multi-institutional series National Marrow Donor Program, USA [169] Nordic Society of Pediatric Hematology and Oncology [164] German [76]

1988–2000

Preparative regimen

Follow-up (years)*

Disease-free survival (%)

CR1 24 CR2 88 >CR2 25

Campath-1G, CY, TBI

3.2 (0.5–8.6)

CR1 10 CR2 34 CR3 10 Relapse 34 CR1 4 CR2 15 CR3 11 Relapse 3 CR1 4 CR2 8 >CR2 7 Relapse 6 CR1 8 CR2 12 CR3 5 Relapse 1

CY/TBI

NS

CY/TBI ± ETOP

2.1 (1–7.3)

CR1 42% CR2 45% >CR2 26% at 5 years CR1 67% CR2 47% CR3 20% BMT in relapse 9% at 3 years CR1/CR2 21% >CR2 42% at 2 years

CY/TBI/ARAC/ ± BU

4.1 (2.3–7.1)

CR1 3 of 4 CR2 5 of 8 >CR2 2 of 13

4 of 25

TBI/ETOP

3.2 (0.3–8.1)

49% overall at 3 years

28%

Relapse (%)

36.5% overall at 5 years

CR1 11% CR2 33% CR3 20% BMT in relapse 50% at 3 years 4 of 35 cases

363

CR2

Multiple; 90% include TBI

2.4 (0–10.4)

36% at 5 years

22%

1990–97

28

CR2

Multiple

4.5 (2–9)

54% at 5 years

40%

1983–2001

81

CR2

TBI/ETOP ± CY (86%)

4.1 (1.1–13.1)

42% at 5 years

25%

* Median and range. ARAC, cytosine arabinoside; BMT, bone marrow transplantation; BU, busulfan; CR, complete remission; CY, cyclophosphamide; ETOP, etoposide; NS, not stated; TBI, total body irradiation.

relapsed ALL remains challenging. Well-matched donors are not available for all children, results from single large centers might not be transferable to multiple smaller centers, and children who do not achieve remission or who die of early chemotherapy toxicity cannot benefit from HCT. Despite a number of attempts, it has proved difficult to perform a randomized trial comparing unrelated donor HCT with chemotherapy to try to translate reports from single centers into a proven therapy. Such a study requires randomization to one of two conceptually very different therapies (HCT versus chemotherapy) that can be difficult for families to accept. In addition, there is a lack of universal acceptance among physicians that equipoise exists in asking this question, with some physicians strongly favoring HCT and others strongly favoring chemotherapy. For example, in the MRC UKALLR1 study of relapsed ALL, children with no matched family donor were randomized to continuation chemotherapy or intensification with autologous HCT. Only 9% of patients eligible for randomization were actually randomized, and even fewer received assigned therapy [167]. As a consequence of these difficulties, there are no available large-scale randomized trials comparing HCT and chemotherapy in childhood ALL. Moreover, an MRC report by Wheeler et al. describing the outcome of 489 children with relapsed ALL reported an

EFS of 3% for children who relapsed in the marrow within 2 years of diagnosis, irrespective of the type of post-relapse treatment (HCT or chemotherapy), suggesting that HCT from any donor source might not be able to rescue this very-high-risk group [123]. A case-control study is an alternative approach to comparison of treatment modalities when a randomized controlled trial is not possible. Supporting possible benefit of unrelated donor HCT for children with the highest risk disease, Borgmann et al. showed improved outcomes with HCT in high-risk children (early relapse or T-cell phenotype), although not intermediate-risk children, in a matched-pair analysis of 81 pairs of transplant and chemotherapy recipients. Of note, none of the high-risk children survived when treated with chemotherapy alone, compared with 44% of the HCT recipients (Fig. 56.4) [168]. While these reports suggest HCT is superior to chemotherapy for children with the most biologically unfavorable disease, outcomes remain suboptimal even in transplanted children, and further improvement is needed. One approach might be the further development of tools to identify high-risk cases likely to fail with chemotherapy prior to relapse. Transplantation in CR1 could then be tested to see if disease control is improved. A challenge to this approach is the likely lack of acceptability of a randomized controlled trial, meaning that all efforts

818

Chapter 56

Disease-free survival (%)

100

Cyclophosphamide/ TBI n = 80 TBI/Ara-C n = 15 Hyperfractionated TBI/Cyclophosphamide n = 28

80 60 40 20 0 0

1

2

3

4

5

6

7

8

9

10

11

12

13

Years post transplant

Fig. 56.4 Comparison of unrelated donor transplantation with chemotherapy in children with acute lymphoblastic leukemia in second remission: probability of event-free survival (pEFS) of matched-pair analysis in the high-risk group is shown. Dashed line: unrelated donor hematopoietic cell transplantation, n = 53, pEFS = 0.44 ± 0.07. Solid line: chemotherapy, n = 53, pEFS = 0.0; p < 0.001.

should be made to select patients for transplantation carefully, and to follow outcomes in children with similar disease characteristics treated with the best contemporary chemotherapy. As larger series of unrelated donor HCTs are being reported, it is now possible to evaluate prognostic factors influencing the outcome of unrelated donor HCT for children with ALL. An analysis of transplants facilitated by the NMDP for children with ALL in second remission showed improved LFS associated with longer duration of first remission, HLA match, and age less than 15 years [169]. Similarly, longer duration of first remission predicted better LFS for children in second remission in the studies from Bristol [160] and Seattle [170]. The Seattle study reported that age less than 10 years and T-cell phenotype were associated with improved LFS. A report from Toronto showed reduced LFS in patients with more advanced disease status prior to HCT and the occurrence of a higher grade of acute GVHD [171]. Dahlke et al. [165] showed improved OS after unrelated donor HCT in younger patients (<18 years) and in patients with donor–recipient sex mismatch. These data indicate that unrelated donor transplantation offers a cure for some children with ALL who are unlikely to be cured with chemotherapy. Challenges that remain include a high rate of TRM associated with GVHD (particularly with unmanipulated grafts), early and late infections (particularly in T-cell-depleted grafts), and relapse. More intensive initial chemotherapy regimens may yield a population of children with highly drug-resistant relapsed disease, requiring novel approaches for cure. UCB transplantation for children with ALL The establishment of banks of cryopreserved UCB for use in transplantation has been an important development in the treatment of children with ALL (see Chapter 39) [173–177]. Potential advantages of UCB include immediate availability of stored units for transplantation once identified (particularly valuable for patients in whom remissions are likely to be short) and reduced GVHD, a major reason for treatment failure in recipients of unrelated donor marrow. The median time needed to identify a suitably matched UCB graft at the University of Minnesota in the year 2001 was 13.5 days, significantly shorter than the 49 days needed to identify a compatible unrelated marrow donor [157]. This is particularly advantageous in children with ALL, in whom achievement of remission can be difficult, and the ability to move to transplantation

as soon as remission is achieved can be valuable. Initial reports of outcomes of UCB transplant procedures have been limited to relatively small series, although larger single-center experiences and registry series are now being reported. The report of successful use of UCB from sibling donors was the basis for development of unrelated donor UCB banks [178]. The first report of 44 sibling donor UCB transplants included eight children with ALL receiving zero- or one-antigen HLA-mismatched grafts, and four receiving two- or three-antigen HLA-mismatched grafts. Two patients with ALL died of graft failure (one transplanted in fifth relapse and one in fourth remission). Actuarial EFS overall for patients with malignant disease was 46% with median follow-up of 1.6 years. Probability of relapse was 49%. A registry report from the European Eurocord group described 143 UCB transplantations from related and unrelated donors performed at 45 different centers [179]. This series included 24 children with ALL receiving related donor UCB transplantation, and 16 receiving unrelated donor UCB. Survival was 63% overall at 1 year for recipients of related donor UCB, and 29% at 1 year for recipients of unrelated donor grafts; separate outcomes were not reported for ALL cases. Relapse was reported in five of 24 related donor UCB transplant recipients, and three of 16 unrelated donor UCB transplant recipients. In a single-center report, Wagner et al. described 102 patients receiving unrelated donor UCB transplantations at the University of Minnesota [180]. Twentyeight patients had ALL, and 55% of standard-risk and 32% of high-risk recipients (defined as short first remission for patients in second remission, or patients beyond second remission) were surviving with a median follow-up of 2.7 years. As experience with UCB transplantations has increased, it has become possible to compare outcomes of UCB transplants with those of other graft sources to assist physicians in donor selection. A registry comparison of unrelated donor marrow (T-cell depleted and T-cell replete analyzed separately) and UCB transplantations in children with leukemia indicated equivalent survival between the three categories. Acute GVHD was reduced in UCB recipients compared with recipients of Tcell-replete marrow, and more relapses occurred in the T-cell-depleted marrow group than in the cord blood grafts [181,182]. Similarly, a single-center matched-pair comparison of unrelated donor UCB and either unmanipulated or T-cell-depleted marrow grafts also indicated at least equivalent survival in UCB recipients [183]. Jacobsohn et al. [184] reported comparable outcomes in children with high-risk ALL when transplanted using UCB grafts or related donor grafts. Eapen et al. [185] compared 503 children with acute leukemia transplanted with UCB with 282 bone marrow recipients between 1995 and 2003. Four hundred ninety-five patients with ALL were included, 186 in the BM group and 309 in the UCB group. Clinical data were obtained from the CIBMTR and the National Cord Blood Program of the New York Blood Center. Recipients of UCB were transplanted with grafts that were HLA matched (n = 35) or HLA mismatched for one (n = 201) or two (n = 267) antigens (typing at antigen level for HLA-A and B, and at allele level for HLA-DRB1). Bone marrow recipients were transplanted with grafts that were allele matched at HLA-A, B, C, and DRB (n = 116), or mismatched at one locus (n = 166). In comparison with allele-matched bone marrow transplantations, 5-year LFS was similar after transplantation of UCB mismatched for either one or two antigens, and possibly higher (numbers were very small) after transplants of HLA-matched UCB. TRM rates were higher after transplantation of two-antigen HLA-mismatched UCB (RR 2.31; p = 0.0003) and possibly after one-antigen HLA-mismatched low cell dose (≤0.3 × 108/kg) UCB transplants (RR 1.88; p = 0.0455). Relapse rates were lower after two-antigen HLA-mismatched UCB transplants (54%; p = 0.0045). These data support the use of HLA-matched and

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children

one- or two-antigen HLA-mismatched UCB with units of adequate size in children with acute leukemia who need transplantation. Greater investment in large-scale banking is needed to increase HLA diversity because better HLA matching and higher cell doses significantly decrease the risk of TRM. Studies that have indicated reduced frequencies of acute GVHD with UCB compared with marrow grafts have raised concern about the possibility of a reduced graft-versus-leukemia (GVL) effect if UCB is used in place of marrow. Current data do not indicate a major difference in relapse frequencies between UCB and marrow grafts in recipients with acute leukemias [181,183]. The recent report from the IBMTR and National Cord Blood Program [185], including large number of patients, showed similar GVHD rates and a lower relapse rate in patients receiving two-antigen-mismatched cord blood transplantation when compared with matched marrow transplantations, possibly indicating a greater, or at least equivalent, GVL effect. Autologous transplantation for children with ALL Autologous transplantation has generally proved disappointing in childhood ALL and is currently infrequently performed. Early, relatively small studies, generally from single centers, described somewhat encouraging results [186–189]. However, these studies commonly included children with both early and late relapses, and those with isolated extramedullary relapses, some of whom would be expected to have a good outcome without transplantation of any kind, making interpretation of the data difficult. A study from Minnesota used a culture technique to identify residual disease pre HCT [190]. The data showed that higher amounts of residual disease predicted increased risk of relapse. It should be recognized, however, that the frequency of relapse in this study was very high, with 83% of children relapsing by 2 years, despite the use of a combination of chemotherapy, antibodies, and immunotoxins to purge the allograft of residual leukemia cells. Only 10 of 83 patients were alive at follow-up of 11–54 months post- transplant at the time of report. Purging of the autograft of contaminating leukemia cells is an attractive concept, and has been attempted by a number of investigators. Overall, the outcomes of these transplantations were disappointing, and there were no clear data that outcomes were superior to those which could be achieved with chemotherapy alone or with unpurged grafts. Increasing availability of unrelated donor marrow and cord blood has significantly reduced the number of children with no available allogeneic stem cell source. In addition, continuing improvements in outcomes of allogeneic transplantations in children, particularly using unrelated donor stem cell sources, with TRM of less than 15% now achievable, leaves little place for autologous transplantation in children with ALL.

Special pediatric considerations Transplantation of infants with ALL Treatment of infant ALL presents particular challenges because of variability in outcomes between the various genetic subtypes, and the susceptibility of very young children to adverse long-term consequences of therapy. Some children with infant ALL have a reasonable prognosis with chemotherapy (those with biologic characteristics similar to older children), while others (with very young age, high WBC count at diagnosis, and the MLL rearrangement) have possibly the most disappointing outcomes in childhood leukemia [191]. In addition, late adverse effects of treatment are a particular concern for this vulnerable group of small children. As a result, transplantation decisions are especially critical in infants.

819

Molecular rearrangements that involve the MLL gene occur in a high proportion of leukemias arising in infants less than 1 year of age (Table 56.1). Fusion between the MLL gene and the AF4 gene as a consequence of a t(4;11)(q21;q23) translocation is the most frequent rearrangement. MLL gene-rearranged infant leukemias frequently have marked leukocytosis and a mixed lineage phenotype (expressing both early B-cell and monocyte markers) [192]. Prognosis in MLL-rearranged ALL in infants is very poor with current chemotherapy, with generally less than 20% 5-year EFS [8–12,191]. Because of the poor response to chemotherapy, infants with MLLrearranged ALL are often considered as candidates for early transplantation. Unfortunately, there are no prospective series of HCT for infants, and outcomes are difficult to compare with those achieved with chemotherapy because of lack of uniform criteria for transplantation eligibility, varying donor sources, and the relatively small numbers of patients transplanted. A consensus has developed that survival is generally poor in infants who are transplanted in relapse or late in their disease course. Sanders et al. analyzed the results of 40 infants diagnosed before age 12 months who received an HCT between July 1982 and February 2003, either in CR1 (n = 17), in CR2/3 (n = 7) or during relapse (n = 16), with a mix of related and unrelated donors [193]. Patients in CR1 had a DFS of 76%, compared with 43% for those transplanted in CR2 and CR3. However, MLL gene status was not known for all children, and of those studied 20% did not have a MLL gene rearrangement. These patients may have had a favorable prognosis with chemotherapy. Only 6% of children transplanted in relapse survived, results in agreement with the dismal results described for transplantation of ALL with active disease in other clinical settings. A Japanese group [194] similarly showed a 3-year post-transplant EFS of 64.4% for 29 patients undergoing HCT in CR1. In the CIBMTR study discussed above, Eapen et al. [166] showed comparable long-term survival after sibling, unrelated donor, and cord blood transplantation for acute leukemia (49% ALL and 51% AML) in children younger than 18 months. These data suggest that if transplantation is indicated, related and unrelated donors can be considered to be equivalent, but they do not address the issue of the relative roles of HCT and chemotherapy in CR1. Establishing without doubt that HCT can improve outcome compared with chemotherapy in high-risk infants in CR1 requires a randomized study, and such a trial is unlikely to take place due to the rarity of this disease and to the logistic challenges of performing such studies, as discussed earlier. A retrospective analysis of outcomes of chemotherapy (n = 103) and transplant (n = 28) in infants with leukemia carrying t(4:11) translocations from 11 institutions concluded that hematopoietic transplantation did not improve clinical outcome [191]. Similarly, Chessells et al. [192] reported outcomes of the United Kingdome Infant 92 study, with a 4-year EFS of 33% overall, compared with 22.5% in the prior study (Infant 87). Only 12 infants were transplanted (three sibling donors and nine unrelated donor) in Infant 92, five of whom survive. The larger and more recent international Interfant-99 study enrolled 482 infants with ALL from 22 countries between 1999 and 2005 [195]. Eligible patients were risk-stratified according to their peripheral blood response to a 7-day prednisone prophase, and then given a hybrid regimen based on the standard protocol for ALL, with some elements designed for treatment of AML. In an intention-to-treat analysis, EFS and OS at 4 years were 47.0% and 55%, respectively. Factors prognostic of poor outcome included MLL gene rearrangements, very high WBC count (>300 × 109/L), age younger than 6 months, and a poor response to the prednisone prophase. Four-year DFS did not differ significantly in high-risk patients treated with chemotherapy alone (37.4%) versus chemotherapy plus HCT (50.2%) when adjusted for time to transplantation (p = 0.19).

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While data from these multicenter reports are perhaps the best available, interpretation is limited by the small number of patients transplanted, multiple donor types, absence of standardized eligibility requirements, and absence of standardized treatments. There is a clear need for further prospective studies to define the role of HCT in infants with genetically defined leukemias that are at high risk for treatment failure. Such studies will need to be multi-institutional and possibly multinational (as was Interfant-99), as infant leukemia is rare. Studies should include evaluation of the late consequences of therapy in survivors, as morbidity can be significant in young children. Transplantation of children with Down’s syndrome and ALL Children with Down’s syndrome have an approximately 20-fold increased risk of leukemia, and now represent around 2% of children enrolled onto phase III cooperative group studies for ALL [196,197]. Initial clinical reports of chemotherapy treatment of leukemia in children with Down’s syndrome indicated increased toxicity in these patients [198]. In support of this finding, in vitro data indicated increased cellular sensitivity to chemotherapeutic agents in cells from children with Down’s syndrome [199]. However, some small recent studies have shown chemotherapy outcomes as good as those seen in children with ALL without Down’s syndrome [200,201]. A recent review of the CCG experience, the largest analysis to date of this population, demonstrated decreased EFS and OS in children with Down’s syndrome and ALL enrolled on CCG trials when compared with children with ALL without Down’s syndrome [202]. Subset analysis revealed that poorer outcomes were restricted to the NCI standard-risk children (OS 70% versus 85%, p = 0.001; EFS 56% versus 74%, p = 0.001). The NCI high-risk subset of ALL and Down’s syndrome patients receiving more intensive therapy fared as well as their non-Down’s syndrome counterparts (OS 63% versus 66%, p = 0.7; EFS 62% versus 59%, p = 0.9), suggesting that, with modern supportive care, children with Down’s syndrome can tolerate intensive therapy. The suggestion of increased therapy-related toxicity with chemotherapy in children with Down’s syndrome may have led to reluctance to offer transplantation to these children in earlier years. In addition, the first report of transplantation in four children with Down’s syndrome indicated poor outcome due to increased toxicity [203]. However, a number of more recent reports have described successful transplantation of children with Down’s syndrome and leukemia using both related and unrelated donor grafts, indicating that these children should be considered as candidates for transplantation [203–206]. Rubin et al. [203] reported 27 patients with Down’s syndrome (18 collected from a survey of bone marrow transplantation centers and nine from the literature). Sixteen received a graft from an HLA-matched sibling, two from nonsibling family members, and four from unrelated donors, and five received autologous grafts. This study included 14 children with ALL, nine in second remission and five in third remission. Five of the 14 ALL patients were surviving 9–60 months post transplantation. The authors did identify a high rate of regimen-related toxicity in these patients (39% cumulative risk of death in remission at 3 years). A high frequency of airway problems and lung problems was noted, perhaps associated with the use of methotrexate. Despite the significant toxicity, relapse-free survival for the whole group was 44% at 3 years, results that are comparable to those seen in children with ALL without Down’s syndrome. Occasionally, the clinical need arises to consider the use of an HLAmatched sibling who has Down’s syndrome as a bone marrow donor. Although data are limited, Barquinero et al. have described four transplants from matched-sibling donors with Down’s syndrome. In only one

of these cases was sustained engraftment achieved [207]. Patton et al. [208] described the occurrence of post-transplant lymphoproliferative disorder after transplantation using a donor with Down’s syndrome, and suggested that this may be reflective of an immune defect in the donor. These reports of poor graft function may be related to poor quality of donor stem cells, and suggest that donors with Down’s syndrome should not be used for transplantation.

Role of second transplant procedures for posttransplant relapse The most important cause of treatment failure following transplantation for childhood ALL is recurrent leukemia. Additional chemotherapy can induce further remission in a proportion of patients, although very few will survive long term. Experience with the use of second transplants following relapse after bone marrow transplantation is limited. The IBMTR reported on 114 recipients of HLA-identical sibling grafts who received a second allogeneic HCT for treatment of relapse between 1978 and 1989 [209]. Twenty-nine of these patients had ALL. LFS for all the ALL cases was 21% (CI 14–30%); however, outcomes were extremely poor in patients who had relapsed less than 6 months after their first transplant, in whom LFS was only 7% (CI 2–19%), largely as a consequence of high TRM. Patients with long remissions after their first transplant, those with good performance status, and those achieving remission after their initial post-transplant relapse had the best outcomes. Three single-center series of second transplantation procedures, each including a small number of ALL cases, also report high early TRM and poor long-term survival [210–212]. A common theme of these reports is the importance of length of remission after transplant, with poor survival after a second HCT in those experiencing early relapse [213,214]. While these procedures remain challenging, some success can, with careful patient selection, be achieved in those with a long remission after the first HCT.

Conclusion The treatment of childhood ALL with chemotherapy has been a story of remarkable success, with survival rates in excess of 70% in the majority of studies. This success is counterbalanced by the fact that current chemotherapy is still inadequate for the almost 30% of cases who will relapse. As ALL is the most frequent malignancy in children, relapsed ALL remains one of the most frequent causes of death from malignant disease in childhood. Determining the appropriate role for HCT in childhood ALL has been a dynamic process, as success with chemotherapy has increased and additional hematopoietic cell sources have become available. Serial, large-scale, essentially population-based randomized studies have led to the improvements in outcomes of chemotherapy for ALL. Application of a similar rigorous approach to evaluation of the role of HCT has not proved possible, and significant uncertainty remains in some areas. Access to HCT has been improved by improvements in the availability and selection of unrelated hematopoietic cell donors and the development of unrelated donor UCB as a hematopoietic cell source. Indeed, outcomes with unrelated donor HCT have improved such that results are now equivalent to sibling donor HCT in well-matched cases. Future improvements in outcome and applicability of HCT for childhood ALL may result from development of methodologies to identify at the earliest possible time children who are destined to relapse, allowing HCT in first remission when outcomes are superior. The detection of MRD in children receiving chemotherapy, either as a harbinger of relapse or as a measure of inadequate early response to therapy, will

Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia in Children

allow early referral for HCT as intensification therapy (see Chapter 26) [115–119]. It is important to know that the significance of MRD varies in different genetic subtypes of leukemia. For example, MRD is cleared more promptly in children with ALL and TEL-AML1 compared with children with favorable trisomies, both groups with an excellent longterm prognosis [215]. Recent studies have also shown association between gene expression profiling of ALL blasts at diagnosis and distinct immunophenotypic/genetic subtypes of ALL, treatment outcomes, and risk of relapse [216–219]. Further validation of these and similar findings in the future could allow immediate referral of children with ALL destined to relapse for HCT in first remission. In recent years, similarly there is growing interest in the role of MRD as a predictive marker for relapse following allogeneic transplantation. Most studies have shown that MRD detection in pre- and/or post-HCT samples predict increased risk of relapse following HCT [220–222]. Different approaches have been suggested to help improve transplantation outcomes using MRD detection to modify either pre- or post-transplant therapy, for example additional cytoreductive therapy pre transplant to reduce the malignant clone and render the patients MRD negative, designing transplant protocols favoring the development of GVHD to increase the GVL effect, the use of donor lymphocyte infusion, etc.

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Some of these interventions are limited by the practicality of these approaches. For example, heavily pretreated patients have limited options for further chemotherapy prior to transplantation, and rapid withdrawal of immunosuppression and donor lymphocyte infusions is associated with the risk of life-threatening GVHD and has very limited efficacy. Thus, although it is possible to identify patients at risk of relapse with MRD detection, strategies to respond to that information are limited. Also, many questions remain unanswered; for example, should MRDnegative status be used to assign patients to nontransplant treatment options to prevent TRM and long-term toxicity? Efforts to assess the clinical significance of pretransplant MRD in children with ALL are hampered by the lack of prospective studies in large cohorts of homogeneously treated patients. Improved strategies for donor selection using molecular methods of HLA typing, or examination of non-HLA loci that influence HCT, also have potential to improve outcomes [223–225]. Inclusion of chemotherapy agents such as etoposide or thiotepa in the conditioning regimen may reduce relapse and improve survival. Lastly, targeting of conditioning therapy with radioimmunoconjugates offers the possibility of reducing radiation toxicity while maintaining the therapeutic effect [226].

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H. Joachim Deeg

Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders

Introduction Myelodysplastic syndromes (MDSs) and myeloproliferative disorders (MPDs) represent the two broad categories of chronic myeloid diseases. Both are clonal diseases of hematopoietic precursors/stem cells. In contrast to acute leukemias, the course of MDS and MPD is generally protracted and may extend over years or even decades. However, both disorders may end in a terminal phase resembling acute leukemia. An alternative scenario is that of progressive peripheral blood cytopenias and hematopoietic failure, which in patients with MPD is associated with severe marrow fibrosis. Marrow fibrosis may also develop in patients with MDS, but is less frequent and generally less severe. Chronic myelomonocytic leukemia (CMML) may have either dysplastic or proliferative features (or both) and, in the new World Health Organization (WHO) classification, has been placed in a separate category, “Myelodysplastic/myeloproliferative disorders” [1]. Whether these diseases evolve towards acute leukemia or severe cytopenias, both paths eventually prove fatal. Chronic myeloid disorders can occur at any age, but the incidence increases progressively with age.

General considerations for hematopoietic cell transplantation for MDS and MPD Hematopoietic cell transplantation (HCT) is the only treatment modality with curative potential for patients with MDS and MPD, but the indications for and timing of HCT are controversial. The relatively chronic course of the disease often leads to reluctance to accept the potential risks associated with HCT. Nonrelapse mortality (NRM) has been in the range of 20–25%, related to organ toxicity, graft-versus-host disease (GVHD), and infectious complications. Particularly in older patients, with reduced biological reserve and an apparently reduced tolerance to certain treatment modalities, those issues are particularly relevant. The high frequency of comorbid conditions in older patients contributes to the problem. Sorror et al. have recently developed an HCT-specific comorbidity index, which provides a useful guide for risk assessment before HCT [2]. Furthermore, Cutler et al. carried out a decision analysis in patients with MDS [3], which showed that the International Prognostic Scoring

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

System (IPSS) was useful in determining the timing of HCT in patients who had human leukocyte antigen (HLA)-identical sibling donors. The results indicate that patients in the intermediate-2 and high-risk groups (by the IPSS) (Table 57.1) who are transplant candidates have the longest life expectancy when transplanted early, as delay of HCT resulted in a loss of life years. In contrast, patients in the low-risk group had the best life expectancy if HCT was delayed until evidence of disease progression. Results for patients in the intermediate-1 risk group were less distinct. Delay of HCT offered some advantage, but it was marginal, presumably related to the fact that the low score that placed patients in this risk group could have been on the basis of different parameters with different impacts on both survival without HCT and outcome after HCT. Results from cytofluorimetric analyses of marrow cells indicate that Int-1 patients can be further subdivided, at least in regards to post-HCT relapse risk, on the basis of phenotypic aberrancy of marrow cells [4]. Different sources of hematopoietic stem cells are discussed elsewhere in this book. Granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells (PBPCs) are currently the most commonly used source of stem cells. Data from several centers, including our own, show decreased rates of graft rejection and relapse in patients with MDS or MPD receiving PBPCs compared with marrow. While the incidence of chronic GVHD tends to be higher with PBPCs, survival rates may not differ significantly between the two groups or may be superior with PBPCs because of lower relapse rates [5,6]. Umbilical cord blood is an alternative source of hematopoietic stem cells, and successful HCT has been reported in small groups of patients with MDS [7,8]. The advantages of umbilical cord blood include the rapid availability of units from storage banks and greater tolerance for HLA disparity, allowing for the use of mismatched units, thereby increasing the potential pool of donors. However, the cell dose in a given cord blood unit is an important determinant of engraftment, and low cell numbers have limited the use of this HCT modality. Recently, the strategy of infusing two units of cord blood has resulted in accelerated engraftment; more studies are needed to examine this modality in patients with MDS [9,10]. The selection of donors on the basis of histocompatibility [6,11,12] is discussed in Chapters 46–48. Relevant for the present discussion is that recent studies in patients with MDS or MPD have shown similar results with HLA-identical sibling donors and with unrelated donors selected on the basis of high-resolution typing. Haploidentical transplants are carried out on an experimental basis. As with other indications, GVHD has remained the most frequent complication after allogeneic HCT for MDS and MPD. Approximately 40–50% of patients with HLA-identical sibling donors and 50–60% with

827

828

Chapter 57

Table 57.1 International Prognostic Scoring System (IPSS) for myelodysplastic syndrome Parameters and severity scores Score value Prognostic parameter

0

0.5

1.0

1.5

2.0†

BM blasts (%) Karyotype* Cytopenias

<5 Good 0 or 1

5–10 Intermediate 2 or 3

– Poor

11–20

21–30

Allogeneic HCT Myelodysplastic syndrome

IPSS risk and outcome Median survival

Risk group Low Intermediate-1 Intermediate-2 High

Composite score 0 0.5–1.0 1.5–2.0 ≥2.5

no significant benefit for RFS [21]. The data suggest that responses to pretransplant chemotherapy may select for good-risk patients who have “chemosensitive” disease and might have done well even without preHCT chemotherapy. Data on the role of debulking chemotherapy in MPD are basically nonexistent. Thus, the disease burden, the pace of disease progression, the presence of comorbidities, the need for pretransplant chemotherapy, the stem cell source, and the type of transplant are all important issues to consider before proceeding with HCT.

All patients

≤60 years

5.7 3.5 1.2 0.4

11.8 5.2 1.8 0.3

Scores for risk groups are as follows: low = 0; intermediate-1 = 0.5–1.0; intermediate-2 = 1.5–2.0; high = ≥2.5 [26]. * Good, normal; −Y, del(5q), del (20q); poor, complex (≥3 abnormalities) or chromosome 7 anomalies; intermediate, other abnormalities. † Shaded column: Based on the World Health Organization, this category of myelodysplastic syndrome no longer exists.

unrelated donors are expected to develop acute GVHD requiring therapy, and about 50–60% will develop chronic GVHD. Calcineurin inhibitors in combination with methotrexate or mycophenolate mofetil have been the most widely used prophylactic agents in recent years (see Chapters 84–87). Conventional high-dose conditioning regimens are generally only offered to patients up to 65 or 60 years of age, with HLA-identical siblings and unrelated donors, respectively [13]. The introduction of so-called reduced-intensity conditioning regimens over the last decade has allowed for expansion of the upper age for HCT, and even patients in their early 70s have been transplanted successfully [14,15]. However, while treatment-related mortality (TRM) is lower, the incidence of relapse tends to be higher than with high-dose regimens. This may be the case particularly in patients with MDS (or MPD) as they generally have not been treated aggressively with cytotoxic therapy before HCT [16–18]. Patients with the lowest disease burden tend to have the best success rates, an observation that has raised the question of a potential benefit of pretransplant debulking chemotherapy [19]. However, this question has remained controversial, and no controlled trials have been conducted. Generally, patients who have an elevated myeloblast count (>5% in the marrow) receive chemotherapy before undergoing HCT with reduced-intensity conditioning. The approach has been less consistent in patients who are prepared for HCT with high-dose regimens. A French study showed that patients with secondary MDS who achieved remissions after pretransplant chemotherapy had a significantly higher probability of relapse-free survival (RFS) after HCT than patients who did not respond [20]. A recent retrospective analysis conducted at our center also suggested that patients with MDS who received pretransplant chemotherapy were less likely to relapse after HCT, although there was

While 15–20% of cases of MDS occur in younger patients who have received chemotherapy or radiation treatment for other cancers or nonmalignant disorders, for example autoimmune diseases, patients are on average in their 60s or 70s at the time of diagnosis, and no specific etiology can be identified. We refer to the latter as primary (or de novo) MDS, and to the former as secondary (or treatment-related) MDS. However, the term “secondary” MDS is also used when the disease develops in patients with an antecedent hematologic diagnosis, for example aplastic anemia. The underlying pathophysiology of MDS is incompletely understood [22]. With low-risk or early-stage disease such as refractory anemia (RA) or refractory cytopenia with multilineage dysplasia (Table 57.2), programmed cell death (apoptosis) is typically upregulated, contributing to the presentation of cellular marrow in association with peripheral blood cytopenias. As the disease advances, clonal precursors become increasingly apoptosis resistant, and proliferative features are prominent. While MDS has long been considered a cellautonomous disease, evidence is mounting for contributions of the microenvironment [23]. Most patients with MDS present with peripheral blood cytopenias, most commonly anemia, and only few patients with isolated neutropenia or thrombocytopenia. About 40–50% of patients with de novo MDS have clonal chromosomal abnormalities that can be detected by karyotype analysis using classic banding techniques or fluorescent in situ hybridization. Some karyotypic abnormalities such as isolated chromosome 5q deletion are associated with a good prognosis, while others, such as abnormalities of chromosome 7, predict a more rapid progression and transformation to acute myeloid leukemia (tAML) [13,24–26]. Several staging systems for MDS have been developed, the most widely used being the French–American–British classification [24]. This system is in the process of being replaced by the WHO classification (Table 57.2) [25]. Modifications include elimination of the RA with excess blasts in transformation (RAEB-T) group (all patients with 20% myeloblasts or more are considered to have AML), and separation of RA into RA (erythroid abnormalities only), refractory cytopenia with multilineage dysplasia, the 5q− syndrome, and MDS unclassified (unilineage dysplasia in granulocytes or megakaryocytes). CMML, as indicated above, has been reclassified in a separate MDS/MPD category [1,25]. These classifications are used in conjunction with the IPSS to stratify patients into prognostic groups (Table 57.1) [26]. The IPSS incorporates three parameters: cytogenetic findings, the myeloblast count in the marrow, and the number of blood cytopenias. Based on the composite score, patients are stratified into four categories: low risk (score 0), intermediate-1 (0.5–1.0), intermediate-2 (1.5–2.0), and high risk (≥2.5), which statistically carry median life expectancies of 5.7, 3.5, 1.2, and 0.4 years, respectively, if patients in all age groups are considered; the median life expectancy is longer in patients less than 60 years of age [26]. Malcovati et al. proposed a modification of the IPSS based on the WHO classification, termed “WPSS” (Table 57.3) [27]. The recommendation for

829

Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders Table 57.2 World Health Organization classification and criteria for myelodysplastic syndrome (MDS) Classification

Peripheral blood

Bone marrow

Refractory anemia (RA)

Anemia No or rare blasts

Erythroid dysplasia only <5% blasts <15% ringed sideroblasts

Refractory anemia with ringed sideroblasts (RARS)

Anemia No blasts

Erythroid dysplasia only <5% blasts ≥15% ringed sideroblasts

Refractory cytopenia with multilineage dysplasia (RCMD)

Bi- or pancytopenia No or rare blasts No Auer rods Monocytes <1000/μL

Dysplasia in ≥10% of cells in two or more myeloid cell lineages <5% blasts No Auer rods <15% ringed sideroblasts

Refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS)

Bi- or pancytopenia No blasts

Dysplasia in ≥10% of cells in two or more cell lineages <5% blasts ≥15% ringed sideroblasts

MDS unclassified (MDS-U)

Cytopenias No or rare blasts No Auer rods

Unilineage dysplasia in granulocytes or megakaryocytes <5% blasts No Auer rods

MDS with del(5q) (5q− syndrome)

Anemia <5% blasts Platelets normal or increased

Normal to increased megakaryocytes with hypolobulated nuclei <5% blasts No Auer rods Isolated del 5q

Refractory anemia with excess blasts 1 (RAEB-1)

Cytopenias <5% blasts No Auer rods Monocytes <1000/μL

Unilineage or multilineage dysplasia 5–9% blasts No Auer rods

Refractory anemia with excess blasts 2 (RAEB-2)

Cytopenias 5–19% blasts Auer rods ± Monocytes <1000/μL

Unilineage or multilineage dysplasia 10–19% blasts Auer rods ±

Table 57.3 World Health Organization (WHO) classification-based prognostic scoring system (WPSS) for myelodysplastic syndrome (MDS)

Parameters and severity scores Score value Prognostic parameter

0

1

2

3

WHO category Karyotype* Transfusion requirement

RA, RARS, 5q− Good No

RCMD, RCMD-RS Intermediate Regular

RAEB-1 Poor –

RAEB-2 – –

WPSS risk and outcome Risk group

Composite score

Survival (years)

AML evolution

Very low Low Intermediate High Very high

0 1 2 3–4 5–6

11.3 5.3 3.7 1.6 0.7

7% @ 10 years – – – 50% at 8 months

See Table 57.2 for abbreviations. AML, acute myeloid leukemia. Scores for risk groups as follows: very low, 0; low, 1; intermediate, 2; high, 3–4; very high, 5–6 [27]. * Good, normal; −Y, del(5q), del (20q); poor, complex (≥3 abnormalities) or chromosome 7 anomalies; intermediate, other abnormalities.

830

Chapter 57

this modification is based on the observation that patients who were transfusion dependent experienced more rapid disease progression. The WPSS incorporates the WHO classification, the karyotype, and transfusion requirements, and risk-stratifies patients into five categories: very low (score 0), low (1), intermediate (2), high (3–4), and very high (5–6), predicting for median survivals of 136, 63, 44, 19, and 8 months, respectively. The WPSS categories are also useful for predicting the probability for leukemia progression: patients in the very low group had a 10-year probability of transforming to tAML of 7%, while those in the very high group had a probability of 50% at 8 months [27]. Until recently the “standard” of therapy for MDS was supportive care; however, several treatment modalities (other than HCT) are now available and are being administered, in part based on the classification schemes discussed above. For patients with a deletion of chromosome 5q, lenalidomide is considered first-line therapy, inducing responses (as defined by transfusion independence) in about two-thirds of patients, for a median duration of 1.5–2 years; many of these patients also experience morphologic and cytogenetic remissions [28]. Two DNA methyltransferase inhibitors, 5-azacytidine and 2-deoxy-5-azacytidine, induce hematologic improvement in about half of the patients in IPSS risk groups intermediate-2 and high, and may delay leukemic transformation and prolong survival by 8–9 months [29–31]. Immunosuppressive therapy with antithymocyte globulin (ATG), alone or in combination with other agents, may induce hematologic responses in 10–30% of patients with low-risk MDS (generally without cytogenetic abnormalities), lasting in some patients for years [32]. A combination of erythropoietin and granulocyte colony-stimulating factor is effective in raising hemoglobin levels in a proportion of patients with low endogenous erythropoietin levels [33]. Various other therapies are being tested in clinical trials. None of these modalities has been shown to have curative potential. However, particularly older individuals and patients with low-risk disease may benefit from these treatment options for variable periods of time and proceed to HCT only if the disease progresses.

Low-risk MDS Some recent reports of allogeneic HCT in patients with MDS are summarized in Tables 57.4 and 57.5. The best results with HCT are achieved in patients with low myeloblast counts in the marrow (<5%) and in patients without high-risk karyotypes; these are generally patients with low IPSS scores (Fig. 57.1). The European Group for Blood and Marrow Transplantation (EBMT) reported results in 131 patients with MDS transplanted from HLA-identical sibling donors after various conditioning regimens. The 5-year RFS was 52%, and the relapse rate was 13% [34]. The same group reported a RFS of 24% at 2 years with a relapse rate of 13% for patients transplanted from unrelated HLA matched donors [35]. The International Bone Marrow Transplant Registry presented results in 452 patients with MDS transplanted from HLA-identical siblings, showing a 3-year RFS of 40% (72% for patients younger than 18 years), although only 140 of these patients (31%) had less than 5% blasts at HCT. TRM was 37%, and the relapse rate was 23%. High marrow blast count and high IPSS score were significantly correlated with relapse [36] (Fig. 57.1). In a cohort of 512 patients with MDS transplanted from unrelated donors, those conditioned with high-dose oral busulfan (BU; 1 mg/kg ×16 doses over 4 days) and cyclophosphamide (CY; 60 mg/kg/day ×2) fared better than patients conditioned with other regimens, in particular regimens containing high-dose total body irradiation (TBI) (e.g. 6 × 200 cGy combined with CY) [37]. We recently reported the Fred Hutchinson Cancer Research Center experience with patients conditioned with a BU/CY regimen in which BU (prescribed dose 16 × 1 mg/kg) was

dose-adjusted based on real-time pharmacokinetic monitoring to achieve target steady state levels, generally of 800–900 ng/mL (targeted BU/CY). The 3-year RFS was 68% with related and 70% with unrelated donors. Among 69 patients with RA/RA with ringed sideroblasts, NRM was 31%, and relapse occurred in 5% of patients [6]. As further discussed below, the use of reduced-intensity regimens, such as fludarabine 3 × 30 mg/m2 intravenously, plus 200 cGy of TBI in a multicenter study resulted in a 2-year survival of about 40% in patients with low-risk MDS [38,39]. As decisions for patients in the IPSS intermediate-1 risk group are often difficult, we attempted to define the impact of one parameter, neutropenia, on HCT outcome. While there were only 16 patients with isolated neutropenia in a cohort of 469 patients, overall 178 patients (61%) had less than 1.5 × 109 neutrophils/L. These patients had a significantly higher NRM (p = 0.01), mostly related to infections, and a lower probability of survival than patients without neutropenia (Deeg et al., unpublished data). Conceivably, earlier HCT, before a severe decline in neutrophil level, would be associated with lower mortality rates. Whether transfusion-dependent patients (see the WPSS classification) have a poorer post-HCT outcome than transfusion-independent patients has yet to be determined. High-risk MDS The success rates with HCT decline as the prognosis by WHO or IPSS criteria worsens (Fig. 57.1), in particular with an increase in the marrow myeloblast count and the presence of high-risk cytogenetics. The decline in the success rate is almost exclusively due to progressively higher rates of relapse. Intensification of conditioning regimens aimed at reducing the relapse risk has been associated with higher rates of NRM and no net improvement in survival [40]. The International Bone Marrow Transplant Registry reported results in 352 patients with high-risk MDS transplanted from HLA-identical sibling donors; most were conditioned with high-dose TBI-based or BU/CY regimens. The 3-year RFS was 63% for patients younger than 18 years and 33% for older patients [36]. The EBMT reported a 5-year RFS for patients transplanted from related donors of 34%, 19%, and 26% for RAEB, RAEB-T, and tAML, respectively; the relapse rate was about 50% for the entire cohort. The 2-year RFS for patients transplanted from HLA-matched unrelated donors was 27%, 8%, and 27% for RAEB, RAEB-T, and tAML, respectively [34,35]. In a study from our center in patients with RAEB transplanted after conditioning with targeted BU/CY, 3-year RFS was 45% with related donors and 40% with unrelated donors. The corresponding figures for RAEB-T/tAML were 33% and 17%, respectively. Our data also indicate that the IPSS correlated strongly with outcome; patients with low IPSS scores had the highest probability of RFS and the lowest rates of relapse [6]. Another trial examined a combination of oral BU (7 mg/kg) and TBI (6 × 200 cGy) without high-dose CY. Among 60 patients with advanced MDS, tAML or CMML transplanted from related (n = 20) or unrelated (n = 40) donors, the 3-year Kaplan–Meier estimate of survival was 26%, and the relapse rate 25% [40]. The reduction in relapse was comparable to an earlier trial using a combination of BU/CY and TBI [41]. However, NRM was 38% at 100 days [40], suggesting that further increases in conditioning intensity (with the aim of reducing relapse frequency) were unlikely to improve overall survival. In an effort to reduce toxicity, several groups have examined the tolerability and efficacy of intravenous fludarabine (at doses of 4 × 30 to 5 × 50 mg/m2) in combination with targeted oral BU [42,43]. In 42 patients with high-risk hematologic neoplasms (38 with MDS) conditioned with fludarabine (4 × 30 mg/m2) plus oral BU (16 × 1 mg/kg with dose adjustments) and transplanted from related (n = 16) or unrelated (n = 26) donors, the probabilities of overall survival and RFS at a median

MDS/AML/MPD RA

131/2–55 (33)

885/NA

109/6–66 (46)

234 4–59 (41) BM 12–60 (47) PB 452/2–64 (38)

42/12–65 (52)

96/19–66 (45)

56/9–65 (50)

27/11–64 (51)

1998

1998

2000

2002

2002

2002

2003

2004

2006

Nevill et al. [119]

Runde et al. [34]

de Witte et al. [120]

Deeg et al. [6]

Guardiola et al. [5]

Sierra et al. [36]

Bornhäuser et al. [42]

de Lima et al. [43]

Deeg et al. [121] 14

30

60

16

452

234

45

885

131

38

Rel

13

26

36

26

0

0

64

0

0

22

URD

0

0

47

0

452

132

81

885

131

60

BM

27

56

49

42

0

102

28

0

0

0

PB

Source of stem cells (number of patients)

tBU/CY

tBU/CY + THY

FLU + IV BU

Various regimens (high-dose TBI 44%) FLU/tBU

Various regimens

tBU/CY

Various regimens

High-dose TBI (70%)

BUCY (38) CY TBI (22)

Conditioning

RA (high risk) RAEB/ RAEB-T CML All patients In CR RAEB AML RA

Good-risk cytology Intermediate Poor RA/RARS RAEB RAEB-T tAML Untreated RA/RARS Treated CR1 Treated No CR Other RA/RARS RAEB RAEB-T/ AML RA RAEB RAEB-T All patients

Disease category

3.7

12.5

0

7

NA

NA

12.5

NA

NA

NA

Day 100

% NRM

22 (1)

23 (2)

4 (1)

24 (1.5)

32 (3)

38 (2) 39

37 (7) 54 68 40 (5) 38 60 48 37 (3) 37 45 52 29 (3)

Years after HCT

Outcome (at years)

29 (1.5) 52 75 43 26 35 (2) 24 0

19 (7) 12 82 13 (5) 44 52 50 13 (3) 30 42 43 5/3 (3) 38/33 33/33 13 (2) 28 38 23 (3)

Related/URD

% Relapse

42 (1.5) 22 0 52 (1) 75 55 (2) 58 78 (1)

51 (7) 40 6 52 (5) 34 19 26 55 (3) 44 32 28 68/70 (3) 45/40 33/17 52 (2) 43 39 40 (3)

Related/URD

% RFS

AML, acute myeloid leukemia; BM, bone marrow; BU, busulfan; CR, complete remission; CY, cyclophosphamide; FLU, fludarabine; IV, intravenous; NA, not available; NRM, nonrelapse mortality; PB, peripheral blood; RA, refractory anemia; RAEB, refractory anemia with excess blasts; RAEB-T, RAEB in transformation; RARS, RA with ringed sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; Rel, related; RFS, relapse-free survival; tAML, transformation to AML; TBI, total body irradiation; tBU/CY, targeted BU/CY; THY, thymoglobulin; URD, unrelated donor; 1°, de novo; 2°, secondary.

MDS/AML

MDS/CML

MDS

MDS

MDS

MDS/tAML (1° = 818; 2° = 67)

MDS/tAML (1° = 116; 2° = 15)

MDS/tAML

60/15–55 (40)

Year

Study

Disease

Number of patients/age (years) range (median)

Donor number of patients)

Table 57.4 Allogeneic hematopoietic cell transplantation (HCT) for myelodysplastic syndrome (MDS) – high-dose regimens

Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders

831

MDS (2° = 41%) MDS (2° = 37%)

94/ 27–74 (61) (FAI) 22–75 (54) (FM) 52/17–71 (52)

75/24–68 (52)

38 RIC/ 27–72 (56)

621 Other/ 18–67 (45)

2003

2004

2005

2006

2006

2006

Kroger et al. [45]

de Lima et al. [122]

Van Besien et al. [48]

Lim et al. [47]

Martino et al. [108]

Scott et al. [39]

MDS/tAML (2° = 27%)

112 Other/ 40–65 (53)

66

26

621

215

0

27

65

19

Rel

46

12

0

0

75

25

29

18

URD

21

2

305

27

28

NA*

53

8

BM

91

36

316

188

47

NA*

41

29

PB

Source of stem cells number of patients)

tBU/CY

FLU + TBI (2–4 Gy) ± ATG/Campath or FLU + alkylator CY + TBI (≥8 Gy) ± ATG; Other or BU/CY FLU (90 mg/m2) + TBI (2 Gy)

NA

RCMD RAEB-1/2 tAML CMML MDS

NA

RA/RARS RAEB RAEB-T/tAML RAEB RA/RARS RAEB-T/tAML

NA

20

MDS

15

NA

NA

NA

Day 100

Standard risk† High risk‡

RA RAEB RAEB-T Other

FLU (120–180 mg/m2) + BU (8 mg/kg p.o.) (or 6.4 IV) + ATG (in 25) FLU/AraC/Ida (FAI) FLU/melphalan (FM) Flu (150 mg/m2 IV) + melphalan (140 mg/m2 IV) + Campath (100 mg) FLU (150 mg/m2 IV) + BU (4 mg/kg p.o.) + Campath (100 mg IV)

Disease category

Conditioning

% NRM

33 (3) 78 31 36 (3) 31 31

32

24 (3) 44 21 65 22 (3)

22 (1) 39

15 (3) 39

12 (Rel) (2)/ 45 (URD)

Years after HCT

Outcome (at years)

33 (3) 22 34 10 22 40

27

45 (3)

43 (3)

16 (1) 39

61 (3) 30

32 (3)

Related/URD

% Relapse

33 (3) 0 35 55 45 29

41

55 (3) 18 47 14 33 (3)

61 (1) 37

30 (3) 35

31/25 (3)

Related/URD

% RFS

AML, acute myeloid leukemia; Ara-C, cytosine arabinoside; ATG, antithymocyte globulin; BM, bone marrow; BU, busulfan; CMML, chronic myelomonocytic leukemia; CR, complete remission; CY, cyclophosphamide; FLU, fludarabine; Ida, idarubicin; IV, intravenous; NA, not available; NRM, nonrelapse mortality; PB, peripheral blood; RA, refractory anemia; RAEB, refractory anemia with excess blasts; RAEB-T, RAEB excess blasts in transformation; RARS, RA with ringed sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; Rel, related; RFS, relapse-free survival; RIC, reduced intensity conditioning; tAML, transformation to AML; TBI, total body irradiation; tBU/ CY, targeted BU/CY; THY, thymoglobulin; URD, unrelated donor; 1°, de novo; 2°, secondary. † Defined as AML in first CR, 2° CR, MSD <5% blasts. ‡ All other patients. * PBs were the stem cell source of choice, but numbers were not provided.

MDS/tAML (2° = 13%)

38 RIC/ 40–72 (62)

MDS

MDS/AML

MDS (26) / AML (68)

MDS/tAML

37/23–72 (55)

Year

Study

Disease (number of patients)

Number of patients/age (years) range (median)

Donor number of patients)

Table 57.5 Allogeneic hematopoietic cell transplantation (HCT) for myelodysplastic syndrome (MDS) – reduced-intensity regimens

832 Chapter 57

Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders (a)

(b)

Fig. 57.1 Impact of International Prognostic Scoring System (IPSS) score on transplant outcome. (a) Relapse-free survival. (b) Cumulative incidence of relapse. +, censored patient. (Reproduced from [6], with permission. Copyright, the American Society of Hematology.)

follow up of 1.5 years were 42% and 35%, respectively. All patients achieved engraftment, and the day 100 mortality was 7% [42]. Similarly, the MD Anderson group used a regimen of intravenous fludarabine (40 mg/m2/day) followed immediately by intravenous BU (130 mg/m2/ day) on days –6 to –3 before HCT to treat patients with advanced myeloid malignancies (22 with MDS). Unrelated HCT recipients also received equine ATG. The 1-year RFS and TRM were 52% and 3%, respectively [43]. Similar data have been reported by Russell et al., who used intravenous BU (3.2 mg/kg/day for 4 days) and added rabbit ATG (Thymoglobulin) at a dose of 4.5 mg/kg to the conditioning regimen [44]. Encouraging studies such as these have stimulated the interest in further modifications of conventional (high-dose) conditioning regimens to reduce toxicity while maintaining efficacy. Kroger et al. reported on 37 patients with MDS or tAML who were transplanted after conditioning with a regimen of fludarabine (120– 180 mg/m2) and reduced-dose BU (8 mg/kg orally or 6.4 mg/kg intravenously) from related (n = 19) or unrelated (n = 18) donors (Table 57.5). TRM was 27%, and the 3-year estimated RFS was 38%, with a relapse rate of 32% [45]. A Spanish trial reported results on 37 patients with MDS or tAML transplanted from HLA-identical siblings following a fludarabine/BU conditioning regimen. The 1-year TRM was 5% and the RFS 66% [46].

833

Several studies have incorporated the anti-CD52 antibody alemtuzumab into reduced-intensity regimens as a method of in vivo T-cell depletion [47,48]. A British study examined the use of a fludarabine/ BU/alemtuzumab regimen and reported results in 75 patients with MDS receiving unrelated donor transplants. The 3-year actuarial survival was 43%, and the cumulative incidence of extensive chronic GVHD was 22%. This analysis also indicated that the disease status at HCT and the comorbidity score were independent risk variables for overall survival; however, patient age and cytogenetic abnormalities did not significantly affect outcomes [47]. A prospective phase II study by van Besien et al. used a reduced-intensity regimen of fludarabine, melphalan, and alemtuzumab in 52 patients with AML and MDS. With a median follow-up of 18 months, the relapse rate was 27%, TRM 33%, and RFS 38%. The cumulative probability of extensive chronic GVHD was 18%. High-risk disease and low performance status were the major adverse factors for outcome [48]. One study combined a reduced dose regimen of fludarabine and 200 cGy of TBI with iodine-131-labeled anti-CD45 monoclonal antibody for targeted delivery of radiation to hematopoietic cells [49]. This strategy clearly represents a “reintensification” of reducedintensity regimens, which may be effective in reducing the relapse rate without unacceptably increasing toxicity. Yet another strategy was chosen by Chan et al., who combined 2 days of photopheresis with pentostatin 4 mg/m2/day on days −5 and −4 before HTC, and TBI 3 × 200 cGy given on days −3 and −2 [50] for conditioning in 18 patients with MDS who were 30–70 years of age. The 1-year survival in remission was 64%. A regimen of 550 cGy of TBI plus CY was used by Hallemeier et al. in 51 patients with MDS or secondary AML [51]. Two-year survival was 37% overall, ranging from 88% in patients with AML in remission to 11% in patients with untreated or refractory disease. Recent studies indicate that risk factors not considered in the past may also impact on HCT outcome. In patients who do not undergo HCT, fibrosis in the marrow is associated with a more rapid progression of MDS. In a retrospective analysis of 471 patients with MDS who underwent HCT, 113 showed evidence of marrow fibrosis of various degrees, while 358 did not. All patients were conditioned with high-intensity regimens. Patients with marrow fibrosis showed a significant delay in neutrophil and platelet recovery (hazard ratio 0.4; p < 0.001). While there were no significant differences in overall survival, RFS, and NRM between the two groups when all patients were considered, patients in IPSS categories intermediate-2 or high risk who also had marrow fibrosis had greater NRM (p = 0.04) and reduced overall survival (p = 0.03) and RFS (p = 0.04). Thus, conceivably, patients with MDS and low-grade disease should be monitored closely, and the progression of fibrosis might be considered an indication for HCT. Another risk parameter may be flow cytometrically defined characteristics of MDS marrow cells. Our initial analyses suggested that patients with severe phenotypic aberrancies of marrow cells had a significantly higher risk of relapse after HCT than patients with minimal abnormalities [4]. As shown in Fig. 57.2, such an impact was seen even in patients who had received induction chemotherapy before HCT. In summary, the success of HCT in patients with MDS is determined primarily by the proportion of myeloblasts in the marrow, the karyotype, and peripheral blood cytopenias. Other factors, such as flow cytometric aberrancies of marrow cells, may also have prognostic relevance. Dependent upon these parameters, as many as 70–80% or as few as 15–20% of patients may be cured by HCT; some patients have been followed now for 25 years following HCT. Recent data show that results with unrelated donors who are HLA matched by high-resolution typing are comparable to those with HLA genotypically identical sibling donors.

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(a)

(b)

aplastic anemia develop MDS within 3 years, and “aplastic anemia” may simply be the initial presentation of MDS. There is agreement that chemotherapy and palliative measures are not useful in pediatric MDS, and HCT should be considered early in the course. Several studies have been reported from Europe, Japan, and the United States. Kardos et al. [53] reported on 67 children, 41 of whom underwent allogeneic HCT. Among children in whom the disease had not progressed, 76% were surviving, compared with 36% with advanced MDS (>5% myeloblasts). Sasaki et al. [54] presented data on 189 patients (122 primary MDS, 24 with constitutional predisposition, and 43 with therapy-related MDS). The 4-year survival among patients with refractory cytopenia was 79%, compared with less than 40% in all other patients. Children who underwent HCT had a significantly higher survival probability. Yusuf et al. reported results in 94 patients with MDS who received allogeneic HCT [55]. The 3-year probabilities of NRM, relapse, and RFS were 28%, 29%, and 41%, respectively. RFS was 74% in patients with refractory cytopenia, 68% with RAEB, and 33% with juvenile myelomonocytic leukemia. Patients with RAEB-T were 5.5 times more likely to relapse than other patients. HCT has also been successful in some children who developed MDS on a background of congenital disorders such as Fanconi’s anemia or Kostman’s syndrome (see Chapters 78 and 79). As in adults, cytogenetic abnormalities (monosomy 7) and myeloblast count are the strongest risk factors for post-HCT failure. Secondary MDS

Fig. 57.2 Marrow cells from patients with myelodysplastic syndrome were characterized by flow cytometry before hematopoietic cell transplantation (HCT) [4]. Patients had not received (a) or had been given (b) induction chemotherapy. Consistently, patients with highly aberrant marrow cells at HCT had a lower probability of survival after HCT, due to an increased incidence of relapse [21]. (Reproduced from [11], copyright [2005], ASBMT [Elsevier].)

MDS in children MDS is rare in children, accounting for 5–7% of hematologic malignancies. There are similarities to but also major differences from MDS in adults, and a separate classification has been proposed (Table 57.6) (the MDS/MPD group is discussed in Chapter 52). Occasional patients with Down’s syndrome who developed leukemia have been treated with HCT. Anecdotal evidence suggests that these patients are prone to experience severe regimen-related toxicity. Experience is limited, and this entity will not be further discussed here. Children with MDS present with thrombocytopenia or neutropenia more frequently than with anemia, and the term “refractory cytopenia” is preferred. Also, the category of RAEB-T has been retained [52]. About half of the patients have clonal cytogenetic abnormalities, the most frequent one being monosomy of chromosome 7 (≈25% of cases). With monosomy 7, the disease progresses faster than with other karyotypes. In secondary MDS, as in adults (see above), about 80% of patients have cytogenetic abnormalities. In contrast to adults, about 75% of children with MDS have hypocellular marrows, and the distinction from aplastic anemia may be difficult. In fact, about 10–15% of children with

As discussed above, we refer to secondary MDS if the disease develops following prior immunosuppressive or cytotoxic therapy, or if it evolves from an antecedent hematologic disorder. Among patients who have undergone an autologous HCT for Hodgkin’s disease or non-Hodgkin’s lymphoma, 1–20% have been reported to develop MDS within 10 years of primary therapy [56,57]. The median lag time varies dependent upon the primary treatment. Friedberg et al. analyzed outcomes in 552 patients after autologous HCT for non-Hodgkin’s lymphoma, 41 of whom showed evidence of MDS at a median follow-up of 47 months, for an actuarial incidence of 19.8% at 10 years [58]. None of the 13 patients who then received an allogeneic HCT survived in the long term, an outcome identical to that observed by the EBMT group [35]. YakoubAgha et al. reported results in 70 patients with secondary MDS or tAML [20]. Among these, 33 had received induction-type chemotherapy; 24 patients were in complete remission at HCT, while 46 had active disease. Most patients were conditioned with high-dose TBI or BU-containing regimens. RFS at 2 years was 28% for the entire cohort, 45% for patients in complete remission, and 18% and 26% for patients with active MDS and tAML, respectively. Other risk factors for poor outcome were older patient age, seropositivity for cytomegalovirus, and more intensive conditioning regimens. Woodard et al. [59] presented results in 38 children given allogeneic HCT for secondary MDS. Overall survival and RFS were similar, at 15.4% at 3 years; NRM was 60% and relapse incidence 24%. The proportion of marrow blasts at the time of HCT was the strongest risk factor for relapse. The number of patients who undergo HCT for secondary MDS has increased considerably, and we recently analyzed results in 257 patients, 3–72 (median 43) years of age with secondary MDS. Patients were conditioned with various intensity regimens including targeted BU/CYand TBI-based regimens. Donors were HLA identical or partially mismatched family members in 135 and unrelated individuals in 122 patients. Five-year RFS was highest (43%) and NRM lowest (28%) among patients conditioned with a targeted BU/CY regimen (Fig. 57.3). Outcomes in patients with secondary MDS were then compared with results in 339 patients with de novo MDS transplanted concurrently. While the nonadjusted data showed a better outcome, that is, long-term

Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders Table 57.6 Classification of myelodysplastic syndrome (MDS) and MDS/myeloproliferative disorder (MPD) of childhood

835

I. MDS/MPD Juvenile myelomonocytic leukemia Chronic myelomonocytic leukemia (secondary only) BCR–ABL-negative chronic myeloid leukemia II. Down’s syndrome disease Transient abnormal myelopoiesis Myeloid leukemia of Down’s syndrome III. MDS Refractory cytopenia (peripheral blood blasts <2% and marrow blasts <5%) Refractory anemia with excess blasts (peripheral blood blasts 2–19% or marrow blasts 5–19%) RAEB in transformation (peripheral blood or marrow blasts 20–29%)

(a)

RFS superior (p = 0.02) when the donors were unrelated. Thus, the data suggest that the overall inferior outcome in patients with secondary MDS was related to the significantly larger proportion of patients with high-risk cytogenetic features. Myeloproliferative disorders

(b)

Fig. 57.3 Probability of relapse-free survival in patients with secondary myelodysplastic syndrome/tAML. (a) By preparative regimen. FLU/TBI, fludarabine and total body irradiation (200 cGy); high-dose TBI, any highdose TBI-containing regimens; BU/CY, busulfan and cyclophosphamide; tBU/CY, busulfan targeted to plasma levels of 800–900 ng/mL and cyclophosphamide; FLU/BU, busulfan targeted to plasma levels of 800– 900 ng/mL and fludarabine. (b) By therapy given for the primary disease. Chemo, chemotherapy; chem/rad, chemotherapy plus radiation therapy; immuno, immunosuppressive treatment; HCT, hematopoietic stem cell transplantation.

survival, in patients with de novo MDS, no significant differences were noted following adjustments for other risk factors. Relapse probability and RFS correlated significantly with disease stage (p < 0.0001) and karyotype (p < 0.0001). Relapse incidence was lower (p = 0.003) and

The second category of chronic myeloid disorders is known by the collective term “MPD.” This includes chronic myeloid leukemia (CML, which is discussed in Chapter 51), polycythemia vera (PV), essential thrombocythemia (ET), and chronic idiopathic myelofibrosis (CIMF). According to the WHO classification, other, rare, disorders such as chronic neutrophilic leukemia, chronic eosinophilic leukemia (and hypereosinophilic syndrome), and chronic myeloproliferative diseases, unclassifiable, are included in the MPD category (mast cell disorders being classified separately) [1]. PV and ET present with increased red blood cell or platelet production, and in many instances, patients have a rather indolent course that may reach over decades. Eventually, however, patients may show severe marrow fibrosis and splenomegaly (spent phase), along with peripheral blood cytopenias, or may develop acute leukemia. Patients with CIMF present up front with marrow fibrosis and may or may not have substantial elevations of white blood cell or platelet counts and generally proceed to develop single or multilineage peripheral blood cytopenias. In 2005, several groups simultaneously identified an activating mutation (V617F) in the JAK-2 kinase in most patients with PV, about half the patients with ET, and 30–40% of patients with CIMF [60,61]. While this mutation contributes to the disease, the exact relationship is a matter of debate, and additional mutations have since been identified, for example in exon 12 of the gene [62]. Over time, patients with MPD develop severe secondary complications including, in addition to marrow fibrosis, hepatic fibrosis, portal hypertension, heart failure, and infectious complications due to cytopenias [63]. Several prognostic scoring systems have been developed for CIMF. Most widely used is the Lille (or Dupriez) score (Table 57.7). This system uses two variables, the hemoglobin level and white blood cell count, to stratify patients into low-, intermediate-, and high-risk groups with median survivals of 93, 26, and 13 months, respectively [64]. More recently, the Mayo Clinic group proposed to add thrombocytopenia as another poor-risk factor to the variables in the Lille system. This system predicts median survivals of 155, 69, and 24 months in the presence of 0, 1, or ≥2 adverse features, respectively (see Table 57.7) [65]. Data from our institution also identified thrombocytopenia in patients with CIMF as an adverse prognostic factor for outcome after HCT [66]. An Italian study identified the peripheral blood CD34+ cell

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Number of risk factors

Risk group

Proportion of patients (%)

Median survival (months)

Lille classification [64] 0 1 2

Low Intermediate High

47 45 8

93 26 13

Mayo Classification [65] 0 1 ≥2

Low Intermediate High

51 23 26

155 69 24

Table 57.7 Scoring systems for myelofibrosis

Risk factors: Hemoglobin <10 g/dL, white blood cell count <4 or >30 × 109/L (Lille); platelet count <100 × 109/L (Mayo only).

count as a prognostic factor associated with leukemic transformation and overall survival in 84 patients with CIMF [67]; however, a follow-up Mayo Clinic study failed to support those results [68]. The exact impact of clonal cytogenetic abnormalities remains to be determined [69], although the presence of deletions of chromosome 13q or 20q appears to be associated with a relatively good prognosis [70]. CIMF, PV, and ET For patients with MPD, few data on the optimal timing of HCT are available. Patients with PV can often be managed with simple phlebotomies for years or even decades. Patients with ET may respond to hydroxyurea or anagrelide, and the addition of aspirin may further reduce the risk of thrombosis. Most of these patients are considered for HCT only when they develop peripheral blood cytopenias or their disease has progressed to myelofibrosis or transformed to AML [13,63]. Patients with CIMF may do well without any therapy for several years; splenomegaly frequently responds to hydroxyurea or steroids, given alone or in combination with thalidomide [71]. Splenectomy does not significantly alter the course of the disease [72]. As with PV and ET, patients generally come to HCT because of peripheral blood cytopenias or leukemic transformation. Whether small molecules directed at the recently identified JAK2 mutation (or other mutations) will alter the natural course of these diseases remains to be determined. Data from patients with PV, ET, and CIMF from our own institution suggest that patient age, Lille score, platelet count, and comorbidity score are useful in arriving at the decision to proceed to HCT [73]. Initially, there was significant concern that patients with myelofibrosis would fail to achieve engraftment of donor cells due to the disturbed marrow architecture. However, several case reports and small series suggested that HCT was feasible and potentially curative therapy [74– 77]. These early studies were recently reviewed by van Besien & Deeg. [78]. Since then, serial skeletal magnetic resonance imaging and histological examination of marrow have shown that myelofibrosis is completely reversible by successful HCT and eradication of the malignant clone [79]. A retrospective study reported results in 55 patients transplanted at multiple institutions, mostly from HLA-identical sibling donors. Thirtyfive of those patients were conditioned with a TBI-based regimen, and 20 with BUCY. The 5-year overall survival was 47%, and NRM at 1 year was 27%. Low hemoglobin levels and the presence of osteosclerosis in the marrow were adverse prognostic markers [80]. We recently presented results in 104 patients with PV, ET or CIMF, 18–70 years of age, who had been transplanted at our center from allogeneic (56 related, 45 unrelated) or syngeneic (n = 3) donors. Most

Fig. 57.4 Survival among 92 allogeneic transplant recipients with chronic idiopathic myelofibrosis, polycythemia vera or essential thrombocythemia, conditioned with targeted busulfan with cyclophosphamide (BU/CY) or other regimens and transplanted from related or unrelated donors (Reproduced from [66], with permission. Copyright [2007], ASBMT [Elsevier].)

patients (n = 73) received targeted BU/CY (combined with ATG in 14) as a conditioning regimen, and 31 were prepared with other regimens, including nine who received a reduced dose regimen of intravenous fludarabine, 3 × 30 mg/m2, and 200 cGy of TBI (see below). Most patients (n = 61) received PBPCs as a source of stem cells, and engraftment was achieved in 101 of 104 patients. Sixty-three patients are surviving, for a 7-year actuarial survival of 61%. Eleven patients experienced relapse or had persistent disease, eight of whom have died. Patients conditioned with targeted BU/CY had a higher probability of survival (68%) than patients conditioned with other regimens (Fig. 57.4). In a multivariable regression model, the use of targeted BU/CY (p = 0.03), high platelet count at HCT (p = 0.01 for PV/ET; p = 0.39 for other diagnoses) (Fig. 57.5), younger patient age (p = 0.04), and low comorbidity score (p = 0.03) remained significantly associated with superior survival. There was no significant difference in outcome between HLAmatched related and unrelated transplants. There were 33 deaths from nonrelapse causes (pulmonary failure, multiorgan failure, GVHD, and invasive aspergillosis plus GVHD), and eight patients died with progressive or recurrent disease. As shown in earlier trials [6,81], the

Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders

837

among nine patients prepared with a reduced-intensity regimen, three died from transplant-related complications. It is also important to note that the actual intensities of the reduced-intensity regimens used in these studies differed. Conceivably, conditioning regimens of intermediate intensity will offer the best outcome [86]. In summary, while most reports present data on small numbers of patients, taken together the results show that HCT provides effective therapy for patients with MPD. The highest success rates have been observed in patients with PV and ET. Once leukemic transformation occurs, the probability of a successful HCT declines significantly. The optimum conditioning regimen remains to be determined. Other MPDs

Fig. 57.5 Impact of primary diagnosis on transplantation outcome in patients with myeloproliferative disorder (same patients as in Fig. 57.4). PV/ET, polycythemia vera/essential thrombocythemia. (Reproduced from [66], with permission. Copyright [2007], ASBMT [Elsevier].)

conditioning regimen had a significant impact on HCT outcome, the highest success rate being achieved with a targeted BU/CY regimen. Overall survival with this regimen in our recent study was not statistically significantly different from that in the originally reported cohort (p = 0.28), even though the median age in the current cohort was almost 1 decade older. Nevertheless, NRM was 13% at day 100 and 34% at 5 years, raising the question as to whether results could be further improved with the use of lower-intensity conditioning regimens. Devine et al. first described four patients, 48–58 years of age, who had been conditioned with a regimen of fludarabine 30 mg/m2/day intravenously for 5 days, and melphalan 70 mg/m2/day for 2 days, followed by PBPCs from HLA-identical siblings. At a median follow-up of 13 months, all patients were alive and had full donor chimerism [82]. Rondelli et al. presented a follow-up, including 21 patients with a median age of 54 (range 27–68) years with intermediate- or high-risk myelofibrosis (based on the Lille score) transplanted at several centers over an extended time interval, using several reduced-intensity regimens. All but one patient engrafted, and at the time of reporting, 18 patients were alive (17 in remission) with a median follow up of 31 months [83]. Kroger et al. presented data from a pilot study in 21 patients with myelofibrosis, 32–63 (median 53) years of age, conditioned with BU 10 mg/kg, plus fludarabine 180 mg/m2 and ATG, and transplanted from related or unrelated donors [84]. With a follow-up of 4–59 months, the 3-year RFS was estimated at 84%. Investigators at City of Hope Medical Center presented results on 9 patients aged 46–68 (median 54) years. Eight patients received fludarabine 25 mg/m2/day for 5 days followed by melphalan 140 mg/m2 for conditioning, while one patient received fludarabine 30 mg/m2 for 3 days followed by 200 cGy of TBI. All patients achieved engraftment, and the probability of 1-year survival was 56% [85]. Similarly, a reduced-intensity regimen of fludarabine 90 mg/m2, plus 200 cGy of TBI, as used in the Seattle study, yielded a survival rate of 56%, which did not differ from that achieved with higher-dose regimens. However, this small cohort consisted of patients up to 70 years, most of whom had comorbid conditions [2] that were not present in the patients reported by Kroger et al. [84]. That comorbid conditions are relevant for the success of HCT is supported by our current analysis, which showed a significant effect of the comorbidity index on survival; even

Experience with HCT for patients with chronic neutrophilic leukemia, chronic eosinophilic leukemia (hypereosinophilic syndrome), and unclassified MPD is limited. The optimal therapy for chronic neutrophilic leukemia is not known. However, because of the potential for blastic transformation, HCT has been considered appropriate therapy, particularly for young patients [87–89]. Similarly, successful HCT has been reported for patients with chronic eosinophilic leukemia [90,91]. However, the recent recognition that a proportion of patients with this disorder have an interstitial deletion on chromosome 4q12 that leads to the expression of a FIP1L1– PDGFRα fusion gene with tyrosine kinase activity has made treatment with tyrosine kinase inhibitors, such as imatinib, an attractive first-line treatment [92]. About 10% of patients are found to have such a gene rearrangement, and about 70–80% of these achieve complete remissions with imatinib [93,94]. Several patients with unclassifiable MPD have also been transplanted successfully [66,77]. Myelodysplastic/myeloproliferative diseases The WHO has listed CMML, atypical CML, juvenile monocytic leukemia, and unclassifiable myelodysplastic/myeloproliferative diseases as a separate category. As implied by the terminology, this category includes diseases with both dysplastic and proliferative features [25]. Peripheral blood monocytosis (>1000 μL) is the hallmark of CMML. Dependent upon the myeloblast count, CMML is divided into CMML-1 (blood <5%, marrow <10%) and CMML-2 (blood 5–19%, marrow 10–19%). The WHO has also defined a separate category of CMML with eosinophilia when there is an accompanying peripheral blood eosinophilia. Patients in this category, representing a small proportion of patients with CMML, often have a t(5;12)(q33;p13) translocation, which results in an abnormal platelet-derived growth factor receptor fusion gene, and those patients are likely to respond to tyrosine kinase inhibitors, such as imatinib [25,95]. Patients with CMML also have other clinical features, including splenomegaly, lymphadenopathy, and skin rashes. The MD Anderson group has developed a scoring system for CMML based on four clinical parameters: hemoglobin less than 12 g/L, the presence of circulating immature myeloid cells, an absolute lymphocyte count of over 2.5 × 109/L, and marrow myeloblasts over 10% predict a poor prognosis (Table 57.8). Based on the presence of one or several of those findings, patients are divided into low, intermediate-1, intermediate-2, and high-risk categories, with median survivals of 24, 15, 8, and 5 months, respectively [96]. Chronic myelomonocytic leukemia As indicated by the MD Anderson classification, CMML can, despite the term “chronic,” show rather rapid progression, generally to an acute leukemia picture. About half of the patients will achieve complete remissions with intensive induction chemotherapy, and, on average,

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Chapter 57

Table 57.8 M.D. Anderson criteria for CMML risk category Number of risk factors

Risk group

Median survival (months)

0–1 2 3 4

Low Intermediate-1 Intermediate-2 High

24 15 8 5

Risk factors: hemoglobin <12 g/L; absolute lymphocyte count >2.5 × 109/L; immature myeloid cells circulating in peripheral blood; ≥10% myeloblasts in bone marrow [96].

While the IPSS and MD Anderson scores showed no correlation with prognosis, there was a trend for a higher relapse incidence among patients with a higher MD Anderson score (Table 57.5) [99]. The available data suggest that HCT is associated with a graft-versus-CMML effect [98,99]. Atypical CML Atypical CML is a rare disorder that has a poor prognosis with conventional chemotherapy. Koldehoff et al. reported results with HCT in nine patients (four transplanted from HLA-identical siblings, four from HLAmatched unrelated donors, and one from an identical twin brother) [100]. One patient died at 9 months from cerebral toxoplasmosis. The remaining patients are alive and in remission (the twin transplant recipient after a second HCT from the same donor following relapse) at a median of 55 months after HCT. These data suggest that HCT offers curative therapy for patients with atypical CML. Juvenile myelomonocytic leukemia Studies in patients with juvenile myelomonocytic leukemia are described in Chapter 52. Systemic mastocytosis

Fig. 57.6 Survival among 42 patients with chronic myelomonocytic leukemia according to the hematopoietic cell transplantation specific comorbidity index. (Reproduced from [99], with permission. Copyright [2005], ASBMT [Elsevier].)

remissions are sustained for 4–5 months. Thus, except for patients with a t(5;12) translocation, the majority of whom will respond to tyrosine kinase inhibitors such as imatinib or desatinib (see above), the only promising therapy is HCT. A large EBMT series reported transplant results in 50 patients. About half of these patients were conditioned with TBI-based regimens (n = 26), and most patients (n = 38) received hematopoietic stem cells from HLA-matched siblings. Forty patients received marrow, nine received PBPCs, and one patient received both. RFS at 2 years was 18%, with an NRM of 55%, and a relapse incidence of 42%. Patients who developed acute GVHD had a lower rate of relapse [97]. The Mayo Clinic group reported results in 17 patients with CMML, the majority receiving TBI-based conditioning; 14 were transplanted from HLA-identical siblings, and three from unrelated donors. TRM was 41%, and seven patients (41%) had persistent disease or relapsed; five of the patients who relapsed received donor lymphocyte infusions, and two of these achieved durable remissions. At the time of reporting and with a median follow-up of 34.5 months, three of the 17 patients (18%) were alive and in remission [98]. At the Fred Hutchinson Cancer Research Center, we have transplanted 50 patients with CMML, and data on 43 patients transplanted from related (n = 21) or unrelated donors (n = 22; 18 HLA matched and four HLA mismatched) have been reported [99]. The 4-year RFS was 41%, with a relapse incidence of 23%. Patients with low comorbidity had a probability of surviving in remission that approached 60% compared with 15% in patients with high co-morbidity scores (Fig. 57.6).

The WHO has classified mast cell disorders in a separate category [1]. However, we will briefly summarize here the limited transplant experience. Patients with indolent mastocytosis have an excellent prognosis. However, patients with aggressive systemic mastocytosis or mast cell leukemia may have a life expectancy of only a year or less [101]. Nakamura et al. reported on three patients who were transplanted from HLAidentical siblings, following conditioning with a reduced-intensity regimen [102]. All patients achieved engraftment (with complete donor CD3 chimerism); however, all experienced disease progression, with the longest response interval being 39 months. Sperr et al. reported a successful HCT following high-intensity conditioning in an 18-year-old boy with myelomastocytic leukemia [103,104]. Another successful HCT was reported by Hennessy et al. [105] and Przepiorka et al. [77]. Optimization of conditioning regimens for allogeneic HCT for MDS and MPD In an attempt to reduce treatment-related toxicity and TRM and to offer the option of HCT to patients who are not considered candidates for high-dose conditioning, reduced-intensity regimens have been developed (discussed in detail in Chapter 71). The basic principle behind this strategy is to provide sufficient immunosuppression to secure donor cell engraftment and rely on the graft-versus-tumor effect mediated by donor-derived cells to eradicate the underlying disease. Initial regimens pioneered by Slavin et al. and by Storb and colleagues consisted of lowdose BU with or without other agents or low-dose (200 cGy) TBI with or without fludarabine [38,106]. Subsequently, many groups have adopted similar regimens or have had stepwise increases in intensity. Conversely, high-dose regimens have been attenuated, leading to a broad spectrum of regimens aimed at eradicating the patient’s disease while minimizing NRM [17]. As illustrated in Fig. 57.7, it appears appropriate to consider the actual transplant strategy in the context of pre-HCT parameters as well as post-HCT options. There is concern that, while reduced-intensity regimens reduce toxicity, there may also be decreased efficacy. Three recent retrospective analyses sought to address this question in patients with MDS and tAML, and all three studies concluded that survival figures for high-dose and reduced-intensity conditioning were comparable [39,107,108].

Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders

Pretransplant Parameters

Conditioning

High?

Comorbidity

Yes

Reduced-intensity

No

Older?

Age

High-dose

Yes

Reduced-intensity

No

Disease stage / induction

839

Treatment response?

High-dose

Yes

Reduced-intensity

No

High-dose

Transplant Approach Source of stem cells

Fig. 57.7 Hematopoietic cell transplantation for patients with myelodysplastic syndrome and myeloproliferative disorders is an evolving field. The algorithm provides some guidance regarding consideration of patient age, comorbid conditions, disease stage (and the possible advisability of induction therapy) when deciding upon a conditioning regimen. For patients with high-risk disease and with reduced-intensity/ nonmyeloablative transplant regimens, granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells (G-PBPC) are currently the preferred source of stem cells. Post-transplant manipulations depend upon the type of transplant, the degree of donor cell chimerism that is achieved, and the remission status. NK, natural killer.

Conditioning intensity

Reduced-intensity

High-dose

Relapse risk

Use G-PBPC

High

Low

Use marrow

Post-transplant Considerations Mixed chimeras

High relapse risk

We analyzed results in patients with tAML and MDS transplanted at our center, 112 of whom received high-dose conditioning with BU/CY, and 38 were conditioned with a reduced-intensity regimen of fludarabine 90 mg/m2 with or without 200 cGy of TBI. Patients receiving reducedintensity conditioning were older, had higher-risk disease by IPSS, had higher comorbidity scores, and, importantly, had enjoyed more durable responses to pre-HCT chemotherapy. The 3-year RFS and overall survival did not differ significantly between the two groups [39]. Alyea et al. carried out a retrospective analysis in 136 patients transplanted for MDS and tAML. Outcomes among 97 patients receiving high-dose conditioning with CY combined with either BU or 1400 cGy TBI were compared with those among 39 patients receiving reducedintensity conditioning with fludarabine and low-dose BU. There was no statistically significant difference in overall survival and RFS between the two groups. Of note was that patients receiving reduced-intensity conditioning had higher relapse rates, but the impact on survival was counterbalanced by a higher rate of TRM in patients receiving high-dose conditioning [107]. Recently, the EBMT reported the largest series to date, including 836 patients with MDS given various high-dose (n = 621) or reduced-inten-

Donor lymphocyte infusion

Selected donor cell infusion (?NK cells)

Reduction of immunosuppression

sity (n = 215) regimens. There was no significant difference in survival between the two groups; relapse rates were significantly lower in patients receiving high-dose conditioning, but, as in the other studies, this advantage was canceled out by a higher TRM in patients conditioned with high-dose regimens [108]. This was true for patients who had not received chemotherapy before HCT, and those who had been given chemotherapy, regardless of whether or not they achieved a remission (Fig. 57.8). Since patients selected for reduced-intensity regimens compared with more conventional regimens were not truly comparable in regards to risk factors in these retrospective studies, conclusions must be drawn cautiously. Only prospective randomized trials will provide definitive answers. Finally, it would be wrong to simply contrast high-dose to reduced-intensity regimens. As emphasized in several recent reviews [17,109], a broad spectrum of conditioning regimens using various modalities has been developed. Investigations at all “intensity levels” have been aimed at minimizing toxicity and optimizing efficacy. It is quite likely that what is “optimal” for one disease category may be “suboptimal” (either too intense or insufficient) for another, and what may yield superior results in patients whose disease is in remission at

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HCT (either with bone marrow or PBPCs) after conditioning with BU/ CY in 24 of 39 patients who achieved complete remissions after induction chemotherapy. Among these, 50% were alive 8–55 months after HCT [111]. De Witte and colleagues presented data on 184 patients with MDS/ tAML who received induction chemotherapy [112]. Among these, 56 had HLA-identical related donors available and 128 did not. One hundred patients achieved remission, and, with or without additional consolidation, 39 were transplanted with allogeneic and 61 with autologous cells. The rate of continuous complete remission was 33% for allogeneic and 31% for autologous transplants. The 4-year RFS (expressed as proportion of the total cohort) was 25% for allogeneic and 15% for autologous HCT [112]. The same authors recently reported results in patients with or without HLA-identical sibling donors on an intent-to-treat basis [113]. There were 159 patients who received remission induction and consolidation chemotherapy. Sixty-five patients had no donor available, and among these, 33 ultimately received autologous HCT. RFS was 23% for patients with, and 21% for patients without, a donor. HCT from alternative donors did not significantly alter the survival of the group without a related donor. This intention-to-treat analysis failed to show a survival advantage for patients with HLA-identical sibling donors compared with those without such a donor. The data indicate, however, that outcome with autologous HCT was superior to that with chemotherapy alone (without HCT). Myeloproliferative disorders

Fig. 57.8 Incidence of relapse (REL) and nonrelapse mortality (NRM) in patients with myelodysplastic syndrome conditioned with conventional regimens (high dose) or reduced-intensity conditioning (RIC) regimens and transplanted from human leukocyte antigen-identical sibling donors. Outcome by pretransplant chemotherapy: no therapy (top panel), therapy but not achieving complete remission (middle panel), and achieving complete remission (bottom panel). (Reproduced from [108], with permission. Copyright, American Society of Hematology.)

the time of HCT may lead to disappointing outcomes in patients with active disease at HCT.

As patients with myelofibrosis typically have high concentrations of CD34+ precursors circulating in the blood, it may be possible to harvest sufficient cells at “steady state.” In a multicenter study, 27 patients with myelofibrosis secondary to CIMF, PV or ET underwent autologous hematopoietic cell collection, and 21 patients underwent conditioning with BU (16 × 1 mg/kg), followed by autologous HCT. The median time to both platelet and neutrophil recovery was 21 days. Clinically significant responses were seen in 10 of 17 patients with anemia, four of eight patients with thrombocytopenia, and seven of 10 patients with symptomatic splenomegaly [114]. However, the study was closed because of graft failure/incomplete hematopoietic recovery in 5 patients (27%). The high graft failure rate was attributed to the fact that autologous hematopoietic cells had been collected late in the disease course, and the investigators speculated that engraftment might be improved if autologous cells were harvested earlier. It appears that the palliative effect achieved with autologous cells was owing to the fact that the growth kinetics of clonal and nonclonal precursor cells differ, providing a temporary advantage to nonclonal cells. To carry out autologous HCT for MPD with curative intent, purging of clonal hematopoietic cells from the harvested cells would be necessary, and currently no effective method to achieve this is available.

Autologous HCT Myelodysplastic syndrome

Relapse after HCT

Autologous HCT generally does not lead to GVHD and is associated with lower TRM than allogeneic HCT. It may hold promise in patients in whom a “pure” population of normal hematopoietic stem cells is obtainable. The EBMT reported results in 79 patients with MDS, showing a 2year RFS of 28% after autologous HCT [110]. NRM was 39% in patients more than 40 years of age. These results were restricted, however, to patients who achieved complete remissions after induction chemotherapy. Wattel et al. prospectively assessed the feasibility of autologous

Post-transplant relapse is a challenging problem. There is evidence that discontinuation of anti-GVHD prophylaxis (or therapy) or the infusion of viable donor lymphocytes can induce remissions in allogeneic recipients. A prospective trial in pediatric patients tested the efficacy of preemptive donor lymphocyte infusion in 65 patients with refractory cytopenia or advanced MDS who showed mixed chimerism with increasing proportions of donor cells after allogeneic HCT [115]. Sequential chimerism testing showed that 48 patients became complete chimeras, and five had increasing proportions of donor cells, while 12 continued

Hematopoietic Cell Transplantation for Myelodysplastic Syndrome and Myeloproliferative Disorders

to show increasing numbers of host cells. RFS was 50%, compared with 0% in historical controls not treated with donor lymphocyte infusions. However, a recent analysis of results in 194 adult patients with MDS or acute or chronic leukemia, originally conditioned with high-dose or reduced-intensity regimens, showed that only 20–30% of patients achieved complete remission following discontinuation of immunosuppression or donor lymphocyte infusion. Further, 2-year survival was 0–4% for patients who had relapsed within 100 days of HCT, and 5–12% if the relapse occurred after day 100 [116]. Second transplants, particularly with reduced-intensity regimens, are an option, not only for allogeneic, but also for autologous transplant recipients (assuming a donor can be found). However, the success rates have been low, related to TRM or refractoriness of the disease [117]. Currently ongoing studies are investigating whether adjuvant or preemptive therapy, for example with DNA methyltransferase inhibitors or lenalidomide, may reduce the relapse probability in high-risk patients. Other strategies include the infusion of donor natural killer cells, T cells sensitized against patient leukemia cells or vaccination approaches.

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MDS by IPSS criteria and patients with secondary MDS should undergo early HCT. Select patients with intermediate-1 MDS are also likely to benefit from early HCT, particularly if their disease shows multilineage cytopenias requiring heavy transfusion support. Patients with low-risk disease by IPSS are likely to have the longest life expectancy if HCT is delayed until there is evidence of disease progression [118]. As new agents for nontransplant therapy become available, the question of timing of HCT will have to be readdressed. Although there are fewer data for patients with MPD, patient age, Lille score, platelet count, and comorbidity score are all useful parameters in making the decision to proceed with HCT [68]. A broad spectrum of conditioning regimens has been used successfully to transplant patients with MDS and MPD. Decreasing conditioning intensity is associated with lower TRM but often at the cost of increased relapse rates. Future studies are needed to define the appropriate conditioning that minimizes toxicity while maximizing efficacy.

Acknowledgments Conclusion HCT is the only current treatment with curative potential for patients with MDS and MPD. The IPSS is helpful in deciding which patients with MDS should be transplanted and when. Patients with high-risk

We thank Joanne Greene and Franchesca Nguyen for maintaining the MDS/MPD database, and Bonnie Larson and Helen Crawford for help with manuscript preparation. This work was supported in part by PHS grants HL36444, CA 15704, CA 18029 and CA119599.

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109. Mohty M, Nagler A, Killmann NM. Reducedintensity conditioning allogeneic stem cell transplantation: hype, reality or time for a rethink? Leukemia 2006; 20: 1653–4. 110. de Witte T, van Biezen A, Hermans J et al. Autologous bone marrow transplantation for patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia following MDS. Blood 1997; 90: 3853–7. 111. Wattel E, Solary E, Leleu X et al. A prospective study of autologous bone marrow or peripheral blood stem cell transplantation after intensive chemotherapy in myelodysplastic syndromes. Leukemia 1999; 13: 524–9. 112. De Witte T, Suciu S, Verhoef G et al. Intensive chemotherapy followed by allogeneic or autologous stem cell transplantation for patients with myelodysplastic syndromes (MDSs) and acute myeloid leukemia following MDS. Blood 2001; 98: 2326–31. 113. Oosterveld M, Suciu S, Verhoef G et al. The presence of an HLA-identical sibling donor has no impact on outcome of patients with high-risk MDS or secondary AML (sAML) treated with intensive chemotherapy followed by transplantation: results of a prospective study of the EORTC, EBMT, SAKK and GIMEMA Leukemia Groups

114.

115.

116.

117.

118.

(EORTC study 06921). Leukemia 2003; 17: 859– 68. Anderson JE, Tefferi A, Craig F et al. Myeloablation and autologous peripheral blood stem cell rescue results in hematologic and clinical responses in patients with myeloid metaplasia with myelofibrosis. Blood 2001; 98: 586–93. Bader P, Niemeyer C, Willasch A et al. Children with myelodysplastic syndrome (MDS) and increasing mixed chimaerism after allogeneic stem cell transplantation have a poor outcome which can be improved by pre-emptive immunotherapy. Br J Haematol 2005; 128: 649–58. Mielcarek M, Martin PJ, Maloney DG et al. Outcomes among patients with recurrent high-risk hematologic malignancy after nonmyeloablative versus myeloablative allogeneic hematopoietic cell transplantation [Abstract]. Blood 2006; 108(Pt 1): 81a, #262. Radich JP, Gooley T, Sanders JE, Anasetti C, Chauncey T, Appelbaum FR. Second allogeneic transplantation after failure of first autologous transplantation. Biol Blood Marrow Transplant 2000; 6: 272–9. Cutler CS, Lee SJ, Greenberg P et al. A decision analysis of allogeneic bone marrow transplantation for the myelodysplastic syndromes: delayed

119.

120.

121.

122.

transplantation for low-risk myelodysplasia is associated with improved outcome. Blood 2004; 104: 579–85. Nevill TJ, Fung HC, Shepherd JD et al. Cytogenetic abnormalities in primary myelodysplastic syndrome are highly predictive of outcome after allogeneic bone marrow transplantation. Blood 1998; 92: 1910–17. de Witte T, Hermans J, Vossen J et al. Haematopoietic stem cell transplantation for patients with myelo-dysplastic syndromes and secondary acute myeloid leukaemias: a report on behalf of the Chronic Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Br J Haematol 2000; 110: 620–30. Deeg HJ, Storer BE, Boeckh M et al. Reduced incidence of acute and chronic graft-versus-host disease with the addition of thymoglobulin to a targeted busulfan/cyclophosphamide regimen. Biol Blood Marrow Transplant 2006; 12: 573–84. de Lima M, Anagnostopoulos A, Munsell M et al. Nonablative versus reduced-intensity conditioning regimens in the treatment of acute myeloid leukemia and high-risk myelodysplastic syndrome: dose is relevant for long-term disease control after allogeneic hematopoietic stem cell transplantation. Blood 2004; 104: 865–72.

58

Muzaffar H. Qazilbash & Sergio A. Giralt

Hematopoietic Cell Transplantation for Multiple Myeloma

Epidemiology and etiology Multiple myeloma (MM) is a clonal disorder of plasma cells affecting approximately 50,000 patients in the United States, with an annual incidence of about 15,000 new cases [1,2]. It affects primarily older patients, with a median age of onset of 72 years, and is rarely seen in patients under the age of 40. Men are slightly more commonly affected than women, and African-Americans have nearly twice the incidence of Caucasians [3]. Although the cause of myeloma is not known, there is evidence that the pathogenesis of myeloma is a multistep process. The disease often evolves from a premalignant condition called monoclonal gammopathy of undetermined significance (MGUS) [4]. It is postulated that clonal plasma cell proliferation in this condition is triggered by translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 [5]. Secondary genetic abnormalities involving other oncogenes and tumor suppressor genes (e.g. ras, p53, and Rb1) and changes in the bone marrow microenvironment are believed to play a role [6,7]. No common molecular pathway for transformation has yet been described [8].

increases the expression of an oncogene that is positioned near a strong Ig enhancer. The malignant clone derived from the transformed cell is capable of further differentiation and division. During the evolution of MM to advanced stages, additional genetic events, including loss of chromosome 13 [26,27], activation of other oncogenes such as Ras [28,29], loss of tumor suppressor gene p53 and Rb (retinoblastoma) [30,31], and dysregulation of the cell cycle may take place [32,33]. Once in the bone marrow, myeloma and stromal cells generate a supportive microenvironment with a network of cytokines and adhesin molecules that promote MM proliferation and perpetuation by promoting growth and preventing apoptosis [7]. Increased osteoclast activation, likely mediated by increased levels of macrophage inflammatory protein1, and receptor activator of nuclear factor-kappa B ligand (RANKL), and decreased levels of osteoprotegerin, the decoy receptor for RANKL, results in lytic bone lesions and osteoporosis [34–36].

Clinical description Clinical manifestations

Molecular and cellular biology The malignant plasma cell migrates from the germinal centers of the lymph nodes to the bone marrow, which provides a microenvironment conducive to terminal plasma cell differentiation [7]. Malignant transformation in MM appears to start with genetic abnormalities late in Bcell differentiation [9–11]. The presence of genetic abnormalities has been demonstrated by fluorescence in situ hybridization (FISH), comparative genomic hybridization, and gene expression profiling in most patients with MM [12–15]. A multistep model has been proposed to explain the events leading to malignant transformation in MM. In an early stage of MGUS or smoldering MM, critical chromosomal abnormalities may occur, leading to one of the five known IgH translocations [5]. The five recurrent IgH translocations seen in MGUS and MM involve the chromosomal partners 4p16, 6p21, 11q13, 16q23, and 20q11. The identified genes include cyclin D1 and other growth factors (11q), the fibroblast growth factor receptor-3 (4p), the basic zipper C-MAF transcription factor (16q), and interferon regulatory factor-4 (6p) [16–25]. The IgH translocation

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Bone pain affects approximately 70% of all myeloma patients [3]. The pain is generally localized to the back and ribs. The bone lesions of myeloma are caused by the proliferation of tumor cells and the activation of osteoclasts that destroy the bone. The bone destruction results in severe bone pain, pathologic fractures, hypercalcemia, and nerve compression syndromes. A unique feature of myeloma bone lesions is that the lesions rarely heal even when the patient is in complete remission (CR) [35,36]. Recurrent bacterial infections constitute another common clinical problem in myeloma. The most common infections are pneumonias and pyelonephritis, and the most frequent pathogens are Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae in the lungs, and Escherichia coli and other Gram-negative organisms in the urinary tract. The susceptibility to infection has several contributing causes, including suppression of normal antibody synthesis, poor antibody response to polysaccharide antigens, and abnormalities in granulocyte and complement functions [37]. Serum creatinine level is elevated in almost 50% of patients, and renal failure is seen in about 25% of patients. Many factors contribute to renal dysfunction, including hypercalcemia, hyperuricemia, amyloid deposition, and recurrent infections. However, tubular damage associated with light chain excretion is the predominant cause [3,38]. Anemia is present in 80% of patients at diagnosis [3]. It is usually normocytic and normochromic, and related both to the replacement of

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normal marrow by expanding tumor cells and to the inhibition of hematopoiesis by factors secreted by the tumor. Diagnosis According to the International Myeloma Working Group, a diagnosis of symptomatic MM requires presence of clonal plasma cells on bone Table 58.1 New International Staging System for myeloma Stage

Criteria

Median survival (months)

I

Serum β2-microglobulin <3.5 mg/L Serum albumin ≥3.5 g/dL Not stage I or III* Serum β2-microglobulin ≥5.5 mg/L

62

II III

44 29

* There are two categories for stage II: serum β2-microglobulin <3.5 mg/L but serum albumin <3.5 g/dL; and serum β2-microglobulin 3.5 to <5.5 mg/L irrespective of serum albumin level. Reproduced with permission from Greipp et al. [46].

marrow examination or biopsy-proven plasmacytoma, monoclonal (M) protein in the serum and/or urine, and evidence of end-organ damage (hypercalcemia, renal insufficiency, anemia or bone lesions) believed secondary to the underlying plasma cell disorder [39]. Conventional radiography shows skeletal abnormalities in approximately 80% of patients. With magnetic resonance imaging, it is possible to determine the extent of marrow infiltration in the axial skeleton despite having normal plain radiologic examinations [40]. The M protein can be detected by serum protein electrophoresis in over 80% of patients, and by immunofixation in over 90% [3,41]. Up to 20% of patients with MM are considered to have light-chain MM. The M protein in these patients is always detected in urine but can be absent in serum, even by immunofixation. Among patients with MM, 3% have no detectable M protein on serum or urine immunofixation and are considered to have nonsecretory MM. In these patients, the serum free light chain assay, which measures the level of free (unbound) κ and λ light chains in the serum, is helpful for diagnosing and monitoring response to therapy. The free light chain κ : λ ratio is used to distinguish polyclonal elevations seen with renal dysfunction from monoclonal elevations that occur in clonal plasma cell disorders [41–43].

Table 58.2 Definitions for response categories Response subcategory

Response criteriaa

CR

Negative immunofixation on the serum and urine and Disappearance of any soft tissue plasmacytomas and ≤5% plasma cells in bone marrowb

sCR

CR as defined above plus Normal FLC ratio and Absence of clonal cells in bone marrowb by immunohistochemistry or immunofluorescencec

VGPR

Serum and urine M-component detectable by immunofixation but not on electrophoresis or 90% or greater reduction in serum M-component plus urine M-component <100 mg per 24 h

PR

≥50% reduction of serum M-protein and reduction in 24-h urinary M-protein by ≥90% or to <200 mg per 24 h If the serum and urine M-protein are unmeasurable,d a ≥50% decrease in the difference between involved and uninvolved FLC levels is required in place of the M-protein criteria If serum and urine M-protein are unmeasurable, and serum free light assay is also unmeasurable, 50% reduction in plasma cells is required in place of M-protein, provided baseline bone marrow plasma cell percentage was 30% In addition to the above listed criteria, if present at baseline, a 50% reduction in the size of soft tissue plasmacytomas is also required

SD (not recommended for use as an indicator of response; stability of disease is best described by providing the time to progression estimates)

Not meeting criteria for CR, VGPR, PR or progressive disease

CR, complete response; FLC, free light chain; PR, partial response; sCR, stringent complete response; SD, stable disease; VGPR, very good partial response. a All response categories require two consecutive assessments made at anytime before the institution of any new therapy; complete and PR and SD categories also require no known evidence of progressive or new bone lesions if radiographic studies were performed. Radiographic studies are not required to satisfy these response requirements. b Confirmation with repeat bone marrow biopsy not needed. c Presence/absence of clonal cells is based upon the k/λ ratio. An abnormal k/λ ratio by immunohistochemistry and/or immunofluorescence requires a minimum of 100 plasma cells for analysis. An abnormal ratio reflecting presence of an abnormal clone is k/λ of >4 : 1 or <1 : 2. Alternatively, the absence of clonal plasma cells can be defined based on the investigation of phenotypically aberrant plasma cell (PC). The sensitivity level is 10−3 (less than one phenotypically aberrant PC within a total of 1000 PCs). Examples of aberrant phenotypes include (1) CD38+dim and CD56+strong and CD19− and CD45−; (2) CD38+dim and CD138+ and CD56++ and CD28+; (3) CD138+, CD19− CD56++, CD117+. d Definitions of measurable disease Response criteria for all categories and subcategories of response except CR are applicable only to patients who have ‘measurable’ disease defined by at least one of the following three measurements: Serum M-protein ×1 g/dl (×10 gm/l)[10 g/l] Urine M-protein ×200 mg/24 h Serum FLC assay: Involved FLC level ×10 mg/dl (×100 mg/l) provided serum FLC ratio is abnormal Reproduced with permission from [48].

Hematopoietic Cell Transplantation for Multiple Myeloma

Staging, prognosis and response Median survival has traditionally been quoted as approximately 3 years. However, improvements in therapies and supportive care have dramatically improved the prognosis [44]. Survival depends on both tumor burden as well as intrinsic biologic features. Tumor burden has traditionally been measured using the Durie–Salmon staging system. However, this staging system has limitations, especially in the categorization of bone lesions [45]. Recently, the new International Staging System, which is a collaborative effort by investigators from 17 institutions worldwide based on data from more than 11,000 patients, has been introduced. The International Staging System overcomes the limitations of the Durie–Salmon staging and divides patients into three distinct stages and prognostic groups on the basis of serum β2-microglobulin and albumin levels (Table 58.1) [46]. Selected clonal chromosomal abnormalities have also emerged as adverse prognostic markers. These include hypodiploidy, deletions involving chromosomes 1, 13, and 17, and translocations involving the IgH locus on chromosome 14 [47]. New uniform response criteria are required to adequately assess clinical outcomes in myeloma. Table 58.2 shows the European Group for Blood and Marrow Transplant/International Bone Marrow Transplant Registry criteria that have been expanded, clarified, and updated to provide a new comprehensive evaluation system [48].

Nontransplantation approaches Patients eligible for autologous hematopoietic cell transplantation In newly diagnosed patients eligible for autologous hematopoietic cell transplantation (HCT), it is important to avoid treatment with alkylating agents like melphalan that are toxic to progenitor cells, and may interfere with adequate hematopoietic cell (HC) mobilization and collection [49,50]. Approximately two to four cycles of induction therapy are administered before HC collection. In patients who will require early autologous HCT, the primary goal of induction therapy is to reduce tumor burden before the procedure. There is no current evidence that the magnitude of the response before autologous HCT affects overall survival (OS). Even patients without an objective response to induction therapy (primary refractory MM) achieve durable responses from autologous HCT [51–53]. In patients who are HCT candidates but want to reserve it as a delayed option for relapsed refractory disease, HC may be collected early in the disease course and cryopreserved [2]. The combination of thalidomide and dexamethasone has emerged as an effective induction regimen with objective response rates of approximately 65–70%. Thalidomide is administered orally at doses of 100– 200 mg/day with dexamethasone at 40 mg/day on days 1 through 4, 9 through 12, and 17 through 20 repeated every 4 weeks [54–56]. The regimen does not interfere with HC collection or engraftment. A prospective randomized study of 207 untreated patients compared thalidomide–dexamethasone with dexamethasone alone. There was a significantly higher response rate with the combination (63% versus 41%; p = 0.001), but it also showed significantly higher grade 3 or greater toxicities (45% versus 21%; p < 0.001), including deep venous thrombosis (17% versus 3%) and peripheral neuropathy (7% versus 3%). Lenalidomide (Revlimid) plus dexamethasone has also shown promise, with response rates exceeding 80% and with lower toxicity than previously observed with thalidomide–dexamethasone [57]. The proteasome inhibitor bortezomib has been used alone or in combination with other agents in previously untreated patients. In a study of 32 consecutive myeloma patients, bortezomib with or without dexamethasone was given to previously untreated patients. A complete or

847

near-complete response of 25% and an overall response rate of 88% were achieved after six cycles of therapy [58]. The regimen does not interfere with HC collection or engraftment. There is suggestion that bortezomib may overcome the poor prognosis conferred by chromosome 13 abnormalities [59]. The intravenous regimen of vincristine, doxorubicin (adriamycin), and dexamethasone (VAD) is no longer recommended as first-line therapy due to the need for a central venous catheter, continuous intravenous infusion with the related increased risk of catheter-related sepsis and thrombosis, and neurotoxicity associated with vincristine, which may limit the future use of thalidomide and bortezomib, both of which are known to be neurotoxic [60,61]. Various retrospective analyses have shown that induction with VAD does not improve outcomes after transplantation when compared with induction therapy using dexamethasone alone or in combination with thalidomide [62,63]. Patients not eligible for autologous HCT Patients who are not candidates for transplantation due to old age, poor performance status or comorbidity are treated with standard alkylating agent therapy [41]. Significant improvements in response rates and time to progression have been reported in one published randomized trial, comparing melphalan, prednisone, and thalidomide with melphalan and prednisone [64]. In the GIMENA trial, 255 untreated patients aged 60–85 years were treated with either melphalan–prednisone– thalidomide or melphalan–prednisone; with a median follow-up of 16 months, the 2-year event-free survival (EFS) favored the thalidomide arm (54% versus 27%; p = 0.006), but the 3-year OS was not significantly different (80% versus 64%; p = 0.19) [63]. Mateos et al. recently reported the results of combining bortezomib with a standard induction therapy of melphalan and prednisone. Sixty patients with a median age of over 75 years were treated. After a median follow up of 16 months, the overall response rate (partial response + complete response) was 89%, and progression-free survival (PFS) and OS were 83% and 90%, respectively. Treatment was well tolerated, with a few patients developing serious adverse events [65]. Thus, bortezomiband thalidomide-based combinations may replace high-dose dexamethasone or melphalan + prednisone as the standard induction treatment for these patients.

Autologous HCT Overview High-dose melphalan without autologous HCT was first reported in 1983 by McElwain and Fowles from the Royal Marsden Hospital [66]. Patients received 100–140 mg/m2 of melphalan, and three previously untreated patients achieved CR. High-dose therapy with autologous HCT to reduce regimen-related toxicity was first reported by Barlogie et al. in 1986 [67]. They used up to 140 mg/m2 of melphalan intravenously. Over the next 20 years, high-dose therapy and autologous HCT evolved into a safe and common therapeutic approach for patients with MM. When compared with chemotherapy alone, intensified chemotherapy followed by autologous HCT has been shown to prolong both EFS and OS in previously untreated patients with myeloma. Various randomized trials and nonrandomized analysis have shown both benefits for survival and PFS in favor of autologous HCT of approximately 12 months [68,69]. These studies are summarized in Table 58.3. Best preparative regimen Many different preparative regimens have been assessed over the last 20 years. Only one prospective, randomized trial by a French coopera-

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Table 58.3 Trials of conventional therapy versus single autologous hematopoietic cell transplantation

Reference

n

Age limit (years)

Complete remission (%)

Median event-free survival (months)

Median overall survival (months)

[68] [69] [90] [91] [92]

200 401 191 164 516

≤65 <65 55–65 ≤65 ≤70

5 versus 22 8 versus 44 5 versus 10 11 versus 30 15 versus 17

18 versus 27 19 versus 31 19 versus 25 33 versus 42 7-year event-free survival: 14 versus 17%

37 versus NR 42 versus 54 47.6 versus 47.8 66 versus 61 7-year overall survival: 38% versus 38%

tive group has directly compared two different preparative regimens. In 282 newly diagnosed symptomatic patients under 65 years of age, they reported that high-dose melphalan at 200 mg/m2 was superior to a combination of melphalan 140 mg/m2 + 8 Gy of total body irradiation (TBI), mainly by reducing toxicity including mucositis and transplant-related mortality (TRM) [70]. Melphalan 200 mg/m2 was associated with a faster hematologic recovery, reduced transfusion requirements, and a shorter hospitalization. While the median duration of EFS was similar in both arms (21 months), survival at 45 months was significantly better in the melphalan-alone arm (66% versus 46%; p = 0.05). As a result, melphalan 200 mg/m2 is the standard conditioning regimen used for autologous HCT in myeloma, with dose reductions based on age and renal function. In two non-randomized studies and a registry analysis, use of more intensive preparative regimens, such as busulfan with melphalan [71], thiotepa, busulfan, and cyclophosphamide [72], or high-dose idarubicin, cyclophosphamide, and melphalan [73], did not result in better outcomes than melphalan at a dose of 200 mg/m2. Patient selection Older age Most centers used to exclude patients over the age of 70, because of the potentially greater risk of regimen-related toxicities. However, advances in supportive care have made autologous HCT feasible in older patients. In a study of patients undergoing tandem autologous HCT, 49 patients aged 65 years or older were compared with 49 pair-mates matched for prognostically relevant disease drawn from 501 younger patients [74]. CR duration, EFS, and OS were comparable in the two groups. The prognosis was primarily determined by presence of unfavorable cytogenetics (11q breakpoints, monosomy 13 or deletion 13q, or any translocation), and serum β2-microglobulin level of over 2.5 mg/L. The authors concluded that age should not constitute a criterion for exclusion from high-dose regimens. Similar results were obtained in two other retrospective analyses [75,76]. Renal failure Although renal failure increases the morbidity and mortality of myeloma, at least two recent reports have shown that high-dose therapy and autologous HCT is feasible in patients with concurrent myeloma and renal failure. Renal failure apparently has no adverse effect on either the quality of HC collection or engraftment following autologous HCT, and the toxicity is acceptable, and survival comparable to that of other patients [77,78]. In a study of patients with MM and a serum creatinine level over 2.0 mg/dL (177 μmol/L) undergoing autologous HCT, median OS for the entire group of 81 patients was longer than 53 months, and was more than 51 months for the 38 patients who were dialysis depen-

Follow up (months) 37–41 42 120 56 76

dent. In this study, only two dialysis patients recovered partial renal function post transplantation [77]. In a second study, four of 17 evaluable patients became dialysis independent at a median time of 5 months post transplantation [78]. High-risk features The presence of deletion 13 [79], deletion 1p [80], and hypodiploidy on conventional karyotyping, or molecular genetic studies showing t(4;14), t(14;16) or –17p, confers a particularly adverse prognosis [81,82]. Recently the Intergroupe Francophone du Myélome (IFM) has demonstrated that abnormalities of chromosome 13 as detected by FISH are only associated with a poor prognosis if β2-microglobulin levels are elevated; likewise, elevated β2-microglobulin levels worsened the prognosis of any chromosomal abnormality [83]. HC source Peripheral blood hematopoietic cells (PBHCs) are preferred over bone marrow cells for transplantation because of rapid and predictable engraftment, and possibly less contamination of the graft with tumor cells [84]. The absolute number of CD34+ cells/kg recipient weight is the most reliable and reproducible method for determining the dose of the HC product. PBHCs should be collected before the patient is exposed to alkylating agents. The current practice at many transplant centers is to harvest enough HCs for two transplantations; a single transplantation is done with one-half of the collected cells, and the other half is cryopreserved. Purging of myeloma cells Autologous HCs harvested from the patients are likely to be contaminated with myeloma cells [85]. This observation is supported by the lower relapse rate seen after syngeneic transplantation when compared with autologous transplantation, and the development of myeloma in a xenotransplant mouse model, where granulocyte colony-stimulating factor-mobilized cells from patients with minimal myeloma resulted in the development of myeloma in the recipients [86,87]. It has been hypothesized that purging of contaminating myeloma cells prior to transplantation might be beneficial. The methods used to purge the HCs include positive selection of CD34+ cells only, and negative selection that involves the elimination of tumor cells by using monoclonal antibodies (see Chapter 42). However, several clinical trials have failed to show any improvement in OS, EFS or relapse rate [88,89]. In the European Group for Blood and Marrow Transplantation (EBMT) multicenter randomized phase III study, 111 patients responsive to initial chemotherapy were randomized to receive CD34+-selected (arm A) or CD34+-unselected (arm B) PBHCs after conditioning with high-dose melphalan and TBI. CD34+ selection gave a median tumor

Hematopoietic Cell Transplantation for Multiple Myeloma

(a)

Survival

1.00

When compared with chemotherapy alone, intensified chemotherapy followed by autologous HCT appears to prolong both EFS and OS in previously untreated patients with myeloma. One comparative study and two randomized trials have shown survival benefits in favor of autologous HCT of approximately 12 months [68,69,90]. In the French IFM 90 trial, high-dose chemotherapy supported by autologous bone marrow transplantation (BMT) was compared with conventional chemotherapy in 200 previously untreated patients with myeloma less than 65 years of age [68]. The results showed a higher CR rate (22% versus 5%), a higher rate of 5-year EFS (28% versus 10%), and greater OS (52% versus 12%) in the autologous BMT group. There was 13 months’ longer median duration of OS in patients assigned to the BMT arm (57 versus 44 months). The Medical Research Council Myeloma VII trial compared conventional-dose chemotherapy with high-dose therapy and autologous HCT in 401 previously untreated patients with myeloma who were less than 65 years of age [69]. The rates of complete response were higher in the autologous HCT group than in the standard-therapy group (44% versus 8%; p < 0.001). Intention-to-treat analysis showed a higher rate of OS (p = 0.04) and PFS (p < 0.001) in the HCT group than in the standardtherapy group (Fig. 58.1). As compared with standard therapy, autologous HCT increased median survival by almost 1 year (54.1 months versus 42.3 months). There was a trend toward a greater survival benefit in the group of patients with a poor prognosis, as defined by a high (more than 8 mg/L) β2-microglobulin level. Three other randomized studies, however, have not shown a survival benefit to autologous HCT [91–93]. Comparison between these trials is difficult due to variability in patient eligibility, including age, induction chemotherapy, the conditioning regimen for the HCT, and definitions of response. In the French study by Fermand et al., with a median follow-up time of approximately 10 years, there was a benefit of autologous HCT in terms of EFS and quality of life, but no evidence for its superiority over conventional therapy in terms of OS, in patients aged 55–65 years with symptomatic newly diagnosed MM [91]. In the Spanish cooperative group PETHEMA (Programa de Estudio y Tratamiento de las Hemopatias Malignas) trial [92], only patients responding to initial chemotherapy were randomized to autologous HCT versus continuing conventional therapy, thus excluding initial nonresponders. This study did not demonstrate a difference in the EFS or OS between conventional treatment and autologous HCT in patients responding to initial chemotherapy. This study excluded patients with primary refractory disease, who can potentially benefit from HCT. In the United States Intergroup Study S9321 [93], newly diagnosed MM patients were, after three to four cycles of induction therapy, ran-

0.50

Standard therapy

Intensive therapy

0.00 0

20

40

60

80

38 30

8 8

Months No. at Risk Intensive therapy 201 Standard therapy 200

(b)

Conventional therapy versus single autologous HCT

P–0.03 by Wilcoxon test P–0.04 by by log-rank test

0.75

0.25

Progression-free Survival

cell depletion of 2.2 (range 0.77–5.96) logs. The five year OS, EFS, and relapse rate were 51%, 20%, and 80% in arm A, and 45%, 18%, and 80% in arm B, respectively, with no significant difference between the two groups. The authors concluded that, despite significant tumor cell reduction, CD34+ selection does not reduce relapse rate but increases the risk of severe post-HCT infections [88]. In a report by Barbui et al., 60 newly diagnosed patients undergoing tandem autologous HCT were randomly assigned to receive either unmanipulated PBHCs or PBHCs purged of tumor cells using a two-step negative selection process [89]. Despite a 3–4 log reduction in tumor cells in vitro, recipients of the purged PBHCs remained polymerase chain reaction positive for minimal residual disease in vivo following transplantation. There was no difference in the estimated OS at 3 years between the two treatment groups.

849

148 129

1.00

79 70

P<0.001 by Wilcoxon test P<0.001 by log-rank test

0.75 0.50

Intensive therapy

0.25 0.00

Standard therapy

0

20

40

60

80

27 9

5 3

Months No. at Risk Intensive therapy 199 Standard therapy 196

124 90

55 25

Fig. 58.1 (a) Kaplan–Meier estimates of overall survival in the intention-totreat population. Overall, there was an improvement in median survival of 11.8 months in the intensive-therapy group (median survival 54.1 months, 95% confidence interval [CI] 44.9–65.2) compared with the standard-therapy group (42.3 months, 95% CI 33.1–51.6; p = 0.04 by the log-rank test and p = 0.03 by the Wilcoxon test). (b) Kaplan–Meier estimates of progressionfree survival. A total of 395 patients could be evaluated. The median duration of progression-free survival was longer in the intensive-therapy group than in the standard-therapy group (31.6 months, 95% CI 27.4–38.0 versus 19.6 months, 95% CI 16.2–21.8; p < 0.001 by the log-rank or Wilcoxon test). (Reproduced from [68], with permission.)

domized to receive melphalan 140 mg/m2 with TBI 1200 cGy or vincristine, BCNU (carmustine), melphalan, cyclophosphamide, and prednisone (VBMCP) for 1 year. At the time of relapse in the VBMCP arm, patients were encouraged to undergo autologous HCT. The VBMCP and early-HCT groups were similar in terms of EFS (21 versus 25 months; p = 0.14). Interpretation of the results for OS was murkier since 52% of patients in the VBMCP arm had a late transplantation at relapse/ progression. Thus, the OS rates (53 versus 62 months; p = 0.87) were not statistically different. This may be a reflection of the suboptimal conditioning therapy. This trial, in effect, showed that up-front and salvage autologous HCT at the time of relapse or progression were comparable. In summary, a single autologous HCT appears to prolong OS and EFS in myeloma by approximately 12 months, but has limitations, and the outcome is affected by patient selection as well as the preparative regimen used [94,95]. Timing of autologous HCT Although comparable OS have been reported for both early and late HCT by the United States Intergroup [93] and the French Myélome

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Table 58.4 Representative trials of tandem autologous hematopoietic cell transplantation

Reference

n

Age limit (years)

[101] Single Tandem

399

60

[102] Single Tandem

220

[99]

231

75

[103] No thalidomide Thalidomide

668

75

Complete remission (%)

Median event-free survival (months)

Median overall survival (months)

42 50

25 30

48 58

35 48

22 35

59 73

41

31

68

144

43 62

60 NR

NR NR

42

Follow up (months) 75

60

55

NR, not reached.

Autogreffe Group trials [91,94], the French trials demonstrated superior quality of life indices in patients receiving early transplantation. However, with the advent of new treatments, particularly new induction regimens associated with high response rates, the role of early HCT for myeloma will need to be redefined in the context of well-designed randomized trials [96,97]. Notwithstanding, many myeloma experts are still recommending early HCT for their patients [95,98].

Single versus tandem autologous transplantation Several nonrandomized trials have suggested that double autologous HCT may be associated with better outcome [98,99]. Barlogie et al. reported on 231 patients with newly diagnosed MM who received tandem autologous HCT. Second transplantations were performed in 71% of the enrolled patients. After the first autologous HCT, 41% achieved CR and 83% achieved CR or partial remission. Those numbers were increased to 51% CR and 95% CR or partial remission in patients completing a second transplantation. Median duration of remission in complete responders was 50 months. In an updated analysis, 15-year OS and disease-free survival were 17% and 7%, respectively [99,100]. A French randomized trial of 399 previously untreated patients less than 60 years of age found significantly improved 7-year EFS (20% versus 10%) and OS (42% versus 21%) in recipients of double versus single autologous HCT [101]. The beneficial effect of the second HCT was mostly seen in patients with less than very good partial response to the first autologous HCT. Cavo et al. reported on 321 patients, randomly assigned to receive either a single course of high-dose melphalan at 200 mg/m2 or melphalan at 200 mg/m2 followed, after 3–6 months, by melphalan at 120 mg/ m2 and busulfan at 12 mg/kg. Patients in the tandem autologous HCT arm had a significantly increased probability of attaining at least a near-complete response (33% versus 47%, respectively; p = 0.008), prolonged relapse-free survival (median, 24 versus 42 months, respectively; p < 0.001), and prolonged EFS (median, 23 versus 35 months, respectively; p = 0.001). Tandem transplantations, however, failed to significantly prolong OS. Administration of a second transplantation and of novel agents for treating sequential relapses in up to 50% of patients randomly assigned to receive a single autologous HCT likely contributed to prolong the survival duration of the whole group, whose 7-year rate (46%) was similar to that of the double autologous HCT group (43%;

p = 0.90). Benefits offered by double autologous HCT were particularly evident among patients who failed at least near-complete response after one autologous HCT [102]. The potential shortcomings of the tandem approach are an increased duration of hospitalization and overall health-care costs, and the potential lack of additional benefit in patients who had already achieved CR with a first autologous HCT. Based on the two randomized trials cited above, a patient who fails to achieve a very good partial response after the first autologous HCT should be offered a second autologous transplantation. The most representative results of tandem transplantation are summarized in Table 58.4.

Lessons from Little Rock The group from the University of Arkansas Medical Center led by Dr Bart Barlogie has over the last two decades made important observations regarding the role of tandem transplantation and intensive therapy in myeloma [98]. This began with Total Therapy I, which explored the concept of intensive induction, followed by tandem high-dose melphalan consolidation and interferon maintenance. This group demonstrated that long-term disease control was feasible in myeloma, and that cytogenetic abnormalities as well as baseline β2-microglobulin levels could predict long-term outcome [99,100]. This initial experience also confirmed observations by others that achievement of a CR was an important surrogate marker for long-term disease control [100]. Their more recent experience with Total Therapy II and Total Therapy III has underscored the feasibility of their initial approach of intensive induction, and consolidation therapy followed by maintenance as a way of achieving high rates of CR. In Total Therapy II, postautologous HCT maintenance with thalidomide was associated with a significant improvement in PFS but a worse survival after relapse, which resulted in similar OS for both groups [103]. Of particular interest has been defining risk groups according to gene expression profiling. Using the extensive, comprehensive database available at University of Arkansas Medical Center, Shaugnessy et al. were able to define poor prognostic groups according to gene expression profiling analysis. If corroborated by others, this will allow a more rational use of therapies in myeloma patients achieving the overarching goal of maximum benefit with minimum burden of therapy [104].

Hematopoietic Cell Transplantation for Multiple Myeloma

Allogeneic HCT in myeloma The curative potential of allogeneic HCT comes not only from a graftversus-tumor effect, but also from the effective delivery of dose-intense therapy rescued with a product that is devoid of tumor contamination. This conclusion is derived from the analysis of the syngeneic transplantation series reported to date. Bensinger et al. reported on the outcomes of 12 patients who underwent syngeneic HCT after a cyclophosphamide and TBI conditioning; two of these patients remain alive and progression free 9 and 16 years after transplantation [105]. Gahrton et al. also reported on three of 25 syngeneic recipients who were alive without progression for more than 8 years after transplantation [106]. These results suggest that high-dose chemoradiotherapy supported by infusion of an uncontaminated HC source can cure patients with MM. Based on the promising results of allogeneic HCT in acute leukemia, allografting for myeloma began to be explored in the 1980s [106]. Since these initial reports, allogeneic HCT has been extensively explored in Europe and in the United States [107–110]. The existence of a graft-versus-myeloma effect was first documented by Tricot et al., and later confirmed in large single- and multiinstitutional series of donor lymphocyte infusion [111–114]. Thus, with the potential benefits of a graft-versus-tumor effect and the use of a clean HC product, it would be expected that high-dose therapy with allogeneic HCT would be extremely useful in the treatment of MM. Unfortunately, that is not necessarily the case. The EBMT case-controlled series reported that the relapse rate after allogeneic HCT was not significantly lower than the relapse rate after syngeneic HCT, suggesting that the graft-versus-tumor effect is not as potent in myeloma as it is in chronic or acute myelogenous leukemia [87]. Results of allogeneic HCT with high-dose preparative regimens A large report from the EBMT registry showed disappointing results for patients undergoing allogeneic HCT. Median survival was only 18 months, and the TRM was prohibitive at 41% [107]. Similar results have been seen in other single institution trials as summarized in Table 58.5. The Dutch Cooperative Group HOVON recently published the results of its initial experience using T-cell-depleted allogeneic HCT after highdose preparative regimens in patients with HLA-identical sibling donors. After initial therapy with the VAD regimen, patients younger than 55 with a human leukocyte antigen (HLA)-identical sibling underwent allogeneic HCT with cyclophosphamide and TBI (n = 47) or cyclophosphamide/TBI plus idarubicin (n = 5). All patients received ex vivo T-cell-depleted transplantation with the goal of reducing TRM. Eleven percent of patients developed grade III acute graft-versus-host disease (GVHD), and 30% developed chronic extensive GVHD. TRM was 34%. Median OS was 25 months from the time of transplantation, and was

851

significantly inferior to survival for patients in other arms of the trial (47 months). Despite T-cell depletion, TRM remained elevated and a graft-versus-myeloma effect was not apparent in this trial [115]. Although high-dose therapy has been associated with high rates of nonrelapse mortality, the achievements of molecular remissions, as well as the lack of relapses in a significant proportion of patients who achieved CR after this therapy, suggest that this treatment is potentially curative [93,116].

Results of allogeneic HCT with reduced-intensity conditioning To try to overcome toxicity from high-dose regimens, and to extend the applicability of this procedure to older patients with significant co-morbidities, allogeneic HCT with reduced-intensity conditioning regimens has been attempted in patients with MM. Several phase II nonrandomized trials, some of them including scheduled donor lymphocyte infusion, have been published over the past few years (Table 58.6). Partial and complete responses were obtained, even though most patients were heavily pretreated and had refractory disease. Responses were evidently related to the development of GVHD, and a small percentage of patients from those series became complete long-term responders [117–122]. More recently, reduced-intensity conditioning followed by allogeneic HCT has been explored as part of a tandem strategy in which patients are treated with autologous transplantation followed by planned allogeneic transplantation. Maloney et al. from the Seattle Group recently published their experience with 54 patients whose median age was 52 years [123]. All patients received autologous transplantation with melphalan (200 mg/m²), and, after a median of 2 months, low-dose TBI (single fraction, 2 Gy) followed by HCT from an HLA-identical sibling. All patients engrafted, and only one patient needed donor lymphocyte infusion. The incidences of acute and chronic GVHD were 39% and 46%, respectively. TRM at 1 year was 15%. Estimated 2-year OS and disease-free survival were 78% and 52%, respectively. Two prospective trials looking at the role of tandem autologous plus a reduced-intensity allogeneic HCT approach as part of the initial therapy for MM have been reported, with conflicting results [124,125]. The IFM group has recently reported on the outcomes of patients with high-risk disease as defined by high levels of β2-microglobulin or deletion of chromosome 13 as detected by FISH. Patients received an initial autologous HCT with melphalan 200 mg/m2. Sixty-five patients had an HLAidentical sibling donor, of which 46 patients received a reduced-intensity conditioning regimen consisting of fludarabine, busulfan, and antithymocyte globulin (ATG). Patients without an HLA sibling donor received a second autologous HCT prepared with melphalan 220 mg/m2. On an intent-to-treat basis, the OS and EFS did not differ significantly in both groups (median 35 and 25 months in the allogeneic HCT patients versus 41 and 30 months in the autologous HCT patients, respectively). There

Table 58.5 Results of allogeneic hematopoietic cell transplantation using high-dose conditioning in multiple myeloma Reference

n

Nonrelapse mortality

3-year overall survival

Median progression-free survival (months)

[107]

334 (1983–93) 356 (1994–98) 18 80 66

46% 30% 16% 44% 24%

35% 55% 77% 24% 39%

9 15 NS NS NS

[108] [109] [110] NS, not significant.

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Table 58.6 Results of allogeneic hematopoietic cell transplantation using reduced-intensity conditioning in multiple myeloma

Reference

n

Regimen

[117] [118]

22 45

[119] [120]

29 41

[121] [122]

64 31

[123] [124]

54 Allogeneic 46 Autologous 166 Allogeneic 60 Autologous 59

FM Mel 100 FM/TBI FM Flu/Bu ATG FM/ATG Mel FM/TBI TBI Flu/Bu Mel 220 Flu/TBI Mel 200

[125]

Response rate (complete response)

Acute graft-versushost disease grade II–IV

Chronic graft-versushost disease

Transplant-related mortality

Disease-free survival

Overall survival at 2 years

72% (32) 61% (31)

46% 42%

27% 58%

40% 38%

19% at 2 years 13% at 3 years

30% 36%

45% 50% (25)

52% 40%

51% 51%

21% 17%

33% at 2 years NR

60% 62%

90% (45) 61%

39% 58%

47% NR

23% 30%

38% at 2 years 31% at 2 years

55% 31%

52% 82%

39% 24%

46% 43%

86% (55) 89% (25)

43%

32%

15% 11% 2% 10% 4%

55% at 2 years 25 months* 30 months 43 months* 33 months

78% 35 months* 41 months NR 56 months*

ATG, antithymocyte globulin; Flu/Bu, fludarabine/busulfan; FM, fludarabine/melphalan; Mel, melphalan; NR, not reported; TBI, total body irradiation. * Median.

(a)

Overall Survival (%)

100

P = 0.01 Sibling HLA identical

50 Sibling not HLA identical

0 0

12

24

36

48

60

72

84

96

14 17

7 8

2 4

0 0

Months No. at Risk Sibling HLA identical 80 82 Sibling not HLA identical (b)

80 78

68 68

51 46

29 28

Event-free Survival (%)

100

P = 0.02

50 Sibling HLA identical

Sibling not HLA identical 0 0

12

24

36

48

60

72

84

96

Months No. at Risk Sibling HLA identical Sibling not HLA identical

80 82

79 76

54 53

30 22

16 9

7 5

3 2

1 1

0 0

Fig. 58.2 Kaplan–Meier estimates of overall and event-free survival from time of diagnosis. (a) Overall survival and (b) event-free survival of the 80 patients with a human leukocyte antigen (HLA)-identical sibling and the 82 patients without an HLA-identical sibling according to intention-to-treat analysis. (Reproduced from [124], with permission.)

Hematopoietic Cell Transplantation for Multiple Myeloma

was a trend for a better OS in patients treated with tandem autologous HCT, with a median of 47.2 versus 35 months (p = 0.07) for patients who actually received a reduced-intensity allogeneic HCT [124]. The Italian Cooperative Group performed a similar study, which was reported by Bruno et al. After a median follow up of 3 years, nonrelapse mortality was 11% for the autologous + allogeneic group versus 4% for the tandem autologous group (p = 0.09). CR was significantly higher in the autologous + allogeneic group compared with the tandem autologous group (46% versus 16%; p = 0.0001)). OS and EFS rates were superior in the autologous + allogeneic group than in the tandem autologous group, at 84% versus 62% (p = 0.003), and 75% versus 41% (p = 0.00008) respectively (Fig. 58.2) [125]. These results coincide with two other retrospective analysis from Carella et al. and Rosinol et al., which also showed improved outcomes for recipients of reduced-intensity allogeneic HCT compared with recipients of tandem autologous HCT [126,127]. A clinical trial sponsored by the Bone Marrow Transplant Clinical Trials Network has recently been concluded which, when reported, should provide valuable information that will allow patients and physicians to make informed decisions regarding this treatment option. The largest analysis of allogeneic HCT for myeloma with reducedintensity conditioning regimens was performed by Crawley et al. for the EBMT. With a total of 229 patients analyzed, the TRM at 1 year was 22%. The 3-year OS and PFS were 41% and 21%, respectively. Adverse OS was associated with chemoresistant disease (relative risk [RR] 2.9), more than one prior transplantation (RR 2.0), and male patients with female donors (RR 1.45). A shorter PFS was associated with chemoresistance (RR 2.4) and alemtuzumab treatment (RR 1.8). TRM was increased with female-to-male donation (RR 2.5) and transplantation more than 1 year from diagnosis (RR 2.3). Chronic GVHD was associated with better OS and PFS, with values for these parameters of 84% and 46% for limited GVHD, 58% and 30% for extensive GVHD, and 29% and 12% for no GVHD, respectively [128].

853

Alternative donor HCT Ballen et al. reported on HCT outcomes for patients with MM undergoing unrelated donor HCT after high-dose conditioning. Nonrelapse mortality was over 50% and PFS at 3 years was 15% [129]. Kroger et al. compared ATG with alemtuzumab in 73 patients with MM who underwent reduced-intensity conditioning with melphalan/fludarabine, followed by allogeneic HCT from HLA-matched or HLA-mismatched unrelated donors [130]. Alemtuzumab resulted in faster engraftment of leukocytes (p = 0.03) and platelets (p = 0.02), and in a lower incidence of acute GVHD grade II–IV (24% versus 47%; p = 0.06). More cytomegalovirus-seropositive patients in the alemtuzumab group experienced cytomegalovirus reactivation (100% versus 47%; p = 0.001). The cumulative incidence of TRM at 2 years was 26% (95% confidence interval 12–37%) for ATG versus 28% (95% confidence interval 15–55%) for alemtuzumab; p = 0.7. There was no significant difference in the estimated 2-year overall and PFS between ATG and alemtuzumab.

Current status and future directions of HCT in myeloma: moving beyond high-dose melphalan MM is currently the most common indication for high-dose therapy with autologous HCT in North America and Europe [131]. In North America alone, more than 2000 transplantations are performed each year. Table 58.7 summarizes the patient characteristics of patients undergoing autologous HCT for myeloma as reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). High-dose melphalan is used in over 90% of patients, with the most common regimen used being single-agent melphalan at a median dose of 180–200 mg/m2 performed as a single transplantation (CIBMTR, unpublished observations).

Table 58.7 Characteristics of patients who underwent autologous hematopoietic cell transplantation for multiple myeloma for consolidation of first remission between 2000 and 2004 in North America as reported to the Centre for International Blood and Marrow Transplant Research Characteristics of patients

n

Number of patients

1371

Age (median, range) (years) Time from diagnosis to stem cell transplantation (months)

Median (range), n (%)

58 (22–80) 7 (1–103)

Conditioning regimen Lpam alone Lpam + TBI ± other Lpam ± other (not TBI) Others

1367 1133 44 130 60

Dose of melphalan 200, median (range) (mg/m2)

1259

Type of second transplant No planned second transplant Planned second autologous Planned second allogeneic

1371 904 (67) 265 (19) 46 (3)

Lpam, melphalan; TBI, total body irradiation. Data courtesy of Giralt, personal communication.

(83) (3) (10) (<1) 187 (0.9–278)

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Table 58.8 Outcomes using intensified conditioning regimens compared with melphalan 200 mg/m2 – trials reporting single autologous hematopoietic cell transplantation

Strategy

Median event-free survival (months)

% Complete remission/% very good partial remission

Melphalan 200 versus Melphalan 140 + TBI Melphalan 200 versus Busulfan–melphalan versus Melphalan + TBI Melphalan 200 versus Melphalan + holmium-DOTMP (<2400 cGy) Melphalan 200 versus Thiotepa–busulfan–cyclophosphamide

20 21 22 32 20 19 30 20 21

35/20 29/14 43/NR 49/NR 31/NR 32/NR 55/NR 16/NR 27/NR

Reference and comments Randomized trial [70] Registry analysis [71]

Retrospective analysis [135] Retrospective analysis [72]

NR, not reported; TBI, total body irradiation.

Improving high-dose melphalan therapy: dose intensification Relapse after autologous HCT remains the single most important cause of treatment failure in patients. Strategies aimed at improving the conditioning regimen have usually focused on intensifying the conditioning regimen either by increasing the dose of melphalan or by adding other alkylating agents, as summarized in Table 58.8. Of these, only tandem high-dose therapy has been reported to improve outcomes in randomized trials [101,102]. This benefit is limited by the fact that many patients do not want to undergo tandem transplantation, and it is possible that only patients failing to achieve a greater than 90% reduction of their myeloma burden after the first high-dose therapy benefit from a second intensification. Various groups have explored dose intensification of melphalan with or without cytoprotectors such as amifostine. Phillips et al. demonstrated the feasibility of administering up to 280 mg/m2 of melphalan with amifostine. Significant mucosal toxicities were observed, and at the higher doses cardiotoxicity was dose limiting [132]. Moreau et al. have explored using melphalan at a dose of 220 mg/m2 as part of a tandem transplantation strategy for patients with high-risk myeloma as defined by cytogenetics and β2-microglobulin. CRs were seen in 30% of patients, with another 18% achieving at least a 90% reduction in tumor burden [133]. Table 58.8 summarizes the results of representative studies looking at intensification of the conditioning regimen as a strategy to improve HCT outcomes. To date, neither melphalan dose intensification nor the addition of other alkylating agents has resulted in large increases in post-transplantation CR rates, and novel conditioning regimens exploring different mechanisms of enhancing the antitumor effect of high dose melphalan need to be explored. Improving high-dose melphalan therapy: novel conditioning regimens The recent expansion of therapeutic options for MM can be partly attributed to a better understanding of the interactions between malignant plasma cells and the bone marrow microenvironment that includes stromal cells, extracellular adhesion molecules, and secreted cytokines. Novel therapies targeting these interactions have shown promising responses and outcomes [134]. Arsenic trioxide, bortezomib, and targeted skeletal radiotherapy with either holmium-DOTMP or samarium-EDMTP have been studied as part of the conditioning regimen, with encouraging phase II results that need to be confirmed in larger phase III trials [135].

Optimizing high-dose melphalan therapy It is a well known fact that patients’ tolerance and response to melphalan at a dose of 200 mg/m2 is extremely heterogeneous, with some patients having no toxicities whatsoever and others having serious life-threatening toxicities due to mucosal damage. To a certain degree, this variability is probably due to genetic polymorphisms among enzymes such as glutathione-S-transferase [136]. However, the practice of dosing melphalan according to body surface area, with arbitrary modifications for overweight patients, also suggests that melphalan dosing may be implicated, as reported by Grazziutti et al. [137]. Lastly, Dimopoulos et al. have demonstrated that the extent of damage and repair seen in the p53 tumor suppressor gene of peripheral blood lymphocytes after exposure to melphalan is an important predictor of outcome, and could serve as a tool for developing a personalized or individually tailored conditioning regimen [138]. Optimizing high-dose therapy also implies minimizing toxicities from this regimen. Gene polymorphisms may predict patients at high risk for developing gastrointestinal or infectious complications, but identification by itself is not an effective intervention strategy. The group at MD Anderson Cancer Center has conducted a series of prospective studies using patient-reported outcomes, and has demonstrated a strong correlation between inflammatory cytokine profiles and symptom burden in myeloma as well as other patients undergoing autologous HCT [139–141].

The emerging role of maintenance therapy The almost universal risk of relapse that occurs after autologous HCT has led many investigators to explore the role of maintenance therapy. Interferon was one of the first drugs used to this effect [142]. Although initial studies demonstrated a potential benefit in regards to EFS, no survival benefit was seen. More recent randomized trials have failed to confirm the initial observations, and few investigators are pursuing this approach today. Bisphosphonate therapy post autologous HCT has been shown to reduce the risks of skeletal events in a series of randomized trials [143]. However, the optimal dose and duration remains uncertain, particularly in light of a recent randomized trial from the IFM group demonstrating no reduction in skeletal events in patients randomized to receive pamidronate monthly versus those receiving no maintenance therapy [144].

Hematopoietic Cell Transplantation for Multiple Myeloma

855

Initial diagnostic features High risk (β2-microglobulin, cytogenetics, ISS stage) Performance status, comorbidities Establish goals of therapy – palliation versus remission induction Encourage participation in clinical trials

High-risk features Encourage participation in clinical trials Induction with novel agents Stem cell collection

High-dose therapy consolidation Assess response Consider for tandem with autologous or allogeneic transplantation

Depending on response, consider for maintenance therapies or observation Chronic bisphosphonate therapies Encourage participation in clinical trials

Standard-risk features – encourage participation in clinical trials Induction with thalidomide/dexamethasone or novel agents (MPT for nontransplant candidates) Stem cell collection

High-dose therapy consolidation Assess response; consider for tandem transplantation

Consider for maintenance therapy including bisphosphonates

72-year-old male professor of romance language with mild coronary heart failure found with hemoglobin 9.7 g/dL, creatinine 2.5 mg/dL, albumin 3.5 g/dL, β2-microglobulin 5.5 mg/dL, serum protein electrophoresis 3.7 g/dL, bone marrow 40% plasma cells. Cytogenetics diploid. Bone survey shows multiple lytic lesions. Initial induction therapy should be?

He opts for an aggressive therapy involving high-dose therapy. Most commonly used induction would be thalidomide-dexamethasone. Bortezomib or lenalidomide combinations would be reasonable choices too

After 4 cycles of induction, he achieves a very good partial response and asks what to do

Based on the retrospective reviews, this patient could benefit from high-dose therapy with autologous SCT, attempting to achieve a complete remission

He opts for a more conservative approach of melphalan, prednisone, and thalidomide based on the IFM and GITMO studies

After 4 cycles of induction, he achieves a very good partial response and asks what to do

Continued treatment with melphalan, prednisone, and thalidomide for 12 months, followed by low-dose thalidomide maintenance, would be an appropriate strategy to achieve long-term disease control

Fig. 58.3 Approach to patients with multiple myeloma – putting it all together. GITMO, Gruppo Italiano Trapianti de Midollo Osseo; IFM, Intergroup Francophone du Myélome; ISS, International Staging System; MPT, melphalan/prednisone/thalidomide. See text for other abbreviations.

Of particular interest is the series of randomized trials studying the use of thalidomide therapy as maintenance therapy post autologous HCT for myeloma. The IFM group conducted a randomized trial of maintenance treatment with thalidomide and pamidronate. Two months after high-dose therapy, 597 patients younger than age 65 years were randomly assigned to receive no maintenance (arm A), pamidronate (arm B) or pamidronate plus thalidomide (arm C). The addition of thalidomide resulted in an increase in major responses (67% versus 55% and 57%, respectively) and improvements in 3-year EFS (52% versus 36% and 37%%, respectively). Thalidomide was also associated with a 4-year post-diagnosis survival benefit, with 87% of patients alive at that time point versus 77% for patients receiving no maintenance and 74% for patients receiving pamidronate alone [144].

Contrasting with the IFM results, Barlogie et al. treated 668 patients with newly diagnosed MM with tandem autologous HCT after intensive induction therapy. A total of 323 were randomly assigned to receive thalidomide from the outset until disease progression. Patients randomized to thalidomide had higher CR rates (62% versus 43%) and improved EFS at 5 years (56% versus 43%). However, 5-year survival rates were similar for both groups since the survival after relapse for the thalidomide arm was significantly worse [103]. Other ongoing randomized trials with thalidomide, lenalidomide, bortezomib, steroids or combinations are being studied as maintenance therapies and, once concluded, should help resolve the issue of optimal post-transplantation therapy in patients with myeloma.

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Summary The treatment for myeloma continues to evolve. Thirty years ago, the standard treatment was melphalan and prednisone, with a response rate of only 40% and an average life expectancy of 3 years. During the last three decades, significant advances in therapy have been made. Today, transplantation-eligible patients receiving a standard induction therapy with thalidomide and dexamethasone followed by consolidation therapy with high-dose melphalan and one autologous HCT have more than an 80% chance of achieving a partial remission or better, and an average life expectancy of more than 5 years. Moreover, approximately one-third of patients treated will achieve CR, of which 30% will remain without disease progression 10 years after HCT, demonstrating the potential curability of this disease [100]. The advent of new agents such as bortezomib, thalidomide, and lenalidomide, and the improved outcomes after induction therapy, should move the field to re-explore the role of high-dose melphalan as consolidation therapy for all patients with MM. Likewise, exploring strategies that could improve on the outcomes of high-dose melphalan are necessary. Post-transplantation therapy is also becoming an important adjuvant to improve outcomes. Two published studies show conflicting outcomes but represent different strategies of thalidomide use. Other studies are ongoing and, at least in preliminary analysis, are showing a potential benefit for post-transplantation thalidomide in regard to EFS

and OS. The role of bisphosphonate therapy post transplantation also needs re-exploration in the context of novel therapies, particularly in the light of the IFM randomized trial showing no improvement in skeletal events with post-transplantation pamidronate [144]. Finally, although both allogeneic and autologous HCT can result in long-term disease control and potential cure, most patients still relapse. Thus, careful consideration needs to be made when recommending allogeneic HCT to patients with myeloma since the risk–benefit ratio of this procedure still makes it difficult to recommend to all patients as up-front therapy. Likewise, recommending maintenance therapy to all patients regardless of their response to therapy or other risk factors will expose a substantial proportion of patients to drug toxicities who may have only a marginal benefit from this strategy. Thus, further delineation of the prognostic value of gene expression profiling as well as other parameters will assist in developing true risk-tailored approaches for this disease, fulfilling the goal of delivering the longest life with the best quality and minimum burden of therapy for the most patients with myeloma (Fig. 58.3).

Acknowledgement We would like to thank Dr Gaurav C. Parikh, MB BS for helping with formatting and reviewing the chapter draft thoroughly.

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Hematopoietic Cell Transplantation for Multiple Myeloma

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Eastern Cooperative Oncology Group Phase III trial. Blood 1996; 88: 2699–706. Corradini P, Inghirami G, Astolfi M et al. Inactivation of tumor suppressor genes, p53 and Rb1, in plasma cell dyscrasias. Leukemia 1994; 8: 758–67. Drach J, Ackermann J, Fritz E et al. Presence of a p53 gene deletion in patients with multiple myeloma predicts for short survival after conventional-dose chemotherapy. Blood 1998; 92: 802– 9. Kulkarni MS, Daggett JL, Bender TP, Kuehl WM, Bergsagel PL, Williams ME. Frequent inactivation of the cyclin-dependent kinase inhibitor p18 by homozygous deletion in multiple myeloma cell lines: ectopic p18 expression inhibits growth and induces apoptosis. Leukemia 2002; 16: 127– 34. Tasaka T, Berenson J, Vescio R et al. Analysis of the p16INK4A, p15INK4B and p18INK4C genes in multiple myeloma. Br J Haematol 1997; 96: 98–102. Choi SJ, Cruz JC, Craig F et al. Macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma. Blood 2000; 96: 671–5. Roodman GD. Pathogenesis of myeloma bone disease. Blood Cells Mol Dis 2004; 32: 290–2. Giuliani N, Bataille R, Mancini C, Lazzaretti M, Barille S. Myeloma cells induce imbalance in the osteoprotegerin/osteoprotegerin ligand system in the human bone marrow environment. Blood 2001; 98: 3527–33. Paradisi F, Corti G, Cinelli R. Infections in multiple myeloma. Infect Dis Clin North Am 2001; 15: 373–84, vii–viii. Winearls CG. Acute myeloma kidney. Kidney Int 1995; 48: 1347–61. International Myeloma Working Group. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol 2003; 121: 749–57. Angtuaco EJ, Fassas AB, Walker R, Sethi R, Barlogie B. Multiple myeloma: clinical review and diagnostic imaging. Radiology 2004; 231: 11– 23. Rajkumar SV, Kyle RA. Multiple myeloma: diagnosis and treatment. Mayo Clin Proc 2005; 80: 1371–82. Abraham RS, Clark RJ, Bryant SC et al. Correlation of serum immunoglobulin free light chain quantification with urinary Bence Jones protein in light chain myeloma. Clin Chem 2002; 48: 655– 7. Katzmann JA, Clark RJ, Abraham RS et al. Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem 2002; 48: 1437– 44. Barlogie B, Zangari M, Bolejack V et al. Superior 12-year survival after at least 4-year continuous remission with tandem transplantations for multiple myeloma. Clin Lymphoma Myeloma 2006; 6: 469–74. Durie BG, Salmon SE. A clinical staging system for multiple myeloma. Correlation of measured myeloma cell mass with presenting clinical features, response to treatment, and survival. Cancer 1975; 36: 842–54.

46. Greipp PR, San Miguel J, Durie BG et al. International staging system for multiple myeloma. J Clin Oncol 2005; 23: 3412–20. 47. Fonseca R, Blood E, Rue M et al. Clinical and biologic implications of recurrent genomic aberrations in myeloma. Blood 2003; 101: 4569–75. 48. Durie BG, Harousseau JL, Miguel JS et al. International uniform response criteria for multiple myeloma. Leukemia 2006; 20: 1467–73. 49. Goldschmidt H, Hegenbart U, Wallmeier M, Hohaus S, Haas R. Factors influencing collection of peripheral blood progenitor cells following high-dose cyclophosphamide and granulocyte colony-stimulating factor in patients with multiple myeloma. Br J Haematol 1997; 98: 736–44. 50. Morris CL, Siegel E, Barlogie B et al. Mobilization of CD34+ cells in elderly patients (>/=70 years) with multiple myeloma: influence of age, prior therapy, platelet count and mobilization regimen. Br J Haematol 2003; 120: 413–23. 51. Alexanian R, Weber D, Delasalle K, Handy B, Champlin R, Giralt S. Clinical outcomes with intensive therapy for patients with primary resistant multiple myeloma. Bone Marrow Transplant 2004; 34: 229–34. 52. Singhal S, Powles R, Sirohi B, Treleaven J, Kulkarni S, Mehta J. Response to induction chemotherapy is not essential to obtain survival benefit from high-dose melphalan and autotransplantation in myeloma. Bone Marrow Transplant 2002; 30: 673–9. 53. Kumar S, Lacy MQ, Dispenzieri A et al. Highdose therapy and autologous stem cell transplantation for multiple myeloma poorly responsive to initial therapy. Bone Marrow Transplant 2004; 34: 161–7. 54. Weber D, Rankin K, Gavino M, Delasalle K, Alexanian R. Thalidomide alone or with dexamethasone for previously untreated multiple myeloma. J Clin Oncol 2003; 21: 16–19. 55. Dingli D, Rajkumar SV, Nowakowski GS et al. Combination therapy with thalidomide and dexamethasone in patients with newly diagnosed multiple myeloma not undergoing upfront autologous stem cell transplantation: a phase II trial. Haematologica 2005; 90: 1650–4. 56. Rajkumar SV, Blood E, Vesole D, Fonseca R, Greipp PR, Eastern Cooperative Oncology Group. Phase III clinical trial of thalidomide plus dexamethasone compared with dexamethasone alone in newly diagnosed multiple myeloma: a clinical trial coordinated by the Eastern Cooperative Oncology Group. J Clin Oncol 2006; 24: 431– 6. 57. Rajkumar SV, Hayman SR, Lacy MQ et al. Combination therapy with lenalidomide plus dexamethasone (Rev/Dex) for newly diagnosed myeloma. Blood 2005; 106: 4050–3. 58. Jagannath S, Durie BG, Wolf J et al. Bortezomib therapy alone and in combination with dexamethasone for previously untreated symptomatic multiple myeloma. Br J Haematol 2005; 129: 776–83. 59. Jagannath S, Richardson PG, Sonneveld P et al. Bortezomib appears to overcome the poor prognosis conferred by chromosome 13 deletion in phase 2 and 3 trials. Leukemia 2007; 21: 151– 7. 60. Anderson H, Scarffe JH, Ranson M et al. VAD chemotherapy as remission induction for multiple myeloma. Br J Cancer 1995; 71: 326–30.

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61. Rajkumar SV. Multiple myeloma: the death of VAD as initial therapy [Editorial]. Blood 2005; 106: 2–3. 62. Anagnostopoulos A, Aleman A, Yang Y et al. Outcomes of autologous stem cell transplantation in patients with multiple myeloma who received dexamethasone-based nonmyelosuppressive induction therapy. Bone Marrow Transplant 2004; 33: 623–8. 63. Kumar S, Lacy MQ, Dispenzieri A et al. Single agent dexamethasone for pre-stem cell transplant induction therapy for multiple myeloma. Bone Marrow Transplant 2004; 34: 485–90. 64. Palumbo A, Bringhen S, Caravita T et al. Oral melphalan and prednisone chemotherapy plus thalidomide compared with melphalan and prednisone alone in elderly patients with multiple myeloma: randomised controlled trial. Lancet 2006; 367: 825–31. 65. Mateos MV, Hernandez JM, Hernandez MT et al. Bortezomib plus melphalan and prednisone in elderly untreated patients with multiple myeloma: results of a multicenter phase 1/2 study. Blood 2006; 108: 2165–72. 66. McElwain TJ, Powles RL. High-dose intravenous melphalan for plasma-cell leukaemia and myeloma. Lancet 1983; 2: 822–4. 67. Barlogie B, Hall R, Zander A, Dicke K, Alexanian R. High-dose melphalan with autologous bone marrow transplantation for multiple myeloma. Blood 1986; 67: 1298–301. 68. Attal M, Harousseau JL, Stoppa AM et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 1996; 335: 91–7. 69. Child JA, Morgan GJ, Davies FE et al. High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med 2003; 348: 1875–83. 70. Moreau P, Facon T, Attal M et al. Comparison of 200 mg/m2 melphalan and 8 Gy total body irradiation plus 140 mg/m2 melphalan as conditioning regimens for peripheral blood stem cell transplantation in patients with newly diagnosed multiple myeloma: final analysis of the Intergroupe Francophone du Myelome 9502 randomized trial. Blood 2002; 99: 731–5. 71. Lahuerta JJ, Martinez-Lopez J, Grande C et al. Conditioning regimens in autologous stem cell transplantation for multiple myeloma: a comparative study of efficacy and toxicity from the Spanish Registry for Transplantation in Multiple Myeloma. Br J Haematol 2000; 109: 138–47. 72. Anagnostopoulos A, Aleman A, Ayers G et al. Comparison of high-dose melphalan with a more intensive regimen of thiotepa, busulfan, and cyclophosphamide for patients with multiple myeloma. Cancer 2004; 100: 2607–612. 73. Fenk R, Schneider P, Kropff M et al. High-dose idarubicin, cyclophosphamide and melphalan as conditioning for autologous stem cell transplantation increases treatment-related mortality in patients with multiple myeloma: results of a randomised study. Br J Haematol 2005; 130: 588– 94. 74. Siegel DS, Desikan KR, Mehta J et al. Age is not a prognostic variable with autotransplants for multiple myeloma. Blood 1999; 93: 51–4. 75. Badros A, Barlogie B, Siegel E et al. Autologous stem cell transplantation in elderly multiple

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myeloma patients over the age of 70 years. Br J Haematol 2001; 114: 600–7. Qazilbash MH, Saliba RM, Hosing C et al. Autologous stem cell transplantation is safe and feasible in elderly patients with multiple myeloma. Bone Marrow Transplant 2007; 39: 279–83. Badros A, Barlogie B, Siegel E et al. Results of autologous stem cell transplant in multiple myeloma patients with renal failure. Br J Haematol 2001; 114: 822–9. Bird JM, Fuge R, Sirohi B et al. The clinical outcome and toxicity of high-dose chemotherapy and autologous stem cell transplantation in patients with myeloma or amyloid and severe renal impairment: a British Society of Blood and Marrow Transplantation study. Br J Haematol 2006; 134: 385–90. Tricot G, Barlogie B, Jagannath S et al. Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotype abnormalities. Blood 1995; 86: 4250–6. Qazilbash MH, Saliba RM, Ahmed B et al. Deletion of the short arm of chromosome 1 (del 1p) is a strong predictor of poor outcome in myeloma patients undergoing an autotransplant. Biol Blood Marrow Transplant 2007; 13: 1066–72. Jaksic W, Trudel S, Chang H et al. Clinical outcomes in t(4;14) multiple myeloma: a chemotherapy-sensitive disease characterized by rapid relapse and alkylating agent resistance. J Clin Oncol 2005; 23: 7069–73. Gertz MA, Lacy MQ, Dispenzieri A et al. Clinical implications of t(11;14)(q13;q32), t(4;14) (p16.3;q32), and –17p13 in myeloma patients treated with high-dose therapy. Blood 2005; 106: 2837–40. Avet-Loiseau H, Attal M, Moreau P et al. Genetic abnormalities and survival in multiple myeloma: the experience of the Intergroupe Francophone du Myélome. Blood 2007; 109: 3489–95. Tricot G, Jagannath S, Vesole D et al. Peripheral blood stem cell transplants for multiple myeloma: identification of favorable variables for rapid engraftment in 225 patients. Blood 1995; 85: 588–96. Zhou P, Zhang Y, Martinez C, Kalakonda N, Nimer SD, Comenzo RL. Melphalan-mobilized blood stem cell components contain minimal clonotypic myeloma cell contamination. Blood 2003; 102: 477–9. Pilarski LM, Hipperson G, Seeberger K, Pruski E, Coupland RW, Belch AR. Myeloma progenitors in the blood of patients with aggressive or minimal disease: engraftment and self-renewal of primary human myeloma in the bone marrow of NOD SCID mice. Blood 2000; 95: 1056–65. Gahrton G, Svensson H, Bjorkstrand B et al. Syngeneic transplantation in multiple myeloma – a case-matched comparison with autologous and allogeneic transplantation. European Group for Blood and Marrow Transplantation. Bone Marrow Transplant 1999; 24: 741–5. Bourhis JH, Bouko Y, Koscielny S et al. Relapse risk after autologous transplantation in patients with newly diagnosed myeloma is not related with infused tumor cell load and the outcome is not improved by CD34+ cell selection: long term follow-up of an EBMT phase III randomized study. Haematologica 2007; 92: 1083–90.

89. Barbui AM, Galli M, Dotti G et al. Negative selection of peripheral blood stem cells to support a tandem autologous transplantation programme in multiple myeloma. Br J Haematol 2002; 116: 202–10. 90. Barlogie B, Jagannath S, Vesole DH et al. Superiority of tandem autologous transplantation over standard therapy for previously untreated multiple myeloma. Blood 1997; 89: 789–93. 91. Fermand JP, Katsahian S, Divine M et al. Highdose therapy and autologous blood stem-cell transplantation compared with conventional treatment in myeloma patients aged 55 to 65 years: long-term results of a randomized control trial from the Group Myelome-Autogreffe. J Clin Oncol 2005; 23: 9227–33. 92. Blade J, Rosinol L, Sureda A et al. High-dose therapy intensification compared with continued standard chemotherapy in multiple myeloma patients responding to the initial chemotherapy: long-term results from a prospective randomized trial from the Spanish cooperative group PETHEMA. Blood 2005; 106: 3755–9. 93. Barlogie B, Kyle RA, Anderson KC et al. Standard chemotherapy compared with high-dose chemoradiotherapy for multiple myeloma: final results of phase III US Intergroup Trial S9321. J Clin Oncol 2006; 24: 929–36. 94. Fermand JP, Ravaud P, Chevret S et al. High-dose therapy and autologous peripheral blood stem cell transplantation in multiple myeloma: up-front or rescue treatment? Results of a multicenter sequential randomized clinical trial. Blood 1998; 92: 3131–6. 95. Hahn T, Wingard JR, Anderson KC et al. The role of cytotoxic therapy with hematopoietic stem cell transplantation in the therapy of multiple myeloma: an evidence-based review. Biol Blood Marrow Transplant 2003; 9: 4–37. 96. Richardson P. Toward a new therapeutic backbone in multiple myeloma. Blood 2007; 109: 2672– 3. 97. Munshi NC, Anderson KC. To transplant or not to transplant. Blood 2005; 106: 3687–8. 98. Barlogie B, Shaughnessy J, Tricot G et al. Treatment of multiple myeloma. Blood 2004; 103: 20–32. 99. Barlogie B, Jagannath S, Desikan KR et al. Total therapy with tandem transplants for newly diagnosed multiple myeloma. Blood 1999; 93: 55– 65. 100. Barlogie B, Tricot GJ, van Rhee F et al. Longterm outcome results of the first tandem autotransplant trial for multiple myeloma. Br J Haematol 2006; 135: 158–64. 101. Attal M, Harousseau JL, Facon T et al. Single versus double autologous stem-cell transplantation for multiple myeloma. N Engl J Med 2003; 349: 2495–502. 102. Cavo M, Tosi P, Zamagni E et al. Prospective, randomized study of single compared with double autologous stem-cell transplantation for multiple myeloma: Bologna 96 clinical study. J Clin Oncol 2007; 25: 2434–41. 103. Barlogie B, Tricot G, Anaissie E et al. Thalidomide and hematopoietic-cell transplantation for multiple myeloma. N Engl J Med 2006; 354: 1021–30. 104. Shaughnessy JD Jr., Zhan F, Burington BE et al. A validated gene expression model of high-risk multiple myeloma is defined by deregulated

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expression of genes mapping to chromosome 1. Blood 2007; 109: 2276–84. Bensinger WI, Demirer T, Buckner CD et al. Syngeneic marrow transplantation in patients with multiple myeloma. Bone Marrow Transplant 1996; 18: 527–31. Gahrton G, Ringden O, Lonnqvist B, Lindquist R, Ljungman P. Bone marrow transplantation in three patients with multiple myeloma. Acta Med Scand 1986; 219: 523–7. Gahrton G, Svensson H, Cavo M et al. Progress in allogeneic bone marrow and peripheral blood stem cell transplantation for multiple myeloma: a comparison between transplants performed 1983– 93 and 1994–8 at European Group for Blood and Marrow Transplantation centres. Br J Haematol 2001; 113: 209–16. Kroger N, Einsele H, Wolff D et al. Myeloablative intensified conditioning regimen with in vivo T-cell depletion (ATG) followed by allografting in patients with advanced multiple myeloma. A phase I/II study of the German Study-group Multiple Myeloma (DSMM). Bone Marrow Transplant 2003; 31: 973–9. Bensinger WI, Buckner CD, Anasetti C et al. Allogeneic marrow transplantation for multiple myeloma: an analysis of risk factors on outcome. Blood 1996; 88: 2787–93. Alyea E, Weller E, Schlossman R et al. Outcome after autologous and allogeneic stem cell transplantation for patients with multiple myeloma: impact of graft-versus-myeloma effect. Bone Marrow Transplant 2003; 32: 1145–51. Tricot G, Vesole DH, Jagannath S, Hilton J, Munshi N, Barlogie B. Graft-versus-myeloma effect: proof of principle. Blood 1996; 87: 1196–8. Lokhorst HM, Schattenberg A, Cornelissen JJ et al. Donor lymphocyte infusions for relapsed multiple myeloma after allogeneic stem-cell transplantation: predictive factors for response and long-term outcome. J Clin Oncol 2000; 18: 3031–7. Salama M, Nevill T, Marcellus D et al. Donor leukocyte infusions for multiple myeloma. Bone Marrow Transplant 2000; 26: 1179–84. Lokhorst HM, Wu K, Verdonck LF et al. The occurrence of graft-versus-host disease is the major predictive factor for response to donor lymphocyte infusions in multiple myeloma. Blood 2004; 103: 4362–4. Lokhorst HM, Segeren CM, Verdonck LF et al. Partially T-cell-depleted allogeneic stem-cell transplantation for first-line treatment of multiple myeloma: a prospective evaluation of patients treated in the phase III study HOVON 24 MM. J Clin Oncol 2003; 21: 1728–33. Corradini P, Cavo M, Lokhorst H et al. Molecular remission after myeloablative allogeneic stem cell transplantation predicts a better relapse-free survival in patients with multiple myeloma. Blood 2003; 102: 1927–9. Giralt S, Aleman A, Anagnostopoulos A et al. Fludarabine/melphalan conditioning for allogeneic transplantation in patients with multiple myeloma. Bone Marrow Transplant 2002; 30: 367–73. Lee CK, Badros A, Barlogie B et al. Prognostic factors in allogeneic transplantation for patients with high-risk multiple myeloma after reduced intensity conditioning. Exp Hematol 2003; 31: 73–80.

Hematopoietic Cell Transplantation for Multiple Myeloma 119. Perez-Simon JA, Martino R, Alegre A et al. Chronic but not acute graft-versus-host disease improves outcome in multiple myeloma patients after non-myeloablative allogeneic transplantation. Br J Haematol 2003; 121: 104– 8. 120. Mohty M, Boiron JM, Damaj G et al. Graftversus-myeloma effect following antithymocyte globulin-based reduced intensity conditioning allogeneic stem cell transplantation. Bone Marrow Transplant 2004; 34: 77–84. 121. Kroger N, Sayer HG, Schwerdtfeger R et al. Unrelated stem cell transplantation in multiple myeloma after a reduced-intensity conditioning with pretransplantation antithymocyte globulin is highly effective with low transplantation-related mortality. Blood 2002; 100: 3919–24. 122. Badros A, Barlogie B, Siegel E et al. Improved outcome of allogeneic transplantation in high-risk multiple myeloma patients after nonmyeloablative conditioning. J Clin Oncol 2002; 20: 1295– 303. 123. Maloney DG, Molina AJ, Sahebi F et al. Allografting with nonmyeloablative conditioning following cytoreductive autografts for the treatment of patients with multiple myeloma. Blood 2003; 102: 3447–54. 124. Garban F, Attal M, Michallet M et al. Prospective comparison of autologous stem cell transplantation followed by dose-reduced allograft (IFM9903 trial) with tandem autologous stem cell transplantation (IFM99-04 trial) in high-risk de novo multiple myeloma. Blood 2006; 107: 3474– 80. 125. Bruno B, Rotta M, Patriarca F et al. A comparison of allografting with autografting for newly diagnosed myeloma. N Engl J Med 2007; 356: 1110– 20. 126. Carella A, Spriano M, Corsetti M. Autografting followed closely by nonmyeloablative allografting as consolidation immunotherapy reduces disease progression compared to tandem autografting in multiple myeloma. Blood 2005; 106: 328a. 127. Rosinol L, Lahuerta J, Sureda A. Feasibility and efficacy of a planned second transplant (“auto” or

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136. Schilthuizen C, Broyl A, van der Holt B, de Knegt Y, Lokhorst H, Sonneveld P. Influence of genetic polymorphisms in CYP3A4, CYP3A5, GSTP1, GSTM1, GSTT1 and MDR1 genes on survival and therapy-related toxicity in multiple myeloma. Haematologica 2007; 92: 277–8. 137. Grazziutti ML, Dong L, Miceli MH et al. Oral mucositis in myeloma patients undergoing melphalan-based autologous stem cell transplantation: incidence, risk factors and a severity predictive model. Bone Marrow Transplant 2006; 38: 501–6. 138. Dimopoulos MA, Souliotis VL, Anagnostopoulos A, Papadimitriou C, Sfikakis PP. Extent of damage and repair in the p53 tumor-suppressor gene after treatment of myeloma patients with high-dose melphalan and autologous blood stemcell transplantation is individualized and may predict clinical outcome. J Clin Oncol 2005; 23: 4381–9. 139. Wang XS, Giralt SA, Mendoza TR et al. Clinical factors associated with cancer-related fatigue in patients being treated for leukemia and nonHodgkin’s lymphoma. J Clin Oncol 2002; 20: 1319–28. 140. Anderson KO, Giralt SA, Mendoza TR et al. Symptom burden in patients undergoing autologous stem-cell transplantation. Bone Marrow Transplant 2007; 39: 759–66. 141. Campagnaro E, Saliba R, Giralt S et al. Symptom burden after autologous stem cell transplant for multiple myeloma. Cancer 2008; 112: 1617–24. 142. Westin J, Rodjer S, Turesson I, Cortelezzi A, Hjorth M, Zador G. Interferon alfa-2b versus no maintenance therapy during the plateau phase in multiple myeloma: a randomized study. Cooperative Study Group. Br J Haematol 1995; 89: 561–8. 143. Berenson JR, Lichtenstein A, Porter L et al. Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma. Myeloma Aredia Study Group. N Engl J Med 1996; 334: 488–93. 144. Attal M, Harousseau JL, Leyvraz S et al. Maintenance therapy with thalidomide improves survival in patients with multiple myeloma. Blood 2006; 108: 3289–94.

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Philip J. Bierman & Auayporn Nademanee

Hematopoietic Cell Transplantation for Hodgkin’s Disease

Epidemiology and etiology Hodgkin’s disease (HD) is a clonal lymphoid disorder of mononuclear Hodgkin’s cells and multinucleated Reed–Sternberg cells, which are felt to be derived from germinal center B-cells [1]. In 2008, it is estimated that there will be 8220 new cases of HD in the United States and that 1350 people will die with this diagnosis [2]. Between 2000 and 2004, the HD incidence rate in the United States was 2.7 per 100,000 [3]. This incidence rate has been stable in recent years. There is a bimodal incidence with one peak in the late teen years and early adulthood, and a second peak that is seen in adults after age 50 years. The incidence rate for HD is higher in men, and higher in white individuals, compared with those who are black, Asians, or Hispanics. The risk factors associated with HD are not as consistent or well understood as those identified for non-Hodgkin’s lymphoma (NHL). Reed–Sternberg cells often express Epstein–Barr virus-associated antigens. The HD incidence rate is higher in patients who have had infectious mononucleosis, although links between Epstein–Barr virus and HD are inconsistent. Patients infected with human immunodeficiency virus (HIV) are more likely to develop HD, and HD has been described following solid organ transplantation. Some studies show a slight familial risk associated with HD, and there appears to be a higher risk in monozygotic twins of affected individuals.

Molecular and clinical biology The World Health Organization classification divides HD into two entities, which have different clinical and biologic characteristics [1]. Approximately 5% of cases are classified as nodular lymphocytepredominant Hodgkin’s lymphoma, which is characterized by neoplastic cells known as popcorn or L&H (lymphocytic and/or histiocytic) cells. The neoplastic cells almost always express CD20. Most patients have classical Hodgkin’s lymphoma, which is subdivided into nodular sclerosis, mixed cellularity, lymphocyte-rich, and lymphocyte-depleted subtypes. The neoplastic Reed–Sternberg cells within classical HD subtypes usually express CD30, and often CD15. Hodgkin’s cells release a variety of cytokines, including various interleukins. This may explain the inflammatory background seen in HD biopsies and explain eosinophilia and some systemic symptoms seen in HD, as well as fibrosis seen in biopsies. No consistent cytogenetic

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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abnormalities are seen in HD, although immunoglobulin gene rearrangements are characteristic. No consistent environmental causes of HD have been identified, although some studies have identified increased risk associated with woodworking.

Clinical description The most common HD symptom is painless lymphadenopathy. The disease spreads contiguously, and patients may have localized or widespread disease. Mediastinal involvement is most common in the nodular sclerosis subtype. Extranodal disease is less common than in NHL. Patients may have systemic or “B” symptoms consisting of unexplained fevers, night sweats or weight loss. Pruritus may be seen and precede the diagnosis of HD for months. Approximately 70% of patients with nodular lymphocytepredominant Hodgkin lymphoma are males in the 30–50-year age range. Patients often have localized disease and may have an indolent disease course. The most common subtype of classical HD is nodular sclerosis, which is more likely to be present in patients with limited-stage disease. The mixed cellularity subtype is more likely to be found in patients with immunodeficiency syndromes.

Nontransplant approaches More than 50% of HD patients, including those with advanced disease, can be cured with a wide variety of chemotherapy regimens [4,5]. This accomplishment is one of the greatest achievements of modern medicine. There has been a dramatic decline in HD mortality, and the 5-year survival rate is now approximately 85%. In the past, limited-stage HD was usually treated with radiation therapy. Limited radiation may be used for low-stage nodular lymphocyte-predominant Hodgkin’s lymphoma. However, concerns about late toxicity associated with extensive radiation fields have led to the common practice of using abbreviated courses of chemotherapy followed by involved-field radiation for most patients. In some cases, chemotherapy may be used, alone. Patients with advanced disease or bulky masses are treated with a full course of chemotherapy and sometimes receive additional radiotherapy. A major focus of new treatment regimens involves the reduction of late toxicities of HD treatment. Although most patients with HD are cured with initial therapy, a significant proportion of patients still fail to attain a complete remission or relapse after attaining a remission. A variety of conventional-dose second-line (salvage) regimens has been used for these patients, but results of treatment with these regimens are disappointing, and few patients achieve long-term disease-free survival.

Hematopoietic Cell Transplantation for Hodgkin’s Disease

861

Table 59.1 Results of autologous hematopoietic cell transplantation for Hodgkin’s disease Reference

Number

Rescue source

Regimen

Median follow-up

Early mortality

Outcome

[12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [9] [23] [24] [25] [26] [27] [28] [29] [30]

73 155 128 85 119 102 280 70 414 40 56 494 104 101 92 102 100 341 141 127

BM, PB BM BM, PB BM, PB BM, PB BM, PB BM, PB BM, PB BM, PB PB PB BM, PB BM, PB BM, PB BM, PB PB BM, PB BM, PB BM, PB BM, PB

VP-16 + MEL BEAM CBV CY + VP-16 + TBI or CBV Various CBV Various BEAC Various BEAM CY + VP-16 + TBI or CBV Various Various BEAM CY + VP-16 + TBI or BU + MEL + T HDS CBV ± Plat Various CBV BU + CY + VP-16

30 months NS 77 months 28 months 40 months 4.1 years 36 months 3.6 years 46 months 28 months 43 months 30.5 months 5.1 years 50 months 6 years 5 years 11.4 years

4% 10% 9% 8% 5% 12% 6% 13% 7% 3% 4% 9% 16%

39% 4-year DFS 50% 5-year DFS 25% 4-year FFS 58% 2-year EFS 48% 4-year EFS 42% 3-year PFS 60% 4-year PFS 32% 5-year EFS 63% 3-year OS 69% 3-year PFS 68% 3-year EFS 45% 5-year EFS 26% 10-year EFS 60% 5-year FF2F 51% 6-year EFS 53% 5-year EFS 51% 15-year PFS 45% 5-year EFS 48% 5-year PFS 48% 5-year PFS

6.3 years 6.7 years

15% 5% 8% 8% 1% 6%

BEAC, carmustine, etoposide, cytarabine, and cyclophosphamide; BEAM, carmustine, etoposide, cytarabine, and melphalan; BM, bone marrow; BU, busulfan; CBV, cyclophosphamide, carmustine, and etoposide; CY, cyclophosphamide; DFS, disease-free survival; EFS, event-free survival; FF2F, freedom from second failure; FFS, failure-free survival; HDS, high-dose sequential chemotherapy; MEL, melphalan; NS, not stated; OS, overall survival; PB, peripheral blood; PFS, progression-free survival; Plat, cisplatin; T, thiotepa; TBI, total body irradiation; VP-16, etoposide.

Hematopoietic cell transplantation The poor results of conventional-dose salvage therapy for relapsed and refractory HD has led to the use of high-dose therapy followed by autologous bone marrow transplantation (BMT) and peripheral blood hematopoietic cell transplantation (PBHCT) for these patients. This approach is based upon the steep dose–response curves exhibited by several drugs, as well as radiation therapy, and the fact that dose-limiting toxicities are often related to myelosuppression. The first attempts at using autologous bone marrow infusions to reduce myelosuppression following radiation and chemotherapy were documented in the 1950s. Later, techniques for bone marrow harvesting and cryopreservation were refined, and results of autologous BMT for HD began to be reported [6]. Since then, the use of high-dose therapy followed by autologous hematopoietic cell transplantation (HCT) for HD has increased dramatically, and this approach has become accepted therapy in a variety of circumstances. In North America, HD is the third most common indication for autologous HCT behind multiple myeloma and NHL. Approximately 1200 autologous HCTs for HD were reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) in 2003 [7]. It is estimated that this total represents approximately 60% of the transplants being performed for this indication in North and South America. Results from the European Group for Blood and Marrow Transplantation (EBMT) are similar [8]. HD is the third most common indication for autologous HCT in Europe, and it was reported that 1677 autologous HCTs for HD were performed there during 2005. Autologous HCT has become easier, safer, and less expensive over the last 20 years. Mortality within 100 days of transplant is generally under 5%, and improved outcomes over time have been demonstrated

[7,9]. These improvements are due to increased experience at individual institutions, improved patient selection, the routine use of hematopoietic growth factors, and the increased use of autologous PBHCT, compared with autologous BMT. Autologous HCT is commonly performed by community oncologists and can be performed on elderly patients, in the outpatient setting [10], and in patients with religious objections to blood transfusion [11]. Results of autologous HCT The results of some of the larger and most recent series of autologous HCT for HD are displayed in Table 59.1. It is difficult to compare results because of differences in selection criteria, patient characteristics, highdose therapy regimens, and supportive care. In addition, the length of follow-up varies considerably, and some reports include patients transplanted before the routine use of autologous PBHCT and hematopoietic growth factors. Nevertheless, these results demonstrate that a significant proportion of patients achieve long-term progression-free survival following transplantation, and that transplantation can be accomplished with low early mortality. It is unknown whether the use of more aggressive and more effective primary chemotherapy regimens will alter the results of transplantation for patients who require salvage therapy [4]. Representative results of high-dose therapy followed by autologous HCT for a large group of patients with relapsed and refractory HD with long follow-up are displayed in Fig. 59.1. The results of autologous HCT for patients with relapsed and refractory HD appear better than those reported with conventional-dose salvage chemotherapy. Randomized trials Two randomized trials have compared the results of conventional-dose salvage chemotherapy with autologous HCT for relapsed and refractory

862

Chapter 59

Overall survival rate (%)

100

p = 0.318

80 BEAM (n = 20) 60 40

Mini-BEAM (n = 20)

20 0

Overall event-free survival rate (%)

(a)

0

Nonrandomized trials Several nonrandomized trials have compared the results of conventional salvage chemotherapy with results of autologous HCT for patients with

Cumulative progression rate (%)

HD. A British National Lymphoma Investigation trial randomized patients with relapsed and refractory HD to receive treatment with miniBEAM (carmustine, etoposide, cytarabine, and melphalan) conventional-dose salvage chemotherapy or treatment with BEAM (the same drugs at higher doses) followed by autologous BMT [31]. This study design tested the role of dose intensity in the treatment of HD since the same drugs were used in each treatment arm. The 3-year event-free survival was estimated to be 53% in transplanted patients, compared with 10% in the mini-BEAM group (Fig. 59.2). The risk of disease progression was also significantly lower in transplanted patients, although no significant differences in overall survival were observed. Some participants preferred treatment with autologous BMT and refused randomization, and the study was terminated early. The German Hodgkin’s Lymphoma Study Group (GHSG) and the EBMT conducted the other randomized trial, which also closed early because of poor accrual [32]. After randomization, patients received two courses of salvage chemotherapy with Dexa-BEAM (dexamethasone, carmustine, etoposide, cytarabine, and melphalan). Responding patients received either two additional courses of Dexa-BEAM, or else high-dose BEAM followed by autologous HCT. The freedom from treatment failure at 3 years was estimated to be 55% for patients treated with autologous HCT, compared with 34% for those who received DexaBEAM, alone (Fig. 59.3). Overall survival rates were estimated to be 71% and 65%, respectively (p = 0.331).

2

3

4

5

100 p = 0.025

80 60 40

BEAM (n = 20) 20 Mini-BEAM (n = 20)

0

(b)

Fig. 59.1 Overall survival (a) and progression-free survival (b) following high-dose chemotherapy and autologous hematopoietic cell transplantation for relapsed and refractory Hodgkin’s lymphoma. (Reproduced from [27], with permission.)

1

0

1

2

3

4

5

100 p = 0.005

80

Mini-BEAM (n = 20)

60 40 BEAM (n = 17) 20 0 0

1

(c)

2

3

4

5

Time (years)

Fig. 59.2 Overall survival, event-free survival, and cumulative progression rate in carmustine, etoposide, cytarabine, and melphalan plus autologous bone marrow transplant (BEAM) and mini-BEAM groups. (Reproduced from [31] ], with permission.)

relapsed and refractory HD. A French registry analysis examined outcomes in patients who failed to respond to treatment or progressed after treatment with a single chemotherapy regimen [24]. The 5-year overall survival was estimated to be 71% for patients who received autologous HCT, compared with 32% for others (p = 0.0001). Freedom from treatment failure was estimated to be 60% and 20%, respectively (p = 0.0001). A multivariate analysis showed that salvage treatment that did not include high-dose therapy was associated with significantly worse overall survival (p = 0.02) and freedom from treatment failure (p = 0.02). Additional trials discussed in later sections include retrospective registry analyses after first relapse [33], single-institution studies of patients with refractory and relapsed disease [34], and registry analyses of patients with primary refractory disease [24,35–37]. Prognostic factors A large number of factors have been identified that are associated with adverse outcomes for patients who undergo autologous HCT for HD. Several prognostic models have been developed that use these factors

Hematopoietic Cell Transplantation for Hodgkin’s Disease

Freedom from treatment failure (%)

Table 59.2 Adverse prognostic factors: autologous hematopoietic cell transplantation for Hodgkin’s disease

BEAM-HSCT Dexa-BEAM

100

80

60

40 p = 0.0187 20

0 0

10

20

30

40

50

60

70

Months after randomization Number of patients BEAM-HSCT 61 Dexa-BEAM 56

43 27

34 20

25 15

863

13 10

8 8

7 5

0 1

Prognostic factor

Reference

Bulky or “nonminimal” disease at transplant Extensive therapy before transplant Poor performance status Short initial remission Extranodal disease at relapse or at transplant Systemic symptoms at relapse Chemotherapy resistance Female gender Male gender Age Advanced stage at relapse Progressive disease at transplant Elevated lactase dehydrogenase level at transplant Anemia at relapse or transplant Hypoalbuminemia at transplant

[12,13,16,19,29,30] [9,13–15,27–29] [14,17,35] [13,18,22,26,38] [15–18,27,38] [16,26,29,34,38] [9,18–21,23–30] [13] [25] [40] [21] [17] [20] [40] [40]

Fig. 59.3 Freedom from treatment failure for patients with relapsed chemosensitive Hodgkin’s disease. (Reproduced from [32], with permission.)

to predict outcome following transplantation. The Vancouver group examined patients undergoing autologous BMT for HD in first relapse [38]. The presence of systemic symptoms at relapse, extranodal disease at relapse, and initial remission duration less than 12 months were associated with shorter progression-free survival. The 3-year progressionfree survival was 100%, 81%, 40%, and 0% for patients with zero, one, two or three risk factors, respectively. Investigators from Memorial Sloan-Kettering Cancer Center found that the same three factors were significant when applied at the time patients received conventional salvage chemotherapy prior to autologous HCT [22]. The event-free survival was estimated to be 83% for those with zero or one risk factor, 27% for those with two risk factors, and 10% for patients with all three risk factors (p < 0.001). The overall survival rates were 90%, 57%, and 25%, respectively (p < 0.001). A model for patients transplanted with relapsed or refractory HD was developed at Stanford University [16]. Bone marrow or pulmonary involvement, systemic symptoms at relapse, and more than minimal disease at transplant (>75% reduction in a bulky mass or no nodes >2 cm, and <10% marrow involvement) was associated with significantly worse overall survival, event-free survival, and freedom from progression. Actuarial freedom from progression at 3 years was estimated to be 85%, 57%, 41%, and less than 20% for patients with zero, one, two and three risk factors, respectively (p = 0.0001). Another model developed in Boston used progressive disease at the time of HCT, more than one extranodal site of relapse, and poor performance status to construct a prognostic index [17]. The 3-year actuarial survival was 82%, 56%, and 19% for patients with zero, one, or two or more risk factors, respectively (p = 0.0001). A French registry analysis evaluated results of autologous HCT for relapsed HD [18]. The presence of extranodal disease at relapse and initial remission duration under 12 months were identified as significant risk factors. The 4-year overall survival was estimated to be 93%, 59%, and 43%, for patients with zero, one or two risk factors, respectively. A GHSG study evaluated patients with relapsed HD and determined that initial remission duration less than 12 months, stage III or IV disease at relapse, and anemia at relapse could be used to construct a prognostic model for patients treated with autologous HCT [39]. An index based upon the

presence of zero, one, two or three of these risk factors was able to identify groups with significantly different rates of survival (p < 0.0001). At the University of Nebraska, a prognostic index was developed based upon factors used in the International Prognostic Factors Project Score for advanced HD [5,40]. Low serum albumin (<4 g/dL), anemia (<10.5 g/dL), age (≥45 years), and lymphocytopenia (<600/mm3 and/or <8% of total white blood cell count) were associated with inferior outcomes. The estimated 10-year overall survival rates were 48%, 35%, 27%, and 20%, for patients with zero, one, two or three or more risk factors, respectively. Another prognostic model was developed at the University of Minnesota [29]. Absence of systemic symptoms at the time of transplant, transplantation in complete remission, and chemotherapy-sensitive disease were independent predictors of improved progression-free survival. The 5-year predicted progression-free survival was 67%, 37%, and 9% for those with zero and one, two or three risk factors, respectively (p < 0.001). The influence of histologic subtype has not been extensively studied, although one study showed that patients with lymphocyte-predominant histology had significantly longer event-free survival, compared with other patients [28]. The use of 18-fluorodeoxyglucose positron emission tomography (FDG PET) scans has been shown to be useful in the management of HD and NHL. Several studies have evaluated the importance of FDG PET scans performed prior to autologous HCT for HD and found that a negative FDG PET scan is associated with improved progression-free survival [41]. The most important risk factors associated with adverse outcome following autologous HCT for HD are displayed in Table 59.2. It should be noted that some of these prognostic factors have not been shown to be important in all studies. This is likely related to differences in patient characteristics, selection bias, and sample size. Nevertheless, most studies show that extensive therapy prior to transplant, poor performance status, chemotherapy resistance, and active disease at the time of transplant are predictive of poorer outcome after autologous HCT for HD. Pretransplant “debulking” chemotherapy Almost all patients with relapsed NHL are treated with conventional-dose salvage chemotherapy to reduce tumor burden prior to treatment with

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Chapter 59

high-dose therapy and autologous HCT. Sensitivity to pretransplant salvage therapy is one of the most important prognostic factors for progression-free survival and overall survival following autologous HCT. The majority of patients with relapsed HD are also treated in this manner, and most studies show that chemotherapy sensitivity is an important prognostic factor for these patients too (Table 59.2). A wide variety of chemotherapy regimens have been used for this purpose, although the ICE (ifosfamide, carboplatin, and etoposide) regimen has become popular [22]. The potential impact of administering pretransplant salvage chemotherapy was evaluated in a study from the Autologous Blood and Marrow Transplant Registry (ABMTR) that compared outcomes of HD patients in first relapse with those in second remission [20]. The actuarial 3-year overall survival was 58% for patients who received autologous HCT in first relapse, compared with 75% for patients transplanted in second complete remission (p <0.001). The Polish Transplant group showed that actuarial 5-year event-free survival was 16% for HD transplanted with active disease, compared with 66% for patients in complete remission (p <0.001) [28]. At the University of Rochester, the 5-year event-free survival was projected to be 46% for patients with minimal disease (all areas ≤2 cm) at the time of transplant, compared with 10% for those with bulky disease (p = 0.0002) [19]. Autologous HCT results from the GEL/TAMO Spanish cooperative group revealed that the failure-free survival was 63.2% at 5 years following HCT for HD patients transplanted in complete remission, compared with 32.2% for those with measurable disease (p = 0.00001) [9]. At Toronto, the 4-year actuarial disease-free survival was 68% for HD patients who had no evidence of disease at the time of transplant, 26% for patients transplanted with nonbulky disease, and 0% for those with bulky disease (p = 0.0002) [12]. At Stanford University, the presence of more than minimal disease (see above) at the time of transplant was associated with significantly worse progression-free survival, event-free survival, and overall survival [16]. The group from Minnesota showed that relapsed and refractory HD patients who underwent autologous HCT with residual disease present had a 2.3-fold relative risk of progressive disease, compared with those who were transplanted while free of disease (p = 0.03) [29]. A similar Cleveland Clinic analysis demonstrated that HD patients transplanted with bulky disease (>10 cm) had significantly poorer survival than other patients (hazard ratio 2.9; p = 0.002) [30]. These results suggest that treatment with conventional salvage chemotherapy to reduce tumor burden prior to autologous HCT improves outcomes. However, the absence of bulk may be a surrogate for chemotherapy sensitivity, and some patients might do well without additional therapy prior to transplant. In addition, some patients with relapsed and refractory HD fail to respond to conventional salvage therapy and never proceed to transplantation. For these reasons, a comprehensive two-step transplant regimen was developed at Memorial Sloan-Kettering Cancer Center so that all patients with relapsed and refractory HD could be evaluated on an intentto-treat basis [22]. This program consists of two cycles of ICE conventional salvage therapy, followed by accelerated fractionation involved-field radiation, and then high-dose therapy and autologous HCT. The rate of response to ICE was 88%, and the overall survival and event-free survival at a median of 43 months were projected to be 73% and 58%, respectively, for all patients. The overall survival and event-free survival for the 86% of patients who proceeded to autologous HCT were 83% and 68%, respectively. This type of intent-to-treat analysis gives a more realistic assessment of the role of transplantation for HD. The use of pretransplant chemotherapy may not be necessary in all circumstances, and may have disadvantages. This approach adds expense and the potential for toxicity. Further, this approach has the theoretical risk of increasing resistance to cells that might still be sensitive to highdose therapy but resistant to conventional-dose salvage chemotherapy.

At University College Hospital in London, United Kingdom, the actuarial 5-year progression-free survival was 78% in a cohort of patients with HD who underwent autologous HCT in “untested” relapse [13]. The 5-year progression-free survival was estimated to be 57% for HD patients in the Spanish registry who were transplanted in untested relapse [9]. At the University of Nebraska, the 5-year overall survival was estimated to be 100% for HD patients who were transplanted without additional therapy after relapse, compared with 45% for patients who received conventional chemotherapy prior to aHCT (p = 0.048) [42]. An EBMT retrospective analysis showed no significant differences in overall survival or progression-free survival when HD patients in untested relapse were compared with those who received conventional salvage therapy prior to autologous HCT [43]. Patients with HD who are transplanted without conventional salvage chemotherapy may do well because they have minimal tumor burden at the time of relapse. Nevertheless, pretransplant debulking chemotherapy may not be necessary for all HD patients, and this practice is potentially harmful for some. It may be possible to eliminate this treatment for patients with minimal tumor burden at the time of relapse, or those without evidence of disease after the site of relapse is resected. The vast majority of patients, however, probably benefit from pretransplant chemotherapy, particularly those who have significant tumor burdens. On rare occasions, surgical debulking prior to transplantation can also be attempted. Pretransplant radiation therapy Radiation is an essential tool in the treatment of HD. At some institutions, radiotherapy is used instead of chemotherapy or in addition to chemotherapy to reduce tumor burden prior to autologous HCT. Other institutions administer radiation as consolidation therapy after transplant (see below). There are several unanswered questions regarding the potential advantages and disadvantages of each approach. The use of radiotherapy prior to transplant can reduce tumor burden and usually does not delay the start of high-dose therapy. Mutiple institutions have used pretransplant involved-field radiation for patients undergoing autologous HCT for HD [12,15,16,27,44–46]. Although this may improve local control and decrease the incidence of relapse in irradiated areas, it has been difficult to demonstrate definite survival advantages associated with this therapy [44–46]. At the Memorial Sloan-Kettering Cancer Center, patients with relapsed and refractory HD are treated with an integrated approach utilizing conventional salvage chemotherapy, 1800–3600 cGy accelerated fractionation involved-field radiation administered twice daily over a 5- to 10 day period, followed by autologous HCT [22]. Pretransplant involved-field radiation has been associated with increased post-transplant complications such as esophagitis and pulmonary toxicity, especially after mediastinal irradiation. An analysis from University of California Los Angeles demonstrated that radiation prior to autologous HCT (using a total body irradiation [TBI]-containing regimen) for HD was associated with significantly higher morbidity and mortality, compared with post-transplant radiation [47]. Similar analyses conducted for patients with NHL suggest that pretransplant radiotherapy may be associated with an increased risk of secondary myelodysplasia or respiratory complications [46]. Thus, pretransplant radiotherapy should be individualized and should be used cautiously, especially when mediastinal irradiation is given before high-dose carmustine. Post-transplant therapy A number of approaches have been utilized to decrease the risk of relapse following autologous HCT for HD. At many institutions,

Hematopoietic Cell Transplantation for Hodgkin’s Disease

involved-field radiation is used following autologous HCT as consolidation therapy or to treat areas of persistent disease [19,24–26,29,32]. Delaying radiation until after autologous HCT may decrease the incidence of transplant-related complications, and may avoid delays in starting high-dose therapy due to toxicities of pretransplant radiation. Post-transplant irradiation can convert a partial remission into a durable complete remission [14]. A retrospective analysis from the University of Rochester demonstrated that actuarial 5-year event-free survival was 44% for HD patients who received post-transplant consolidative radiotherapy, compared with 26% for patients who did not receive radiation (p = 0.0056) [19]. A retrospective analysis from the University of Maryland also demonstrated significantly improved event-free survival among HD patients who received additional radiotherapy following autologous HCT [48]. Potential advantages attributed to post-transplant radiation therapy have been seen in other analyses, although randomized trials have not been conducted [45]. Issues relating to selection bias make it difficult to evaluate the advantages of involved-field radiation therapy following autologous HCT for HD, because patients with early relapse, poor performance status, and delayed engraftment may not be able to be treated. In addition, the use of radiation with primary therapy makes many patients ineligible for additional radiotherapy. Post-transplant radiation may also lead to significant hematologic and nonhematologic toxicity, and it is unclear whether it is more advantageous to administer the radiation prior to autologous HCT. There are also other unresolved issues related to the timing and schedule of treatment, treatment fields, and radiation dose. In addition to post-transplant involved-field radiation therapy, several groups have conducted phase I–II trials with interferon, interleukin-2, and interleukin-2 with lymphokine-activated killer cells to decrease relapse rates after autologous HCT for HD [49,50]. A retrospective analysis from the Polish Lymphoma Research Group suggested that the use of immunotherapy following autologous HCT for primary refractory HD was associated with significantly better progression-free survival and overall survival [49]. Although the use of post-transplant immune modulation is feasible and augmentation of various parameters of immune function can be demonstrated, there are no studies that conclusively show a benefit from this approach. A number of other approaches have been used to decrease relapse rates following autologous HCT for HD. Investigators in Baltimore used consolidation chemotherapy administered at 3, 6, 9, and 12 months after transplant [48]. Perhaps the most aggressive form of post-transplant therapy involves the use of a planned second transplant. The group from Valhalla, New York, performed tandem transplants with a regimen containing thiotepa, mitoxantrone, and carboplatin for the first transplant, and ifosphamide, carboplatin, and etoposide for the second [51]. An HD tandem transplant protocol developed at the City of Hope utilized melphalan for the first transplant and TBI or carmustine, combined with etoposide and cyclophosphamide, for the second [52]. Other investigators have performed a planned reduced-intensity conditioning (RIC) followed by allogeneic HCT following autologous HCT [53]. Timing of transplantation An important issue dealing with autologous HCT for HD relates to the appropriate time to perform this procedure during the course of disease. Some patients with relapsed HD may be cured with conventional salvage chemotherapy or radiotherapy, and it may be appropriate to delay transplantation until after these approaches fail. Conversely, transplantation should not be delayed to a point when resistance to chemotherapy or poor performance status makes cure unlikely with any approach. Potential times when autologous HCT for HD can be performed are displayed

865

Table 59.3 Potential timing of autologous hematopoietic cell transplantation for Hodgkin’s disease Failure to attain initial complete remission Primary refractory First partial remission First relapse Short versus long initial remission Second or subsequent relapse First remission

Fig. 59.4 Overall survival (OS) and progression-free survival (PFS) following autologous hematopoietic cell transplantation for primary refractory Hodgkin’s disease. The y-axis denotes actuarial percentage survival. (Reproduced from [54], with permission.)

in Table 59.3. Although data from randomized trials are lacking, results in each situation can be compared with results of conventional salvage therapy. Failure to attain initial complete remission HD patients who do not achieve complete remission with initial chemotherapy have a poor prognosis [4], and autologous HCT may be the only curative treatment for such patients. Several cooperative groups and institutions have reported results of autologous HCT for these patients. The EBMT identified 175 HD patients who failed to achieve an initial complete or partial remission after one or more regimens [54]. The 5year actuarial overall survival and progression-free survival were 36% and 32%, respectively (Fig. 59.4). Overall survival was significantly better for patients who were transplanted within 18 months of diagnosis. The ABMTR analyzed autologous HCT results in 122 HD patients who never achieved remission after completing at least one combination chemotherapy regimen [55]. The 3-year overall survival and progression-free survival were estimated to be 50% and 38%, respectively. Overall survival was significantly worse in patients with systemic symptoms at diagnosis and in patients with a poor performance status at the time of transplant. Other reports of autologous HCT for primary refractory HD are displayed in Table 59.4. No randomized trials have compared results of autologous HCT with conventional salvage chemotherapy for patients with primary refractory HD, although other comparisons have been performed. At Stanford

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Table 59.4 Results of autologous hematopoietic cell transplantation for primary refractory Hodgkin’s disease

Table 59.5 Results of autologous hematopoietic cell transplantation for Hodgkin’s disease after first relapse

Reference

Number

Outcome

Reference

Number

Outcome

[54] [55] [35] [56] [37] [57] [49] [9] [13] [58] [59] [36] [29] [34]

175 122 86 75 70 62 65 49 46 30 28 27 21 13

32% 5-year PFS 38% 3-year PFS 25% 5-year EFS 45% 10-year EFS 31% 5-year PFS 15% 5-year TTF 36% 3-year PFS 13% 5-year PFS 33% 5-year PFS 42% 5-year PFS 26% 3-year EFS

[65] [20] [33] [43] [29] [42] [27] [26] [38]

357 295 187 139 120 85 60 59 58

49% 46% 36% 45% 45% 40% 62% 63% 64%

45% 10-year PFS 52% 4-year PFS

36% 5-year OS 50% 5-year OS 35% 5-year OS 48% 10-year OS 43% 5-year OS 26% 5-year OS 55% 3-year OS

60% 5-year OS 34% 5-year OS 81% 4-year OS 48% 10-year OS 44% 4-year OS

EFS, event-free survival; OS, overall survival; PFS, progression-free survival; TTF, time to treatment failure.

University, induction failure was defined as progression during initial therapy or relapse within 2 months [34]. Results of autologous HCT for these patients were compared with historical controls who received conventional salvage therapy. The event-free survival at 4 years was projected to be 52% and 19%, respectively (p = 0.01). However, there were no significant differences in overall survival (44% versus 38%, respectively; p = 0.32). A case–control study from the French database examined the outcome of HD patients who progressed during first-line treatment or within 3 months of completing treatment [35]. Overall survival 6 years from the time of diagnosis was 38% for transplanted patients, compared with 29% for patients who received conventionally salvage therapy (p = 0.058). A retrospective analysis from Cologne, Germany, examined outcomes in 67 patients with primary progressive HD, defined as progression during first-line therapy or within 3 months after initial therapy [60]. The mean survival was 56.2 months for patients treated with autologous HCT, compared with 11.2 months for those who received conventional salvage therapy. The actuarial 5-year overall survival rates were 53% and 0%, respectively. The use of autologous HCT was identified as a significant factor associated with improved freedom from treatment failure (p = 0.031) and overall survival (p = 0.039). A retrospective analysis from the Gruppo Italiano per lo Studio dei Linfomi reviewed the results of therapy in 72 patients with HD who progressed or had minimal response to initial chemotherapy [36]. Highdose therapy followed by autologous HCT was attempted in 27 of these patients. The actuarial 4-year overall survival was 81% for this group, compared with 38% in patients who received chemotherapy alone (p = 0.019). Interpretation of results of autologous HCT in these patients is difficult because of different definitions of “primary refractory” and “induction failure.” Definitions include patients with disease progression during initial therapy, stable disease after initial therapy, progression within 90 days of completing initial therapy, and partial remission with initial therapy. Patients who progress on primary therapy, and those who have no response, are likely to have a different prognosis to patients who have a partial remission or near-complete remission. This is supported by French registry data demonstrating significantly worse overall survival and progression-free survival when autologous HCT results for

5-year TTF 3-year DFS 5-year FF2F 5-year PFS 10-year PFS 5-year FFS 15-year DFS 5-year EFS 3-year PFS*

57% 5-year OS 58% 3-year OS 54% 5-year OS 49% 5-year OS 47% 10-year OS 51% 5-year OS 67% 15-year OS 77% 5-year OS 72% 3-year OS*

DFS, disease-free survival; EFS, event-free survival; FF2F, freedom from second failure; FFS, failure-free survival; OS, overall survival; PFS, progression-free survival; TTF, time to treatment failure.

* Median follow-up 2.3 years.

primary refractory HD patients were compared with results from patients in first partial remission [59]. Another French study identified 27 HD patients with stage IV disease and/or large mediastinal masses who were in an incomplete response (≥50%) following three cycles of ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) chemotherapy [61]. This group was treated with autologous HCT, and the 5-year event-free survival was estimated to be 87%, compared with 61% for control patients treated with conventional therapy (p = 0.02). The actuarial 5-year overall survival rates were 92% and 77%, respectively (p = 0.2). Other investigators have also reported good outcomes following autologous HCT for HD patients in first partial remission [62–64]. It would seem that patients who attain a good partial remission following primary therapy can do well following autologous HCT, although reports may classify these patients in the category of primary refractory disease. Interpretation of results is also hampered by the fact that patients may not have biopsy-proven residual disease before transplantation, and that some merely had residual radiographic abnormalities without active disease. It is hoped that the use of modalities such as PET scans will help with this difficulty. Finally, it is important to recognize that a significant proportion of patients with primary refractory disease will not be able to proceed to transplant, and that patients reported in these series may be selected [24,37]. Nevertheless, the results in Table 59.4 appear superior to those attainable with conventional salvage chemotherapy, and autologous HCT may be the best option for patients who fail to respond to initial chemotherapy. Additional chemotherapy or radiotherapy may be considered prior to high-dose therapy for patients with significant tumor burden, while other patients with minimal disease remaining after primary therapy should proceed directly to transplantation [54]. First relapse High-dose therapy followed by autologous HCT for HD is most often considered for patients who relapse following their initial chemotherapy-induced complete remission. Several reports of autologous HCT in this situation have been published, and allow comparison with results of conventional salvage therapy. Selected results of autologous HCT for HD after first relapse are displayed in Table 59.5, which includes series that specifically address this population of patients [20,32,33,38,42,43,65]. Some of these trials have compared the results of autologous HCT with those of conventional salvage chemotherapy. The duration of initial

Hematopoietic Cell Transplantation for Hodgkin’s Disease

remission is an important prognostic factor for outcome with conventional salvage chemotherapy for HD [4,39]. In the randomized GHSG trial, HD patients in first relapse were divided into those with early (<12 months) or late (>12 months) relapses [32]. The freedom from treatment failure at 3 years was estimated to be 41% for early relapse patients treated with autologous HCT, compared with 12% for those who received conventional salvage chemotherapy (p = 0.008). The freedom from treatment failure at 3 years was also significantly better for patients with long first remissions given autologous HCT (75%) than for those who received conventional salvage chemotherapy (44%; p = 0.025). Overall survival rates were 93% and 75%, respectively (p = 0.088). An analysis from Stanford University compared the outcome of autologous HCT for patients after first relapse of HD with the results of conventional salvage chemotherapy in historical controls [34]. The 4year event-free survival for patients with initial remissions of 12 months or less was estimated to be 56% following autologous HCT compared with 19% for those who received conventional salvage chemotherapy (p <0.01). Overall survival rates were 58% and 38%, respectively (p = 0.15). No significant differences in overall survival or event-free survival were seen in patients with longer initial remissions. A retrospective French–Belgian analysis of HD treatment after first relapse found that overall survival was 69% following autologous HCT, compared with 55% for patients treated with conventional chemotherapy (p = not significant) [33]. Among patients with initial remissions less than 12 months in duration or with stage III–IV disease at relapse, the freedom from treatment failure was estimated to be 64% following autologous HCT and 40% for those who received conventional chemotherapy. The overall survival rates were estimated to be 55% and 30%, respectively. A subsequent analysis demonstrated that the actuarial 4year overall survival following autologous HCT was 81% for patients with late relapses, compared with 65% for patients with shorter initial remissions (p < 0.05) [18]. Other groups have also analyzed transplant outcome in relation to the duration of the first HD remission. A report from the Spanish registry demonstrated that 5-year time to treatment failure was estimated at 57% following autologous HCT after a first remission of at least 12 months, compared with 43% for those with shorter remissions (p = 0.0014) [65]. The overall survival rates were 67% and 50%, respectively (p = 0.001). The group from Minnesota showed that 5-year overall survival following aHCT was estimated to be 60% for those with long initial remission (>12 months), compared with 44% for those with shorter remissions (p = 0.04) [29]. At Vancouver the progression-free survival was estimated to be 85% for HD patients treated with autologous HCT after an initial remission of at least 1 year, compared with 48% for those with shorter remissions (p = 0.016) [38]. In contrast, other reports have failed to note statistically significant differences in outcome related to the duration of initial remission [13,17,26,42,43]. Prognostic factors for autologous HCT after first HD relapse are similar to those reported in Table 59.2. Results from the Spanish registry revealed that the actuarial 5-year overall survival was 75% for patients who achieved a second remission with conventional salvage therapy prior to autologous HCT, 43% for patients who were sensitive to salvage therapy but did not attain a second remission, and 19% for resistant patients [65]. Results from the EBMT and ABMTR also demonstrated prolonged overall survival in patients who were transplanted in second complete remission, compared with patients who had sensitive relapse, but did not achieve a second remission [20,43]. Although overall survival advantages have not been demonstrated, most investigators would recommend autologous HCT for HD patients who relapse within 1 year of attaining remission with initial chemotherapy. There is more controversy regarding management of patients

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with longer remissions, although transplantation regardless of length of initial remission is usually advocated [4,32,39,43]. Second or subsequent relapse Prolonged disease-free survival is unusual following treatment with third-line chemotherapy regimens for HD. No significant differences in time to treatment failure or overall survival were identified when autologous HCT and conventional salvage chemotherapy were compared in patients with multiple relapses in the randomized GHSG trial [32]. However, only 24 patients were included in the comparison, and most investigators would recommend transplantation for HD patients following a second relapse from chemotherapy. First remission Some trials have demonstrated that NHL patients with adverse prognostic characteristics benefit from strategies that employ autologous HCT in first remission or as part of primary therapy. There is less experience with early autologous HCT for HD, although a number of variables have been identified that might be used to identify potential candidates [5]. Italian investigators reported results of high-dose chemotherapy followed by autologous HCT in 22 HD patients in first remission [66]. All patients had adverse characteristics at diagnosis and had been treated with MOPP/ABVD (mechlorethamine, vincristine, procarbazine, and prednisone/ABVD). Their outcome was compared with concurrently treated patients with similar characteristics who also achieved complete remission but did not desire transplantation. Eighteen patients were alive at the time of publication, and only one had relapsed. The actuarial survival of transplanted patients was 80% with a median follow-up of 83 months. The actuarial progression-free survival was estimated to be 77%. Only 8 of 24 patients who were not consolidated with autologous HCT were alive and free of disease. A prospective European intergroup trial also evaluated the role of early autologous HCT for HD [67]. Patients with adverse prognostic characteristics were treated with ABVD or similar regimens. Those in complete or partial remission after four cycles of therapy were randomized to treatment with four additional cycles of therapy or to treatment with high-dose therapy followed by autologous HCT. The 5-year failurefree survival was estimated to be 75% for patients randomized to transplantation, compared with 82% for patients who continued on chemotherapy (p = 0.4). The 5-year overall survival rates were estimated at 88% in each arm (p = 0.99). Results of other phase II trials and retrospective analyses examining results of autologous HCT for patients with HD in first remission are displayed in Table 59.6. Results suggest that progression-free survival may be prolonged by early transplantation

Table 59.6 Results of autologous hematopoietic cell transplantation for Hodgkin’s disease in first remission Reference

Number

Outcome

[9] [59] [66] [62] [64] [63]

57 45 22 20 16 13

70% 5-year TTF 70% 5-year EFS 77% PFS (median follow-up 86 months) 100% CCR (13.2–149.2 months)* 87% 5-year DFS 77% 5-year DFS

CCR, continuous complete remission; DFS, disease-free survival; EFS, event-free survival; PFS, progression-free survival; TTF, time to treatment failure. * Some patients in first partial remission.

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for poor-prognosis HD. However, there is little evidence that overall survival is prolonged, since transplantation or other salvage therapies can be used at relapse. In addition, most series only report outcomes for the selected poor-prognosis patients who are able to be transplanted. It is likely that many patients with adverse prognostic features do not attain remission or may not be transplanted for other reasons, and this “denominator” is rarely reported [63]. Until prospective trials demonstrate significant advantages for patients undergoing autologous HCT for HD in first remission, this approach should not be considered standard therapy.

results are comparable to those in adults. Although results of conventional salvage chemotherapy are better in this population, the use of autologous HCT appears to be justified, particularly in patients with refractory disease and those with multiple relapses. Long-term growth and development are normal, although delayed onset of ovarian function has been observed [69,72]. Of greater concern are reports of respiratory complications [70], and myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) following autologous HCT for HD in children [69,72]. Transplantation for elderly patients

Autologous HCT for children The prognosis for children and adolescents with HD is better than for adults, and most are cured with primary therapy. Nevertheless, approximately 10–15% of these patients are not cured, and autologous HCT has been extended to the pediatric population. A case-matching study from the EBMT compared the results of autologous BMT in 81 HD patients who were under the age of 16 at diagnosis with results from 81 adults aged 16 or more years [68]. The case-matching was performed following selection of the main prognostic factors for progression-free survival by multivariate analysis. Sex and status at transplant were found to be the two statistically significant prognostic factors for both pediatric and adult patients; therefore, these two factors were used for matching. The progression-free survival (median follow-up 36 months) for pediatric patients was 39%, compared with 48% for adults (median follow-up 34 months) (p = 0.64). The patterns of relapse and the incidence of transplant-related toxicities were also similar. A retrospective study from the University of Nebraska analyzed the results of autologous HCT in 53 children and adolescents (age ≤ 21) with relapsed and refractory HD [69]. The 5-year failure-free survival and overall survival were estimated to be 43% and 31%, respectively. No significant differences in outcome were observed when younger pediatric patients were compared with adolescents, and no significant differences in outcome were observed when these patients were compared with a historical control group of patients over the age of 21 who were transplanted during the same time period. A similar analysis from Stanford University examined the results of autologous HCT in 41 pediatric patients with relapsed and refractory HD [70]. The median age was 18 years. The actuarial 5-year progression-free survival and overall survival were 63% and 68%, respectively. Results of salvage therapy for pediatric patients with relapsed and refractory HD following treatment on German–Austrian trials were analyzed [71]. The median age at the time of relapse or progression was 14.7 years. The actuarial 10-year overall survival was 51% for patients treated with autologous HCT following first recurrence. These results were similar to children who received conventional salvage chemotherapy. However, the 6-year overall survival of children who were transplanted after a second recurrence was estimated to be 52%, compared with 29% for those receiving conventional treatment (p = 0.04). A retrospective study from the United Kingdom Children’s Cancer Study Group also examined results of autologous HCT for childhood HD [72]. The median age of transplanted patients was 12 years. When results of patients treated with autologous HCT were compared with controls who received conventional salvage chemotherapy after first relapse, there were no significant differences in survival. However, patients with primary refractory disease had significantly longer survival following autologous HCT. Prolonged progression-free survival can be seen after autologous HCT for children and adolescents with relapsed and refractory HD, and

Data from the CIBMTR indicate that 26% of patients treated with autologous HCT between 2001 and 2004 were older than 60 years [7]. Age was not a significant prognostic factor in the majority of trials evaluating results of autologous HCT for HD, although relatively few patients over the age of 60 were included, and they may be highly selected. However, in one report, patients who were 45 years of age or more had significantly worse event-free survival and overall survival [40]. Most reports of autologous HCT in the elderly have included patients with a variety of hematologic malignancies and have not reported separate results for HD patients. Results from Stanford showed that regimenrelated toxicity and event-free survival were worse in patients over the age of 50 who underwent autologous HCT [73]. Similar results have been reported for older patients with NHL [74]. Patients over the age of 60 can have prolonged failure-free survival following autologous HCT for relapsed and refractory HD. Although toxicity may be greater, patients should not be excluded from treatment based upon age alone. It may be appropriate to perform more extensive pretransplant tests of cardiac, pulmonary, and renal function in these patients [74]. Transplantation for HIV-associated HD Although HD is not an acquired immune deficiency syndrome (AIDS)defining illness, the incidence of HD is increased 8- to 10 fold in patients infected with HIV. Highly active antiretroviral therapy (HAART) has changed the natural history of HIV infection, and it is now possible for patients with HIV-related HD to receive the same primary chemotherapy treatment that HIV-negative individuals receive. Investigators from Paris reported the results of autologous HCT in four patients with relapsed HIV-associated HD [75]. All patients received HAART before transplantation and were continued on HAART during hematopoietic cell collection, and during and after transplantation. Three patients were alive in complete remission at 4, 13, and 15 months after transplant. An Italian group described results of autologous HCT in four patients with relapsed and refractory HIV-associated HD [76]. All patients received HAART during treatment. Their outcomes were not reported separately from a cohort of patients with NHL, but it is important to note that four additional patients with HIV-associated HD could not be transplanted because of difficulties with hematopoietic cell mobilization. Two patients with relapsed HIV-associated HD were treated with autologous HCT at the City of Hope [77]. Patients received HAART and had to be free of opportunistic infections for 1 year. The patients were reported to be alive and in remission at 56 and 61 months following HCT. These results demonstrate that peripheral blood hematopoietic cells can be collected in HIV-infected individuals on HAART, and that autologous HCT can be performed in selected individuals. Potential candidates for autologous HCT should be entered on clinical trials or referred to centers with experience in this population of patients.

Hematopoietic Cell Transplantation for Hodgkin’s Disease

Late events Increasing numbers of people are long-term survivors after autologous HCT for HD and other hematologic malignancies. There are several reports on quality of life after HCT for hematologic malignancies, although no reports have dealt exclusively with outcomes following autologous HCT for HD alone. Long-term follow-up of HD patients treated with autologous HCT in British Columbia demonstrated that 89% had a performance level of at least 90% [27]. Nevertheless, some patients remained on disability, and others described significant levels of anxiety and depression. A survey conducted by the International Bone Marrow Transplant Registry (IBMTR) and the ABMTR showed that transplant survivors were more likely to report poorer health than age- and sex-matched healthy controls, and reported more problems with fatigue, sleep, and sexual function [78]. Survivors were also more likely to report problems with anxiety, depression, and social functioning. Patients with HD who had been treated with autologous HCT formed a relatively small percentage of patients in this cohort, however. A similar survey of long-term function following autologous HCT was conducted in Germany [79]. Patients with HD represented only a small percentage of patients in this survey, also. Most patients (67%) rated their global quality of life as good to excellent, and scores improved over time. The majority of patients had returned to their former employment, although difficulty with holding a job has been reported in other studies [80]. Sexual dysfunction was commonly observed. Patients who undergo autologous HCT for HD are at risk for late relapse. Although some series have not reported late relapses, others have demonstrated that patients continue to be at risk of relapse for long periods of time. Relapse more than 7 years following autologous HCT has been reported [9,27]. These results indicate that lifelong follow-up is necessary after autologous HCT for HD. The Bone Marrow Transplant Survivor Study (BMTSS) examined long-term outcomes in patients who had survived at least 2 years after autologous HCT [80]. Patients transplanted for HD had a 28.2-fold risk of late death in comparison with the general population. This risk included relapse and nonrelapse causes of death. A nationwide study of patients undergoing autologous HCT in Finland noted that patients who had received autologous HCT for HD had an 8.1% incidence of nonrelapse mortality [81]. A large number of reports have described the occurrence of secondary MDS and AML following autologous HCT for HD as well as NHL [82]. The 5-year risk of developing AML or MDS following autologous HCT for HD has ranged between 4% and 15%. This complication has been observed within months of the transplant. Risk factors that have been reported to be associated with the development of post-transplant MDS/ AML include age, extent of prior therapy with alkylating agents, use of TBI conditioning or prior use of radiotherapy, history of splenectomy, and use of autologous PBHCT compared with autologous BMT. There is evidence that the risk of secondary MDS/AML following autologous HCT for HD is largely related to treatment administered prior to transplant rather than the high-dose conditioning regimen, itself. An analysis from Vancouver did not find an increased risk of secondary AML or other malignancies when HD patients who had received autologous HCT were compared with others who received only conventional chemotherapy [83]. Investigators have also used fluorescence in situ hybridization analysis to demonstrate that the cytogenetic abnormalities present in post-transplant MDS/AML can be detected in stored pretransplant blood or marrow samples from patients with HD [84]. This observation has led to the recommendation that cytogenetic analysis should be performed on bone marrow from all patients prior to

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hematopoietic stem cell collection. In addition, most studies of posttransplant MDS/AML have identified abnormalities of chromosomes 5 and 7 that are associated with typical alkylating agent-based therapy administered prior to autologous HCT. The short latency period between autologous HCT and the development of MDS/AML in many cases also suggest that malignancy results from prior therapy and not the high-dose chemotherapy regimen. The occurrence of solid tumors has also been described following autologous HCT for HD. The 15-year cumulative risk of developing a solid tumor was estimated to be 3.2% following autologous HCT for HD in the analysis from British Columbia [83]. Other late complications of autologous HCT for HD include infections [9,13,20,43], cardiac complications [13,15], and pulmonary complications [9,15,20]. The BMTSS found that the risk of late mortality from cardiac dysfunction was increased 4.3-fold and the risk from pulmonary compromise was increased 29.1-fold in survivors of autologous HCT for HD [80]. Virtually all women will develop amenorrhea following autologous HCT for HD, although return of ovarian function is not unusual. Successful pregnancies, both assisted and unassisted, have been described following autologous HCT for HD [13,14], and patients should be counseled about the need for contraception. Management of relapse A significant proportion of patients will relapse following autologous HCT for HD. The median survival following disease progression after autologous HCT has been reported to be less than 12 months in some series, although recent reports show a better prognosis [85,86]. Treatment of HD patients who relapse after autologous HCT must be individualized. Options are frequently limited because of prior therapy, low blood counts, and poor performance status. Radiation therapy should be considered for patients with localized relapse. High response rates and occasional long-term remissions have been noted with single-agent vinblastine [13,87], and patients will often respond to other conventional salvage regimens for HD [88]. Surgical resection is occasionally useful and some cases can be managed with observation. Some patients may be candidates for a second transplant. The EBMT reported that 50% of patients were alive and in remission 2 years after a second autologous HCT for HD [89]. Others have reported similar results [88]. A report from the IBMTR analyzed results of allogeneic HCT in 114 patients, including 35 with HD, who relapsed after autologous HCT for lymphoma [90]. All patients received high-dose conditioning, and some received unrelated or mismatched transplants. The 100-day mortality rate was 21%, and the 5-year cumulative incidence of disease progression was 70%. The 5-year actuarial survival was 24%, and no evidence of a survival plateau was noted. The 5-year actuarial progression-free survival was 5%, although outcomes for HD were not reported separately. RIC followed by allogeneic HCT has also been used to decrease the transplant-related mortality of allogeneic HCT for HD patients with a prior autologous HCT (see below). At M.D. Anderson Cancer Center, the 18-month overall survival and progression-free survival were estimated to be 61% and 32%, respectively, in a cohort of 30 HD patients (75% with a prior autologous HCT) who received reduced-intensity allogeneic HCT [91]. The 3-year probability of progression-free survival was estimated to be 8% for patients with HD in a series of patients from several institutions who were treated with a reduced-intensity allogeneic HCT after relapsing from a prior transplant [92]. A prospective trial from Spain examined the use of reduced-intensity allogeneic HCT in 29 HD patients who had progressed after autologous HCT [93]. The actuarial 2-year overall survival and event-free survival were 52% and 23%, respectively. These values were 75% and 50% for

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patients whose remission duration following autologous HCT was at least 12 months. Source of hematopoietic cells The vast majority of autologous HCT procedures in North America and Europe are now performed with mobilized peripheral blood hematopoietic cells, and autologous BMT is rarely performed [7,8]. Hematopoietic recovery is more rapid following autologous PBHCT, and this approach allows cells to be collected without general anesthesia. Most important, this approach allows the use of high-dose therapy in patients whose marrow cannot be harvested due to tumor contamination or prior radiation. Results from the series of autologous HCT for HD displayed in Table 59.1 have not shown differences in survival when results of autologous PBHCT and autologous BMT were compared. A Spanish registry analysis showed that the 5-year time to treatment failure was estimated to be 41% for HD patients treated with autologous BMT, compared with 55.3% for patients who were treated with autologous PBHCT (p = 0.007) [9]. These differences were not significant in the multivariate analysis, however. In contrast, the analysis from Vancouver showed that first-relapse HD patients treated with autologous PBHCT had inferior progression-free survival compared with recipients of autologous BMT (p = 0.04) [27]. Again, these differences were not significant in the multivariate analysis. An EBMT matched-pair analysis compared outcomes of autologous PBHCT and autologous BMT for HD patients [94]. The actuarial 4-year progression-free survival was 52% following BMT, compared with 38% following PBHCT (p = 0.0082). The actuarial overall survival rates were 65% and 53%, respectively (p = 0.0198). A single-center matched-pair analysis from University College Hospital in London, United Kingdom, also compared outcomes of autologous BMT and PBHCT for HD [95]. The 3-year progression-free survival was estimated to be 59% following autologous BMT, and 58% for those who received autologous PBHCT (p = 0.255). The 3-year overall survival rates were 69% and 78%, respectively (p = 0.078). Prospective trials in cohorts containing patients with both HD and NHL have also compared outcomes in relation to hematopoietic cell source [96–98]. No significant differences in survival were noted, although improvements in secondary outcomes such as engraftment rate, duration of hospital stay, costs, and quality of life have been described in patients treated with autologous PBHCT. Therefore, the use of autologous PBHCT is now preferred due to secondary advantages associated with ease of collection and engraftment rate. Allogeneic HCT Between 1998 and 2004, approximately 5% of HD transplants registered with the CIBMTR were performed using human leukocyte antigen (HLA)-identical sibling donors. In 2005, approximately 6% of HD transplants in Europe used HLA-identical sibling donors [8]. Smaller numbers of transplants were performed using unrelated donors. Allogeneic HCT may be considered for patients with inadequate hematopoietic cell collections, or for patients with dysplastic bone marrow or cytogenetic bone marrow abnormalities. It has also been suggested that allogeneic HCT might correct the lifelong immunologic defects seen in HD patients. Allogeneic HCT may also result in a graft-versus-Hodgkin’s effect similar to the graft-versus-leukemia effect observed after allogeneic HCT for leukemia. Although it is unknown whether infused Reed– Sternberg cells are clonogenic, the use of allogeneic donors also eliminates the risk of reinfusing malignant cells. The role of allogeneic HCT for HD is controversial. Results of highdose allogeneic HCT are poor, although most series have been composed

of patients who were extensively pretreated and those with advanced chemoresistant disease [99–101]. The actuarial 3-year disease-free survival was 15% in 100 patients with advanced HD reported to the IBMTR who underwent allogeneic BMT from matched siblings [100]. The 3year risk of treatment-related mortality was 61%. Factors associated with treatment failure include older age, poor performance status, and recent infection. The 4-year actuarial progression-free survival and nonrelapse mortality were 15% and 48% respectively, following allogeneic HCT in a series of 45 patients reported to the EBMT [101]. The results of allogeneic and autologous HCT for HD have been compared retrospectively. A case-matching analysis from the EBMT identified no significant differences in progression-free survival, overall survival, relapse or transplant-related mortality when autologous and allogeneic HCT for HD were compared [101]. At Johns Hopkins Oncology Center, patients with relapsed and refractory HD were preferentially assigned to receive allogeneic HCT if they had a matched sibling donor and were under the age of 56 years [23]. Patients were otherwise treated with autologous HCT. A retrospective comparison revealed no significant differences in relapse, event-free survival or overall survival between groups. Investigators from Seattle compared the outcome of allogeneic HCT and autologous HCT for HD [99]. The relapse rate following allogeneic HCT was lower (45% versus 76%; p = 0.05), although overall survival differences were not observed because of higher transplant-related mortality in allogeneic HCT. A more recent EBMT analysis evaluated the outcome of 167 patients with HD who were treated with allogeneic HCT [102]. The donor was an HLA-identical sibling in 81.5% of cases. The 4-year progression-free survival and overall survival were estimated to be 15.5% and 24.7%, respectively. The procedure-related mortality was estimated to be 51.7% at 4 years. A matched-pair analysis demonstrated that autologous HCT was associated with superior progression-free survival, compared with allogeneic HCT. Graft-versus-Hodgkin’s effect Although no definite evidence of a graft-versus-Hodgkin’s effect has been demonstrated, clinical studies provide some evidence to support this concept [103,104]. Some comparisons have demonstrated that the rate of relapse is lower after allogeneic HCT for HD when compared with autologous HCT [23,99,101]. There is also evidence that relapse rates may be lower in patients who develop graft-versus-host disease (GVHD) [101]. The existence of a graft-versus-Hodgkin’s effect is also supported by reports of response to donor leukocyte infusions following allogeneic HCT [103–105]. The high mortality rate following allogeneic HCT for HD and the frequent lack of donor availability limits the applicability of this approach. It is unknown whether this toxicity is disease related or whether the high mortality results from adverse characteristics of patients selected for this form of treatment. Results of allogeneic HCT for HD may be better if performed earlier in the course of disease, although it is not known which patients are candidates for this procedure. RIC followed by allogeneic transplantation Several groups of investigators have explored the use of RIC allogeneic HCT for HD to exploit potential graft-versus-Hodgkin’s effects and to reduce transplant-related mortality [91,93,105–109]. These studies have shown the feasibility of this approach in patients who have relapsed after autologous HCT, and in patients who are not candidates for allogeneic HCT using conventional myeloablative regimens. The largest series of RIC and allogeneic HCT for HD was first reported by the EBMT [108]. Fifty-two patients were conditioned with a variety of regimens, although the majority received fludarabine-based treatments. The 2-year progression-free survival and overall survival

Hematopoietic Cell Transplantation for Hodgkin’s Disease

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Fig. 59.5 Overall survival of patients with chemosensitive or chemoresistant Hodgkin’s disease following nonmyeloablative conditioning. The y-axis denotes percentage survival. (Reproduced from [106], with permission.)

were estimated to be 42% and 56.3%, respectively. The 1-year transplant-related mortality was 17.3%. Patients with HD had better outcomes than those with high-grade NHL or mantle cell lymphoma. Updated results in 94 patients have been published [106]. Approximately 50% had progressed after prior treatment with autologous HCT, and 31% had primary refractory disease or were resistant to conventional salvage chemotherapy. The 2-year actuarial progression-free survival and overall survival were approximately (estimated from curves) 38% and 47%, respectively. The only factor that was significantly associated with disease-free survival and overall survival was chemosensitivity prior to transplant (Fig. 59.5). Encouraging results of RIC and allogeneic HCT for HD have been reported elsewhere. At M.D. Anderson Cancer Center, 40 patients with relapsed and refractory HD were treated with RIC allogeneic HCT (matched sibling n = 20, matched unrelated donor n = 20) [91]. The conditioning regimens consisted of fludarabine and cyclophosphamide (±antithymocyte globulin), and fludarabine plus melphalan. The actuarial progression-free survival and overall survival at 18 months were 32% and 61%, respectively. The cumulative incidence of progressive disease was 55%. The overall survival at 18 months was estimated to be 73% for patients who received fludarabine–melphalan compared with 39% for patients treated with fludarabine–cyclophosphamide (p = 0.03). Investigators from the United Kingdom reported results of RIC allogeneic HCT (matched sibling n = 31, matched unrelated donor n = 18) in 49 patients with multiply relapsed HD [105]. The majority (90%) had progressed after a previous autologous HCT. The conditioning regimen consisted of fludarabine, melphalan, and alemtuzumab. The 4-year progression-free survival and overall survival were estimated to be 39% and 55.7%, respectively. Patients in complete remission at the time of transplant had significantly better progression-free survival (p = 0.0389) and overall survival (p = 0.0398), compared with patients in partial remission or with refractory disease. Progression-free survival was also poorer in patients with a history of autologous HCT (p = 0.0267). Baron et al. (see above) reported results of RIC allogeneic HCT in 35 HD patients who had a failed previous transplant [92]. The conditioning regimen consisted of fludarabine and 2 Gy TBI in the majority of patients. The actuarial 3-year progression-free and overall survival were 8% and 35%, respectively.

Fig. 59.6 Influence of chronic graft-versus-host disease (cGVHD) on the outcome after allogeneic hematopoietic cell transplantation (allo-SCT). Cumulative incidence of (a) relapse or progression, (b) nonrelapse mortality, and (c) Kaplan–Meier estimation of progression-free survival. RR, relative risk. (Reproduced from Sureda et al. [109], with permission.)

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Investigators from Spain have also reported on the results of RIC allogeneic HCT (matched sibling n = 37) in 40 patients with relapsed or refractory HD [93]. The majority (n = 29) had been treated previously with autologous HCT. Conditioning consisted of fludarabine and melphalan. The 2-year progression-free survival and overall survival were estimated to be 32% and 48%, respectively. Resistant disease at the time of transplant was associated with significantly worse progression-free survival and overall survival. Similar outcomes, including the adverse influence of chemotherapy resistance, have been reported by other groups using RIC allogeneic HCT for HD [107]. The comparative analysis of allogeneic HCT after RIC or conventional high-dose conditioning in patients with relapsed or refractory HD was recently reported by the EBMT [109]. In this study, the clinical outcomes of 89 patients who underwent RIC allogeneic HCT were compared with the outcomes of 79 patients who underwent conventional high-dose allogeneic HCT. The two groups were comparable except that more patients in the high-dose group were transplanted in earlier years and had received bone marrow. RIC patients were more heavily pretreated, and more patients had relapsed after a prior autologous HCT (61.8% versus 40.5%; p = 0.005). There was no difference in hematopoetic recovery or the incidence of GVHD between the two groups. However, the nonrelapse mortality was significantly decreased in the RIC group (hazard ratio 2.85; p <0.001). The 5-year overall survival was also better, and there was a trend for better 5-year progression-free survival in the RIC group. Patients who developed chronic GVHD had a significantly decreased risk of relapse, which translated into a trend for a better progression-free survival (Fig. 59.6). These results support the use of RIC followed by allogeneic HCT in patients with relapsed or refractory HD. Moreover, this study confirms the existence of a graftversus-Hodgkin’s effect associated with the development of chronic GVHD. Results of allogeneic HCT for HD are displayed in Table 59.7. The results of allogeneic HCT for HD using RIC appear to be better than those reported with full-intensity myeloablative conditioning, especially in patients who have relapsed after a prior autologous HCT. This approach should be considered for patients who relapse after autologous HCT, although mortality rates are high, and risks from relapse and GVHD are considerable. Allogeneic HCT with RIC may not benefit

patients with resistant disease and alternative treatment strategies should be explored in these patients. Additional studies are required to compare results with other approaches, determine the appropriate timing of transplantation, develop optimal RIC regimens, and design strategies to decrease toxicity and the risk of GVHD. Other investigators have studied the strategy of autologous HCT followed by RIC allogeneic HCT for HD (see above). This approach is designed to exploit a graft-versus-Hodgkin’s effect in patients who have achieved a minimal disease state following a high-dose therapy regimen. Carella et al. have demonstrated the feasibility of this approach [53]. Ten patients with relapsed and refractory HD received high-dose therapy followed by autologous HCT. At a median of 84 days after autologous HCT, patients underwent allogeneic HCT after conditioning with fludarabine and cyclophosphamide. Five patients were alive between 210 and 430 days after the second transplant. Additional studies are required to assess the curative potential of this approach.

Umbilical cord transplantation The majority of candidates of allogeneic HCT will not have an HLAmatched sibling donor. Matched unrelated donors may be used, although many patients will not have suitable donors, and this option may be associated with a higher risk of GVHD and transplant-related mortality. The use of unrelated umbilical cord blood (UCB) has the advantages of rapid availability and lower risk of GVHD despite donor–recipient HLA disparity. Investigators from Minnesota compared the results of RIC allogeneic HCT with UCB in 9 patients, with the results of transplantation in 12 patients with HLA-matched siblings [110]. The cumulative incidences of acute and chronic GVHD, as well as treatment-related mortality, were similar in both groups. The 2-year progression-free survival was estimated to be 25% following UCB allogeneic HCT, compared with 20% for patients with matched sibling donors (p = 0.67). The actuarial 2-year progression-free survival was 0% for patients with primary refractory disease and those who relapsed within 1 year of autologous HCT. These results demonstrate that allogeneic HCT using UCB may be an alternative for patients without matched sibling donors or fully matched unrelated donors.

Table 59.7 Results of allogeneic hematopoietic cell transplantation for Hodgkin’s disease Reference

Number

Regimen

Mortality

Outcome

[100] [23] [101] [99] [102] [106] [91] [105] [93] [92] [91] [109] [107] [109]

100 53 45 53 167 94 40 49 40 35 58 21 14 168

Myeloablative Myeloablative Myeloablative Myeloablative Myeloablative RIC (55% prior aHCT, 10 URD) RIC (75% prior aHCT, 20 URD) RIC (90% prior aHCT, 18 URD) RIC (73% prior aHCT, 2 URD) RIC RIC (83% prior aHCT, 33 URD) Myeloablative, RIC (66% prior aHCT, 9 UCB) RIC (50% prior aHCT) RIC = 89, 61.8% prior aHCT Myeloablative = 79, 41% prior auto

61% 3-year TRM 43% 65% 4-year TRM 53% 5-year TRM 51.7% 4-year TRM 20% 22% 18 months TRM 16% 730 days NRM 25% 1-year TRM 32% 3-year NRM 15% 2-year TRM 11% UCB, 17% MSD 100 days TRM None 23% at 1 year 46% at 1 year

15% 3-year DFS 27% 10-year EFS 15% 4-year PFS 22% 5-year EFS 15.5% 4-year PFS 39% 2-year PFS 32% 18 months PFS 39% 4-year PFS 32% 2-year PFS 8% 3-year PFS 32% 2-year PFS 25% 2-year PFS UCB, 20% 2-year PFS MSD 36% 1-year PFS 28% 5-year OS 22% 5-year OS

aHCT, autologous hematopoietic cell transplant; DFS, disease-free survival; EFS, event-free survival; MSD, matched sibling donor; NRM, nonrelapse mortality; OS, overall survival; PFS, progression-free survival; RIC, reduced intensity conditioning regimen; TRM, transplant-related mortality; UCB, umbilical cord blood; URD, unrelated donor.

Hematopoietic Cell Transplantation for Hodgkin’s Disease Table 59.8 Commonly used high-dose therapy regimens for Hodgkin’s disease Regimen

Total dose administered

CBV

Cyclophosphamide, 4.5–7.2 g/m2 or 100 mg/kg Carmustine, 300–600 mg/m2 or 15 mg/kg Etoposide, 750–2400 mg/m2 or 60 mg/kg

BEAC

Carmustine, 200–400 mg/m2 Etoposide, 600–1200 mg/m2 Cytarabine, 800–1200 mg/m2 Cyclophosphamide, 140–180 mg/kg or 6–7.5 g/m2

BEAM

Carmustine, 300–400 mg/m2 Etoposide, 400–1600 mg/m2 Cytarabine, 400–1600 mg/m2 Melphalan, 140 mg/m2

CY–TBI

Cyclophosphamide, 120–200 mg/kg Total body irradiation, 800–1320 cGy

VP-16, CY, TBI

Etoposide, 30–60 mg/kg Cyclophosphamide, 60–120 mg/kg Total body irradiation, 1200–1375 cGy

BU, CY

Busulfan 16 mg/kg Cyclophosphamide 120–200 mg/kg

High-dose therapy regimens A wide variety of high-dose therapy regimens has been used with autologous HCT for HD (Table 59.8). No prospective trials have been performed, although regimens have been compared retrospectively. High-dose therapy regimens are frequently divided into those that use TBI and those that contain only drugs. Use of radiation with prior treatment often eliminates the possibility of using TBI. Investigators from Seattle examined the influence of the preparative regimen in a cohort of HD patients who were treated with both autologous and allogeneic HCT [99]. No significant differences in event-free survival were observed when TBI-containing and drug-only regimens were compared, although 5-year overall survival rates were 28% and 14%, respectively (p = 0.081). The actuarial 5-year survival was 25% for patients who received cyclophosphamide and TBI, compared with 11% for patients who received busulfan and cyclophosphamide (p = 0.061). Patients treated with cyclophosphamide and TBI had similar outcomes compared with patients treated with cyclophosphamide, carmustine, and etoposide. A retrospective analysis of HD patients undergoing autologous or allogeneic HCT at the Johns Hopkins also failed to note differences in outcome when TBI-containing regimens were compared with regimens using chemotherapy alone [23]. Patients at City of Hope Hospital received a regimen of TBI, etoposide, and cyclophosphamide, or else carmustine, etoposide, and cyclophosphamide if they had received prior radiation [15]. No significant differences in response rate, disease-free survival, overall survival or toxicity were observed. Similar results were reported by Stanford University [16], and from an ABMTR analysis [20]. An EBMT analysis also failed to show significant differences in progression-free survival or overall survival associated with the use of TBI [54]. At Memorial Sloan-Kettering Cancer Center, there were no significant differences in event-free survival and overall survival when HD patients were treated with a regimen using accelerated-fraction total lymphoid

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irradiation combined with cyclophosphamide and etoposide, compared with a high-dose chemotherapy regimen [22]. The use of TBI may be associated with increased pulmonary toxicity, particularly in patients who have received prior mediastinal irradiation. Spanish registry results showed that the use of TBI-containing regimens prior to autologous HCT for HD was associated with a significantly higher risk of early transplant-related mortality (relative risk 2.3; p = 0.02) [9]. A multivariate analysis of another Spanish registry study of autologous HCT for HD identified the use of TBI-containing regimens to be associated with a 6.09-fold risk of nonrelapse mortality (p = 0.0001) [65]. The actuarial 5-year overall survival was 33% for patients transplanted with TBI-containing regimens, compared with 60% for those transplanted with chemotherapy regimens (p = 0.01), although this difference was not significant in the multivariate analysis. One of the most common high-dose chemotherapy regimens used prior to autologous HCT for HD is the CBV (cyclophosphamide, carmustine [BCNU], and etoposide) regimen developed at the M.D. Anderson Cancer Center. The original CBV regimen has been modified and individual institutions have used widely differing schedules (Table 59.8). No prospective trials have examined whether variations in the CBV regimen lead to different outcomes, although higher doses of carmustine have been associated with an increased risk of pulmonary toxicity, particularly in patients who have received prior chest irradiation [99,111]. The addition of cisplatin to the CBV regimen did not result in significant differences in outcome, although this modification allowed the dose of carmustine to be reduced [27,58]. Another high-dose chemotherapy regimen for HD autologous HCT is the BEAM regimen, which was developed by investigators from London and has also been widely modified (Table 59.8) [13]. Retrospective analyses have failed to show significant differences in survival when CBV-type and BEAM-type regimens were compared [18,28]. In one retrospective comparison, CBV was associated with significantly higher rates of transplant-related morbidity and mortality, although an unusually high dose of carmustine (800 mg/m2) was used [112]. In one study, escalation of the total dose of etoposide in the BEAM regimen to 1600 mg/m2 did not significantly improve survival [113]. A variety of other high-dose therapy regimens has also been used prior to autologous HCT for HD. In Seattle, a retrospective analysis compared results of a regimen containing busulfan, melphalan, and thiotepa with results from the combination of etoposide, cyclophosphamide, and TBI for patients undergoing autologous HCT for HD [25]. No significant differences in toxicity, event-free survival or overall survival were observed. Other high-dose therapy regimens used prior to autologous HCT for HD include busulfan, etoposide, and cyclophosphamide [30], carmustine, etoposide, and cisplatin [114], thiotepa, mitoxantrone, and carboplatin [115], and high-dose sequential chemotherapy regimens [26]. It is unclear from existing data that any high-dose therapy regimen is superior for patients with HD. Prospective trials will be required to prove the superiority of any regimen, and it is difficult to recommend any single regimen, although TBI and higher doses of carmustine should be avoided in patients with pulmonary disease or a history of chest irradiation. Several institutions have tested the use of double (tandem) autologous HCT for HD (see above). This approach is feasible and good outcomes are possible, although a significant fraction of patients are not able to receive the second planned transplants [51,52].

Summary and future directions Over the last 20 years, the use of high-dose therapy followed by autologous HCT has become the treatment of choice for most patients with

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relapsed and refractory HD. The outcomes are better in chemotherapysensitive patients and those with good prognostic factors at relapse. New approaches are needed for patients with primary refractory HD since the chance for long-term remission remains poor even with autologous HCT. Tandem autologous HCT or autologous HCT followed by reducedintensity allogeneic HCT is being explored in these patients. Long-term survivors of autologous HCT for HD are at higher risk of nonrelapse mortality due to prior exposure to multiple chemotherapies and radiotherapy. New strategies to reduce long-term complications are needed. Incorporation of early interim FDG PET during and at completion of

therapy may help indentify patients who may not be curable with conventional therapy and thus benefit from early transplant. Despite the lower incidence of nonrelapse mortality, the role of reduced-intensity allogeneic HCT for relapsed and refractory HD requires further studies. At present, only patients with chemosensitivity benefit from allogeneic HCT, and the existence of graft-versusHodgkin’s effect has clearly been demonstrated. The optimal choices of RIC regimens and the best GVHD prophylaxis have not yet been defined; therefore, allogeneic HCT should be performed in prospective clinical trials.

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refractory pediatric Hodgkin’s disease: results and prognostic indices. J Clin Oncol 2004; 15: 4532– 40. Schellong G, Dörffel W, Claviez A et al. Salvage therapy of progressive and recurrent Hodgkin’s disease: results from a multicenter study of the Pediatric DAL/GPOH-HD study group. J Clin Oncol 2005; 25: 6181–9. Stoneham S, Ashley S, Pinkerton CR, Wallace WH, Shankar AG. Outcome after autologous hemopoietic stem cell transplantation in relapsed or refractory childhood Hodgkin disease. J Pediatr Hematol Oncol 2004; 26: 740–5. Kusnierz-Glaz CR, Schlegel PG, Wong RM et al. Influence of age on the outcome of 500 autologous bone marrow transplant procedures for hematologic malignancies. J Clin Oncol 1997; 15: 18–25. Jantunen E. Autologous stem cell transplantation beyond 60 years of age. Bone Marrow Transplant 2006; 38: 715–20. Gabarre J, Azar N, Autran B, Katlama C, Leblond V. High-dose therapy and autologous haematopoietic stem-cell transplantation for HIV-1-associated lymphoma. Lancet 2000; 355: 1071–2. Re A, Cattaneo R, Michieli M et al. High-dose therapy and autologous peripheral-blood stemcell transplantation as salvage treatment for HIVassociated lymphoma in patients receiving highly active antiretroviral therapy. J Clin Oncol 2003; 21: 4423–7. Krishnan A, Molina A, Zaia J et al. Durable remissions with autologous stem cell transplantation for high-risk HIV-associated lymphomas. Blood 2005; 105: 874–8. Andrykowski MA, Bishop MA, Hahn EA et al. Long-term health-related quality of life, growth, and spiritual well-being after hematopoietic stemcell transplantation. J Clin Oncol 2005; 23: 599– 608. Hensel M, Egerer G, Schneeweiss A, Goldschmidt H, Ho AD. Quality of life and rehabilitation in social and professional life after autologous stem cell transplantation. Ann Oncol 2002; 13: 209– 17. Bhatia S, Robison LL, Francisco L et al. Late mortality in survivors of autologous hematopoietic-cell transplantation: report from the Bone Marrow Transplant Survivor Study. Blood 2005; 105: 4215–22. Jantunen E, Itälä M, Siitonen T et al. Late nonrelapse mortality among adult autologous stem cell transplant recipients: a nation-wide analysis of 1482 patients transplanted in 1990–2003. Eur J Haematol 2006; 77: 114–19. Metayer C, Curtis RE, Vose J et al. Myelodysplastic syndrome and acute myeloid leukemia after autotransplantation for lymphoma: a multicenter case-control study. Blood 2003; 101: 2015– 23. Forrest DL, Hogge DE, Nevill TJ et al. High-dose therapy and autologous hematopoietic stem-cell transplantation does not increase the risk of second neoplasms for patients with Hodgkin’s lymphoma: a comparison of conventional therapy alone versus conventional therapy followed by autologous hematopoietic stem-cell transplantation. J Clin Oncol 2005; 23: 7994–8002. Abruzzese E, Radford JE, Miller JS et al. Detection of abnormal pretransplant clones in progenitor cells of patients who developed

85.

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myelodysplasia after autologous transplantation. Blood 1999; 94: 1814–19. Paltiel O, Rubinstein C, Or R et al. Factors associated with survival in patients with progressive disease following autologous transplant for lymphoma. Bone Marrow Transplant 2003; 31: 565–9. Kewalramini T, Nimer SD, Zelenetz AD et al. Progressive disease following autologous transplantation in patients with chemosensitive relapsed or primary refractory Hodgkin’s disease or aggressive non-Hodgkin lymphoma. Bone Marrow Transplant 2003; 32: 673–9. Little R, Wittes RE, Longo DL, Wilson WH. Vinblastine for recurrent Hodgkin’s disease following autologous bone marrow transplant. J Clin Oncol 1998; 16: 584–8. van Besien K, Smith S, Lazarus HM. Therapeutic options for patients with Hodgkin’s disease and non-Hodgkin lymphoma who relapse after autologous transplant. Curr Treat Options Oncol 2005; 6: 279–87. Vandenberghe E, Pearce R, Taghipour G, Fouillard L, Goldstone AH. Role of a second transplant in the management of poor-prognosis lymphomas: a report from the European Blood and Bone Marrow Registry. J Clin Oncol 1997; 15: 1595–600. Freytes CO, Loberiza FR, Rizzo JD et al. Myeloablative allogeneic hematopoietic stem cell transplantation in patients who experience relapse after autologous stem cell transplantation for lymphoma: a report from the International Bone Marrow Transplant Registry. Blood 2004; 104: 3797–803. Anderlini P, Saliba R, Acholonu S et al. Reducedintensity allogeneic stem cell transplantation in relapsed and refractory Hodgkin’s disease: low transplant-related mortality and impact of intensity of conditioning regimen. Bone Marrow Transplant 2005; 35: 943–51. Baron F, Storb R, Storer BE et al. Factors associated with outcomes in allogeneic hematopoietic cell transplantation with nonmyeloablative conditioning after failed myeloablative hematopoietic cell transplantation. J Clin Oncol 2006; 24: 4150–7. Alvarez I, Sureda A, Caballero MD et al. Nonmeloablative stem cell transplantation is an effective therapy for refractory or relapsed Hodgkin lymphoma: results of a Spanish prospective cooperative protocol. Biol Blood Marrow Transplant 2006; 12: 172–83. Majolino I, Pearce R, Taghipour G, Goldstone AH. Peripheral-blood stem-cell transplantation versus autologous bone marrow transplantation in Hodgkin’s and non-Hodgkin lymphomas: a new matched-pair analysis of the European Group for Blood and Marrow Transplantation Registry Data. J Clin Oncol 1997; 15: 509–17. Perry AR, Peniket AJ, Watts MJ, Leverett D, Goldstone AH, Linch DC. Peripheral blood stem cell versus autologous bone marrow transplantation for Hodgkin’s disease: equivalent survival outcome in a single-centre matched-pair analysis. Br J Haematol 1999; 105: 280–7. Weisdorf DJ, Verfaillie CM, Miller WJ et al. Autologous bone marrow versus non-mobilized peripheral blood stem cell transplantation for lymphoid malignancies: a prospective, comparative trial. Am J Hematol 1997; 54: 202–8.

97. Kanteti R, Miller KB, McCann JC et al. Randomized trial of peripheral blood progenitor cell vs bone marrow as hematopoietic support for highdose chemotherapy in patients with non-Hodgkin lymphoma and Hodgkin’s disease: a clinical and molecular analysis. Bone Marrow Transplant 1999; 24: 473–81. 98. Kottaridis PD, Peggs K, Schmitz N et al. Survival and freedom from progression in autotransplant lymphoma patients is independent of stem cell source: further follow-up from the original randomized study to assess engraftment. Leuk Lymphoma 2002; 43: 531–6. 99. Anderson JE, Litzow MR, Appelbaum FR et al. Allogeneic, syngeneic, and autologous marrow transplantation for Hodgkin’s disease: the 21-year Seattle experience. J Clin Oncol 1993; 11: 2342– 50. 100. Gajewski JL, Phillips GL, Sobocinski KA et al. Bone marrow transplants from HLA-identical siblings in advanced Hodgkin’s disease. J Clin Oncol 1996; 14: 572–8. 101. Milpied N, Fielding AK, Pearce RM, Ernst P, Goldstone AH. Allogeneic bone marrow transplant is not better than autologous transplant for patients with relapsed Hodgkin’s disease. J Clin Oncol 1996; 14: 1291–6. 102. Peniket AJ, Ruiz de Elvira MC, Taghipour G et al. An EBMT registry matched study of allogeneic stem cell transplants for lymphoma: allogeneic transplantation is associated with a lower relapse rate but a higher procedure-related mortality rate than autologous transplantation. Bone Marrow Transplant 2003; 31: 667– 78. 103. Porter DL, Stadtmauer EA, Lazarus HM. ‘GVHD’: graft-versus-host disease or graft-versus Hodgkin’s disease? An old acronym with new meaning. Bone Marrow Transplant 2003; 31: 739–46. 104. Grigg A, Ritchie D. Graft-versus-lymphoma effects: clinical review, policy proposals, and immunobiology. Biol Blood Marrow Transplant 2004; 10: 579–90. 105. Peggs KS, Hunter A, Chopra R et al. Clinical evidence of a graft-versus-Hodgkin’s lymphoma effect after reduced-intensity allogeneic transplantation. Lancet 2005; 365: 1934– 41. 106. Schmitz N, Sureda A, Robinson S. Allogeneic transplantation of hematopoietic stem cells after nonmyeloablative conditioning for Hodgkin’s disease. Semin Oncol 2004; 31: 27– 32. 107. Todisco E, Castagna L, Sarina B et al. Reducedintensity allogeneic transplantation in patients with refractory or progressive Hodgkin’s disease after high-dose chemotherapy and autologous stem cell infusion. Eur J Haematol 2007; 78: 322–9. 108. Robinson SP, Goldstone AH, Mackinnon S et al. Chemoresistant or aggressive lymphoma predicts for a poor outcome following reduced-intensity allogeneic progenitor cell transplantation; an analysis from the Lymphoma Working Party of the European Group for Blood and Bone Marrow Transplantation. Blood 2002; 100: 4310– 16. 109. Sureda A, Robinson S, Canals C et al. Reducedintensity conditioning compared with conventional allogeneic stem-cell transplantation in

Hematopoietic Cell Transplantation for Hodgkin’s Disease relapsed or refractory Hodgkin’s lymphoma: an analysis from the Lymphoma Working Party of the European Group for Blood and Marrow Transplantation. J Clin Oncol 2008; 26: 455–62. 110. Majhail NS, Weisdorf DJ, Wagner JE, Defor TE, Brunstein CG, Burns LJ. Comparable results of umbilical cord blood and HLA-matched sibling donor hematopoietic stem cell transplantation after reduced-intensity preparative regimen for advanced Hodgkin lymphoma. Blood 2006; 107: 3804–7. 111. Wheeler C, Antin JH, Churchill WH et al. Cyclophosphamide, carmustine, and etoposide with autologous bone marrow transplantation in refractory Hodgkin’s disease and non-Hodgkin

lymphoma: a dose finding study. J Clin Oncol 1990; 8: 648–56. 112. Puig N, de la Rubia J, Remigia MJ et al. Morbidity and transplant-related mortality of CBV and BEAM preparative regimens for patients with lymphoid malignancies undergoing autologous stem-cell transplantation. Leuk Lymphoma 2006; 47: 1488–94. 113. Martín A, Caballero MD, Simón-Pérez JA et al. Results of autologous transplantation in lymphoma are not improved by increasing the dose of etoposide in the BEAM regimen: a single-centre sequential-cohort study. Bone Marrow Transplant 2004; 34: 675–82.

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114. Wadhwa PD, Fu P, Koc ON et al. High-dose carmustine, etoposide, and cisplatin for autologous stem cell transplantation with or without involved-field radiation for relapsed/refractory lymphoma: an effective regimen with low morbidity and mortality. Biol Blood Marrow Transplant 2005; 11: 13–22. 115. Waheed F, Kancherla R, Seiter K, et al. High dose chemotherapy with thiotepa, mitoxantrone and carboplatin (TMJ) followed by autologous stem cell support in 100 consecutive lymphoma patients in a single centre: analysis of efficacy, toxicity and prognostic factors. Leuk Lymphoma 2004; 45: 2253–9.

60

Laura J. Johnston & Sandra J. Horning

Non-Hodgkin’s Lymphoma

Introduction Non-Hodgkin’s lymphoma (NHL) is a lymphoid malignancy projected to afflict more than 60,000 people in the United States in 2007 [1]. Although the 5-year survival rate of NHL has steadily increased from 48% in 1975 to 63% in 2003, it is estimated to have been responsible for over 18,000 deaths in 2007. Soon after its introduction into the medical field, hematopoietic cell transplantation (HCT) has been utilized as a mode of prolonging disease-free survival (DFS) and overall survival (OS) for NHL patients. In 2005, there were over 4000 HCTs for the diagnosis of NHL in North America, of which approximately 80% were autologous HCT [2]. This chapter will review the use of autologous as well as allogeneic HCT for NHL, concentrating on the pertinent clinical data available. The authors refer you to the alternate sections within the book for complete and related discussions of preparative regimens (see Chapter 22), radioimmunotherapy (see Chapter 24), minimal residual disease (see Chapter 26), hematopoietic cell sources (see Section 4), complications after HCT (see Section 7) and reduced intensity regimens (see chapter 71).

NHL classification The classification of lymphoma continues to evolve, with more than 30 subtypes described in the new World Health Organization Lymphoma Classification [3]. The subtypes are categorized based on B-cell or T-cell and natural killer cell lineages, and are further characterized by their histologic, immunologic, genetic, and clinical features. The major diagnoses and their frequency using the World Health Organization nomenclature are detailed in Table 60.1. The majority of lymphoid malignancies are of B-cell lineage with diffuse large B-cell lymphoma (DLCL) and follicular lymphoma (FL) comprising over half of all lymphomas. This chapter will focus on the common NHL subtypes but also include the less common entities in which HCT has been utilized.

Clinical features DLCL is the most common subtype of NHL. The median age of onset is 64 years; however the age range is broad, from children to the elderly. Patients present with an equal distribution of limited (stage I–II) or dis-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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seminated (stage III–IV) disease, based on the Ann Arbor staging system. Historically, a significant proportion of patients with limitedstage disease were cured with cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) chemotherapy and irradiation, whereas the majority of patients with advanced-stage disease relapsed [4]. The International Prognostic Index (IPI) introduced in 1993 has proven a robust approach to predicting outcome for patients with DLCL treated with doxorubicin-containing chemotherapy [5]. The age-adjusted IPI (aa-IPI) identified lactate dehydrogenase (LDH), performance status, and stage of disease as risk factors in patients under 60 years of age. The IPI for an individual, reflecting the number of adverse prognostic factors present at diagnosis, correlated with chemotherapy response, the durability of the response and OS. Patients identified at a high risk for treatment failure based on their IPI have been targeted to receive more intensive treatment, including high-dose therapy (HDT) and autologous HCT to consolidate remission. The recent incorporation of anti-CD20 chimeric monoclonal antibody rituximab to CHOP (cyclophosphamide, vincristine, and prednisone)like regimens in the elderly (>60 years) as well as the younger population has been shown to improve progression-free survival (PFS) and OS of DLCL by about 10–15% compared with CHOP-like therapy, based on well-controlled, randomized trials in Europe and North America [6–9]. These remarkable improvements have been validated in practice, as evidenced by the outcome of 292 newly diagnosed DLCL patients with either limited or advanced disease treated in the pre- and postrituximab era in British Columbia [10]. Regardless of age, the 2-year OS was improved for patients treated with rituximab and CHOP (RCHOP) compared with CHOP. Although the British Columbia investigators have proposed a revision to the IPI, data from the French, German, and United States trials, as well as biologic correlative studies, indicate that the IPI and its components remain prognostic [7–9,11]. Table 60.2 summarizes the outcomes based on the established IPI in nonrituximab induction therapies [5] and in R-CHOP as published by the British Columbia group [12]. Available results regarding the use of rituximab in HCT will be reviewed below. FL is the second most common NHL subtype, with an onset during the middle-age years [3]. The majority of patients present with disseminated disease that is highly responsive to a variety of initial therapies including chemotherapy, radiation therapy, and biologic therapy. Despite the characteristic pattern of multiple relapses, the median OS of FL has improved in the past decade according to several sources [13– 15]. The availability of multiple therapies, particularly rituximab, has likely contributed to this advance. Two meta-analyses of completed phase III clinical trials investigating rituximab as well as

Non-Hodgkin’s Lymphoma

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Table 60.1 Major categories of lymphoma WHO classification [3] B-lineage Large B-cell lymphoma Follicular lymphoma

Small lymphocytic lymphoma/chronic lymphocytic leukemia MALT (mucosa-associated lymphoid tissue) lymphoma Mantle cell lymphoma Marginal zone lymphoma Primary mediastinal large B-cell lymphoma Burkitt-like lymphoma Burkitt’s lymphoma T-lineage Peripheral T-cell lymphoma Anaplastic large cell lymphoma Lymphoblastic lymphoma Mycosis fungoides

Table 60.2 Outcome and prognostic factors

Risk category

Distribution of Patients (%)

n risk factors*

% Survival at 5 years

Disease-free Overall

International Prognostic Factors Index in 2031 patients, nonrituximabcontaining induction [5] Low 35 0, 1 70 73 Low intermediate 27 2 50 51 High intermediate 22 3 49 43 High 16 4, 5 40 26 Outcome based on International Prognostic Index (IPI) in 365 patients treated with rituximab–CHOP in British Columbia [12] Standard IPI Low 22 0, 1 85 82 Low intermediate 32 2 80 81 High intermediate 32 3 57 49 High 14 4, 5 51 59 * Risk factors include: elevated serum lactate dehydrogenase, nonambulatory performance status, Ann Arbor stage III–IV disease, >2 extranodal sites and age >60 years.

interferon-alpha (IFN-α) showed improved remission duration and OS in patients receiving either agent with their chemotherapy regimens [16,17]. Two additional abstract reports of completed phase III trials from the Groupe d’Etude des Lymphomes de Adulte–Groupe Ouest-Est des Leucémies et des Autres Maladies du Sang (GELA–GOELAMS) and German Lymphoma Study Group also demonstrate the benefits in event-free survival (EFS) and OS from rituximab included in the firstline treatment of FL [18,19]. The IPI is a means of identifying FL patients with a poor outcome; however, very few patients are in the IPI high-risk group at diagnosis, thus limiting the IPI’s utility for this disease [20]. In 2004, a Follicular Lymphoma International Prognostic Index (FLIPI) was created by an international group from a retrospective review of 4167 FL patients [21].

Working formulation [106]

Frequency (%)

Diffuse large cell (G) Follicular small cleaved (B) Follicular mixed (C) Follicular large cell (D) Small lymphocytic (A) – –

30.6 22.0

Diffuse large cell (G) Small noncleaved, non-Burkitt (J) Small noncleaved, Burkitt (J)

6.7 7.6 6.0 6.0 2.4 2.1 <1.0

– – Lymphoblastic lymphoma Mycosis fungoides (“miscellaneous”)

7.0 2.4 1.7 <1.0

The FLIPI included age 60 years or over, Ann Arbor stage III–IV, hemoglobin less than 12 g/L, abnormal LDH, and more than four nodal sites of involvement. Table 60.3 summarizes the outcome and relative risk of death according to the risk group identified by the FLIPI. Based on the FLIPI score, FL patients were equally subdivided within the low-, intermediate-, and high-risk groups. Although based on OS, the FLIPI has demonstrated predictive value for both PFS and OS in multiple settings, including rituximab-containing chemotherapy regimens [22–24]. Despite these advances, therapy for FL is not considered to be curative. A proportion of FL undergoes transformation to DLCL. The outcome for these patients is consistently poorer than for nontransformed FL or de novo DLCL, and has been shown to be dependent on the extent of disease at transformation and the exposure to prior chemotherapy, with a prolonged survival in selected complete remission (CR) patients [25,26]. There are a number of less common lymphoid malignancies listed in Table 60.1, based on B- and T-cell phenotype. Mantle cell lymphoma (MCL) presents as disseminated disease, predominantly in older males [27]. Although initially responsive to cytotoxic therapy, MCL patients are not cured with current approaches, and median survival is less than 5 years [27,28]. Rituximab has had a less beneficial impact in MCL than FL or DLCL [17]. Peripheral T-cell lymphomas (PTCLs) are a heterogeneous, rare group of malignancies with the majority of patients having an incurable disease and a median OS of 2–3 years [29,30]. Anaplastic large cell lymphoma (ALCL) is a unique T-cell lymphoma expressing the CD30 (Ki-1) antigen [31] and is commonly associated with anaplastic lymphoma kinase (ALK) protein expression (30–60% of cases) [32]. The 5-year PFS and OS exceed those of all other lymphoma subtypes. The absence of ALK protein expression has been associated with a later age of onset and a poorer survival, more similar to PTCL [32,33]. The majority of patients with lymphoblastic lymphoma and Burkitt’s lymphoma comprise children, adolescents, and young adults. Lymphoblastic lymphoma overlaps with acute lymphoblastic leukemia of T-cell lineage, and is best approached with therapy directed toward that disorder [34]. Mediastinal disease is common at diagnosis, and involvement of the bone marrow and central nervous system (CNS) occurs early in the disease course. Burkitt’s lymphoma is a rapidly progressive disorder that requires intensive multiagent chemotherapy. Treatment is extremely

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Chapter 60

Table 60.3 Outcome and relative risk of death according to risk group as defined by the Follicular Lymphoma International Prognostic Index (FLIPI) Risk group

Number of factors*

Distribution of patients (%)

5-year OS

10-year OS

Relative risk of death

Low Intermediate High

0–1 2 ≥3

36% 37% 27%

90.6 77.6 52.5

70.7 50.9 35.5

1.0 2.3 4.3

n = 1795. OS, overall survival. * Factors adversely affecting survival in the FLIPI include age above 60 years, Ann Arbor stage III–IV, more than 4 nodal sites; serum lactate dehydrogenase level higher than the upper limit of normal, and hemoglobin level less than 120 g/L. Data from [21].

Gene profiling in NHL DLCL was among the first human tumors in which gene expression analysis by complementary DNA microarray was explored [42]. This landmark work demonstrated two DLCL subtypes, germinal center-like and activated B-cell-like, with gene expression patterns related to their cell of origin and, importantly, differences in prognosis tracked with molecular subtype [42,43]. Attempts to translate these molecular subtypes with fidelity using other platforms such as immunohistochemistry and expression of a limited number of genes by reverse-transcriptase polymerase chain reaction (PCR) are in progress. Meanwhile, molecular profiling has been used to identify another unique subset of DLCL, primary mediastinal large cell lymphoma and established Burkitt’s lymphoma as a distinct molecular entity [44–47]. Similarly, molecular profiling has demonstrated distinctions among the T-cell lymphomas [48]. Another significant advance attributed to profiling has been an increased understanding of the interplay between lymphoma cells and their microenvironment. Prognosis in FL, for instance, appears to be governed by two immune response signatures within the host cell infiltrate [49]. The

100

Disease-free Survival (% of patients)

successful in both children and adults with appropriate management, even in patients with marrow and CNS disease [35,36]. Expert pathologists often have difficulty in distinguishing high-grade Burkitt-like lymphoma from DLCL and Burkitt’s lymphoma [37]. Despite this histopathologic uncertainty, a highly curable subgroup can be defined on the basis of prognostic factors. Primary CNS lymphoma (PCNSL) comprises 1–2% of all NHL, the vast majority being of DLCL histology. The incidence of PCNSL steadily increased from the mid 1980s to 1995, most notably in young and middle-aged males related to human immunodeficiency virus (HIV) infection, but also in individuals 60 years or more of age. In more recent years, the incidence appears to be declining in the younger age group with improved HIV treatments, but seems to have stabilized in the elderly population [38]. (See Chapter 68 for a discussion of HIVassociated NHL and HCT.) In immunocompetent individuals, the use of methotrexate (MTX)-based induction therapy with whole-brain radiotherapy (WBRT) has shown the most promising results, with median survival ranging from 30 to 60 months [39]. Unfortunately, continued relapse of disease and late neurotoxicity remain significant. The neurotoxicity may manifest as a fatal leukoencephalopathy, occurring in up to one-third of patients receiving WBRT and/or MTX-based regimens, with the onset at 3 months to 5 years post therapy [39–41]. The risk of neurotoxicity appears greatest in the patient population 60 or more years of age [40]. Historically, the prognosis for patients with relapsed or refractory PCNSL has been poor. Recent non-HCT strategies utilizing high-dose MTX (HD MTX) in patients failing WBRT, WBRT in patients failing HD MTX, as well as novel chemotherapeutic agents or combinations, have shown greater than 50% response rates and median OS of 11–60 months [39].

80

60

40

Chemotherapy-sensitive relapse

20

Chemotherapy-resistant relapse

No remission 0 1

2

3

4

5

6

7

8

9

10

11

12

Years after Transplantation

Fig. 60.1 Updated disease-free survival rates after high-dose therapy and autologous bone marrow transplantation for relapsed or refractory intermediate- and high-grade non-Hodgkin’s lymphoma. Patients are segregated according to chemotherapy-sensitive relapse (n = 44), chemotherapy-resistant relapse (n = 22), and failure to achieve a complete remission with primary treatment (n = 34). Tick marks represent censored data. (Reproduced from [50], with permission.)

significance of increased understanding of the underlying molecular biology of the NHL for HCT is several-fold, including improved diagnostic accuracy, increased ability to assign risk for primary and secondary treatment outcomes, and application of more targeted therapies based on signaling pathways and other survival mechanisms employed by different NHLs that are currently grouped by histopathologic nomenclature.

Hematopoietic cell transplantation Diffuse aggressive lymphoma Autologous HCT HDT and autologous HCT has been utilized to improve PFS and OS in patients with relapsed or refractory diffuse aggressive lymphoma for more than two decades [50]. In 1987, in an international group effort, Philip et al. reported on 100 patients treated with HDT and autologous HCT. DFS was 36% for patients with chemotherapy-sensitive disease compared with 14% for patients with chemotherapy-resistant disease (Fig. 60.1). Subsequently, the landmark Parma trial proved the superiority of autologous HCT for patients with chemotherapy-sensitive, diffuse

Non-Hodgkin’s Lymphoma

Fig. 60.2 Event-free survival rates for chemotherapy-sensitive patients with recurrent intermediate- or high-grade non-Hodgkin’s lymphoma treated with high-dose therapy and autologous bone marrow transplantation (n = 49) and patients treated with conventional chemotherapy (n = 54). The data are based on an intent-to-treat analysis. Tick marks represent censored data. (Reproduced from [51], with permission.)

aggressive lymphoma compared with standard decadron, cytarabine, and cisplatin (DHAP) therapy [51]. Following a documented response to DHAP, patients were randomized to receive either additional DHAP and radiotherapy, or high-dose carmustine (BCNU), etoposide, cytarabine, and cyclophosphamide and autologous HCT. The patients treated on the autologous HCT arm had a significantly prolonged 5-year EFS (46% versus 12%; p = 0.0001) and OS (53% versus 32%; p = 0.038) compared with the DHAP arm, respectively (Fig. 60.2). This trial included patients below 60 years of age without bone marrow or CNS disease, with randomization offered only to those patients with chemotherapy-sensitive disease. Hence, approximately half of the patients meeting initial eligibility were randomized. Subsequent, retrospective analyses of the Parma trial identified relapse less than 12 months from diagnosis and elevated LDH at relapse as predictors of poorer survival in patients in the trial [52]. The IPI score at the time of relapse was predictive of outcome in patients receiving additional DHAP therapy, but not for patients receiving HDT and autologous HCT [53]. The incorporation of rituximab, as pre-HCT therapy, mobilization, conditioning, and/or maintenance therapy, has been explored with the safety, feasibility, and “in vivo” purging capabilities of rituximab evident from multiple phase II clinical trials [54–59]. The majority of these initial studies included FL and MCL patients and will be reviewed later in this chapter. Subsequent single-institution, phase II studies employed variable approaches with rituximab in the relapsed or refractory aggressive NHL setting, and reported encouraging results compared with historical expectations [60–63]. While potential benefits were observed in the aggressive B-cell lymphoma patients who were rituximab naïve, this patient population is no longer relevant given the new standard of R-CHOP. In an ongoing multicenter, intergroup European, phase III trial (CORAL), relapsed or refractory DLCL patients are randomized to three cycles of rituximab with ifosfamide, carboplatin, and etoposide or DHAP followed by a second randomization after autologous HCT with BEAM (BCNU, etoposide, cytarabine, and melphalan) to maintenance rituximab or observation. Although maintenance rituximab did not improve outcomes in primary DLCL after R-CHOP in the US Intergroup Study, there are theoretical reasons to support its efficacy post autologous HCT in this high-risk population [7]. A preliminary report of the

881

progress of the CORAL trial begun in 2003 has shown that a larger number of patients with primary refractory disease had prior rituximab (69%) compared with the relapsed patients (15%) [64]. Despite a good overall response to reinduction therapy, as expected, the primary refractory patients previously exposed to rituximab were less likely to proceed to the second randomization. Thus, the overall risks and benefits of rituximab in this trial will be challenging to interpret and extrapolate to current practice. Transient neutropenia, usually without significant infection, was seen in a subset of patients treated with rituximab in the peritransplant setting. Delayed immunoglobulin recovery and impaired vaccine responses were reported in the Stanford University series [60]. Of greater concern are case reports of progressive multifocal leukoencephalopathy in remission after high-dose chemotherapy and peritransplant rituximab [65], with progressive multifocal leukoencephalopathy following rituximab linked to immunosuppression. In addition, hepatitis B reactivation has been reported with rituximab in the non-HCT setting [66,67]; hence close monitoring and/or antiviral prophylaxis should be instituted for patients at risk. As appreciated from the Parma trial, a significant proportion of patients with relapsed or refractory diffuse aggressive lymphoma will not proceed to autologous HCT, most often due to chemotherapyresistant disease, but also due to intolerance of salvage therapy or ineligibility due to comorbidities, age, bone marrow or CNS involvement. An Italian Intergroup evaluation of risk assessment for patients with first relapse of DLCL indicated that only 20% of relapsed patients received HDT and autologous HCT [68]. Efforts to improve the outcome of the relapsed, refractory diffuse aggressive lymphoma patient include the application of immunotherapy in the peritransplant period, such as radiolabelled antibodies, more effective salvage chemotherapy, mobilization of tumor-free grafts, more effective preparatory regimens, posttransplant immunotherapy, and allogeneic HCT, as discussed elsewhere in this and other chapters. Utilizing autologous HCT earlier in the disease course as part of primary therapy has been an additional approach to augmenting the survival of patients with “high-risk,” diffuse, aggressive lymphoma. Prior to the recognition of the IPI in 1993, investigators identified a number of different prognostic factors including LDH, stage of disease, number of extranodal sites, and tumor dimensions [69]. Phase II autologous HCT studies were conducted in patients with these high-risk features who achieved CR with chemotherapy. Initial results were encouraging, with Gulati et al. and Nademanee et al. reporting improved survival compared with historical controls treated with standard chemotherapy [70,71]. Subsequently, multiple randomized trials have explored the use of HDT and autologous HCT as primary therapy for patients with highrisk, diffuse, aggressive NHL, as outlined in Table 60.4 [72–81]. Several of these trials were initiated prior to the identification of the aa-IPI with retrospective analysis of the IPI risk factors in two of the initial trials [73,74]. The results are inconsistent in this collection of trials, with great variability in the study population, including the definition of “high-risk” features, the disease status of the patient at time of enrollment (i.e. enrolling all patients versus responding patients versus complete responders only), and the inclusion of other B-cell lymphoma or T-cell lymphoma with DLCL. The standard and HDT arms also varied broadly, with standard, novel, and abbreviated courses of induction therapy, as well as novel HDT. The majority of these studies suffered from attrition, with one-fifth to one-third of patients not completing the assigned treatment, thus reducing sample size and eroding their power to observe significant differences. A recent meta-analysis of randomized trials evaluating the use of HDT and autologous HCT as first-line treatment for patients with

First CR; aggressive NHL

Aggressive NHL

B-cell diffuse large cell lymphoma

Aggressive NHL 29–36% HI-H IPI

Aggressive NHL Abnormal LDH ~75% HI-H IPI

Aggressive NHL HI-H IPI

Aggressive NHL HI-H IPI

Aggressive NHL I-HI IPI; 50% HI IPI

Aggressive NHL I-HI-H IPI

GELA/subset [73] retrospective HI-H IPI

Italian Coop/subset [74] retrospective HI-H IPI

Milano [75]

EORTC [76]

German HGLSG [77]

GELA [78] Prospective IPI

Italian Coop [79] Prospective IPI

GOELAMS [80] Prospective IPI

Multinational [81] Prospective IPI

Study entry

CR/PR after CHOPx4 or CEEP × 2

CR/PR/MR after MACOP-B

Study entry

CR/PR/MR after 2 cycles

CR/PR after 3 cycles

Study entry

Study entry

CR

Time of randomization

50 70

99 98

75 75

181 189

154 158

96 98

50 48

36 34

111 125

n

56 61 49 59 (EFS) 51 39 49 61 28 56 44 39 (EFS)

CHVmP/BV × 8 CHVmP/BV × 6 + BEAC and AHCT CHOEP × 5 CHOEP × 3 + BEAM and AHCT ACVB × 4 + Sequential CT CEOP × 1, ECVBP × 2 + BEAM and AHCT MACOP-B × 12 weeks MACOP-B × 8 weeks and BEAC and AHCT CHOP × 8 CEEP × 2/Ara-C-MTX/ BEAM and AHCT CHOP × 6–8 (88% HI-H IPI) High-dose sequential and AHCT (72% HI-H IPI)

37 65

VACOP-B × 12 weeks VACOP-B × 8 weeks and AHCT 49 76 (EFS)

39 55

A/NCVB × 4 + sequential CT A/NCVB × 4, MTX + CBV and AHCT

MACOP-B (76% HI-H IP) High-dose sequential and AHCT (94% HI-H IP)

(%)

Therapy

n.s.

0.003

n.s.

0.01

n.s.

n.s.

0.004

0.08

0.02

p

Progression-free survival

53 46

44 74

65 64

60 46

63 62

77 68

55 81

55 68

49 64

(%)

Overall survival

n.s.

0.001

n.s.

0.007

n.s.

n.s.

0.09

0.7

0.04

p

3

4

5

5

3

5

7

6

8

Time (years)

ACVB, doxorubicin, cyclophosphamide, vindesine, and bleomycin; AHCT, autologous hematopoietic cell transplantation; BEAC, carmustine, etoposide, cytarabine, and cyclophosphamide; BEAM, carmustine, etoposide, doxorubicin and melphalan; CBV, cyclophosphamide, carmustine, etoposide; CEEP, cyclophosphamide, epirubicin, vindesine, and prednisone; CEOP, cyclophosphamide, etoposide, vincristine, and prednisone; CHOEP, cyclophosphamide, doxorubicin, vincristine, etoposide, and prednisone; CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisone; CHVmP/BV, cyclophosphamide, doxorubicin, teniposide, prednisone, bleomycin, and vincristine; CR, complete remission; CT, chemotherapy; ECVBP, epirubicin, cyclophosphamide, vindesine, bleomycin, and prednisone; EFS, event-free survival; EORTC, European Organization for Research and Treatment of Cancer; GELA, Groupe d’Etude des Lymphomes de Adulte; HGLSG, High Grade Lymphoma Study Group; GOELAMS, Groupe Ouest-Est des Leucémies et des Autres Maladies du Sang; I-HI-H IPI, intermediate, high-intermediate, and high-risk international prognostic index; LDH, lactate dehydrogenase; MACOP-B, methotrexate, doxorubicin, cyclophosphamide, vincristine, and prednisone; MR, minimal response; MTX, methotrexate; NCVB, mitoxantrone, cyclophosphamide, vindesine, and bleomycin; NHL, non-Hodgkin’s lymphoma; n.s., not significant; PFS, progression-free survival; PR, partial remission; VACOP-B, etoposide, doxorubicin, cyclophosphamide, vincristine, and prednisone.

Patient population

Group/analysis

Table 60.4 Autologous transplantation in first remission for diffuse aggressive lymphoma

882 Chapter 60

Non-Hodgkin’s Lymphoma

aggressive NHL, comprised mainly of the studies summarized in Table 60.4, showed no conclusive benefit in EFS or OS for patients receiving HDT and autologous HCT [82]. In evaluating the good-risk (≤1 IPI risk factor) versus the poor-risk patients (>1 IPI risk factor) based on aa-IPI, there was a possible detrimental effect of autologous HCT in the goodrisk group, and a beneficial effect of autologous HCT in the poor-risk group, on OS. Interestingly, a subsequent cohort study of the patients undergoing autologous HCT (including patients in CR and “undefined” CR) from the relatively large GELA trials [73,78] identified non-anaplastic T-cell histology, one or more extranodal sites, and bone marrow involvement, and not the aa-IPI criteria, as poor prognostic factors for patients receiving front-line autologous HCT [83]. As in relapsed and primary refractory DLCL, rituximab with autologous HCT regimens has been and is being explored for patients with “high-risk” disease as well [84,85]. The benefit of consolidation with HDT and autologous HCT after R-CHOP chemotherapy is the subject of the Southwest Oncology Group-led S9704 phase III trial. This study is limited to intermediateand high-risk IPI patients after a full course of R-CHOP. Several of the trials discussed above utilizing HDT and autologous HCT as primary therapy focused on patients considered “slow responders” or patients in partial remission (PR). Historically, patients not achieving a CR to primary therapy have a poor prognosis with conventional therapy, a finding that is supported by retrospective studies as well as a meta-analysis of phase II studies in intermediate-grade and highgrade lymphomas [86–88]. Phase III trials targeting patients with a variable PR status documented at mid-course of induction therapy have not shown a clear benefit to HDT and autologous HCT compared with continued standard therapy [89,90]. The modality utilized to identify the poorly responding or PR patient in these previous studies was conventional computed tomography. More recently, positron emission tomography (PET) using fluoride-18 fluorodeoxyglucose (FDG) has emerged as a more accurate radiologic tool for assessment of lymphoma [91]. Juweid et al. reported that the 3 year PFS in 54 CHOP-treated patients was significantly different based on the presence or absence of FDG uptake of the residual disease (33% and 89%, respectively) and a PR defined by FDG uptake was more prognostic than a PR defined by the older computed tomography-based criteria [92]. Accordingly, a “Revised Response Criteria for Malignant Lymphoma” incorporating PET results into the definitions of disease response is now recommended [93]. PET results obtained at the end of chemotherapy as well as early in the course of therapy for aggressive NHL are predictive for remission duration, EFS, and/or OS [94]. PET positivity was an independent predictor regardless of IPI, use of rituximab with induction or other prognostic factors in prospective studies evaluating PET scans early in the course of therapy. Multiple reports in the autologous HCT literature have also shown a correlation between outcome and PET results after salvage therapy prior to autologous HCT for patients with relapsed or refractory lymphoma [94]. Although PET restaging better defines disease status and prognosis, there are challenges to prospective evaluation of the role of PET in clinical trials. The Imaging Subcommittee of the International Harmonization Project in Lymphoma has recently published guidelines for performing and interpreting PET imaging, likely improving the reproducibility and accuracy of PET results within and across medical centers [91]. Despite compelling prognostic data, altering the therapeutic approach for patients based on PET status, either early in the course of treatment or prior to autologous HCT, is yet to be broadly and systematically tested. Radioimmunotherapy, discussed in greater detail in Chapter 24, has been explored in conjunction with autologous HCT utilizing a radioisotope linked to a monoclonal antibody, targeting the tissue of interest and, theoretically, sparing normal tissues. The majority of clinical trials incorporating the radiolabeled antibody therapies, such as iodine-131

883

(131I) tositumomab (Bexxar) and yttrium-90 (90Y) ibritumomab tiuxetan (Zevalin), have included multiple lymphoma histologies [95–100]. After demonstrating the safety and efficacy of 131I linked to the antiCD20 antibody tositumomab, Press et al. [95] combined the radioimmunoconjugate with etoposide and cyclophosphamide followed by autologous HCT in a phase I–II study [96]. This and subsequent trials included patients with relapsed or refractory DLCL, FL, and MCL with 131 I-labeled tositumomab administered alone, or with etoposide and cyclophosphamide or BEAM [97,98,100,101]. An additional report of the use of 131I-tositumomab alone followed by autologous HCT included 24 patients 60 or more years of age with relapsed B-cell lymphoma, 54% with chemotherapy-resistant disease [101]. This study included an equal number of DLCL, FL, and MCL patients. The 3-year OS and PFS rates were 59% and 51%, respectively, with a median follow-up of 2.9 years and no treatment-related deaths. Vose et al. treated 40 patients with relapsed or refractory, chemotherapy-sensitive DLCL, 88% having had prior rituximab therapy, with 131I -tositumomab combined with BEAM as the preparative regimen [102]. At a median follow-up of 28 months, the 3-year PFS and OS were 70% and 81%, respectively. These phase II results have led to a multi-institutional study conducted by the Bone Marrow Transplant Clinical Trials Network randomizing relapsed or refractory DLCL patients between rituximab–BEAM and 131Itositumomab–BEAM as the autologous HCT preparative regimen. Investigators have also successfully escalated doses of 90Y chelated with ibritumomab, a murine anti-CD20 antibody, in combination with etoposide and cyclophosphamide or the BEAM regimen [99,103,104]. In the failed HCT setting, Vose et al. have recently reported the feasibility of single agent 90Y-ibritumomab for the treatment of patients relapsing after autologous HCT [105]. Allogeneic HCT The benefits of allogeneic HCT for the treatment of NHL may include a “disease-free” graft, a decreased risk of secondary myelodysplasia or acute myeloid leukemia, and a graft-versus-lymphoma effect enhancing long-term disease control. Allogeneic HCT has typically been pursued for the patient with relapsed or refractory lymphoma not considered an appropriate autologous HCT candidate. The bulk of the published literature pertaining to allogeneic HCT for NHL includes phase II clinical trials, retrospective review of registry data, and historical comparisons to autologous or alternative allogeneic preparative regimens. The patient populations within each report are often heterogeneous, including multiple lymphoma histologies, disease stages, and remission states. Historical comparisons of high-dose conditioning and allogeneic, autologous, and/or syngeneic HCT conducted by the European Bone Marrow Transplantation Lymphoma Registry (EBMTR) as well as the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation (IBMTR/EBMT) included subset analyses based on the histologic lymphoma subtypes of the Working Formulation [106]. In the EBMTR analysis of 1185 lymphoma patients receiving high-dose conditioning allogeneic HCT as their first HCT, 147 patients were identified as intermediate-grade lymphoma [107]. The 4-year PFS and OS were 34.6% and 38.3%, respectively, with a 4-year nonrelapse mortality (NRM) of 41.8%. A matched comparison with autologous HCT recipients found a decreased relapse rate coincident with an increased NRM in the intermediate-grade lymphoma patients receiving allogeneic HCT. Although PFS was the same for low- and intermediategrade lymphoma patients regardless of type of HCT, the OS was superior for all lymphoma subtypes undergoing autologous HCT. The IBMTR/EBMT undertook a comparison of autologous (purged and unpurged) versus syngeneic versus high-dose conditioning allogeneic (T-cell-depleted and T-cell-replete) HCT in an attempt to clarify the benefits of a graft-versus-lymphoma effect from allogeneic HCT as

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well as a “tumor-free” graft [108]. There were 193 allogeneic and 1563 autologous HCT recipients with a diagnosis of intermediate-grade lymphoma. There were no survival advantages between an autologous and syngeneic HCT or a purged and unpurged autologous graft. Patients receiving a T-cell-replete allogeneic graft had a significantly poorer DFS and OS compared with a syngeneic graft (relative risk [RR] 1.85, p = 0.03; and RR 2.19, p = 0.008, respectively) as well as a poorer OS than patients receiving a T-cell-depleted graft (RR 1.18, p = 0.03). The allogeneic HCT cohorts in these comparative studies had a significantly greater proportion of patients with higher clinical stage of disease, more pre-HCT therapy, and/or more chemotherapy resistance. In addition, chemotherapy sensitivity was identified as a significant predictor of a superior outcome. A notable limitation to the possible benefit of allogeneic HCT for DLCL has been the increased NRM compared with autologous HCT. Investigators have been exploring the utility of reduced-intensity (RI) preparative regimens in many diseases, as reviewed in Chapter 71. NRM may be reduced compared with conventional high-dose conditioning regimens, but the risks of acute and chronic graft-versus-host disease (GVHD) continue to be problematic. The current literature on the use of RI allogeneic regimens for lymphoma is more heterogeneous than the literature on the use of high-dose regimens for lymphoma. Preliminary data have shown poor disease control with DLCL compared with more indolent histologies, with chemotherapy sensitivity remaining a significant predictor of outcome [109–112]. Currently, one of the most intriguing aspects of RI regimens is the apparent feasibility of embarking on a second HCT for a patient having failed a prior high-dose HCT regimen. The majority of the above references of RI transplantation, in fact, included 30–50% of patients that had failed prior autologous HCT. The MD Anderson Cancer Center reported 20 lymphoma patients (DLCL, MCL, and FL) with chemotherapy-sensitive relapse treated with RI allogeneic HCT [113]. At a median follow-up of 25 months, one patient died of infection, with the remaining 19 alive and disease free. Baron et al. analyzed data from 147 patients with hematologic malignancies relapsing after high-dose autologous, allogeneic or syngeneic HCT who received RI HCT with a median follow-up of 27.1 months [114]. Fifty patients had lymphoma diagnoses, with 3-year OS of 64% for MCL (n = 14), 56% for FL (n = 12), and 31% for aggressive lymphoma (n = 24). The 3-year NRM was 32% for the entire cohort of transplanted patients. This preliminary information is in remarkable contrast to previous grim expectations for lymphoma patients receiving high-dose conditioning and allogeneic HCT after failing prior autologous HCT [115]. Prospective evaluation of RI regimens for relapse after HCT, including larger numbers of well-

characterized patients and extended follow-up, will be required to determine the promise of these encouraging results. Summary on HCT for DLCL It must be realized that the projected outcome of patients with diffuse, aggressive B-cell lymphoma, either relapsed, refractory or newly diagnosed, has likely been reset with the routine incorporation of rituximab to induction as well as second-line therapies. Molecular markers, such as bcl-2 and bcl-6, and the genotypic subtypes in germinal centers and activated peripheral blood identified by microarray analysis considered to have prognostic significance in DLCL prior to the advent of rituximab, may have a less dramatic influence than previously found [42,84,116,117]. The impact of rituximab must be considered as we determine appropriate current therapy for the patient with diffuse aggressive B-cell lymphoma. In addition, continued investigation of modalities including molecular diagnostics, genetic profiling, and PET may be important in aptly identifying the patient in need of more aggressive measures for primary or secondary treatment of their lymphoma. Meanwhile, autologous HCT remains the standard of care for the patient with chemotherapy-sensitive relapsed or refractory DLCL. The role of HDT and autologous HCT is less clear in the setting of the “highrisk” DLCL patient in first CR, with the subset of patients with more than one IPI risk factor more likely to benefit after a nonabbreviated induction regimen prior to HCT. The ideal approach to the use of rituximab or radiolabeled antibody therapy for the patient proceeding to HCT is yet to be determined, emphasizing the continued need for welldesigned and appropriately conducted clinical trials. As evidenced from above, the data regarding allogeneic HCT in DLCL are limited, with an absence of completed or planned phase III trials comparing allogeneic HCT with nontransplant therapies or autologous HCT. Clearly, a definitive answer regarding the utility of allogeneic HCT for patients with DLCL requires further investigation. Follicular lymphoma Autologous HCT Prior to the recent decade, the majority of clinical data available regarding autologous HCT and FL in the relapsed, refractory as well as first remission setting were from phase II clinical trials, with particular investigators conducting formal comparisons to matched, historical controls [118–126]. The studies differed in their preparative regimens, status of disease at the time of autologous HCT, inclusion of other lymphoma histologies, and use of purged or unpurged hematopoietic cells. Table 60.5 summarizes results of HDT and autologous HCT in a select series

Table 60.5 Autologous transplantation in follicular lymphoma, beyond first remission

Group

n

High-dose regimen

Stem cell source

% Survival Disease-free

Overall

Years

% 2nd CA

DFCC [127]

153

CY/TBI

Purged marrow

42

66

8

12

99

CY/TBI

Purged marrow

63

69

5

12

Nebraska [126]

100

CY/TBI BEAC

Unpurged marrow

44

65

4

2

Stanford [120]

92

TBI/VP/CY BCNU/VP/CY

Purged PBPCs

44

60

4

3

St Bartholomew’s [121]

2nd CA, secondary myelodysplasia or acute myelogenous leukemia; BEAC, carmustine, etoposide, cytarabine, and cyclophosphamide; CY/TBI, cyclophosphamide and total body irradiation; DFCC, Dana Farber Cancer Center; PBPC, peripheral blood progenitor cells; VP, etoposide.

Non-Hodgkin’s Lymphoma (a)

1.0

CY + TBI SBH control group

Survival (%)

0.7

0.5 χ21 = 25.67 Pr > χ2 = 0.00 0.2

0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 Time (years)

(b) 1.00 χ21 = 5.72 Pr > χ2 = 0.02 0.7 Survival (%)

of phase II clinical trials in relapsed or refractory FL patients with median follow-up of 4 or more years [120,121,126,127]. In these relapsed/refractory studies, Dana Farber Cancer Center and St Bartholomew’s Hospital patients were required to be in a minimal disease state (≤2 cm residual disease and <20% bone marrow involvement) at the time of HDT, Stanford University required chemotherapysensitive patients, whereas the Nebraska group included patients with resistant and untested relapse but no bone marrow involvement. In these four studies, the DFS was 42–63% and OS was 60–69% at 4–8 years. The GELA group conducted a retrospective review of 372 patients with FL in first relapse after previous enrollment in the phase III Groupe d’Etude des Lymphomes Folliculaires (GELF) 86 trial for newly diagnosed FL [122]. Relapsed patients receiving autologous HCT had improved 5-year PFS (42% versus 16%; p = 0.0001) and OS (58% versus 38%; p = 0.0005) compared with patients receiving conventional salvage therapy, respectively. In an attempt to clarify the benefit of autologous HCT in relapsed FL, a randomized trial was conducted by the EBMTR Lymphoma Working Party [128]. The trial design included randomization of relapsed or progressive FL patients to one of three treatment arms after a documented CR or PR following three cycles of CHOP chemotherapy: Chemotherapy alone with an additional three cycles of CHOP (C) versus unpurged autologous HCT (U) versus purged autologous HCT (P). After slow accrual of 140 of the planned 250 patients, the trial was closed early for final analysis, with a median follow-up of 69 months at time of final publication. Of the 140 patients enrolled, 89 patients were randomized to one of the three treatment arms (24–33 patients per arm), with approximately two-thirds of enrolled patients in first relapse and the remainder beyond first relapse. Two-year PFS was significantly longer in patients receiving autologous HCT (C 26% versus U 58% versus P 55%; p = 0.0037). The survival difference at 4 years did not reach statistical significance (C 46% versus U 71% versus P 77%; p = 0.079); however, there was a significant reduction in hazard ratios for OS when the combined transplantation arms were compared with the chemotherapy arm (hazard ratio 0.4; p = 0.026). The limited sample size precluded definitive comments regarding the impact of purging. Of note, there were no reported cases of secondary myelodysplasia or acute leukemia during the follow-up period. Given the poor accrual and eventual small number of patients enrolled in this randomized EBMTR trial, its instructive value is limited. Possibly a more informative surrogate to the available randomized trial is the recently reported, mature, retrospective comparison of ageand remission-matched controls including FL patients beyond first remission treated similarly at Dana Farber Cancer Center and St Bartholomew’s Hospital [125]. With a median follow-up of 13.5 years in the transplanted patients, there was evidence of a plateau in the remission duration curve at 12 years of 48%, with a 10-year OS of 54%. Patients receiving autologous HCT in second remission at St Bartholomew’s had significantly improved remission duration and OS compared with patients receiving standard therapy (Fig. 60.3). Although the phase II and III trials reviewed above did not include patients with exposure to rituximab, there are now multiple phase II reports confirming the feasibility of rituximab’s use in the HDT setting, as reviewed by Gisselbrecht and Mounier. [129]. The majority of the trials include small numbers of patients with relapsed and refractory FL as well as other B-cell histologies, with rituximab administered with mobilization, conditioning, and/or maintenance. The evidence of “in vivo purging” with rituximab based on PCR analysis of bcl-2 in the hematopoietic cell product has been apparent, with more complete discussions of the clinical significance of minimal residual disease and purging techniques in Chapters 26 and 42, respectively. A multicenter, randomized trial was begun via the EBMTR (GELA, GOELAMS, and

885

0.5

0.2 CY + TBI SBH control group 0

2

4

6

8

10 12 14 16 18 20 22 24 26 28 30 Time (years)

Fig. 60.3 (a) Duration of remission with comparison of outcome between patients treated with cyclophosphamide plus total-body irradiation (CY + TBI) and a control group from St Bartholomew’s Hospital (SBH). (b) Overall survival with comparison of outcome between patients treated with CY + TBI and the control group from SBH. (Reproduced from [125], with permission.)

Spanish Lymphoma and Autologous Transplant Group [GEL/TAMO]; Lym-1 study) to evaluate the impact of rituximab with in vivo purging and/or post-HCT rituximab maintenance therapy on the outcome of relapsed FL patients in very good PR or CR after reinduction treatment [129]. The Lym-1 study closed prematurely, with 280 of a target 460 patients enrolled. Patients were randomized to one of four arms: (1) in vivo purging and maintenance; (2) purging without maintenance; (3) maintenance without purging; and (4) no rituximab. As mentioned above, radiolabeled antibody therapy has been explored for patients with relapsed or refractory lymphomas, including FL. Gopal et al. conducted a historical comparison of 27 FL patients receiving 131 I-tositumomab alone and autologous HCT with 98 disease-matched controls receiving standard autologous HCT preparative regimens [98]. Patients receiving 131I-tositumomab had improved 5-year PFS (53% versus 29%; p = 0.03, respectively) and OS (67% versus 48%; p = 0.004, respectively) and no difference in secondary myelodysplastic syndrome or NRM. Phase II trials of autologous HCT for FL patients in first remission have shown compelling results, with a median follow-up of 4–5 years

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in a select group of studies showing a PFS of 59–76% and an overall OS of 84–92% [119,123,130]. Three randomized trials were completed and published in recent years to address the question of a true benefit of HDT compared with conventional therapy for the FL patient in first remission [131–133]. These three randomized trials are summarized in Table 60.6 and included untreated, adult FL patients. All three trials compared an IFN-containing maintenance therapy arm with a non-IFNcontaining autologous HCT arm, limiting the post-induction treatment to patients with chemotherapy responsive or stable disease only. The German Lymphoma Study Group delivered similar induction therapy to each arm with randomization to IFN maintenance versus autologous HCT [131]. The French group, GOELAMS, delivered a different induction regimen to patients dependent on their randomization arm, with chemotherapy and IFN administered as maintenance versus autologous HCT with a CD34-selected or monoclonal antibody-purged hematopoietic cell product [132]. The GELA included IFN in the induction and maintenance therapy of the non-HCT arm, with chemotherapy and IFN administered as maintenance as well [133]. The benefit of autologous HCT in EFS or PFS was consistent across these three trials without an impact on OS at the time of publication. As with autologous HCT in primary therapy of DLCL, the utility of HCT in first remission of FL may be better defined by delineating the high-risk patient. The GOELAMS and GELA trials retrospectively identified the FLIPI scores of enrolled patients, finding the benefit of HCT in terms of EFS, PFS or OS to be more evident in or limited to the intermediate- or high-risk groups of patients. The Italian Bone Marrow Transplant Group (GITMO) recently reported on the results of the GITMO-IIL trial comparing rituximab–HDT (R-HDT) therapy and autologous HCT with R-CHOP in high-risk FL [134]. The 3-year EFS and PFS were superior in the patients receiving R-HDT (R-HDT 66% and 72%, respectively, versus R-CHOP 36% and 38%, respectively) with no difference in OS (83% in both arms). This trial allowed crossover, with 69% of relapsed R-CHOP patients receiving R-HDT. Molecular response was the strongest predictor of outcome regardless of treatment, with a higher number of patients achieving a molecular response with R-HDT compared with R-CHOP (80% versus 44%; p < 0.001).

Transformation to a diffuse aggressive lymphoma occurs over the course of FL and portends a grave prognosis [26]. Selected patients with transformation may benefit from HDT and autologous HCT. Friedberg et al. reported a 46% DFS at 36 months in transformed lymphoma patients, with superior results for patients who had transformed within 18 months of diagnosis [135]. Similar DFS at 4 years (49%) was reported in 17 transformed lymphoma patients transplanted at Stanford University [120]. In a series of 27 transformed patients from St Bartholomew’s Hospital, 19 were alive and disease free at 2.4 years after HCT [136]. Relapses occurred with FL as well as transformed lymphoma. Williams et al., reporting from the EBMTR, found that outcomes with autologous HCT in transformed lymphoma patients were not different from those reported for de novo DLCL in relapse [137]. In this report, as well as that from Chen et al. [138], there was a relatively high transplant-related mortality related to advanced age and previous therapy. Together, these data indicate that selected patients with transformed disease have curative potential with HDT and autologous HCT. Allogeneic HCT Allogeneic HCT has been a potential curative therapy for FL, with evidence of a decreased relapse rate compared with autologous HCT. Unfortunately, the benefit of disease control has been offset by the increased NRM and morbidities of acute and chronic GVHD. Table 60.7 summarizes select trials conducting historical and/or matched comparisons of high-dose conditioning with allogeneic or autologous HCT for relapsed FL. The IBMTR study included only FL patients, with the patients receiving allogeneic HCT having more advanced disease, more chemotherapy resistance or unknown response prior to HCT [139]. The relapse rate was 54% lower with allogeneic HCT versus unpurged autologous HCT (p < 0.001), and the NRM was 4.4 times higher with allogeneic versus autologous HCT (p < 0.001). However, there was evidence of decreasing NRM as survival improved after allogeneic HCT over the time periods from 1990–93 to 1997–99 (Fig. 60.4). There were rare relapses after 1 year from allogeneic HCT, and an eventual, similar 5-year OS between purged autologous, unpurged autologous, and allogeneic HCT.

Table 60.6 Randomized trials of autologous transplantation in follicular lymphoma (FL), first remission

Reference

Patient population (n)

Consolidation (n)

EFS (5 year)

Overall survival

Median follow-up

Lenz [131] GLSG

Responders (307 FL) <60 years; ITT

CHOP/MCP × 4–6 CHOP/MCP × 4–6 DexaBEAM mob.

IFN maintenance (126) TBI/CY AHCT (114)

33.3% 64.7 (PFS) p < 0.001

NA NA

4.2 years

Deconinck [132] GOELAMS

Stable or responding ≥PR patients (172) <61 years

CHVP × 6 VCAP × 2–3, IMVP-16
CHVP maintenance and IFN (80) TBI/CY AHCT (86) CD34 or MoAb purged

48% 60% p = 0.05

84% 78% p = 0.49

5 years

Sebban [133] GELA

Responders (339 FL) <61 years; ITT

CHVP–IFN × 6

CHVP-IFN (165) (129 completed maintenance) CY/VP/TBI (150) (131 pts completed HDT)

29%

NA

7.6 years

40% (7 year) p = 0.05

NA p = 0.4

Induction

CHOP × 4

AHCT, autologous hematopoietic cell transplantation; CHOP/MCP, cyclophosphamide, doxorubicin, vincristine, prednisone, mitoxantrone, chlorambucil, and prednisone; CHVP, cyclophosphamide, low-dose doxorubicin, teniposide, and prednisone; CY/VP/TBI, cyclophosphamide, VP-16, and TBI; DexaBEAM, dexamethasone, BCNU, etoposide, cytarabine, and melphalan; DHAP, dexamethasone, high-dose cytarabine, and cisplatin; GELA, Groupe d’Etude des Lymphomes de l’Adulte; GLSG, German Lymphoma Study Group; GOELAMS, Groupe Ouest-Est des Leucémies et des Autres Maladies du Sang; IFN, interferon; IMVP-16, ifosfamide, methotrexate, and VP-16; ITT, intention-to-treat analysis; MoAb, monoclonal antibody; NA, not available; PFS, progression-free survival; PR, partial remission; TBI/CY, total body irradiation and cyclophosphamide; VCAP, cyclophosphamide, high-dose doxorubicin, prednisone, and vincristine.

887

Non-Hodgkin’s Lymphoma Table 60.7 Selected studies in follicular lymphoma comparing high-dose conditioning with allogeneic or autologous transplantation

Author

Graft

n

Median age (years)

% NRM

% Relapse

% OS

Median follow-up

van Besien [139]

Allo-MRD

176

42

30 (5 year)

21 (5 year)

51 (5 year)

36 months

IBMTR

Auto-purged Auto-unpurged

131 567

49 49

14 4

43 58

62 55

49 months 41 months

Peniket [107]

Allo-84% MRD

231

42

38 (4 year)

25* (4 year)

51

60 month

EBMTR

Auto

693*

NA

10*

55*

65*

NA

Hosing [140]

Allo-89% MRD

44

43

27 (100 day)

19

45 (EFS)

49

53 months

MDACC

Auto

68

42

74 p < 0.001

17 p = 0.01

34 p > 0.05

71 months

6

% PFS

42.7

Allo, allogeneic; Auto-unpurged, autologous unpurged; EBMTR, European Blood and Marrow Transplantation; EFS, event-free survival; IBMTR, International Bone Marrow Transplantation Registry; MDACC, MD Anderson Cancer Center; MRD, matched related donor; NRM, nonrelapse mortality; OS, overall survival; PFS, progression-free survival. * Extracted from the published Kaplan–Meier curves and manuscript.

1.0 Allogeneic, 1997–99 (n = 62)

80 60

Allogeneic, 1994–96 (n = 64)

40 Allogeneic, 1990–93 (n = 50) 20 0 0

1

2

3

4

5

Years

Fig. 60.4 Probabilities of survival after human leukocyte antigen-identical sibling high-dose transplantation by year of transplantation. (Reproduced from [139], with permission.)

A matched comparison of lymphoma patients receiving autologous and allogeneic HCT was conducted by the EBMTR [107]. The cohort included 231 patients with FL receiving related and unrelated donor allogeneic HCT. As in the IBMTR report, the allogeneic HCT recipients had more advanced disease at diagnosis and at time of HCT, as well as exposure to more prior therapies. Recipients of autologous HCT had improved OS with a lower NRM and a higher relapse rate compared with those receiving allogeneic HCT. The status of disease at time of HCT was predictive of outcome. The MD Anderson Cancer Center reported a historical comparison of indolent lymphoma patients receiving high-dose conditioning and allogeneic versus autologous HCT [140]. Recipients of autologous HCT were more likely to have chemotherapysensitive disease and to achieve a CR prior to HCT than were the allogeneic recipients. As in the above registry data, there was improved EFS but increased NRM with allogeneic HCT, and hence no difference in OS between allogeneic and autologous HCT. At 44 months post allogeneic HCT, there was a plateau in OS, whereas there was a continuous pattern of relapse after autologous HCT (Fig. 60.5). RI regimens have been utilized in FL with encouraging results in comparison to more aggressive lymphoma subtypes, such as DLCL. Table 60.8 summarizes selected studies of RI allogeneic HCT for relapsed or refractory FL [110,111,141–143]. Of particular interest is the proportion of patients having failed prior autologous HCT, ranging from

Cumulative proportion surviving

Survival probability, %

100

0.9 0.8 0.7 0.6 Allogeneic

0.5 0.4 0.3

Autologous

0.2 0.1 0.0

0

20

40 60 80 100 Months after transplantation

120

140

Fig. 60.5 Kaplan–Meier estimates of overall survival after high-dose therapy followed by autologous or allogeneic hematopoietic cell transplantation for refractory or recurrent indolent non-Hodgkin’s lymphoma. (Reproduced from [140], with permission.)

17% to 34% of the patients in each series. The majority of these studies also included patients having had multiple prior regimens as well as chemotherapy-resistant or refractory disease. The 2–3-year PFS of chemotherapy-sensitive patients ranged from 51% to 65%, with an NRM of 11–34%. Figure 60.6 illustrates the dramatic difference in survival between patients with chemotherapy-sensitive and chemotherapy-refractory disease as seen by Vigouroux and colleagues of the French Society of Bone Marrow Graft Transplantation and Cellular Therapy [143]. Rodriguez et al. performed a retrospective analysis of 88 lymphoma patients receiving conventional high-dose (n = 48) and RI (n = 40) allogeneic HCT [112]. Patients receiving the RI regimen were older (50 years versus 45 years), had more often failed prior autologous HCT, and were more likely to receive unrelated donor and peripheral blood grafts. In the subset of FL patients (18 conventional and 16 RI), there was no difference in relapse rate, PFS or OS. NRM and GVHD incidence were also similar between regimens in the entire cohort of lymphoma patients

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Table 60.8 Selected studies utilizing reduced-intensity preparative regimens and allogeneic transplantation for relapsed follicular lymphoma

Author

n

Median age (years)

Graft source

% PFS

% OS

AGVHD (grade II–IV)

% NRM

Median follow-up

Prior auto

Morris [110]

41

48

Alemtuzumab Flu/Mel URD

Related

65* (3 year)

73 (3 year)

15%

11 (3 year)

36 months

37%

Maris [141] (includes 5 SLL, 2 MZL)

45

54

2 Gy TBI ± Flu

Related URD

51 (2 year)

58 (2 year)

60%

34 (2 year)

23.8 months

27%

Khouri [142]

47

53

Flu/Cy HD rituximab

Related

85 (2 year)

88 (3 year)

NA

11 (cumul.)

34 months

17%

Kusumi [111]

45

48

Flu-based

Related URD

83 CT-sens. 64 CT-refr.

76 (3 year)

49%

18 (cumul.)

23.9 months

22%

Vigouroux [143]

73

51

Flu/BU/ATG Flu/TBI URD

Sibling

66† CR (3 year) 64 PR 32 NR

66 (3 year)

34%

32 (3 year)

37 months

34%

Regimen

52 32

28 63

AGVHD, acute graft-versus-host disease; ATG, antithymocyte globulin; BU, busulfan; CR, complete remission; CT-refr., chemotherapy refractory; CT-sens., chemotherapy sensitive; cumul., cumulative; CY, cyclophosphamide; Flu, fludarabine; HD, high dose; Mel, melphalan; MZL, mantle zone lymphoma; NR, no response; NRM, nonrelapse mortality; OS, overall survival; PFS, progression-free survival; PR, partial remission; SLL, small lymphocytic lymphoma; TBI, total body irradiation; URD, unrelated donor. * Includes patients with response to donor lymphocyte infusions. † Event-free survival.

Overall survival

Event-free survival

Survival

0.8 0.6 0.4

Event-free survival

CR PR Refractory

1.0

1.0

CR PR Refractory

0.8 0.6 0.4 0.2

0.2 0.0 0.0 0

20

40

60

80

0

20

Months

analyzed. The IBMTR reported retrospective data on 205 FL patients receiving matched related donor allogeneic HCT with high-dose (n = 120) and RI (n = 85) regimens [144]. There was not a statistically significant difference in NRM, PFS, OS or GVHD incidence between the high-dose and RI regimens. This account illustrated the notable increase in the use of RI regimens for allogeneic HCT in FL in the recent decade, with fewer than 10% of allogeneic recipients receiving RI preparative regimens in 1997, and 80% in 2002. The MD Anderson Cancer Center reported on 68 consecutive patients with chemotherapy-sensitive, relapsed FL receiving autologous versus RI matched related donor HCT [142]. Patients were eligible for a related donor HCT if they had a donor identified, were beyond first relapse or had failed a prior autologous HCT. Forty-seven patients received RI allogeneic and 21 received autologous HCT. As the eligibility criteria for RI would dictate, there were more patients having failed prior autologous HCT, beyond first relapse, and having received more than three prior regimens in the RI HCT cohort. The 3-year OS was 88% after RI allogeneic HCT and 84% after autologous HCT.

40 Months

60

80

Fig. 60.6 Overall survival and event-free survival according to the status of disease at the time of reduced-intensity conditioning allogeneic hematopoietic cell transplantation. CR, complete response group (n = 21); PR, partial response group (n = 33); refractory, stable + progressive disease group (n = 19). (Reproduced from [143], with permission.)

Allogeneic HCT has been utilized for transformed FL, with many of the trials reviewed above including small numbers of these patients in the “aggressive” or “high-grade” histology categories. The incomplete and variable data available pertaining specifically to transformed FL patients in the high-dose and RI setting prevents conclusive statements to be made. Summary of HCT for FL Autologous HCT extends the PFS of patients with relapsed FL compared with conventional therapy, but its impact on OS is less evident. The plateau in the OS curve seen in the historical comparison from St Bartholomew’s Hospital supports the possible curative potential of autologous HCT for relapsed FL early in the disease course [125]. The use of autologous HCT in FL in first remission has likely been redefined with rituximab improving the initial and subsequent outcome of FL patients. The most concerning limitations of OS for the FL patient receiving autologous HCT are not only the risk of relapsed lymphoma, but also the risk of secondary malignancies [125,132,145,146]. Allogeneic HCT

Non-Hodgkin’s Lymphoma

has had an impact on reducing the relapse risk for patients with relapsed FL, with evidence of long-term disease control but with a coincident increase in early and late NRM. How the advent of RI regimens may alter these expectations is yet to be seen. Meanwhile, there is an expanding repertoire of non-HCT as well as HCT alternatives available that include the encouraging results seen with radiolabeled antibody therapies and long-term benefits of rituximab. HCT should therefore target individuals expecting limited survival with standard therapies. As in diffuse aggressive lymphoma, chemosensitivity is an important predictor of outcome, certainly in the autologous and RI allogeneic HCT settings, and therefore chemoresponsive disease should be a prerequisite prior to these HCT approaches. As always, defining the role of HCT, whether high-dose allogeneic, RI allogeneic or autologous HCT, begs the support and participation of the medical community for comparative clinical trials. Mantle cell lymphoma Autologous HCT MCL continues to be characterized by a poor prognosis with a short time to progression and a median OS of 3–4 years [147]. Selected earlier studies of autologous HCT in MCL are summarized in Table 60.9 [148–155]. These trials included patients with variable disease states, depicting the notable difference in outcome in the patients transplanted in first remission versus those beyond first remission. The PFS ranged from 38% to 77% versus 17% to 36%, and OS ranged from 75% to 100% versus 25% to 64% at 2–5 years for patients in first remission and beyond first remission, respectively. Regardless of disease status at the time of autologous HCT, there was a pattern of continued relapse. The grim prognosis for patients beyond first remission has led investigators in more recent autologous HCT studies to target the newly diagnosed patient with advanced disease [149,155–158]. The European Mantle Cell Network completed the singular randomized autologous HCT trial for MCL in first remission. The study randomized newly diagnosed patients to high-dose radiochemotherapy and autologous HCT versus IFN-α maintenance after achieving a CR or PR to CHOPlike induction [159]. Although 296 patients were enrolled, only 122 patients were randomized and assessable for response due to ineligibility, lack of response, and patient refusal. Sixty-two patients received autologous HCT, and 60 patients received IFN-α maintenance. At a

889

median follow-up of 34 months, the 3-year PFS was significantly better after autologous HCT than IFN-α (54% versus 25%; p = 0.01). The 3year OS, however, was similar, at 83% with autologous HCT versus 77% with IFN-α (p = 0.18). Twelve patients relapsed after IFN-α maintenance, eventually receiving autologous HCT, with eight of 12 alive. The exclusion of these patients from the intention-to-treat analysis did not alter the lack of significance between the OS after autologous HCT and IFN-α. The use of more aggressive induction regimens as well as the inclusion of rituximab in induction may improve initial disease control in MCL, although long-term disease control has not been demonstrated [17,147]. The University of Nebraska reported that the use of the “hyperCVAD” (fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone plus high-dose MTX–cytarabine) rather than a CHOPlike induction regimen, with or without rituximab, was associated with improved survival after autologous HCT in MCL patients in first remission [160]. Gianni et al. supplemented a high-dose sequential autologous HCT regimen with rituximab in 28 patients with untreated, advancedstage MCL [155]. The EFS and OS at 54 months were 79% and 89%, respectively. A historical comparison to 35 age-matched controls treated with standard-dose, nonrituximab-containing chemotherapy had an EFS rate of 18% (p < 0.0001) and an OS rate of 42% (p = 0.027). Dreger et al. have most recently reported on the experience in 34 patients with newly diagnosed MCL with rituximab and sequential HDT [158]. At a median follow-up of 33 months, the 4-year EFS was 83%, and OS was 87%. A historical comparison to conventionally, nonrituximab-treated patients was conducted, with an EFS of 47% (p = 0.036) and an OS of 77% (nonsignificant). These historical comparisons are of limited value as the majority of patients will now receive rituximab as part of their induction therapy. The use of high-dose 131I-tositumomab with autologous HCT rescue in relapsed and refractory MCL patients has shown intriguing preliminary results [97]. Sixteen heavily pretreated patients with MCL received tositumomab followed by etoposide and cyclophosphamide and autologous HCT. At a short median follow-up of 19 months, the 3-year OS was 93% and PFS was 61%, without therapy-related deaths. Allogeneic HCT The available literature on high-dose conditioning and allogeneic HCT in MCL is minimal at best, likely due to the usual age of onset of the

Table 60.9 Autologous transplantation for mantle cell lymphoma Author

n

Disease status

Regimens

% Progression-free survival

% Overall survival

Years

Khouri [148,149]

33 20 8 20 40 27 42 34 12 42 152

First remission >CR/PR1 CR1 ≥CR1 >CR1 CR1 >CR1 First remission >First remission CR1 >CR1

HyperCVAD HyperCVAD CY/TBI

43 17 38 18* 36 77 39 77 30 55 35(PR1); 20(>PR1)

77 25 NA NA 65 93 64 100 54 75 50

5 3 4

Freedman [150] Vose [151] Molina [152] Dreger [153] Vandenberghe [154]

CY/TBI, BEAC, BEAM FTBI or BCNU and VP-16/CY CY/TBI FTBI or non-TBI based

2 3 2 5

BEAC, carmustine, etoposide, cytarabine, and cyclophosphamide; BEAM, carmustine, etoposide, cytarabine, and melphalan; CR, complete remission; CY/TBI, cyclophosphamide and total body irradiation; FTBI, fractionated TBI; HyperCVAD, cyclophosphamide, vincristine, doxorubicin, dexamethasone, and methotrexate–cytarabine; NA, not available; PR, partial remission; VP-16/CY, etoposide and cyclophosphamide. * Estimated from Kaplan–Meier curve in publication.

890

Chapter 60

Table 60.10 Selected studies utilizing reduced-intensity preparative regimens and allogeneic transplantation for mantle cell lymphoma

Author

Median age (years)

n

Regimen

Graft source

% PFS (2 year)

% OS (2 year)

AGVHD (grade II–IV)

% NRM

Prior auto

26 months

28%

Khouri [149]

18

56.5

FCR-13 Flu/cisp/Ara-c

Sibling Unrelated

82 (3 year)

85.5 (3 year)

17%

Maris [164]

33

53.5

2 Gy TBI/Flu

Sibling Unrelated

60

65

57%

24 (2 year)

24 months

42%

Avivi [165]

66

50

67% RI

Unrelated

28

42

35%

27 (1 year)

15 months

53%

Sibling

26

31

36%

35 (1 year)

9 months

42%

EBMT Robinson [166]

0 (100 day)

Median Follow-up

33% myelo. 144

49

EBMT

varied RI

Unrelated

2 Gy TBI/Flu, 2 Gy + total body irradiation + fludarabine; AGVHD, acute graft-versus-host disease; auto, autologous hematopoietic cell transplantation; EBMT, European Blood and Marrow Transplantation; FCR, fludarabine, cyclophosphamide, and rituximab; Flu/cisp/Ara-c, fludarabine + cisplatin + cytarabine; NRM, nonrelapse mortality; OS, overall survival; PFS, progression-free survival; RI, reduced intensity.

disease in the seventh decade of life. The initial evidence of a possible graft-versus-lymphoma effect in MCL was from a report of 16 patients receiving allogeneic HCT for relapsed (n = 11) and newly diagnosed (n = 5) MCL [161]. Fourteen patients received high-dose and two patients received RI regimens. At a median follow-up of 24 months, the 3-year failure-free survival and OS were both 55%, with a cumulative NRM of 38%. Patients with chemotherapy-sensitive disease at time of HCT had significantly improved one year failure-free progression and OS (both 90%) compared with patients with chemotherapy-refractory disease (44%; p = 0.04). The achievement of a CR in the setting of GVHD, and the conversion of positive detection of bcl-1 by PCR to negative over time post HCT, supported the graft-versus-lymphoma effect. Other investigators have reported on their experience of autologous and highdose allogeneic HCT for patients with MCL, albeit with small numbers and variable patient characteristics to date [162,163]. As with other comparisons of allogeneic versus autologous HCT in lymphoma, the NRM may obviate the benefit of reduced relapse. The use of RI regimens has been more broadly investigated than highdose regimens for MCL, again likely due to the advanced age of the MCL patient at diagnosis. The majority of clinical trials include relatively small numbers of patients, with Table 60.10 summarizing selected phase II studies [149,164–166]. The remission stages of patients included relapsed and refractory MCL with exposure to multiple prior regimens, although the majority of patients were chemotherapy sensitive prior to HCT. As in all lymphoma histologies described in this chapter, sensitivity to chemotherapy and/or disease state at the time of HCT was predictive of outcome. RI and high-dose allogeneic HCT regimens have not been formally compared; however, Roderiguez et al. conducted a retrospective analysis of 88 patients with multiple lymphoma histologies receiving conventional high-dose or RI regimens, including 15 patients with MCL [112]. Chemotherapy-sensitive disease and absence of a prior autologous HCT were associated with improved survival, whereas highdose conditioning and absence of prior autologous HCT were associated with a lower relapse rate. Summary on HCT for MCL Autologous HCT has limited benefit in the setting of relapsed or refractory MCL, although the investigation into more aggressive induction and preparative regimens including rituximab or radiolabeled antibodies

may alter these expectations. The role of autologous HCT in first remission of MCL may be evolving, with the more intensive, sequential regimens including rituximab showing remarkable disease control with the preliminary results available. Although the data on allogeneic HCT in MCL are limited to date, a graft-versus-tumor effect appears to exist, with extended follow-up required to determine the duration of disease control and the impact of the increased NRM and comorbidities associated with allogeneic HCT. Appropriately, investigation of RI regimens continues at a brisk pace as this approach reaps the benefits of the graft-versus-lymphoma effect at an apparent lower risk of NRM and morbidity. Clinical trials in MCL are sorely lacking, with the near-absence of randomized studies to investigate newer therapies, including autologous and allogeneic HCT approaches. Given the fairly rare occurrence of this disease and lack of a definitive curative therapy, all patients receiving primary and subsequent therapy for MCL should be enrolled in investigational studies. T-cell lymphomas There is a paucity of data regarding the efficacy of HCT in T-cell lymphomas, which represent only about 15–20% of NHL in Western countries (see also Chapter 70). The majority of reports are of autologous HCT, often combining multiple T-cell subtypes, including lymphoblastic, angioimmunoblastic, and the more favorable ALCL. Retrospective reviews of autologous HCT in relapsed or refractory T-cell lymphoma including 24–64 patients per report have shown 3–5year PFS of 24–50% and OS of 33–53% [167–169]. These results were quite favorable given the poor prognosis for PTCLs [170]. The poor-risk features identified in the above and other studies are the pre-HCT aa-IPI as well as chemotherapy sensitivity prior to HCT. Variably reported in the past, recent historical and case-matched comparisons have shown similar outcomes for patients with relapsed or refractory DLCL and Tcell lymphoma following autologous HCT [167,168]. Song et al., however, found the subtype of PTCL designated “not otherwise specified” had an inferior survival compared with B-cell lymphoma (EFS 23% versus 42%, p = 0.028) [167]. Autologous HCT has been utilized as consolidation in first remission T-cell lymphoma with a PFS of 59– 63% and an OS of 62–68% [169,171].

Non-Hodgkin’s Lymphoma

GEL/TAMO investigators demonstrated an improved PFS and OS in poor-risk patients with T cell lymphoma receiving autologous HCT compared with conventionally treated control patients. The GEL/TAMO group retrospectively reviewed the efficacy of autologous HCT in 35 patients with a poor response to initial therapy [172]. Partially responsive patients had a 5-year EFS of 36% and an OS of 37%, with the pre-HCT IPI predictive of survival. Blystad et al. reported a series of 41 chemosensitive T-cell lymphoma patients who received autologous HCT in Norway and Sweden [173]. Seventeen patients were in first PR or CR. OS at 4 years was 79% for the 14 ALCL patients and 44% for the remaining 27 patients. These data confirm other reports of successful autologous HCT in T-cell ALCL in relapse or initial remission [174,175]. However, the prognosis for this subtype is known to be heterogeneous, with a particularly favorable outlook for cases that express the ALK protein. Information specifically pertaining to ALK− ALCL and HCT is sparse, with one report from Zamkoff et al. describing poor survival in ALK− ALCL despite autologous HCT [176]. High-dose conditioning and allogeneic HCT in T-cell lymphoma has been explored with the minimal data available documenting decreased relapse rates but at the expense of significant NRM as in other lymphoma subtypes [169,177]. RI regimens are beginning to be explored, with encouraging preliminary results from Italy in heavily pretreated PTCL patients, with a 3-year OS of 81% and a PFS of 64%, associated with NRM of 6% at 2 years [178].

High-grade lymphomas Due to the rare occurrence of lymphoblastic and Burkitt’s lymphoma in adults, the reports of HCT have generally included only small numbers of patients. Consolidation of first remission in lymphoblastic lymphoma with autologous HCT or further chemotherapy was studied in a randomized trial in Europe in which there was a trend toward longer relapse-free survival with autologous HCT but no difference in OS [179]. Registry data were reported in lymphoblastic lymphoma patients, 128 treated with autologous HCT and 76 treated with high-dose, matched sibling donor HCT [180]. At 5 years, the leukemia-free survival was similar between allogeneic and autologous HCT, at 36% and 39%, respectively (p = 0.82). There were fewer relapses after allogeneic transplantation, but OS was inferior to autologous HCT at 6 months and not significantly different thereafter. As anticipated, regardless of type of transplantation, patients with marrow involvement and those transplanted at relapse had a less favorable outlook. The selection of appropriate induction chemotherapy and prognostic factors influence the rate of cure with primary chemotherapy as well as the transplant results. This impact is evidenced by a single-institution study by Bouabdallah et al. reporting superior outcomes with leukemia induction therapy prior to either autologous or allogeneic HCT [181]. Fewer data are available for Burkitt’s lymphoma and HCT. An OS of 72% at 3 years was observed in registry data compiled on 70 patients receiving autologous HCT in first remission [182]. These data compare favorably with conventional treatment, but excellent survival (73%) at 2 years has been reported with the use of intensive pediatric induction protocols in adult Burkitt’s lymphoma patients [36]. With this intensive induction approach, the poorest-risk patients maintained an estimated survival of 59% at 2 years. The report from the EBMTR conducting a matched comparison of 71 Burkitt’s lymphoma patients receiving allogeneic or autologous HCT showed no difference in relapse rate and improved OS in autologous HCT recipients [107]. Autologous as well as allogeneic transplantation in recurrent Burkitt’s lymphoma has been generally unsuccessful and is definitely not recommended for chemoresistant patients [35].

891

NHL of the CNS Primary CNS lymphoma One of the first reports of autologous HCT in PCNSL was in the relapsed or refractory setting including 22 patients with PCNSL or intraocular lymphoma [183]. Twenty of the patients received autologous HCT, with a 3-year OS of 60% and an EFS of 53% at a median follow-up of 41.5 months. Seven patients developed clinically significant neurologic toxicity, of whom two died. Six of these seven had prior cranial radiotherapy or were 60 or more years of age at the time of HCT. The concern for persistent relapse as well as late neurotoxicity in patients with PCNSL has led investigators to explore autologous HCT earlier in the course of treatment. Preliminary reports of several phase II trials including 13–30 patients utilizing autologous HCT as first-line therapy for PCNSL have shown DFS and OS ranging from 43% to 79% and 60% to 77%, respectively, via intention-to-treat analyses [41,184– 186]. In patients receiving HCT, the DFS and OS ranged from 43% to 90% and 60% to 87%, respectively, with approximately one-half to three-fourths of initially enrolled patients completing HCT. Illerhaus et al. reported the longest median follow-up of 63 months in 30 patients, with a stable relapse rate and OS at 3 and 5 years, but with a 16.7% incidence of late neurotoxicity [185]. In their subsequent trial, WBRT was limited to patients without a CR (four of 11). At a short median follow-up of 25 months, the 3-year DFS and OS were similar at 77% without late neurotoxicity. The use of HD MTX-containing induction regimens as well as other chemotherapeutic agents with greater penetration through the blood–brain barrier (such as high-dose cytarabine, carmustine, thiotepa, and etoposide) included in the mobilization and preparatory regimens may offer increased initial response rates and improved DFS and OS after autologous HCT. Limiting the use of WBRT while maximizing the use of the above-mentioned agents may also minimize the risk of severe late neurotoxicity that currently limits the benefits of non-HCT as well as HCT approaches. In summary, the use of autologous HCT for PCNSL for the newly diagnosed as well as relapsed or refractory patient should be limited to formal clinical trials investigating the appropriate use of drug combinations as well as the incorporation of WBRT and/or intrathecal chemotherapy in the induction, mobilization, preparative, and post-HCT regimens. There have been no notable reports of allogeneic HCT for PCNSL. Secondary CNS lymphoma Secondary CNS lymphoma refers to patients with NHL and CNS involvement as a secondary site. The incidence of secondary CNS lymphoma occurs in 5–9% of advanced lymphoma, and has been associated with poor survival after standard therapy. The impact secondary CNS lymphoma may have on the outcome of HCT has been addressed via multiple retrospective reviews [187–189]. Consistently, there has been no significant difference in outcome in patients with or without a prior history of CNS disease at the time of autologous HCT, and no clear benefit to allogeneic versus autologous HCT [189]. The EBMTR review of 62 patients with prior CNS involvement receiving autologous HCT identified active CNS disease as a predictor of poor survival [187]. The PFS of patients with cleared CNS disease at the time of HCT, compared with patients with active CNS disease at the time of HCT, was 42% versus 9%, respectively (p = 0.001). Although intrathecal chemotherapy and/or craniospinal irradiation was commonly included in these retrospective studies, there are no definitive recommendations for the use of these modalities in conjunction with HCT. To conclude, patients with a prior history of CNS involvement of advanced NHL are appropriate candidates for autologous HCT if there is no active CNS disease at the time of HCT.

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Chapter 60

Summary HCT remains a frequently utilized therapeutic modality for patients with NHL. The trend in recent years has shown a continual increase in the number of patients 50 years of age or older receiving autologous as well as allogeneic HCT due to improved supportive care and the widespread use of RI preparative regimens [2]. Consequently, the median age of the NHL patient receiving HCT is nearing the median age of onset of the majority of lymphomas. The advent of rituximab has dramatically altered the outlook of the B-cell lymphoma patient, with its true impact on the survival of these patients yet to be determined, in the HCT as well as non-HCT setting. Radiolabeled antibody therapies have also shown promise, with their feasibility and effectiveness demonstrated with autologous HCT. The investigation of novel chemotherapeutic and immunologic strategies

such as other monoclonal antibodies and vaccines in HCT may improve outcomes. Alternative allogeneic HCT regimens have shown evidence of alleviating the deleterious effects of high-dose conditioning regimens while maintaining the graft-versus-lymphoma response. The routine use of PET imaging, molecular diagnostics, and genomic profiling in the diagnosis, response assessment, and surveillance of patients may lead to more risk-adapted or targeted therapeutic approaches. Much of the available data regarding HCT in NHL are heterogeneous due to variable lymphoma histologies, disease states, and treatment modalities, with a paucity of well-controlled clinical trials. The development of cooperative groups and their subsequent coordination of clinical trials to investigate novel therapies can lead to more effective and prompt completion of clinical studies, and hence a more direct path to improving our patient’s clinical outlook.

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32.

33.

34. 35.

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41.

42.

43.

44.

45.

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152. Molina A. Autologous stem cell transplantation for mantle cell lymphoma: a report of 69 patients from City of Hope and Stanford. Blood 2002; 100: 182a. 153. Dreger P, Martin S, Kuse R et al. The impact of autologous stem cell transplantation on the prognosis of mantle cell lymphoma: a joint analysis of two prospective studies with 46 patients. Hematol J 2000; 1: 87–94. 154. Vandenberghe E, Ruiz de Elvira C, Loberiza FR et al. Outcome of autologous transplantation for mantle cell lymphoma: a study by the European Blood and Bone Marrow Transplant and Autologous Blood and Marrow Transplant Registries. Br J Haematol 2003; 120: 793–800. 155. Gianni AM, Magni M, Martelli M et al. Longterm remission in mantle cell lymphoma following high-dose sequential chemotherapy and in vivo rituximab-purged stem cell autografting (RHDS regimen). Blood 2003; 102: 749–55. 156. Lefrere F, Delmer A, Levy V, Delarue R, Varet B, Hermine O. Sequential chemotherapy regimens followed by high-dose therapy with stem cell transplantation in mantle cell lymphoma: an update of a prospective study. Haematologica 2004; 89: 1275–6. 157. Mangel J, Leitch HA, Connors JM et al. Intensive chemotherapy and autologous stem-cell transplantation plus rituximab is superior to conventional chemotherapy for newly diagnosed advanced stage mantle-cell lymphoma: a matched pair analysis. Ann Oncol 2004; 15: 283–90. 158. Dreger P, Rieger M, Seyfarth B et al. Rituximabaugmented myeloablation for first-line autologous stem cell transplantation for mantle cell lymphoma: effects on molecular response and clinical outcome. Haematologica 2007; 92: 42–9. 159. Dreyling M, Lenz G, Hoster E et al. Early consolidation by myeloablative radiochemotherapy followed by autologous stem cell transplantation in first remission significantly prolongs progressionfree survival in mantle-cell lymphoma: results of a prospective randomized trial of the European MCL Network. Blood 2005; 105: 2677–84. 160. Vose J, Loberiza F, Bierman P, Bociek G, Armitage J. Mantle cell lymphoma (MCL): induction therapy with HyperCVAD/high-dose methotrexate and cytarabine (M-C) (±rituximab) improves results of autologous stem cell transplant in first remission. J Clin Oncol 2006; 24: 7511a. 161. Khouri IF, Lee MS, Romaguera J et al. Allogeneic hematopoietic transplantation for mantle-cell lymphoma: molecular remissions and evidence of graft-versus-malignancy. Ann Oncol 1999; 10: 1293–9. 162. Kasamon YL, Jones RJ, Diehl LF et al. Outcomes of autologous and allogeneic blood or marrow transplantation for mantle cell lymphoma. Biol Blood Marrow Transplant 2005; 11: 39–46. 163. Ganti AK, Bierman PJ, Lynch JC, Bociek RG, Vose JM, Armitage JO. Hematopoietic stem cell transplantation in mantle cell lymphoma. Ann Oncol 2005; 16: 618–24. 164. Maris MB, Sandmaier BM, Storer BE et al. Allogeneic hematopoietic cell transplantation after fludarabine and 2 Gy total body irradiation for relapsed and refractory mantle cell lymphoma. Blood 2004; 104: 3535–42. 165. Avivi I, Canals C, Taghipour G et al. Matched unrelated donor stem cell transplantation for relapsed or refractory mantle cell lymphoma. A

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retrospective analysis from the EBMT Lymphoma Working Party. ASH Annual Meeting Abstracts 2006; 108: 3123a. Robinson SP, Schmitz N, Taghipour G, Sureda A. Reduced intensity allogeneic stem cell transplantation for mantle cell lymphoma is associated with substantial late transplant related mortality and a poor outcome in patients with chemoresistant disease. Blood 2004; 104: 2260a. Song KW, Mollee P, Keating A, Crump M. Autologous stem cell transplant for relapsed and refractory peripheral T-cell lymphoma: variable outcome according to pathological subtype. Br J Haematol 2003; 120: 978–85. Kewalramani T, Zelenetz AD, Teruya-Feldstein J et al. Autologous transplantation for relapsed or primary refractory peripheral T-cell lymphoma. Br J Haematol 2006; 134: 202–7. Feyler S, Prince HM, Pearce R et al. The role of high-dose therapy and stem cell rescue in the management of T-cell malignant lymphomas: a BSBMT and ABMTRR study. Bone Marrow Transplant 2007; 40: 443–50. Vose J. International Peripheral T-Cell Lymphoma (PTCL) Clinical and Pathologic Review Project: poor outcome by prognostic indices and lack of efficacy with anthracyclines. Blood 2005; 106: 811a. Rodriguez J, Conde E, Gutierrez A et al. The results of consolidation with autologous stem-cell transplantation in patients with peripheral T-cell lymphoma (PTCL) in first complete remission: the Spanish Lymphoma and Autologous Transplantation Group experience. Ann Oncol 2007; 18: 652–7. Rodriguez J, Caballero MD, Gutierrez A et al. High dose chemotherapy and autologous stem cell transplantation in patients with peripheral T-cell lymphoma not achieving complete response after induction chemotherapy. The GEL-TAMO experience. Haematologica 2003; 88: 1372–7. Blystad AK, Enblad G, Kvaloy S et al. High-dose therapy with autologous stem cell transplantation in patients with peripheral T cell lymphomas. Bone Marrow Transplant 2001; 27: 711–16. Fanin R, Ruiz de Elvira MC, Sperotto A, Baccarani M, Goldstone A. Autologous stem cell transplantation for T and null cell CD30-positive anaplastic large cell lymphoma: analysis of 64 adult and paediatric cases reported to the European Group for Blood and Marrow Transplantation (EBMT). Bone Marrow Transplant 1999; 23: 437–42. Deconinck E, Lamy T, Foussard C et al. Autologous stem cell transplantation for anaplastic largecell lymphomas: results of a prospective trial. Br J Haematol 2000; 109: 736–42. Zamkoff KW, Matulis MD, Mehta AC, Beaty MW, Hutchison RE, Gentile TC. High-dose therapy and autologous stem cell transplant does not result in long-term disease-free survival in patients with recurrent chemotherapy-sensitive ALK-negative anaplastic large-cell lymphoma. Bone Marrow Transplant 2004; 33: 635–8. le Gouill S, Milpied N, Buzyn A et al. Allogeneic stem cell transplantation (allo-SCT) in T-cell lymphomas: a French national survey from the Societe Francaise de Greffe de Moelle et de Therapie Cellulaire (SFGM-TC). J Clin Oncol 2007; 25(Suppl): 8095. Corradini P, Dodero A, Zallio F et al. Graftversus-lymphoma effect in relapsed peripheral

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T-cell non-Hodgkin’s lymphomas after reducedintensity conditioning followed by allogeneic transplantation of hematopoietic cells. J Clin Oncol 2004; 22: 2172–6. Sweetenham JW, Santini G, Qian W et al. Highdose therapy and autologous stem-cell transplantation versus conventional-dose consolidation/ maintenance therapy as postremission therapy for adult patients with lymphoblastic lymphoma: results of a randomized trial of the European Group for Blood and Marrow Transplantation and the United Kingdom Lymphoma Group. J Clin Oncol 2001; 19: 2927–36. Levine JE, Harris RE, Loberiza FR Jr. et al. A comparison of allogeneic and autologous bone marrow transplantation for lymphoblastic lymphoma. Blood 2003; 101: 2476–82. Bouabdallah R, Xerri L, Bardou VJ et al. Role of induction chemotherapy and bone marrow transplantation in adult lymphoblastic lymphoma: a report on 62 patients from a single center. Ann Oncol 1998; 9: 619–25. Sweetenham J, Pearce R, Taghipour G, Blaise D, Gisselbrecht C, Goldstone A. Adult Burkitt’s and Burkitt-like non-Hodgkin’s lymphoma – outcome for patients treated with high-dose therapy and autologous stem-cell transplantation in first remission or at relapse: results from the European Group for Blood and Marrow Transplantation. J Clin Oncol 1996; 14: 2465–72. Soussain C, Suzan F, Hoang-Xuan K et al. Results of intensive chemotherapy followed by hematopoietic stem-cell rescue in 22 patients with refractory or recurrent primary CNS lymphoma or intraocular lymphoma. J Clin Oncol 2001; 19: 742–9. Colombat P, Lemevel A, Bertrand P et al. Highdose chemotherapy with autologous stem cell transplantation as first-line therapy for primary CNS lymphoma in patients younger than 60 years: a multicenter phase II study of the GOELAMS group. Bone Marrow Transplant 2006; 38: 417–20. Illerhaus G, Marks R, Ihorst G et al. High-dose chemotherapy with autologous stem-cell transplantation and hyperfractionated radiotherapy as first-line treatment of primary CNS lymphoma. J Clin Oncol 2006; 24: 3865–70. Illerhaus G, Muller F, Feuerhake F, Schafer AO, Ostertag C, Finke J. High-dose chemotherapy and autologous stem-cell transplantation without consolidating radiotherapy as first-line treatment for primary lymphoma of the central nervous system. Haematologica 2008; 93: 147–8. Williams CD, Pearce R, Taghipour G, Green ES, Philip T, Goldstone AH. Autologous bone marrow transplantation for patients with non-Hodgkin’s lymphoma and CNS involvement: those transplanted with active CNS disease have a poor outcome – a report by the European Bone Marrow Transplant Lymphoma Registry. J Clin Oncol 1994; 12: 2415–22. Alvarnas JC, Negrin RS, Horning SJ et al. Highdose therapy with hematopoietic cell transplantation for patients with central nervous system involvement by non-Hodgkin’s lymphoma. Biol Blood Marrow Transplant 2000; 6: 352–8. Kasamon YL, Jones RJ, Piantadosi S et al. Highdose therapy and blood or marrow transplantation for non-Hodgkin lymphoma with central nervous system involvement. Biol Blood Marrow Transplant 2005; 11: 93–100.

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Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia

Introduction

Reduced apoptosis and overexpression of BCL2

Chronic lymphocytic leukemia (CLL) is the most common leukemia and predominately a disease of the elderly. Many patients with CLL have an indolent clinical course managed with expectant observation and occasional therapies for symptom management. However, these conventional chemotherapies are not curative. Targeted immunologic therapies including rituximab (anti-CD20 humanized monoclonal antibody) and alemtuzumab (anti-CD52 monoclonal antibody) have added benefit in combination with chemotherapy [1,2]. Nonetheless, some patients exhibit rapidly progressive disease, respond poorly to chemotherapy, and stand to benefit most from an aggressive approach including hematopoietic cell transplantation (HCT) if they could be identified early in their care. Fortunately, recent studies have identified molecular prognostic factors such as 17p and 11q chromosomal deletions, unmutated status of the variable heavy region of the immunoglobulin heavy chain gene (IgG VH), and cell immunophenotypes including C38 expression and zeta-associated protein 70 (ZAP-70) [3–6] that predict for patients who will have poor outcomes with conventional chemotherapy. Recent advances in HCT, including the use of reduced-intensity conditioning (RIC) followed by allogeneic HCT, have markedly decreased transplantrelated morbidity and mortality. Approximately 50% of patients with CLL undergoing RIC allogeneic HCT are achieving durable complete remissions, possibly cures.

CLL is characterized by the progressive accumulation of morphologically mature appearing small B lymphocytes, but their immature immunophenotype (CD5+CD19+CD23+) and heterogeneous heavy chain immunoglobulin rearrangements do not readily suggest a unifying etiology. Most CLL cells are nonproliferating cells arrested in G0/G1 phase of the cell cycle and are resistant to apoptosis, suggesting that an accumulation of CLL cells result from decreased apoptosis rather than proliferation. Consistent with this hypothesis, CLL B cells overexpress the antiapoptotic protein BCL2 [9]. The overexpression of the BCL2 gene has been reported in many other hematologic malignancies, and is often the result of the chromosomal translocation t(14;18)(q32;q21) that places the heavy chain immunoglobulin enhancers ahead of BCL2 [10]. However, the t(14;18) translocation of BCL2 is present in less than 5% of all patients with CLL [11].

Incidence and etiology of CLL CLL is the most common leukemia, and estimates suggest that over 15,000 men and women in the United States were diagnosed with CLL in 2007 [7]. Based on rates from 2002–04, 0.45% of men and women born today will be diagnosed with CLL some time in their lifetime [8]. While CLL is predominately a disease of the elderly, onethird of patients are under the age of 65, and over 10% of newly diagnosed CLL patients are under the age of 55. As such, there are many young CLL patients seeking curative therapy. The failure to elucidate the etiology of the disease and the variable clinical course have hampered the identification of patients who would benefit most from HCT.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

MicroRNAs MIRN15A and MIRN16-1 While chromosomal translocations are associated with many leukemias and lymphomas, no such unifying pathogenic translocation(s) have been identified in CLL. The most common cytogenetic abnormality associated with CLL is a deletion at 13q14.3 that develops in more than 50% of cases followed longitudinally [3]. Two microRNAs (miRNAs), MIRN15A and MIRN16-1, cluster within 200 kilobases of each other at 13q14.3 [12]. They are ubiquitously expressed as noncoding RNAs, with highest expression in normal CD5+ lymphocytes. miRNAs are a class of small noncoding RNAs that are similar to but distinct from small interfering RNAs, which regulate messenger RNA translation and decay [13]. The majority of CLL patients have decreased MIRN15A and MIRN16-1 expression that correlates with loss of heterozygosity in 68% of informative cases. It is proposed that MIRN15A and MIRN16-1 bind upstream of BCL2, and their overexpression causes decreased BCL2 expression, leading to increased apoptosis [14]. In vitro experiments confirm this, showing that overexpression of MIRN15A and MIRN16-1 is associated with decreased BCL2 expression and increased apoptosis in the human leukemia cell line MEG01, which is deleted at 13q14 [15]. miRNA expression studied across hundreds of probes using an miRNA microarray chip identified a unique signature of 13 miRNAs that distinguished CLL cases according to other molecular risk factors, IgG VH gene status, and ZAP-70 expression levels in association with disease progression [16]. This microarray study confirmed the importance of MIRN16-1 and MIRN15A, showing their low expression in good-risk CLL analogous to the previously described 13q14.3 deletion.

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The role of antigen stimulation in CLL

Summary of CLL etiology

B lymphocytes develop in the bone marrow, and their maturation is characterized by the rearrangement of their immunoglobulin variable (V) gene segments to create the antigen-binding domain of the B-cell receptor. When an antigen of appropriate affinity binds to the receptor, the normal B cell enters a germinal center in lymphoid follicles, where it rapidly divides and its V genes undergo somatic hypermutation [17]. This process leads to mutations in the variable heavy (VH) and variable light (VL) gene segments that encode the antigen-binding domain. This process usually requires T-cell help within germinal centers, but can occur without T cells outside the germinal centers [18]. Since B-cell leukemias are derived from the clonal expansion of a single B cell with a unique IgG VH and VL gene rearrangement, these can be used as clonal markers for an individual’s tumor population. Mutations of the V genes are detected by comparing the genes in the CLL B cells with corresponding genes in the patient’s germline. A sequence that differs from germline by more than 2% is defined as mutated and separates CLL into two groups: mutated and unmutated CLL. This has prognostic importance, as discussed later. Besides this distinction, the B-cell receptors of CLL patients have been shown to share structural similarities, suggesting that CLL B-cell receptors bind common antigens and may be relevant to their pathogenesis [19,20]. The VH1–69 gene was the first to be reported overrepresented in CLL patients [21]. The binding specificities for these overrepresented VH are mostly unknown, but an antibody using the VH1-69/VKA27 genes binds a variety of self-antigens including human immunoglobulin, myoglobulin, thyroglobulin, actin, and single-stranded DNA [22]. This and other cases of shared B-cell receptor antigen binding suggest that a limited set of endogenous autoantigens [22,23] or exogenous environmental/infectious antigens could promote CLL, similar to gastric lymphomas that develop in response to Helicobacter pylori [24].

Numerous genes have been implicated in the development of CLL, but no single unifying model supports all observations. As such, it remains probable that CLL is a heterogeneous disease with many underlying molecular defects. This heterogeneity implies that CLL patients will have variable disease courses and mixed clinical responses to available therapies. Nonetheless, researching the cause of CLL has identified useful molecular prognostic tests as follows.

The importance of microenvironment Isolated CLL cells are unable to grow in vitro, but interactions with bone marrow stromal cells [25], CD31 (the ligand for CD38) [26], and activated T cells expressing CD40 ligand [27] support the growth of CLL cells in vitro. This growth can be enhanced by the addition of stromal cell-derived factor-1 [28] and vascular endothelial growth factor [29]. These factors prevent CLL from undergoing apoptosis, and their variable expression in bone marrow and lymph nodes may account in part for interpatient variation with respect to predominate leukemic versus lymph node disease, as in small lymphocytic leukemia. Familial CLL Siblings have been noted to have clonal B cell proliferations in 8–10% of families of CLL patients [30–32]. The overall risk for a relative to develop CLL is two to seven times higher for first-degree relatives than for the general population [33]. This high familial incidence has special significance in allogeneic HCT when these siblings could be used as donors, and supports the screening of human leukocyte antigen (HLA)identical siblings for clonal B-cell proliferation during donor work-up. Recently, death-associated protein kinase-1 (DAPK1) has been implicated as a CLL tumor suppressor and was shown to be downregulated in the germline cells of affected individuals, providing a pathogenic mechanism for familial CLL; it may also be important in sporadic CLL [34]. DAPK1 is an actin filament-associated, calcium calmodulin-dependent serine/threonine kinase that promotes apoptosis in response to Fas, interferon-gamma, and tumor necrosis factor-alpha [35]. Loss of DAPK1 in CLL makes the cells resistant to apoptosis [36].

Clinical presentation, diagnosis, and staging CLL is often diagnosed in asymptomatic individuals as a result of routine blood count testing, but some patients present with profound lymphadenopathy or constitutional symptoms associated with rapidly expanding malignant cells, that is, fever, night sweats, and weight loss. Other patients present with fatigue secondary to anemia resulting from either bone marrow infiltration with CLL cells or autoimmune hemolytic anemia. The diagnosis of CLL is suggested by peripheral blood lymphocytosis and confirmed by flow cytometry of blood or marrow revealing a clonal B-cell population expressing the mature B-cell marker CD19, with lowlevel CD20 and high-level CD5 and CD23 expression [37,38]. In the World Health Organization classification, CLL is always a neoplastic B-cell disease, while the entity previously called T-CLL is now called T-cell prolymphocytic leukemia [39]. CLL expresses low-level immunoglobulin, and is monoclonal with evidence of either lambda or kappa light chain restriction. The National Cancer Institute Working Group (NCI-WG) diagnostic criteria for CLL [40] were established in 1988, with revisions in 1996, and 2008 [40,41]: 1 A lymphocyte count of over 5 × 109/L of small mature lymphocytes in the peripheral blood for more than 1 month. 2 Immunophenotype as detected by flow cytometry showing: a. light chain restriction; b. coexpression of CD19 and CD5, together with CD23 expression; c. low expression of surface immunoglobulin and the absence or low expression of CD79b. A bone marrow biopsy is not required for diagnosis but is recommended before initiating therapy [41]. A bone marrow biopsy is required to assess a complete remission and is often useful when evaluating factors that may contribute to cytopenias, including autoimmune responses, leukemia infiltration, or therapy-related causes. Computed tomography scans are not required for diagnosis, and staging relies on physical examination and blood counts only. There are two staging systems used to define CLL disease extension. The Modified Rai Staging System classifies CLL into three groups [42,43]: 1 Low-risk patients: have only peripheral blood or bone marrow lymphocytosis. 2 Intermediate-risk patients: have lymphocytosis and lymphadenopathy (stage I) and/or hepatosplenomegaly (stage II). 3 High-risk patients: have lymphocytosis as well as anemia (hemoglobin level <11 g/dL) (stage III) and/or thrombocytopenia (platelet count <100 × 109/L) (stage IV). The Binet Staging System is based on the number of lymphoid sites involved and hemoglobin and platelet values [44]: 1 Stage A: patients with hemoglobin over 10 g/dL, platelets over 100 × 109/L, and up to two lymph node sites involved. 2 Stage B: patients with hemoglobin above 10 g/dL, platelets over 100 × 109/L, and more than two lymph node sites involved. 3 Stage C: patients with hemoglobin less than 10 g/dL, and/or platelets less than 100 × 109/L regardless of the number of lymph node sites involved.

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Prognosistic markers The natural history of CLL is heterogeneous, with survival ranging from months to decades, and overall survival (OS) is grossly predicted by the Binet and Rai staging systems, which only require a physical examination and a complete blood count. Patients with low-risk disease have a median survival of 15 years, patients with intermediate-risk disease survive 5–7 years, and patients with high-risk disease have a life expectancy less than 3–4 years [45]. However, these staging systems have limitations. While low-risk patients have expected survival close to 15 years, over 25% of these low-risk patients die of causes related to CLL, 40% progress to advanced stages, and 50% ultimately require treatment [46]. This suggests that neither the Rai nor the Binet staging system can accurately predict which patients among the low-risk prognosis group will ultimately develop progressive disease [47]. Similarly, these clinical staging systems do not predict an individual’s rate of clinical progression when diagnosed with early-stage disease, or the likelihood of response to a treatment in a patient with advanced-stage disease. Markers of CLL tumor burden The lymphocyte count, lymphocyte doubling time, serum lactate dehydrogenase level, and bone marrow infiltration pattern have prognostic relevance in CLL. A lymphocyte doubling time longer than 12 months predicts long treatment-free survival and prolonged OS [48]. The bone marrow CLL histology pattern also matters, with diffuse bone marrow infiltration having a worse prognosis than a nodular pattern [49]. Relevant serologic measurements include β2-microglobulin, thymidine kinase, and soluble CD23. β2-Microglobulin is an extracellular protein that associates with the α chain of the class I major histocompatibility complex, with high values predicting poor responses to chemotherapy and shortened survival [50]. High levels of soluble CD23 at initial diagnosis predict early disease progression [51].

Fig. 61.1 The prognostic relevance of genomic alterations in chronic lymphocytic leukemia (CLL). The estimated overall survival probabilities from date of diagnosis in 325 CLL patients divided by fluorescence in situ hybridization cytogenetic results. The median survival times for the 17p deletion (n = 223), 11q deletion (n = 56), 12q trisomy (n = 47), normal karyotype (n = 57), and 13q deletion (as a single abnormality; n = 117) groups were 32, 79, 114, 111, and 133 months, respectively. (Reproduced from [3], with permission. © 2000 Massachusetts Medical Society.)

Cytogenetic prognostic markers Approximately 80% of CLL patients have cytogenetic abnormalities detected by fluorescence in situ hybridization [3]. These genomic abnormalities provide pathogenic insights as they identify candidate genes (17p13: p53; 11/q22–q23: ATM) and have prognostic significance, as shown in Fig. 61.1. The median survival times for the 17p deletion, 11q deletion, 12q trisomy, normal karyotype, and 13q deletion groups were 32, 79, 114, 111, and 133 months respectively [3]. Interphase cytogenetic abnormalities, 17p deletion, and 11q deletion have been confirmed as high-risk CLL features predicting shortened progression-free survival (PFS) and OS for untreated CLL patients treated with combined fludarabine and rituximab initial therapy [52], or in a phase III comparison of either fludarabine alone or fludarabine and cyclophosphamide (CY) initial therapy [53]. In these studies, high-risk cytogenetic features did not identify who would respond to these initial therapies, but predicted who would experience disease progression more quickly and have shortened survival. Detection of these high-risk cytogenetic features could influence initial treatment decisions because CLL patients who have CLL with deletion of chromosome 17p respond poorly to fludarabinebased therapy, and may respond better to alemtuzumab-based therapy [54–56]. As such, interphase cytogenetic assessment is recommended for patients being treated on all clinical trials, including those considering HCT [41]. Prognostic predictor: VH gene mutation status Another important prognostic indicator for CLL is VH gene mutation status [57,58]. VH IgG is considered unmutated when the CLL VH is

Fig. 61.2 Unmutated VH gene status (VH homology ≥98%) predicts shortened overall survival. Evaluating 300 patients with chronic lymphocytic leukemia (CLL), the median overall survival of 168 patients with unmutated VH gene status was 70 months, compared with 132 patients with mutated VH gene status having a median survival of 152 months. (Reproduced from [59], with permission. © American Society of Hematology.)

greater than 98% identical to germline VH immunoglobulin genes. Approximately one-half of CLL expresses unmutated VH genes and is thought to originate from postgerminal center B cells. CLL patients with unmutated VH have relatively poor clinical outcomes with rapid disease progression, shortened time to treatment, and shortened OS. Figure 61.2 shows the OS of 300 patients with CLL separated by VH mutation status, the median survival times being 79 months for the unmutated VH group

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versus 152 months for the mutated VH group [59]. Interestingly, a recent prospective analysis of 64 CLL patients over a minimum of 3 years time identified 11 patients undergoing cytogenetic clonal evolution, all 11 of whom had unmutated gene status, suggesting that unmutated VH CLL may have chromosome instability as a pathogenic mechanism [60]. While VH mutation status determination is labor-intensive and expensive, the clonal VH DNA sequence can be used to develop an allelespecific oligonucleotide (ASO) reverse transcriptase polymerase chain reaction (RT-PCR) assay to quantify minimal residual disease (MRD) with a sensitivity of detecting one CLL cell in 100,000 normal blood cells [61]. Nonetheless, to make prognostic markers more accessible to clinical laboratories, prognostic immunophenotypes detectable by flow cytometry have been sought. Prognostic predictors: CD38 and ZAP-70 Unmutated VH status was first shown to associate with CD38 expression on the CLL cell surface [57]. Subsequent genome-wide gene expression studies of CLL characterized as either mutated or unmutated VH showed a close association with the presence of ZAP-70 [62]. ZAP-70 is highly expressed in natural killer and T cells, while normal B cells have low or no ZAP-70 expression. ZAP-70 detected on B cells by flow cytometry strongly correlated with VH unmutated gene status [6,26]. However, subsequent measurements of both ZAP-70 and CD38 showed interlaboratory variations, making their validity as a surrogate for unmutated gene status or as independent poor-prognosis markers controversial. Eventually, it became clear that approximately 10–30% of CLL clones are discordant between ZAP-70 and VH [5,26,59,63,64]. This led to additional independent cytogenetic risk factors being sought in ZAP-70negative, VH unmutated poor-risk CLL patients and identified 11q deletion, 17p deletion, and V3-21 usage [63]. Together, these data suggest that a prognostic model for CLL can be based on either genomic alterations and ZAP-70 expression or VH mutation status, high-risk genomic aberrations, and V3-21 usage. This need for multiple independent prognostic predictors suggests again that CLL is a heterogeneous disease with multiple pathogenic mechanisms. Prognostic index for OS in previously untreated CLL patients In order to improve upon the Rai and Binet staging systems, Wierda et al. evaluated 1674 previously untreated CLL patients managed at M.D. Anderson Cancer Center from 1981 to 2004, and developed a weighted prognostic index and nomogram to predict 5- and 10-year survival [65]. The six prognostic factors included: age, β2-microglobulin, absolute lymphocyte count, sex, Rai stage, and number of involved nodal groups. This model has not been externally validated, and does not consider molecular assessments that are independently shown to be powerful predictors. Further, Wierda’s model was developed evaluating patients whose treatment spanned the last 20 years of the 20th century, and did not account for their therapies ranging from observation to allogeneic HCT. At this time, a prognostic index for CLL that omits consideration of cytogenetic abnormalities or VH mutation status could be predicted to have limited utility.

Response criteria Assessment of response includes a careful physical examination and evaluation of blood and marrow. According to the revised NCI-WG guidelines, a complete response (CR) requires a normal physical examination, lymphocytosis of 5 × 109/L or less, the absence of clonal lymphocytes, a hemoglobin level of over 11 g/dL, and a platelet count of

greater than 100 × 109/L. Patients with residual CLL cells by conventional flow cytometry or immunohistochemistry are defined as having a partial response (PR) [41].

Methods for detecting MRD in CLL patients The National Cancer Institute-sponsored working group guidelines (NCI-WG) for response to CLL were an adequate tool for measurement of the therapeutic response when conventional alkylating therapies were used [40]. The introduction of fludarabine provided more effective therapies than alkylating agents alone [66,67]. Likewise, alemtuzumab can result in dramatic eradication of CLL from both blood and bone marrow [68–70], and when rituximab or alemtuzumab is used in combination with fludarabine and alkylating reagents, many patients achieve complete remission testing with conventional flow cytometry. For example, combination chemotherapy incorporating fludarabine, CY, and rituximab (FCR) achieved a 70% complete remission rate [1]. However, the NCI-WG criteria do not require four-color flow cytometry or ASO VH IgG PCR assessment for MRD. Nonetheless, MRD assessment may be necessary to distinguish these improved CLL disease responses, and MRD development following HCT supports their added utility. These molecular techniques can detect a single CLL cell in 10,000–1,000,000 normal cells [71]. The potential advantages of MRD assessment include: (1) more discriminating prognostic prediction; (2) more sensitive quantification of complete remission; and (3) potentially a surrogate for complete disease eradication and cure. The malignant CLL clone can be identified by its unique IgG VH rearrangement. Two PCR strategies can be used to detect the CLL heavy chain gene sequence: consensus primer PCR and allele specific oligonucleotide (ASO) ASO-PCR. Consensus PCR is less sensitive and amplifies the third complimentary-determining region of the IgH gene using a standard set of universal oligonucleotide primers [72]. Consensus primers may fail due to mutations in the IgH gene, and sensitivity is reduced by the presence of normal polyclonal B-cell products. Consensus primer PCR can detect CLL when it represents 1–2% of total B cells [73]. A more sensitive PCR strategy termed ASO-PCR uses oligonucleotide primers specific for the individual patient’s malignant clone in the PCR amplification step and thereby overcomes the interference of normal B cells seen with consensus primers. ASO-PCR is currently the most sensitive technique for MRD detection of CLL, with reported sensitivity as low as 1 CLL cell in 106 leukocytes [61]. However, the design of ASO primers is time-consuming, labor-intensive, expensive, and not routinely available. Flow cytometry MRD relies on multiparameter antibody detection. The initial MRD flow assay used a combination of four antibodies (CD5, CD19, CD20, and CD79B) with a sequential gating strategy to identify CLL cells from normal B cells [74]. Recently, a group of laboratories with an interest in MRD detection in CLL have collaborated under the auspices of the European Research Initiative on CLL to develop a consensus for MRD assessment [75]. Their assessment of 50 CLL-specific antibody combinations identified three with low interlaboratory variation: CD38/CD19/CD5, CD22/CD81/CD19/CD5, and CD43/CD79b, CD19/CD5. Two research groups have compared methods of MRD detection following HCT. Bottcher et al. prospectively compared MRD assessment in 74 CLL patients after HCT using four-color flow cytometry (CD19, CD5, CD43, and CD20) in parallel with consensus IgH PCR and ASOPCR. MRD flow cytometry was shown to be more sensitive than consensus IgH PCR since 23.6% of the samples that were MRD flow positive were polyclonal by consensus PCR. However, ASO-PCR was more sensitive than MRD flow cytometry, with 15% MRD samples CLL positive by ASO-PCR and negative by flow cytometry [76]. Similar results are reported by Moreno et al. studying 40 patients with CLL after

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HCT [77]. In 254 samples from 30 patients, MRD was simultaneously evaluated by ASO-PCR and flow cytometry, with good correlation (r = 0.826). In 103 paired peripheral blood and bone marrow samples from 28 patients analyzed by ASO-PCR, a concordance between results in both sample sites was observed (r = 0.786), suggesting that peripheral blood MRD assessment reflects bone marrow CLL disease status. However, the CLL MRD consensus group cautions that marrow analysis is necessary to detect MRD within 3 months of alemtuzumab therapy [75].

Relapses and refractory disease

CLL therapy

Autologous HCT

Figure 61.3 shows a schema for the diagnosis, prognostic evaluation, and therapeutic options in CLL. For patients with low-risk CLL (Rai stage 0–1/Binet A, 13q14 cytogenetics, and VH mutated gene status), observation with clinical follow up every 3–6 months is usually acceptable. Symptomatic disease progression from observation or intermediate-risk disease at diagnosis may be managed with standard therapy. For decades, the standard therapy was chlorambucil, but this approach is now primarily used in patients with comorbidities. Three randomized trials have demonstrated significantly higher CR rates and longer PFS using fludarabine-based therapy compared with alkylator-based therapies [66,67,78]. A subsequent randomized trial has demonstrated that fludarabine plus CY combined therapy is superior to single-agent fludarabine as initial therapy, providing improved overall response rates, CR, and PFS [79]. Phase II trials of monoclonal antibodies directed against B-cell surface antigens suggest improved benefit. Rituximab, a chimeric humanized anti-CD20 antibody, and alemtuzumab, a humanized rat antiCD52 antibody, are effective as single agents or in combination therapy. Combining purine analogs with an alkylating agent and rituximab (i.e. FCR) has been reported to provide overall response rates of 95% and CR rates of 70% [1]. In addition, these combination regimens can achieve molecular remissions. However, higher response rates have not yet demonstrated improved OS, and most patients treated with FCR will eventually relapse.

As the purpose of this text is to summarize the results of HCT for CLL, this chapter will provide a detailed review of high-dose conditioning and autologous HCT as it has been used since the late 1980s to treat patients with CLL. It is important to emphasize that no randomized controlled trials comparing autologous HCT with either conventional chemotherapy or chemotherapy combined with immunotherapy have been reported. Nonetheless, clinical and molecular remissions have been reported using autologous HCT even in patients with poor prognostic features [82,83].

The retreatment of relapsed CLL with combination therapy using purine analogs, alkylating agents, and rituximab can be effective, but typically has lower response rates and a shorter duration of response [7]. Patients who are refractory to purine analog therapy can be salvaged using alemtuzumab alone or in combination [80,81]. Patients being treated with alemtuzumab should receive Pneumocystis jiroveci and varicella zoster prophylaxis, and cytomegalovirus reactivation should be monitored.

Autologous HCT is feasible for patients with relapsed CLL Relative to other hematologic malignancies, autologous HCT was introduced to CLL care relatively late, presumably due to concern that persistent CLL would contaminate the graft. Addressing this concern, the first clinical trial of autologous HCT for CLL was performed at the Dana-Farber Cancer Institute (DFCI) employing in vitro “purging” of the bone marrow graft with anti-CD20, anti-CD10, B5 monoclonal antibodies via complement depletion. Rabinowe et al. reported an early 12-patient pilot study of a preparatory regimen using total body irradiation (TBI) and CY chemotherapy employing “purged” autologous bone marrow grafts [84]. All patients achieved timely hematologic

CLL evaluation and treatment

CLL diagnosis 1) Physical exam and CBC 2) Blood flow cytometry

Fig. 61.3 Diagnosis, prognostic evaluation, and treatment options for chronic lymphocytic leukemia (CLL). Risk-stratified CLL disease assessment guides treatment. As shown in bold, high-risk patients are predicted by cytogenetic abnormalities 11q22− and 17p13−, VH unmutated gene status, and IgG VH 3-21 usage. High-risk patients follow a treatment course that begins with standard combination therapy to cytoreduce their disease, and once high-risk patients achieve adequate disease control, they should immediately be considered for reduced-intensity conditioning allogeneic hematopoietic cell transplantation (HCT). CBC, complete blood count; FISH, fluorescence in situ hybridization. See text for other abbreviations.

Prognostic evaluation 1) Rai/Binet Clinical Stage 2) FISH cytogenetics 3) IgG VH mutation status 4) Consider ZAP70 and CD38

Low risk Rai stage 0-1, Binet A FISH: 13q14 or 13qIgG VH mutated (except VH 3-21) Zap 70–, CD38–

Intermediate risk Raistage 0-II, Binet A-B FISH: normal, 12+ IgG VH mutated (except VH 3-21) ZAP 70–, CD38+ or–

Therapeutic options

Observation

Good response

progression

Standard treatments FR, FCR, PCR, CHOPR

Poor response or fludarabine refractory

High-risk and CR

Salvage therapies or research protocols High risk Rai stage 0-IV, Binet A-C FISH: 11q22–, 17p13– IgG VH unmutated IgG VH 3-21 ZAP 70+

Reduced intensity conditioning allogeneic HCT

902

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reconstitution and 11 achieved clinical CR. Six of the 12 obtained molecular remissions by clonal IgG gene rearrangement PCR detection studies. The DFCI continued this approach for over 10 years and recently reported the mature results of 137 CLL patients [85]. Retrospective VH IgG sequencing showed that 90% of the patients had poor-risk unmutated VH IgH gene status. The six-year OS was 58%, but there was no plateau evident on the survival curve, suggesting autologous HCT is probably not curative. Khouri et al. reported a similar 11-patient early feasibility study using TBI–CY conditioning with anti-CD19 immunomagnetic bone marrow graft purging [86]. Six patients achieved CR, and five had no detectable MRD by VH IgG PCR analysis. These early feasibility studies treated CLL patients who were relatively young, with a median age of 45 and 48, respectively. Nonetheless, these studies demonstrated the autologous HCT was relatively safe and frequently achieved CR, including some molecular remissions, supporting additional autologous HCT studies. Prospective single-arm studies evaluating the efficacy of autologous HCT in patients with relapsed/refractory CLL continued to predominately use TBI–CY conditioning throughout the 1990s. Pavletic et al. treated 16 CLL patients who had a median age of 49 years using autologous mobilized peripheral blood progenitor cells (PBPCs), reporting a remarkable 100% CR attainment [87]. The projected 3-year OS and failure-free rate were 68% and 37%, respectively. Dreger et al. evaluated chemotherapy conditioning using sequential dexa-BEAM (dexamethasone, carmustine, etoposide, cytarabine, and melphalan) with granulocyte colony-stimulating factor (G-CSF)-mobilized PBPCs in 18 young patients with a median age of 49 years. These PBPC grafts were purged by immunomagnetic B-cell depletion using CD19, CD20, CD23, and CD37 monoclonal antibodies [88]. Sutton et al. treated CLL patients with ESHAP (etoposide, methylprednisolone, high-dose cytarabine, and cisplatin), salvage chemotherapy followed by TBI–CY conditioning, and unmanipulated PBPC graft infusion. Of the 20 patients enrolled, sufficient hematopoietic cells to proceed to autologous HCT were harvested in only eight patients, raising the concern that successful hematopoietic cell harvest might be impeded by aggressive salvage chemotherapy [89]. Schey et al. used CD34selected PBPC grafts to treat 10 patients following fludarabine induction treatment followed by TBI–CY conditioning. All 10 patients achieved CR, and seven achieved molecular remissions 3 months after HCT, but subsequent molecular relapses occurred at 6, 9, 12, and 24 months post transplant [90]. These molecular disease assessment studies have shown that MRD following autologous transplant may not be sustained in most CLL patients, again suggesting that autologous HCT is not curative. Table 61.1 provides a comprehensive summary of all reported autologous HCT studies showing encouraging clinical and molecular responses with low transplant-related mortality ranging from 0% to 6%. Unanswered questions derived from these initial feasibility studies include those relating to the timing of CLL, “purging” benefit, the relative efficacy of conditioning regimens, and molecular risk assessment for both patient selection and post-transplant molecular surveillance. Up-front autologous HCT following induction chemotherapy Throughout the 1990s multiple groups had performed small trials demonstrating that autologous HCT for CLL patients was feasible, but only the United Kingdom Medical Research Council (MRC) performed a prospective clinical trial to determine the benefit of autologous HCT following initial induction chemotherapy [91]. One hundred fifteen CLL patients aged 60 years or younger with previously untreated CLL requiring treatment for Binet stage B or C or progressive stage A disease were

enrolled in the years 1996–2001. Eligible patients were treated with single-agent fludarabine 25 mg/m2 for 5 days. The aim of the study was to achieve a major response before proceeding to hematopoietic cell harvest, but only 22 (19%) attained CR. Forty-four (38%) required further treatment with alternative agents before PBPC mobilization. The addition of these agents only increased the number of patients in CR to 23%, but the overall response rate was 83%. CY and G-CSF mobilization was attempted in 88 patients (77%), and in only 49 (66%) were at least 2.0 × 106 CD34+ cells/kg body weight collected. Thus, 33% failed to mobilize adequate PBPCs following fludarabine induction. Overall, only 65 of 115 enrolled patients (56%) proceeded to autologous HCT after receiving at least three cycles of fludarabine. However, the autologous HCT provided significant clinical benefit; disease status improved from 37% CR before mobilization to 74% after autologous HCT. Similar improvements were measured using molecular assessments: none of the CLL patients tested MRD negative before autologous HCT, but of 20 patients tested, 16 (80%) achieved molecular remission in the first 6 months following transplantation. Evaluating all 115 enrolled patients, the 5-year DFS and OS were 52% and 78%, respectively, and if only the 65 patients who received autologous HCT are considered, the DFS and OS were 64.5% and 88.6%, respectively. Since the MRC trial was a single-arm prospective trial, it was unable to demonstrate any therapeutic benefit for autologous HCT following induction chemotherapy in comparison to conventional chemotherapy, but the high CR and MRD rates were encouraging. Three potential problems for up-front autologous HCT were identified by the MRC trial: (1) few CLL patients achieved CR following fludarabine induction; (2) PBPC collection was frequently inadequate following fludarabine therapy despite waiting 12 weeks to begin mobilization; and (3) secondary malignancies may limit the benefits of autologous HCT [84]. While single-agent fludarabine as initial therapy provides overall response rates of 50–80%, most large trials report CR rates of 20–40% [66,67,92]. However, current induction therapy might use fludarabine in combination with CY and rituximab (FCR), which has a reported CR rate of 70% [1]. Thus, the benefit of autologous HCT following a more effective induction therapy, such as FCR, may differ significantly from that in the MRC study. However, the one-third failure rate for PBPC mobilization does highlight mobilization problems following fludarabine. A French multisite prospective study evaluated the success of G-CSFor glycosylated G-CSF (lenograstim)-primed PBPC mobilization in 38 CLL patients in first remission after oral front-line therapy with fludarabine and CY [93]. Despite delaying initiation of mobilization until 2 months after the last chemotherapy, only 17 (45%) of the patients had apheresis attempted with the first mobilization, and only six (16%) achieved a target CD34 cell collection. Univariate analysis showed that a low platelet count immediately prior to mobilization predicted failure (p = 0.009). Further, all patients with platelet counts less than 150 × 109/L failed to mobilize adequately. Similarly, other studies have reported poor PBPC mobilization results following fludarabine therapy [94,95], and attemping PBPC collection following fludarabine therapy warrants caution. The development of secondary myelodysplastic syndrome/acute myelogenous leukemia (MDS/AML) after TBI-based regimens has been a concern for all patients undergoing autologous HCT. Gribben et al. reported an MDS/AML incidence of 9% in their recent long-term followup report of patients transplanted with TBI–CY conditioning in the early 1990s [85]. The median time from transplant to diagnosis of MDS/AML was 35 months, with a range of 1–138 months. The MRC experience with TBI–CY conditioning resulted in a similar rate of MDS/AML complications [91]. Subsequent analysis of the MRC study reports the 5-year actuarial risk of developing MDS/AML post autologous graft was

11

16

8 10

137

Rabinowe 1993 [84]

Khouri 1994 [86]

Dreger 1998 [88]

Pavletic 1998 [87]

Sutton 1998 [89] Schey 1999 [90]

Gribben 2005 [85]

51

54 51

49

49

48

45

TBI/CY

TBI/CY TBI/CY

TBI/CY

TBI/CY

TBI/CY

TBI/CY

Conditioning regimen

179

Dreger, 2004 [83]

51

51 TBI/CY

TBI/CY

4 BM, 16 PBPC unmanipulated PBPC CD34+ selected, and antibody depleted Immunomagnetic bead purge 83%

16 BM +/− PBPC 49 PBSC; no purges

3 BM, ex vivo antibodies 10 PBPC purged 3 BM, 13 PBPC No graft purge 8 PBSC No graft purge 8 PBPC CD34+ selected, 2 PBPC no selection BM, ex vivo antibodies

BM, ex vivo antibodies

BM, ex vivo antibodies

Graft purge

78% 3 months

85% 20 months

5% 5%

65% 25 months

74%

93% 6 months

100% 100% 3 months

–

83% 5 months follow-up 55% CR 36% nCR 92% 4 years

Complete response

10%

1.5%

16.7% (6 yrs)

0% 0%

6%

0%

9%

8%

Transplant-related mortality

–

10 MRD–

–

85% (20 months)

13/16 MRD− –

75% (25 months)

64.7% (5 years)

30% (6 years)

75% (2.5 years) –

15/19 MRD−

16/20 MRD−

–

– 7/8 MRD–

37% (3 years)

55%

5 MRD–

–

–

–

MRD

Disease-free survival (median follow-up)

84% (4 years)

95% (20 months)

90% (25 months)

88.6% (5 years)

58% (6 years)

–

68% (3 years)

–

–

–

Overall survival (median followup)

1 myelodysplastic syndrome

–

–

5/65 (8%)

16 (12%) (8 years)

– –

–

–

–

–

Second malignancy incidence

n, number of patients undergoing conditioning and receiving an autologous graft. BEAM, BCNU, etoposide, cytarabine, and melphalan; BM, bone marrow; CR, complete response; CY, cyclophosphamide; MRD, minimal residual disease; PBPC, peripheral blood progenitor cell; TBI, total body irradiation.

20

Dreger, 2000 [99]

Upfront autologous HCT for high-risk CLL Meloni, 2000 [143] 20 47 BEAM

Autologous HCT as postinduction consolidation Milligan 2005 [91] 65 49 49 TBI/CY 11 BEAM

12

13

n

Study

Median age

Table 61.1 Selected studies of autologous hematopoietic cell transplantation (HCT) for patients with chronic lymphocytic leukemia (CLL)

Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia

903

904

Chapter 61

even higher at 12.4%, with a median time to development post transplant of 39 months [96]. The risk of MDS/AML seems to be associated with the use of TBI, and the 8–12% observed incidence of MDS following TBI–CY autologous HCT must be offset by a benefit in terms of OS that remains to be demonstrated.

Autologous HCT for patients with high-risk CLL To improve the prognosis of patients with aggressive CLL, Dreger et al. implemented a sequential treatment strategy combining intensive chemotherapy mobilization using one or two cycles of dexa-BEAM, which achieved CR in 16 of 18 patients. Grafts were successfully collected in 14 recipients (three bone marrow and 11 PBPC) and were then purged using immunomagnetic methods and infused following TBI–CY conditioning [88]. With a median follow-up of 20 months, 17 patients remained in CR. Sixteen patients were molecularly analyzed by VH IgG-specific PCR, with 50% of the patients remaining MRD negative 6 months after transplant. However, there is no long-term follow-up to confirm the benefits of early transplant in high-risk patients. The German CLL study group conducted a prospective multicenter trial (GCLLSG CLL3) to assess the efficacy of early autologous HCT in patients with poor-risk CLL, enrolling 179 patients from 1997 to 2002. Pre-transplant cytoreduction was varied, and patients’ best documented CR and PR was 28% and 64%, respectively. Grafts purged by immunomagnetic beads were obtained in 83% of the patients, and 98 of 128 (77%) patients who underwent mobilization proceeded to transplant. Three months post transplant, CR was reported in 78% and PR in 21% of patients. With follow-up of 36 months, an intent-to-treat analysis showed that the median PFS of all 128 patients was 59 months [83]; however, no long-term outcomes have yet been published.

Is autologous HCT superior to conventional chemotherapy? There are no randomized studies that compare autologous HCT with conventional chemotherapy, but such a trial has already accrued patients in the European intergroup effort coordinated by the European Group for Blood and Marrow Transplantation (EBMT). Two hundred twenty CLL patients in first or second remission after conventional chemotherapy were randomized to consolidation autologous HCT or observation. The primary endpoint is event-free survival from the time of randomization, but analysis will not be available before 2009 [97]. In the absence of randomized results, Dreger et al. performed a riskmatched comparison of 66 CLL patients who had undergone autologous HCT with control patients gathered from a database of 291 CLL patients conventionally treated matching for age, Binet disease stage, IgG VH gene mutation status, and lymphocyte count [82]. Forty-four pairs were matched for all four variables, and patients were well matched at other risk factors including genomic abnormalities and CD38 expression. With a median follow-up in excess of 70 months, survival was significantly longer for the autologous HCT patients than for the conventionally treated patients when calculated from either time of diagnosis or study entry. The benefit remained significant when analysis was restricted to the 29 pairs with unmutated IgG VH gene status. One potential bias to the superior autologous HCT results was that the median observation time from diagnosis was shorter for the autologous HCT patients than for the conventional therapy patients (70 months versus 86 months; p = 0.03), providing a possible lead time bias. However, in the absence of a randomized control trial, this case-matched comparison provides evidence that autologous HCT may prolong survival in high-risk CLL patients beyond conventional therapy.

A similar case-matched comparison of autologous HCT and conventional therapy conducted by the EBMT and the French CLL cooperative group was reported at the 2002 American Society of Hematology meeting, but publication has not followed [98]. Their analysis compared 621 autologous HCT patients from the EBMT database with 630 conventionally treated patients from the French CL90 trial (which compared fludarabine treatment and two alkylator–anthracycline regimens) [78]. The primary endpoint was OS at 60 months, and autologous HCT was inferior (p = 0.0003). As such, the results of the ongoing randomized trials comparing autologous HCT with conventional therapy will be required to clarify the potential benefits of autologous HCT for patients with CLL.

Ex vivo monoclonal antibody purging of autologous grafts Autologous HCT provides high response rates, but most patients eventually relapse, suggesting that either grafts are contaminated with CLL or the HCT conditioning is insufficient to eradicate CLL disease. To address the “graft contamination” concern, numerous ex vivo purging strategies have been developed using monoclonal antibodies to eradicate CLL from the autologous graft before reinfusion. In the two original autologous HCT studies, Rabinowe et al. depleted bone marrow cells using anti-CD20, anti-CD10, and B5 antibodies followed by complement cytotoxicity [84], while Khouri et al. used anti-CD19 immunomagnetic bead separation [86]. Later, Dreger et al. combined positive and negative selection employing ISOLEX technology to positively select CD34 hematopoietic stem cells, followed by combined CD19, CD20, CD23, and CD37 monoclonal antibody depletion [99]. All of these strategies provided rapid durable engraftment with high rates of CR and MRD. However, excessive depletion of lymphocytes from the graft can result in poor immune reconstitution following HCT, especially when patients have already been heavily pretreated with chemotherapy including lymphocyte-depleting agents, fludarabine, and alemtuzumab. For example, Altes et al. employed a two-step positive ISOLEX CD34 selection followed by anti-CD19 monoclonal antibody immunomagnetic bead depletion strategy to treat 13 peripheral blood hematopoietic cell grafts collected from six CLL patients and seven other patients with non-Hodgkin’s lymphoma [100]. The ex vivo purge effectively removed disease from each graft. All 13 PBPC grafts measured MRD positive prior to manipulation, but 12 (92%) became MRD negative with purging. Ten patients underwent autologous HCT, and were in CR testing MRD negative 1 and 3 months post transplant. However, three of the 10 patients died 9, 10, and 13 months post transplant, due to infection, and two others suffered herpes simplex infections despite acyclovir prophylaxis. The overall nonrelapse mortality (NRM) rate was 30%, and the authors cautioned that they observed delayed immune reconstitution of peripheral B and T cells [100]. Ex vivo purging strategies provided rapid durable engraftment with high rates of CR and molecular remission. However, in the absence of randomized clinical trial data, their overall benefit remains unclear. Purging strategies require sufficient PBPC mobilization to allow for graft manipulation losses, and may be precluded in heavily pretreated patients or those receiving fludarabine [91]. Finally, excessive lymphocyte depletion can cause delayed immune reconstitution and excessive infectious complications. Evaluating “purging” through syngeneic HCT In essence, syngeneic twin studies reveal the maximum benefit that graft purging might provide, and the 19-patient Center for International Blood and Marrow Transplant Research (CIBMTR) identical twin

Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia

transplantation outcomes for CLL showed little benefit [101]. These 19 patients with CLL were transplanted between 1980 and 2001 using genetically identical twin grafts and predominately TBI–CY conditioning. With 89-month follow-up, the 5-year relapse rate was 50%, and the estimated 5-year survival was 61%. Interestingly, there was molecular evidence that one patient developed CLL 6 years after HCT that differed from the original pretransplant CLL but was identified as the CLL that subsequently also developed in the twin donor. This observation unfortunately confirms the familial risk for CLL in sibling donors and emphasizes the need for careful donor screening. The 5-year relapse rate of 50% was thought to be consistent with the overall autologous transplant experience in CLL, arguing that bone marrow in vitro purging is not the limiting factor for CLL treatment improvement. Thus, investigators have moved their attention to improved conditioning with in vivo purging using targeted immunotherapy. In vivo purging and autologous HCT Relapse following syngeneic HCT suggests that CLL recurrence following autologous HCT is mostly due to malignant cells surviving highdose conditioning. The first reported in vivo purge following transplant used a monoclonal anti-CD19 antibody called B4 conjugated to the protein toxin “blocked ricin” (anti-B4-bR). Grossbard et al. treated 12 patients following TBI–CY autologous HCT with dose-escalating antiB4-bR via 7-day continuous infusion dosing [102]. All patients were in CR at the time of transplantation, and 11 of the 12 remained in complete remission between 13 and 26 months after transplant, but repeat dosing was precluded in most by human antimouse antibody development, and disease recurred [103]. CLL expresses low-level CD20+ [104], but single-agent rituximab (anti-CD20 humanized monoclonal antibody) provides only brief partial responses against CLL in the peripheral blood, and bone marrow CLL persists [105,106]. In contrast, CD52 is abundantly expressed on CLL [107], and alemtuzumab as a single agent provides effective CLL depletion from blood and bone marrow [68–70]. Alemtuzumab treatment frequently achieves molecular remission in patients who have been fludarabine refractory, and remains one of the few effective therapies for p53-deficient CLL [108]. These results provided the rationale to use alemtuzumab for in vivo purging prior to hematopoietic cell collection and autologous HCT. In a phase II study by Montillo et al., 34 patients who had had a clinical response to fludarabine-based therapy received alemtuzumab 10 mg subcutaneously three times a week for 6 weeks [109]. PBPCs were collected after mobilization with cytarabine and GCSF. CR rates improved from 35% after fludarabine induction to 79% after alemtuzumab consolidation, including 19 patients (56%) who achieved MRD negativity. PBPC collection was successful in 24 of 26 patients (92%), and 18 patients underwent autologous HCT. The most common side-effects were alemtuzumab injection site reactions, fever, and cytomegalovirus reactivation. This study showed that alemtuzumab does not compromise G-CSF mobilization of PBPC and demonstrated promising efficacy: 17 of the 18 patients remain in remission with a follow-up of 14.5 months after autologous HCT [109]. Subsequently, Zenz et al. reported a skin rash consistent with autologous graft-versus-host disease (GVHD)-like syndrome that developed in seven (58%) patients after alemtuzumab was incorporated in the autologous HCT regimen [110]. In this German CLL3C trial, 16 patients received six doses of alemtuzumab on days 10 through day 2 prior to TBI–CY conditioning for autologous HCT. Hematopoietic cells had been collected following dexa-BEAM mobilization. Twelve of the 16 patients who received alemtuzumab, TBI, and CY developed unexplained skin rashes between 43 and 601 days after transplant. Eleven of these patients were treated with corticosteroids. In seven patients, a

905

clinical diagnosis of autologous GVHD was made, and histologic findings were compatible with grades I and II GVHD in the five patients in whom a skin biopsy was performed. It is striking that alemtuzumab provided as a single-agent therapy before hematopoietic cell mobilization is well tolerated, but when incorporated into a TBI–CY conditioning regimen results in unacceptable cutaneous toxicity most consistent with autologous GVHD syndrome. Ironically, preliminary studies suggest that the addition of alemtuzumab to RIC regimens reduces the severity of GVHD [111]. Additional studies are necessary to clarify the utility of alemtuzumab in autologous HCT, but in vivo purging before mobilization looks like a promising strategy. Significance of MRD after autologous HCT Autologous HCT was the first treatment modality that frequently provided CR rates exceeding the NCI-WG criteria for which MRD assessment became necessary. Gribben and co-workers at the DFCI first implemented MRD assessment in CLL management by developing a PCR-based method using CDR III consensus primers [112]. With follow-up ranging from 2 months to 5 years after autologous HCT, they initially reported that MRD negativity persisted, suggesting that their in vitro bone marrow graft purging strategies combined with high-dose chemotherapy might be curative. However, longer follow-up showed that their CLL patients did relapse following autologous HCT [85], and others have shown the loss of MRD predicted relapse [91,113,114]. Rawstron et al. made similar conclusions for assessment of MRD by flow cytometry by studying 104 patients treated with either alemtuzumab or autologous HCT. Twenty-five of these patients achieved complete remission by NCI-WG criteria, but six of these 25 patients had residual CLL in their bone marrow detected at over 0.05% [74]. These authors concluded that patients who have detectable MRD at any time following monoclonal antibody therapy or autologous HCT will experience progressively increasing levels of CLL in their peripheral blood and eventually relapse. In summary, MRD studies following autologous HCT suggest that detectable MRD by any method is highly predictive of eventual clinical disease recurrence. If a patient is destined to become MRD negative, this occurs immediately after autologous HCT, and no further fall in detectable disease occurs after 3 months post transplant [115]. While early series of autologous HCT reported that 61–76% of CLL patients achieved MRD-negative status [74,114], high-risk CLL patients rarely achieved MRD-negative status following autologous HCT. Ritgen et al. reported that PCR negativity was achieved in only six of 26 (23%) patients with unmutated CLL following autologous HCT, and their MRD was not durable. In contrast, the kinetics of MRD after allogeneic HCT differ strikingly from those after autologous HCT. MRD persistence early after allogeneic HCT is not associated with increased risk of relapse, and delayed development of molecular remission 6–12 months after transplant has been described [114,115]. Figure 61.4 provides an example of delayed attainment of MRD following allogeneic HCT from our own Stanford University experience. A 48-year-old man with high-risk CLL (del 17p and unmutated VH IgG) underwent RIC allogeneic HCT using total lymphoid irradiation and antithymocyte globulin (ATG) with rituximab infused 2 months after HCT as per the current Stanford CLL protocol [116,117]. This patient’s bone marrow had 10% CLL infiltration before HCT, and his peripheral blood measured 75,000 VH IgG copies/µg DNA by ASO quantitative PCR measurement. His CLL disease persisted early after allogeneic HCT but then became MRD negative by 150 days post HCT, and molecular remission persisted. Rituximab infusion (375 mg/m2) infused 56, 63, 70, and 77 days after HCT may provide improved disease control early post RIC allogeneic

VH Ig copies/mg DNA

906

Chapter 61

150000 100000 50000

Rituximab

8000 6000 4000 2000 0 0

100

200

300

400

500

Time (days) Fig. 61.4 Minimal residual disease (MRD) assessment of a patient with high-risk chronic lymphocytic leukemia (CLL). A 48-year-old male with del17p and unmutated VH immunoglobulin G (IgG) underwent reducedintensity conditioning allogeneic hematopoietic cell transplantation (HCT) using total lymphoid irradiation and antithymocyte globulin with rituximab infused 2 months after HCT at Stanford University [116,117]. This patient’s bone marrow had 10% CLL infiltration before HCT, and his peripheral blood measured 75,000 VH IgG copies/µg DNA by VH IgG allele-specific oligonucleotide quantitative polymerase chain reaction. CLL disease persists early after allogeneic HCT but then becomes MRD negative by 150 days post HCT, and molecular remission persists. (Miklos, unpublished data.)

HCT, as suggested by this patient’s MRD assessment. We hypothesize that anti B-cell therapy (rituximab) may prevent or diminish chronic GVHD [118], and this chronic GVHD prophylaxis trial is ongoing. Figure 61.4 shows that allogeneic HCT can provide durable MRDnegative clinical remissions that might indicate complete disease eradication, that is, cure. Since there are no randomized control trials of either autologous or allogeneic HCT, MRD remains a useful surrogate for disease response.

Allogeneic HCT using high-dose conditioning regimens Allogeneic HCT offers the benefits of high-dose chemotherapy conditioning, a tumor-free graft, and a potential for immune-mediated graftversus-leukemia (GVL) benefit. Many other forms of leukemia have been cured following allogeneic HCT; for example, genetic randomization of high-dose conditioning and allogeneic HCT versus chemotherapy for acute lymphoblastic B-cell leukemia favors allogeneic HCT treatment [119]. Nonetheless, the GVL benefits of allogeneic HCT could be offset by high transplant-related morbidity and mortality. An early EBMT registry study of 54 patients, median age 41 years, with CLL reported a transplant-related mortality of 46% and a 3-year probability of survival of 46%. Nearly all of the patients received TBI–CY conditioning and HLA-identical sibling bone marrow grafts. Ten-year clinical outcomes were reported for the entire 54 patient CLL cohort, demonstrating 41.2% OS and 36.6% leukemia-free survival [120]. This first long-term registry study suggested that CLL can be cured by conventional allogeneic HCT with long-term continuous complete remission observed up to 16 years after transplantation. Table 61.2 summarizes the high-dose allogeneic HCT studies for CLL. The published high-dose allogeneic HCT experience is a compilation of small, single-institution patient series including 23–30 patients and a few larger registry studies. Although the CIBMTR registry study by Horowitz et al. has yet to be published beyond abstract form [121],

it is evident that high-dose conditioning and allogeneic HCT provides long-term OS ranging from 33% to 60% with 5 years of follow-up. However, despite the inclusion of relatively young patients, the overall NRM rates remain a daunting 24–50%. Doney et al. reported the early Seattle experience showing a 40% NRM, but six of the seven patients who received busulfan and CY developed sinusoidal obstructive syndrome (SOS), and all seven patients treated with busulfan and CY died within 3 years [122]. However, this experience preceded pharmacokinetically targeted dosing with busulfan. Importantly, the Seattle study demonstrated that beneficial GVL responses often developed beyond 90 days post transplant. Pavletic et al. reported clinical outcomes for 38 patients with CLL receiving unrelated donor bone marrow grafts with predominant TBI– CY conditioning regimens [123]. Fifty-five percent of patients were chemorefractory, and 89% had received prior fludarabine-containing regimens. Of note, 10 of the 38 donors were HLA mismatched, and seven (18%) of the 38 patients had graft failure. Three developed secondary graft failure, and all three were HLA mismatched. Nonetheless, the 5-year OS was 33%, with significant losses due to the 38% NRM [123]. Moreno et al. addressed the significance of unmutated VH gene status following high-dose conditioning allogeneic HCT. The risk of relapse at 5 years in 20 CLL patients who had autologous HCT or 14 who underwent allogeneic HCT was 66% and 17%, respectively (p = 0.01), again suggesting that allogeneic HCT can more effectively treat poor-risk CLL [124]. An overall review of high-dose conditioning allogeneic HCT for patients with CLL confirms that longstanding disease-free survival (DFS) is possible, including the use of unrelated donors, but was limited by NRM. In order to overcome treatment-related complications, investigators moved to RIC to improve the tolerability of allogeneic HCT while taking advantage of the potent GVL effect. Promising RIC allogeneic HCT regimens Regimen-related toxicity, GVHD, and infections were perceived to be the main cause of treatment failure after high-dose conditioning allogeneic HCT for CLL, yielding 24–50% NRM (Table 61.2), but RIC could potentially decrease NRM and maintain the GVL benefit. While autologous HCT and high-dose allogeneic HCT trials uniformly applied the same TBI–CY conditioning regimen, RIC regimens for CLL have so far varied widely between research centers. Nonetheless, these varied concurrent trials provide similar OS and PFS, suggesting that the allogeneic GVL response is the critical component. Another difference distinguishing the RIC approach is that it no longer limits therapy to a single conditioning regimen and PBPC infusion. Rather, the initial RIC and graft infusion often follow an integrated customized pretransplant chemotherapy regimen intended to debulk the patient’s CLL, and then the evaluations following HCT combined clinical assessment along with molecular disease quantification and donor chimerism determination. This strategy enables customized immune suppression adjustments and targeted immune therapies including rituximab or donor lymphocyte infusions (DLIs) to mange early disease progression following HCT. DLI both boosts donor chimerism and improves disease control through increased GVL allogeneic immunity. This monitored allogeneic RIC HCT strategy is predicted to be most effective treating slowly growing hematologic malignancies, allowing time enough for the donor’s alloimmune response to develop an effective GVL effect before the disease progresses. This presumed benefit of RIC allogeneic HCT for CLL has been recently confirmed when Kahl et al. analyzed the single-institution relapse rates of 834 patients undergoing RIC allogeneic HCT using 2 GY TBI with or without fludarabine. This analysis included 82 CLL patients,

Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia

907

Table 61.2 Selected studies of high dose conditioning and allogeneic HCT for patients with CLL

Study (location) Michallet, 1996 [120] (IBMTR, EBMT registry study) Pavletic, 2000 [144] (Omaha, singleinstitution series) Horowitz, 2000 [121] (IBMTR registry study) Doney, 2002 [122] (Seattle, singleinstitution series) Khouri, 2002 [145] (Houston, singleinstitution series) Toze, 2005 [146] (Canadian, two institution series) Gribben, 2005 [85] TCD (Boston, singleinstitution series) Pavletic, 2005 [123] NMDP/IBMTR registry study)

Conditioning regimen

Graft source

NRM (%)

Acute GVHD grade ≥II (%)

39

51 CY/TBI 3 BU/CY

Sib: 100% BM: 100%

50

31

31

41% (10 year)

37% (10 year)

46

61

16 VP/CY/TBI 6 CY/TBI

Sib: 88% BM: 70%

35

54

68

65% (5 year)

62% (5 year)

242

47

50

42% CY/TBI 33% CY/TBI+

Sib: 78% BM: 75%

30

NR

NR

44% (3 year)

45% (3 year)

25

47

64

17 CY/TBI 7 BU/CY

Sib:100% BM: 88%

40

56

40

NR

32% (5 year)

28

43

68

27 CY/TBI 1 BEAM

Sib: 79% BM: 82%

32

49

64

45% (5 year)

42% (5 year)

30

48

47

15 CY/TBI 15 BU/CY

Sib: 79% BM: 82%

47

52

65

39% (5 year)

39% (5 year)

25

47

NR

25 CY/TBI

Sib: 100% BM: 100%

24

NR

NR

24% (6 year)

55% (6 year)

38

45

55

29: CY/TBI 6: TBI + other

Sib: 0% BM: 100%

38

45

85

30% (5 year)

33% (5 year)

Median age

Refractory patients (%)

54

41

23

Patients (n)

Chronic GVHD (%)

Percentage progression-free survival (years of projection)

Percentage overall survival (years of projection)

BEAM, BCNU, etoposide, cytarabine, and melphalan; BM, bone marrow; BU, busulfan; CY, cyclophosphamide; VP, etoposide; GVHD, graft-versus-host disease; IBMTR, International Bone Marrow Transplant Registry; NMDP, National Marrow Donor Program; NR, not reported; NRM, nonrelapse mortality; OS, overall survival; PFS, progression-free survival; Sib, sibling; TBI, total body irradiation; TCD, T-cell depleted.

showing that their relapse rate was 0.26 per patient year follow-up, and was lower than for any other type of hematologic malignancy [125]. Table 61.3 summarizes the results of published allogeneic HCT using RIC regimens to treat patients with CLL. As is evident from Table 61.3, the use of RIC has decreased the NRM to 15–20%, comparing favorably with the 40% NRM associated with high-dose allogeneic HCT. This NRM reduction was primarily accomplished by reducing the regimenrelated organ toxicities and improving infection control. Rates of acute and chronic GVHD have remained unchanged. Most RIC allogeneic HCT reports are limited to projected 2-year clinical outcomes. The RIC regimens vary widely. Schetelig et al. used a combination of fludarabine, busulfan, and ATG, while Khouri et al. employed fludarabine and CY and the latter added rituximab [126,127]. Sorror et al. used 2 GY TBI and fludarabine, while Delgado et al. incorporated fludarabine, melphalan, and alemtuzumab [128,129]. Brown et al. used fludarabine and busulphan alone [130], and Dreger et al. reported the EBMT registry experience of 77 patients with CLL transplanted following a variety of RIC regimens [131]. Schetelig et al. reported the first RIC allogeneic HCT experience treating 30 patients with fludarabine, busulfan, and ATG conditioning followed by PBPC graft infusions from equal numbers of related and unrelated donors [126]. Twelve of the 30 (40%) achieved a CR, and late CRs developed up to 2 years after HCT, suggesting that the GVL effect persisted post HCT and might even be curative. MRD was monitored in

eight patients, and six have persistent MRD-negative status with 7 months to 3 years of follow-up. With a median observation of 24 (range 7–43 months) months, 23 patients (72%) are alive, and the NRM was 15% [126]. Khouri et al. employed an RIC regimen using a combination of fludarabine and CY to treat 17 patients with CLL whose disease was refractory or recurred after prior fludarabine response. Their strategy maximized beneficial immune responses by tapering immune suppression within 100 days of HCT and then incorporating either DLI or rituximab therapy for patients with progressive disease. DLI was not given if the patient had already developed GVHD, but these patients with GVHD were treated with rituximab and sometimes attained molecular remission. The benefits of rituximab led the investigators to incorporate rituximab into the RIC regimen itself, with 10 patients receiving rituximab therapy on day −13 (375 mg/m2) and on days −6, +1, and +8 (1000 mg/m2) relative to transplantation. A significant survival difference was observed in this group of 10 patients receiving rituximab along with fludarabine and CY conditioning (FCR), attributed to markedly decreased GVHD with rituximab treatment. A recent update of this FCR RIC regimen treating 39 patients with advanced CLL assessed the impact of ZAP-70 expression, and reported an OS of 48% with 27 months of follow-up [132]. Interestingly, patients who were ZAP-70+ had an equivalent 56% OS rate, and DLI increased their disease-free status from 30% to 53%. By multivariate analysis,

54

53

41

46

57

31

87

53

37

53

33

46

FLU/BU

Alemtuzumab FLU/MEL

2 Gy TBI/FLU as above

2 Gy TBI/FLU

Mixed 11 20 FLU/ MEL

7 FC, 10 FCR

Mixed

FLU/BU, ATG

Conditioning regimen

Sib: 33% PBPC: 98%

Sib: 59% PBPC: 85%

As above

Sib: 69% PBPC:100%

Sib: 100% PBPC:100%

Sib: 81% PBPC: 91% Sib: 100% PBPC: 94%

Sib: 50% PBPC: 90%

Graft source

17

26

23 (5 years)

22

22

22

18

15

NRM (% at 2 years)

34

41, any grade

55 As above

55

40

29

34

56

Acute GVHD grade ≥II (%)

53

33

50

50

65

6

57

75

Chronic GVHD (%)

34

45

39 (5 year)

52

70 (5 year)

60

56

67

Percentage progressionfree survival (2 years)

54

51

50% (5 year)

60

72 (5 year)

80

72

72

Percentage overall survival (2 year)

ATG, antithymocyte globulin; BM, bone marrow; BU, busulfan; FC, fludarabine and cyclophosphamide; FCR, fludarabine, cyclophosphamide, and rituximab; FLU, fludarabine; GVHD, graft-versus-host disease; NRM, nonrelapse mortality; PBPC, peripheral blood progenitor cell; Sib, sibling; TBI, total body irradiation.

56 as above

64 as above

53

30

56

54

17

64

54

77

Sorror, 2005 [128] (Seattle, single-institution study) Update of Sorror, 2008 [133] (Seattle, single-institution study) Delgado, 2006 [129] (London, multi-institution study) Brown, 2006 [130] (Boston, single-institution study)

50

30

Schetelig, 2003 [126] (German multi-institution study) Dreger, 2005 [131] (EBMT registry study) Khouri, 2004 [127] (Houston, single-institution series) Caballero, 2005 [147] (Spain, multi-institution study)

Median age

Patients (n)

Study (location)

Refractory patients (%)

Table 61.3 Selected studies of reduced-intensity conditioning and allogeneic HCT for patients with CLL

908 Chapter 61

Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia 100

100

90

90

80

80

Probability, %

70 60 Allogeneic, Myeloablative Conditioning (N=290)

50

Allogeneic, Reduced Intensity Conditioning (N=317)

909

70

Autologous (N=342)

60 50

40

40

30

30

20

20

10

10

P < 0.001

0

0 0

1

2

3

4

5

6

Years Fig. 61.5 Probability of survival after autologous and human leukocyte antigen-identical sibling transplants for chronic lymphocytic leukemia (CLL). Overall survival of 949 CLL patients who underwent hematopoietic cell transplantation (HCT) for CLL were reported by the Center for International Blood and Marrow Transplant Research (CIBMTR) [134]. This registry analysis includes many different conditioning regimens classified using the CIBMTR operational definition of myeloablative (high-dose conditioning) including total body irradiation doses of ≥500 cGY single dose or ≥800 cGY fractionated, busulfan of more than 9 mg/kg, and melphalan of over 150 mg/m2 given as single agents or in combination with other agents. Reprinted with the permission of CIBMTR Director, Dr Mary Horowitz.

chemorefractory disease at transplantation and mixed donor T-cell chimerism at day 90, but not ZAP-70 status, were correlated with the risk of progression after HCT. This observation argues strongly that a GVL effect following allogeneic RIC HCT can overcome poor-prognosis refractory CLL. Sorror et al. treated 64 chemotherapy refractory CLL patients with RIC using 2 GY TBI with (n = 53) or without (n = 11) fludarabine [128]. With two-thirds unrelated donors, their NRM was relatively low at 22% despite an acute GVHD (grade II–IV) rate of 55%. Overall, the 2-year incidence of relapse/progression was only 26%, and 11 patients who attained CR remained MRD negative. The 2-year OS and DFS were 60% and 52%, respectively. An update of these same 64 patients with a median of 5 years of follow-up was recently reported, showing that the clinical and molecular remissions achieved in this poor-prognosis CLL patient population were durable, with only 8% of the 25 patients in CR at 2 years later progressing, and 10 patients remain MRD negative [133]. At 5 years, their OS and DFS were 50 and 39%, respectively, but 24% of the living patients remain on immune suppression for chronic GVHD management. In multivariate analysis, lymphadenopathy in excess of 5 cm, but not cytogenetic abnormalities, predicted relapse, and patients with lymphadenopathy less than 5 cm and no comorbidities had a 5-year OS of 71% [133]. This result emphasizes the importance of aggressive pretransplant cytoreduction for patients with persistent bulky lymphadenopathy. Overall, the data on RIC allogeneic HCT support the following recommendations. First, pretransplant cytoreductive therapy should be aggressively pursued to decrease CLL bone marrow involvement to less than 5% [130] and lymphadenopathy to less than 5 cm [133]. Second, the incorporation of rituximab into the RIC regimen or early postallogeneic HCT is well tolerated, providing targeted antitumor therapy and possibly reduced GVHD [116,127]. Third, post-HCT disease control is associated with attainment of full donor chimerism [130,133]. Finally, allogeneic RIC HCT should be pursued earlier in poor-risk patients to avoid patients developing comorbidities or excessive refractory disease burden [130,133]. In total, these RIC results provide overwhelming

evidence of robust anti-CLL GVL responses in poor-risk CLL patients, and support continued trials with RIC. The CIBMTR published the OS rates for 949 CLL patients who underwent HCT for CLL (autologous HCT, high-dose conditioning allogeneic HCT, and RIC allogeneic HCT), shown in Fig. 61.5. This registry analysis includes many different conditioning regimens classified using the CIBMTR operational definition of myeloablative (high-dose conditioning), including TBI doses of 500 cGY or more as a single dose or 800 cGY or more fractionated, busulfan over 9 mg/kg, and melphalan greater than 150 mg/m2 given as single agents or in combination with other agents [134]. While allogeneic HCT patients have increased early NRM, their survival curves appear to plateau, suggesting that the GVL response may ultimately cure these patients. In contrast, patients with CLL who were treated with autologous HCT experienced much less early NRM, but their sloping survival curve suggests that CLL disease frequently recurs and autologous HCT is not curative. One hopes that improvements in RIC allogeneic HCT regimens will further decrease early NRM and further increase OS rates, providing more CLL patients with curative therapy.

HCT recommendations for CLL This chapter has reviewed the response of CLL to autologous and allogeneic HCT. In the process, detailed molecular and clinical prognostic markers including the Rai and Binet staging systems, cytogenetic abnormalities (Fig. 61.1), IgG VH mutation status (Fig. 61.2), and associated immunophenotype markers ZAP-70 and CD38 have been associated with poor outcome following conventional chemotherapy treatment. Numerous CLL treatment guidelines [135–139] have been developed that allow for risk-stratified CLL disease management. Figure 61.3 presents a flow diagram incorporating clinical and molecular prognostic evaluation guiding risk-stratified treatment recommendations. It is clear that GVL allogeneic immunity following RIC allogeneic HCT can provide durable disease control even for patients with poor-risk CLL, and RIC allogeneic HCT potentially cures CLL.

910

Chapter 61

Low-risk patients have early-stage disease, with good prognosis cytogenetic findings (13q−), and mutated VH status. At the time of diagnosis, most patients have low-risk disease, and can be reassured that they are likely to have an initial asymptomatic period, and can be expectantly observed off therapy. They should be educated to recognize signs of disease progression including lymph node enlargement, constitutional symptoms, and rapid lymphocytosis. Their clinical evaluations, including complete blood count checks, should be obtained every 3–6 months, with repeat prognostic evaluations performed when disease clinically progresses. The progression of cytogenetic and immunophenotype characteristics suggests that CLL disease undergoes clonal evolution in association with more aggressive disease behavior, and supports repeating a patient’s CLL molecular assessment when their disease progression or treatment failure necessitates new treatments. Intermediate-risk patients have normal or trisomy 12 cytogenetics as well as mutated VH gene status. Intermediate disease is also defined by clinical progression scored by the Rai and Binet intermediate stages, and their symptomatic problems warrant standard combination immunotherapy and chemotherapy treatments including purine analog therapy combined with monoclonal antibodies such as FCR therapy or others. The goal of this combination therapy is symptomatic relief. Those patients who do not respond to initial therapy should be considered for more aggressive salvage therapies, autologous HCT, experimental research protocols or RIC allogeneic HCT since fludarabine-refractory patients have a 10-month median survival and only 22% respond to first-line salvage chemotherapy [140]. Fortunately, newer combination therapies incorporating alemtuzumab have reported overall response rates for salvage approaching 50% in fludarabine-refractory CLL patients [80,141]. Once a CLL patient achieves CR, he or she should be closely followed with routine clinical and complete blood count assessment for evidence of disease progression every 3–6 months.

High-risk patients are not defined by their clinical stage, but are strongly predicted by cytogenetic abnormalities 11q22− and 17p13− [3], VH unmutated gene status, and IgG VH 3-21 usage [59]. After patients with CLL receive treatment, they can demonstrate additional poor prognostic factors defined by their clinical response, including fludarabine resistance [140], a short time from diagnosis to needing first therapy (<12 months) [142], and an incomplete response to fludarabine [1]. Thus, in Fig. 61.3, high-risk patients follow a treatment course shown in bold that begins with standard combination therapy to cytoreduce their disease, and once high-risk patients achieve adequate disease control, they should immediately be considered for RIC allogeneic HCT. RIC allogeneic HCT does not include high-dose conditioning, and highrisk patients who do not achieve required disease control should proceed to additional salvage therapies including autologous HCT or alemtuzumab-containing regimens to gain better disease control before proceeding to RIC allogeneic HCT. RIC regimens developed for the treatment of patients with CLL vary widely, and yet all provide acceptable disease control, further emphasizing the importance of GVL alloimunity in providing durable remission and possible cures. Nonetheless, RIC regimens need to be compared, which will require uniform pretransplant molecular risk assessment and CLL disease quantification to provide accurate efficacy comparisons. Clearly, the gold standard for outcomes remains OS, but the intermediate goal is attainment of CR and MRD negativity, measured by either ASO PCR quantification or multiparameter flow cytometry. Until the optimal RIC conditioning regimen, graft source, and immune prophylaxis regimen have been determined, poor-risk CLL patients should receive RIC allogeneic HCT on prospective clinical trials. These patients’ cases should be discussed with a transplant center as early as possible to avoid extensive cytotoxic pretreatment or disease transformation, and to identify potential donors.

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Hematopoietic Cell Transplantation for Chronic Lymphocytic Leukemia 117. Lowsky R, Takahashi T, Liu YP et al. Protective conditioning for acute graft-versus-host disease. N Engl J Med 2005; 353: 1321–31. 118. Cutler C, Miklos D, Kim HT et al. Rituximab for steroid-refractory chronic graft-versus-host disease. Blood 2006; 108: 756–62. 119. Goldstone AH, Richards SM, Lazarus HM et al. In adults with standard-risk acute lymphoblastic leukemia, the greatest benefit is achieved from a matched sibling allogeneic transplantation in first complete remission, and an autologous transplantation is less effective than conventional consolidation/maintenance chemotherapy in all patients: final results of the International ALL Trial (MRC UKALL XII/ECOG E2993). Blood 2008; 111: 1827–33. 120. Michallet M, Archimbaud E, Bandini G et al. HLA-identical sibling bone marrow transplantation in younger patients with chronic lymphocytic leukemia. European Group for Blood and Marrow Transplantation and the International Bone Marrow Transplant Registry. Ann Intern Med 1996; 124: 311–15. 121. Horowitz M, Montserrat E, Sobocinski K et al. Hematopoietic stem cell transplantation (SCT) for Chronic Lymphocytic Leukemia (CLL). American Society of Hematology, San Francisco, CA. Blood; 2000: 522a. 122. Doney KC, Chauncey T, Appelbaum FR. Allogeneic related donor hematopoietic stem cell transplantation for treatment of chronic lymphocytic leukemia. Bone Marrow Transplant 2002; 29: 817–23. 123. Pavletic SZ, Khouri IF, Haagenson M et al. Unrelated donor marrow transplantation for B-cell chronic lymphocytic leukemia after using myeloablative conditioning: results from the Center for International Blood and Marrow Transplant research. J Clin Oncol 2005; 23: 5788–94. 124. Moreno C, Villamor N, Colomer D et al. Allogeneic stem-cell transplantation may overcome the adverse prognosis of unmutated VH gene in patients with chronic lymphocytic leukemia. J Clin Oncol 2005; 23: 3433–8. 125. Kahl C, Storer BE, Sandmaier BM et al. Relapse risk in patients with malignant diseases given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2007; 110: 2744–8. 126. Schetelig J, Thiede C, Bornhauser M et al. Evidence of a graft-versus-leukemia effect in chronic lymphocytic leukemia after reduced-intensity conditioning and allogeneic stem-cell transplanta-

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62

Raymond L. Comenzo & Morie A. Gertz

Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis

Introduction Amyloidosis is the term for a group of diseases in which abnormal proteins self-assemble to form extracellular deposits of insoluble nonbranching linear fibrils that are 7–10 nm in width, vary in length and resist proteolysis [1–4]. Medical investigators at autopsy recognized such deposits over three centuries ago and thought the material was a fatty substance, hence the term “lardaceous disease” [5]. In 1854, the pathologist Virchow described the deposits in post mortem liver tissue with the botanical term “amyloid,” meaning starch, because of their starch-like affinity for iodine. Under the light microscope, the deposits appear amorphous and hyalinized, eosinophilic with hematoxylin and eosin staining. When viewed in polarized light after being stained with Congo red, however, they exhibit a characteristic color change from hyaline pink to flaky yellow/green, a dichromic feature first reported in 1927 (Plate 62.1) [3]. A century after Virchow, the characteristic βpleated sheet configuration and fibrillar nature of amyloid were identified by electron microscopy (Plate 62.1) [6,7]. In the 1970s, researchers established that all amyloid variants have fibril-precursor proteins that self-assemble and become first filaments and then fibrils [8–11]. In patients with light chain (AL-) amyloidosis the fibril-precursors are usually free light chains (FLCs) with an abnormal tertiary structure. These FLCs aggregate into oligomers that may be toxic to cellular metabolism and then assemble as filaments of β-pleated sheets, four or six of which coil together to form relatively insoluble fibrils. In patients with hereditary amyloidosis, the fibril-precursor protein is often a mutant form of the protein transthyretin, although other mutant proteins such as fibrinogen Aα, apolipoproteins I and II, and lysozyme can occur. In amyloidosis associated with chronic inflammation (so-called “secondary” amyloidosis), the fibril-precursor protein is an acute phase protein called serum amyloid A (SAA). There is also a form of amyloidosis that occurs almost exclusively in elderly men and is caused by fibrils formed from normal transthyretin. The etiology of this form of amyloid, called senile systemic amyloidosis, is poorly understood; often this form involves the heart (senile cardiac amyloidosis) and, in contrast to the rapidly progressive cardiac amyloidosis due to the AL type, can cause cardiac arrhythmias and heart failure that worsens gradually over several years. The term “primary amyloidosis” is of historic interest only and was used when the patient did not have a secondary cause or a positive family

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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history [12]. In that older nomenclature, primary actually referred to amyloid as being idiopathic. Until recently, it was also used to refer to AL-amyloidosis, a form almost always associated with a monoclonal plasma cell disorder that is most commonly not multiple myeloma. It is uncommon for the clinical course of a patient with AL-amyloidosis to be significantly impacted by associated myeloma. Patients with ALamyloidosis who do not have myeloma at presentation have a risk of only 0.5% of subsequent evolution to multiple myeloma. Currently, the classification system designates the amyloid-forming protein by the first letter A for amyloid and subsequent letter(s) for the fibril-precursor protein: L for light chain and TTR for transthyretin, for example. The type of amyloidosis also designates the disease. In AL-amyloidosis, nearly three-quarters of the pathologic clones secrete λ light chains, while, in contrast, in the normal repertoire and in multiple myeloma the κ-to-λ ratio is 3:2. This difference speaks to an intrinsic amyloid-forming propensity of λ light chains. AL-amyloidosis is of particular interest to specialists in plasma cell diseases because it is a clonal plasma cell disease [13–15]. AL-amyloidosis is a disorder both of protein conformation and of clonal plasma cells [16]. In theory, the treatment of AL-amyloidosis could be directed at various aspects of its pathogenesis (Fig. 62.1). Most frequently, as in multiple myeloma, therapies have aimed to reduce or eliminate clonal plasma cells, the source of the aberrant FLCs. By the mid-1990s, limited progress had been made with standard chemotherapy in reversing the inexorable progression of systemic ALamyloidosis, not surprisingly in view of the visceral dysfunction it causes. At that time, because the efficacy of autologous hematopoietic cell transplantation (HCT) had been demonstrated in myeloma, HCT was tested in systemic AL-amyloidosis. When HCT was successful, the production of deposits was halted and prior amyloid deposits were slowly resorbed. Organ function, performance status, and quality of life could improve [17–20]. However, in the early clinical trials, transplantrelated mortality was high because the organ reserve of patients was compromised by cardiac, gastrointestinal and neuropathic amyloid involvement. This made patients with AL-amyloidosis distinct from other stem cell transplant candidates [21–23]. Not surprisingly then, the evolution of HCT for patients with AL-amyloidosis has been characterized by refinement of patient selection and improvement of peritransplant clinical management [21,24,25]. In this chapter, we describe the epidemiology, pathogenesis, and distinctive clinical aspects of this disease and offer an approach to the evaluation of patients with AL-amyloidosis. We summarize recent clinical research in HCT for AL-amyloidosis and identify novel emerging therapies.

Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis

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P

P P

Fig. 62.1 Theoretical remedies for AL-amyloidosis. There are a number of points at which intervention could, in theory, control AL-amyloidosis: (1) reducing the number of plasma cells with cytoreductive therapy; (2) reducing the secretion of clonal free light chains with drugs; (3) impairing the selfassembly and aggregation of light chains; (4) inhibiting fibril formation in tissues; and (5) dissolving or enhancing the proteolysis of amyloid fibrils. Remedies that reduce the number of plasma cells include standard and highdose chemotherapy. Although it is currently not available, 4-iodo-4-deoxydoxorubicin (IDOX) may enhance the proteolysis of fibrils. The other possible remedies remain theoretical.

Epidemiology The epidemiology of AL-amyloidosis is difficult to define because the disease often goes undiagnosed. Data from tertiary referral centers are not necessarily representative. AL-amyloidosis is a rare disorder with an age-adjusted incidence estimated to be 5.1–12.8 per million person– years, resulting in approximately 1275–3200 new cases annually in the United States and 255–640 cases in the United Kingdom, an incidence similar to that of chronic myelogenous leukemia or Hodgkin’s disease [26]. AL-amyloidosis appears to be more common in men, but the difference may be due to self-selection and referral bias. There have been no links with race, occupation or environmental exposures. Sixty percent of patients with AL are between 50 and 70 years of age at diagnosis [26]. The median age of patients with amyloidosis is 63 years, with 2% under 40 years of age. We have seen patients as young as 26 and as old as 89 years of age. AL-amyloidosis is approximately one-fifth as common as multiple myeloma, but it confers a worse prognosis since the median survival of patients within 1 month of tissue diagnosis is 13.2 months without treatment [26].

Pathogenesis The final pathway in the pathologic process of amyloidosis is the formation and deposition of fibrils [2,4,27–30]. When the amyloid subunit proteins are solubilized and deciphered by amino acid sequencing, the dominant constituent of deposits is the light chain variable region, and less frequently part of the constant region or the whole immunoglobulin [31–35]. Although the reason why some FLC-form amyloid remains unknown, comparisons of the primary structures of normal and amyloidforming FLCs have provided a partial answer based on the aberrant

915

properties of the pathologic FLCs [36–41]. The basis for light chain amyloid formation appears to be an effect of primary structure on conformation [36,37,42–44]. Analyses of primary structure have emphasized the critical contribution of uncommon amino acid substitutions to the stability and interactive properties of misfolded or partially folded light chains [39,40]. Such partially folded intermediate forms may be prone to self-assemble. The identification of potentially destabilizing uncommon amino acid substitutions at unique positions in the variable regions of amyloid-forming FLCs has supported this hypothesis [45,46]. Other modifications of light chain conformation such as post-translational glycosylation may also predispose FLCs to self-assemble [32]. Purified urinary light chains from patients with AL-amyloidosis reproduce fibrillar disease when injected into mice, while those from myeloma patients without AL-amyloidosis do not [47]. If the pathogenesis of AL-amyloidosis were a function of fibrillar amyloid burden, however, patients with minimal tissue deposits would be relatively asymptomatic. But this is not always the case. A patient’s clinical status is not determined simply by amyloid burden. Patients with peripheral nervous system or cardiac involvement can have minimal tissue amyloid but experience symptomatic organ-system failure. Conversely, a significant fraction of patients who respond to treatment with elimination of the monoclonal FLCs do not show a reduction in amyloid burden despite clinical improvement and abatement of symptoms, when amyloid burden is evaluated with serial 123-iodine-labeled serum amyloid P protein (SAP) total body scintiscans [48]. Such observations have led to the view that amyloid fibrils per se may not be the sole pathologic basis of amyloid disease [2,4,49–52]. Experiments in vitro with several fibril-precursor proteins have shown that intermediate forms or oligomeric aggregates may be toxic to cells [53–57]. This has led some to suggest that fibrils may represent a more benign form of the precursor protein and may provide relative protection from cellular toxicity until the burden of fibrils becomes overwhelming [2,58]. The relationship between intermediate forms and fibrils in the various types of amyloidosis remains undefined, and the mechanistic role of intermediates in disease is unclear. However, if experimental systems can be developed to elucidate these mechanisms, we may improve our understanding of disease pathogenesis and be able to pursue novel protective therapies. How amyloid fibrils cause organ dysfunction is not known, and how fibrils are metabolized and organs improve is no less a mystery. The liver, for example, can tolerate a large burden of amyloid without failing, and, once treatment eliminates the supply of FLCs, the liver can respond with restoration of normal synthetic function and dramatic regression to normal size. These changes can happen over months. This is attributed to the regenerative capacity of the liver but begs the question as to how the fibrils are removed. Removal, when it occurs, may be related to the presence of other components of the fibrillar deposits. The interactions between amyloid-forming FLCs and the passenger constituents of amyloid deposits, such as glycosaminoglycans and apolipoproteins, may be related to the amino acid variations noted earlier, and may influence the course of disease by stabilizing filament and fibril formation and by modifying the sensitivity of fibrils to proteolysis [32]. SAP, a member of the pentraxin protein family, is a major component of amyloid deposits, one that is highly resistant to proteolysis and may act as a pathologic chaperone for fibrils [59,60]. SAP also employs a calcium-dependent binding mechanism in its interactions with amyloid fibrils and may contribute to the occurrence of deficiencies in calcium-binding vitamin K-dependent coagulation factors, most notably factor X, a clinical feature associated with massive hepatosplenic amyloidosis. Monoclonal FLCs and minimal bone marrow plasmacytosis are detected in the majority of patients with AL-amyloidosis, often in association with suppression of noninvolved immunoglobulin production

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[61–63]. The levels of monoclonal protein and plasma cell infiltration in the marrow do not increase over time, as occurs in multiple myeloma. Sixty percent of patients have marrow biopsies showing 10% or fewer clonal plasma cells [26]. Eleven percent of patients with amyloid have over 20% plasma cells with no overt signs of multiple myeloma. When a clonal immunoglobulin heavy chain is detectable in the serum, it is immunoglobulin G (IgG) in 58%, IgA in 10%, and IgM in 8%. The presence of IgM amyloidosis is not well recognized; these patients usually do not have Waldenström’s macroglobulinemia [64]. The clones that cause AL-amyloidosis are distinct from myeloma clones with respect to their repertoire of immunoglobulin light chain variable germline (Ig VL ) genes, but similar in that their immunoglobulin genes are highly mutated (i.e. they are antigen-driven or post-germinal center clones) [65–72]. The repertoire of Ig VL genes in AL is skewed, unlike that in myeloma, which is similar to the normal expressed repertoire. Over half of all AL clones employ one of three immunoglobulin λ light chain variable region germline genes: 2a2 (λII), 3r (λIII) or 6a (λVI). AL clones using genes derived from the 3r germline donor have a significantly higher divergence from the germline sequence than those using the 6a donor, a difference possibly related to both antigen selection and the inherent amyloid-forming propensity of 6a light chains [66,68,69]. A puzzling feature of AL-amyloidosis is the variety of organs that can be affected [2,62]. Although the basis for this tropism is unknown, evaluations of the immunoglobulin light chain genetics underlying AL suggest that some light chain variable region germline donor genes are more likely to give one pattern of organ involvement than another [66,68,69]. Using the reverse transcriptase polymerase chain reaction to clone amyloid-forming FLC genes, and available databases for germline gene identification, several investigators have sought to test the hypothesis that the light chain variable region genes used by AL-amyloidosis plasma cell clones influenced organ tropism. In one series, light chain variable region germline genes were identified from 60 patients with AL-amyloidosis. Dominant renal involvement was linked to clones whose FLCs were derived from the 6a germline gene, while dominant cardiac and multisystem disease was linked to those with FLCs derived from the 2a2 and 3r genes [66]. Despite these suggestive data, we do not know how the assembly of abnormal FLCs causes progressive organ dysfunction and clinical disease, often at a rapid tempo, nor do we know how partially folded light chain intermediates may be more toxic to cellular metabolism than fibrillar deposits [73]. Interestingly, cytogenetic abnormalities commonly seen in myeloma clones are also found in AL-amyloidosis [74,75]. Trisomies of chromosomes 7, 9, 11, 15, and 18 were reported in 33–52% of patients, and trisomy X in 13% of women and 54% of men. The aneuploidy seen in the monoclonal plasma cell population in AL-amyloidosis supports a neoplastic nature for the disorder even when the plasma cell fraction is low. Immunoglobulin heavy chain translocations have been reported in 55% of patients, and abnormalities of chromosome 13 are frequent [75]. AL-amyloidosis is then a plasma cell dyscrasia similar to monoclonal gammopathy of undetermined significance except that the monoclonal FLCs cause clinical symptoms and fibrillar deposition disease.

Diagnosing AL-amyloidosis The most common symptoms of amyloidosis are fatigue, weight loss, dyspnea, edema, and paresthesias. The fatigue, generally caused by cardiac involvement, may be misdiagnosed as functional or stress related since the usual features of congestive heart failure are absent. Lightheadedness is common, and occurs in patients with nephrotic syndrome with hypoalbuminemia and contraction of plasma volume. Patients early in the course of cardiac amyloidosis have poor diastolic filling and a

Table 62.1 Seven presenting syndromes of systemic AL-amyloidosis Infiltrative cardiomyopathy Nephrotic-range proteinuria Demyelinating peripheral neuropathy Hepatomegaly Carpal tunnel syndrome Tongue enlargement Intestinal symptoms of steatorrhea or pseudo-obstruction

reduced stroke volume leading to orthostatic hypotension, but a normal echocardiographic ejection fraction. Amyloidosis patients with autonomic neuropathy can also have orthostatic hypotension, frequently associated with syncope. Amyloidosis should be considered when patients with a monoclonal gammopathy complain of fatigue, weight loss, and vague constitutional symptoms. The physical findings of amyloidosis are specific but not sensitive. Purpura are seen in only one patient in six, most commonly on the eyelids, upper face, and webbing of the neck [61]. A palpable liver is present in one-fourth of patients and may be due to infiltration of the liver or congestion related to elevated right-sided cardiac pressures. Enlargement of the tongue is the most specific finding, but is seen in only one patient in 10. It is easily recognized because it results in dental indentations on the tongue laterally and inferiorly, and enlargement of the submandibular salivary glands, often misdiagnosed as enlarged lymph nodes. An occasional patient will present because of vascular claudication due to small vessel occlusion with amyloid deposits [76]. Because the symptoms and physical findings are not diagnostic of amyloidosis, it is important to recognize the clinical syndromes associated with AL-amyloidosis. The seven most common presentations are described in Table 62.1. When one of these clinical syndromes is seen, the next step is to obtain serum FLCs and immunofixation studies of serum and urine. A screening serum protein electrophoresis is inadequate because of the high prevalence of light chain-only disease. Since many patients have significant degrees of proteinuria, a peak may be obscured in the urine protein electrophoresis, necessitating immunofixation [14,15]. Serum FLCs and/or immunofixation studies will be abnormal in over 95% of patients. The serum FLC assay has demonstrated abnormal circulating FLCs even when the serum and urine immunofixation studies are negative [77,78]. Since AL-amyloidosis is usually a plasma cell dyscrasia, the finding of a monoclonal light chain in a patient with a consistent clinical syndrome is an indication to obtain a tissue biopsy for diagnosis. Rarely, AL-amyloidosis can be associated with lymphoplasmacytic lymphomas with elevated FLCs [79]. FLCs unassociated with heavy chains are produced in slight excess by normal plasma cells, and in significant excess at times by clonal plasma cells. FLCs circulate in normal individuals and can be measured with the FLC assay. This assay is quantitative, unlike immunofixation, and is a more sensitive measure of the presence of clonal disease than immunofixation in many cases. FLC levels and ratios are abnormal in more than 95% of patients with AL-amyloidosis, and the elevated FLCs usually are the precursor protein [63,80]. When the diagnostic evaluation includes serum FLCs, serum and urine immunofixation, and clonal analysis of marrow biopsy for plasma cells, all patients with AL-amyloidosis should be recognized. If one of these features of an immunoglobulin light chain disorder cannot be detected, the type of amyloidosis remains in question, and one should consider forms of non-AL amyloidosis such as localized, senile or hereditary [1–4]. The diagnosis of amyloidosis must be confirmed by biopsy, as is standard for patients with malignancy. Patients with visceral involvement of the kidneys, heart, liver or nervous system may have the

Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis

diagnosis confirmed by involved organ biopsy. The nature of amyloid, however, is that there is widespread deposition in blood vessels at diagnosis, and less invasive biopsies will readily establish the diagnosis. The abdominal fat will contain Congophilic amyloid deposits in 80% of patients, although false positives can sometimes be seen due to overstaining [81,82]. A bone marrow biopsy will demonstrate Congophilic deposits in 56% of patients [83]. The bone marrow will also allow assessment of the percentage of plasma cells in order to determine whether myeloma coexists and to establish clonality [18]. Other sites that can be safely biopsied include the skin, minor salivary glands, and rectum. Visualizing amyloid in visceral biopsies with both Congo red staining and electron microscopy enhances diagnostic confidence [84,85]. With respect to the typing of amyloid, extracting and sequencing the amyloid protein is technically arduous and requires ample tissue deposits of fibrils [86,87]. It remains a research technique. Although immunohistochemical techniques have been described, such approaches with tissue other than renal biopsies remain unreliable and controversial [88,89]. Immunogold electron microscopy is a highly sensitive and specific technique, but is only available at a few centers globally [85]. The process of typing amyloid then remains a function of clinical acumen in combination with serologic and marrow studies and genetic tests for hereditary variants. The evaluation at diagnosis includes testing for organ involvement and type of amyloid (Table 62.2). Concerns about whether a patient has AL-amyloidosis or another type are warranted because myelotoxic chemotherapies have no place in the treatment of non-AL amyloidosis, and also because failure to diagnose a hereditary disease has consequences [89,90]. Family history is not a useful guide for hereditary disease, and, with the exception of renal biopsies, tissue staining for amyloid typing Table 62.2 Distribution of amyloid-related organ involvement at diagnosis Involved organ

Frequency (%)

Symptoms

Heart

37

Heart failure Fatigue Arrhythmias Syncope

Kidneys

30

Nephrotic-range proteinuria Lower extremity edema Hypoalbuminemia

Liver

16

Hepatomegaly Early satiety Right upper quadrant abdominal discomfort Factor X deficiency

Gastrointestinal tract

7

Bleeding Pseudo-obstruction Diarrhea

Nervous system

20

Paresthesias Orthostasis Weakness Urinary retention Impotence

Other

12

Soft tissue Tongue Pulmonary

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is unreliable. Investigators at two centers have reported patient series (n = 178, n = 350) in which 2–10% of patients presumed to have ALamyloidosis had both a monoclonal gammopathy and a hereditary mutant protein, either transthyretin or fibrinogen Aα. Of note, a hereditary mutant transthyretin (the valine-122-isoleucine variant) occurs in 4% of African-Americans, a racial group that also has a higher incidence of monoclonal gammopathy [91,92]. Therefore, African-Americans presenting with amyloid need to be tested for both sources. We also recommend testing patients presenting with peripheral neuropathy for a hereditary source of disease. Patients with two possible sources should be referred to centers specializing in amyloidosis for typing and treatment. The practitioner who rarely sees patients with amyloidosis must also be sensitive to the occurrence of localized light chain and systemic senile amyloidosis. The larynx, tracheobronchial tree, lungs, duodenum, and bladder are the most common sites of localized light chain amyloidosis. This rarely progresses to systemic disease. Senile systemic amyloidosis usually presents with cardiac involvement in men over 70 years of age, although the lungs can sometimes be symptomatically involved, and progression can be rapid in rare cases [93–95]. Patients who may have localized or senile systemic amyloidosis should also be referred to centers specializing in amyloidosis for diagnostic confirmation.

Clinical presentations Heart The distribution of involved organs is depicted in Table 62.2. The heart is a commonly involved organ in patients with AL-amyloid, and heart involvement carries the most serious prognosis because its extent directly impacts on outcome and survival [96]. The pathophysiology is infiltration of the myocardial wall leading to restriction of ventricular filling; patients may present with disabling fatigue and unexplained weight loss as the only symptoms. The electrocardiogram can show low voltage with no prior history of an ischemic event. The echocardiogram shows thickening of the wall, commonly misinterpreted as concentric left ventricular hypertrophy rather than infiltration. The ejection fraction is usually preserved. Thickening of the mitral and tricuspid valves due to amyloid infiltration is common and an important clinical clue [97]. In patients who have a ventricular thickness of 15 mm or more, the median survival is less than 1 year. If the septal thickness is less than 15 mm, the median survival approaches 4 years. Exercise-induced syncope has been associated with a median survival of 2 months [98]. Any patient with intractable fatigue or an echocardiogram showing a thickened wall needs to have studies for the presence of a monoclonal protein in the serum or urine. A role for cardiac biomarkers has been established in AL-amyloidosis [99,100]. A staging system using both N-terminal fragment of pro-brain natriuretic peptide (NT-proBNP) and troponin T has been developed [101]. Cardiac involvement is prevalent; therefore, these biomarkers are useful in screening. Patients with pronounced elevations of both BNP and troponin have a poor prognosis [102]. These biomarkers are also of use in the management of patients. BNP can decrease in cardiac patients when the precursor FLCs are reduced with therapy; however, these tests are nonspecific, and can vary as the result of factors other than progression or hiatus of cardiac amyloid [103]. The mainstay of the treatment of cardiac amyloidosis is diuretics [104]. Many patients will have associated orthostatic hypotension and intravascular volume contraction secondary to nephrotic syndrome, making diuretic therapy difficult. The use of diuretics may precipitate syncope and can reduce renal blood flow, with a resultant rise in the serum creatinine level. The high frequency of hypotension in AL-amyloidosis can make challenging the use of angiotensin converting-enzyme inhibitors and receptor blockers. Also, stasis frequently leads to thrombi

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in the right and left ventricles, potential sources of embolism that may complicate the course of HCT. These thrombi can occur even in patients in normal sinus rhythm. Vascular deposits in small coronary arterioles can be demonstrated on endomyocardial biopsy and can produce angina [105]. Not all patients with cardiac amyloidosis have the AL type. In the hereditary and senile cardiac forms of amyloidosis, the cardiac syndromes are indistinguishable from AL-amyloidosis except that the tempo of disease progression is much more gradual, and therefore overall survival is longer [94]. Kidneys The most common presentation of renal amyloidosis is proteinuria. Thirty to 40% of patients with AL-amyloidosis will have renal involvement [106–108]. Patients with AL-amyloidosis and renal involvement usually have both albuminuria and free monoclonal light chains in the urine. The differential diagnosis is amyloidosis, heavy or light chain deposition or crystalline inclusion disease, myeloma cast nephropathy or cryoglobulinemia [109]. Immunofixation of the urine in patients with proteinuria is an important aspect of the diagnostic evaluation. Amyloidosis is demonstrated in 3% of renal biopsies [110]. Survival, both with standard therapy and with HCT, is impacted by the serum creatinine at diagnosis [111,112]. With standard chemotherapy, patients presenting with a creatinine level less than 1.3 mg/dL have a median survival of 25.6 months. Those with an elevated creatinine have a median survival of 14.9 months [113]. The level of urine protein excretion per day has no impact on survival, but patients with higher levels of proteinuria have greater morbidity associated with pretransplant conditioning, including increased fluid retention and transient acute renal failure. Thirty percent of all patients will have proteinuria above 3 g/day [26]. Light chains are found in the urine of 86% of patients with renal AL-amyloid when the daily urinary protein loss exceeds 1 g/day. The major clinical consequences of renal amyloid are severe serum hypoalbuminemia and eventual renal failure. The loss of albumin results in reduced intravascular oncotic pressure, and edema of the lower extremities and presacral area. In severe cases, it can lead to anasarca, ascites, and pleural effusions. The edema generally requires diuretics for control, but diuresis may aggravate intravascular volume contraction and exacerbate hypotension. The principal long-term complication of continuous urinary protein loss in renal amyloid is tubular damage that results in endstage renal failure. One of the goals of HCT is to eliminate the high urinary protein loss, and therefore delay or prevent the need for dialysis. The presenting 24hour urine protein and serum creatinine predict which patients will ultimately develop endstage renal disease in the absence of HCT. The median time from diagnosis of AL-amyloidosis to dialysis is 15 months [114]. Median survival from the start of dialysis is 8 months [114]. This poor outlook justifies the use of HCT in patients with single-organ renal involvement. The extent of amyloid deposits seen on a renal biopsy does not correlate with the severity of proteinuria and hypoalbuminemia. Limited amyloid deposits can be associated with advanced nephrotic syndrome. The kidneys are normal in size in amyloid. If amyloid does not involve the kidneys at presentation, it rarely occurs during follow-up. Renal transplantation has been performed in patients with AL-amyloidosis, but recurrence is common [115]. HCT for AL-amyloidosis has been performed successfully both before and after renal transplantation [116,117]. If renal transplant is undertaken, strong consideration should be given to subsequent HCT. Without HCT, it is estimated that renal transplant recipients have at least a 20% chance of amyloid recurrence at 1 year.

Liver Hepatomegaly is found by physical examination in one-fourth of patients with AL-amyloidosis associated with an unexplained elevation of the serum alkaline phosphatase level and early satiety. The majority of patients are symptomatic [118]. Hepatomegaly may be due to amyloidinduced heart failure or direct infiltration of the hepatic parenchyma. When two organs are involved with amyloid, the most common presentation is combined hepatic and renal. Half of the patients with hepatic amyloid have greater than 1 g of daily proteinuria. The clues that allow the recognition of hepatic amyloid are hepatomegaly out of proportion to the degree of abnormal liver function tests, a monoclonal protein in the serum or urine, Howell–Jolly bodies in the peripheral blood film (due to splenic infiltration), and proteinuria. Most patients will have only alkaline phosphatase elevation with normal transaminases. Elevation of the bilirubin level is generally a preterminal finding. Rarely, a patient can present with splenic or hepatic rupture. At diagnosis in patients with hepatic involvement, the liver descends a median of 7 cm below the right costal margin; splenomegaly is seen in 11% and nephrotic syndrome in 36% of patients with hepatic involvement. Ten percent of patients, however, are diagnosed because of a significant elevation of the serum alkaline phosphatase without hepatomegaly on examination. The median elevation of the serum alkaline phosphatase value in AL patients with hepatic involvement is 2.3 times the upper limit of normal. Portal hypertension with varices and bleeding has been reported but is rare [119]. Ascites is usually due to associated nephrotic syndrome, hypoalbuminemia, and congestive heart failure, but is not generally associated with portal hypertension. The median survival following diagnosis is 1 year. Radionuclide scintigraphy does not produce specific findings in hepatic amyloid and is not considered useful; the diagnosis is easily confirmed with liver biopsy with a low complication rate. The presence of hyposplenism on the peripheral blood film is specific for splenic amyloid. Technetium scanning will demonstrate a marked reduction in splenic blood flow due to amyloid infiltration.

Gastrointestinal tract Most patients with amyloidosis will have deposits seen in gastrointestinal tract biopsies [120]. Usually, these are vascular deposits only and do not produce symptoms. Fewer than 5% of patients with AL-amyloidosis will present with symptoms referable to the gastrointestinal tract. The presence of anorexia and weight loss does not correlate with the presence of gastrointestinal amyloid deposits. Malabsorption with steatorrhea is seen in less than 5% of patients. When symptoms are present, they can include pseudo-obstruction [121]. Patients with advanced gastrointestinal tract involvement can require long-term total parenteral nutrition for management. These patients have intractable nausea and vomiting, and do not respond to enteral feeding or pharmacologic interventions. Small bowel biopsy proof of amyloidosis is obtained in only 1% of patients. The most common presenting symptoms are diarrhea, anorexia, dizziness, and abdominal pain. The median weight loss is 30 pounds (13.6 kg), and 50% of patients have orthostatic hypotension. A quarter of them will have vitamin K deficiency due to malabsorption. Factor X levels are reduced in one-quarter of the patients. X-ray studies of the gastrointestinal tract are generally not helpful. Small bowel dilatation can be seen as can thickening, nodularity, and delayed transit. Computed tomography scanning demonstrates thickening of the bowel wall. There is usually a significant delay from the onset of symptoms to the recognition of amyloid. In our experience, the median time from symptoms to histologic diagnosis is 7 months; a laparotomy may be required to establish the diagnosis.

Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis

Nutritional failure is the cause of death in over half of the patients; an additional quarter die of cardiac amyloid. The diarrhea of amyloid is difficult to treat [122]. Loperamide, diphenoxylate, octreotide, and tincture of opium have all been tried with limited success. Amyloidosis can present as ischemic colitis with deposits obstructing the vessels of the lamina propria and muscularis mucosa, and leading to chronic mucosal sloughing and hemorrhage. There is a risk for gastrointestinal bleeding in patients who undergo HCT. Presumably, high-dose chemotherapy leads to denuding of the intestinal mucosa, exposing amyloid-involved vessels that can rupture easily and lead to massive bleeding. Gastrointestinal bleeding has been associated with an inferior outcome in HCT [123]. Nervous system Amyloid involvement of the peripheral nerves was first described in 1938 [124]. Peripheral neuropathy is present in one patient in five. When patients present with dominant neuropathy, consideration needs to be given to the possibility of hereditary amyloidosis. The finding of a monoclonal protein in the serum or urine suggests that the amyloidosis is AL type. The most common symptoms are paresthesias, muscle weakness, numbness, pain, orthostasis, urinary retention, and impotence. Syncope is seen in 12% of patients. In a quarter of patients, dysesthesias and distal burning accompany the peripheral neuropathy. The lower extremities are involved before the upper extremities in 90% of patients, and two-thirds of patients have autonomic symptoms. Cranial nerve involvement is rare. Carpal tunnel syndrome is seen in half of patients with amyloid peripheral neuropathy, and a third have significant weight loss. When patients present with amyloid peripheral neuropathy, the echocardiogram is abnormal in 44%, while renal involvement occurs in only 5%. Sural nerve biopsy may demonstrate deposits in endoneurial capillaries. Standard chemotherapy treatment rarely results in clinical improvement in neuropathic symptoms. The neuropathy is progressive over time. Marked restriction of mobility ultimately develops in three-quarters of patients, and a third are ultimately bedridden. There is often a significant diagnostic delay with amyloid neuropathy. Symptoms precede the diagnosis by a median of 29 months. Patients with a peripheral neuropathy should have serum FLC and serum and urine immunofixation studies performed. Finding a monoclonal light chain limits the differential diagnosis to gammopathy-related neuropathy, POEMS syndrome ( Polyneuropathy, Organomegaly, Endocrinopathy, M protein, Skin changes), cryoglobulinemia and AL-amyloidosis. Since amyloidosis preferentially causes loss of small unmyelinated fibers, an electromyogram can be normal early in the disease course. Because amyloid is often deposited at the level of the nerve root, causing distal demyelination, the sural nerve biopsy is often negative. Respiratory tract Involvement of the respiratory tract is usually asymptomatic. Almost 40% of patients with amyloid deposits in the lungs have localized forms and do not have systemic amyloidosis [125–127]. In patients who have histologic evidence of systemic amyloidosis and lung involvement, the symptoms may be overshadowed by concomitant cardiac involvement. Even when deposits are present, gas exchange in the lungs is preserved until late in the disease. Pulmonary amyloidosis presents radiographically as an interstitial or reticulonodular infiltrate with or without pleural effusion [128]. Bronchoscopic lung biopsy is safe and is not associated with an increased risk for bleeding. The chest X-ray is not specific, demonstrating an interstitial process that can be misinterpreted as fibrosis. In patients who have dyspnea due to interstitial lung disease, low doses of prednisone may produce symptomatic benefit. Pleural

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infiltration with amyloid can result in pleural effusions. Rarely, pulmonary hypertension develops in association with right-sided heart failure [129]. Hemostasis Bleeding can be a serious complication of amyloidosis. The most common manifestation of bleeding is skin purpura. Deficiency of factor X is seen in less than 5% of patients and is associated with hepatic involvement. Normalization of factor X levels following transplantation for hepatic amyloidosis has been reported. Bleeding associated with factor X deficiency is generally seen only when the level falls below 25% [130]. Severe factor X deficiency is associated with increased mortality with HCT. The management of factor X deficiency prior to myeloablative chemotherapy has included splenectomy and the use of recombinant human factor VIIa [131]. Rupture of liver or spleen can also occur in a small fraction of patients with hepatosplenic AL-amyloidosis. Rarely, thromboembolic disease is observed in patients with ALamyloidosis. At the Mayo Clinic, Minnesota, 40 patients with AL-amyloidosis and documented thromboembolic disease have been reported [132]. In half of the cases, thromboembolism was seen more than 1 month following the diagnosis of AL-amyloidosis. Twenty-nine of the 40 events were venous and 11 arterial. Risk factors for thrombosis included nephrotic syndrome, immobilization, tobacco use, heart failure, and disseminated intravascular coagulation. Five of the 40 patients had activated protein C resistance. The mortality associated with thromboembolism was 20%. Forty-five percent of the patients died within a year of the thrombotic event.

Prognosis In the pre-HCT era, the median survival of patients treated with oral chemotherapy was 20 months, with a 5-year survival of 20% [133]. Patients with congestive heart failure had a median survival of 8 months and a 5-year survival of 2.4%. Median survival of patients whose amyloidosis was diagnosed by liver biopsy was 9 months, with a 5-year survival of 13%. The best outcome was seen in patients with amyloid neuropathy as the sole manifestation of the disease, with a median survival of 40 months and a 5-year survival of 32%. Using clinical prognostic classifications, heart failure and orthostatic hypotension are both associated with a median survival of less than 1 year. At some centers, the presence of orthostatic hypotension is an exclusion criterion for HCT. The most common cause of death in patients with AL-amyloidosis is cardiac, and can be either congestive cardiomyopathy or sudden death caused by arrhythmias, pulseless electrical activity or asystole. Clinical outcome is largely determined by the extent of cardiac involvement, and echocardiography and cardiac biomarkers are useful in assessing patients with AL-amyloidosis with regard to their suitability for HCT. In early cardiac amyloid, relaxation is abnormal, and BNP and troponin levels are minimally abnormal. In advanced cardiac amyloid, there is restricted filling with a shortened deceleration time, and both BNP and troponin levels can be markedly elevated. Diastolic function in cardiac amyloid can be monitored serially using Doppler echocardiography. Patients can be divided into two groups on the basis of their deceleration time. At 150 ms or less, the 1-year survival is 49%, compared with 92% in those with longer than 150 ms deceleration time [134]. The presence of heart failure, a urinary monoclonal light chain, hepatomegaly, and multiple myeloma were all adverse factors affecting survival during the first year after diagnosis. After the first year, an increase in serum creatinine, the presence of multiple myeloma, orthostatic

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hypotension, and a monoclonal serum protein were associated with poor survival. Recognizing these prognostic factors is important when comparing studies of therapy. Because of the morbidity associated with myeloablative chemotherapy, patients with these features may be high-risk candidates for HCT. An elevated creatinine has an important impact on the morbidity of HCT, and consideration of melphalan dose reduction must be given to patients with creatinine elevation. The time between histologic diagnosis and referral for evaluation to a medical center is also an important prognostic variable. When all patients with amyloidosis seen are analyzed, the median survival is 2 years. However, when the analysis is limited to those patients seen within 30 days of diagnosis, the median survival is 13 months. This difference suggests that there is a referral bias that favors those patients who are physically able to come to a major treatment center. This

information is important when interpreting the results of clinical trials originating from a single center compared with those performed in a cooperative group setting.

Therapy Principles and endpoints of therapy The only effective therapeutic approach to systemic AL-amyloidosis remains reduction of amyloid-forming FLCs while providing best supportive care. Treatments that gradually reduce FLCs are likely to be ineffective because progression of amyloid organ disease continues. The FLC assay provides a direct quantitative measure of the fibril-precursor protein and of response when used serially during the course of therapy (Fig. 62.2). The FLC assay is less subject to artifactual errors than

Free light chain serum λ (mg/dL)

1000

100

10

1

0.1 0.1

1

10

100

1000

Free light chain serum k (mg/dL)

(a)

Free kappa

mg/dL

Free kappa (mg/dL) 450 400 350 300 250 200 150 100 50 0

H L

2005

2006

2007

Free kappa (mg/dL) 50

mg/dL

40 30 20 10 H

0 (b)

L

2005

2006

2007

Fig. 62.2 Serum free light chain values in amyloidosis. (a) Graph depicting the serum free light chain (FLC) values in newly diagnosed patients with amyloidosis seen over a 2-year period at Memorial Sloan-Kettering Cancer Center (n = 110; 93 with systemic AL [䊉], 11 with localized AL [䊊], and six with ATTR [ⵧ; see text]). The axes are logarithmic, and the oval shows the normal range. The serum FLC assay is a sensitive measure of the fibril-precursor protein in systemic AL-amyloidosis, but is occasionally minimally abnormal in localized and hereditary disease [80,90]. (b) Response of the pathologic FLCs to hematopoietic cell transplantation in two cases is depicted; note the prompt and stable reduction of the pathologic FLCs.

Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis

traditional measures such as immunofixation studies of urine. In addition, the cardiac biomarkers, particularly BNP (or NT-proBNP), have provided practitioners with an objective measure of cardiac stress, albeit one requiring interpretation. The FLC assay has significantly changed the way that patients with AL-amyloidosis are monitored during therapy. In a retrospective study, decreases in the pathologic FLCs of 50% or more with treatment were associated with significantly better survival [63]. In a prospective phase II trial using HCT and adjuvant therapy, patients achieving a normal serum FLC κ-to-λ ratio at 3 months postHCT had significantly better overall and progression-free survival [135]. In numerous studies, hematologic response to treatment has predicted overall survival [21,136,137]. The importance of a complete hematologic response as a major contributor to overall survival has been and continues to be documented (Fig. 62.3). A complete hematologic response requires that, at evaluation after therapy, evidence of the mono-

100 CR (n=41)

OS (%)

75 p<<0.001 PD (n=43)

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clonal protein be absent. The serum and urine immunofixation studies must not show the baseline monoclonal protein, the ratio of the serum free light chains should normalize, and bone marrow plasma cells should be normal in number and show no evidence of light chain restriction. Criteria for assessing organ responses have been defined and are in widespread use (Table 62.4) [62]. Organ responses in patients with complete hematologic responses are compared with those in patients with persistent plasma cells in Table 62.5 [136]. Prior to the use of the FLC assay, it was difficult to gauge hematologic response and titrate therapy. The serologic markers of clonal disease were often difficult to interpret, and the response of amyloid organ disease lags the reduction in fibril-precursor protein, making clinical correlation a poor way to assess the need for continued therapy. Integration of the quantitative FLC assay into clinical practice allows the titration of therapy in anticipation of response before evidence of organ improvement may be available. However, despite this advance, we do not yet have a test that can tell us the threshold below which the pathologic FLCs must be reduced (short of a complete hematologic response) in order to maximize the likelihood of stopping the disease process. We continue to follow markers of organ disease such as BNP and daily proteinuria to make that inference, although the criteria for hematologic and organ responses make the design and comparison of clinical trials considerably more rational [62]. The major endpoints of clinical research in systemic AL-amyloidosis remain overall survival, hematologic response, particularly the complete hematologic response rate, progression-free survival, and organ response rate. New agents, new era

25

0 0 (a)

5

10 15 20 25 30 35 40 45 50 55 60

Months

Therapy for systemic AL-amyloidosis has undergone a remarkable transformation over the past 4 decades. Because colchicine was useful in treating patients with familial Mediterranean fever (about a third of whom acquire “secondary” amyloidosis due to SAA protein), it was used in the 1970s to treat patients with AL-amyloidosis for whom no standard effective therapy existed [138,139]. Its use was not debunked until the 1990s when treatment arms containing colchicine were shown to be ineffective in phase III trials, the same trials in which oral melphalan + prednisone (MP) was shown to prolong survival from 8 to 18 months [113,140]. The most significant side-effects of melphalan were and are myelotoxicity and myelodysplasia. In patients who survived for more than 3.5 years, there was a 20% risk of myelodysplasia, often leading to secondary leukemia [141]. Approximately 5% of patients treated with alkylating agents survived for 10 years or more [142]. These long-term survivors were predominantly patients without symptomatic cardiac involvement or peripheral neuropathy, and with relatively Table 62.3 Types and frequencies of selected treatment-related toxicities (South-West Oncology Group [SWOG] > grade 2) in hematopoietic cell transplantation (HCT) for AL-amyloidosis

Fig. 62.3 Importance of achieving a hematologic complete response. In (a), survival as a function of complete response (CR) or persistent disease (PD) is shown for the first 84 patients who underwent hematopoietic cell transplantation (HCT) at Boston Medical Center from 1994 to 1998. (Reproduced from [21], with permission.) In (b), survival is shown as a function of response as scored by the current consensus criteria for 270 patients who underwent HCT at Mayo Clinic [62]. (Reproduced from [137], with permission.)

Toxicity

200 mg/m2 (n = 23) Frequency (% (n)) [18]

100 mg/m2 (n = 27) Frequency (% [n]) [117]

Nausea/vomiting Diarrhea Mucositis Nongastrointestinal bleeding Gastrointestinal bleeding

83 (19) 65 (15) 91 (21) 17 (4) 22 (5)

52 (14) 48 (13) 37 (10) 0 (0) 7 (2)

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Table 62.4 Criteria for amyloid-related organ responses to treatment Improved

Worsened

Heart • Decrease of ≥2 mm in mean left ventricular wall thickness • Two-class improvement in NYHA class without an increase in diuretic use

Heart • Increase ≥2 mm in wall thickness • Two-class worsening in NYHA class

Kidneys • 50% decrease (at least 0.5 g/day) in daily proteinuria without progressive renal insufficiency

Kidneys • >50% increase in daily proteinuria • Progressive renal insufficiency

Liver • A decrease in liver span of ≥2 cm • A decrease of abnormal alkaline phosphatase by 50%

Liver • An increase in liver span of ≥2 cm • An increase of alkaline phosphatase by 50%

Nervous system • Autonomic: normalization of orthostatic vital signs and symptoms, resolution of gastric atony • Improvement in electromyogram • Peripheral neuropathy: resolution of symptoms

Nervous system • Autonomic: worsening of orthostatic vital signs and symptoms, worsening of gastric atony • Peripheral neuropathy: worsening of symptoms

If organ function neither improves nor worsens, it is graded as stable. NYHA, New York Heart Association. Adapted with permission from [62].

Table 62.5 Organ responses to treatment

Organ system

Improvement in patients with complete hematologic response (n = 73)*

Improvement in patients with noncomplete hematologic response (n = 108)*

P value

Cardiac Renal Gastrointestinal and liver Neuropathy Soft tissue Performance status Factor X Patients with 1 or more improvement†

% (n/n) 27 (6/22) 63 (29/46) 57 (26/46) 47 (17/36) 11 (1/9) 53 (38/72) 40 (2/5) 66 (48/73)

% (n/n) 17 (6/36) 11 (7/68) 30 (13/44) 29 (13/45) 0 (0/17) 22 (23/106) 38 (3/8) 30 (32/108)

>0.2 <0.001 0.010 0.090 0.161 <0.001 >0.2 <0.001

* n/n, number improved/number involved at baseline. † Improvement relates to five organ systems: cardiac, renal, gastrointestinal and liver, neuropathy, and soft tissue. Reproduced with permission from [136].

normal renal function. By the late 1990s, oral melphalan and prednisone had become the standard treatment for patients with AL-amyloidosis. At that time, and not without controversy, high-dose melphalan with HCT was shown in phase III trials to provide a significant survival benefit to patients with multiple myeloma [143]. With the high hematologic response rate noted in myeloma, phase II trials for HCT in ALamyloidosis began. Early trials were characterized by high hematologic and organ response rates, and evidence of regression of amyloid in patients achieving complete hematologic responses [23]. The complete hematologic response rates in these early phase II trials of HCT in ALamyloidosis were five to 10 times higher than those seen with oral melphalan and prednisone [18]. But these trials also had excessive treatment-related mortality (TRM) due to sudden cardiac death, intractable hypotension, and gastrointestinal bleeding [117,144]. The 100-day mortality ranged from 20% to 40%, the latter in a multicenter setting, and deaths were noted during stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) alone, an unusual happenstance [23,145,146].

In the first decade of the second millennium came the advent of new agents such as thalidomide, lenalidomide, and bortezomib, all of which are now approved for use in myeloma. A combination of the first of these agents, thalidomide, with melphalan and prednisone (MPT) was compared on the phase III level with both MP and intermediate-dose melphalan with HCT in the treatment of myeloma, and shown to be superior [147]. At last the threshold was passed: a combination of agents had superseded MP for the treatment of myeloma patients not undergoing HCT. Phase II trials in AL-amyloidosis using a variety of combinations of traditional and new agents have also produced higher response rates than those seen with MP. Notably, phase II trials of dexamethasone combined in one instance with interferon-alpha and in another with oral melphalan, showed high partial and complete response rates [148,149]. Indeed, oral melphalan and dexamethasone has become a standard therapy in ALamyloidosis because of its effectiveness and convenience (one 4-day course per month) despite the risk of myelodysplasia and secondary leukemia. There have also been several trials of thalidomide in AL-

Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis

amyloidosis, including one in which it was used in the adjuvant setting after HCT. The drug is active in AL-amyloidosis but difficult for patients to tolerate. Recently phase I–II trials of lenalidomide and bortezomib for AL-amyloidosis have been reported, with encouraging results [150– 152]. We have entered a new era in the treatment of clonal plasma cell diseases. The integration of traditional therapies and novel agents will be a complex process. Clearly, MP can no longer be considered the standard therapy for AL-amyloidosis, or HCT as investigational. Moreover, we now can distinguish front-line and second-line options for patients, although there is as yet no consensus on how to combine, sequence or customize traditional and novel approaches. This is particularly important with respect to HCT. A case-cohort analysis has shown a survival advantage in AL-amyloidosis for good-performance status patients treated with HCT, and a second study has shown that the quality of life improves significantly in patients who respond to HCT [153,154]. The difference between a year of oral melphalan and dexamethasone on the one hand, and HCT with high-dose melphalan on the other, is that, as in myeloma, the latter provides a platform for therapy with a low risk of myelodysplasia and secondary leukemia. Viewing HCT as such a treatment platform, we conducted a phase II trial combining risk-adapted high-dose melphalan and HCT with adjuvant thalidomide and dexamethasone. As discussed below, we showed that HCT for AL-amyloidosis can be performed with a TRM of less than 5% and that post-HCT adjuvant therapy significantly and safely improves hematologic response rates [155]. In contrast, French investigators in a phase III multicenter trial that compared HCT with oral melphalan and dexamethasone failed to show a difference between the two treatments [156]. Many sites had difficulty managing patients with AL-amyloidosis undergoing HCT, based on the frequency of stem cell mobilization failure and treatment-related deaths (>20%), while the sites with experience in HCT showed lower TRM and a trend in overall survival favoring HCT. Therefore, the determination that HCT for AL-amyloidosis is a superior therapy for those patients well enough to undergo it remains to be demonstrated on the phase III level. 12/95 Pre-SCT

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HCT: clinical experience The experience with HCT for AL-amyloidosis can be divided into reports of patient series and reports of prospective clinical trials. The dose-intensive regimens used to treat patients with AL-amyloidosis have been melphalan based, and the experience parallels that in myeloma. Melphalan alone is preferred to melphalan plus total body irradiation. In the mid-1990s, phase II trials were performed giving patients with AL-amyloidosis dose-intensive intravenous melphalan and autologous HCT [17,19–21,23]. Investigators at numerous centers documented the effectiveness of HCT in two-thirds of surviving patients, and SAP scintiscans demonstrated resorption or stabilization of deposits after the clonal plasma cells were reduced or eliminated (Fig. 62.4) [22,145]. As the circulating light chain burden and the deposition process are halted, both the performance status and the quality of life of patients can improve [18,154]. Historically, the most useful marker of response of the plasma cell dyscrasia to HCT has been the complete hematologic response [21,137]. Amyloid-related organ disease improves in the majority of patients who achieve complete hematologic response post HCT [136]. The experience with HCT was initially controversial because of TRM [24,25]. In four single-center studies, the average 100-day mortality of HCT was 21%, and in two multicenter studies it was 39%, making HCT in this population particularly morbid [18,22,23,117,157]. Deaths were also reported during stem cell mobilization, highlighting the fact that patients with AL-amyloidosis undergoing HCT were unusually prone to adverse events [117,144]. TRM was high in these early studies because the visceral reserve of patients was compromised by amyloid burden. Therefore, refinement of patient selection became a priority. The extent of amyloid organ involvement clearly accounted for much of the TRM. In two single-center trials, patients with amyloidosis involvement of two or fewer major organ systems (of heart, kidney, liver/gastrointestinal tract, and peripheral nervous system) had significantly superior 100-day survival (81%; 25 of 31 patients); in contrast to those who had more than two organ systems involved (25%; four of 12 patients; p < 0.01, Fisher’s exact test) [18,157]. Typical post-HCT survival as a function of number 2/97 Post-SCT

Grams 15

12

Fig. 62.4 Responses of amyloid organ involvement to hematopoietic cell transplantation (HCT). The panel of nuclear scans on the left in (a) are scintiscans employing iodine-131-labeled serum amyloid P (SAP). (Courtesy of Dr Philip Hawkins.) The black areas show uptake consistent with amyloid deposits that are extensive pre HCT and markedly improved 14 months later. The graph in (b) depicts the responses in 50 consecutive AL patients spilling more than 1 g/day of albumin in the urine. Patients who achieved a complete response (CR, left panels) had significantly diminished daily albuminuria (UTP24) and increased serum albumin levels 1 year post HCT ( p < 0.05 by paired t-test). (Adapted from [173], with permission.) Those with persistent disease (PD; right panels) were basically unchanged. Mean values are shown and the whisker-lines represent the standard deviations.

9

6 *

*

3

0

UTP24

Albumin

CR (n = 21) *p <0.05 (a)

(b)

UTP24

Albumin

PD (n = 29)

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Fig. 62.5 Factors influencing survival after hematopoietic cell transplantation (HCT). These survival curves describe outcomes in 270 patients with systemic AL-amyloidosis who underwent HCT at the Mayo Clinic. In (a), survival is shown as a function of number of organs involved, and in (b), as a function of cardiac involvement at baseline based on NT-proBNP levels. BNP, brain natriuretic peptide. (Reproduced from [137], with permission.)

of involved organs and relative cardiac involvement is shown in Fig. 62.5. The causes of TRM included cardiac arrhythmias, sepsis, intractable hypotension, gastrointestinal bleeding. and multiorgan failure. Toxic responses to HCT occurred more frequently in patients with AL-amyloidosis than in those who receive HCT for other indications. Although it is difficult to distinguish amyloid-related from treatmentrelated toxicities, the frequency and grade of strictly defined treatmentrelated toxicities appear to be a function of the dose of intravenous melphalan. This is indicated by the lower-grade toxicities experienced by HCT patients with AL-amyloidosis treated at 100 as opposed to 200 mg/m2 of melphalan, as shown in Table 62.3 [18,117]. Of particular note, the gastrointestinal toxicity with 200 mg/m2 of melphalan is striking, as are the higher rates of edema and bleeding. It is reasonable to conclude that amyloidosis confers on HCT patients an increased risk; however, the toxicities per se are regimen related, and are amenable to clinical management by patient selection and dose reduction (riskadapted melphalan dosing). In a series of 92 patients transplanted in the United Kingdom over a 10-year period, the overall TRM was 23%, but it was 13% over the second half of that period [22]. Median survival was 5.3 years, and more than 80% of patients had a greater than 50% reduction of the pathologic FLCs. On multivariate analysis, TRM was associated with the number

of major organ systems involved, cardiac involvement, age, performance status, and albumin level. At 1 year post HCT, the complete hematologic response rate was 21% by intent-to-treat (19 of 92), being 36% in evaluable patients. At 18 months post HCT, the organ response rate was 48%. Whole-body amyloid load by SAP-scintigraphy was reduced in 65% of patients evaluated pre and post HCT. In a report from the Center for International Blood and Marrow Transplant Registry, a series of 107 patients from 48 transplant centers was described [158]. The 30-day TRM was 18%, and median progression-free survival 4 years. The key predictor of survival was the experience of physicians and caregivers at the transplant center. In a series of 270 patients transplanted at the Mayo Clinic for AL-amyloidosis, melphalan dosing was risk adapted [137]. Two-thirds of the patients received melphalan at 200 mg/m2, over four-fifths had one or two organ systems involved, and the overall TRM was 11%. In a proportional hazards model, predictors of outcome included number of organ systems affected and the baseline FLC level. Patients achieving a complete or partial response had significantly better survival than those with no response (median survival <12 months) (Fig. 62.3). The complete hematologic response rate was 33%. Progression-free survival and relapse rate were not described. In a series of 312 patients transplanted at Boston Medical Center, melphalan dosing was risk adapted, and the TRM was 15.7% with 4.2% during or just after mobilization [136]. The median overall post-HCT survival was 4.6 years. For those without cardiac involvement, the median survival was 6.4 years, with 60% 5-year survival, while for those with cardiac involvement, median survival was 1.6 years, with 29% 5-year survival. The complete hematologic response rate in evaluable patients (i.e. not intent-to-treat) was 40%, while on an intent-to-treat basis it was 27% (73 of 273; 39 patients were less than a year post HCT). Complete hematologic response was associated with an 82% 5-year survival, compared with 55% for those not achieving complete hematologic response. At 1 year post stem cell transplantation, 44% of evaluable patients achieved organ responses. Curiously, patients with kappa light chain disease had a higher complete response rate than those with lambda light chain disease. HCT: clinical trials Many of the patients in the series recounted above had been diagnosed more than 12 months prior to stem cell transplantation and had been previously treated. A more relevant picture is obtained in clinical trials of newly diagnosed untreated patients. In a multicenter phase II trial conducted by the Eastern Cooperative Study group, the TRM was 10%, suggesting that treatment-related complications need not occur at a higher rate in the multicenter setting [159]. In a single-center, large randomized prospective phase II clinical trial, 100 patients were stratified based on organ involvement and time from diagnosis, and then randomized to initial HCT or HCT after two cycles of oral melphalan and prednisone [146]. The 100-day TRM was 20%, and 12% of patients died in association with stem cell mobilization. There were no significant differences between the two arms with respect to overall survival at a median of 4 years follow-up. At 5 years post HCT, the overall survival was 50% for immediate and 39% for delayed HCT. The complete response rates were 21% and 17%, and 42% of patients achieved an organ response at 1 year. In this trial, fewer patients randomized to initial oral therapy received HCT because of progression of disease making them ineligible for HCT. This affected patients with cardiac involvement disproportionately. Newly diagnosed untreated patients with AL-amyloidosis eligible for HCT did not benefit from two cycles of MP. Indeed, there is no evidence to indicate that preHCT induction therapy helps patients with AL-amyloidosis. The number of organ systems affected and the presence of cardiac involvement were predictors of outcome in that large phase II trial. For patients with one or two major organ systems affected of heart, kidneys, liver/gastrointestinal tract, and peripheral nervous system, and for those

Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis

without cardiac involvement, median survival had not been reached at a median of 4 years follow-up. For the others, median survivals in the two groups were 9.3 and 5.2 months for patients with more than three organs involved, and 9.6 and 4.8 months for cardiac patients. This trial demonstrated that HCT could be safely applied to patients with ALamyloidosis who do not have symptomatic three or four organ system involvement or advanced cardiac amyloid characterized by recurrent pleural effusions, cardiac syncope or symptomatic arrhythmias. These results made clear that patients with more than two major organs involved and those with advanced cardiac involvement are at a high risk of dying within the peritransplant period. Such patients are poor candidates for HCT utilizing high-dose regimens. French investigators conducted a randomized prospective multicenter phase III trial in which stem cell transplant with high-dose melphalan was compared with oral melphalan and dexamethasone given continuously for up to 18 months [156]. In the HCT group, there was a 44% early failure rate due largely to treatment-related deaths and progression of disease. TRM was 24%. Analysis of outcomes between the groups demonstrated a center effect in that patients treated at the majority of centers (27 of 29) had far better survival if they received oral therapy (p < 0.01), while patients in the two centers in which the largest fraction of HCT were performed had a trend to better survival with HCT. Comparisons of response rates and survival between those alive at least 3 months post HCT and those who completed at least 3 months of oral melphalan and dexamethasone showed no difference. For both groups, the hematologic response rates were 65%. The organ response rate was higher in the HCT group (52% versus 40%). Median survival was 48 months for HCT and 58 months for oral therapy. Surprisingly, no cases of myelodysplasia were reported in the oral melphalan group. Despite the apparent center effect and the need to be cautious in interpreting results with such a high TRM, this phase III trial did not define a standard therapy for systemic AL-amyloidosis. In a phase II trial for newly diagnosed untreated patients conducted at Memorial Sloan-Kettering Cancer Center, 34% of the 170 patients with AL-amyloidosis seen between September 2002 and June 2005 were untreated and eligible for HCT and the trial [155]. Thirteen of the eligible patients declined treatment or were treated elsewhere for insurance reasons, leaving 45 patients who enrolled and were treated on study. Treatment on study was in two phases: initial risk-adapted melphalan-based HCT, and at 3 months post HCT adjuvant therapy with thalidomide and dexamethasone for patients failing to achieve complete hematologic response. Primary endpoints were overall and progression-free survival at 2 years post HCT, and secondary endpoints were hematologic and organ responses. Median age was 57 years (range 34–73 years; 23 men and 22 women). Median time from diagnosis to enrollment was 1.5 (range 0.1–7.5) months. Thirty-one patients (69%) had single organ involvement, and 14 (31%) had two organ systems involved. Renal impairment (creatinine clearance <51 mL/min) was present in 7 of 45 (16%) patients. Based on criteria of age and organ involvement, six patients were assigned to mephalan 100 mg/m2, 24 to melphalan 140 mg/m2, and 15 to melphalan 200 mg/m2. In this trial, TRM was 4.4% (2 of 45), with both deaths in patients with cardiac involvement. There were no deaths during adjuvant therapy. At 3 months post HCT, response rate was 63% with 21% complete hematologic response and no difference in response rate or complete hematologic response based on the dose of melphalan. Thirty-one patients received adjuvant therapy, but only 52% (n = 16) completed all 9 months as planned, with 16% and 32% discontinuing for progression of disease and toxicity respectively. Median dose of thalidomide tolerated was 150 (range 50–200) mg/day, and the majority of patients received one or two dexamethasone pulses per month. Despite the high discontinuation rate, 42% of patients receiving adjuvant therapy had an improved hematologic response at 1 year post HCT. Overall response rate was 78% (95% confidence interval [CI] 65–90%) with 39% achiev-

925

ing a complete hematologic response (95% CI 24–53%). By intent-totreat, these rates are 71% (95% CI 57–84%) and 36% (95% CI 21–50%), respectively, again with no significant difference seen based on melphalan dosing. Forty-four percent of patients had improvement in their primary involved organ at 12 months post HCT. Sixty-nine percent of patients with complete hematologic response at 12 months had organ improvement, compared with 50% of those with a partial response, and 11% with stable or progressive disease (p = 0.005). As of April 2007, the median follow-up for surviving patients is 31 (range 20–49) months. One- and 2-year overall survivals were 91% (95% CI 83–98%) and 84% (95% CI 73–94%), respectively. For patients with cardiac involvement, estimates of 1- and 2-year overall survivals were 63% at both times (95% CI 34–91%). Median progression-free survival is 40 months with 74% (95% CI 61–87%) of patients remaining progression-free at 2 years. Three of 16 patients with complete hematologic response had relapses at 12, 19, and 24 months after stem cell transplantation. All three had achieved complete hematologic response 3 months post HCT (two with melphalan 200 mg/m2 and one with melphalan 140 mg/m2) and had not received adjuvant therapy. In univariate analysis, the baseline characteristics associated with overall survival were the number of organ systems involved and the baseline troponin I level. In multivariate analysis, having a normal serum FLC κ-to-λ ratio at 3 months post HCT and achieving a hematologic response at 12 months post-HCT were associated with improved overall and progression-free survival. This trial clearly demonstrates that risk-adapted melphalan dosing achieves a low TRM and, when combined with adjuvant therapy, high hematologic response rates. It also shows that both survival and organ response depend on hematologic response, as many earlier studies had indicated, making improvement or optimization of hematologic response a rational goal. Blood hematopoietic cell mobilization Mobilized hematopoietic stem cells collected by apheresis of blood have provided the marrow rescue products for patients with AL-amyloidosis receiving high-dose melphalan. In an early trial, two-thirds of patients had amyloid identified in the bone marrow, but this did not impair mobilization [18]. Contamination with clonotypic plasma cells has been demonstrated in these stem cell products collected after growth factor priming [160,161]. CD34+ cell selection is possible with adequate yields, but the effect on the disease course of having a positively selected apheresis product remains unknown [160]. Currently, G-CSF mobilization is used at the majority of centers transplanting AL patients regularly, and can be considered the standard approach to mobilization in this population. Given the impaired visceral reserve, vasculopathy, and coagulopathies associated with AL-amyloidosis, it was predictable that treatment-related toxicity would be more prominent in these patients. It was not expected, however, that there would be significant toxicity associated with the mobilization and collection of blood stem cells [18,144]. Deaths have been reported during the mobilization of patients with symptomatic cardiac amyloid or multisystem disease both at centers employing moderate doses of cyclophosphamide (e.g. 2.5 g/m2) and at those using growth factors alone. During mobilization with G-CSF (16 mg/kg/day × 5 days) on rare occasions we have observed a sometimes fatal although unexplained syndrome associated with hypoxia and hypotension, which was unresponsive to supportive measures. It can occur in patients without cardiac involvement and may be due to a combination of the effects of G-CSF, activated platelets returned during leukapheresis, pulmonary shunting, cytokines or mediators of septic hemodynamics [117,162]. Currently, in an attempt to minimize the risk of such toxicities, we recommend that G-CSF dosing for mobilization be given twice a day in lower doses (6 mg/kg every 12 hours), with collection beginning on day 5 [163]. Immune recovery after autologous HCT for AL has been studied prospectively in a cohort of patients with AL-amyloidosis [164]. At 3

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months following HCT, CD4+ cells were significantly decreased and T-cell function was depressed. In contrast, CD8+ cells, monocytes, natural killer cells and B-cell number and functional activity (measured by both quantitative immunoglobulin levels and the proliferative response to staphylococcal antigen) had returned to the normal range. These outcomes are similar to those reported for patients with other hematologic diseases after autologous HCT, although the prompt recovery of humoral immunity is somewhat atypical [165]. Opportunistic infections, including Pneumocystis iiroveci (formerly Pneumocystis carinii) pneumonia and herpes zoster, were seen within the first year post HCT, and several deaths due to sepsis occurred between 6 and 12 months post HCT. In the 8-year experience at Boston Medical Center, of those patients who survived 100 days post HCT, 10% died between 100 days and 1 year post HCT, half of sudden cardiac causes and the remainder of infectious causes, including opportunistic infections such as cytomegalovirus [136]. This experience in the year post HCT clearly supports regular cardiology consultation as well as the use of pre-HCT trimethoprim–sulfamethoxazole and post-HCT acyclovir prophylaxis. Peritransplant management Cardiac complications, gastrointestinal bleeding, and multiorgan failure with hypotension have been significant causes of early mortality with HCT in AL-amyloidosis [23,24,123]. These complications have usually occurred within the first 14 days of HCT; the degree to which sepsis, autonomic dysfunction, occult chronic infections, immunologic impairments, and amyloid vasculopathy contribute to these complications is unknown. The risk profile for these complications has centered on the extent of amyloid-related organ involvement, as noted above, and not on specific physiologic sequelae of organ involvement. In doing so, we should not overlook the fact that our understanding is limited despite this attribution. In one series, 20% (nine of 45) of patients had gastrointestinal bleeding, nearly half of whom had both upper and lower gastrointestinal bleeding [123]. The most common findings on endoscopy were diffuse esophagitis and gastritis. Age, platelet nadir, and CD34 cell count of the graft did not correlate with the risk of bleeding. Women, patients with multiorgan involvement, and those on hemodialysis or with slow platelet engraftment were more likely to have gastrointestinal bleeding. Patients with gastrointestinal bleeding were hospitalized a median of 37 days compared with 14.5 days for those without gastrointestinal bleeding ( p < 0.01). Five of the nine patients died during the post-HCT period, one directly of gastrointestinal bleeding. Gastrointestinal bleeding is unusual after autologous HCT, and in frequency and severity is unique to patients with AL-amyloidosis. If amyloid extensively infiltrates the submucosa of the stomach or lower tract, the potential for severe mucositis with hemorrhage clearly must be anticipated, while neuropathic compromise of the enteric plexus often results in atony, persistent post-transplant nausea, and failure to thrive. For these reasons, pretransplant planning and peritransplant supportive care become critical elements of the care plan. Recommendations with respect to pre- and peritransplant management have been described in detail, and include stool guaiac testing for occult blood [25]. Pretransplant patient evaluation should also include a detailed review of gastrointestinal signs and symptoms, endoscopic studies to define pathology when indicated by history or the presence of occult blood in stool, and a complete assessment of coagulation status. Peri transplant, proton pump inhibitors should be used for gastrointestinal prophylaxis, and, because dose-intensive intravenous melphalan can cause delayed emesis, an antiemetic regimen may be particularly useful beginning the day after the infusion of the graft. The combination of dexamethasone, lorazepam, and prochlorperazine seems to be very useful. If breakthrough nausea and vomiting occur, daily granisetron is often useful.

It is important to note that major gastrointestinal bleeding can present atypically as, for example, new-onset atrial fibrillation or supraventricular tachycardia. In HCT patients with known gastrointestinal amyloid, if stool guaiac results are positive, the hemoglobin should be kept above 9 or 10 g/dL and platelets maintained over 50,000/μL. It is also important to keep in mind that visceral rupture (e.g. splenic rupture) can also occur acutely in patients with AL-amyloidosis. During the early postHCT period, vague or atypical left-sided abdominal or shoulder pain should raise a concern about splenic hemorrhage and lead to consideration of imaging the abdomen. Splenic rupture occurring during this period has been successfully managed surgically. Other viscera, such as the esophagus or small bowel, can also perforate and present life-threatening challenges [166]. In our experience, the peri-HCT mortality rate in patients with cardiac amyloid and congestive heart failure, or with a history of cardiac arrhythmias, syncope or recurrent pleural effusions, approaches 100%, while patients with uncomplicated or well-compensated cardiac amyloid and no other symptomatic organ involvement have a low peri-HCT mortality and 65% 1-year survival. In patients with dominant cardiac amyloid with minimal symptoms, preserved left ventricular function usually assures diuretic responsiveness. Maintenance of normal electrolyte levels in cardiac patients receiving diuretic therapy is an obvious requirement. The mortality associated with cardiac amyloid in HCT recipients is due to sudden cardiac death and to cardiopulmonary failure, resulting in hypotension and hypoxia. Patients rarely, if ever, survive after ventricular arrhythmias, or hypotensive bradycardic episodes occur peri HCT despite the addition of appropriate medications and use of advanced life support measures. Hemodynamically stable tachycardias, on the other hand, occur with some frequency and are usually well tolerated. Use of β-blockade simply for control of sinus tachycardia should not be routine. Whether prophylactic pacemakers, implanted automated defibrillators or antiarrhythmic agents, such as low-dose β-blockers or amiodarone, may impact peri-HCT mortality in these patients remain matters for further clinical investigation. Clearly, the management of intravascular volume and hypotension is a critical aspect of care. Nephrotic syndrome causes salt avidity and hypoalbuminemia, and therefore often leads to significant edema. The risk of overdiuresis, however, may be greater than the risk of allowing some peripheral edema in the setting of clinical euvolemia. Nevertheless, maintaining a diuresis concurrently with melphalan administration and graft infusion is reasonable. However, even mild intravascular volume depletion may exacerbate nausea and emesis; therefore, twice daily orthostatic vital signs and normal saline support when indicated are recommended. Since a major factor causing pulmonary and peripheral edema is hypoalbuminemia, albumin infusions should be used throughout the treatment period to maintain a serum level above 2.0 g/dL. At the Mayo Clinic, post-HCT G-CSF is not used to support hematopoietic recovery because of the morbidity, particularly the fluid retention, associated with its use. In the series of 270 patients from that institution, median time to recovery of an absolute neutrophil count over 500/μL is 14 days, while in contrast the median time at other major centers is 10 days [137]. A detailed cost–benefit comparison has not been performed, but now with the availability of pegfilgrastim (Neulasta), a long-acting preparation requiring a single administration and hence minimal nursing and pharmacy costs, such a comparison would be welcome and useful. It is likely that long periods of neutropenia result in an increased frequency of fevers that require laboratory evaluation and empiric antibiotic use, and in inadvertent side-effects. Cytokine measurements that enable transplant physicians to distinguish engraftment fever from infection have been developed but are not in widespread use [167].

Autologous Hematopoietic Cell Transplantation for Systemic Light Chain (AL-) Amyloidosis

The multiorgan failure syndrome and intractable hypotension seen as a toxicity of HCT in patients with AL-amyloidosis likely reflect the limitation on visceral reserve imposed by fibril deposition. Volume depletion, bleeding, sepsis, and hypoadrenalism, followed by worsening autonomic neuropathy, are the most likely causes of hypotension post HCT. The cellular and hormonal aspects of the syndrome remain poorly characterized. Use of morphine or fentanyl to treat mucositis can affect both blood pressure and urine output, and can complicate acyclovir prophylaxis. When renal perfusion and urine output are reduced, risk of acyclovir toxicity increases. Midodrine and fludrocortisone are useful treatments for orthostasis but do not work reliably in the transplant setting. At the time of neutrophil recovery or myeloid engraftment, it is not uncommon for patients to experience orthostasis requiring more aggressive hydration. Emerging therapies and approaches Although the repertoire of agents available to treat AL-amyloidosis has never been as robust as it is at this time, there have been several notable failures in drug development. 4′-Iodo-4′-deoxy-doxorubicin (IDOX) was an anthracycline anticancer drug, initially given serendipitously to a patient with AL-amyloidosis who surprisingly experienced rapid clinical improvement [168,169]. Subsequently, this observation led to investigations into the mechanism of action of IDOX, further tests of its clinical activity, and a phase II trial showing a modest hematologic response rate [170]. CPHPC, a novel agent that inhibited SAP dimerization by reversing the inner and outer faces of the dimer and enhancing clearance, was shown to be safe at low doses and effective at depleting serum SAP, but drug development in that case also met obstacles of trial design and the waning interest of pharmaceutical companies [171]. Neither of these agents is available for further multicenter testing. The combination of new agents such as lenalidomide and bortezomib with traditional treatments for AL-amyloidosis will likely result in improvements in initial therapy, particularly if the use of oral melphalan

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and other alkylating agents in initial therapy can be avoided or minimized. Agents in preclinical development include monoclonal antibodies that target the clonal plasma cells in AL-amyloidosis. The Fcγ-receptor IIB (CD32B) is present in high levels on clonal plasma cells from patients with AL-amyloidosis at diagnosis and relapse, and provides a potential target for monoclonal antibody therapy [172]. If such therapy proves useful, optimizing the combination and sequencing of the many active agents, and customizing approaches to individual patients based on organ involvement, will require focused multicenter clinical research efforts.

Conclusion AL-amyloidosis remains a disease for which our tests, treatments, and knowledge continue to evolve. Survival remains the primary endpoint for patients as well as clinical investigators. The FLC assay provides the most important tool for diagnostic testing and for monitoring the course of therapy. Although the response rates with HCT are higher than those with standard therapies, HCT for AL-amyloidosis remains most effective in patients who are younger or have limited disease. By combining HCT with novel agents the overall progression-free survival of patients may improve over the near term, and, with the promise of monoclonal antibody therapy, the possibility exists that HCT may become secondline therapy in the future. As we come to understand more fully the basis of the organ disease that amyloid-forming light chains cause, novel cytoprotective pathways may be identified and simple and effective drug treatment developed. The ideal treatment for AL-amyloidosis in the future will likely involve a combination of approaches aimed at eliminating the fibril-precursor light chains, inhibiting their self-assembly and enhancing resorption of fibrillar deposits (Fig. 62.1). In the future, such combinations will likely include minimally toxic cytoreductive HCTbased therapies that will enhance the recovery and extend the survival of most, if not all, patients with AL-amyloidosis.

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91. Jacobson DR, Pastore RD, Yaghoubian R et al. Variant-sequence transthyretin (isoleucine 122) in late-onset cardiac amyloidosis in black Americans. New Engl J Med 1997; 336: 466–73. 92. Landgren O, Gridley G, Turesson I et al. Risk of monoclonal gammopathy of undetermined significance (MGUS) and subsequent multiple myeloma among African American and white veterans in the United States. Blood 2006; 107: 904–6. 93. Westermark P, Sletten K, Johansson B, Cornwell GG 3rd. Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc Natl Acad Sci U S A 1990; 87: 2843–5. 94. Ng B, Connors LH, Davidoff R, Skinner M, Falk RH. Senile systemic amyloidosis presenting with heart failure: a comparison with light chain-associated amyloidosis. Arch Intern Med 2005; 165: 1425–9. 95. Matsutani H, Hoshii Y, Setoguchi M et al. Vascular amyloid of unknown origin and senile transthyretin amyloid in the lung and gastrointestinal tract of old age: histological and immunohistochemical studies. Pathol Int 2001; 51: 326–32. 96. Falk RH. Diagnosis and management of the cardiac amyloidoses. Circulation 2005; 112: 2047–60. 97. Elliott PM, Mahon NG, Matsumura Y, Hawkins PN, Gillmore JD, McKenna WJ. Tissue Doppler features of cardiac amyloidosis. Clin Cardiol 2000; 23: 701. 98. Chamarthi B, Dubrey SW, Cha K, Skinner M, Falk RH. Features and prognosis of exertional syncope in light-chain associated AL cardiac amyloidosis. Am J Cardiol 1997; 80: 1242–5. 99. Takemura G, Takatsu Y, Doyama K et al. Expression of atrial and brain natriuretic peptides and their genes in hearts of patients with cardiac amyloidosis. J Am Coll Cardiol 1998; 31: 754–65. 100. Palladini G, Campana C, Klersy C et al. Serum N-terminal pro-brain natriuretic peptide is a sensitive marker of myocardial dysfunction in AL amyloidosis. Circulation 2003; 107: 2440–5. 101. Dispenzieri A, Gertz MA, Kyle RA et al. Serum cardiac troponins and N-terminal pro-brain natriuretic peptide: a staging system for primary systemic amyloidosis. J Clin Oncol 2004; 22: 3751–7. 102. Dispenzieri A, Gertz MA, Kyle RA et al. Prognostication of survival using cardiac troponins and N-terminal pro-brain natriuretic peptide in patients with primary systemic amyloidosis undergoing peripheral blood stem cell transplantation. Blood 2004; 104: 1881–7. 103. Palladini G, Lavatelli F, Russo P et al. Circulating amyloidogenic free light chains and serum N-terminal natriuretic peptide type B decrease simultaneously in association with improvement of survival in AL. Blood 2006; 107: 3854–8. 104. Nash KL, Brij SO, Clesham GJ. Cardiac amyloidosis and the use of diuretic and ACE inhibitor therapy in severe heart failure. Int J Clin Pract 1997; 51: 384–5. 105. Al Suwaidi J, Velianou JL, Gertz MA et al. Systemic amyloidosis presenting with angina pectoris. Ann Intern Med 1999; 131: 838–41. 106. Steensma DP, Kyle RA. A history of the kidney in plasma cell disorders. Contrib Nephrol 2007; 153: 5–24. 107. Dember LM. Emerging treatment approaches for the systemic amyloidoses. Kidney Int 2005; 68: 1377–90.

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108. Gertz MA, Lacy MQ, Dispenzieri A. Immunoglobulin light chain amyloidosis and the kidney. Kidney Int 2002; 61: 1–9. 109. Picken MM. Immunoglobulin light and heavy chain amyloidosis AL/AH: renal pathology and differential diagnosis. Contrib Nephrol 2007; 153: 135–55. 110. Yahya TM, Pingle A, Boobes Y, Pingle S. Analysis of 490 kidney biopsies: data from the United Arab Emirates Renal Diseases Registry. J Nephrol 1998; 11: 148–50. 111. Gertz MA, Lacy MQ, Dispenzieri A. Myeloablative chemotherapy with stem cell rescue for the treatment of primary systemic amyloidosis: a status report. Bone Marrow Transplant 2000; 25: 465–70. 112. Osawa Y, Kawamura K, Kondo D et al. Renal function at the time of renal biopsy as a predictor of prognosis in patients with primary AL-type amyloidosis. Clin Exp Nephrol 2004; 8: 127–33. 113. Kyle RA, Gertz MA, Greipp PR et al. A trial of three regimens for primary amyloidosis: colchicine alone, melphalan and prednisone, and melphalan, prednisone, and colchicine. New Engl J Med 1997; 336: 1202–7. 114. Gertz MA, Kyle RA, O’Fallon WM. Dialysis support of patients with primary systemic amyloidosis. A study of 211 patients. Arch Intern Med 1992; 152: 2245–50. 115. Gillmore JD, Madhoo S, Pepys MB, Hawkins PN. Renal transplantation for amyloid end-stage renal failure – insights from serial serum amyloid P component scintigraphy. Nucl Med Commun 2000; 21: 735–40. 116. Leung N, Griffin MD, Dispenzieri A et al. Living donor kidney and autologous stem cell transplantation for primary systemic amyloidosis (AL) with predominant renal involvement. Am J Transplant 2005; 5: 1660–70. 117. Comenzo RL, Sanchorawala V, Fisher C et al. Intermediate-dose intravenous melphalan and blood stem cells mobilized with sequential GM+G-CSF or G-CSF alone to treat AL (amyloid light chain) amyloidosis. Br J Haematol 1999; 104: 553–9. 118. Gertz MA, Kyle RA. Hepatic amyloidosis: clinical appraisal in 77 patients. Hepatology (Baltimore, Md) 1997; 25: 118–21. 119. Itescu S. Hepatic amyloidosis. An unusual cause of ascites and portal hypertension. Arch Intern Med 1984; 144: 2257–9. 120. Friedman S, Janowitz HD. Systemic amyloidosis and the gastrointestinal tract. Gastroenterol Clin N Am 1998; 27: 595–614, vi. 121. Hayman SR, Lacy MQ, Kyle RA, Gertz MA. Primary systemic amyloidosis: a cause of malabsorption syndrome. Am J Med 2001; 111: 535– 40. 122. Guirl MJ, Hogenauer C, Santa Ana CA et al. Rapid intestinal transit as a primary cause of severe chronic diarrhea in patients with amyloidosis. Am J Gastroenterol 2003; 98: 2219–25. 123. Kumar S, Dispenzieri A, Lacy MQ, Litzow MR, Gertz MA. High incidence of gastrointestinal tract bleeding after autologous stem cell transplant for primary systemic amyloidosis. Bone Marrow Transplant 2001; 28: 381–5. 124. Rajkumar SV, Gertz MA, Kyle RA. Prognosis of patients with primary systemic amyloidosis who present with dominant neuropathy. Am J Med 1998; 104: 232–7.

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125. Berk JL, O’Regan A, Skinner M. Pulmonary and tracheobronchial amyloidosis. Semin Resp Crit Care Med 2002; 23: 155–65. 126. Utz JP, Swensen SJ, Gertz MA. Pulmonary amyloidosis. The Mayo Clinic experience from 1980 to 1993. Ann Intern Med 1996; 124: 407–13. 127. Lachmann HJ, Hawkins PN. Amyloidosis and the lung. Chron Respir Dis 2006; 3: 203–14. 128. Gillmore JD, Hawkins PN. Amyloidosis and the respiratory tract. Thorax 1999; 54: 444–51. 129. Dingli D, Utz JP, Gertz MA. Pulmonary hypertension in patients with amyloidosis. Chest 2001; 120: 1735–8. 130. Choufani EB, Sanchorawala V, Ernst T et al. Acquired factor X deficiency in patients with amyloid light-chain amyloidosis: incidence, bleeding manifestations, and response to highdose chemotherapy. Blood 2001; 97: 1885–7. 131. Boggio L, Green D. Recombinant human factor VIIa in the management of amyloid-associated factor X deficiency. Br J Haematol 2001; 112: 1074–5. 132. Halligan CS, Lacy MQ, Vincent Rajkumar S et al. Natural history of thromboembolism in AL amyloidosis. Amyloid 2006; 13: 31–6. 133. Gertz MA, Kyle RA, Greipp PR. Response rates and survival in primary systemic amyloidosis. Blood 1991; 77: 257–62. 134. Klein AL, Hatle LK, Taliercio CP et al. Prognostic significance of Doppler measures of diastolic function in cardiac amyloidosis. A Doppler echocardiography study. Circulation 1991; 83: 808–16. 135. Cohen AD, Zhou P, Chou J et al. Risk-adapted autologous stem cell transplantation with adjuvant dexamethasone +/− thalidomide for systemic light-chain amyloidosis: results of a phase II trial. Br J Haematol 2007; 139: 224–33. 136. Skinner M, Sanchorawala V, Seldin DC et al. High-dose melphalan and autologous stem-cell transplantation in patients with AL amyloidosis: an 8-year study. Ann Intern Med 2004; 140: 85–93. 137. Gertz MA, Lacy MQ, Dispenzieri A, Hayman SR, Kumar S. Transplantation for amyloidosis. Curr Opin Oncol 2007; 19: 136–41. 138. Shirahama T, Cohen AS. Blockage of amyloid induction by colchicine in an animal model. J Exp Med 1974; 140: 1102–7. 139. Skrinskas G, Bear RA, Magil A, Lee KY. Colchicine therapy for nephrotic syndrome due to familial Mediterranean fever. CMAJ 1977; 117: 1416–7. 140. Skinner M, Anderson J, Simms R et al. Treatment of 100 patients with primary amyloidosis: a randomized trial of melphalan, prednisone, and colchicine versus colchicine only. Am J Med 1996; 100: 290–8. 141. Gertz MA, Kyle RA. Acute leukemia and cytogenetic abnormalities complicating melphalan treatment of primary systemic amyloidosis. Arch Intern Med 1990; 150: 629–33. 142. Kyle RA, Gertz MA, Greipp PR et al. Long-term survival (10 years or more) in 30 patients with primary amyloidosis. Blood 1999; 93: 1062–6. 143. Attal M, Harousseau JL, Stoppa AM et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Français du Myelome. New Engl J Med 1996; 335: 91–7. 144. Saba N, Sutton D, Ross H et al. High treatmentrelated mortality in cardiac amyloid patients undergoing autologous stem cell transplant. Bone Marrow Transplant 1999; 24: 853–5.

145. Perz JB, Rahemtulla A, Giles C et al. Long-term outcome of high-dose melphalan and autologous stem cell transplantation for AL amyloidosis. Bone Marrow Transplant 2006; 37: 937–43. 146. Sanchorawala V, Wright DG, Seldin DC et al. High-dose intravenous melphalan and autologous stem cell transplantation as initial therapy or following two cycles of oral chemotherapy for the treatment of AL amyloidosis: results of a prospective randomized trial. Bone Marrow Transplant 2004; 33: 381–8. 147. Facon T, Mary JY, Hulin C et al. Major superiority of melphalan and prednisone (MP) + thalidomide (THAL) over MP and autologous stem cell transplantation in the treatment of newly diagnosed elderly patients with multiple myeloma. Blood 2005; 106: 230a. 148. Dhodapkar MV, Hussein MA, Rasmussen E et al. Clinical efficacy of high-dose dexamethasone with maintenance dexamethasone/alpha interferon in patients with primary systemic amyloidosis: results of United States Intergroup Trial Southwest Oncology Group (SWOG) S9628. Blood 2004; 104: 3520–6. 149. Palladini G, Perfetti V, Obici L et al. Association of melphalan and high-dose dexamethasone is effective and well tolerated in patients with AL (primary) amyloidosis who are ineligible for stem cell transplantation. Blood 2004; 103: 2936–8. 150. Dispenzieri A, Lacy MQ, Zeldenrust SR et al. The activity of lenalidomide with or without dexamethasone in patients with primary systemic amyloidosis. Blood 2007; 109: 465–70. 151. Sanchorawala V, Wright DG, Rosenzweig M et al. Lenalidomide and dexamethasone in the treatment of AL amyloidosis: results of a phase 2 trial. Blood 2007; 109: 492–6. 152. Reece DE Sanchorawala SV, Hegenbart U, Merlini G et al. Phase I/II study of bortezomib (B) in patients with systemic AL-amyloidosis (AL). J Clin Oncol 2007; 25: 453s. 153. Dispenzieri A, Kyle RA, Lacy MQ et al. Superior survival in primary systemic amyloidosis patients undergoing peripheral blood stem cell transplantation: a case-control study. Blood 2004; 103: 3960–3. 154. Seldin DC, Anderson JJ, Sanchorawala V et al. Improvement in quality of life of patients with AL amyloidosis treated with high-dose melphalan and autologous stem cell transplantation. Blood 2004; 104: 1888–93. 155. Cohen AD, Zhou P, Chou J et al. Risk-adapted autologous stem cell transplantation with adjuvant dexamethasone +/− thalidomide for systemic light-chain amyloidosis: results of a phase II trial. Br J Haematol 2007; 139: 224–33. 156. Jaccard A, Moreau P, Leblond V et al., Myélome Autogreffe (MAG) and Intergroupe Francophone du Myélome (IFM) Intergroup. High-dose melphalan versus melphalan plus dexamethasone for AL amyloidosis. N Engl J Med 2007; 357: 1083–93. 157. Gertz MA, Lacy MQ, Gastineau DA et al. Blood stem cell transplantation as therapy for primary systemic amyloidosis (AL). Bone Marrow Transplant 2000; 26: 963–9. 158. Vesole DH, Perez WS, Akasheh M, Boudreau C, Reece DE, Bredeson CN. High-dose therapy and autologous hematopoietic stem cell transplantation for patients with primary systemic amyloidosis: a Center for International Blood and Marrow Transplant Research Study. Mayo Clinic Proc 2006; 81: 880–8.

159. Gertz MA, Blood E, Vesole DH, Abonour R, Lazarus HM, Greipp PR. A multicenter phase 2 trial of stem cell transplantation for immunoglobulin light-chain amyloidosis (E4A97): an Eastern Cooperative Oncology Group Study. Bone Marrow Transplant 2004; 34: 149–54. 160. Comenzo RL, Michelle D, LeBlanc M et al. Mobilized CD34+ cells selected as autografts in patients with primary light-chain amyloidosis: rationale and application. Transfusion 1998; 38: 60–9. 161. Perfetti V, Ubbiali P, Magni M et al. Cells with clonal light chains are present in peripheral blood at diagnosis and in apheretic stem cell harvests of primary amyloidosis. Bone Marrow Transplant 1999; 23: 323–7. 162. Gertz MA, Lacy MQ, Bjornsson J, Litzow MR. Fatal pulmonary toxicity related to the administration of granulocyte colony-stimulating factor in amyloidosis: a report and review of growth factorinduced pulmonary toxicity. J Hematother Stem Cell Res 2000; 9: 635–43. 163. Arbona C, Prosper F, Benet I, Mena F, Solano C, Garcia-Conde J. Comparison between once a day vs twice a day G-CSF for mobilization of peripheral blood progenitor cells (PBPC) in normal donors for allogeneic PBPC transplantation. Bone Marrow Transplant 1998; 22: 39–45. 164. Akpek G, Lenz G, Lee SM et al. Immunologic recovery after autologous blood stem cell transplantation in patients with AL-amyloidosis. Bone Marrow Transplant 2001; 28: 1105–9. 165. Avanzini MA, Locatelli F, Dos Santos C et al. B lymphocyte reconstitution after hematopoietic stem cell transplantation: functional immaturity and slow recovery of memory CD27+ B cells. Exp Hematol 2005; 33: 480–6. 166. Schulenburg A, Kalhs P, Oberhuber G, Reiter E, Base W, Greinix HT. Gastrointestinal perforation early after peripheral blood stem cell transplantation for AL amyloidosis. Bone Marrow Transplant 1998; 22: 293–5. 167. Tuma R, Almyroudis N, Sohn S et al. The serum IL-12:IL-6 ratio reliably distinguishes infectious from non-infectious causes of fever during autologous stem cell transplantation. Cytotherapy 2006; 8: 327–34. 168. Gianni L, Bellotti V, Gianni AM, Merlini G. New drug therapy of amyloidoses: resorption of ALtype deposits with 4′-iodo-4′-deoxydoxorubicin. Blood 1995; 86: 855–61. 169. Merlini G, Ascari E, Amboldi N et al. Interaction of the anthracycline 4′-iodo-4′-deoxydoxorubicin with amyloid fibrils: inhibition of amyloidogenesis. Proc Natl Acad Sci U S A 1995; 92: 2959–63. 170. Gertz MA, Lacy MQ, Dispenzieri A et al. A multicenter phase II trial of 4′-iodo-4′deoxydoxorubicin (IDOX) in primary amyloidosis (AL). Amyloid 2002; 9: 24–30. 171. Pepys MB, Herbert J, Hutchinson WL et al. Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis. Nature 2002; 417: 254–9. 172. Zhou P, Comenzo RL, Olshen AB et al. CD32B is highly expressed on clonal plasma cells from patients with systemic light-chain amyloidosis and provides a target for monoclonal antibodybased therapy. Blood 2008; 111: 3403–6. 173. Dember LM, Sanchorawala V, Seldin DC et al. Effect of dose-intensive intravenous melphalan and autologous blood stem-cell transplantation on AL amyloidosis-associated renal disease. Ann Internal Med 2001; 134: 746–53.

63

Yago Nieto & Elizabeth J. Shpall

Hematopoietic Cell Transplantation for Breast Cancer

Epidemiology and etiology Breast cancer is the most common cancer in Western women, with a lifetime risk in the United States of one in eight. It is the second cause of cancer-related death in US women after lung cancer, and the leading cause among women ages 20–59 [1]. Its incidence dropped in the United States by almost 7% from 2002 to 2003, leveling off by mid-2003 [2]. The decrease was evident only in women who were 50 years of age or older and for tumors that were estrogen receptor (ER) positive. The decrease was temporally related to the drop in the use of hormone replacement therapy that followed the first report of the Women’s Health Initiative randomized study, showing an increase in breast cancer (and in coronary heart disease) associated with the use of estrogen– progesterone replacement therapy [3]. Factors associated with increased risk of breast cancer include its prior diagnosis in a first-degree relative, early menarche, late menopause, radiation exposure, no prior deliveries, and age over 30 years at first pregnancy. The risk is especially high in carriers of mutated BRCA1 or BRCA2 genes or in the Li-Fraumeni multiple familial cancer syndrome. Gene mutations should be suspected if both a mother and a sister had breast or ovarian cancer, or if either one had bilateral or premenopausal breast cancer. Current surveillance guidelines for women with no risk factors include yearly bilateral mammograms and clinical breast examination for women after the age of 40, and a monthly breast self-examination after the age of 18 [4]. Digital mammography improves accuracy in younger women and those with dense breasts [5]. Women at higher risk should start their screening at an earlier age (e.g. at age 25–30). Women at risk for hereditary breast cancer should undergo surveillance with breast magnetic resonance imaging, which appears to be a better screening tool than mammograms in this group [6]. Current prophylactic options for women at high risk include bilateral simple mastectomy, which has been shown to prevent almost completely breast cancer in carriers of BRCA1 or BRCA2 mutation [7], and the use of tamoxifen [8] or raloxifene [9] for 5 years.

Molecular and cellular biology A gene-based taxonomic classification of breast cancer using DNA microarray technology has been recently developed. Listed from best to

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

worst prognosis, the five new molecularly defined subtypes are: (1) normal breast-like, (2) luminal A (high expression of ER and estrogen-regulated genes, such as progesterone receptor [PR], and expression of cytokeratins 8/18); (3) luminal B (low-to-moderate expression of ER and estrogen-regulated genes); (4) HER2+ and ER−; and (5) basal like (HER2−, ER−, PR−, and expression of cytokeratins 5 and 17 and epidermal growth factor receptor [EGFR]) [10]. Several other genetic signatures have been described in patients with early node-negative disease with a prognostic impact, most notably the 21-gene recurrence score [11] and the 70-gene profile [12]. Although their gene contents are largely nonoverlapping, there is a high degree of concordance between both signatures [13]. Trials in the United States and Europe are underway to test their clinical applicability in patients with node-negative disease.

Clinical description Breast cancer spreads to regional lymph nodes, mainly those in the axilla but also to the internal mammary chain, and infraclavicular and supraclavicular basins, and can metastasize to almost any organ in the body, most frequently to bone, lungs, and liver. A new American Joint Committee on Cancer staging system of breast cancer was adopted for use in tumor registries in January 2003 [14]. The major changes introduced by the new system were: 1 a size-based discrimination between: nodal macrometastases (>2 mm in diameter), micrometastases (>0.2 and ≤0.2 mm), and isolated tumor cells (≤0.2 mm), the latter group considered N0; 2 classification of lymph nodal status by number of involved axillary nodes: one to three, N1; four to nine, N2; 10 or more, N3; 3 new classifications for metastases to the: internal mammary (N1 if not apparent clinically, or N2 if clinically apparent), infraclavicular (N3), and supraclavicular (N3) lymph nodes. Local treatment with breast-conserving surgery followed by radiation offers the same local control as mastectomy [15,16], and is, therefore, the preferred option in most cases. Preoperative chemotherapy does not improve outcome compared with the same regimen administered postoperatively, but allows a higher rate of breast-conserving surgery by downsizing the tumor [17]. The likelihood of achieving a pathologic complete remission (pCR) to preoperative chemotherapy is inverse to the tumor size. Overall, a pCR is obtained in 15–25% of cases, which is associated with improved outcome. Thus, pCR is currently used as an important intermediate endpoint, a surrogate of final outcome, in trials of preoperative chemotherapy.

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Nontransplant approaches Nonmetastatic breast cancer Relapse rates, particularly at distant sites, increase proportionally to the number of positive axillary lymph nodes, and can be estimated to be approximately 20% at 5 years for patients with no involved nodes, 40% for patients with one to three positive nodes, 45–65% for patients with four to nine involved nodes, and over 70% for those with 10 or more positive nodes [18]. There have been recent important advances in postoperative standarddose chemotherapy (SDC) for patients with node-positive disease, affecting mostly the group with one to three positive nodes, compared with the group with high-risk primary breast cancer (HRPBC), defined by four or more nodes involved. Large studies have shown the benefit of a minimum of six to eight cycles (compared with the old standard of four cycles of adriamycin and cyclophosphamide) of anthracyclines combined with paclitaxel or docetaxel, either in sequential [19–21] or concurrent [22,23] fashion. The use of a dose-dense schedule of anthracyclines administered every 2 weeks with granulocyte colonystimulating factor (G-CSF) support also seems to improve the outcome of patients with node-positive disease [24]. Several trials enrolling patients with HER2+ tumors have demonstrated a large benefit at shortterm follow-up by adding trastuzumab to postoperative chemotherapy, either concurrently with taxanes [25,26] or upon completion of all chemotherapy [27]. Likewise, the addition of trastuzumab to preoperative chemotherapy improves pCR rate (greater than 50%) and outcome in HER2-overexpressing tumors [28]. The superiority of the new aromatase inhibitors over tamoxifen as adjuvant hormonal therapy for postmenopausal women with hormone receptor-positive tumors has been demonstrated. Several trials have shown improved outcomes using anastrazole [29], letrozole [30,31] or exemestane [32]. Although these three agents vary in their chemical properties and potency, it is unclear whether one is superior. It is also unclear which of the different schedules tested in these trials is best: use up front for 5 years, treatment for 3 years after 2 years of tamoxifen (“early switch”), or a “late switch” after 5 years of tamoxifen for an additional 5 years.

Metastatic breast cancer Since most patients receive an anthracycline regimen in the adjuvant setting, nonanthracycline regimens, with docetaxel, paclitaxel or capecitabine, have been developed for use as first-line treatment for metastatic disease. Several such combinations have been shown to improve survival, such as docetaxel–capecitabine [33], gemcitabine– paclitaxel [34], and gemcitabine–docetaxel [34]. The triweekly schedule of paclitaxel, previously considered standard, has been shown to be inferior to newer paclitaxel schedules or formulations, such as its weekly administration [35], or to nanoparticle albuminbound paclitaxel [36]. As in the adjuvant setting, several trials have demonstrated a benefit from combining trastuzumab with anthracyclines (although frequent clinical cardiac toxicity prevents the broad use of this combination with doxorubicin) [37], paclitaxel [37] or docetaxel [38] in patients with HER2+ tumors. Recently, the new dual EGFR–HER2 tyrosine kinase inhibitor lapatinib has been shown to improve significantly event-free survival (EFS) and response rate, but not overall survival (OS), in combination with capecitabine when compared with capecitabine alone, in patients with advanced tumors co-overexpressing both receptors, even after failure of prior trastuzumab [39]. Similarly, the addition of the anti-vascular endothelial growth factor antibody bevacizumab to paclitaxel has been shown

to extend EFS and increase response rate, but not OS, over paclitaxel alone, as initial treatment for metastatic breast cancer (MBC) [40]. The new aromatase inhibitors have shown superiority over tamoxifen for postmenopausal women with metastatic ER+ tumors [41]. The new bisphosphonates pamidronate [42] and zoledronic acid [43] have proven efficacy in preventing bone fractures and other skeletally related events in patients with bone metastases.

Hematopoietic cell transplantation for breast cancer Autologous hematopoietic progenitor cell support (AHPCS), derived from bone marrow or from peripheral blood progenitor cells (PBPCs), allows for the dose escalation of chemotherapy up to 10-fold, exploiting the dose–response effect of many drugs. While AHPCS circumvents myelosuppression, extramedullary organ toxicities become dose limiting. These substantial dose increments clearly increase the antitumor activity of high-dose chemotherapy (HDC) compared with SDC, with the hope that it will translate into improvements in outcome. Widespread substitution of PBPCs for bone marrow support and other improvements in patient care have increased the safety of HDC to current treatmentrelated mortality (TRM) rates below 5%. Despite the recent advances in standard therapy, more than 50% of patients with HRPBC and virtually all patients with MBC receiving SDC experience treatment failure and ultimately die from their disease. The initial prospective phase II trials of HDC in breast cancer, dating back to the late 1980s, were followed by randomized studies in both settings. Some argued that the impressive results of the phase II trials could be explained by patient selection, staging biases or short follow-up. Thus, controversy has surrounded HDC for breast cancer for more than a decade [44]. HDC trials in MBC Prospective HDC trials in MBC targeted in a sequential fashion patients with refractory [45,46], untreated [47], and responding disease [48–50]. It soon became clear that HDC produced not only the highest response rate and complete response (CR) rates ever reported in MBC, but also a consistent long-term EFS rate of 10–25% in patients transplanted after response to first-line chemotherapy, which appeared unprecedented (Fig. 63.1(a)). These results triggered phase III evaluation of HDC, employing a variety of regimens, for MBC. In the “Philadelphia” study, 184 patients responding to first-line therapy were randomized to receive HDC with cyclophosphamide, thiotepa, and carboplatin (STAMP-V) or maintenance cyclophosphamide, methotrexate, and fluorouracil (CMF) for 18 months or until progression [51]. In its latest update at a median follow-up of 67 months, there were no differences in EFS (4% versus 3%) or OS (14% versus 13%) [52]. A significant interaction was detected between age and treatment, with young and older patients apparently benefiting from HDC and CMF, respectively. Only 45 patients in CR were treated in study, which conferred only a 20% power to detect a 20% absolute OS difference in this subset. Strikingly, the partial remission to CR conversion rate was higher after CMF than after STAMP-V (9% and 6%, respectively). The low CR conversion rate in the transplant arm seems much lower than in most HDC trials, where CR conversion rates of 20–50% are typically reported. In the National Cancer Institute of Canada (NCIC) trial, 224 responsive MBC patients were randomized to additional SDC or HDC with cyclophosphamide, mitoxantrone, and etoposide [53]. In this trial, 20% of patients randomized to the HDC arm were never transplanted. Transplant-related mortality was 6%. At median follow-up of 48 months, significant differences in favor of transplantation were observed in EFS

1.0

1.0

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0.9

0.8

0.8 Cumulative proportion

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Hematopoietic Cell Transplantation for Breast Cancer

0.7 0.6 0.5 0.4

OS

0.3 0.2

p = 0.001

0.7 0.6 0.5

Soft tissue (n = 46)

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Fig. 63.1 High-dose chemotherapy for metastatic breast cancer (MBC). (a) Long-term disease-free survival (DFS) and overall survival (OS) of prospective phase II trials with cyclophosphamide, cisplatin, and BCNU (STAMP-I) as first-line therapy for MBC patients at the University of Colorado. (b) Event-free survival of STAMP-I trials according to specific metastatic sites. BM, bone marrow.

(median 11 versus 8 months; p = 0.006), but not OS (median 24 versus 28 months; p = 0.4). In the French PÉGASE 03 study, 180 patients with responsive tumors were randomized to HDC with cyclophosphamide and thiotepa, or observation [54]. The CR rate increased from 11% to 24% after HDC (p = 0.0002). At a median follow-up of 48 months, a significant EFS advantage was observed in favor of HDC (27% versus 10%; p = 0.0005), without significant OS differences in this first analysis (38% versus 30%; p = 0.7). Investigators from the International Breast Cancer Dose Intensity Study group treated 110 patients with four cycles of doxorubicin and docetaxel, followed by either six cycles of CMF or sequential high-dose cycles of ifosfamide, carboplatin, and etoposide and cyclophosphamide/ thiotepa with AHPCS [55]. The TRM rates in both arms were 4% and 9%, respectively. The HDC arm produced higher response rate (71% versus 44%) and CR (29% versus 6%) rates. At a median follow-up of 42 months, the study was positive for its primary endpoint, EFS (HDC 16% versus SDC 9%; p = 0.01). There was a trend for an OS advantage in the HDC arm in an intent-to-treat analysis (45% versus 37%; p = 0.1), which reached significance in an analysis based on actual treatment received (49% versus 35%; p = 0.02). In the PÉGASE 04 trial, 61 responding MBC patients were randomized to additional SDC or HDC with cyclophosphamide, mitoxantrone, and melphalan [56]. In its preliminary analyses, nonsignificant differences favoring HDC in EFS and OS were noted [56]. However, in its final analysis at a median follow-up of 7.5 years, significant benefits from HDC in progression-free survival (PFS) (18.7% versus 0%; p < 0.005) and OS (36.8% versus 13.8%; p = 0.02) were demonstrated, with median PFS times of 12 versus 6 months, and median OS times of 44 versus 19 months [57]. In the German Breast Cancer Dose Intensity Study group trial, 92 untreated MBC patients were randomized to receive six to nine cycles of adriamycin and paclitaxel or tandem cycles of high-dose cyclophosphamide, mitoxantrone, and etoposide, 6 weeks apart with AHPCS [58]. At a median follow-up of 52 months, there were no significant differences between the high-dose and control arms in PFS (27% versus 20%; p = 0.5) or OS (18% versus 21%; p = 0.75). Investigators at Duke University conducted two randomized trials with a crossover design, comparing early versus late use of HDC, using

cyclophosphamide, cisplatin, and BCNU (carmustine) (STAMP-I). In the first trial, 100 hormone-refractory MBC patients in first CR were randomized to immediate HDC or observation with HDC at subsequent relapse [59]. At a median follow-up of 10.6 years, EFS was superior in the immediate transplant arm (26% versus 10%; p = 0.03), with a median EFS of 10 versus 4 months (p < 0.006). Six-year OS rates were 26.5% versus 35% (p = 0.69), with median OS times of 2.1 versus 3.3 years (p = 0.2). The combined TRM from transplant in both arms was high (8.6%). In the second Duke trial, 69 patients with hormone-refractory boneonly MBC were randomized after first-line SDC to immediate HDC and radiotherapy of all bony metastases, or to radiotherapy and observation, again with HDC upon progression [60]. At a median follow-up of 8.1 years, EFS was superior in the immediate transplant arm (17% versus 0%; p < 0.0001) with median EFS times of 12 versus 4 months (p < 0.0001). The OS rates were not significantly different between the immediate and late transplant arms (17% versus 9%; p = 0.1) with median OS times of 3 versus 1.8 years. Interestingly, all three survivors in the observation arm received a transplant after progression, two of them subsequently remaining event-free. As in the other Duke trial with STAMP-I, overall transplant TRM was high (9.7%). In summary, a significant benefit in EFS in favor of HDC has been noted in six of the eight trials, but only one study (PÉGASE 04) shows an advantage in OS (Table 63.1). Longer follow-up is clearly needed in the NCIC and PÉGASE 03 trials to determine whether the early HDC advantage in EFS ultimately translates into an OS benefit. The lack of a direct comparison between HDC and non-HDC arms in the two Duke crossover trials obviates OS analyses and ultimately complicates their interpretation. The 2004 Cochrane Collaboration review of this issue included six trials (excluding the two Duke crossover studies) and a total of 850 patients [61]. A significant overall EFS benefit from HDC was detected at 1 year (risk ratio 1.81, 95% [confidence interval] CI 1.39–2.34; p < 10−5), and at 5 years (risk ratio 2.84, 95% CI 1.07–7.5; p = 0.04). While there was no OS difference at 1 year (risk ratio 0.99, 95% CI 0.91–1.08; p = 0.9), it approached statistical significance at 5 years (risk ratio 1.5, 95% CI 0.96–2.34; p = 0.08). Of note, at the 5-year time point analysis of this review, the final analysis of PÉGASE 04 reported in 2005, which was significant for OS, was not included. The overall TRM rates in the

934

Chapter 63

1.0

1.0

0.9

0.9 OS

0.7 0.6

RFS

0.5 0.4 0.3 0.2

0.8 Cumulative proportion

Cumulative proportion

0.8

4–9+

0.7 0.6 IBC

0.5

≥10+

0.4 0.3 0.2

0.1

p = 0.39

0.1

0.0

0

1

2

(a)

3

4

5

6

7

8

9

0.0

10 11 12 13

Years

0 (b)

1

2

3

4

5

6

7

8

9

10

11

12

13

Years

Fig. 63.2 High-dose chemotherapy for high-risk primary breast cancer. (a) Long-term relapse-free survival (RFS) (67%) and overall survival (OS) (70%) of 264 patients enrolled in prospective phase II trials of cyclophosphamide + cisplatin + carmustine at the University of Colorado, at a median follow-up of 7 years. (b) RFS in the different populations enrolled in those trials: four to nine positive nodes (4–9+), 10 or more positive nodes (≥10+), and inflammatory breast cancer (IBC).

HDC and SDC groups were 3.4% and 0.4%, respectively (risk ratio 4.07, 95% CI 1.39–11.88; p = 0.01). HDC in HRPBC The groups at Duke University [62] and the National Tumor Institute in Milan [63] pioneered the evaluation of HDC in patients with HRPBC. Long-term results of the Duke phase II trial in patients with 10 or more involved (+) axillary nodes showed a 10-year EFS rate of 72% [64]. In the Milan study, which explored a sequential high-dose single-agent approach, a 57% EFS rate was reported at median follow-up of 4 years [63]. The encouraging prospective phase II data of HDC in HRPBC (Fig. 63.2) prompted randomized trials across the world. Two very small randomized phase II trials were first reported in 1998. In a study conducted at the MD Anderson Cancer Center, 78 patients with 10 or more positive nodes after up-front surgery, or four or more positive nodes after preoperative chemotherapy, received eight cycles of fluorouracil, adriamycin, and cyclophosphamide, followed by either two cycles of dose-intense cyclophosphamide, etoposide, and cisplatin (a regimen that is not currently considered HDC) or no further therapy [65]. At latest follow-up of 11.8 years, there were no significant differences between the control and experimental arms in EFS (40% versus 26%; p = 0.1) or OS (47% versus 42%; p = 0.1) [66]. The other early study was a small Dutch pilot trial enrolling 81 patients with axillary level III involvement, who were treated with neoadjuvant 5-fluorouracil, epirubicin, and cyclophosphamide (FEC), surgery, and one postoperative cycle of FEC, and were then randomized to high-dose cyclophosphamide, thiotepa, and carboplatin (CTC; administered at higher doses and an optimized schedule, compared with STAMP-V) or observation [67]. The high-dose arm had a 15% drop-out rate. The final analysis of this trial, at median follow-up of 7 years, did not show differences between the HDC and SDC arms in EFS (49% versus 47.5%; p = 0.3) or OS (61% versus 62.5%; p = 0.8) [68]. Of note,

this trial employed a nonstandard infraclavicular lymph node biopsy, and not a standard axillary node dissection, to determine eligibility. Both of these trials were clearly underpowered to detect realistic differences. It is worth noting that the minimum detectable benefit in either trial would have been required to be of greater magnitude than the overall impact of adjuvant chemotherapy, versus no postoperative treatment at all, for breast cancer. Subsequent to their pilot trial, Rodenhuis and colleagues conducted a larger population-based phase III study that enrolled 885 patients with four or more positive lymph nodes, randomized to receive four cycles of FEC followed by one more cycle of FEC or high-dose CTC [69]. At its latest analysis at a median follow-up of 84 months, a trend for an EFS advantage was noticed in favor of HDC (64.3% versus 59%, hazard ratio 0.84; p = 0.07), with no detectable OS differences (73% versus 70%; p = 0.2) [70]. Prospectively planned subset analyses showed a substantial improvement in EFS among patients with 10 or more positive nodes (68% versus 49%; p = 0.01). In the group with four to nine positive nodes, EFS rates were 72% (HDC) versus 65% (SDC) (p = 0.5). Other subgroup analyses, which were unplanned, suggested that young patients and those with HER2− or with lower-grade tumors might benefit from HDC. Specifically, patients with HER2− disease (n = 621) experienced a marked benefit from HDC in EFS (72% versus 59%; p = 0.002) and OS (78% versus 71%; p = 0.02), with a hazard ratio for relapse decreased by about one-third after HDC [70]. In Cancer and Leukemia Group B (CALGB) 9082 trial, Peters and colleagues enrolled 785 patients with 10 or more positive nodes, who received four cycles of cyclophosphamide, doxorubicin, and 5fluorouracil (CAF) and were randomized to HDC with STAMP-I or to one additional cycle of those drugs at intermediate dose (ID) with GCSF support [71]. At a median follow-up of 7.3 years, there were no significant differences between the HDC and ID arms in EFS (61% versus 58%; p = 0.2) or OS (71% for both groups; p = 0.7). There was a 31% reduction in the number of relapses in the HDC arm, particularly among women younger than 50, which is certainly consistent with a

Hematopoietic Cell Transplantation for Breast Cancer

dose–response effect. However, the high TRM rate of 8.1% in the HDC arm (versus 0% in the ID arm) offsets this benefit. In the Anglo-Celtic trial, 605 patients with four or more positive nodes were randomized to four cycles of doxorubicin followed by either highdose cyclophosphamide and thiotepa or eight cycles of CMF [72]. At median follow-up of 6 years, there were no differences in EFS (57% and 54%, respectively; p = 0.7) or OS (62% and 64%, respectively; p = 0.4). Investigators from the Eastern Cooperative Oncology Group (ECOG) randomized 540 patients with 10 or more positive nodes to receive six cycles of CAF, with or without high-dose cyclophosphamide and thiotepa, administered with AHPCS from bone marrow or, towards the end of the study, PBPCs [73]. At a median follow-up of 6.1 years, there were no significant differences between HDC and SDC in EFS (55% versus 48%; p = 0.1) or OS (58% versus 62%; p = 0.3). Among patients meeting strict protocol eligibility, EFS appeared higher in the HDC arm (55% versus 45%; p = 0.04). In the transplant arm, there were nine toxic deaths (eight of the patients having received bone marrow support) and nine cases of secondary leukemia/myelodysplastic syndrome. The Southwestern Oncology Group (SWOG) trial randomized 539 patients with four or more positive nodes (92% of them with four to nine positive nodes) to receive conventional treatment with sequential dosedense adriamycin, paclitaxel, and cyclophosphamide, or four cycles of doxorubicin and cyclophosphamide (AC) followed by HDC (using STAMP-V in 86% and STAMP-I in 14% of patients) [74]. At a median follow-up of 70 months, there were no differences in EFS (78% versus 73%; p = 0.35) or OS (87% versus 83%; p = 0.4). In the Scandinavian trial, 525 patients with eight or more positive nodes (or five or more positive nodes plus an ER− and high S-phase fraction tumor) received nine cycles of individually tailored doseintensive FEC, or three cycles of conventional FEC followed by HDC with STAMP-V [75]. The tailored FEC regimen resulted in fewer breast cancer relapses than STAMP-V, but, probably due to its high cumulative epirubicin dose, it induced a relatively high 4% incidence of secondary leukemia/myelodysplastic syndrome. In the latest update of this trial at a median follow-up of 5 years, there was a trend in favor of tailored FEC in EFS (51% versus 45%; p = 0.07), with no significant differences in OS (60% versus 56%; p = 0.3) [76]. A major problem in interpreting this study is the fact that the control arm received higher cumulative doses of chemotherapy than the transplant arm. Nitz and colleagues from the West German Study Group (WSG) randomized 403 patients with 10 or more positive nodes to a dose-dense arm with four cycles of epirubicin and cyclophosphamide (EC) followed by six cycles of CMF, all every 2 weeks with G-CSF, or a transplant arm with two initial cycles of EC followed by tandem cycles of highdose epirubicin, cyclophosphamide, and thiotepa administered 4 weeks apart with AHPCS [77]. Neither arm had any TRM. At a median followup of 48 months, there was significant superiority of transplant in EFS (60% versus 44%; p < 0.001) and in OS (75% versus 70%; p = 0.02). An unplanned retrospective analysis showed a large benefit from HDC for tumors with a basal-like phenotype (ER/PR negativity, HER2 negativity, and basal cytokeratin positivity), with a hazard ratio for relapse of 3.06 (95% CI 1.41–6.06) if receiving dose-dense chemotherapy compared with tandem HDC [78]. The Michelangelo study compared the Milan high-dose sequential single-agent approach to SDC in 382 patients with four or more positive nodes [79]. At its most recent analysis at a median follow-up of 11 years, nonsignificant differences in favor of the high-dose arm in EFS (52% versus 44%) and OS (60% versus 51%) were noted. In the International Breast Cancer Study Group (IBCSG) study, 344 patients with 10 or more positive nodes (or five to nine positive nodes and an ER− or a T3 tumor) received standard four cycles of AC/EC fol-

935

lowed by three cycles of CMF, or a more dose-intense arm of three cycles of EC above conventional doses, every 3 weeks with AHPCS [80]. At a current follow-up of 5.8 years, the EFS rates were 52% and 43% for the dose-intense and standard arms, respectively (p = 0.07), with OS rates of 70% versus 61% (p = 0.1). In the PÉGASE 01 trial, 314 patients with seven or more positive nodes were randomized to receive SDC followed by high-dose cyclophosphamide, mitoxantrone, and melphalan or observation [81]. The TRM in the HDC arm was 0.6%. At a short median follow-up of 33 months, there were significant differences in EFS (71% versus 55%; p = 0.002), without an OS advantage (84% versus 85%; p = 0.3). Zander and colleagues of the German Autologous Bone Marrow Transplant Group (GABG) randomized 307 patients with 10 or more positive nodes to receive four cycles of EC followed by HDC with cyclophosphamide, thiotepa, and mitoxantrone, or three cycles of CMF [82]. Its most recent update at a median follow-up of 6.1 years showed nonsignificant trends in favor of HDC in EFS (51% versus 41%; p = 0.1) and OS (62.5% versus 57%, hazard ratio 0.84; p = 0.3) [83]. Bliss et al. randomized 281 patients with four or more positive nodes to receive six cycles of FEC, or three cycles of FEC followed by HDC with STAMP-V [84]. At a median follow-up of 68 months, there were no differences in EFS (59% versus 57%; p = 0.76) or OS (67% versus 66%; p = 0.4). In summary, at least 15 randomized trials, 11 of them enrolling over 300 patients, have compared diverse forms of HDC with lower doses of chemotherapy for HRPBC. Their results, in most cases still preliminary, appear contradictory at this point (Table 63.2). There are clearly negative trials, such as the ECOG, Scandinavian, CALGB, SWOG, and Anglo-Celtic studies. In contrast, other studies, such as WSG or PÉGASE 03, show significant superiority of HDC. Other trials, such as the National Dutch, GABG, IBCSG, and Michelangelo studies, show nonsignificant trends in favor of transplant at this point. The 2006 Cochrane review on HDC for HRPBC analyzed 13 of those trials, totaling 5111 patients [85]. Mature results, defined as all study participants followed for a minimum of 3 years, were available for only the small MD Anderson and Dutch pilot trials, and the CALGB study. A significant EFS benefit in favor of HDC was detected at 3 years (risk ratio 1.19, 95% CI 1.06–1.19), 4 years (risk ratio 1.24, 95% CI 1.16– 1.45), and 5 years (risk ratio 1.06, 95% CI 1–1.13), despite not including the positive PÉGASE 01 and WSG trials in this last time point analysis. There were no significant OS differences at any time point. Overall TRM rates were 2.5% and 0.1% in the transplant and conventional treatment groups, respectively (risk ratio 8.58, 95% CI 4.13–17.8). Quality of life scores were significantly worse immediately after HDC, but with no differences after 1 year. The recently reported MD Anderson Cancer Center/European Group for Blood and Marrow Transplantation meta-analysis of individual patient data from 15 randomized trials, including 6102 patients, has detected an overall absolute EFS benefit of 13% in favor of HDC (p = 0.0001) at an overall median follow-up of 6 years, with separation of the curves only appreciable after the second year of follow-up (Fig. 63.3(a)) [86]. The difference in OS (which, not surprisingly, was not noticeable until later than 5 years of follow-up) did not reach statistical significance (p = 0.16) (Fig. 63.3 (b)). Critical commentary on the available results in MBC and HRPBC Adequate length of follow-up is critical. Many of the results reported to date are still preliminary. An overall EFS benefit for HDC is apparent in both HRPBC and MBC. As has been pointed out [87], EFS is clinically relevant in the adjuvant setting, and has become a valid endpoint

Responsive

Responsive Untreated Untreated

CR

Untreated

HR, Bone only Responsive

NCIC [53]

Philadelphia [51,52] PÉGASE 03 [54] IBDIS-1 [55]

Duke crossover1(Patients in CR) [59] GEBDIS [58]

Duke crossover-2 (Bone only) [60] PÉGASE 04 [57] CPA/Mito/Mel

61

69

Tandem CPA/Mito/Eto ×2 STAMP-I

CPA/TT/Cb(STAMP-V) CPA/TT Sequential Ifos/Cb/Eto → CPA/TT CPA/CDDP/BCNU (STAMP-I)

CPA/Mito/Eto

HDC regimen

92

100

184 180 110

224

n

90

97

52

127

67 48 42

48

27% Median 11 months 17% Median 12 months 19% Median 12 months

NR Median 11 months 4% Median 10 months 27% 25% Median 14 months 25% Median 10 months

HDC

Event-free survival rates

0% Med: 6 months

0% Median 4 months

20% Median 11 months

10% Median 4 months

3% Median 9 months 10% 20% Median 9 months

NR Median 9 months

Control

18% Median 27 months 17% Median 3 years* 37% Median 44 months

<0.0001 <0.005

26.5%*

<0.006

0.7

14% Median 26 months 38% 39% Median 32 months

37% Median 24 months

HDC

0.3 0.0002 0.01

0.006

p

Overall survival rates

14% Median 19 months

9% Median 1.8 years*

21% Median 23 months

38%*

13% Median 24 months 30% 35% Median 23 months

38% Median 28 months

Control

Cb, carboplatin; CDDP, cisplatin; CPA, cyclophosphamide; CR, complete response; Eto, etoposide; GEBDIS, German Breast Cancer Dose Intensity Study; HR, hormone refractory; IBDIS, International Breast Cancer Dose Intensity Study; Ifos, ifosfamide; Mel, melphalan; Mito, mitoxantrone; NCIC, National Cancer Institute of Canada; NR, not reported; PÉGASE, Programme d’Étude de la Greffe Autologue dans les Cancers du Sein; TT, thiotepa. *Not evaluable for a direct comparison of overall survival, due to crossover design.

Population

Trial

Follow-up (months)

Table 63.1 Randomized trials of high-dose chemotherapy (HDC) in metastatic breast cancer

0.02

0.1*

0.77

0.7*

0.6 0.7 0.1

0.4

p

936 Chapter 63

885 785 605 540 523 525 403 382 340 314 302 281 95 81 78 6,210

≥4 ≥10 ≥4 ≥10 ≥4 >5 to 8 ≥10 ≥4 ≥10 >7 ≥10 ≥4 ≥10 Axillary level III involvement ≥10, or ≥4 after chemo ≥4

NWAST [69,70] CALGB [71] Anglo-Celtic [72] ECOG [73] SWOG [74] SBCG [75,76] WSG [77] Michelangelo [79] IBCSG [80] PÉGASE 01 [81] GABG [82,83] ICCG [84] JCOG [140] Dutch pilot [67,68] MDACC [65,66] MDACC/EBMT meta-analysis [86]

CPA/TT/Cb (CTC) CPA/CDDP/BCNU (STAMP-I) CPA/TT (CT) CT CPA/TT/Cb (STAMP-V) STAMP-V Epi/CPA/TT × 2 Sequential single agents Epi/CPA × 3 CPA/Mito/Mel CPA/TT/Mel STAMP-V CT CTC CPA/Eto/CDDP × 2 (DICEP) Various

HDC regimen 7 5.1 6 6.1 NR 5 4 11 5.8 2.75 6.1 5.6 5.25 6.9 12 6

Median follow-up (yrs)

Control

64 59 61 58 57 54 55 48 75 78 45 51 60 44 52 44 52 43 71 55 51 41 57 59 52 37 49 47 26 40 Hazard ratio 0.87 in favor of HDC

HDC

Event-free survival rates

0.07 0.2 0.7 0.1 NS 0.07 0.0006 NS 0.07 0.002 0.1 0.7 0.1 0.37 0.1 0.0001

p

73 71 62 58 84 56 75 60 70 84 62 66 63 61 42

HDC

70 71 64 62 87 60 70 51 61 85 57 67 62 62 47 HR 0.89 (HDC)

Control

Overall survival rates

0.2 0.75 0.4 0.3 NS 0.3 0.02 NS 0.1 0.33 0.3 0.4 0.7 0.85 0.1 0.16

p

CALGB, Cancer And Leukemia Group B; EBMT, European Blood and Marrow Transplant Group; ECOG, Eastern Collaborative Oncology Group; Epi, epirubicin; GABG, German Autologous Bone Marrow Transplant Group; IBCSG, International Breast Cancer Study Group; ICCG, International Collaborative Cancer Group; JCOG, Japan Clinical Oncology Group; MDACC, MD Anderson Cancer Center; NS, not significant; NWAST, Netherlands Working Party on Autologous Transplantation in Solid Tumors; SBCG, Scandinavian Breast Cancer Group; SWOG, South Western Oncology Group; WSG, West German Study Group. See Table 63.1 for other abbreviations.

n

Population (number of positive nodes)

Trial

Table 63.2 Randomized trials of high-dose chemotherapy (HDC) in high-risk primary breast cancer

Hematopoietic Cell Transplantation for Breast Cancer

937

938

Chapter 63

1.0

1.0

HDC SDC

0.8

Overall survival

Disease-free survival

0.8

It would seem unlikely that the old all-alkylator regimens, not specifically breast cancer directed, which were developed two decades ago and employed in many randomized studies, would end up as the optimal stem-cell supported high-dose combinations for this disease. While STAMP-I appears to decrease the incidence of relapses compared with SDC, its potential for serious toxicity hampers its broad applicability. On the other hand, cyclophosphamide and thiotepa, and STAMP-V are both safe, but clearly do not seem to be the most active combinations that could be currently administered with AHPCS.

HDC SDC

0.6

0.4

0.6

0.4

Can we select patient populations that might selectively benefit from HDC?

0.2

0.2 n = 6192 3048 events p = 0.0001

0.0 0

5

(a)

10

n = 6192 2424 events p = 0.16

0.0 15

Years

0

5

(b)

15

10 Years

Fig. 63.3 MD Anderson Cancer Center/European Group for Blood and Marrow Transplantation meta-analysis of randomized trials of high-dose chemotherapy (HDC) for high-risk primary breast cancer. (a) Comparison of event-free survival (p = 0.0001). (b) Comparison of overall survival (p = 0.16). SDC, standard-dose chemotherapy.

35 31 Cumulative proportion

30 25 20

16

15 10 10

8

5

2

5

5th year

>5th year

0 1st year

2nd year

3rd year

4th year

Fig. 63.4 Time distribution of relapses after high-dose chemotherapy for high-risk primary breast cancer at the University of Colorado (n = 264). The number above each bar corresponds to the number of relapses during each year of follow-up.

for new drug approval in the adjuvant setting for breast and colorectal cancer. It can be argued that EFS differences in MBC are of little benefit unless they translate into an improved quality of life or prolonged OS. However, it is obvious that trials showing significant EFS differences in their first analyses, such as the NCIC or PÉGASE 03 studies, need longer follow-up. It is important to consider the different natural history of breast cancer after SDC and HDC. Median time to relapse of HRPBC patients after SDC is generally between 2 and 3 years. In contrast, the majority of relapses after HDC for HRPBC occur within the first 2 years following treatment, and very few patients experience recurrence after the fifth year (Fig. 63.4) [88,89]. Observations from other settings, such as lymphoma [90] or myeloma [91], where survival benefits were only noticed after mature analyses, may be also applicable to breast cancer, and longterm follow-up and second publication of major trials will be important for definitive conclusions. While we tend to discuss HDC as a single approach, there are substantial differences in the HDC regimens used in the randomized trials.

We can now predict which patients with MBC are most likely to achieve long-term remissions after HDC, such as those with low tumor burden, in CR after SDC, or without liver involvement (Fig. 63.1(b)) [92,93]. Prospective studies of conventional multimodal treatment for patients with isolated relapses had shown long-term EFS rates of 32–39% [94–96]. The hypothesis that these MBC patients with low tumor burden might attain major benefit from HDC early in the course of their disease was prospectively tested at the University of Colorado [97]. A phase II study of four cycles of adriamycin-based induction therapy followed by HDC with STAMP-I, as first-line therapy for oligometastatic disease, was conducted in 60 consecutive patients. Oligometastases were defined as one or more sites of macroscopic tumor that could be either resected en bloc and/or encompassed within a single radiotherapy field, and/or less than 5% of bone marrow involvement. Most patients had received previous adjuvant chemotherapy. At a median post-transplant follow-up of 5 years, the EFS and OS rates were 52% and 62%, respectively, with median EFS and OS times of 4.3 years and 6.7 years, respectively (Fig. 63.5(a)). HER2− status and a single metastatic site were independent favorable predictors of outcome (Fig. 63.5 (b)) [98]. Likewise, retrospective analyses have identified prognostic factors for HDC for HRPBC. Somlo et al. identified PR positivity as an independent favorable predictor of EFS in 114 patients enrolled in their studies using two different HDC regimens at City of Hope National Medical Center [99]. In an analysis of 176 patients treated at the University of Colorado with STAMP-I, tumor size, presence of either or both of ER and PR (ER/PR), and nodal ratio (number of positive nodes to number of sampled nodes) were independently associated with relapse [100]. Those three variables formed the basis for the following scoring system, which can assign a relapse risk to each patient before HDC: Score = (Nodal ratio × 3.05) + (Tumor size × 0.15) – (ER/PR × 1.15) In this formula, tumor size is entered in centimeters, and ER/PR is assigned “1” if positive (i.e. ER and/or PR are positive) and “0” if negative (if both are negative). Scores of 2.41 or more, and less than 2.41, allocate patients to a high- or low-risk category, with risks of relapse of 65% and 11%, respectively. The differences in EFS (p < 0.000001) and OS (p < 0.00001) were highly significant (Fig. 63.6(a)). This predictive model was subsequently validated both externally and prospectively [89]. The prognostic value of the nodal ratio, probably superior to that of the absolute number of positive nodes, has been subsequently confirmed by other authors in HDC-treated patients [101,102], and is being actively investigated in breast cancer populations treated with SDC [103]. HER2 overexpression has been shown to have major prognostic implications after HDC in HPRBC (Fig. 63.6 (b)) [104,105,106] and MBC (Fig. 63.5(b)) [97,107]. Additionally, HER2− status may predict benefit from HDC compared with SDC [70]. Other molecular independent adverse prognosticators include EGFR overexpression (Fig.

Hematopoietic Cell Transplantation for Breast Cancer

Cumulative proportion surviving relapse free

Cumulative proportion surviving

1.0 0.9 0.8 0.7 OS

0.6 0.5 0.4

RFS

0.3 0.2 0.1

1.0 0.9 0.8

HER2– and 1 site

0.7 0.6 0.5 HER2+ or >1 site 0.4 0.3 0.2 HER2+ and >1 site 0.1 0.0

0.0 0 (a)

939

20

40

60

80

100

120

140

Months

0 (b)

20

40

60

80

100

120

140

Months

Fig. 63.5 High-dose chemotherapy for oligometastatic disease. (a) Relapse-free survival (RFS) and overall survival (OS) results of a prospective trial of STAMP-I. (b) RFS curves in this trial according to HER2 status and number of metastatic sites.

63.6(c)), particularly when co-overexpressed with HER2 (Fig. 63.6(d)) [108], and increased tumor angiogenesis (Fig. 63.6(e)) [109]. Future comparative trials could focus on the populations most likely to benefit from HDC. Purging of progenitor cell grafts Although tumor contamination may be lower in PBPC than in bone marrow fractions [110], breast cancer cells in PBPCs can still be detected in substantial fractions of MBC and HRPBC patients. Most post-HDC relapses in patients with MBC occur in sites of prior disease, suggesting an insufficient cytoreductive capacity of HDC, rather than a direct effect from tumor cells contaminating the graft. Recent analyses have shown a significant, but not independent, adverse effect from tumor contamination of PBPC products in MBC patients receiving HDC (Fig. 63.7(b)) [111,112], which suggests that tumor cells may constitute a marker of systemic micrometastases, rather than being directly responsible for failure of transplant. In contrast, the presence of occult tumor cells in apheresis products constitutes an independent adverse prognostic factor for recurrence in recent analyses of HRPBC patients (Fig. 63.7(a)), providing indirect proof of a role of contaminating tumor cells in relapse in this population [111,113]. While direct evidence for a contribution of contaminating tumor cells to relapse after autografting, as demonstrated for other tumors [114,115], is lacking, purging the graft of tumor cells holds promise, particularly for HRPBC patients. Negative purging, which eliminates contaminating cells by pharmacologic or immunologic methods, achieves 2–4-log tumor cell depletion, albeit with marked engraftment delays [116–118]. Positive selection targets the CD34 antigen, expressed on 0.5–3% of normal cells in the bone marrow and PBPC fractions, but not on breast cancer cells [119]. Shpall and collaborators tested CD34 selection in 155 patients, showing a mean 2-log tumor cell depletion, with normal post-transplant neutrophil and platelet recovery and immune reconstitution [120]. In a separate trial, 92 HRPBC patients were randomized to receive HDC with CD34selected or unselected PBPCs [121]. No short-term differences in EFS or OS were noticed in this small trial. Since most patients with contaminated products still have detectable cancer cells present in their stem

cell grafts following CD34 selection, effective purging may require a combination of different procedures [122,123]. Other potential ways of future progress There is a pressing need to improve HDC for breast cancer. It appears unlikely that first-generation high-dose regimens developed two decades ago would end up being the optimal stem cell-supported, high-dose combinations. It is necessary to develop new synergistic HDC combinations of more active drugs showing a dose–response effect, such as docetaxel [124] or gemcitabine [125]. In parallel, the traditional use of a late single HDC cycle after long SDC induction does not appear to constitute optimal treatment, and earlier use of sequential HDC cycles after shorter induction SDC courses [126], or used up front as in the randomized WSG trial in HRPBC [77], needs to be further explored. Independent of whether the superior tumor debulking capacity of HDC translates into improved outcomes, post-transplant minimal residual disease provides an optimal scenario for application of novel therapies. HDC and novel therapeutics targeting signal transduction pathways are not mutually exclusive. In solid tumors, available data suggest that these promising new agents may help improve outcome when combined with chemotherapy, but they are unlikely to have a substantial impact alone. The anti-HER2 monoclonal antibody trastuzumab or other biologic agents can be easily administered after transplant, or perhaps even integrated into the high-dose regimen. A pilot study has shown the safety of concurrent administration of trastuzumab with HDC, exploiting their in vitro synergy [127]. While immune therapies for breast cancer are still in development, their testing post HDC has a robust rationale. Retrospective analyses of the post-transplant recovery of the absolute lymphocyte count suggest a major role of the immune system in controlling post-HDC residual tumor, particularly in MBC (Fig. 63.8) [93,128]. A post-transplant immune role appears to be more important for the subset of memory T cells [129]. Combination of HDC with immune therapies holds substantial promise. In summary, achievement of a minimal residual disease status with HDC may allow institution of innovative therapies post transplant to prevent recurrence by such immunologic approaches, or novel targeted

940

Chapter 63

1.0 1.0

0.9

LOW SCORE

0.9 Cumulative proportion

Cumulative proportion

0.8 0.7 0.6 0.5 0.4

HIGH SCORE

0.3 0.2

p = 0.0001

HER2– 0.8 0.7 0.6 0.5

HER2+

0.4 0.3 0.2

0.1

p = 0.001

0.1

0.0 0

1

2

3

4

5

6

(a)

7

8

9

10

11

12

0.0

13

0

1

2

3

4

5

6

(b)

Years

7

8

9

10

11

12

13

Years

1.0 1.0

0.9

0.9 0.8

EGFR–

Cumulative proportion

Cumulative proportion

EGFR and HER2 negative

0.8

0.7 0.6 0.5 0.4 EGFR+ 0.3

0.7 EGFR or HER2 positive 0.6 0.5 0.4

EGFR and HER2 positive

0.3 p < 0.0001

0.2

0.2

p = 0.003

0.1

0.1

0.0

0.0 0

2

4

6

8

10

12

14

Years

(c)

0 (d)

2

4

6

8

10

12

14

Years

1.0

Cumulative proportion

0.9 0.8

Low MVD

0.7 0.6 0.5 0.4 High MVD

0.3 0.2 0.1

p < 0.001

0.0 0 (e)

2

4

6

8

10

12

14

Years

Fig. 63.6 Effect of prognostic factors on event-free survival (EFS) of patients with high-risk primary breast cancer after high-dose chemotherapy. (a) Effect of the clinical score (tumor size, hormone receptor status, and nodal ratio). (b) Effect of HER2 overexpression. (c) Effect of endothelial growth factor (EGFR) expression. (d) Combined effect of HER2 and EGFR expression. (e) Effect of intratumor microvessel density (MVD).

Hematopoietic Cell Transplantation for Breast Cancer

1.0

1.0

0.9

0.9 0.8

–

OTC

Cumulative proportion

Cumulative proportion

941

0.7 0.6 0.5 0.4 OTC+

0.3 0.2

0.8 0.7 0.6 0.5

p = 0.04

0.4 OTC–

0.3 0.2

p = 0.007

0.1

OTC+

0.1

0.0

0.0 0

1

2

3

4

5

(a)

6

7

8

9

10

11

12

13

0

1

2

3

4

(b)

Years post HDC

5

6

7

8

9

10

11

12

Years post HDC

Fig. 63.7 Prognostic effect of detection of occult tumor cells (OTC) by immunocytochemistry in the apheresis products on event-free survival. (a) High-risk primary breast cancer. (b) Metastatic breast cancer. HDC, high-dose chemotherapy.

1.0

1.0

0.9

0.9 0.8 High ALC

0.7

Cumulative proportion

Cumulative proportion

0.8

0.6 Low ALC

0.5 0.4

FFR

0.3 0.2

FFR

0.6 0.5 0.4

High ALC

0.3 0.2

p = 0.54

0.1

Low ALC

p = 0.007

0.1

0.0

0.0 0

(a)

0.7

10

20

30

40

50

60

70

80

90

100

110

0

120

Months post transplant

(b)

1

2

3

4

5

6

7

8

9

10

11

12

13

Years

Fig. 63.8 Effect of immune recovery, determined by the absolute lymphocyte count (ALC) on day 15 after transplantation (cut-off 500/mm3), on freedom from relapse (FFR) after high-dose chemotherapy. (a) No effect in high-risk primary breast cancer. (b) Significant effect in metastatic breast cancer.

agents. All new therapies will require the scrutiny of controlled clinical trials.

Allogeneic transplantation for MBC Allogeneic hematopoietic cell transplant has been shown to confer an immune graft-versus-tumor (GVT) effect against hematologic malignancies. Following anecdotal reports suggesting the existence of a potential GVT effect in breast cancer [130,131], Ueno and colleagues at MD Anderson Cancer Center treated 10 MBC patients with high-dose cyclophosphamide, thiotepa, and BCNU, followed by allogeneic PBPC transplantation from a matched sibling [132]. Four patients who experienced tumor progression after transplant had their immunosuppression reduced, and one received a donor lymphocyte infusion. Two of those patients experienced regression of liver metastases in association with exacerbation of acute graft-versus-host disease (GVHD).

Allogeneic transplantation using HDC is associated with significant morbidity and mortality. The introduction of reduced-intensity conditioning (RIC) regimens, generally based on fludarabine or lower doses of busulfan, which can provide enough immunosuppression to allow allogeneic stem cell engraftment, produced a substantial reduction in TRM. This strategy relies most of its antitumor efficacy on a GVT effect. The demonstration of a therapeutic effect of RIC allogeneic transplantation in metastatic renal cell carcinoma [133] led to trials of this approach at different institutions in MBC (Table 63.3). Bishop and colleagues employed an elegant design to differentiate the antitumor effects of the conditioning regimen from a GVT effect in 16 patients with MBC involving in most cases the liver or lungs, pretreated with a median of four prior regimens [134]. Patients first received fludarabine and cyclophosphamide to attain tumor control and sufficient immunosuppression (estimated as a CD4+ count <50/μL) to allow engraftment of T-cell-depleted PBPCs from a matched sibling donor,

16

12

10

8

2

6

Bishop [134]

Blaise [141]

Rizzieri [142]

Ueno [143]

Pedrazzoli [144]

Bregni [145]

2–5

3 (including prior HDC/AHPCS in both patients) 3 (including prior HDC/AHPCS)

4 (including prior HDC/AHPCS in 25% patients)

N/A

2 (including prior HDC/AHPCS in 23% pts)

5 (including prior HDC/AHPCS in 33% patients)

3

Flu/CPA/TT

Flu/CPA

Flu/Mel

Flu/CPA/alemtuzumab

Flu/Bus/ATG

T-cell replete

T-cell replete

T-cell replete

T-cell depleted

T-cell depleted

T-cell depleted

T-cell replete

Tandem HDC → Flu/CPA

Flu/CPA

Allograft

Conditioning regimen

6–43

14

10

16

6

16

19

43

Median follow-up (months)

13%

1

0

0

2

1

2

3

Treatment-related mortality (number)

26%

2 (2 PR)

2 (2 CR with limited or no GVHD: 1 in regional lymph nodes, 1 in bones) 2 (2 PR)

2 (1 CR in bones without concurrent GVHD, 1 PR in visceral metastases after DLI) 3 (3 PR)

2 (2 PR in visceral metastases)

4

15%

1

0

4

N/A

1

0

3

Event-free survival Responses (number) (number)

31%

3

0

4

N/A

2

4

5

Overall survival (number)

Most patients in PD at RIC All patients experienced further PD after RIC 5 patients received DLI, 2 responded

Both patients had visceral metastases

No post-NST immunosuppression No patient in remission at RIC Low-risk population with mostly nonvisceral metastases in PR/ SD at RIC 1 patient alive in CR 3 years after RIC

Responses associated with GVHD. 3 patients alive in CR 3.5–6 years after RIC Planned DLIs from day +42. Overall 37.5% rate of tumor regression (including minor responses), mostly associated with GVHD Most patients in PD at RIC

Comments

AHPCS, Autologous hematopoietic progenitor cell support; ATG, antithymoglobulin; Bus, busulfan; CPA, cyclophosphamide; CR, complete response; DLI, donor lymphocyte infusion; Flu, fludarabine; GVHD, graft-versus-host disease; HDC, high-dose chemotherapy; Mel, melphalan; NST, nonmyeloablative stem cell transplantation; PD, progressive disease; PR, partial response; SD, stable disease; TT, thiotepa.

71

17

Carella [135]

Aggregate results

n

Author

Median number of prior chemotherapy regimens

Table 63.3 Reported clinical studies of reduced-intensity conditioning (RIC) allogeneic transplantation for metastatic breast cancer

942 Chapter 63

Hematopoietic Cell Transplantation for Breast Cancer

with prophylactic cyclosporine through to day 40 after transplantation. On day 28 after transplantation, tumor restaging tests provided a baseline for evaluation of subsequent GVT-related responses. This was followed by planned donor lymphocyte infusions (DLIs) in 11 patients at increasing doses between 1 and 3 months post transplantation. Nine patients developed grade II–IV acute GVHD, and two patients developed extensive chronic GVHD. Two patients died from transplant complications on day 3 after transplantation and at 6 months, respectively. Six patients (40%) experienced tumor regression, with two partial responses and four minor responses. These responses were observed 42 days to 13 months post transplant after DLI in five patients, and after cyclosporine discontinuation in one patient. They occurred after establishment of full donor chimerism, and in association with clinical GVHD in five of the six cases. Unfortunately, they seemed to be quickly abrogated by the steroids needed to treat GVHD, and lasted for a median of only 4 (range 1–7) months. At a median follow-up of 2 years, four patients remained alive with disease, with a median OS time of 10 months. In an attempt to cytoreduce tumors before transplantation, Carella and colleagues evaluated a tandem auto/allogeneic transplant approach [135]. These authors treated 17 patients with heavily pretreated and largely refractory MBC with high-dose mitoxantrone and thiotepa with AHPCS, followed 1–3 months later by an allogeneic transplant from a matched sibling donor, using an RIC regimen of fludarabine and cyclophosphamide, and cyclosporine and methotrexate as GVHD prophylaxis. Eleven patients received a median number of three DLIs for disease progression or mixed chimerism. Only two patients developed acute grade III GVHD, with no case of grade IV, and five patients developed extensive chronic GVHD. There was no TRM after the autologous transplantation and no TRM in the first 100 days after the allogeneic procedure, although three patients died of late complications related to extensive chronic GVHD. Interestingly, the response rate was higher after the RIC plus allogeneic transplant than after the preceding HDC and AHPCS (24% and 21%, respectively). Responses to the allogeneic transplant were gradual and occurred after GVHD. At median follow-up of 3.6 years, five patients (29%) were alive, three of them in CR. In summary, this early and limited experience of allogeneic transplant using RIC in heavily pretreated patients with MBC suggests the following: 1 This treatment is feasible for breast cancer, with an overall TRM rate below 15%. 2 This procedure is largely devoid of regimen-related toxicity, with acute and chronic GVHD being its main toxicities, in many cases elicited to treat persistent or progressive disease. 3 RIC allows attainment of full donor chimerism in most patients.

943

4 There is convincing evidence of a clinical GVT effect in breast cancer, with an overall response rate of 26%. 5 Withdrawal of immunosuppression and/or DLI seems necessary for tumor response. 6 Most, but not all, responses have been observed in association with GVHD. 7 Responses have been short lived in many cases, probably due to the treatment required to control GVHD. 8 In contrast to HDC, where responses are usually quick, GVT-mediated responses are usually delayed until 3 or 4 months after RIC. 9 In many cases, patients experience tumor progression after transplant and may respond to subsequent immune maneuvers. 10 Rapidly growing tumors are unlikely to respond. 11 Tumor burden before RIC has a critical effect on outcome. In this regard, the strategy of Carella and collaborators of a sequential approach using HDC with AHPCS to cytoreduce tumors before RIC is promising [135]. This strategy has been tested with success in multiple myeloma [136,137]. There remain questions about the donor cells that mediate the GVT effects or their target antigens. Current evidence suggests that donor T-cell populations are responsible for the antitumor activity. Experiments in murine breast cancer transplant models show that donor CD8+ cells of type 1 cytokine phenotype mediate greater GVT and GVHD effects than those with a type 2 polarization [138]. Consistent with this, clinical responses in renal cell carcinoma patients appear mediated by CD8+ T cells producing the type 1 cytokine interferon-gamma [139]. Less is known about the antigenic targets of these cells. Whether clinical responses result from the interaction of donor T cells with host antigens that are largely restricted to the tumor, or whether they are the result of nonspecific generation of inflammatory cytokines that accompany GVHD, remains to be elucidated. While different antigens mediate GVHD and GVT in preclinical models, the early clinical experience seems to indicate that occurrence of GVHD is necessary for the GVT effect, which suggests that both events may be mediated by the same alloreactive T cells. Efforts to purge allogeneic T cells targeting antigens that are broadly expressed on normal tissues, while expanding immune cells with tumor specificity, could potentially enhance GVT effects and reduce GVHD. In summary, in contrast to hematologic malignancies, it appears unlikely that RIC and allogeneic transplantation by itself will be capable of complete eradication of metastatic solid tumors. More experience with this approach is necessary to determine whether it can be considered a therapeutic option for patients with advanced, but not bulky or progressive, disease.

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54.

55.

56.

57.

58.

59.

60.

61.

62.

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64.

breast cancer: NCIC MA.16. J Clin Oncol 2008; 26: 37–43. Biron P, Durand M, Roché H et al. High dose thiotepa (TTP), cyclophosphamide (CPM) and stem cell transplantation after 4 FEC 100 compared with 4 FEC alone allowed a better disease free survival but the same overall survival in first line chemotherapy for metastatic breast cancer. Results of the PEGASE 03 French protocol. Proc Am Soc Clin Oncol 2002; 21: abstract 42. Crown, J, Perey L, Lind M et al. Superiority of tandem high-dose chemotherapy (HDC) versus optimized conventionally-dosed chemotherapy (CDC) in patients (pts) with metastatic breast cancer (MBC): the International Breast Cancer Dose Intensity Study (IBDIS 1). Proc Am Soc Clin Oncol 2003; 22: abstract 23. Lotz J-P, Curé H, Janvier M et al. High-dose chemotherapy (HD-CT) with hematopoietic stem cells transplantation (HSCT) for metastatic breast cancer (MBC): results of the French Protocol PEGASE 04. Proc Am Soc Clin Oncol 1999; 18: abstract 43. Lotz JP, Cure H, Asselain B et al. High-dose chemotherapy with haematopoietic stem cell transplantation for metastatic breast cancer patients: final results of the French multicentric randomised CMA/PEGASE 04 protocol. Eur J Cancer 2005; 41: 71–80. Schmid P, Schippinger W, Nitsch T et al. Upfront tandem high-dose chemotherapy compared with standard chemotherapy with doxorubicin and paclitaxel in metastatic breast cancer: results of a randomized trial. J Clin Oncol 2005; 23: 432– 40. Vredenburgh J, Coniglio D, Broadwater G et al. Consolidation with high-dose combination alkylating agents with bone marrow transplantation significantly improves disease-free survival in hormone-insensitive metastatic breast cancer in complete remission compared with intensive standard-dose chemotherapy alone. Biol Blood Marrow Transplant 2006; 12: 195–203. Vredenburgh J, Madan B, Broadwater G et al. A randomized phase III comparative trial of immediate consolidation with high-dose chemotherapy and autologous peripheral blood progenitor cell support compared to observation with delayed consolidation in women with metastatic breast cancer and only bone metastases following intensive induction chemotherapy. Bone Marrow Transplant 2006; 37: 1009–15. Farquhar C, Marjoribanks J, Basser R et al. High dose chemotherapy and autologous bone marrow or stem cell transplantation versus conventional chemotherapy for women with metastatic breast cancer. Cochrane Database Syst Rev 2005 (3): CD003142. Peters WP, Ross M, Vredenburgh JJ et al. Highdose chemotherapy and autologous bone marrow support as consolidation after standard-dose adjuvant therapy for high-risk primary breast cancer. J Clin Oncol 1993; 11: 1132–43. Gianni AM, Siena S, Bregni M et al. Efficacy, toxicity and applicability of high-dose chemotherapy as adjuvant treatment in operable breast cancer with 10 or more involved axillary nodes: five-year results. J Clin Oncol 1997; 15: 2312– 21. Nikcevich DA, Vredenburgh JJ, Broadwater G et al. Ten year follow-up after high-dose chemo-

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Christie J. Moore, Brandon Hayes-Lattin & Craig R. Nichols

Hematopoietic Cell Transplantation in Germ Cell Tumors

Introduction

Initiation of salvage therapies in germ cell tumors

Malignant testicular tumors are the most common solid tumors among young men, with a rising annual incidence of approximately 9000 new cases in the United States. Ninety-five percent of malignant testicular tumors are germ cell tumors [1]. Germ cell tumors can also arise in extragonadal sites such as the retroperitoneum or mediastinum, often portending a poorer prognosis. Staging is based on pattern of spread, and initial therapy is guided by the presenting stage (Tables 64.1 and 64.2) [2]. The importance of germ cell tumors in the field of oncology is highlighted by the fact that standard therapies for early stage disease routinely cure more than 95% of patients. Current research goals focus on limiting therapy while preserving high cure rates for these patients. Before the advent of modern cisplatin-based chemotherapies, metastatic germ cell tumors were almost always fatal, but today the cure rate for metastatic disease reaches 70–80% (Tables 64.3 and 64.4) [3,4]. Unfortunately, 20–30% of patients with advanced disseminated disease require additional therapy after primary cisplatin-based treatments, including salvage surgery or chemotherapy. In an effort to better care for and study patients with metastatic disease, many prognostic models based on the pattern of disease spread and serum tumor markers at disease presentation have been employed [5–14]. In 1995, a uniform system was developed based on 5202 patients with nonseminomatous germ cell tumors and 660 patients with seminomas treated with cisplatin-containing regimens, to guide decisionmaking and standardize patient enrolment in clinical trials (Table 64.5) [15]. Additional poor prognostic factors may include a high proliferative index or the presence and number of cytogenetic abnormalities such as an isochromosome of the short arm of chromosome 12 [16,17]. Conventional-dose salvage approaches to patients with poor risk, recurrent or refractory germ cell tumors lead to remission in 30–60% of patients, but only 20% of patients gain long-term survival. Such a relatively low rate of cure with salvage therapy spurred investigation of alternate therapies including high-dose chemotherapy with autologous hematopoietic cell transplantation (HCT) (Table 64.6). Herein, we will review the management of the small fraction of patients for whom initial treatments are not fully effective, with emphasis on the role of high-dose chemotherapy.

The decision to pursue salvage therapy for germ cell tumors is important, and consultation with experts in the intricacies of managing this disease is strongly recommended. Several clinical situations may mimic progressive or recurrent disease and lead to inappropriate initiation of salvage therapy.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Tumor markers Management of testicular cancer has come to depend on the accurate determination and clinical interpretation of serum tumor markers. Lactate dehydrogenase is a nonspecific marker that probably correlates with disease bulk. The most sensitive and specific markers are alphafetoprotein (AFP) and β-subunit human chorionic gonadotropin (HCG). Among patients with disseminated testicular or primary retroperitoneal nonseminomatous germ cell tumors, HCG will be elevated in 75% and AFP in 40–60% of patients. HCG is elevated in 15–20% of patients with metastatic seminoma [1]. AFP is a glycoprotein normally produced by the fetal yolk sac or embryonal carcinoma elements of germ cell cancers, and is not detectable in normal adults. The half-life of AFP in the serum is about 5 days. False-positive elevation of AFP is quite rare, with differential considerations including laboratory error, liver inflammation, and other tumor types such as hepatoma, pancreatic, biliary, and gastric cancers. Additionally, a hereditary persistence of AFP has been documented in rare cases [18]. Persistent elevation of AFP in a patient with a seminoma is viewed as evidence of nonseminomatous elements, and management should proceed as such. β-Subunit HCG is a smaller glycoprotein that is normally produced by trophoblastic tissues, such as the syncytiotrophoblastic components of germ cell tumors. The HCG protein comprises antigenically distinct α and β subunits, and the serum half-life of the entire protein is 18–24 hours. False elevations may occur in patients who use marijuana, and there is some cross-reactivity in the radioimmunoassay with luteinizing hormone. In cases of persistently elevated HCG, patients should be asked about marijuana use, and testosterone should be given to ensure that a hypogonadal state with resultant high levels of luteinizing hormone is not interfering with the measurement. If the level remains elevated, restaging procedures and investigation of sanctuary sites are in order. Pure seminoma is often associated with normal AFP and HCG levels, but some patients, particularly with advanced disease, may have lowlevel elevation of HCG (usually <100 mIU/mL). The rate of disappearance of elevated tumor markers is very useful in determining response to treatment. A 10-fold decrease in serum HCG

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Table 64.1 American Joint Commission on Cancer (AJCC) staging system [2]. Tumor, node, metastasis (TNM) definitions Primary pTX pTO pTis pTl pT2 pT3 pT4

tumor (T) Cannot be assessed No evidence of tumor Intratubular neoplasia (carcinoma in situ) Limited to testis and epididymis without lymphatic/vascular invasion Limited to testis and epididymis with lymphatic/vascular invasion, or tumor extending through the tunica albuginea with involvement of the tunica vaginalis Invades the spermatic cord with or without lymphatic/vascular invasion Invades the scrotum with or without lymphatic/vascular invasion

Regional NX N0 N1 N2 N3

lymph nodes (N) Cannot be assessed No regional lymph node metastases Metastases in a single lymph node, ≤2 cm or less in greatest dimension Metastases in a single lymph node, >2 cm but not >5 cm in greatest dimension, or multiple lymph nodes, none >5 cm in greatest dimension Metastases in a lymph node, >5 cm in greatest dimension

Distant metastasis (M) MX Cannot be assessed M0 No distant metastasis M1 Distant metastasis: M1a: nonregional nodal or pulmonary metastasis M1b: distant metastasis other than nonregional nodes and lungs Serum Markers (S) SX Not available or not performed S0 Marker study levels within normal limits S1 LDH <1.5 times the upper limit of normal AND HCG (mIU/mL) <5000 AND AFP (μg/mL) <1000 S2 LDH 1.5–10.0 times the upper limit of normal OR HCG (mIU/mL) 5000–50,000 OR AFP (μg/mL) 1000–10,000 S3 LDH >10 times the upper limit of normal OR HCG (mIU/mL) >50,000 OR AFP (μg/mL) >10.000 AFP, α-fetoprotein; HCG, human chorionic gonadotropin; LDH, lactate dehydrogenase.

Table 64.2 American Joint Commission on Cancer (AJCC) staging system [2] Stage groupings Stage 0 Stage I Stage IA Stage IB Stage IS Stage II Stage IIA Stage IIB Stage IIC Stage III Stage IIIA Stage IIIB Stage IIIC

pTis, N0, M0, S0 pTI–4. N0, M0, SX pT1, N0, M0, S0 pT2–3, N0, M0, S0 Any pT, N0, M0, S1–3 Any pT, Nl–3, M0, SX Any pT, NI, M0, S0–1 Any pT, N2, M0, S0–1 Any pT, N3, M0, S0–1 Any pT, any N, M1, SX Any pT, any N, M1a, S0–1 Any pT, N1–3, M0, S2 Any pT, any N, M1a, S2 Any pT, N1–3, M0, S3 Any pT, any N, M1a, S3 Any pT, any N, M1b, any S

level over a 3-week period is consistent with disease eradication. Less steep declines of HCG levels usually correlate with residual disease post surgery or the emergence of drug resistance to chemotherapy. The reappearance of a marker elevation often precedes radiographic appearance and is an invaluable method of detecting early relapse. However, particularly among patients with levels of HCG over 50,000 mIU/mL, the disappearance of HCG from the serum may be unpredictable and occasionally quite prolonged. In a retrospective review of 41 patients with presenting HCG levels of over 50,000 mIU/mL, less than 10% had a normal HCG at the institution of the fourth course of chemotherapy [19]. However, 22 of 41 (53.7%) were continuously without evidence of disease despite no further therapy. We feel that the optimal strategy for such patients is close observation with initiation of salvage therapy if and when there is serologic progression. Importantly, salvage therapy should not be initiated on the basis of a persistent HCG elevation alone, but rather reserved for the clinical setting of a well-documented rise in the markers. In cases of rising markers after standard therapy, investigation of sanctuary sites such as the central nervous system or the contralateral testis should be performed before initiating salvage therapy.

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Table 64.3 Conventional primary therapy results Stage

Treatment

Disease-free survival

I II, III

Radical orchiectomy followed by active surveillance and appropriate treatment at time of relapse Radical orchiectomy plus bleomycin–etoposide–cisplatin (BEP) × 3–4 cycles, plus retroperitoneal lymph node dissection for partial response, plus BEP × 2 cycles for residual disease

~100% 80% (good risk) 60% (poor risk)

Table 64.4 Conventional-dose chemotherapy regimens

Indication

Regimen

Primary treatment of disseminated disease (stage II–III)

BEP [52]: Bleomycin 30 units IV on days 2, 9, and 16 Etoposide 100 mg/m2/day IV on days 1–5 Cisplatin 20 mg/m2/day IV on days 1–5 Repeat cycle at 21-day intervals

Primary salvage treatment of relapsed disease

VeIP [18]: Vinblastine 0.11 mg/kg/day IV on days 1–2 Ifosfamide 1.2 g/m2/day IV on days 1–5 Cisplatin 20 mg/m2/day IV on days 1–5 Repeat cycle at 21-day intervals VIP [18]: Etoposide 75 mg/m2/day IV on days 1–5 Ifosfamide 1.2 g/m2/day IV on days 1–5 Cisplatin 20 mg/m2/day IV on days 1–5 Repeat cycle at 21-day intervals TIP [57]: Paclitaxel 250 mg/m2 IV continuous infusion on day 1 Ifosfamide 1500 mg/m2/day IV on days 2–5 Cisplatin 25 mg/m2/day IV on days 2–5

IV, intravenously.

Table 64.5 International Germ Cell Consensus Classification prognostic staging system Non-seminoma Good prognosis

Intermediate prognosis

Poor prognosis

Seminoma

• Testis or retroperitoncal primary and • No non-pulmonary visceral metastases and • Good markers (AFP <1000 ng/mL and HCG <5000 IU/L and LDH <1.5 × upper limit of normal)

56% of patients PFS 89% @ 5 years

• Testis or retroperitoncal primary and • No non-pulmonary visceral metastases and • Intermediate markers (AFP 1000–10,000 ng/mL or HCG 5000–50,000 IU/L or LDH 1.5–10 × upper limit of normal)

28 % of patients PFS 78% @ 5 years

• Mediastinal primary or • Non-pulmonary visceral metastases or • Poor markers (AFP >10,000 ng/mL or HCG >50,000 IU/L or LDH >10 × upper limit of normal)

16% of patients PFS 41% @ 5 years OS 48% @ 5 years

OS 92% @ 5 years

OS 80% @ 5 years

• Any primary site and • No non-pulmonary visceral metastases and • Normal AFP, any HCG, any LDH

90% of patients PFS 82% @ 5 years

• Any primary site and • Non-pulmonary visceral metastases and • Normal AFP, any HCG, any LDH

10% of patients PFS 67% @ 5 years

AFP, α-fetoprotein; HCG, human chorionic gonadotropin; LDH, lactate dehydrongenase; OS, overall survival rate; PFS, progression-free survival rate.

OS 86% @ 5 years

OS 72% @ 5 years

Hematopoietic Cell Transplantation in Germ Cell Tumors

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Table 64.6 Possible indications for high-dose chemotherapy in the management of germ cell tumor (GCT) Salvage therapy for multiply relapsed or refractory GCT Initial salvage for relapsed GCT Primary treatment for poor-risk GCT

A small proportion of (otherwise incurable) patients will be cured Large single-center studies suggest there may be a role. However, recent European prospective randomized trial failed to show any effect on treatment outcomes Routine inclusion of high-dose chemotherapy in first-line treatment did not improve treatment outcome in a prospective randomized North American trial. Not indicated outside of a clinical trial

Radiographic abnormalities Depending on the stage at the time of diagnosis, 20–50% of patients who undergo induction chemotherapy for disseminated germ cell tumor will have significant residual radiographic abnormalities. Postchemotherapy surgical resection should be considered only in the setting of normalization or continuing decline of serum tumor markers. Resection of residual abnormalities is rarely urgent, and repeat imaging of the areas of abnormality should be performed prior to surgery, as often tumors will continue to involute. Patients with residual abnormalities and persistent elevations of AFP or HCG often have unresectable, viable tumor, and should therefore be considered for salvage chemotherapy regimens rather than surgical debulking [20]. Other clinical situations may mimic progressive or recurrent disease. For example, in patients with an appropriate fall in serologic markers but radiographic progression during induction chemotherapy, enlarging benign teratomatous portions of the tumor may be mistaken for progressive disease [21]. Appropriate management of such a patient includes completion of induction chemotherapy with subsequent surgical resection of residual radiographic abnormalities and not the administration of salvage therapies. The development of nodular lesions on chest imaging at the end or soon after completion of chemotherapy may represent bleomycininduced pulmonary injury rather than recurrent disease. These nodules are characteristically located in a subpleural region, and the diagnosis should be considered in a patient who is otherwise responding to therapy [22].

Conventional salvage therapies for germ cell tumors A unique feature in the management of germ cell tumors is the ability to achieve long-term disease-free survival with secondary chemotherapies after initial treatment failures. The standard for comparison for initial salvage therapies in germ cell tumors is the combination of vinblastine, ifosfamide, and cisplatin (VeIP) [23]. In one series, 135 patients with progressive disease after cisplatin- and etoposide-based chemotherapy were treated with VeIP regardless of metastatic site or performance status [24]. A 50% complete response (CR) rate after chemotherapy with or without surgical resection of residual disease was seen, with a long-term overall survival rate of 32% and a disease-free survival rate of 24%. Importantly, none of the 32 patients with extragonadal nonseminomatous disease was continuously disease free. Subsequently, paclitaxel was found to have significant single-agent activity in patients with refractory germ cell tumors, leading to its incorporation into salvage combination chemotherapy regimens [25–28]. Kondagunta et al. published promising results with the combination of paclitaxel, ifosfamide, and cisplatin (TIP) as second-line salvage therapy in patients with relapsed testicular germ cell tumors [29]. Forty-six patients with progressive germ cell tumor were given up to four cycles of TIP. Of note, all treated patients had prior treatment limited to six or fewer cycles of cisplatin, a gonadal primary tumor site, normal postchemotherapy markers, and either a CR or a partial response (PR) for

more than 6 months after completion of initial chemotherapy. CR was achieved with chemotherapy alone in 63%, and an additional 7% achieved CR with surgical resection of residual mass containing viable malignancy after chemotherapy. A durable CR rate of 63% was observed at a median follow-up of 69 months. In this highly selected group of patients with favorable prognostic factors for response to standard-dose salvage chemotherapy, TIP is an active salvage regimen and can be considered as an alternative to VeIP. However, these salvage regimens have never been compared in a phase III trial of unselected relapsed or refractory patients. Salvage surgery may play a significant role in the treatment of patients with chemorefractory germ cell tumors. Murphy et al. retrospectively reviewed all patients felt to have progressive chemorefractory yet resectable disease who underwent salvage surgery at Indiana University from 1977 to 1990 [30]. The majority underwent isolated retroperitoneal lymphadenectomy (69%). Thirty-eight of 48 patients (79%) were grossly disease free after surgery, and 29 (60%) obtained a serologic remission. Ten patients (21%) achieved event-free survival for 31–89 months. Six additional patients who relapsed after surgery achieved disease-free survival after additional surgery (four patients) or high-dose chemotherapy and autologous HCT (two patients). Notably, no patients with more than one site of metastatic disease, even when resectable, achieved long-term disease control. This series confirmed many of the conclusions drawn by Wood et al. regarding the experience with surgical salvage at the Memorial SloanKettering Cancer Center [31]. Surgical salvage was performed on 15 patients with chemorefractory disease and persistently elevated serum markers. The entire residual mass or solitary metastasis was entirely resected in each case. Twelve of 15 patients achieved normalization of serum markers after resection, and 7 of these patients remained without evidence of disease on follow-up 3–53 months after resection. Five of the 12 patients achieving CR with salvage surgery relapsed within 2 months, and 2 of them were rendered disease free with further chemotherapy. Of the three patients who did not achieve CR with salvage surgery, two achieved disease-free status with postoperative salvage chemotherapy. Overall, 11 of 15 patients (73%) were rendered disease free after salvage surgery with or without additional therapy. Isolated AFP elevation and retroperitoneal site of disease correlated with favorable outcome. These series and others point to a definite potential for cure with salvage surgery in selected patients with recurrent chemotherapyrefractory germ cell tumor.

High-dose chemotherapy for recurrent germ cell tumors An active strategy to improve outcomes for patients with relapsed or refractory germ cell tumors is the use of high-dose chemotherapy with autologous hematopoietic cell rescue. Initial attempts at cisplatin dose escalation (without hematopoietic stem cell support) increased toxicity without a survival advantage [32]. However, several advances led to

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substantial progress towards defining the role of high-dose therapy for germ cell tumors. Drugs such as carboplatin, etoposide, and alkylating agents such as thiotepa or the oxazaphosphorines (ifosfamide and cyclophosphamide) demonstrated antitumor activity, dose responsiveness, and a wide dose range between dose-limiting myelotoxicity and doselimiting extramedullary toxicities. Additional improvements in transplant procedures (hematopoietic cells from peripheral blood rather than bone marrow), supportive care (hematopoietic growth factors and selective antibiotics), and patient selection have advanced the field. The first investigations of high-dose chemotherapy with autologous HCT for germ cell tumors involved single-agent etoposide or etoposide and cyclophosphamide [33–35]. Although some patients were refractory to these agents in standard doses, 20–40% response rates and rare longterm cures were observed.

I–II study and none of 11 patients in the phase II study achieving a complete remission. In an update of this series, there were no failure-free survivors at 2 years in 13 patients with primary mediastinal nonseminomatous germ cell tumor treated with tandem high-dose chemotherapy [39]. However, in an international multicenter analysis of second-line chemotherapy in patients with relapsed extragonadal nonseminomatous germ cell tumor, 48 patients received high-dose chemotherapy followed by autologous bone marrow transplant at the time of relapse [40]. Ten of those patients (21%) were continuously disease free; seven of the patients had retroperitoneal germ cell tumor, and the other three had primary mediastinal disease. This is a population for whom conventional salvage therapies also have poor outcomes, and such patients should be the focus of investigational approaches [41,42]. Dose escalation and additional agents

Early trials Most modern phase I–II trials of high-dose chemotherapy have included carboplatin and etoposide, sometimes with ifosfamide, cyclophosphamide, or thiotepa. Several centers intended high-dose therapy to be repeated once after recovery (tandem transplants) to maximize potential benefit. Initial investigations focused on patients with either relapse after best standard salvage chemotherapies or cisplatin-refractory germ cell tumors that were felt unlikely to be cured by any available treatment. Nichols and colleagues at Indiana University pioneered this field with the first phase I–II study of two courses of high-dose carboplatin and etoposide in patients with germ cell tumors refractory to cisplatin (defined as progression on or within 4 weeks of the last cisplatin dose) or recurrent after primary cisplatin-based therapy and a salvage regimen containing ifosfamide and cisplatin [36]. The results were updated after the first 40 patients [37]. Over half of the patients had received three or more prior regimens, and 70% were considered cisplatin refractory. Therapy consisted of carboplatin 900–2000 mg/m2 and etoposide 1200 mg/m2. Ifosfamide was added to the regimen in three patients. Twenty-six of 40 (65%) received both courses of high-dose therapy. Overall, 7 of 40 (18%) died as a consequence of therapy, primarily due to infection. There were 12 patients (30%) who attained a CR, and 14 (35%) who achieved a PR, for an overall response rate of 65%. Six patients (15%) were long-term disease-free survivors, and a seventh patient died at 22 months free of germ cell tumor from a therapy-related acute myeloid leukemia. These results were confirmed in a phase II multi-institutional trial of 38 similar patients receiving tandem high-dose carboplatin and etoposide, where 58% completed both transplants, a 13% treatment-related mortality was observed, 9 patients (24%) achieved a CR (2 after posttransplant surgical resections), and 5 patients (13%) were alive and free of disease with a minimum of 18 months follow-up [38]. These initial phase I and II trials of high-dose chemotherapy and HCT provided multiple insights. First, these trials clearly demonstrated that a portion of patients (perhaps 13–24%) with multiply recurrent germ cell tumors could achieve long-term disease-free survival. Second, nearly all relapses occurred in the first 18 months post transplant, and disease status after high-dose therapy predicted long-term outcome. Third, the tandem transplant approach was feasible. In the phase I–II study, all of the long-term disease-free survivors received both transplants, and 8 of 12 patients in partial remission after the first transplant achieved complete remission after the second. Fourth, surgical salvage remained an important treatment modality after transplant, leading to complete remissions and likely contributing to cures in several patients. Lastly, patients with primary mediastinal germ cell tumors failed to respond, with none of 8 patients in the phase

Subsequent trials of high-dose therapy began to enroll less heavily pretreated patients, and incorporated the use of peripheral blood hematopoietic cells and growth factors, which all served to shorten the duration of cytopenias. These improvements led investigators to attempt further dose escalations or add additional agents such as ifosfamide or cyclophosphamide. One trial of dose escalation involved treating 33 patients with relapsed or refractory germ cell tumors with carboplatin 1650– 2100 mg/m2 and etoposide 1200–2250 mg/m2 for tandem cycles [43]. The dose-limiting toxicities for this regimen were mucositis and peripheral neurotoxicity, and reversible transaminase abnormalities were common. Results were similar to earlier studies, with 20 of 33 patients (61%) completing both transplant procedures. Treatment-related mortality was 18%, including four patients who died prior to response evaluation. Four of the remaining 29 evaluable patients (14%) achieved complete remission, and 8 of 33 (24%) became long-term survivors. This trial served to establish the maximum tolerated doses of carboplatin (2100 mg/m2) and etoposide (2250 mg/m2), which continue to represent the current standard of care conditioning regimen. The German Testicular Cancer Study Group added ifosfamide 0– 10 g/m2 to carboplatin 1500–2000 mg/m2 and etoposide 1200–2400 mg/ m2 as a transplant regimen in 74 patients with relapsed or refractory germ cell tumors [44,45]. Therapy was delivered regardless of response to two preceding cycles of reinduction standard-dose cisplatin, etoposide, and ifosfamide (VIP). Treatment-related mortality was only 3%, but late toxicities included renal toxicity (21%), paresthesias (29%), and ototoxicity (18%). Approximately 50% of patients achieved a complete remission with this therapy, and 28 of 74 (38%) were alive from 3.2 to 5.6 years post treatment. In an attempt to decrease the toxicities observed with ifosfamide, investigators at Memorial Sloan-Kettering Cancer Center studied the addition of cyclophosphamide to carboplatin and etoposide in patients with cisplatin-refractory germ cell tumors [46]. In this series, 58 patients with an incomplete response to initial conventional-dose cisplatin-containing chemotherapy, or incomplete response or relapse from CR with a cisplatin-based salvage regimen, were enrolled. Patients received carboplatin 1500 mg/m2, etoposide 1200 mg/m2, and a range of 60–150 mg/ kg cyclophosphamide followed by infusion of bone marrow-derived hematopoietic stem cells. Thirty-one patients (53%) received one cycle of therapy, and 27 (47%) received two. CR was achieved by 23 patients (40%); 20 of these were treated with two cycles of therapy. With a median follow-up of 28 months, 12 patients (21%) were alive and free of disease. There were seven (12%) treatment-related deaths, the majority of which were related to bleeding or sepsis with multiorgan failure. Hepatic and renal toxicities were observed in 26% of patients. Two independently predictive variables for survival were identified: pretreatment HCG level and presence of retroperitoneal metastases.

Hematopoietic Cell Transplantation in Germ Cell Tumors

Several investigators have revisited the incorporation of newer and older drugs in an attempt to improve the results of high-dose chemotherapy. A phase II multicenter French study (TAXIF) treated 45 patients with relapsed poor-prognosis germ cell tumors with two cycles of epirubicin and paclitaxel, followed by three consecutive high-dose chemotherapy cycles (cyclophosphamide 3g/m2 plus thiotepa 400 mg/m2 followed by two cycles of ifosfamide 10 g/m2, carboplatin area under the curve 20, and etoposide 1500 mg/m2) supported by stem cell transplantation [47]. Twenty-five patients died from disease progression, and five patients from toxicity. Of the 22 patients who received the complete course, the overall response rate was 37.7%, with 8.9% achieving a CR. Median overall survival was 11.8 months, with a 3-year survival and progression-free survival rate of 25.3%. Notably, patients with adverse prognostic factors (absolute cisplatin refractoriness, or HCG >1000 mIU/ mL before transplant, or a combination of progressive disease before transplant, cisplatin-refractoriness prior to transplant, or primary mediastinal tumor) did not benefit from the approach. At Memorial Sloan-Kettering Cancer Center, 47 patients germ cell tumor patients with cisplatin resistance and one or more unfavorable prognostic features for achieving CR with conventional-dose salvage therapy (including extragonadal primary site, progressive disease after an incomplete response to first-line therapy, and poor or lack of response to prior treatment with cisplatin plus ifosfamide conventional-dose therapy) were treated with two cycles of paclitaxel plus ifosfamide, followed by three cycles of carboplatin and etoposide with peripheral blood stem cell support [48]. Etoposide was administered at a total dose of 1200 mg/m2 and carboplatin was dose escalated according to target area under the curve. Three of the 47 patients were treated with paclitaxel– ifosfamide but did not receive carboplatin–etoposide. Of the 44 patients, 77% received all three planned cycles of high-dose chemotherapy. All 47 patients were evaluated for response and after completion of highdose chemotherapy; 23 patients (49%) achieved a CR. Three additional patients (6%) achieved a CR to chemotherapy and surgery, for an overall CR rate of 55%. With a median follow-up time of 40 months, 24 patients (51%) were alive and free of disease. When the results of this study were combined with the results of a prior phase I–II trial of similar design [49], an overall CR rate of 56% was reported, with 50% of patients alive with no evidence of disease. Finally, the value of sequential high-dose chemotherapy in patients with relapsed or refractory germ cell tumors was addressed in a prospective randomized multicenter trial of the German Testicular Study Group [50]. Two hundred and sixteen patients were randomized to either one cycle of conventional-dose VIP followed by three sequential cycles of high-dose carboplatin and etoposide followed by autologous stem cell transfusion, or three conventional-dose cycles of VIP followed by a single cycle of high-dose carboplatin, etoposide, and cyclophosphamide followed by autologous stem cell reinfusion. There was no statistically significant difference in event-free, progression-free or overall survival between the two arms. However, treatment-related deaths were less frequent in patients receiving sequential high-dose chemotherapy

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(p <0.1). This study is somewhat difficult to interpret given that the conditioning regimens differed in the two arms. As evidenced in previous trials, survival outcomes appear to be improved in patients who receive sequential high-dose chemotherapy compared with those receiving a single cycle. Prognostic models In reviewing these trials, it is important to recognize the large variability in inclusion criteria, making comparisons of results difficult. A series from Margolin et al. with a long-term disease-free survival of 45% included 40% of patients treated during initial salvage [51]. Ayash et al. reported a 17% long-term disease-free survival but included only 10% of patients treated during initial salvage, and included 48% of patients with cisplatin-refractory disease, including 34% who were absolutely refractory (having never achieved even stable disease on a cisplatincontaining regimen) [52]. This heterogeneity led investigators to compile pooled data to identify prognostic variables for response and survival in male patients with relapsed or refractory germ cell tumors treated with high-dose chemotherapy [53]. Three hundred and ten patients from four centers in the United States and Europe were retrospectively evaluated, and data on 283 patients were complete. Overall, the treatment-related mortality was 8%. Fifty-five percent of patients achieved a favorable response (complete remission or partial remission with negative tumor markers), and 47% of those patients later relapsed. The actuarial overall survival rate was 51% at 1, 36% at 2, and 30% at 3 years, respectively. Multivariate analysis identified progressive disease before transplant, mediastinal nonseminomatous primary tumor, disease that was refractory or absolute refractory to conventional-dose cisplatin, and HCG levels greater than 1000 IU/L before transplant as independent adverse prognostic factors. Importantly, response to first-line therapy did not predict outcomes. These variables were used to identify patients with good, intermediate or poor prognoses, with predicted failure-free survival rates at 2 years after transplantation of 51%, 27%, and 5%, respectively (Table 64.7). Of note, most of these patients had received only one cycle of high-dose chemotherapy, as the enrollment period captured patients who were largely treated without the benefit of peripheral hematopoietic stem cells and growth factor support. A comprehensive retrospective review of the Indiana experience with stem cell rescue for metastatic testicular cancer was reported in 2007 (Fig. 64.1) [54]. Between 1996 and 2004, 184 patients were treated with two cycles of carboplatin 700 mg/m2 plus etoposide 750 mg/m2, both given 5, 4, and 3 days prior to infusion of peripheral blood stem cells. A total of 135 (73%) of these patients had received one previous chemotherapy regimen, and 49 (27%) had received more than two previous regimens. With a median follow-up of 48 months, 116 of 184 patients (63%) were continuously disease free. This included 94 (70%) of the 135 patients for whom high-dose chemotherapy represented second-line treatment, and 22 (45%) of 49 patients who received treatment as third-

Table 64.7 Beyer Prognostic Score for survival after high-dose salvage chemotherapy Factor

Score

Risk

2-year failure-free survival

2-year overall survival

Progressive disease before transplant Primary mediastinal tumor Cisplatin refractory before transplant Absolute cisplatin refractory before transplant Human chorionic gonadotropin >1000 IU/L before transplant

1 1 1 2 2

Good (score 0) Intermediate (score 1–2) Poor (score >2)

51% 27% 5%

61% 34% 8%

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Summary 100

Overall survival rate (%)

80 60 40 20 0 0

10 20 30 40 50 60 70 80 90 100 110 120 Months since first day of high-dose chemotherapy

No. at risk

184 161 128 103 80 61 49 27 23 17

7

4

In summary, the use of high-dose chemotherapy with autologous HCT for the treatment of relapsed or refractory germ cell tumors can produce long-term survivors in 15–50% of patients. The principal toxicities include nephrotoxicity, peripheral neurotoxicity, reversible transaminase elevations, and gonadal toxicity. Treatment-related mortality rates ranged from 0% to 18%, with most recent series reporting 3–5%. Patients with primary mediastinal tumors only rarely benefit from salvage highdose therapy. Treatment earlier in the course of relapsed or refractory disease appears to be associated with better outcomes. Current standard of care at most high-volume centers consists of a tandem transplant strategy utilizing the conditioning regimen of carboplatin 2100 mg/m2 and etoposide 2250 mg/m2. The addition of a third agent requires dose reductions of these two most active drugs, and it is unlikely that an adequately powered randomized trial to determine the benefit of adding a third agent will ever be performed given the rarity of this clinical circumstance.

Fig. 64.1 Kaplan–Meier estimates of overall survival. The top and bottom lines show the 95% confidence interval.

Initial salvage with high-dose chemotherapy for germ cell tumors

Overall survival rate (%)

100 Low risk (0 points)

80 60

Intermediate risk (2–3 points) 40 High risk (4–7 points) 20 0 0

10 20 30 40 50 60 70 80 90 100 110 120 Months since first day of high-dose chemotherapy

No. at risk Low risk Intermediate risk High risk

73 63 59 44 34 26 21 10 9 64 45 37 35 28 22 19 12 10

7 7

47 23 17 13 10

1

7

5

2

1

4 3

2 2

Fig. 64.2 Disease-free survival according to the Indiana Prognostic Scoring System. The prognostic scoring algorithm, based on the three-variable model, assigned a score of 3 points for third-line chemotherapy, 2 points for platinum refractoriness, and 2 points for advanced International Germ Cell Cancer Collaborative Group stage. High scores indicated a low probability of disease-free survival.

line or later therapy. Notably, 18 (45%) of 40 patients with cisplatinrefractory disease achieved disease-free status. In patients with cisplatin-sensitive disease, 98 (68%) of 144 were disease free. Three drug-related deaths (2%) were reported, and three additional patients developed acute leukemia after therapy. Based on this retrospective review of the Indiana University experience, a three-variable prognostic model was elucidated that included timing of high-dose chemotherapy, platinum sensitivity or refractoriness, and International Germ Cell Cancer Collaborative Group stage (Fig. 64.2). However, this scoring system was not validated by outside groups, and there currently is no universally accepted prognostic score for patients at initial relapse [55,56]. An international effort to elucidate prognostic factors for first-salvage treatment is currently under way.

The modest results of standard-dose salvage chemotherapies (the 24% long-term disease-free survival with VeIP discussed above), combined with the improving rates of durable responses and decreasing morbidity and mortality associated with high-dose therapy, have led to the investigation of high-dose therapy for initial salvage of relapsed germ cell tumors. Indiana University published one of the first series to incorporate high-dose chemotherapy as initial salvage [57]. Sixty-five patients with relapsed or persistent testicular cancer were given initial salvage treatment with tandem courses of carboplatin 2100 mg/m2 and etoposide 2250 mg/m2 followed by autologous hematopoietic cell rescue. All patients had primary testicular tumors. Fifty-six of the 65 patients (86%) received at least one cycle of standard-dose salvage chemotherapy (usually VeIP) prior to transplant. Ten of 65 (15%) received ex vivo cytokine-stimulated, mdr-1-transduced autologous CD34+ cells, and 26 of 65 (40%) received post-transplant maintenance oral etoposide. Using the Beyer Prognostic Score for outcomes with high-dose therapy (Table 64.7), 61% were good risk, 31% intermediate risk, and 8% poor risk. There were no treatment-related deaths. Complete remission was achieved in 28 patients (43%) with high-dose chemotherapy, and in another patient with additional VeIP after only one transplant, and 13 patients (20%) with post-transplant surgical resections. With follow-up ranging from 16 to 91 months, 37 patients (57%) were continuously disease free, and 40 (60%) remain disease free after definitive therapy. Only one phase III randomized prospective trial of high-dose chemotherapy in the initial salvage setting has been reported [58]. In this study, 280 patients with relapsing germ cell tumors who had achieved complete or partial remission with first-line platinum combination chemotherapy were randomized to either four cycles of VIP or VeIP (arm A) or three cycles of VIP or VeIP followed by high-dose carboplatin, etoposide, and cyclophosphamide with hematopoietic stem cell support (arm B). Overall CR was observed in 51 (42%) patients in arm A and 53 (43%) patients in arm B. Additionally, 21 (17%) versus 23 (18%) patients achieved a tumor marker-negative partial remission. There was no clinical benefit to the replacement of a fourth cycle of VIP or VeIP by high-dose chemotherapy in terms of either 3-year event-free survival (35% versus 42%; p = 0.16) or overall survival (53%; 95% confidence interval 46–59%). In a subgroup analysis of the 104 patients achieving a CR, the 3-year disease-free survival rates were 55% and 75% (p = 0.04) in favor of the high-dose chemotherapy arm.

Hematopoietic Cell Transplantation in Germ Cell Tumors

This study was criticized for being underpowered (only 80% of patients in arm A and 73% of patients in arm B receiving the fourth cycle of chemotherapy) to detect a significant difference in 3-year eventfree survival. Additional sources of criticism included the high degree of variability in initial treatment regimens, and the single-transplant approach. In light of the previously discussed superiority of sequential high-dose versus single-dose chemotherapy, it is difficult to conclude from this trial alone that there is no role for high-dose chemotherapy in the initial salvage setting.

Primary treatment with high-dose chemotherapy for poor-prognosis germ cell tumors Incorporating high-dose chemotherapy as a first-line strategy for the treatment of patients with poor-risk germ cell tumors at presentation has also been investigated. Two randomized trials of high-dose chemotherapy with autologous transplant support for the treatment of patients with poor-risk germ cell tumors have now failed to show a benefit [59–61]. In a French trial, Droz reported 115 patients deemed poor risk by Institut Gustave Roussy criteria (an older prognostic model) who were randomized to receive either four cycles of cisplatin 200 mg/m2, vinblastine, etoposide, and bleomycin (PVeVB), or two cycles of PVeVB plus one cycle of high-dose therapy including cisplatin 200 mg/m2 followed by autologous transplant. With a median follow-up of 9.7 years, 31 and 27 patients have continuously shown no evidence of disease in the respective arms. There was no significant difference between the overall survival curves (p = 0.167). This study was criticized because the four-drug regimen is not considered a standard therapy, the dose intensity and total cisplatin dose were lower in the “high-dose” arm, and a substantial number of patients randomized to the high-dose arm did not receive the assigned therapy. Additional criticisms included the extensive accrual period, and significant variability in initial treatment regimen [59,60]. The question was more definitively answered by a United States trial reported in early 2007 [61]. In this randomized Intergroup (Cancer and Leukemia Group B–South-Western Oncology Group–Eastern Cooperative Oncology Group) phase III study, 219 previously untreated patients with intermediate- or poor-risk germ cell tumors were randomized to either four cycles of standard bleomycin, etoposide, and platinum (BEP) or two cycles of BEP followed by two cycles of carboplatin 600 mg/m2, etoposide 600 mg/m2, and cyclophosphamide 50 mg/kg administered on days 1–3 before transplantation followed by autologous stem cell infusion. The trial was designed to detect a 20% improvement in durable CR at 1 year. CR was achieved in 61 of 111 patients (55%) on the BEPalone arm, and in 61 of 108 patients (56%) on the BEP + high-dose chemotherapy arm. With a median follow-up of 51 months, the 1-year durable CR rate was 48% after BEP alone and 52% after BEP + highdose chemotherapy (p = 0.53). The median time to treatment failure was 11.3 months in the BEP-alone arm, and 23.2 months in the BEP + highdose chemotherapy arm (p = 0.40). No difference in survival was seen between the two arms (p = 0.94). Rate of tumor marker decline was correlated with treatment outcome as a secondary endpoint. In a subset analysis, 67 patients with unsatisfactory marker decline were identified. In this subset, the proportion achieving 1-year durable CR was 61% for patients receiving high-dose chemotherapy versus 34% for those on the BEP-alone arm (p = 0.03). The authors concluded that routine inclusion of high-dose chemotherapy in the first-line treatment of germ cell tumor patients with metastases and poor predicted outcome to chemotherapy did not improve treatment outcome. The subset analysis of patients with unsatisfactory marker decline is provocative and will likely be incorporated into future trials analyzing

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the role of high-dose chemotherapy. With collaborative international clinical trial efforts, it may be possible to adequately power studies to detect smaller yet clinically relevant differences in outcome in this rare group of patients.

Surgical management after high-dose chemotherapy The role of surgical resection after high-dose chemotherapy was assessed in a retrospective review of the German experience [62]. This retrospective review identified 216 patients treated on three consecutive high-dose chemotherapy trials between 1989 and 1999. Partial remissions with negative or positive tumor markers after high-dose chemotherapy were achieved in 128 patients (59%). Of these patients, 57 (45%) proceeded to residual tumor resection; the remaining 71 (55%) could not be operated on because of progressive disease, inoperability due to tumor mass or poor performance status, or patient refusal. With a median follow-up of 87 months, 37 (65%) of 57 patients were alive, and 34 (59%) of 57 patients remained continuously disease free. Viable cancer was documented in the resected specimen of 26 (46%) of the 57 patients. The projected overall and event-free survival rates after 5 years in patients with viable cancer were 42% and 38% (p < 0.01). The projected overall and event-free survival rates after 5 years in patients without viable cancer were 84% and 77% (p < 0.01). Of note, all patients in this series had been treated with a single cycle of high-dose chemotherapy, which likely influenced the incidence of viable cancer in resected specimens. However, this series confirmed that postchemotherapy resection contributed to overall treatment outcome, and thus residual tumor resection continues to be offered to patients with partial remission after high-dose chemotherapy.

Management of relapse post high-dose chemotherapy Outcomes have been unfavorable in patients who experience relapse after high-dose chemotherapy with stem cell support. A retrospective review of the Indiana University experience identified 186 patients with relapsed germ cell tumors who were treated with high-dose chemotherapy and peripheral blood stem cell transplantation between 1986 and 1997 [63]. Of these, 101 patients relapsed after high-dose chemotherapy after a median interval of 10 months. Forty-seven of these patients received further chemotherapy, and seven patients underwent surgery alone. A total of 66 chemotherapy regimens were administered to 47 patients, with an overall response rate of 18.2% and a CR rate of 5%. Of the patients who received surgery alone, only one patient was a longterm survivor. Finally, two recent trials have examined the use of additional salvage chemotherapy for patients relapsing after high-dose chemotherapy. In a phase II trial at Indiana University, 32 patients with progressive disease after two cycles of high-dose carboplatin and etoposide were treated with paclitaxel 100 mg/m2 and gemcitabine 1000 mg/m2 intravenously on days 1, 8, and 15 every 4 weeks for a maximum of six cycles [64]. Patients who had received either agent prior to high-dose chemotherapy were ineligible. An objective response was achieved by 10 patients (31%); this included six CRs and four PRs (of 2–6 months’ duration). Of the six CRs, four patients (12.5%) were continuously disease free with paclitaxel and gemcitabine alone at more than 20, 40, 44, and 57 months from treatment. One additional CR patient was rendered disease free after two subsequent resections of carcinoma. There was no treatment-related mortality. The German Testicular Study Group treated 32 patients with relapse after high-dose chemotherapy and nine patients with cisplatin-refractory disease with gemcitabine 800 mg/m2 and oxaliplatin 130 mg/m2 on day

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1, and paclitaxel 80 mg/m2 on day 8, of a 3-week cycle for a minimum of two cycles [65]. Of the 41 patients, 2 patients (5%) achieved a CR, 34% achieved a marker-negative PR, and 12% achieved marker-positive PR, for an overall response rate of 51%. With a median follow-up of 5 months, 15% of patients remained in complete remission after gemcitabine, oxaliplatin, and paclitaxel chemotherapy with or without residual tumor resection. There were no toxic deaths with this regimen. These trials point to some possibility of chemotherapeutic salvage even in heavily pretreated and cisplatin-refractory patients.

receiving high-dose carboplatin and etoposide, three patients (2.6%) developed secondary leukemias [71]. This was not significantly different from the expected rate of secondary leukemia in patients receiving additional cycles of standard-dose etoposide as salvage chemotherapy. Advances in adjuvant or maintenance therapies with oral etoposide or immunmodulators such as interleukins have been considered [72]. Development of an international prognostic scoring system for firstsalvage treatment is currently under way. Ultimately, advances in the molecular understanding of germ cell malignancies may lead to targeted therapies.

Ongoing studies and future directions Newer compounds with single-agent response rates are being incorporated into trials including bexarotene, paclitaxel, gemcitabine, bendamustine, and oxaliplatin [66–68]. Unique dosing strategies such as multiple cycles or alternating agents may be incorporated in future highdose algorithms. Adjunctive agents may reduce the toxicity of high-dose therapy such as amifostine or keratinocyte growth factor [69]. Concern has been raised over the generation of secondary hematologic malignancies, particularly with the use of high-dose etoposide. However, in one series, the incidence of secondary leukemia among 302 recipients of autologous HCT with cumulative doses exceeding 2 g/m2 of etoposide was only 1.3% [70]. In another series of 113 patients

Conclusion High-dose carboplatin and etoposide-based chemotherapy with autologous HCT is accepted as a standard therapy for patients with germ cell tumors who have failed two prior standard-dose regimens. Prospective randomized trials have thus far failed to support the use of high-dose chemotherapy as primary treatment or initial salvage for poor-prognosis patients. Due to the complexity of clinical decision-making, it is of paramount importance that patients in this rare clinical situation be treated at high-volume referral centers. Clinical trial participation should be encouraged for all patients requiring high-dose chemotherapy.

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Richard W. Childs & Ramaprasad Srinivasan

Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors

Introduction Our perception of the mechanisms through which malignant cells are eradicated following allogeneic hematopoietic cell transplantation (HCT) has evolved substantially over the past four decades. No longer merely thought of as a means of rescuing hematopoietic function following high-dose conditioning, allogeneic transplantation is now known to be a powerful type of immunotherapy capable of curing patients with otherwise fatal malignant diseases. This conceptual evolution has translated into a diversification of the indications for allogeneic HCT and led to the development of reduced-intensity transplant approaches whose beneficial antineoplastic effects can be attributed largely to the transplanted donor immune system. Recently, investigators have begun to test whether nonhematologic malignancies might likewise be susceptible to allogeneic immune attack. In this chapter, we highlight recent work that has improved our awareness and understanding of the graftversus-tumor (GVT) effect, and discuss the preliminary results of its application as an investigational therapeutic modality in the treatment of metastatic solid tumors.

Allogeneic transplantation as immunotherapy: the GVT effect Acute leukemias have traditionally been viewed as “chemosensitive” malignancies. The inability to “cure” the majority of adults with leukemia following conventional chemotherapeutic regimens was attributed at least in part to a failure to deliver the sufficiently high doses of antineoplastic agents required to eradicate all malignant cells. Dose-limiting toxicities, including prolonged delays in lymphohematopoietic recovery, significantly hindered efforts to intensify such regimens. Pioneering work by E.D. Thomas and others led to the development of allogeneic HCT as a method to allow for accelerated recovery of lymphohematopoietic function following the administration of high-dose chemotherapy [1,2]. For the next two decades, allogeneic HCT became increasingly used as a therapeutic option to treat patients with a variety of different treatment-resistant hematologic malignancies. Initially, the curative

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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potential of the procedure was ascribed solely to the cytotoxic effects of the conditioning regimen, be it chemotherapy alone or in conjunction with total body radiation. It was not until years following its inception that investigators first documented the very important and powerful immune mechanisms that contributed to the curative potential of the approach in humans. Throughout the 1980s, several landmark observations provided insight into this phenomenon known as the “graft-versus-tumor” effect. First, in an analysis of over 200 patients with hematologic malignancies undergoing human leukocyte antigen (HLA)-matched HCT from a sibling donor, investigators noted that patients who experienced moderateto-severe acute graft-versus-host disease (GVHD) or chronic GVHD had a significantly lower incidence of leukemic relapse compared with patients with little or no GVHD [3]. Similar benefits in disease-free survival were observed in recipients of HLA-identical (nontwin) sibling donor allografts compared with those receiving syngeneic allografts [4], as well as in recipients of T-cell-replete grafts versus those receiving T-cell-depleted transplants [4–7]. These observations suggested a very important role for donor T cells in mediating this effect, and also implicated the targets of GVT as falling outside the realm of antigens encoded by the major histocompatibility complex (MHC). Definitive evidence that GVT effects were clinically meaningful came from studies in patients with chronic myelogenous leukemia who relapsed posttransplant and were subsequently induced back into remission following a donor lymphocyte infusion (DLI) [8]. Over the last decade, numerous investigators have successfully exploited GVT effects against a variety of malignancies of hematopoietic origin, including both acute and chronic leukemias, lymphomas, multiple myeloma, and Epstein–Barr virus (EBV)-related lymphoproliferative disorder [9–17]. The ability of GVT to induce remission varies among these malignancies, with the critical determining factors dictating neoplastic susceptibility to GVT being not entirely understood.

The immune system and solid tumors Tumors such as metastatic melanoma and renal cell carcinoma (RCC), with their ability to undergo occasional spontaneous regressions or maintain prolonged periods of stable disease, have long fascinated investigators and led to the concept that the immune system might play a role in the regulation or control of malignant cells. Indeed, the first spontaneous regression of metastatic RCC after nephrectomy was described in 1928 and was attributed to an antibody-mediated immune response [18].

Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors

The propensity of nude (athymic) mice to develop lymphoreticular tumors, and the increased incidence of malignancies in patients with congenital or acquired immunodeficiency states, lent further credence to the notion that the immune system might play a role in the control of cancer [19–21]. The development of immune-based therapeutic strategies against metastatic solid tumors was a logical consequence of these observations. At the National Cancer Institute (NCI), pilot immunotherapy trials in patients with advanced solid tumors – particularly metastatic RCC and metastatic melanoma – were conducted in the early 1980s by Rosenberg and colleagues [22]. The earliest trials were intended to nonspecifically stimulate innate immune responses against tumors using cytokines such as interleukin-2 (IL-2). In their preliminary experience using IL-2 in patients with either metastatic RCC or melanoma, overall response rates of 20% (RCC) and 17% (melanoma) were observed. Importantly, some patients achieved complete and durable remission of disease, providing valuable proof of concept of the therapeutic potential of immune-based therapeutic strategies. Subsequent studies conducted at the NCI as well as by the Cytokine Working Group employed adoptive transfer of lymphokine-activated killer cells or ex vivo-expanded tumor-infiltrating lymphocytes in conjunction with IL-2. Although response rates in some trials approached 35%, it remains unclear if tumor-infiltrating lymphocyte therapy provided any additional therapeutic benefit over cytokine treatment alone [23–29]. The low response rate and significant morbidity that was frequently associated with such therapy (particularly highdose IL-2) provided the impetus for the exploration and development of immune strategies that would selectively target the tumor. The foundation for tumor-specific cellular immunotherapy was laid following the identification of a number of different tumor-associated antigens (TAAs) in melanoma and other solid tumors [30–32]. Several clinical trials have evaluated the safety and efficacy of cancer vaccines designed to enhance immune responses to TAAs. Although immune correlative studies have shown that some vaccines significantly expand TAA-reactive T-cell populations in vivo, early clinical results from such trials have been modest. Several factors likely contribute to the poor clinical efficacy of conventional vaccine strategies designed to enhance “self” immunity against TAAs. To date, the vast majority of trials have used an MHC class Irestricted peptide-based approach. As a consequence, resultant immune responses are limited to a single antigenic epitope devoid of a critical CD4+ helper T-cell component [33]. Furthermore, such vaccine strategies may favor the selection of tumor cells lacking the targeted antigen, a phenomenon described as “antigen escape.” Modifying this approach by simultaneously immunizing with multiple tumor antigens that are both MHC class I and II restricted, or by using the tumor itself as a vaccine (tumor lysates, tumor apoptotic bodies, etc.), may partly overcome this limitation [34]. It has also been suggested that tumors may evade cytotoxic T lymphocytes (CTL) by downregulating expression of accessory molecules required by CTLs to mediate lysis. Perhaps one of the greatest limitations of conventional immunotherapy is that the host immune system it attempts to enhance has intrinsic functional compromise. A number of lines of evidence support this concept. First, the host immune system is often rendered incompetent by prior chemotherapy and/or by tumor-related factors. Second, longstanding immune tolerance to tumor antigens, including TAAs targeted by conventional cancer vaccines, may exist. Theoretically, these limitations could be circumvented by allogeneic HCT, a procedure that culminates in complete host immune replacement. Perhaps more importantly, unlike innate host immunity, an allogeneic immune system would have the capacity to mount immune responses to polymorphic variants of tumor-specific or broadly expressed minor histocompatibility antigens (mHAs) [35–37].

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Solid tumors as a GVT target Animal models exploring graft-versus-solid tumor effects The notion that immune responses following allogeneic HCT can be directed against solid tumors was first tested in animal models in the early 1980s. Using a mouse model, Moscovitch and Slavin showed that the high incidence of spontaneous lymphosarcomas in NZ B/W hybrids could be significantly diminished by the transplantation of allogeneic hematopoietic stem cells from BALB/c mice [38]. Subsequently, this group demonstrated the ability of minor and major histocompatibility antigen-mismatched transplants to protect mice from developing metastatic disease following mouse mammary adenocarcinoma (4T1) tumor challenge [39,40]. These experiments showed that alloimmune responses against mHAs that were expressed on tumors could be generated following hematopoietic stem cell transplantation. Early clinical data suggesting GVT effects against solid tumors in humans Compelling clinical evidence supporting the therapeutic utility of the GVT effect in hematologic malignancies prompted investigators to explore allogeneic HCT as a therapeutic tool against metastatic solid tumors in the late 1990s. The first report suggesting a possible GVT effect against a tumor of epithelial origin noted the incidental regression of a metastatic breast adenocarcinoma lesion following allogeneic HCT for relapsed acute myeloid leukemia [41]. Subsequently, Eibl et al. [42] reported tumor regression coincident with acute GVHD in a patient with chemotherapy-resistant metastatic breast cancer following a high-dose transplant from an HLA-identical sibling. The ability to expand mHAspecific CTLs (obtained from the patient at the time of clinical response) that lysed breast cancer cell lines in vitro lent credence to the argument that the tumor regression was at least in part alloimmune mediated. Investigators at the M.D. Anderson Cancer Center reported their experience in 10 patients with metastatic breast cancer who received an allogeneic HCT from an HLA-matched sibling following high-dose conditioning [43]. One complete response (CR) and five partial responses (PR) were noted. GVT effects were implicated in at least two patients whose tumor regression was temporally associated with the onset of acute GVHD and withdrawal of immunosuppression. Delayed regression of metastatic lesions following high-dose allogeneic HCT in a woman with ovarian cancer offered further evidence that some tumors of epithelial origin were indeed susceptible to GVT effects [44].

Use of reduced-intensity conditioning in allogeneic transplantation for solid tumors By the late 1990s, there was sufficient interest in investigating for GVT effects against solid tumors based on available preclinical and clinical data. The major factor limiting the initiation of pilot trials was the significant morbidity and mortality associated with conventional allogeneic HCT. Dose-intensive conditioning used to provide both tumor cytoreduction and a means to allow donor engraftment contributed in part to the morbidity and mortality associated with HCT. Subsequently, it was recognized that reducing the intensity of the preparative regimen might translate to a reduction in the risk of early, conditioning-related morbidity and mortality. In the late 1990s, transplant regimens using reduced-intensity conditioning (RIC) were designed by a number of investigators, and were evaluated for their engraftment potential and toxicity profile [45–49]. Two major factors impacted on the design and development of these dose-reduced conditioning regimens. First was the recognition that GVT

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effects alone might be sufficient to eradicate some hematologic malignancies in the absence of dose-intensive therapy. Second was the realization that the primary role of the conditioning regimen might be limited to preparing the recipient for engraftment by inducing adequate host immunosuppression. Thus, RIC regimens were designed using agents that would induce adequate immunosuppression to facilitate donor immune engraftment while maintaining a low toxicity profile. Such “low-intensity” regimens were first evaluated in hematologic malignancies known to be responsive to allogeneic HCT. Pilot trials utilizing RIC HCT demonstrated that such regimens were generally well tolerated, having a decreased incidence of transplant-related morbidity and mortality, while achieving sufficient donor immune engraftment to induce sustained remissions of some hematologic malignancies [45–49]. One of the major contributions of RIC was that it allowed for the extension of allogeneic HCT to debilitated and older patients who, because of the high risk of treatment-related mortality (TRM) with a high-dose transplant, would ordinarily have been denied the benefit of a procedure with curative potential. Importantly, investigators anticipated that the overall risk of TRM would be lower with a reducedintensity approach, a notion which emboldened them to explore for GVT effects against solid tumors.

Clinical results of reduced-intensity allogeneic HCT transplantation in solid tumors RCC: the National Institutes of Health experience The current worldwide clinical experience of RIC HCT for solid tumors is limited, and the approach continues to be experimental. Because there was no convincing evidence to support the existence of a GVT effect in solid tumors, and given the potential for considerable morbidity with HCT, early pilot trials were restricted primarily to terminally ill patients with advanced treatment refractory metastatic disease. At present, RCC remains the solid tumor in which allogeneic antitumor responses have been best characterized. The following factors provided an incentive to study the susceptibility of this malignancy to a GVT effect. First, metastatic RCC is a uniformly fatal cancer in which the majority of patients succumb to their disease within a year of diagnosis. Second, despite the recent introduction of targeted agents against the vascular endothelial growth factor (VEGF), therapeutic options are extremely limited, with conventional chemotherapy and radiotherapy being largely ineffective [50]. Third,

RCC is considered an “immunoresponsive” tumor based on its susceptibility to cytokine therapy [22,51,52], reports of occasional spontaneous regression [18,53], and the existence of tumor-infiltrating T lymphocytes in regressing metastatic lesions [23,25]. These considerations led to the development of a clinical protocol at the National Institutes of Health (NIH) that sought to test the safety and efficacy of RIC HCT in patients with cytokine-refractory metastatic RCC [48,54]. The primary considerations in the design of the allogeneic HCT trial for patients with metastatic kidney cancer were the following: 1 devising a conditioning regimen that would ensure maximal host immunosuppression to favor rapid and complete donor immune engraftment with relative sparing of recipient myeloid progenitors; 2 devising a post-transplant immunosuppressive approach that would provide optimal prophylaxis for GVHD without precluding the ability to initiate a donor immune mediated GVT effect; 3 judicious use of DLIs to break donor tolerance to host antigens for the promotion of a GVT effect; 4 appropriate patient selection: choosing patients with cytokinerefractory metastatic disease who would be anticipated to survive at least as long as would be required for a delayed GVT effect to occur (i.e. 4–6 months). Therefore, the presence of evaluable metastatic disease, a good performance status (Eastern Cooperative Oncology Group grade 0–1), adequate organ function, and a life expectancy of at least 3 months were prerequisites for patient enrolment. Cyclophosphamide and fludarabine have profound immunosuppressive effects and are well tolerated when given in combination to treat patients with low-grade lymphoproliferative disorders [55,56]. Accordingly, we devised an RIC HCT approach in which patients received a conditioning regimen consisting of cyclophosphamide (60 mg/kg × 2 days) and fludarabine (25 mg/m2 × 5 days), followed by infusion of an unmanipulated, granulocyte colony-stimulating factor-mobilized peripheral blood hematopoietic cell graft from a six of six or five of six HLA antigen-matched sibling donor (Fig. 65.1). Lineage-specific engraftment of donor myeloid (CD14+/CD15+) and T cells (CD3+) was quantitated from post-transplant peripheral blood lymphocyte samples using a polymerase chain reaction-based analysis of either variable number tandem repeats or short tandem repeats polymorphic between patient and donor (Fig. 65.2). Based primarily on animal data showing a decreased risk of GVHD following RIC HCT, we chose to use single-agent cyclosporine (CSP)

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Fig. 65.1 Cyclophosphamide- and fludarabine-based reduced-intensity conditioning hematopoietic cell transplantation protocol for renal cell carcinoma. CSP, cyclosporine; DLI, donor lymphocyte infusion; G-CSF, granulocyte colony-stimulating factor.

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Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors

as GVHD prophylaxis in our initial patient cohort. Much to our surprise, a high probability of grade II–IV acute GVHD (actuarial probability 56%) was observed in the first 25 patients with RCC treated (which was lethal in three cases). Consequently, mycophenolate mofetil (MMF) was added to CSP as GVHD prophylaxis in the second cohort of RCC patients. Gradual withdrawal of CSP/MMF was initiated on day 30 or 60 after transplantation, with the timing and rapidity of immunosuppression tapering being dictated by the rate of tumor progression, degree of donor T-lymphoid chimerism, and presence or absence of GVHD. One or more dose-escalated DLIs were administered to all patients with mixed T-cell chimerism, progressive disease or partial remissions in the absence of acute or chronic GVHD. Since interferon (IFN) has been shown to upregulate MHC expression on RCC tumor cells in vitro (Fig. 65.3), we hypothesized that treating with this cytokine post

Fig. 65.2 Example of polymerase chain reaction-based analysis of two different minisatellites polymorphic between patient and donor. Patient (lane 1) and donor (lane 5) lanes show two patient-specific and two donor-specific bands. Also shown are: control mixtures of donor-into-patient DNA representing 30% (lane 2), 50% (lane 3), and 90% donor chimerism (lane 4); and post-transplant blood sample sorted into lineages using magnetic beads showing 100%, 60%, and 99% donor chimerism in CD2+/CD3− natural killer cell (lane 6), CD14+/15+ myeloid cell (lane 7), and CD3+ T-cell fractions (lane 8), respectively.

transplant might make the tumor a better target for a GVT effect. Therefore, patients without GVHD who failed to respond to DLI were eligible to receive post-transplant cytokine therapy with IFN-α, either alone or in combination with IL-2. The initial experience of RIC HCT in metastatic RCC was published in 2000 [48]. Ten of the first 19 patients treated with this transplant approach had tumor shrinkage, including three who had a CR and seven patients who had a PR. At present, 74 patients have undergone RIC HCT for RCC at the NIH. Of these, 73 patients demonstrated durable engraftment, achieving 100% donor T-cell chimerism by day 100 post transplant. Twenty-nine of 74 (39%) patients have had a disease response, including seven CRs and 22 PRs. Five patients who were deemed to be “nonresponders” had radiographic evidence for a mixed response. Disease regression was temporally associated with acute and chronic GVHD in many patients, was typically delayed in onset and did not occur until CSP was tapered, consistent with an alloimmune-mediated GVT effect. Several patients have proven to have a durable disease response, including the first complete responder, who remains without evidence of metastatic disease now over 9 years post transplant (Fig. 65.4). Regression of disease in multiple metastatic foci has been observed, although pulmonary responses appear to occur most frequently. On occasion, disease responses have been dramatic and have included complete resolution of large pulmonary metastases with bulky adenopathy (Fig. 65.5). Preliminary data would suggest that disease response following RIC HCT is a clinically meaningful phenomenon since regression of metastatic RCC appears to be associated with a trend towards improved survival. Survival in nonresponders has been less than 6 months in contrast to those achieving a PR, who survived a median 2.5 years post transplant. In general, patients with metastatic kidney cancer have tolerated this conditioning well. Although virtually all patients have developed febrile neutropenia, we observed no sinusoidal obstructive syndrome of the liver or chemotherapy-associated mucositis. Twentyeight percent (21 of 74) of patients developed cytomegalovirus antigenemia, with only one case of cytomegalovirus disease (esophagitis) that was responsive to gancyclovir therapy. Mortality associated with transplant-related complications occurred in 8 of 74 (11%) patients, with either infection or acute or chronic GVHD being the major cause of TRM. Since kidney cancer frequently metastasizes to the lungs, some patients with RCC may be at high risk for the development of postobstructive pneumonia following transplantation. Such a complication occurring in the profoundly immunocompromised can lead to disastrous infectious sequelae, as was observed in two of our RCC patients who

[A] FL2 Log

[A] FL2 Log 14

+ Interferon-alpha

14

No interferon-alpha

B

Fig. 65.3 Upregulation of human leukocyte antigen (HLA) class I expression (both percent positive and mean channel fluorescence) on renal cell carcinoma cells 24 hours after exposure to interferon-alpha (10,000 U/mL).

100

101

102

HLA class 1 PE FL2 Log

B

103

100

101

102

HLA class 1 PE FL2 Log

103

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Fig. 65.4 Renal cell carcinoma metastatic to the lungs (pretransplant images A1 and A2). There was no change in metastatic disease 30 days following conditioning (B1 and B2). Following cyclosporine withdrawal, metastasis regressed completely by 110 days post transplant (C1 and C2). The patient remains without evidence of disease over 9 years after transplantation.

Fig. 65.5 Delayed regression of renal cell carcinoma. Pretransplant images showing bulky anterior mediastinal (A1) and hilar (A2) adenopathy. Stable disease seen 6 months post transplant (B1 and B2). Regression of bulky adenopathy was observed 9 months post transplant (C1 and C2).

died from bacterial sepsis related to obstructive bronchial pneumonia. Most deaths related to acute GVHD occurred during the first 2–3 years following inception of the trial; more recently, advances in our ability to manage severe or steroid-refractory GVHD have translated into a greatly reduced risk of mortality from this complication [57]. Acute GVHD has been the greatest contributor to morbidity and mortality in our RCC patients undergoing RIC HCT. Because MMF is a potent inhibitor of inosine monophosphate and has been shown in vitro to block proliferative responses of cytotoxic T cells, we added MMF to CSP as prophylaxis for GVHD in our second cohort of patients with RCC. Unfortunately, an interim analysis of the first 30 patients treated with both drugs revealed no difference in the incidence of grade II–IV or grade III–IV acute GVHD between the CSP alone and the CSP + MMF cohorts (actuarial probability of acute grade II–IV GVHD 56% and 61%, respectively). Based on these observations, we have withdrawn MMF from the transplant regimen and are currently investigating the effect of adding low-dose methotrexate (MTX) to CSP as GVHD prophylaxis. A recent analysis of 230 patients with a variety of malignancies and nonmalignant hematologic disorders undergoing RIC HCT with cyclophosphamide and fludarabine conditioning at our institution revealed that the incidence of acute GVHD grade II–IV and III–IV was

significantly less in those patients who received CSP and MTX as GVHD prophylaxis compared with those who received either CSP alone or CSP plus MMF. Importantly, the addition of MTX to CSP did not appear to abrogate the ability to induce GVT effects against RCC, with four of 18 patients who received this regimen demonstrating an objective response (including one patient with a CR). Assessment of donor chimerism following RIC HCT is important not only to document the establishment of donor engraftment, but also to allow for post-transplant manipulation of the donor immune system to optimize the chances of inducing a GVT effect. In our experience, GVT effects following RIC HCT typically do not occur until the immune system has converted from mixed to predominantly donor T-cell chimerism. As a consequence, detailed serial measurements of donor engraftment in both lymphoid and myeloid lineages are performed on all patients undergoing this approach. Representative lineage-specific engraftment profiles and their relationship to clinical outcome are shown in Fig. 65.6. Although donor T-cell engraftment typically precedes myeloid engraftment in patients receiving cyclophosphamide- and fludarabinebased conditioning, engraftment patterns may vary considerably among individual patients [58]. Several factors influence the degree and rapidity of donor engraftment, including the agents used in the conditioning

Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors Progression GVHD

Regression Taper CSP

Donor chimerism (%)

100

75

50 T-cell chimerism Myeloid chimerism

25

0

963

Importantly, however, tumor shrinkage has also been observed to occur in the absence of or temporally distant from GVHD, an observation which might imply that tumor cells are particularly sensitive to allogeneic immune attack or that tumor-specific immune effectors might be involved in mediating RCC regression in some patients. Finally, regression of RCC following a DLI or after treatment with low-dose subcutaneous IFN-α has also been observed. Disease regression after DLI suggests that the mediators of the GVT effect may be analogous to those mediating leukemia regression. Interestingly, some patients who had failed to respond to IFN-α before transplantation had disease regression when the drug was given post transplant. This observation suggests that the mechanism by which IFN-α promotes tumor regression in the post-transplant setting relates to the ability of the drug to make the tumor a better target or enhance the allogeneic immune system rather than to a direct antineoplastic effect.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Weeks post transplant

Fig. 65.6 Representative lineage-specific engraftment profile following cyclophosphamide- and fludarabine-based conditioning. The percentage donor chimerism in T cells (䊉) and myeloid cells (䉱) is shown in a patient who had a disease response 17 weeks post transplant. CSP, cyclosporine; GVHD, graft-versus-host disease.

Table 65.1 Tumor response patterns consistent with a graft-versus-tumor effect after reduced-intensity conditioning hematopoietic cell transplantation • • • • •

Delayed onset of response (>100 days post transplant) Response following withdrawal of immunosuppression Response following or concomitant with graft-versus-host disease Response following donor lymphocyte infusions Prolonged duration of response in tumors previously treatment refractory

regimen, the prevailing state of host immunity, and allograft cell doses. In a multivariate analysis of 36 patients with metastatic solid tumors who underwent RIC HCT at the NIH, pretransplant exposure to chemotherapy and increased allograft CD34+ cell dose were found to significantly facilitate the engraftment of donor myeloid and T cells [59]. Therefore, tailoring the intensity of RIC in solid tumor patients based on prior chemotherapy exposure would seem worthy of exploration. While the exact mechanisms underlying the regression of metastatic RCC following allogeneic HCT are yet to be unraveled, several observations (both laboratory and clinical) would suggest an alloimmune effect mediated at least in part by donor T cells is at work (Table 65.1) [48]. The majority of patients who ultimately achieved a disease response showed early tumor growth in the first few months after transplantation, a time period when the newly engrafted donor immune system was kept in check by immunosuppressive therapy or when mixed T-cell chimerism prevailed, leading to “tolerance” of host tissues (including the tumor). Tumor regression was typically delayed (4–8 months) and followed conversion to predominantly donor T-cell chimerism after immunosuppression had been withdrawn or was being tapered. These observations highlight the importance of utilizing a transplant approach that favors rapid donor T-cell engraftment and incorporates judicious and timely withdrawal of GVHD prophylaxis. As had previously been described in patients with hematologic malignancies, a prior history of acute GVHD was associated with an increased probability of having a tumor response. One might speculate that disease regression in this setting could be the consequence of alloreactive T cells targeting mHAs that are broadly expressed on both normal tissues and tumor cells.

Emerging role of RIC HCT for RCC Although allogeneic HCT is clearly an investigational approach for the treatment of metastatic RCC, several other investigators have begun to report promising results in this disease (Table 65.2). Investigators at the University of Chicago initially reported on 15 patients undergoing HCT following a fludarabine- and cyclophosphamide-based conditioning regimen [60]. The first four patients treated were conditioned with an extremely low-intensity regimen consisting of fludarabine (90 mg/m2) and cyclophosphamide (2 g/m2) given at doses that failed to achieve adequate levels of host immunosuppression. As a consequence, three of the four patients (75%) failed to achieve stable donor T-cell engraftment and ultimately rejected their transplant. Subsequent patients received higher doses of the same conditioning agents (fludarabine 150 mg/m2 and cyclophosphamide 4 g/m2), and achieved successful and sustained donor engraftment. Of their 12 patients who engrafted and were evaluable for a disease response, four (33%) had radiographically documented disease regression consistent with a PR, including one patient who had tumor regression in his primary kidney tumor. These data have since been updated to include the first 19 patients transplanted, four of whom achieved a PR. Although all patients have relapsed at a median of 609 days following HCT, all responders were alive with a median follow-up of 41 months (compared with a median overall survival of 14 months for the entire cohort) [61]. As with other trials of RIC HCT for solid tumors, a dominant donor immune system (determined by T-cell chimerism studies) was a prerequisite for the generation of a meaningful GVT effect. Only those patients who had stable and predominantly donor immune engraftment demonstrated a disease response. In fact, one patient with graft rejection (a nonresponder) underwent a second transplant with the intensified conditioning regimen, achieved complete donor engraftment, and subsequently had a PR. Only two of 12 (17%) patients experienced grade II or greater acute GVHD, perhaps the consequence of a more gradual termination of GVHD prophylaxis. Whether delaying the withdrawal of post-transplant immunosuppression will decrease the incidence of acute GVHD without negating beneficial GVT effects will need to be determined from larger studies. A Cancer and Leukemia Group B (CALGB) intergroup trial evaluated the feasibility of performing HCT for metastatic RCC in a multiinstitutional setting in the United States [62]. Twenty-two patients underwent HCT from an HLA-matched sibling donor following cyclophosphamide plus fludarabine-based conditioning. Seventeen of nineteen evaluable patients achieved greater than 90% donor chimerism by day 120 after transplantation. Acute (grade II–IV) GVHD was seen in 50% of patients, while 23% of patients experienced chronic GVHD. There were no objective responses seen, and median overall survival

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Table 65.2 Summary of clinical trials of reduced-intensity conditioning hematopoietic cell transplantation in metastatic renal cell carcinoma

Investigators

Conditioning agents

GVHD prophylaxis

Childs et al. [48] and unpublished data

CY + FLU

Rini et al., Artz et al. [60,61] Bregni et al. [67] Pedrazzoli et al. [87] Blaise et al. [88] Nakagawa et al. [89] Ueno et al. [90] Hentschke et al. [91] Massenkeil et al. [92] Tykodi et al. [93] Barkholt et al. [63] Rini et al. [62] Peres et al. [94]

CY + FLU CY + FLU + thiotepa CY + FLU FLU + BU + ATG FLU /CLA + BU + ATG FLU + MEL FLU + TBI ± ATG FLU + CY + ATG FLU + TBI Multiple FLU-based regimens FLU + Cy FLU + CY or FLU + TBI

CSP (first 25 patients) CSP + MMF (subsequent patients) Tacro + MMF CSP + MTX CSP + MTX CSP CSP Tacro + MTX CSP + MMF CSP ± MMF CSP + MMF CSP ± MMF or MTX Tacro + MTX CSP + MMF

Acute GVHD (II–IV)

Chronic GVHD

Treatment-related mortality

Response (partial or complete)

53%

21%

11%

53%

22% 86% 0% 42% 44% 47% 50% 29% 50% 40% 32% 44%

39% 71% N/A 60% 44% 27% 30% 57% 50% 33% 23% 38%

14% 0% 29% 9% 0% 33% 40% 14% 13% 16% 9% 12%

22% 57% 0% 8% 11% 27% 0% 29% 13% 32% 0% 32%

ATG, antithymocyte globulin; BU, busulfan; CLA, cladribine; CSP, cyclosporine; CY, cyclophosphamide; FLU, fludarabine; GVHD, graft-versus-host disease; MEL, melphalan; MMF, mycophenolate mofetil; MTX, methotrexate; N/A, not available; Tacro, tacrolimus; TBI, total body irradiation.

was only 5.5 months, with most patients dying from disease progression (with a median time to progression of 3 months). Inclusion of a number of patients with multiple adverse prognostic factors, sparing use of DLI (only two of 22 patients receiving DLIs despite disease progression in the majority), and inclusion of patients with nonclear cell histology are some of the factors that may account for the poor outcome observed in this trial. This trial clearly highlights the importance of appropriate patient selection and the need for identifying prognostic factors likely to predict for a favorable outcome. The European Group for Blood and Marrow Transplantation (EBMT) reported on their experience in 124 patients with metastatic RCC undergoing HCT at multiple centers from an HLA-identical (n = 106) or partially matched (n = 5) family donor, or from an HLA-matched unrelated donor (n = 13) (Table 65.2) [63]. A variety of fludarabine-based RIC regimens were utilized. CSP alone or in combination with MMF or MTX was used for GVHD prophylaxis in the majority of patients. Durable engraftment was achieved in 121 patients. The incidences of acute grade II–IV and chronic GVHD were 40% and 33%, respectively. Twenty-eight of ninety-eight evaluable patients had an objective tumor response including four patients with a CR. Responses were typically delayed, with PRs and CRs occurring a median of 135 days and 265 days, respectively. A short time interval from diagnosis of RCC to HCT, the presence of acute GVHD, and the use of an HLA-mismatched donor were each associated with a higher probability of response. Transplantrelated mortality was 16% at 1 year, while overall survival at 2 years was 30%. In a multivariate analysis, chronic GVHD, good performance status (Karnofsky score ≥80), DLI administration, and fewer than three sites of metastatic disease were identified as factors favorably impacting survival.

Toxicities and limitations of RIC HCT in metastatic RCC Although there is ample evidence now to support the susceptibility of metastatic RCC to an allogeneic GVT effect, there are a number of potentially life-threatening toxicities of RIC HCT that currently limit the

Table 65.3 Limitations of reduced-intensity conditioning hematopoietic cell transplantation in solid tumors Incidence (%) HLA-matched sibling donor available Acute GVHD Chronic GVHD Cytomegalovirus reactivation Graft rejection Treatment-related mortality

25–30% 30–60% 40–70% 20–40% 5–10% 10–20%

GVHD, graft-versus-host disease; HLA, human leukocyte antigen.

broader application of this approach (Table 65.3). While the incidence of early TRM appears to be less with RIC, overall 5–20% of patients will ultimately die from a transplant-related cause. Infection and complications related to acute GVHD are the greatest risks associated with the procedure. In fact, three of the first 25 patients with RCC transplanted at the NIH died from GVHD-related causes [48]. Unfortunately, the addition of MMF to CSP as GVHD prophylaxis did not reduce the incidence of grade II–IV or grade III–IV acute GVHD. However, new second-line therapies for severe steroid refractory GVHD such as daclizumab (monoclonal antibody directed against the alpha chain of the IL-2 receptor) and infliximab (monoclonal antibody directed against the tumor necrosis factor-alpha receptor) have shown early promise [64,65]. Our preliminary experience suggests that these monoclonal antibodies are effective in reducing mortality from steroidrefractory GVHD, particularly when given with lipid-complexed amphotericin B or voriconazole as Aspergillus prophylaxis, concomitant with a rapid reduction in corticosteroid dose once steroid-resistance is apparent [57]. Larger studies will be required to determine whether these agents will ultimately result in a reduction in the risk of GVHD-related mortality without compromising the ability to generate a GVT effect.

Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors Table 65.4 Patient characteristics likely to predict a favorable outcome after reduced-intensity conditioning hematopoietic cell transplantation for solid tumors • • • • • •

Good performance status (Eastern Cooperative Oncology Group grade 0–1) Younger patient age (i.e. <65 years) Small tumor volume Slow tumor growth kinetics Absence of central nervous system metastatic disease Evidence (clinical or in vitro) that tumor is susceptible to immunemediated attack

Careful patient selection, with particular attention to performance status, medical comorbidities, and tumor growth kinetics may further improve transplant-related outcome. Although the use of RIC HCT for solid tumors is presently limited, preliminary clinical experience provides insight into patient characteristics that would be expected to impact favorably on transplant outcome (Table 65.4).

RIC HCT in melanoma Few tumors are associated with a more abysmal prognosis than metastatic melanoma. At present, because of a paucity of efficacious treatments, there exists no accepted “standard therapy” for those who have developed metastatic disease. Nevertheless, along with kidney cancer, malignant melanoma has long shared the reputation of being an “immunoresponsive” tumor. In fact, more immunotherapy trials have been targeted at melanoma than any other solid tumor. It would seem logical, therefore, to investigate whether this tumor might be a target for a GVT effect following allogeneic HCT. Unfortunately, and rather surprisingly, the results of RIC HCT for melanoma at the NIH and at other centers worldwide have been far from inspiring. We reviewed the outcome of 25 patients with metastatic melanoma treated at four different institutions using one of three different RIC HCT conditioning regimens: cyclophosphamide (120 mg/kg) + fludarabine (125 mg/m2) (n = 18), busulfan (8 mg/kg) + fludarabine (150 mg/m2) + antithymocyte globulin (n = 5), or 200 cGy total body irradiation + fludarabine (90 mg/m2) (n = 2) [66]. Engraftment was documented in 24 of 25 patients. Twelve patients (48%) developed grade II–IV acute GVHD, including 11 who had GVHD involving the skin. In 11 patients (92%), GVHD responded to immunosuppressive therapy, while one died from grade IV steroid-refractory liver GVHD. Five patients (20%) had radiographically documented disease regression consistent with a disease response (all PRs); four responses occurred in the immediate posttransplant period, likely the consequence of a chemotherapy effect, while one response was delayed in onset and occurred in association with chronic skin GVHD consistent with a GVT effect. None of the disease responses were durable, with melanoma progression occurring within 1–3 months in all responders. Six patients were treated with a DLI for tumor progression without evidence for a response. None of the 25 patients transplanted survive. Two (8%) died from transplant-related complications (acute GVHD and idiopathic encephalitis), and 23 (92%) died from progression of metastatic disease. Median survival was only 100 (range 7–660) days, with no survival difference seen between the five responding patients (median survival 114 days) and the 20 nonresponding patients (median survival 93 days). The disappointing outcome of RIC HCT in this tumor is perplexing, particularly when clinically meaningful GVT effects in RCC have been demonstrated. Rapid tumor kinetics and bulky metastatic disease may

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explain at least in part why some patients failed to manifest a GVT effect. Of more concern, however, was the observation that some patients appeared to have acceleration in the pace of tumor growth in the immediate post-transplant period following RIC HCT (Niederweiser, personal communication). While the description of this phenomenon is admittedly subjective and could simply reflect the natural evolution of tumor growth kinetics, it is tempting to speculate that transplant-induced immunosuppression could promote tumor progression as a consequence of the destruction of innate host immune surveillance. These preliminary data demonstrate that transient conditioning-related regression of metastatic melanoma may occur following RIC HCT, although the likelihood of a clinically meaningful GVT effect appears low. The high risk of death from rapid disease progression has discouraged investigators from pursing similar RIC HCT studies in melanoma patients. Preclinical studies of “tumor-targeted” allogeneic transplant approaches are needed and, if successful, could lead to future “second-generation” RIC HCT trials in metastatic melanoma.

RIC HCT in other solid tumors The collective global experience with RIC HCT in solid tumors other than RCC and melanoma is limited to a handful of publications, all with small patient numbers. However, the important observation of GVT effects in patients with RCC will likely lead to a rapid rise in the number of experimental transplants conducted for other types of treatmentrefractory solid tumor. Indeed, as previously discussed, GVT effects against metastatic breast carcinoma following high-dose conditioning and allogeneic HCT have been described in some patients in association with acute GVHD [42,43]. The high prevalence of breast carcinoma in the general population, as well as the disappointing results of autologous transplantation trials, has inspired a number of investigators to explore RIC HCT in patients with this malignancy. Bregni and colleagues treated six patients with metastatic breast cancer with an RIC HCT following conditioning with cyclophosphamide, fludarabine, and thiotepa [67]. Two patients had a PR that was delayed and did not occur until several months following transplantation. In both cases, responses were preceded by DLIs and GVHD, consistent with a GVT effect. At the NCI, 16 patients with treatment-refractory metastatic breast cancer received a T-cell-depleted peripheral blood hematopoietic cell graft from an HLA-matched sibling following fludarabine and cyclophosphamide conditioning; escalating doses of DLIs were then administered on days 42, 70, and 98 post transplant. All 15 evaluable patients achieved complete donor T-cell chimerism. Delayed responses (beyond day 28 after transplantation) consistent with a GVT effect were seen in six of 16 patients, including two patients who had experienced early disease progression following transplantation [68]. A joint International Bone Marrow Transplant Registry/EBMT registry review identified 76 patients with metastatic breast cancer undergoing allogeneic HCT between 1992 and 2000 [69]. As would be expected from the timeframe of the study, a variety of both high-dose and reducedintensity conditioning regimens were employed. While no engraftment data were provided, approximately 51% of patients developed acute GVHD, and 25% of patients had chronic GVHD. With a median followup of approximately 25 months, progression-free survival in this cohort was 9%, and overall survival at 2 years was only 22%. The median time to progression and median overall survival were 8 months. Carella and colleagues recently described a novel approach of evaluating allogeneic immunotherapy in 17 patients with breast cancer whose tumors were first maximally debulked with intensive chemotherapy, followed by autologous HCT [70]. Four patients (24%) had delayed regression of metastatic breast cancer as the consequence of a GVT effect. However, in contrast to studies of allogeneic HCT alone, where only

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partial regression of metastatic breast cancer was typically observed, 18% of patients in this study undergoing tandem autologous/allogeneic HCT achieved long-term disease-free survival. Remarkably, three patients had delayed and complete tumor regression associated with the development of acute GVHD, and remained in remission 3.6, 4.2, and 5.9 years after transplantation. Of note, three of five patients who had partial disease regression after autografting subsequently experienced a sustained CR as a result of a GVT effect, in contrast to no CRs after allogeneic HCT in the 11 patients who failed to respond to high-dose chemotherapy. The results presented here suggest that a tandem transplant approach can induce long-term disease-free survival in a minority of patients with metastatic breast cancer. More importantly, they highlight the importance of reducing pretransplant tumor burden to facilitate and enhance allogeneic graft-versus-solid tumor effects. A small number of RIC HCTs have also been performed in patients with advanced ovarian cancer. One group reported delayed tumor regression in a patient with metastatic ovarian carcinoma who received an HLA-matched transplant from her sibling donor [44]. A small series of patients (n = 5) with treatment-refractory ovarian carcinoma who underwent HCT following either high-dose (one of five) or reduced-intensity (four of five) conditioning was reported by Bay et al. [71]. Three patients had regression of metastasis and/or a decrease in disease-related serum markers (i.e. CA125) early in the post-transplant period. The proximity of tumor shrinkage to chemotherapeutic conditioning makes it unclear whether these responses were truly related to an alloimmune effect. Longer follow-up and additional patients should clarify whether this tumor is indeed responsive to a GVT effect. Another group reported the results of RIC HCT that used either cladribine or fludarabine in combination with busulfan and antithymocyte globulin in seven patients with a variety of different solid tumors [72]. Partial disease regression ascribed to a GVT effect was reported in a patient with osteosarcoma and two patients with RCC. In summary, insufficient data are available at this time to comment on the efficacy of RIC HCT in the vast majority of solid tumors. Interpreting these limited data is made even more difficult as it is often not clear whether disease regression is related to chemotherapeutic agents in the conditioning regimen or is truly the sequela of a donor immunemediated GVT effect. Strict adherence to criteria such as those outlined in Table 65.1 may help substantiate that a GVT effect has indeed occurred and should minimize the risk of inadvertent overinterpretation of clinical data. The efficacy of GVT in terms of its ability to induce remission varies among these malignancies, with the critical determining factors dictating neoplastic susceptibility to GVT not entirely understood.

Mechanisms underlying GVT effects in solid tumors Disease regression following allogeneic HCT in some patients with treatment refractory RCC has stimulated interest in exploring the mechanisms through which GVT effects occur. A better understanding of both the targets and effectors of the GVT response could lead to the development of safer and more efficacious transplant regimens. Based on clinical data as well as our knowledge of the mediators of the GVT effect, T lymphocytes (both CD8+ and CD4+) as well as natural killer (NK) cells probably play a role in mediating immune responses against solid tumors [73–77]. The observation that disease regression is (1) associated with an increase in the percentage of circulating CD8+ T cells with an activated phenotype (DR+CD38+CD57+), (2) does not occur until T-cell chimerism is predominantly donor, and (3) sometimes follows a DLI provides strong evidence to support this hypothesis [48,78]. What, if any, role allogeneic NK cells play in mediating GVT effects in solid tumors is currently unknown. Researchers have shown that NK

cells may play a critical role in mediating GVT effects in patients with acute and chronic myelogenous leukemia undergoing transplants from haploidentical donors with killer immunoglobulin-like receptor (KIR) incompatibility in the GVHD direction (i.e. patient Cw allele mismatched to donor and therefore unable to function as a ligand for donor NK cell KIR) [79,80]. Recently, we have conducted in vitro experiments showing that NK cell clones generated from KIR-mismatched allogeneic donors can kill both melanoma and RCC tumor cells preferentially through KIR incompatibility [81]. These preliminary data would suggest that the antitumor effects seen against acute myelogenous leukemia following KIR-incompatible allogeneic HCT might also occur against select solid tumors in the setting of haplotransplantation. A recent retrospective analysis in a subset of patients with metastatic RCC who underwent a T-cell-replete RIC HCT from an HLA-matched sibling donor at the National Heart, Lung, and Blood Institute provides preliminary clinical evidence in support of this hypothesis [82]. In this study, KIR incompatibility (defined as the presence of one or more KIR genotypes in donor cells for which the corresponding inactivating HLAB or -C ligand is missing from the recipient) appeared to predict for higher objective response rates as well as improved overall survival, an effect most pronounced in patients lacking HLA-Bw4 who received a transplant from donors with genotypic evidence of KIR3DL1. The identification of target antigens involved in GVT effects is an area of intense investigation, and recent findings suggest that both lineage-restricted and broadly expressed mHAs are potential targets. In an attempt to clarify the role of mHAs in alloimmune T-cell responses against RCC, previously characterized mHA-specific CTL clones were tested for cytotoxicity against EBV-transformed B cells (EBV-LCLs) (HLA matched for the appropriate mHA-restricting allele) to first identify RCC patients expressing the relevant antigen. Subsequently, CTLs recognizing patients’ EBV-LCLs were then tested for their ability to recognize mHAs present on patient RCC cells. In the majority of cases, CTLs recognizing patient EBV-LCLs also recognized patients’ RCC cells, suggesting that broadly expressed mHAs could serve as targets for immune-mediated tumor regression in the setting of acute GVHD [83]. Preliminary data obtained from patients with RCC regressing after allogeneic transplantation suggest that the target antigens may differ in those who experience tumor regression in the context of GVHD as opposed to those without. T-cell clones that specifically lysed patient RCC cells or both RCC and patient hematopoietic cells were isolated and expanded from one responder where a GVT effect occurred in the absence of GVHD. In contrast, we could only expand T-cell clones that reacted against both tumor and hematopoietic cells in a patient who had disease regression that occurred in the setting of acute GVHD [78]. Thus, it is conceivable that cellular immune responses directed against antigens shared by tumor and normal tissues would result in GVHD as well as GVT, while GVT without GVHD would be expected to occur in those having a cellular immune response against tumor-restricted antigens (Fig. 65.7). The identity of antigens that serve as targets for a GVT effect, specifically those that appear to be restricted to the tumor, could provide clues to the intricate mechanisms of GVT and provide valuable insight that might aid in the development of future tumor-targeted immunotherapeutic approaches. Using T cells isolated from a patient who had regression of metastatic RCC following allogeneic transplantation, we recently identified a new solid tumor antigen that appears to be overexpressed in the majority of kidney tumors. CD8+ T-cell clones isolated from lymphocytes obtained from this patient after transplantation and following tumor regression lysed the patient’s RCC cells in vitro. Using complementary DNA expression cloning, the target antigen of these allogeneic T cells (donor in origin) was found to be a 10-mer peptide

Hematopoietic Cell Transplantation for Renal Cell and other Solid Tumors GVT mediators

Tumor cell

CD 4+ T cells

TAA mHA

mHA

Fig. 65.7 Immune populations hypothesized to contribute to graft-versus-tumor (GVT) effects following reduced-intensity conditioning hematopoietic cell transplantation. Minor histocompatibility antigens (mHAs) are either restricted to the tumor or broadly expressed on normal and malignant tissues. Tumor cells need to express a dominant natural killer (NK) cell receptor-activating ligand (i.e. MICA) to initiate NK cell-mediated killing. GVHD, graft-versus-host disease; MHC, major histocompatibility complex; NKL, natural killer ligand; TAA, tumor-associated antigen.

NK cells

tumor antigen (called CT-RCC-1) that was recognized in the context of HLA-A11. The genes encoding this antigen were found to be derived from a human endogenous retrovirus that was found to be overexpressed in more than 50% of clear cell carcinomas but not in any normal tissues [84]. We believe that this is the first solid tumor antigen identified using allogeneic T cells from a patient undergoing HCT, and could provide a potential target for cellular immunity both within and outside the context of allogeneic HCT. At present, responses following RIC HCT have only been observed in patients presenting with the common clear cell form of RCC. Since clear cell RCC is typically associated with a mutation in the von Hippel–Lindau (VHL) tumor suppressor gene, mutant VHL protein or antigens that are upregulated as a consequence of the absence of functional VHL protein (carbonic anhydrase-9, platelet-derived growth factor, VEGF, etc.) have received considerable scrutiny as possible TAAs. Whether any of these antigens are targets for the GVT effect in RCC has not yet been elucidated. Interestingly, preliminary data from our group suggest that the CT-RCC antigen (derived from the aforementioned human endogenous retrovirus) overexpressed in RCC may have expression limited to clear cell RCC (unpublished data).

Future directions The lack of efficacy of chemotherapeutics, radiotherapy, and cytokinebased immunotherapy for many patients with metastatic cancer has catalyzed enthusiasm for exploring allogeneic immunotherapy against solid tumors. While there is little doubt about the potential for its application in oncology, extensive use of RIC HCT in the treatment of solid tumors will likely remain limited until both the safety and efficacy of the approach are improved. Further progress in the fields of tumor vaccination and adoptive “tumor-targeted” T-cell infusion are needed to

Tumor restricted = No GVHD Tumor restricted = No GVHD

Broadly expressed = GVHD

NK cell receptor-activating ligand (non-MHC restricted killing)

NKL

mHA

CD 8+ T cells

967

Broadly expressed = GVHD

Tumor restricted = No GVHD

mHA

TAA

Tumor restricted = No GVHD

address these problems. Recently, murine models have demonstrated that tumor-specific immunity can be boosted following post-transplant tumor immunization. In an MHC-matched but mHA-disparate allotransplant model, immunization of the recipient post transplant against either leukemia or fibrosarcoma resulted in enhanced antitumor activity without exacerbating GVHD [85]. A similar strategy could be used in humans for those patients who fail to achieve a CR after RIC HCT. Likewise, adoptive transfer of ex vivo-expanded tumor-specific CTLs that are generated from the donor could be used to enhance a GVT effect. Recent developments in our understanding of the role played by NK cells in both enhancing GVT effects and minimizing GVHD offer the prospect of incorporating “KIR-incompatible” or “KIR-mismatched” NK cell therapy into allogeneic immunotherapy strategies. In vitro, allogeneic KIR-incompatible NK cells have enhanced cytotoxicity against RCC cells compared with autologous or KIR-matched NK cells [81]. The adoptive infusion of alloreactive donor NK cells was recently found to significantly reduce the risk of acute GVHD and potentiate graft-versus-RCC effects in a murine model of HCT for kidney cancer [86]. Trials evaluating adoptively infused allogeneic NK cells and other novel methods to direct or target alloimmune cells against RCC in humans undergoing HCT for metastatic RCC will likely be explored in the near future. A variety of novel “targeted” agents have recently been approved for the treatment of several malignancies. Inhibitors of the VEGF pathway such as sunitinib and sorafenib are now available for the treatment of metastatic RCC. While unlikely to be curative, these agents have been shown to prolong progression-free survival. Since heavy disease burden and rapidly progressive tumors can significantly detract from the ability to generate clinically meaningful GVT responses, incorporating the aforementioned disease-stabilizing antiangiogenic agents into HCT strategies might be beneficial and should be explored.

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Jason Law & Katherine K. Matthay

Hematopoietic Cell Transplantation for Neuroblastoma

Introduction Neuroblastoma is a malignancy of young children derived from embryonic neural crest cells of the peripheral sympathetic nervous system. It is the most common extracranial solid tumor of childhood, with approximately 650 new cases per year in the United States, and accounts for 15% of cancer-related deaths in children [1]. Some of its unique features, such as the ability to secrete and store catecholamines, the genetic heterogeneity, and the propensity to undergo differentiation either spontaneously or with various stimuli, have led to novel diagnostic and therapeutic approaches. As the therapy has become more tailored to biologic and clinical risk groups, a higher proportion of children with neuroblastoma are surviving. However, nearly half of the children with neuroblastoma present after 1 year of age with metastatic disease, of whom less than 40% will survive more than 5 years, even with aggressive combination therapeutic approaches [2]. The goals of future therapy are to increase the specificity of treatment in order to improve the survival of children with advanced disease with less therapy-induced toxicity.

Clinical presentation and staging Neuroblastoma can originate from any site in the sympathetic nervous system, presenting as a mass in the abdomen, mediastinum, neck or pelvis. The most common primary site is the adrenal gland or other abdominal sites (70%), while mediastinal tumors are more common in infants than in older children. The signs and symptoms of neuroblastoma depend on the location and extent of the primary tumor, as well as the presence of metastatic disease. Large abdominal masses may cause complaints of fullness, discomfort, vomiting or anorexia. Masses arising from the organ of Zuckerkandl in the pelvis can cause constipation and bladder dysfunction. High thoracic or cervical masses can present with unilateral ptosis, meiosis, and anhidrosis (Horner’s syndrome) [3]. Epidural or intradural extension of neuroblastoma occurs in 5–16% of cases of neuroblastoma. These patients may have symptoms associated with spinal cord compression including pain, bladder or bowel dysfunction, paraparesis or paraplegia. Prompt administration of chemotherapy appears to be an effective therapy for treating intraspinal neuroblastoma without the long-term sequelae associated with either radiation or surgical resection and laminectomy [4]. Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Metastatic neuroblastoma can classically present with proptosis and periorbital ecchymoses, and bone pain resulting in irritability and limp. Rapid enlargement of the liver metastases can result in respiratory compromise, particularly in neonates [5]. Skin lesions, seen almost exclusively in infants, have a bluish hue and have been given the nickname “blueberry muffin” lesions. Rarely, neuroblastoma will present with a paraneoplastic syndrome, including either watery diarrhea caused by secretion of vasoactive intestinal peptide, or opsoclonus–myoclonus– ataxia syndrome, characterized by jerky multidirectional eye movements, loss of balance, and intermittent muscle jerks [6]. The latter syndrome is thought to be brought about by production of antineuronal antibodies that cross-react with normal brain tissue [7]. Multiple staging systems have been used for neuroblastoma, but currently most centers use the International Neuroblastoma Staging System (INSS). The INSS is a surgical staging that is quite prognostic and combines the two most common previous systems utilized by the United States pediatric cooperative groups, the Pediatric Oncology Group (POG) and the Children’s Cancer Group (CCG) (Table 66.1) [8]. Nonmetastatic tumors are classified as INSS 1, 2A, 2B or 3, depending on the amount of gross residual tumor after surgery, the extent of lymph node involvement, and whether the tumor is invasive across the vertical midline of the body. INSS 4S is a special designation developed only for neuroblastoma, because of the unusually favorable course in infants under 1 year of age with metastases limited to the liver, skin, and bone marrow. Such tumors are often characterized by spontaneous differentiation and regression, and may be treated more than half the time with simple observation and supportive care. However, the majority of patients have stage 4 disease at diagnosis, with large primary tumors and disease that is widely metastatic, with the most frequent sites of involvement being bones and bone marrow, seen in 60–80% of cases. Lung metastases are very rare in neuroblastoma, either at diagnosis or relapse, in contrast to other pediatric solid tumors [9]. Overall survival by INSS stage is excellent for patients with localized disease, with more than 95% survival at 5 years for children with stage 1, 2A, and 2B with surgery as the primary treatment [10]. Children with INSS stage 3 disease also have a survival of 80–90%, although primary treatment includes chemotherapy as well as surgery and, in some cases, local radiation. This group is actually biologically heterogeneous, and patients with favorable biologic factors have nearly 100% survival, while those with unfavorable biology have only 50% survival [11]. Patients with INSS 4 are also biologically heterogeneous, with infants aged under 1 year at diagnosis achieving 70% survival, compared with 40% for those aged over 1 year at diagnosis. Furthermore, within the infant group, patients whose tumors bear a single copy of MYCN

Hematopoietic Cell Transplantation for Neuroblastoma Table 66.1 International Neuroblastoma Staging System Stage Definition 1

2A 2B

3

4 4S

Localized tumor with complete gross excision, with or without microscopic residual disease; representative ipsilateral lymph nodes negative for tumor microscopically Localized tumor with incomplete gross excision; representative ipsilateral lymph nodes negative for tumor microscopically Localized tumor with or without complete gross excision, with ipsilateral lymph nodes positive for tumor. Enlarged contralateral lymph nodes must be negative microscopically Unresectable unilateral tumor infiltrating across the midline, with or without regional lymph node involvement; or localized unilateral tumor with contralateral regional lymph node involvement; or midline tumor with bilateral extension by infiltration (unresectable) or by lymph node involvement Any primary tumor with dissemination to distant lymph nodes, bone, bone marrow, liver and other organs (except as defined for stage 4S) Localized primary tumor (as defined as stage 1, 2A or 2B), in patient <1 year, with dissemination limited to skin, liver and/or bone marrow (marrow involvement should be minimal with malignant cells <10% of total nucleated cells)

Reproduced with permission from [8].

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extensive amplification may be found in neuroblastomas in 50–60% of cases at chromosome 17q, and may portend a worse outcome [17,18]. The most common area of gain is at 17q22, and multiple genes have been suggested from this area as possibly responsible for the aggressive behavior [18]. Neuroblastomas may be diploid or hyperdiploid, and chromosomal content by measurement of DNA index has established that near-diploid DNA content is less favorable and associated with other adverse prognostic markers [19]. Allelic loss of chromosome 1p and 11q are common in neuroblastoma, and both predict an adverse outcome [20]. Whereas 1p deletions, seen in 30% of tumors, are closely associated with MYCN amplification, 11q losses, seen in 20% of tumors, usually occur in tumors without MYCN amplification, but are associated with older age and unfavorable pathologic features. Other genetic deletions are less common, but losses at chromosome 3p, 4p, 9p, 14q, and 19q have also been described, and some are associated with a poorer prognosis [21]. Aberrant expression of multiple genes, and in particular the family of nerve growth factor receptors, may also be prognostic. High levels of TrkA mRNA are present in tumors from patients with favorable-stage disease, while low-to-undetectable levels are observed in MYCN-amplified tumors. In contrast, coexpression of full-length NTRK2 (TrkB) and its ligand BDNF is highly associated with MYCN amplification and may represent an autocrine survival pathway [22,23]. Recent studies of risk assessment by gene expression arrays have validated the current risk classification and further refined possible treatment groups [24,25].

Risk classification and treatment approach oncogene have a greater than 90% event-free survival (EFS), compared with 10% for those with amplified MYCN tumors [12]. A new International Neuroblastoma Risk Group Classification schema is under development, taking into account the recently refined optimal age cut off of 18 months, stage, MYCN amplification, tumor differentiation, ploidy, and genetic aberrations of chromosome 11q. In order to use a clinical risk grouping that does not depend upon surgical approach, image-defined risk factors will be used to determine if the tumor is localized, localized but not safely resectable, or metastatic [13]. Patients will then be separated into 15 pretreatment groups ranging from low to intermediate to high and ultrahigh risk groups. This will facilitate comparisons and collaborations between international studies [14].

Tumor molecular biology The perinatal onset of neuroblastoma and the extreme heterogeneity in behavior, ranging from the spontaneous differentiation and regression of metastatic disease to the inexorable and rapid progression seen in other infants with metastatic disease, stimulated the cytogenetic characterization of tumor-derived cell lines. Early studies showed double minute chromosomes, homogeneously staining regions representing DNA amplification, and deletions of the short arm of chromosome 1. This demonstrated the importance of both genetic gains and losses in this tumor, consistent with theories of a role for oncogenesis and for tumor suppressor genes in the pathogenesis and behavior of neuroblastoma. Amplification of the MYCN oncogene on chromosome 2p24, found in 25% of neuroblastomas, is the most consistent unfavorable genetic change, independent of age, stage, and other biologic characteristics, and is an essential component of risk assessment, which can now be easily accomplished by fluorescent in situ hybridization on primary tissue or bone marrow tumor [15]. Targeted overexpression of MYCN in the transgenic mouse initiates neuroblastomas that mimic the human tumor, suggesting a role in pathogenesis [16]. Chromosomal gain without

Risk groups with suggested treatment assignment based on recent cooperative clinical trials have been adopted by the Children’s Oncology Group (COG), with similar guidelines followed by other cooperative groups internationally. Although a myriad of clinical, laboratory, and genetic characteristics have demonstrated prognostic import, current risk classification uses only a few of the most widely tested and established, readily available markers [3,15]. The risk assignment depends on age, INSS stage, MYCN gene copy number, histopathology, and, for infants, tumor cell DNA index [8,26–28]. The risk classification is shown in Table 66.2 as incorporated into current COG protocol assignment for trials opening in 2007. This reflects more recent data that advocate for an adjustment of the criteria of age at diagnosis and incorporate chromosome 11q aberrations into the reduction of therapy plan for intermediate-risk patients [20,29,30]. In the future, the new recommendations from the International Neuroblastoma Risk Groups, and eventually possible genomic DNA signatures that may predict relapse in low- and intermediate-risk patients, will be used for treatment assignment [20,25,29,30]. Low-risk tumors are managed with surgery alone, unless symptomatic cord compression or respiratory compromise necessitates a short course of chemotherapy. Patients in the low-risk group with stage 1 or 2 disease have an expected 4-year survival of over 95% with surgery alone [10], while infants with INSS grade 4S have over 90% survival with supportive care or a short course of chemotherapy [31]. The smaller intermediate-risk group is comprised of infants with more advanced disease (but no tumor MYCN amplification), favorable biology stage 3 or INSS stage 4S disease with unfavorable histology or a diploid DNA index. Patients in this group are expected to have an estimated survival of over 80% with standard doses of chemotherapy for 2–8 months and primary tumor resection. The high-risk group in neuroblastoma traditionally is comprised of patients with stage 4 disease who are over 1 year of age at diagnosis regardless of MYCN status, but also includes those with stage 3 disease either with tumor MYCN amplification or over 1 year with unfavorable

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Chapter 66

Table 66.2 Current Children’s Oncology Group neuroblastoma risk classification

Stage

Age

MYCN

Ploidy

Shimada histopathologic risk classification

1

Any*

Any

Any

Any

2A/2B 2A/2B

Any Any

Non-amplified Non-amplified

Any Any

Any Any

Resection ≥50% Resection <50%

2A/2B 2A/2B

Any Any

Non-amplified Amplified

Any Any

Any Any

Biopsy only Any degree of resection

3 3 3 3

<547 days ≥547 days Any ≥547 days

Non-amplified Non-amplified Amplified Non-amplified

Any Any Any Any

Any Fav Any Unfav

Intermediate Intermediate High High

4 4 4 4 4 4 4

<365 days <365 days 365 to <547 days 365 to <547 days 365 to <547 days 365 to <547 days ≥547 days

Amplified Non-amplified Amplified Any Any Non-amplified Any

Any Any Any DI = 1 Any DI > 1 Any

Any Any Any Any Unfav Fav Any

High Intermediate High High High Intermediate High

4S 4S 4S 4S 4S 4S

<365 days <365 days <365 days <365 days <365 days <365 days

Non-amplified Non-amplified Missing Non-amplified Non-amplified Amplified

DI > 1 DI = 1 Missing Any Any Any

Fav Any Missing Any Unfav Any

Other

Risk group Low

Asymptomatic Asymptomatic or symptomatic Too sick for biopsy Symptomatic Asymptomatic or symptomatic Asymptomatic or symptomatic

Low Intermediate† Intermediate High

Low Intermediate Intermediate Intermediate Intermediate High

DI, DNA index; Fav, favorable histology; Unfav, unfavorable histology. * The term “Any” does not imply that the data are not important. Even though certain variables may not influence current risk assignment within particular patient subsets, these data are being used to assign treatment duration (intermediate-risk patients) and/or to attempt refinement of the risk classification system. Every attempt should be made to obtain all biological data whenever possible. † Treatment of intermediate-risk group patients is intensified by loss of heterozygosity at loci 1p and 11q, and by unfavorable pathologic classification or DNA index.

histopathology, stage 2 disease patients over 1 year of age with MYCN amplification, and stage 3, 4, and 4S infants with MYCN amplification. Recent analysis of the 3666 patients enrolled in the POG and CCG studies from 1986 to 2001 demonstrates that the contribution of age to risk stratification is a continuous variable. Using a multivariate Cox model to adjust for MYCN and stage, the optimal age cut-off that would predict outcome was found to lie between 15 and 20 months of age. If the cut-off were raised above the current 365 days of age, up to 11% of high-risk patients (excluding those with MYCN amplification) would shift to a lower risk classification, saving them more intensive treatment without compromising EFS [29]. Additional evidence is provided by a report on the outcome of the CCG study CCG-3891 with respect to age. Here, the EFS for MYCN-non-amplified patients aged 12 to 18 months old at diagnosis (6 year EFS 87 ± 8.8%) was found to be significantly greater than that for patients 18–24 months of age (6 year EFS 36 ± 14.5%) despite receiving similar therapy [20,25,29,30]. With increasingly aggressive combined-modality treatments, and increased remission rates and durations, the long-term survival for INSS stage 4 disease in children who are more than 1 year of age at diagnosis has only recently risen above 15% [32] to a 3-year EFS of 30–40%. Despite advances, much still needs to be done to improve outcome (Fig. 66.1) [2]. The rest of this chapter focuses on the treatment of these

patients, which has become increasingly dependent on dose-intensive high-dose conditioning regimens.

Treatment of high-risk neuroblastoma Therapy for high-risk neuroblastoma is currently divided in three phases: intensive induction treatment, marrow ablative therapy, and management of minimal residual disease. The goal of induction therapy is to achieve maximum reduction of the tumor burden, including reduction of bone marrow tumor (in vivo purging), within a timeframe that will minimize the risk of developing resistant tumor clones and clinical progression. Subsequently, very high-dose marrow ablative therapy may be used to try to overcome residual and potentially resistant tumor, followed by hematopoietic cell transplantation (HCT). The relapse rate of greater than 40% even after such treatment [33] has led to the approach of using tumor-targeted therapies following high-dose conditioning to try to eliminate microscopic resistant clones (minimal residual disease).

Induction therapy The introduction of platinum drugs into the combination chemotherapy may be largely responsible for improving the remission induction rate

Hematopoietic Cell Transplantation for Neuroblastoma

ators and a platinum derivative, along with surgery to bulky tumor sites. While current and future regimens seek to improve outcome, often at the cost of more intensive therapy, the risk of second malignancies is steadily increasing. Because induction therapy contains drugs with potential late toxicity as well as the usual acute toxicities, new concerns regarding quality of life and organ function (cardiac, renal, and hearing) have arisen as some children survive many years post transplantation. These toxicities are further potentiated by the common use of more platinum and alkylating agents during the myeloablative consolidation phase (see “Acute and late complications of HCT in neuroblastoma” below).

1

Probability

0.8

0.6

0.4 1986–1995 n = 675 0.2

1978–1985 n = 507

0 0

1

2

3

4

5

6

7

8

973

9

10

Years

Fig. 66.1 Improving survival of children aged over 1 year with stage 4 neuroblastoma [32].

in recent years, but other important components of the induction regimen include combinations with other active drugs, such as cyclophosphamide, doxorubicin, etoposide, vincristine, and ifosfamide. Induction regimens used in recent large cooperative studies have shown overall response rates, including complete remission and partial remission (CR and PR), ranging from about 60% to 90% at the end of 5–6 months of treatment [2,34,35]. Most regimens also include surgery to residual disease, although the overall impact of complete resection on survival in stage 4 disease is still uncertain. Some studies have also investigated newer single agents in newly diagnosed neuroblastoma, using the “up-front phase 2 window.” Following two courses of single-agent therapy prior to induction treatment, response rates (CR + PR) of over 30% were seen for ifosfamide, carboplatin, and iproplatin [34] and topotecan [36]. Preliminary data from pilot studies incorporating topotecan into induction regimens demonstrate that it is well tolerated, paving the way for future phase III studies. Two agents that were less effective in this setting were epirubicin [34] and taxol [36]. There was no evidence that the window design adversely affected patients’ outcome when compared with patients treated with similar induction without the phase 2 window. Single-institutional studies have also suggested that further dose intensifications of alkylating agents and cisplatin have improved CR and very good PR rates up to 85% [37,38]. The induction regimen consisted of cyclophosphamide, doxorubicin, and vincristine, alternating with cisplatin and etoposide. Response by iodine-131 (131I) metaiodobenzylguanidine (MIBG) scintigraphy revealed 21 of 24 patients with at least a very good partial response. These excellent results, however, could not be reproduced by a UK pilot study of 47 patients [39] nor by the French Society of Pediatric Oncology, who have built their NB 97 protocol upon this regimen [40]. From 1998 to 1999, only 17 of 47 patients enrolled showed CR at the end of seven courses of induction, not a significantly better outcome than that for the their previous shorter, less-intense induction regimen. This more intensive regimen did produce greater bone marrow remissions (82%) than the prior regimens. A large cooperative COG trial A3973 using the same regimen in 489 patients also resulted in a CR/very good PR rate of only 52%, with a PR of 26% for overall response rate being similar to that of previous protocols [41]. Future attempts to improve the induction response may include the use of biologic or targeted agents, such as tyrosine kinase inhibitors, 131IMIBG therapy [42] or anti-GD2 monoclonal antibodies [43]. Current standard induction chemotherapy usually includes five to seven cycles of combination chemotherapy, including high-dose alkyl-

Local control Recurrence in the local or regional area of primary disease is a component of relapse in a large proportion of children with high-risk neuroblastoma, in rates ranging from 20% to 80% in reports that often include local radiotherapy and myeloablative therapy [2,33,44,45]. There are both single-arm studies and one randomized study that demonstrate the benefit of local control measures for children with advanced but nonmetastatic neuroblastoma [11,46–48], but the impact of resection in stage 4 disease has been mixed [49–51]. It is possible that problems with control of metastatic disease have obscured the potential value of local resection. The role of radiation therapy is similarly not well established as to the best timing and optimal dose. The small patient numbers and variable rates of resection have made a randomized study difficult to perform. Only one such study has been carried out that showed better local control with the addition of 24–30 Gy of radiation in patients with stage 3 disease, but this took 8 years to complete, used suboptimal chemotherapy, and was begun before the refinement of biologic staging had been implemented [52]. More recent pilot studies in stage 4 disease utilizing myeloablative consolidation with radiotherapy administered to the primary tumor bed either pre or post high-dose conditioning suggest a lower local recurrence rate using radiotherapy in retrospective analysis, but the variability in extent of resection makes these hard to interpret [45,53]. Pilot studies have also been reported using higher focal radiation via intraoperative radiotherapy in order to spare normal organs [54]. One retrospective study has recently lent support to the therapeutic value of intraoperative radiotherapy while avoiding the associated toxicities of external beam radiotherapy [55]. However, this benefit must be balanced by the occasional occurrence of hypertension and vascular stenosis. While the particular modality may be debated, the current standard therapy of high-risk neuroblastoma continues to include radiotherapy to the primary tumor bed and residual metastatic sites [56,57].

Evolution of high-dose conditioning The observed linear-log relationship between drug dose and tumor cell cytotoxicity for alkylating agents suggested that if drug dose could be increased without increasing toxicity, a multiple log increment in tumor cell killing could be achieved. The demonstration that hematopoiesis could be restored with autologous hematopoietic cells allowed the use of much higher doses of chemotherapy with autologous bone marrow support for treatment of solid tumors. The demonstration that bone marrow tumor cells could be eliminated using immunomagnetic purging [58] gave credence to the use of autologous marrow support in neuroblastoma, a tumor which is metastatic to bone marrow in 80% of children with high-risk stage 4 disease [9]. Early pilot studies showed that responses were seen in children with resistant neuroblastoma after highdose chemoradiotherapy and bone marrow reinfusion [59]. Comparison of long-term survival for children over 1 year of age with stage 4 neu-

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Chapter 66

roblastoma in the premyeloablative era, and after 1985, a time when high-dose therapy with HCT became the routine for many of these patients, showed a significant difference in outcome (Fig. 66.1) [32]. Subsequent single-arm studies in the United States and Europe verified an apparent improvement in outcome for purged or nonpurged autologous bone marrow transplantation (BMT) compared retrospectively with results for chemotherapy [33,60–64]. The single-arm studies containing more than 20 patients that have been reported are summarized in Table 66.3. The 3-year EFS in these studies varied from 24% to 50%. The comparison between studies in this table is fraught with pitfalls, because EFS is sometimes estimated from time of transplant, which differs from 4 to 10 months from diagnosis, and in a few studies from time of diagnosis. The patient groups differ in whether they were comprised of only stage 4 patients over 1 year at diagnosis or also included other high-risk patients. Some studies included all patients, while others only included those who achieved CR or PR. In cases where the 3-year EFS was not stated in the text, it was estimated from the Kaplan–Meier curves if available; if not, another time point was reported. Finally, although we tried to avoid repetition of data, a few studies may include patients already reported in other studies, such as the overall European Group for Blood and Marrow Transplantation (EBMT) report [65].

Nonrandomized comparisons of HCT to chemotherapy Several cooperative pediatric groups have attempted statistical nonrandomized comparisons of outcome for groups of patients treated with either conventional doses of chemotherapy or high-dose conditioning chemotherapy, total body irradiation (TBI), and purged autologous BMT, with differing conclusions [66–69]. Philip et al. [67] compared the LMCE1 protocol (1983–88) with the previous Lyon cooperative study, LMCE (1978–83), and showed a difference in 2-year progressionfree survival of 39% for patients treated with myeloablative therapy and autologous BMT versus 12% for standard chemotherapy. In order to remove the bias introduced by comparing two different time periods, other reports compared concomitant groups of patients treated with either autologous BMT or chemotherapy by investigator or institutional choice. On the basis of two POG studies, one a surgery plus conventional chemotherapy study (POG 8441) and the other an elective autologous BMT pilot protocol (POG 8340), there was no significant prognostic benefit of switching in remission from the chemotherapy protocol to the transplant protocol (p = 0.91). The analysis was based on 116 patients achieving a CR or PR, 32 of whom received transplants on the pilot protocol [68].

Table 66.3 Event-free survival (EFS) for high-risk neuroblastoma in first remission using myeloablative therapy and autologous hematopoietic cell transplantation for studies involving more than 20 patients. Unless otherwise stated, EFS is measured from time of transplantation Reference

Date

Myeloablative regimen

[156] [93] [69,115]

1987 1991 1991, 1996

[67] [60] [157] [158]

1991 1991 1991 1995

[92] [66] [148] [2] [45] [76] [159] [79]

1996 1997 1998 1999 1999 1999 2001 2002

[71] [73] [81]

2005 2005 2006

BCNU, teniposide, melphalan† Melphalan, TBI Cisplatin, teniposide, doxorubicin, melphalan, TBI Cisplatin, etoposide, melphalan, TBI Carboplatin, etoposide melphalan, TBI Vincristine, melphalan, TBI Vincristine, melphalan, TBI Cisplatin, BCNU, melphalan (or thiotepa), etoposide Etoposide, melphalan or cisplatin, etoposide, THP-adriamycin, melphalan, with (n = 6) or without TBI Teniposide/etoposide, thiotepa, TBI Melphalan ± etoposide, vincristine, cisplatin, BCNU Cyclophosphamide, thiotepa Carboplatin, etoposide, melphalan, TBI Carboplatin, etoposide, melphalan, local radiation Busulfan, melphalan Cyclophosphamide, carboplatin Triple tandem: Carboplatin + etoposide Carboplatin + etoposide Thiotepa + etoposide‡ Melphalan Melphalan, carboplatin§ Double tandem: etoposide + carboplatin + cyclophosphamide Melphalan + TBI¶

Toxic deaths

3-Year EFS (%)

33 54 45 54 48 62 34 25 31

7 7 5 4 13 1 6 3

49 (2 years)* 32 42 50 41 30* 29 40 50

27 39 51 129 77 116 49 25

4 7 1 12 4 7 4 1

41 35* 48 34* 62 47* 33* 57*

32 75 97

2 5 5

38 (5 years) 56* 55*

n

TBI, total body irradiation. * Denotes EFS is calculated from time of diagnosis. All others are from time of transplantation. † One course only n = 15, two courses n = 18. ‡ Single transplant n = 2, double transplant n = 2, triple transplant n = 17. § Variations in myeloablative therapy: melphalan alone n = 3, Melphalan + cyclophosphamide n = 3, melphalan + busulfan n = 9. ¶ Single transplant n = 7, double transplant n = 82. Adapted with permission from [155].

Hematopoietic Cell Transplantation for Neuroblastoma

The CCG examined the outcome of stage 4 patients over 1 year of age treated with identical induction chemotherapy on a CCG pilot protocol, who then either continued on the same chemotherapy (n = 73) for 1 year (CCG-321P2) or proceeded to trial CCG-321P3, with myeloablative chemotherapy, TBI, and purged autologous BMT (n = 94) [69]. The decision to use autologous BMT was nonrandom and depended on parental, investigator, and institutional choice. The analysis was performed using Cox regression for censored failure–time data, treating time to autologous BMT as a time-varying covariate, and also by Kaplan–Meier analysis comparing EFS from time of autologous BMT with EFS from 8 months after diagnosis for chemotherapy patients. The advantage for autologous BMT over chemotherapy was significant for the group as a whole, with a respective 3-year EFS of 40% versus 19% [69], different from the finding of Shuster et al. [68]. The advantage for autologous BMT was greatest for certain very high-risk subgroups, including those over 2 years of age at diagnosis, those with bone or bone marrow metastases, those with MYCN gene amplification, and those who had only a partial rather than complete response to the first four to six cycles of induction chemotherapy [69]. A smaller study by the German cooperative group evaluated 39 patients undergoing megatherapy and HCT with a variety of conditioning regimens and either allogeneic or autologous bone marrow, all with a melphalan “backbone,” compared with 49 patients receiving continued chemotherapy by investigator choice. All were patients who achieved CR or PR. EFS was significantly better in the transplanted patients compared with the chemotherapy group by log-rank analysis (p = 0.005), although the curves nearly converge by 6 years [66]. These studies, and the overall improvement in survival for high-risk neuroblastoma seen in the era after the introduction of HCT, led to the randomized comparisons discussed below.

Randomized comparison of autologous BMT to standard-dose chemotherapy The European Neuroblastoma Study Group (ENSG) performed the first randomized study comparing high-dose conditioning treatment with conventional chemotherapy [70]. This study, open from 1983 to 1985, was actually a comparison of high-dose melphalan compared with no further therapy after induction, and showed a significant advantage in progression-free survival for those patients with stage 4 disease aged 1 year of age at diagnosis who were undergoing high-dose therapy (5-year EFS 33% versus 17%, p = 0.01; 5-year overall survival 46% versus 21%, p = 0.03). However, there are several problems in the interpretation of these results. First, randomization was performed only for those patients achieving CR or good PR, and it was not performed until approximately 10 months from diagnosis, after 10 cycles of chemotherapy. Of 84 eligible stage 4 patients, only 50 (59%) were randomized, for a variety of reasons ranging from toxic death to parental or physician preference. Thus, the applicability of these results is limited because the group as a whole excluded the highest-risk patients, including the 15% who were expected to progress early in the course of induction and those who showed a lesser response at the end of induction (perhaps another 20%). Also, bias may have been introduced by the high proportion of nonrandomized patients. Long-term follow up of the ENSG-1 was published in 2005 for a median of 14.3 years from randomization [71]. The median time to relapse for the melphalan versus “no melphalan” group was unaltered at 18 months compared with 3 months. Most relapses occurred within 2 years of randomization, and most deaths occurred within 5 years. There were four, late deaths beyond 60 months occurring evenly in each group. The authors also analyzed survival by treatment arm and included those not randomized when eligible in an attempt to address the suspi-

975

cion of selection bias. Despite the limitations of the study the survival improvement achieved at 5 years appears sustained at 14 years (Fig. 66.2). At the time of publication of the initial promising European results, the CCG launched the first large randomized study in the United States comparing high-dose chemoradiotherapy with purged autologous BMT against a new intensive chemotherapy intensification [2]. This study differed from that of the ENSG by performing the randomization much earlier in the course, after only two cycles of chemotherapy, at a time when 95% of the patients were still eligible. The study was also much larger, with 190 patients in each randomized group. However, it still had the problem of refusal of randomization, with a randomization rate of 70%. As autologous BMT was considered to be the experimental arm, patients who refused randomization were assigned to the chemotherapy arm but analyzed separately. The results clearly showed a significant improvement in EFS for the patients randomly assigned to autologous BMT, both by an intent-to-treat analysis and also by treatment received (Fig. 66.2). As in the previous CCG nonrandomized comparison, the highest-risk patients, those with MYCN amplified tumors or those aged over 2 years at diagnosis, had the most significant benefit. In addition, there was no significant difference in toxic deaths for patients randomized to the two arms, and the hospital days were identical, helping to validate the cost-effectiveness of this treatment. Recent analysis of continued follow-up of a median of 7.7 years on this study has shown a significant advantage in 5-year survival for the patients randomized to the autologous BMT arm (p = 0.004) [72]. In contrast to the reported CCG regimen, ongoing studies both in Europe and the United States have sought to eliminate TBI and further increase chemotherapy dose intensity and the use of local irradiation instead [45]. The most recent and only other randomized study to compare myeloablative therapy against continued chemotherapy in fact did not include scheduled radiotherapy at all [73]. The Society of German Pediatric Oncology and Hematology enrolled 339 patients from 1997 to 2002. Patients (n = 295) were randomized to either “megatherapy” followed by autologous stem cell transplant or maintenance chemotherapy. Patients were initially treated with 5–7 months of cisplatin, etoposide, and vindesine alternating with vincristine, ifosfamide, doxorubicin, and dacarbazine. The myeloablative regimen consisted of melphalan, etoposide, and carboplatin. Maintenance chemotherapy was only oral cyclophosphamide. Immunotherapy followed with either chimeric anti-GD2 monoclonal antibody given for 1 year or oral 13-cisretinoic acid for 6 months. In accordance with the CCG study, EFS was significantly increased in the megatherapy group. Overall survival failed to show significance when analyzed with intention-to-treat. Several potential confounding variables exist in this study. First, randomization was allowed to occur anywhere from 7 to 224 days after diagnosis. Variations in treatment were also tolerated, including a preferential treatment of patients with 131 I-MIBG therapy prior to transplant. In addition, surgical timing and the timing of hematopoietic cell collection was left unspecified and varied substantially. Some patients also had variations in the actual regimen, with some receiving 131I-MIBG and others receiving external beam radiotherapy. Finally, this study was plagued with high crossover rates of 23% for those assigned megatherapy and 30% for those assigned maintenance chemotherapy (Fig. 66.2). Despite the limitations of these studies, improvement of outcome is consistent in all three randomized studies for those undergoing highdose conditioning regimens compared with traditional chemotherapy. Current investigations are now pursuing the benefit of repetitive highdose regimens with hematopoietic cell rescue or of novel conditioning regimens.

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Chapter 66 100 Event-free survival by treatment arm (n = 65)

1.0

80 70

0.8 % EFS

Probability of Event-free Survival

90

0.6

60 50 40

0.4

HDM

30

Bone marrow transplantation

NFT 20

0.2 Continuation chemotherapy

10

p = 0.08, Log-rank test

0

0.0 0

1

3

2

4

5

6

7

0

8

2

4

6

8

10

12

14

16

18

20

Years since randomization

Years after first randomization (a)

(b) Event-free survival

100 Intention to treat

80

Survival (%)

p = 0.0221

60 Megatherapy 40

Maintenance therapy 20

0 0 (c)

1

2

3

4

5

6

Time from diagnosis (years)

High-dose conditioning regimens The very first high-dose regimen used in the treatment of neuroblastoma was single-agent, high-dose melphalan. This progressed to various combinations of other agents, including cisplatin, etoposide, and doxorubicin, melphalan with busulfan, and melphalan with carboplatin and etoposide. Other centers used a thiotepa base, coupled with cyclophosphamide or etoposide, or busulfan combined with cyclophosphamide. More recently, attempts have been made to incorporate topotecan into the conditioning regimen in combination with thiotepa and etoposide [74]. Few studies have tried to compare two high-dose regimens in a randomized fashion. However, the retrospective analysis by the EBMT failed to show any difference in EFS using different high-dose regimens [61,65,75]. On the other hand, an analysis from the Institut GustaveRoussy showed that patients treated with busulfan and melphalan appeared to have better EFS than those on other conditioning regimens at a single institution [76]. These data may depend on the chronology of the protocols as well as the fact that the busulfan regimen was com-

Fig. 66.2 Outcomes of randomized trials comparing hematopoietic cell transplantation to no further therapy or chemotherapy for high-risk neuroblastoma. (a) High-dose regimen: carboplatin, etoposide, melphalan, total body irradiation. Transplant group: n = 189 as randomized with a 3-year event-free survival (EFS) of 34% (±4% standard error). Continued chemotherapy group: n = 190 as randomized with a 3-year EFS of 22% (±4%); p = 0.034. (Reproduced from [2], with permission.) (b) High-dose regimen: high-dose melphalan. Transplant group: n = 32 with a 5-year EFS of 38% (95% confidence interval [CI] 21–54%). No melphalan group: n = 33 with 5year EFS of 27% (95% CI 12–42%); p = 0.08, (Reproduced from [71], with permission.) (c) High-dose regimen: melphalan, etoposide, and carboplatin. Transplant group: n = 118 as randomized with a 3-year EFS of 47% (95% CI 38–55). Maintenance chemotherapy group: n = 107 as randomized with a 3year EFS of 31% (95% CI 23–39); p = 0.0221. (Reproduced from [73], with permission.)

pared with a combination of a variety of other regimens. The ENSG is currently conducting a large, multinational cooperative randomized study, comparing the COG regimen of melphalan, etoposide, and carboplatin with the busulfan and melphalan regimen.

Tandem transplants Other investigators are pursuing the strategy of trying to benefit from the further increase in dose intensity obtainable with repetitive high-dose therapies with hematopoietic cell rescue, but a randomized study is required to verify whether this approach truly improves EFS or survival [77–80]. The earlier French study of this approach included patients both in relapse and in partial response, and utilized immunomagnetic purging of autologous bone marrow and conditioning regimens of VM26, carmustine (BCNU), and cisplatin (or carboplatin), followed by a conditioning regimen of vincristine, melphalan, and TBI, with a high toxic death rate of eight of 33 patients. Only survival figures were given, with

Hematopoietic Cell Transplantation for Neuroblastoma

36% survival for the group treated in first PR [80]. A second study was for patients with a less than complete or very good partial response to induction, and utilized first high-dose therapy with BCNU, VM26, and carboplatin in 25 patients, and then a final consolidation with vincristine, melphalan, and TBI. There were four toxic deaths and a rather long time to neutrophil engraftment of 31 days. Only 20 patients were able to receive both autografts. Progression-free survival at 3 years was 28% [77]. The more recent pilot studies in the United States utilized peripheral blood hematopoietic cells, either CD34-selected or unpurged. Grupp et al. [78] reported 39 evaluable patients who completed 70 cycles of high-dose therapy with hematopoietic cell rescue. The first high-dose therapy employed a regimen of etoposide, carboplatin, and cyclophosphamide, while the second included melphalan and TBI. An average of 7.2 × 106 CD34+ cells/kg were available to support each cycle. Engraftment was rapid with a median time to neutrophil engraftment of 11 days. Four patients who completed the first high-dose therapy course did not complete the second, and there were three toxic deaths. With a median follow-up of 22 months, the 3-year EFS rate from diagnosis was 58%, an apparent improvement from previously reported studies. However, the follow-up is still short and the curve does not appear to have reached a plateau. The pilot study reported by Kletzel et al. [79] has taken this strategy one step further, with a small group of patients undergoing three successive high-dose therapies followed by HCT. Preparative regimens for the first two high-dose courses with HCT consisted of carboplatin and etoposide, with a final third high-dose course of thiotepa and cyclophosphamide. Of the initial 26 patients registered at diagnosis, 22 survived induction therapy without progression, with successful hematopoietic cell harvest, and were able to proceed to high-dose therapy; of these, 19 were able to proceed to cycle 2, and only 17 to cycle 3, because of intervening toxicity. Three children had failure to engraft after the third myeloablative therapy, but the progression-free survival from diagnosis at 3 years was 57%. All patients on both of these tandem transplant studies received therapy for minimal residual disease with 13-cis-retinoic acid and, in some cases, anti-GD2 monoclonal antibody. More recently, George et al. reported an updated compilation of the limited institution experience with tandem transplantation initially reported by Grupp et al. [81]. Ninety-seven high-risk patients were treated with five cycles of chemotherapy followed by two courses of myeloablative therapy and hematopoietic cell rescue. Induction consisted of alternating courses of cisplatin and etoposide with doxorubicin, vincristine, and cyclophosphamide. Progression free survival at 3, 5, and 7 years for all patients was 55% (95% confidence interval [CI] 44–64%), 47% (95% CI 36–56%), and 45% (95% CI 34–55%) respectively. Overall survival, however, fell slightly from 3, 5, and 7 years from transplantation at 72% (95% CI 62–80%), 60% (95% CI 48–69%), and 53% (95% CI 40–64%), respectively. While the majority (93%) underwent both courses of ablation, seven patients received only one course. Data for patients who received at least one transplant and those who completed both were provided and appeared similar. The design of the study does not determine if two myeloablative courses are better than one. The outcome appears promising, but a large cooperative randomized study will be required to answer this question. The COG has designed such a study to compare the outcomes of a single versus double high-dose regimens, which opened in 2007. Comparison of the multiple transplant outcomes from different studies is difficult, because of differing time to transplant, different hematopoietic cell sources, and differing patient populations. The earlier studies included patients with more resistant disease, either by response status or because of previous relapse. The two most recent US reports use an

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intent-to-treat analysis of EFS from diagnosis for all high-risk patients, which is harder to compare with studies that evaluate EFS from time of transplantation. However, these studies do show that repetitive transplants are feasible in the majority of patients who would be eligible for single myeloablative therapy, and suggest that EFS is not likely to be lower than in the previous studies. The possible added immunosuppressive effects, as well as the additional toxicity, hospitalization, and cost, will have to be evaluated and eventually justified by a prospective randomized comparison.

Targeted radionuclides as part of high-dose therapy The use of targeted radionuclides allows the delivery of very large radiation doses to the tumor with less radiation to normal organs. Targeted radioisotope therapy using anti-GD2 antibody or MIBG for delivery of radiation in the form of 131I has been tested extensively in clinical trials in relapsed neuroblastoma. Cheung et al. [82] have reported on the use of 131I-3F8 (anti-GD2 antibody) for treatment of refractory neuroblastoma with documented responses. They have also treated newly diagnosed patients using 131I-3F8 in high doses followed by bone marrow rescue and further treatment with cold antibody after transplant [37]. 131 I-MIBG has been widely shown in European and US studies to elicit about a 30–40% response rate for refractory neuroblastoma [83– 85]. It has also elicited responses as initial therapy for patients with regional disease [86]. A phase I dose escalation trial of 131I-MIBG reported by Matthay et al. [85] determined the maximal marrow nonablative dose (444 MBq/kg), and the maximal practical high dose with hematopoietic cell rescue (666 MBq/kg). In 30 patients, there was a 37% response rate and no significant toxicity other than hematologic. A follow-up phase II study of 164 patients who failed to achieve a partial or complete response with prior therapy or who relapsed after high-dose therapy has since been completed [87]. Use of 18 mCi/kg (666 MBq/kg) of 131I-MIBG supported by autologous HCT produced an overall response of 37% in this heavily pretreated cohort. In fact, the majority received a median of three prior regimens, and 78% had received prior high-dose therapy. A retrospective analysis of 28 patients treated with multiple courses of 131I-MIBG was attempted to discern if the benefits of 131I-MIBG therapy were additive [88]. The results suggest that it is feasible with an overall response rate of 39%, comparable to results obtained with a single infusion. The frequency of administration was limited by hematologic toxicity, particularly thrombocytopenia that did not always respond to hematopoietic cell infusion, likely because of disease involvement of the bone marrow. Despite this, 25% of the patients did show successive response with additional infusions, so a study of two consecutive doses of 131I-MIBG therapy administered in rapid succession followed by HCT has recently closed. Conducted by the New Approaches to Neuroblastoma Therapy (NANT) consortium, the report showed minimal toxicity and thus the ability to intensify 131I-MIBG dosing. The results in these relapsed patients argue strongly for the incorporation of 131I-MIBG therapy into regimens that include chemotherapeutic agents. A NANT phase I study combining irinotecan, vincristine, and escalating doses of 131I-MIBG is currently underway. Given the demonstration that haematopoiesis can be successfully restored after high-dose 131 I-MIBG therapy, the combination of 131I-MIBG therapy with highdose chemotherapy has been tested in several pilot studies, providing evidence of feasibility and engraftment with this approach. Subsequently, a phase I study incorporated escalating doses of 131I-MIBG followed by carboplatin, etoposide, and melphalan with hematopoietic stem cell infusions [89]. The combination appeared feasible and effective at a dose of 12 mCi/kg 131I-MIBG, and chemotherapy doses minimally decreased from those feasible without the MIBG. Dose adjustments

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were required for patients with decreased renal function, associated with an increased risk of sinusoidal obstructive syndrome (SOS). A phase II study is now underway in NANT and a pilot study for newly diagnosed patients in the COG.

Allogeneic transplantation Bone marrow involvement is present in over 80% of patients at diagnosis, and residual tumor cells can still be detected in bone marrow samples by sensitive immunodetection methods even after several cycles of induction therapy. For this reason, allogeneic HCT has been proposed as an alternative to autologous HCT. There has also been hope that the allogeneic cells will provide a further “graft-versus-neuroblastoma” effect, although the rationale for this concept is weak because neuroblastoma cells express little class I human leukocyte antigen (HLA). To date, lack of evidence of any immunologic benefit, coupled with the problems of frequent lack of an HLA-compatible sibling donor and the significantly higher toxic death rates, have discouraged the extensive use of allogeneic transplantation. Most reports consist of fewer than 10 patients included in large groups of autologous transplants [59,90–93]. Nonrelapse death rates were generally higher than those reported for autologous transplants, being in the range of 15–30%. Two reports were found that directly compared allogeneic and autologous transplantation in neuroblastoma. The first was the CCG study from Matthay et al. [94] comparing two concomitant groups of patients in a pilot study, receiving the same induction and conditioning regimen, including 36 patients receiving an autologous purged BMT and 20 similar patients with HLA-compatible sibling donors who received allogeneic BMT. There was no significant difference in relapse rate and an apparently higher toxicity in the allogeneic group. There were four of 20 deaths from causes other than relapse in the allogeneic group, compared with three of 36 in the autologous group (nonsignificant); the estimated progression-free survival was 25% for the allogeneic group versus 49% for the autologous group (p = 0.051). Ladenstein et al. [95] performed a case-control study using the EBMT Solid Tumor Registry to investigate the potential advantage of allogeneic BMT in high-risk neuroblastoma patients without prior disease progression. Seventeen allogeneic and 34 autologous BMT cases were matched based on a number of prognostic factors including age, sex, prior treatment duration, pregraft response status and bone and bone marrow involvement before BMT. The progression-free survival was not significantly different: 35% for autologous and 41% for allogeneic BMT at 2 years, respectively. Nine of the allogeneic BMT patients had developed graft-versus-host disease: seven with grade 1–2 and only two with grade 4. The median donor age was very young at 74 months (range 20–240 months), which might explain the low graft-versus-host disease rate. Currently, autologous HCT continues to be the preferred method of support for high-dose therapy, while allogeneic HCT is reserved for specific clinical trials testing immunomodulatory effects.

Hematopoietic cell source and purging Bone marrow involvement by neuroblastoma is extremely common in children with metastatic disease, being present by light microscopy in 60–80% of children with INSS stage 4 disease at diagnosis [9]. Detection of tumor by immunocytology using a mixture of monoclonal antibodies reactive at the cell surface has recently been shown to reliably detect tumor with a sensitivity that may vary from 1 : 104 to 1 : 106 nucleated bone marrow cells, depending on the method used [58,96–103]. Evaluation of the efficacy of in vivo purging of tumor cells in bone marrow or blood may be a very important component of response evaluation. Assessment of tumor content may also be critical for the evalu-

ation of hematopoietic cell products, because the use of high-dose therapy followed by HCT has been shown to be beneficial to EFS. Furthermore, because bone marrow or peripheral blood hematopoietic cells are necessary to reconstitute patients after high-dose tumor therapy, an induction regimen capable of efficient tumor cell reduction in bone marrow may be important. The ability of reinfused occult tumor cells to contribute to disease relapse has been demonstrated by the report that, after infusion of unpurged autologous bone marrow marked with a transduced neomycinresistance gene, tumor cells in the recurrent neuroblastoma in all three cases showed the genetic marker [104]. The occasional reports of miliary lung relapse after autologous HCT, the site one would expect to be involved after intravenous infusion of tumor cells, supports the importance of tumor-free hematopoietic cell grafts [105]. Circulating tumor cells can also be detected in the blood of up to 50% of children with INSS stage 4 neuroblastoma at diagnosis [91,99]. Furthermore, immunocytology has demonstrated that although a marked reduction occurs in both bone marrow tumor and circulating tumor cells, contamination of bone marrow can be detected in 25% of bone marrow samples at the end of 3 months of induction chemotherapy and in 7% of blood samples [103]. The efficacy of in vivo purging has been shown to correlate with EFS, such that patients with more than 0.1% tumor in bone marrow at the end of induction have a very poor outcome. Measurement of residual tumor cells in both blood and bone marrow by immunocytology and by the possibly more sensitive technique of reverse transcription-polymerase chain reaction (RT-PCR) may prove a useful surrogate marker of response and also predict impending relapse [103,106–108]. Various methods tested for ex vivo tumor cell removal from bone marrow in neuroblastoma patients include physical methods (sedimentation and filtration) [109], chemical purging with 6-hydroxydopamine [103,104], desferal [110] or mafosfamide [111], or immunologic methods with direct antibody plus complement [112,113] or immunomagnetic beads [58,114]. The most widely tested and validated method capable of 4–6 logs of tumor cell removal and no impairment of engraftment is immunomagnetic purging. The methodology was developed in the 1980s [58,114] and then tested in pilot studies shortly thereafter, demonstrating good engraftment [33,64,115]. This technique has been utilized in multi-institutional cooperative studies in Europe and the United States [2,116]. The large CCG study showed that bone marrow can be successfully harvested, shipped at room temperature overnight, purged using sedimentation, filtration, and immunomagnetic bead separation, and then cryopreserved without injury and with successful tumor cell removal and engraftment. More recent studies have shown the feasibility of leukapheresis for peripheral blood hematopoietic cell harvest in young children, and more rapid engraftment using this hematopoietic cell source rather than bone marrow. There is an attraction to using peripheral blood hematopoietic cells for HCT for solid tumors that infiltrate bone marrow, because one would expect fewer contaminating tumor cells in peripheral blood than bone marrow. None the less, up to 50% of neuroblastoma patients have circulating tumor cells at diagnosis [103], and pilot testing of peripheral blood hematopoietic cell collections have, using RT-PCR, demonstrated tumor cell contamination in patients who had been treated with multiple cycles of chemotherapy [96,108,117,118]. Although it is not clear what level of tumor contamination in infused grafts will cause a regrowth of tumor cells, the study from the UK suggests that low levels of tumor in peripheral blood detected by RT-PCR do predict relapse [106]. Therefore, purging of peripheral blood hematopoietic cells is being studied, either by positive selection of CD34+ cells or by immunomagnetic purging using a similar method to that in bone marrow. Several

Hematopoietic Cell Transplantation for Neuroblastoma

studies have documented the feasibility and good engraftment after transplantation for neuroblastoma using CD34-selected hematopoietic cells [78,119,120]. There have also been several small pilot studies documenting tumor cell depletion using RT-PCR techniques [120,21], despite some initial concerns that some neuroblastoma cell lines expressed the CD34 antigen [122]. However, even after CD34 cell selection of hematopoietic cells, there have been reports of residual neuroblastoma tumor cells detected by immunocytology [123]. CD34 cell selection provides a relatively simple commercial method that can be utilized in most transplant centers. However, the frequency and clinical significance of tumor cell contamination must be closely monitored and studied. The other potential problem with CD34 cell selection is the occasional immune dysregulation, with reports of post-transplant lymphoproliferative disorders, otherwise rarely reported after autologous HCT [124]. The other approach under investigation for purging of hematopoietic cells uses a modified immunomagnetic purging method, which, after pilot testing in 20 patients with successful engraftment, has been tested in a group-wide phase III COG study (A3973, 2001–06) for high-risk neuroblastoma. Protocol therapy consists of induction chemotherapy, hematopoietic cell harvest, radiation to the primary tumor bed, and highdose therapy with HCT, followed by 13-cis-retinoic acid. Patients were randomized at the time of study registration to either purged or unpurged peripheral blood hematopoietic cells as a stem cell source for high-dose consolidation. Hematopoietic cells were harvested after the first two cycles of induction chemotherapy. For purging, the cells were sedimented with hetastarch, excess phagocytic cells were removed with carbonyl iron, and this was then followed by treatment with monoclonal antibodies attached to magnetic immunomagnetic beads followed by magnetic separation and cryopreservation. An aliquot of the treated product was analyzed to determine if all detectable neuroblastoma cells were removed and to quantify viable cells, colony-forming unit-granulocyte–macrophage and CD34+ cells (before and after freezing). If no tumor cells were detectable and if adequate normal cells were present, the hematopoietic cells were used for reinfusion; if not, the product was not used and a second harvest or alternative therapy was necessary. Endpoints include EFS, time to engraftment, and tumor content by RTPCR analysis before and after purging. A preliminary report has thus far shown the feasibility of this approach and high success in harvesting peripheral blood hematopoietic cells after two cycles. [41]

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patients [128]. These data indicate that 13-cis-retinoic acid is well tolerated after intensive chemoradiotherapy and may have efficacy against minimal residual disease. A subsequent phase III randomized trial by the CCG of children with high-risk neuroblastoma completing consolidation chemotherapy or autologous BMT showed that the use of oral 13-cis-retinoic acid following intensive therapy improves outcome. The 3-year EFS from time of randomization was significantly better for the patients randomized to 13-cis-retinoic acid (46 ± 6%) compared with those randomized to no further therapy (29 ± 5%; p = 0.03) (Fig. 66.3) [2]. It is interesting to note the apparently contradictory report from the ENSG showing no advantage for using 13-cis-retinoic acid to treat minimal residual disease after myeloablative therapy [129]. The most likely reason for the lack of efficacy in the ENSG trial is the low dose employed for 13-cis-retinoic acid. The study began in 1989, prior to publication of the data from the in vitro studies and the phase I trial that led to the CCG randomized study. The ENSG study was designed using a dose that was approximately 15% of that shown to be the maximally tolerated dose in the phase I study by Villablanca and colleagues [128] and of the subsequent randomized CCG phase III trial [2]. At that low dose, drug levels would be far below those shown to be effective for sustained growth arrest of neuroblastoma cell lines [125]. Other retinoids are currently under investigation for use in minimal residual disease, such as fenretinide. In contrast to cis- and trans-retinoic acid, fenretinide does not induce maturational changes, but is cytotoxic and induces both apoptosis and necrosis [130]. Phase I studies of fenretinide in refractory neuroblastoma have been completed in the CCG and in Europe. From the phase I CCG trials of fenretinide in children with high-risk solid tumors, effective plasma levels can be achieved with minimal toxicity [131]. A phase II COG trial has been completed and results are pending. Another approach to minimal residual disease post transplant is the use of antibody-targeted therapy. Antibody therapy in relapsed neuroblastoma using murine, chimeric, and humanized antibodies against the membrane ganglioside GD2 has provided promising response and toxicity profiles that warrant the further investigation of these agents in

Treatment of minimal residual disease Despite improvements in EFS using high-dose therapy, the relapse rate, even for patients transplanted in complete response, remains high[2,33]. For this reason, it has become increasingly important to find new approaches to eliminate minimal residual disease with agents that will be tolerable following high-dose therapy. The time immediately after HCT, when disease is likely to be minimal, provides the ideal window to eradicate resistant clones that are still present using novel therapies not dependent upon standard cytotoxic mechanisms. In vitro, both all-trans-retinoic acid and 13-cis-retinoic acid cause decreased proliferation and differentiation in neuroblastoma cell lines, including some established from refractory tumors [125,126]. A phase II trial in children with relapsed neuroblastoma using 13-cis-retinoic acid on a single daily administration schedule of 100 mg/m2 showed responses in only two of 22 patients [127]. However, based on in vitro experiments with higher intermittent dosage, a phase I trial in children with high-risk neuroblastoma post HCT determined that a high-dose intermittent schedule of 13-cis-retinoic acid following BMT had minimal toxicity, achieved levels that were effective against neuroblastoma cell lines in vitro, and resulted in complete bone marrow responses in three of 10

Fig. 66.3 Treatment of minimal residual disease with 13-cis-retinoic acid. Use of 13-cis-retinoic acid in the treatment of minimal residual disease improves event-free survival (EFS) after hematopoietic cell transplantation for high-risk neuroblastoma. Follow-up began at the time of randomization, 34 weeks after diagnosis, with a significant difference in 3-year EFS; p = 0.027 [2].

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randomized studies [132–134]. With granulocyte–macrophage colonystimulating factor (GM-CSF) or interleukin-2 (IL-2), anti-GD2 seems to be tolerated in patients who have undergone autologous HCT [133,135]. A new randomized prospective trial of the use of chimeric anti-GD2 antibody with GM-CSF and IL-2 is underway in the COG. Patients who are in remission after high-dose therapy and HCT are randomized to receive either 13-cis-retinoic acid alone or the retinoid along with chimeric anti-GD2 antibody (Ch14.18) and GM-CSF and IL-2. Further improvements are being tested in phase I trials using a fusion protein of the humanized form of the antibody, Hu14.18, and IL-2. This immunocytokine has the advantage of working simultaneously through both antibody-dependent cytotoxicity and natural killer cell mechanisms. A murine neuroblastoma model showed superior activity of the immunocytokine to the physical mixture of the antibody and cytokine [136]. Phase I and II trials have since been completed, with demonstration of activity in bone marrow disease [137]. Another intense avenue of study includes the inhibition of angiogenesis in the progression of embryonal tumors including neuroblastoma. Angiogenesis appears to play a major role in the progression of disease as multiple proteins associated with angiogenesis have been shown to be associated with more advanced disease or worse prognosis [138,139]. Testing in animal models suggests responsiveness to angiogenic inhibitors, particularly in minimal disease states [140]. Much investigation has focused upon new and novel antiangiogenic factors and antibodies, with promising results in cell lines, in vitro models, and xenografts. Vascular endothelial growth factor inhibitors alone and in combination with chemotherapeutic agents seem to be promising [141,142]. Early studies of multitargeted tyrosine kinase inhibitors to vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor are also underway [143]. The well-known association between MYCN amplification and poor prognosis appears also to correlate with downregulation of activin A, a protein with antiangiogenic properties. Deficiency of activin A appears linked to tumor progression [144]. In the midst of all these new therapeutics being studied are traditional wellknown agents such as thalidomide, imatinib, cyclophosphamide, and topotecan. Delivering these agents in particular schedules and combinations may harness their antiangiogenic properties [145]. Other approaches to minimal residual disease in the future may utilize genetically engineered vaccines to generate an immune response [146,147].

Acute and late complications of HCT in neuroblastoma The complications of high-dose therapy and HCT for neuroblastoma are similar to those of other high-dose regimens and vary with the particular conditioning regimen. These are discussed in detail in Section VII of this book. The most common acute complications with most preparative regimens for neuroblastoma include the toxic effects of the high-dose therapy, with frequent mucositis, VOD, fever, and infection. Potentially fatal complications include gastrointestinal hemorrhage, pneumonitis, and acute respiratory distress syndrome, severe VOD with renal failure or, rarely, intracranial hemorrhage or cardiac failure. Toxic death rates generally have ranged from 5% to 20% on autologous HCT protocols (Table 66.3). With increasing expertise, more rapid engraftment with peripheral blood hematopoietic cells, and the removal of TBI from most regimens, the acute toxic death rate appears to be decreasing to about 5% [45,76,148]. With more patients surviving, some of the later complications of highdose therapy are becoming more troublesome, and new studies examining quality of life in survivors will assume greater importance. One common complication particular to neuroblastoma is the frequent occurrence of significant hearing loss post transplantation, seen in 10–20% of

children. These patients receive large doses of platinum compounds during induction chemotherapy and again with many of the preparative regimens. They also are exposed to other ototoxins, including aminoglycoside antibiotics, diuretics, and noise exposure, all of which may exacerbate the effects of carboplatin [149]. Other late effects are attributable to the high-dose chemotherapy and/or TBI, and are common to many pediatric regimens. A retrospective analysis of the late effects of TBI was conducted comparing the children treated with megatherapy requiring transplantation and TBI by the LMCE group (Lyon, Marseille, Curie, East of France) and those treated by the Institut Gustave Roussy group, of whom none received TBI [150]. These studies demonstrate the consequences for children with neuroblastoma treated often at a young age. These include hormonal deficiencies resulting in growth retardation, abnormal dental development, thyroid dysfunction, sterility or delayed onset of puberty, osteochondromas, and secondary malignancies. This is supported by a US institutional study that found the most common complications to be hearing loss, ovarian failure, primary hypothyroidism, and musculoskeletal problems [151]. In contrast to the French study, however, hearing loss was found to be more severe in more patients (49% requiring hearing aids) and did not appear related to platinum dosage. A rate of hypothyroidism was also found commensurate with TBI therapy, presumably related to the prevalence of targeted radioiodinated antibody therapy at that institution. In another study of long-term outcome, the use of laminectomy and local spinal radiotherapy were found to be significant risk factors for scoliosis [152]. Although no patient received asymmetric vertebral body irradiation, 21% of patients developed scoliosis at a median of 51 months from diagnosis. The dose of radiotherapy was also found to be significant, with 50–60% of patients developing scoliosis having received more than 1750 cGy versus 10% for those receiving less than 1750 cGy. Earlier studies of transplant in neuroblastoma reported a low incidence of second malignancies, in only one patient in 129 in the CCG study [2] and two of 509 patient in the EBMT Registry report [61]. However, secondary malignancies may become a more vexing problem in children transplanted for neuroblastoma, as the high doses of alkylators and etoposide in both the induction and conditioning regimens have been shown to result in secondary leukemia [153]. Some children have now been reported to develop secondary leukemia after radiotargeted therapy for neuroblastoma [78,149]. In addition, some quite unusual second malignancies have been reported after transplantation for neuroblastoma, such as renal cell carcinoma [154].

Conclusion The use of high-dose chemotherapy has been shown to increase the response rate and to improve EFS and survival in children with advanced neuroblastoma. It is likely that this approach has also contributed to the significant overall increase in survival for children diagnosed at over 1 year of age with stage 4 neuroblastoma that has been seen over the last two decades. The tumor reduction during the transplant conditioning regimen may be further improved by eliminating TBI and using radiotargeted therapy and new non-cross-resistant agents. Second, it has been shown that therapy for minimal residual disease post transplantation is also a critical component of the improvement in outcome for these patients. New targeted approaches to minimal residual disease deserve study, including genetic targeting, differentiating agents, and antiangiogenic and immunologic pathways. Third, with this improved outcome, it is critical to refine the therapy of these children to reduce the serious late complications of treatment and to study health-related quality of life in the survivors.

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David M. Loeb & Allen R. Chen

Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors

Introduction Chemosensitivity As a result of ongoing improvements in diagnosis, multimodal therapy, and supportive care, the treatment outcome of children with cancer has steadily improved since 1960. For instance, the 5-year survival of children with Wilms’ tumor has risen from under 45% to over 90%, for rhabdomyosarcoma from under 20% to 70%, and for Ewing’s sarcoma from 10% to 60% (reviewed in [1,2]). This is in part because the most frequent pediatric solid tumors (neuroblastoma, Wilms’ tumor, rhabdomyosarcoma, retinoblastoma, germ cell tumor, osteosarcoma, and Ewing’s sarcoma) are all chemosensitive. However, subsets of patients have been defined who face a poor prognosis despite having initially chemoresponsive disease. Examples include patients over 1 year of age with metastatic neuroblastoma (see Chapter 66), and patients with metastatic Ewing’s sarcoma (see below). For such patients treated conventionally, the prognosis for disease-free survival (DFS) 3 years from diagnosis remains less than 20%. In addition, the prognosis for recurrent or refractory pediatric solid tumors is dismal, and few patients have experienced prolonged survival. Most pediatric solid tumor patients are treated with intensive chemotherapy, and most recurrences occur during treatment or within 1 year of its completion. Rapid recurrence indicates the presence of tumor cells resistant to the chemotherapeutic agents used. Tumor cells often manifest substantial crossresistance to multiple antineoplastic agents, including drugs to which the cells were never exposed. Dose–response For radiation and most classes of chemotherapeutics, including alkylators, anthracyclines, vinca alkaloids, and antimetabolites, there is a steep linear relationship between drug concentration and the log tumor cell kill assessed by in vitro colony and limiting dilution assays [3]. This steep log-linear dose–response relationship translates into important effects of chemotherapy dose on tumor response and survival in experimental tumor-bearing animals. In studies of treatment of animals with osteosarcoma and mammary adenocarcinoma, sharp reductions in complete response (CR) and partial response (PR) rates accompany small (~15%) reductions in the dose of single agents of all classes, including

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

antimetabolites (5-fluorouracil and 6-mercaptopurine), anthracyclines (doxorubicin and daunorubicin), antitubule agents (vincristine), and alkylators (melphalan and cyclophosphamide [CY]). In this system, the only curative single agent is CY, given at the dose that causes lethal toxicity in 30% of animals (LD30). Various combinations of chemotherapy can produce cures at lower than LD10, but, even so, modest (~33%) dose reductions eliminate cures [4]. Of great relevance to pediatric oncology is a rhabdomyosarcoma xenograft model demonstrating a strong effect of the dose of melphalan on tumor response of six primary tumor cell lines, including one from a CY-resistant tumor. Importantly, melphalan at adequate doses is much more active than the standard chemotherapy combination for rhabdomyosarcoma, vincristine, actinomycin D, CY, and doxorubicin [5]. Modeling of experimental data indicates that the likelihood of cure correlates best with dose intensity, while the duration of PR correlates best with the total dose of chemotherapy. The maximum cumulative dose of alkylators and anthracyclines that can be administrated safely does not increase when dose intensity is reduced. It is therefore advantageous to maximize dose intensity; the maximum dose intensity is achieved by giving a single very large course of drug [6]. Dose-limiting myelotoxicity For agents such as melphalan, carmustine (see Chapter 21) (reviewed in [7]), and carboplatin [8], whose major dose-limiting toxicity is myelosuppression, hematopoietic cell transplantation (HCT) may permit a three- to 10-fold dose escalation. Given a steep dose–response relationship, this approach may translate into several logs more cell kill and improved durability of response.

Indications and outcomes Ewing’s sarcoma family of tumors Epidemiology and etiology The Ewing’s sarcoma family of tumors (ESFT) encompasses Ewing’s sarcoma, primitive neuroectodermal tumor (PNET), Askin tumor, and peripheral neuroepithelioma, diseases previously considered distinct. The recognition that these tumors are all characterized by a reciprocal chromosomal translocation involving a gene on chromosome 22 known as EWS with a fusion partner that is an ets family member transcription factor has resulted in their reclassification as a distinct, molecularly defined tumor type. Although clearly a single group of tumors, there is a spectrum of differentiation within this tumor group: the typical

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undifferentiated Ewing’s sarcoma lies at one end of the spectrum, and PNET with neural differentiation lies at the other. The ability to characterize these tumors molecularly and the growing reliance upon evaluation of chromosomal abnormalities as the definitive means of establishing a diagnosis are emblematic of the era of molecular oncology. ESFT is the second most common type of malignant bone tumor, behind osteosarcoma. These tumors account for 2% of childhood cancers, according to data from the Surveillance, Epidemiology and End Results (SEER) section of the National Cancer Institute. SEER data, however, are stratified for age, and thus make epidemiologic understanding of these tumors that affect both adolescents and young adults somewhat difficult. Moreover, SEER data do not break down adult sarcoma incidence by histology, making a true understanding of the epidemiology of ESFT in the United States impossible. A review of the M.D. Anderson Cancer Center tumor registry evaluated the epidemiology of sarcomas in the adolescent and young adult (AYA) population without restrictions based on age or histologic subtype [9]. In this study, ESFT accounted for 25% of the bone sarcomas in the AYA population, whereas osteosarcoma accounted for 57%. These data allow an estimation of new cases in the United States every year of 390 ESFT of bone and 170 of soft tissue. ESFT incidence peaks in the second decade of life, and is predominantly a disease of the AYA population. Sixty-four percent of cases occur in the second decade of life, compared with 27% in the first decade and only 9% in the third [10]. The M.D. Anderson data corroborate that finding, as chondrosarcoma was the second most frequent bone sarcoma histology in the population as a whole (28% of cases), but ESFT was more frequent in the AYA group. Interestingly, ESFT is overwhelmingly a disease of Caucasian children. In New York, ESFT represents 22% of primary bone cancers in whites, but only 7% in blacks [11]. A report from Nigeria similarly shows that ESFT accounts for only 1.8% of bone tumors in their national registry [12]. ESFT is not commonly associated with other congenital diseases, nor with skeletal abnormalities or other cancers. No constitutional chromosomal abnormalities have been reported in patients with ESFT, and the concordance rate in siblings is very low. ESFT is rarely reported as second malignant neoplasms in patients previously treated for cancer. Molecular and cellular biology These tumors are defined molecularly by the presence of a reciprocal translocation t(11;22)(q24;q12) that fuses a gene known as EWS with the ets family transcription factor Friend leukemia virus integration-1 (fli1) and is found in 85% of such tumors [13]. Alternative reciprocal translocations, such as t(21;22)(q22;q12), t(7;22)(p22;q12), t(17;22)(q12;q12), and t(2;22)(q33;q12), all of which result in a fusion between EWS and an ets family transcription factor, have been reported in the remaining 15% [14]. Several studies have suggested that EWSFLI1 is oncogenic, and is sufficient to initiate and maintain the transformed phenotype [15,16]. In fact, inhibition of the fusion gene in cell lines inhibits growth in vitro [17,18]. In addition to the defining translocation, numerous other cytogenetic abnormalities have been described in ESFT, the most common of which is der(16)t(1;16) [19]. Sandberg and Bridge provide a comprehensive overview of the cytogenetic abnormalities reported in these tumors [14]. Controversy still surrounds the most basic cellular biologic question related to ESFT – the cell of origin. There is cytologic and ultrastructural evidence of neural maturation in the more differentiated ESFT tumors [20] that were formerly classified as PNET. An attempt to establish the histogenesis of ESFT cell lines by analysis of intermediate filament expression, however, suggested an epithelial phenotype, on the basis of expression of cytokeratins 8 and 18 [21]. More recently, DNA microarrays have been employed to attempt to address this question. Gene

expression profiling experiments have shown that these tumors are related to both neuronal and endothelial lineages [22]. Interestingly, expression of EWS-FLI1 in human embryonic kidney cells is insufficient to induce the entire ESFT gene expression profile, suggesting that the cell being tested impacts the process of differentiation. In contrast, Riggi and colleagues demonstrated that forced expression of EWS-FLI1 in bone marrow-derived mesenchymal progenitor cells induces the formation of tumor cells with all of the essential hallmarks of ESFT [16]. Thus, support can be found in the literature for a neuronal, epithelial, endothelial or mesenchymal origin of these tumors. Future work will be required to clarify the histogenesis of ESFT. Clinical description Pain and swelling are the most common presenting symptoms of ESFT, occurring in 84% and 63% of patients, respectively [23]. These symptoms are often longstanding, with a median delay between onset of symptoms and diagnosis in excess of 6 months [24]. While tumors can arise in any bone in the body and in soft tissue, these tumors show a predilection for the lower extremity (46%) and pelvis (20%) [25]. ESFT is a very aggressive malignancy, with 25% of patients presenting with overt metastases. Of these, half have metastases to the lungs, one-quarter to other bones, and one-fifth to bone marrow [23]. Metastases to other sites are rare, except in late-stage disease. Nontransplant approaches For these tumors, the primary adverse risk factor is the presence of metastatic disease. A more recently recognized prognostic factor is histologic response to chemotherapy. Data from the Rizzoli Institute showed 95% DFS for patients with localized disease and complete tumor necrosis, compared with 34% for patients with macroscopic viable tumor at the time of definitive surgery. Pelvic primary disease or a large tumor size >8 cm in maximal diameter or >100 mL in volume historically carried a poor prognosis for survival. Intensification of therapy by the addition of ifosfamide and etoposide improved event-free survival (EFS) from 54% to 69% for patients with localized disease, and eliminated pelvic primary location and large tumor size as prognostic factors [26]. Unfortunately, their use did not improve the EFS of patients with metastatic disease, which was 22% regardless of treatment regimen. The recently completed Children’s Oncology Group study tested increasing dose intensity by randomizing patients to receive this same treatment regimen with cycles given at 2-week intervals versus the standard 3-week intervals. Four-year EFS was 65% for patients with localized disease treated at 3-week intervals, but 76% for patients treated with the time-compressed regimen (a statistically significant difference; Womer, personal communication), lending further support to the importance of dose and time intensity for the cure of pediatric solid tumors. In addition to systemic chemotherapy, adequate local control is essential to eradicate the disease. Local control measures include radiation therapy and surgery. The relative efficacy of radiotherapy compared with surgery remains controversial. The most recent studies, utilizing dose-intensive chemotherapy, show little difference in survival between patients treated with surgery and patients treated with radiation [27], with respect to both local recurrence and overall survival (OS). In a single-institution study of 76 patients with localized disease, the local failure rate was identical in the patients treated with radiation, surgery or both [28]. Interestingly, in a multivariate analysis, only the use of chemotherapy was a prognostic factor for local control (the 10-year local control rate for patients treated with chemotherapy being 83.7%, compared with 51.1% for patients who did not receive chemotherapy), supporting the concept that systemic treatment contributes to local control.

Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors

Hematopoietic cell transplantation It is clear that patients with high-risk ESFT, including patients with metastatic or recurrent disease, have a very poor prognosis. Doseintensive chemotherapy has not improved the outcome for patients with metastatic disease, and patients with recurrent disease are not cured with standard chemotherapy. For these patient populations, HCT has been investigated as a way to improve outcomes. One of the earliest and largest reports of high-dose therapy investigated the use of total body irradiation (TBI) with autologous peripheral blood progenitor cell (PBPC) support as consolidation therapy for high-risk ESFT patients. Over a 5-year period, 91 patients were treated on three successive chemotherapy protocols at the National Institutes of Health, and remissions were consolidated in 65 of the patients with 8 Gy of TBI [29]. Nineteen patients were not given TBI because they failed to achieve remission or relapsed prior to treatment, and seven refused. Twenty of the 65 treated patients (31%) became long-term survivors, a higher rate than expected with chemotherapy alone in this group of patients, and superior to a group of contemporary patients treated without high-dose therapy. However, only patients who did not progress after chemotherapy were eligible for HCT, so OS for the total patient population was only 22%, no better than results with chemotherapy alone. These results do suggest, however, that a select group of patients might benefit from HCT. At the same time, the groups in Vienna and Dusseldorf reported very impressive results using a combination of TBI, melphalan, and etoposide for poor-risk patients with ESFT. These patients had either multifocal disease (seven of 17 patients) or early or multiple relapses (10 of 17 patients). The dose of TBI was 12 Gy – higher than utilized by the National Institutes of Health. Relapse-free survival for these patients was 45% at 6 years [30]. Unfortunately, these results did not hold up with additional enrollment and follow-up. Updated results, analyzing 36 patients with up to 139 months of follow-up, showed an EFS of only 24% [31]. Similar results were reported from the UK, where a group of 18 patients with poor-risk ESFT (defined as metastatic at diagnosis, very large primary tumors or a patient in second complete remission) were treated with high-dose busulfan and melphalan and HCT. OS in this group was good (13 of 18 patients, EFS of 72%, with a range of followup from 2 months to 7 years), but OS of the patients with metastatic disease was only 25% [32]. More promising results have been reported from a review of European Bone Marrow Transplant Registry (EBMTR) data. While the DFS rate for patients with ESFT metastatic to bone or bone marrow who underwent HCT between 1982 and 1992 was only 21% [33], results were superior for the subgroup of patients treated with the combination of busulfan and melphalan, and for the patients who did not receive TBI. An updated review of these data reported 5-year OS of 44% for the patients treated with a busulfan-containing regimen, compared with 23% for patients treated without this drug [34]. These results have been confirmed in two smaller studies. The group at the University of Washington reported their results utilizing triple alkylator therapy (busulfan, melphalan, and thiotepa) followed by total marrow irradiation with HCT after each of these treatments for 16 patients with poor-risk ESFT. Six of their patients (36%) survived for 27–66 months [35]. Davies et al. have reported 62% DFS at 3 years for a similar group of 11 patients also treated with the same triple alkylator therapy (but no irradiation) [36]. Further support for a possible benefit from alkylator-intensive therapy comes from two other European groups. The group in London recently reported results of HCT for 33 patients with recurrent or progressive ESFT between 1992 and 2004 [37]. The 22 patients treated with busulfan and melphalan had OS of approximately 50%, with follow-up as long as 12 years. Similarly, the Société Française des Cancers de l’Enfant

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treated 97 newly diagnosed patients with metastatic ESFT using doseintensive chemotherapy and consolidation with busulfan and melphalan for patients with a complete or very good partial remission. Five-year EFS for all 97 patients was 37%, and EFS after HCT was 47% (52% for patients with metastases limited to the lungs) [38]. Although none of these reports presents results of a randomized trial, the data suggest a survival advantage for patients treated with regimens incorporating high doses of alkylating agents, especially busulfan. A recent publication from the University of Washington looked specifically at patients with relapsed ESFT to determine the benefit of high-dose therapy and HCT for this particular subset of patients. Patients were treated with a variety of salvage induction chemotherapy regimens, and patients who responded to this therapy were offered high-dose therapy with busulfan, melphalan, and thiotepa with HCT support, some with total marrow irradiation as discussed above. While the 5-year OS for this group of patients was poor (23%), the subgroup of patients with metastases limited to the lungs who had chemosensitive disease and were treated with HCT had 62% OS (eight of 13 patients). In fact, among patients who responded to their retrieval therapy, progression-free survival and OS were superior with HCT compared with chemotherapy alone (PFS 61% versus 21%, and OS 77% versus 21%) (Fig. 67.1). Thus, for a subset of patients with chemosensitive recurrent disease, HCT appears to offer a survival advantage compared with chemotherapy alone [39]. All of the studies summarized above share the same weakness – none is prospective and randomized. This limits the confidence with which HCT can be recommended as “standard of care” for ESFT patients. An ongoing study, Euro-Ewing 99, was designed to address this deficiency. In this study, patients are assigned to one of three risk groups, based on the presence or absence of metastases, the size of the primary tumor, and the response to neoadjuvant therapy. Patients in the lowest-risk group are treated with 14 cycles of chemotherapy. Patients judged to have intermediate risk disease (PR rather than CR to neoadjuvant therapy, a tumor >200 mL or pulmonary metastases) are randomized to consolidation with chemotherapy versus high-dose therapy with busulfan and melphalan, and patients with the highest risk (extrapulmonary

Fig. 67.1 Survival from time of first recurrence for patients with a recurrent Ewing’s sarcoma family tumor who responded to salvage induction chemotherapy, by use of high-dose therapy (HDT). (Reproduced from [39], with permission.)

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metastases) are randomized between two different preparative regimens for their HCT. This study is expected to resolve some of the controversies surrounding the utility of HCT for patients with ESFT. Rhabdomyosarcoma Epidemiology and etiology Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood, accounting for 5–8% of childhood cancer [10]. About 250 new cases of rhabdomyosarcoma are diagnosed in the United States annually, slightly more frequently in boys than in girls. Little is known about the etiology of rhabdomyosarcoma. These tumors sometimes arise as second malignant neoplasms in patients previously treated with ionizing radiation, but radiation exposure accounts for a very small minority of cases. Molecular and cellular biology There are two major histologic variants of rhabdomyosarcoma: embryonal and alveolar. Embryonal rhabdomyosarcoma, named for its resemblance to immature skeletal muscle, has been subdivided into solid and botryoid variants. Embryonal histology accounts for 57% of cases of rhabdomyosarcoma and usually affects children between 3 and 12 years of age. Alveolar histology, named for its resemblance to normal lung parenchyma, accounts for 19% of cases of rhabdomyosarcoma, and usually strikes patients between 6 and 21 years old [10]. Embryonal rhabdomyosarcoma carries a more favorable prognosis than alveolar. As in ESFT, recurrent translocations have been identified in rhabdomyosarcoma, although only in the alveolar subtype. In 55% of cases, a translocation between chromosomes 2 and 13, t(2;13)(q35;q14), is seen, and in 22% of cases a similar translocation involving chromosome 1, t(1;13)(p36;q14), is identified [40]. These translocations involve related transcription factor genes, paired box 3 (PAX3) and 7 (PAX7), respectively. In each case, the DNA-binding domain of the PAX gene is fused to the transactivation domain of the forkhead box (FKHR) gene. Disruption of PAX genes leads to abnormal muscle development [41], suggesting a direct etiologic relationship between the translocation and the development of malignancy. Moreover, ectopic expression of these chimeric genes can transform cells. The PAX3-FKHR translocation appears to carry a poorer prognosis than PAX7-FKHR [42]. Clinical description Signs and symptoms present at diagnosis depend on the site of the primary tumor, the involvement of surrounding normal organs, and the presence of distant metastases. Typically, patients present with an asymptomatic mass, although involvement of cortical bone may cause pain, and genitourinary disease may cause hematuria or urinary retention. Different subtypes of rhabdomyosarcoma arise in different sites. Embryonal rhabdomyosarcoma typically arises in the head and neck region or the genitourinary tract, while alveolar rhabdomyosarcoma tends to arise in the extremities and the trunk [10]. Head and neck sites, including orbit and parameningeal primaries, account for 40% of cases; trunk and extremity sites account for 25–30% of cases; and genitourinary sites account for 20% [40]. The most common sites of metastasis are the lungs, lymph nodes, cortical bone, and bone marrow. Nontransplant approaches Rhabdomyosarcoma is treated with multimodality therapy, including chemotherapy, radiation therapy, and surgery. Surgery alone is insufficient to cure even localized disease. In the era before the development of effective systemic chemotherapy, the OS for patients with localized disease was 25% or less [43]. With current multimodality therapy,

Table 67.1 Clinical group definitions for rhabdomyosarcoma patients Group

Definition

Group I Group IIa Group IIb

Localized disease completely resected Gross total resection with microscopic residual disease Regionally involved lymph nodes, completely resected with the primary Regional disease with involved nodes, totally resected with either microscopic residual disease or histologic evidence of involvement of the most distant lymph node in the dissection Incomplete resection Distant metastases

Group IIc

Group III Group IV

failure-free survival rates for patients with localized disease are as high as 90% [44]. The most important predictor of treatment failure is the clinical group identified in early Intergroup Rhabdomyosarcoma Study Group studies (Table 67.1). In addition to classification by clinical group, rhabdomyosarcoma is also staged using a specific tumor–node–metastasis staging system [44] that not only takes into account tumor size, lymph node status, and distant metastases, but also incorporates information regarding the site of the primary tumor, reflecting prognostic differences between favorable and unfavorable anatomic locations. Patients are then classified as high, intermediate or low risk based on clinical group and stage, and treatment is based on this assessment of risk. The principles of nontransplant therapy for rhabdomyosarcoma, as established by the Intergroup Rhabdomyosarcoma Study Group clinical trials, include local control (consisting of surgery, radiation therapy or both) and systemic therapy to address either gross or microscopic metastatic disease. Primary surgical resection is indicated, unless the tumor is deemed unresectable. In this case, radiation therapy is employed and can provide definitive local control for patients with unresectable rhabdomyosarcoma [45]. The specifics of adjuvant systemic chemotherapy vary depending on risk stratification. In general, the chemotherapy used for low-risk patients is significantly less intensive than the therapy for intermediate- or high-risk patients. Unlike osteosarcoma or ESFT, patients with rhabdomyosarcoma undergo surgical resection prior to the institution of systemic therapy, as reflected in the staging system, which takes into account the extent of resection. Upon recovery from surgery, patients are treated with chemotherapy with or without radiation, which is usually delivered relatively early in the treatment regimen. Hematopoietic cell transplantation The role of high-dose therapy and HCT for high-risk rhabdomyosarcoma is not established. Several retrospective analyses have been reported. Between 1982 and 1994, the EBMTR received reports of 98 transplants performed in children and young adults with relapse or progression of initially localized rhabdomyosarcoma. The proportion surviving diseasefree is approximately 20%, and median survival remains short, at only 8.3 months from HCT [46]. The German–Austrian Pediatric BMT Group in 1997 published the results of 36 transplants performed for metastatic or recurrent rhabdomyosarcoma between 1986 and 1994. Thirty-four of the 36 patients received high-dose melphalan-based regimens, usually augmented with etoposide and carboplatin. The estimated EFS at 2 years after HCT was 36% (standard error [SE] 7%) [47]. This rate is better than expected, but the small number of patients and possible effects of selection bias preclude conclusions about the efficacy of HCT for rhabdomyosarcoma.

Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors

The European Collaborative MMT4–91 trial was the first prospective trial to evaluate the efficacy of HCT as consolidation therapy in first remission for patients who presented with metastatic rhabdomyosarcoma. This study resulted from the amendment of MMT4–89 for patients with newly diagnosed metastatic rhabdomyosarcoma, so that institutions could choose to substitute HCT with high-dose melphalan for the fourth and final 9-week cycle of chemotherapy in patients who had achieved CR before the third cycle of therapy. Thus, there was a nearly contemporaneous control population of patients either enrolled on MMT4–89 or enrolled on MMT4–91 at centers not participating in HCT. Fifty-two patients in CR after six courses of chemotherapy were treated with highdose melphalan, either alone or in combination with other agents such as carboplatin, etoposide, thiotepa, and/or busulfan. Outcomes were compared with those of 44 patients who were also in CR after six identical cycles of chemotherapy but went on to receive further conventional dose chemotherapy rather than HCT. Although patients were not randomized to HCT or conventional therapy, decisions to administer HCT consolidation were made at the center level, reducing the potential for selection bias. Unfortunately, the patient population treated with HCT (n = 52) in fact had poorer prognostic factors: lymph node involvement was more common (56% versus 34%), alveolar histology was more common (44% versus 30%), fewer patients were less than 10 years old (60% versus 68%), and more patients had large tumors over 5 cm in diameter (73% versus 61%) than in the control population (n = 44). Despite these differences and the small sample size, the median time from the end of therapy to relapse was significantly longer for the HCT group than the conventional chemotherapy group (168 versus 104 days; p = 0.05), but there was no difference in EFS or OS [48]. Single-institution protocols for high-risk rhabdomyosarcoma that incorporate HCT consolidation show similar results, with DFS for stage IV patients ranging from 14% to 28% at 3 years or more [49–52].

Wilms’ tumor Epidemiology and etiology Wilms’ tumor is the most common renal malignancy of childhood, with an annual incidence in the United States of 8.1 cases per million children under age 15 years, with a slight female predominance [53]. Children with bilateral disease have an earlier age of onset, 23.5 months for boys and 30.5 months for girls, than those with unilateral disease, 36.5 months for boys and 42.5 months for girls [54]. This age difference was a key observation in the development of Knudson’s classic two-hit model of tumorigenesis [55]. The additional variation in age of onset by gender and ethnicity suggests heterogeneity in the pathogenesis of Wilms’ tumor [56]. Tumors associated with intralobular nephrogenic rests have an earlier onset [57], while those with perilobular nephrogenic rests are associated with a higher birth weight and a later age of onset [58]. Molecular and cellular biology Molecular correlates of these epidemiologic observations have recently been identified. In most normal tissue, imprinting of the insulin-like growth factor-2 (IGF2) gene silences the maternal allele, so only the paternal allele is expressed. In a study of 36 informative Wilms’ tumor specimens, loss of imprinting that led to expression of the normally silent maternal allele was strongly associated with perilobular nephogenic rest histology and a 2.2-fold increased expression of IGF2 [59]. Conversely, mutation of the Wilms’ tumor 1 (WT1) suppressor gene is associated with earlier age at onset, stroma-predominant histology, intralobular nephrogenic rests, and poorer response to therapy [60].

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Clinical description Children with Wilms’ tumor typically present with a painless abdominal mass or abdominal distention. Other findings include gross hematuria and fever. Hemorrhage into the tumor can result in a rapidly expanding abdominal mass and anemia. Compression of renal vasculature can produce hypertension. Radiographic findings can usually distinguish Wilms’ tumor as an intrinsic renal mass from neuroblastoma arising in the adrenal gland and displacing the kidney. Patterns of spread include local extension beyond the tumor pseudocapsule into the renal sinus, the renal vasculature, and lymphatics. The tumor can be locally aggressive, violating the renal capsule, and can disseminate through lymphatics. Hematogenous dissemination is primarily to the lungs, and less commonly to the liver. Nontransplant approaches Wilms’ tumor is the exceptional childhood solid tumor that is often curable by conventional chemotherapy when metastatic at diagnosis, and even after relapse. However, high-risk groups can be defined. The National Wilms Tumor Studies, NWTS-2 and NWTS-3, accrued 2757 untreated patients age 15 years or younger, with stage I–IV disease. The prognosis of the 367 patients (14%) who relapsed after achieving an initial CR was analyzed in 1989. Histology was an important predictor of outcome, with unfavorable histology defined by the presence of diffuse anaplasia (Fig. 67.2) [61]. In NWTS-3, 3-year survival after relapse was 42% with favorable histology versus 16% with unfavorable histology. Similar numbers of patients relapsed at less than 6 months, 6–11 months, and over 12 months from diagnosis. Their respective 3year survival was 18%, 30%, and 41%. Among patients with favorable histology, the 3-year survival by initial stage was 57% for stage I, 36% for stage II/III, and 17% for stage IV; for patients with unfavorable histology, 3-year survival by stage was 17% for stage I, 14% for stage II/III, and 7% for stage IV. Among stage II/III patients with favorable histology, those randomized to receive three-drug initial therapy had 16% 3-year survival from relapse, compared with 42% among those initially treated with two-drug regimens. Intra-abdominal relapse was unfavorable when this field had been previously irradiated, precluding further radiation therapy to the site of relapse [62]. An analysis of the long-term survival outcome of patients relapsing on the United Kingdom Children’s Cancer Study Group Wilms Tumor 1 trial, which accrued 381 patients from 1980 to 1986, confirmed the negative prognostic importance of unfavorable histology, high initial stage, and early relapse, given salvage with ifosfamide or cisplatin and etoposide [63,64]. Taken together, these results have led to the accepted definition of a high-risk subgroup of relapsed Wilms’ tumor patients whose expected 3-year survival is under 20%, including those with any of the following features: unfavorable histology, relapse within 6 months of diagnosis, failure of a three-drug regimen, and involvement of sites other than lung and abdomen, or involving the abdomen after irradiation. However, NWTS-5 treated patients who relapsed after vincristine and dactinomycin therapy without prior irradiation on a uniform regimen consisting of vincristine, doxorubicin, CY, and etoposide, combined with radiation to involved organs. Of 72 patients enrolled, 68 were evaluable, and the 58 patients age 18 and younger with unilateral disease at initial diagnosis were analyzed. Only 10 of the patients had no adverse prognostic features, 32 had one, and 16 had two. Nevertheless, 4-year EFS was 71% and 4-year OS 82% [65], showing that aggressive conventional therapy can overcome adverse prognostic factors in minimally pretreated patients. In contrast, results of the NWTS-5 study for patients who relapsed after initial therapy with vincristine, dactinomycin, and doxorubicin were less favorable. One hundred three patients were enrolled in this stratum, and 91 were evaluable. The 60 patients with initially unilateral

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Fig. 67.2 Top panel: Patterns of anaplasia within Wilms’ tumor that constitute diffuse anaplasia. Bottom panel: Anaplastic focus on the right, sharply demarcated from adjacent nonanaplastic Wilms’ tumor on the left. (Reproduced from [61], with permission.)

disease who relapsed after achieving a CR were analyzed. For these 60 patients, 4-year EFS was 42% and 4-year OS was 48%. Twenty-five patients had only one adverse prognostic factor, 22 had two or three adverse prognostic factors, and 13 had four or more adverse prognostic factors. More adverse prognostic factors, male gender, and sites of relapse other than the lungs conferred significantly poorer EFS [66]. In summary, there is room for improvement over the results of nontransplant chemotherapy in patients with high-risk relapses following threedrug initial therapy. HCT for high-risk relapse of Wilms’ tumor Limited published information is available regarding high-dose therapy for patients with high-risk relapse of Wilms’ tumor. The experience collected by the European Group for Blood and Marrow Transplant (EBMT) has been reviewed [67]. Twenty-five patients with Wilms’ tumor received high-dose therapy over a 7-year period from 1984 to 1991. Twenty-one of these children had had one to four relapses, and four patients had

stage IV disease refractory to first-line therapy. Of 11 patients transplanted in second CR (CR2), 10 had one or more high risk features as defined above. Twenty of the 25 patients received high-dose melphalanbased regimens, although seven different regimens were used. Eight of 17 children transplanted in CR became long-term disease-free survivors. Of patients with measurable disease, five of eight achieved CR and one of eight achieved PR to high-dose therapy, demonstrating the activity of these regimens in refractory Wilms’ tumor. However, only one of eight children with measurable disease at the time of HCT became a long-term survivor [67]. Small case series have suggested that patients with multifocal, generally multiply recurrent Wilms’ tumor may benefit from high-dose thiotepa-based preparatory regimens. In addition, a single institution treated 13 patients with recurrent Wilms’ tumor with HCT from 1991 to 2001. Twelve of 13 had at least one adverse prognostic factor. Four different preparative regimens were used, and four of the patients received tandem HCT. There was no transplant-related mortality, and 4-year EFS was 60% [68]. These results are sufficiently encouraging to warrant prospective evaluation, first in phase II trials to identify active regimens, and then in a randomized comparison with chemotherapy for patients with highrisk disease. The French Society of Pediatric Oncology (Société Française de l’Oncologie Pédiatrique [SFOP]) has completed the first prospective trial of HCT for high-risk relapsed Wilms’ tumor [69]. From 1988 to 1994, 31 patients underwent HCT, including 29 relapsed (16 in CR2, four in PR2, three in CR3, five in PR3, and one in CR5) and two patients with stage IV anaplastic Wilms’ tumor transplanted in CR1. All patients had at least one high-risk feature, and were heavily pretreated with five or six chemotherapy drugs before HCT. The preparatory regimen consisted of melphalan 180 mg/m2, etoposide 1000 mg/m2, and carboplatin, dosed to achieve an area under the curve of 20 mg–min/mL over 5 days. Radiation therapy was delivered to sites of bulky metastatic disease after recovery from HCT. Seven patients sustained renal tubular damage, one developed sinusoidal obstruction syndrome of the liver but recovered fully, and three developed interstitial pneumonitis. Only one of nine evaluable patients failed to achieve CR after HCT. Sixteen patients relapsed at a median of 8.5 (range 3–53) months after HCT, and 12 patients remained in continuous CR a median of 48.5 (range 36–96) months after HCT. DFS was 50% (SE 17%) and OS 60% (SE 18%) at 3 years [69]. The only statistically significant prognostic factor was the number of disease progressions before HCT: in second response, DFS was 63.1 +/− 20%, compared with 22.2 +/− 24% in third response or beyond. The results are better than historical results for high-risk patients with such advanced disease. However, over the same period, 15 patients in the participating centers with at least one adverse prognostic factor failed to undergo consolidation because of uncontrolled progressive disease, and they would need to be included in an intent-to-treat analysis to estimate survival from relapse in an unselected population. The German Cooperative Wilms Tumor Studies confirmed these results, treating 23 patients with HCT from 1992 to 1998 because of defined high-risk criteria including relapse, progression or incomplete response of initial stage IV disease (12), second or subsequent relapse (nine), relapse within a radiation field (four), bone or brain metastases (three), relapse at less than 6 months after nephrectomy (three), or relapse of a tumor with unfavorable histology (one). The preparative regimen was identical to that of the SFOP in 19 patients. One patient developed renal failure requiring dialysis for 1 month, and six developed tubular dysfunction. For the entire cohort, EFS was 48% and OS 61%, but disease status at the time of HCT strongly predicted outcome: 11 of 13 patients in CR but only three of 10 patients in PR survived at a median follow-up of 58 months [70].

Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors

The recently completed NWTS V Pediatric salvage protocol was initially designed to incorporate HCT for patients who failed to achieve a CR after two courses of salvage induction chemotherapy. However, the study was amended to eliminate HCT. Although the results of this salvage protocol are not yet mature, the St. Jude institutional experience and the results of the Children’s Cancer Group pilot salvage protocols suggest that new chemotherapy approaches have improved the outcome of patients with high-risk relapse even without HCT [71,72], and it would therefore be appropriate to launch an international trial to assess the value of HCT compared with conventional-dose chemotherapy in high-risk recurrent Wilms’ tumor, with the greatest benefit expected in patients in second response (Fig. 67.3). HCT as consolidation for Wilms’ tumor Because of the excellent curability of Wilms’ tumor with conventional chemotherapy, few patients have been treated with HCT for Wilms’ tumor in first response, all those who have having stage IV disease. All

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five patients reported by EBMT, including two with unfavorable histology, survive disease free at a median of 62 (range 14–67) months after HCT [67]; both patients with stage IV anaplastic Wilms’ tumor transplanted by the SFOP died of progressive disease [69], while the one similar patient treated by the German Cooperative Wilms Tumor Studies survived [70].

Osteosarcoma Epidemiology and etiology Osteosarcoma is the most common primary bone tumor of childhood and adolescence, and usually involves the long bones [10]. The incidence of osteosarcoma is three new cases per million per year, or 5.6 cases per million Caucasian children younger than 15 years in the United States [73]. The peak age of onset is during the second decade of life, during the adolescent growth spurt [74], somewhat earlier in girls than in boys. Boys are affected more frequently than girls, a finding speculated to reflect the larger bone volume in boys compared with girls [75]. A second incidence peak after the age of 50 years [76] may reflect tumors induced by environmental exposures. Because of its association with the adolescent growth spurt, the relationship between height and the risk of developing osteosarcoma has been investigated. Several contradictory reports on this subject have been published, most having significant methodological flaws. A recent population-based study from the United Kingdom analyzed 364 patients with osteosarcoma and confirmed that patients with osteosarcoma were significantly taller than the general population [77]. Interestingly, although these findings were later confirmed in an even larger study from Italy, the latter group found that this difference was limited to patients diagnosed while still growing [78]. They found that in patients diagnosed while still growing, height at the time of diagnosis exceeded age-matched controls, but that patients diagnosed after completing their growth were the same height as their peers. These findings support the hypothesis that growth of long bones is etiologically involved in the development of osteosarcoma, but the precise relationships remain unclear. The only proven exogenous risk factor for the development of osteosarcoma is exposure to ionizing radiation [79]. Unlike ESFT, however, osteosarcoma has been associated with several inherited syndromes, including bilateral retinoblastoma, Li–Fraumeni syndrome, Bloom’s syndrome, Rothmund–Thomson syndrome, Werner’s syndrome, and multiple exostoses. Osteosarcoma is also associated with Paget’s disease. These findings have led to the identification of numerous genetic changes involved in the development of this tumor. Thus, a good deal is known about the molecular biology of osteosarcoma. Molecular and cellular biology

Fig. 67.3 Metastatic recurrent Wilms’ tumor. Computed tomography scans showing first chemoresponsive relapse subsequently consolidated with autologous HCT.

The recognition that patients with bilateral retinoblastoma are at increased risk of developing osteosarcoma implicates the RB1 gene in the pathogenesis of osteosarcoma. As expected from this observation, loss of RB1 at chromosomal locus 13q14 is a frequent finding in sporadic osteosarcoma [80]. Osteosarcoma is one of the defining tumors of Li– Fraumeni syndrome, and this has led to investigation of mutations in p53 in spontaneous osteosarcoma. Mutations in p53 occur in 40–60% of cases [81]. Both of these genes, TP53 and RB1, are implicated in cell-cycle regulation and proliferation. Numerous other cell-cycle regulatory genes have also been implicated in this disease, including MYC, CDKNA2, CDKN2A, CDKN2B, and MDM2 [81–83]. Chromosomal instability appears to play a key role in the molecular pathogenesis of osteosarcoma. No recurrent chromosomal translocation has been identified, but numerous loci with amplification, point mutation or loss of heterozygosity have been reported (reviewed in [84]). This is

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consistent with the observation that osteosarcomas are diagnosed with increased frequency in patients with Bloom’s syndrome, Rothmund– Thomson syndrome, and Werner’s syndrome, disorders caused by germline mutations in RecQ helicases, which are involved in maintaining genomic stability [85]. In addition to these general regulatory genes, a loss of function of genes implicated in bone differentiation has also been seen in osteosarcoma. Deregulated bone differentiation is commonly seen in osteosarcoma [86]. Runt-related transcription factor-2 is a primary regulator of bone development [87] and is upstream of other regulatory genes, including OSX [88] and TWIST [89]. The TWIST locus, 7p21, was found to be rearranged in 31 cases of osteosarcoma, deleted in 22, and amplified in nine out of a total of 68 cases examined [90]. Additionally, a high-expression ezrin has been correlated with a poor outcome in osteosarcoma patients [91], ezrin being a protein implicated predominantly in metastatic disease [92]. Further investigation into the molecular biology of osteogenesis will undoubtedly uncover further genes that contribute to the development of osteosarcoma. Clinical description The most common presenting complaint of osteosarcoma is pain [10]. Swelling and pathologic fracture are often also seen. Radiographic findings typically include a mixed osteolytic/sclerotic lesion with disorganized soft tissue calcification. At diagnosis, the disease is localized in 80% of cases, with metastases, most commonly to lung, seen in the other 20%. Bone is the other common site of metastatic disease [93], with metastases rarely seen in lymph nodes. Osteosarcoma arises most frequently in the limbs (80% of cases), the femur being the most common bone [94]. It occurs predominantly in the metaphysis of long bones, arising from the medullary cavity and invading into the epiphysis, even in the presence of a growth plate. Nontransplant approaches The mainstays of therapy for osteosarcoma remain surgery and chemotherapy. Until the 1970s, surgery was the only available therapy, but complete resection cured only 20% of patients. Three out of four patients died within 2 years of diagnosis, almost exclusively due to pulmonary metastases. The introduction of adjuvant chemotherapy has increased OS to almost 70% for patients with localized disease [95], although the cure rate for patients with metastatic disease remains unacceptably low. The first combination chemotherapy regimens consisted of methotrexate, doxorubicin, and cisplatin, and this combination remains the standard 30 years later. Neoadjuvant chemotherapy was introduced in the late 1970s to facilitate limb salvage surgery [96], and led to the recognition that histologic response to therapy was a strong prognostic feature. Patients with a good histologic response (>90% tumor necrosis) had 68% OS at 5 years, compared with 52% survival in poor responders [97]. Based on this finding, there have been several attempts to improve survival by augmenting chemotherapy for the poor responders. No such attempt has shown a statistically significant benefit [98]. Thus, standard therapy for osteosarcoma remains the same today as it was in 1980 – surgical resection and a three-drug regimen of methotrexate, doxorubicin, and cisplatin – and is the same for patients with localized or metastatic disease, despite the vast difference in prognosis between these groups. Hematopoietic cell transplantation HCT is not commonly used to treat osteosarcoma, despite the lack of effective therapy for relapsed patients and the poor prognosis of patients who present with metastases. In fact, the EBMT received reports of only seven patients transplanted for osteosarcoma as of 1992. Of the five patients with measurable disease, one died of toxicity, three patients had no response, and one transplanted in refractory relapse achieved a PR

that was improved to CR by surgical excision of lung metastases, and remains in CR2 18 months post HCT. The two patients transplanted in CR3 relapsed 8 and 11 months after HCT [99]. The Cooperative German–Austrian–Swiss Osteosarcoma Study Group retrospectively analyzed 15 patients who received HCT for recurrent osteosarcoma. These patients had all achieved a CR when initially treated. Their sites of relapse were lung in nine patients, lung plus local in two, mediastinal in two, and local only in two. All underwent resection of their recurrent disease before HCT; only two had macroscopic residual disease, and neither responded to the high-dose preparative regimen. The preparative regimens all incorporated a combination of etoposide with melphalan or carboplatin, or both. Six patients received two courses of HCT, first with thiotepa and CY and then with melphalan, but two of these patients died of toxicity. The 3-year OS was 29%, and DFS was 20%. Both disease-free survivors received melphalan-based preparative regimens [100]. The Italian Sarcoma Group reported the results of a trial of surgery and tandem autologous HCT for patients with relapsed osteosarcoma. Thirty-two patients were treated. PBPCs were mobilized with CY and etoposide. Fourteen patients had surgery before chemotherapy, and 11 had surgery at the completion of all therapy. Eleven of 32 patients were in CR before their first course of high-dose therapy, while 21 were treated with gross disease. At the end of treatment, 25 patients (78%) were in CR, six were in progressive disease, and one had died of toxicity. Despite this excellent response rate, the 3-year OS was only 20%, and 3-year DFS was only 12% [101]. These few studies suggest that there is little if any benefit to high-dose chemotherapy with HCT for relapsed osteosarcoma. In light of the lack of benefit to treatment intensification for patients with a poor response to neoadjuvant therapy, this suggests that future improvements in the treatment of patients with relapsed or refractory osteosarcoma will not come from treatment intensification, but that new approaches are required. Retinoblastoma Epidemiology and etiology Retinoblastoma affects one child of every 18,000 live births in the United States in the first 5 years of life [102]. Approximately 25–30% of cases are bilateral and follow an autosomal dominant inheritance pattern; in addition, approximately 10–15% of unilateral cases are hereditary. The observation that hereditary cases occur earlier in life and are bilateral or multifocal was key in the development of Knudson’s two-hit model of carcinogenesis [103]. Knudson proposed that patients who inherit one mutation need only a single somatic mutation in a given cell for malignant transformation, while sporadic cases required the accumulation of two independent somatic mutations to transform a single cell. Molecular and cellular biology The postulated inactivation of a tumor suppressor gene was confirmed by the demonstration of loss of heterozygosity at 13q14 [104]. The cloning of the RB1 tumor suppressor gene [105,106] was a pivotal step in understanding the pathogenesis of retinoblastoma as well as its role as a tumor suppressor gene involved in many other malignancies. The RB1 gene product is commonly absent in both sporadic and heritable retinoblastoma, as predicted by the two-hit model. The messenger RNA is ubiquitously expressed and encodes a nuclear protein with DNAbinding sequences [107]. The protein is unphosphorylated in quiescent cells, but is phosphorylated in proliferating cells [108,109], and regulates the cell cycle in its unphosphorylated form by binding the E2F1 transcription factor [110]. In addition to loss of function of both alleles of the RB1 gene, amplification of MDMX (65%) or MDM2 (10%) occurs

Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors

in the majority of human retinoblastomas, inactivating p53 and promoting the development of retinoblastoma [111]. Clinical description Retinoblastoma typically presents as leukocoria when the tumor becomes visible through the pupil or causes retinal detachment or vitreous hemorrhage. Patients may lose central vision, leading to strabismus. Less commonly, heterochromia can result from neovascularization of the iris. In developing countries, the diagnosis may not be made until the eye becomes enlarged or the orbit is invaded, but extensive disease is uncommon in the United States. In patients with germline RB1 mutations, new primary tumors may appear in the retinas for the first 3–5 years of life. In addition to bilateral retinoblastoma, these patients are at risk for primitive neuroectodermal tumors in the pineal and suprasellar regions, a presentation termed trilateral retinoblastoma. Nontransplant approaches Early detection of retinoblastoma is the norm, and the treatment outcome of patients with localized disease is excellent. Enucleation is curative for early solitary unilateral lesions, while adjuvant chemotherapy is indicated for patients with pathologic risk factors such as extension beyond the cribriform plate. Orbital extension is curable in 60–85% of patients with multimodal therapy, including external beam irradiation and systemic chemotherapy. In patients with bilateral disease, additional methods are used in an effort to preserve useful vision, including brachytherapy, cryotherapy, laser photocoagulation, and systemic as well as subconjunctival chemotherapy. In contrast, advanced central nervous system (CNS) involvement and “trilateral retinoblastoma” [112] are incurable: multimodal therapy including systemic and intrathecal chemotherapy and cranial irradiation has produced remissions which, however, have not been durable [113]. Hematogenous dissemination to bone, marrow, and viscera, and involvement of soft tissue and lymph nodes carries an extremely poor prognosis. Only ifosfamide and CY have efficacy as single agents, and have produced CRs when used in two- and three-drug combinations [113]. There is a single case report of long-term survival of hematogenously disseminated retinoblastoma in a patient treated aggressively with MAD-DOC (mechlorethamine, doxorubicin, cisplatin, dacarbazine, vincristine, and CY); however, 2 years later, the patient developed secondary myelodysplasia requiring allogeneic bone marrow transplantation [114]. The patient became a long-term survivor without recurrent retinoblastoma or myelodysplasia. HCT as consolidation for disseminated disease There are several individual case reports and small case series [115–119] of HCT for patients in CR2 after metastatic recurrence in bone or marrow. These reports describe a total of 25 patients, of whom only five experienced further progression of their disease. The remaining patients had no evidence of disease 18–107 months after relapse. The SFOP formally evaluated HCT for patients with high-risk retinoblastoma, using a single-arm protocol active from 1989 to 1994 [120]. The target population was patients with extraocular disease or histologic evidence of tumor at the cut end of the optic nerve or its subarachnoid space. Measurable extraocular disease had to be chemosensitive in order for patients to be eligible to proceed to HCT. During the study period, 34 high-risk patients were identified: eight with microscopic residual optic nerve involvement, 10 with extraocular disease confined to the orbit, 11 with extraocular involvement including distant bones or marrow, and five with CNS involvement. Nine patients did not proceed to HCT because of CNS progression (six), parental refusal (two), or toxicity (one). Therefore, 25 patients (six with optic nerve disease, seven with orbital disease, eight with distant bone or marrow disease,

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and four with CNS involvement) proceeded to HCT, with a preparatory regimen of carboplatin 1250–1750 mg/m2, etoposide 1750 mg/m2, and CY 3200 mg/m2. Five of six patients who had measurable disease at the time of HCT achieved CR. On an intent-to-treat basis, five of eight patients with optic nerve involvement, six of 10 patients with orbital involvement, five of 11 patients with bone or marrow involvement, and one of five patients with CNS involvement survived with no evidence of disease. Compared with historical results using much longer courses of conventional chemotherapy, this approach produced comparable survival for patients with optic nerve or orbital involvement, and was superior for patients with distant bone or marrow involvement [120].

Desmoplastic small round cell tumor Epidemiology and etiology Desmoplastic small round cell tumor (DSRCT) is a highly malignant abdominal small round blue cell tumor with epithelial, mesenchymal, and neural characteristics, initially described in 1991 [121]. It occurs in children and young adults and is probably not rare; over 101 cases were reported between 1989 and 1996. There is a strong male predominance, and a median age at diagnosis of 25 years [122]. There are no known associations with exposure to carcinogens [123]. Molecular and cellular biology A balanced translocation, t(11;22)(p13;q12) results in EWS-WT1 fusion transcripts considered diagnostic of DSRCT [124]. As in EWS-FLI1 fusion transcripts in Ewing’s sarcoma, these fusion transcripts retain the amino-terminal effector domain of EWS and replace its DNA-binding domain with that of its fusion partner. The fusion transcripts are heterogeneous both within and between tumors with respect to the use of specific EWS and WT1 exons, the presence of internal deletions, and the insertion of heterologous DNA. However, the splice variants are in-frame and the chimeric proteins bind WT1 response elements [125]. The transcriptional regulatory activity of EWS-WT1 differs from that of WT1. The platelet-derived growth factor A chain is directly induced by EWS-WT1, and may contribute the stromal content of DSRCT [126]. In addition, expression of IGF1 receptor, which has mitotic and antiapoptotic functions, is normally repressed by WT1, but is activated by EWS-WT1 chimeras [127]. The interleukin-2/15 (IL-2/15) receptor β chain promotes cell growth by downstream activation of signal transducer and activator of transcription-3 (STAT3) and STAT5 [128]. The brain-specific angiogenesis inhibitor-1-associated protein-3, when expressed ectopically in tumor cells, enhances anchorage-independent and low-serum growth, and may assist the secretion of fibroblast growth factor-1 (FGF-1) and FGF-2 to produce the desmoplastic reaction characteristic of the tumor [129]. The leucine-rich repeat containing 15 gene normally expressed only in placenta, in the trophoblast cell layer responsible for implantation of the embryo, is hypothesized to contribute to the invasiveness of DSRCT [130]. Clinical description Patients typically present with abdominal distention and pain, with constipation a common associated finding [122,131]. DSRCTs most commonly grow in the peritoneal cavity, with large masses and multiple implants along serosal surfaces, and may metastasize to lymph nodes, liver, lung, bone, spleen, kidney, and pleura. Characteristic computed tomography findings are of bulky intra-abdominal soft tissue masses without a distinct organ of origin [132]. Unusual sites of primary involvement include kidney, lung, bone, and pancreas. Exploratory laparotomy reveals large, firm, multinodular tumors with smooth surfaces. Conventional microscopy reveals sharply demarcated clusters of

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small round tumor cells embedded in a dense desmoplastic stroma. Immunohistochemical staining reveals expression of epithelial (cytokeratin and epithelial membrane antigen), mesenchymal (vimentin), myogenic (desmin), and neural (neuron-specific enolase and CD56) markers [122,133]. WT1 immunoreactivity results from expression of the EWS-WT1 fusion transcript. CD99 (MIC2), commonly used to identify Ewing’s family tumors, is also commonly positive [134]. Nontransplant approaches This tumor is often responsive to chemotherapy such as combinations of doxorubicin, cisplatin, CY, etoposide, and fluorouracil. However, the outcome has been dismal, with median survival of only 17 (range 3–72) months; as of 1996, only seven of 101 reported patients were alive a median of 24 (range 4–48) months from diagnosis [135]. More extensive series have been reported from single institutions. A series of 10 previously untreated patients has been reported from the Memorial SloanKettering Cancer Center. Two of these patients underwent initial gross total resection, and one died early of disease. Chemotherapy consisted of CY, doxorubicin, and vincristine alternating with ifosfamide and etoposide. All seven evaluable patients achieved PR. After second-look surgery, there were seven individuals in CR and two in PR [136]. The Washington Hospital experience included seven patients with intra-abdominal DSRCT, treated aggressively with debulking surgery and intraperitoneal chemotherapy. Two showed objective responses to chemotherapy, including cisplatin/etoposide and dacarbazine as a single agent, and these two patients experienced extended survival of 58 and 102 months from diagnosis, although all seven patients eventually died of progressive disease [123]. A series of 11 patients treated at St. Jude Children’s Research Hospital, Memphis, similarly included only two whose tumors were resectable at presentation, both of whom received adjuvant chemotherapy and are long-term survivors 8 and 10 years from diagnosis. All 11 patients received chemotherapy with the same agents as the Memorial Sloan-Kettering Cancer Center series, and seven of eight evaluable patients responded to chemotherapy [131]. In summary, complete resection of desmoplastic small cell tumors contributes to long-term survival, but these tumors are generally not resectable at initial presentation; they are usually chemosensitive, but the short duration of responses warrants attempts at consolidation with intensified therapy. Hematopoietic cell transplantation There are a few reported cases of HCT as salvage therapy for recurrent or refractory desmoplastic small cell tumor, but none has resulted in long-term survival [135,137,138]. In the Memorial Sloan-Kettering Cancer Center series, the intent was to consolidate first responses of patients with initially unresectable disease with HCT, but two of five patients did not proceed to HCT because of toxicity of initial chemotherapy. Three patients with initially unresectable disease received HCT with thiotepa and carboplatin in first response, two in CR, and one in PR. The patient transplanted in PR progressed at 15 months. The patients transplanted in CR were alive without evidence of disease 13 and 34 months from initiation of therapy [136]. The St. Jude DSRCT series included four patients who underwent autologous HCT. Two patients who received HCT after radiation therapy died of toxicity. Of the two other patients, the one transplanted in CR and treated with radiation after HCT remains alive without disease 16 months after completion of therapy. In an adult population, entered on a protocol with intent to transplant in Milan, only five of 10 patients with DSRCT achieved PR to induction therapy with ifosfamide, vincristine, and etoposide, and proceeded to HCT with a melphalan-based preparative regimen. None of the patients were converted to CR or cleared their EWS-WT1 fusion transcripts after

high-dose melphalan, and all progressed within 12 months [139]. One of these patients subsequently received allogeneic HCT after reducedintensity conditioning, and cleared his fusion transcript after developing grade III acute graft-versus-host disease (GVHD) [139], suggesting the presence of a graft-versus-DSRCT effect. Success has been achieved in small series exploring more intensive alkylator-based approaches. All four patients with DSRCT treated in first PR (n = 2) or CR (n = 2) with busulfan, melphalan, and thiotepa, with or without amifostine, remained alive after 1.1–6.4 (median 3.2) years follow-up, of whom only one had progressive disease [140]. Three patients with DSRCT treated with the VACIME (vincristine, adriamycin, CY, ifosfamide, mesna, and etoposide) regimen with repeated PBPC support became long-term survivors with no evidence of disease 25–66 months from diagnosis [141]. In summary, the role of HCT remains to be defined for patients with DSRCT, but the few long-term survivors have obtained complete surgical resection, and intensified chemotherapy may contribute to survival. Case reports of HCT for pediatric solid tumors Pleuropulmonary blastoma is a rare tumor, approximately 100 cases of which have been reported. The primary therapy is surgical excision, without adjuvant therapy. Because of the histologic resemblance to soft tissue sarcoma and Wilms’ tumor, chemotherapy regimens designed for these entities have been used. Such combination chemotherapy has produced 30–40% DFS [142]. Three patients with pleuropulmonary blastoma have received melphalan-based HCT for progressive disease [143], for chemotherapy-sensitive microscopic residual disease [142], and for refractory metastatic disease [144]. The patients with bulky refractory disease progressed soon after HCT, but the patient with microscopic residual disease was alive in continuous CR at 12 months after HCT. Esthesioneuroblastoma is a rare tumor, arising from olfactory epithelium; of over 240 cases in the literature, only 21% are pediatric. The behavior of this tumor may be more aggressive in childhood. It has been suggested that esthesioneuroblastoma may be a member of the EWST family on the basis of a t(11;22) in two of three cell lines derived from metastatic esthesioneuroblastoma cases. However, unlike Ewing’s tumors, esthesioneuroblastomas do not express MIC2, and primary esthesioneuroblastomas are characterized by trisomy 8, not t(11;22) [145]. In a single-institution series, long-term survival was achieved in three of five adult patients salvaged with CY-containing HCT preparatory regimens versus four of 17 salvaged with conventional chemotherapy [146]. One adolescent was treated for a cervical nodal recurrence with modified radical neck dissection, and consolidation with high-dose carboplatin, etoposide, melphalan, and HCT, and has no evidence of disease 1 year later [147].

Future directions Patterns of failure After HCT for Ewing’s tumors, the majority of relapses are metastatic, most commonly in lung, and secondly in bone [148,149]. A more recent analysis indicates that bony failures often occur in involved bones outside the local radiation field, and in lesions detected by magnetic resonance imaging (MRI) or positron emission tomography (PET) scanning, but not bone scans [150]. Possible solutions to this problem include irradiating the lungs and the entire involved bone system prophylactically and incorporating the use of MRI and/or PET scans in staging before HCT. In contrast, failures after HCT for rhabdomyosarcoma tend to occur early, at a median of 4 months after HCT, almost always (23 of 26 cases)

Hematopoietic Cell Transplantation for Other Pediatric Solid Tumors

in previously known sites [47]. This observation argues for the use of local radiation to all previously known sites of disease.

Regimens Role of TBI For all but the most radiosensitive tumors, the dose of radiation therapy that can be delivered as TBI is inadequate for control of bulky tumors. Doses of adjuvant radiation demonstrated to control the majority of microscopic residual disease (typically 106–108 cells) exceed the maximum tolerable doses of TBI [151]. However, the relationship between the dose of radiation and the percent reduction in risk of recurrence appears to be linear. Extrapolating from these dose–risk data, zero reduction in risk occurs at a dose between 0 and 5 Gy, indicating that there is a low threshold, if any, for tumor cell killing by radiation [152]. Therefore, the modest doses of radiation feasible as TBI may be expected to control very small micrometastases (perhaps 102–104 cells). The question of whether TBI is a necessary or desirable component of the high-dose cytoreductive regimen has been addressed retrospectively by the EBMT. For rhabdomyosarcoma patients, addition of TBI to melphalan appears only to increase toxicity [153]. A retrospective analysis of the German–Austrian Pediatric BMT Group also found no benefit of 12 Gy hyperfractionated TBI added to melphalan, etoposide, and carboplatin [47]. For Ewing’s sarcomas, the EBMTR observed that patients receiving TBI fared worse (n = 30, 19% EFS) than those receiving high-dose chemotherapy alone (n = 33, 34% EFS); the best results were obtained with chemotherapy combinations that included busulfan (51% EFS) [154]. Tandem transplants A substantial number of tandem transplants, in which two or more HCTs are performed with minimal recovery time between transplants, have already been performed in an attempt to improve tumor control. This approach is most likely to be effective if there is more than one active and noncrossresistant cytoreductive regimen, without cumulative toxicity. Even if a single regimen must be used twice, repeated application may, if a similar log cell kill may be obtained in a second course, overcome problems of delivery of drug to the core of a bulky tumor. Whether tandem HCTs as delivered so far have been beneficial is controversial. The results of several institutional series indicate longer responses in patients receiving multiple courses of high-dose therapy [155–157]. However, the cumulative experience of EBMT provides no convincing evidence in favor of double transplants in Ewing’s sarcoma [154]. In addition, at least with carboplatin and etoposide, no patient improved his or her response with a second course [158]. The use of PBPCs instead of bone marrow may improve the efficacy of tandem transplants because faster hematopoietic recovery may allow greater treatment intensity. Two courses of melphalan 100 mg/m2 could consistently be administered with PBPC rescue within 21–34 (median 24) days with no change in pharmacokinetics or pharmacodynamics [159]. A limited institution pilot study demonstrated the feasibility of tandem PBPC transplant for metastatic neuroblastoma or sarcoma, delivering CY 3600 mg/m2, etoposide 2400 mg/m2, and carboplatin 2000 mg/ m2 followed within 28–42 days by melphalan 180 mg/m2 and TBI 12 Gy in 46 of 51 eligible patients [160,161]. Of the five patients who received only one HCT, three were by patient request and two were ineligible to proceed because of liver toxicity. There were four toxic deaths (9%). Three courses of HCT have been administered to 17 of 22 patients, with one toxic death in the acute transplant period [162]. Although short-term toxicity may be acceptable, this pilot trial shows the importance of longterm follow-up, because three patients have developed pancytopenia as

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a late complication, two with graft failure and one with monosomy 7 associated myelodysplastic syndrome. A novel variation is the use of double high-dose therapy with only one HCT procedure [163]. Because the dose-limiting toxicity of melphalan and thiotepa in the setting of hematopoietic cell rescue is mucosal, and the affected tissues recover relatively quickly, two cycles can be given 1 week apart, and patients rescued with a single PBPC product after the second cycle. This strategy allows the safe delivery of thiotepa 1000 mg/m2 and melphalan 280 mg/m2 over 9 days. The indications for HCT were heterogeneous in this phase I–II trial, but 10 of 13 patients entering HCT in CR and six of 13 patients entering HCT with residual disease are alive with no evidence of disease 15–59 (median 35) months after transplantation. Of particular interest is that all three patients with hepatoblastoma who entered HCT with persistent alphafetoprotein elevation achieved long-term CR [163]. Targeted therapy Targeted therapeutics refers to treatments designed to exploit a biologic feature of the tumor to eradicate it. Examples of targeted therapies include tyrosine kinase inhibitors such as imatinib mesylate, and ligandtargeted monoclonal antibodies such as rituximab. There are two significant ongoing applications of targeted therapy for pediatric solid tumors that involve HCT: 131I-metaiodobenzylguanidine for the treatment of neuroblastoma (see Chapter 66) and 153Sm-ethylenediaminetetramethylene phosphonic acid (EDTMP) for the treatment of osteosarcoma. Each of these treatments utilizes a fundamental characteristic of the tumor to direct a radiopharmaceutical agent specifically to the tumor, and ameliorates the myelosuppression with autologous hematopoietic cell support. 153 Sm-EDTMP consists of a radioisotope (153Sm) conjugated to a tetraphosphonate compound that localizes to sites of bone turnover, such as a metastatic bone lesion. Because 153Sm-EDTMP targets bone lesions by a mechanism similar to the radiotracer used in a bone scan, lesions that are visible on bone scan, including both primary osteosarcoma and metastatic deposits, are targeted by this agent. Radioactive decay of the 153Sm yields a medium energy electron (β particle) and a lowenergy photon. The electron has a very short path length (1–2 mm), thus providing exquisitely precise targeting of the cytotoxic energy. The photon can be detected by the same instrument used for a diagnostic bone scan, allowing uptake at a target tumor to be confirmed and the amount of radiation delivered to the tumor to be measured. The half-life of 153Sm is very short (46 hours), facilitating handling. This compound was originally tested in adult patients with bone metastases from carcinomas, such as breast and prostate cancer, and is approved by the United States Food and Drug Administration for palliation of painful metastases. In 2002, Anderson and colleagues published the results of a phase I study of 153Sm-EDTMP for the treatment of high-risk osteosarcoma patients [164]. Thirty patients were treated with 1–30 mCi/kg of 153SmEDTMP. Because this compound significantly depresses bone marrow function, patients were rescued with an infusion of autologous PBPCs 14 days after treatment. Other than low blood counts, the only side-effect was transient hypocalcemia in patients treated with the highest dose. All of the patients began the trial requiring narcotics for pain relief, and every patient experienced a decrease or elimination of their narcotic requirement. In an attempt to improve efficacy, this group subsequently treated patients with gemcitabine, a chemotherapeutic agent that is thought to also act as a radiation sensitizer [165]. Fourteen patients were treated with high-dose 153Sm-EDTMP followed 1 day later by gemcitabine. After 2 weeks, autologous PBPCs were reinfused. At 6–8 weeks of follow-up, there were six partial remissions and two mixed responses, but none of these responses was durable. Thus, 153Sm-EDTMP

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shows promise for treating osteosarcoma, but further work will be required to determine its optimal use. Hematopoietic cell grafts The source of the graft: peripheral blood hematopoietic cells versus bone marrow It is now recognized that mobilizing and collecting PBPCs is feasible in children despite their small size and the intensity of the pediatric chemotherapy regimens, and PBPCs produce faster engraftment [166,167] and immune reconstitution [168] than marrow. Although tumor cells can frequently be detected in mobilized PBPC grafts [169,170], the question of whether the purging of autologous hematopoietic cells can improve outcome remains to be answered. Few data are available regarding the relationship between tumor contamination of the graft with survival outcomes, but long-term DFS can occur in patients with Ewing’s sarcoma who receive PBPC grafts containing detectable fusion transcript [171]. In rhabdomyosarcoma, the major determinant of long-term survival after HCT was whether CR was achieved prior to HCT [153], and 23 relapses occurred in previously known sites, compared with only three in new metastatic sites [47], suggesting that tumor control in the body, not reinfusion of tumor cells in the graft, is the major problem. Allogeneic HCT The role of allogeneic HCT is being explored in pediatric solid tumors. Allogeneic transplantation carries the risk of GVHD, but its potential benefits include the absence of tumor cells in the graft and perhaps an immunologic antitumor effect. For rhabdomyosarcoma, the German– Austrian Pediatric BMT Group found no evidence of benefit from allogeneic HCT, as none of five allogeneic HCT recipients survived [47]. In addition, donor lymphocyte infusion was reported to be ineffective in a case of relapsed rhabdomyosarcoma [172], but since the patient did not have measurable disease at the time of donor lymphocyte infusion and did not develop GVHD, this case did not address the activity of an effective immune response. A CR after reduced-intensity matched sibling peripheral blood HCT for a patient who had failed two prior lines of therapy including autologous HCT suggests benefit from an allogeneic effect [173]. For Ewing’s tumors, there was a trend toward better outcome with allogeneic HCT (50% survival versus 23% survival of the entire group of 27 allografted patients at a median of 56 months) in the singleinstitution experience with (TB)I + melphalan + etoposide with or without carboplatin of Vienna, from 1984 to 1996 [174]. With longer follow-up, incorporating the experience from Düsseldorf, DFS after allogeneic HCT was not better than after autologous HCT for Ewing’s tumors because of higher rates of nonrelapse mortality [31]. However, there are informative case reports of patients with recurrent, metastatic Ewing’s sarcoma who underwent allogeneic HCT and whose disease responded to withdrawal of immune suppression [173] or institution of IL-2 therapy [175] with subsequent development of acute GVHD. However, few data are available regarding allogeneic HCT for pediatric solid tumors. This probably results largely from the role of neuroblastoma as the prototypical poor-prognosis pediatric solid tumor, and the clear consensus that there is no role for allogeneic transplantation in neuroblastoma [176,177]. The lack of benefit from allogeneic HCT would indicate that tumor contamination of autologous grafts is not a major source of relapse, and that alloreactive cytotoxic T-lymphocytes (CTLs) are not substantially effective against high-risk pediatric solid tumors. However, a potentially important biologic difference between neuroblastoma and sarcomas is that, in vitro, neuroblastoma cells are poor targets for CTLs, whereas sarcoma cells are excellent CTL targets [178].

Immunotherapy Rhabdomyosarcoma and Ewing’s tumors are targets both for natural killer cells and for CTLs [179], and tumor-infiltrating lymphocytes isolated from osteosarcomas can lyse allogeneic osteosarcoma cell lines [180]. The possibility that these immunologic effector mechanisms may be clinically important in high-risk Ewing’s tumors was suggested in the pilot trial of hyperfractionated TBI with melphalan and etoposide conducted in Vienna and Düsseldorf. In their initial cohort of 17 patients with very high-risk Ewing’s tumors, three of four allograft recipients were disease-free survivors, along with three of five autograft recipients treated with IL-2 versus two of eight autograft patients without IL-2 [181]. The advantage of IL-2 after autografting persisted in a larger European Intergroup Cooperative Ewing’s Sarcoma Study cohort treated using the same approach: DFS was 52% for patients receiving IL-2 after HCT versus 22% without IL-2 (p < 0.05) [150]. The possible effector mechanisms involved in patients receiving IL-2 after HCT have been explored, and expansion of T and natural killer (NK) cell numbers, but not those of NK/T cells was demonstrated. NK activity was enhanced, in spite of increased activation of the CD94 inhibitor receptor [182]. The National Cancer Institute has conducted a pilot trial of tumorspecific vaccination for Ewing sarcoma and alveolar rhabdomyosarcoma, based on pulsing dendritic cells with the unique EWS-Fli1 and PAX3-FKHR fusion peptides associated with the t(11;22) and t(2;13) translocations characteristic of these tumors [183]. Sixteen patients were enrolled, and 15 received at least one vaccination. Unfortunately, most patients experienced tumor progression during the first 6-week vaccination cycle, so only four patients received two or more cycles of vaccination. One of these four patients had a mixed response, and another had a fusion peptide-specific lymphocyte proliferative response. This study is important not only because of the encouraging suggestion of some immunologic activity against tumor, but also because it identifies several pitfalls of immunotherapy. These patients had severely impaired immunity and large tumor burdens, and the immature circulating dendritic cells used may actually suppress immune responses [183]. Moreover, a single peptide antigen was used, with uncertain human leukocyte antigen restriction and cell surface expression, and the tumor itself may have induced tolerance or anergy in autologous lymphocytes. An alternative strategy of generating anti-tumor CTL cell lines ex vivo has been explored in Ewing’s sarcoma patients. Specific antitumor CTLs were generated from three patients, all of whom had undergone prior autologous HCT, after two or more cycles of stimulation of autologous peripheral blood mononuclear cells with autologous peripheral bloodderived dendritic cells, CD4+ lymphocytes, and irradiated early-passage autologous tumor cells [184]. Although the approach is labor intensive, potential advantages include the response to unknown as well as previously identified tumor antigens, and the reduced potential for escape by loss of antigen expression. The clinical utility of such cell lines has yet to be demonstrated. The development of reduced-intensity allogeneic HCT offers an alternative avenue to immunotherapy for pediatric solid tumors, but novel approaches will be required to demonstrate activity if this approach is beneficial only in minimal residual disease.

Conclusion The prognosis of patients with metastatic, recurrent or refractory pediatric solid tumors is dismal. High-dose preparatory regimens with HCT have demonstrated activity against a broad spectrum of the pediatric solid tumors, and HCT has produced promising results in the treatment of Ewing’s tumors. Prospective randomized trials of HCT versus con-

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ventional chemotherapy are in progress for patients with poor-prognosis Ewing’s tumors and are being considered for high-risk recurrent Wilms’ tumor. For less common indications, phase I and II trials are appropriate.

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Approaches now being investigated to improve the efficacy of HCT for pediatric solid tumors include tandem HCT, targeted therapy, chemoprotection, and immunotherapy, possibly via allogeneic HCT.

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114. Petersen RA, Friend SH, Albert DM. Prolonged survival of a child with metastatic retinoblastoma. J Pediatr Ophthalmol Strabismus 1987; 24: 247– 8. 115. Ekert H, Tiedemann K, Waters KD, Ellis WM. Experience with high dose multiagent chemotherapy and autologous bone marrow rescue in the treatment of twenty-two children with advanced tumors. Austr Paediatr J 1984; 21: 195–201. 116. Dunkel IJ, Aledo A, Kernan NA et al. Successful treatment of metastatic retinoblastoma. Cancer 2000; 89: 2117–21. 117. Rodriguez-Galindo C, Wilson MW, Haik BG et al. Treatment of metastatic retinoblastoma. Ophthalmology 2003; 110: 1237–40. 118. Kremens B, Wieland R, Reinhard H et al. Highdose chemotherapy with autologous stem cell rescue in children with retinoblastoma. Bone Marrow Transplant 2003; 31: 281–4. 119. Matsubara H, Makimoto A, Higa T et al. A multidisciplinary treatment strategy that includes high-dose chemotherapy for metastatic retinoblastoma without CNS involvement. Bone Marrow Transplant 2005; 35: 763–6. 120. Namouni F, Doz F, Tanguy ML et al. High-dose chemotherapy with carboplatin, etoposide and cyclophosphamide followed by a haematopoietic stem cell rescue in patients with high-risk retinoblastoma: a SFOP and SFGM study. Eur J Cancer 1997; 33: 2368–75. 121. Gerald WL, Miller HK, Battifora H, Miettinen M, Silva EG, Rosai J. Intra-abdominal desmoplastic small round-cell tumor. Report of 19 cases of a distinctive type of high-grade polyphenotypic malignancy affecting young individuals. Am J Surg Pathol 1991; 15: 499–513. 122. Lae ME, Roche PC, Jin L, Lloyd RV, Nascimento AG. Desmoplastic small round cell tumor: a clinicopathologic, immunohistochemical, and molecular study of 32 tumors. Am J Surg Pathol 2002; 26: 823–35. 123. Gil A, Gomez Portilla A, Brun EA, Sugarbaker PH. Clinical perspective on desmoplastic small round-cell tumor. Oncology 2004; 67: 231–42. 124. Ladanyi M, Gerald W. Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor. Cancer Res 1994; 54: 2837–40. 125. Liu J, Nau MM, Yeh JC, Allegra CJ, Chu E, Wright JJ. Molecular heterogeneity and function of EWS-WT1 fusion transcripts in desmoplastic small round cell tumors. Clin Cancer Res 2000; 6: 3522–9. 126. Lee SB, Kolquist KA, Nichols K et al. The EWSWT1 translocation product induces PDGFA in desmoplastic small round-cell tumour. Nat Genet 1997; 17: 309–13. 127. Finkeltov I, Kuhn S, Glaser T et al. Transcriptional regulation of IGF-I receptor gene expression by novel isoforms of the EWS–WT1 fusion protein. Oncogene 2002; 21: 1890–8. 128. Wong JC, Lee SB, Bell MD et al. Induction of the interleukin-2/15 receptor beta-chain by the EWSWT1 translocation product. Oncogene 2002; 21: 2009–19. 129. Palmer RE, Lee SB, Wong JC et al. Induction of BAIAP3 by the EWS–WT1 chimeric fusion implicates regulated exocytosis in tumorigenesis. Cancer Cell 2002; 2: 497–505. 130. Reynolds PA, Smolen GA, Palmer RE et al. Identification of a DNA-binding site and tran-

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John A. Zaia, Amrita Krishnan, & John J. Rossi

Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection

Introduction The acquired immune deficiency syndrome (AIDS) is in many ways analogous to a neoplastic disorder because it is a complex disease with multiple pathogenic features, is treated with modalities that permit at least temporary clinical remission but that fail to cure without eradication of latent virus, and is subject to development of resistance to chemotherapy. Thus, similar to certain cancer therapies, it is not unusual to propose that hematopoietic cell transplantation (HCT) could have a role in the management of AIDS. Several features intrinsic to human immunodeficiency virus type 1 (HIV-1) (Fig. 68.1) infection contribute to the complex pathogenicity of this viral infection. These include an early infiltration and seeding of the hematopoietic tissue with HIV-infected cells, a virus replication cycle that occurs predominantly within this tissue and is coupled to cell activation and function, and virus replication that occurs in multiple cell types and tissues (e.g. thymus, brain, and gut) at all stages of the disease [1]. There is gradual destruction of lymph nodes during the asymptomatic phase of infection and eventual loss of function of the immune system [2]. Confounding the pathogenic features of the disease is the genetic instability of the virus that results in HIV-1 variants in the infected individual [3], a potential constellation of drug-related adverse effects, and the difficulty of adherence to drug regimens that require significant adjustments in lifestyle and daily activity [4]. Two factors, in particular the emergence of drug resistance and the continued replication of drugresistant HIV-1 variants in tissue, undermine the clinical benefit of antiretroviral chemotherapy. In this setting, the potential for cell-based therapy to re-establish an immune system resistant to HIV-1 has been suggested. Because HIV-1 is dependent on a variety of well-described molecular events for either infection or replication, gene-based strategies that could inhibit these events are being assessed as potentially complementary therapies for HIV-1 infection. It is the purpose of this chapter to describe the experience with HCT in AIDS and to view the current status of therapeutic approaches for HIV-1 treatment using gene-based approaches to HIV-1 infection.

Epidemiology and etiology HIV-1 spreads via contact with infectious body fluids and gains entry into susceptible cells following interaction of the viral envelope proteins

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

and a primary cellular receptor, CD4 (Fig. 68.2) [5]. Of hematopoietic interest, chemokine receptors, primarily CXCR4 and CCR5, are coreceptors required for viral entry [6,7]. Chemokines are a diverse family of peptides that bind to receptors and activate immune and/or inflammatory responses [8]. The chemokines with two consecutive cysteines are C-C chemokines, and those with an intervening amino acid are C-X-C chemokines. Natural ligands for CCR5, such as RANTES and macrophage inflammatory protein-1 alpha, can compete with HIV-1 for binding to this receptor and inhibit HIV infection [9]. The ligand for CXCR4 is stromal-derived factor 1, which can also inhibit HIV-1 infection [10]. A meta-analysis of patient cohorts has shown that only the CCR5-Δ32 and the CCR2-64I co-receptor mutations are associated with strong protective effects from progression of HIV-1 infection [11]. As a lentivirus, a subgroup of the retrovirus family, HIV-1 shares major genetic features common to all retroviruses [12]. Based on related findings, a model has emerged that describes the sequence of events leading to HIV-1 entry. Initial virus–cell interaction occurs between the HIV-1 envelope protein (gp120) and the CD4 receptor. The gp120–CD4 complex then engages one of the chemokine receptors, depending on the specific HIV-1 isolate (CCR5 for macrophage-tropic isolates called “R5 strains;” CXCR4 for T lymphocyte tropic or X4 strains) [6,7]. Many other chemokine co-receptors have subsequently been identified that can fulfill this co-receptor function in vitro, but CCR5 and CXCR4 appear to be most critical in vivo [13]. Following formation of the HIV envelope–CD4 receptor–chemokine receptor heterotrimer complex, there is an alteration of the membrane that leads to exposure of a fusion domain contained within the HIV-1 fusion protein (gp41) [14]. The engagement of gp41 with the target cell membrane culminates in penetration of HIV1 into the cell, with subsequent movement into the nucleus. Chemokine receptors have a role in leukocyte trafficking and in inflammatory responses, raising the legitimate concern that therapeutic anti-HIV manipulations, designed to interfere with the receptors’ normal cellular functions, might impinge on cellular homeostasis. However, the findings that these receptors are minimally polymorphic and that individuals with homozygous mutations exhibit no ill effects, yet display relative resistance to HIV-1 infection [10,11,15–18], have fueled the search for strategies that block this very first event in HIV-cell interaction. Based on the crystal structure of HIV envelope [19], specific compounds that inhibit the envelope–CCR5 interaction [20], as well as the gp41-mediated fusion reaction [21], have been designed; one of these fusion inhibitors, enfuvirtide (Fuzeon), has been approved in the United States for use in patients with AIDS. It is likely that additional compounds that inhibit these early steps in the viral life cycle will be designed and developed for clinical applications.

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Chapter 68

Nef Ltr

Y

Vpu

Vif

Gag Pol

Vpr

Ltr

Env Tat

Tat Rev

Rev

Fig. 68.1 Schematic structure of human immunodeficiency virus type 1 genome. env, envelope; Gag, group antigen; Ltr, long-terminal repeat; Pol, polymerase; Rev, regulator of expression of viral proteins; Tat, transactivator of transcription; Vif, Vpu, Vpr, Nef, accessory proteins. (Reproduced from Berkhout et al. [150] with permission.)

Chemokine receptor

Infectious virion CD4

Uncoating Viral RNA RT RNaseH Circular DNA

Integrase

Unintegrated linear DNA

Tat/Rev

Integrated proviral DNA

mRNA

Protein synthesis

Genomic RNA Protease

Assembly and budding

Mature virion

Fig. 68.2 Life cycle of human immunodeficiency virus type 1 (HIV-1). HIV-1 binds to susceptible CD4+ T lymphocytes via the cellular CD4 receptor, and enters the cell via a fusion process facilitated by a chemokine receptor. Following penetration, the viral RNA is reverse transcribed by the viral enzyme reverse transcriptase (RT) to an RNA–DNA duplex that is degraded by viral ribonuclease H (RNaseH). Second-strand complementary DNA (cDNA) is completed by the viral RT. The cDNA then migrates to the nucleus, where it integrates into cellular chromosomes. Following activation by either viral regulatory proteins or cellular factors, viral transcription commences, producing Tat and Rev. Under this regulation, an internal circuit exists in which polypeptide precursors are processed by a virus-encoded protease, as shown. The mature peptides and two copies of the genomic RNA eventually migrate to the cell membrane, assemble together, and finally bud from the cell as a mature virion. All of these complex steps are potential targets for gene therapy strategies. mRNA, messenger RNA.

Molecular and clinical biology Lentiviruses, as a group, exhibit genomic complexity far beyond that of a prototype retrovirus, which consists of only the gag, pol, and env genes [12]. Six additional regulatory genes (Fig. 68.1) with a spectrum of biologic function have been identified in HIV-1 (with seven identified in simian immunodeficiency virus, the simian homolog of HIV). Two of these genes, tat and rev, are required for virus replication. The Tat protein is involved early in the virus life cycle, with a primary role in transcriptional activation of the viral promoter, and the HIV requirement

for Tat-induced activation appears to depend on the type and activation state of the infected cell. In contrast, Rev appears to be absolutely required for HIV-1 replication and acts as a temporal gatekeeper later in the replication cycle, allowing transport of late messenger RNAs from the nucleus to the cytoplasm, where they are translated into structural proteins for assembly of progeny virus [22]. There currently are no antiTat and anti-Rev small-molecule compounds in clinical trials, but these have been the target for gene transfer studies. The remaining four unique genes in HIV-1 are the “accessory” genes nef, vif, vpr, and vpu (reviewed Derdeyn et al. in [23]). These genes are

Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection

termed “accessory” based on early studies suggesting that they are dispensable for virus growth in vitro. However, more comprehensive studies performed in vitro or in experimental models that simulate in vivo conditions (e.g. primary T lymphocytes and macrophages infected with clinical HIV-1 isolates) identified several functions for the Nef, Vif, Vpr, and Vpu proteins that are important in HIV-1 replication and pathogenesis. Moreover, each of these regulatory proteins is known to be multifunctional, having distinct roles at different stages of the virus life cycle and in pathogenic events. Replication and T-lymphocyte homeostasis Critical to the understanding of HIV-1 molecular biology and pathogenesis is the mechanism by which CD4+ lymphocyte reduction and eventual loss of immune function occurs. Proposed mechanisms of pathogenesis have evolved as the understanding of HIV-1 infection has increased. Early in the epidemic, much of the immunologic damage of AIDS was proposed to be a manifestation of autoimmune disease [24]. Later studies led to the “sink and drain” model, which uses as its basis the dynamics of HIV-1 RNA levels in the blood (viral load) and CD4+ lymphocyte turnover following the initiation of highly active antiretroviral therapy (HAART) [25,26]. Studies leading to this hypothesis demonstrated that a rise in CD4+ lymphocyte count correlated with a reduction in HIV-1 replication, and that HIV-1 infection appeared to directly destroy the CD4+ T-lymphocyte compartment. The sink and drain model postulated the direct killing (“drain”) of CD4+ lymphocytes and elimination of the CD4+ lymphoid compartment (“sink”) by HIV-1 infection. In this model, a hypothetical hematopoietic “faucet” dynamically replenishes this loss until the putative supply of T cells becomes exhausted, thereby resulting in overt immunodeficiency. Based on this hypothesis, antiviral agents are expected to reverse the loss of CD4+ lymphocytes and restore immune function. However, at the early stages of HIV-1 infection when CD4+ lymphocytes are in decline and the memory CD8+ lymphoid compartment is relatively increasing, both naïve CD4+ and CD8+ T lymphocytes decline at the same rate. Because naïve CD8+ lymphocytes are not usually infected with HIV-1, and naïve CD4+ lymphocytes are relatively resistant to HIV-1 infection, these declining numbers cannot be easily explained by direct killing of cells by HIV-1 [27]. Rather, HIV-1 infection appears to result in a dysregulation of Tlymphocyte homeostasis involving the peripheral pools of these cells and their altered thymic output [28]. These observations have been consolidated into a “redistribution” hypothesis, which postulates that several cytokines are released coincident with active HIV-1 infection and the ensuing lymphoid cell activation [29]. In turn, these cytokines cause trapping of T lymphocytes in peripheral lymphatic sites, where they are efficiently infected by HIV-1 [30,31]. With potent anti-HIV-1 therapy, the viral load is diminished and T lymphocytes are spared from infection. As the levels of the cytokines decline, uninfected cells are released from the lymphatic tissue and enter the peripheral blood, resulting in an increase in CD4+ lymphocytes, albeit a moderate one. This model also predicts that the redistribution of CD4+ and CD8+ lymphocytes will increase as AIDS progresses. This prediction is borne out in clinical studies, demonstrating that the initial response to potent antiHIV-1 chemotherapy is greater in patients with advanced disease compared with that in patients at earlier stages of the disease [32]. Immune reconstitution during potent antiviral therapy With continued chemotherapy and repression of HIV-1, naïve T lymphocytes slowly increase [32]. The degree to which naïve T

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lymphocytes can be restored and their repertoire expanded will most likely determine the success of such treatment. Reports that pretreatment perturbations in the CD4+ lymphocyte immune repertoire are gradually alleviated during potent anti-HIV-1 chemotherapy [33] have not been confirmed in all studies [34]. While it appears that certain recall immune responses lost during HIV-1 disease progression may be reestablished following initiation of anti-HIV-1 chemotherapy, responses to other antigens, particularly those encoded by HIV, do not appear to be reestablished as readily. This variable recovery may be explained in part by the observation that HIV-specific CD4 cells are preferential targets for HIV infection [28]. In order to return to a situation in which T-lymphocyte homeostasis and normal immune function exist, strategies that directly influence immune reconstitution will most likely be required to effectively complement anti-HIV-1 chemotherapy. The ability of HAART to restore antigen-specific immune functions will undoubtedly depend on a number of factors, including the time at which therapy is initiated after HIV-1 infection and the extent and duration of disease progression. Indeed, recent clinical findings indicate that the strength of immune responses to both HIV and non-HIV antigens may be proportional to the CD4+ cell nadir before HAART initiation [35]. Restoration of immune functions will be strongly affected by the ability of the patient to generate new populations of naïve T lymphocytes, a process believed to be largely dependent on the presence of a functional thymus. Thymic function can be affected not only by HIV-1 infection, but also by age, stress, and underlying medical conditions [36,37]. Understanding such person-to-person heterogeneity might shed light on what appear to be conflicting results concerning immune system restoration in different patient populations following potent antiviral therapy [33,34,38,39].

Cellular and anatomic reservoirs of HIV It is now well established that a reservoir of latently infected cells (resting memory CD4+ lymphocytes) persists for prolonged periods of time, even in patients on potent antiviral therapy whose viral loads have decreased to undetectable levels [40–42]. These cells could form a pool from which HIV-1 replication could be rekindled following drug withdrawal or HIV-1 gene activation. In cases where anti-HIV-1 chemotherapy has been suspended during structured treatment interruption or following prolonged use, a rebound of replicating virus has occurred in the majority of patients studied within a few weeks [43,44]. This rapid reappearance of virus indicates that HIV-1 eradication has not occurred and implies that continuous combination therapy must be maintained at all times, possibly for the life of the patient, in order to keep HIV-1 at bay. In contrast to the demonstrated requirement for continuous drug therapy for most patients, a few reports have suggested that, in certain rare circumstances, patients can control viral rebound after withdrawal of the drugs [45,46]. In addition, in rare HIV-infected persons, there is control of HIV viremia for long periods without treatment [47–49]. These persons have CD8-specific adaptive immunity to HIV, which appears to be human leukocyte antigen restricted and is quantitatively different from what is seen in those with progressive HIV infection [50,51]. The confirmation of these observations and the mechanisms responsible for long-term viral suppression in these patients are subjects of intense investigation [43,50]. Encouragingly, a recent report estimates that the half-life of the latent viral reservoir is approximately 4.6 years, which suggests that, if a patient is on continual antiretroviral therapy from the early onset of infection, it may be possible to completely eradicate the latent reservoir in 7.7 years [52].

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Clinical description Antiretroviral therapy Combination antiretroviral therapy using reverse transcriptase and protease inhibitors has resulted in dramatic reductions in HIV-1 blood levels [25,26,53] and in prolonged survival [54]. Current standards of care involve the use of multiple drugs that target both HIV-1 reverse transcriptase and protease proteins [4]. In optimal circumstances, and with careful adherence to the rigid schedule of drug administration demanded by this therapy, many patients achieve dramatic reductions in plasma viral load, often to undetectable levels. It is not clear how long these levels can remain suppressed, but many patients have maintained viral suppression for years while on continuous antiviral therapy. However, these treatments are not without associated complications, which have resulted in current guidelines that these therapies not be initiated in asymptomatic patients until the level of CD4+ cells has declined to 350 cells/mL [4]. Further, the risk of generating drug-resistant variants increases with time on treatment. Because the presence of even low levels of HIV-1 replication in persons receiving anti-HIV-1 treatment is associated with drug resistance [55,56], questions of longterm efficacy of these treatments remain unknown. Nevertheless, as improvements have occurred in antiretroviral chemotherapies, AIDS is becoming more of a chronic disease, with the need to manage the infection in the setting of advancing age [57]. From a hematologic standpoint, with this improved long-term survival, the risk of developing lymphoma is a concern, and an understanding of cancer treatment in the AIDS patient is important. Lymphoma and AIDS The presence of HIV-1 infection and aggressive B-cell lymphomas (specifically Burkitt’s lymphoma, immunoblastic lymphoma, and lymphoma of the central nervous system) was made part of the definition of AIDS in 1985 [58]. The two other cancers associated diagnostically with AIDS are Kaposi’s sarcoma and cervical cancer. A meta-analysis of the incidence of cancer in persons with HIV/AIDS (n = 444,172), compared with iatrogenically immunosuppressed transplant recipients (n = 31,977), found that both immunosuppressed groups share an increased risk for the three types of cancer linked by definition to AIDS, namely Kaposi’s sarcoma, non-Hodgkin’s lymphoma (NHL), and cervical cancer, as well as for all human papilloma virus-related cancers, for Hodgkin’s lymphoma (HL), liver cancer, and stomach cancer [59]. The lymphomas associated with Epstein–Barr virus infection, namely HL and NHL, or with Kaposi’s sarcoma-associated herpesvirus infection, namely primary effusion lymphoma and plasmablastic lymphoma, have a much higher incidence in HIV/AIDS than in other immunosuppressed groups [59]. Thus, the World Health Organization classifies AIDSrelated lymphoma (ARL) into three groups: those lymphomas that occur in immunocompetent patients (e.g. Burrkitt’s lymphoma, diffuse large cell lymphoma of centroblastic or immunoblastic types, HL, T-cell lymphoma, and extranodal mucosa-associated marginal zone B-cell lymphoma), those lymphomas occurring in HIV/AIDS patients (e.g. primary effusion lymphoma and plasmablastic lymphoma), and lymphomas occurring in other immunodeficiency states (e.g. polymorphic B-cell lymphoma) [60]. Effect of HAART on incidence of AIDS-related lymphoma Prior to the availability of HAART, patients with HIV infection had a 60- to 600-fold increased risk of developing NHL compared with the general population and a 7 to 20-fold increased rate of HL [61–63]. With

the availability of HAART, there has been a reduced incidence of opportunistic infections and Kaposi’s sarcoma, increased CD4 counts, and improved overall survival [64,65]. Of the many studies that have assessed the impact of HAART on the incidence of AIDS lymphoma, there are paradoxical changes due to the fact that the proportion of HIV-infected persons progressing to AIDS has declined, the absolute number of ARL cases has decreased, but the relative incidence of some lymphomas has actually increased [66,67]. A meta-analysis of the incidence of ARL in 47,936 HIV-infected persons showed that the incidence rate for NHL declined from 6.2 to 3.6 per 1000 person-years with largest declines in primary central nervous system lymphoma and immunoblastic lymphoma [68]. Yet the occurrence of NHL increased from 3.6% of AIDSdefining illnesses in 1994 to 5.4% in 2000, a change that was significant (p <0.001) [67]. Similar increases in the proportion of AIDS-defining NHL have occurred in the United States and Australia [60]. ARL can occur as a relatively early or as a late manifestation of HIV-1 infection [67,69] and can occur in individuals with CD4 cell counts of greater than 200/mm3 and without prior histories of opportunistic infection [70]. Of interest, the incidence of ARL remained unchanged in the pre- and post-HAART eras for certain groups with similar CD4 counts, and the decline in ARL in the post-HAART era was due to the decreased proportion of persons with very low CD4 counts [71]. Within ARL, the major histologic lymphoma types are Burkitt’s lymphoma (ARL-BL) and diffuse large cell lymphoma (ARL-DLCL). Since the advent of HAART, the relative proportion of cases of ARL-BL has declined from approximately 24% in the pre-HAART era to approximately 10% of ARL in the post-HAART era [72,73]. In addition, since the widespread availability of HAART in 1996, the risk for developing HL has increased [74]. In a large multicenter study of the relationship of HIV, HAART, CD4 counts, and HL, analysis of cancer registry data revealed an incidence of HL highest in moderately immunosuppressed patients and lower in the more severely immunodeficient patients with advanced HIV infection. It is postulated that this is due to the requirement for healthy non-neoplastic cells to enhance the growth of Reed– Sternberg cells in the development of HL [74].

Nontransplant approaches to AIDS-related lymphoma treatment Given the widespread disease presentation of AIDS lymphoma, systemic therapy is the treatment of choice [60,68,75]. Initial studies to treat NHL, comparing standard-dose methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine, and dexamethasone (m-BACOD) with reduced dose m-BACOD, produced different response rates (52% versus 41%, respectively) and median survival (6.8 months versus 7.2 months, respectively), but with less hematologic toxicity and fewer opportunistic infections in the reduced-dose regimen [76]. With the availability of HAART, more aggressive chemotherapy was evaluated. The current recommendations for antineoplastic chemotherapy during concomitant anti-HIV chemotherapy have been reviewed [60,68,69,75]. In brief, HAART therapy is usually continued during antilymphoma chemotherapy, but because of its myelosuppressive potential, zidovudine, a frequent component of HAART, is usually avoided in such regimens. For first-line therapy of NHL, however, the EPOCH regimen (etoposide, vincristine, and doxorubicin for 96 hours with bolus doses of cyclophosphamide and oral prednisone) has been used without HAART during the period of chemotherapy. The response rate was 74% overall, and median overall survival at 56 months varied based on CD4 count greater than or less than 100/mm3 (87% versus 16%, respectively) [77]. This dose-adjusted EPOCH regimen has been recommended with or without HAART as first-line therapy for ARL [60]. There has been no randomized study of m-BACOD versus the CHOP

Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection

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1

Fig. 68.3 Progression-free survival in patients with acquired immune deficiency syndrome (AIDS) lymphoma. Fifteen patients with AIDS lymphoma were treated with high-dose chemotherapy and hematopoietic cell transplantation as described by Krishnan et al. [90] ASCT, autologous stem cell transplantation. (Reproduced from Krishnan et al. [90] with permission.)

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(cyclophosphamide, Adriamycin [doxombicin], vincristine, and prednisone) regimen, but, based on the experience in a single institution, there appears to be no significant difference in survival rates with these two treatment regimens. Of interest, however, the median survival of ARLDLCL improved in the post-HAART era from 8.3 months to 43.2 months, while the median survival of ARL-BL remained unchanged (6.4 and 5.7 months, respectively), suggesting that more dose-intense regimens are needed for ARL-BL [72]. Whether using CHOP, m-BACOD, or other regimens, treatment is usually modified based on the patient’s AIDS status. In a large multicenter study, a risk-adapted approach assigned treatment based on the presence of three risk factors: prior AIDS, CD4 count less than 100/mm3, and Eastern Cooperative Oncology Group performance status 2–4. Those with no risk factors received doxorubicin, cyclophosphamide, vindesine, bleomycin, and prednisolone (ACVBP) or CHOP, and there was no difference in 5-year overall survival rates (51% versus 47%). Those with one risk factor received either CHOP or low-dose CHOP, and 5-year survival was the same (28% versus 24%, respectively) but less than that of the no-risk group. Those with two or three risk factors received either low-dose CHOP or vincristine and prednisolone, the 5year survival being 11% versus 3%. The significant factors for overall survival were HAART therapy, HIV score, and International Prognostic Index [75]. The use of rituximab in AIDS-related NHL is controversial, and, because it has been associated with increased infectious deaths in subjects with CD4 counts less than 50/mm3, it should be used based on HIV/AIDS status [60,78]. AIDS-related HL generally responds well to conventional therapy such as the Stanford V regimen, with overall responses of 89% and 3-year disease-free survival of 68% [79]. Although several conventional regimens of lymphoma chemotherapy result in remission rates that are increased in patients since the advent of HAART [60], the outcome of treatment of relapsed or partially responsive disease remains very poor, with median survival remaining less than 1 year with salvage therapy [69,72,80,81]. Salvage therapy with either dexamethasone, cytosine arabinoside, and cisplatin (DHAP) or etoposide, methylprednisolone, cytosine arabinoside, and cisplatin (ESHAP) have shown good response rates in HIV-negative patients with relapsed lymphomas, but these regimens have not produced improved results in relapsed ARL [80]. Nevertheless, ESHAP is currently a recommended salvage therapy for this patient group [60,80]. For the high-risk patient, however, there is a need for alternative, more effective treatments, and thus high-dose therapy with HCT has been a method used for treatment of AIDS lymphoma.

12

16

20 24 28 32 36 40 Surv time (months) post-ASCT

44

48

52

56

60

HCT for AIDS lymphoma The superiority of high-dose chemotherapy and autologous HCT over salvage chemotherapy for both relapsed lymphoma and primary refractory lymphoma in the non-HIV-1 setting has been demonstrated [82–84]. This approach has now been extended to patients with “high-risk” lymphomas in first complete remission and has shown a benefit for patients in the International Prognostic Index high–intermediate- and high-risk groups in nonrandomized single-institution trials [85,86]. As the majority of AIDS-related NHL falls into this category, the use of high-dose regimens in HIV-infected patients has been studied. Gabarre et al. [87] reported the first treatment of a patient with ARL using high-dose chemotherapy and autologous HCT. This patient was treated prior to the general availability of HAART and died of opportunistic infection. With the introduction of HAART, the ability to treat AIDS lymphoma with high-dose therapy has significantly improved. Molina et al. [88] and Krishnan et al. [89,90] have described a method of high-dose therapy and autologous HCT for high-risk ARL with very promising early outcomes (Fig. 68.3). Additional successful experiences have been reported by Gabarre et al. and Re et al. [91,92]. The City of Hope group updated their results on 28 patients with high-risk ARL treated with either a radiation-based or high-dose chemotherapy-based conditioning regimen. All patients engrafted white blood cells at a median of 11 days post HCT, and this is similar to that observed in non-HIV-1 transplant recipients [93]. Procedure-related toxicity consisted of mild hepatic toxicity and mucositis in most patients, but three patients developed BCNU (carmustine)related pneumonitis. One patient aged 68 years died because of cardiomyopathy and multiorgan failure. Opportunistic infections included Pneumocystis jiroveci pneumonia in two patients not compliant with prophylaxis, cytomegalovirus blood infection in three, and varicellazoster in two patients. The median CD4 count at the time of transplant was 164 cells/μL, and this rose to a median of 263 cells/μL at 2 years post HCT. One patient, transplanted for relapsed lymphoma, died of therapy-related myelodysplastic syndrome while still in remission for lymphoma. With a median follow-up of 41 months, the 2-year overall survival was 78% [93]. In the series of 16 patients reported by Re et al., the myeloid cell engraftment time was 10 days, also similar to that of HIV-negative patients [92]. Thus, high-dose chemotherapy and HCT appear to be a promising therapy for high-risk ARL. Hematologic toxicity is similar to that for HIV-negative patients, and infectious complications can be managed with appropriate prophylaxis and surveillance.

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two patients with AIDS and hematologic malignancies underwent reduced-toxicity HCT without significant procedure-related toxicity. One patient died of relapse at 12 months post HCT, and the other remained well and in remission, with rising CD4 counts and wellcontrolled HIV-1 levels. This observation clearly indicates that nonmyeloablative allogeneic HCT is feasible in patients having HAART-responsive HIV infection. The study of Kang et al. [99] also introduced genetically modified hematopoietic stem cells (HSCs) into the patient with the HCT. After 2 years, there was only evidence of lowlevel transgene expression, and this finding raises the possibility that an improved method of HSC transduction or selection post HCT could eventually produce successful gene therapy of AIDS using HCT. The potential for genetic approaches to control HIV-1 rests in the understanding of HIV-1 infection and replication, and several methods have been proposed for control of HIV-1 using gene transfer.

Allogeneic HCT and AIDS It has been recognized since the 1980s that persons with AIDS can also undergo allogeneic HCT with rapid engraftment of blood cells [94–96]. In the pre-HAART era, the use of allogeneic HCT for the treatment of AIDS lymphoma was unsuccessful [95]. With the availability of HAART, it may be feasible to treat these patients with an allogeneic HCT, as illustrated by a few case reports describing the use of high-dose conditioning and allogeneic bone marrow or peripheral blood progenitor cell transplantation. In one report, the patient was not found to be HIV positive until several days after busulfan and cyclosphosphamide conditioning had been administered [97]. For more than 2 years of post-transplant followup, the patient’s HIV load fluctuated around 105 genome copies/mL despite HAART, and his CD4 counts remained relatively constant between 100 and 200 CD4 cells/μL [97]. Another case report of a known HIV-positive patient with acute myeloid leukemia and hepatitis C described the use of busulfan and cyclophosphamide conditioning with concomitant HAART [98]. HAART had to be suspended between days 5 and 15 post HCT due to severe mucositis and, when it was restarted, rising levels of cyclosporine occurred due to interactions between cyclosporine and ritonavir, requiring dose adjustments of the cyclosporine. At 39 months of follow-up, the patient remained in remission with a normal CD4 count [98]. Kang et al. [99] have described the use of a reduced-intensity regimen, consisting of cyclophosphamide and fludarabine, for the transplantation of genetically modified cells using reduced-intensity HCT. In this study,

Strategies for cell-based gene therapy of AIDS General rationale Because of the genetic instability of HIV-1 and the progressive destruction of the immune system that occurs from onset of infection, there is a need for new therapeutic strategies that are multipronged and comprehensive, attack both active and quiescent forms of HIV, and have longterm therapeutic outcome. Among such strategies are cell- and gene-based therapeutic approaches that can be used in concert with conventional antiretroviral drugs. Gene-based strategies aim to target viral elements

T T

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Fig. 68.4 Gene therapy strategies in clinical trials. This schema illustrates strategies for genetic manipulation of two types of cell for inhibition of human immunodeficiency virus type 1 (HIV-1) infection: T lymphocytes and hematopoietic stem cells (HSCs). (a) The selection and expansion of T lymphocytes. T lymphocytes are selected from peripheral blood mononuclear cells. Transduction can be performed with vectors containing either a candidate anti-HIV-1 transgene or a control transgene. Transduced cells are then expanded in vitro, combined, and infused. After infusion, the survival of transduced lymphocytes can be compared as a ratio of genetically marked cells. (b) The method used for gene therapy with HSC transplantation. Progenitor cells must be mobilized from marrow using a cytokine such as granulocyte colony-stimulating factor (G-CSF) and collected by leukocyte apheresis. CD34+ peripheral blood progenitor cells are then selected using immunoabsorption methods and cryopreserved. When stem cell transplantation is ready to proceed, a portion of the stored HSCs is thawed, transduced with appropriate vectors encoding candidate and control transgenes, and transplanted into an autologous recipient with the remaining untransduced HSCs. As before, progeny cells in the peripheral blood can be analyzed for relative frequency of genetically marked cells. An effective anti-HIV-1 transgene would be one that promotes enhanced survival of genetically marked cells compared with controls.

Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection

in the anatomic compartments and reservoirs where the virus takes hold in the body, to protect CD4+ T lymphocytes and other susceptible cells against HIV-1 infection or replication, and to restore immune function. Highlighted here are those strategies undergoing clinical evaluation and those in advanced preclinical development. Approaches Gene therapy involves the transfer of genetic information (transgene) into humans to replace a missing or defective function or to produce an intracellular molecule (RNA, DNA or protein) for therapeutic purposes. This approach was made possible by the ability to copy human genes in vitro and then insert them into target cells of interest. While simple at first glance, this process is highly complex. First, the transgene must be cloned into a delivery vehicle (vector) that also provides the transcriptional regulatory elements required for gene expression. The delivery vehicle may be a viral or nonviral vector. Second, the vector must be delivered efficiently into the target cells (Fig. 68.4). Most delivery methods employ a process whereby cells are removed from the patient and the transgene is delivered to the target cells ex vivo. The modified cells are then expanded in culture and reinfused into the patient. Several approaches for direct delivery of genes to target cells in vivo, bypassing the need for expensive and timeconsuming ex vivo cell manipulations, are also in clinical studies, but at this point these are restricted to local tissue delivery such as skin, muscle, and lung. Third, long-term stable expression of the transgene in modified cells is essential. Both integrating and nonintegrating vectors are being developed, with each having its own unique advantages and disadvantages. Vectors that can integrate the transgene into host DNA promise to produce long-lived expression, whereas nonintegrating vectors can be expected to have a shorter period of expression but will minimize the theoretical risk of insertional mutagenesis by the integrated DNA. Nonintegrated vectors replicate autonomously with every cell division and partition equally to each daughter cell. Efforts to improve the technologies are directed toward the three critical problems facing this approach: the design of more efficient, safe, and cost-effective gene delivery vehicles; the design of delivery vehicles that do not induce immune responses that could result in the clearance of the engineered cells in vivo; and the design of vectors that can be targeted to specific cells or tissues for direct in vivo delivery into patients.

Molecular targets for HIV-1 gene therapy A diverse array of transgenes to suppress HIV-1 functions or block the infectious cycle have been designed and tested in vitro (Table 68.1). These can be broadly categorized into two groups: those coding for protein-based suppressors, and those coding for nucleic acid-based suppressors. As in drug-based therapy, the rationale for antiviral gene therapy is that inhibition of the virus at several critical steps of virus replication is likely to result in more complete and effective suppression. Also, as in drug-based therapy, there are concerns over the emergence of HIV-1 variants that resist or escape the gene-based strategy. For these reasons, current gene therapy approaches use combinations of different types of transgene with different viral and cellular targets that aim to completely neutralize HIV-1 functions at different points in the infection cycle. There are two general strategies for genetic targeting of HIV-1: RNA-based inhibitors, such as antisense, ribozyme, and small inhibitory RNA, and protein inhibitors such as dominant negative mutants. A list of such genetic inhibitors is shown in Table 68.1, and for a complete review of these molecular targets for gene therapy of HIV-1 see [100].

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Table 68.1 Transgenes for human immunodeficiency virus type 1 gene therapy Protein based Transdominant negative mutant Rev (RevM10) Tat Tat/Rev fusion (Trev)

[95,151] [94] [96]

Intrabodies (intracellular single chain antibodies) Anti-Tat Anti-Env Anti-Rev Anti-Gag (MA) Anti-RT

[99] [137,152] [153] [154] [155]

Intrakines Antichemokine receptor Toxins Diphtheria toxin HSV tk gene (followed by ganciclovir administration)

[156,157] [158,159] [160,161]

RNA based Antisense Anti-tat Anti-rev Anti-gag

[162] [103] [163]

Ribozymes Hairpin Hammerhead

[164,165] [166]

Small inhibitory RNA Rev Tat, Rev vif, nef, TAR, LTR

[104] [105] [106]

Decoys TAR or poly TAR RRE

[108,167,168] [107,110,169]

Others Targeted cytolytic viruses CD4 and CD8 zeta chain-modified T-cell receptors Herpes simplex virus host shut-off protein Interferon

[109,170] [111] [171] [172]

RNA-based suppressors The binding of newly transcribed RNA antisense molecules to messenger RNAs sequences prevents translation of the encoded protein, thereby resulting in loss of gene function [101]. The first lentivirus vector-based gene transfer in humans encoded an antisense to HIV env and, in the setting of T-cell immunotherapy, has been shown to be safe [102]. RNA decoy strategies attempt to compete with specific HIV-1 RNA elements that bind viral proteins as part of the replication cycle by overexpressing their RNA homologs. TAR (Tat-responsive) and RRE (Rev-responsive) are two such cis-acting RNA elements that are required for the proper function of Tat and Rev, two key HIV-1 regulatory proteins [103]. Together, these and related studies provided the conceptual basis for a clinical trial of transduced and transplanted marrow-derived HSCs in HIV-1-infected children [104].

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Ribozymes are RNA molecules that can cleave RNA targets at specific sequences, a property that is being exploited to cleave and thus inactivate HIV-1 RNAs [105]. The first in vitro study suggesting the use of a “hammerhead” ribozyme to suppress HIV-1 was directed against the HIV-1 gag RNA sequence [106]. A “hairpin” ribozyme that cleaved the leader sequence at the 5′ end of the HIV-1 genome confers resistance to several HIV-1 isolates, and considerable inhibitory activity was observed for up to 35 days in vitro [107,108]. Clinical trials using ribozymes are currently active. RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces a post-transcriptional degradation of homologous transcripts, and RNAi has been observed in a variety of organisms including fungi, plants, insects, protozoa, and mammals [109–111]. RNAi is initiated by exposing cells to dsRNA via either transfection or endogenous expression of viral or transposon RNAs. The dsRNAs are processed by the RNAse III-like enzyme, called dicer, into 21–23-nucleotide doublestranded fragments known as short interfering RNAs (siRNAs). These siRNAs form a complex known as the RNA-induced silencing complex, which leads to cognate target RNA destruction [112]. Chemically synthesized 21–23-length siRNA nucleotide duplexes with short 3′ overhangs have been synthesized and are capable of mediating RNAi in vivo and in vitro [113]. Several studies have demonstrated that the functional unit of RNAi, the siRNA, can elicit sequence-specific target downregulation either by introduction of preformed siRNAs via transfection or by endogenous expression of 21–23 base-paired RNAs using polymerase III promoter systems [114–116]. This enabling of expression of siRNAs in mammalian cells has opened up new possibilities for therapeutic applications of siRNAs. RNAi can be applied as a potent mechanism for inhibition of HIV-1 infection. The siRNA component of RNAi can circumvent the nonsequence-specific interferon pathways. Thus, RNAi represents an exciting, new, and potentially powerful tool for anti-HIV therapy [115,117–120]. The recent publications demonstrating that HIV replication can be inhibited by using either synthetic or expressed siRNAs, targeting the virus, its receptor, or its co-receptor, have generated a great amount of interest and enthusiasm for further testing of the anti-HIV therapeutic potential of siRNAs. There are at least two different modalities by which siRNA can be used as an antiviral: transfection of the preformed siRNAs, or intracellular expression of siRNAs. The latter approach may potentially be utilized in a gene therapy setting, providing intracellular immunity to hematopoietic cells susceptible to HIV-1 infection.

Protein-based suppressors Transdominant mutant proteins act as competitors of cognate HIV-1 proteins and thus suppress normal viral functions. The most experimentally advanced transdominant protein to date is a mutant Rev protein (RevM10) [121]. RevM10 retains two Rev functions: the ability to bind RRE on the viral genome and the ability to form Rev multimers. However, it is incapable of exerting its regulatory role in transporting unspliced or singly spliced RNAs from the nucleus to the cytoplasm. Thus, susceptible cell lines that express RevM10 exhibit long-term (>30 days) resistance to HIV-1 replication [122]. Similarly, human CD34+ blood progenitor cells transduced with RevM10 gave rise to T lymphocytes that exhibited significant resistance to challenge with HIV1 [123]. Based on these and other pivotal studies, clinical studies were undertaken to assess the safety of the transgene and its protective effect on transduced cells measured in cell survival relative to unprotected cells. The first human clinical study examined the potential of RevM10 to prolong the survival of transduced CD4 T lymphocytes in vivo after ex vivo transduction, expansion, and reinfusion into HIV-1-seropositive

subjects [124]. In this study, the duration of engraftment was limited, and transient, but genetically modified cells were detected in one subject up to 2 months after gene transfer, although the recombinant gene was not detectable in two subjects after 2 weeks [124]. In a subsequent attempt to achieve more durable engraftment, a retrovirus delivery vector was used in lieu of plasmid DNA, and, as before, the cells transduced with RevM10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in three HIV-positive subjects tested [125]. Intracellular HIV-specific single-chain antibodies (intrabodies; SFv) can sequester or redirect HIV-1 proteins away from their normal subcellular compartment. Combinations of SFv targeted to reverse transcriptase, integrase, and CCR5 have been described by Strayer et al. [126]. Additional transdominant protein transgenes are listed in Table 68.1. Intracellular toxins and conditionally toxic molecules can be used to preferentially eradicate HIV-1 infected cells [127–132]. A major deficiency of this approach is that these gene-modified cells do not possess a selective survival advantage and therefore cannot be enriched relative to nonmodified cells. It is expected that repeated infusions of ex vivomodified cells will be required for therapeutic benefits. Unless an in vivo vector delivery system is available, the aforementioned requirement is likely to render this strategy impractical when the large HIV-infected population is considered. Strategies based on preventing the expression of chemokine coreceptors at the cell surface are particularly attractive primarily because they block the very first step in HIV-1 infection of target cells rather than blocking a viral event that occurs after the establishment of the proviral DNA in the cellular genome. Intrakine-modified cells should also have survival advantages in vivo in HIV-infected individuals because of their resistance to infection by certain HIV-1 isolates. Moreover, a gene-based approach that utilizes a nonpolymorphic human protein is unlikely to induce an immune response against the expressed protein. Such an undesired outcome could lead to clearance by the immune system of transduced cells expressing the foreign protein. Because HIV-1 replication can be influenced by a variety of cellular perturbations, there are many other molecular strategies for inhibiting this virus (Table 68.1), and these strategies will increase as more is known about the biology of HIV-1.

Current application of cell-based gene therapy to AIDS Current gene therapy strategies make extensive use of an ex vivo approach in which cells are taken from a donor, modified with the transgene of interest, expanded, and then returned to the donor. Early studies used a marker gene that allows detection of the modified cells; however, gene transfer has the potential for lethal toxicity, and future gene transfer trials will not be able to ethically use marker genes owing to the potential toxicity of cells expressing foreign proteins [133] or to the induction of integrational mutagenesis [134].

T lymphocytes as targets for gene therapy This approach uses autologous T lymphocytes as the target cells for gene modification, thereby eliminating complications caused by hostversus-graft rejection or graft-versus-host disease that would otherwise occur with grafts from nonidentical individuals. Using this strategy, mature CD4+ lymphocytes have been genetically modified and reinfused into the patients [109,124,135]. A similar strategy, in which mature CD8+ lymphocytes have been modified to kill HIV-infected cells, has also been reported [102,136–138]. In either case, mature T lymphocytes have attributes that make them attractive targets for gene therapy: they

Hematopoietic Cell Transplantation for Patients with Human Immunodeficiency Virus Infection

are easily obtained from the donor’s peripheral blood, and they can be expanded to large numbers in vitro. Indeed, a method of cell expansion that involves cell surface stimulation, using antibodies to CD3 and CD28, promotes the expansion of cells to very high numbers [139]. Because these stimuli also transiently downregulate the expression of the chemokine receptor CCR5 [140], these cells are at a minimum temporarily resistant to infection with macrophage-tropic strains of HIV, adding another advantage for their immediate survival in an HIVinfected host. Targeting mature T lymphocytes for gene therapy has the added advantage that the effect of the therapeutic gene can be rapidly monitored for effects on cell survival, viral load, and other parameters. The transduced cells can also be selected in vitro, using the marker gene included in the vector, before reinfusion in the host, thereby generating a highly enriched population of genetically modified cells. For these reasons, gene therapy approaches that target mature lymphoid populations may be the method of choice for initial evaluation of new gene therapy strategies. Using a lentivirus vector to transfer an antisense RNA target to HIV-1 envelop, CD4 lymphocytes have been safely infused into AIDS patients who were resistant to antiretroviral therapy [102]. A negative aspect of using mature lymphocytes is that they are terminally differentiated and have limited in vivo growth potential and finite life span. Moreover, CD4+ T lymphocytes represent only one of the cell types susceptible to HIV-1 in vivo. In addition, techniques used to expand CD4+ lymphocytes in vitro can affect the expression of various surface markers and result in altered homing properties when the cells are reinfused to the host. Intense efforts are therefore under way to develop gene transfer protocols for HSCs that can give rise to all cells of the lymphoid and myeloid lineages. Conceptually, HSCs transduced with a potent anti-HIV-1 gene should confer anti-HIV-1 protection to all hematopoietic cells lineages, including those susceptible to HIV-1 infection (CD4+ T lymphocytes, monocytes, Langerhans’ cells, dendritic cells, and others) for the life of the infected individual.

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instead of stromal cells [141,142], and the combination of recombinant Flt3-ligand [143,144], thrombopoietin, and stem cell factor [145]. The critical issue is whether the vector can transduce a pluripotent stem cell, and to conclusively demonstrate that transduced CD34+ cells are in fact pluripotent, it is necessary to show that cultures derived from a single transduced cell can differentiate into all hematopoietic lineages (T and B lymphocytes, monocytes, erythrocytes, and others), and can result in reconstitution in vivo of a surrogate host. Studies in non-human primates indicate that lentivirus-based gene transfer, combined with a selection system that is toxic to nontransduced HSCs, can result in high levels of gene-marked blood progeny [146]. The ability of HSCs to engraft in patients who have not been “preconditioned” with cytoreductive or high-dose preparative regimens is an important issue in HSC-based gene therapy. The first successful retrovirus-based gene therapy was performed in patients with severe combined immunodeficiency [147,148]. The initial successful outgrowth of T lymphocytes was demonstrated to contain the transgene and to be functional [148]. This clinical trial provided the first demonstration that gene transfer could cure a genetic disease, and it is a model on which anti-HIV-1 gene therapy using genetically modified HCT has been based. However, the initial successes in patients with severe combined immunodeficiency have yielded retrovirus-related insertional mutagenesis with late T-cell leukemia [149]. Evidence is convincing that this leukemoid reaction was brought about by the interaction of the insertional site and the interleukin γ-chain transgene, and adverse events such as these further define the problems that must be understood before stem cell-based gene therapy can be safe and efficacious. It is arguable that the setting of autologous HCT for AIDS lymphoma is a clinical setting in which these new methods can be ethically evaluated [90]. The comparative value of cytoreduction versus reduced-intensity regimens, the evaluation of new vectors and stability of transgene expression, and the development of selection strategies for expansion of transduced cells in vivo all need to be studied.

Conclusion HSCs as targets of gene transfer While HSCs are an attractive target for gene therapy, they present a unique set of issues that are not encountered when mature T lymphocytes are used as the target cells. In addition to their low frequency, HSCs are quiescent. This relative paucity of cell replication may affect transduction strategies that rely on retroviral vectors, a class of vectors that require cell division for efficient gene transfer (see Chapter 11). As a result, current transduction methods often stimulate cell division, which may compromise the undifferentiated status, functional activity, and subsequent cell lineage commitment of the transduced progenitor cells. Over recent years, newer methods of transduction have resulted in incremental improvements of vector-mediated gene transfer into CD34+ cells. These include the use of recombinant fibronectin fragment CH-296

HCT currently has application to the treatment of relapsed of refractory AIDS-related lymphoma. Patients tolerate dose-intense chemotherapy, and the timing of engraftment is similar to that observed in non-HIV lymphoma patients undergoing the same treatment. Based on this, it has been proposed to use HCT as a delivery method for genetically modified hematopoietic progenitor cells having resistance to HIV-1 infection. Assuming that cell-based gene therapy becomes optimized, specific uses for gene therapy are likely to be identified, and the test-of-concept for this approach could be established. The treatment of relapsed ARL provides the setting for testing these novel therapies and, if gene transfer is ever shown to be effective in this setting, the potential will exist for eventual treatment of the HIV-1 infected person who has no cancer, with a goal of decreasing the virus reservoir and delaying disease progression.

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125. Ranga U, Woffendin C, Verma S et al. Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals. Proc Natl Acad Sci USA 1998; 95: 1201–6. 126. Strayer DS, Branco F, Landre J et al. Combination genetic therapy to inhibit HIV-1. Mol Ther 2002; 5: 33–41. 127. Brady HJ, Miles CG, Pennington DJ et al. Specific ablation of human immunodeficiency virus Tat-expressing cells by conditionally toxic retroviruses. Proc Natl Acad Sci USA 1994; 91: 365– 9. 128. Caruso M. Gene therapy against cancer and HIV infection using the gene encoding herpes simplex virus thymidine kinase. Mol Med Today 1996; 2: 212–17. 129. Caruso M, Klatzmann D. Selective killing of CD4+ cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population. Proc Natl Acad Sci USA 1992; 89: 182–6. 130. Curiel TJ, Cook DR, Wang Y et al. Long-term inhibition of clinical and laboratory human immunodeficiency virus strains in human T-cell lines containing an HIV-regulated diphtheria toxin A chain gene. Hum Gene Ther 1993; 4: 741–7. 131. Dinges MM, Cook DR, King J et al. HIV-regulated diphtheria toxin A chain gene confers longterm protection against HIV type 1 infection in the human promonocytic cell line U937. Hum Gene Ther 1995; 6: 1437–45. 132. Smith SM, Markham RB, Jeang KT. Conditional reduction of human immunodeficiency virus type 1 replication by a gain-of-herpes simplex virus 1 thymidine kinase function. Proc Natl Acad Sci USA 1996; 93: 7955–60. 133. Su L, Lee R, Bonyhadi M et al. Hematopoietic stem cell-based gene therapy for acquired immunodeficiency syndrome: efficient transduction and expression of RevM10 in myeloid cells in vivo and in vitro. Blood 1997; 89: 2283–90. 134. Woods NB, Bottero V, Schmidt M et al. Gene therapy: therapeutic gene causing lymphoma. Nature 2006; 440: 1123. 135. Walker RE. A phase I/II pilot study of the safety of the adoptive transfer of syngeneic gene-modified cytotoxic T lymphocytes in HIV-infected identical twins. Hum Gene Ther 1996; 7: 367–400. 136. Mitsuyasu RT, Anton PA, Deeks SG et al. Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood 2000; 96: 785–93. 137. Riddell SR, Elliott M, Lewinsohn DA et al. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nat Med 1996; 2: 216–23. 138. Riddell SR, Greenberg PD, Overell RW et al. Phase I study of cellular adoptive immunotherapy using genetically modified CD8+ HIV-specific T cells for HIV seropositive patients undergoing allogeneic bone marrow transplant. The Fred Hutchinson Cancer Research Center and the University of Washington School of Medicine, Department of Medicine, Division of Oncology. Hum Gene Ther 1992; 3: 319–38. 139. Levine BL, Bernstein W, Craighead N et al. Ex vivo replicative potential of adult human peripheral blood CD4+ T cells. Transplant Proc 1997; 29: 2028.

140. Carroll RG, Riley JL, Levine BL et al. Differential regulation of HIV-1 fusion cofactor expression by CD28 costimulation of CD4+ T cells. Science 1997; 276: 273–6. 141. Hanenberg H, Xiao XL, Dilloo D et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells. Nat Med 1996; 2: 876– 82. 142. Moritz T, Dutt P, Xiao X et al. Fibronectin improves transduction of reconstituting hematopoietic stem cells by retroviral vectors: evidence of direct viral binding to chymotryptic carboxy-terminal fragments. Blood 1996; 88: 855–62. 143. Kiem HP, Andrews RG, Morris J et al. Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor. Blood 1998; 92: 1878–86. 144. Shah AJ, Smogorzewska EM, Hannum C et al. Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38– cells and maintains progenitor cells in vitro. Blood 1996; 87: 3563–70. 145. Dao MA, Hannum CH, Kohn DB et al. FLT3 ligand preserves the ability of human CD34+ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviralmediated transduction. Blood 1997; 89: 446– 56. 146. Neff T, Beard BC, Peterson LJ et al. Polyclonal chemoprotection against temozolomide in a largeanimal model of drug resistance gene therapy. Blood 2005; 105: 997–1002. 147. Aiuti A, Vai S, Mortellaro A et al. Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement. Nat Med 2002; 8: 423–5. 148. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669–72. 149. French gene therapy group reports on the adverse event in a clinical trial of gene therapy for Xlinked severe combined immune deficiency (XSCID). Position statement from the European Society of Gene Therapy. J Gene Med 2003; 5: 82–4. 150. Berkhout B, Silverman RH, Jeang KT. Tat transactivates the human immunodeficiency virus through a nascent RNA target. Cell 1989; 59: 273–82. 151. Krishnan A, Molina A, Zaia J et al. Durable remissions with autologous stem cell transplantation for high-risk HIV-associated lymphomas. Blood 2005; 105: 874–8. 152. Rosenberg SA, Blaese RM, Brenner MK et al. Human gene marker/therapy clinical protocols. Hum Gene Ther 1996; 7: 1621–47. 153. Crooks ET, Moore PL, Richman D et al. Characterizing anti-HIV monoclonal antibodies and immune sera by defining the mechanism of neutralization. Hum Antibodies 2005; 14: 101–13. 154. Bagarazzi ML, Boyer JD, Ayyavoo V et al. Nucleic acid-based vaccines as an approach to immunization against human immunodeficiency virus type-1. Curr Top Microbiol Immunol 1998; 226: 107–43.

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Richard A. Nash

Hematopoietic Cell Transplantation for Autoimmune Diseases

Introduction An autoimmune disease is characterized by organ damage resulting from an immunologic response to self-antigens. Autoimmunity, on the other hand, is an immune response to self-antigens but without the implication that this has resulted in organ damage. Autoimmunity may be present spontaneously in normal individuals or after an infectious or other disease-related event which results in tissue damage (e.g. infarction). Criteria which support a disease having an autoimmune mechanism are: (1) humoral or cellular self-reactivity; (2) disease-related autoantibody or lymphocytic infiltrate in a pathologic lesion without any other identified cause; and (3) autoantibodies or T cells that cause tissue pathology or cell dysfunction by transplancental transmission, adaptive transfer into animals or in vitro experiments. An autoimmune disease develops in approximately 3–8% of the population [1]. Autoimmune diseases may involve single organs or multiple systems and may or may not have defined antigenic targets. Most autoimmune diseases are not life-threatening and can be effectively treated even if not curable. However, some autoimmune diseases may be life-threatening or damage critical organs and become refractory to conventional treatments. This then would be the candidate population for consideration of an approach requiring autologous or allogeneic hematopoietic cell transplantation (HCT).

Proposed mechanisms for autoimmunity and autoimmune diseases To better understand how an approach using HCT might be effective for treatment of an autoimmune disease, an understanding of the pathogenic mechanisms of autoimmune disease is required. There is a marked diversity of the B- and T-cell receptor specificity that results from somatic genome modification in lymphocytes. This allows for an effective immune response to foreign antigens. The unique specificities of these receptors are generated during B- and T-cell differentiation by recombination of the variable (V), diversity (D), and joining (J) genes, and then, later in B cells, somatic hypermutation generates further diversity. These random processes of recombination and somatic hypermutation result in a significant percentage of lymphocytes (up to 50%) with B- and T-cell receptors that have an affinity for self-antigens [2].

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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It has been proposed that there are four cellular strategies to prevent the development of autoimmunity and autoimmune diseases from lymphocytes with self-reactive receptors. First, lymphocytes with highaffinity, self-reactive receptors can be clonally deleted. Clonal deletion occurs in central lymphoid organs during development. Second, a selfreactive receptor can be edited by further V(D)J recombination or somatic hypermutation to display a different receptor that is not selfreactive [3]. Receptor editing induced by self-reactivity can occur in both B and T cells. For B-cell receptor editing, new rearrangements at L chain loci and less commonly at H chain loci are induced. Third, downregulation of cell surface self-reactive receptors and intracellular changes, including those which affect signal pathways, decrease responsiveness to the binding of self-reactive receptors, resulting in clonal anergy [4]. Last, extrinsic factors such as cytokine-dependence for proliferation and regulatory mechanisms, including regulatory T cells, may limit the activity of lymphocytes with self-reactive receptors. In B cells with self-reactive receptors, B-cell activating factor receptor is poorly induced and therefore may be more susceptible to competitive regulation by their requirement for B-cell activating factor than other B cells with non-self-reactive receptors. Some T cells with self-reactive receptors are positively selected in the thymus after T-cell receptor engagement by self-peptide–major histocompatibility complex (MHC) class II complexes. They develop into CD25+CD4+FoxP3+ regulatory T cells which suppress proliferation and the interleukin-2 (IL-2) production of autoreactive CD25−CD4+ and CD8+ T cells [5,6]. Suppression of proliferation by regulatory T cells is contact dependent. Regulatory T cells are IL-2 dependent with constitutively expressed CD25 (IL-2 receptor) and IL-7 independent without CD127 (IL-7 receptor). The transcription factor foxP3 may endow regulatory function to thymic T cells in mice, but this is less clear in humans [7]. Natural killer-T cells and other T-cell subsets producing transforming growth factor-beta and IL-10 have also been associated with immune regulation.

The genetics of autoimmune disease Autoimmune disease can be associated with either single-gene defects or susceptibility genes as part of a complex multigenic trait. Four inherited disorders from single-gene defects have been described which have contributed further insights into autoimmune disease mechanisms in humans (Table 69.1) [8–11]. These include defects in: (1) autoimmune regulator-dependent negative selection in the thymus; (2) cytotoxic T lymphocyte antigen-4-dependent inhibition of T cell function; (3) Fas/

Hematopoietic Cell Transplantation for Autoimmune Diseases Table 69.1 Single-gene defects associated with autoimmunity Autoimmune disease

Gene defect

Mechanisms of autoimmunity

Autoimmune polyglandular syndrome type I [8] Grave’s disease, type I diabetes mellitus, others [9] Autoimmune lymphoproliferative syndrome [10] IPEX [11]

AIRE

Defective expression of thymic self-antigens

CTLA4

Defect in inhibitory signaling

FAS, FASL

Defect in apoptosis

FOXP3

Decreased regulatory T cells

IPEX, immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome.

Table 69.2 Susceptibility genes for common autoimmune diseases

Autoimmune disease

Major histocompatibility complex association (class II)

Candidate susceptibility genes

Multiple sclerosis [15] Rheumatoid arthritis [12]

DR2 DR4

Systemic lupus erythematosus [12] Systemic sclerosis [14] Crohn’s disease [12] Type 1 diabetes mellitus [12]

DR2/DR3

IL-7R, IL-2R PADI4, PDCD-1, PTPN22, FCRL3 PTPN22, PDCD-1

DR11 DRB3*0301 DR3/DR4

PTPN22 NOD2, IBD5 CTLA-4, PTPN22, INS

CTLA-4, cytotoxic T-lymphocyte antigen-4; FCRL3, Fc receptor-like 3; IBD5, inflammatory bowel disease-5 (susceptibility locus on chromosome 5q31); IL-7R, interleukin-7 receptor; INS, insulin (disease-associated polymorphism of the insulin gene); NOD2, nucleotide oligomerization domain containing-2 (also known as caspase recruitment domain family, member 15 [CARD15]); PADI4, peptidyl arginine deiminase type IV; PDCD-1, programmed cell death-1; PTPN22, nonreceptor type 22 protein tyrosine phosphatase.

FasL-dependent apoptosis of B and T cells that recognize self-antigens; and (4) FoxP3-dependent development of regulatory T cells [8–11]. The most common autoimmune diseases are considered to result from alleles of susceptibility genes at multiple loci, environmental triggers, and stochastic events [12,13]. The disease-associated alleles of susceptibility genes have been shown to confer only modest risk for the development of an autoimmune disease and were first identified among the MHC genes (Table 69.2) [12,13]. Most correlations of autoimmune diseases were with MHC class II rather than class I molecules [13]. Other susceptibility genes affect, in general, pathways for activation/ inhibition of T cells (IL-7R, IL-2R, PDCD-1, PTPN22, and CTLA-4) or presentation of self-antigens (insulin, and PADI4) [12,14,15]. It is unclear to what degree environmental factors contribute to risk of developing autoimmune disease. Environmental factors including hydralazine, contaminated rapeseed oil, L-tryptophan, and Borrelia burgdorferi infections in association with specific alleles of the MHC loci have been shown to increase the risk of an autoimmune disease.

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Family or twin studies may provide further information about the contribution of genetic factors to the onset or presentation of autoimmune diseases. In general, the disease concordance rates among monozygotic twins was threefold greater or more than in dizygotic twins for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type I diabetes mellitus (DM) and multiple sclerosis (MS) [16,17]. The average concordance rates reported for monozygotic twins ranged from 15–30% for DM, MS, SLE, and RA. It has also been reported that there was a higher risk for many autoimmune diseases in other family relatives. Although genetic factors appear to play a role in the development of autoimmune diseases, the relatively low penetrance as noted in the monozygotic twins indicates that nongenetic factors are also important.

Preclinical models of autoimmunity and HCT There are two potential approaches utilizing HCT to consider for the treatment of autoimmune diseases (also see Chapter 20). The first approach is with autologous HCT after high-dose immunosuppressive therapy (HDIT). The therapeutic effects derive from the high-dose cytotoxic immunosuppression. Although it has been proposed, there is no evidence that the infusion of autologous hematopoietic cells has any benefit other than to shorten the period of pancytopenia [18]. Allogeneic HCT, as the second approach, can be done after either high-dose or reduced-intensity conditioning regimens. The therapeutic effect of this approach likely derives from the eradication of autoreactive immune effector cells when complete donor hematopoietic chimerism is established. However, remissions of autoimmune diseases have been reported after establishing only mixed hematopoietic chimerism. Therefore, it is likely that the immunomodulation from establishing the donor graft, including effects on regulatory mechanisms, may have a self-tolerizing effect even if there are persistent host autoreactive immune cells. There are two types of model for autoimmune disease in which studies of autologous and allogeneic HCT have been conducted: (1) a spontaneously developed autoimmune disease, such as a lupus-like systemic condition in NZB mice, or an organ-specific condition, such as DM in NOD mice; and (2) antigen-induced autoimmune diseases, such as adjuvant arthritis and experimental allergic encephalomyelitis (EAE). In the spontaneous autoimmune disease models, high-dose cytotoxic therapy and syngeneic bone marrow transplantation early in life did not prevent the development of the clinical or pathologic autoimmune process, whereas allogeneic marrow transplants from an autoimmune-resistant strain prevented and, when given early after disease onset, ameliorated the autoimmune manifestations (Fig. 69.1) [19,20]. In contrast to the experience in models of spontaneous-onset autoimmune diseases, studies of antigen-induced autoimmune diseases have shown therapeutic responses in recipients after high-dose cytotoxic immunosuppressive treatments and syngeneic or pseudoautologous HCT. Pseudoautologous donors were syngeneic donors with the same disease severity as the recipients at the time of HCT. EAE, an experimental model for MS, may be induced in Buffalo rats and SJL/J mice by active immunization with myelin basic protein. In this model, highdose therapy with total body irradiation (TBI) or cyclophosphamide followed by allogeneic, syngeneic or autologous marrow rescue can prevent or ameliorate disease activity [21,22]. Allogeneic HCT was superior in preventing spontaneous and induced relapses from reimmunization of myelin basic protein. In rats, after immunization with a unique peptide fragment of myelin, the CD4+ T lymphocyte cell initiating EAE is skewed towards overutilization of the Vβ 8.2 T-cell receptor. When analyzed by reverse transcriptase polymerase chain reaction and in situ hybridization, Vβ 8.2 T lymphocytes were not detected in the central nervous system (CNS) after HDIT and syngeneic HCT [23]. Therefore, high-dose therapy was able to eliminate the effector lympho-

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Fig. 69.1 Survival curves of BXSB mice (spontaneous lupus-like syndrome) after treatment with 9.5 Gy of TBI (caesium-137 irradiation, 0.70 Gy/min) and transplantation with T-cell-depleted marrow cells intravenously at 16 weeks of age from BXSB or normal BALB/c donors after the development of the autoimmune disease. The transplant groups were: BALB/c + BXSB → BXSB group (䊐; group I; n = 18), BALB/c → BXSB group (䉭; group II, n = 8), and BXSB → BXSB group (䊊; group III; n = 7). Untreated BXSB mice served as controls (䉬; n = 8). Compared with untreated controls or group III (pseudoautologous), survival was improved in recipients of the allogeneic marrow grafts. (Reproduced from [19], with permission. Copyright [1999], National Academy of Sciences, USA.)

cytes responsible for EAE from the CNS in this model. Similarly, in adjuvant-induced arthritis, the prevention of relapses after HDIT, syngeneic HCT, and repeat immunization is consistent with a tolerizing effect to the inciting antigens (Fig. 69.2). Increased intensity of the highdose cytotoxic immunosuppression resulted in a more effective induction of remissions and fewer relapses [24]. Observations in preclinical models of spontaneous-onset autoimmune diseases would support the use of allogeneic HCT for those human autoimmune diseases related to single-gene disorders with high penetrance (e.g. IPEX syndrome). However, for the other more common human autoimmune diseases, in which environmental or stochastic factors may play a larger role for triggering disease onset, the preclinical data would support the investigation of HDIT and autologous HCT.

Outcomes in patients with autoimmune diseases transplanted for another primary disease Observations in patients with hematologic disorders and a concomitant autoimmune disease gave us our first understanding that the response of human autoimmune diseases to allogeneic HCT could be comparable to the experience in the preclinical models (Table 69.3) [25–42]. Baldwin et al. first reported sustained remissions in patients who were transplanted with severe aplastic anemia but also had a diagnosis of RA. Follow-up was short, and there were early transplant-related deaths. Subsequent case reports of patients transplanted with a concomitant diagnosis of RA confirmed that remissions could be achieved and be

Fig. 69.2 Response of adjuvant-induced arthritis after high-dose immunosuppressive therapy with various doses of cyclophosphamide (CY) and combinations of CY with sublethal total body irradiation (TBI) in Buffalo rats. Adjuvant-induced arthritis is a chronic progressive disease and a preclinical model for rheumatoid arthritis. For this study, arthritis was induced by an intradermal injection of Freund’s complete adjuvant, and the severity of the arthritis was expressed as an arthritic score (increases with increased arthritic severity). Remissions were greater and more durable in recipients which received a more intensive regimen containing TBI, although some benefit was observed with all of the regimens. BMT, bone marrow transplantation. (Reproduced from [24], with permission. Copyright [2000], Macmillan Publishers Ltd.)

sustained for over 20 years [30]. It was also noted that three of the nine patients with RA relapsed after allogeneic HCT. One of the relapses was transient with a subsequent treatment-free remission of 11 years. In one of the two sustained relapses for which data was available, the recipient had complete donor hematopoietic chimerism, but the human leukocyte antigen (HLA)-identical donor was serologically positive for rheumatoid factor but without clinical disease [26]. Sustained remissions and some relapses were also observed in patients with other concomitant autoimmune diseases after allogeneic HCT (Table 69.3). One patient who relapsed with Crohn’s disease had mixed hematopoietic chimerism detected at 3 months after HCT. There were insufficient data from the case reports to develop an understanding of why relapses occurred after allogeneic HCT. In all but one of the cases reporting the use of allogeneic HCT for concomitant autoimmune diseases, the donor was an HLA-identical sibling. Therefore, one possible

Hematopoietic Cell Transplantation for Autoimmune Diseases

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Table 69.3 Outcomes in patients with autoimmune diseases transplanted with an allogeneic hematopoietic cell graft for another primary disease

Autoimmune disease

Hematologic disease

Evaluable patients (total) (n)

Rheumatoid arthritis [25–31]

SAA (n = 8), 1 MM

9

Systemic lupus erythematosus [32] Psoriatic arthritis [29,33–35] Ulcerative colitis [33] Crohn’s disease [36] Multiple sclerosis [37–40] Autoimmune hepatitis [41] Lupus anticoagulant [42]

SAA AML, CML (n = 3) AML CML CML (n = 2), LGL leukemia, AML ALL CML

1 4 1 5 4 1 1

Remission of autoimmune disease (after HCT) (n) 7 (2 relapses, 1 transient relapse) 1* (ANA titer +) 3 (1 relapse) 1 4† 3 1 1

Outcome (alive at last follow-up) (n)

Follow-up

6

2 months–21 years

1 3 1 5 3 1 1

15 years 1, 3, 5, 5 years 4 years 4.5–15.3 years 1, 2, 3, 4 years 4 years 5 years

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANA, antinuclear antibody; CML, chronic myelogenous leukemia; HCT, hematopoietic cell transplantation; LGL, large granular lymphocytes; MM, multiple myeloma; SAA, severe aplastic anemia. * Clinical remission but ANA remained positive. † Relapse occurred in patient with mixed chimerism.

explanation for recurrence of the autoimmune diseases may be the presence of shared genetic factors between the related donors and recipients. Another possible explanation is the persistence of host immune cells, resulting in recurrence of disease activity. In the late 1990s, a series of case reports was published on the outcomes of concomitant autoimmune diseases after high-dose cytotoxic therapy and autologous HCT [43,44]. These case reports were of interest since the clinical trials of HDIT and autologous HCT were just being initiated at that time. Most of the reported cases had non-Hodgkin’s lymphoma as the indication for high-dose cytotoxic therapy. Remissions of the concomitant autoimmune diseases were observed in the majority of patients early after treatment, but only five of 15 cases had sustained responses at last follow-up. This experience reflects the risks of progression of autoimmune disease after high-dose cytotoxic therapy. Clinical trials of HDIT and autologous HCT specifically for autoimmune diseases were designed to intensify the immunosuppressive effect compared with standard cytotoxic regimens for treating hematologic malignancies. This was done by depleting T cells from the autologous hematopoietic cell graft and adding other noncytotoxic immunosuppressive agents to the HDIT regimen for in vivo T-cell depletion. The intensity of the HDIT regimen may be important in achieving sustained remissions of autoimmune diseases [45]. These case reports, although small in number, were important in establishing our understanding of the potential role of allogeneic HCT and HDIT with autologous HCT for the treatment of patients with severe autoimmune diseases.

HDIT and autologous HCT for autoimmune diseases: clinical experience HDIT has been performed most commonly for MS, systemic sclerosis (SSc), SLE, RA, and juvenile idiopathic arthritis (JIA) [45]. HDIT regimens which have been investigated have had varying intensities. High-dose single-agent cyclophosphamide has been considered a lowintensity regimen [45]. It is highly immunosuppressive but is not myeloablative. Clinical trials of high-dose cyclophosphamide have been conducted with and without the support of autologous HCT. Regimens which included TBI or high-dose busulfan (BU) were considered high intensity and required support with autologous HCT.

Patient selection Patients eligible for clinical trials of HDIT were generally those with severe autoimmune disease (life-threatening or threatening the function of critical internal organs) and had failed conventional therapy. Although the criteria for selecting patients depended upon the type of autoimmune disease, it was usually required that patients have a poor prognosis based on the activity and severity of the disease.

Collection and processing of the hematopoietic cell graft Although some of the earliest adult cases were transplanted with marrow grafts, most of the later patients were transplanted with hematopoietic cell grafts harvested from the peripheral blood [46]. Marrow was the preferred source of hematopoietic stem cells for many pediatric patients [47]. The optimal dose of peripheral blood CD34+ cells for successful engraftment was 2–5 × 106/kg in patients with hematologic malignancies, and therefore this was the number targeted for collection in patients with autoimmune diseases. Eight of 173 patients (4.6%) failed to mobilize sufficient numbers of CD34+ cells to allow proceeding to HDIT [46]. Five of these eight patients eventually had adequate numbers of cells harvested by either a supplemental marrow harvest (n = 2) or successful second mobilization (n = 3). Early in the clinical trial experience of HDIT for autoimmune diseases, it was observed that some patients had disease flares during mobilization. In one review, five of 56 patients mobilized with granulocyte colony-stimulating factor (G-CSF) alone had exacerbations of the autoimmune disease (MS n = 2; RA n = 3) [46]. There have been other reports of exacerbations during mobilization in MS and RA patients with G-CSF alone [48,49]. Most exacerbations were transient, but one patient with MS died from the development of severe brainstem dysfunction. As a result of these early flares, most patients have now been mobilized with the combination of cyclophosphamide and G-CSF. In 117 patients mobilized with the combination of G-CSF and cyclophosphamide, no exacerbations were observed. In fact, after mobilization with cyclophosphamide, an improvement was noted in the underlying autoimmune disease. The addition of cyclophosphamide to G-CSF also improved the yield of progenitor cells after mobilization [46]. However, there were

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Chapter 69

three patient deaths in this report from either cyclophosphamide-related toxicity or infections. In another report of 126 patients from a single center who were mobilized with the combination of G-CSF and cyclophosphamide, there was one patient death from infection [50]. Four mild exacerbations of disease and seven infectious events were also observed during mobilization with a cyclophosphamide-based regimen. Another mobilization strategy that has been used in 22 MS patients without disease exacerbations or infections is the combination of prednisone and G-CSF [51]. G-CSF alone has been used to mobilize 34 patients with SSc without any significant disease exacerbations except for a few cases in which there was an increase in telangiectasias or skin tightness [52]. The risk of exacerbations during mobilization using GCSF alone may be related to the type of autoimmune disease. The use of cyclophosphamide as part of the mobilization regimen should be carefully considered, possibly by the type of autoimmune disease, because of the potential risks associated with the resulting toxicity. Many of the reported clinical trials used T-cell-depleted autologous grafts as a strategy to prevent recurrence of the autoimmune disease. This strategy was based on observations in preclinical models in which the autoimmune disease could be adoptively transferred with T cells. An adoptive transfer of autoimmune disease to human recipients has also been observed after allogeneic HCT [53]. In most of the clinical trials, CD34 selection was performed and resulted in a 3–4-log depletion of T cells for the autologous graft. Further T- and B-cell depletion was done in some clinical trials after the CD34 selection. The importance of Tcell-depletion of the graft was addressed in a retrospective analysis of registry data from the European Group for Blood and Marrow Transplantation (EBMT). No strong beneficial effect or risk of relapse was identified [45]. Although the risk of infection may be increased with T-cell depletion of the autologous graft, the incidence of infectious disease is low if these patients are managed with the same infection prophylaxis strategies used for patients after allogeneic HCT [47,54]. The contribution of T-cell depletion of the autologous hematopoietic cell graft to the prevention of relapse remains to be determined in future clinical trials. HDIT regimens The early rationale for HDIT was to systemically deplete autoreactive immune effector cells. Cytotoxic agents (at conventional doses) had already been observed to have a beneficial effect in most autoimmune diseases, but not all patients had a clinical response and remissions were mostly not sustained. With follow-up of the pilot studies of HDIT with autologous HCT now extending to 7 and 8 years after treatment, it has been observed that there is a high initial response rate, and a significant proportion of patients have achieved sustained remissions [47,52,55– 57]. The clinical response early after HDIT likely resulted from the overall reduction in number of immune effector cells responsible for the autoimmune disease. The sustained response observed after recovery of the lymphocyte counts at 2 years may have resulted from a late immunomodulatory effect of the HDIT regimen [54,58,59]. Randomized clinical trials need to be completed to confirm that the clinical responses after HDIT are an improvement over what might have been expected from conventional management strategies. The intensity of the HDIT regimen was important for the prevention of disease progression. In a report of 473 patients from the EBMT registry, sustained responses were observed in 78% of patients after a regimen with high-intensity conditioning compared with 68% with an intermediate and 30% with a low-intensity conditioning regimen (p = 0.0001) [45]. Regimens that included BU or TBI in combination with cyclophosphamide were placed in the high-intensity conditioning group, and cyclophosphamide alone was placed in the low-intensity conditioning group. All other regimens were considered of intermediate intensity.

The analysis of the registry data had some limitations, including variability of the diagnoses, patient selection criteria, and treatments (as well as having imbalances between diagnosis and type of HDIT regimen), but a dose intensity–response effect on treatment outcomes was evident. One of the most frequently used HDIT regimens was high-dose, single-agent cyclophosphamide (200 mg/kg) followed by autologous HCT for hematopoietic support [45]. A small study compared 100 with 200 mg/kg of cyclophosphamide followed by HCT in patients with RA. Remissions were longer with the higher dose of cyclophosphamide, but all patients relapsed [60]. Relapse rates were also high in a study of early SSc in which patients were treated with high-dose cyclophosphamide (200 mg/kg) alone. Four of 11 patients died by 18 months after treatment (three from progression), and another four patients had progressed requiring secondary treatment [61]. The addition of antithymocyte globulin (ATG) to cyclophosphamide may improve the response rate and duration. Since it is not myeloablative, high-dose cyclophosphamide (200 mg/ kg) as a single agent without autologous HCT has been reported for treatment of autoimmune diseases or immune-mediated diseases such as aplastic anemia [18,62]. The median time to recovery of neutrophils counts was 2–3 days longer without the support of an autologous hematopoietic cell graft, but the upper limit of the range was 7 days longer [18,55]. The relapse rate was also high in SLE patients (n = 14) after high-dose cyclophosphamide without HCT, but a significant proportion (36%) had durable complete remissions. In this small experience, there was no apparent benefit on relapse rate by withholding the infusion of an autologous hematopoietic cell graft. High-dose combination chemotherapy with BCNU, etoposide, cytosine arabinoside, and melphalan (BEAM) with or without ATG was considered an intermediate-intensity regimen [45]. For autoimmune diseases, this regimen has most commonly been used for the treatment of MS [63]. It has been well tolerated in this group of patients, and no significant data exist for other autoimmune diseases. Regimens with either TBI or BU in combination with cyclophosphamide were considered to be high intensity [47,51,52,64–66]. A pilot study of BU in combination with cyclophosphamide has been reported for the treatment of MS [66]. The use of TBI in combination with cyclophosphamide has been reported in clinical trials of HDIT for MS, SSc, and JIA. TBI is highly immunosuppressive and was shown to be effective in preclinical models of autoimmune disease [24,67]. Organs can be shielded with lead during TBI to protect them from the potential adverse effects of this form of cytotoxic therapy, which is not possible with systemic chemotherapy. TBI has the potential for killing cycling and noncycling stem cells as well as immune effector cells. Support with an infusion of autologous hematopoietic stem cells is required after both intermediateand high-intensity HDIT regimens to prevent prolonged pancytopenia or lack of hematopoietic recovery. From the EBMT registry, treatment-related mortality was 14% and 3% in the groups who received the high- and low-dose conditioning intensity regimens, respectively [45]. However, there was no difference in overall survival based on the intensity of the HDIT regimen. Treatment-related mortality resulted primarily from infections and severe organ toxicity. Several fatal cases and one nonfatal case of Epstein–Barr virus-associated posttransplant lymphoproliferative disorder have been reported [65,68]. In patients who have had a high-intensity immunosuppressive regimen, early detection of Epstein–Barr virus reactivation and treatment with rituximab may prevent the clinical manifestations of posttransplant lymphoproliferative disorder. The risks of infectious complications and organ toxicities after HDIT are closely related to the type of autoimmune disease being treated. Patients with MS and RA have had a lower treatment-related mortality than patients with SSc and

Hematopoietic Cell Transplantation for Autoimmune Diseases

1019

SLE who have had significant internal organ involvement at the time of transplant. Prolonged or intensive pretransplant treatment with immunosuppressive agents including corticosteroids, as in the management of patients with severe SLE, likely predisposes to infectious complications after HDIT. Better patient selection and modifications to the treatment regimen appear to have reduced the risks of treatment-related mortality in recent years [47,56,69]. Long-term complications after high-dose cytotoxic therapy include the risk for secondary myelodysplastic syndrome (MDS) and leukemia [52,65]. In one of the three cases of MDS reported, evidence of clonal abnormalities was found in cryopreserved pretransplant hematopoietic cells. The intensity and duration of the pretransplant chemotherapy is known to be an additional risk factor for the development of secondary MDS/leukemia. Secondary autoimmune diseases (other than the one for which patients were transplanted) have been described late after HDIT and autologous HCT [52,65,70]. The type of conditioning agent included in the HDIT regimen may increase the risk of this occurring [70]. Immune reconstitution after an HDIT regimen Natural killer cell counts recovered by 1 month and were followed by recovery of B- and CD8+ T-cell counts by 6–12 months after HDIT. There was a slower recovery of CD4+ T-cell counts, which were lownormal by 2 years [54]. Immune recovery at 2 years after HDIT was associated with increasing thymic-derived naïve CD4+ T cells (Fig. 69.3) [58]. It was also observed that there was an increase in T-cell receptor excision circles (a marker for recent thymic emigrants) in CD4+ T cells at 1 and 2 years after HDIT. There was a steady decrease over time in CD4+ central memory T cells. CD4+ effector memory cells were relatively increased at 6 months after HDIT, likely from homeostatic proliferation, but had recovered to normal levels by 2 years. There were no significant changes in the CD8+ T cell subsets. There was an increase in regulatory T cells, and broader clonal diversity than was present before HDIT (Fig. 69.4) [58,59]. In association with the increased levels of naïve CD4+ T cells, there was hypertrophy of the thymus at 1 and 2 years compared with baseline, especially in the younger patients (less than 43 years of age). This evidence suggested a thymic origin for the recovery of the CD4+ T-cell repertoire after HDIT and autologous HCT. Even though B-cell counts were very low in the first 3 months after HDIT, median serum levels of immunoglobulin G specific for tetanus toxoid, Haemophilus influenzae and Streptococcus pneumoniae, were normal. The clinical responses to HDIT which have persisted for 2 or more years may have resulted from these late immunomodulatory effects, especially evident in the CD4+ T-cell compartment. Specific autoimmune diseases and HCT Phase II clinical trials of HDIT and autologous HCT have been conducted for specific autoimmune diseases, and in some cases the results from long-term follow-up evaluations are available. Multiple sclerosis MS is an autoimmune inflammatory disorder of the CNS manifesting as acute focal demyelination and axonal loss with limited remyelination and focal sclerotic changes. Recurrent inflammatory events result in the development of chronic multiple sclerotic plaques in the brain and spinal cord. This disease has a lifetime risk of 1 in 400, affects 350,000 people in the United States, and is potentially the most common cause of neurologic disability in young adults [71]. Seven of 27 monozygotic pairs (25.9%) and one of 43 dizygotic pairs (2.3%) were concordant for MS, which suggests there is a genetic predisposition for the development of MS with a contribution of environmental factors [17]. Similar to other

Fig. 69.3 Naïve CD4+ T cells increased and central memory CD4+ T cells decreased at 2 years after high-dose immunosuppressive therapy (HDIT) and autologous hematopoietic cell transplantation (HCT) for multiple sclerosis. In a longitudinal analysis of T-cell subsets after HDIT and autologous HCT, a trend towards reduction of naïve (CD45RA+/CD45RO−/CD27+) CD4+ T cells was observed at 6 months, consistent with an increase of effector memory (EM) phenotypes resulting from homeostatic proliferation in a lymphopenic environment. Central memory (CM; CD45RA−/CD45RO+/ CD27+) CD4+ cells decreased steadily during the follow-up. The proportion of EM (CD45RA−/CD45RO+/CD27−) CD4+ T cells rose significantly at 6 months after HSCT and later declined toward baseline levels after the gradual recovery of the absolute CD4+ T-cell numbers. At the 2-year followup, the frequency of naïve CD4+ T cells had increased 118% compared with pretherapy (p = 0.032). Correspondingly, CM CD4+ T cells had decreased 38% at 2 years after therapy (p = 0.008). The frequencies of the EM CD4+ T cells did not change significantly at the 2-year follow-up compared with the baseline. These data support the concept that there is significant immunomodulation at 2 years after HDIT and autologous HCT, and this may explain the observed durable clinical remissions in a significant proportion of patients with autoimmune disease. Tx, treatment. (Reproduced from J Exp Med 2005; 201: 805–16. Copyright [2005], Rockefeller University Press.)

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Chapter 69 (a)

(b)

Fig. 69.4 Recovery of CD4+CD25bright T-cell frequency after high-dose immunosuppressive therapy (HDIT) and autologous hematopoietic cell transplantation (HCT). (a) The relative number of CD4+CD25bright T cells in 25 healthy controls, eight systemic juvenile idiopathic arthritis (JIA) patients on conventional therapy (Syst-co), and 12 children who received autologous stem cell transplantation (ASCT) for refractory JIA was measured by fluorescence-activated cell sorter staining. The patient represented by open triangles suffered a complete relapse 6 months after ASCT. Since it has been shown that the regulatory CD4+ T cells preferentially reside within the CD4+CD25bright population, only the CD4+CD25bright T cells were analyzed. There is a significant increase in CD4+CD25bright T cells after HDIT and autologous HCT (*p = 0.05; **p = 0.06). (b) Mean and standard error of the mean for the absolute number of CD4+CD25bright T cell counts per microliter (and other CD4+ T cell subsets) in 12 children before and after HDIT and autologous HCT for refractory JIA. An increase in the peripheral blood of CD4+CD25bright T cells after HDIT and autologous HCT is consistent with restoration of the immune regulatory network as a possible mechanism for sustained remission of JIA in a significant proportion of patients. Bef, before; CY, cyclophosphamide; PE, phycoerythrin. (Reproduced from [59], with permission. Copyright [2006], American Society of Hematology.)

suspected autoimmune disorders, subjects with MS have an increased frequency of certain HLA haplotypes that code for the class II molecules (particularly for HLA-DR2) involved in antigen recognition by CD4+ T cells. It is postulated that myelin proteins, such as myelin basic protein, proteolipid protein, and perhaps myelin oligodendrocyte glycoprotein, are targeted by pathogenic CD4+ T cells in patients with MS [72]. The pathology shows a predominant T-cell response both in perivascular spaces and in the demyelinated lesion. Axonal injury is evident not only in the acute inflammatory and the chronic nonenhancing lesions, but also in the normal-appearing white matter. The clinical manifestations of the disease may include loss of vision from optic neuritis, diplopia, sensory loss and paresthesias, vertigo, fecal and/or urinary incontinence, impotence, intellectual decline, paroxysmal pain, recurrent infections, loss of coordination or paralysis. Clinical diagnosis depends on two or more attacks involving noncontiguous anatomic regions separated by at least 1 month or progression of symptoms or signs for more than 6 months [71,73]. About 85% of patients with MS present with relapsing-remitting disease, and over 10 years, about 50% of these patients will develop secondary progressive disease

Table 69.4 Types of clinical course for multiple sclerosis (MS) Relapsing-remitting MS

Secondary progressive MS

Primary progressive MS Progressive relapsing MS

Acute episodes of worsening with recovery and stable periods between relapses; loss of neurologic function is associated with relapse and may or may not recover Previous relapsing-remitting disease with gradual neurologic deterioration with or without acute relapses Gradual continuous neurologic deterioration from onset Progressive disability from symptomatic onset but with superimposed relapses

with cumulative disability (Table 69.4). The other 15% of patients have progressive disease from the onset (primary). The standard for measuring outcome in studies of MS is the Kurtzke Expanded Disability Status Scale and, more recently, the Multiple Scle-

Hematopoietic Cell Transplantation for Autoimmune Diseases

rosis Functional Composite. There is little influence of MS on survival until at least 15 years after onset. At 20 years after onset, patients with MS had 85% of the expected survival. Prognostic factors for worse outcome have been identified and include increased age, short intervals between relapses, a high frequency of relapses after disease onset, and early significant disability. Despite responses to immunomodulating agents, no standard therapy is curative or has been demonstrated to prevent development of a progressive clinical course. Agents that have been demonstrated to be effective as disease-modifying therapies in relapsing-remitting MS include interferon-beta 1a (IFN-β1a), IFN-β1b, glatiramer acetate, mitoxantrone, and natalizumab [74]. Treatment reduced clinical relapse rates by 30–68%, with mitoxantrone and natalizumab being more effective than IFN or glatiramer acetate. These agents, however, remain inadequate to completely prevent relapses and progression. There is no effective therapy for primary or secondary progressive MS. The results of eight clinical trials of HDIT and autologous HCT for MS have been reported from North American and European centers (Table 69.5) [51,63–66,75–77]. Although there was variability in the design of each of these clinical trials, they all included patients with advanced MS and the progressive type of the disease. All patients received high-dose combination chemotherapy or TBI and cyclophosphamide. In seven of the eight clinical trials, the hematopoietic cell grafts were mobilized from the peripheral blood, and all but one of the trials included T-cell depletion with CD34 selection. Overall treatmentrelated mortality in these clinical trials was 2.8% (4 of 145) and the progression-free survival or rate of neurologic stability reported in the individual clinical trials ranged from 36% to 95% at 2–3 years after treatment. Five (3.5%) patients died after progression of disease and further loss of neurologic function or other complications. Although no direct comparisons can be made between the clinical trials regarding outcomes, there did not appear to be any advantage to including TBI in the HDIT regimen. In a report by Saccardi et al., 19 MS patients with high levels of disease activity based on magnetic resonance imaging (MRI) of the brain and sustained clinical deterioration were treated with high-dose combination chemotherapy and ATG (Table 69.5) [63]. MRI of the brain was then done on a scheduled basis every 3–6 months for 2 years. There was a marked reduction of gadolinium-enhancing lesions in the brain after the HDIT regimen, which was sustained to 5 years after treatment (Fig. 69.5). Progression-free survival at 6 years was 95%. After treatment, a statistically significant improvement was achieved in measures of quality of life. The overall results of these clinical trials were comparable to what was reported from the EBMT registry in which treatment-related mortality after HDIT and autologous HCT in 178 patients was 5.3%, and neurologic stability or improvement was observed in 64% of patients at a median follow-up of 42 months [56]. Although MRI after transplant showed a marked reduction in gadolinium-enhancing brain lesions in all the groups studied compared with baseline, it is still uncertain if continued loss of neurologic function observed in some of these progressive patients is the result of a degenerative process or a failure to completely control inflammation related to the autoimmune disease. Randomized clinical trials need to be completed to confirm if there is a therapeutic benefit of HDIT and autologous HCT in this patient population of advanced MS. Clinical trials would also be of interest in patients with less-advanced relapsing-remitting MS to determine whether outcomes could be improved and the progressive type of the disease could be prevented. Systemic sclerosis SSc is an uncommon disabling autoimmune disease that is associated with a high mortality. In studies done in different geographic settings,

1021

the prevalence has ranged from 28 to 286 per million, and the incidence has been reported as ranging from 3.7 to 19.2 per million [78]. SSc is characterized by two major clinical features including a non-inflammatory small vessel vasculopathy, and fibrosis of the skin and multiple internal organs [78]. There is evidence of immune dysregulation with SSc-specific autoantibodies, mononuclear cell infiltrates in several affected organs, and elevated cytokine levels in tissue and serum. The anticentromere antibody is found in the group with limited cutaneous SSc, and the antitopoisomerase I antibody (Scl-70) is found in 30–40% of subjects with diffuse cutaneous SSc. Antinuclear antibodies occur in 95% of subjects overall. Diffuse cutaneous SSc is a more severe manifestation of disease than limited cutaneous SSc. It has a higher mortality and is associated with substantial morbidity as a consequence of digital ischemia/skin ulcerations from the vasculopathy, both truncal and acral scleroderma, interstitial lung disease, hypertensive renal crisis, diffuse gastrointestinal disease, and myocardial involvement. In one study of the natural history of SSc, survival was less than 80% at 2 years, less than 50% at 8.5 years, and under 30% at 12 years [79]. Other studies have confirmed the poor survival and poor-risk features of SSc, especially when there is internal organ involvement [80,81]. Two validated tools for evaluating the degree of scleroderma and measuring the effect of disease on overall function are the modified Rodnan Skin Score (mRSS) and the modified Health Assessment Questionnaire Disability Index (mHAQ) [82]. Immunosuppressive therapies investigated for severe SSc have been inadequate or ineffective. A recent study reported that 12 months of daily oral cyclophosphamide was superior to placebo in slowing the rate of progression of SSc lung disease when evaluated at 12 and 24 months after treatment [83]. Patients also had a modest improvement in the mRSS compared with the placebo group. Cyclophosphamide may be considered a standard of care for individuals with early and severe SSc, particularly those with pulmonary involvement, but the therapeutic benefits are very modest. There are no controlled, prospective clinical trials that demonstrate the efficacy of any other immunomodulatory agents. Important supportive care measures for patients with SSc include angiotensin-converting enzyme inhibitors for the management of renal crisis, and bosentan or other agents for the management of pulmonary hypertension. A limited number of clinical trials of HDIT and autologous HCT for SSc have been conducted. Patients selected for these clinical trials had in general a poor prognosis based on the presence of diffuse cutaneous disease and internal organ involvement. In a single-center study of high-dose therapy with a low-intensity regimen consisting of a single cytotoxic agent only (cyclophosphamide n = 10, melphalan n = 1) and autologous HCT, major or partial responses were observed in eight of 11 patients, but at a median of 18 months, eight patients had not responded or had relapsed (Table 69.6) [61]. Four (36%) patients had died by 18 months after HDIT. In a later study of patients (n = 26) who had already survived at least 6 months after high-dose cyclophosphamide only and HCT, survival was 96% and event-free survival was 64% at 5 years [57]. However, about half of the patients included in this study had diffuse cutaneous disease but without internal organ involvement. In a multicenter study of a more intensive HDIT regimen consisting of TBI and cyclophosphamide in 34 patients with diffuse cutaneous disease and internal organ involvement, 17 of 27 (63%) evaluable patients who survived at least 1 year after HDIT had sustained responses (without progression or disease activation) at a median follow-up of 4 years [52]. Patients with sustained responses had required no immunebased treatment after HDIT. There was a major improvement in the degree of scleroderma as measured by the mRSS, and in overall function as measured by the mHAQ at final evaluation (Fig. 69.6). Skin biopsies confirmed that the improvement in skin score was associated with a

14

19

26

21

14

Saiz [76]

Saccardi [63]

Nash [51]

Burt [64]

Samijn [65]

SP (9) RR (5) SP (15) RR (4) SP (17) PP (8) RR (1) SP (14) PR (6) RR (1) SP

SP

SP (19) PP (14) RR (2) SP

6.0 (5.0–6.5)

7.0 (3.0–8.5)

6.5 (5.5–7.5) 6.5 (6.0–7.5) 6.0 (4.5–6.5) 6.5 (5.0–6.5) 7.0 (5.0–8.0)

6.0 (4.5–8.0)

Median EDSS* (range)

CD34 selection

BEAM + ATG TBI/CY + ATG

TBI/CY

TBI/CY + ATG

G-CSF + CY G-CSF + prednisone

G-CSF + CY

None (marrow)

CD34 selection

BCNU/CY + ATG

G-CSF + CY

CD34 selection

None

CD34 selection

CD34 selection + monoclonals

BEAM ± ATG

G-CSF + Cy

CD34 selection

CD34 selection

BU/CY

BEAM + ATG

G-CSF/GM-CSF + CY

T-cell depletion

G-CSF only

High-dose therapy

Mobilization

36 (7–36)

24 (12–60)

27 (2–47)

36 (12–72)

37 (19–55)

8 (1–18)

22 (17–30)

35 (3–67)

Follow-up median months (range)

0/1

0/2

1/1

0/0

0/0

0/0

2/0

1/1†

Treatment/disease-related mortality (n)

PFS 36%

62% stable or improved

PFS 95% (6 years) 76% stable or improved

PFS 86%

90% stable or improved

PFS 40%

PFS 81% at 5 years

Clinical result

ATG, antithymocyte globulin; BEAM, BCNU, etoposide, cytosine arabinoside, melphalan; BU, busulfan; CY, cyclophosphamide; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; PFS, progression-free survival; PP, primary progressive; PR, progressive relapsing; RR, relapse-remitting; SP, secondary progressive; TBI, total body irradiation. * In brief, the functional levels of the Expanded Disability Status Scale (EDSS) are graded from 0 to 10 points and include changes in increments of 0.5 points. An EDSS score of 0 indicates a normal neurologic examination in all functional systems. An EDSS score of 10 indicates death from MS. Most of the MS patients who entered these clinical trials of HDIT and autologous HCT had an EDSS score of 5.0–8.0. In general, the function of patients at these different EDSS scores are: 5.0 Ambulant 200 m without aids, difficulty to work full day, functional systems grade 5. 6.0 Intermittent or unilateral walking aid for 100 m. 7.0 Wheelchair-bound (walking <5 m with assistance). Able to transfer and use a wheelchair alone. Sometimes severe pyramidal grade 5. 8.0 Bed- and chair-bound, self-care functions retained (arm function retained), sitting out of bed most of the day. † One patient with progression of MS was started on interferon-beta after a relapse, developed a factor VIII inhibitor at 14 months, and died at 28 months after HDIT and autologous HCT.

10

Kozak [77]

5

35

Fassas [75]

Openshaw [66]

Patients (n)

Author

MS type (n)

Table 69.5 Clinical trials of high-dose immunosuppressive therapy (HDIT) and autologous hematopoietic cell transplantation (HCT) for multiple sclerosis (MS)

1022 Chapter 69

Hematopoietic Cell Transplantation for Autoimmune Diseases

Fig. 69.5 Gadolinium-enhancing lesions in the brain after high-dose immunosuppressive therapy (HDIT) (BEAM) and autologous hematopoieitic cell transplantation (HCT): magnetic resonance imaging results. The mean numbers of gadolinium-enhancing lesions in the brain per month per patient are reported at baseline during the 3-month period before mobilization (−3– 1), after mobilization (Cy), and after autologous stem cell transplantation (ASCT) to last follow-up. The duration of gadolinium enhancement of a new lesion is approximately 4 weeks so the presence of gadolinium-enhancing lesions in the central nervous system indicates recently active disease. After HDIT and autologous HCT, there is a marked reduction in enhancing lesions for the duration of the follow-up. (Reproduced from [63], with permission. Copyright [2005], American Society of Hematology.)

decrease of the dermal fibrosis. Lung, heart, and kidney function, in general, remained clinically stable. The treatment-related mortality was 23% and the estimated progression-free survival was 64% at 5 years. Better patient selection and modifications to the treatment regimen may reduce treatment-related mortality. Other smaller studies have also suggested a major therapeutic effect of HDIT on skin and function [84,85]. In a report from the EBMT on 57 patients in their registry with a median follow-up of 20 months, the cumulative probability of disease progression at 5 years was 48%, and the cumulative probability of survival at 5 years was 72% [69]. The responses observed in skin and overall function seem to be greater than what has been previously described with standard therapy, but randomized clinical trials are required to determine if HDIT improves overall outcome. Systemic lupus erythematosus SLE is an autoimmune disorder that is characterized by the presence of antinuclear antibodies and immune complexes [86]. The incidence and prevalence have been estimated at 5.1 and 52.2 per 100,000, respectively. Numerous organ systems can be involved with SLE, and disease severity may vary from mild to life-threatening. Antinuclear antibodies, antidouble-stranded DNA antibodies, and anti-Smith antibodies are present in 98%, 70%, and 25% of SLE patients. Overall, the 10-year survival of patients with SLE has been reported as 75–85%, with more than 90% surviving at 5 years [87]. In the group of SLE patients with severe disease who are refractory to standard treatment, survival is expected to be much less. Standard treatment options are not curative, and complete sustained remissions are rare. Some rheumatologists would therefore recommend a strategy in which treatment was sufficiently intense to prevent organ damage and suppress symptoms to an acceptable level. Antimalarials

1023

like hydroxychloroquine reduce the frequency of disease flares. Lowdose corticosteroids are used for patients in whom disease symptoms have not been controlled by more conservative measures, and disease flares are treated with high-dose corticosteroids. Agents such as cyclophosphamide, azathioprine, rituximab, and mycophenolate mofetil have also been found effective for controlling disease activity [88]. The SLE Disease Activity Index is a validated tool for following disease activity over time [89]. Clinical trials of HDIT with and without HCT were conducted on patients with SLE who were refractory to standard therapies. In a singlecenter study (n = 50) of HDIT and HCT, patients were mobilized with G-CSF and cyclophosphamide, and the autologous graft was T-cell depleted by CD34 selection [55]. The HDIT regimen included high-dose cyclophosphamide (200 mg/kg) and ATG. The probability of overall and disease-free survival at 5 years was 84% and 50%, respectively. There was a significant improvement in the SLE Disease Activity Index score and renal function stabilized (Fig. 69.7). Titers for antinuclear and antidouble-stranded antibodies and complement levels improved after HDIT. Treatment-related mortality was 4%, and both deaths occurred before HDIT. In comparison, in a study of HDIT without HCT in patients with SLE, outcomes after single-agent, high-dose cyclophosphamide (200 mg/kg) (n = 14) were similar at a median follow-up of 27 months, with five (36%) patients reported as having durable complete remissions [18]. This approach avoids the reinfusion of cells which has the potential for causing a relapse. However, as might be expected, time to recovery of blood counts is longer, and this may contribute to increased morbidity from the procedure. There was no treatment-related mortality. The EBMT registry reported the experience with HDIT and autologous HCT for SLE and observed significant disease responses in 31 of 50 patients [90]. However, 10 of these 31 patients relapsed, and some of the other patients remained on immunomodulatory therapy after HDIT to prevent relapse. Treatment-related mortality was 16%. Although the experience is limited, HDIT can induce a high percentage of disease responses in patients with SLE who have otherwise been refractory to standard therapy, and remissions have been durable in a significant proportion of patients. Rheumatoid arthritis RA is a chronic multisystem disease characterized by synovial inflammation, cartilage damage, and bone erosions [91]. The criteria for determining response to treatment have been defined by the American College of Rheumatology [92]. The disease affects about 0.8–1% of the population, and the incidence is 25 and 54 per 100,000 persons in men and women, respectively. The pathologic hallmark of RA is the synovial inflammation with proliferation of macrophages and fibroblasts. If the condition is severe, the inflamed synovium develops into an invasive pannus which destroys cartilage and bone. The disease course can be mild with minimal damage of only a few joints or severe with many inflamed, damaged joints. Other complications associated with RA are a vasculitis, cervical spine disease, lung nodules or interstitial fibrosis, and cardiac complications including pericarditis. Risk of progression can be predicted by prognostic factors such as increased number of affected joints, high level of C-reactive protein, presence of rheumatoid factor, and extra-articular features of the disease [91]. The risk of mortality is increased with more severe disease activity or one or more extra-articular disease manifestations [93–95]. There is a mortality of approximately 30% at 5 years for patients with the highest disease activity. Many anticytokine or immunomodulatory agents are approved for RA, including methotrexate, hydroxychloroquine, sulfsalazine, leflunamide, infliximab, adalimumab, etanercept, abatacept, and anakinra. B-cell-targeted therapy with

57 (29–86)

47 (25–60)

55 (21–100)

61 (40–83)

67 (48–80)

30 (4–41)

26 (15–32)

32 (9–51)

30 (3–48)

29 (13–36)

BL median mRSS (range)

CD34 selection

TBI CY + ATG

CY

CY

CY + ATG

G-CSF

G-CSF + CY

G-CSF + CY

G-CSF + CY

None

CD34 selection

CD34 selection

CD34 selection (n = 9)

CY (n = 10) or Mel (n = 1)

G-CSF + CY

T-cell depletion

High-dose therapy

Mobilization

25 (5–40)

20 (13–33)

63 (12–90)

48 (12–96)

18 (1–26)

Follow-up median months (range)

0/1

0/0

2/2*

8/4

1/3

Treatment-related/ disease-related mortality

3 patients alive in remission (27%) 4 patients alive with no response or progression PFS – 64% Sustained response (evaluable n = 27) in 63% Improvement in skin score (p = 0.001) + stable lung function overall EFS (evaluable n = 26) 64% at 5 years and 57% at 7 years Improvement in skin score (p = 0.05) and lung function PFS 70%

Clinical results

* There was one additional death at 5 years after HDIT from lung cancer. ATG, antithymocyte globulin; BL, baseline; CY, cyclophosphamide (mobilization: 2 or 4 g/m2; treatment: 120 mg/kg in combination with TBI or 200 mg/kg if single cytotoxic agent); DLCO, diffusion capacity of the lung for carbon monoxide; EFS, event-free survival; G-CSF, granulocyte colony-stimulating factor; Mel, melphalan; mRSS, modified Rodnan Skin Score (ranges from 0 to 51; increasing score indicates worsening scleroderma); PFS, progression-free survival; TBI, total body irradiation.

10

28

Vonk [57]

Oyama [85]

34

Nash [52]

6

11

Farge [61]

Tsukamoto [84]

Patients (n)

Author

BL median DLCO% (range)

Table 69.6 Clinical trials of high-dose immunosuppressive therapy (HDIT) and autologous hematopoietic cell transplantation (HCT) for systemic sclerosis

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(a)

(b)

Fig. 69.7 Systemic Lupus Erythematosus (SLE) Disease Activity Index (SLEDAI) improves after high-dose immunosuppressive therapy and autologous hematopoietic cell transplantation. In patients with severe refractory SLE, there was a rapid decrease in the SLEDAI (indicating a decrease in disease activity) which was sustained in a substantial proportion of patients for more than 2 years after treatment. (Reproduced from [55], with permission. Copyright [2006], American Medical Association.)

Fig. 69.6 Change in the modified Rodnan Skin Score (mRSS) and the modified Health Assessment Questionnaire (mHAQ) score after high-dose immunosuppressive therapy (HDIT) and autologous hematopoietic cell transplantation. A determination was made as to whether a parameter value was statistically significantly increasing or decreasing over time using a generalized estimating equation (GEE) model. The bold black dotted line represents an estimate of the modeled linear relationship between the parameter value and time, and summarizes the results of the GEE models. The bold black solid line represents the mean value over time for the parameter of interest. The gray solid lines are parameter values for individual patients. The mean mRSS and mHAQ values statistically significantly decreased with time after HDIT as objective markers of less scleroderma and improved function (both p < 0.001; panels (a) and (b), respectively). (Reproduced from [52], with permission. Copyright [2007], American Society of Hematology.)

decrease in progression of joint damage compared with baseline [101]. No mortality was observed in any of the clinical trials. Seventy-six patients with RA who underwent HDIT and autologous HCT were available for analysis in the EBMT registry, and the outcomes were comparable to the experience reported from the clinical trials [102]. Cyclophosphamide alone was used as the HDIT regimen in 82%, and the combination of cyclophosphamide and ATG was used in 9% of the patients. Major responses were observed in 67% of patients, and there was a significant reduction in the measures of disability. Most patients had restarted immunomodulatory treatment by 6 months after HDIT for persistent or recurrent disease activity. No treatment-related mortality was observed. The apparent difference in response of RA to HDIT compared with other autoimmune diseases might be related to immune or nonimmune biologic factors. More durable responses might be obtained with more intense HDIT regimens, but this may further increase the risk for treatment-related mortality. Another strategy to decrease relapses or progression may be to add immunomodulatory therapy after HDIT. Juvenile idiopathic arthritis

rituximab has recently been shown to be effective as well. The goal of standard therapy is to relieve the signs and symptoms of the disease since none of these treatments is curative. Four small clinical trials have been reported of HDIT for patients with RA who had failed standard treatment [60,96–100]. All patients received high-dose cyclophosphamide with or without ATG. The initial reports described early major responses in the majority of the patients. On longterm follow-up, it was noted that all 32 patients accrued to these clinical trials relapsed and required further treatment. Many of these relapsed patients who had been refractory were responsive to treatment after HDIT. Moreover, one study showed that, after HDIT, there was a

JIA is a heterogeneous group of chronic inflammatory diseases involving the joints and extra-articular tissue that begins before 16 years of age [103]. The incidence and prevalence have been estimated as 10 and 65 per 100,000 children, respectively. Patients with unremitting systemic JIA are considered to have the worst prognosis. Severe disease has an effect on bone and joint development, resulting in overgrowth or undergrowth of juxta-articular bone and therefore in limb deformities. The macrophage activation syndrome is a potentially life-threatening complication in which there is uncontrolled activation and proliferation of macrophages and T cells, as well as a marked increase in serum cytokine levels such as those of IFN-γ and granulocyte–macrophage colony-

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stimulating factor. This complication may occur in 5–8% of patients with systemic JIA, and close monitoring and early treatment are required for the best outcomes. The mortality in this disease is less than 1% and is mostly in the systemic JIA subtype [104]. One clinical trial of HDIT and autologous HCT in patients with treatment-refractory JIA (n = 22) has been reported with a median follow-up of 80 months [47]. Eighteen patients had systemic JIA and four had polyarticular JIA. The HDIT regimen included TBI (400 cGy), cyclophosphamide, and ATG. Early in the clinical trial, two patients developed macrophage activation syndrome less than 1 month after HDIT and died. Precautionary measures added to the treatment may have reduced this risk in later patients. Two other patients died after relapsing and restarting immunosuppressive treatment at more than 1 year after HDIT. Overall and disease-free survival were 82% and 36%, respectively. There were significant improvements in disease activity in the group based on disability and active joint scores. In a report from the EBMT registry on 34 JIA patients, 18 (53%) patients were in complete remission without additional therapy at 12–60 months after HDIT [105]. In a small subset of poor-prognosis JIA patients who fail standard treatment, HDIT and autologous HCT may be of benefit. Other autoimmune diseases Investigations of HDIT and autologous HCT have also been conducted in patients with other autoimmune diseases, but long-term follow-up is still not reported and the experience is still very limited. In all these clinical trials, the HDIT regimen was high-dose cyclophosphamide with or without ATG. In a clinical trial of patients with treatment-refractory Crohn’s disease, 11 of 12 patients achieved a sustained remission at a median of 18 (7–37) months after HDIT without significant treatmentrelated toxicity or mortality [106]. Six of 14 (43%) patients with refractory chronic thrombocytopenia obtained durable complete responses (platelet count >100,000/mm3), and two patients obtained a partial response at a median of 27 (9–41) months after HDIT [107]. An interesting clinical trial was performed in patients with recent-onset DM. Fourteen of 15 patients had prolonged periods of insulin independence after HDIT [108]. The durability of these responses on longer follow-up and safety of the procedure will determine if this approach with its potential for more toxicity could be considered as an acceptable treatment for DM.

Allogeneic HCT for autoimmune diseases: clinical experience Allogeneic HCT may cure severe autoimmune diseases by replacing the “diseased” autoreactive immune compartment with one that is not autoreactive (although potentially alloreactive). The primary risks of allogeneic HSCT are the morbidity and morality associated with delayed immune reconstitution and graft-versus-host disease (GVHD). Although the risk of complications and treatment-related mortality is greater than with autologous HCT, there may be a greater potential for sustained remissions of severe autoimmune diseases after allogeneic HCT, and therefore improved overall outcome. Survival after allogeneic HCT for nonmalignant hematologic disorders or good-risk hematologic malignancies like chronic myelogenous leukemia in the chronic phase has ranged from 85% to 95%. The best outcomes were observed in young patients transplanted from an HLA-matched donor. No clinical trials have yet been conducted of allogeneic HCT for which an autoimmune disease was the primary indication, but a small number of cases have been reported. The largest experience has been in patients with autoimmune cytopenias [109–112]. Of the seven evaluable patients reported from the EBMT registry, five were alive and in remission at a median follow-up of 41

months [109]. One patient with Evan’s syndrome in the report from the EBMT registry had disease progression and died. The second patient died from treatment-related complications after HCT from an HLAhaploidentical donor. Of the three case reports of Evan’s syndrome in the literature, one patient relapsed and died, and the other two died of transplant-related complications in complete remission [110–112]. There are six cases reported of allogeneic HCT for connective tissue diseases such as SSc, overlap syndrome, and RA [113–117]. Two patients with SSc had high-dose conditioning, one of whom was alive at 5 years in disease remission with resolution of the dermal fibrosis and scleroderma lung. Four patients received reduced-intensity conditioning regimens. All patients were alive at last follow-up and in remission. Three of these patients had stable mixed hematopoietic chimerism and no history of GVHD. Regulatory immune mechanisms associated with the establishment of mixed hematopoietic chimerism may be responsible for inducing remission of the autoimmune disease. Conclusions regarding the therapeutic role of allogeneic HCT for severe autoimmune diseases await the results from clinical trials conducted in a carefully selected group of patients.

Autoimmunity after HCT Autoimmune disorders have been observed to occur infrequently after both autologous and allogeneic HCT for hematologic malignancies. This risk needs to be considered for those patients for whom the primary indication for HCT is an autoimmune disease. The mechanisms for autoimmune disorders after HCT are not well understood but may relate to the disruption of immune regulatory pathways as a result of induction to a lymphopenic state with conditioning and then homeostatic expansion of lymphocytes during reconstitution of the immune system [118]. The development of an autoimmune disease after HCT likely depends on predisposing factors and the balance between reconstituting autoreactive and regulatory lymphocytes. The incidence of an autoimmune hemolytic anemia was observed to be 2.3% and 4.4% with onset up to 18 months after allogeneic HCT in two case series of 299 and 272 patients, respectively. There have been additional case reports of autoimmune disorders after both autologous and allogeneic HCT including other cytopenias, thyroid disease, acquired hemophilia, and myasthenia gravis [119]. For patients treated for an existing autoimmune disorder, there may be a predisposition for a new secondary autoimmune disorder to occur [52,70]. After allogeneic HCT, there have also been case reports of adoptive transfer of autoimmune disorders [53].

Conclusion The common autoimmune diseases likely result from a genetic predisposition and an environmental trigger. There is a high rate of disease remissions after HDIT and autologous HCT, and a significant proportion are sustained after 4–5 years. The rate and durability of response may depend on the intensity of the HDIT regimen and the type of autoimmune disease being treated. Randomized clinical trials comparing HDIT and autologous HCT to standard therapy or “best clinical practice” are now being conducted in specific autoimmune diseases. The experience with allogeneic HCT as a treatment for autoimmune diseases is still limited but promises to be highly effective. The risks of increased morbidity and mortality from GVHD have delayed clinical trials of this approach. Carefully selected patients with active autoimmune disease that is life-threatening or threatening critical organs and refractory to standard treatment should be considered as candidates for clinical trials of allogeneic HCT or HDIT followed by autologous HCT.

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tory juvenile idiopathic arthritis: analysis of clinical effects, mortality, and transplant related morbidity. Ann Rheum Dis 2004; 63: 1318–26. Oyama Y, Craig RM, Traynor AE et al. Autologous hematopoietic stem cell transplantation in patients with refractory Crohn’s disease. Gastroenterology 2005; 128: 552–63. Huhn RD, Fogarty PF, Nakamura R et al. Highdose cyclophosphamide with autologous lymphocyte-depleted peripheral blood stem cell (PBSC) support for treatment of refractory chronic autoimmune thrombocytopenia. Blood 2003; 101: 71–7. Voltarelli JC, Couri CE, Stracieri AB et al. Autologous nonmyeloablative hematopoietic stem cell transplantation in newly diagnosed type 1 diabetes mellitus. JAMA 2007; 297: 1568–76. Passweg JR, Rabusin M, Musso M et al. Haematopoetic stem cell transplantation for refractory autoimmune cytopenia. Br J Haematol 2004; 125: 749–55. Oyama Y, Papadopoulos EB, Miranda M et al. Allogeneic stem cell transplantation for Evans syndrome [Review]. Bone Marrow Transplant 2001; 28: 903–5. Marmont AM, Gualandi F, Van Lint MT, Bacigalupo A. Refractory Evans’ syndrome treated with allogeneic SCT followed by DLI: demonstration of a graft-versus-autoimmunity effect. Bone Marrow Transplant 2003; 31: 399–402. Raetz E, Beatty PG, Adams RH. Treatment of severe Evans syndrome with an allogeneic cord blood transplant. Bone Marrow Transplant 1997; 20: 427–9.

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113. Nash RA, McSweeney PA, Nelson JL et al. Allogeneic marrow transplantation in patients with severe systemic sclerosis: resolution of dermal fibrosis. Arthritis Rheum 2006; 54: 1982– 6. 114. Khorshid O, Hosing C, Bibawi S et al. Nonmyeloablative stem cell transplant in a patient with advanced systemic sclerosis and systemic lupus erythematosus. J Rheumatol 2004; 31: 2513– 16. 115. Jones OY, Good RA, Cahill RA. Nonmyeloablative allogeneic bone marrow transplantation for treatment of childhood overlap syndrome and small vessel vasculitis. Bone Marrow Transplant 2004; 33: 1061–3. 116. Burt RK, Oyama Y, Verda L et al. Induction of remission of severe and refractory rheumatoid arthritis by allogeneic mixed chimerism. Arthritis Rheum 2004; 50: 2466–70. 117. Loh Y, Oyama Y, Statkute L et al. Nonmyeloablative allogeneic hematopoietic stem cell transplantation for severe systemic sclerosis: graft-versus-autoimmunity without graft-versushost disease? Bone Marrow Transplant 2007; 39: 435–7. 118. King C, Ilic A, Koelsch K, Sarvetnick N. Homeostatic expansion of T cells during immune insufficiency generates autoimmunity. Cell 2004; 117: 265–77. 119. Daikeler T, Tyndall A. Autoimmunity following haematopoietic stem-cell transplantation. Baillieres Best Pract Clin Haematol 2007; 20: 349– 60.

70

Vinod Pullarkat & Stephen J. Forman

Hematopoietic Cell Transplantation for Rare Hematologic Malignancies

Introduction In this chapter, we review the results of autologous and allogeneic hematopoietic cell transplantation (HCT) for a variety of rare hematologic malignancies that, in our opinion, would be rarely encountered outside of specialized centers, and for which HCT has been performed (Table 70.1). As would be expected with rare disorders, there is a paucity of prospective well-designed trials, and most published literature is limited to single case reports, small case series or retrospective analyses that include heterogeneous groups of patients. We have attempted to review the published literature and make recommendations based on the preponderance of evidence. Recognizing the bias towards reporting only positive results that is inherent in single case reports, we have used case series and retrospective analysis of registry data rather than single case reports as the basis for our recommendations whenever possible. It is unlikely that prospective studies to test the role of HCT that contain adequate numbers of patients will ever be performed for the extremely rare entities discussed below. However, there is a need for such trials preferably performed by large cooperative groups for the less uncommon malignancies discussed here.

Rare myeloid malignancies Langerhans cell histiocytosis Langerhans cell histiocytosis (LCH) refers to a group of clinical syndromes characterized by clonal Langerhans cell proliferation. These syndromes have historically been designated by different names based on the pattern of organ involvement. These include histiocytosis X, Letterer–Siwe disease, and Hand–Schüller–Christian disease [1]. Although typically a disorder of infants and children, LCH can also occur in adults. Age, pattern of organ system involvement, and organ dysfunction are the most important prognostic factors. In general, children under 2 years of age and adults over 65 years of age do poorly [2–4]. While single-system LCH usually involving the skeleton or skin is an indolent disease, chemotherapy-resistant multisystem LCH (MSLCH) especially involving organs like the bone marrow, liver, spleen, and lungs has an extremely poor prognosis [3,5]. Various case reports have described successful allogeneic HCT for patients with MS-LCH [6–10]. One of the initial reported patients who underwent allogeneic HCT for chemotherapy-resistant disease remained

in complete remission (CR) 12 years later, showing that long-term disease-free survival (DFS) is possible using this approach [11]. The French Langerhans Cell Study Group has reported results of eight patients with refractory MS-LCH transplanted at six institutions. Three patients underwent autologous HCT while five underwent allogeneic HCT. Of the three patients who underwent autologous HCT, two died of relapse and one was alive and in CR 7 years later. Of the five patients who underwent full-intensity allogeneic HCT, two died of regimenrelated toxicity and one died of sepsis 23 months after HCT, while two were alive and in CR 12 years and 21 months, respectively, after HCT, again demonstrating the ability of allogeneic HCT to achieve durable remissions in refractory MS-LCH [12]. The transplant-related mortality of full-intensity allogeneic HCT is high due to the heavy prior exposure of these patients to chemotherapy, as well as involvement of vital organs like the lungs and liver [13]. In an attempt to decrease the treatment-related mortality of allogeneic HCT, Steiner et al. have reported the results of reduced-intensity allogeneic HCT using fludarabine-based conditioning in nine pediatric patients with high-risk LCH. Graft sources included matched siblings, matched unrelated donors, mismatched unrelated donors, and haploidentical parental donors. The three patients who underwent haploidentical HCT underwent second transplants for graft failure. After a median follow-up of 390 days, seven of the nine patients were alive without evidence of disease, which is remarkable for this group of heavily treated high-risk patients [13]. Interestingly, one patient who had engraftment failure with autologous reconstitution after a second allogeneic haploidentical HCT remained in remission 770 days after the first HCT [13], suggesting that autologous HCT may be a viable option for patients who lack suitable donors or are not candidates for allogeneic HCT. Two patients with MS-LCH who underwent successful autologous HCT have been reported. One remained in CR over 4 years and the other at 10 months after HCT [9,14]. Based on the above data and given the poor prognosis of refractory MS-LCH, it seems preferable to perform allogeneic HCT early in the course of disease in patients who have matched donors. Reducedintensity conditioning may be better in patients who have impairment of vital organ function or heavy prior exposure to chemotherapy. Autologous HCT may be an option for patients who lack matched donors. HCT is a promising treatment modality for treatment of MS-LCH, and large clinical trials testing both autologous and allogeneic HCT early in the disease course are warranted. Systemic mastocytosis

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Systemic mastocytosis (SM) is characterized by infiltration of the bone marrow and other organs by neoplastic mast cells. Activating mutations

Hematopoietic Cell Transplantation for Rare Hematologic Malignancies Table 70.1. Selected rare hematologic malignancies for which HCT has been performed • • • • • • • • • • • • • • • •

Langerhans cell histiocytosis Systemic mastocytosis Hypereosinophilic syndrome Acute megakaryoblastic leukemia Acute erythroleukemia Chronic myelomonocytic leukemia Adult T-cell leukemia/lymphoma Natural killer cell leukemia/lymphoma Mycosis fungoides Sézary syndrome Angioimmunoblastic T-cell lymphoma Primary central nervous system lymphoma Hepatosplenic T-cell lymphoma (gamma/delta T-cell lymphoma) T-cell prolymphocytic leukemia Subcutaneous panniculitic T-cell lymphoma Hematodermic neoplasm (blastic natural killer cell lymphoma)

in the KIT gene (most commonly D816V) are almost invariably present in these neoplastic mast cells. The World Health Organization (WHO) classification recognizes various clinicopathologic SM syndromes, including indolent SM, SM with associated clonal hematologic nonmast cell lineage disease (SM-AHNMD), aggressive SM, and mast cell leukemia (MCL) among others [1,15]. Aggressive SM and MCL have a dismal prognosis [15,16]. While indolent SM has a prolonged clinical course as its name suggests, the clinical course in the case of SMAHNMD is dictated by the underlying malignancy, which most often is acute myeloid leukemia (AML), myelodysplasia or a myeloproliferative disorder [17,18]. A specific association exists between SM and t(8;21) AML [18–20]. The HCT experience in SM is very limited. Nakamura et al. have transplanted three SM patients from human leukocyte antigen (HLA)identical siblings using reduced-intensity conditioning with fludarabine and cyclophosphamide (CY). One patient had MCL, another had SM with myelodysplasia (SM-AHNMD), and the third had aggressive SM. All patients achieved complete T-cell engraftment. Although there was clinical and pathologic evidence of a graft-versus-mast cell effect, this did not translate into durable remissions, and all three patients eventually relapsed. In one patient, there was significant morbidity from mast cell mediator release at day 3 after transplantation and concurrent with flareups of acute graft-versus-host disease [21]. Another report describes a patient with MCL who underwent a matched unrelated donor HCT after standard busulfan and CY (BU/CY) conditioning. The number of residual recipient bone marrow mast cells after HCT could be lowered by administration of a donor lymphocyte infusion (DLI), thereby suggesting a graft-versus-mast cell effect. This patient remained in clinical remission over 3 years after HCT [22]. Patients with SM-AHNMD fare poorly compared with patients with the underlying hematologic disease alone [17]. This is especially true of cases where the associated hematologic disorder is t(8;21) AML, an otherwise low-risk AML subset. However, this is not surprising given the fact that KIT mutations are associated with a poor prognosis in t(8;21) AML [23,24]. Durable remissions have been described in SM– AML with t(8;21) translocation after allogeneic HCT even though recipient bone marrow mast cells carrying t(8;21) were detected a year after allogeneic HCT [20,25]. Prolonged remission after a second allogeneic HCT following high-dose conditioning has also been reported in a case of SM with t(8;21) AML that relapsed after an initial reduced-

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intensity sibling donor HCT [19]. In a reported case of SM associated with myelodysplastic syndrome, mast cell infiltration of bone marrow and liver persisted at 8 months following allogeneic HCT, even after normal hematopoiesis had been restored. This patient also developed venous thrombosis in the post-transplant period, which was attributed to mast cell mediator release [26]. Another patient who was alive 6 years after allogeneic HCT for SM with associated chronic myelomonocytic leukemia (CMML) has also been reported [18]. Given the extremely grim prognosis of patients with aggressive SM and MCL and some evidence of graft-versus-mast cell effect, it appears reasonable to test the efficacy of allogeneic HCT for these subsets of SM, preferably in the context of a clinical trial. Newer kinase inhibitors like dasatinib are capable of inhibiting the D816V KIT and mutation may have a role in conjunction with allogeneic HCT [27]. It is possible that more intense conditioning may also be beneficial [19]. Special approaches may be needed to manage morbidity from mediator release during the peritransplant period in patients with a large mast cell burden [21,26]. For patients with SM and t(8;21), it appears that allogeneic HCT in first CR (CR1) is the best option if a suitable donor is identified based on the limited data available. Recipient-derived bone marrow mastocytosis may persist after allogeneic HCT and does not appear to have clinical significance [20,25]. The efficacy of HCT for SM associated with other hematologic malignancies is unknown at present. It is unlikely that HCT will have a role in indolent SM, especially with development of targeted therapies that inhibit D816V KIT mutation. Hypereosinophilic syndrome Hypereosinophilic syndrome (HES) refers to a heterogeneous group of disorders that are characterized by peripheral blood eosinophilia and a propensity to cause organ damage, especially cardiac dysfunction from endomyocardial fibrosis. Criteria for diagnosis of HES have been defined [28,29]. Myeloproliferative and lymphoproliferative variants of HES are now recognized. A subset of patients, almost exclusively male, with the myeloproliferative variant carry an interstitial deletion resulting in a FIP1L1-PDGFRa (FIP1-like 1–platelet-derived growth factor receptoralpha) fusion and are exquisitely sensitive to imatinib [30–32], while the lymphoproliferative variant is associated with clonal T-cell expansion [33]. The major cause of morbidity and mortality in HES is cardiac dysfunction [28]. The results of allogeneic HCT for HES are limited to case reports. Hence the overall results of allogeneic HCT for this disorder cannot be determined. A number of these reports have been summarized by Cooper et al. [34]. The majority of transplants have been performed from HLA-identical siblings after full-intensity conditioning. A few cases have undergone reduced-intensity conditioning [34,35]. DFS after up to 6 years of follow-up and reversal of myelofibrosis have been reported [34,36]. These reports of long-term remission after allogeneic HCT may be suggestive of the curative potential of allogeneic HCT for this disorder. An interesting observation is that eosinophilia can recur in some cases even when 100% of the bone marrow and peripheral blood cells are of donor origin [34,37,38]. This cannot be considered failure of HCT and usually resolves spontaneously. It is not clear if these cases had the lymphoproliferative variant of HES with clonal T-cell expansion. High interleukin-5 levels were detected in two patients, which suggests that abnormal T cells may persist in lymphoid organs (despite full donor marrow engraftment) and lead to eosinophilia, which probably resolves with complete donor engraftment in lymphoid organs [34,38]. Our understanding of the biology of HES is rapidly evolving. Newer classification based on cytogenetic and molecular abnormalities and

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improved definition of prognostic factors may help better predict patients who are not imatinib responsive and who are more likely to develop end-organ damage. The results of imatinib therapy for FIP1L1PDGFRa-positive patients are excellent, while FIP1L1-PDGFRanegative cases rarely have durable responses to imatinib [39]. Prognosis for FIP1L1-PDGFRa-negative patients who develop end-organ dysfunction is poor, and allogeneic HCT should be strongly considered in suitable patients who have early signs of end-organ damage and have failed prednisone and a second-line agent such as hydroxyurea or alpha-interferon. Although there are reports of improvement in cardiac function after allogeneic HCT in patients with HES and cardiac dysfunction [40], the nonrelapse mortality (NRM) in this situation is likely to be high. Acute megakaryoblastic leukemia Acute megakaryoblastic leukemia (AMKL) accounts for about 1% of AML in adults and about 5–10% of AML in children, with a higher incidence in children with Down’s syndrome [41–43]. The disease has a bimodal age distribution with peaks in early childhood (under 3 years) and adulthood [42]. Bone marrow fibrosis is a prominent feature of this disorder, making AMKL the most common cause of “acute myelofibrosis” [1,41]. AMKL may occur de novo or arise from an underlying myeloproliferative or myelodysplastic syndrome. Although 50% of adult patients achieve a CR with standard AML induction chemotherapy, long-term survival rates are dismal with conventional consolidation chemotherapy [41,44–46]. Hence HCT has been used as consolidation therapy in an attempt to improve the outcome. Single case reports have been published describing reversal of bone marrow fibrosis and durable remissions after allogeneic HCT [47–50]. One of the early reports of allogeneic HCT for AMKL describes a 28year-old woman with “acute malignant myelosclerosis” who was transplanted from her histocompatible brother (Fig. 70.1) [49]. This patient continues to be followed at the City of Hope National Medical Center and remains in remission over 27 years after HCT. A retrospective analysis of the results of HCT for de novo AMKL in CR1 has been performed by the European Group for Blood and Marrow Transplantation (EBMT). At 3 years after allogeneic HCT, the results in children and adults respectively were: leukemia-free survival (LFS) of 66% and 46%; overall survival (OS) of 82% and 43%; and NRM of 0% and 26%. The results of autologous HCT (children and adults respectively) were: LFS of 52% and 27%; OS of 61% and 30%; and NRM of 3% and 8% [51] (Fig. 70.2). Athale et al. have reported their single-institution experience treating 41 patients with childhood AMKL that included six patients with secondary AMKL and six with Down’s syndrome. The 2-year event-free survival (EFS) was 26% for allogeneic HCT compared with 0% for chemotherapy alone. The results of allogeneic HCT were significantly better when performed in remission than with persistent disease (2-year EFS 46% versus 0%). In both of the above-mentioned studies, the outcomes were better for children with Down’s syndrome, who had an estimated 2-year EFS of 83% in the latter study [45]. Based on above data, allogeneic HCT from matched sibling or matched unrelated donors should be the consolidation treatment of choice in adults with AMKL who achieve CR. In children, autologous transplant in CR1 is an option in patients who lack a matched sibling donor, and there are reports of prolonged survival after this approach. The outcome of autologous transplant in adults is very poor. At present, allogeneic HCT cannot be recommended for patients with resistant disease, who should be treated with investigational approaches. There appears to be no advantage for allogeneic HCT over autologous HCT in patients with Down’s syndrome and AMKL.

Acute erythroleukemia Acute erythroleukemia (AEL) is an uncommon form of AML accounting for about 4.5% of all cases of AML [52]. AEL may occur de novo or secondary to an underlying myelodysplastic or myeloproliferative disorder, or after previous chemotherapy [53,54]. AEL has a poor prognosis compared with other AML subtypes, with a median OS of only 9 months in one retrospective study [54]. Like AMKL, AEL has a dismal prognosis with conventional chemotherapy [54,55]. The EBMT has reported the results of 103 autologous and 104 HLAidentical sibling allogeneic HCTs done for de novo AEL in CR1. For allogeneic HCT, the 5-year LFS, relapse incidence, and NRM were 57 ± 5%, 21 ± 5% and 27 ± 5%, respectively, while the corresponding figures for autologous HCT were 26 ± 5%, 70 ± 6% and 13 ± 4%. In multivariate analysis, age remained the most important prognostic factor for both allogeneic and autologous HCT [56]. It must be noted that the median age of patients in this study (40 years for autologous HCT and 36 years for allogeneic HCT) is lower than the median age of patients with AEL, which was 59 years in one series [54]. In a smaller study, Killick et al. have reported the outcome of 27 patients with AEL, which included eight adults with secondary AEL and five children. Among patients with de novo AEL, 95% achieved CR with induction chemotherapy compared with only 57% of patients with secondary AEL. Eighteen of 22 (82%) patients achieving CR underwent consolidation by HCT (15 allogeneic, and three autologous). The median survival of 2.9 years for de novo AEL was not different from that of their matched controls with other forms of AML. The overall relapse rate of only 35% suggests a benefit for HCT consolidation in CR1 [57]. Therefore, allogeneic HCT is presently the treatment of choice for patients with AEL who are in CR1. Autologous HCT may be an option for patients in CR1 who lack matched donors. The results of allogeneic HCT for refractory and relapsed disease remain unknown at the present time.

Chronic myelomonocytic leukemia CMML is a clonal stem cell disorder characterized by peripheral blood monocytosis that shares features with both myelodysplastic and myeloproliferative disorders. The median age at diagnosis is 65–75 years, with a male predominance. Criteria for diagnosis of CMML have been defined in the WHO classification [1]. Since CMML was previously grouped with the myelodysplastic syndromes, patients who underwent allogeneic HCT for CMML were included in reports of HCT results for myelodysplastic syndromes. However, CMML has distinct clinicopathologic features that warrant its consideration as a separate entity. Although CMML includes a heterogeneous group of patients with varying rates of disease progression, the overall prognosis is poor, with a median survival of only 12 months in one study [58]. CMML is not curable with conventional chemotherapy, and the response to targeted therapies is poor except in the small subset of patients who carry a PDGFRb fusion oncogene and are sensitive to imatinib [59]. Recognizing this dismal prognosis, allogeneic HCT has been tried as a potentially curative option. The EBMT has reported the outcome of 50 patients who underwent allogeneic HCT for CMML. Forty-three patients received transplants from related donors, and seven patients received unrelated donor transplants. The median age of the patients was 44 years. Conditioning was total body irradiation (TBI) based in 26 patients and chemotherapy based in the rest. After a median follow-up of 40 months, the 5-year estimated OS and DFS and probability of relapse were 21%, 18%, and 49%, respectively [60]. A report from the Mayo Clinic describes 17 patients

Hematopoietic Cell Transplantation for Rare Hematologic Malignancies

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Fig. 70.1 (a–d) Hematoxylin and eosin stains of bone marrow biopsies from diagnosis and days 15, 100, and 360, respectively, after allogeneic hematopoietic cell transplantation (HCT) in a patient with “acute myelofibrosis.” Progressive restoration of normal hematopoiesis is seen. (e–h) Corresponding reticulin stains show total clearing of reticulin fibrosis after allogeneic HCT. (Reproduced from [49], with permission.)

who underwent allogeneic HCT (14 related and three unrelated) for CMML. Eighteen percent of patients were alive and disease free at a median follow up of 34.5 months, and the NRM at 3 years was 41%. CRs lasting 15 months could be induced with DLI in one relapsed patient and in another with mixed chimerism, suggesting a graft-versusleukemia (GVL) effect [61]. Kerbauy et al. have reported better results for a cohort of 43 patients transplanted at the Fred Hutchinson Cancer Research Center in Seattle. Twenty-two of these patients received HCT from unrelated donors. The median age of the patients was 48 years. BU/CY was the most common conditioning regimen used (15 patients). The estimated 4-year OS and RFS was 41%, and relapse incidence was 23%. Although these authors applied various prognostic-scoring systems, including the MD Anderson prognostic score [58], in an attempt to predict HCT outcome, comorbidity was the only significant predictor for OS (Fig. 70.3) [62]. Laport et al. have recently reported their experience with seven patients with CMML who underwent allogeneic HCT after conditioning with fludarabine and a single 2 Gy dose of TBI [63]. Six of these seven patients have subsequently died of relapsed disease (Laport, personal communication).

Allogeneic HCT from matched siblings or matched unrelated donors should be an early consideration for patients with CMML who are suitable candidates. It must be noted that although the median age of patients in the above studies is much lower than the median age at diagnosis of CMML patients as a whole, the NRM for allogeneic HCT is quite high. Comorbidity is a strong predictor of outcome, and must be an important consideration in selecting patients for allogeneic HCT. The majority of reported patients have undergone full-intensity conditioning, which most likely contributed to the high NRM. Despite the initial discouraging result with reduced-intensity conditioning [63], the optimum conditioning intensity needs to be further explored in order to improve the results of allogeneic HCT as a curative option in CMML. HCT for CMML is also discussed in Chapter 57.

Rare lymphoid malignancies Adult T-cell leukemia/lymphoma Adult T-cell leukemia/lymphoma (ATLL) refers to a specific clinicopathologic syndrome characterized by prominent skin involvement,

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Chapter 70

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Fig. 70.2 (a) Leukemia-free survival (LFS) after autologous hematopoietic cell transplantation (HCT) in adults and children with de novo acute megakaryoblastic leukemia (AMKL) in first complete remission (CR1). (b) LFS after allogeneic HCT in adults and children with de novo AMKL in CR1. (Reproduced from [51], with permission).

hypercalcemia, and an acute leukemic or lymphomatous presentation. ATLL is specifically associated with infection with type I human T-cell lymphotropic retrovirus (HTLV-1), which is clonally integrated into the genome of the malignant T cells [1]. Although ATLL is endemic in parts of Japan and the Caribbean, cases also occur in the United States, especially in the south-east [1,64]. Chronic or smoldering forms of the disease do occur and have a more indolent course compared with the acute or lymphoma types [1,65]. Not surprisingly, most of the experience in treating ATLL comes from Japan. The results of intensive combination chemotherapy are poor, with a median survival of only 8.5 months in one study and 13 months in another [66,67]. The results of autologous HCT are similarly poor. In a review of eight reported cases, there were no long-term survivors [68]. Hence, allogeneic HCT has been tried with some success. In the largest series reported, Fukushima et al. have reported results of 40 patients with acute or lymphoma types of ATLL who underwent allogeneic HCT at seven institutions in Japan. All but one patient received high-dose conditioning. Twenty-seven patients were transplanted from matched related donors, five from mismatched related donors, and eight from matched unrelated donors. Of the 36 donors whose HTLV-1 status was known, nine were HTLV-1 positive. The estimated 3-year OS, relapse-

Fig. 70.3 (a) Relapse-free survival of 43 patients with chronic myelomonocytic leukemia (CMML) who underwent allogeneic hematopoietic cell transplantation (HCT). Eighteen patients were alive without relapse; five of the 18 had been followed up for 9.6–14.1 years after transplantation and are indicated as censored at 9 years (censored patients are indicated by tick marks). (b) Probability of survival according to the HCTspecific comorbidity index. Patients with scores of 0–2 (solid line) were compared with patients with scores of 3–6 (dotted line). One patient could not be scored because of incomplete data. Four patients with scores of 0–2 were alive at 9.6–14.1 years, and one patient with a score of 3–6 was alive at 12.2 years. Patients indicated as censored at 9 years. (Reproduced from [62], with permission.)

free survival (RFS), and disease relapse rate were 45.3%, 33.8%, and 39.3%, respectively (Fig. 70.4). Of 10 patients that relapsed, three were able to achieve remission by reduction in immunosuppression, suggesting a GVL effect [69]. Another study by Kami et al. with shorter follow up and smaller number of patients showed similar overall results. These investigators performed allogeneic HCT in 11 patients with ATLL. Ten patients received grafts from related donors and one from an unrelated donor. Two donors were seropositive for HTLV-1. Nine patients received highdose conditioning. Four patients remained alive and disease free at a median follow up of 25 months, and the rest succumbed to HCT-related complications. In two patients who had recurrence, CR could be reinduced by withdrawal of immunosuppression and DLI [70]. In another series of 10 cases who underwent high-dose conditioning and allogeneic HCT (nine sibling donors and one unrelated donor), five remained disease free at a median follow-up of 31.5 months [71]. Allogeneic HCT

Hematopoietic Cell Transplantation for Rare Hematologic Malignancies

appears to be the only currently available therapy for ATLL that offers a prospect of long-term survival. Donor-derived ATLL has been reported in a patient who underwent allogeneic HCT from his HLA-matched brother who was an HTLV-1 carrier [72]. Using a tumor-specific polymerase chain reaction technique, it was later shown that the relapse originated from donor T cells that were already infected in the donor [73]. Thus, the immunosuppression of the recipient appears to have facilitated the transformation of HTLV-1-infected donor T cells to frank T-cell leukemia/lymphoma. The precise incidence of donor-derived relapse is not known, and therefore it is not clear at present if HTLV-1-seronegative unrelated donors should be preferred over seropositive matched siblings.

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Fig. 70.4 (a) Estimated 3-year overall survival (OS) of 40 patients who underwent allogeneic hematopoietic cell transplantation (HCT) for adult Tcell leukemia/lymphoma (ATLL). (b) Estimated 3-year relapse-free survival (RFS). (c) Estimate 3-year relapse risk (RR). (Reproduced from [69], with permission.)

Natural killer (NK) cell neoplasms include the extranodal NK/T-cell lymphoma, nasal type, and aggressive NK-cell leukemia subtypes described in the World Health Organization classification. Extranodal NK/T-cell lymphomas have a predilection to arise in the upper aerodigestive tract, but may also involve other sites such as the skin, gastrointestinal tract, and testes. Cases with disseminated disease often have significant comorbidity from cytokine-mediated phenomena like hepatic dysfunction and hemophagocytic syndrome (HPS). Rare cases may have a cytotoxic T-cell phenotype and hence the designation NK/T-cell lymphoma. NK-cell neoplasms are strongly associated with Epstein–Barr virus (EBV), which may have a role in their pathogenesis. There is marked geographic variation in the incidence of NK-cell neoplasms due to their strong predilection for Asian individuals and the indigenous populations of Mexico, Central and South America [1,74]. The so-called blastic NK-cell lymphoma (CD4+CD56+ hematodermic neoplasm) is now known to be a malignancy of plasmacytoid dendritic cells and therefore is not a true NK-cell neoplasm [75]. In the case of extranodal NK/T-cell lymphoma, only about a third of patients with localized disease become long-term survivors with the conventional approach of radiation and combination chemotherapy [76]. Locoregional and systemic failures are common, and patients with relapsed disease or systemic disease at presentation have a dismal outcome [77,78]. The prognosis is even worse for aggressive NK leukemia, with a median survival of only 2 months in one study [79]. Hence, there has been interest in using HCT to improve the poor outcome of NK-cell neoplasms. Despite not being an extremely rare neoplasm in some geographic areas, systematic prospective trials to evaluate the efficacy of HCT for NK-cell neoplasms have not been performed. Anecdotal reports describe success with autologous and allogeneic HCT even for advanced disseminated disease [80–84]. In the largest series of autologous HCTs reported, Au et al. have reported their experience with 18 consecutive cases of nasal type NK/T-cell lymphoma treated at two institutions in Hong Kong. Eight cases were treated in CR1, five in second CR (CR2), and five with refractory disease. Five patients had extranasal disease before autologous HCT. The most common initial treatment used was combination chemotherapy and radiotherapy. Two patients died of treatment-related causes, nine patients relapsed, and seven remained in CR. All patients with active or disseminated disease at time of HCT died early of relapse or disease progression. The actuarial survival for the whole cohort was 39% after 6 months. Disease relapse beyond 6 months was not seen. These results suggest that prolonged survival is possible with autologous HCT in some patients with extranodal NK/T-cell lymphoma if transplanted in CR [85]. The Japanese NK-cell Tumor Study Group has analyzed outcome of 40 patients who underwent HCT from among 228 patients who were diagnosed with NK-cell lymphoma between 1994 and 1998. This was a

Chapter 70

Mycosis fungoides and Sézary syndrome Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma, is a neoplasm of CD4+ T cells that home to the skin. The initial stages of MF are characterized by patch or plaque lesions and typically last for many years. In the late stages, patients develop skin tumors and extracutaneous dissemination to lymph nodes and visceral organs [1,89,90]. Although patients with the early stages of MF often

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heterogeneous patient group including patients with nasal-type NK lymphoma (n = 22), blastic NK-cell lymphoma (n = 11), aggressive NK-cell leukemia (n = 3), and NK-cell precursor acute leukemia (n = 4). Twentyfive patients underwent autologous HCT, and the remaining 15 underwent high-dose conditioning and allogeneic HCT, all except one from matched sibling donors. Overall, there was a highly significant survival advantage for patients who received HCT compared with patients who did not undergo HCT: 40% versus 25% with a median follow up of 51 months and 32 months, respectively. The outcome of HCT was better for patients who were transplanted in CR. More importantly, during the follow-up period, no patient died later than 2 years after HCT, in contrast to patients who did not receive HCT, thereby demonstrating the durability of HCT-induced remissions. The long-term survival and relapse probabilities for autologous versus allogeneic HCT were 47% versus 29% and 17% versus 29%, respectively. However, these differences were not statistically significant. There was significantly higher NRM in allografted patients compared to those who received autologous HCT (48% versus 4%). The OS was better for HCT compared with conventional therapy, both for extranodal NK/T-cell lymphoma and for the other subtypes included in this study. As the authors themselves have cautioned, being a retrospective analysis, it is quite possible that patients with better performance status may have been selected to undergo HCT and thereby account for the better results of HCT [86]. Murashige et al. have reported results of allogeneic HCT for 28 patients with NK-cell neoplasms. The majority (n = 22) had extranodal NK/T-cell lymphoma. Twelve patients were chemosensitive and 16 were chemorefractory at the time of transplant. Twenty-three patients received grafts from matched related donors, and 23 underwent high-dose conditioning. With a median follow-up of 34 months, the 2-year progressionfree survival (PFS) and OS were 34% and 40%, respectively (Fig. 70.5). Patients who did not relapse within 10 months after HCT remained free of disease. In multivariate analysis, hematopoietic cell source (bone marrow versus peripheral blood) and histology (extranodal NK/T-cell lymphoma versus others) were risk factors for PFS (relative risk 3.03 and 3.94, respectively). These results are good considering the fact that 19 of the 28 patients had active disease at the time of HCT [87]. A similar OS rate was seen for 12 patients with NK-cell lymphoma who underwent unrelated donor HCT through the Japan Marrow Donor Program [88]. Based on the above evidence, for extranodal NK/T-cell lymphoma with localized disease, autologous HCT can be recommended as a consolidation therapy for patients who enter CR after up-front radiation therapy. Long-term survival has been reported with this approach, and NRM is expected to be low. Autologous HCT has no role in refractory or disseminated nasal-type extranodal NK/T-lymphoma or aggressive NK-cell leukemia, and these patients should be strongly considered for allogeneic HCT since this is the only therapy known to result in longterm survival in this group of patients. In the case of systemic NK-cell neoplasms, the major obstacle to allogeneic HCT appears to be the comorbidity from paraneoplastic phenomena such as hepatic dysfunction. Therefore, better strategies for the initial management are needed to realize the full curative potential of allogeneic HCT. The existence of a GVL effect after allogeneic HCT has been suggested [80].

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Fig. 70.5 (a) Progression-free survival (PFS) and (b) overall survival (OS) of 28 patients with natural killer cell neoplasms who underwent allogeneic hematopoietic cell transplantation (HCT). PFS was 34% and OS was 40% at 2 years. Median follow-up was 34 months. Broken lines indicate 95% confidence intervals. (Reproduced from [87], with permission.)

respond to current therapies that include retinoids, alpha-interferon, photopheresis, monoclonal antibodies, and chemotherapy among others, these therapies have no curative potential, and the disease ultimately progresses to its more advanced stages [90]. In one study, median survival of stage IV patients was only 13 months from initiation of therapy [91]. Sézary syndrome (SS) is an aggressive variant of MF and is characterized by erythroderma, lymphadenopathy, circulating neoplastic T cells, and a much more aggressive clinical course [1,89,90]. From the limited experience available from two small studies, it can be concluded that autologous HCT is not capable of inducing durable remissions when performed in the advanced stages of MF in heavily pretreated patients. In the first study, Bigler et al. performed autologous HCT in six patients with advanced-stage MF using a variety of conditioning regimens, including total skin electron beam radiation in four patients. Although four of the six patients achieved an initial CR, PFS was short (between 64 days and 1 year), with three of the six patients relapsing within 3 months [92]. In the second study, Olavarria et al. performed T-cell-depleted autologous HCT in nine patients with tumorstage MF. One patient died of sepsis before engraftment, and the remaining eight patients achieved CR. With a median follow-up of 29 months,

Hematopoietic Cell Transplantation for Rare Hematologic Malignancies

all but one patient had relapsed, and the median duration of CR was 7 (range 2–14) months. In the four patients who relapsed, the disease appeared to be more indolent than before HCT [93]. Whether autologous HCT early in the disease course, possibly in combination with posttransplant maintenance therapy with currently available active agents, will produce durable remissions remains to be determined. Given this grim prognosis of patients with advanced stage MF and SS, allogeneic HCT has been attempted to harness a potential GVL effect. Molina et al. have reported the results of a series of eight patients treated at the City of Hope National Medical Center with allogeneic HCT. Three patients had tumor-stage MF, one had erythrodermic MF, and the remaining three had SS. All patients were heavily pretreated and had failed a median of seven prior therapies. Graft source was from a matched sibling in four patients and matched unrelated donor in the other four. Conditioning regimens were fractionated TBI/CY (three patients), BU/CY (one patient), and fludarabine–melphalan (four patients). All patients achieved CR, and their original T-cell clones disappeared from the blood by 60 days after HCT. Although two patients had new T-cell clones detectable in the peripheral blood on follow-up analysis, these did not have any clinical impact, and these patients remained disease free. With a median follow-up of 56 months, six patients remained disease free (Plate 70.1) [94]. Three other reports, containing three patients in each who received HLA-matched sibling HCT for advanced, refractory MF have been published. Guitart et al. transplanted three patients with TBI/CY conditioning. Two of these patients remained in CR at 4.5 years and 15 months, respectively, while the third patient had cutaneous relapse that responded to withdrawal of immunosuppression and DLI [95]. Herbert et al. have reported three patients who received fludarabine–melphalan conditioning. All three patients had relapse or persistent disease after HCT. However, all responded partially to withdrawal of immunosuppression or DLI, and the disease appeared to be more indolent than before HCT [96]. Of the three patients reported by Soligo et al., all patients achieved CR after HCT. While two patients remained in durable CR 18 and 24 months, respectively, after HCT, one died of bacterial sepsis on day 73 post transplantation [97]. Collectively, these reports are evidence for the feasibility as well as the curative potential of allogeneic HCT, even in this group of advancedstage, high-risk patients. Firm conclusions about the optimum conditioning intensity cannot be drawn from the limited data available, although the reduced-intensity regimens are better tolerated. Clear evidence of a potent GVL effect has been demonstrated in MF by the regression of persistent lesions by withdrawal of immunosuppression or DLI [95,96,98]. Viral infections appear to be a major contributor to NRM, no doubt resulting from extensive prior use of therapies with profound immunosuppressive effects. Considering the considerable impact of MF on quality of life and the inability of current treatments to prevent progression, consideration should be given to allogeneic HCT after failure of two or three initial therapies, at least in patients who are under 60 years of age and have matched sibling donors.

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motherapeutic agents, the overall prognosis for AITL is poor [1,99,100]. In one study that analyzed the outcome of 33 patients treated with chemotherapy, the OS at 5 years was 36%. Notably, two of the long-term survivors in this study had received autologous HCT [101]. Although there are some patients with AITL included in various reports of HCT for T-cell lymphomas, two reports have analyzed the outcome of autologous HCT for AITL as a separate cohort. In the first of these reports, Schetelig et al. have analyzed data on 29 patients from 16 EBMT centers. The median age of these patients was 51 years. Twenty-eight patients had stage III or IV disease, and 79% had high intermediate- or high-risk International Prognostic Index scores, clinical features that are typical of patients with AITL. Extranodal disease was present in 17 patients, of whom 12 had bone marrow involvement. Combination chemotherapy was the most common therapy administered prior to HCT. Autologous HCT was part of first-line therapy in 14 patients (48%), and the remaining patients received HCT later in the disease course. Thirteen patients (45%) were in CR at time of HCT, and 22 patients (76%) achieved CR after HCT. The 5-year probabilities of OS and EFS were 44% and 37% respectively. There was a suggestion that patients who received HCT as part of first-line therapy did better than those who received it later. Five patients were in continuous CR for over 5 years, showing that long-term survival is possible with autologous HCT in selected patients [102]. In the second study, the Spanish Lymphoma/Autologous Bone Marrow Transplant Study Group has reported the outcome of 19 patients who underwent autologous HCT. Again, the median age of 46 years of these patients is low for AITL. Eleven patients were in a first or later CR. Fifteen patients (79%) achieved CR after HCT. The OS and PFS at 3 years were 60% and 55% for the whole group (Fig. 70.6). Predictably, bone marrow involvement, refractory disease, and more than one factor of the age-adjusted International Prognostic Index were associated with poor outcome [103]. Although the above two studies undoubtedly represent a selected group of patients who were able to undergo autologous HCT, these results appear to compare favorably with that of conventional therapy. Patients with AITL are at risk for EBV-positive as well as EBV-negative secondary B-cell lymphomas [104,105]. Both EBVpositive and EBV-negative lymphoproliferative disorders can occur after autologous HCT, and these appear to respond to rituximab-based therapy [106,107]. The allogeneic HCT experience in AITL is limited to patients who are included in reports of allogeneic HCT for T-cell lymphomas. In a phase II study of salvage chemotherapy followed by reduced-intensity allogeneic HCT for relapsed peripheral T-cell lymphomas, four patients (age range 39–50 years) with AITL were included. Three of these patients who were in PR at time of HCT were alive at 12.8, 6.8, and 60 months after allogeneic HCT, while one patient died early of sepsis [108]. The advanced age of patients with AITL and the significant tumorassociated comorbidities are significant hurdles for successful allogeneic HCT. However, allogeneic HCT should be considered in suitable patients who have primary or relapsed refractory disease since these patients do not benefit from autologous HCT.

Angioimmunoblastic T-cell lymphoma Angioimmunoblastic T-cell lymphoma (AITL) is a rare lymphoma accounting for 1–2% of non-Hodgkin’s lymphomas. AITL is strongly associated with EBV, which can be detected within the B cells in involved tissues in the majority of cases. Systemic symptoms, skin rash, immunodeficiency, and autoimmune phenomena such as hemolytic anemia are characteristic features of AITL. Patients with AITL are usually middle-aged or elderly and often present with advanced disease. Although anecdotal reports describe responses to a variety of agents including immunosuppressants, biologic response modifiers, and che-

Primary central nervous system lymphoma Primary central nervous system lymphoma (PCNSL) comprises about 1–2% of non-Hodgkin’s lymphoma cases. Its incidence is rising both in immunocompromised and immunocompetent individuals [109]. PCNSL typically presents as unifocal or multifocal periventricular parenchymal masses. Ocular involvement, either clinical or asymptomatic, may be present in 10–20% of patients at diagnosis. Other systemic involvement is extremely rare at presentation. The median age at diagnosis is 60 years. Over 90% of PCNSLs have a diffuse large B-cell histology, while

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Chapter 70

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Fig. 70.6 (a) Overall survival (OS) and (b) progression-free survival (PFS) of 19 patients who underwent autologous hematopoietic cell transplantation (HCT) for angioimmunoblastic T-cell lymphoma (AITL). (Reproduced from [103], with permission.)

2–4% of cases are of T-cell origin. Unlike the case in immunocompromised patients, EBV does not play a role in the pathogenesis of PCNSL in immunocompetent patients [110]. Although data from randomized trials are lacking, current therapy for PCNSL involves chemotherapy with high-dose methotrexate (MTX), alone or in combination, followed by whole-brain radiotherapy (WBRT). The long-term outcome of PCNSL is poor. Although a 2-year PFS survival of 57% and OS of 67% were reported in a recent study [111], the 10-year OS of patients under 60 years of age in another trial was only 32% [112]. Notably, in the latter trial, there were no long-term survivors among patients who were over 60 years of age at diagnosis. Neurotoxicity resulting from WBRT and chemotherapy is a major cause of morbidity in survivors, especially in those above 50 years of age who comprise the majority of PCNSL patients [113]. Therefore, high-dose chemotherapy with autologous hematopoietic cell support after initial chemotherapy has been tried as a strategy to minimize neurotoxicity by eliminating or reducing WBRT dose and improve the long-term outcome in PCNSL. The small sizes of reported studies as well as the marked variations in the initial and high-dose chemotherapy regimens make it difficult to draw firm conclusions regarding the efficacy of the autologous HCT approach as front-line therapy of PCNSL. In general, these studies have selected chemotherapeutic agents for conditioning based on their ability to cross the blood–brain barrier. Illerhaus et al. have reported results of 30 patients younger than 65 years of age with PCNSL who were enrolled

in a trial of initial chemotherapy with three cycles of high-dose MTX followed by a cycle of cytarabine and thiotepa, following which peripheral blood stem cells were collected. High-dose therapy consisted of carmustine and thiotepa. Fractionated radiotherapy (45 Gy, given as two doses of 1 Gy/day) was given as consolidation. Twenty-three patients were able to undergo autologous HCT, 15 of whom achieved CR and eight achieved partial remission. All 21 patients who underwent WBRT achieved CR. With a median follow-up of 63 months, the 5-year OS probability was 69% for the whole group and 87% for patients who received HCT. Leukoencephalopathy developed in 17% of patients, all of whom had received WBRT [114]. In another recently reported phase II study, Colombat et al. treated 25 patients below 60 years of age with two cycles of high-dose MTX-based chemotherapy. Seventeen of the responding patients then received high-dose therapy with carmustine, etoposide, cytarabine, and melphalan (BEAM) and autologous HCT followed by 30 Gy of WBRT. With a median follow-up of 34 months, the estimated 4-year OS and EFS were 64% and 46%, respectively. Although follow-up is short, no leukoencephalopathy has developed in evaluable patients who received the whole planned therapy [115]. In another small study of six patients treated with the same regimen as the above study, four patients were alive without evidence of disease with a median follow-up of 41.5 months [116]. WBRT was eliminated from two studies of high-dose therapy for newly diagnosed PCNSL. In the larger study, 28 patients received induction with high-dose MTX and cytarabine followed by high-dose therapy with BEAM in chemosensitive patients. Fourteen patients underwent autologous HCT. With a median follow-up of 28 months, median EFS was 5.6 months for all patients, and 9.3 months for the 14 patients who underwent HCT. Six (43%) of these 14 patients were disease free at the time of the report [117]. Cheng et al. treated seven patients with highdose MTX induction followed by high-dose therapy with thiotepa, BU, and CY. Two patients in this study received autologous HCT as sole therapy for progressive disease after initial chemotherapy. Five of the seven patients remained disease free at 5–42 months from diagnosis, including the two patients with progressive disease after initial chemotherapy. These authors attributed their good result in these poorprognosis patients to the use of agents like thiotepa and BU that have better CNS penetration than the BEAM regimen [118]. No neurotoxicity was observed in these two studies during the short follow-up. High-dose chemotherapy with autologous hematopoietic cell support has also been tried as salvage therapy for patients with refractory or recurrent PCNSL who had chemosensitive disease or intraocular lymphoma that was refractory to high-dose MTX. Soussain et al. have reported results for 22 such patients, 20 of whom received autologous HCT after high-dose therapy with thiotepa, BU, and CY. Sixteen of these 20 patients achieved CR after autologous HCT, and 14 remained alive with a median follow up of 41.5 months. The 3-year OS probability was 63.7%. Seven patients suffered neurologic adverse events, which were lethal in two patients [119]. A single case report describes a patient with refractory PCNSL who underwent an allogeneic sibling HCT after reduced-intensity conditioning. This patient remained disease free 30 months after HCT, suggesting that a graft-versus-lymphoma effect may be operational in the CNS [120]. In summary, high-dose chemotherapy with autologous HCT is feasible as front-line therapy for chemosensitive PCNSL and carries a low treatment-related mortality, at least in patients less than 65 years of age. A reduced dose of WBRT can be given after autologous HCT, with low short-term neurotoxicity, although it is not clear if high-dose chemotherapy can allow for complete elimination of WBRT. There is a suggestion that using agents such as thiotepa and BU that have better CNS penetration for high-dose therapy is superior to conventional lymphoma

Hematopoietic Cell Transplantation for Rare Hematologic Malignancies

regimens like BEAM. Autologous HCT may have a role in salvaging patients who are refractory to initial chemotherapy or who have relapsed after chemoradiotherapy, although neurotoxicity appears to be high in this setting. Other rare lymphoid malignancies Hepatosplenic T-cell lymphoma is an aggressive T-cell lymphoma characterized by marked hepatosplenomegaly and almost invariable involvement of the bone marrow. The vast majority are of the gamma/delta T-cell type (WHO). Various reports describe durable remissions after allogeneic HCT for hepatosplenic T-cell lymphoma even in chemotherapy-refractory cases [121–124]. T-cell prolymphocytic leukemia (T-PLL) is an aggressive neoplasm with a poor response to chemotherapy [1,125]. De Lavallade et al. have reported a patient who had relapsed after an autologous HCT but continued to remain in CR at 38 months after matched sibling HCT with reduced-intensity conditioning, thereby suggesting a GVL effect [126]. Kruspe et al. have recently reviewed the literature on allogeneic HCT for T-PLL. Among 13 published case reports, including a case of their own, there were 10 patients who remained in CR for periods ranging from 2 to 48 months after HCT. Notably, only four of these 13 were in CR at the time of HCT [127]. Subcutaneous panniculitic T-cell lymphoma (SPTL) mostly affects young adults, who present with subcutaneous nodules. A hemophago cytic syndrome may occur [1]. In a review of 21 patients treated with a variety of modalities, including five patients who received HCT, the OS from diagnosis was only 15 months [128]. Alpha/beta (AB) and gamma/ delta (GD) subtypes (the latter also called cutaneous gamma/delta T-cell lymphoma) of SPTL are now recognized and have distinct clinical features and markedly different prognoses. In a study of 83 patients with SPTL, the 5-year OS was 82% for the AB subtype versus only 11% for the GD subtype. Among patients with SPTL-AB, the presence of HPS was the most important prognostic factor, with a 5-year OS of only 46% for patients with HPS compared with 91% for those without [129]. There are single case reports of prolonged survival after autologous HCT even in patients who have relapsed disease and other adverse prognostic factors, such as elevated lactate dehydrogenase, HPS, and bone marrow involvement [130–134]. Allogeneic sibling HCT has been reported in a patient with progressive disease who remained in CR 31 months after HCT [135].

1039

CD4+/CD56+ hematodermic neoplasm (termed blastic NK-cell lymphoma in the initial WHO classification) [1] is a malignancy of plasmacytoid dendritic cells with a marked predilection for skin involvement. Bone marrow involvement is common [75,136]. Although there are some reports of durable CR after combination chemotherapy [137], the overall outcome with conventional combination chemotherapy alone is considered poor [75]. Eleven cases of hematodermic neoplasm have been included in a report of HCT outcome for NK-cell neoplasms. Of the six patients who underwent autologous HCT, three achieved durable CR, while among the five patients treated with allogeneic HCT, two were in CR at 11 and 43 months after HCT, suggesting benefit for both approaches in selected patients [86]. There are single case reports of durable CR after autologous HCT in patients with widely disseminated disease, including bone marrow involvement [138,139]. One patient had progressive disease on conventional chemotherapy prior to autologous HCT, thereby suggesting a benefit for dose intensification of chemotherapy in this disorder [138]. Yoshimasu et al. have reported a case of relapsed hematodermic neoplasm that was successfully treated with unrelated cord blood allogeneic HCT. This case and other reports of durable CR after allogeneic HCT (reviewed by these authors) suggest a role for allogeneic HCT in the treatment of this disorder [140,141]. HCT should be considered early in the treatment plan for the four extremely rare neoplasms discussed in this section. For hepatosplenic T-cell lymphoma and T-PLL, allogeneic HCT up front appears to be the treatment of choice, preferably after inducing remission with chemotherapy or antibody-based therapy. There is currently no evidence to suggest a benefit for autologous HCT for these two disorders. In the case of SPTL and hematodermic neoplasm, it appears reasonable to perform autologous HCT to consolidate an initial CR or at chemosensitive relapse. Allogeneic HCT could be reserved for primary refractory disease or relapsed cases that are chemorefractory, and for patients who have poor prognostic factors like extensive bone marrow involvement. Although no conclusions about the overall effectiveness of HCT can be drawn from the reported cases, at present HCT is the only therapy known to produce durable remissions in these aggressive lymphoid malignancies. Exceptions may be hematodermic neoplasm, where the curative potential of modern intensive chemotherapy regimens remains to be fully defined, and indolent cases of SPTL-AB without HPS, for which local treatments, steroids or immunosuppressive therapy may suffice [129].

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Brenda M. Sandmaier & Rainer Storb

Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies

Introduction Allogeneic hematopoietic cell transplantation (HCT) has an important role in the treatment of malignant and nonmalignant hematological diseases. Successful allogeneic HCT must overcome two immunologic barriers: graft-versus-host (GVH) and host-versus-graft (HVG) reactions. The conventional strategy to overcome this bidirectional barrier has relied upon three elements. First, intensive conditioning regimens are delivered with the dual purposes of immunoablation and disease eradication, which consist of otherwise supralethal doses of irradiation and/or chemotherapy. Second, donor hematopoietic cells are given to rescue patients from lethal myeloablation. Third, T-cell depletion or post-grafting immunosuppression is used to control GVH disease (GVHD) and establish long-term graft–host tolerance. Toxicities related to the conditioning regimens are a major limitation in the application of high-dose HCT. Toxicities include pancytopenia, which sets the stage for life-threatening infections, and damage to the liver, kidneys, and lungs, which may impair the ability to deliver the doses of immunosuppression necessary for control of GVHD. For these reasons, high-dose allogeneic HCTs have been carried out in specialized hospital wards and their use restricted to relatively young patients at most transplant centers. The concept that conditioning dose intensification was the only approach for eradicating malignancy came under question early in the history of clinical HCT. Findings in the late 1970s and early 1980s drew attention to graft-versus-tumor (GVT) effects, as evidenced by better relapse-free survival in patients with acute or chronic GVHD [1–6]. Additional evidence supporting GVT effects included lower relapse rates among patients who received unmodified allogeneic HCT compared with T-cell-depleted allografts [6], autologous grafts [7,8] or syngeneic grafts [9], and the induction of durable remissions with donor lymphocyte infusions (DLIs) in patients who had relapsed after allogeneic HCT [10–15]. These observations led to the hypothesis that GVT effects might be exploited in patients thought to be too old or medically infirm to tolerate high-dose conditioning regimens. Accordingly, several groups of investigators have developed reduced-intensity regimens that minimize the regimen-related toxicities and rely on GVT effects to treat the underlying malignancies.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

The least intensive regimens rely on pre- and post-transplant immunosuppression to establish the allografts. It is important to remember that both HVG and GVH reactions are mediated by T cells of host and donor origin, respectively, when transplants are carried out across minor histocompatibility barriers. From these facts follows the hypothesis that HVG reactions can, to some extent, be controlled through novel and effective agents that at the same time serve to control GVHD. Consequently, much of the toxic high-dose pretransplant therapy previously employed for HVG control can be replaced by relatively nontoxic immunosuppression. Once established, grafts would create marrow space through subclinical GVH reactions, and cytotoxic and myeloablative doses of total body irradiation (TBI) or busulfan (BU) would not be needed for that purpose. Ideally, control of HVG and GVH reactions results in mutual graft–host tolerance and initial mixed donor–host hematopoietic chimerism. While mixed chimerism is likely to correct phenotypic expression of certain genetic diseases, in patients with hematologic malignancies full donor chimerism may be required to control disease through GVT effects. Thus, novel transplantation programs can be designed that are safer than high-dose regimens and have the potential to be administered in the ambulatory care setting rather than on intensive care wards. This chapter discusses the results of clinical studies with reduced-intensity conditioning for hematological malignancies, including the preclinical studies that served as their basis.

Preclinical studies Murine studies A number of successful preclinical studies have involved inbred strains of mice. Most have in vivo depletion of host T cells using antibodies, followed by TBI at high but, at least for mice, sublethal doses and dose rates. In other cases, TBI was delivered along with thymic irradiation and postgrafting cyclosporine (CSP) or high-dose cyclophosphamide (CY). A murine study by Cobbold et al. [16] combined pretransplant treatment with anti-CD4 and anti-CD8 monoclonal antibodies (mAbs) for in vivo T-cell depletion and 6–8.5 Gy TBI delivered at 0.35 Gy/min. In other studies, nonobese diabetic mice received ≥7.5 Gy TBI followed by infusion of very large numbers of allogeneic marrow cells, ≥30 × 106/mouse (≥1.5 × 109/kg) [17,18]. In one report, mice were conditioned for successful allografts with 5 Gy TBI and given additional immunosuppression with 200 mg/kg CY after transplantation [19]. Other investigators combined anti-CD4 and anti-CD8 mAbs with 3 Gy TBI and 7 Gy thymic irradiation to ensure engraftment [20]. They reported that the need for thymic irradiation could be avoided by anti-

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CD4 and anti-CD8 mAb injections after transplantation, thereby depleting host T cells [21,22]. The need for TBI could be eliminated by pretreatment with anti-CD4 and anti-CD8 mAbs, thymic irradiation, and the injection of the equivalent of 8.7 × 109 marrow cells/kg, suggesting that TBI provided immunosuppression [23]. Yet another study, using A/JÆB10 and B10.AÆB10 murine marrow grafts, showed persistent chimerism when recipients were given anti-CD4 and anti-CD8 mAbs and 3 Gy TBI administered at 1 Gy/min before and antibodies against natural killer (NK) cells after transplantation for up to 16 weeks [24]. Thymic irradiation could be replaced by the injection of one co-stimulatory blocker (anti-CD154 mAb or cytotoxic T lymphocyte antigen-4 [CTLA-4] immunoglobulin [Ig]) [25], whereas both thymic irradiation and host T-cell-depleting mAbs could be eliminated by single injections each of anti-CD154 mAb and CTLA-4 Ig [26]. Koch & Korngold [27] obtained prolonged chimerism in major histocompatibility complex (MHC)-mismatched murine allograft recipients when a synthetic CD4–CDR3 peptide analog was used for immunosuppression in combination with TBI. In mice sensitized to donor cells, treatment with the peptide was more effective than anti-CD4 mAb therapy for enhancing donor chimerism, suggesting this approach might be valuable in obviating rejection after MHC-mismatched HCT. Seledtsov et al. [28] used 200 mg/kg CY to condition mice bearing P815 leukemia for allografts and observed mixed chimerism and prolonged survival compared with syngeneic controls, raising the possibility of using nonlethal cytoreductive therapy in combination with allogeneic cells for a GVT effect. Slavin et al. used fractionated irradiation (34 Gy) targeted to the lymphoid tissues, including the spleen, lymph nodes, and thymus (total lymphoid irradiation) to condition mice for bone marrow transplants from H-2-disparate donors. The mice became chimeras, and none of the recipients developed clinical evidence of GVHD, with 80% of mice surviving beyond 110 days after HCT [29]. Subsequent studies in the same MHC-disparate model using TBI demonstrated that depletion of NK1.1+ TCRαβ+ cells from the marrow inoculum resulted in development of lethal GVHD, suggesting a “suppressor” function of these NK T cells [30]. More recent work showed that the cells that suppressed GVHD after total lymphoid irradiation and H-2-incompatible HCT were CD4+CD25+ “naturally occurring” regulatory T cells [31]. Canine studies MHC-matched grafts A clinically successful protocol employing 2 Gy TBI before and immunosuppression with mycophenolate mofetil (MMF)/CSP after HCT has been developed in a canine model. The following describes the development of the regimen. Marrow toxicity of TBI While previous studies have shown 2 Gy TBI (given at 0.07 Gy/min) to be sublethal and 4 Gy to be uniformly lethal [32], more recent studies, using more intensive antibiotics, parenteral fluid, and transfusion support, showed that dogs given 7 Gy TBI survived with endogenous marrow recovery, while 8 Gy was lethal. TBI doses needed for marrow engraftment Table 71.1 shows results of unmodified dog leukocyte antigen-identical marrow allografts following increasing single doses of TBI delivered at 0.07 Gy/min [33]. At a dose of 4.5 Gy, only 13% of dogs showed alldonor-type engraftment, and 28% were stable mixed donor–host chimeras, while 59% of dogs rejected those grafts and either died of marrow aplasia (23%) or survived with eventual autologous recovery of hematopoiesis (36%). Survival of the latter dogs was probably because of the

extended hematopoietic support provided by the transient allograft. Host immunosuppression sufficient for sustained engraftment of 95% of dogs required 9.2 Gy TBI. Low-dose TBI before and immunosuppressive drug treatment after marrow transplantation Two drugs that had been widely used in GVHD prevention, CSP and prednisone, were evaluated in dogs conditioned with 4.5 Gy TBI [34]. Postgrafting immunosuppression in this and subsequent experiments was discontinued at the latest by day 35. Prednisone, administered in extremely high doses patterned along a regimen used by Kernan et al. [35] along with antithymocyte globulin (ATG) to condition patients for second marrow grafts, failed to enhance engraftment after 4.5 Gy TBI. By comparison, all dogs given CSP were stably engrafted and none developed GVHD. This result was significantly better than that seen in controls and consistent with the theory that postgrafting immunosuppression could control both HVG and GVH reactions. A comparable rate of engraftment in the absence of CSP was seen only after conditioning with 9.2 Gy (Table 71.1). It follows that the immunosuppression accomplished by postgrafting CSP was similar to that achieved by 4.7 Gy TBI. In subsequent experiments, the TBI dose was decreased to 2 Gy [36]. CSP alone proved ineffective: all four dogs treated rejected their allografts after 4 weeks but survived with autologous recovery. Six dogs were given methotrexate along with CSP, a drug combination previously shown to be synergistic in preventing GVHD both in dogs and humans [37,38]. Three of five evaluable dogs became stable mixed chimeras, and two rejected their grafts. MMF, whose metabolite, mycophenolic acid, blocks de novo purine synthesis by binding to inosine monophosphate dehydrogenase and interferes with de novo purine synthesis required for lymphocyte replication, was next combined with CSP. The two drugs had been found to be synergistic and superior to methotrexate/CSP for GVHD prevention in the canine model [39]. Only one of 12 dogs rejected the allograft at week 12, while 11 dogs remained stable mixed chimeras for up to 130 weeks after HCT without clinical evidence of GVHD. Further reducing the TBI dose to 1 Gy resulted in only transient engraftment in MMF/CSP-treated dogs, suggesting a delicate balance of host and donor immunities. Similar results were seen by combining rapamycin and CSP [40]. Marrow space versus immunosuppression In order to clarify whether the major role of low-dose TBI before transplantation was to create marrow space or provide host immunosuppression, six canine recipients were given 4.5 Gy (2 Gy/min) pretransplant irradiation to the cervical, thoracic, and upper abdominal lymph node

Table 71.1 Doses of total body irradiation (TBI) needed for the engraftment of dog leukocyte antigen (DLA) identical marrow in the absence of postgrafting immunosuppression

TBI dose (Gy)* 9.2 8 – myeloablative and supralethal 7 6 4.5

Number of dogs studied

% with sustained engraftment

21 5

95 80

0 0

5 23 39

60 52 41

0 17 36

* Delivered at 0.07 Gy/min.

% with autologous recovery

Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies

1045

CD45, respectively, could be substituted for low-dose TBI. α-Emitters were an attractive alternative to β-emitting radionuclides for targeting hematopoietic cells given that their high energy is deposited over only a few cell diameters (40–90 μm), sparing surrounding normal tissue. 213 Bi has a half-life of 46 minutes and delivers a short-range high-energy radiation, leading to a high relative biologic effectiveness. Studies showed that 213Bi labeled anti-TCRαβ and anti-CD45 mAb resulted in stable engraftment of dog leukocyte antigen-identical littermate marrow [44,45]. Selectively ablating T cells or ablating all nucleated hematopoietic cells in this way allowed transplantation with very low toxicity, making it suitable both for patients with nonmalignant and with malignant hematologic disorders. Further, mAbs against the panhematopoietic marker CD45 would be useful in patients with MHCnonidentical donors where additional host immune cells, such as NK cells, play a major part in graft rejection. Based on results in both the murine and large animal models, clinical studies with β-emitting radionuclides have been initiated in patients. Data from ongoing preclinical studies with α-emitters will allow us to refine future approaches for our patients.

chain. This was followed by dog leukocyte antigen-identical littermate marrow grafts and MMF/CSP post transplant [41]. In all six, mixed chimerism was present in peripheral blood granulocytes, T cells and monocytes, and nonirradiated marrow and lymph node spaces. Two of the six rejected their grafts after 8 and 18 weeks, and four remained stable mixed chimeras. When, more than 1 year after HCT, T cells from the marrow donors were infused which had been sensitized to recipient minor antigens, conversion to all donor chimerism occurred. While other explanations were possible, results seemed consistent with the hypothesis that pretransplant irradiation provided host immunosuppression and grafts could create their own marrow space. Therefore, conceivably, the small doses of either lymphoid radiation or TBI currently used for establishing mixed chimerism could ultimately be replaced by more specific and less toxic means of inducing HVG anergy, including mAbs to T-cell surface determinants. These efforts were prompted by concerns that even low doses of radiation or chemotherapy might increase the risk of secondary malignancies. Subsequent studies showed that the equivalent of 1 Gy TBI could be replaced by co-stimulatory signal blockade with either CTLA-4 Ig or a mAb directed against CD154 [42,43]. Recipient T cells were activated with intravenous injections of donor peripheral blood mononuclear cells before 1 Gy TBI, with concurrent administrations of either the fusion peptide CTLA-4 Ig, which blocks T-cell co-stimulation through the B7: CD28 signal pathway, or the mAb against CD154, which blocks the signal from CD40 to CD154. Most dogs so treated showed mixed chimerism lasting for the observation periods of 2 years. Control dogs not experiencing T-cell co-stimulatory blockade showed transient mixed chimerism lasting for 3–12 weeks.

Clinical results Several groups of investigators have explored the feasibility of reducedintensity HCT with a variety of regimens to minimize regimen-related toxicities while exploiting GVT effects. Figure 71.1 outlines the spectrum of commonly used regimens relative to their hypothetical immunosuppressive and myelosuppressive effects. In some cases, drugs were chosen with proven activities against the targeted malignancies for disease control while waiting for GVT effects to occur. With the minimally myelosuppressive regimens, engraftment was ensured by innovative postgrafting immunosuppression that also controlled GVHD, and disease control was more dependent on GVT effects. Other differences

Radiolabeled mAb In other studies, we investigated whether radioimmunotherapy with an α-emitting radionuclide, bismuth-213 (213Bi) and mAbs to TCRαβ and

Conditioning regimens Reduced intensity

Myeloablative Cy120/TBI 12 Gy Cy120/TBI 5.5 Gy Bu16/Cy120

Cy200/ATG

HLA-matched unrelated

HLA-identical sibling

Immunosuppression

Fig. 71.1 Commonly used nonmyeloablative or reduced-intensity conditioning regimens in relation to their immunosuppressive and myelosuppressive properties. Ale, alemtuzumab; ATG, antithymocyte globulin; Bu8, busulfan 8 mg/kg; Bu16, busulfan 16 mg/kg; Cy, cyclophosphamide; Cy120, cyclophosphamide 120 mg/kg; Cy200, cyclophosphamide 200 mg/kg; F, fludarabine; FlagIda, fludarabine/cytosine arabinoside/idarubicin; M, melphalan; M 140, melphalan 140 mg/m2; M 180, melphalan 180 mg/m2; TBI, total body irradiation; TLI, total lymphoid irradiation; TT, thiotepa, (Adapted from [113], with permission. Copyright, American Society of Hematology.)

Genetic disparity

HLA-haploid related/ T-cell depleted

F/Cy/TBI 2 Gy

Ale/F/M 140

F/M 180

F/TBI 2 Gy

Bu8/F/ATG

F/M 140 TLI ATG

F/Cy

Flag-Ida TBI 2 Gy

TT-Cy

Myelosuppression Aggressiveness of malignancy

F/Bu16

1046

Chapter 71

among the reduced-intensity regimens included the degrees of marrow aplasia and the tempo of establishing complete donor chimerism. In the reduced-intensity regimens that have more myelosuppressive doses of chemotherapy, recipients uniformly become severely hypoplastic before graft function occurs. Complete donor engraftment occurs rapidly. By contrast, recipients of a more immunosuppressive regimen show only moderate declines of peripheral blood cell counts before recoveries are seen, and all patients are initially mixed donor–host hematopoietic chimeras. In these patients, it may take 6–12 months before donor engraftment is complete. The comparative benefit of one over another reduced-intensity regimen may depend on the underlying diseases, stage of diseases, and clinical status of the patients. In diseases presumed to be more sensitive to GVT effects, such as chronic myelogenous leukemia, chronic lymphocytic leukemia, and low-grade lymphomas, immunosuppressive regimens may be sufficient to ensure engraftment because there is time for GVT effects to become operative. In rapidly progressive diseases or diseases where GVT is less potent or less able to keep ahead of growth of the underlying malignancy, such as high-grade lymphomas, Hodgkin’s lymphoma, multiple myeloma, and acute myeloid leukemia (AML) beyond first remission, a certain amount of cytoreduction may be necessary to minimize residual disease. In this chapter, we discuss strategies of reduced-intensity transplants, with a review of the initial results and current approaches, and with a focus on minimally myelosuppressive TBI-based regimens. HLA-matched related HCT While many studies of reduced-intensity hematopoietic transplants have been performed in elderly and medically infirm patients unable to tolerate a high-dose preparative regimen, some studies have included younger patients who were otherwise eligible for high-dose transplants with standard eligibility criteria. These differences in patient selection generally preclude a meaningful comparison among regimens. Table 71.2 outlines the initial publications regarding reduced conditioning regimens currently being investigated. Initial studies at the MD Anderson Cancer Center in Houston, Texas, utilized purine nucleoside analog-based regimens for treatment of both myeloid and lymphoid malignancies. Giralt et al. [46] combined fludarabine with idarubicin and cytarabine or melphalan, or cladribine and cytarabine before HCT from HLA-identical or single HLA antigenmismatched sibling donors for treatment of AML or myelodysplastic syndromes. Of 15 patients treated, four failed to engraft, and one treatment-related death occurred before HCT. With a median follow-up of 100 days, six patients were alive and two were disease-free. Khouri et al. [47], also at MD Anderson, treated 15 patients with lymphoid malignancies using a regimen of fludarabine/CY or fludarabine/cytarabine/cisplatin. Eleven of 15 patients attained durable engraftment, and eight of 11 engrafted patients achieved complete remissions. Three nonrelapse deaths occurred. With a median follow-up of 180 days, five of six patients with chemosensitive disease were alive compared with two of nine with refractory or untreated malignancies. Giralt et al. [48] subsequently reported on a study combining purine analogs (fludarabine or cladribine) with melphalan in patients with hematologic malignancies. It was hypothesized that these drug combinations would have enhanced abilities to achieve remissions given the antitumor effects of high-dose melphalan and the observation that purine analogs have been shown to inhibit DNA repair after alkylator-induced damage. Seventy-eight of 86 patients received fludarabine in combination with high-dose melphalan, and eight received cladribine/melphalan. The day 100 nonrelapse mortality (NRM) rate was 37% for the fludarabine/melphalan combination and 88% for the cladribine/melphalan com-

bination. Both regimens were sufficiently immunosuppressive to allow for engraftment of both HLA-matched unrelated and related, and one HLA antigen-mismatched related donor grafts. Fifty-seven percent of patients achieved complete remissions, illustrating the beneficial effect of the cytoreductive conditioning therapy. Two-year overall and diseasefree survivals were 28% and 23%, respectively. Slavin et al. [49] at Hadassah University in Israel used a regimen that consisted of fludarabine, BU (8 mg/kg) and ATG in a younger group of patients (median 34 years, range 1–61 years) with both hematologic malignancies and genetic diseases. All patients achieved either partial or complete donor chimerism with this regimen, with four patients developing moderate to severe hepatic sinusoidal obstructive syndrome, suggesting that this regimen was still quite toxic. This same regimen was used in another group of heavily treated high-risk lymphoma patients with grafts from HLA-matched related and unrelated donors [50]. Again, there was consistent engraftment with disease-free survival of 40% at 37 months. Childs et al. [51] at the National Cancer Institute combined CY and fludarabine to treat patients both with hematologic malignancies and with solid tumors. One of 15 patients rejected the graft, and two patients died of transplant-related causes. Ten of 14 patients surviving more than 30 days had disease regression, suggesting GVT effects. The observation was made that full donor T-cell chimerism preceded both acute GVHD and disease regression. Investigators at the Massachusetts General Hospital in Boston evaluated the use of CY, ATG, and thymic irradiation (among patients without previous mediastinal radiotherapy) in patients given HLA-matched transplants [52]. Eighteen of 20 evaluable patients developed persistent mixed hematopoietic chimerism. Ten of the 20 received prophylactic DLI to convert mixed to full donor chimerism and to optimize GVL effects. Six of eight patients receiving prophylactic DLI converted to full donor chimerism. Transplant complications included CY-induced cardiac toxicity. One patient died from transplant complications and one patient died from GVHD after two prophylactic DLIs. Overall, 11 of 21 patients survived; four had continued complete responses with a median follow-up of 14.6 months. In an attempt to reduce the incidence of GVHD, Campath (anti-CD52 mAb) was added to fludarabine and melphalan conditioning, in recipients of both HLA-matched sibling and unrelated donor grafts [53]. Forty-two of 43 evaluable patients had sustained engraftment. Eighteen of 31 patients studied were full donor chimeras, while 13 patients were mixed chimeras. The regimen prevented grade III–IV acute GVHD, and only two patients developed grade II GVHD. At a median follow-up of 9 months, 33 of the 44 patients remained alive either in complete remission or without disease progression. While the chemotherapy-based reduced-intensity regimens had significantly fewer toxicities compared with conventional high-dose allogeneic HCT, patients still experienced profound pancytopenias, and most required hospitalizations of durations that were similar to those for high-dose transplants. In order to further reduce the regimen-related toxicities and thereby perform allogeneic HCT in the outpatient setting, the low-dose (2 Gy) TBI conditioning regimen developed in the dog model was evaluated in a multi-institutional clinical study [54]. TBI of 2 Gy was given as a single fraction on day 0, with postgrafting immunosuppression of CSP from day −1 to day 35, and MMF given twice a day from day 0 to 27. Granulocyte colony-stimulating factor-mobilized peripheral blood hematopoietic cells (PBHCs) collected over 2 days from HLA-identical related donors were used as sources of hematopoietic cells. Trial eligibility criteria required patients to be either too old to be eligible for a high-dose HCT, or, if younger, to have medical contraindications to high-dose HCT. Overall, the HCT regimen was well tolerated, with the majority of eligible patients having their transplants in the outpatient clinic setting. Most patients did not develop severe

50 (23–68)

44 (22–62)

15

86

26

23

15

21

426

37

44

19

5

68

Houston [46]

Houston [47]

Houston [48]

Jerusalem [49]

Jerusalem [50]

NIH [51]

Boston [52]

Seattle [112]

Stanford [57]

London [53]

Freiburg [65]

Boston [58]

Baltimore and Seattle [61]

46 (1–71)

30 (20–51)

64 (60–70)

41 (18–56)

52 (28–66)

55 (9–74)

41 (13–63)

34 (1–61)

52 (22–70)

55 (47–71)

59 (27–71)

15

Transplant center [Reference]

Median age in years (range)

Number of patients studied

Haplo

Haplo

MRD MURD

MRD MURD

MRD MURD

MRD

MRD

MRD

MRD

MURD MRD 5/6 RD MRD

MRD

MRD 5/6 RD

Donor

BM

BM

PBHC BM

PBHC BM

PBHC

PBHC

BM

PBHC

PBHC

PBHC

BM PBHC

PBHC BM

PBHC BM

Stem cell source

4

CSP

CSP + MMF T + MMF CSP + MMF

CSP ± MTX

CSP + MMF

CY + ATG ± TI 2 Gy TBI ± F TLI + ATG

F+M+ Campath F + BCNU +M+ ATG CY + ATG ± TI

HM

HM

NHL

AML MDS

HM

HM

HM

CY + F + 2 Gy TBI

Cy + T + MMF

CSP

24

CSP

F + CY

HM ST

13

0

0

2

16

7

0

0

CSP

F+B+ ATG F+B+ ATG

L

HM GD

HM

2

27

±T ± MTX

T + MTX T + MP CSP + MP CSP

27

CSP + MP

F+I+A F+I+M 2-CDA + A F + CY F+C+A

Rejections (%)

Postgraft immunosuppression

Conditioning regimen

F+M 2-CDA + M

CLL NHL

AML MDS

Diagnosis

Table 71.2 Nonmyeloablative conditioning regimens for patients with hematologic malignancies

35

100

59

5

3

47

29

60

35

38

40

7

20

Acute grade II–IV

GVHD* (%)

Cy1: 25% Cy2: 5%

NA

65

2

27

50

NA

27

9

27

24

13

0

Chronic

OS: 40% DFS: 20% Median F/U: 103 days 2-year OS: 36% 2-year PFS: 27%

OS: 40% DFS: 13% Median F/U: 100 days OS: 47% DFS: 33% Median F/U: 180 days 2-year OS: 28% 2-year DFS: 23% 2-year NRM: 45% OS: 85% DFS: 81% Median F/U: 240 days OS: 43% DFS: 43% Median F/U: 675 days OS: 53% PFS: 53% Median F/U: 200 days OS: 52% DFS: 33% Median F/U: 445 days 3-year OS: 51% 3-year PFS: 38% OS: 73% DFS: 62% Median F/U: 446 days OS: 82% PFS: 75% Median F/U: 270 days 1-year OS: 68% 1-year PFS: 61%

Outcome(s)

Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies

1047

16

42

47

52

89

103

Transplant center [Reference]

Jerusalem [62]

Dresden [63]

London [64]

Leipzig [67]

Seattle [68]

Seattle [69]

54 (17–70)

53 (4–69)

48 (6–65)

44 (18–62)

47 (16–65)

17 (8–48)

Median age in years (range)

MURD

MURD

MURD MMURD MURD MMURD

MURD MMURD

MURD

Donor

PBHC

BM PBHC

BM PBHC

BM

PBHC BM

BM

Stem cell source

HM

HM

HM

HM

HM

HM

Diagnosis

CSP + MMF

CSP + MMF

2 Gy TBI + F

5

21

12

CSP + MMF

2 Gy TBI + F

4

CSP

21

CSP + MMF CSP ± MTX

F+M+ Campath 2 Gy TBI + F

0

CSP

F+B+ ATG F+B+ ATG

Rejections (%)

Postgraft immunosuppression

Conditioning regimen

53

52

63

21

26

44

Acute grade II–V

GVHD* (%)

49

37

25

6

38

0

Chronic

3-year OS: 75% 3-year DFS: 60% OS: 36% DFS: 26% Median F/U: 390 days 1-year OS: 75.5% 1-year PFS: 61.5% OS: 35% DFS: 25% Median F/U: 570 days 1-year OS: PBHC: 57%; BM: 33% 1-year PFS: PBHC: 44%; BM: 17% 2-year OS: 58% 2-year PFS: 49%

Outcome(s)

* GVHD before donor lymphocyte infusion. 2-CDA, cladribine; A, ara-C; AML, acute myeloid leukemia; ATG, antithymocyte globulin; B, busulfan; BCNU, carmustine; BM, bone marrow; C, cisplatin; Campath, Alemtuzumab; CLL, chronic lymphocytic leukemia; CSP, cyclosporine; CY, cyclophosphamide; DFS, disease-free survival; F, fludarabine; F/U, follow-up; GD, genetic diseases; Haplo, haploidentical related donor; HM, hematologic malignancies; I, idarubicin; L, lymphoma; M, melphalan; MDS, myelodysplastic syndrome; MMF, mycophenolate mofetil; MMURD, mismatched unrelated donor; MP, methylprednisolone; MRD, matched related donor; MTX, methotrexate; MURD, matched unrelated donor; NA, not available; NHL, non-Hodgkin’s lymphoma; NRM, nonrelapse mortality; OS, overall survival; PBHC, peripheral blood hematopoietic cells; PFS, progression-free survival; RD, related donor; ST, solid tumors; T, tacrolimus; TBI, total body irradiation; TI, thymic irradiation; TLI, total lymphoid irradiation.

Number of patients studied

Table 71.2 (Continued)

1048 Chapter 71

Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies n = 212

100,000

Neutrophils/μL

10,000

Max Median

1000

tion of regulatory NK/T cells [56], consisted of total lymph node irradiation (8 Gy total lymphoid irradiation) and ATG (Thymoglobulin, 7.5 mg/kg total dose) in combination with MMF and CSP for postgrafting immunosuppression. Of the first 37 patients reported [57], two developed acute GVHD, and nine of 33 evaluable developed chronic GVHD.

Min

100

HLA-mismatched related donor allografting

10

1 0

10

20

30

40

50

60

10,000

Platelets × 1000/μL

1049

1000 Max 100

Median

Min

10

1 0

10

20

30

40

50

60

Days after HCT

Fig. 71.2 Neutrophil and platelet changes among 212 patients given 2 Gy total body irradiation ± fludarabine before and mycophenolate mofetil/ cyclosporine after human leukocyte antigen-matched related hematopoietic cell transplantation (HCT). (Reproduced from [114], with permission. Copyright, American Society of Clinical Oncology.)

neutropenia or thrombocytopenia (Fig. 71.2.) While all patients had initial donor engraftment, nine (20%) of the first 45 patients experienced graft rejection between 2 and 4 months after transplant, a timing that coincided with discontinuation of the MMF/CSP immunosuppression [54]. Lack of intensive preceding chemotherapy before HCT predicted rejections, and rejecting patients had low-level donor T-cell chimerism on day 28. The rejections were nonfatal, and all patients had autologous reconstitution of peripheral blood counts. To prevent graft rejection, the conditioning regimen was modified by adding 30 mg/m2/day fludarabine on days −4, −3, and −2 to the 2 Gy TBI, with a subsequent reduction of rejection rate to 3%. The outcomes of different durations of CSP administration were analyzed in 185 patients who received grafts from HLA-matched donors and CSP for 56, 77 or 180 days after HCT in combination with MMF for 28 days. While the duration of CSP prophylaxis did not significantly influence the overall rate of acute GVHD (grade II–IV), chronic GVHD, or NRM, prolonged administration of CSP (180 days) was associated with a significantly decreased hazard of grades III–IV acute GVHD and an increased likelihood of discontinuing all systemic immunosuppression when compared with the shortest course of CSP (35 days) [55]. Only 3.7% of patients receiving CSP through day 180 developed grades III–IV acute GVHD. The Stanford group took a different approach to reduce the incidence of GVHD. The reduced-intensity regimen, which was adapted from murine studies that were designed to favor the presence of a high propor-

In a report by Sykes et al. [58], five patients with refractory non-Hodgkin’s lymphoma underwent marrow transplantation from HLA-haploidentical related donors, sharing at least one HLA-A, B or DR allele on the mismatched haplotypes after conditioning with high-dose CY (200 mg/kg), 7 Gy thymic irradiation, and ATG (Table 71.2). Four evaluable patients showed engraftment with predominantly donor lymphoid cells and varying degrees of myeloid donor chimerism. All five patients developed grades II–III acute GVHD, with three patients dying between days 12 and 108 from pulmonary hemorrhage, progressive lymphoma, and aspergillosis. At the time of the report, one patient was alive at day 103 with a partial response, and one was alive at day 416 in continuous complete remission. The Johns Hopkins’ group developed a conditioning regimen for HLA-haploidentical related recipients based on a murine model whereby high-dose CY administered after HCT inhibited both graft rejection and GVHD [59]. The regimen for patients consisted of pretransplant CY 29 mg/kg, fludarabine 150 mg/m2, and TBI 2 Gy followed by an infusion of marrow and post-transplant CY 50–100 mg/kg, and tacrolimus and MMF (three times a day). A recent study expanded on the initial observations [60] in two groups of patients at two centers (Johns Hopkins and Fred Hutchinson Cancer Research Center [FHCRC]), which differed by the regimens of post-transplantation immunoprophylaxis [61]. On day 3 (Seattle group) or days 3 and 4 (Hopkins group), 50 mg/kg/day Cy was administered. All patients received tacrolimus and MMF beginning on the day after the last dose of Cy. Sixty-eight patients received these regimens, of whom 67 had hematologic malignancies and one had paroxysmal nocturnal hemoglobinuria. Thirteen percent of evaluable patients had graft failure, 35% and 6% of patients had grades II–IV and III–IV acute GVHD, respectively. The only difference seen between the two groups of patients was that the rate of extensive chronic GVHD in the group that received one dose of post-transplant Cy was 25% compared with 5% in the group that received two doses post transplant (p = 0.05). The overall and event free survivals at 2 years after HCT were 36% and 27%, respectively. HLA-matched and mismatched unrelated donor allografting Nagler et al. [62] reported the results of HLA-matched unrelated marrow transplantation following conditioning with fludarabine and BU (8 mg/ kg) and ATG. Fifteen of 16 patients achieved 100% donor chimerism, and one patient became a mixed chimera. Seven patients developed grades II–IV acute GVHD, with one patient dying from complications related to acute GVHD. The overall and disease-free survival estimates at 36 months were 75% and 60%, respectively, with this reduced-intensity conditioning regimen in a relatively young population of patients (median age 17 years, range 8–48 years). Using a similar regimen of fludarabine, BU, and ATG, Bornhauser et al. [63] treated 42 patients with hematologic malignancies with HLAmatched or single HLA antigen-mismatched unrelated grafts. In this older population group (median 47 years, range 16–65 years), diseasefree survival was 64% for patients with lymphoid malignancies, 38% for patients with standard-risk leukemias, and 14% for patients with

Chapter 71

high-risk diseases. Nine patients (21%) had either primary or secondary graft failures. In patients with stable engraftment, the probability of acute GVHD grades II–IV was 32%, with one patient dying from grade IV GVHD. In a similarly aged group of patients with hematologic malignancies using a different conditioning regimen consisting of Campath mAb, fludarabine, and melphalan [64], Chakraverty et al. described results in 47 patients who received HLA-matched or mismatched unrelated donor transplants. Primary graft failure occurred in two patients, and three patients developed grades III–IV acute GVHD. The overall and progression-free survivals at 1 year were 75.5% and 61.5%, respectively. Bertz et al. reported on 19 patients (ages 60–70 years) with myeloid malignancies who received conditioning with fludarabine, melphalan, and carmustine before HLA-matched related (n = 7) or unrelated (n = 12) HCT [65]. With a median follow-up of 825 days, the 1-year NRM and survival rates were 22% and 68%, respectively. These results were confirmed in a subsequent update from this group on 34 patients receiving unrelated HCT using this regimen [66]. Using the low-dose TBI (2 Gy) and fludarabine (90 mg/m2) conditioning regimen, Niederwieser et al. [67] reported on 52 patients (median age 48 years, range 6–65 years) with advanced hematologic malignancies given unrelated grafts. Seventy-one percent were HLA matched at the antigen (serologic) level, and 29% were one HLA antigen mismatched. Additionally, 15 patients had allele level mismatches for class I HLA antigens. Durable donor engraftment was attained in 88% of patients. Grades II–IV acute GVHD occurred in 63% of patients, with 9% of patients having fatal GVHD. With a median follow-up of 19 months, 35% of the patients were alive and 25% were in remissions of their underlying malignancies. In a subsequent study, Maris et al. [68] reported on 89 patients with unrelated donor grafts. Early data on 22 of the patients were included in the report by Niederwieser et al. [67]. Eligibility criteria for this trial included serological matching for HLA-A, B, and C, and allele-level matching for HLA-DRB1 and DQB1. The median patient age was 53 (range 5–69) years. While the preferred source of stem cells was PBHCs, these were available in only 71 patients, while 18 patients received marrow. Initial donor T-cell engraftment was documented in 87% of patients, with median donor T-cell chimerisms on days 28, 56, and 84 being higher in PBHC recipients than marrow recipients (p = 0.02, 0.01, and 0.08, respectively). The peripheral blood granulocyte and marrow donor chimerism values were not significantly different between the two groups of recipients at the various time points. Sustained donor engraftment occurred in 79% of patients. By multivariate analysis, graft rejection was more frequent in marrow recipients (p = 0.003) and in patients without preceding chemotherapy (p = 0.003). Donor T-cell chimerism values of less than 50% on day 28 were highly associated with eventual graft rejection (p = 0.001). Preceding conventional HCT or preceding chemotherapy reduced the risk of rejection, presumably because the prior cytotoxic therapy increased pretransplant immunosuppression of the host. Of the 85 patient–donor pairs with complete 10/10 HLA antigen sequencing, HLA class I allele-level mismatch (16 of 85 pairs) was associated with a trend towards increased risk of graft rejection (p = 0.13). With a median follow-up of 13 (range 0.6–28) months, the cumulative probabilities of NRM were 11% at 100 days and 16% at 1 year. The Kaplan–Meier estimates for 1-year survival were 57% versus 33%, and for progression-free survival were 44% versus 17% for recipients of PBHCs versus marrow, respectively. A high degree of day 28 CD3 chimerism (>50%) was associated with less relapse (p = 0.05) and a trend towards improved progression-free survival (p = 0.10). By multivariate analysis, better survival was conferred to patients with low-risk diseases (p = 0.04), with no prior transfusions (p = 0.003), with less than 5% blast cells in the marrow before HCT

100

Donor chimerism (%)

1050

CSP MMF

80

60

40 CD3 Gran BM

20

0 0

50

100

150

Days after HCT

Fig. 71.3 Donor chimerism (fluorescence in situ hybridization) in a patient with Philadelphia chromosome-negative myeloproliferative disorder after unrelated hematopoietic cell transplantation (HCT). BM, bone marrow; CSP, cyclosporine; Gran, granulocytes; MMF, mycophenolate mofetil.

(p = 0.002), with HCT from a female donor (p = 0.01), and with CD3+ cell dose greater than or equal to the median (p = 0.0006). Better progression-free survival was identified in a multivariate analysis in patients with less than 5% blasts in the marrow prior to HCT (p = 0.0001) and those who received PBHCs as a hematopoietic stem cell source (p = 0.006). Figure 71.3 illustrates the donor chimerism changes in a 65-year-old patient with Philadelphia chromosome-negative myeloproliferative disorder whose disease was characterized by high white blood cell counts, very high platelet counts (>1 million/mL), and high basophil counts. The patient’s T-cell chimerism increased to 80% by day 84, whereas the marrow and granulocyte donor chimerism declined from 40–45% on day 28 to 10% in the marrow on day 84. The patient’s platelet and basophil counts increased to 900,000/mL and 1500/mL, respectively, consistent with disease progression after an initial response. CSP was rapidly tapered to induce a GVT response. The marrow and granulocyte donor chimerism rose to nearly 100% by day 150, and both basophilia and thrombocytosis resolved. The patient also developed mild signs of chronic GVHD which responded to CSP. The patient is surviving over 7 years after HCT with complete donor engraftment and no evidence of recurrent myeloproliferative disorder. This clinical case illustrates the relationship between postgrafting immunosuppression, GVHD, and GVT effects. It also emphasizes the importance of lineage-specific chimerism evaluations in monitoring patients. The study suggested that PBHCs were the preferred source of grafted hematopoietic cells in unrelated transplantation with a fludarabine/2 Gy TBI conditioning regimen. Given the importance of postgrafting immunosuppression to both facilitate engraftment and reduce the risk of GVHD, our attention was drawn to another finding of the study, the short (3.5-hour) serum half-life of the active metabolite of MMF, mycophenolic acid. This finding resulted in a modification of the post-transplant immunosuppression: an increase in the dosing of MMF to three times daily in the hope of minimizing the risk of late graft rejections. Among 103 patients so treated, graft rejections occurred in 5%, while acute GVHD remained at 53%, with 2-year NRM and overall and progressionfree survival of 19%, 58%, and 49%, respectively [69]. Because three times daily compared with twice-daily MMF diminished the incidence of graft rejection, MMF should be administered three times a day in unrelated donor recipients receiving this regimen. In a subsequent study of 71 patients who received an extended course of MMF until day 150 and tapered through day 180, and a shortened course of CSP through

Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies

day 80, there was an incidence of 77% acute and 47% chronic GVHD [70] compared with 52% (p = 0.03) and 49% (p = 0.43), respectively, in patients who had MMF until day 40 with a taper-through day 96 and CSP until day 100 with a taper-through day 180 [69]. Chimerism and engraftment kinetics Donor chimerism after allogeneic HCT has been evaluated by several means in the past. Currently, fluorescent in situ hybridization of sex chromosomes and amplified fragment length polymorphism analysis are the most commonly used methods (reviewed in [71] and see Chapter 25). Amplified fragment length polymorphism-based techniques utilize the polymerase chain reaction for analysis of polymorphic DNA sequences such as variable number of tandem repeats or short tandem repeats. Additionally, real-time polymerase chain reaction of single nucleotide polymorphisms is being evaluated for this purpose. Reduced-intensity conditioning usually leads to an initial state of mixed chimerism [51,72–74], although some patients given grafts after more myelosuppressive regimens achieve full donor chimerism in cellular subsets as early as day 14 after HCT [75]. Childs et al. [51] evaluated the kinetics of engraftment after conditioning with fludarabine (125 mg/m2) and CY (120 mg/kg). Most frequently after that regimen, full donor chimerism was achieved earlier in peripheral blood T cells than in myeloid cells. The NK-cell chimerism correlated with the T-cell chimerism, whereas the B-cell chimerism was distinct from the T-cell and myeloid lineages. Bornhauser et al. observed that patients with NK chimerism below 75% in the first month after unrelated donor HCT had a higher risk of graft failure after conditioning with fludarabine 150 mg/ m2, BU 6.6 mg/kg intravenously, and ATG [63]. The kinetics of chimerism was also evaluated in patients who received conditioning with 2 Gy TBI with or without fludarabine (90 mg/m2) [74]. Most patients remained mixed donor–host chimeras for up to 3–6 months after HCT. Patients given preceding chemotherapy or peripheral blood stem cell grafts had the highest degrees of donor chimerism. High levels of donor T- or NKcell chimerism (>50%) 28 days after HCT have each been associated with a reduced risk of graft rejection [54,74]. There was also an association with higher donor T-cell chimerism and an increased risk of grade II–IV acute GVHD [74]. An association between the kinetics of engraftment and GHVD was observed by Childs et al. [51]. The achievement of full donor T-cell chimerism preceded the development of grades II–IV GVHD in patients who received conditioning with fludarabine and CY. The studies by Mattsson et al. [76], by contrast, found that 82% of patients had mixed donor–host T-cell chimerism at the time of the development of acute GVHD. This is similar to the results observed after conditioning with TBI with or without fludarabine, in which 83% of the patients were mixed donor–host T-cell chimeras at the time of the development of GVHD [74]. However, when chimerism was analyzed as a continuous linear variable, patients with higher levels of donor T-cell chimerism on day 28 after HCT were at higher risk for grade II–IV acute GVHD [54,68,74], with a corresponding reduced risk of relapse in those patients who achieved full donor T-cell chimerism [74]. GVHD and GVT effects Reconstitution of donor-derived immunity after reduced-intensity conditioning differed from what occurred after high-dose conditioning. First, as mentioned above, there was an initial state of mixed donor–host chimerism in the recipients of reduced-intensity conditioning that changed the balance of both GVH and HVG tolerance, which may affect the onset of GVHD. Second, high-dose conditioning regimens may contribute to the pathophysiology of GVHD via tissue damage causing the release of cytokines, the so-called “cytokine storm” [77]. However,

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the number of host-derived antigen-presenting cells may be higher after reduced-intensity conditioning, thus contributing to acute GVHD given their putative role in the initiation of GVHD [78]. In order to address the relationship between conditioning intensity and GVHD, several retrospective studies have been carried out. Most have shown lower incidences of acute and similar or lower incidence of chronic GVHD [79–82]. One age-matched retrospective analysis was carried out comparing the incidence of GVHD in recipients of reducedintensity versus high-dose conditioning. The cumulative incidence of grades II–IV acute GVHD was lower in reduced-intensity HCT (64% versus 85%; p = 0.001), resulting in the use of less systemic immunosuppression during the first 3 months after transplant [79]. There was no difference in the cumulative incidence of chronic GVHD requiring treatment. Of note, reduced-intensity HCT was associated with a syndrome of acute GVHD occurring beyond day 100 in a subset of the related recipients, and this should be taken into consideration in the design of prospective studies comparing reduced-intensity and high-dose HCT. The prognostic relevance of early-onset GVHD was evaluated in 395 patients who had HCT from HLA-matched related (n = 297) or unrelated (n = 98) donors after conditioning with fludarabine and 2 Gy TBI [83]. The cumulative incidence of grades II–IV acute GVHD and chronic GVHD were 45% and 47%, respectively, with related donors, and 68% and 68%, respectively, with unrelated donors. The median days of highdose corticosteroid use for treatment of acute or chronic GVHD (used as a marker for the onset of clinically relevant GVHD) were 79 (range 8–799) days and 30 (range 5–333) days after HCT for related and unrelated recipients, respectively. The cumulative incidence of NRM among patients with GVHD was 55% at 4 years when corticosteroids were started before day 50 as compared with 29% when started after day 50 in related recipients, suggesting that early-onset GVHD may benefit from intensified primary immunosuppressive therapy. There was no association of NRM with early-onset GVHD seen in unrelated recipients. Historically, the occurrence of GVHD was associated with GVT effects in patients receiving high-dose conditioning regimens [1,2]. Several retrospective analyses have been carried out to evaluate the impact of GVT on outcomes after reduced-intensity conditioning regimens since the regimens themselves have less of a role in disease control. Martino et al. demonstrated a reduced risk of relapse in patients with AML and myelodysplastic syndrome who experienced acute and/or chronic GVHD compared with those who did not [84]. Kroger et al. found that while there was no impact of acute GVHD on the risk of relapse in patients with multiple myeloma, there was a significantly lower risk of relapse in patients who had chronic GVHD [85]. These outcomes were also observed in an analysis of multiple myeloma patients by Crawley et al. for the European Group for Blood and Marrow Transplantation [86]. Blaise et al. performed a landmark analysis starting on day 100 in patients with AML and observed a lower risk of relapse and higher leukemia-free survival in the patients that developed chronic GVHD [87]. GVT effects were analyzed in a cohort of 322 patients (related donor n = 192, unrelated donor n = 130) with hematologic malignancies following conditioning with fludarabine and TBI [88]. Fifty-seven percent of patients with measurable disease (n = 221) achieved complete (n = 98) or partial (n = 28) remission. The achievement of full donor chimerism was associated with a decreased risk of relapse/progression. Grades II–IV GVHD did not impact progression/relapse but were associated with an increase in NRM and decreased progression-free survival. Conversely, chronic GVHD was associated with a decrease in progression/ relapse and an increase in progression-free survival without an increase in NRM. This suggests that protocols designed to reduce acute GVHD might improve survival while allowing GVT to occur later.

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Toxicities In a retrospective cohort study, recipients of reduced-intensity conditioning had significantly reduced transfusion requirements compared with conventional HCT recipients, with only 23% of the patients requiring platelet transfusions and 63% requiring red blood cell transfusions compared with 100% of high-dose HCT recipients (p ≤ 0.0001). Recipients of reduced-intensity HCT who did require transfusion used significantly fewer units of platelets and red blood cells (p ≤ 0.0001) [89]. Recipients of reduced-intensity conditioning had shorter periods of neutropenia than recipients of myeloablative conditioning (p < 0.0001), and this was associated with fewer episodes of bacteremia during the first 30 days (p = 0.01) [90]. Similarly, there was a trend to less cytomegalovirus antigenemia, viremia, and disease in reduced-intensity HCT recipients compared with recipients of myeloablative conditioning, with a significant reduction when more serious manifestations (cytomegalovirus viremia and disease) were combined during the first 100 days (p = 0.01) [91]. The cumulative incidence of proven or probable invasive mold infections in the first year after reduced-intensity HCT was 15%, which was similar to that for myeloablated recipients [90,92]. Severe acute GVHD and cytomegalovirus disease were the primary risk factors associated with invasive mold infections after reduced-intensity conditioning, with high-dose corticosteroid therapy on diagnosis of invasive mold infections being associated with an increased risk for death related to this cause. The incidence of idiopathic pneumonia syndrome was significantly lower following reduced-intensity conditioning compared with “conventional” conditioning regimens (2.2% versus 8.4%; p = 0.003) [93]. Liver injury is a frequent, serious complication of high-dose preparative regimens. The frequency and severity of hepatic injury after reduced-intensity conditioning was evaluated in 193 consecutive patients [94]. No patients developed sinusoidal obstructive syndrome. Twentysix percent of patients developed hyperbilirubinemia of 4 mg/dL or more, most commonly resulting from cholestasis due to GVHD or sepsis. Overall survival was superior for patients who had maximum bilirubin in the normal or minimally elevated (1.3–3.9 mg/dL) ranges compared with those in the elevated (≥4 mg/dL) ranges. Incidences of acute renal failure (ARF) among 140 recipients of highdose and 129 of reduced-intensity conditioning were compared [95]. Severity of ARF was classified into four grades based on serum creatinine increases in the first 100 days after HCT. Recipients of reducedintensity conditioning were significantly older and had greater pretransplant comorbidities. Despite this, high-dose patients had greater incidences of severe ARF (73% versus 47%; p < 0.001) and dialysis (12% versus 3%; p < 0.001) compared with those receiving reducedintensity regimens. NRM was also greater in the high-dose group at 100 days and 1 year. The incidences of chronic kidney disease after reducedintensity HCT were analyzed in 122 patients from three centers [96]. Multivariate analysis revealed that ARF in the first 100 days was associated with chronic kidney disease. Previous autologous HCT, long-term calcineurin use, and chronic GVHD were independently associated with chronic kidney disease. Future research should focus on preventing ARF and decreasing the use of calcineurin inhibitors.

Comparing morbidity and mortality: influence of pretransplant comorbidities Until recently, allogeneic HCT has been reserved for young patients in good medical condition. Comorbidities, defined as any additional clinical entity in a patient with an index disease, affect therapeutic plans and outcomes after treatment for the index disease. Many patients with hematologic malignancies either have pre-existing comorbidities or

develop them during the course of therapy for their malignancies. In clinical trials in hematology and oncology, patients with comorbidities are often excluded if it is thought that a specific comorbidity may dramatically impact outcomes. This is also true in clinical trials of HCT. With the advent of reduced-intensity conditioning regimens, HCT is being carried out in older patients and those with considerable comorbidities. Objective measures of patients’ underlying health are required in order to study and disseminate transplantation approaches in an older and medically less fit population. While efforts to analyze the impact of comorbidities on outcomes after HCT are still in the early stages, initial data have suggested that comorbidity indices are useful tools in predicting outcomes that can be used in the design of inclusion/exclusion criteria for clinical trials. One of the indices used was the Charlson Comorbidity Index (CCI), which was originally developed in a cohort of patients at a general medical center [97]. The CCI was applied to patients with hematologic malignancies after HCT following both high-dose and reduced-intensity conditioning regimens. In both related [98] and unrelated [99] recipients, the adapted CCI successfully predicted NRM. Yet the CCI had limitations. Some comorbidities were rarely found among HCT recipients due to existing exclusion criteria, particularly in patients conditioned with highdose regimens, and the CCI did not capture other frequent comorbidities. Further, the CCI lacked sensitivity. Therefore, a study was designed to better define previously identified comorbidities by utilizing pretransplant laboratory data and, additionally, investigating additional HCTrelated comorbidities so as to develop a scoring system more suited to HCT. Data were collected from 1055 patients and randomly divided into training and validation sets [99]. Based on the prognostic significance, various weights were assigned to the individual comorbidities and then validated. The new HCT-Comorbidity Index (HCT-CI) was more sensitive than the CCI since it captured more than 62% of patients with a score of over 0 compared with 12%, respectively. The HCT-CI additionally showed a better survival prediction than the CCI (Fig. 71.4). Pretransplant score assignments with the index were simple to accomplish and should be highly objective. This tool has been validated in patients with myelodysplasia or AML, where similar outcomes have been found after highdose and reduced-intensity conditioning after stratification of patients into low- and high-risk disease categories and low and high HCT-CI scores [100]. This suggests that patients with low HCT-CI scores could be candidates for randomized trials comparing conditioning intensity. Currently, studies are ongoing comparing outcomes using this scoring system in patients treated at other transplant centers, and early results suggest similar utility in predicting outcome [101]. The development of the HCT-CI sets the stage for prospective studies such as a riskstratification system that could discriminate which patients could be enrolled in prospective randomized studies comparing high-dose and reduced-intensity regimens to evaluate which patients benefit from either kind of conditioning. Relapse risk for hematologic malignancies Reduced-intensity allogeneic HCT relies on GVT effects for the eradication of malignancy. An analysis was carried out in 834 patients who had received conditioning with 2 Gy TBI with or without fludarabine to get an estimate of relapse risk according to disease characteristics [102]. Median patient age was 55 (range 5–74) years, and recipients received either related (n = 498) or unrelated (n = 336) donor grafts. Relapse rates per patient–year at risk for the first 2 years after HCT were calculated for 29 different diagnosis and disease stages. Excluding multiple myeloma patients who had a planned autologous transplant followed by an allogeneic HCT, there was no evidence that a history of

Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies (a)

(b)

(c)

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Fig 71.4 Nonrelapse mortality (NRM) as stratified by the HCT-Comorbidity Index (HCT-CI) (a) compared with the Charlson Comorbidity Index (CCI) (b), and Kaplan–Meier estimates of survival as stratified by the HCT-CI (c) and CCI (d). HCT, hematopoietic cell transplantation.

a failed autologous transplant was associated with a higher risk of relapse. The overall risk of relapse per patient–year was 0.36 and was 0.35 among patients with related donors, and 0.37 among those with unrelated donors. Patients were grouped into low-, standard-, and highrisk groups (Table 71.3). The survivals, cumulative relapse rates, and cumulative NRM rates according to risk groups are shown in Fig. 71.5. Patients with low-grade lymphoproliferative diseases experienced the lowest relapse rates, whereas patients with advanced myeloid and lymphoid malignancies had the highest relapse rates after reduced-intensity HCT. In order to improve patient outcomes in the high-risk group, the option of allografting should be considered earlier in the disease course when the tumor burden is lower. Alternatively, the use of debulking chemotherapy prior to HCT, or targeted therapies with limited systemic toxicities, should be added to the conditioning. Consolidative reduced-intensity allografts following planned autografts In patients with aggressive malignancies, the GVT effect following a reduced-intensity allograft may not be sufficiently fast enough to eradicate large-volume disease. Carella et al. adopted the strategy of using debulking autologous HCT followed by reduced-intensity allogeneic HCT in patients with refractory Hodgkin’s and non-Hodgkin’s lymphomas [103]. Patients (n = 15) were first given high-dose therapy with carmustine, etoposide, cytarabine, and melphalan, with reinfusion of autologous PBHCs. At a median of 61 days after engraftment, patients were given fludarabine 90 mg/m2 and CY 900 mg/m2 followed by PBHCs from HLA-matched related donors. With a median follow-up of 337 days, 10 patients were alive, with five patients in remission. This approach was evaluated in 17 patients with multiple myeloma by Kroger et al. [104]. After autografting with melphalan (200 mg/m2), the patients

received a dose-reduced regimen consisting of fludarabine (180 mg/m2), melphalan (100 mg/m2), and ATG (3 × 10 mg/kg) followed by allografting. After a median follow-up of 13 months after allogeneic HCT, 13 patients were alive, 12 of whom were free of relapse or progression. A similar approach was evaluated by Maloney et al. in patients with multiple myeloma (n = 54) who were given a cytoreductive autograft with melphalan conditioning (200 mg/m2) followed by an allograft from an HLA-identical sibling after conditioning with 2 Gy TBI [105]. One patient died before day 100 following the allograft because of disease progression. With a median follow-up of 550 days after allograft in surviving patients, the overall survival rate was 78%. The overall disease response rate was 83%, with 57% of patients achieving complete remission and 26% partial remission. Based on this study, a large phase III trial comparing tandem autologous transplant with tandem autologous/ allogeneic transplant has been carried out by the Bone Marrow Transplant–Clinical Trials Network. All patients have been enrolled and are currently in follow-up. Another trial based on this approach has been recently reported by Bruno et al., comparing allografting with autografting for newly diagnosed multiple myeloma [106]. Patients (n = 162) were enrolled at the time of diagnosis and treated with vincristine, doxorubicin, and dexamethasone followed by melphalan and autologous PBHC infusion. Patients with an HLA-identical sibling then received 2 Gy TBI and PBHCs from the sibling donor, whereas those without an HLA-identical sibling received a second autograft. The median overall survival was longer in the patients who had an HLA-identical sibling than those who did not (80 versus 54 months, respectively). Among patients who completed their treatment protocols, the disease-related mortality was significantly higher in the tandem autologous group (43%) compared with the autologous–allogeneic group (7%), without a significant difference in NRM.

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Table 71.3 Relapse rates in 29 diagnosis and disease stage groups Number of patients Low risk CLL NHL – low grade NHL – low grade Waldenström’s macroglobulinemia MM NHL – mantle cell NHL – mantle cell MPD NHL – high grade ALL Standard risk CLL MM MDS AML CML AML High risk MDS AML NHL – high grade HD MDS HD AML CML CML Renal cell ALL ALL CMML

CR Not in CR CR

Relapse rate per patient–year

7 34 9 9 29 16 25 19 26 19

8.2 40.8 11.1 10.8 42.8 15.7 30.4 18.6 31.0 21.0

0.00 0.15 0.18 0.19 0.19 0.19 0.20 0.21 0.23 0.24

75 136 20 80 26 59

89.0 174.8 18.2 78.3 32.8 62.6

0.26 0.27 0.33 0.33 0.34 0.37

23 42 36 13 18 38 13 14 7 18 8 3 12

19.2 29.1 26.3 9.6 14.4 30.6 9.2 10.1 3.8 10.6 4.6 2.2 6.3

0.52 0.55 0.57 0.62 0.70 0.72 0.87 0.99 1.05 1.23 1.29 1.35 1.42

CR† CR Not in CR CR 1st CR Not in CR Not in CR RA/RARS 1st CR 1st CP ≥2nd CR

Patient–years of follow-up*

RAEB/RAEB-t Evolved from MDS Not in CR CR Secondary Not in CR Not in CR AP/BC 2nd CP ≥2nd CR Not in CR

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; AP, accelerated phase; BC, blast crisis; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; CP, chronic phase; CR, complete remission; HD, Hodgkin’s disease; MDS, myelodysplastic syndrome; MM, multiple myeloma; MPD, myeloproliferative disease; NHL, non-Hodgkin’s lymphoma; RA, refractory anemia; RAEB, refractory anemia with excess blasts; RAEB-t, refractory anemia with excess blasts in transformation; RARS, refractory anemia with ringed sideroblasts. * Patient–years of follow-up are the total number of person–years of observation time from transplant until death, relapse/progression, last contact, or 2 years. † The criteria for complete remission from multiple myeloma were absence of monoclonal immunoglobulin and of discernible light chains in the urine by standard electrophoresis, absence of visible monoclonal bands on immunofixation, fewer than 1% plasma cells in marrow aspirates, absence of evidence of clonal disease according to flow cytometry of marrow cells, and absence of an increase in the size or number of osteolytic lesions. Adapted with permission from [102]. Copyright, American Society of Hematology.

Reduced-intensity allografting after failed high-dose transplants Use of conventional high-dose conditioning for allografts following failed autologous or allogeneic HCT has resulted in poor outcomes in adult patients, primarily related to early fatal regimen-related toxicities [107]. A number of investigators have therefore evaluated reducedintensity regimens to condition patients for second HCT. Investigators in Jerusalem conditioned patients with fludarabine, BU, and ATG, with CSP as GVHD prophylaxis [108]. Among 12 relatively young patients transplanted (median age 33 years, range 8–63 years), one nonrelapse death occurred and six patients were disease-free with a median followup of 23 months. Actuarial overall and disease-free survivals were 56% and 50%, respectively, at 34 months. Investigators at Massachusetts

General Hospital described their results with 13 patients (median age 38 years) who relapsed following autologous HCT and received HLAmatched related donor allografts following conditioning with CY and ATG with or without thymic irradiation [109]. DLI was administered at 5–6 weeks post transplant to facilitate full donor chimerism. One nonrelapse death occurred, and 2-year estimates of overall and disease-free survivals were 45% and 38%, respectively. Investigators at the City of Hope National Medical Center have described their results in 28 patients who failed previous autologous transplants or developed myelodysplastic syndrome after autografts [110]. The median interval from the failed autologous HCT and allogeneic HCT was 15 months. The median age of the patients was 47 years, and patients were treated with either fludarabine plus melphalan (n = 24) or fludarabine plus 2 Gy TBI (n = 4). Patients received HCT from

Reduced-intensity Conditioning Followed by Hematopoietic Cell Transplantation for Hematologic Malignancies (a)

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Using the 2 Gy TBI-based regimen (with or without fludarabine), the Seattle Consortium reported on 147 patients (median age 46 years, range 9–73 years) who failed conventional autologous (n = 135), allogeneic (n = 10) or syngeneic (n = 2) HCT and subsequently received HLAmatched related (n = 62) or unrelated (n = 85) allografts [111]. The 3year NRM was 32% in related recipients and 28% in unrelated recipients with a median follow-up of 813 (range 84–1925) days. The Kaplan– Meier estimates of relapse and overall survival were 48% and 32%, respectively, for related recipients, and 44% and 44%, respectively, in unrelated recipients. The best outcomes were observed in patients with non-Hodgkin’s lymphoma, whereas patients with multiple myeloma and Hodgkin’s lymphoma had the poorest outcomes due to high incidences of relapse and progression. A lower risk of relapse or progression was seen in patients in partial or complete remission at the time of HCT and in patients who developed chronic GVHD. Factors associated with better overall survival were the partial or complete remission of underlying diseases and lack of comorbidity at the time of HCT. Thus, allogeneic GVT effects can be used with some success in patients who have exhausted other treatment options, including high-dose therapy with stem cell rescue (either autologous or allogeneic). Outcomes using related or unrelated donors were comparable.

Conclusion

(c)

Fig 71.5 (a) Overall survival rates, (b) cumulative relapse rates, and (c) cumulative nonrelapse mortality rates according to risk groups. HCT, hematopoietic cell transplantation.

either HLA-matched related (n = 14) or unrelated (n = 14) donors. The day 100 mortality and NRM were 25% and 21%, respectively. With a median follow-up of 24 months in surviving patients, the 2-year probabilities of overall survival, event-free survival, and relapse rate were 56.5%, 41%, and 41.9%, respectively.

The use of reduced-intensity or minimally myelosuppressive preparative regimens has resulted in durable donor engraftment and the development of GVT effects in the setting of underlying malignant diseases. The regimens have been relatively nontoxic in older patients and have been successfully applied to younger patients with comorbid conditions that had excluded them from high-dose HCT. The optimal regimen may ultimately be defined by the nature of the diseases being treated. In aggressive lymphomas, acute leukemias not in remission, and other fastgrowing malignancies, cytoreduction by either preceding chemotherapy with or without autologous stem cell rescue, or the use of a reducedintensity regimen with disease-specific chemotherapy may be necessary to allow time for adequate GVT responses to develop. For low-grade lymphomas, acute leukemias in first complete remission, chronic leukemias, and other more indolent diseases, minimal reduced-intensity regimens may be sufficient for successful outcomes by allowing the donor T cells to generate sufficient GVT responses and eradicate the underlying malignancies. The role of DLI for the achievement of full donor chimerism and specific GVT effects needs to be explored, and current ongoing research is aimed toward those goals. Other current issues include minimizing GVHD, which may result in prolonged immunosuppression, thereby placing patients at risk for infections that, in turn, contribute significantly to NRM in all regimens. Additionally, prolonged immunosuppression may blunt optimal GVT effects. Ultimately, reduced-intensity HCT might be used to establish mixed donor–host chimerism that can serve as a platform for adoptive immunotherapy with the infusion of tumor-specific cytotoxic T cells that would exert GVT effects without causing GVHD. In the future, reduced-intensity transplants may become the procedure of choice also for younger patients with either malignant or nonmalignant diseases. Towards that end, phase III studies are needed to determine both immediate- and long-term outcomes in different disease categories and age groups.

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53. Kottaridis PD, Milligan DW, Chopra R et al. In vivo CAMPATH-1H prevents graft-versus-host disease following nonmyeloablative stem cell transplantation. Blood 2000; 96: 2419–25. 54. McSweeney PA, Niederwieser D, Shizuru JA et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versustumor effects. Blood 2001; 97: 3390–400. 55. Burroughs L, Mielcarek M, Leisenring W et al. Extending postgrafting cyclosporine decreases the risk of severe graft-versus-host disease after nonmyeloablative hematopoietic cell transplantation. Transplantation 2006; 81: 818–25. 56. Lan F, Zeng D, Higuchi M, Huie P, Higgins JP, Strober S. Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: “natural suppressor” cells. J Immunol 2001; 167: 2087–96. 57. Lowsky R, Takahashi T, Liu YP et al. Protective conditioning for acute graft-versus-host disease. N Engl J Med 2005; 353: 1321–31. 58. Sykes M, Preffer F, McAfee S et al. Mixed lymphohaemopoietic chimerism and graft-versuslymphoma effects after non-myeloablative therapy and HLA-mismatched bone-marrow transplantation. Lancet 1999; 353: 1755–9. 59. Luznik L, Jalla S, Engstrom LW, Iannone R, Fuchs EJ. Durable engraftment of major histocompatibility complex-incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, and posttransplantation cyclophosphamide. Blood 2001; 98: 3456–64. 60. O’Donnell PV, Luznik L, Jones RJ et al. Nonmyeloablative bone marrow transplantation from partially HLA-mismatched related donors using posttransplantation cyclophosphamide. Biol Blood Marrow Transplant 2002; 8: 377– 86. 61. Luznik L, O’Donnell PV, Symons HJ et al. HLAhaploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, post-transplantation cyclophosphamide. Biol Blood Marrow Transplant (in press). 62. Nagler A, Aker M, Or R et al. Low-intensity conditioning is sufficient to ensure engraftment in matched unrelated bone marrow transplantation. Exp Hematol 2001; 29: 362–70. 63. Bornhauser M, Thiede C, Platzbecker U et al. Dose-reduced conditioning and allogeneic hematopoietic stem cell transplantation from unrelated donors in 42 patients. Clin Cancer Res 2001; 7: 2254–62. 64. Chakraverty R, Peggs K, Chopra R et al. Limiting transplantation-related mortality following unrelated donor stem cell transplantation by using a nonmyeloablative conditioning regimen. Blood 2002; 99: 1071–8. 65. Bertz H, Potthoff K, Finke J. Allogeneic stem-cell transplantation from related and unrelated donors in older patients with myeloid leukemia. J Clin Oncol 2003; 21: 1480–4. 66. Spyridonidis A, Bertz H, Ihorst G, Grullich C, Finke J. Hematopoietic cell transplantation from unrelated donors as an effective therapy for older patients (≥60 years) with active myeloid malignancies. Blood 2005; 105: 4147–8.

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67. Niederwieser D, Maris M, Shizuru JA et al. Lowdose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases. Blood 2003; 101: 1620–9. 68. Maris MB, Niederwieser D, Sandmaier BM et al. HLA-matched unrelated donor hematopoietic cell transplantation after nonmyeloablative conditioning for patients with hematologic malignancies. Blood 2003; 102: 2021–30. 69. Maris MB, Sandmaier BM, Storer BE et al. Unrelated donor granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell transplantation after nonmyeloablative conditioning: the effect of postgrafting mycophenolate mofetil dosing. Biol Blood Marrow Transplant 2006; 12: 454–65. 70. Baron F, Sandmaier BM, Storer BE et al. Extended mycophenolate mofetil and shortened cyclosporine failed to reduce graft-versus-host disease after unrelated hematopoietic cell transplantation with nonmyeloablative conditioning. Biol Blood Marrow Transplant 2007; 13: 1041–8. 71. Baron F, Sandmaier BM. Chimerism and outcomes after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning [Review]. Leukemia 2006; 20: 1690–700. 72. Dey BR, McAfee S, Colby C et al. Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 2003; 9: 320–9. 73. Carvallo C, Geller N, Kurlander R et al. Prior chemotherapy and allograft CD34+ dose impact donor engraftment following nonmyeloablative allogeneic stem cell transplantation in patients with solid tumors. Blood 2004; 103: 1560–3. 74. Baron F, Baker JE, Storb R et al. Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2004; 104: 2254–62. 75. Ueno NT, Cheng YC, Rondon G et al. Rapid induction of complete donor chimerism by the use of a reduced-intensity conditioning regimen composed of fludarabine and melphalan in allogeneic stem cell transplantation for metastatic solid tumors. Blood 2003; 102: 3829–36. 76. Mattsson J, Uzunel M, Brune M et al. Mixed chimaerism is common at the time of acute graftversus-host disease and disease response in patients receiving non-myeloablative conditioning and allogeneic stem cell transplantation. Br J Haematol 2001; 115: 935–44. 77. Mohty M, Blaise D, Faucher C et al. Inflammatory cytokines and acute graft-versus-host disease after reduced-intensity conditioning allogeneic stem cell transplantation. Blood 2005; 106: 4407– 11. 78. Shlomchik WD, Couzens MS, Tang CB et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science 1999; 285: 412–15.

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72

Ginna G. Laport & Robert S. Negrin

Management of Relapse after Hematopoietic Cell Transplantation

Introduction Disease relapse is an ominous clinical event following either autologous or allogeneic hematopoietic cell transplantation (HCT). Following autologous HCT, relapse is the major cause of treatment failure. Although disease recurrence is less common after allogeneic HCT, it remains a frequent clinical event. In both settings, patients are highly sensitized to the risk of return of the underlying malignancy. As such, great care is necessary when discussing and investigating signs and symptoms of potential disease that requires radiographic and often pathologic investigation before a specific diagnosis is made. In this chapter, we will review efforts to reduce the risk of disease recurrence and the management of patients who suffer a relapse following HCT.

Management of relapse after allogeneic HCT Allogeneic HCT is an accepted curative treatment modality for patients with malignant and nonmalignant diseases. In contrast to autologous HCT, which relies solely on chemotherapy and/or total body irradiation (TBI) to eradicate disease, allogeneic HCT is associated with a graftversus-tumor (GVT) effect mediated by alloreactive donor T and B cells. Despite this added benefit of adoptive immunotherapy, relapse remains one of the leading causes of treatment failure after allogeneic HCT and typically portends a very poor prognosis. Historically, salvage chemotherapy, withdrawal of immunosuppression, and/or a second HCT were the only available options. However, additional options are now available that include various forms of cellular adoptive immunotherapy such as donor leukocyte infusions (DLIs), cytokine-induced killer (CIK) cells, natural killer (NK) cells, and antigen-specific cytotoxic T lymphocytes (CTLs). These interventions attempt to harness the powerful therapeutic benefits of the donor’s immune system to eliminate residual host tumor. For patients with relapsed chronic myelogenous leukemia (CML), additional options include the tyrosine kinase inhibitors such as imatinib to reinduce remissions or to eradicate persistent disease following allogeneic HCT. The following section of this chapter will discuss the above-mentioned approaches, with an emphasis on the utility of adoptive immunotherapy.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Withdrawal of immunosuppressive therapy Withdrawal of immunosuppressive medications is usually the initial step taken when relapse occurs after allogeneic HCT, and typically precedes subsequent interventions such as DLI or palliative chemotherapy. Immunosuppression withdrawal is intended to allow for maximal exertion of the GVT effect to induce remission, which unfortunately is rarely successful. In addition, graft-versus-host disease (GVHD) remains a serious complication. Scattered case reports in almost all hematologic malignancies have been published, with occasional durable remissions seen. The highest response rates have been observed in patients with CML and indolent lymphomas, with less favorable results in the acute leukemias and aggressive lymphomas. The more rapid proliferation rates of these diseases may simply outpace the slower kinetics of the potential GVT effects. The largest series comes from Germany, in which cyclosporine was withdrawn from 42 patients with CML and acute leukemia [1]. The 4-year probability of achieving a durable remission was 84% among the CML patients, 10% for the patients with acute myelogenous leukemia (AML) at 3 years, and 0% for those with advanced CML or acute lymphoblastic leukemia (ALL). These results underscore the marked sensitivity of early-phase CML to the GVT effect, which appears to be much weaker against the acute leukemias, especially ALL. In most cases of relapse, discontinuation of immunosuppressive agents is a reasonable initial intervention in the absence of active GVHD, but unfortunately rarely induces remissions except in cases of CML. Donor leukocyte infusions DLI confers a direct graft-versus-malignancy effect by infusion of alloreactive donor lymphocytes. Purposes of DLI include conversion of mixed donor chimerism to full donor chimerism after HCT, as preemptive therapy to prevent relapse or for the treatment of relapse. Table 72.1 summarizes published trials highlighting the clinical results of DLI in selected reports. The use of DLI was first reported by Kolb et al. in 1990, in which three patients with relapsed CML achieved long-term remissions with the use of interferon (IFN) and donor buffy coat cells [2]. Since then, numerous reports with related and unrelated donors have confirmed the efficacy of DLI in reinducing remissions in patients with hematologic malignancies, with the highest responses observed in patients with CML [3–6]. Disease status at the time of DLI and disease type (acute versus chronic leukemia) appear to be the most important predictors of response. The application of DLI, however, is not without toxicity and carries a mortality rate of 3–10%, with acute GVHD and marrow aplasia being

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Table 72.1 Selected studies of the use of donor leukocyte infusions for relapse after allogeneic hematopoietic cell transplantation

Group/year

n

Disease

Donor

Complete remission

EBMT, 2007 [25] European, 2006 [23] Hopkins, 2006 [3]

171 63 83

AML MM Various

MRD/URD MRD/URD MRD

EBMT, 2002 [11] Hammersmith, 2000 [6]

298 66

CML CML

MRD/URD MRD/URD

34% 19% CML 71% AML 31% Others <10% 70%* 67% MR

NMDP, 2000 [5] IBMTR, 2000 [28] US multicenter, 1997 [8]

58 44 140

Various ALL Various

URD MRD/URD MRD/URD

42% 25% AML 46% ALL 18% CML* Cytogenetic relapse 100% Hematologic relapse 74% Accelerated phase 33% Blast crisis 16%

Overall survival

Grade II–IV graft-versus-host disease

Treatment-related mortality

Relapse mortality

Follow-up

21% NR NR

43% 22% 35%

11% 11% 11%

71% 21% 60%

27 months 14 months 857 days

72% 95%† 33%‡ NR 13% NR

32% NR

5–22% NR

21% NR

48 months 29 months

37% 40% 46%

22% 23% 18%

45% 59% 50%

66 weeks 1 year 506 days

ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; EBMT, European Bone Marrow Transplant Group; IBMTR, International Bone Marrow Transplant Registry; MM, multiple myeloma; MR, molecular remission; MRD, matched related donor; NMDP, National Marrow Donor Program; NR, not reported; URD, unrelated donor. * Complete responses defined as absence of t(9;22) translocation or bcr/abl negativity by molecular analysis. † Overall survival for patients achieving MR. ‡ Overall survival for patients not achieving MR.

the leading causes of death [6,7]. The incidence of both acute and chronic GVHD after DLI is approximately 40–60%, with over half of the patients who develop chronic GVHD having extensive disease [7,8]. The onset of acute GVHD typically occurs 32–42 days after DLI. The incidence and severity of GVHD does not appear to be affected by donor source (related versus unrelated donor). Factors that predict for the development of GVHD after DLI include recipient–donor sex mismatch, patient–donor cytomegalovirus seropositivity, DLI administration in bulk dosing, and use of a T-cell-depleted graft at the time of HCT [9]. The incidence of marrow aplasia has ranged from 18% to 50%, although sustained pancytopenia occurs in fewer than 5% of patients [7,8]. Marrow aplasia is thought to occur when donor lymphocytes ablate recipient cells but donor hematopoietic cells are unable to maintain adequate hematopoiesis, especially when there is little or no donorderived hematopoiesis at the time of DLI. Therefore, the use of DLI is cautioned in patients who relapse and have mostly recipient-type hematopoiesis. DLI for CML Most published reports regarding DLI involve patients with relapsed CML, which is the hematologic malignancy known to be the most sensitive to the GVT effects (see Chapter 51). Disease status at the time of DLI is the most important predictor of response, with patients in chronic phase showing the highest sustained response rates. Results from two large retrospective registry analyses and numerous single-center reports have been remarkably consistent. For patients with relapsed chronicphase CML, complete remissions (CRs) approach 80%, with most patients also achieving molecular remissions (MRs). In a retrospective

Fig. 72.1 Disease-free survival for chronic myelogenous leukemia (CML) after donor lymphocyte infusion. (Reproduced from [10], with permission.)

analysis from the North American registry, only two of 32 patients (6%) who achieved remission after DLI for relapsed early-phase CML had disease recurrence, and the estimated probability of event-free survival (EFS) from the time of remission was 73% at 3 years (Fig. 72.1) [10]. In a series of 66 patients with relapsed CML from the Hammersmith

Management of Relapse after Hematopoietic Cell Transplantation

group, 44 patients (67%) achieved an MR, with survival being superior for patients who achieved an MR compared with those who did not (95% versus 53% at 3 years; p = 0.0001) [6]. Factors predicting for a lower probability of achieving an MR included advanced-phase disease at relapse, a short interval between HCT and relapse (<9 months versus >9 months). These results and others confirmed that the majority of MRs after DLI are durable. For patients with advanced CML, DLI has been less effective, with only 12–28% of patients in accelerated or blast phase achieving a remission, with most responses being transient [3,6,8]. The prognostic relevance of cell dose, cell administration, and cell composition of DLI administration has also been explored. The European Group for Blood and Marrow Transplantation (EBMT) retrospectively analyzed the effect of the initial cell dose on outcome in 298 patients with CML who relapsed after allogeneic HCT [11]. Three groups of patients were stratified by initial cell dose of 108 mononuclear cells/kg: less than 0.2, 0.21–2.0, and more than 2.0. Results showed that the group who received the lowest cell dose of less than 0.2 × 108 cells had less GVHD, less myelosuppression, similar response rates, better overall and failure-free survival, and less DLI-related mortality compared with the higher cell doses. A recent report from the Hammersmith group, however, did not find an association between cell dose and incidence of GVHD [9]. By multivariate analysis, the only factor that emerged as a predictor for GVHD in this latter study was infusion of DLI from a female donor into a male recipient, which underscores the fundamental role of minor histocompatibility antigens (mHAs) in posttransplant allogeneic immune responses. Investigators from the Johns Hopkins University also concurred and found that T-cell dose did not impact GVHD incidence [3]. Interestingly, this group also reported that disease responses were strongly associated with the occurrence of GVHD in all diseases studied except for CML, where CRs were independent of GVHD; this is in agreement with the above data from the Hammersmith group. Also worth noting from the Johns Hopkins report was the impact of donor chimerism percentage at the time of DLI. Patients with greater than 50% donor chimerism before DLI were nearly five times more likely to achieve a CR than patients whose donor chimerism was less than 50%. Further, patients who attained full donor chimerism after DLI were 22 times more likely to obtain a CR than patients who did not, which suggests that donor chimerism may be a reliable predicator of GVT reactions. Manipulation of certain cell populations in the DLI has been undertaken with the goal to reduce toxicity, mainly GVHD. Investigators from the Dana Farber Cancer Institute (DFCI) and M.D. Anderson Cancer Center (MDACC) performed ex vivo depletion of CD8+ lymphocytes, with the aim of reducing acute GVHD incidence. Results from small numbers of patients suggested that the GVT effect was not compromised, with an apparent reduction in acute GVHD [12,13]. Imatinib. Tyrosine kinase inhibitors such as imatinib, dasatinib, and nilotinib have dramatically altered the treatment of newly diagnosed CML patients. Until recently, DLI was the treatment of choice for CML patients with recurrent disease after allogeneic HCT; however, the introduction of the tyrosine kinase inhibitors is also changing this practice. The complications of DLI such as GVHD and pancytopenia, the lack of availability of the original donor especially for unrelated donors, and the presence of GVHD at the time of relapse are factors that deem tyrosine kinase inhibitors more appealing or perhaps even safer compared with traditional DLI. The EBMT has reported the largest series to date of 128 patients with CML who received imatinib in various phases of relapse ranging from patients in only a molecular relapse to patients with blast crises [14]. Of these 128 patients, 50 had failed treatment with DLI. After imatinib administration, the overall hematologic response rate was

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84% but was as high as 98% for the chronic-phase patients. As observed with DLI, the stage of disease at relapse was a strong predictor for outcome. The complete cytogenetic response was 58%, 48%, and 22% for patients in chronic phase, accelerated phase, and blast crisis respectively at the time of imatinib administration, with the estimated 2-year overall survival (OS) being 100%, 86%, and 12% respectively. Complete molecular responses were attained in 26% of patients, most of whom were in chronic phase. Only three of 30 patients with GVHD at the initiation of imatinib experienced exacerbation of this condition, with no new cases of GVHD reported. Investigators from the MDACC reported similar results in 28 CML patients who relapsed after allogeneic HCT [15]. The complete hematologic response rate was 74% for all patients and reached 100% for the chronic-phase patients. Similar to the above-mentioned EBMT report, cytogenetic responses correlated with disease phase. Complications included five recurrences of GVHD and reversible severe granulocytopenia and thrombocytopenia. The cytopenias resolved with imatinib dose adjustments. An Italian group detailed the treatment course of 16 CML patients also in various stages of relapse who received imatinib, with all patients except for one achieving cytogenetic and molecular responses [16]. All responses were associated with re-establishment of full donor chimerism without GVHD exacerbation. It is worth mentioning that one patient with blast crisis obtained a complete MR that was sustained for 45 months after combined therapy with imatinib and DLI. When imatinib is concomitantly administered with DLI, responses appear to be faster, and this dual strategy may be more effective for relapsed patients with advanced disease. A report from the National Institutes of Health compared the approaches of DLI alone, imatinib alone or imatinib with DLI in 37 patients with CML who had relapsed following allogeneic HCT [17]. Patients who received the combined regimen achieved a molecular response more rapidly than the other two groups. Also of note, the four patients with accelerated phase who received the combination therapy achieved an MR and remained disease free at a range of 249–1247 days. The proposed basis of synergy of DLI and imatinib is that imatinib may effectively reduce the disease burden to levels below molecular detection but cannot completely eliminate leukemic progenitors. Thus, DLI can eradicate residual disease via the immune-mediated GVT effect. Although the high response rates conferred by imatinib in this setting are encouraging, there is no clear evidence that imatinib is superior to DLI regarding long-term survival. A recent retrospective analysis from Germany compared the outcomes of 21 relapsed CML patients who received DLI and 10 patients who received imatinib [18]. Although this study was very small, the imatinib group had a higher incidence of relapse (60% versus 14%; p = 0.006) and inferior leukemia-free survival, although OS was comparable (100% versus 76%; p = 0.18). Three patients relapsed after discontinuation of imatinib, suggesting that imatinib cannot completely eradicate residual disease. Dasatinib is an oral, multitargeted, kinase inhibitor of bcr-abl and src kinases that is approximately 300 times more potent in vitro than imatinib, and has been shown to be safe and effective in CML patients resistant or intolerant to imatinib [19]. The efficacy of dasatinib was demonstrated in three large multicenter trials that accrued imatinibresistant or imatinib-intolerant CML patients in various disease phases including blast crises and in patients with Philadelphia-positive (Ph+) ALL [20–22]. Dasatinib conferred major hematologic and cytogenetic responses, with the highest responses seen in the chronic-phase CML patients. Myelosuppression and pleural effusions were the most common serious toxicities reported. Thus, longer follow-up is necessary to determine the durability of responses conferred by tyrosine kinase inhibitors for patients who relapse after allogeneic HCT. It should be emphasized,

Chapter 72

however, that most of the above-mentioned studies involved patients who were imatinib naïve or still imatinib sensitive prior to HCT. With the more widespread use of imatinib up front, the majority of patients with CML who now proceed to HCT will be imatinib resistant or intolerant, and thus imatinib may be less efficacious in that group. DLI for AML and myelodysplastic syndrome In contrast to the notable positive response rates for early-phase CML relapse, DLI is much less successful for patients with relapsed acute leukemia and myelodysplastic syndrome (MDS) (see Chapters 53 and 57). For patients with AML, the response rates range from 15% to 47%, but most of the responses are not durable (Fig. 72.2) [8,23,24]. The growth of leukemic blasts may simply outpace the GVT effect imparted by DLI. The EBMT recently analyzed the outcomes of 171 patients with AML who relapsed after allogeneic HCT [25]. Thirty-four percent obtained a CR, but most responses were not durable as the 2-year OS was 21% with a relapse mortality of 71%. Not surprisingly, factors found to predict for a more favorable outcome were disease status, low tumor burden, and favorable cytogenetics (Fig. 72.3). A report from the Johns Hopkins University of 83 patients included 13 patients with relapsed AML. With a median follow-up of around 4 years, the complete response after DLI for this subset of patients was 46%, but only 31% experienced a sustained remission [3]. A French group published their long-term outcomes of 30 patients with relapsed hematologic malignancies after allogeneic HCT, including nine patients with AML and one with MDS [26]. With a median follow-up of nearly 5 years, the 3-year OS was 80% for the CML group and only 48% for all the non-CML patients. The diagnosis of acute leukemia predicted for a worse survival in a multivariate analysis.

In a prospective German study, investigators administered imatinib pre-emptively to 27 patients with Ph+ ALL upon detection of molecular relapse after either allogeneic or autologous HCT [29]. Fifty-two percent of patients showed a complete molecular response at a median of 1.5 months. For patients who achieved an early molecular response, diseasefree survival (DFS) and OS at 1 year were 91% and 100% compared with only 8% and 23%, respectively, in the patients who retained detectable bcr-abl transcripts (p < 0.001). At a median follow-up of 8 months, the estimated probability of relapse was 52%. These results are considerably more encouraging than the results reported when imatinib is given at the time of hematologic relapse as detailed in a European multicenter

1.0

Patients receiving DLI Patients not receiving DLI

0.8 Probability of Survival

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0.6

0.4

0.2

0

1

DLI for ALL

2

3

Time After Relapse (years)

DLI has limited activity in ALL as long-term responses for relapsed ALL are uncommon. The European registry reported a 5-year survival of 0% for ALL patients after DLI, while the North American registry with 44 ALL patients reported a 13% OS (Fig. 72.4) [27,28]. For patients with Ph+ ALL, relapse after allogeneic HCT portends a particularly dismal prognosis (see Chapter 55). Imatinib may salvage a subset of these patients, especially when utilized at the time of molecular relapse.

Fig. 72.3 Unadjusted survival of patients with first hematologic relapse of acute myelogenous leukemia after allogeneic hematopoietic cell transplantation. Upper curve: patients receiving donor lymphocyte infusion (DLI) with an estimated 20-year overall survival (OS) from relapse of 21% (standard deviation ± 3%). Lower curve: patients not receiving DLI, estimated 2-year OS 9% (standard deviation ± 2%); p < 0.0001. (Reproduced from [25], with permission from the American Society of Clinical Oncology.)

1.0 1.0 0.8

0.8 Probability of Survival

Probability of Survival

Chronic phase (n=51)

ACC. phase (n=31) 0.6 0.4 Blastic phase (n=46)

0.2 P < 0.0001 0.0 0

3

6

0.6

0.4

0.2 9

12

15

18

21

24

27

30

33

36

Months Fig. 72.2 Kaplan–Meier survival curves for patients treated with imatinib for relapse of chronic myelogenous leukemia (CML) after allogeneic hematopoietic cell transplantation (HCT) according to disease phase. Survival is calculated from the start of treatment with imatinib. Acc. phase, accelerated phase. (Reproduced by permission from Macmillan Publishers Ltd: Leukemia 2003; 17: 1707–12 [14].)

0 0.0

0.5

1.0

1.5 2.0 Years post DLI

2.5

3.0

Fig. 72.4 Actuarial survivals with 95% confidence intervals in 44 patients with acute lymphoblastic leukemia treated with donor lymphocyte infusion (DLI). (Reproduced by permission from Macmillan Publishers Ltd: Bone Marrow Transplantation 2000; 26: 511–16 [28].)

Management of Relapse after Hematopoietic Cell Transplantation

report of 56 patients with relapsed or refractory Ph+ ALL or lymphoid blast crisis CML [30]. For the 48 patients with Ph+ ALL, imatinib induced a 29% hematologic response rate, but responses were shortlived, with a median time to progression and OS of 2.2 months and 4.9 months, respectively. Ten patients had failed a prior HCT. The discouraging lack of response of relapsed Ph+ ALL to salvage therapy has led to studies in which imatinib is provided either as scheduled maintenance therapy immediately after allogeneic HCT or as part of front-line therapy prior to HCT to eradicate minimal residual disease (MRD) as early as possible [31,32]. One such report included a phase I feasibility study in which 22 patients with either CML or Ph+ ALL were to receive imatinib for 1 year after allogeneic HCT. Eighteen patients tolerated full-dose imatinib for the planned year of administration, with reversible nausea and hepatotoxicity being the most common adverse events [31]. Longer follow-up is necessary to determine whether such early interventions will positively affect survival, but early results are encouraging. DLI for multiple myeloma DLI for relapsed multiple myeloma (MM) after allogeneic HCT has yielded response rates ranging from 30% to 50%, with the occurrence of GVHD consistently correlating with response (see also Chapter 58) [23,33–36]. However, despite these moderate response rates, long-term survival has been the exception rather than the rule. A European multicenter group reported their initial results in 2000 of one of the larger series consisting of 27 patients with MM who relapsed following highdose conditioning [37]. Approximately half of the patients received salvage therapy just prior to DLI. The overall response rate was 52% and the CR rate was 22%, with a median OS of 11 months after DLI. These results were recently updated with 54 patients [34]. The response rate remained at 52%, with progression-free survival (PFS) and OS being 19 and 23 months, respectively. Predictive factors for response to DLI were occurrence of acute and chronic GVHD, which suggested that mHAs expressed on both recipient normal and myeloma cells were the targets of the donor T cells known to confer alloreactivity. However, investigators from the Dana Farber group reported that the complete responses to DLI in MM patients correlated with sustained high-titer antibody responses to various highly expressed myeloma-associated antigens, which speaks for the role of humoral immunity and a coordinated response from B cells in mediating the graft-versus-myeloma effect [38]. A recent retrospective study described the effect of DLI for relapsed or persistent disease in patients with MM after reduced-intensity conditioning (RIC) [23]. The overall response rate was 38%, with a median OS of 24 months. When only the responding patients were separately analyzed, the median OS had not been reached at the time of publication. Once again, the occurrence of GVHD was the only significant factor associated with response, although GVHD was also the predominant cause of treatment-related mortality (TRM). With the goal of reducing the incidence and severity of GVHD in this setting, low-dose escalating DLI was administered to 21 myeloma patients with persistent or relapsed disease after reduced-intensity HCT [39]. The response rate was 38%,with the incidence of acute and chronic extensive GVHD being 14% and 5%, respectively, with one death directly related to GVHD. The feasibility of pre-emptive DLI after T-cell-depleted allogeneic HCT has also been studied, with promising results [40]. Novel agents such as thalidomide, lenolidomide, and bortezomib have recently been incorporated with DLI to enhance the antimyeloma effect and to possibly reduce GVHD owing to the potential immunomodulatory effects of these agents. Eighteen patients with relapsed or persistent MM after allogeneic HCT received low-dose thalidomide followed by DLI, resulting in a 67% response rate with a 22% CR rate [41]. The

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major adverse events were thalidomide-related such as weakness and neuropathy. Notably, no case of grade II–IV GVHD or extensive chronic GVHD was observed, and the 2-year PFS and OS were 84% and 100%, respectively. These outcomes compare very favorably with previously published reports of DLI alone, especially considering that 12 patients had failed DLI prior to thalidomide administration. Also of note, both thalidomide and bortezemib have induced responses as single agents in patients who failed DLI and conferred responses without increasing GVHD risk [42]. The role of DLI as a single agent after relapse in myeloma now becomes questionable, with the preliminary favorable observations regarding both efficacy and toxicity when novel immunomodulators are added to a DLI regimen. DLI for lymphoid malignancies There are a paucity of data regarding the effect of DLI in non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma (HL), and chronic lymphocytic leukemia, primarily because autologous HCT is offered more often than allogeneic HCT, especially for the lymphomas. However, small series and case reports have reported partial remissions (PRs) and CRs in these lymphoid malignancies when DLI is administered for relapse [43–46]. With the increasing number of RIC before allogeneic HCT offered to patients with lymphoid malignancies, the use of DLI will most likely also increase allowing for further assessment of efficacy. Second allogeneic HCT Second allogeneic HCT after an initial allogeneic HCT is not commonly offered owing to substantial nonrelapse mortality (NRM) in previous reports [47,48]. However, this option may be an appropriate salvage therapy in selected patients. All published reports detailing second allogeneic HCT are retrospective, with the three largest series comprising registry data from the EBMT, Center for International Blood and Marrow Transplant Research (CIBMTR), and SFGM (Société Française de Greffe de Moelle) [49–51]. The CIBMTR study described the outcomes of 279 patients with acute and chronic leukemia who underwent second allogeneic HCT after relapsing from a prior myeloablative allogeneic HCT [50]. The 5-year OS and leukemia-free survival were both 28%. The cumulative incidences of TRM and relapse at 5 years were 30% and 42%, respectively. Length of remission after the first HCT was the most relevant variable predicting outcome as relapse; treatment failure and mortality were higher in patients with early relapse (defined as length of remission less than 6 months after first HCT). The significant impact of remission length after the first HCT was also confirmed in the SFGM and EBMT reports. Interestingly, use of a matched related donor other than the original related donor did not improve risk of relapse. Other factors predicting for more favorable outcomes in the above-mentioned series were disease status at the time of second HCT, younger age, and presence of chronic GVHD. Additionally, relapse rates were higher after reduced-intensity regimens compared with myeloablative regimens in the CIBMTR report. Novel approaches for adoptive immunotherapy Allogeneic co-stimulated donor T cells Ex vivo activated T cells, or “activated DLI,” has been explored as a means of ameliorating the disease resistance that occurs despite the presence of GVT effects after conventional DLI. Investigators at the University of Pennsylvania conducted a phase I trial with “activated” DLI for patients with relapsed disease other than chronic-phase CML [52]. Donor T cells underwent ex vivo co-stimulation and expansion by exposure to magnetic beads coated with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs). It was hypothesized that these “activated”

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Chapter 72

T cells could overcome disease-induced anergy, augment CD4+ T-cell function, and enhance the GVT effect. Eighteen patients with relapsed disease after allogeneic HCT received DLI followed by escalated doses of ex vivo co-stimulated donor T cells. Eight patients achieved a CR, with four patients alive in continued remission with a median follow-up of 23 months, with an estimated 2-year OS of 51%. The actuarial probability of developing grade II–IV acute GVHD was 33%, with no deaths related to GVHD. Further studies will include higher dose escalation and repetitive dosing to minimize late recurrence. CIK cells CIK cells are cytotoxic effector cells expressing the T-cell marker, CD3+, and NK-cell markers, such as CD56+. CIK cells are generated by the in vitro culture of peripheral blood lymphocytes with IFN-γ, interleukin-2 (IL-2), and anti-CD3. T-cell expansion and activation occurs, resulting in cytolytic cells which recognize targets through NKG2D [53]. NKG2D is an activating receptor expressed on all NK cells (see Chapter 13), and also serves as a T-cell co-stimulatory molecule which augments the cytotoxic and proliferative responses of T cells upon encountering antigen [54]. CIK cell-mediated cytotoxicity is major histocompatibility antigen (MHC) unrestricted and T-cell receptor independent as target killing occurs through NKG2D-mediated recognition. In preclinical studies, CIK cells have shown potent activity against several tumor cell lines, with a markedly reduced capacity of inducing GVHD in murine models [53,55]. CIK cells generated from patients with AML have demonstrated cytotoxic activity against both autologous and allogeneic leukemic blasts [56]. Two studies have reported the feasibility of allogeneic CIK cells in patients relapsing after allogeneic HCT. In a phase I trial, investigators from Stanford University administered CIK cells generated from the matched sibling donors of 10 patients with relapsed acute leukemia, myeloma, and lymphoma who received cytoreductive chemotherapy prior to CIK infusion [57]. In vitro culturing yielded nearly a 100-fold expansion of the CD3+CD56+ population. The median EFS and OS were 3 months and 22 months, respectively, for this very high-risk patient cohort. One patient experienced grade I acute GVHD, and one patient developed limited chronic GVHD. An Italian group performed a similar study in 11 patients with relapsed disease after allogeneic HCT from related and unrelated donors [58]. Six patients had failed conventional DLI infusion prior to CIK cell infusions. As seen in the Stanford study, only mild acute GVHD was reported, with two patients developing extensive chronic GVHD. Four patients showed either a PR or CR, and six patients died from progressive disease. These results are encouraging considering that responses were seen in DLI-resistant patients. Clinical trials continue in both the autologous and allogeneic setting to determine the optimal dose of CIK cells to maximize efficacy with reduced GVHD incidence. Antigen-specific CTLs The detection, quantification, and purification of antigen-specific T cells has improved our ability to identify and track T-cell immune responses in vivo, thus facilitating the design of adoptive immunotherapeutic strategies (see Chapter 18). A number of potential target antigens have emerged which are expressed on hematologic malignancies. Proteinase1 (PR1) is a human leukocyte antigen (HLA)-A2 restricted, leukemiaassociated peptide derived from proteinase-3, an aberrantly expressed protein in myeloid leukemia cells [59]. PR1-specific CTLs have shown preferential killing towards allogeneic HLA-A2 myeloid leukemia cells compared with normal hematopoietic progenitor cells [60]. Early clinical results demonstrated a strong correlation between the presence of PR1-specific T cells and clinical responses after IFN-α and allogeneic HCT [61]. Additionally, PR1 peptide vaccination has been safely shown

to elicit both immunologic and clinical responses in patients with refractory and relapsed myeloid leukemias, including patients who failed either allogeneic or autologous HCT. Thirty-five patients with either AML, MDS or CML were vaccinated with the PR1 peptide [62]. Immune responses were measured by PR1/HLA-A2 tetramer staining and intracellular IFN-γ production by CTLs. Results showed clinical, cytogenetic, and molecular responses primarily in the AML and CML patients with superior PFS and OS among patients who developed an immune response compared with those who did not (6 months versus 2 months, p = 0.003; and 4 years versus 0 years, p < 0.0001, respectively). Another class of candidate antigens for immunotherapy of leukemia are mHAs, which are short peptides derived from endogenous proteins and presented by MHC molecules on recipient cells [63]. Several features of mHAs make them attractive target antigens for antitumor therapy such as recognition by donor T cells, derivation from proteins that differ between donor and recipient, high immunogenicity, and the high avidity possessed by mHA T-cell clones for their cognate antigen [64]. T-cell responses to mHAs are capable of inducing both GVHD and a GVT effect, but it is possible to identify and select mHAs with expression restricted to hematopoietic tissue, making them highly attractive targets. CD8+ CTL clones specific for mHAs presented by class I MHC molecules have been shown to lyse leukemic cells in vitro and inhibit the growth of clonogenic myeloid leukemic progenitors in culture [65– 68]. Two reports have detailed the adoptive transfer of T-cell clones specific for mHAs for patients with relapsed leukemia and myeloma after allogeneic HCT, with durable remissions seen [69,70]. The most frequent toxicities observed have been fever and chills. However, the broad application of this immunotherapeutic intervention has been limited by the few hematopoietic lineage-restricted mHAs that have been molecularly characterized thus far. The feasibility of generating and administering leukemia-reactive CTL lines for patients who relapsed after allogeneic HCT has recently been described [71]. In 16 of 27 donor–recipient pairs, a CTL line was produced using limiting dilution in which donor T cells were stimulated with HLA-identical leukemic antigen-presenting cells (APCs). In eight patients who relapsed, a CTL line was generated that showed cytotoxic activity against recipient leukemic cells in vitro. These eight patients received anywhere from one to multiple CTL lines, with one patient entering a CR with CTLs alone, and another CR being seen after combined CTL and DLI infusion. The logistically complex and laborintensive requirements of this intervention, however, limit the wider application of this technology. Memory CD4+ T cells Prevention of relapse through enhancement of GVT obviously represents a high priority in the field of allogeneic HCT. However, transferring allogeneic T-cell immunity without causing GVHD is a major challenge. Selective depletion of naïve CD4+ T cells with infusion of CD4+ memory T cells appears to be a possible method to circumvent GVHD and also to potentially enhance alloreactivity against tumor antigens. Peripheral T cells can be broadly divided into naïve T cells (CD44− CD62L+) that have never encountered antigen, and memory/activated T cells (CD44+CD62L−) that have previously been exposed to their corresponding antigens [72,73]. If a donor has never encountered the host alloantigens, GVHD-inducing host-reactive T cells should be segregated in the naïve T-cell compartment. Since the donor has not encountered host alloantigen, the population of memory T cells will not be host alloantigen specific and will therefore be incapable of causing GVHD. Naïve cells appear to be more potent inducers of GVHD because they express CD62L+ and CCR7, which promotes T-cell trafficking into secondary lymphoid tissues that encounter APCs presenting alloantigens. Several groups have now shown that infusion of CD62L− T cells, which

Management of Relapse after Hematopoietic Cell Transplantation

are devoid of naïve T cells and represent a subset of memory/activated T cells, does not induce GVHD in murine models [74–77]. Findings suggest that the inability of memory T cells from unprimed donors to induce GVHD may be a result of “abortive” alloresponses mediated by nonalloantigen-specific memory T cells. Regulatory T cells do not appear to modulate this effect as depletion of this subset of cells does not increase GVHD incidence. Also of note, when the memory cells were immunized against BCL1 (a leukemia/lymphoma cell), they retained the ability to proliferate and to protect the mouse against a challenge of tumor cells without causing GVHD in third-party recipients [74]. Important clinical implications of this novel technique include adoptive transfer of allogeneic memory antitumor immunity without causing GVHD. Clinical translation of this concept is ongoing. NK cells Human NK cells comprise about 10–15% of peripheral blood lymphocytes and express the phenotype CD3+CD56+. NK cell killing is MHC unrestricted, and NK cells kill tumor and virus-infected cells without previous stimulation or antigen recognition. Normal tissues are protected from NK-cell killing by recognition of their killer immunoglobulin-like receptors (KIRs) that interact with target cell MHC class I molecules. The KIRs recognize various self-MHC class I alleles, and engagement of these NK-cell receptors results in inhibition of NK-cell effector function (see Chapter 13). In the haploidentical HCT setting, NK cells facilitate both engraftment and GVT, and may temper GVHD by targeting hematopoietic cells of recipient origin [78]. Successful manipulation of alloreactive NK cells can potentially prevent leukemia or possibly treat relapse. Investigators from the University of Minnesota have performed adoptive transfer of NK cells in both the autologous and the allogeneic setting. In the autologous setting, NK cells were infused into patients with NHL and HL, with the goal of preventing relapse. These efforts were generally unsuccessful and were later attributed to inhibitory KIR receptors that recognized “self” MHC-expressing tumor cells and thus led to blocked lysis of tumor targets. In contrast, more optimistic results were reported using in vivoexpanded haploidentical, related-donor NK-cell infusions to treat 43 patients with melanoma, renal cell carcinoma, refractory HL, and refractory AML. Three different preparative chemotherapy regimens of varying intensity were administered in an effort to lymphodeplete the recipient. Interestingly, successful NK-cell expansion was seen only in the AML patients who received the fully lymphodepleting cyclophosphamide and fludarabine regimen. NK-cell infusions were administered after chemotherapy, followed by subcutaneous low-dose IL-2 administered over 2 weeks. This combination of fludarabine and cyclophosphamide was the only regimen that induced pancytopenia, and was associated with a surge of endogenous IL-15 after chemotherapy. IL-15 is required for NK-cell differentiation and for in vivo expansion and survival of NK cells. Five of the 19 patients with AML achieved CR, with this cohort of patients achieving the highest levels of circulating NK cells. Additionally, patients with a KIR ligand mismatch experienced significantly higher CR rates compared with KIR ligand-matched patients. The benefits of NK alloreactivity are under further investigation in HCT patients with infusion of NK cell products either prior to or during the early recovery phase, with other groups using NK-cell DLI after haploidentical HCT to consolidate engraftment and attempt to reduce disease recurrence.

Management of relapse after autologous HCT High-dose chemotherapy with autologous HCT is the standard of care for most patients with relapsed or refractory NHL, HL, MM, germ cell tumors, selected solid tumors, and selected acute leukemias. Relapse,

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however, remains the major cause of treatment failure and typically portends a poor prognosis. For patients with hematologic malignancies who eventually relapse, approximately 75% relapse within 1 year, and over 95% of patients who ultimately relapse will do so within 3 years. Since disease recurrence is an ominous clinical event, prevention of relapse should be the highest priority. Such pre-emptive strategies have included the use of novel conditioning agents such as radioimmunoconjugates, in vivo and ex vivo purging of the autograft, post-HCT maintenance therapy, and involved field radiation therapy. Immunologic interventions also being explored include the use of cytokines and cellular therapies. After relapse, options include treatment with agents such as chemotherapy, immunomodulators, and mAbs. In some cases, a second HCT is a feasible option, with allogeneic RIC transplantation being increasingly offered in this setting. High-dose conditioning followed by transplantation after failure of autologous HCT is challenging due to the prohibitive NRM, which ranges from 50% to 90% [79]. However, there are a few published studies that have reported the feasibility of a second high-dose HCT. For patients with relapsed MM after autologous HCT, lenalidomide has shown particularly encouraging results. The results of two large phase III multicenter trials, one conducted in North America and the other in Europe, demonstrated the efficacy of lenalidomide plus dexamethasone versus dexamethasone alone in patients with relapsed MM, including patients who relapsed after autologous HCT [80,81]. The patients who received lenalidomide showed a significantly higher response rate, time to progression, and OS. This section of the chapter will discuss the above-mentioned strategies for prevention and treatment of disease relapse. Second HCT after failure of autologous HCT Allogeneic high-dose HCT As previously mentioned, undergoing a second high-dose regimen is associated with a high NRM, although a few series have demonstrated the feasibility of this approach using either an allograft or autograft. A report from the Fred Hutchinson Cancer Research Center (FHCRC) detailed the results of 59 patients with various hematologic malignancies who underwent high-dose conditioning and allogeneic HCT after failure of autologous HCT [82]. The median age was 28 years, age ranging from 1 to 57 years. The 2-year DFS was 23% for all patients, with a dismal 0% DFS for the 18 lymphoma patients in the series. The NRM was 51% for all patients, but reached as high as 71% when the lymphoma patients in the group were analyzed separately. Age was a significant factor as the NRM was 12% versus 71% for the under-17-year-old group compared with the over-17-year-old group, respectively. A series from Spain reported the results of 14 patients who also received high-dose conditioning and allogeneic HCT as salvage therapy for patients who had suffered a relapse, and reported a TRM of 71% with only a 16% 1-year OS [83]. One of the largest series to date comprised 114 patients with lymphoma from the CIBMTR [84]. One hundred fourteen patients with either NHL or HL underwent high-dose conditioning and allogeneic HCT after failing a prior autologous HCT. The median age was 34 years. With a median follow-up of 43 months, the 5-year OS, PFS, and TRM were 24%, 5%, and 25%, respectively. CR at the time of allogeneic HCT and use of TBI in the NHL patients were associated with lower rates of disease progression and a higher incidence of OS. Recipients of HLAmatched siblings also had less TRM compared with recipients of unrelated donors (URD) and haploidentical donors. The reported TRM of 25% is much lower than comparable series, and was attributed to the fact that most patients underwent second HCT after 1990, and half of

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the patients underwent HCT between 1997 and 1999 when supportive care measures and donor selection strategies were much improved compared with earlier in the decade. Methodology may also result in a lower TRM as TRM was calculated using cumulative incidences versus Kaplan–Meier estimates. The EBMT performed a retrospective analysis of 164 patients with acute leukemia who received either a second autologous HCT (n = 74) or a high-dose allogeneic HCT (n = 90) after relapse from a previous autograft [85]. These results were compared with those of 2584 patients who received only palliative chemotherapy. The 2-year OS was 32%, 42%, and 11% for the allogeneic HCT, autologous HCT, and chemotherapy recipients respectively, with recurrent leukemia being the most common cause of death in all three groups. By multivariate analysis, factors associated with increased OS were age under 26 years old, CR at time of second HCT, and longer than a 5-month interval from first autologous HCT to relapse. Second autologous high-dose HCT The use of a second autologous HCT after a failed prior autologous transplant is limited to a handful of small series. The MDACC group summarized the results of 14 myeloma patients who underwent a second autologous HCT [86]. With a median follow-up of 18 months, the median OS and PFS were 29 months and 7 months, respectively, with an NRM of 14%. Although the low NRM was impressive for such a heavily pretreated group, most patients eventually relapsed. A French group performed second autologous HCT in 14 NHL patients who relapsed after a prior autologous transplant [87]. Results were moderately impressive with a 5-year OS and PFS of 27% and 36%, respectively, with a 22% incidence of TRM. Thus, these retrospective studies indicate that second autologous HCT occasionally enables long-term remissions but at the cost of higher NRM. The concept of a planned second autologous HCT in case of relapse after an initial autologous HCT has also been explored for patients with MM (see Chapter 58).

Reduced-intensity regimens followed by allogeneic HCT The prohibitive NRM associated with a second high-dose regimen has fueled the increasing utilization of allogeneic RIC HCT in this setting. The obvious advantages of this strategy include introducing a GVT effect and lessened up-front toxicity with reduced doses of chemotherapy and/or radiation. A Spanish prospective trial accrued 29 patients with relapsed HL after prior autologous HCT [88]. The conditioning regimen was fludarabine/melphalan, and the majority of patients received allografts from matched related donors. All patients except for one showed sustained engraftment. The 2-year OS and PFS were 52% and 34%, respectively, with a TRM of 24% at 1 year. For patients who had remained in CR for at least 1 year after the first autologous HCT, the OS and PFS increased to 75% and 70%, respectively. Interestingly, of the 11 patients who received DLI for persistent disease or progression, a 54% overall response rate was seen, which supports the existence of a graft-versus-Hodgkin’s effect. The FHCRC group reported the outcome of 31 patients with recurrent/refractory myeloma, including 14 patients who had failed prior autologous HCT [89]. These 14 patients received a conditioning regimen of fludarabine and low-dose TBI (200 cGy), and all received unrelated donor allografts. The 3-year OS and PFS were 42% and 17%, respectively for these 14 patients. The fludarabine/TBI was well tolerated, with an NRM of 21% for the entire group. The results are similar to a report from the City of Hope in which 28 patients with various recurrent lymphoid and myeloid malignancies after failed autologous HCT subsequently underwent RIC allogeneic HCT using either matched related donors or unrelated donor allografts [90]. With a median follow-up of 24 months, the TRM was 23%, which was similar to the FHCRC findings mentioned above. The 2-year OS and EFS were 56% and 41%, respectively, with a relapse rate of 42%. Not surprisingly, EFS was superior among patients who had chemosensitive disease at the time of second HCT. Table 72.2 summarizes the above results and also includes other similar series.

Table 72.2 Reduced-intensity allogeneic hematopoietic cell transplantation (HCT) after relapse from prior autologous HCT

Group/year

n

Disease

Italian multicenter 2007 [153] Spanish multicenter 2006 [88]

14

Hodgkin’s lymphoma Hodgkin’s lymphoma

29

Spanish multicenter 2002 [154] City of Hope 2003 [90]

46

Various

28

Various

United Kingdom 2002 [155]

38

Lymphoid

Chronic graft- Treatmentversus-host related disease mortality

Follow-up 21 months

Overall survival

63%* 0%† 23%

43%

61%

0%

MRD/URD

83%* 44%† 52%

10%

47%

25%

260 days

MRD

63%

57%

42%

30%

24%

450 days

MRD/URD

56%

41%

54%

67%

32%

24 months

MRD

53%

50%

21%‡

15%

20%

14 months

Conditioning regimen

Donor

Fludarabine based

MRD/URD

Fludarabine/ melphalan ± ATG Fludarabine based Fludarabine/ melphalan, TBI ± fludarabine Fludarabine/ melphalan/ alemtuzumab

Grade II–IV acute graftversus-host disease

Progressionfree/eventfree survival

ATG, antithymocyte globulin; MRD, matched related donor; TBI, total body irratiation; URD, unrelated donor. * Chemosensitive disease. † Chemoresistant disease. ‡ Grade I–II only.

Management of Relapse after Hematopoietic Cell Transplantation

Taken together, these data demonstrate the feasibility of RIC allogeneic HCT after a prior failed autologous HCT, and speaks for the efficacy of a GVT effect even in patients who progressed after high-dose chemotherapy. However, the widespread application of this salvage intervention is limited by the availability of a suitable donor and the chemosensitivity of the malignancy at the time of HCT. Patients who receive a RIC regimen ideally should have minimal disease bulk when proceeding to HCT, since this type of HCT relies more on the adoptive immunotherapeutic effect of the donor T cells rather than the cytoreduction of the conditioning regimen. Vaccination of donors to induce tumor-specific immunity Vaccination of donors pre HCT has been explored as a novel strategy to enhance the specific antitumor effect of the allograft without increasing the risk of GVHD. This approach involves vaccinating the donor with a defined, patient tumor-derived antigen prior to graft harvest to induce antigen-specific immunity, followed by transference of this immunity to the recipient after HCT. After demonstrating the feasibility of this approach in murine models, investigators sought to induce tumor-specific T-cell immunity in five sibling donor–recipient pairs by vaccinating donors with myeloma idiotype (Id) proteins conjugated to keyhole limpet hemocyanin (KLH), a highly immunogenic carrier protein [91]. The myeloma Id protein was isolated from recipient plasma prior to HCT. Recipients then received booster Id immunizations following allogeneic HCT. In three of the five donor–recipient pairs that were evaluable, Id and carrier-specific T-cell responses were detected in all three patients post HCT but not pre HCT, and persisted for 18 months. No adverse effects were seen, and all three patients converted from PR to CR following HCT. Following these results, extending this strategy to solid tumors or other hematologic malignancies with defined viable antigens such as PR-1 may also be feasible and aid in optimizing GVT effects. Strategies to prevent relapse after autologous HCT: enhanced preparative regimens Since the outcome for patients with relapsed disease after autologous HCT is poor, investigators have explored ways to augment the conditioning regimen for improved cytoreduction. Tandem transplant procedures such as two planned successive autologous HCTs or an autologous HCT followed by RIC allogeneic HCT have been explored. For patients with MM, the results of a large, randomized French study showed the superiority of tandem autologous HCT compared with single autologous HCT, and there is an ongoing study within the Southwest Oncology Group (SWOG) assessing the efficacy of tandem autologous HCT for patients with relapsed HL [92]. Current trials are assessing the efficacy of autologous HCT followed by RIC allogeneic HCT for various hematologic malignancies [93,94]. The premise of these studies is that the autologous HCT will provide up-front cytoreduction with high-dose chemotherapy, and that the allogeneic HCT will confer a GVT effect to eradicate MRD. Radioimmunoconjugates Radioimmunoconjugates are ideal agents to incorporate into the autologous HCT setting for patients with lymphoma due to the inherent radiosensitivity of lymphoid malignancies (see Chapter 24). Radioimmunoconjugates deliver locoregional radiation via radioisotopes with the targeting effect of mAbs while limiting exposure to uninvolved organs. Lymphoma cells within bulky nodal masses may be shielded from unconjugated antibody binding and thus escape killing. However, radioimmunotherapy can deliver penetrating ionizing radiation to these

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shielded tumor cells via a cross-fire effect. Myelosuppression is the major dose-limiting toxicity of radioimmunotherapy which can be reversed by autologous HCT, which makes this approach highly attractive in the HCT setting. Iodine-131 tositumomab (Bexxar) and yttrium90 ibritumomab tiuxetan (Zevalin) are two radioimmunoconjugates currently approved in the United States. Both are murine mAbs that bind to CD20. This novel approach was first reported by FHCRC investigators in a landmark phase I study in which 24 patients with lymphoma received 131 I-tositumomab as a single agent followed by autologous HCT. The median OS was 21 months, and the procedure was well tolerated. Subsequent trials from the FHCRC have combined 131I-tositumomab with high-dose chemotherapy for patients with mantle cell NHL, those with follicular NHL, and elderly patients with the goal of eliminating TBI in the conditioning regimen [95–97]. Three-year OS and PFS have ranged from 67% to 93% and 48% to 61%, respectively, with TRM being remarkably low, ranging from 0% to 3.7%. The City of Hope National Medical Center group summarized their results combining Y90 ibritumomab tiuxetan with high-dose etoposide and cyclophosphamide in 31 patients with poor-risk or relapsed NHL [98]. With 22 months of followup, the OS and PFS were an impressive 92% and 78%, respectively with a 3% TRM, which is comparable to the TRM seen after conventional autologous HCT. Also worth noting, two small series have reported the feasibility and efficacy of administering radioimmunoconjugates as salvage therapy in patients with relapsed NHL after failure of autologous HCT [99,100]. Clinical trials incorporating radioimmunoconjugates in the autologous HCT setting continue, but widespread use is limited by the high cost of these chimeric antibodies and the complex logistics required to administer these radioactive agents. Involved field radiotherapy after autologous HCT The rationale for the addition of involved field radiation therapy (IFRT) in the HCT setting stems from the observations that patients frequently fail in sites of previous bulk disease. IFRT has unequivocally demonstrated a profound effect on local control in numerous published series, with the sites at greatest risk of failure being those failing to achieve a CR to salvage therapy prior to HCT [101]. Little consensus exists regarding the optimal timing of IFRT in relation to HCT. Advantages of IFRT pre HCT include the debulking effect prior to HCT and the ability to deliver IFRT fairly quickly without waiting for engraftment. Disadvantages include the possibility of increasing peritransplant morbidity by increasing the risk of pneumonitis, radiation recall, etc. Delaying IFRT until after HCT may allow a smaller, tailored radiation field, thus limiting toxicity to surrounding normal tissues and facilitating the conversion of patients with persistent disease to CR [102]. The survival benefit of IFRT after HCT, however, has been equivocal despite the clear benefit of local control. A small series from the University of Chicago found that adjuvant IFRT after autologous HCT improved the 4-year local control at all irradiated sites, persistent sites following induction prior to HCT, and persistent sites after transplantation [101]. The sample size, however, was too small to assess the impact of IFRT on OS. The University of Utah group retrospectively compared the records of 53 patients with relapsed/refractory NHL who received IFRT with 144 patients who did not receive IFRT in the peritransplant period [103]. After a median follow-up of over 4 years for both groups, no differences were found in OS or PFS. However, the IFRT group had more adverse clinical features such as bulky or extranodal disease, which lends support that perhaps IFRT was beneficial since the patients in the high-risk IFRT group would have been predicted to fare worse. A Stanford University series of 24 patients with HL indicated that either cytoreductive or consolidative IFRT relative to autologous HCT

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improved 3-year freedom from relapse with a trend towards improved OS compared with non-IFRT patients [104]. The number of serious adverse events was similar in both groups. Another report compared the outcomes of 41 patients with relapsed/refractory HL and NHL who received IFRT in the peritransplant period to 265 HCT patients who did not undergo IFRT [105]. By multivariate analysis, the non-IFRT patients were over twice as likely to die of disease progression compared with the patients treated with IFRT (p = 0.06). Timing of IFRT delivery either prior to or following autologous HCT did not affect OS. Therefore, despite the lack of randomized trials, IFRT is routinely recommended due to its benefit in achieving local control and its ability to convert patients with persistent disease to CR after HCT. Graft purging Relapse after autologous HCT has been attributed to both incomplete eradication of the underlying disease by the preparative regimen and/or contamination of the graft by clonogenic tumor cells. Such concerns have led to the development of various techniques to purge tumor cells from the graft, including in vitro methods such as complement-mediated purging and immunomagnetic purging (see Chapter 42). In vivo purging entails removal of circulating malignant cells before hematopoietic cell harvest either with multiple cycles of chemotherapy or with mAbs. One of the larger single-center studies exploring the utility of purging comes from the DFCI group, in which 113 patients with relapsed follicular NHL received bone marrow grafts purged ex vivo with mAbs. With a median follow-up of 61 months, patients whose marrow autograft was polymerase chain reaction (PCR) negative after purging experienced longer freedom from recurrence than those whose marrow remained PCR positive (p < 0.0001) [106]. Registry data from the CIBMTR analyzed the outcomes of 597 patients with follicular NHL who received purged autografts (n = 131) and found that these patients had a 26% lower chance (p = 0.04) of relapsing and 33% less risk of death (p = 0.03) compared with recipients of unpurged autografts [107]. More recently, rituximab has been increasingly utilized for the purpose of in vivo purging. Several groups have reported the ability of rituximab to yield PCR-negative grafts in patients with mantle cell and follicular NHL without compromising engraftment [108–110]. However, there is no definitive evidence that B-cell purging actually improves OS in NHL patients, although PFS seems to be consistently superior in patients who receive PCR-negative grafts. With regards to myeloma, purging is less efficacious, as three multicenter randomized trials failed to show benefit in patients who received CD34+-selected autografts compared with patients who received unselected grafts [111–113]. The role of purging in acute leukemia is unsettled, with most centers administering unpurged grafts. A more complete review regarding the role of purging is discussed in Chapter 42 of this book. Post-HCT maintenance therapy Post-HCT maintenance therapy represents an attractive method to reduce relapse through elimination of persistent MRD following high-dose chemotherapy, and to convert partial clinical responses to full complete responses. For patients with B-cell lymphomas, rituximab has been increasingly utilized for this purpose, especially in patients with mantle cell and follicular NHL. The Stanford University group administered weekly infusions of rituximab post HCT to 35 NHL patients with relapsed and refractory disease and to patients in first CR1 [114]. The 2-year EFS and OS were 83% and 88% for all patients, and 85% and 81% for the patients with relapsed/refractory diffuse large cell lymphoma. These data compared quite favorably with a comparable historical cohort at Stanford who showed a 2-year EFS and OS of 58% and 62%, respectively. Grade 3–4 neutropenia occurred in 54% of patients, but no serious infections were observed.

Similar to the Stanford schedule of administration, Brugger et al. infused rituximab weekly for four doses, with the first dose given 3 weeks post HCT [115]. Leukopenia was reported in only 3.6% of patients, and, impressively, 100% of the 31 patients with mantle cell and follicular NHL were PCR negative after rituximab, whereas only 22% of the patients were PCR-negative prior to commencing the HCT process. Utilizing a long-term maintenance schedule of monthly rituximab infusions post HCT, another study from Germany of 27 NHL patients reported neutropenia in 19% resulting in two serious infections [116]. Taken together, all of the above data show that rituximab is effective in eradicating MRD, but adverse events such as leukopenia must be closely monitored. For patients with MM, there are several published reports investigating the use of thalidomide and IFN as maintenance therapy following autologous HCT. Registry data from the EBMT demonstrated an improved OS and PFS in 473 autologous HCT patients who received maintenance IFN-α compared with a similar cohort who did not take IFN-α [117]. However, data were not provided regarding patient tolerability. The use of IFN-α in a randomized study was shown to exert antimyeloma activity but was poorly tolerated by patients and without a significant impact in OS [118]. Thalidomide has also been the subject of more recent reports. In a prospective phase II trial, the City of Hope National Medical Center group prescribed thalidomide to 29 patients after a single autologous HCT, with a target dose of 400 mg/day [119]. After a median follow-up of 28 months, the median tolerated dose was 200 mg/day, with a 2-year OS and PFS of 83% and 49%, respectively. Neurotoxicity and fatigue were the most common reported adverse events. In another phase II trial, thalidomide was given to 17 patients after tandem autologous HCT, but nearly 80% of patients failed to tolerate the drug due to side-effects [120]. Thalidomide exhibited antitumor activity as the conversion rate from PR to CR or near-CR was 47% to 77%. The most convincing evidence demonstrating the value of post-HCT maintenance therapy in MM comes from a large randomized trial in which patients were randomized to undergo tandem autologous HCT versus single autologous HCT followed by thalidomide 100 mg daily as maintenance therapy [121]. The thalidomide patients experienced a significantly superior OS and EFS compared with the control group (Fig. 72.5). The lower target dose was well tolerated as only 9% of patients discontinued thalidomide due to neurotoxicity. In summary, α-IFN and thalidomide both demonstrate clinical activity post HCT, but the toxicities associated with their use impede long-term administration. Lenalidomide, an immunodulator that appears to be better tolerated, is currently the subject of active investigation in the post-HCT setting. Post-transplantation cytokine administration The administration of cytokines post HCT initially appeared to be a highly promising strategy to deter relapse due to their powerful ability to stimulate and expand immune effector cells. However, results from clinical trials to date have been inconsistent and somewhat disappointing. IL-2, in particular, has been extensively evaluated in the post-HCT setting due to its broad antitumor cytolytic activity in both animal and in vitro models. Recombinant IL-2 has been shown to stimulate both NK expansion and cytotoxic activity against breast and lymphoma tumor targets in vivo and in vitro, and also to potentiate a graft-versusmalignancy effect by lysis of malignant human hematopoietic cells via activated NK cells [122,123]. A phase II study from the City of Hope National Medical Center administered IL-2 after autologous HCT upon hematologic recovery to 39 patients with AML in CR1 [124]. The 2-year DFS was 79% for all 39 patients, with the most frequently seen toxicities being hematologic along with fever and fluid retention. These results

Management of Relapse after Hematopoietic Cell Transplantation

B

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These interventions, however, require that the desired cell population will persist, expand, traffic to the tumor sites, and mediate the desired effector function. Fortunately, the available expertise for cell manipulation has been rapidly growing within the HCT field, with several innovative strategies currently under development. Some of these immunotherapeutic interventions have already been described above, but the following text will further elucidate their role in the autologous HCT setting.

A

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0

10

20 30 Months from randomization

40

50

Fig. 72.5 Overall survival according to treatment arm. The solid line (arm A) denotes single autologous hematopoietic cell transplantation (HCT) followed by maintenance thalidomide. The dashed line (arm B) denotes second autologous HCT in case of relalpse/progression. The probability (95% CI) of overall survival for arm A versus arm B is 65% (49–80%) versus 85% (76–95%), respectively; p = 0.04. (This research was originally published in Blood. Abdelkefi A, et al. Blood 2008; 111: 1805–10 © the American Society of Hematology [121].)

were quite favorable considering that even patients with poor-risk cytogenetics fared reasonably well. Results appeared less promising for ALL patients as the addition of IL-2 after autologous HCT did not improve remission rates compared with a control group who did not receive adjuvant IL-2 [125]. IL-2 has also been administered to patients with NHL and breast cancer after autologous HCT [126]. In a two-part trial, investigators from the University of Minnesota concomitantly administered either ex vivo IL-2-activated NK cells (n = 34) or IL-2 boluses (n = 23) to patients receiving planned daily IL-2 subcutaneously. Lytic function of peripheral blood mononuclear cells obtained on day 1 post infusion from both treatment groups was found to be markedly enhanced. The patients who received the IL-2 boluses also showed transient increases in levels of IL-6, IFN-γ, tumor necrosis factor-alpha, and IL1-β. However, despite these observations of increased lytic activity and cytokine production, relapse and survival rates were not improved when outcomes were compared with case-matched controls. A recently completed large phase III trial from SWOG also failed to show efficacy of IL-2 in the post-HCT setting [127]. Three hundred ninety-four patients with relapsed B-cell NHL were to be randomized to IL-2 or no IL-2 post autologous HCT, but only 204 patients actually proceeded to randomization. The 4-year relapse and survival incidences did not significantly differ between the treatment and control groups. In summary, published studies to date have shown discouraging results with IL-2 despite the promising results seen in preclinical studies. Ongoing studies in the HCT setting of other cytokines such as IL-7, IL-15, and keratinocyte growth factor appear encouraging but show more utility in improving immune reconstitution rather than deterring relapse [128]. Cellular therapy following autologous HCT The expansion and activation of effector cell populations is an attractive approach when patients are in an MRD state after autologous HCT.

CIK cells. As mentioned earlier in this chapter, CIK cells are efficient effector cells that mediate cytotoxicity in a non-MHC-restricted fashion. CIK cells produce several T helper cell type 1 (Th1)-type cytokines, such as IFN, tumor necrosis factor-alpha, and granulocyte–macrophage colony-stimulating factor, and upregulate key molecules required for effector function, such as perforin, granzymes, NKG2D, and Fas ligand. CIK cells have been expanded from granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells of heavily pretreated patients with various malignancies undergoing apheresis prior to autologous HCT, with the expanded cells exerting cytolytic activity against autologous tumor cells [129]. This observation was followed by a phase I clinical trial including patients with NHL and HL who had relapsed after autologous HCT and received autologous ex vivo-expanded CIK cells in a dose-escalation format starting at a cell dose of 1 × 109 with the maximum dose of 1 × 1010 cells [130]. The planned cell dose was attained in 19 of 21 planned infusions, and toxicity was minimal. Two patients of the six patients treated attained partial responses and two patients showed stable disease, but all responses were transient. A similar strategy was employed in a trial with hepatoma patients who underwent surgical resection followed by randomization to receive or not receive autologous expanded cells [131]. The patients who received the cellular therapy showed a superior relapse-free survival compared with the untreated controls. A recent study in the pediatric setting explored the efficacy of autologous CIK cells against autologous Epstein–Barr virus-transformed lymphoblastoid cell line (EBV-LCL) cells generated from six pediatric patients who underwent allogeneic HCT [132]. The expanded cells showed significant in vitro cytotoxicity against the EBV-LCLs without significant toxicity. NK cells. Although early results utilizing the role of NK cells in the allogeneic HCT setting have been encouraging, the results in autologous HCT patients have been disappointing. Some of the earlier adoptive immunotherapy trials tested autologous lymphokine-activated killer cells by stimulating peripheral blood mononuclear cells ex vivo with IL-2. The stimulated cells were then reinfused with high-dose IL-2 in patients with melanoma, lymphoma, and renal cell cancer, but little clinical benefit was observed [133]. The University of Minnesota group employed a similar approach with low-dose subcutaneous IL-2 to circumvent the severe toxicities associated with high-dose IL-2 in patients with lymphoma and breast cancer who received autologous NK cells as adjuvant therapy after autologous HCT [126]. The infused cells led to increased serum cytokine levels and generated peripheral blood mononuclear cells with enhanced cytotoxicity against NK-resistant targets, but failed to improve clinical outcomes. Several recent observations may help explain the failure of autologous NK-cell-based therapies, including the expresssion of inhibitory KIRs recognizing self-MHC-expressing tumor cells which block NK lysis of tumor targets [134,135]. Therefore, further testing with NK cells is ongoing primarily in the allogeneic HCT setting. Cytotoxic T lymphocytes. Adoptive immunotherapy using antigenspecific T cells is based on the premise that tumors are immunogenic yet employ various means to evade immune-mediated elimination. Thus,

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it is assumed that the transferred T cells have the capability to recognize tumor cells and elicit an immune response causing specific tumor cell killing. The main disadvantage of antigen-specific approaches is the required identification of tumor-associated antigens, which is difficult for most tumors and thus has limited the widespread applicability of this strategy. However, several groups have successfully identified and expanded CTL populations capable of recognizing and lysing virusinfected cells and tumor cell targets. The use of virus-specific CTLs was first described by the FHCRC group, who prophylactically administered cytomegalovirus-specific CD8+ T cell clones to allogeneic HCT patients to reconstitute cytomegalovirus-specific immunity [136].The Baylor University Medical Center group has administered autologous EBV-specific CTLs to treat or prevent EBV-associated lymphoproliferative disorders. Clinical responses have been observed with regression of tumors in the treatment setting and a noted reduction in EBV-associated lymphoproliferative disorders when CTLs were administered as prophylaxis [137,138]. As opposed to EBV+ lymphoproliferative disorders, which arise in patients with T-cell dysfunction, EBV+ HL occurs in immunocompetent hosts. EBV-encoded RNA is detected in the Reed–Sternberg cells in 40% of patients with HL and evades host immune surveillance through multiple mechanisms including downregulation of immunodominant EBV nuclear antigens. However, the tumor cells express subdominant viral latent membrane proteins such as LMP1 and LMP2, with the latter protein being a more ideal target due to more consistent expression by Reed–Sternberg cells. LMP2-specific CTLs have been administered to relapsed HL patients after both conventional therapy and failed prior autologous or allogeneic HCT, resulting in observed durable clinical responses [139]. The number of tumor-reactive T cells within the EBVspecific T-cell lines was increased by using dendritic cells (DC) and LCLs transduced with an LMP2-expressing adenovirus vector as APCs. This pulsing of DCs with tumor-specific antigens such as peptides, proteins, RNA or whole tumor lysates is the most widely used method for the generation of CTLs. DCs are potent, effective APCs that can be directly isolated from the blood and expanded from mononuclear or CD34+ cells by the addition of various cytokines such as IL-4 and granulocyte–macrophage colony-stimulating factor. Much interest has been generated in the adoptive transfer of DC populations that are primed with specific antigens in vitro [140]. Whole tumor cells have been used for this purpose since the identification of the immunogenic peptide need not be known, but this approach has been restricted due to the limited availability of tumor tissue and the secretion of immunosuppressive cytokines by the tumor cells. Transforming growth factor-beta (TGF-β) has been identified as the most potent and widely employed immunosuppressive cytokine that exerts devastating effects on CTL proliferation and function. Strategies under evaluation to overcome this cytokine include generation of CD4+ and CD8+ T cells expressing dominant negative TGF-β receptor type II, which renders these effector cells insensitive to TGF-β suppression and allows the T cells to maintain proliferation and antigen-specific cytolysis [141]. The use of lymphodepleting agents such as CD45 mAbs is under study to deplete TGF-βsecreting regulatory T cells prior to cell-based therapies [142]. The lack of appropriate co-stimulatory molecules on tumor cells also facilitates the impaired recognition by the immune system. Two phase I trials attempted to address this issue in the autologous HCT setting by ex vivo expansion through polyclonal activation of peripheral blood progenitor cells with a combination of anti-CD3 and anti-CD28 mAbs. The aims of these clinical trials were to augment or enhance T-cell function against MRD after autologous HCT. One trial involved infusion of the expanded co-stimulated T cells to patients with refractory/relapsed NHL after receiving a CD34+-selected autograft [143]. Infusion of the

autologous expanded T cells resulted in rapid reconstitution of lymphocyte numbers with the expanded cells found to be functionally superior to those directly obtained from the patients as demonstrated by their ability to secrete IFN-γ when stimulated by tumor cells in vitro. This approach was subsequently combined with a heptavalent pneumococcal conjugative vaccine in myeloma patients undergoing autologous HCT [144]. A four-way randomization assigned patients to receive T-cell infusions either 14 days or 100 days after HCT, and two doses of pneumococcal vaccine starting 1 month after HCT. Patients who received vaccine-primed T cells early after HCT showed faster T-cell recovery with better antipneumococcal humoral responses and CD4+ proliferative responses than the other treatment groups. Thus, these results demonstrated the applicability of combining vaccination and adoptive T-cell transfer to ameliorate post-transplant lymphopenia and to circumvent immune evasion mechanisms. Id protein is a tumor-specific antigen found in patients with B-cell malignancies such as NHL and MM. In MM patients, the Id protein can be readily isolated from the peripheral blood, thus making it a particularly attractive target antigen. After Id pulsing of DCs in myeloma patients, CTLs have been generated capable of lysing the pulsed DCs and autologous MM targets [145]. In another study, 26 patients with MM underwent autologous HCT followed by two infusions of Id-pulsed DCs and subcutaneous boosts of Id coupled to KLH [146]. Twenty-four of the 26 patients generated a KLH-specific immune response, but only four patients developed an Id-specific immune response. A similar strategy was administered to 12 NHL patients after autologous HCT [147]. Id was isolated from the patient’s tumor tissue, coupled to KLH, and then pulsed with autologous DCs followed by reinfusion into the patients 2–12 months after HCT. Notably, 10 of the 12 patients developed either a humoral or cellular anti-Id response, with seven patients experiencing prolonged remissions. In summary, all of the above results confirm that T-cell adoptive therapy can induce antitumor responses in humans and restore viral immunity. Optimization of these responses and demonstration of clinical benefit are the major goals of future studies. Genetic modification to redirect T-cell specificity. The weak antigenicity of tumor cells represents a principal limitation of adoptive T-cell therapy. Genetic modification of T cells to engineer improved antitumor activity and enhance immune reconstitution represents a novel, potential solution. Earlier trials in patients with congenital and acquired immunodeficiencies have shown that the genetically modified T cells can persist for years in humans after adoptive transfer [148]. One strategy includes the grafting of T cells with novel receptors via introduction of chimeric antigen receptors (CARs) also known as “T bodies.” CARs are chimeric immunoreceptors containing antibodybased external receptor structures and cytosolic domains encoding signal transduction of the T-cell receptor. These constructs facilitate “redirecting” of T cells in vitro in an MHC-unrestricted manner to attack the tumor while retaining MHC-restricted specificity for the endogenous T-cell receptor. In first-generation trials, investigators from the City of Hope National Medical Center generated CD8+ CTLs specific for tumor cells that expressed the B-cell lineage markers CD19 and CD20 [149]. Generation of the cellular product involved the genetic modification of CTLs to express a chimeric immunoreceptor composed of CD19or CD20-specific single-chain immunoglobulin extracellular targeting domain molecularly fused to the T-cell receptor’s complex zeta chain cytoplasmic tail. Preclinical observations with the redirected CD20specific CTLs demonstrated the ability of these clones to proliferate in the presence of lymphoma stimulators and to eliminate tumors in murine models [149]. The CD19-specific CTLs (CD19R+) displayed potent CD19-specific lytic activity including the ability to lyse B-cell ALL blasts [150]. However, most tumors do not express co-stimulatory

Management of Relapse after Hematopoietic Cell Transplantation

molecules necessary for optimal T-cell activation. Thus, second-generation T cells expressing CARs have incorporated signaling domains from a co-stimulatory molecule such as CD28, resulting in co-stimulationindependent cytokine secretion and proliferation in response to tumor stimulation [151]. More recently, an immunocytokine was created by fusing IL-2 to a CD20-specific mAb with the goal of targeted delivery of an immunostimulatory cytokine such as IL-2 to the tumor microenviroment [152]. In summary, all of the above results confirm the capability of T-cell adoptive therapy to induce antitumor responses in some patients and restore viral immunity. However, the potential pitfalls of human adoptive immunotherapy trials include the facts that (1) lack of CD4+ T-cell help or cytokine dependence, (2) the ex vivo expansion process may render the cells immunogenic to the host, thus triggering rejection or apoptosis, and (3) ex vivo-expanded human T cells may have reached replicative senescence. Additionally, practical limitations include the expense, labor intensity, the requirements for Good Manufacturing Practices-certified cell processing facilities, the scant availability of clinical grade reagents, and the individual patient specificity of cellular products.

Conclusion Disease recurrence is arguably the most feared clinical outcome following HCT. Multiple strategies have been developed or are under investigation which could impact relapse rates. Following HCT is an ideal time

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Table 72.3 Adoptive immunotherapy for malignant disease Following relapse after allogeneic HCT Donor leukocyte infusions Reduced-intensity allogeneic HCT Ex vivo activated cellular therapy Natural killer cells Cytokine-induced killer cells Antigen-specific cytotoxic T lymphocytes Genetically modified T cells Idiotype vaccinations Co-stimulated CD3+/CD28+ T cells Memory CD4+ T cells Tumor-infiltrating lymphocytes HCT, hematopoietic cell transplantation.

in which to explore these approaches since patients are commonly rendered to a state of MRD, where interventions may be more effective (Table 72.3). Prevention of disease recurrence in high-risk patients holds the most promise by defining those patients with greatest risk through the use of clinical, genetic or molecular strategies. In the future, careful prospective clinical trials using the most promising approaches discussed in this chapter will hopefully help reduce the risk of disease relapse.

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relapsed or refractory B-cell lymphoma. J Clin Oncol 2007; 25: 1396–402. Gopal AK, Rajendran JG, Petersdorf SH et al. High-dose chemo-radioimmunotherapy with autologous stem cell support for relapsed mantle cell lymphoma. Blood 2002; 99: 3158–62. Nademanee A, Forman S, Molina A et al. A phase 1/2 trial of high-dose yttrium-90-ibritumomab tiuxetan in combination with high-dose etoposide and cyclophosphamide followed by autologous stem cell transplantation in patients with poor-risk or relapsed non-Hodgkin lymphoma. Blood 2005; 106: 2896–902. Vose JM, Bierman PJ, Loberiza FR Jr., Bociek RG, Matso D, Armitage JO. Phase I trial of (90)Yibritumomab tiuxetan in patients with relapsed B-cell non-Hodgkin’s lymphoma following highdose chemotherapy and autologous stem cell transplantation. Leuk Lymphoma 2007; 48: 683– 90. Jacobs SA, Vidnovic N, Joyce J, McCook B, Torok F, Avril N. Full-dose 90Y ibritumomab tiuxetan therapy is safe in patients with prior myeloablative chemotherapy. Clin Cancer Res 2005; 11: 7146s–50s. Mundt AJ, Williams SF, Hallahan D. High dose chemotherapy and stem cell rescue for aggressive non-Hodgkin’s lymphoma: pattern of failure and implications for involved-field radiotherapy. Int J Radiat Oncol Biol Phys 1997; 39: 617– 25. Mundt AJ, Sibley G, Williams S, Hallahan D, Nautiyal J, Weichselbaum RR. Patterns of failure following high-dose chemotherapy and autologous bone marrow transplantation with involved field radiotherapy for relapsed/refractory Hodgkin’s disease. Int J Radiat Oncol Biol Phys 1995; 33: 261–70. Wendland MM, Smith DC, Boucher KM et al. The impact of involved field radiation therapy in the treatment of relapsed or refractory nonHodgkin lymphoma with high-dose chemotherapy followed by hematopoietic progenitor cell transplant. Am J Clin Oncol 2007; 30: 156–62. Poen JC, Hoppe RT, Horning SJ. High-dose therapy and autologous bone marrow transplantation for relapsed/refractory Hodgkin’s disease: the impact of involved field radiotherapy on patterns of failure and survival. Int J Radiat Oncol Biol Phys 1996; 36: 3–12. Kahn ST, Flowers CR, Lechowicz MJ, Hollenbach K, Johnstone PA. Refractory or relapsed Hodgkin’s disease and non-Hodgkin’s lymphoma: optimizing involved-field radiotherapy in transplant patients. Cancer J 2005; 11: 425–31. Freedman AS, Neuberg D, Mauch P et al. Longterm follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 1999; 94: 3325–33. van Besien K, Loberiza FR Jr., Bajorunaite R et al. Comparison of autologous and allogeneic hematopoietic stem cell transplantation for follicular lymphoma. Blood 2003; 102: 3521–9. Galimberti S, Guerrini F, Morabito F et al. Quantitative molecular evaluation in autotransplant programs for follicular lymphoma: efficacy of in vivo purging by rituximab. Bone Marrow Transplant 2003; 32: 57–63. Gianni AM, Magni M, Martelli M et al. Longterm remission in mantle cell lymphoma following high-dose sequential chemotherapy and in

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vivo rituximab-purged stem cell autografting (RHDS regimen). Blood 2003; 102: 749–55. Lazzarino M, Arcaini L, Bernasconi P et al. A sequence of immuno-chemotherapy with rituximab, mobilization of in vivo purged stem cells, high-dose chemotherapy and autotransplant is an effective and non-toxic treatment for advanced follicular and mantle cell lymphoma. Br J Haematol 2002; 116: 229–35. Bourhis JH, Bouko Y, Koscielny S et al. Relapse risk after autologous transplantation in patients with newly diagnosed myeloma is not related with infused tumor cell load and the outcome is not improved by CD34+ cell selection: long term follow-up of an EBMT phase III randomized study. Haematologica 2007; 92: 1083–90. Stewart AK, Vescio R, Schiller G et al. Purging of autologous peripheral-blood stem cells using CD34 selection does not improve overall or progression-free survival after high-dose chemotherapy for multiple myeloma: results of a multicenter randomized controlled trial. J Clin Oncol 2001; 19: 3771–9. Lemoli RM, Martinelli G, Zamagni E et al. Engraftment, clinical, and molecular follow-up of patients with multiple myeloma who were reinfused with highly purified CD34+ cells to support single or tandem high-dose chemotherapy. Blood 2000; 95: 2234–9. Horwitz SM, Negrin RS, Blume KG et al. Rituximab as adjuvant to high-dose therapy and autologous hematopoietic cell transplantation for aggressive non-Hodgkin lymphoma. Blood 2004; 103: 777–83. Brugger W, Hirsch J, Grunebach F et al. Rituximab consolidation after high-dose chemotherapy and autologous blood stem cell transplantation in follicular and mantle cell lymphoma: a prospective, multicenter phase II study. Ann Oncol 2004; 15: 1691–8. Neumann F, Harmsen S, Martin S et al. Rituximab long-term maintenance therapy after autologous stem cell transplantation in patients with B-cell non-Hodgkin’s lymphoma. Ann Hematol 2006; 85: 530–4. Bjorkstrand B, Svensson H, Goldschmidt H et al. Alpha-interferon maintenance treatment is associated with improved survival after high-dose treatment and autologous stem cell transplantation in patients with multiple myeloma: a retrospective registry study from the European Group for Blood and Marrow Transplantation (EBMT). Bone Marrow Transplant 2001; 27: 511–15. Cunningham D, Powles R, Malpas J et al. A randomized trial of maintenance interferon following high-dose chemotherapy in multiple myeloma: long-term follow-up results. Br J Haematol 1998; 102: 495–502. Sahebi F, Spielberger R, Kogut NM et al. Maintenance thalidomide following single cycle autologous peripheral blood stem cell transplant in patients with multiple myeloma. Bone Marrow Transplant 2006; 37: 825–9. Martino M, Console G, Callea V et al. Low tolerance and high toxicity of thalidomide as maintenance therapy after double autologous stem cell transplant in multiple myeloma patients. Eur J Haematol 2007; 78: 35–40. Abdelkefi A, Ladeb S, Torjman L et al. Single autologous stem cell transplantation followed by maintenance therapy with thalidomide is superior

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to double autologous transplantation in multiple myeloma: results of a multicenter randomized clinical trial. Blood 2008; 111: 1805–10. Miller JS, Tessmer-Tuck J, Pierson BA et al. Low dose subcutaneous interleukin-2 after autologous transplantation generates sustained in vivo natural killer cell activity. Biol Blood Marrow Transplant 1997; 3: 34–44. Fierro MT, Liao XS, Lusso P et al. In vitro and in vivo susceptibility of human leukemic cells to lymphokine activated killer activity. Leukemia 1988; 2: 50–4. Stein AS, O’Donnell MR, Slovak ML et al. Interleukin-2 after autologous stem-cell transplantation for adult patients with acute myeloid leukemia in first complete remission. J Clin Oncol 2003; 21: 615–23. Attal M, Blaise D, Marit G et al. Consolidation treatment of adult acute lymphoblastic leukemia: a prospective, randomized trial comparing allogeneic versus autologous bone marrow transplantation and testing the impact of recombinant interleukin-2 after autologous bone marrow transplantation. BGMT Group. Blood 1995; 86: 1619– 28. Burns LJ, Weisdorf DJ, DeFor TE et al. IL-2based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release: a phase I/II trial. Bone Marrow Transplant 2003; 32: 177–86. Thompson JA, Fisher RI, LeBlanc ML et al. Total body irradiation, etoposide, cyclophosphamide and autologous peripheral blood stem cell transplantation followed by randomization to therapy with interleukin-2 versus observation for patients with non-Hodgkin’s lymphoma: results of a phase III randomized trial by the Southwest Oncology Group (SWOG 9438). Blood 2006; 108; 326a. Goldberg GL, Zakrzewski JL, Perales MA, van den Brink MR. Clinical strategies to enhance T cell reconstitution. Semin Immunol 2007; 19: 289–96. Alvarnas JC, Linn YC, Hope EG, Negrin RS. Expansion of cytotoxic CD3+ CD56+ cells from peripheral blood progenitor cells of patients undergoing autologous hematopoietic cell transplantation. Biol Blood Marrow Transplant 2001; 7: 216–22. Leemhuis T, Wells S, Scheffold C, Edinger M, Negrin RS. A phase I trial of autologous cytokineinduced killer cells for the treatment of relapsed Hodgkin disease and non-Hodgkin lymphoma. Biol Blood Marrow Transplant 2005; 11: 181–7. Takayama T, Sekine T, Makuuchi M et al. Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial. Lancet 2000; 356: 802–7. Petvises S, Pakakasama S, Wongkajornsilp A, Sirireung S, Panthangkool W, Hongeng S. Ex vivo generation of cytokine-induced killer cells (CD3+ CD56+) from post-stem cell transplant pediatric patients against autologous-Epstein–Barr virustransformed lymphoblastoid cell lines. Pediatr Transplant 2007; 11: 511–17. Rosenberg SA, Lotze MT, Muul LM et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high-dose interleukin-2 alone. N Engl J Med 1987; 316: 889– 97.

134. Klebanoff CA, Khong HT, Antony PA, Palmer DC, Restifo NP. Sinks, suppressors and antigen presenters: how lymphodepletion enhances T cellmediated tumor immunotherapy. Trends Immunol 2005; 26: 111–17. 135. Barao I, Hanash AM, Hallett W et al. Suppression of natural killer cell-mediated bone marrow cell rejection by CD4+CD25+ regulatory T cells. Proc Natl Acad Sci U S A 2006; 103: 5460–5. 136. Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME, Greenberg PD. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 1992; 257: 238–41. 137. Gottschalk S, Heslop HE, Roon CM. Treatment of Epstein–Barr virus-associated malignancies with specific T cells. Adv Cancer Res 2002; 84: 175–201. 138. Rooney CM, Smith CA, Ng CY et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein–Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92: 1549–55. 139. Kennedy-Nasser AA, Bollard CM, Rooney CM. Adoptive immunotherapy for Hodgkin’s lymphoma. Int J Hematol 2006; 83: 385–90. 140. Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997; 90: 3245–87. 141. Bollard CM, Rossig C, Calonge MJ et al. Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity. Blood 2002; 99: 3179–87. 142. Krance RA, Kuehnle I, Rill DR et al. Hematopoietic and immunomodulatory effects of lytic CD45 monoclonal antibodies in patients with hematologic malignancy. Biol Blood Marrow Transplant 2003; 9: 273–81. 143. Laport GG, Levine BL, Stadtmauer EA et al. Adoptive transfer of costimulated T cells induces lymphocytosis in patients with relapsed/refractory non-Hodgkin lymphoma following CD34+selected hematopoietic cell transplantation. Blood 2003; 102: 2004–13. 144. Rapoport AP, Stadtmauer EA, Aqui N et al. Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer. Nat Med 2005; 11: 1230–7. 145. Wen YJ, Barlogie B, Yi Q. Idiotype-specific cytotoxic T lymphocytes in multiple myeloma: evidence for their capacity to lyse autologous primary tumor cells. Blood 2001; 97: 1750–5. 146. Liso A, Stockerl-Goldstein KE, AuffermannGretzinger S et al. Idiotype vaccination using dendritic cells after autologous peripheral blood progenitor cell transplantation for multiple myeloma. Biol Blood Marrow Transplant 2000; 6: 621–7. 147. Davis TA, Hsu FJ, Caspar CB et al. Idiotype vaccination following ABMT can stimulate specific anti-idiotype immune responses in patients with B-cell lymphoma. Biol Blood Marrow Transplant 2001; 7: 517–22. 148. Mitsuyasu RT, Anton PA, Deeks SG et al. Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood 2000; 96: 785–93. 149. Jensen M. Strategies to enhance the therapeutic efficacy of autologous hematopoietic stem cell

Management of Relapse after Hematopoietic Cell Transplantation transplantation by posttransplantation adoptive transfer of T cells with engineered graft-versustumor activity. Biol Blood Marrow Transplant 2005; 11: 34–9. 150. Cooper LJ, Topp MS, Serrano LM et al. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-Blineage leukemia effect. Blood 2003; 101: 1637– 44. 151. Kowolik CM, Topp MS, Gonzalez S et al. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persis-

tence and antitumor efficacy of adoptively transferred T cells. Cancer Res 2006; 66: 10995–1004. 152. Singh H, Serrano LM, Pfeiffer T et al. Combining adoptive cellular and immunocytokine therapies to improve treatment of B-lineage malignancy. Cancer Res 2007; 67: 2872–80. 153. Todisco E, Castagna L, Sarina B et al. Reducedintensity allogeneic transplantation in patients with refractory or progressive Hodgkin’s disease after high-dose chemotherapy and autologous stem cell infusion. Eur J Haematol 2007; 78: 322–9.

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154. Martino R, Caballero MD, de la Serna J et al. Low transplant-related mortality after second allogeneic peripheral blood stem cell transplant with reduced-intensity conditioning in adult patients who have failed a prior autologous transplant. Bone Marrow Transplant 2002; 30: 63– 8. 155. Branson K, Chopra R, Kottaridis PD et al. Role of nonmyeloablative allogeneic stem-cell transplantation after failure of autologous transplantation in patients with lymphoproliferative malignancies. J Clin Oncol 2002; 20: 4022–31.

Section 6 Hematopoietic Cell Transplantation for Inherited Diseases

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Guido Lucarelli & Javid Gaziev

Hematopoietic Cell Transplantation for Thalassemia

Introduction The thalassemias refer to a diverse group of hemoglobin disorders characterized by a reduced synthesis of one or more of the globin chains (α, β, γ, δβ, γδβ, δ, and εγδβ) and are the most common monogenic disorders to cause a major public health problem worldwide [1]. Globally, it is estimated that there are 270 million carriers of hemoglobin disorders, of which 80 million are carriers of β-thalassemia. In most countries where the transfusion and iron chelation treatment is given irregularly, mainly due to the high cost, the majority of patients with homozygous β-thalassemia die in childhood from chronic anemia and its complications, and from the organ iron overload. Currently recommended nontransplant therapy is available in most countries, and consists of transfusions to maintain hemoglobin levels between 9 and 10 g/dL, together with chelation therapy aimed at preventing iron accumulation as a consequence of the transfusion therapy. To be effective, chelation therapy should consist of continuous subcutaneous infusion for at least 8–12 hours daily. The successful implementation of this treatment regimen has improved life expectancy up to and beyond the second decade of life, and has dramatically improved the quality of life for children with thalassemia. Future treatment of the thalassemic patient may be easier and improved if orally administered iron chelators become available. Two potentially useful oral chelators are now available, but their efficacy compared with desferrioxamine (DFO) in the long term has not yet been determined. Hematopoietic cell transplantation (HCT) has been used in attempts at curing thalassemia. The first successful marrow transplant for thalassemia was reported in 1982 by Thomas and his colleagues [2], and the first reports of series of transplants were by Lucarelli et al. [3,4]. Some discussion of the epidemiology, molecular biology, pathophysiology, clinical features, and supportive therapy is necessary in an attempt to define how, when, and for whom to pursue bone marrow transplantation (BMT) for thalassemia.

Epidemiology and etiology Thalassemia is a growing global public health problem worldwide. Even though the relationship has not been proven conclusively for β-thalas-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

semia, the geographic distribution coincides historically with malarial areas, and there is more convincing evidence for β-thalassemia and sickle cell disease. Because of a selective advantage of heterozygotes against the severe form of malaria, the frequencies of β-thalassemia are particularly high in the malarial tropical and subtropical regions of the Mediterranean area, the Indian subcontinent, Africa, and South and East Asia [1]. It is rare in people of Northern European origin, around one in 1000 being carriers, while in the Northern Mediterranean countries (south Europe) the carrier prevalence is 1–19%. In the Arab world, the carriage rate is around 3%, while in central Asia the prevalence is 4– 10%. From the Indian subcontinent to South-east Asia, β-thalassemia coexists with hemoglobin E (HbE) in carrier rates that range from 1% to 40% [5]. Worldwide, about 60,000 children with a major thalassaemia and 250,000 with a sickle cell disorder are born annually, giving a rate of more than 2.4 affected children per 1000 births [6]. The epidemiology of the disease is changing due to recent population migrations. In Europe, the United States, and Australasia, thalassemia has become an important part of clinical practice.

Molecular and clinical biology More than 200 mutations affecting the β-globin gene are known to result in a phenotype of β-thalassemia. With the exception of the 619 base pair deletion, which accounts for 20% of the β-thalassemias in Asian Indians [7], the common mutations of β-thalassemias are caused by point mutations as single-base substitutions, minor insertions or deletions of a few bases within the gene or its immediate flanking sequences, and are classified according to the mechanism by which they affect gene regulation: transcription, RNA processing or RNA translation [8]. These genetic defects lead to a variable reduction in β-globin production ranging from a minimal deficit (β+-thalassemia) to complete absence (β0-thalassemia). Mutations affecting transcription generally result in a mild-to-minimal deficit of β globin and can be silent in carriers. Mutations that affect RNA processing can involve the splice junction, in which case normal splicing is completely abolished, with resulting β0-thalassemia phenotype, or can occur within the consensus sequences at the splice junction, reducing the efficacy of normal splicing to varying degrees and producing a β+-thalassemia phenotype that ranges from mild to severe. Approximately half of the β-thalassemia alleles affect the different stages of RNA translation, which leads to a lack of β-globin production, resulting in β0-thalassemia. Most of these defects result from the introduction of premature termination codons due to frameshifts or nonsense mutations [8]. The pathophysiology of β-thalassemia relates to a quantifiable deficiency of functional β globin, which leads to imbalanced globin chain

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production and an excess of α-globin chains [1,9]. The latter are not able to form viable tetramers and instead precipitate in the red cell precursors, forming inclusion bodies that cause mechanical damage and the premature destruction of red cell precursors in the bone marrow, leading to ineffective erythropoiesis. The red cells that survive to reach the peripheral circulation are prematurely destroyed in the spleen, which becomes enlarged, eventually leading to hypersplenism. Thus, anemia in β-thalassemia results from a combination of ineffective erythropoiesis, peripheral hemolysis, and an overall reduction in hemoglobin synthesis, leading to intense proliferation and expansion of the bone marrow, with the resulting skeletal deformities. These secondary complications of bone disease, splenomegaly, and endocrine and cardiac damage can be related to the severity of anemia and the iron loading that results from increased gastrointestinal iron absorption and blood transfusions.

Clinical features The clinical manifestations of β-thalassemia are extremely diverse, ranging from the transfusion-dependent state of thalassemia major to the slightly less severe transfusion-dependent state of thalassemia intermedia or to the asymptomatic state of thalassemia trait. The common βthalassemia alleles that are prevalent in malarial regions are inherited typically in a Mendelian recessive manner, although some forms of βthalassemia are dominantly inherited. The most severe form of disease is characterized by the complete absence of β-globin production and results from the inheritance of two β0-thalassemia alleles, homozygous or compound heterozygous states. These combinations usually result in β-thalassemia major, and patients present within 6 months of birth with severe anemia; if not treated with regular blood transfusions, they die within the first 2 years of life. The anemia is due to a combination of ineffective erythropoiesis, excessive peripheral red blood cell hemolysis, and progressive splenomegaly. The red cells are microcytic with marked anisochromasia. The bone marrow shows marked erythroid hyperplasia, and the serum ferritin level is elevated. The hemoglobin pattern of patients with β-thalassemia major consists of a variable increase in HbF, which can account for 8–90% of the total hemoglobin concentration and show a variable quantity of HbA2 up to 6% [1]. In children and young adults, the expansion of hyperplastic bone marrow results in skeletal deformities and bone disease. Because of chronic anemia and iron overload, endocrinopathies such as hypopituitarism, hypothyroidism, hypoparathyroidism, diabetes mellitus, cardiomyopathy, and testicular and ovarian failure become common as the child with thalassemia grows older [1,10]. Chronic lung disease and pulmonary hypertension are other complications that are being increasingly recognized in older β-thalassemic patients, and are thought to be related to the increased tendency for thrombosis and small pulmonary emboli [11].

Nontransplant approaches The current conventional treatment of β-thalassemia major consists of lifelong regular blood transfusions combined with daily subcutaneous iron chelation therapy with DFO. In 1964, Wolman reported that patients maintained at a higher baseline hemoglobin level had less severe complications and were in better health than those maintained at lower hemoglobin levels [12]. Stimulated by this observation, Piomelli and his colleagues studied a transfusion regimen designed to prevent the baseline hemoglobin from falling below 10 g/dL. When this hypertransfusion regimen was instituted in infancy and rigorously implemented, cardiomegaly and bone malformation were greatly reduced, although normalization of growth was not achieved and the development of hypersplenism was not prevented [13]. The target hemoglobin level of

10 g/dL was designed to depress endogenous erythropoiesis and thereby avoid marrow hypertrophy with accompanying marrow space extension. This objective was partially achieved, but complete suppression of endogenous red cell production requires that the hemoglobin be maintained at levels greater than 13 g/dL [14]. There are major obstacles to achieving such levels. Raising the baseline hemoglobin level from 10 to 11 g/dL requires a 20% increase in requirements for red cell transfusion [15], and there are many reasons for minimizing transfusion in patients with thalassemia. Hypertransfusion regimens have a major impact on iron traffic and distribution in patients with severe hemolytic anemia. The complications of iron overload, such as endocrine deficiency, hepatic and pancreatic damage, and heart disease, are the principal causes of morbidity and mortality in patients with thalassemia receiving this form of therapy. The transfusion of 165 mL of packed red cells per kilogram per year is associated with an annual iron intake of approximately 180 mg/kg [16]. More recently, clinical experience suggested that a moderate transfusion regimen maintaining a hemoglobin level of 9–10 g/dL throughout life may reduce iron loading in patients with β-thalassemia major without producing an excessive expansion of erythropoiesis [17]. Iron accumulation resulting from both repeated blood transfusions and increased gastrointestinal absorption in β-thalassemia major patients may cause serious organ damage and ultimately death. Effective removal of this excess iron can improve the survival of these patients. In the last 30 years, DFO was the only effective iron-chelating drug in patients with β-thalassemia major. DFO has a strong and selective affinity for iron and does not appear to have any major side-effects. However, DFO displays poor bioavailability when given orally and has a rapid plasma clearance. Therefore, DFO is efficient only when administered parenterally over several hours in order to keep a constant serum level. Since the maintenance of steady plasma levels of this agent is essential for the effective management of iron traffic, it must be continuously administered either intravenously or subcutaneously. This has led to the now standard therapeutic approach, that is, administration of DFO by slow infusion overnight (8–12 hours for 5–7 nights) using a special pump. Intensive long-term chelation with DFO has proved beneficial for a large number of patients, as evidenced by the gradual decrease of their serum ferritin and liver iron, with improved survival [18]. DFO therapy, when rigorously adhered to, substantially reduces, but does not eliminate, the iron overload of patients on hypertransfusion therapy. There are two common reasons for the failure of chelation therapy with DFO. In emerging countries where thalassemia is a substantial public health problem, the high cost of the drug and of the supplies for its administration make it unavailable for most patients. Despite the wide availability of DFO in Western countries, some patients are unable to comply sufficiently with the prescribed treatment because the effective chelation is unpleasant and cumbersome, requiring daily subcutaneous or intravenous administration, which leads to poor compliance with therapy. It is therefore obvious that there is a pressing need for an orally administered chelator. Two oral iron chelators are currently available for clinical use. Deferiprone or L1 (DFP) is a bidentate chelator which forms a chelator–iron complex excreted mainly in the urine [19]. At a standard dose (75 mg/ kg/day in three doses), iron stores may decrease in some patients, remain stable in others, and increase in still others [20]. DFP is approved in Europe and many other countries, often as a second-line treatment for iron overload. In general, DFP is less effective in removing liver iron, although great individual variations occur. Toxic effects include agranulocytosis, neutropenia, and arthropathy, which demand close monitoring. Studies in patients treated with DFP have shown significant improvement in cardiac magnetic resonance imaging, consistent with a reduction in cardiac iron overload and improved cardiac function, in

Hematopoietic Cell Transplantation for Thalassemia

comparison with DFO, supporting a potential cardioprotective role of DFP [21,22]. Another oral chelator is now available – deferasirox (ICL670), which is a member of a new class of tridentate iron chelators [23]. It is orally bioavailable with a terminal elimination half-life between 8 and 16 hours, allowing for once-daily administration. Deferasirox–iron complexes are excreted in the stools. A recent phase III randomized study comparing deferasirox with DFO in pediatric and adult patients with β-thalassemia major receiving regular blood transfusions showed that, at 20–30 mg/kg/day, deferasirox can keep most but not all patients in even or negative iron balance in rough equivalence to moderate doses of DFO. The most common adverse events included rash, gastrointestinal disturbances, and mild nonprogressive increases in serum creatinine [24]. While the efficacy and limits of DFO therapy in transfusion-dependent β-thalassemia major patients are well known, long-term data on these two new oral chelators in treating iron overload are not yet available, and they should be investigated in large-scale prospective randomized studies. The beneficial effect of high levels of HbF in β-thalassemia and sickle cell anemia has been recognized for many years. Pharmacologically, three classes of agents have been shown to be capable of inducing HbF to therapeutic levels: erythropoietins, short-chain fatty acid derivatives, and chemotherapeutic agents. However, the sustained pharmacologic induction of HbF to therapeutic levels in β-thalassemia has been disappointing. Despite addressing the ineffective erythropoiesis, correction of the anemia has been difficult because of suppression of erythropoiesis by the currently available pharmacologic agents. Gene therapy for β-thalassemia requires gene transfer into hematopoietic stem cells (HSCs) using integrating vectors that direct the regulated expression of β globin at therapeutic levels. Early attempts using conventional oncoretroviral vectors carrying the human β-globin gene and portions of the locus control region have suffered from problems of vector instability, low titers, and variable expression [25]. Recently, human immunodeficiency virus-based lentiviral vectors have been shown to stably transmit the human β-globin gene and a large locus control region element, resulting in correction of the mouse thalassemia intermedia phenotype, with variable levels of β-globin expression [26]. The levels of β-globin expression achieved from insulated self-inactivating lentiviral vectors were sufficient to phenotypically correct the thalassemia phenotype from four patients with thalassemia major in vitro, and this correction persisted long term for up to 4 months in xenotransplanted mice in vivo [27]. Despite promising results of gene therapy in animal models, its clinical potential remains uncertain, and the safety of these vectors to use for gene therapy of hemoglobinopathies remains to be seen. Programs for prevention of thalassemia are presently based on prospective carrier screening and prenatal diagnosis. Although traditional prenatal diagnosis of β-thalassemia has been highly effective in some countries, the fact that affected pregnancies must be terminated to avoid the birth of affected children makes it unacceptable to most people. Recently developed preimplantation genetic diagnosis with in vitro fertilization has already become an alternative to traditional prenatal diagnosis, allowing the establishment of only unaffected pregnancies and avoiding the risk for pregnancy termination [28]. Preimplantation genetic diagnosis completed with human leukocyte antigen (HLA) typing allows selection of healthy and compatible embryos in families with affected children with the certainty that a source of stem cells will be available for transplantation. However, this technique, apart from raising bioethical concerns due to selecting the “HLA-compatible and healthy” embryo and discarding other (equally not sick) embryos just because they are not HLA compatible with the affected child, requires a high degree of technical expertise and is expensive, making it difficult for large application.

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Bone marrow transplantation At present, allogeneic HCT is the only rational therapeutic modality for the eradication of β-thalassemia major [29]. It is a form of gene therapy that uses allogeneic stem cells as vectors for genes essential for normal hematopoiesis. Eventually, the vector may well be autologous stem cells transformed by the insertion of normal genes, but there is no indication that this approach will be a clinical option in the foreseeable future. All of the transplant procedures in Pesaro discussed in this chapter were performed with bone marrow, and the term “bone marrow transplantation” is used for these procedures. There are no controlled trials of HCT versus medical treatment for thalassemia major, and few studies comparing quality of life. Regular blood transfusion and iron chelation have improved both the survival and quality of life of patients with thalassemia, and have changed a previously fatal disease with early death to a chronic, although progressive, disease compatible with prolonged survival [6,30]. Despite the prolonged life expectancy, a recent study from the UK Thalassemia Registry has shown a steady decline in survival starting from the second decade, with fewer than 50% of patients remaining alive beyond 35 years, mainly because of poor compliance with chelation therapy [31]. In the developing world, where thalassemia is more common, most children die before the age of 20 years because of the unavailability of safe blood products and/or expensive iron-chelating drugs. The two recently developed oral iron chelators could allow better compliance with chelation, and therefore could have a favorable impact on the survival of patients with thalassemia. However, even an ideal iron chelator with rigorous adherence only substantially reduces, but does not eliminate, the iron overload of patients on lifelong transfusions. The acute toxicities of transplantation are challenged by the observation that, in the developed world, patients with thalassemia are well served by medical treatment, and in some countries patients receiving regular blood transfusion and chelation could have a better survival rate [18,32]. Unfortunately, these same patients, with increasing age, currently experience poor outcome on conventional treatment, even in well-resourced countries with universal access to good medical treatment [10,31]. The first two transplant procedures for the treatment of thalassemia with marrow from matched related donors were performed in December 1981, in Seattle, WA, and in Pesaro, Italy. The Seattle approach was based on the assumption that the risks associated with BMT would be increased by the iron overload and by sensitization to HLAs induced by hypertransfusion. Therefore, it was decided that early clinical studies would be conducted in very young patients who had received very few transfusions. On December 3, 1981 a 14-month-old child with β-thalassemia major who had been transfused with a total of 250 mL of packed red blood cells received BMT from his HLA-identical sister in Seattle [2]. The treatment was completely successful. The Pesaro approach was based on an assessment that restricting transplants to untransfused patients was impracticable. On December 17, 1981, the Pesaro team performed a transplant in a 16-year-old thalassemic patient who had received 150 red blood cell transfusions, using marrow from his HLA-identical brother. This patient rejected the graft and was the first of an extensive series of transplants for thalassemia that provides most of the data supporting this chapter. Preparatory regimens Preparatory regimens for HCT in patients with diseases other than aplastic anemia must achieve two objectives. One is elimination of the (disordered) marrow, and the other is establishment of a tolerant environment that will permit transplanted marrow to survive and thrive. Total body irradiation (TBI) can accomplish both these objectives, but there are many reasons to avoid the use of this marrow-ablative modality. These

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include the known growth-retarding effects of TBI in young children and the increased risk of secondary malignancies, which has been reported in patients treated for leukemia [33], lymphoma, and aplastic anemia [34,35]. These hazards are particularly objectionable in very young patients with potential for a long life span. The risk of these toxicities has not yet been fully explored for cytotoxic regimens that do not involve TBI. There is a considerable body of experience with the use of busulfan (BU) and its derivatives in ablating marrow in patients undergoing HCT for the treatment of nonmalignant conditions such as the Wiskott–Aldrich syndrome [36,37] and inborn errors of metabolism [38]. Cyclophosphamide (CY) is an agent that is well established as providing immunosuppression adequate for allogeneic engraftment of patients with aplastic anemia [39,40]. Experience in the use of chemotherapy-only transplant regimens for the treatment of malignancy [41– 46] has been pivotal in developing regimens appropriate for the treatment of thalassemia by transplantation. BU is an alkylating agent with exquisite specificity for the most primitive precursors of the myeloid–erythroid axis. It has been used in low doses for more than 30 years for the treatment of patients with chronic myeloid leukemia, and its toxicity and effectiveness have been well documented in that setting. Studies in rodents demonstrated that marrow-lethal doses of BU have minor toxicity for the lymphoid system and cause little immunosuppression [47]. Canine studies of transplants between dog leukocyte antigen-identical littermates demonstrated 50% engraftment after BU alone and 95% engraftment when antithymocyte serum was added to the conditioning regimen [48]. Clinical experience with the use of BU in very high doses was delayed due to the lack of an acceptable preparation suitable for intravenous use. Santos et al. reported the first clinical trials of very high-dose BU in the context of BMT [43]. In these studies, patients with acute myeloid leukemia received allogeneic marrow transplants after immunosuppression with CY (200 mg/kg over 4 days), and oral BU (16 mg/kg over 4 days) was administered as additional antitumor therapy. Early results with this therapy were encouraging, and in successful attempts to reduce early transplant-related toxicity, Tutschka et al. reduced the CY dose to 120 mg/kg over 2 days [49]. Further, CY has been a component of most conditioning regimens for transplanting patients with hematologic malignancies. Santos et al. reported on its use in high doses (200 mg/kg over 4 days) as the sole antitumor agent in patients receiving allogeneic transplants for leukemia and demonstrated that it was sufficiently immunosuppressive to permit sustained allogeneic engraftment [41]. Also, CY is most commonly employed for the treatment of lymphoid malignancies and solid tumors, and it has been used as a component of combination chemotherapy for the treatment of acute leukemia. It is not considered a highly effective agent against myeloid malignancies, although single-drug studies are not available in this context. The dose-limiting toxicity of CY is to the heart and not to the marrow. Mice, monkeys, and humans recover hematopoiesis promptly after the highest doses of CY because CY does not eliminate HSCs. Therefore, CY alone is not an appropriate conditioning modality for BMT for the treatment of thalassemia because the thalassemic marrow would recover rapidly. On the other hand, BU is an agent that has a good possibility of eradicating a diseased erythron but when used alone is not likely to be sufficiently immunosuppressive to permit sustained allogeneic engraftment. In summary, a combination of BU and CY can eradicate the thalassemia and facilitate sustained allogeneic engraftment.

marrow hyperplasia with aggressive extension of a rapidly proliferating erythron into intra- and extramedullary areas not usually occupied by marrow. This results in major bone remodeling together with marked hepatomegaly and splenomegaly. By analogy with the behavior of malignant tissue, it might be hypothesized that this large mass of rapidly proliferating hematopoietic tissue would be more difficult to eradicate than normal hematopoietic tissue, and more likely to recur after transplantation. Although post-transplant thalassemic recurrence is a problem, it occurs in circumstances that differ from those usually observed with leukemic relapse. The most common presentation of leukemic relapse is a return of host-type leukemia in the presence of a persisting immune system of donor origin. In contrast, the recurrence of thalassemia usually occurs in the context of a return of host-type immune and hematologic reconstitution. Thus, this event has aspects of both relapse and rejection, and it is customary to speak of this phenomenon as rejection. Engraftment Most patients undergoing transplantation for the treatment of thalassemia have been transfused repeatedly. Experience with BMT for the treatment of aplastic anemia in heavily transfused patients demonstrated that a history of many pretransplant transfusions increased the probability of graft rejection [50,51]. There is a substantial incidence of graft rejection in thalassemia patients after most preparatory regimens for marrow grafting, and this seems to be related to the stage of disease at the time of transplant. As discussed above, in most cases of graft rejection the thalassemic marrow will regrow and subsequent survival will be long (albeit with thalassemia). Occasionally, patients will reject grafts without recurrence of thalassemia. Unless rescued by a second transplant, such patients will die from the consequences of marrow aplasia. The treatment of patients after graft rejection will differ depending on whether the rejection is accompanied by regeneration of host-type hematopoiesis or by marrow aplasia. The Pesaro experience with this situation is discussed below. Transplant-related morbidity and mortality HCT is a dangerous undertaking. Regimens capable of eradicating a diseased marrow and facilitating persistent engraftment are necessarily toxic, and the consequences of successful allogeneic marrow engraftment include acute and chronic graft-versus-host disease (GVHD), syndromes associated with severe immune incompetence. Both prophylaxis against GVHD and methods for its treatment are immunosuppressive. Transplant-associated toxicity may be aggravated by GVHD and by measures aimed at the prevention of this complication. Such toxicity can be categorized either as regimen-related toxicity or as GVHD. In studies of HCT for the treatment of hematologic malignancies, regimen-related toxicity from the preparative regimens has been well described [52,53]. The lungs and liver are the organs most at risk for toxicity induced by TBI and BU, while the heart is the main site of CYinduced damage. Increasing patient age, previous exposure to cytotoxic agents, and the presence of latent viruses such as hepatitis C and cytomegalovirus adversely influence these toxicities. Patients transplanted for the treatment of thalassemia derive benefit from the fact that they are usually young and without prior exposure to cytotoxic agents. However, because of previous intensive transfusion therapy, they will have a high probability of carrying harmful viruses and have organ damage induced by extreme iron overload. Clinical transplant experience

Disease eradication

The Pesaro experience

Many of the hematopoietic manifestations of thalassemia resemble those of hematopoietic malignancy. The disease is characterized by extreme

By far the largest body of experience in the treatment of thalassemia with BMT has been accumulated in Pesaro, Italy. From December 1981

Hematopoietic Cell Transplantation for Thalassemia

to June 2001, 915 patients with homozygous β-thalassemia received marrow transplants. The thalassemia-free survival of the entire group of patients (aged 1 through 35 years at the time of the transplantation) is 69%. For 880 patients, the donors were HLA identical (853 siblings and 27 parents), 29 were HLA partially matched relatives, and six were HLA-identical unrelated donors. Most patients were treated with one of two types of regimen: one, used for 547 patients, prescribed BU 14 mg/ kg and CY 200 mg/kg, and the other used BU with a lower dose of CY for patients believed to be especially susceptible to the toxic complications associated with this high dose of CY. In early experience, the best results were obtained in younger patients [4,54,55]. Of the first six patients older than 16 years, four died of GVHD-related causes within the first 100 days, one died of infection on day 235, and one had recurrence of thalassemia on day 48 and died of consequent cardiac damage more than 6 years after transplantation. In view of this experience, early studies concentrated on patients under the age of 17 years.

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had all three, and class 2 patients had one or two adverse risk factors. This analysis confirmed the prognostic significance for transplant outcome of risk class and the strong influence of the quality of chelation therapy. By the end of December 2003, 524 patients under 17 years of age had received HLA-identical transplants using regimens containing BU 14 mg/kg and CY 200 mg/kg. There were 145 class 1 patients, 333 class 2 patients, and 46 class 3 patients. The probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality for class 1 patients were 90%, 87%, 3%, and 10%, respectively, and for class 2 patients they were 87%, 85%, 3%, and 13%, respectively (Table 73.1). The probabilities of thalassemia-free survival for class 1 and for class 2 patients are reported in Fig. 73.1. The probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality for the 46 patients in class 3 were 53%, 51%, 7%, and 42%, respectively, calculated at 15 years. In an attempt to improve results in class 3 patients, new treatment regimens were devised using BU 14 mg/kg and lower doses of CY (160 or 120 mg/kg). These regimens improved the probability of survival in class 3 younger (age <17 years) patients from 53% to 79%, but were associated with an increase of rejection probability from 7% to 30%, probably due to inadequate immunosuppression and failure to eradicate the massive erythroid hyperplasia characteristic of these patients [57]. In April 1997, a new preparative regimen (Protocol 26) was adopted for class 3 patients under 17 years of age in an attempt to decrease the 30% rejection rate without a corresponding increase in reappearance of the thalassemic clone. This protocol involved an intensified preparation with 3 mg/kg of azathioprine and 30 mg/kg hydroxyurea daily from day 2 45 before the transplant (day −45), fludarabine 20 mg/m from day −17 through day −13, followed by the administration of BU 14 mg/kg total dose and CY 160 mg/kg total dose. Prophylaxis against GVHD consisted of CY 7.5 mg/kg intravenously on day 1, methotrexate (MTX) 10 mg/m2 intravenously on days 3 and 6, and cyclosporine (CSP) 5 mg/ kg intravenously daily from day −2 through day 5, reduced to 3 mg/kg/ d intravenously until an oral administration of 12.5 mg/kg/day could be tolerated. Continuous 24-hour infusions of 40 mg/kg of DFO via central venous catheter were initiated on day −45, and a regimen of hypertransfusion with red blood cells was used to keep the level of hemoglobin between 14 and 15 g/dL. During this time interval, patients received growth factors, granulocyte colony-stimulating factor, and erythropoietin twice weekly to maintain stem cell proliferation in the face of hypertransfusion, thereby facilitating the effect of the hydroxyurea. Protocol 26 was devised on the assumption that preparation with BU 14 mg/kg and CY 160 mg/kg was inadequate to eradicate thalassemic hematopoiesis in class 3 patients under 17 years of age. The probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality in 33 such patients treated with Protocol 26 were 93%, 85%, 8%,

Children and adolescents. Results in young patients were very encouraging, and in 1990 Lucarelli and his colleagues reported their experience through August 1988 in treating 222 consecutive patients under the age of 16 years [4]. All these patients received HLA-identical marrow, in 10 cases from parents and in the other cases from siblings, after conditioning with regimens containing BU 14 mg/kg and CY 200 mg/kg. One hundred and forty-one donors were heterozygous for β-thalassemia, and 81 donors were normal homozygotes. Five different regimens were used to prevent acute GVHD. Analysis of the influence of pretransplant characteristics on the outcome of transplantation was conducted in 116 patients who were all treated with exactly the same regimen. It was demonstrated that hepatomegaly and portal fibrosis were associated with a significantly reduced probability of survival. In multivariate analysis, a history of poor compliance with the chelation regimen could not be distinguished from hepatomegaly as a predictor of survival and rejection-free survival. The influence of pretransplant characteristics on the outcome of transplantation was re-examined in late 1989 [56], by which time 161 patients less than 17 years of age had been treated with the same regimen. The quality of chelation was characterized as regular when DFO therapy was initiated no later than 18 months after the first transfusion and was administered subcutaneously for 8–10 hours continuously for at least 5 days each week. The chelation variable was defined as irregular for any deviation from this criterion. The degree of hepatomegaly (greater than or not greater than 2 cm), the presence or absence of portal fibrosis in the pretransplant liver biopsy, and the quality of chelation (regular or irregular) given through the years before transplant were identified as variables permitting the categorization of patients into three risk classes. Class 1 patients had none of these adverse risk factors, class 3 patients

Table 73.1 Pesaro experience of bone marrow transplantation for thalassemia from human leukocyte antigen-matched related donors between May 1985 and December 2003: protocols in use

Patients (n) Protocols Survival (%) Thalassemia-free survival (%) Death (%) Rejection (%)

Class 1

Class 2

Class 3 (age <17 years) (since 1997)

Adult (age >17 years) (since 1997)

145 6 90 87 10 3

333 6 87 85 13 3

33 26 93 85 6 8

15 26 65 65 28 7

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Chapter 73

1 Class 1

Probability

0.8

87% 85%

Class 2

0.6 0.4 0.2 0 0

5

10

15

20

25

Years

Fig. 73.1 Estimates of thalassemia-free survival for class 1 and class 2 patients younger than 17 years and treated with busulfan 14 mg/kg and cyclophosphamide 200 mg/kg ± thiotepa 10 mg/kg between May 1985 and December 2003.

and 6%, respectively (Table 73.1) [58]. Interestingly, this regimen improved the Kaplan–Meier probability of thalassemia-free survival for patients with class 3 thalassemia aged younger than 17 years at the time of the transplantation from 58% to 85%, together with a reduction of the probability of rejection from 30% to 8% compared with previous preparative regimens. The immunosuppressive qualities of fludarabine may have contributed to the effectiveness of this regimen. Adult patients. Adult thalassemia patients have more advanced disease with both disease- and treatment-related organ complications mainly due to prolonged exposure to iron overload. From November 1988 through September 1996, 107 patients older than 16 years received transplants from matched donors. The median age for this population was 20 (range 17–35) years. Eighteen patients were in class 2, and these received BU 14 mg/kg and CY 200 mg/kg. The other patients were in class 3; 15 of these received BU 14 mg/kg and CY 120 mg/kg, 13 received BU 16 mg/ kg and CY 120 mg/kg, 18 received BU 16 mg/kg and CY 160 mg/kg, and 43 received BU 14 mg/kg and CY 160 mg/kg. The probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality for the entire group of these patients were 66%, 62%, 4%, and 37% respectively [59,60]. For the 18 class 2 adults, the probabilities of survival and of thalassemia-free survival were both 76%. From April 1997 all adult patients were prepared for transplantation according to Protocol 26, with the only difference that the dose of CY was reduced to 90 mg/ kg total dose. The probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality in 15 high-risk group patients were 65%, 65%, 7%, and 28% respectively [61]. Transplantation provided what is probably a permanent cure for the marrow defect in nearly all these patients, but prolonged follow-up is necessary to determine the long-term outcome. It is reasonable to hope that removal of the continuing cause for extramedullary organ damage will modify disease progression and permit healing of the damaged organs. Management of patients with rejection or disease recurrence. Patients with engraftment failure and without functioning marrow have a bleak prospect because an early second transplant with a second course of conditioning is usually not a reasonable option. However, occasionally patients have late graft failure without thalassemia recurrence, and in this situation second transplant attempts with intensive conditioning may provide the only treatment option to offer a chance of prolonged survival.

Patients who reject their grafts and have a return of host hematopoiesis do not have an urgent need for second transplants, and such interventions can be delayed until the toxic effects of the conditioning regimen for the first transplants have resolved. At least a year should be allowed to elapse between the first and second transplant. The Pesaro team has performed second transplants in 21 patients who had complete thalassemia recurrence, and in 11 patients who developed irreversible aplasia following the first transplant. Patients with thalassemia recurrence received BU/CY alone or in association with total lymphoid irradiation or antilymphocyte globulin, while most patients with aplasia were given CY with or without total lymphoid irradiation [62]. The probabilities of survival and thalassemia-free survival were 55% and 29% in patients with thalassemia recurrence, and 41% and 41% in patients with aplasia, respectively. Survival and thalassemia-free survival were better in patients who had late graft failure (over 60 days) compared with patients developing graft failure within 60 days after the first transplant. Mixed chimerism after BMT in thalassemia. Ablation of all host HSCs is usually considered necessary to establish conditions for stable and complete marrow engraftment of donor stem cells (complete chimerism). However, we have observed that mixed chimerism is not unusual in transplanted thalassemic patients. Further, mixed chimerism may be transient when it evolves into complete chimerism or graft rejection, or persistent (PMC) when the coexistence of donor and recipient cells is longer than 2 years, with hemoglobin levels sufficient for a good quality of life without red blood cell transfusion [63]. When PMC was detected using sensitive techniques [64], it could be demonstrated in all the nucleated hematopoietic cell subpopulations of the marrow and peripheral blood. In informative situations, all circulating mature red blood cells were of the same blood group as the donor, even when 70% of burst forming units-erythroid were of host origin. In a group of 335 mostly consecutive patients, all with 2 or more years of post-transplant follow-up, the incidence of mixed chimerism was 32.2% at 2 months after HCT. This proportion decreased to 10.1% at 2 years after transplantation. Within this group, there were 185 class 1 or 2 patients, all treated with the same pretransplant conditioning regimen (BU 14 mg/kg and CY 200 mg/kg). For these patients, the incidence of mixed chimerism at 2 months after the transplant was 21%, while it was 54.9% in the group of the remaining 150 patients (who were treated with BU 14 mg/kg and CY 120 mg/kg or 160 mg/kg). The incidence of PMC 2 years after transplant was 10.1% in each of the two groups of patients, although they were not homogeneous for the preparative regimen. Graft rejection did not occur in any of the 227 patients with complete chimerism early after HCT, whereas graft rejection occurred in 35 of the 108 patients (32.4%) with mixed chimerism detected during the first 2 months after HCT. Rejection was related to the number of residual host cells (RHCs) present in patients with transient mixed chimerism early after HCT. Andreani and colleagues reported a classification scheme for mixed chimerism [64] in which the presence of less than 10% RHCs was designated as level 1, RHCs between 10% and 25% was categorized as level 2, and RHCs of over 25% was categorized as level 3. Of the 108 patients who at 2 months after the transplant were in mixed chimerism, 61 patients had level 1, 27 patients had level 2, and 20 patients had level 3 chimerism. In the group of level 1 patients, 57% eventually developed complete chimerism, 13% rejected the transplant, and 30% achieved PMC. In the group of level 2 patients, 44% showed complete chimerism, 41% rejected the transplant, and 15% remained in PMC. Eighteen of the 20 patients with level 3 mixed chimerism rejected the transplant, while only two developed PMC. It is interesting to observe that, of the 227 patients with complete chimerism 2 months after BMT, 4% showed the

Hematopoietic Cell Transplantation for Thalassemia

importantly, showed the reproducibility of the Pesaro experience in other centers. The use of alternative related donors. Between 60% and 70% of patients with hemoglobinopathies lack matched sibling donors. The curative potential of transplantation for hemoglobinopathies has encouraged the use of hematopoietic cell donations from donors other than matched related donors (alternative donors). Recently, the Pesaro team analyzed the results of transplantation from alternative related donors for 29 patients with thalassemia major [66]. Six of the donors were relatives and HLA phenotypically identical with the recipients, two were mismatched relatives, 13 were mismatched siblings, and eight were mismatched parents. Of the mismatched donors, 15 were mismatched for one antigen, five for two antigens, and three for three antigens. Most patients in this study received antilymphocyte globulin or irradiation in addition to BU and CY in an attempt to improve the prospect for successful engraftment. Unfortunately, the patients receiving these transplants had a high incidence of graft failure (55%), with a consequent low probability of thalassemia-free survival. No relationship between survival and the degree of HLA disparity could be demonstrated in this small series of cases.

Survival

100% 94%

1 Thalassemia-free survival 0.8 Probability

presence of PMC at 2 years after the transplant. This may be because the number of RHCs early after HCT in these patients was below the limit of sensitivity of the test. Thirty-four “ex-thalassemic” patients after transplant have maintained PMC for 2–13 years and are transfusion independent with hemoglobin levels ranging from 8.3 to 14.7 g/dL [63]. Fifteen of these patients showed level 3 persistence with large numbers of recipient precursor cells. In some patients, the proportion of donor engrafted cells decreased to levels usually predictive of complete rejection. One of these 15 patients who received BMT in 1998 showed a progressive increase of RHCs on annual follow-ups, although her hemoglobin level was maintained between 10 and 11 g/dL until late 2005, when the patient became anemic with a percentage of donor cells of 5%. The patient became again transfusion dependent, and a successful second transplant from the same donor was performed in May 2006 [65]. This is the latest rejection with autologous reconstitution ever documented after BMT for thalassemia, which demands lifelong monitoring of the chimerism status in patients with PMC. It is not known why mixed chimerism is transient in some patients and remains persistent in others. Clones of regulatory T cells may develop in some patients with PMC and establish a state of reciprocal tolerance. Further studies are needed of the mechanisms underlying this state of tolerance to permit the design of protocols that will produce it predictably. The discovery of this phenomenon has potential importance for the future use of gene therapy and for the adoption of less toxic conditioning regimens as preparation for transplantation. The finding that patients can have major clinical benefit in the presence of a high level of thalassemic hematopoiesis should be of great interest to students of gene therapy. Also, these findings suggest that patients with a high risk of rejection can be identified soon after transplantation, permitting the application of pre-emptive treatment strategies aimed at preventing rejection.

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0.6 0.4 0.2 Rejection

The Rome experience

6%

0 0

0.5

1

1.5 Years

2

2.5

3

Fig. 73.2 Estimates of survival, thalassemia-free survival, nonrejection mortality, and rejection for 32 class 1 and class 2 patients younger than 17 years who were treated with busulfan 14 mg/kg, cyclophosphamide 200 mg/ kg, and ± thiotepa 10 mg/kg.

1 Survival

87%

0.8

80%

Thalassemia-free survival Probability

In 2004, the authors of this chapter and two other colleagues from the Pesaro Group moved to Rome and started a new transplant program at the International Centre for Transplantation in Thalassemia and Sickle Cell Anemia in the Mediterranean Institute of Hematology. While the Pesaro experience was predominantly with an Italian patient population, the Rome experience is characterized by two peculiarities: the patients were ethnically very heterogeneous, and the vast majority of these patients were not regularly transfused/chelated, and therefore were highly sensitized due to red blood cell transfusions without leucodepletion filters. Therefore, these patients could have a higher risk of graft rejection as a result of sensitization to HLA antigens. From June 2004 through February 2007, 32 class 1 and class 2 patients with median age of 5 (range 2–16) years, and 31 class 3 patients with median age of 10 (range 5–16) years were given BMT from HLAmatched related donors in our center. Class 1 and class 2 patients under 4 years of age were given a total dose of BU 14 mg/kg plus CY 200 mg/ kg and thiotepa 10 mg/kg, while patients aged 4 years or over received BU 14 mg/kg plus CY 200 mg/kg as a preparatory regimen. Prophylaxis against GVHD consisted of CSP, a low dose of methylprednisolone and a short course of MTX. Class 3 patients were prepared for BMT according to Protocol 26. Since June 2006, all patients have been given a targeted dose of intravenous Busilvex instead of oral BU. The probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality of class 1 and class 2 patients aged less than 17 years are 100%, 94%, 6%, and 0% respectively (Fig. 73.2). The probabilities of survival, thalassemia-free survival, nonrejection mortality, and rejection for class 3 patients were 87%, 80%, 12%, and 10% respectively (Fig. 73.3). The Rome experience confirmed the results obtained in Pesaro and, most

0.6 0.4 0.2

Transplant-related mortality

12% 10%

Rejection

0 0

0.5

1

1.5

2

2.5

3

Years

Fig. 73.3 Estimates of survival, thalassemia-free survival, nonrejection mortality, and rejection for 31 class 3 patients younger than 17 years who were treated with the regimen prescribed by Protocol 26 (four out of 31 patients who received a transplant from a human leukocyte antigen phenotypically identical donor were given busilvex, cyclophosphamide 200 mg/kg, thiotepa 10 mg/kg, and antithymocyte globulin 12 mg/kg).

3.5

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Haploidentical T-cell-depleted HCT from mother to child using a megadose of CD34 cells is under study at our center. The use of unrelated donors. Results of HCT from unrelated donors for the treatment of malignant disease have improved steadily, mainly due to the introduction of high-resolution molecular techniques for histocompatibility testing, and improvements in the management of posttransplant complications. These results have stimulated the use of HCT from unrelated donors for the treatment of hemoglobinopathies. The Italian cooperative group for BMT (Cagliari, Pavia, Pesaro, and others) has recently reported results obtained in 32 thalassemic patients (aged 2–28 years) who received bone marrow from unrelated donors selected for similarity with the recipients for extended HLA haplotypes [67]. Four patients were class 1, 11 were class 2, and 17 were class 3 candidates with respect to the outcome of BMT. Twenty-eight patients received BU 14 mg/kg and thiotepa 10 mg/kg. Of these 28 patients, the class 1 and class 2 patients received BU 14 mg/kg, thiotepa 10 mg/kg, and CY 200 mg/kg, and the class 3 patients received BU 14 mg/kg, thiotepa 10 mg/kg and CY 160 mg/kg, or CY 120 mg/kg if they were over 16 years of age. Prophylaxis against GVHD was with CSP and a short course of MTX. The probability of thalassemia-free survival was 66% for the entire group, and the mortality from causes other than rejection was 25%. Although the difference was not significant, five of the six patients who died belonged to class 3. The incidences of grade II–IV acute and of chronic GVHD were 41% and 25%, respectively. Patients who shared at least one extended haplotype had less acute or chronic GVHD and better survival. Updating the observations of these three centers to 44 patients, the probabilities of survival, thalassemia-free survival, rejection, and nonrejection mortality are 79%, 68%, 12%, and 23%, respectively. These data are encouraging and show that the results of HCT from well-selected unrelated donors might be comparable to those of HLA-identical sibling transplants, especially in patients with less advanced disease. Recently, this group has reported data on 27 adult thalassemia patients (aged 17–37 years) transplanted from unrelated donors selected by highresolution HLA molecular typing [68]. Fifteen patients received BU 14 mg/kg, thiotepa 10 mg/kg and CY 160 mg/kg (nine patients) or CY 120 mg/kg (six patients). In view of the high mortality in these patients, the subsequent 12 patients were given BU 14 mg/kg and CY 120 mg/kg. GVHD prophylaxis consisted of CSP and a short course of MTX. Three patients were also given antithymocyte globulin. The probabilities of survival, thalassemia-free survival, transplant-related mortality, and rejection were 70%, 70%, 30%, and 4% respectively. The incidences of grade II–IV acute and chronic GVHD were 37% and 27%, respectively. Although limited, these data show that unrelated marrow transplantation not only in younger patients, but also in adult patients from wellselected donors may offer a success rate similar to that obtained from HLA-identical sibling transplantation. The main limitation of the experience with unrelated BMT for thalassemia is that, using such stringent criteria, only about one-third of thalassemia patients who started the search found a suitable donor in a median time of 3–4 months. One possibility to increase the donor pool could be adopting less stringent criteria of HLA matching for donor selection, allowing one- or two-allele disparity, but the results of such transplants remain to be determined. HSC transplantation from an unrelated cord blood (UCB) donor is now standard practice for the treatment of hematologic malignancies. The number of UCB transplants has increased dramatically, because several studies have demonstrated that results from HLA-mismatched UCB transplants were comparable to those from HLA-matched unrelated bone marrow transplants in children [69]. Advantages such as

faster availability, tolerance of one to two HLA mismatches, and a low incidence of acute GVHD have made UCB transplants attractive for patients with some nonmalignant diseases [70]. Five patients (aged 2.3–11.4 years) with β-thalassemia major were given unrelated cord blood transplantation in Taiwan [71]. Cord blood units were mismatched for two loci in two recipient–donor pairs, and the remaining three pairs had mismatchs at one locus. Patients were given BU 14 mg/kg, CY 200 mg/kg, and antithymocyte globulin 30 mg/kg as conditioning, and CSP and methylprednisolone as GVHD prophylaxis. Nucleated cell dose ranged from 3.25 to 11.8 × 107/kg. All patients had sustained engraftment with complete donor chimerism. Three patients had grade II–III acute GVHD responsive to steroid treatment. Updating the observations from this group to 36 patients with transfusion-dependent thalassemia and two with sickle cell disease who received unrelated cord blood transplants in 14 transplant centers the probabilities of survival and thalassemia-free survival are 77 ± 7% and 65 ± 8%, respectively [72]. Results were better in experienced centers (more than five UCB transplants for hemoglobinopathies), with overall survival and thalassemia-free survival of 87 ± 7% and 77 ± 9%, respectively. These data are very encouraging and show that patients who need transplantation and do not have a suitable either related or unrelated matched donor can benefit from unrelated cord blood transplantation. Use of reduced-intensity HSC transplantation. Unlike hematologic malignancies, where mixed chimerism may predict relapse, the development of stable mixed chimerism in nonmalignant disorders has a potential for an ameliorative effect that has been well documented for β-thalassemia major and sickle cell anemia [63,73]. Stable mixed chimerism is not uncommon in thalassemia major patients after transplantation. It appears that when stable mixed chimerism is established, even a minority of donor cells is sufficient to overcome an underlying genetic defect. These observations have provided the rationale for using reducedintensity preparatory regimens in patients with thalassemia or sickle cell anemia, which might reduce the toxicity of transplantation, especially in patients with advanced disease. Reduced-intensity conditioning followed by allogeneic transplantation has been tested in limited clinical series that rely upon immunosuppressive rather than ablative preparatory regimens to facilitate donor cell engraftment. Recently, there has been a report on seven patients (aged 3–20 years) with sickle cell anemia or thalassemia who were given stem cell transplantation following a preparative regimen consisting of fludarabine, low-dose (i.e. 200 cGy) TBI, and antithymocyte globulin (in two patients) [74]. Although transplant-related toxicity was minimal, and many patients had transient engraftment, all eventually rejected their donor graft. In a recent study, results of reduced-intensity HCT using peripheral blood stem cells from siblings or unrelated matched donors after preparatory regimen with fludarabine (180 mg/m2 total dose), intravenous BU (6.4 mg/kg total dose) and an equine antithymocyte globulin (160 mg/kg total dose) in patients with sickle cell anemia (three patients) or β-thalassemia (one patient) were not encouraging: only one out of four patients had sustained engraftment [75]. These results indicate that stable donor engraftment after reducedintensity HSC transplantation is difficult to achieve among immunocompetent patients with hemoglobinopathies. Therefore, patients with thalassemia or sickle cell disease need more intensive myeloablative conditioning regimens because graft failure in these patients is not a negligible problem even when a conventional myeloablative preparative regimen is administered [3,4,67]. Reduced-intensity conditioning regimens should be studied in selected higher-risk thalassemia patients with advanced disease along with organ damage, who might have a higher transplant-related toxicity after conventional myeloablative transplantation.

Hematopoietic Cell Transplantation for Thalassemia Table 73.2 Transplants for thalassemia: reports from centers other than Pesaro Center

Patients

Survival

Disease-free survival

Pescara [76] Cagliari [77] USA [78] UK [79] Tehran [80] Vellore [81] Malaysia [82] Hong Kong [83] Bangkok [84] Thailand [85]

102 37 68 54 60 50 28 44 21 28

0.91 0.88 0.94/0.81* 0.95 0.83 0.76 0.86 0.86 0.76 0.92

0.87 0.88 0.81/0.57* 0.82 0.73 0.68 0.75 0.82 0.53 0.82

* By risk category, survival = best risk/worst risk.

Experience at other transplant centers Many reports describe the experience of other groups in transplanting patients for the treatment of thalassemia (Table 73.2) [76–85]. In Italy, the Pescara group has been particularly active, reporting 102 patients transplanted through August 1996 [76].

Management of the ex-thalassemic patient after HCT After transplantation, ex-thalassemic patients still carry the clinical complications acquired during years of transfusion and chelation therapy. Among the issues requiring long-term management in such patients are iron overload, chronic hepatitis, liver fibrosis, and endocrine dysfunction. There is no reason to expect that BMT will eliminate the excess iron acquired during years of thalassemia, since spontaneous iron elimination occurs very slowly. In our experience, serum ferritin and transferrin saturation spontaneously return to normal levels only in class 1 patients [86]. Persisting tissue iron overload can cause significant morbidity and mortality similar to that seen in hereditary hemochromatosis [87]. Thus, iron removal is indicated in all transplanted thalassemic patients who have evidence of persisting iron overload, and initiating a sequence of phlebotomies or restarting chelation with DFO at 18 months post HCT achieves this best. Excess iron can be completely removed from the body, and a body iron content within the normal range can be achieved. The necessary duration of treatment is directly correlated with the magnitude of the iron overload, and ranges from a few months to several years [88]. In most ex-thalassemics, reduction or normalization of the iron pool results in marked improvement in serum levels of liver enzymes and in the histologic activity index [89]. The serum aminotransferases and histologic activity index normalized after iron depletion in about half of the patients who were seropositive for hepatitis C virus (HCV), suggesting that iron is a cofactor of HCV for liver disease [89]. Ex-thalassemics with early cardiac involvement, characterized by systolic and/or diastolic dysfunction, show complete regression of these subclinical cardiac abnormalities after iron depletion [90]. Infection with HCV is common in thalassemic patients, particularly in those transfused before second-generation enzyme-linked immunosorbent assay tests became available for detecting HCV in donated blood. In thalassemia, liver damage due to HCV infection is exacerbated by iron overload, and liver disease is a recognized cause of mortality and morbidity. In fact, analysis of the natural history of liver fibrosis following BMT for thalassemia showed that iron overload and HCV

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infection are independent risk factors for progression of liver fibrosis, and their concomitant presence results in a striking increase in risk [91]. All the donors in this series of patients were HCV seronegative. After HCT, approximately 10–15% of HCV-infected patients become HCV seronegative [92]. This may reflect complete elimination of the virus, but we cannot be sure of this. However, seropositivity has not returned in these patients. The chronic risk of conditions associated with HCV infection, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma, persists in the remaining patients. The precise risk of developing one of these complications in ex-thalassemic patients is uncertain. Transplanted thalassemics have a long, probably normal life expectancy, and mild chronic liver disease has to be considered in this perspective. Chronic HCV infection and transplant-related complications are probably the only factors that may limit survival in ex-thalassemics. Thus, avoidance of progression of liver damage to cirrhosis must be a primary goal. Treatment for chronic hepatitis C has improved over the last two decades. Present standard therapy is peginterferon alfa/ribaverin for adults and children without thalassemia [93]. Concern about ribaverin-induced hemolysis and worsening of the anemia and iron overload limit the use of this therapy in patients with thalassemia. Ex-thalassemics with complete donor chimerism and chronic hepatitis C will experience a spectrum of side-effects similar to that of patients without thalassemia. Therefore ex-thalassemic patients should be offered treatment with the peginterferon alfa/ribaverin combination after they have completed their iron depletion program. It is well known that growth failure and endocrine dysfunction are common in thalassemia major patients treated by conventional treatment. While the endocrine dysfunction is usually due to iron overload as a result of chronic transfusions, lifelong DFO treatment also plays a negative role in growth failure in these patients. Although the role of BU-containing regimens on growth velocity after transplant remains controversial, the negative effect of this regimen on gonadal damage is well documented. Age at the time of transplant in β-thalassemia patients is an important predictor of growth. In fact, children receiving a transplant before 8 years of age showed a normal growth rate, while older children, class 3 patients, and patients who developed chronic GVHD had impaired growth [94]. Gonadal damage is a common side-effect of BU/CY conditioning. Indeed, approximately one-third of boys and twothirds of girls failed to spontaneously enter puberty following transplantation [95]. Nevertheless, some patients can restore their fertility after transplant, which is supported by our observations of five successful pregnancies and three spontaneous paternities in our patients. These data demonstrate that patients exposed to the BU/CY regimen are not inevitably infertile. One of the major late complications of BMT is chronic GVHD, which is the principal cause of morbidity and nonrelapse mortality. Most of our patients developed a limited form of chronic GVHD, and the probability of the moderate or severe form was only 8% and 2%, respectively [96]. With the increasing number of transplant survivors, there is a risk for secondary malignancies. Patients who received BMT for β-thalassemia and sickle cell disease in Pesaro had a low (0.8%) incidence of malignancies. The types of malignancy observed in our patients consisted of three early and one late non-Hodgkin’s lymphoma, and four solid tumors (squamous cell carcinoma, Kaposi’s sarcoma, melanoma, and colon cancer, respectively). Four of these patients are alive and well.

The role of HCT in the treatment of thalassemia HCT is the only treatment able to definitively cure thalassemia. The results of transplantation from HLA-identical family members are clear.

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Chapter 73

Class 1 patients have a very high probability of cure, with a very low early and late morbidity and mortality. There is no reason to deny these patients the advantages of a life free from daily tedious, expensive, and uncomfortable therapy. We still do not know the probability that a patient receiving conventional therapy will deteriorate into a worse risk category, but the fact is that transplant centers are often confronted with patients in risk classes 2 and 3 who represent failures of conventional

treatment. Delaying transplantation until the patient is in a risk category beyond class 1 substantially reduces the probability of transplant success and jeopardizes the reversibility of liver and cardiac damage. We therefore believe that all patients with β-thalassemia who have HLA-identical related donors should be transplanted as soon as possible. Patients without such donors who have well-selected unrelated donors should also be considered for HCT.

References 1. Weatheral DJ, Clegg JB. The Thalassemia Syndromes, 4th ed. Oxford: Blackwell Science; 2001. 2. Thomas ED, Buckner CD, Sanders JE et al. Marrow transplantation for thalassaemia. Lancet 1982; ii: 227–9. 3. Lucarelli G, Galimberti M, Polchi P et al. Marrow transplantation in patients with advanced thalassemia. N Engl J Med 1987; 316: 1050–5. 4. Lucarelli G, Galimberti M, Polchi P et al. Bone marrow transplantation in patients with thalassemia. N Engl J Med 1990; 322: 417–21. 5. Angastiniotis M, Modell B. Global epidemiology of hemoglobin disorders. Ann NY Acad Sci 1998; 850: 251–69. 6. Modell B, Khan M, Darlison M et al. A national register for surveillance of inherited disorders: β thalassaemia in the United Kingdom. Bull World Health Organ 2001; 79: 1006–13. 7. Clark BE, Thein SL. Molecular diagnosis of hemoglobin disorders. Clin Lab Haem 2004; 26: 159– 76. 8. Thein SL. Genetic modifiers of β-thalassemia. Haematologica 2005; 90: 649–60. 9. Schrier SL. Pathophysiology of thalassemia. Curr Opin Hematol 2002; 9: 123–6. 10. Cunningham MJ, Macklin EA, Neueld EJ, Cohen AR. Thalassemia Clinical Research Network. Complications of beta-thalassemia major in North America. Blood 2002; 99: 36–43. 11. Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood 2002; 99: 36–43. 12. Wolman IJ. Transfusion therapy in Cooley’s anemia. Growth and health as related to long-range hemoglobin levels, a progress report. Ann NY Acad Sci 1964; 119: 736–47. 13. Piomelli S, Danoff SJ, Becker MH, Lipera MJ, Travis SF. Prevention of bone malformations and cardiomegaly in Cooley’s anemia by early hypertransfusion regimen. Ann NY Acad Sci 1969; 165: 427–36. 14. Piomelli S, Hart D, Graziano J, Karpatkin M, McCarthy K. Current strategies in the management of Cooley’s anemia. Ann NY Acad Sci 1985; 445: 256–67. 15. Piomelli S, Graziano J, Karpatkin M et al. Chelation therapy, transfusion requirement, and iron balance in young thalassemic patients. Ann NY Acad Sci 1980; 344: 409–17. 16. Gabutti V, Piga A. Results of long-term iron-chelating therapy. Acta Haematol 1996; 95: 26–36. 17. Cazzola M, Borgna-Pignatti C, Locatelli F, Ponchio L, Beguin Y, De Stefano P. A moderate transfusion regimen may reduce iron loading in beta-thalassemia major patients without producing excessive expansion of erythropoiesis. Transfusion 1997; 37: 135–40. 18. Borgna-Pignatti C, Rugolotto S, DeStefano P et al. Survival and complications in patients with thalassemia major treated with transfusions and deferrioxamine. Haematologica 2004; 89: 1187–93.

19. Kontoghiorghes GJ, Aldouri MA, Hoffbrand AV et al. Effective chelation of iron in beta thalassemia with the oral chelator 1,2-dimethyl-3-hydroxypyri4-1. Br Med J 1987; 295: 1509–12. 20. Fischer R, Longo P, Nielsen P et al. Monitoring long-term efficacy of iron chelation therapy by deferiprone and desferrioxamine in patients with beta-thalassemia major: application of SQUID biomagnetic liver susceptometry. Br J Haematol 2003; 121: 938–48. 21. Anderson LJ, Wonke N, Prescott E, Holden S, Walker JM, Pennel DJ. Comparison of effects of oral deferiprone and subcutaneous desferrioxamine on myocardial iron concentrations and ventricular function in β-thalassemia major. Lancet 2002; 360: 516–20. 22. Pennell DJ, Berdoukas V, Karagiorga M et al. Randomized controlled trial of deferiprone or deferoxamine in beta-thalassemia major patients with asymptomatic myocardial siderosis. Blood 2006; 107: 3738–44. 23. Nick H-P, Acklin P, Lattmann R et al. Development of tridentate iron chelators: from desferriothiocin to ICL670. Curr Med Chem 2003; 10: 1065– 76. 24. Cappellini MD, Cohen A, Piga A et al. A phase 3 study of deferasirox (ICL670), a once-daily oral iron chelator, in patients with beta-thalassemia. Blood 2006; 107: 3455–62. 25. Pavlec I, Papayannopoulou T, Maury C, Meyer F. A human beta-globin gene fused to the human beta globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells. Blood 1993; 81: 1384–92. 26. May C, Rivella S, Callegari J et al. Therapeutic haemoglobin synthesis in beta-thalassemic mice expressing lentivirus-encoded human beta-globin. Nature 2000; 406: 82–6. 27. Malik P, Arumugam PI. Gene therapy for β-thalassemia. Hematology Am Soc Hematol Educ Program 2005: 45–50. 28. Kuliev A, Recitsky S, Verlinsky O et al. Preimplantation diagnosis and HLA typing for haemoglobin disorders. Rep Bio Online 2005; 11: 362–70. 29. Slavin S, Or R, Cividalli G et al. Bone marrow transplantation in β-thalassemia with prevention of graft-vs.-host disease. In: Fucharoen S, Rowley PT, Paul NW, editors. Birth Defects: Original Article Series. Thalassemia: Pathophysiology and Management. Vol. 23, Part 5B. New York: Alan R Liss; 1988. pp. 313–16. 30. Piga A, Longo F, Consolati A. Mortality and morbidity in thalassemia with conventional treatment. Bone Marrow Transplant 1977; 19(Suppl 2): 11–13. 31. Modell B, Khan M, Darlison M. Survival in betathalassemia major in the UK: data from the UK Thalassemia Register. Lancet 2000; 355: 2051– 2.

32. Olivieri NF, Nathan DG, MacMillan JH et al. Survival in medically treated patients with homozygous beta-thalassemia. N Engl J Med 1994; 331: 574–8. 33. Witherspoon RP, Fisher LD, Schoch G et al. Secondary cancers after bone marrow transplantation for leukemia or aplastic anemia. N Engl J Med 1989; 321: 784–9. 34. Socié G, Henry-Amar M, Cosset JM, Devergie A, Girinsky T, Gluckman E. Increased incidence of solid malignant tumors after bone marrow transplantation for severe aplastic anemia. Blood 1991; 78: 277–9. 35. Witherspoon RP, Storb R, Pepe M, Longton G, Sullivan KM. Cumulative incidence of secondary solid malignant tumors in aplastic anemia patients given marrow grafts after conditioning with chemotherapy alone [Letter]. Blood 1992; 79: 289–92. 36. Parkman R, Rappeport J, Geha R et al. Complete correction of the Wiskott-Aldrich syndrome by allogeneic bone-marrow transplantation. N Engl J Med 1978; 298: 921–7. 37. Kapoor N, Kirkpatrick D, Oleske J et al. Reconstitution of normal megakaryocytopoiesis and immunologic functions in Wiskott-Aldrich syndrome by marrow transplantation following myeloablation and immunosuppression with busulfan and cyclophosphamide. Blood 1981; 57: 692–6. 38. Hobbs JR, Hugh-Jones K, Shaw PJ, Downie CJC, Williamson S. Engraftment rates related to busulphan and cyclophosphamide dosages for displacement bone marrow transplants in fifty children. Bone Marrow Transplant 1986; 1: 201–8. 39. Thomas ED, Buckner CD, Storb R et al. Aplastic anaemia treated by marrow transplantation. Lancet 1972; i: 284–9. 40. Storb R, Champlin RE. Bone marrow transplantation for severe aplastic anemia. Bone Marrow Transplant 1991; 8: 69–72. 41. Santos GW, Sensenbrenner LL, Burke PJ et al. Marrow transplantation in man following cyclophosphamide. Transplant Proc 1971; 3: 400–4. 42. Tutschka PJ, Elfenbein GJ, Sensenbrenner LL et al. Preparative regimens for marrow transplantation in acute leukemia and aplastic anemia. Baltimore experience. Am J Pediatr Hematol Oncol 1980; 2: 363–70. 43. Santos GW, Tutschka PJ, Brookmeyer R et al. Marrow transplantation for acute nonlymphocytic leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med 1983; 309: 1347–53. 44. Appelbaum FR, Storb R, Ramberg RE et al. Allogeneic marrow transplantation in the treatment of preleukemia. Ann Intern Med 1984; 100: 689–93. 45. Tutschka PJ, Copelan EA, Kapoor N. Replacing total body irradiation with busulfan as conditioning of patients with leukemia for allogeneic marrow transplantation. Transplant Proc 1989; 21: 2952–4. 46. Tutschka PJ, Copelan EA, Kapoor N, Avalos BR, Klein JP. Allogeneic bone marrow transplantation

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48.

49.

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62.

63.

for leukemia using chemotherapy as conditioning. Six-year results of a single institution trial. Transplant Proc 1991; 23: 1709–10. Floersheim GL, Elson LA. Restoration of hematopoiesis following a lethal dose of dimethyl myleran by isologic bone marrow transplantation in mice. Experiments on modification of intolerance to homologous bone marrow by 6-mercaptopurine, amino-chlorambucil and cortisone. Acta Haematol 1961; 26: 233–45. Storb R, Weiden PL, Graham TC, Lerner KG, Nelson N, Thomas ED. Hemopoietic grafts between DLA-identical canine littermates following dimethyl myleran. Evidence for resistance to grafts not associated with DLA and abrogated by antithymocyte serum. Transplantation 1977; 24: 349–57. Tutschka PJ, Copelan EA, Klein JP. Bone marrow transplantation for leukemia following a new busulfan and cyclophosphamide regimen. Blood 1987; 70: 1382–8. Storb R, Thomas ED, Buckner CD et al. Marrow transplantation in thirty “untransfused” patients with severe aplastic anemia. Ann Intern Med 1980; 92: 30–6. Storb R, Prentice RL, Thomas ED et al. Factors associated with graft rejection after HLA-identical marrow transplantation for aplastic anaemia. Br J Haematol 1983; 55: 573–85. Bearman SI, Appelbaum FR, Buckner CD et al. Regimen-related toxicity in patients undergoing bone marrow transplantation. J Clin Oncol 1988; 6: 1562–8. Bearman SI, Appelbaum FR, Back A et al. Regimenrelated toxicity and early posttransplant survival in patients undergoing marrow transplantation for lymphoma. J Clin Oncol 1989; 7: 1288–94. Lucarelli G, Polchi P, Izzi T et al. Allogeneic marrow transplantation for thalassemia. Exp Hematol 1984; 12: 676–81. Lucarelli G, Polchi P, Galimberti M et al. Marrow transplantation for thalassemia following busulphan and cyclophosphamide. Lancet 1985; i: 1355–7. Lucarelli G, Galimberti M, Polchi P et al. Bone marrow transplantation in thalassemia. Hematol Oncol Clin North Am 1991; 5: 549–56. Lucarelli G, Andreani M, Angelucci E. The cure of thalassemia by bone marrow transplantation. Blood Rev 2002; 16: 81–5. Sodani P, Gaziev J, Polchi P et al. New approach for bone marrow transplantation in patients with class 3 thalassemia aged younger than 17 years. Blood 2004; 104: 1201–3. Lucarelli G, Galimberti M, Polchi P et al. Bone marrow transplantation in adult thalassemia. Blood 1992; 80: 1603–7. Lucarelli G, Clift RA, Galimberti M et al. Bone marrow transplantation in adult thalassemic patients. Blood 1999; 93: 1164–7. Gaziev J, Sodani P, Polchi P, Andreani M, Lucarelli G. Bone marrow transplantation in adults with thalassemia. Treatment and long-term follow-up. Ann NY Acad Sci 2005; 1054: 196–205. Gaziev D, Polchi P, Lucarelli G et al. Second marrow transplants for graft failure in patients with thalassemia. Bone Marrow Transplant 1999; 24: 1299–306. Andreani M, Nesci S, Lucarelli G et al. Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation. Bone Marrow Transplant 2000; 25: 401–4.

64. Andreani M, Manna M, Lucarelli G et al. Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia. Blood 1996; 87: 3494–9. 65. Giardini C, Visani G, Lucesole M et al. Unusual late rejection of the first graft seven years after allogeneic transplant for thalassemia successfully treated with second BMT. Bone Marrow Transplant 2007; 39(Suppl 1): S191. 66. Gaziev D, Galimberti M, Lucarelli G et al. Bone marrow transplantation from alternative donors for thalassemia: HLA-phenotypically identical relative and HLA nonidentical sibling or parent transplants. Bone Marrow Transplant 2000; 25: 815–21. 67. La Nasa G, Giardini C, Argiolu F et al. Unrelated donor bone marrow transplantation for thalassemia: the effect of extended haplotypes. Blood 2002; 99: 4350–6. 68. La Nasa G, Caocci G, Argiolu F et al. Unrelated donor stem cell transplantation in adult patients with thalassemia. Bone Marrow Transplant 2005; 36: 971–5. 69. Rocha V, Cornish J, Sievers EL, Filipovich A, Locatelli F, Peters C. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001; 97: 2962–71. 70. Wagner JE, Barker JN, DeFor TE, Bker KS, Blazar BR, Edie C. Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival. Blood 2002; 100: 1611–18. 71. Jaing T-H, Hung I-J, Yang CH-P, Chen S-H, Sun C-F, Chow R. Rapid and complete donor chimerism after unrelated mismatched cord blood transplantation in 5 children with β-thalassemia major. Biol Blood Marrow Transplant 2005; 11: 349–53. 72. Jaing T-H, Tan P, Rosenthal J et al. Unrelated cord blood transplantation (CBT) for thalassemia. Blood 2006; 108: 11. 73. Walters M, Ptience M, Leisenring W et al. Multicenter investigation of bone marrow transplantation for sickle cell disease. Stable mixed haematopoietic chimerism after bone marrow transplantation for sickle cell anemia. Biol Blood Marrow Transplant 2001; 7: 665–73. 74. Iannone R, Casella JF, Fuchs EJ et al. Results of minimally toxic nonmyeloablative transplantation in patients with sickle cell anemia and beta-thalassemia. Biol Blood Marrow Transplant 2003; 9: 519–28. 75. Jacobsohn DA, Duerst R, Tse W, Kletzel M. Reduced intensity haemopoietic stem-cell transplantation for treatment of non-malignant diseases in children. Lancet 2004; 364: 156–62. 76. Di Bartolomeo P, Di Girolamo G, Olioso P et al. The Pescara experience of allogeneic bone marrow transplantation in thalassemia. Bone Marrow Transplant 1997; 19(Suppl 2): 48–53. 77. Argiolu F, Sanna MA, Cossu F et al. Bone marrow transplant in thalassemia. The experience of Cagliari. Bone Marrow Transplant 1997; 19(Suppl 2): 65–7. 78. Clift RA, Johnson FL. Marrow transplants for thalassemia. The USA experience. Bone Marrow Transplant 1997; 19(Suppl 2): 57–9. 79. Lawson SE, Roberts IAG, Amrolia P, Dokal I, Szydlo R, Darbyshire PJ. Bone marrow transplantation for β-thalassemia major: the UK experience in two paediatric centers. Br J Haematol 2003; 120: 289–95.

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80. Ghavamzadeh A, Bahar B, Djahani M, Kokabandeh A, Shahriari A. Bone marrow transplantation of thalassemia, the experience in Tehran (Iran). Bone Marrow Transplant 1997; 19(Suppl 2): 71–3. 81. Dennison D, Srivastava A, Chandy M. Bone marrow transplantation for thalassaemia in India. Bone Marrow Transplant 1997; 19(Suppl 2): 70. 82. Lin HP, Chan LL, Lam SK, Ariffin W, Menaka N, Looi LM. Bone marrow transplantation for thalassemia. The experience from Malaysia. Bone Marrow Transplant 1997; 19(Suppl 2): 74–7. 83. Li CK, Shing MK, Chik KW et al. Haematopoietic stem cell transplantation for thalassaemia major in Hong Kong: prognostic factors and outcome. Bone Marrow Transplant 2002; 29: 101–5. 84. Issaragrisil S, Suvatte V, Visuthisakchai S et al. Bone marrow and cord blood stem cell transplantation for thalassemia in Thailand. Bone Marrow Transplant 1997; 19(Suppl 2): 54–6. 85. Hongeng S, Pakakasama S, Chuansumrit A et al. Outcomes of transplantation with related and unrelated-donor stem cells in children with severe thalassemia. Biol Blood Marrow Transplant 2006; 12: 683–7. 86. Lucarelli G, Angelucci E, Giardini C et al. Fate of iron stores in thalassemia after bone marrow transplantation. Lancet 1993; 342: 1388–91. 87. Niederau G, Fischer R, Purschel A, Stremmel W, Haussinger D, Strohmeyer G. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996; 110: 1107–19. 88. Angelucci E, Muretto P, Lucarelli G et al. Phlebotomy to reduce iron overload in patients cured of thalassemia by bone marrow transplantation. Italian Cooperative Group for Phlebotomy Treatment of Transplanted Thalassemia Patients. Blood 2002; 90: 994–8. 89. Angelucci E, Muretto P, Lucarelli G et al. Treatment of iron overload in the “ex-thalassemic”. Report from the phlebotomy program. Ann NY Acad Sci 1998; 30: 288–93. 90. Mariotti E, Angelucci E, Agostini A, Baronciani D, Sgarbi E, Lucarelli G. Evaluation of cardiac status in iron-loaded thalassemia patients following bone marrow transplantation: improvement in cardiac function during reduction in body iron burden. Br J Haematol 1998; 103: 916–21. 91. Angelucci E, Muretto P, Nicolucci A et al. Effects of iron overload and hepatitis C virus positivity in determining progression of liver fibrosis in thalassemia following bone marrow transplantation. Blood 2002; 100: 17–21. 92. Erer B, Angelucci E, Lucarelli G et al. Hepatitis C virus infection in thalassemia patients undergoing allogeneic bone marrow transplantation. Bone Marrow Transplant 1994; 14: 369–72. 93. Strader DB, Wright T, Thomas DL, Seef LB. Diagnosis, management, and treatment of hepatitis C. Hepatology 2004; 39: 1147–71. 94. Gaziev D, Galimberti M, Giardini C, Baronciani D, Lucarelli G. Growth in children after bone marrow transplantation for thalassemia. Bone Marrow Transplant 1993; 12(Supp l1): 100–1. 95. De Sanctis V, Galimberti M, Lucarelli G et al. Growth and development in ex-thalassemic patients. Bone Marrow Transplant 1997; 19(Suppl 2):126–7. 96. Gaziev D, Polchi P, Galimberti M et al. Graftversus-host disease after bone marrow transplantation for thalassemia: an analysis of incidence and risk factors. Transplantation 1997; 63: 854–60.

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Mark C. Walters

Hematopoietic Cell Transplantation for Sickle Cell Disease

Introduction Sickle cell disease (SCD) is caused by a single point mutation in codon 6 of the β-globin chain. This mutation directs an amino acid substitution of valine for glutamic acid which promotes the formation of long hemoglobin polymers under hypoxic conditions [1–3]. This propensity for polymer formation deforms the red blood cell (RBC) and causes significant alterations in red cell integrity, rheologic properties, and life span [4,5]. It is probably directly or indirectly responsible for the vasculopathy that forms in virtually all body organs. This heritable disorder of hemoglobin affects one in every 375 African-Americans in the United States. Worldwide, an estimated 350,000 infants are born annually with a clinically significant hemoglobinopathy, 75% of whom inherit SCD. Ever since the initial report in 1984 of successful hematopoietic cell transplantation (HCT) in a child who had acute myelogenous leukemia and SCD who was cured of both, hematologists and transplantation physicians have struggled with how to make this curative therapy available to a wider group of affected individuals [6,7]. During the intervening years, a better understanding of the natural history of SCD has emerged, and views about who might benefit most from this intensive procedure have evolved to include more, not fewer, individuals [8,9]. To illustrate how progress in understanding the pathophysiology of vasoocclusion and the response of clinical complications such as stroke to conventional therapy have helped clarify who might best benefit from HCT, updates in the areas of pulmonary and central nervous system disease are provided here, as are updates in the supportive management of other clinical vaso-occlusive events. This acts as a preamble for the application of HCT for the indications of stroke, recurrent vasoocclusive events, and other clinically significant complications that impact life span and quality of life. Ultimately, as most patients who might otherwise be suitable candidates for HCT lack a sibling donor, the most important progress must occur in the arena of alternative donor HCT for SCD, which remains in a nascent phase of development. Thus, this chapter concludes with reference to future clinical trials that might expand HCT to include more of those who might benefit.

Epidemiology and etiology Although first reported in the medical literature in 1910 as a disorder characterized by its peculiar alteration in RBC morphology, SCD was Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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recognized many generations earlier in Western Africa, where it was called by various local names that reflected its most common feature, which is recurrent pain. Thus, well-established tribal names for the disorder including “The Ga Chwechweechwe, the Fante Nwiiwii, the Ewe Nuiduidui, the Akan Ahotutuo all reflect the onomatopoeia of the repetitive gnawing pains characteristic of sickle cell crisis” [10]. The path of discovering the molecular determinant of SCD focused on the most likely suspect, hemoglobin, the most abundant protein in the misshapen RBC. In 1949, Pauling showed that the sickle RBC contained hemoglobin that had an altered electrophoretic mobility, and reasoned that SCD must be a disorder of hemoglobin [1]. Shortly thereafter, Neel and his colleagues showed that RBCs from patients with SCD could be induced to sickle, and also showed that this phenomenon was inherited as an autosomal recessive disorder [11]. Finally, in 1957, Ingram showed that the cause of SCD was an amino acid substitution of valine for glutamic acid at the β-6 position of the globin chain, which subsequently was determined to follow a single base pair change in the coding DNA [3]. Based upon estimates, it appears that the sickle mutation independently originated from regional populations in Africa and Saudi Arabia approximately 70,000–150,000 years ago [12]. It is generally believed that the persistence of the sickle gene in the African and Mediterranean regions follows from its conferring a reduced risk of infection and mortality from infection by Plasmodium falciparum, the malaria parasite [13]. This is true also of thalassemia and glucose-6-phosphate dehydrogenase deficiency traits, whose demographics mimic that of SCD, with a frequency that is greatest in those areas of the world where malaria is endemic. However, infants and young children with sickle cell anemia (homozygous HbSS) in particular are at high risk of dying from malaria, which accounts in part for the significant early mortality of sickle cell anemia in certain regions of Africa [14,15]. Nonetheless, protection from malaria appears to be conferred in a dose-dependent manner relating to the HbS content in the RBC, with those having sickle cell anemia conferring the greatest natural resistance to malaria infection. It is estimated that between 55,000 and 80,000 persons in the United States are affected by SCD, and that 2000 new births occur annually [16]. The frequency of the sickling hemoglobin disorders, as illustrated by the experience of the California Newborn Screening Program, is presented in Table 74.1, and indicates that the majority of affected individuals in the United States are of African descent [17]. However, individuals of Mediterranean, Caribbean South and Central American, Arab, and East Indian descent also are at risk for SCD. Together, these disorders also contribute to a larger national and worldwide public health concern, by virtue of the impact of SCD on individuals and health-care

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Table 74.1 Birth prevalence of β-globin gene variants in a California newborn population, by ethnicity, 1990–96 Gene variant Ethnicity

Hb SS

Hb AS

Hb S-β thalassemia

Hb SC

Hb SD

Hb SE

Asian Asian Indian Black Hispanic Middle Eastern Native American White

0/207,551 0/15,843 1/700 1/45,622 0/21,677 1/16,529 1/158,127

1/1,336 1/725 1/14 1/183 1/360 1/176 1/625

0/207,551 1/15,843 1/4,056 1/729,953 0/21,677 0/16,529 1/553,447

0/207,551 0/15,843 1/1,297 1/364,976 0/21,677 0/16,529 0/1,106,895

0/207,551 0/15,843 1/63,885 1/1,459,900 0/21,677 0/16,529 0/1,106,895

0/207,551 0/15,843 1/63,885 1/1,459,900 0/21,677 0/16,529 0/1,106,895

Reproduced with permission from Lorey et al. [17]. Hb, hemoglobin; Hb S, sickle hemoglobin. Gene variants: Hb SS, sickle cell anemia (includes Hb S/β0-thalassemia); Hb AS, sickle cell trait; Hb SC, sickle hemoglobin C disease; Hb SD, sickle hemoglobin D disease; Hb SE, sickle hemoglobin E disease.

Fig. 74.1 Representation of the change in human globin production, indicating the sites of hematopoiesis, size of the erythrocytes, and erythrocyte morphology during ontogeny. (Reproduced from [125], with permission.)

systems. In 2004, there were an estimated 113,098 hospital stays during which SCD was noted [18]. The total hospital costs in 2004 for hospitalizations principally for SCD were approximately $488 million. Although even, today, HCT is considered in very few affected individuals, it is quite likely that a broader application of this treatment early in the course of the disease might generate considerable health-care cost savings over the long term.

Molecular and cellular biology The genes that encode the α- and β-globin polypeptides that together account for the hemoglobin tetramer reside on human chromosomes 16 and 11, respectively [19]. Expression of the α- and β-globin proteins is coordinately regulated to avoid polypeptide chain imbalance, the proximate cause of ineffective erythropoiesis in profound inborn examples of

chain imbalance such as thalassemia major. The globin loci contain arrays of genes that are expressed sequentially during the course of erythroid ontogeny, and this regulation includes a developmental switch at the transcriptional level of control, which coincides with an alteration in the site of erythropoiesis during embryonic development and in RBC morphology, common characteristics of erythropoiesis in vertebrate animals (Fig. 74.1). With regard to SCD, the most clinically important developmental switch occurs during the transition from fetal γ globin to adult β globin at birth, at which time the antisickling fetal hemoglobin is replaced by the polymer-prone HbS, and the clinical features of SCD begin to emerge. Elucidating the molecular mechanism of the γ to β globin switch has been the focus of intense investigation yet remains elusive. There is general agreement that γ-globin transcription is silenced in erythroid progenitor cells developing in the postnatal marrow, due to either the

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absence of a specific transcriptional activator(s), the presence of a transcriptional repressor(s), the establishment of chromatin conformation that does not support γ-globin transcription, or, combination of some or all these factors. Regardless of the mechanism, the abrogation of this switch with persistent γ-globin transcription has an ameliorative effect on the clinical expression of SCD as occurs when hereditary persistence of fetal hemoglobin (HPFH) is co-inherited with SCD [20]. Accordingly, the γ to β molecular switch has been the target of pharmacologic discovery for drugs that might elicit persistent HbF expression, and of replacement gene therapy to express γ globin or other antisickling globins in gene delivery systems that ensure long-term expression for a potent and durable antisickling effect [21,22]. In the absence of an antisickling hemoglobin, there is a strong propensity for sickle hemoglobin polymerization, and consequently vasoocclusion, which is the clinical hallmark of this disorder. The phenomenon of vaso-occlusion originates in sickle hemoglobin, which, in the deoxygenated state, tends to form polymers that deform the RBC and diminish its biochemical and structural integrity [23]. While the causal relationship between sickle hemoglobin and vaso-occlusion is not disputed, the contributing factors and sequence of events that ultimately impair the flow of blood in both small and large blood vessels are complex but are being elucidated. The deoxygenated sickle polymer induces cellular potassium and water efflux, caused both by acidification and cellular swelling, which activates the potassium–chloride cotransport efflux pathway [24], and by cellular membrane distortion, which, with the accompanying transient calcium influx, activates the calcium-dependent Gardos channel pathway of potassium and water efflux [25,26]. The resulting cellular dehydration increases the cell density and sickle hemoglobin concentration, thereby promoting sickle hemoglobin polymerization. Sickle hemoglobin polymerization also induces perturbations in erythrocyte cellular membranes, which promote adhesive interactions with vascular endothelial cells [27]. These interactions impact factors that control the vascular tone of blood vessels, and generally elicit vasoconstriction [28]. Other cellular blood components are affected by these events, importantly granulocytes and activated platelets that interact with adhesive sickle erythrocytes and endothelial cells [24,29,30]. Reticulocytes released prematurely from marrow also express membrane proteins that promote erythrocyte–endothelial cell interactions [31]. These cellular interactions ultimately generate the release of cytokines that promote an inflammatory response and amplify the cascade of vaso-occlusion and tissue injury. None of these factors alone can explain vaso-occlusion, although they may contribute singly or in a combinatorial manner. As a result, the cause and severity of vaso-occlusion may vary from event to event, and from individual to individual. Another consequence of sickle hemoglobin polymerization is hemolysis, and considerable evidence supports the notion that the most important consequence of hemolysis is vaso-occlusion. This is due to impaired nitric oxide (NO) bioavailability, which appears to be the central feature of endothelial dysfunction [32]. There is resistance to replacement of NO by exogenous NO donors such as sodium nitroprusside and nitroglycerin, and this resistance is correlated with plasma hemoglobin levels [33]. Intravascular hemolysis releases hemoglobin and erythrocyte arginase into blood plasma, where the former scavenges NO, and the latter depletes plasma of L-arginine, the obligate substrate for NO synthase; together, these processes severely reduce NO bioavailability (Fig. 74.2) [34]. Ordinarily, the gas exchange reaction of NO with oxyhemoglobin is inhibited by the diffusion constraints created by an intact RBC membrane. However, intravascular oxyhemoglobin abrogrates this constraint and causes a 1000-fold increased rate of NO destruction [35,36]. As a result, vasodilation is impaired because guanyl cyclase is not activated. An important consequence of this intravascular cascade is pulmonary

hypertension, a common clinical condition in adults with SCD that is associated with a very high risk of mortality in the short-term (see “Clinical description” below) [37]. Platelet activation is another important contributor to vaso-occlusion in SCD [38]. NO synthesized by the endothelium and platelets promotes vasodilation, but inhibits platelet activation, platelet aggregation, and endothelial adhesion in a normal vascular environment [39]. However, this process is abrogated in SCD, in part because NO is depleted. In addition, elevated levels of endothelial activation markers, including soluble vascular cell adhesion molecule-1 (sVCAM-1), have been observed, which also can promote platelet activation [40]. Recently, it was shown that platelet activation is linked to hemolytic rate, suggesting that free hemoglobin might also trigger platelet activation by blocking the inhibitory effect of NO on platelet function [41]. Of interest, there appears to be an association between the severity of pulmonary hypertension and platelet activation, which then might help explain the underlying pathophysiology of this condition, which includes increased vascular tone, vascular proliferation, and in situ thrombosis, all of which might be precipitated by hemolysis-associated NO depletion [42]. A similar pathophysiology occurs in the cerebral arteries after stroke in SCD, which also includes intimal proliferation and arterial thrombosis [43]. Thus, a common thread in the development of sickle vasculopathy has been suggested by these observations in disparate target organs, which is characterized by vasoconstriction, intimal proliferation with smooth muscle hyperplasia, and thrombosis in situ, even though the clinical manifestations of vascular injury in these organs remain quite distinct.

Clinical description A comprehensive discussion of all the clinical complications of SCD is beyond the scope of this chapter; however, this brief description supports the notion that SCD is a serious, life-threatening hematologic disorder that significantly reduces the quality of life for most individuals and is associated with, on average, a 30-year decrement in life span [44]. These clinical observations provided the rationale to conduct clinical trials of HCT as a curative intervention for SCD, with the judgment that the risks inherent to HCT are balanced by the considerable difficulties of living with SCD. Today, the clinical severity of SCD remains an important stimulus for supporting the development of novel transplantation and other approaches that might transform the outlook for individuals who inherit this disease. Pain The most common feature of SCD is pain, which varies by frequency and intensity both within and between individuals and sickle genotypes [45,46]. Febrile illnesses, dehydration, stress, and exposure to extreme temperatures can trigger a painful episode, but more typically an inciting cause is not apparent. In the Cooperative Study of Sickle Cell Disease (CSSCD), a prospective observational study that enrolled 3578 patients with SCD between 1978 and 1988 in an attempt to define sickle-related complications and their rates, 50% of children with sickle cell anemia (HbSS) had had a painful episode, defined as lasting longer than 2 hours and requiring clinical attention in a medical setting, before 4.9 years of age. A small group (5.2%) of individuals had more than 10 painful episodes per year, which accounted for 39% of all the episodes. Adults who had three or more painful episodes during three consecutive years had a higher risk of early mortality compared with adults who had less frequent pain. In addition, very young children who developed dactylitis, which is a painful episode in infancy and early childhood involving the hands and feet, in combination with baseline leukocytosis and

Hematopoietic Cell Transplantation for Sickle Cell Disease

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Sources of Plasma L-Arginine Endogenous Synthesis in Kidney From Citrulline Protein Turnover Diet

VASCULAR COMPARTMENT

CELLULAR COMPARTMENT Decreased L-Arginine Available for Cellular Uptake

Competes With L-Arginine for Cellular Uptake Increased L-Ornithine

Increased Ornithine Synthesis

Decreased Plasma L-Arginine

Plasma L-Arginine

L-Arginine

Urea

Plasma Arginase

Uncoupled Reaction

Release of RBC Arginase

O2 Hemolysis

Cell Free Hemoglobin L-Ornithine

L-Citruline

LUNGS NO Scavenging

Polyamines

NOS

Decreased NO Synthesis

Proline

NO

Decreased NO Superoxide Peroxynitrite

Smooth Muscle Proliferation

Collagen Production and Deposition

Airway Remodeling

Pulmonary Hypertension

Fig. 74.2 Role of hemolysis in nitric oxide (NO) bioavailability. Arginine is synthesized endogenously from citrulline primarily via the intestinal–renal axis. The bioavailability of arginine and NO are decreased by several mechanisms linked to hemolysis in sickle cell disease. The release of erythrocyte arginase during hemolysis increases plasma arginase levels, thereby decreasing the amount available for NO production. Despite a compensatory increase in NO synthase (NOS), NO bioavailability is reduced due to substrate constraints and by NO scavenging by cell-free hemoglobin released during hemolysis. Endothelial dysfunction resulting from NO depletion appears to contribute to the pathogenesis of lung injury and pulmonary hypertension. RBC, red blood cell. (Reproduced from [34], with permission.)

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profound anemia, were more likely to have a morbid outcome, although this experience predated the widespread use of prophylactic pencillin to prevent overwhelming pneumococcal sepsis [47]. Nonetheless, frequent painful episodes, particularly when unresponsive to supportive therapy, have been employed as an indication for HCT due to the diminished quality of life and risk of early mortality these episodes portend. Pulmonary complications Acute chest syndrome and pulmonary hypertension Pulmonary complications, including acute chest syndrome (ACS), pulmonary hypertension, and pulmonary fibrosis, conservatively have been linked as a cause of death in 20–30% of patients with SCD [44,48]. The most frequent and clinically dramatic expression of pulmonary injury in SCD is ACS, a clinical triad of fever, chest pain or other respiratory symptoms, and the appearance of a new radiographic infiltrate. The etiology of ACS is multifactorial, but as often as not, an etiologic agent can be identified if rigorously pursued [49]. In childhood, most episodes are associated with infection, while in adults, ACS generally follows acute painful episodes. Secretory phospholipase A2 is an enzyme that cleaves fatty acids, and this enzyme may be a biomarker for impending ACS [50]. The release of free fatty acids and the proinflammatory arachidonic acid in the lungs likely contributes to the pathogenesis of ACS, especially in cases of pulmonary fat embolism. Transfusions can prevent ACS when administered during a painful episode accompanied by an elevated serum secretory phospholipase A2 level [51]. For purposes of prognosis, children who experience episodes of ACS early are more likely to experience recurrent episodes, which, if not associated with a higher risk of early mortality, certainly portend morbidity that makes interventions such as hydroxyurea and HCT worthy of consideration [9]. In the long term, adult patients in the CSCCD who had recurrent episodes of ACS experienced restrictive pulmonary disease and abnormal diffusing capacity, which are the most frequent abnormalities noted on pulmonary function testing in affected adults [52,53]. Still, the global effect of SCD is expressed best in the finding that 90% of individuals had abnormal pulmonary function test results. The recognition of pulmonary hypertension as a significant pulmonary complication postdates ACS, but now it is more commonly recognized as a clinically significant complication of SCD. Thirty-three percent of adults with SCD screened by Doppler echocardiography have a tricuspid regurgitant jet velocity that exceeds 2.5 m/s, indicative of pulmonary hypertension [37]. Pulmonary hypertension in these patients is associated with a 10-fold increased risk for early mortality, with a mortality rate of at least 40% at 40 months [54]. Of interest, elevated pulmonary arterial pressure does not appear to be associated with recurrent ACS, leukocytosis or frequent vaso-occlusive pain episodes [37]. This lack of association with selected vaso-occlusive events is consistent with a similar high prevalence of pulmonary hypertension in other hereditary hemolytic diseases, such as thalassemia intermedia and paroxysmal nocturnal hemoglobinuria, which lack sickle hemoglobin, vasoocclusive events, and ACS [55,56]. As noted above, hemolysis appears to be mechanistically linked with this clinical complication, a notion that is supported by evidence that elevated serum lactate dehydrogenase (released during hemolysis) also is associated with a high risk of early mortality in adults with SCD [57]. In addition, brain natriuretic peptide, a hormone that is released in response to cardiomyocyte stretch, also provides diagnostic information about the development of pulmonary hypertension and identifies patients at the highest risk of death [58]. Together, these advances are very likely to help identify a new group of patients for whom HCT might provide a protective and curative effect in the long term, and for whom risk–benefit considerations are balanced acceptably.

Stroke The consequence of vaso-occlusion in cerebral arteries is stroke, a leading cause of morbidity and disability in SCD. The risk of stroke in children with SCD in increased by more than 300-fold, making SCD the most common cause of childhood stroke [59]. Clinically overt stroke was observed in 11% of SCD patients by 20 years of age, while another 17–35% had subclinical evidence of “silent” cerebral infarction on brain magnetic resonance imaging (MRI), which is defined as a radiographic abnormality that is not associated with neurologic symptoms or a clinical event [59,60]. The majority of strokes occur in patients with homozygous HbS, and many fewer occur in other SCD genotypes. Co-inherited α-thalassemia appears to confer protection from stroke by possibly improving RBC deformability and decreasing hemolysis [61]. However, previous studies found that α-thalassemia was not a significant risk factor after adjustment for hemoglobin concentration [59,62]. There is also increasing recognition about immune regulation and inflammation with regard to human leukocyte antigen (HLA) genes in diseases such as atherosclerosis and aortic aneurysm [63,64]. There is a predisposition to Moyamoya disease among Japanese children who inherit the class II HLA allele DQB1*0502 [65]. There was also a strong association with stroke in SCD children who inherited this allele [66]. Likewise, unique HLA alleles DPB1*0401 and DPB1*1701 were associated with increased susceptibility to and protection from small cerebral vessel stroke, respectively [66]. With respect to large cerebral vessel disease, HLA A*0102, A*2612, and A*3301 are distinct alleles associated with stroke among SCD patients, and may indicate distinct genetic etiologies for stroke in SCD. In addition, homozygosity at the HLA loci is associated with increased stroke risk, perhaps related to a modulated immunogenic response to foreign antigens. VCAM-1, a gene responsible for expression of adhesion molecules regulating the attachment and migration of leukocytes, is upregulated in response to sickle erythrocytes and appears to be involved in the pathophysiology of SCD-related microvascular occlusion [31]. The VCAM-1 (–1594)C variant was associated exclusively with small-vessel stroke risk in a cohort from the CSSCD [67]. Another group has documented a protective association between the VCAM-11238C variant and stroke [68]. Other single-nucleotide polymorphisms in linkage disequilibrium may be present at this locus and suggest a stroke “haplotype.” To understand better the relationship between these epistatic associations and stroke that have been reported in pilot retrospective analyses, one group recently analyzed 108 single-nucleotide polymorphisms in 39 candidate genes involved in vasoregulation, inflammation, cell adhesion, coagulation, hemostasis, and cell proliferation. Among 1398 SCD patients evaluated, 31 single-nucleotide polymorphisms were identified in 12 genes that interact with fetal hemoglobin to modulate the risk of stroke. A combinatorial association of polymorphisms with stroke successfully predicted the occurrence of stroke in seven of 114 SCD patients with 98.2% accuracy in a validation of this model [69]. Further evaluation and confirmation in larger prospective studies is required to document their utilization as a predictor of stroke risk in SCD. However, these may offer utility also in screening those who might benefit from HCT before the onset of central nervous system injury.

Therapeutic alternatives to HCT RBC transfusions and stroke prevention It is possible to determine the risk for clinical stroke in children with SCD by measuring the flow velocity in the intracranial arteries by a noninvasive method termed transcranial Doppler ultrasonography (TCD) [70]. The risk of stroke is increased when the time-averaged mean of the maximum (TAMM) flow velocity in the distal internal carotid

Hematopoietic Cell Transplantation for Sickle Cell Disease

artery or middle cerebral artery (MCA) exceeds a threshold observed in stenosis. The association of increasing TCD TAMM velocity and increasing risk for stroke formed the basis of the primary stroke prevention trial, the Stroke Prevention Trial in Sickle Cell Anemia (STOP) [71]. The STOP trial tested the hypothesis that periodic blood transfusions would lower the risk of stroke compared with the risk while receiving standard care. The STOP trial was halted prematurely when 11 strokes were observed in the standard supportive care arm and only one in the transfusion arm, indicating a 92% relative risk reduction over 2 years of follow-up on stroke risk with chronic transfusion therapy [71]. The impact of the STOP trial on public health among children in California with SCD was revealed in the prevalence data indicating a dramatic decreased rate of symptomatic stroke largely due to the widespread implementation of TCD screening and prophylactic intervention of chronic transfusion therapy [72]. To extend these findings, the STOP investigators conducted a second randomized, controlled trial, the Optimizing Primary Stroke Prevention in Sickle Cell Anemia (STOP 2) trial, to determine whether it was possible to limit the duration of transfusion prophylaxis [73]. Children whose Doppler studies normalized after 30 or more months of transfusion in the STOP trial were eligible for enrollment in STOP 2, and were randomized to halt or to continue regular RBC transfusions. Among the 41 children in the transfusion-halted group, high-risk Doppler findings developed in 14 and stroke in two others by a mean duration of 4.5 (standard deviation ±2.6; range 2.1–10.1) months after the last transfusion. Neither of these events occurred in the 38 children who continued to receive transfusions, so this trial was terminated early. The authors concluded that discontinuation of transfusion for the prevention of stroke in high-risk children with SCD results in a high rate of reversion to abnormal blood flow velocities on TCD and stroke. These conclusions were supported more recently by extended follow-up observations that included additional strokes in six children during the post-trial follow-up period [74]. It is unclear how long transfusion therapy should be continued as a means of preventing stroke in children with SCD, a question that was not formally resolved by the STOP 2 trial [73]. In addition, while transfusion therapy reduces the risk of acute ischemic stroke, its effect on the development of subclinical small vessel disease associated with subtle neurocognitive and psychologic deficits is still unproven. However, among children enrolled in the STOP trial who had a silent infarction noted by baseline MRI, the risk of progressive silent infarction was reduced in the transfusion therapy arm compared with the standard care arm [75]. A trial similar in design to the STOP study has been initiated to determine whether transfusion is warranted on the basis of MRI lesions in clinically asymptomatic children with SCD [76]. Together, these trials are very likely to define a subgroup of children with SCD who appear to benefit from prevention of neurologic injury by receiving regular RBC transfusions. Thus, like those who have stroke, those at risk for stroke and other brain injury are very likely also to be suitable candidates for HCT, as an alternative to long-term RBC transfusions. Hydroxyurea Hydroxyurea is a potent inhibitor of ribonucleotide reductase, which also stimulates fetal hemoglobin production, which in turn inhibits the formation of the sickle hemoglobin polymer. The laboratory effect of hydroxyurea in SCD is to raise the HbF fraction, serum hemoglobin, and the red cell mean corpuscular volume [77]. Hydroxyurea also decreases the white blood cell, reticulocyte, and platelet counts while increasing NO production [78], decreasing RBC intracellular dehydration [79], and decreasing red cell adhesiveness to endothelium. Together,

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these laboratory effects translate into a clinical benefit of decreased rates of pain and ACS events in severely affected adults and children [80,81]. A long-term follow-up of the Multicenter Study of Hydroxyurea (MSH) showed that, at 9 years, the mortality rate among patients who took hydroxyurea was reduced by 40% compared with the rate among patients who did not take the drug [48]. The ready availability of hydroxyurea therapy and its favorable safety profile make it an appropriate intervention in children and adults who are experiencing frequent painful vasoocclusive events and ACS. Unfortunately, the beneficial effect of hydroxyurea is neither universal nor curative, as adult patients with SCD die of disease complications while receiving hydroxyurea therapy [82]. In addition, 81 of 225 children in one series had hydroxyurea therapy withdrawn, primarily due to a lack of clinical response or difficulty with adherence, although there were also failures due to stroke (n = 2) and the development of a TCD velocity greater than 200 cm/s (n = 3) [81]. Investigation of the quality of life showed that hydroxyurea improved some measures of quality of life, including general health perception and pain recall [83]. However, there was no effect of hydroxyurea on other quality of life measures such as physical functioning or physical role. Even though many experienced an average 50% reduction in acute pain episodes, subjects still had frequent acute pain episodes and high levels of chronic pain. Thus, while hydroxyurea is the sole Food and Drug Administration-approved drug for SCD and its use when indicated should be applied more broadly, it does not appear to be a substitute for the curative potential offered by HCT. To expand the indications for hydroxyurea, prospective studies have focused on the efficacy of hydroxyurea therapy on recurrent stroke risk and on TCD velocities in children with SCD. The effect of hydroxyurea on hemoglobin levels also makes it feasible to conduct phlebotomy to reduce iron overload in patients who have received chronic transfusions to prevent recurrent stroke. In one series of 35 patients, secondary stroke prevention and reduction of iron stores was facilitated by hydroxyurea, although seven patients experienced a second infarctive stroke after transfusions were replaced by hydroxyurea [84]. However, the stroke rate of 5.7 per 100 patient–years was not significantly different from historical rates observed during chronic transfusion therapy. To explore the efficacy of hydroxyurea for the prevention of primary stroke, a registry report from Belgium suggested a beneficial effect of hydroxyurea in 11 children who had abnormal TCD and serial measurements, with a significant decrease observed in TCD velocities [85]. In another study, 37 pediatric patients with increased TCD flow velocities of 140 cm/s or more had significantly lowered TCD values, and this effect was sustained for at least 1 year after reaching a maximum tolerated dose of hydroxyurea [86]. Thus, a prospective trial is underway to determine whether hydroxyurea might be applied in lieu of RBC transfusions in stroke prevention.

HCT for SCD Current indications for HCT In standard practice, HCT for SCD currently is reserved almost exclusively for patients with clinical features that portend a poor outcome or significant sickle-related morbidity, in part due to the toxicity of this intensive therapy [87]. These clinical indications, which were adapted from the multicenter investigation of bone marrow transplantation for SCD, are listed in Table 74.2. In addition, these criteria have been applied almost exclusively to children, where the risk–benefit ratio is most advantageous in terms of years of life gained among those who survive with sustained engraftment of donor cells. Less certain is how to apply inclusion criteria to adults with SCD, where the experience of

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HCT is limited, but for whom the risk of significant transplantationrelated toxicity remains substantial. For all patients, clinicians must carefully weigh therapeutic alternatives to HCT, with particular attention to safety, efficacy, availability, and the cost of intervention [18,88,89]. Current results of HCT for SCD The worldwide experience of transplantation for SCD is summarized in Table 74.3 [90–102]. In the collective experiences of these studies, the transition of HCT from an experimental intervention reserved for severely affected patients, to one in which younger children with early signs of sickle-related morbidity are targeted, has been observed. Several series in Europe and North America have reported very similar results after HLA-identical sibling transplantation in which bone marrow is the source of hematopoietic cells [91,92,100,101]. The principal aim of these multicenter phase I–II clinical studies was to define more completely the risks and benefits of this therapy, and to characterize the Table 74.2 Indications of hematopoietic cell transplantation for sickle cell disease Patients with sickle cell disease (HbSS or HbSβ°-thalassemia) less than 16 years of age One or more of the following complications: Stroke or central nervous system event lasting longer than 24 hours Impaired neuropsychologic function with abnormal cerebral magnetic resonance imagaing and angiography Recurrent acute chest syndrome Stage I or II sickle lung disease Recurrent vaso-occlusive painful episodes or recurrent priapism Sickle nephropathy (glomerular filtration rate 30–50% of predicted normal) Other indications to consider: Abnormal transcranial Doppler scan Pulmonary hypertension Silent cerebral infarction

Height

natural history of those surviving free of SCD. The results of transplantation were best when performed in children with SCD who had HLAidentical sibling donors. Even though many children who received allografts had significant sickle-related complications such as stroke and recurrent episodes of ACS, the disease-free survival varied from 80% to 85% in several series. However, 5–10% of patients died of complications related to transplantation, with graft-versus-host disease (GVHD) and its treatment as the leading cause of death. As virtually all clinical investigations of HCT for SCD have targeted enrollment of children in lieu of adults, the application of HCT in adults with SCD remains uncertain. While case reports of successful outcome after HCT in adults suggest that HCT is feasible, toxicity related to GVHD in particular has not permitted its routine use in adult individuals; thus, HCT for adults with SCD remains limited and investigational [87,96]. In the multicenter investigation of HCT for SCD, 59 children who ranged in age between 3.3 and 15.9 (median 9.9) years received HLAidentical sibling marrow allografts between September 1991 and April 2000 [102]. Patients received a myeloablative combination of busulfan (BU), cyclophosphamide, and equine antithymocyte globulin (ATG), and most received a combination of methotrexate and cyclosporine after HCT to prevent GVHD. Fifty of 59 children survive disease free after HCT. The Kaplan–Meier probabilities of survival and disease-free survival are 93% and 84%, respectively (Fig. 74.3). A recent publication from the Center for International Blood and Marrow Transplantation Research reported very similar results [100]. Outcomes after HLAmatched sibling HCT in 67 patients with SCD transplanted between 1989 and 2002 were described, in which the leading indications for transplantation were stroke and recurrent vaso-occlusive crisis occurring in 38% and 37% of patients, respectively. The median age at transplantation was 10 years. Ninety-two percent received BU and cyclophosphamide in the conditioning regimen, and bone marrow was the predominant source of hematopoietic cells. Most patients achieved hematopoietic recovery, and there were no deaths during the early post-transplant period. Rates of acute and chronic GVHD were 10% and 22%, respectively. Sixty-four of 67 patients are alive; 5-year probabilities of diseasefree and overall survival are 85% and 97%, respectively. The outcomes after HCT for SCD in Europe have also been reported. Results among 101 patients who received conventional myeloablative

Table 74.3 p-values from each comparison of the hematopoietic cell transplantation (HCT) cohort to the Cooperative Study of Sickle Cell Disease (CSSCD) and HUG-KIDS groups

Weight

Baseline

Velocity

Baseline

Velocity

Comparison Group

Male

Female

Male

Female

Male

Female

Male

Female

CSSCD HUG-KIDS (Pre) HUG-KIDS (HU)

0.37 0.06(1) 0.15

0.22 0.72 0.87

0.01(4) 0.15 0.68

0.12 0.72 0.25

0.30 0.89 0.01(2)

0.02(3) 0.66 0.51

0.0004(5) 0.73 0.30

0.08 0.53 0.92

(1) The males in the HUG-KIDS (Pre) group tended to be approximately 4.0 cm taller than the males in the HCT group, regardless of age. (2) The males in the HUG-KIDS (HU) group tended to be 2.7 kg heavier than the males in the HCT group, regardless of age. (3) Three females, older than 11 years, in the HCT group had low baseline weight, creating an illogical decreasing trend in the baseline weight curve. (4) Between the baseline ages of 3.3 years and 11.6 years, the linear height velocity was greater for the HCT group than the predicted velocity for the CSSCD group. (5) Until the baseline age of 12.1 years, the linear weight velocity during follow-up in the HCT group was greater than the predicted linear velocity during follow-up for the CSSCD group. Reproduced with permission from [115].

Percent

Hematopoietic Cell Transplantation for Sickle Cell Disease

100

Survival (93%)

80

Event-free survival (84%)

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60

40

20 Rejection or recurrence (9%) 0 0

2

4

6

8

10

Time (years) after transplantation

Fig. 74.3 Survival and event-free survival after hematopoietic cell transplantation (HCT) for sickle cell disease (SCD). Kaplan–Meier estimates of survival and event-free survival after HCT for sickle cell anemia are shown. Events were defined as death, SCD recurrence, and graft rejection. A cumulative incidence curve for graft rejection and return of SCD is also shown.

HLA-identical sibling HCT in Europe were remarkably similar to North American reports, with an overall survival probability of 88% and disease-free survival of 80% [92]. A recent update included 87 consecutive patients treated between November 1988 and December 2004 at 14 centers in France [101]. The median age was 9.5 years, and the leading indication for HCT was stroke or transient ischemic attack (n = 36) and the development of abnormal cerebral arterial flow velocity as detected by TCD (n = 8). Five of the 18 patients who did not receive ATG had graft rejection. One patient with 50% mixed chimerism developed aplastic anemia 15 months post transplant and received a second successful allograft, while the other four patients had autologous reconstitution and recurrent SCD. The rate of graft rejection declined from 22.6% to 2.9% after the addition of ATG to the conditioning regimen (Fig. 74.4(a)). The Kaplan–Meier estimate of event-free survival at 5 years was 86.1%. Multivariate analysis showed that the date of transplantation was the only significant risk factor. The 5-year event-free survival rate was 95.3% in patients treated after January 2000, compared with 76.7% in patients treated earlier (Fig. 74.4(b)). A retrospective review of 24 Belgian patients with SCD analyzed the impact of pretransplant ATG and hydroxyurea on outcome [103]. The administration of hydroxyurea before HCT was associated with a significantly lower rate of graft rejection, compared with patients who did not receive hydroxyurea, but the administration of ATG before transplantation did not affect the rate of engraftment. Overall 20 of 24 patients had stable engraftment of donor cells after HCT, but none of those who received hydroxyurea before transplantation with ATG in the conditioning regimen had graft rejection or a late recurrence of SCD. This suggests that the augmentation of pretransplantation immunosuppression might be necessary to overcome the immunologic barrier of tolerance to donor histocompatibility antigens. The propensity for graft rejection even after HLA-identical sibling HCT follows the hypothesis that multiple transfusion exposures before HCT induce sensitization to minor histocompatibility antigens expressed on the surface of transfused cells. It appears possible that leukocytes present in blood products are not alone in this ability to cause sensitization, as RBCs might also present histocompatibility antigens via the HLA class I pathway to elicit expansion and activation of host CD8+ T cells [104].

Fig. 74.4 (a) Comparison of the cumulative incidence of rejection in patients conditioned with and without antithymocyte globulin (ATG). (b) Comparison of event-free survival in patients transplanted before (n = 43) and after (n = 44) January 2000.

Stable donor–host chimerism after high-dose HCT for SCD The observation of donor–host hematopoietic chimerism after conventional high-dose HCT has lent substantial support to the notion that persistence of even a fraction of normal erythropoiesis might elicit a curative clinical effect [93,102]. The basis of this effect appears to be twofold: improved survival of healthy donor erythrocytes in the blood compared with sickle erythrocytes, and ineffective erythropoiesis in the SCD marrow that lends a competitive advantage to donor erythroid progenitors [105]. Approximately 25% of children with SCD developed stable mixed chimerism after HLA-identical sibling HCT [102]. In the multicenter investigation of bone marrow transplant for SCD, 13 of 50 patients with clinically successful allografts developed stable mixed chimerism, which is defined as the durable coexistence of donor and host hematopoietic cells well after the discontinuation of postgrafting immunosuppression. The level of donor chimerism, measured in peripheral blood 6 or more months after transplantation, varied between

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90% and 99% in eight patients who had normal hemoglobin levels. Five additional patients had a lower proportion of donor cells (range 11– 74%). Among these five, hemoglobin levels varied between 11.2 and 14.2 (median 11.3, mean 12.0) g/dL. In the patients whose donors had a normal hemoglobin genotype, the sickle hemoglobin (HbS) fractions were 0%, 0%, and 7%, corresponding to donor chimerism levels of 67%, 74%, and 11%, respectively. In the patients whose donors had sickle trait, the HbS fractions were 36% and 37%, corresponding to donor chimerism levels of 25% and 60%, respectively. Thus, allograft recipients with stable mixed chimerism had HbS levels similar to donor levels; only one patient required RBC transfusion beyond 90 days after transplantation. None of the patients experienced painful events or other clinical complications related to SCD after HCT. One patient, who had a stroke before transplantation, had no further strokes after HCT and had stable appearance by cerebral MRI, despite having as few as 10% donor cells detectable in the peripheral blood. In the French series, low levels of residual host hematopoiesis were observed significantly more frequently in patients who did not receive ATG before HCT (p = 0.04 at 12 months and p = 0.002 at 24 months post transplant) [101]. A higher fraction of residual host cells was observed in patients who received ATG (p = 0.03 at 12 months and p = 0.001 at 24 months post transplant), and this level of donor–host chimerism remained stable in the long term. These observations are consistent with the idea that chimerism, even with a minority of donor cells, can have a significant ameliorative effect so that full engraftment of donor cells is not a requirement for successful HCT, and that the donor chimerism fraction remains stable 12 months and longer after HCT.

immunosuppression of the regimens and an accompanying risk of opportunistic infection. A cohort of older SCD patients who received such “reduced-intensity” conditioning regimens also received augmented pregrafting immunosuppression to facilitate donor cell engraftment. Nevertheless, three of 12 recipients experienced graft rejection (Table 74.4). Acute and chronic GVHD were also more frequent (four of 12 patients) in this group, and GVHD was fatal in two cases. Thus, in older recipients, the problem of transplant-related mortality was not eliminated by the reduced-intensity conditioning regimen. In contrast, donor chimerism was successfully established in children who received a reduced-intensity regimen consisting of BU, fludarabine, ATG, and total lymphoid irradiation (500 cGy) [95]. Five patients who were between 6 and 18 years of age received HLA-identical sibling bone marrow transplantation with cyclosporine and mycophenolate mofetil for postgrafting immunosuppression. Treatment-related toxicity was minimal, and all the patients had evidence of stable donor engraftment. Another study that included 16 children with nonmalignant disorders included one patient with SCD [99]. The patients received melphalan, fludarabine, and an anti-T cell antibody, Campath-1, before transplantation, and included seven patients who received unrelated or HLAmismatched related donor allografts. Twelve of the 16 (75%) patients survive disease free after transplantation, and none of the surviving patients had graft rejection. These encouraging results in younger patients suggest that the future application of reduced-intensity conditioning regimens should focus on children who already have or are at risk for symptomatic disease. This particular approach is being studied in a multicenter clinical trial of unrelated donor HCT for children with symptomatic SCD in the United States.

Reduced-intensity HCT for SCD Due in part to the observations discussed above, it was reasoned that pretransplantation therapy might be adjusted to promote stable engraftment of a threshold fraction of donor cells that would be sufficient to prevent vaso-occlusion, and that this regimen could be carried out more safely than conventional high-dose conditioning, particularly in adult patients. Toward this end, several groups have attempted to apply reduced-intensity HCT to SCD, utilizing conditioning regimens of varying intensity [93,95–99]. Although these investigations are continuing, it has been difficult to identify a regimen that is sufficiently immunosuppressive to ensure stable engraftment of donor cells from HLA-identical siblings, yet also meets the objective of “reduced toxicity” with a risk that is distinguishable from conventional allografting. A minimally toxic regimen was first developed in a large animal model and translated successfully into human trials for older adult patients with hematologic malignancies [106]. When applied to SCD, this approach was safe, generated little or no acute GVHD, and in most cases was associated with an initial period of donor engraftment. Unfortunately, in nearly all cases, withdrawal of postgrafting immunosuppression was followed by graft rejection with disease recurrence (Table 74.4). In another patient series of reduced-intensity HCT, there was a twofold higher expression of donor β-globin RNA compared with total genomic DNA in the blood after nonmyeloablative HCT. Direct bone marrow analysis revealed ineffective erythropoiesis of native HbSS erythroblasts, with a progressive increase in representation of donor RBCs during erythrocyte maturation [105,107]. These findings were associated with clinical benefit after transplantation, and with improvements in hemolysis, endothelial function, and NO bioavailability; however, they did not persist after graft rejection. An increasing number of patients have received regimens that are less intense than a high-dose regimen, but retain a moderate degree of the myelosuppressive effect in order to suppress the host-versus-graft reaction and promote engraftment. These regimens require hospitalization, but their risk of regimen-related toxicity is related to the profound

Effect of donor hematopoiesis on vaso-occlusion After successful HCT for SCD, patients with stable engraftment of donor cells have not experienced clinical complications or required transfusions, even when there was stable mixed donor–host hematopoietic chimerism [91,92,101]. In addition, investigators have reported improvements in splenic function and osteonecrosis [108,109]. Patients treated by HCT also had stabilization of the underlying cerebral vascular disease. Forty-six of 55 patients who were enrolled in the multicenter study had a cerebral MRI performed a median of 25.4 months after transplantation. These studies were compared with pretransplantation baseline examinations in all but four of the patients. Among those 29 who had a history of stroke before transplantation, one patient with graft rejection had a second stroke and 28 survive stroke free. Of the 28 patients with stroke studied after transplantation, all but the patient with a second stroke had a stable or improved appearance by cerebral MRI. Ten patients had evidence of silent cerebral infarction before transplantation, and of these, all eight with post-HCT studies had stable and four patients an improved appearance. There were no strokes after HCT in this group. Sixteen patients had no documented central nervous system disease before bone marrow transplantation, and all 10 studied in this group had a normal MRI appearance after HCT, with none having a stroke after HCT. Together, these observations show that there is stabilization of cerebral vasculopathy after HCT. These data also suggest that, in those with silent cerebral infarction who are at risk to develop stroke, there is protection from disease progression and a first stroke. A similar experience was reported from the French group [101]. Among the 36 patients with a history of stroke before HCT, two had a recurrent stroke. One patient experienced a transient ischemic attack 10 days after HCT, while the other, who had severe cerebrovascular injury with Moyamoya disease, had fatal intracranial hemorrhage 32 days after HCT. With a median follow-up of 6 years, the risk of recurrent stroke was 5.6%, which mirrors the rate of second stroke observed in SCD patients treated by regular RBC transfusion therapy after a first stroke.

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Table 74.4 Hematopoietic cell transplantation (HCT) for sickle cell disease: published reports [90–94,96–102,105,124] Conventional myeloablative conditioning regimen

Number of patients Patient age (median, in years) Conditioning regimen (dose) {number of patients}

Minimal-toxicity conditioning regimen

Reduced-intensity conditioning regimen

US collaborative

11* 10 (range 3–28)

18† 12.4 (range 1.8–56)

Flu (90–150 mg/ m2)/TBI (200 cGy) {5}; Flu (125–150 mg/ m2)/ATG/TBI (200 cGy) {6}

Flu (175 mg/m2)/BU (8 mg/kg)/ ATG/TLI (500 cGy) {5}; Flu (120 mg/m2)/Mel (140 mg/ m2)/ATG {2}; Flu (120 mg/ m2)/CY (120) {1}; Flu (120 mg/m2)/Mel (140/70 mg/m2)/Campath {2}; Flu (120 mg/m2)/BU (3.2 mg/kg) {2}; Campath/ FLU (150 mg/m2)/Mel (140 mg/m2) {2}; Flu (180 mg/m2)/BU (6.4 mg/ kg)/ATG {4}‡ Marrow (8)§; CBSC (1); PBHC (10)

French

Belgian

CIBMTR

59 9.9 (range 3.3–15.9) BU/CY/ATG {55}; BU/ CY/Campath {4}

87 9.5 (range 2–22) BU/CY {12}; BU/CY/ATG {65}

50 7.5 (range 0.9–23) BU/CY {30}; BU/CY/TLI {6}; BU/CY/ ATG {14}

67 10 (range 2–27) BU + CY ± other {63}; other {4}

Marrow

Marrow (48); CBSC (2)

Marrow (54); PBHC (9); CBSC (4)

Yes

Yes

Source of stem cells (number of patients)

Marrow (9); PBHC (2)

Induction of mixed chimerism Number with graft rejection/disease recurrence Number with graftversus-host disease

Yes (transient in 10) 10 (91%)

Yes

Yes

Marrow (74); CBSC (10); CBSC + marrow (2); PBHC (1) Yes

5 (28%)

5 (8.5%)

7 (8%)

5 (10%)

9 (13%)

Acute 1 (grade I), chronic none

Acute 5 (grade II–IV), chronic 4 (3 fatal)

Acute 25%, chronic 12%

Acute 20%, chronic 12.6%

Acute 40%, chronic 20%

1 (after a second HCT) (9%) 1 (9%)

4 (22%)

4 (7%)

6 (7%)

2 (4%)

Acute 10%, chronic 22% 3 (4.5%)

9 (50%)

50 (85%)

74 (85%)

43 (86%)

55 (82%)

Number of deaths Number with eventfree survival

ATG, antithymocyte globulin; BU, busulfan; CBSC, umbilical cord blood stem cell; CIBMTR, Center for International Blood and Marrow Transplantation Research; CY, cyclophosphamide; Flu, fludarabine; Mel, melphalan; PBHC, peripheral blood stem cell; TBI, total body irradiation; TLI, total lymphoid irradiation. * Includes two patients with thalassemia major. † Includes three patients with thalassemia major. ‡ Human leukocyte antigen-matched unrelated donor PBHCs were used for HCT. § One patient received a combination of marrow and umbilical cord blood from the same donor.

However, vascular occlusions tended to persist after HCT, and the longterm outcome varied somewhat: in five, the stenoses resolved, in 16 there was no change, and in two cases, the stenoses showed progression. However, none of the patients with durable donor engraftment had a clinical stroke or silent ischemic lesion, including the two patients who had progressive cerebrovascular narrowing. Cortical atrophy worsened in two cases. In addition, cerebral artery velocity was significantly decreased (p < 0.001) by 1 year after HCT in the 49 patients who had a pre-HCT TCD examination performed (the TCD velocity declining from 138 (range 88–188) before HCT to 100 (range 66–134) cm/s as measured in the right MCA, and from 138 (range 92–184) to 103 (range 63–143) cm/s as measured in the left MCA). The TCD velocity also

normalized by 3 months after HCT in two patients who had abnormal measurements before HCT, even though both had been treated by regular RBC transfusions before HCT. In other series, patients with stroke before HCT have also experienced transient ischemic attacks and intracranial hemorrhage in the era before measures to prevent these adverse events were instituted [110,111]. After the institution of preventive measures, neurologic complications still occurred after transplantation, but these tended to be self-limited events such as seizures with no long-term sequelae. More recently, one group of investigators observed radiographic changes in some patients with SCD who underwent HCT for stroke [112]. Five of nine patients had either new or slightly increased size of cerebral lacunae or leukoen-

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cephaly that stabilized 2–7.5 years after HCT. Thus, it is possible that these observations reflect the evolution of cerebrovascular disease that existed before bone marrow transplantation. Of interest, these cerebral MRI changes were not associated with progressive neurocognitive deficits. Growth and development after HCT for SCD There remains concern about possible growth impairment after HCT for SCD. The potential for growth impairment stems, in part, from the gonadal toxicity associated with high doses of BU, particularly in females. While it is difficult to predict the impact of BU on linear growth in patients with SCD, there is also the possibility that, by removing the energy requirements associated with the hemolytic anemia of SCD, growth might improve after transplantation [113,114]. A recent report compared four groups of children with SCD in an analysis of growth after HCT. Children who were enrolled in the CSSCD study comprised one supportive therapy comparison group. Children who were enrolled in the HUG-KIDS trial made up the remaining two comparison groups [115]. These two groups included children who had pretreatment growth measurements (HUG-KIDS Pre) and the same children who again had measurements for at least 1 year after achieving the maximum tolerated dose of hydroxyurea (HUG-KIDS HU). Using two-level hierarchical linear models, there were no statistically significant differences in the estimated height (weight) velocity curves between the HCT and the three comparison groups among females; however, there were significant differences between males in the HCT and CSSCD groups (Table 74.3, Fig. 74.5). There appeared to be an age-dependent growth effect in the secondary analysis of the CSSCD/ HCT height and weight comparisons in males. The predicted height velocity during follow-up in the HCT group was greater than the velocity in the CSSCD group between 3.3 years and 11.6 years of age. After this period of 8.4 years, there was no apparent growth benefit associated with HCT. As a result, the predicted height velocity in HCT males who were greater than 12 years of age at transplantation was less than the velocity in CSSCD males. Similarly, until the baseline age of 12.1 years, the linear weight velocity in the HCT group was greater than in the CSSCD group. In the HUG-KIDS Pre and HCT male height comparison, secondary analysis showed a statistically significant group effect on linear height velocity during follow-up after adjusting for estimated baseline height (p = 0.04). In summary, growth after transplantation for SCD was not impaired, except in older males. These data show that conventional high-dose HCT generally had no adverse effect on height or weight gain in young children. Gonadotropin and sex hormone levels of surviving patients have also been monitored, and these confirm the toxic effect of BU on gonadal function. Among seven surviving females in the multicenter study who were older than 13 years after HCT, an interim analysis showed that five had primary amenorrhea and five had corresponding elevated LH and FSH levels that were associated with decreased serum estradiol levels in four [91]. One individual receiving hormonal replacement therapy had elevated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels and a normal serum estradiol. One postpubertal female had normal serum FSH and estradiol levels. Among six evaluable prepubertal girls in the Belgian cohort, five had primary amenorrhea with elevated serum LH and FSH [92]. Two postpubertal females developed secondary amenorrhea. In the French series, seven postpubertal females (ranging from 13 to 22 years of age) developed amenorrhea after HCT with decreased serum estradiol and elevated LH and FSH levels, and received hormone replacement therapy [101]. In addition, most females who were prepubertal before HCT received hormone replacement therapy to promote the development of secondary sexual characteristics after they reached

Fig. 74.5 (a) Height velocity during follow-up. The height velocity in cm/ year is depicted as a function of the baseline age during the follow-up period. The open circles represent height velocities as determined by a hierarchical linear model (HLM) for the male Cooperative Study of Sickle Cell Disease (CSSCD) comparison group, and the accompanying dashed line shows the predicted velocity curve. The solid circles represent height velocities as determined by a HLM for the male hematopoietic cell transplantation (HCT) group, and the accompanying solid line represents the predicted velocity curve. (b) Weight velocity during follow-up. The weight velocity in kg/year is depicted as a function of the baseline age during the follow-up period. The open circles represent weight velocities as determined by a HLM for the male CSSCD comparison group, and the accompanying dashed line shows the predicted velocity curve. The solid circles represent weight velocities as determined by a HLM for the male HCT group, and the accompanying solid line represents the predicted velocity curve. (Reproduced from [115], with permission.)

Hematopoietic Cell Transplantation for Sickle Cell Disease

a bone age of 13 years. However, two younger girls (6.4 and 8.3 years of age) experienced spontaneous pubertal development, raising the possibility that HCT performed in very young patients might not be associated with delayed puberty. Nonetheless, it is anticipated that many if not most of the females will require hormonal replacement therapy after HCT with high-dose BU. However, males may be somewhat more resistant to the adverse effect on gonadal function, although the long-term outcome remains somewhat uncertain. Of seven males from the multicenter study of HCT for SCD who were more than 13 years of age, none of the four tested had elevated serum LH/FSH levels [91]. Two males who were 14 and 16 years of age had low testosterone levels that were correlated with gonadotropin levels in the prepubertal range. Testicular function was also adversely affected in four of six evaluable boys from the Belgian cohort who demonstrated decreased testosterone and elevated FSH levels [92]. However, all the male recipients from the French series had testosterone, FSH, and LH levels in keeping with their bone age and pubertal status [101]. Unfortunately, none of these series is sufficiently mature to have definitive information about the impact of treatment on fertility, which is a key factor in decision-making about whether or not to pursue HCT among families with this disorder. Thus, long-term follow-up studies continue to have importance with regard to when and if to pursue this interventional treatment. Alternative sources of donor hematopoietic cells The use of umbilical cord blood (UCB) in lieu of bone marrow in transplantation for SCD is of interest, due primarily to a lowered risk of GVHD after UCB transplantation. However, this benefit is balanced by a higher rate of graft rejection and a longer duration until hematologic recovery that accompanies UCB transplantation [116,117]. Thus, to overcome these risks, investigators have modulated pre- and postgrafting immunosuppression and intensified supportive care to ensure favorable outcomes. In a report of 44 patients that included 11 with SCD who received HLA-identical or one-antigen HLA-mismatched sibling UCB allografts, the overall 2-year SCD-free survival rate was 91%, with one patient experiencing recurrent disease [118]. However, among patients who received thiotepa or fludarabine in addition to the BU/CY backbone and who did not receive methotrexate with postgrafting immunosuppression, the outcome after sibling donor UCB transplantation was superior, particularly among recipients with thalassemia major. In addition, only four of 44 patients experienced acute, and two chronic, GVHD after UCB transplantation, and there were no deaths related to GVHD. In another series of related donor UCB transplantation for hemoglobinopathies, 18 of 22 patients survive event free after transplantation [119]. Thus, it is possible that UCB might be utilized as an effective source of hematopoietic cells in hemoglobinopathies. The current experience of unrelated donor UCB transplantation for SCD is quite limited, but it illustrates the potential for success and the challenges yet to be investigated. In one series, seven patients were conditioned with high dose (n = 4) or reduced-intensity preparative regimens [120]. The indication for cord blood transplantation was stroke, and all received cord blood grafts that were mismatched at two HLA antigens. Five of seven patients (71%) experienced significant adverse events of graft rejection or death. Currently, 43% survive disease free after UCB transplantation due to a successful second cord blood transplantation in one patient, and one of seven (14%) subjects died of transplant-related causes. These preliminary findings suggest the possibility of success after unrelated donor cord blood transplantation in high-risk patients. It is the introduction of transplantation in patients like these at risk of progressive neurovascular injury, and who are also very likely to need long-term

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transfusion support with its attendant risk of life-threatening transfusional iron overload, where risk–benefit considerations might achieve a suitable balance. But to tip the balance in favor of transplantation, the problems of graft rejection and GVHD after unrelated donor cord blood transplantation will require better control. That the broader application of HCT for SCD is limited is best illustrated by the observation that only 14% of SCD recipients are likely to have a HLA-identical sibling donor [7]. Not surprisingly, lacking a suitable donor is the major barrier to HCT for SCD. An expanded availability of HLA-compatible unrelated donor sources is necessary to pursue HCT as a therapeutic option in most patients with SCD. Donors who are matched at six (HLA-A, B, and DRB1 loci) to 10 HLA antigens (including HLA-C and DQB1 loci) can be identified via the National Marrow Donor Program in approximately 80% of Caucasian recipients; however, the likelihood of identifying a similarly HLA-matched unrelated donor is less likely in other ethnic groups due to underrepresentation of these groups in the volunteer donor pool and greater HLA diversity. The reduced likelihood of identifying a suitable donor is a difficult problem in HCT for SCD and can limit the application of HCT among those who might benefit most [121–123]. However, the feasibility of identifying suitable donors was suggested by surveys in which 60% of SCD patients had at least one potential suitable unrelated donor, and 26% had three or more potential donors. The challenge of identifying a donor might also be mitigated by the possibility of UCB transplantation. An unrelated donor search for 40 SCD patients showed that 100% and 50% had HLA four of six and five of six antigen-matched unrelated donor cord blood units, respectively [123]. Thus, it appears feasible to pursue alternate donors suitably matched at HLA alleles in the treatment of individuals with severe SCD. It is quite possible and even likely that the successful translation of unrelated donor HCT for SCD will hinge on our ability to establish bidirectional donor–host immunologic tolerance, and that this will involve the administration of more intensive immunosuppression than we are accustomed to using in this clinical setting. This represents a shift from the notion that marrow ablation and elimination of sickle cell progenitor cell populations are the principal objectives in carrying out a successful HCT. There are many reasons why this might be, not least of which is that the immunologic milieu of SCD is one of inflammation, where innate stimulation of graft-versus-host and host-versus-graft reactions is very likely to occur. In the course of inhibiting these reactions by pharmacologic means, controlling the emergence of opportunistic viral and other infections will take on greater importance than has been appreciated previously after transplantation for hemoglobin disorders. These considerations have been taken into consideration in the design and implementation of a national United States trial to study unrelated donor HCT in children with severe SCD.

Conclusion The track record of HCT from HLA-identical sibling donors for clinically significant hemoglobinpathies is among the best of disorders currently treated by HCT. Most patients with SCD survive after HCT with no evidence of the characteristic clinical sequelae of vaso-occlusion. Moreover, as new indications for HCT emerge and the possibility of alternate donor HCT is expanded, there is likely to be a possibility of HCT for individuals where none existed previously. Nonetheless, very few individuals currently are offered this therapy. Decisions about treatment options in the future are very likely to be influenced by perceptions about the nature of SCD as a chronic illness, and whether the short-term risk of mortality inherent to HCT is balanced by the possibility of survival without symptoms or treatment. While HCT is the only curative therapy for SCD, other treatments such as hydroxyurea and regular RBC

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transfusion may pose fewer short-term risks and should be considered in selected children who lack suitable donors or in those who exhibit favorable-risk characteristics. Controlled clinical trials comparing

chronic transfusions, hydroxyurea, and HCT should be conducted to determine the relative values of these treatments and ultimately provide the optimal care for the SCD patient.

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70. Adams RJ, McKie VC, Carl EM et al. Long-term stroke risk in children with sickle cell disease screened with transcranial Doppler. Ann Neurol 1997; 42: 699–704. 71. Adams RJ, McKie VC, Hsu L et al. Prevention of a first stroke by transfusions in children with sickle cell anemia and abnormal results on transcranial Doppler ultrasonography. N Engl J Med 1998; 339: 5–11. 72. Fullerton HJ, Adams RJ, Zhao S, Johnston SC. Declining stroke rates in Californian children with sickle cell disease. Blood 2004; 104: 336–9. 73. Adams RJ, Brambilla D. Discontinuing prophylactic transfusions used to prevent stroke in sickle cell disease. N Engl J Med 2005; 353: 2769–78. 74. Lee MT, Piomelli S, Granger S et al. Stroke Prevention Trial in Sickle Cell Anemia (STOP): extended follow-up and final results. Blood 2006; 108: 847–52. 75. Pegelow CH, Wang W, Granger S et al. Silent infarcts in children with sickle cell anemia and abnormal cerebral artery velocity. Arch Neurol 2001; 58: 2017–21. 76. Kirkham FJ, DeBaun MR. Stroke in children with sickle cell disease. Curr Treat Options Neurol 2004; 6: 357–75. 77. Charache S, Dover GJ, Moore RD et al. Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia. Blood 1992; 79: 2555–65. 78. Gladwin MT, Shelhamer JH, Ognibene FP et al. Nitric oxide donor properties of hydroxyurea in patients with sickle cell disease. Br J Haematol 2002; 116: 436–44. 79. Orringer EP, Blythe DS, Johnson AE, Phillips G Jr., Dover GJ, Parker JC. Effects of hydroxyurea on hemoglobin F and water content in the red blood cells of dogs and of patients with sickle cell anemia. Blood 1991; 78: 212–16. 80. Charache S, Terrin ML, Moore RD et al. Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. N Engl J Med 1995; 332: 1317– 22. 81. de Montalembert M, Brousse V, Elie C, Bernaudin F, Shi J, Landais P. Long-term hydroxyurea treatment in children with sickle cell disease: tolerance and clinical outcomes. Haematologica 2006; 91: 125–8. 82. Bakanay SM, Dainer E, Clair B et al. Mortality in sickle cell patients on hydroxyurea therapy. Blood 2005; 105: 545–7. 83. Ballas SK, Barton FB, Waclawiw MA et al. Hydroxyurea and sickle cell anemia: effect on quality of life. Health Qual Life Outcomes 2006; 4: 59. 84. Ware RE, Zimmerman SA, Sylvestre PB et al. Prevention of secondary stroke and resolution of transfusional iron overload in children with sickle cell anemia using hydroxyurea and phlebotomy. J Pediatr 2004; 145: 346–52. 85. Gulbis B, Haberman D, Dufour D et al. Hydroxyurea for sickle cell disease in children and for prevention of cerebrovascular events: the Belgian experience. Blood 2005; 105: 2685–90. 86. Zimmerman SA, Schultz WH, Burgett S, Mortier NA, Ware RE. Hydroxyurea therapy lowers transcranial Doppler flow velocities in children with sickle cell anemia. Blood 2007; 110: 1043– 7.

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Trudy N. Small, Wilhelm Friedrich, & Richard J. O’Reilly

Hematopoietic Cell Transplantation for Immunodeficiency Diseases

Introduction Multiple advances in the diagnosis and treatment of severe combined immunodeficiency syndrome (SCID) and other congenital lymphoid immunodeficiencies have occurred since the third edition of this book was published in 2004 [1]. Since the first description of SCID in 1950 by Glanzmann and Riniker [2] and of the Wiskott–Aldrich syndrome (WAS) by Wiskott in 1937 [3] and Aldrich et al. in 1954 [4], the genetic causes of over 120 primary immunodeficiency diseases have been elucidated (reviewed in [5–8]). Transplantation for SCID and WAS continues to improve, with current survival rates of 80–100% observed following HLA-matched related hematopoietic cell transplantation (HCT) and 50–80% following an alternative donor transplant (reviewed in [1]). In this chapter, the results of HCT for SCID, WAS, and combined lymphoid immunodeficiency syndromes (CIDs) will be updated, the genetic mutations giving rise to SCID and CID summarized, and the problems still facing physicians caring for these children analyzed. In addition, the emerging role of gene therapy in the treatment of these diseases will be discussed.

Severe combined immunodeficiency syndrome SCID refers to a heterogeneous group of lethal congenital disorders which result in the absence of antigen-specific T and B lymphocyte responses (reviewed in [1,8,9]). It is estimated to occur in 1/50,000– 100,000 live births [5]. In Athabascan-speaking Native Americans, a higher incidence (52/100,000) has been reported [10]. In the absence of a positive family history, children with SCID generally reach medical attention at 3–8 months of age, coincident with waning protection afforded by passively transferred maternal antibodies. Most children with SCID present with failure to thrive, refractory thrush, persistent diarrhea, recurrent otitis media, and/or interstitial pneumonia secondary to Pneumocystis carinii, cytomegalovirus (CMV), or common respiratory viruses, such as respiratory syncytial virus, parainfluenza, adenovirus or influenza [9,11]. Some genetic causes of SCID are associated with distinctive clinical presentations (Table 75.1). Children with genital and oral ulcers usually have Artemis mutations [10,12,13]. Infants with Omenn’s syndrome (scaling erythroderma, lymphadenopathy, organomegaly, eosinophilia,

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

and high immunoglobulin E (IgE) levels [14,15]) most frequently have defects within recombinase activating genes (RAG1/RAG2) [16–19] and less commonly within the Artemis [20] or interleukin-7R-alpha (IL7Rα) [21] gene. Microcephaly, developmental delay, and photosensitivity are common in SCID syndromes associated with genes involved in repair of DNA double-strand breaks by nonhomologous end joining, such as DNA ligase IV [22–24]. Adenosine deaminase (ADA) deficiency [25–27] can be associated with skeletal, renal, hepatic, and neurologic defects [28], which may persist despite successful correction of SCID by HCT [29]. Rare children with SCID present with short-limbed dwarfism and ectodermal dysplasia [30,31], neutropenia with sensorineural deafness (reticular dysgenesis) [32–34] or cartilage–hair hypoplasia [35,36]. The latter disease is associated with mutations in the RNA component of the mitochondrial RNA processing RNase complex, which maps to chromosome 9 [37]. These and other distinctive features of the known genetic variants of SCID are summarized in Table 75.1. Prior to the identification of the multiple genetic defects which can give rise to SCID, affected children were classified according to the phenotype of their circulating lymphocytes, the mode of inheritance, and/or the presence or absence of ADA, an enzyme deficiency known to be associated with SCID since 1972 [25]. Three major forms of SCID were identified, consisting of the most common form of SCID (B+ SCID with absent T cells (T−B+), classical SCID (T−B−), and ADA-deficient SCID, which may present with either phenotype (reviewed in [27]). Currently, mutations in over 15 genes are known to give rise to SCID (reviewed in [5–9,22–24,38,39]). The currently known genetic defects resulting in SCID affect four major aspects of lymphoid development: (1) premature death of lymphoid precursors due to toxic metabolites (ADA deficiency); (2) defective development resulting from abnormal cytokine signaling (γc, IL-7Rα, Janus kinase [JAK] deficiency, IL-2Rα or IL-2R/IL-15β); (3) defective VDJ gene rearrangement in T-cell and B-cell precursors (recombinase activating gene defects (AG1/AG2), DNA repair defects (Artemis or DNA ligase IV); and (4) abnormal signaling through the T-cell receptor (CD3/TCR subunit defects and CD45) (reviewed in [5–9,38]). Elucidation of many of these defects has shown that different mutations within the same gene (null, hypomorphic) may result in different clinical syndromes and presenting lymphoid phenotype [18,19]. For example, RAG1/RAG2 [16] mutations can be associated with classical (T−B−NK+) SCID, Omenn’s syndrome or a syndrome characterized by oligoclonal T cells, autoimmune cytopenias, and increased risk of severe CMV infections [16–19,40]. Identical mutations in a sibship can also result in diverse presentations [18,41]. Furthermore, mutations in more than one gene (RAG1/RAG2 [16–19], Artemis [20], IL-7Rα [21]) can result in Omenn’s syndrome.

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Table 75.1 Mutations associated with severe combined immunodeficiency syndrome (SCID) Defect

Function

Common phenotype

Chromosomal location

Distinctive presentations

References

Adenosine deaminase

Purine salvage enzyme

T−B−NK+

20q12–q13

[23–26]

CD3 invariant chains γ δ ε γc

T-cell signaling

Tlow−nlB+NK+ TloB+NK+ TloB+NK+

11q23

Skeletal, renal, hepatic, deafness, mental retardation, motor dysfunction –

Common cytokine receptor γ chain (IL-2, -4, -6, -15, -21) Tyrosine kinase Tyrosine kinase, signaling through γc Recombinase activating genes: initiation of VDJ recombination

T−B+NK−

Xq13

–

[43–45]

CD8 deficiency T−B+NK−

2q12 19p13

– –

[60–62] [47,48]

T−B−NK+ T+B−NK+

11p13

Omenn’s syndrome (scaling erythroderma, organomegaly, lymphadenopathy) – Common: oral and genital ulcers Less common: Omenn’s syndrome – – – Microcephaly, developmental delay, photosensitivity –

[16–19]

ZAP-70 JAK-3 RAG-1, RAG-2 Omenn’s syndrome

IL-7Rα Artemis

Cytokine receptor DNA repair enzyme

T−B+NK+ T−B−NK+

5p13 10p

p56lck CD45 IL-2R/IL-15b subunit DNA ligase IV

– Tyrosine phosphatase Cytokine receptor signaling DNA repair enzyme

CD4 deficiency TloB+NKlo TloB+NK− T−B−NK+

– 1q31–q32 Unknown 13q22–q34

CD3ζ

TCR complex subunit: TCR expression and signaling

Partial deficiency: TloB+NK+ Complete absence: T−B+NK+

–

[52,54]

[42] [10,12,13]

[64] [56] [49] [20–22]

[53,55]

IL, interleukin; NK, natural killer; TCR, T-cell receptor.

Deficiency of ADA, an enzyme involved in purine catabolism, was identified as the first known cause of SCID by Giblett et al. in 1972 [25]. In 1993, the etiology of X-linked (T−B+NK−) SCID, which accounts for approximately 50% of all cases, was found to arise from mutations within the γ chain of the common cytokine receptor, γc [42–44]. Infants with an autosomal recessive form of T−B+NK− SCID usually have mutations within the JAK3 gene [45–47]. A rare variant of (T−B+) SCID with absent circulating natural killer (NK) cells is caused by mutations within the IL-2R/IL-15β subunit [48]. Infants with T−B+ SCID who have circulating NK cells may have defects within the α chain of the IL-7 receptor [49,50] or null mutations within the δ, ε or ζ invariant chains of the CD3 subunit of the CD3–TCR complex [51–54]. Children with mutations within genes encoding CD45 [55] or hypomorphic mutations within the invariant chains of the CD3 receptor (reviewed in [51]) or IL-2Rα [56] generally present TloB+NK+ SCID. T−B− SCID can occur in patients with defects in RAG1 or RAG2 [16–19], DNA repair enzymes (Artemis [12,13], DNA ligase IV [22–24], Cernunnos-XRCC4-like factor [39]) or the gene encoding ADA (reviewed in [25]). ZAP-70 [57–62] and p56lck [63] defects result in patients with T cells but absent CD8 or CD4 cells, respectively. Capping defects [64] and abnormal CD7 [65] expression have also been associated with SCID, but the specific gene mutations involved have not yet been identified.

While ADA deficiency (reviewed in [25]), short-limbed dwarfism [30,31], Omenn’s syndrome [18,19], capping defects [64], CD8 deficiency [57–62] and SCID associated with mutations within the Artemis gene (reviewed in 5) or the IL-7R [49,50] are inherited as autosomal recessive disorders, classical SCID [5–8], SCID with B cells (reviewed in [5]), and reticular dysgenesis [32,33] can be inherited as either an autosomal recessive or X-linked disease. In 2004, Buckley [38] analyzed the genetic cause of 174 consecutive patients with SCID evaluated at Duke University Medical Center. Seventy-two percent of cases were due to defects within three genes: γc (46%), ADA (16.1%), and the IL-7Rα (10.1%) genes. Defects in JAK3, RAG1/2, and the Artemis gene accounted for 6.9%, 3.4%, and 1.1% of cases, respectively, with less than 1% being due to CD3ε or δ mutations. In only 14% of 174 patients was the genetic defect unknown. Analysis of transplantation outcomes on the basis of genetic mutation in 89 infants who received an unmodified (n = 12) or soy bean agglutinin and E-rosette depletion (SBA−E−) (E−) T-cell-depleted (TCD), mismatched-related (n = 77) bone marrow transplantation (BMT) at Duke University Medical Center [66] showed similar survival rates (79–100%) regardless of SCID mutation. In contrast, a European study analyzing the impact of SCID phenotype on outcome following HLAmismatched, TCD parental BMT demonstrated superior disease-free

Hematopoietic Cell Transplantation for Immunodeficiency Diseases

survival in patients with B-cell-positive variants of SCID compared with B-negative SCID (64% versus 36%, respectively) [67,68]. A trend toward better outcome in B(−) SCID patients was also observed in those patients who received pretransplant cytoreduction [68]. Elucidation of many of the genetic mutations which give rise to SCID has increased the proportion of infants who can be diagnosed in utero [69–74]. Early identification and transplantation in the neonatal period has been associated with survival rates of 90% or greater, reflecting transplantation prior to the development of severe opportunistic infections and more rapid immunologic reconstitution [75]. In a study by Myers et al. [75], long-term survival was observed in 21 of 22 of infants transplanted within the first 28 days of life, compared with 51 of 67 children transplanted at later time points. In the future, implementation of universal newborn screening [76,77] by quantitation of T-cell receptor excision circles (TRECs), the nonreplicative episomal DNA formed during intrathymic maturation of single positive mature α/β T-cell receptor-containing T cells, or other techniques, will likely lead to improved outcomes for affected infants by permitting diagnosis and earlier HCT prior to the development of infectious complications. Newborn screening will also allow a more precise determination of the incidence of SCID/CID.

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previous studies from the Hôpital Necker-Enfants Malades and the Children Hospital of Los Angeles (reviewed in [1]). Earlier diagnosis and improved methods for the detection, treatment, and prevention of opportunistic infections have contributed to the improved outcome of SCID transplants from all donor sources. Following infusion of bone marrow from an HLA-matched sibling, expansion of memory T cells infused with the graft results in normal numbers of circulating T cells within 2–4 weeks post HCT and rapid restoration of T-cell mitogen-induced and antigen-specific responses [11,66,83,84]. Circulating naïve CD45RA+ T cells generated from bone marrow-derived lymphoid precursors that have undergone thymic education are usually detected 3–4 months post HCT. Following an unmodified HLA-matched related HCT, specific B-cell function usually develops by 1 year post transplant, allowing cessation of monthly intravenous gammaglobulin therapy and subsequent reimmunization [11,66,84]. In our series of 10 consecutive recipients of an unmodified HLA-matched sibling BMT administered without preparatory cytoreduction between 1987 and 2005, all have specific antibody production following vaccination with routine childhood immunizations, and none remains on intravenous gammaglobulin. Alternative donor transplants for SCID

HCT for SCID Unmodified human leukocyte antigen-matched related HCT The first successful human allogeneic HCT was reported in 1968 by Gatti et al. in a patient with X-linked SCID [78], who currently remains alive and well almost four decades later. Transplantation of T-cell-replete HCT from a human leukocyte antigen (HLA) genotypically identical sibling remains the treatment of choice for infants with SCID, resulting in long-term survival of over 80% (reviewed in [1]). In most of these patients, including those with functional NK cells or a marginal in vitro T-cell proliferative response to phytohemagglutinin, pretransplant cytoreduction is not necessary to ensure engraftment. However, in patients with reticular dysgenesis (SCID with severe neutropenia), administration of cytoreduction is indicated to ensure engraftment of hematopoietic as well as lymphoid progenitors. Following HLA-matched HCT for SCID, prophylaxis for graft-versus-host disease (GVHD) is usually not given due to the low incidence of significant GVHD in these infants [66]. Of 10 consecutive recipients of an unmodified HLA-matched sibling BMT for SCID at Memorial Sloan Kettering Cancer Center (MSKCC) since 1987, none has developed GVHD despite lack of prophylaxis and infusion of unmanipulated bone marrow containing a median of 7.18 × 108 (range 2.5–10.9 × 108) total nucleated cells/kg. The basis for the low incidence of acute and chronic GVHD may be the absence of tissue injury due to lack of cytoreduction [79], as well as the young age of the donor–recipient pair. Results of HLA-matched transplants for SCID have improved from the 48% long-term survival reported in 1979 [80] to 80–100% in current series [66,67,81]. An update of the long-term results of transplants for SCID in Europe from 1968 to 1999 demonstrated an 81% 3-year survival in 104 recipients of an HLA-matched sibling BMT [67]. Patients with ADA deficiency or reticular dysgenesis had a 3-year survival of 81% and 75%, respectively [67]. In 2006, Grunebaum et al. reported a 92% long-term survival in 13 infants with SCID after HLA-matched sibling BMT [81]. Notarangelo et al. demonstrated an over 80% longterm survival in 35 infants with SCID who received an HLA-matched sibling HCT at the University of Brescia [82]. At the University of Ulm, Germany, 17 of 21 consecutive recipients of an HLA-matched family member HCT for SCID are alive and well. These results are similar to

In 1973, Keightly et al. [85] reported the successful use of a fully mismatched fetal liver graft to reconstitute a patient with ADA deficiency. A 1983 review of this modality by O’Reilly et al. [86] demonstrated that only 22 of 105 fetal liver transplants resulted in a durable engraftment of fetal lymphoid cells. Only 20% of patients survived more than 1 year following fetal liver transplants. Results with this approach are clearly inferior to those achieved with TCD, haplotype-disparate parental HCT and T-replete transplants from histocompatible unrelated adult or cord blood donors. However, they provided the first human evidence that allogeneic hematopoietic cells lacking mature T cells could be transplanted without risk of GVHD. Transplants of TCD, HLA-disparate related marrow are now the most common type of transplant administered to patients lacking a matched related donor. However, the success of TCD, HLA-mismatched related transplants for SCID depends on the degree and method of T-cell depletion used (reviewed in [87]), variables which affect the risk of graft failure, GVHD, and post-transplant complications, such as Epstein–Barr virus-associated lymphoproliferative disorders (EBV-LPD). The techniques for T-cell depletion that have been utilized for transplantation of infants with SCID include soy bean agglutination followed by rosetting with sheep red blood cells (SBA−E−) [88,89], E-rosetting alone [90], antibody-mediated techniques, such as treatment of the graft with OKT3 [91] or Campath-1 [92,93], and positive selection of CD34+ progenitors from granulocyte colony-stimulating factor mobilized peripheral blood hematopoietic cells, with or without further T-cell depletion by Erosetting or monoclonal antibody treatment [94]. The first successful corrections of SCID by TCD, mismatched-related BMT were reported by our center in three infants in 1983 [88], transplanted with haploidentical parental SBA−E− bone marrow. The children did not receive pretransplant cytoreduction or post-transplant GVHD prophylaxis. Long-term stable donor T-cell chimerism with normal Tcell function was achieved in all three infants, without the development of acute or chronic GVHD. Currently, for the 71 children who have received an SBA−E− (E−), mismatched parental BMT at MSKCC from 1980 through 2006, the 10 year survival is 72% (Fig. 75.1). Disease-free survival was 79% in the 20 children transplanted at less than 3 months of age, and 66% in the 51 infants transplanted at over 3 months of age (p = 0.2) (Fig. 75.1). The incidence of grade II acute or chronic GVHD has been less than 10%. Similar results with haploidentical SBA−E−

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1 0.9 ≤3 months = 79.33% n = 20

0.8

>3 months = 66.67% n = 51

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Fig. 75.1 Overall survival at 15 years of SBA−E−-mismatched T-celldepleted bone marrow transplantation for severe combined immunodeficiency syndrome performed 1980–2006 at the Memorial Sloan Kettering Cancer Center (n = 71) on the basis of age at transplant (≤3 months or >3 months).

BMT for the treatment of SCID have been reported from other centers, including the University of Ulm [95], the University of California Medical Center at San Francisco [96], and the Duke University Medical Center, NC [66]. In each of these series, patients did not receive any post-transplant drug prophylaxis against GVHD. In contrast, HLA-mismatched related HCT depleted of T cells by Erosetting alone or by treatment with Campath-1M have been administered together with post-transplant prophylaxis with cyclosporine. Such transplants have achieved long-term survival rates ranging from 52% to 70% [67,11]. Acute GVHD requiring therapy has occurred in 35–40% of cases and has been the major predictor of mortality [67]. For example, in a report of the European Group for Blood and Marrow Transplantation and European Society for Immunodeficiencies, summarizing results for 267 patients with SCID who had received HLA-disparate, TCD grafts after cytoreduction with, busulfan (BU; 8 mg/kg) and cyclophosphamide (CY; 200 mg/kg) [67], outcomes further improved to a 70% survival for transplants administered from 1996 to 1999, a majority of which were depleted of T cells by positive selection of CD34+ progenitor cells. With this approach, doses of T cells could be reduced from a 5 × 105 T cells/ kg upper limit selected by the European Society for Immunodeficiencies in 1986 to 1 × 104 T cells/kg body weight. Transplant outcomes are also significantly influenced by the status of the patient at the time of the procedure. In the European series, a multivariate analysis revealed that infants with B-cell deficient variants of SCID and those with an antecedent pulmonary infection were at significant risk for inferior outcome [67]. As noted in the European Group for Blood and Marrow Transplantation report [67], centers with greater experience in the use of HLAnonidentical TCD grafts also achieved significantly better outcomes (57% versus 43%). In fact, the Hospital Necker-Enfants Malades in Paris has reported a 70% survival for children with SCID who received transplants of marrow depleted by E-rosetting alone [11]. Similarly, of 17 infants with SCID at the Royal Victoria Infirmary in Newcastle, UK, between 1986 and 1998 who received HLA-disparate grafts depleted of T cells by treatment with Campath-1M and complement, 11 (64%) achieved long-term survival with reconstitution [93]. Recently, several studies have evaluated the results of unmodified HLA-matched and TCD HLA-haploidentical HCT in the rarer forms of SCID. Results from these small series underscore the differences in graft

resistance exhibited by specific variants of SCID. For example, Roberts et al. [97] recently reported results of HCT in 10 patients with JAK-3deficient SCID. Two of these patients engrafted following an HLAmatched, unmodified graft without conditioning. Of eight patients who received an SBA−E− BMT from an HLA-haploidentical related donor without conditioning, six achieved engraftment and functional reconstitution of donor T cells. However, no patient showed engraftment of donor B cells. Furthermore, two patients failed to engraft and reconstitute despite multiple SBA−E− BMT; one of these patients was ultimately reconstituted following high-dose conditioning and a cord blood graft. In contrast, in a series of 16 patients with SCID associated with an IL7Rx deficiency, Giliani et al. [98] reported that each of six recipients of TCD, HLA-haplotype-disparate HCT achieved engraftment, reconstitution and long-term survival when cytoreduced pre transplant with BU and CY. However, despite conditioning, B-cell engraftment was not detected in these patients and was only detected in two of nine recipients of HLA-matched marrow grafts, of whom three had received pretransplant conditioning. Overall, 13 of 16 patients in this series survive with reconstitution. For patients with SCID associated with either Omenn’s syndrome or reticular dysgenesis, cumulative evidence indicates that high-dose conditioning, or at least reduced-intensity conditioning, prior to transplant is likely indicated to secure engraftment and functional reconstitution following a TCD, HLA-haploidentical, related HCT [99]. In patients with Omenn’s syndrome, early transplants administered without cytoreduction were associated with a high incidence of graft failure. In contrast, such patients, when transplanted with HLA-haplotype-disparate, TCD HCT after cytoreduction with BU and CY, have ultimately achieved full engraftment and reconstitution in seven of nine patients reported [100]. Thus, such cytoreduction can overcome the graft resistance thought to be mediated by the small numbers of autoactivated T cells and NK cells consistently detected in these patients. Similarly, in a series of 10 patients with reticular dysgenesis transplanted without high doses of BU and CY, none survived with sustained engraftment, while each of five patients treated with this high-dose combination engrafted, three of whom are long-term survivors [101]. There are only a limited number of studies published on the specific use of TCD peripheral blood hematopoietic cells transplantation for the treatment of SCID. Lanfranchi et al. [94] evaluated the use of frozen TCD peripheral blood hematopoietic cells plus TCD bone marrow in nine patients with primary immunodeficiency disease, including four with SCID, three with Omenn’s syndrome, and two with CID. This approach was undertaken primarily to determine whether high cell doses could overcome the increased incidence of graft failure observed in patients with CID, who possess abnormally low but residual T-cell function. Patients were cytoreduced with BU 16 mg/kg, CY 200 mg/kg, and thiotepa 10 mg/kg. In the majority of cases, the bone marrow was T-cell depleted by Campath-1M, and peripheral blood hematopoietic cells by CD34 selection followed by E-rosetting. Although the follow-up is short (mean 7.5+ months), six of the seven patients transplanted for SCID or Omenn’s syndrome survive. Although all four patients with SCID engrafted, one died of a disseminated CMV infection. Dupuis-Girod et al. [102] recently reported successful transplantation of HLA-mismatched, maternal, TCD peripheral blood HCT (PBHCT) in a 12-month-old child with ectodermal dysplasia and SCID, following cytoreduction with BU 5 mg/kg per day for 4 days, followed by 4 days of CY 50 mg/kg/day. This patient engrafted on day 25 after transplantation, had no evidence of GVHD, and survives over 7 years post HCT, with full chimerism and a normal T-cell response to phytohemagglutinin, tetanus, and polio, as well as antibody responses to immunization with tetanus and Haemophilus influenzae vaccines. Other than recurrent plantar warts, the child is clinically well.

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Hematopoietic Cell Transplantation for Immunodeficiency Diseases 1.0

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Fig. 75.2 Overall survival of CD34+ peripheral blood hematopoietic cell transplantation for severe combined immunodeficiency syndrome, with (䉬) or without (䊏) conditioning, performed in Ulm, Germany from 1995 to 2004 (n = 53).

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From 1995 to 2006, 53 patients received a CD34-selected, rigorously TCD mismatched, parental peripheral blood hematopoietic cells graft at the University of Ulm for the treatment of ADA-SCID (n = 2), B−T− SCID (n = 13), Omenn’s syndrome (n = 3), B+ SCID (n = 29), B+T+ (n = 3), bare lymphocyte syndrome (n = 2), and reticular dysgenesis (n = 1). The majority of patients received cytoreduction (41 of 53 patients), primarily with BU/CY. Again, no drug prophylaxis has been administered post transplant to prevent GVHD. To date, 38 (72%) patients are alive and well, at a median of 80 (range 14–144) months post TCD PBHCT, none of whom has GVHD. There were two graft failures, one in a patient with an Artemis mutation, the other in one with B+T+ SCID/ CID. Neither child was given pre-transplant cytoreduction. There were 15 deaths, all due to infection, one following graft failure. Neither de novo acute nor chronic GVHD was observed in any of the surviving patients. The overall survival rate in this group of SCID patients following TCD, mismatched, CD34+-selected transplants (Fig. 75.2) is similar to the experience in SCID patients who have received rigorously TCD BMT. As with marrow grafts, complete engraftment and reconstitution by donor cells remained highly dependent on prior use of myelosuppression. Infusion of purified CD34+ cells in significant numbers did not facilitate full donor chimerism. Time to develop effective T-cell immunity post TCD, mismatched PBHCT ranged from 3 to 6 months and was similar to that observed after TCD BMT. Thus, the use of purified blood CD34 cells in high numbers does not appear to accelerate the functional maturation of donor T cells. Following an SBA−E− mismatched, related BMT, the majority of patients will develop normal numbers of CD3 (Fig. 75.3(a)), CD8, and CD4 T cells by 3–6 months post HCT, irrespective of whether or not cytoreduction is employed [1,66,103]. Approximately 50% of patients will have a normal phytohemagglutinin response by 6 months post transplant, and 80% will have normal T-cell mitogen and antigenspecific responses by 6–12 months (reviewed in [1,66,67]). In a large European study of mismatched, TCD HCT recipients using a variety of TCD techniques, the median time to develop normal T-cell proliferative responses to tetanus toxoid was 8.7 months [103]. Of the 116 patients in this study who survived at least 6 months post HCT, only 43% of those with absent T-cell function at 6 months (n = 21) ultimately developed normal T-cell responses, compared with 71% of those patients with normal or near-normal T-cell function 6 months post transplant [103].

Months post TCD MM related BMT for SCID, 2007

Fig. 75.3 (a) 10%, 50%, and 90% of CD3 recovery following SBA−E−-Tcell-depleted (TCD) human leukocyte antigen (hla)-mismatched bone marrow transplantation (BMT) for severe combined immunodeficiency disease (SCID) with and without cytoreduction. (b) Median CD4 : CD8 ratio following SBA−E−- CD HLA-mismatched (MM) BMT for SCID on the basis of cytoreduction.

The quality and kinetics of T-cell reconstitution in the European series correlated with SCID phenotype, with a higher percentage of patients with B+ SCIDs exhibiting normal T-cell function at all time points later than 6 months post HCT [103] compared with patients with B− SCID. Several studies have evaluated factors that influence long-term survival and immune reconstitution following TCD, mismatched, related HCT for SCID. Younger age at the time of transplant has been associated with a better outcome, with particularly good results achieved in patients transplanted in the neonatal period [66,67,75,104–108]. In a study by Myers et al. [75], long-term survival was observed in 21 of 22 infants transplanted within the first 28 days of life, compared with 51 of 67 children transplanted at later time points. As seen in earlier studies [108], interstitial pneumonia [66,67,103,108], particularly antecedent CMV or adenoviral infections, was associated with an increased mortality. Failure to develop T-cell function by 6 months post transplant or the presence of chronic GVHD for more than 6 months post HCT also confers an increased risk of mortality [103]. In the combined MSKCC and Ulm series (reviewed in [1]), NK+ SCID patients who did not receive pretransplant cytoreduction had a poorer outcome primarily due to graft failure. Although not all studies have shown an association between NK function and graft resistance [66], this may relate to differences in the genetic basis giving rise to NK+ SCID in the transplant population evaluated.

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In comparative studies of the quality and kinetics of immune reconstruction, Muller et al. [104] did not find any differences in the pattern of naïve CD4+CD45RA+ T-cell reconstitution in patients who received an unmodified HLA-matched sibling graft versus TCD, mismatched, related HCT for SCID. Furthermore, in patients who received no pretransplant cytoreduction prior to SBA−E−, mismatched, parental HCT, there was a dramatic increase in the percentage and absolute numbers of CD3+CD45RA, TREC-bearing T cells within the first year following HCT [104]. Myers et al. [75] also demonstrated that infants who receive an SBA−E−, mismatched, parental BMT within 28 days of birth develop higher numbers of CD45RA+ and TREC+ T cells, and more robust T-cell proliferative responses within the first 3 years post transplant, than infants transplanted at later time points. Recently, evaluations of patients with SCID 10–15 years after successful HLA-haplotype-disparate TCD grafts have focused attention on those characteristics of host and HCT that sustain thymic output and normal T-cell function. Initially Patel et al. [105] showed that the average number of naïve T cells and T-cell mitogen responses decreased with time after such transplants. Data from the MSKCC (Fig. 75.3(b)) also demonstrate that, in the absence of cytoreduction, the CD4 : CD8 ratio inverts in a percentage of patients following TCD SBA−E− BMT, paralleling a decline in phytohemagglutinin response in this subset of patients. However, these alterations are not seen in cytoreduced transplant recipients. Similarly, Borghans et al. [106] evaluated predictors of long-term T-cell reconstitution in a heterogeneous group of 19 SCID patients evaluated 5–32 years post HCT. Unlike the patients in the study of Patel et al. [105], who received SBA−E− HCT without prior conditioning, 74% of patients in this study were cytoreduced, and 14 of 19 patients received transplants that were T-cell depleted by E-rosetting and concomitant GVHD prophylaxis. In 11 of 19 patients, there was no evidence of impaired T-cell reconstitution, naïve T cell production or reduced telomere length post HCT when compared with age-matched controls. In the eight patients with impaired T-cell reconstitution late after transplant, these parameters were already abnormal early after HCT, suggesting that poor engraftment rather than reduced thymic output with time was the etiology of the poor T-cell reconstitution. However, two more recent studies have shown that those patients with SCID that have received haplotype-disparate, TCD HCT who have donor cells in their peripheral myeloid lineages or among the CD34+ progenitor cells in the marrow late after transplant also have sustained normal levels of TREC+ T cells without evidence of a decline in T-cell number or function [107,108]. The study of Friedrich et al. [108] is particularly informative, since 25 of the 31 patients studied had received HLA-haplotype-disparate, SBA−E− TCD marrow grafts, either with (n = 13) or without (n = 12) pretransplant cytoreduction. Sustained engraftment of donor-type CD34+ progenitor cells was regularly observed in the cytoreduced group and was directly correlated with persistently normal numbers of TREC+ and CD45RA+ T cells. Donor CD34+ cells were not detected in the noncytoreduced group. These patients also exhibited significant reduction of CD45RA+ T cells 15 years post transplant. Although these studies demonstrate the ability of the epithelium of the SCID thymus to support robust thymopoiesis in most patients, the declining generation of naïve thymus-dependent T cells observed in some patients, particularly those who received no cytoreduction prior to transplant, remains of concern [105]. Thymopoiesis may not be sustained if long-term engraftment of hematopoietic stem cells capable of continuously providing thymic precursors is not achieved, a scenario that is common in noncytoreduced patients [107,108]. Unfortunately, in patients with poor T-cell reconstitution, Slatter and colleagues demonstrated that boosts of TCD HCT following initial transplant, in the absence of cytoreduction, were not associated with improved function [109].

Reconstitution of specific antibody formation following SBA−E−, mismatched, related HCT, as with other TCD techniques, is generally observed only in those patients given pretransplant cytoreduction, and is correlated with the degree of donor B-cell engraftment (reviewed in [1,66,67,109]). In the MSKCC series, normal antibody responses following immunization were observed in all patients engrafted with donor B cells, compared with less than 10% of patients with exclusively host B cells (reviewed in [1]). Forty-five of 72 infants transplanted at Duke University Medical Center [66] required prolonged gammaglobulin therapy, the majority of whom received an SBA−E− TCD, mismatched, related marrow graft without prior cytoreduction. This contrasts to the smaller study by Dror et al. [96] which showed that, despite absence of donor B cells, six patients developed specific antibody responses 1.5–10.0 years following an SBA−E−, mismatched, related BMT. In a single-center study by Haddad et al. [110], donor B-cell engraftment was documented in four of five and three of 17 recipients of unmodified HLA-matched or TCD, mismatched, related BMT, respectively. Ten of 18 patients with host B cells remained on intravenous Ig more than 2 years post BMT. In this study, in contrast to the combined MSKCC–ULM experience, cytoreduction with BU 8 mg/kg and CY 200 mg/kg did not promote donor B-cell engraftment or the development of specific antibody production [110]. In patients with B− Artemis mutations, B-cell engraftment was observed only if autologous precursor B cells in the bone marrow were eradicated [111]. Despite the success of TCD, mismatched, related HCT for patients lacking an HLA-matched sibling donor, several problems clearly remain, namely: 1 graft failure in some series in those patients with NK-cell function or minimal T-cell function (reviewed in [1]); 2 GVHD following less rigorous TCD techniques [67,90,91]; 3 EBV-LPD, due to uncontrolled proliferation of transformed B cells prior to T-cell development [112,113]; 4 lack of donor NK-cell engraftment, which has been associated with epidermodysplasia verruciformis in both MSKCC and other series [114,115]; 5 autoimmune disorders due to dysregulated B-cell function [116,117]; 6 hypogammaglobulinemia, despite successful T-cell reconstitution in patients not given cytoreduction [66,68,110] who fail to develop donor B cells; such patients require regular infusions of Ig to avert recurrent sinopulmonary infections, bronchiectasis, and respiratory insufficiency; 7 diminished long-term capacity to produce naïve T cell populations [104,105]. An ongoing debate at many centers performing alternative donor transplants for SCID is whether all clinically stable children should receive pretransplant cytoreduction, even in the absence of factors known to inhibit T-cell engraftment. Universal cytoreduction would increase the proportion of children who engraft not only donor T cells, but also donor B and NK cells, thus avoiding continued need for intravenous Ig, risk of recurrent sinopulmonary infections despite intravenous Ig, the potential of diminished thymic output with time, and complications associated with lack of NK cells [114,115]. However, adoption of this approach obviously needs to be weighted against the long-term side-effects of cytoreduction in sick infants, particularly in newborns diagnosed antenatally or with newborn screening techniques. Furthermore, while most infants will develop at least partial donor B-cell chimerism and specific B-cell function with reduced doses of BU and CY, this is not true in all patients [110]. It is possible that reduced-intensity conditioning, for example with BU combined with fludarabine, followed by TCD HCT, will be more readily tolerated than BU/CY and will still allow the devel-

Hematopoietic Cell Transplantation for Immunodeficiency Diseases

opment of donor-derived T- and B-cell reconstitution. This is currently being explored.

1.0 0.9 0.8

Unrelated HCT for the treatment of SCID was first performed in 1973 by O’Reilly and colleagues [118]. The stem cell source was bone marrow derived from a one-antigen HLA class I-disparate female donor and administered to an HLA-A1-homozygous male infant. After multiple transplants from this donor, lymphoid engraftment and full immunologic reconstitution were achieved, complicated by the development of extensive chronic GVHD. This patient ultimately died 9 years post HCT of metastatic squamous cell carcinoma of the skin. In 1992, Filipovich et al. [119] reported on eight children who received unmodified, unrelated HCT. All but one child was cytoreduced with BU/CY and antithymocyte globulin (ATG). All patients received GVHD prophylaxis. Six of eight children survived with immunologic reconstitution between 1.0 and more than 3.8 years after BMT. Only one patient developed significant GVHD. In 1996, an update of unrelated BMT for SCID (n = 24) was published [120]. In this series, children with X-linked or ADA+ SCID were not given cytoreduction. Of those given cytoreduction, most received BU, CY, ATG, and GVHD prophylaxis with cyclosporine (CSP) and methotrexate. Four patients died less than 28 days post HCT and could not be evaluated. Of the 20 remaining evaluable patients, graft failure occurred in two. The estimated 3-year survival was 61%, with grade II–IV GVHD occurring in 17% of engrafted patients [120]. Nine children with SCID received an unmodified, unrelated BMT at the Hospital for Sick Children at Toronto, Canada, between December 1987 and August 1997 [121] following cytoreduction with BU/CY. CSP, methylprednisolone, with or without short-course methotrexate, was given to prevent GVHD. Donors were identified at a mean of 4.7 (range 1–13) months. Despite antecedent pneumonia in four of the nine SCID patients, six survive a median of 54.5 (range 18–72) months post unrelated transplant. Death in the other three patients was due to graft failure (n = 1) or grade III GVHD (n = 2). T-cell reconstitution occurred in all six patients. B-cell reconstitution was documented in four patients. Grunebaum et al. [81] reported on the results of unrelated HCT performed at the University of Brescia in Italy and the Hospital for Sick Children in Canada between 1990 and 2004. Thirty-three of 41 (80.5%) patients who received an unmodified unrelated graft survive. Acute GVHD was observed in 71% of patients. In the 2003 update of the European experience of HCT for SCID, the 3-year survival was 63% in the 28 recipients of an unrelated HCT [67]. Results of unrelated HCT (bone marrow or peripheral blood) reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) from 1990 through 2004 are shown in Fig. 75.4. The probability of overall survival after unrelated allogeneic donor HCT for SCID at 5 year (including unrelated bone marrow and peripheral blood) is 60% (95% confidence interval [CI] 52–67%). Reduced-intensity conditioning and HCT for SCID A subset of infants with SCID present with comorbidities which prohibit the use of high-dose conditioning. Several groups have explored the use of reduced-intensity conditioning for such patients [122,123]. Rao et al. [122] compared survival in 13 patients with SCID, of whom seven received BU (16 mg/kg) and CY (200 mg/kg), and six received reducedintensity conditioning with fludarabine, melphalan, and either alemtuzumab or ATG. The former group received unrelated bone marrow depleted of T cells with Campath and donor complement. Recipients of reduced-intensity conditioning received unmodified bone marrow. Five of seven patients with SCID who received high-dose conditioning sur-

Overall survival, 61% (95% CI 52-70) @ 5 years

0.7 Probability

Unmodified, unrelated BMT for SCID

1111

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Fig. 75.4 Probability of overall 5-year survival following unrelated bone marrow or peripheral blood transplantation for patients with severe combined immunodeficiency disease performed from 1990 to 2004, as reported to the Center for International Blood and Marrow Transplant Research (n = 129). (Reproduced with permission from the CIBMTR.)

vived, compared to five of six patients who received reduced-intensity conditioning [122]. Umbilical cord blood cell transplants for SCID In 2000, Knutsen and Wall [124] described eight infants with severe T-cell immunodeficiency disease who received an unrelated cord blood cell transplant, seven of whom engrafted. Graft failure occurred in one infant with reticular dysgenesis given BU/CY who subsequently engrafted following a cord blood transplant from a second unrelated donor after total body irradiation conditioning. Despite significant HLA mismatching between the cord blood donor and recipient in this group of eight patients, five patients developed grade I acute GVHD, and only one patient developed severe GVHD involving the skin and gut. Tand B-cell immunity recovered at 60–100 days post-transplant [123]. Buckley et al. have successfully used unrelated cord blood transplantation to rescue three children with X-linked SCID who rejected a TCD, mismatched, related BMT given in the absence of cytoreduction. In two of these cases, BU/CY was given prior to cord blood transplantation [66]. Successful correction of Omenn’s syndrome (n = 1) and X-linked SCID (n = 2) by unrelated cord blood cell transplant have been reported, as well as correction of T−B−NK+ SCID by HLA-matched sibling cord blood cell transplant [124–128]. Bhattacharya and colleagues transplanted eight patients with SCID with unrelated (n = 7) or related (n = 1) cord blood transplant [128]. Infants were treated for ADA deficiency (n = 4), reticular dysgenesis (n = 1), T−B+NK+ SCID (n = 2), and one undefined SCID syndrome. Conditioning consisted of BU (16 mg/kg)/ CY (200 mg/kg) in four patients, and no conditioning in three of four patients with ADA deficiency, one of whom received a matched sibling cord blood transplant. Seven of eight patients engrafted, and six of eight survive. One patient developed severe sinusoidal obstruction syndrome, which was successfully treated with defibrotide. Two deaths occurred. One patient with ADA deficiency died of respiratory failure secondary to parainfluenza-3 which antedated transplant. The other death was due to multiorgan failure. The results of a CIBMTR analysis of 77 cord blood transplants performed for SCID between 1990 and 2004 are shown in Fig. 75.5. The probability of overall survival at 5 years was 57% (95% CI 44–69%) [129].

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Chapter 75 Years

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Fig.75.5 Probability of overall 5-year survival following unrelated donor cord blood transplants, 1990–2004, reported to the Center for International Blood and Marrow Transplant Research (n = 77). (Reproduced with permission from the CIBMTR.)

In utero HCT for SCID In 1996, Flake et al. reported the first successful correction of SCID by a TCD, haploidentical parental marrow graft administered by in utero transplantation [130]. This report was followed closely by similar case reports of Wengler et al. in 1996 [131], and Gil and colleagues in 1999 [132]. The patient reported by Flake et al. had X-linked SCID that was diagnosed during the first trimester [130]. This patient received three infusions of TCD bone marrow cells containing 114.0, 8.9, and 6.2 × 106/kg CD34+ cells and less than 5.2 105 CD3+ cells/kg fetal weight. The cells were injected into the abdomen of the fetus between 15.0 and 18.5 weeks gestation. Circulating donor T cells were observed at birth and at 3 and 6 months thereafter. However, B cells, NK cells, monocytes, and granulocytes remained of host origin. The engrafted paternal T cells were tolerant to the patient and demonstrated normal in vitro T-cell proliferative responses. Subsequent analysis of this patient has demonstrated persistent engraftment of donor T cells with specific T- and B-cell function 4 years post HCT [130,133,134]. Muench [135] summarized the results of in utero transplants in 10 patients performed between 1996 and 2005 to correct γc (n = 5) or IL7Rα deficient (n = 1) SCID, Omenn’s syndrome (n = 1) or genetically uncharacterized T−B+ (n = 1), T−B− (n = 1) or T−B+NK+ (n = 1) SCID. Of the 10 patients with SCID and absent T cells, nine received TCD bone marrow or peripheral blood; one patient received fetal liver and thymus antenatally and additional fetal liver and thymus, as well as TCD HCT, postnatally. This latter patient lived for 9 years post HCT, but succumbed after a liver transplant performed at 9.5 years of age. Engraftment of T lymphocytes with or without donor NK cells was observed in eight of ten patients. Although these cases demonstrate the feasibility of in utero transplants to induce T-cell reconstitution, comparison of this treatment with other modalities is difficult due to the small numbers of such transplants. To date, B-cell reconstitution has been achieved in only one patient [136]. Conclusion As summarized above, the results of related HLA-disparate, TCD HCT, which date from the early 1980s and are applicable to all patients with SCID, continue to improve and are now associated with a low risk of graft failure and a very low incidence of severe GVHD [1,67]. Centers with large series of patients so treated report disease-free survival rates

with sustained engraftment and reconstitution of T-cell immunity in over 70% of cases [1,66,80,93,95,96]. Similar survival rates are now also being achieved with unmodified grafts from HLA-matched unrelated donors, albeit with higher rates of both acute and chronic GVHD [81,119]. While a European retrospective multicenter analysis has reported significantly better overall results with unrelated transplants, analysis of patients transplanted since 1987, when unrelated donor identification became practicable, did not indicate a significant advantage [67]. A more recent retrospective comparison of transplant results in Brescia and Toronto [81] also suggested an advantage to the use of a matched unrelated donor. However, in that study, the vast majority of clinically unstable patients referred for transplant received a TCD HLAhaplotype-disparate parental graft. This analysis also did not account for the clinical consequences of the excess time required to identify an HLA-identical unrelated donor. More importantly, the survival rates recorded for recipients of unrelated transplants were comparable to those for recipients of HLA-nonidentical TCD grafts but markedly inferior to those currently reported from other large centers that regularly employ HLA-haplotype-disparate TCD grafts [1,66,90,93,95,96]. At this time, only a prospective study comparing outcomes of alternative donor transplants in terms of survival, graft failure, GVHD, infectious complications, and long-term immune reconstitution is likely to determine whether and to what degree one or the other approach is more beneficial. Further, from a clinical prospective, the optimal alternative donor, stem cell source, and cytoreductive regimen to be selected is unlikely to be uniform for this heterogeneous population. The clinical status of the affected infant, the variant of SCID requiring correction, the availability of a suitable HLA-matched related or unrelated donor, and the T-cell-depletion techniques available to an individual transplant center all need to be considered to determine how best to transplant the child. Many children with SCID who lack a matched sibling donor present with severe pneumonias caused by viruses or other pathogens and are too ill to be transferred to a transplant center experienced in the use of TCD, HLA-haplotype-disparate, parental grafts for SCID. While donor marrow grafts can be depleted of T cells at a specialized center and transported back for transplantation into such patients, this approach is at present logistically difficult. In such circumstances, an unmodified, HLA-matched marrow or cord blood graft could be the better option, but only if the identification and procurement of such a transplant can be achieved promptly. If the time needed to identify a suitable unrelated donor exceeds 3–4 weeks, the added infectious morbidity and risk of fatality imposed on an infant with SCID is not justified. Conversely, a matched unrelated marrow or cord blood graft would be preferred if a graft from a haplotype-matched parental or unrelated donor would impose an undue risk of infection, such as active CMV or hepatitis B or C infection. Transplantation for other CIDs and lethal congenital disorders The CIDs, including Nezelof syndrome [137], include a heterogeneous group of rare diseases in which patients present with recurrent severe acute and chronic infections associated with profound but not absolute deficiencies of T- and B-cell function (reviewed in [6,7,137]). In these disorders, T-cell numbers may be normal or reduced. Although antigenspecific responses to pathogens are not detectable, proliferative responses to nonspecific mitogens and allogeneic cells, while markedly abnormal, are not absolutely deficient as they are in patients with SCID. In several CID syndromes, B cells are prominent and Ig classes may be normal or increased. However, specific antibody production is usually absent (reviewed in [6,7,137,138]).

Hematopoietic Cell Transplantation for Immunodeficiency Diseases

The genetic mutations responsible for many of the CID syndromes are known, permitting definitive diagnosis. Genetic analysis is also important to distinguish these patients from a subset of patients with variants of SCID including Omenn’s syndrome [14,15], ADA deficiency [27], CD3 invariant chain mutations [51], and defects of DNA ligase IV [22–24], and Cernnunos [39] expression who may present with partial rather than complete absence of T- and B-cell function. A second set of CID syndromes are the result of defects in cytokines or their receptors or lack of cell surface molecules involved in cell contact, antigen presentation, activation, and/or proliferation. These include: • defective cytokine production or responsiveness; • defective IL-1 responsiveness [139]; • defective transcription of multiple cytokine genes [140]; • abnormal IL-2 production [141,142]; • nucleoside phosphorylase deficiency [143,144]; • CD8 deficiency caused by mutations in ZAP-70 [58]; • bare lymphocyte syndromes [6,7,145–147], a series of genetic disorders resulting in defective transcription and expression of HLA class I and/or class II determinants on the surface of lymphoid and hematopoietic cells; • deficiency of leukocyte function-associated antigen-1 (LFA-1) [148,149]; • CD40 ligand and CD40 deficiency [150–154]; • common variable immunodeficiency disease [6,7,155]. A third set of disorders for which transplantation may be curative includes a series of genetically defined diseases that cause either lymphoproliferative disease or severe and uncontrolled autoimmune disorder. These include: • X-linked lymphoproliferative syndrome [156,157]; • abnormalities of T cell regulation: IPEX, autoimmune lymphoproliferative syndrome (ALPS: ALPS1a, ALPS1b, ALPS2, and ALPS3) (reviewed in [6,7,158]). Each of these disorders of immunity can be corrected by a marrow allograft. However, because these disorders are so rare, experience with allogeneic HCT in these diseases has been largely limited to transplants of from one to three patients at any given center. In aggregate, the results of transplants applied to patients with these disorders have been distinctly inferior to those reported for transplants applied to patients with SCID or WAS. For example, in the European experience of HCT for non-SCID patients with T-cell immunodeficiency performed between 1968 and 1993, 3-year overall survival was 63% for 47 recipients of an HLA-matched sibling graft, and only 35% in 72 recipients of an HLAmismatched, TCD graft [67]. For comparison, survival rates for patients with SCID receiving such grafts were 81% and 54%, respectively. The inferior results achieved in these disorders reflect in part the higher incidence of graft rejections in these patients, indicating the need for effective immunoablation, and possibly myeloablation, to achieve consistent engraftment. They also reflect the increase in transplant-related mortality due to toxicity and infection in these children, who are often chronically infected and debilitated at the time of diagnosis and referral for transplantation. However, based on analysis of recent experience in certain more common CID diseases, over the last decade, an improved diagnosis of these disorders, recognition of the need for effective pretransplant cytoreduction, and improvements in the identification of compatible donors and in the development of less toxic cytoreductive regimens for transplantation are likely resulting in better outcomes [67,98,159–169]. Recent results of transplants applied to patients with hyper IgM syndrome caused by CD40 ligand deficiency are a case in point. For example, Gennery et al. [167] updated the European experience of transplants for X-linked hyper IgM syndrome (n = 38) performed from

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1993 through 2002. Donors included an HLA-identical sibling (n = 14), a phenotypic HLA-matched family member (n = 4), and a volunteer unrelated donor (n = 22). Thirty-four of thirty-eight patients engrafted, and 26 patients survived. Twenty-two patients developed normal T-cell function, and 18 developed specific antibody following vaccination. One patient who engrafted but subsequently developed impaired T-cell reconstitution had autologous reconstitution, 22 (58%) were cured, and one other patient engrafted but had poor T-cell immune reconstitution. Twelve patients (32%) died from infection, which was associated with grade II–IV GVHD in six. Poor prognostic factors in this study included pre-existing liver and pulmonary disease. Recently, however, Tsuji et al. [165] reported the results of eight consecutive patients with hyper IgM syndrome transplanted from 1998 to 2004. The median age of the patients was 12.75 (range 3.9–19.9) years. Six patients received a transplant from an HLA-matched sibling, and two patients received an unrelated donor transplant. Of the eight patients, six received conditioning with BU/CY. Six patients remain alive at 350–2190 days post HCT, including both recipients of an unrelated HCT. Two patients died, one of sepsis and one of an Aspergillus infection. In 2004, Tomizawa et al. [166] reported the outcome of seven patients with hyper IgM syndrome, four of whom received an HLA-matched sibling BMT and three unrelated HCT. The median age of these patients was 11.3 (range 3–19) years. Cytoreduction consisted of BU/CY in six of seven patients with CSP and methotrexate for GVHD prophylaxis. Five of the seven patients are alive with normal expression of CD40L, four no longer remain on intravenous Ig. Two patients with hyper IgM syndrome received unmodified, HLA-matched sibling HCT following cytoreduction with a reduced-intensity regimen consisting of low-dose BU, fludarabine, ATG, and CSP [167]. Both children had significant liver disease documented by liver biopsy prior to transplant, which resolved after HCT. Both children are alive more than 2 years post HCT, one a mixed chimera, the other a full chimera. Normal T-cell function and freedom from intravenous Ig have been achieved in both patients. The impact of reduced-intensity conditioning, initially applied to other combined immune deficiencies by Amrolia et al. [159], is increasingly reflected in recent results from several centers. Rao et al. [122], in an update of the Great Ormond Street series, reported a 1-year survival rate of 95% for 27 patients with non-SCID congenital immune deficiencies who received unmodified, HLA-matched or one-alleledisparate transplants from unrelated donors after conditioning with fludarabine, melphalan, and either alemtuzumab or ATG. Reduced-intensity conditioning was also used by Cohen et al. [169] in eight patients with various CID syndromes who developed an EBVLPD pre HCT. This study included patients transplanted from an unrelated donor (n = 6), an HLA-matched sibling (n = 1), and a phenotypically HLA-matched family member (n = 1). All patients received rituximab prior to transplant. Patients were transplanted for intractable diarrhea of infancy (n = 1), Chediak–Higachi syndrome (n = 1), ALPS-like syndrome (n = 2), undefined CID (n = 2), X-linked lymphoproliferative syndrome (n = 1), and WAS (n = 1). Seven of eight patients were cytoreduced with fludarabine (150 mg/m2 over 5 days), melphalan (140 mg/m2), and alemtuzumab (1 mg/m2 in five divided doses). Patients received CSP alone (n = 5) or combined with mycophenolic acid (n = 3). All patients are currently alive without evidence of EBV-LPD between 1 and more than 7 years post HCT. Reduced-intensity conditioning has also been used successfully as preparation for successful transplants for patients with purine nucleoside phosphorylase deficiency [163] and IPEX syndrome [164]. While the results of transplants from matched related, unrelated, and HLA-disparate related donors have continued to improve in the treatment of most CID syndromes, patients with HLA class II deficiency

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remain a major challenge. In the initial European survey of transplants for immunodeficiencies conducted between 1968 and 1985 [161], only one of six patients with bare lymphocyte syndrome, who received an HLA-matched transplant, and none of four recipients of HLAnonidentical grafts survived reconstitution. However, despite advances in transplantation medicine and improvements in diagnosis and supportive care, recent reports from centers with the largest experience with such patients attest to continuing major impediments to the success of transplants in this patient group [162]. Of particular concern are the high incidence of graft rejection despite use of high-dose conditioning and the strikingly high mortality resulting from viral infections, of which the majority have antedated the transplant period. In the series from London [167], two of four recipients of HLAmatched and the one recipient of an HLA-nonidentical partial graft rejected their transplants despite preparative cytoreduction with BU and CY or melphalan and fludarabine. Similarly, in the Paris experience [162], one of six recipients of unmodified, HLA-matched and eight of 12 recipients of HLA-disparate, TCD transplants rejected their transplants despite preparative cytoreduction with BU and CY with or without anti LFA-1. In both series, viral infections were a major cause of morbidity and mortality. In aggregate, only four of the 11 recipients of HLA grafts and seven of 14 recipients of HLA-nonidentical transplants survive with reconstitution in these two series. In part, the complications observed may reflect the activation of quiescent and partially resistant host lymphocytes responding to HLA alloantigen newly presented by transplanted donor cells as well as the inability of donor T cells to interact effectively with infected host tissue that do not express HLA [170,171]. Both series indicate that the probability of a successful transplant in these disorders is greatly impaired by the presence of an active viral infection.

Wiskott–Aldrich syndrome WAS is a rare X-linked disorder characterized by thrombocytopenia, immunodeficiency, and eczema that is estimated to occur in between 1 and 10 per million live male births [3,4,172–175]. Affected individuals also have an increased risk of lymphomas, particularly EBV-LPD [176,177], and autoimmune disorders [178,179]. The mutated gene responsible for WAS and the milder disorders of X-linked thrombocytopenia and X-linked neutropenia was discovered in 1994 by Derry and colleagues [179]. The normal gene, which maps to Xp11.22–p11.23, encodes a 501 amino acid, 53 kDa intracellular protein (WAS protein or WASp), expressed in most hematopoietic cells [179]. WASp is involved in cytoskeletal organization (reviewed in [174]) and plays an essential role in T- and NK-cell activation [174,180–182]. Derry and colleagues identified three separate mutations in four patients with WAS, two of whom were related [174]. Since then, over 150 unique mutations in more than 300 families worldwide have been described [174,183–185]. In 124 patients with WAS and 116 patients with X-linked thrombocytopenia, the most common mutations found in the former group were deletions (30 of 124 patients). In a recent study by Proust et al. [184], 28 new mutations were reported using multiplex polymerase chain reaction [185]. In 2006, the mutation responsible for WAS in the three brothers described by Wiskott in 1937 was identified – a deletion of two nucleotides at positions 73 and 74 in exon 1 of the WAS gene – which resulted in a frameshift mutation [186]. Recently, several females with WAS have also been described (reviewed in [174]). Although all patients with WAS are thrombocytopenic, the level of T- and B-cell immunodeficiency, and the subsequent risk of autoimmune phenomena (nephropathy and vasculitis) and lymphoid malignancies can be quite variable. Scherbina et al. [187] demonstrated that the

clinical heterogeneity observed in WAS patients is due to differences in the expression of WASp in lymphoid cells. Patients could be divided into four clinical groups on the basis of WASp expression, ranging from group A – patients with mild clinical disease associated with low levels of WASp in their peripheral blood mononuclear cells and higher levels in EBV-immortalized B-lymphoblastoid cell lines, together with near normal levels of WASp RNA – to group D, patients who manifested severe disease in which peripheral blood mononuclear cells and EBVimmortalized B-lymphoblastoid cell lines lacked WASp expression. Knowledge of the pattern of WASp expression in individual patients [187–189] is used to determine which patients lacking an HLA-matched, related donor should be treated with supportive care alone [175], and which should be early candidates for HCT from alternative donors or, potentially, gene therapy [190,191, reviewed in 192]. Several other studies have shown a strong genotype–phenotype correlation in this disorder. Imai et al. studied the outcome of 50 Japanese patients with WAS/XLT from 43 unrelated families [188]. All patients with missense mutations had detectable levels of WASp. In contrast, patients with nonsense mutations, large deletions, small deletions or small insertions were WASp negative and had more severe disease. A separate study evaluated the correlation of genotype with phenotype in 262 patients from 227 unrelated families with WAS/XLT followed in two centers, one in Seattle, the other in Italy [189]. As in the previous study, the best predictor of clinical manifestations was determined by WASp expression. The majority of patients in this study who had WASpnegative lymphoid cells had deletions or insertions which resulted in a frameshift, nonsense or splice-site mutations. Seventy-four percent of patients in this study with XLT had missense mutations. HCT for WAS In 1968, Bach et al. [193] reported correction of the immunologic abnormalities associated with WAS following HLA-matched sibling transplantation. Despite the use of high-dose CY, this patient did not demonstrate engraftment of hematopoietic elements and remained thrombocytopenic. In 1978, Parkman et al. [194] corrected the immunologic and hematologic defects in a child with WAS by administering an HLA-matched marrow graft following myelosuppressive and immunosuppressive cytoreduction consisting of total body irradiation, procarbazine, and ATG. These authors hypothesized that myeloablation as well as immunosuppression was required to correct disorders involving nonlymphoid lineages by providing “space” in which progenitor cells could proliferate and develop. Subsequently, Kapoor et al. [195] demonstrated that BU/CY provided a myelosuppressive and immunosuppressive regimen adequate to insure full hematopoietic chimerism, correcting both the lymphoid and platelet defects in a series of children with WAS. A worldwide review from 1968 through 1997 indicated that 57 of 65 children with WAS survived in the long term after HLA-matched BMT [196]. Single-center studies of HLA-identical, related BMT from Brochstein et al. [197], Ozsahin et al. [198], Rimm and Rappeport [199], and Pai [200] have recorded long-term survival rates of 80% (eight of 10), 91% (10 of 11), 88% (seven of eight), and 100% (four of four), respectively. In the study by Imai et al., all of the 11 patients with WAS who received HLA-matched sibling HCT are surviving [188]. Nine of 11 recipients of HLA-matched, related HCT reported by Kobayashi et al. are surviving, compared with five of 10 mismatched related, and 17 of 21 unrelated, transplant recipients for WAS [201]. For patients without an HLA-matched sibling, transplants from closely matched, unrelated donors have provided excellent results. Of 30 patients with WAS who received unrelated, unmodified BMT prior to July of 1994, the overall actuarial survival was 67% [202]. Analysis

Hematopoietic Cell Transplantation for Immunodeficiency Diseases (a)

1.0 0.9 0.8 Overall survival, 65% (95% CI 57-73) @ 5 years

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of these transplantation results demonstrated that children under 2 years of age at the time of transplant had a much better prognosis, with 21 of 23 surviving (actuarial survival at 3 years 91%). Acute grade II–IV GVHD occurred in 50% of patients, with the highest incidence and severity observed in patients over 5 years of age at HCT. All four children who received an unmodified, unrelated BMT reported by Lenarsky et al. [203] survived between 3 to over 17 months post BMT. In 2001, Filipovich et al. [204] summarized and compared the results of 170 transplants reported to the International Bone Marrow Transplant Registry and/or the National Marrow Donor Registry (1968–96), including 58 patients transplanted from HLA-matched siblings, 48 from other relatives, and 67 patients from unrelated donors. Transplant outcome differed according to donor type, with a probability of 5-year survival of 87% (74–93%) with an HLA-identical sibling donor, 52% (37–65%) with partially compatible related donor, and 71% (58–80%) with unrelated donors [204]. For recipients of unrelated HCT, the best outcomes were observed in patients transplanted at less than 5 years of age. Pai reviewed 16 unrelated cases of HCT for WAS performed at the University of Brescia from 1990 through 2005 [200]. All patients received cytoreduction with oral BU (16 mg/kg) and intravenous CY. Thirteen patients were treated with ATG. All patients received unmodified bone marrow. Seven patients received additional CD34+-selected bone marrow. Donors were 6/6, 8/8 or 10/10 HLA-matched donors in 2, 5, and 4 cases, respectively. Donors for four patients were HLA mismatched at one (n = 3) or two (n = 1) loci. In one case, HLA typing was not stated. GVHD prophylaxis consisted of oral CSP 5 mg/kg twice daily. Of the 16 patients, 13 (81.2%) are surviving a median of 6 (range 0.6–9) years post HCT. Cause of death has been EBV-LPD and CMV (n = 1), Pneumocystis carinii pneumonia (n = 1), and postsplenectomy infection (n = 1). The Committee for Stem Cell Transplantation of the Japanese Society of Pediatric Haematology reported that 80% of WAS patients who received an unrelated HCT from an adult volunteer donor (n = 21) have survived [205]. Prognosis was poorer in patients who were transplanted at over 5 years of age and in those who received cytoreduction with a regimen other than BU/CY. Of the 156 unrelated BMTs or PBHCTs from unrelated donors reported to the CIBMTR from 1999 through 2004 [129], the overall survival at 5 years was 65% (95% CI 57–73%) (Fig. 75.6(a)). In the last 5 years, the results of cord blood transplantation for WAS have been reported from several centers, including results of double cord blood transplants [201,206,207]. The majority of patients received cytoreduction with BU and CY with or without ATG. Of the 21 unrelated cord blood transplants performed, the majority in Japan [201], 17 resulted in long-term survival. Figure 75.6(b) demonstrates the results of 54 cord blood HCTs reported to the CIBMTR from 1990 through 2004, showing an overall survival of 70% (95% CI 56–82%) at 5 years. For patients who lack a suitable cord blood or unrelated donor, TCD HCT has been utilized with variable success. In a review of 15 recipients of TCD HCT, only six patients have become long-term survivors [195]. In the initial MSKCC series, only one of six patients survived long term. In the recent update of the European experience, 3-year survival for WAS was 45% in 43 recipients of an HLA-mismatched, related HCT compared with 81% in 32 recipients of an HLA genotypically matched transplant [67]. Failures in these series were primarily due to EBV-LPD and GVHD [194]. However, with the recent development of therapies such as rituximab and EBV-specific T cells that are effective in treating or preventing EBV-LPD in these patients [208–215], the success of such transplants may significantly improve. For example, two patients with WAS have recently been successfully transplanted at MSKCC from their TCD, HLA-mismatched mothers following cytoreduction with hyperfraction-

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Fig. 75.6 (a) Probability of overall 5-year survival following unrelated donor bone marrow or peripheral blood transplants, 1990–2004, for Wiskott– Aldrich syndrome (WAS) reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). (b) Probability of overall 5-year survival following unrelated donor cord blood transplantation, 1990–2004, for WAS reported to the CIBMTR. (Reproduced with permission from the CIBMTR.)

ated total body irradiation, thiotepa, and fludarabine followed by a CD34+E− PBHCT. At 96 and 56.9 months post HCT, both children are alive and fully reconstituted with complete donor chimerism and without GVHD. Both children received post-transplant rituximab to prevent EBV-LPD. One child required prolonged treatment due to recurrence of an autoimmune hemolytic anemia post HCT. However, late autoimmune disorders have been noted in up to 20% of patients transplanted for WAS, particularly in patients with sustained mixed chimerism [216].

Gene therapy for SCID and other lethal disorders of immunity The identification and subsequent cloning of genes responsible for many of the lethal disorders of hematopoiesis and immunity, coupled with the development of increasingly efficient methods for the isolation of hematopoietic stem cells, and the genetic modification of hematopoietic stem cells derived from a diseased subject to express a normal gene product, both in vitro and in animal models, in vivo, has spawned a series of clinical trials exploring the potential of gene therapy to correct these diseases. The severe combined immunodeficiency caused by ADA [25,217] was initially selected as a target for clinical trials of gene therapy for several reasons [218]. First, it was recognized that this disorder, as well as other variants of SCID, could be corrected by HLA-matched, related marrow grafts, and, further, that T-cell chimerism alone regularly induced

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full functional reconstitution [219]. Second, in vitro studies [220] as well as small clinical trials had demonstrated that transfusions of normal red cells [221] or treatment with polyethylene glycol-conjugated ADA (PEG-ADA) [222,223] could provide sufficient ADA activity to reduce toxic levels of deoxyadenosine triphosphate in lymphocytes, and thereby to permit the generation of T cells and at least a partial correction of functional deficiencies in immunity [224]. Finally, the group of Bordignon [225] established that human ADA− lymphocytes, transduced to express the normal ADA gene, exhibited a long-term survival advantage over ADA cells and developed significant immune reactivity after adaptive transfer into immunodeficient mice. Initially, it was not expected that the introduction of a normal gene into hematopoietic progenitors bearing one of the mutations causing other lethal immunodeficiencies would also confer a survival advantage to the genetically modified cells. However, over the past 13 years, several groups have reported patients with immunodeficiencies who have developed spontaneous somatic mutations in clones of lymphoid cells resulting in correction of the underlying defect, followed by preferential expansion and long-term survival of these normal, functional clones [226–233]. Such spontaneous lymphoid revertants have now been demonstrated in rare cases of the ADA-deficient and X-linked γc deficient variants of SCID [226–230], the Nemo variant of combined immunodeficiency [231], type 1 leukocyte adhesion deficiency [232], and a significant proportion of patients with WAS [233]. Such cases have provided compelling evidence supporting the possibility that in vivo transfer of a limited population of genetically corrected lymphoid progenitor cells could also result in significant and sustained partial or complete reconstitutions of immunity in patients with these disorders. The evolution of gene therapy as a clinically feasible therapeutic strategy for these immune deficiencies derives from a series of advances in the design of vector constructs inducing sustained high-level constitutive expression of the normal human gene in transduced cells [234–239], the isolation of hematopoietic progenitor cells targeted for genetic modification [240], and the development of systems for efficient transduction and integration of the normal gene constructs into hematopoietic progenitor cells [241,242]. So far, the clinical trials that have been conducted have employed replication of incompetent retroviruses as the vectors because of their high efficiency for gene transfer, their capacity to integrate into the genomic DNA of dividing somatic cells, and their anticipated safety [243]. The vectors most frequently used are constructed from a replication- incompetent Moloney leukemia virus lacking gag, pol, and env sequences but retaining the packaging signal (psi), the viral long tandem repeat (LTR), and sequences essential for reverse transcription and integration. The normal human gene construct is inserted under the transcriptional control of the viral LTR, which acts as the promoter/enhancer for gene expression [234,235,243–245]. In order to generate infectious virus, these constructs are introduced into packaging cells which generate gag, pol, and env proteins from independent gene inserts to produce infectious virus in response to the vector construct’s packaging signal, psi [239,246]. Cells targeted for genetic correction have included patient-derived peripheral blood lymphocytes and CD34+ hematopoietic progenitor cells isolated from the bone marrow or cytokine-mobilized peripheral blood hematopoietic cells, and cord blood cells. Following infection of cells with the retroviral vector, the vector RNA is reverse-transcribed and forms a preintegration complex which can only be transferred into the nucleus and integrated into the DNA of cells during mitosis [247,248]. Activated human T lymphocytes, which divide at significant frequency, are readily transduced at high efficiency with retroviral vectors. In contrast, self-renewing human hematopoietic stem cells, which normally divide at very low frequency, are markedly less susceptible to the successful transduction and integration of genes when

retroviral vectors are employed. As a consequence, although 30–40% of the CD34+ cells infected may express vector-encoded genes, the proportion of stem cells capable of multilineage differentiation that are transduced may represent only 0.1–1% of the population. Thus, in the absence of a selective growth or survival advantage, such cells would be expected to develop into no more than a minor population of the host’s hematopoietic progenitor pool. Because of this limitation, the initial trial of gene therapy for the treatment of ADA-deficient SCID [249,250] explored the therapeutic potential of peripheral blood T lymphocytes transduced with the Moloney leukemia virusbased vector LASN, which contained the human ADA complementary DNA under the transcriptional control of the retroviral LTR and a neomycin phosphotransferase gene controlled by an SV40 promoter for the selection of transduced cells. The two patients in this trial received 11 and 12 infusions, respectively, of vector-treated T cells, providing, in aggregate, approximately 1011 total lymphocytes, of which 18–53% (patient 1) and 0.9–3% (patient 2), respectively, expressed the vector. Both patients were also treated with PEG-ADA both prior to and continuously following infusion with transduced cells. Concurrent treatment with PEG-ADA has made ascertainment of the effects of gene transfer on immune function difficult, and likely reduced or eliminated the potential growth advantage of the transduced cells. Nevertheless, patient 1 still has a significant proportion of vector-modified lymphocytes over 16 years post initiation of therapy [250,251]. Subsequently, Bordignon et al. [252,253] treated two patients with deteriorating immune function despite treatment with PEG-ADA with infusions of autologous T cells and bone marrow progenitors that were transduced with separate ADA gene-containing vectors. This study again demonstrated long-term survival of transduced cells, but also showed that the proportion of marrow-derived transduced T cells greatly exceeded that of the T cells derived from transduced peripheral blood at times later than 1 year post treatment. At 16 months post infusion, 2–4.8% of circulating T cells and 17.25% of clonable marrow cells expressed the vector genes. Both patients have exhibited sustained improvements in T-cell function following these infusions. Again, however, coadministration of PEG-ADA in both patients has limited accurate estimates of the contribution of gene therapy to the functional reconstitution of immunity observed. Because hematopoietic stem cells in human cord blood replicate at significant frequency and are capable of self-renewal [254], Kohn et al. [255] used the LASN vector to transduce autologous cord blood-derived CD34+ cells from three neonates with ADA− SCID and evaluated their growth after reinfusion. Initially, each of these patients achieved sustained but low levels of the genetically modified peripheral blood lymphocytes (0.01–0.3%) in the blood, and higher frequencies of clonogenic myeloid progenitors (4–6% colony-forming myeloid cells) in the marrow. These patients were also being treated with PEG-ADA, which again prevented assessment of the effects of gene therapy on immune function. Nevertheless, over the subsequent 3 years of observation, the proportion of gene-transduced T cells gradually increased in each patient to 1–10% of the circulating T-lymphocytes. To test whether these ADA+ T cells would preferentially expand in an ADA-deficient host, PEGADA treatment was stopped in one of these patients. Although the proportion of transduced ADA+ T cells in the blood increased, progressive lymphopenia and impaired T-cell function necessitated resumption of PEG-ADA [256]. Taken together, these early trials demonstrated that peripheral blood T cells or CD34+ bone marrow progenitor transduced to express a normal human ADA gene could be safely transferred into patients with ADA− SCID, and that populations of such cells could survive and continuously express the transduced gene for periods extending to 16 years post infusion without deleterious complications. However, the proportional

Hematopoietic Cell Transplantation for Immunodeficiency Diseases

representation of transduced marrow progenitor cells and peripheral blood T cells remained low, and their contribution to the overall immune system of the host uncertain. This could reflect either the lack of a growth or survival advantage for ADA+ over ADA− marrow stem cells or a diminished capacity of ADA+ lymphoid progenitors to compete with ADA− progenitors in the presence of normal tissue levels of ADA provided by concurrent treatment of the patients with PEG-ADA. Recently, Aiuti et al. [257] directly addressed both of these possibilities in a trial in which children with ADA-deficient SCID were treated with transduced autologous marrow CD34+ cells containing 25% cells expressing the GIADA-1 vector encoding human ADA, after preparatory myelosuppression with BU 2 mg/kg/day for 2 days. Of critical importance, these patients received no PEG-ADA prior to or following this treatment. The two patients initially reported exhibited sustained full (patient 1) and partial (patient 2) reconstitution of normal T-, B-, and NK-cell counts, with 70–100% of circulating T cells expressing vectorencoded ADA. Expression of the vector in B lymphocytes, and in 20% of myeloid, megakaryocytic, and erythroid progenitors, suggested durable engraftment of transduced stem cells. Increases of TREC+ and CD45RA T lymphocytes in the circulation indicated effective thymopoiesis. The ADA+ T cells generated responded to mitogens and antigens normally. Further, Ig levels increased to normal and antibody responses to vaccination were recorded. Since that report, an additional 11 patients have been treated on this trial. Of the 13 evaluable cases, 12 have achieved full functional reconstitution of T- and NK-cell populations, of which 70–100% contain the vector. B-cell numbers and IgM levels have normalized. In 11 patients now off intravenous Ig, IgG levels are also sustained; five of these patients have generated antibody responses following tetanus vaccination. All patients are surviving in good health (Aiuti, personal communication). Confirmation of the potential of this approach has also been provided by Gaspar et al. [258], who treated a patient with ADA-deficient SCID with autologous CD34+ marrow cells transduced with a γ retroviral vector (SFUDA/W) containing the human ADA gene under the transcriptional control of a spleen focus-forming virus LTR after myelodepletion with melphalan 140 mg/m2/day for 1 day. This patient had previously received PEG-ADA, but this treatment had been stopped 1 month prior to cytoreduction and the infusion of transduced cells. Following a 2–3-week period of melphalan-induced pancytopenia, the patient experienced recovery of hematopoiesis followed by rapid recovery of CD4+ and CD8+ T-cell populations, expansion of TREC+ naïve T cells, and recovery of cell-mediated immune function. These latter studies demonstrated the potential of hematopoietic cells transduced to express ADA to repopulate a myelodepleted ADA− SCID and for ADA+ lymphoid cells to preferentially expand and restore immune function in an ADA-deficient host. While these studies do not formally establish a need to induce myelodepletion to secure a high level of engraftment of transduced progenitors, emerging data from a trial in which transduced marrow has been administered without either myelosuppressive or PEG-ADA treatment suggest that, in the absence of such preparative cytoreduction, lymphopenia persists and lymphoid reconstitution is more limited [259].

X-linked γc-deficient SCID In 2000, Cavazzana-Calvo et al. [260] reported the first clear demonstrations of the potential of gene therapy to correct a lethal congenital immunodeficiency. They used an MFG retroviral vector containing the normal human gene encoding the IL-2 γ chain under the transcriptional control of the viral LTR to transduce autologous purified CD34+ marrow cells from two children with X-linked γc-deficient SCID. These trans-

1117

duced progenitors were then administered to each patient without preparatory conditioning. Each patient achieved rapid reconstitution of γc+ T-cell populations bearing the vector, with full recovery of proliferative responses to mitogens and antigens and T-cell receptor diversity. In these patients, approximately 0.1% of the myeloid cells and 1% of the B cells tested over extensive follow-up have also demonstrated that they contain the vector, indicating durable engraftment of transduced pluripotent stem cells and again demonstrating the growth advantage of transduced immunocompetent T cells in these T-lymphopenic hosts [261,262]. At 8–9 months post infusion, and 5–6 months following cessation of monthly intravenous gammaglobulin, Ig levels had increased and the patients generated responses to tetanus diphtheria and polio virus vaccinations. Since this report, a total of 10 patients with X-linked SCID have been accrued to this trial [262,263]. Of these, eight have achieved reconstitution of T cells and their function, as well as at least partial recovery of B-cell function. While the number of NK cells has transiently increased early after treatment, long-term reconstitution of NK cells and their function has been poor. The two patients who failed to achieve sustained recovery of T cells and their functions had received the lowest doses of transduced cells (1 × 106/kg versus >3 ×106/kg), suggesting that a high level of engraftment of the transduced stem cells may be required to sustain normal levels of lymphopoiesis [263]. Subsequently, in a separate trial, Gaspar et al. [264] treated another four children with X-linked γc− SCID with autologous CD34+ marrow cells transduced with the same γc gene-containing vector construct packaged in a gibbon ape leukemia virus envelope. Three of the four patients subsequently achieved full, and one patient a partial, reconstitution of γc+ T-cell populations. All patients exhibit normal T-cell proliferative responses to mitogen, antigens, and allogeneic cells. Two patients who were taken off intravenous Ig replacement generated antibodies to polio and H. influenzae B vaccinations. However, as also observed in the series from Paris, recovery of NK-cell populations and functions has been poor. Since this report, an additional six patients have been treated successfully by this group using the same approach, but the level of reconstitution achieved has not been reported. Taken together, these two series have clearly demonstrated the potential of lymphoid cells derived from progenitor cells transduced to express a normal IL-2R γ chain to provide sustained and complete corrections of the T-cell defects associated with X-linked SCID and to induce recovery of B cells capable of producing antibodies in response to microbial immunogens. Whether the restoration of humoral immune responses observed reflects the activity of the small number of transduced B cells detected or unmodified host B cells responding to transduced γc+ T helper cells, however, is as yet unclear. Similarly, the basis for the failure of this approach to induce sustained corrections of NK cells and their function remains to be determined. Nevertheless, the sustained clinical responses of these children and their normal responses to infection have been striking. Recently, however, the success of this approach has been counterbalanced by the late (>30 months) development of T-cell acute lymphoblastic leukemia (ALL) in four of the original 10 children with X-linked SCID treated (Fischer, personal communication) [265]. Although these patients exhibited a prompt response to chemotherapy and achieved remissions, one subsequently relapsed and died of leukemia. In each case, the cloned leukemia cells have exhibited a mature T-cell phenotype not normally seen in children. Molecular analyses have demonstrated vector sequences in each of these leukemias. In each case, the transduced provirus has been found to be inserted within or in proximity to a known proto-oncogene involved in the control of early T-cell differentiation, specifically the LMO-2 gene in three cases and the cyclin D2 gene in one patient (Fischer, personal

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communication) [262,265]. Subsequent studies have demonstrated that, contrary to initial expectations, retroviral vector insertions are not random, but tend to cluster in transcription start sites of genes highly expressed in the targeted CD34+ cells [266–272]. Further, a comparison of insertion sites in CD34+ cells in nine patients of the original series, both immediately after transduction and in the marrow of patients late after treatment, demonstrated a different and highly skewed spectrum of insertion sites in CD34+ cells isolated from the patients many months after treatment, strongly suggesting that cells with these insertions may have a selective growth advantage [270]. Of the common insertion sites detected in clones of the CD34+ cells, insertions in LMO-2 and CCND2 were significantly increased in frequency [270]. A significant role for vector insertion in the promoter region or first intron of LMO-2 in the pathogenesis of T-cell ALL in three of these patients is inferred from the fact that a translocation affecting LMO-2 is associated with one variant of T-cell ALL affecting children [273,274], and, further, that its aberrant expression in mice is associated with late development of a T-cell leukemia [275]. However, it remains unclear in these patients whether altered expression of LMO-2 is causal or whether the T-cell ALL develops preferentially in T-lymphocyte progenitors possessing the selective growth advantage provided by altered LMO-2 expression. In the clinical experience reported to date, none of the ADA-deficient SCID patients who have received retroviral vector-transduced peripheral blood lymphocytes of marrow CD34+ cells and remain engrafted have developed a leukemia over up to 16 years of follow-up. Further, while an evaluation of vector insertion sites in transduced CD34+ cells in ADA-deficient patients has exhibited a pattern of common insertion sites similar to that exhibited in the initial series of X-linked SCID patients treated, clonal selection of cells bearing these inserts has not been observed, nor have the cells bearing LMO-2 inserts exhibited increased expression of LMO-2 [271]. This finding, coupled with studies in γc knockout mice [275], has raised the possibility that the γc-deficient Xlinked SCID may be particularly prone to retrovirally mediated mutagenesis. However, it is also striking that none of the 10 X-linked SCID patients treated by Gaspar et al. [264] have developed a leukemia, suggesting the possibility that other factors, such as the vector used or the conditions used for marrow cell transduction, may also play an important or determining role. However, longer follow-up will be required before the risk of leukemogenesis in the second trial can be assessed, since the leukemias have developed more than 30 months after treatment with transduced cells, and most of the patients in the second series have yet to reach this milestone. Because of these serious adverse events, all trials evaluating gene therapy for X-linked SCID were initially put on hold. However, trials incorporating the gibbon ape leukemia virus packaging vector employed by Gasper et al. [264] have recently been reopened for patients lacking suitably matched related or unrelated donors. Further, trials employing self-inactivating vectors that delete the retroviral LTR [276] and vectors that include insulation sequences to reduce activation of genes neighboring insertion sites [277,278] are being developed.

Other lethal immunodeficiencies The striking level of immune reconstitution achieved in recent trials of gene therapy in the treatment of ADA-deficient and X-linked γcdeficient SCID has stimulated extensive preclinical exploration of this approach in the treatment of other lethal congenital immunodeficiencies, particularly those disorders which, unlike X-linked SCID, require preparation with high-dose chemotherapy to secure consistent engraftment of an HLA-matched related graft, or a transplant from a matched, unrelated or HLA-haplotype-disparate, related donor.

In murine models, infusions of marrow progenitors transduced with retroviral vectors encoding a normal gene have corrected the immune deficiencies observed in JAK-3[279], RAG-2−/− [280], Artemis−/− [281] mice, and Wiskott–Aldrich−/− mice [282–285]. In vitro corrections of the phenotypic and functional deficiencies of human T cells generated from patients with ZAP-70 deficiency [286] and WAS [282–285] have also been achieved by transducing lymphocytes from those patients with retroviral or lentiviral vectors encoding the normal human gene. Clinical trials evaluating the effects of CD34+ cells transduced with a retroviral vector encoding a normal WASp gene, when administered to patients with high risk factors for WAS after low-dose conditioning, have been initiated (Klein, personal communication). However, these studies are too early to permit evaluation.

Conclusion Allogeneic HCT transplantation from a normal, matched related donor remains the treatment of choice for each of the different genetic variants of SCID. Currently, HLA-matched, unmodified grafts from unrelated donors administered with post-transplant drug prophylaxis to prevent GVHD and rigorously TCD, HLA-haplotype-disparate, related transplants administered without post-transplant GVHD prophylaxis yield equivalent results, with long-term disease-free survival rates of approximately 70% overall and approximately 80% for patients transplanted in the first 3 months of life. The relative advantages of each approach will require careful prospective analyses of large series. Certain types of SCID, particularly Omenn’s syndrome, ADA-deficient SCID and NK+ variants, exhibit graft resistance and require preparative cytoreduction, particularly when the donors are other than HLA-matched siblings. Preparatory cytoreduction with myelosuppressive agents such as BU, thiotepa or melphalan may also be preferable to secure consistent engraftment of donor B cells and NK cells, as well as T cells, in all variants with reconstitution of both cell-mediated and humoral immunity. For patients with other combined immunodeficiency diseases and WAS, preparative cytoreduction with a myelosuppressive agent together with immunoablation with CY or fludarabine is required to achieve consistent engraftment and reconstitution. For these indications, transplants of unmodified, HLA-matched, related and unrelated HCT have thus far been associated with better outcomes than TCD, HLA-disparate grafts. Certain of these disorders, particularly the bare lymphocyte syndrome associated with HLA class II deficiency, exhibit excessive rates of graft failure despite use of these regimens. For the future, the development of well-tolerated preparative regimens and transplant approaches which secure consistent engraftment and full chimerism is a high priority. In the future, introduction of selected cytokines or specific cell therapies at the time of transplant may foster full engraftment and enhance recovery of immune function. While treatment with genetically modified autologous hematopoietic stem cells has recently shown great promise in the treatment of children with ADA-deficient SCID when administered after myelosuppression, the proportion of patients achieving full reconstitution is thus far not different from that already achievable with matched, unrelated or TCD, HLA-nonidentical grafts. Longer follow-up will be required to determine whether and to what degree gene therapy will supplant HCT as a treatment of choice for this disease. For patients with X-linked γcdeficient SCID, gene-modified autologous HCT can reconstitute both T- and B-cell immunity without preparative cytoreduction. However, improved vectors are needed to reduce or eliminate the risk of late leukemic transformation of transduced cells in this patient group.

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Peter F. Coccia

Hematopoietic Cell Transplantation for Osteopetrosis

Introduction Osteopetrosis is a rare inherited disorder characterized by generalized skeletal sclerosis that occurs in various mammals including man [1–3]. Osteopetrosis is a result of the dysfunction of osteoclasts, the multinucleated giant cells that resorb bone and mineralized cartilage [4,5]. The osteoclast is a specialized macrophage polykaryon derived from the hematopoietic stem cell (HSC) [4–6]. Some variants of osteopetrosis in laboratory animals and man can be corrected by hematopoietic cell transplantation (HCT) [1,3,7–9]. In the past several years, remarkable advances have been made in the understanding of osteoclast biology, in the elucidation of the osteoclast resorption defects that result in the various clinical phenotypes of human osteopetrosis, and in the identification of multiple genetic mutations in patients with osteopetrosis. In this chapter, the origin and function of the osteoclast will be described briefly. Studies of HCT in laboratory animals with congenital osteopetrotic mutations and transgenic mutations will be reviewed. The clinical and mutational classification of the various types of osteopetrosis in humans will be detailed. The role of HCT and other therapeutic modalities in the treatment of infantile malignant osteopetrosis will be discussed, and future directions will be considered.

The osteoclast and bone resorption There is extensive information in the literature concerning the origin, structure, and function of the osteoclast and its role in bone resorption [4,5,10]. A brief overview will be provided to clarify issues important to an understanding of the pathophysiology of osteopetrosis and the role of HCT in its correction. The cell of origin of the osteoclast is the pluripotent HSC. The colonyforming unit-granulocyte–macrophage is likely the committed progenitor cell of origin of the osteoclast, as well as the circulating monocyte, the tissue macrophage, and the foreign body giant cell [5,11]. Osteoclasts are thought to form from repeated asynchronous fusions of postmitotic mononuclear precursors that migrate to skeletal sites via vascular pathways. Bone-resorbing osteoclasts have from one to over 20 nuclei. Differentiation, fusion, maturation, activation, inhibition, and apoptosis are controlled by multiple lymphokines, monokines, and hormones.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B (NFκB) ligand (RANKL) promote differentiation of bone marrow macrophages into multinucleated mature osteoclasts. A soluble protein, osteoprotegrin, inhibits bone resorption by binding RANKL. Osteoprotegrin appears to inhibit osteoclast maturation. Both osteoblasts, the bone-forming cells of mesenchymal origin that synthesize bone matrix, and marrow stromal cells express M-CSF and RANKL on their cell surface and have important roles in the development, activation, and regulation of osteoclasts. Bone formation and remodeling are intimately linked and interdependent. Degradation of bone mineral complexes and collagenous bone matrix requires activation of the osteoclast. Activation involves elaboration of cytoplasmic infoldings of the plasma membrane next to the bone surface that is known as the ruffled border. Once the ruffled border is formed, plasma and lysosomal membranes fuse, and lysosomal contents including cathepsin K and other proteases, carbonic anhydrase isoenzyme II (CAII), and acid hydrolases are released to the extracellular space. Acidification requires the influx of hydrogen ions produced by CAII by the vacuolar H+ ATPase proton pump and the exchange of chloride/ bicarbonate mediated by a chlorine channel pump (ClCN7). Essentially, all reported mutations described in human osteopetrosis are a result of defects in acidification: T-cell immune regulator-1 (TCIRG1), which is also known as ATP6i; ClCN7; osteopetrosis transmembrane protein-1 (OSTM1), or gl/gl; and CAII (see below). Bone resorption is confined to a small area of osteoclast–bone interface by a circumferential seal of the plasma membrane that is defined as the clear zone. Hydroxyapatite crystals are first solubilized by hydrochloric acid at a pH of 4.5, and then the organic matrix is digested by the proteases. The products of resorption are then endocytosed for additional intracellular processing.

Osteopetrotic mutations in laboratory animals Congenital osteopetrosis is a well-known mutation in a variety of mammalian species [1,3,7]. There are eight well-studied mutations in common laboratory animals, including the mouse, rat, and rabbit (Table 76.1). In all variants, the mode of inheritance is autosomal recessive. All variants demonstrate osteoclast hypofunction, generalized skeletal sclerosis, absent or poorly developed marrow cavities, delayed tooth eruption, reduced size of osseous foramina, defective bone modeling and remodeling, and weak bone susceptible to pathologic fracture. All mutants studied are resistant to the hypercalcemic effects of parathyroid hormone and 1,25 dihydroxyvitamin D, and all have increased blood levels of 1,25 dihydroxyvitamin D.

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Table 76.1 Mammalian osteopetrosis. Osteoclasts Mutation

Symbol

Gene identified

Number

Size

Ruffled borders

Survival

Effective treatment

Mouse Grey lethal Microphthalmia Osteosclerosis Osteopetrosis

gl/gl mi/mi oc/oc op/op

OSTM1 ? TCIRG1 M-CSF

N, ↑ N ↑ ↓↓

↑ ↓↓ ↓ ↓

± − − +

Lethal Normal Lethal Normal

HCT HCT HCT M-CSF

Rat Incisors absent Osteopetrosis Toothless

ia/ia op/op tl/tl

PLEKHM1 ? M-CSF

↑↑ ↓ ↓↓

N ↑↑ ↓

− ± ±

Normal Lethal Reduced

HCT HCT M-CSF

Rabbit Osteopetrosis

os/os

?

↓

N

−

Lethal

None

Modified, expanded, and updated from numerous literature reports and reviews [1,3,7]. Bibliography available upon request. Increases or decreases with respect to normal littermates/control subjects are indicated by arrows. N, same as normal littermate/control; + and −, presence or absence of ruffled borders; ?, not reported/unknown. HCT, hematopoietic cell transplantation; M-CSF, macrophage colony-stimulating factor.

An additional mutation in the Norway rat, microphalmia blanc (mib/ mib), has been characterized [12,13]. Affected mutants have a mild transient osteopetrosis at birth with reduced osteoclast numbers. Skeletal sclerosis gradually resolves, osteoclast numbers increase, and survival is normal. Gene mapping reveals a mutation at 4q34–q41 that produces a nonfunctional microphthalmia-associated transcription factor gene. The major differences among the variants that have been described in the current literature are compiled in Table 76.1. Included are remarkable differences in osteoclast numbers from virtually absent in the tl/tl rat to significant hyperplasia in the ia/ia rat; differences in osteoclast size from primarily mononuclear forms in the mi/mi mouse to very large with many nuclei in the op/op rat; and differences in the appearance of the ruffled border at the osteoclast–bone interface from absent to normal in the various mutants. Reported gene mutations are included for mutants if they have been elucidated. Disease severity as measured by survival past infancy and known effective treatment are also detailed. The development of osteopetrosis is an in utero event. The defect may be intrinsic to the osteoclast or in the microenvironment supporting the development and activation of the osteoclasts. In all cases, the failure of the osteoclast to resorb calcified cartilage during bone development leads to persistent primary spongiosa characterized by cores of calcified cartilage within the bone. This resorption failure prevents or delays development of the bone marrow cavity and is responsible for the distinctive radiographic appearance of homogeneously dense bones. The severity of disease and the potential therapeutic interventions depend on the etiology of the osteoclast dysfunction in the various osteopetrotic mutations. In addition to spontaneous mutations, various induced mutations have produced transgenic mice by targeted disruptions of proto-oncogenes [1,3,7]. These loss of function, or “knockout,” experiments have resulted in abnormalities in either the production or function of osteoclasts, and the mutant mice usually have severe osteopetrosis. Examples of induced mutations that result in abnormal osteoclast production or maturation include the targeted disruption of c-fos that yields mice with abundant bone marrow macrophages but no osteoclasts, and PU.1, a hematopoietic transcription factor, that produces mice deficient in both macrophages and osteoclasts that die within 24–48 hours

of birth of septicemia. Additional mutants that fail to generate osteoclasts include those with deficient NFκB1 plus NFκB2 and with deficient RANKL. Mutations that result in abnormal osteoclast function include the disruption of c-src, a tyrosine kinase, yielding mice with normal osteoclast numbers, absent ruffled borders, and defective bone resorption. Mice produced with a deficiency in tumor necrosis factor receptor-associated factor 6 have impaired osteoclast function. Transgenic mice with specific resorption defects have also been created. Cathepsin K-deficient mice lack a cysteine proteinase necessary to degrade bone matrix. TClRG1 (Atp6i) deficient mice have an extracellular acidification defect and cannot solubilize bone mineral [14]. HCT in animal models In a series of elegant experiments, Walker [15] demonstrated that osteopetrosis can be cured in the gl/gl and mi/mi mouse mutants by either temporary parabiosis or HCT. Infusions of marrow or spleen cells from normal littermates into lethally irradiated osteopetrotic mice resulted in complete correction of osteopetrosis and normal survival. Conversely, infusion of spleen cells from osteopetrotic mice to lethally irradiated normal littermates led to the development of osteopetrosis [16]. Similar experiments have demonstrated complete correction after HCT in the ia/ia [17] and op/op [18] rat mutations. Correction or induction of osteopetrosis in these four strains by HCT suggests defects intrinsic to the osteoclast progenitor or the osteoclast itself. Studies in the mi/mi mouse which demonstrate that the bone resorbing osteoclasts are of donor origin after HCT support this viewpoint [19,20]. Beige mice, which have giant lysosomes in their hematopoietic cells, were used as hematopoietic cell donors for HCT into irradiated mi/mi mice. After HCT, giant lysosomes were present in the functional osteoclasts of the recipients. Experimental HCT in animal models has been very consistent in its approach. In most reported studies, the preparative regimen has utilized a single fraction of total body irradiation (TBI) of 600 cGy for recipient osteopetrotic mutants or 900 cGy for recipient normal littermates delivered by a cobalt-60 source. This dose is considered marrow lethal for each group. Marrow or spleen cells at 10 × 106–50 × 106 nucleated

Hematopoietic Cell Transplantation for Osteopetrosis

cells/animal are given intravenously or intraperitoneally 2 hours later. Donors are highly inbred littermates, full engraftment is routine, and graft-versus-host disease (GVHD) has not been reported. HCT from normal littermates has also been reported to cure osteopetrosis in transgenic mutants [1,3,7]. Complete correction has been reported in the c-src, c-fos, NFκB1 plus NFκB2, and PU.1 variants. In the newborn PU.1– mutant, HCT is performed soon after birth utilizing 4-week-old PU.1+ donor animals in germ-free environments. HCT should be successful in all mutants that have osteopetrosis secondary to an induced mutation that results in a defect in the HSC, but not in mutants that have defects in the microenvironment. In three other spontaneous mutants (op/op mouse, tl/tl rat, and os/os rabbit), HCT is not able to correct osteopetrosis. This observation suggests that the defects in these mutants are not intrinsic to the osteoclast progenitor or the osteoclast. The lethal mutation in the os/os rabbit has not been identified [7]. Both the op/op mouse and the tl/tl rat have been found to be severely deficient in M-CSF, and daily treatment with recombinant human MCSF remarkably improves bone resorption in both mutants [21]. The op/op mouse has a nonlethal point mutation within the coding region of the M-CSF gene, absent M-CSF, and markedly reduced monocytes, macrophages, and osteoclasts. The osteopetrosis progressively corrects over time. Treatment with M-CSF results in normalization of monocytes, macrophages, and osteoclasts, and rapid correction of osteopetrosis. Granulocyte–macrophage colony-stimulating factor treatment restores monocytes, and macrophages, but fails to stimulate osteoclast development or correct osteopetrosis. The tl/tl rat also has decreased monocytes, macrophages, and osteoclasts, but does not demonstrate skeletal improvement with age and has a reduced life span. Bioassay reveals low M-CSF activity and treatment with M-CSF improves osteopetrosis and prolongs survival. The genetic defect in the oc/oc mouse spontaneous mutation has been identified [14,22]. The defective gene encodes an H+ ATPase necessary for acidification, and appears to be a defect similar to that of the TCIRG1 (Atp6i)-deficient transgenic mouse model [22]. While this lethal mutation is intrinsic to the osteoclast, successful HCT had not been reported until recently. The oc/oc mouse has been studied extensively as it has the identical mutation found in 50–60% of children with infantile malignant osteopetrosis (see below). Previous attempts to transplant oc/oc mice have been unsuccessful as the animals have only a 3-week life expectancy. HCT in 1-day-old oc/oc mice results in long-term survival [23]. The same group also has reported successful HSC-targeted gene therapy with oc/oc fetal liver cells transduced with a retroviral vector expressing TCIRG1 in irradiated neonatal oc/oc mice [3,24]. A cell line derived from oc/oc mice has been developed which differentiates into murine osteoclasts but lacks resorption activity [25].

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order rarely have dense bones. Most patients are asymptomatic and have a normal life expectancy. Minimal disability has been reported, secondary to an increased susceptibility to fractures, and occasionally, bone pain, osteomyelitis of the mandible, and cranial nerve palsies [28]. This autosomal dominant form has been extensively studied, and two distinct types (types I and II) have been described based on clinical, radiologic, histologic, and biochemical criteria. Type I is rarer; fractures are uncommon, bones are uniformly dense, and osteoclasts are reduced in number and size. Recently, the defect in autosomal dominant type I disease has been shown to be due to an osteoblast defect, a mutation in low-density lipoprotein receptor-related protein-5 (LRP5), which mediates increased bone formation [29,30]. As the LRP5 gain-of-function mutations do not cause an osteoclastic resorption defect, most authors suggest excluding this diagnosis from the osteopetrosis family of disorders [1–3]. More families with type II disease have been reported. Fractures are more frequent, bone density is more variable, and osteoclasts are increased in number, large, and highly multinucleated. Type II autosomal dominant osteopetrosis has been mapped to chromosome 16p13.3 [31]. Heterozygous mutations of ClCN7 have been found in almost all patients with type II disease [32–37]. Clinical manifestations have ranged from entirely asymptomatic in one-third of subjects to the more typical misshaped, brittle bones with increased fractures and occasionally other manifestations of osteopetrosis. There is no genotypic–phenotypic correlation, and families frequently have both unaffected gene carriers and even severely affected individuals [35–37]. A number of variants with autosomal recessive inheritance in addition to the infantile malignant variant, which is the subject of this chapter, have been described. CAII deficiency, a syndrome with mild osteopetrosis, renal tubular acidosis, and cerebral calcification, has been documented in multiple kindreds [38,39]. Patients have mutations of the CAII gene resulting in a profound deficiency of CAII. Transient infantile osteopetrosis has been reported [40–42] and is speculated to be similar to the mib/mib mutation in the Norway rat [12,13]. Intermediate autosomal recessive and mild autosomal recessive osteopetrosis continues to be reported [2,33,35,43,44]. Usually, it represents patients with dense bones in the first year of life without significant hematologic problems, but authors have presented variable findings to define the condition. Likely, it most frequently represents early onset of type II autosomal dominant disease with ClCN7 mutations, compound heterozygous mutations of ClCN7 or mild phenotypes of autosomal recessive osteopetrosis with homozygous ClCN7 mutations. Other obscure variants of osteopetrosis [1,2,6] and other disorders that cause increased skeletal mass [6,26,45] continue to be described. Infantile malignant osteopetrosis

Osteopetrosis in humans Historically, two major forms of osteopetrosis have been described. The benign or “adult” form is inherited in an autosomal dominant pattern, and is associated with minimal disability and a normal life expectancy. The malignant or “infantile” form is inherited in an autosomal recessive pattern and has widespread systemic manifestations. However, both forms have been found to be clinically heterogeneous, and other specific variants of osteopetrosis have been reported [2,6,26]. The adult benign autosomal dominant form was first described by the German radiologist Albers-Schönberg in 1904 [27] by roentgenographic demonstration of the characteristic dense, radiopaque bones (AlbersSchönberg syndrome or marble bone disease). Diagnosis is usually made in late childhood or adulthood by roentgenographic demonstration of diffuse skeletal sclerosis that increases over time. Infants with this dis-

The classic form of infantile malignant osteopetrosis is characterized by autosomal recessive inheritance and is almost always diagnosed in early infancy [2,8,35,46–48]. The inability to resorb and remodel bone due to osteoclast dysfunction, in the presence of normal bone formation by osteoblasts, results in the deposition of excessive mineralized osteoid and cartilage. All bones are uniformly dense, sclerotic, and radiopaque. Medullary cavities are absent on long bone radiographs [45,49]. Bone biopsies reveal encroachment of medullary cavities by bone and mineralized cartilage, thick trabeculae, and decreased medullary spaces. The residual medullary cavities are occupied with large numbers of nonfunctional osteoclasts, and there is usually increased marrow fibrosis [47,50]. Osteoclasts are reported to be normal in size in some patients and increased in size in others, with the large osteoclasts having increased numbers of nuclei. Electron microscopy of osteoclasts has been reported

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to demonstrate normal ruffled borders at the bone junction in some cases and decreased or complete absence of ruffled borders in other cases. Encroachment of marrow spaces leads to extramedullary hematopoiesis, progressive hepatosplenomegaly, and hypersplenism. The result is anemia with reticulocytosis, leukoerythroblastosis, and thrombocytopenia. Encroachment of cranial nerve foramina leads to retinal atrophy which progresses to blindness [48,51,52], auditory nerve damage [53], and oculomotor and facial nerve palsies [48]. Defective bone resorption also leads to progressive macrocephaly, frontal bossing, hypertelorism, exophthalmos, and other craniofacial abnormalities. Hydrocephalus, ventricular enlargement, increased intracranial pressure, cerebral vascular occlusive complications and seizures are also reported [47,48]. Nasal stuffiness is a common presenting symptom that is secondary to progressive narrowing and obstruction of the nasal airways [46]. Obstructive sleep apnea has also been described. Excessive tearing may occur secondary to nasolacrimal duct stenosis. Seizures and tetany in the neonatal period secondary to hypocalcemia have been reported in a few cases [47]. Rarely, diagnosis may be obscured in the neonate secondary to maternal vitamin D deficiency. Other reported complications are osteomyelitis of the mandible and maxilla, retarded tooth eruption [54], and rampant caries. Sclerotic bones are brittle, and pathologic fractures occur frequently. Linear growth is retarded, with dwarfism reported in survivors to the second decade. Neuropsychologic and developmental evaluation reveals a wide range of cognitive, adaptive, and language skills with routinely delayed gross motor development [48]. Infections are common in children with infantile malignant osteopetrosis. Various subtle defects in neutrophil and monocyte function have been reported. Included are defects in phagocytosis, decreased intracellular killing, decreased natural killer cell function, abnormal nitro blue tetrazolium reduction by both neutrophils and monocytes, and decreased response to stimuli of neutrophil activation. The children become progressively severely debilitated, and in the past survival beyond the first decade was not reported. Infection, bleeding, and severe anemia were the usual reported causes of death. Improvements in supportive care, antibiotics, and a ready availability of blood components have prolonged survival [47]. Affected children are at high risk for anesthetic morbidity and mortality [55]. A variant of infantile malignant osteopetrosis has been described that is associated with neuronal storage of ceroid lipofuscin. Children with this disorder have features of infantile malignant osteopetrosis but, in addition, are severely retarded and demonstrate chorioretinal degeneration and progressive generalized neurodegeneration. Findings include cerebral atrophy, ventricular dilatation, hypotonia or hypertonia, central apnea, and seizures. The disorder is invariably fatal before 2 years of age, and after HCT, while bone changes are improved, the disease progresses [47,48,56–60]. Gerritsen and co-workers [47] reported on 33 patients in 26 sibships seen in Paris, France and Leiden, Holland between 1972 and 1988, and summarized the major findings of an additional 59 cases reported in the literature to 1990. About 30% of children survived to 6 years of age, with survivors to the second or third decade having poor quality of life. About 75% developed visual impairment secondary to optic atrophy in the first year of life, most in the first 3 months. About 75% also developed hematologic abnormalities in the first year of life, which was an unfavorable prognostic sign, especially when associated with early visual impairment. The eight children with both visual and hematologic impairment before the age of 3 months died before 1 year of age. An important caveat to consider in evaluating the above descriptions of complications in patients with infantile malignant osteopetrosis is that some reported series may include patients with the variant with neuronal

storage disease, patients with CA II deficiency, and patients with the autosomal dominant and intermediate forms of osteopetrosis. Molecular genetics of osteopetrosis Until the advent of widespread molecular genetic analysis [32–37,56– 59,61–65], the etiology of the bone resorption defect in infantile malignant osteopetrosis was a subject of speculation. The finding of increased numbers of multinucleated osteoclasts in essentially all patients studied suggested the disorder was not a defect in stem cell differentiation, maturation or cell fusion. Osteoclast hyperplasia suggested a disorder of function supported by the observed increase in humoral stimulators of bone resorption. Possible etiologies were speculated to include defective activation due to abnormal cell surface receptors, defective recognition of effete bone, and digestive incompetence secondary to abnormal intracellular enzymes. Utilization of gene-linkage analysis and mutational analysis has resulted in the identification of five mutations that explain the etiology of about 80% of the cases of autosomal recessive osteopetrosis. Gene mapping in two consanguineous Bedouin kindreds to 11q13 [66] correlated to the position predicted by comparative mapping of the oc/oc murine mutation [14,22]. The Israeli team identified 13 children from four related families belonging to one Bedouin tribe and performed HCT on nine patients [67]. Prenatal linkage analysis identified three fetuses with osteopetrosis, and two received HCT as infants [68]. Concurrent reports [69,70] described mutations in the a3 subunit of the vacuolar H+ ATPase and defects in the ATP6i subunit of the vacuolar protein pump – the same defect that is now designated TCIRG1. This is the same protein pump mutation reported in the oc/oc mouse [14,22]. Subsequent studies [33,61,63,71] have demonstrated that 50–60% of all patients with infantile malignant osteopetrosis have mutations in TCIRG1. TCIRG1 mutations appear only to cause an acidification defect in osteoclasts and not cause defects in other cell types. ClCN7 [32–37] mutations also cause acidification defects. Homozygous mutations result in severe osteopetrosis similar to the phenotype seen with TCIRG1. ClCN7 mutations are found in about 15% of all patients with infantile malignant osteopetrosis. A ClCN7 knockout model in mice demonstrated the expected severe osteopetrosis, but in addition neural degeneration and retinal degradation secondary to deficient ClCN7 expression in the central nervous system [72]. The TCIRG1 natural mutation in the oc/oc mouse and a TCIRG1 knockout did not demonstrate neural or retinal defects [72]. It is speculated that children with ClCN7 homozygous mutations may be at an increased risk for retinal problems in addition to the expected optic nerve compression [3,48]. OSTM1 mutations [56–60] are responsible for the osteopetrotic variant with neuronal storage of ceroid lipofuscin [47,48]. The mutation is also present in the gl/gl mouse, and is rapidly fatal in both mouse and man. OSTM1 protein is thought to function as a subunit of ClCN7 and regulate hydrochloric acid secretion. OSTM1 mutations have been identified in eight patients [60] and represent 1–3% of autosomal recessive osteopetrosis. The fourth mutation was recently reported [64] as a pleckstrin homology domain-containing family M member-1 (PLEKHM1), which is present in the ia/ia rat model. PLEKHM1 protein is essential for bone resorption. Two siblings were identified with a mild form of osteopetrosis with dense deformed bones and no other symptoms. The carrier parents were related. The most recently described variant is the first human variant with a phenotype that does not represent a resorption defect [65,73]. It is described as osteoclast-poor human osteopetrosis due to RANKL deficiency. Patients who were negative for the above four mutations and

Hematopoietic Cell Transplantation for Osteopetrosis

who on bone biopsy had decreased or absent osteoclasts were screened for other mutations. Six affected children were identified, and three had correction of bone resorption by cultured osteoclasts when exposed to RANKL, as previously shown in the mouse RANKL knockout model. The other three children had genetic testing after HCT and could not be studied in vitro. Occasional patients have been reported previously with osteoclast-poor osteopetrosis [2,62,74]. The above five defects and CAII deficiency account for about 80% of human autosomal recessive patients. Potentially, the remaining patients may have a few or many more mutations. Most patients who have no currently identifiable mutations are phenotypically similar to infants with TCIRG1 and ClCN7 mutations. It is speculated [2,3] that most will also have acidification defects, as other mutations would likely involve many other cell types and lead to embryonic lethality. In addition to the remarkable progress in molecular genetics that the above studies represent, advances in cell culture have allowed the defects to be studied and elucidated in vitro. Investigators had attempted to culture functional osteoclasts for over 20 years from bone marrow and blood cells [8], but succeeded only in producing multinucleated giant cells which did not resorb bone. Methods have been developed to generate large numbers of both normal and defective osteoclasts in vitro in both laboratory animals [75] and humans [76–78]. Osteoclasts can now be obtained in large numbers from cultures of peripheral blood monocytes (enriched CD14 cells) and bone marrow macrophages stimulated with both M-CSF and RANKL. Bone resorption can be studied in vitro, as can the various gene products involved in acidification and matrix degradation [4,25,30,34–36,62,64,65].

HCT for infantile malignant osteopetrosis Historically, treatment of infantile malignant osteopetrosis consisted of supportive care measures and attempts to control mineral intake. Anemia and thrombocytopenia were treated with transfusions and occasionally splenectomy. Dietary manipulations to reduce calcium intake, increase phosphate intake or both were employed in an attempt to mobilize bone calcium without success. Attempts to induce bone resorption with infusions of parathyroid hormone, vitamin D, and calcitonin were also unsuccessful. Treatment with high-dose corticosteroids resulted in decreased hepatosplenomegaly, decreased leukoerythroblastosis, increased hemoglobin and platelet counts, and decreased need for transfusion. However, there was no significant effect on the underlying process, and patients became profoundly Cushingoid. When corticosteroids were tapered or discontinued, the patients’ condition deteriorated. Other than HCT, no other curative therapies have been reported for infantile malignant osteopetrosis. Treatment with high-dose calcitriol (1,25 dihydroxyvitamin D) may stabilize disease findings [79]. Recombinant human interferon-gamma (IFN-γ) [79] has been utilized to treat children with infantile osteopetrosis. IFN-γ increases superoxide production that is reduced in granulocytes, transformed B lymphocytes, and osteoclasts of osteopetrotic patients. Patients treated for 6–18 months demonstrate increases in hemoglobin concentration and platelet counts, and transfusion requirements decrease. However, no changes in bone density are observed on radiographs. All patients have biochemical evidence of increased bone resorption, and bacterial infections decrease. Two patients receiving IFN-γ have subsequently been reported to develop fatal acute respiratory distress syndrome after receiving a platelet transfusion [80]. A subsequent clinical trial of IFN-γ1b plus calcitriol demonstrated that patients with infantile osteopetrosis experienced fewer infections and had a 50% reduction in bone mass, and increases in the size of both the optic nerve foramina and auditory canals [79]. While children receiving calcitriol and IFN-γ

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demonstrate clinical improvement, the underlying disease process remains unchanged. Treatment of infantile malignant osteopetrosis with M-CSF has been suggested in view of the observations in the op/op mouse [21,81] and tl/tl rat [21,82]. Measurement of M-CSF in 13 patients with osteopetrosis demonstrated normal-to-increased levels in all patients [83]. Further, in six patients studied, the M-CSF present was biologically active [83]. In patients undergoing evaluation for HCT, treatment with combinations including low-dose corticosteroids, high-dose calcitriol, IFN-γ, and possibly M-CSF may be beneficial, especially if there are delays in finding a suitable donor [79,83]. Reports [15,17,18] of successful correction of osteopetrosis in mouse and rat mutants in the mid 1970s by HCT led to attempts to utilize allogeneic HCT to treat children with infantile malignant osteopetrosis. In 1977, Ballet et al. [84] reported the first HCT procedure for osteopetrosis. A 3-month-old girl was transplanted without immunosuppression with marrow from a human leukocyte antigen (HLA)-identical 2-yearold sister. Although durable engraftment was not demonstrated, radiologic and other evidence for significant bone resorption was documented. Of interest, the family was highly inbred and later found to be a CAII deficiency kindred [38]. In 1980, Coccia et al. [8] reported a 5-month-old girl who received an HCT from her HLA-identical, mixed leukocyte culture-compatible brother after preparation with cyclophosphamide (CY; 200 mg/kg) and modified TBI (400 cGy with head and lung shielding). Engraftment was documented by chromosomal analysis. Anemia, thrombocytopenia, leukoerythroblastosis, and metabolic abnormalities corrected within 12 weeks of HCT. Comparison of bone biopsies before HCT and at 13 weeks after HCT revealed complete correction (Fig. 76.1). Serial X-rays demonstrated bony remodeling and new nonsclerotic bone formation (Fig. 76.2). Fluorescent Y-body analysis after HCT showed that nuclei were of donor origin (male) in osteoclasts (Fig. 76.3), but nuclei in osteoblasts remained of recipient origin (female). Subsequent follow-up showed progressive loss of her graft. Dense new bone formation can be appreciated on the last three panels of Fig. 76.2 but with preservation of bone marrow cavities. Since 18 months after HCT, no male karyotypes have been detected in her peripheral blood or marrow cells. The recipient is now more than 29 years after HCT. Her bones are extremely sclerotic and she is short, but no other evidence of osteopetrosis remains. While her visual acuity is diminished, she has normal hearing, intelligence, and pubertal development. She is a college graduate with a Master’s degree and is gainfully employed. She has no hepatosplenomegaly, and blood counts and chemistries are normal. In this patient, it appears that once marrow cavities and foramina were remodeled, the correction was permanent in spite of sclerotic new bone formation. Fifty percent of the patients treated by our HCT group are long-term survivors who have engrafted promptly, have normal bones, and are fully chimeric; some are blind, but all have normal development and intelligence. Over 100 case reports and small series of patients detailing HCT for osteopetrosis have been published between 1981 and 2007. Most patients are represented in the large studies which are reviewed below, or their data are in HCT databanks. Analysis of these individual reports is beyond the scope of this chapter. Gerritsen et al. [9] for the European Group for Blood and Marrow Transplantation (EBMT) have reported a detailed summary of the outcome of 69 patients receiving HCT for osteopetrosis between 1976 and 1994 in Europe, Saudi Arabia or Costa Rica. The 69 children received 83 HCTs at 17 centers. Median age was 3 (range 1–81) months. Four patients had HCT without conditioning. Of the remaining 65 patients, 19 had matched sibling donors, nine had five- or six-antigen

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Fig. 76.1 Bone histology before bone marrow transplantation (BMT) (left) and 13 weeks after BMT (right). The biopsy specimen studied before BMT had the characteristic features of osteopetrosis. The trabecular matrix mass was markedly increased and composed of mineralized cartilage (C) as well as bone (white arrow). Numerous nonresorbing osteoclasts (black arrows) were present in the residual marrow space and contained few hematopoietic precursors (undecalcified; modified Masson stain, magnification: ×100). The biopsy specimen obtained 13 weeks after BMT revealed virtual normalization of the bone and marrow. The trabecular matrix mass was markedly reduced, and there were few residual cartilaginous bars. The marrow space contained abundant normal hematopoietic precursors, and the osteoclasts, the number of which was markedly reduced, were actively resorbing bone, as evidenced by their presence in resorption bays (insert) (undecalcified; modified Masson stain, magnification: ×100; insert, ×450). (Reproduced from [8], with permission.)

Fig. 76.2 Representative roentgenograms of the left leg obtained before bone marrow transplantation (BMT) and 10–69 weeks after BMT. Before BMT, the bones were very dense. The fraying of the ends of the long bones is typical of rachitic change. Ten weeks after transplantation, metaphyseal and periosteal remodeling and new bone formation of nearly normal density were seen. The rachitic changes were resolving. The provisional zone of calcification was reestablished. At 18 and 29 weeks, there was a marked increase in the thickness of normal-appearing new bone as a result of longitudinal and appositional growth. Metaphyses and epiphyses had a normal appearance. No evidence of rickets was seen. Dense remnants of the original bone were seen within the normal-appearing new bone. At 40, 49, and 69 weeks, new bone formation is again of increased density, suggesting recurrence of the osteopetrotic process. Marrow cavities have not been replaced by dense bone. The small round hole seen in the proximal tibia within the dense remnant of original bone is at the site of a trephine needle biopsy performed 6 weeks after BMT.

HLA-matched family donors, seven had six-antigen HLA-matched unrelated donors, and the remaining 30 had family donors with two or more nonidentical HLA antigens. Most patients were prepared with busulfan (BU) and CY. Many of the patients with HLA-disparate donors received additional immunosuppression. All 19 children with matched sibling donors engrafted, while only 30 of 42 with other donors engrafted. Engraftment was not evaluable in the remaining patients because of early death. In engrafted patients who were evaluable, persistent osteoclast function was docu-

mented by serial radiographs in 37 of 41 cases. Severe hypercalcemia developed after HCT in seven of 29 evaluable recipients (discussed below). Six of seven with hypercalcemia underwent HCT after 2 years of age. At the time of the report, 39 of 69 patients reported had died. Of the 30 survivors, 25 have osteoclast function, and most are healthy aside from visual disability in some. The authors conclude that HCT is the treatment of choice in children with matched sibling donors, that early HCT is important if vision is to be preserved [47], and that hypercalcemia is a worrisome complication,

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Fig. 76.3 Osteoclasts with fluorescent Y bodies after bone marrow transplantation (BMT). The osteoclasts were stained with quinacrine dihydrochloride. Arrows indicate the Y bodies in nuclei that are seen in this focal plane (fluorescence microscopy, magnification: ×100; right panel enlarged for clarity). (Reproduced from [8], with permission.)

especially in older recipients. The authors express concern with the poor outcome (13%) in patients with family donors with two or more disparate antigens and recommend search for an unrelated phenotypically HLA-identical donor. However, in children with early hematologic and visual impairment [47], they recommend HCT from the best available family donor as these children may not survive the search process to identify a matched unrelated donor. A German team [85] reported five of seven infants alive with complete resolution of osteopetrosis at a median of 4 years after HCT utilizing purified CD34+ T-cell depleted, mobilized peripheral blood from HLA-haploid identical parents. This encouraging result is a remarkable improvement in the outcomes reported by the EBMT group [9] and the Center for International Blood and Marrow Transplant Research (CIBMTR) [86] for HLA-haplotype mismatched HCT. The EBMT report [9] has been updated and expanded [87]. From 1980 to January 2001, 122 patients received 141 HCTs for autosomal recessive osteopetrosis in 20 centers, including the 65 conditioned patients in the previous report [9]. Median age was 6 (range 1–105) months. Visual impairment was severe in 42% and mild in 23%. Forty had matched sibling donors (group A); 20 had matched unrelated donors (two cord blood) (group B); 21 had six- or five-antigen HLA-matched family donors (group C); and 41 had family donors with two or more nonidentical HLA antigens (group D). Engraftment was documented in 95%, 79%, 52%, and 61% in groups A–D respectively. Survival with osteoclast function is show in Fig. 76.4. Actuarial 5-year disease-free survival were 73%, 40%, 43%, and 24% for groups A–D respectively. Group D patients included the seven cases previously reported [85]. Six of 19 patients retransplanted engrafted. At last evaluation, 56 of 122 patients (46%) were alive with osteoclast function at a median of 5 (range 0.5–16.5) years. Six survived with persistent osteopetrosis, and 62 died. Most deaths were related to graft failure or myeloablative conditioning toxicity. In 116 evaluable patients, 17 developed acute GVHD, four with grade III–IV, and six developed chronic GVHD. Conservation of vision was better if HCT occurred before age of 3 months of age. There were 272 patients registered through 2006 with CIBMTR, who had HCT for osteopetrosis between 1978 and 2006. Year of HCT was between 1978 and 1989 in 37, 1990–1999 in 137, and 2000–2006 in 98. The group includes 155 males and 118 females. Age at HCT was 1–136 (median 6) months. Donors were 36% matched siblings, 24% other related, and 40% unrelated. Of the 109 unrelated donor HCTs, 47 were umbilical cord blood grafts. Most patients (66%) were prepared with BU- and CY-containing regimens. Three-year probability of survival was 55 ± 9% for matched siblings, 49 ± 12% for alternate related donors,

Fig. 76.4 Survival with osteoclast function. Kaplan–Meier curves of diseasefree survival after first or subsequent hematopoietic stem cell transplantation (HSCT) in relation to human leukocyte antigen (HLA) compatibility with the HSC donor. A, genotypically HLA-identical sibling donor; B, matched unrelated donor; C, related donor phenotypically HLA identical or with one nonidentical HLA antigen; D, related donor with two or more nonidentical HLA antigens (haploidentical). Survival of group A is significantly better compared with that of groups B, C, and D (all p < 0.03). The differences between B, C, and D are not significant. (Reproduced from [87], with permission of Nature Publishing Group; Copyright © 2003, Nature Publishing Group.)

and 44 ± 9% for unrelated donors. When the CIBMTR registry data were reviewed in 2001, the 3-year probabilities were 54%, 47%, and 35%, respectively. (It should be noted that the data presented here are preliminary and were obtained from the Statistical Center of the CIBMTR. The analysis has not been reviewed or approved by the Advisory or Scientific Committee of the CIBMTR.) Recently, Tolar et al. [88] reported HCT for osteopetrosis using a reduced-intensity conditioning regimen including BU, fludarabine, and total lymphoid irradiation. Six patients received bone marrow or peripheral stem cell grafts, and all engrafted with over 75% chimerism. All five recipients of umbilical cord blood first grafts failed to develop donor chimerism. Four of 11 survive with donor engraftment at 1 year.

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Transplantation issues In general, HCT for osteopetrosis is similar to HCT for other nonmalignant inherited disorders detailed in this book. However, there are certain unique issues that should be understood to select appropriate patients and donors and to improve outcomes. The typical infant with osteopetrosis will present with anemia and thrombocytopenia. Transfusion may be necessary, and red cell and platelet survival may be short secondary to hypersplenism. A course of corticosteroid therapy may stabilize the patient hematologically and temporarily reduce transfusion requirements. Some patients will present with, or develop, severe bone pain, progressive debilitation, and poor oral intake, requiring narcotic analgesics and hyperalimentation prior to HCT. Evaluation for optic atrophy [48], including fundoscopic examination, visual evoked-response electroencephalograms, and optic foramina imaging, should be completed quickly as early optic nerve decompression may preserve vision [51,52,89]. Early correction of hypocalcemia may prevent or resolve seizures [47]. Baseline imaging studies should include long bone radiographs, measurements of liver and spleen size, and cranial computed tomography or magnetic resonance imaging scans [45,48,49]. It is recommended that an open-wedge bone biopsy from the anterior or posterior iliac crest be obtained to confirm the diagnosis [8]. Trephine needle biopsies may cause fractures, and are usually not adequate for evaluation due to crush artifact. Marrow aspiration is rarely successful. Light microscopic analysis of osteoclast number, size, nucleation, and morphology should be performed on touch preparations, and histologic sections examined to detect the rare osteoclast-poor phenotype [38,62,74]. Marrow cellularity and the degree of myelofibrosis should be determined. In patients over 2 years of age, fewer osteoclasts are identified due to increased fibrosis. Recipient and donor cytogenetic or molecular studies to assess the degree of hematopoietic chimerism after HCT are essential. Molecular genetic studies should be sent to a laboratory that can screen for known and potential mutations. Infants with early neurologic degeneration may have the syndrome of osteopetrosis associated with neuronal storage of ceroid lipofuscin [2,3,47,48,56–60]. They should not be considered as candidates for HCT as the storage disease progresses in spite of correction of the osteopetrosis. HCT in other variants of osteopetrosis is controversial. Dini and coworkers [43] report HCT in two children at 5 and 12 years of age with a variant of mild autosomal recessive osteopetrosis. Both had severe optic atrophy and dense bones but no significant hematologic or other complications of osteopetrosis. They each engrafted, developed severe hypercalcemia, had subsequent normalization of bone density but remained blind without catch-up growth, and are alive and well 5 and 6 years after HCT, respectively. They may represent one of the variants of ClCN7 mutations [44]. CAII deficiency syndrome [38,39] produces mild osteopetrosis, renal tubular acidosis, cerebral calcification, mental retardation, growth failure, and dental complications. Hematologic complications are not reported, and cranial nerve compression is usually mild. While HCT should correct bone manifestations, it will not improve renal insufficiency or central nervous system complications [38]. McMahon et al. [90] recently reported HCT in two children aged 9 months and 4 months from related Irish families with severe progressive visual and hearing loss. Both successfully engrafted, normalized bone density, and stabilized vision and hearing. HCT did not correct the metabolic acidosis, the renal lesions or the developmental delays. HCT may have delayed the onset of cerebral calcifications. In both mild and CAII osteopetrosis, it remains questionable whether correction of only the skeletal problems warrants the risks and morbidity of HCT.

An HLA-matched nonaffected sibling is the preferred donor. If a matched sibling is unavailable, it is possible that a matched or minimally mismatched parental or extended family donor can be identified. Obligate heterozygote relatives are suitable donors. Since progression of the disease is relatively slow in many cases, unrelated donor search and unrelated marrow or cord blood (see Chapters 39 and 47) HCT are reasonable options. However, if infants have early visual and hematologic impairment, rapid identification of a donor is essential [9,47,87]. Cord blood HCT with high-dose conditioning [88] and haploid-identical parent donors are reasonable options if a closely matched family donor is unavailable. Failure of engraftment [47,87] is a serious concern with mismatched donors, T-cell-depleted grafts, and reduced-intensity conditioning followed by cord blood HCT [88]. Additional immunosuppression is probably advisable in these situations. Second HCT is frequently necessary [47,87]. Serial re-evaluations to document engraftment and bone resorption are important to assess efficacy of therapy. Studies to assess the extent of hematopoietic chimerism and, in cases of mixed chimerism, to document sustained engraftment or late graft failure are vital for long-term patient management. Marrow aspirations and biopsies at 3–6 months after HCT are valuable to assess bone resorption, marrow cavity formation, and marrow cellularity. Serial long bone radiographs are the best indicators of sustained correction of osteopetrosis in most cases. The Ulm transplant team [91] reported that seven of 11 children after HCT between 1996 and 2001 developed sinusoidal obstructive syndrome (SOS). Three cases were severe, and one child died. Between 2001 and 2005, nine additional patients had HCT with defibrotide prophylaxis. One of the nine developed moderate SOS. Two groups have reported a high incidence of pulmonary hypertension after HCT for osteopetrosis. Steward et al. [92] for the EBMT reported that eight of 28 patients who received HCT between 1996 and 2002 at three institutions developed severe pulmonary hypertension at day 2 before HCT to day 89 after. Six required ventilator support, and five died. Three children had a sustained improvement after prostacyclin administration. Kasow et al. [93] reported five of 12 children at four institutions from 1997 to 2003 who developed primary pulmonary hypertension. Four died, and one dramatically improved. An extensive literature review has not revealed other reports with excessive morbidity or mortality from either SOS or pulmonary hypertension. An unusual but not necessarily surprising complication after HCT, referable to osteopetrosis, is hypercalcemia secondary to excessive donor osteoclast resorptive activity after HCT. A 30-month-old male child (Gluckman, personal communication) received a marrow graft in 1980 from his HLA-identical sister after preparation with CY and 400 cGy modified TBI. He engrafted rapidly and developed mild acute GVHD. The child demonstrated both histologic and radiologic improvement and grew 5 cm in the first 2 months after HCT. Severe hypercalcemia developed on day 60 after HCT, and was refractory to saline diuresis, Lasix, corticosteroids, calcitonin, and dialysis. Death with hypercalcemia occurred on day 165 after HCT. In the recent EBMT update, Driessen et al. [87] report severe hypercalcemia in eight of 50 evaluable patients. Two of 40 patients aged 2 years or less, and six of 10 older than 2, developed the complication. The current availability of agents that selectively inhibit calcium resorption from bone should provide effective options to control this serious complication. Bisphosphonate derivatives [94] selectively inhibit osteoclast-mediated bone resorption. These agents also can induce osteoclast apoptosis in murine osteoclasts [5]. Side-effects of concern are myelosuppression and nausea. Titration of these agents should allow controlled resorption of sclerotic bone without severe life-threatening hypercalcemia.

Hematopoietic Cell Transplantation for Osteopetrosis

An illustrative example is a 9-month-old male (Chang and Coccia, personal observation) who received a graft from an HLA-matched sibling donor for infantile osteopetrosis, rapidly developed complete hematopoietic chimerism without GVHD, and promptly demonstrated new bone formation of normal density. At 6 months after HCT, his serum calcium was 12.7 mg/dL. He responded poorly to saline diuresis and furosemide, and 4 weeks later his serum calcium was 13.8 mg/dL. He received pamidronate at 0.5 mg/kg intravenously, with a rapid decline to 12.3 mg/dL. Four weeks later, his serum calcium was 14.8 mg/ dL, and after 0.75 mg/kg intravenous pamidronate, it was 10.8 mg/dL within 3 days. Four weeks later, he received 1.5 mg/kg intravenous pamitronate for a serum calcium of 14.0 mg/dL. All subsequent serum calcium determinations have been normal. The child remains fully chimeric with complete resolution of all manifestations of osteopetrosis 8 years after HCT.

Future directions In July 1978, a 3-month-old female infant was seen in a St Paul, Minnesota emergency room with a stuffy nose and fever. On examination, she had impressive hepatosplenomegaly. Her complete blood count revealed anemia, marked leukoerythroblastosis, and thrombocytopenia. A chest X-ray was reported as normal but with dense bones consistent with “osteopetrosis.” In 1978, there were two types of osteopetrosis – the “good” autosomal dominant and the “bad” autosomal recessive. Donald Walker (1925–79) thought it would be exciting to translate his mouse experiments to a human. The patient and her only sibling, a 5-year-old brother, matched at the A and B loci and were nonreactive in mixed lymphocyte culture. Osteoclasts were the huge cells with all the nuclei rarely seen in marrow aspirates. Thus began the odyssey.

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Today, there are numerous subtypes of osteopetrosis, and the gene defects have been characterized in mouse and man for almost all patients with dominant and about 80% with recessive osteopetrosis. Acidification defects at the osteoclast–bone interface is likely the etiology of over 90% of inherited osteopetrotic disorders in humans. After more than two decades of failed attempts to culture osteoclasts, abundant osteoclasts can now be cultured in vitro by stimulating CD14+ cells from patients and controls with M-CSF and RANKL. This breakthrough allows the study of digestion and indigestion of bone fragments. Yet, only 50% of infants are cured with a toxic, expensive, and frequently unreliable therapy. This is about the same percentage reported in the mid-1980s [95]. No other effective therapies have yet been developed. Certainly, the rapidly increasing understanding of osteoclast bone biology is being translated into improved therapies for osteoporosis, Paget’s disease of bone, periodontal disease, abnormal fracture healing, and other disorders of excessive bone resorption. The development of bisphosphonates [94], which mediate osteoclasts apoptosis, have led to improved therapy for osteoporosis and multiple myeloma. The remarkable progress that continues to be made in our understanding of the osteopetrotic syndromes, the immunodeficiencies, storage diseases, and other inherited disorders of the HSC will eventually lead to in utero HCT (see Chapter 40), gene therapy (see Chapter 10) [3,24], reduced-intensity conditioning [88], and other strategies to increase the effectiveness and decrease the toxicity of HCT. Antiresorptive medications based on gene products are in clinical trials, and other gene products identified in the molecular biologic studies will potentially have a role in the development of therapeutic agents for various subtypes of osteopetrosis [3]. Many of the children cured of infantile malignant osteopetrosis by HCT are productive, contributing members of society.

References 1. Tolar J, Teitelbaum SL, Orchard PJ. Osteopetrosis. N Engl J Med 2004; 351: 2839–49. 2. Balemans W, Van Wesenbeeck L, Van Hul W. A clinical and molecular overview of the human osteopetroses. Calcif Tissue Int 2005; 77: 263–74. 3. Askmyr MK, Fasth A, Richter J. Towards a better understanding and new therapeutics of osteopetrosis. Br J Haematol 2008; 140: 597–609. 4. Abu-Amer Y. Advances in osteoclast differentiation and function. Curr Drug Targets Immune Endocr Metabol Disord 2005; 5: 347–55. 5. Teitelbaum SL. Osteoclasts: what do they do and how do they do it? Am J Pathol 2007; 170: 427– 35. 6. Helfrich MH. Osteoclast diseases. Microsc Res Tech 2003; 61: 514–32. 7. Van Wesenbeeck L, Van Hul W. Lessons from osteopetrotic mutations in animals: impact on our current understanding of osteoclast biology. Crit Rev Eukaryot Gene Expr 2005; 15: 133–62. 8. Coccia PF, Krivit W, Cervenka J et al. Successful bone-marrow transplantation for infantile malignant osteopetrosis. N Engl J Med 1980; 302: 701– 8. 9. Gerritsen EJ, Vossen JM, Fasth A et al. Bone marrow transplantation for autosomal recessive osteopetrosis. A report from the Working Party on Inborn Errors of the European Bone Marrow Transplantation Group. J Pediatr 1994; 125: 896–902. 10. Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone 2007; 40: 251–64.

11. Udagawa N, Takahashi N, Akatsu T et al. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci U S A 1990; 87: 7260–4. 12. Opdecamp K, Vanvooren P, Rivièr M et al. The rat microphthalmia-associated transcription factor gene (Mitf) maps at 4q34-q41 and is mutated in the mib rats. Mamm Genome 1998; 9: 617–21. 13. Weilbaecher KN, Hershey CL, Takemoto CM et al. Age-resolving osteopetrosis: a rat model implicating microphthalmia and the related transcription factor TFE3. J Exp Med 1998; 187: 775–85. 14. Li YP, Chen W, Liang Y, Li E, Stashenko P. Atp6ideficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification. Nat Genet 1999; 23: 447–51. 15. Walker DG. Bone resorption restored in osteopetrotic mice by transplants of normal bone marrow and spleen cells. Science 1975; 190: 784–5. 16. Walker DG. Spleen cells transmit osteopetrosis in mice. Science 1975; 190: 785–7. 17. Marks SC Jr. Studies of the cellular cure for osteopetrosis by transplanted cells: specificity of the cell types in ia rats. Am J Anat 1978; 151: 383– 7. 18. Milhaud G, Labat ML, Graf B et al. Démonstration cinétique, radiographique et histologique du la guérison de l’osteopetrose congenitale du rat. C R Acad Sci Hebd Seances Acad Sci D 1975; 280: 2485–8.

19. Ash P, Loutit JF, Townsend KMS. Osteoclasts derived from haematopoietic stem cells. Nature 1980; 283: 669–70. 20. Marks SCJ, Walker DG. The hematogenous origin of osteoclasts: experimental evidence from osteopetrotic (microphthalmic) mice treated with spleen cells from beige mouse donors. Am J Anat 1981; 16: 1–10. 21. Shalhoub V, Jackson ME, Paradise C, Stein GS, Lian JB, Marks SC Jr. Heterogeneity of colony stimulating factor-1 gene expression in the skeleton of four osteopetrotic mutations in rats and mice. J Cell Physiol 1996; 166: 340–50. 22. Scimeca JC, Franchi A, Trojani C et al. The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants. Bone 2000; 26: 207–13. 23. Johansson M, Jansson L, Ehinger M, Fasth A, Karlsson S, Richter J. Neonatal hematopoietic stem cell transplantation cures oc/oc mice from osteopetrosis. Exp Hematol 2006; 34: 242–9. 24. Johansson MK, de Vries TJ, Schoenmaker T et al. Hematopoietic stem cell targeted neonatal gene therapy reverses lethally progressive osteopetrosis in oc/oc mice. Blood 2007; 109: 5178– 85. 25. Blin-Wakkach C, Breuil V, Quincey D, Bagnis C, Carle GF. Establishment and characterization of new osteoclast progenitor cell lines derived from osteopetrotic and wild type mice. Bone 2006; 39: 53–60.

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26. Cohen MM Jr. The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A 2006; 140: 2646–706. 27. Albers-Schönberg H. Roentgenbilder einer seltenen Knochener-krankung. Münch Med Wochenschr 1904; 51: 365–8. 28. Johnston CC Jr., Lavy N, Lord T, Vellios F, Merritt AD, Deiss WP. Osteopetrosis: a clinical, genetic, metabolic, and morphologic study of the dominantly inherited, benign form. Medicine 1968; 47: 149–67. 29. Van Wesenbeeck L, Cleiren E, Gram J et al. Six novel missense mutations in the LDL receptorrelated protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet 2003; 72: 763–71. 30. Henriksen K, Gram J, Hoegh-Andersen P et al. Osteoclasts from patients with autosomal dominant osteopetrosis type I caused by a T253I mutation in low-density lipoprotein receptor-related protein 5 are normal in vitro, but have decreased resorption capacity in vivo. Am J Pathol 2005; 167: 1341– 8. 31. Benichou O, Cleiren E, Gram J, Bollerslev J, de Vernejoul M-C, Van Hul W. Mapping of autosomal dominant osteopetrosis type II (AlbersSchönberg Disease) to chromosome 16p13.3. Am J Hum Genet 2001; 69: 647–55. 32. Kornak U, Kasper D, Bosl MR et al. Loss of the CIC-7 chloride channel leads to osteopetrosis in mice and man. Cell 2001; 104: 205–15. 33. Frattini A, Pangrazio A, Susani L et al. Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis. J Bone Miner Res 2003; 18: 1740–7. 34. Henriksen K, Gram J, Schaller S et al. Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II. Am J Pathol 2004; 164: 1537–45. 35. Del Fattore A, Peruzzi B, Rucci N et al. Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment. J Med Genet 2006; 43: 315–25. 36. Chu K, Snyder R, Econs MJ. Disease status in autosomal dominant osteopetrosis type 2 is determined by osteoclastic properties. J Bone Miner Res 2006; 21: 1089–97. 37. Waguespack SG, Hui SL, DiMeglio LA, Econs MJ. Autosomal dominant osteopetrosis: clinical severity and natural history of 94 subjects with a chloride channel 7 gene mutation. J Clin Endocrinol Metab 2007; 92: 771–8. 38. Sly WS, Shah GN. The carbonic anhydrase II deficiency syndrome: osteopetrosis with renal tubular acidosis and cerebral calcification. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill; 2001. pp. 5331–43. 39. Shah GN, Bonapace G, Hu PY, Strisciuglio P, Sly WS. Carbonic anhydrase II deficiency syndrome (osteopetrosis with renal tubular acidosis and brain calcification): novel mutations in CA2 identified by direct sequencing expand the opportunity for genotype-phenotype correlation. Hum Mutat 2004; 24: 272–81. 40. Monaghan BA, Kaplan FS, August CS, Fallon MD, Flannery DB. Transient infantile osteopetrosis. J Pediatr 1991; 118: 252–6. 41. Iacobini M, Migliaccio S, Roggini M et al. Apparent cure of a newborn with malignant osteopetrosis

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60. Alroy J, Pfannl R, Ucci A, Lefranc G, Frattini A, Megarbane A. Electron microscopic findings in skin biopsies from patients with infantile osteopetrosis and neuronal storage disease. Ultrastruct Pathol 2007; 31: 333–8. 61. Scimeca JC, Quincey D, Parrinello H et al. Novel mutations in the TCIRG1 gene encoding the a3 subunit of the vacuolar proton pump in patients affected by infantile malignant osteopetrosis. Hum Mutat 2003; 21: 151–7. 62. Blair HC, Borysenko CW, Villa A et al. In vitro differentiation of CD14 cells from osteopetrotic subjects: contrasting phenotypes with TCIRG1, CLCN7, and attachment defects. J Bone Miner Res 2004; 19: 1329–38. 63. Ogbureke KU, Zhao Q, Li YP. Human osteopetroses and the osteoclast V-H+-ATPase enzyme system. Front Biosci 2005; 10: 2940–54. 64. Van Wesenbeeck L, Odgren PR, Coxon FP et al. Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans. J Clin Invest 2007; 117: 919– 30. 65. Frattini A, Vezzoni P, Villa A, Sobacchi C. The dissection of human autosomal recessive osteopetrosis identifies an osteoclast-poor form due to RANKL deficiency. Cell Cycle 2007; 6: 3027–33. 66. Heaney C, Shalev H, Elbedour K et al. Human autosomal recessive osteopetrosis maps to 11q13, a position predicted by comparative mapping of the murine osteosclerosis (oc) mutation. Hum Mol Genet 1998; 7: 1407–10. 67. Kapelushnik J, Shalev C, Yaniv I et al. Osteopetrosis: a single centre experience of stem cell transplantation and prenatal diagnosis. Bone Marrow Transplant 2001; 27: 129–32. 68. Shalev H, Mishori-Dery A, Kapelushnik J et al. Prenatal diagnosis of malignant osteopetrosis in Bedouin families by linkage analysis. Prenat Diagn 2001; 21: 183–6. 69. Kornak U, Schulz A, Friedrich W et al. Mutations in the a3 subunit of the vacuolar H(+)-ATPase cause infantile malignant osteopetrosis. Hum Mol Genet 2000; 9: 2059–63. 70. Frattini A, Orchard PJ, Sobacchi C et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet 2000; 25: 343–6. 71. Sobacchi C, Frattini A, Orchard P et al. The mutational spectrum of human malignant autosomal recessive osteopetrosis. Hum Mol Genet 2001; 10: 1767–73. 72. Kasper D, Planells-Cases R, Fuhrmann JC et al. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J 2005; 24: 1079–91. 73. Sobacchi C, Frattini A, Guerrini MM et al. Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL. Nat Genet 2007; 39: 960–2. 74. Nicholls BM, Bredius RG, Hamdy NA et al. Limited rescue of osteoclast-poor osteopetrosis after successful engraftment by cord blood from an unrelated donor. J Bone Miner Res 2005; 20: 2264– 70. 75. David JP, Neff LCY, Rincon M, Horne WC, Baron R. A new method to isolate large numbers of rabbit osteoclasts and osteoclast-like cells; application to the characterization of serum response element binding proteins during osteoclast differentiation. J Bone Miner Res 1998; 13: 1730–8.

Hematopoietic Cell Transplantation for Osteopetrosis 76. Teti A, Migliaccio S, Taranta A et al. Mechanisms of osteoclast dysfunction in human osteopetrosis: abnormal osteoclastogenesis and lack of osteoclastspecific adhesion structures. J Bone Miner Res 1999; 14: 2107–17. 77. Flanagan AM, Sarma U, Steward CG, Vellodi A, Horton MA. Study of the nonresorptive phenotype of osteoclast-like cells from patients with malignant osteopetrosis: a new approach to investigating pathogenesis. J Bone Miner Res 2000; 15: 352– 60. 78. Helfrich MH, Gerritsen EJA. Formation of nonresorbing osteoclasts from peripheral blood mononuclear cells of patients with malignant juvenile osteopetrosis. Br J Haematol 2001; 112: 64– 8. 79. Key LLJ, Ries WL. Osteopetrosis. Principles of Bone Biology. San Diego: Academic Press; 2002. 80. Madyastha PR, Jeter EK, Key LL Jr. Cytophilic immunoglobulin G binding on neutrophils from a child with malignant osteopetrosis who developed fatal acute respiratory distress mimicking transfusion-related acute lung injury. Am J Hematol 1996; 53: 196–200. 81. Umeda S, Takahashi K, Naito M, Shultz LD, Takagi K. Neonatal changes of osteoclasts in osteopetrosis (op/op) mice defective in production of functional macrophages colony-stimulating factor

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(M-CSF) protein and effects of M-CSF on osteoclast development and differentiation. J Submicrosc Cytol Pathol 1996; 28: 13–26. Seifert MF. Abnormalities in bone cell function and endochrondral ossification in the osteopetrotic toothless rat. Bone 1996; 19: 329–38. Orchard PJ, Dahl N, Aukerman SL, Blazar BR, Key LL Jr. Circulating macrophage colony-stimulating factor is not reduced in malignant osteopetrosis. Exp Hematol 1992; 20: 103–5. Ballet JJ, Griscelli C, Coutris C, Milhaud G, Maroteaux P. Bone marrow transplantation in osteopetrosis. Lancet 1977; 2: 1137. Schulz AS, Classen CF, Mihatsch WA et al. HLAhaploidentical blood progenitor cell transplantation in osteopetrosis. Blood 2002; 99: 3458–60. Fasth A, Porras O. Human malignant osteopetrosis: pathophysiology, management and the role of bone marrow transplantation. Pediatr Transplant 1999; 3(Suppl 1): 102–7. Driessen GJ, Gerritsen EJ, Fischer A et al. Longterm outcome of haematopoietic stem cell transplantation in autosomal recessive osteopetrosis: an EBMT report. Bone Marrow Transplant 2003; 32: 657–63. Tolar J, Bonfim C, Grewal S, Orchard P. Engraftment and survival following hematopoietic stem cell transplantation for osteopetrosis using a

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reduced intensity conditioning regimen. Bone Marrow Transplant 2006; 38: 783–7. Hwang JM, Kim IO, Wang KC. Complete visual recovery in osteopetrosis by early optic nerve decompression. Pediatr Neurosurg 2000; 33: 328– 32. McMahon C, Will A, Hu P, Shah GN, Sly WS, Smith OP. Bone marrow transplantation corrects osteopetrosis in the carbonic anhydrase II deficiency syndrome. Blood 2001; 97: 1947–50. Corbacioglu S, Honig M, Lahr G et al. Stem cell transplantation in children with infantile osteopetrosis is associated with a high incidence of VOD, which could be prevented with defibrotide. Bone Marrow Transplant 2006; 38: 547–53. Steward CG, Pellier I, Mahajan A et al. Severe pulmonary hypertension: a frequent complication of stem cell transplantation for malignant infantile osteopetrosis. Br J Haematol 2004; 124: 63–71. Kasow KA, Bonfim C, Asch J et al. Malignant infantile osteopetrosis and primary pulmonary hypertension: a new combination? Pediatr Blood Cancer 2004; 42: 190–4. Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S. Bisphosphonate-induced osteopetrosis. N Engl J Med 2003; 349: 457–63. Coccia PF. Cells that resorb bone. N Engl J Med 1984; 310: 456–8.

77

Charles Peters

Hematopoietic Cell Transplantation for Storage Diseases

Introduction The storage diseases are a diverse group of disorders arising from singlegene defects that often involve a lysosomal hydrolytic enzyme or vital peroxisomal function, causing devastating systemic diseases that can affect the brain (Tables 77.1–77.3). Progressive losses of neurodevelopmental milestones and neurologic function are common. Cardiopulmonary disease and compromise lead to shortened life expectancies. Diseases with onset during infancy or childhood are characterized by rapid deterioration. Treatment recommendations and clinical decision making must take account of the disorder, age at onset, rate of progression, presence of clinical signs and symptoms, family values, and risks and benefits associated with available therapies, which include hematopoietic cell transplantation (HCT), enzyme replacement therapy (ERT), substrate depletion, and gene transfer. Lysosomes are cellular organelles that contain hydrolytic enzymes; peroxisomes are subcellular organelles catalyzing metabolic functions primarily related to lipid metabolism. In 1968, Fratantoni and Neufeld established the concept of transferable lysosomal enzymes through metabolic cross-correction of defects in co-cultured fibroblasts of patients with Hurler and Hunter syndromes [1]. Metabolic correction of lysosomal storage diseases is due to the mannose-6-phosphate receptor-mediated endocytosis of secreted enzyme, or by direct transfer of enzyme from adjacent cells [2–4]. It is likely that both mechanisms occur following HCT (Fig. 77.1). Variability in receptor-mediated enzyme endocytosis in various cells and tissues could affect hydrolase uptake after HCT [5]. For example, monocytes and macrophages have receptors for N-acetylglucosamine and mannose; glial cells have receptors for sialic acid [6]. X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder of very long chain fatty acid (VLCFA) β-oxidation. The likely mechanism by which HCT halts cerebral demyelination is perhaps threefold: immunosuppression, replacement with metabolically normal cell populations leading to decreased perivascular inflammation, and metabolic correction. The impact of normalizing VLCFAs in plasma is unclear. A critical question in HCT for storage diseases with central nervous system (CNS) involvement is whether donor-derived cells enter the CNS and achieve metabolic correction or halt inflammation. Microglia are mononuclear phagocytes in the CNS, which are hematopoietically derived [7,8]. They account for 5–10% of non-neuronal cells in brain. Activated microglia participate in antigen presentation, inflammatory

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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responses, infection, and CNS injury [9,10]. In humans, repopulation with donor-derived microglia can take a year. The kinetics of microglial replacement after HCT is slower than that of other tissue macrophages (e.g. alveolar macrophages or Kupffer cells) [11]. This observation explains, in part, the inability of HCT to stabilize effectively the CNS and neurologic aspects of a rapidly progressing disorder.

HCT for storage diseases History On April 8, 1980, a 9-month-old boy received the first bone marrow transplant for Hurler syndrome [12]. Today, fully engrafted with maternal marrow, he uses a computer as a self-reliant employee. Despite genotype analysis revealing homozygous W402X mutations indicative of severe Hurler syndrome (mucopolysaccharidosis IH [MPS IH]) phenotype, he has stable, normal intelligence [12]. In 1982, the first child with Maroteaux–Lamy syndrome, a 13-year-old girl, was successfully transplanted from her human leukocyte antigen (HLA)-matched, enzymatically normal sister, and experienced resolution of hepatosplenomegaly and normalization of cardiopulmonary function [13]; she survives and functions independently. In the late 1980s and into the 1990s, successful transplants were performed for each of the leukodystrophies, including cerebral X-ALD [14], globoid cell leukodystrophy (GLD) [15], and metachromatic leukodystrophy (MLD) [16]. In the mid-1990s to the mid-2000s, the first large multicenter reports for HCT were published on Hurler syndrome [17–19] and cerebral X-ALD [20]. Sources of hematopoietic cells for transplantation included bone marrow [15,16,20], peripheral blood [21], and umbilical cord blood (UCB) [20,22,23]. In addition to high-dose regimens, reduced-intensity conditioning regimens were employed effectively [21]. Successful use of combination therapy, ERT, and HCT for patients with Hurler syndrome has been described [24,25]. Finally, participants in the field have recognized the importance of a comprehensive approach to caring for these patients, which is described below. General considerations The following elements are critical to the success of HCT in treating patients with storage disorders: a multidisciplinary and multispecialty approach to evaluation and follow-up; careful attention to optimizing HLA typing, and donor and hematopoietic cell selection; the risks and benefits of high-dose versus reduced-intensity conditioning regimens, supportive care measures, and long-term post-HCT evaluations. Several sets of evaluation and follow-up guidelines are provided later in this chapter (Tables 77.4 and 77.5).

Autosomal recessive Autosomal recessive Autosomal recessive

Autosomal recessive 248/242 2111/726 422/285

β-Glucuronidase Phosphotransferase

128/169 235/230 1407/593 1056/514 201/206

162/136,000

111/148

Galactose 6-sulfatase β-Galactosidase Arylsulfatase B

Heparan-N-sulfatase N-acetylglucosaminidase AcetylCoA : N-acetyltransferase N-acetylglucosamine 6-sulfatase

Iduronate-2-sulfatase

X-linked

Autosomal recessive

α-L-Iduronidase

Enzyme/protein

Autosomal recessive

Genetics

ERT, enzyme replacement therapy; GT, gene therapy; HCT, hematopoietic cell transplantation; SD, substrate depletion. Data from [170].

Mucolipidoses (ML) ML-II (I-cell) ML-III (pseudo-Hurler polydystrophy)

Sly (MPS VII)

Mucopolysaccharidoses (MPS) MPS I Hurler (MPS IH) Hurler/Scheie (MPS IH/S) Scheie (MPS IS) Hunter MPS II Severe (MPS IIA) Attenuated (MPS IIB) Sanfilippo (MPS III) MPS IIIA MPS IIIB MPS IIIC MPS IIID Morquio (MPS IV) MPS IV A MPS IV B Maroteaux–Lamy (MPS VI)

Disorder

Incidence in thousands/ carrier frequency (estimates)

/ / / /

− − − − / / / /

− − − − / / / /

− − − −

HCT in a very limited number of a cases Significant clinical challenges to performing HCT primarily due to airway and pulmonary issues

+/−/−/−

ERT is the preferred therapy; however, HCT can be considered

Severe skeletal deformities preclude HCT

No evidence of neurocognitive efficacy following successful, timely HCT

ERT is currently offered to patients with Hunter

HCT or HCT + ERT ERT is considered the standard therapy

Comments

+/−/−/−

−/−/−/− −/−/−/− +/+/−/−

− − − −

−/+/−/− −/+/−/−

+/+/−/− +/+/−/− +/+/−/−

Treatment options: HCT/ERT/SD/GT

Table 77.1 The mucopolysaccharidoses and mucolipidoses: mode of inheritance, enzyme/protein deficiency, incidence and carrier frequencies, treatment options, and comments

Hematopoietic Cell Transplantation for Storage Diseases

1137

Autosomal recessive

Autosomal recessive X-linked Autosomal recessive

Galactocerebrosidase

Autosomal recessive Autosomal recessive

Proteolipid protein Mutations in translation initiation factor eIF2B and possibly others Several, including accumulation of VLCFA, marked deficiency of plasmalogens

Arylsulfatase-A

ALD protein (ALDP)

Enzyme/protein

X-linked

Genetics

Long-term follow-up of infants transplanted with UCB is needed The extended time to CNS stabilization and the inability of HCT to favorably affect the PNS dampens enthusiasm regarding quality of life outcomes in late-onset patients. ERT may be effective for treating the PNS; however, further study is needed

+/−/−/− +/+/−/−

−/−/−/− −/−/−/−

−/−/−/−

?/?

−/−/−/−

HCT is the only effective long-term therapy offering the possibility of stabilization of the cerebral disease course. Lorenzo’s oil appears to decrease the likelihood of developing cerebral disease in selected boys with biochemically diagnosed X-ALD

+/−/−/+ +/−/−/− +/−/−/− ?/−/−/? −/−/−/−

?/? ?/?

?/?

121/152

201/188

19/19,000

Comments

ERT, Enzyme replacement therapy; GT, gene therapy; PNS, peripheral nervous system; SD, substrate depletion; ?, unknown. See text for other abbreviations. Data from [170].

Zellweger syndrome

Pelizaeus–Merzbacher disease Vanishing white matter disease

Alexander

Metachromatic leukodystrophy

Leukodystrophies and other white matter diseases X-ALD phenotypes Childhood-onset cerebral X-ALD Adolescent-onset cerebral X-ALD Adult-onset cerebral X-ALD AMN with or without cerebral X-ALD Addison disease only Globoid-cell leukodystrophy

Disorder

Incidence in thousands/ carrier frequency Treatment options: (estimates) HCT/ERT/SD/GT

Table 77.2 The leukodystrophies: mode of inheritance, enzyme/protein deficiency, incidence and carrier frequencies, treatment options, and comments [170]

1138 Chapter 77

Hematopoietic Cell Transplantation for Storage Diseases

1139

Table 77.3 The glycoprotein disorders and miscellaneous lysosomal storage diseases and inborn errors of metabolism: mode of inheritance, enzyme/protein deficiency, incidence and carrier frequencies, treatment options, and comments [170]

Disorder

Genetics

Enzyme/protein

Fucosidosis

Autosomal recessive Autosomal recessive

Fucosidase

Gaucher disease I (non-neuronopathic) II (acute neuronopathic) III (subacute neuronopathic)

α-Mannosidosis Aspartylglucosaminuria Cerebrotendinous xanthomatosis Fabry disease Farber lipogranulomatous disease Gangliosidoses (GM1 and GM2 including Tay–Sachs, Sandhoff, GM2 activator deficiency) GM1 gangliosidoses GM2 gangliosidoses – Tay–Sachs – Sandhoff – GM2 activator deficiency Glycogen storage disease II (Pompe) Neuronal ceroid lipofuscinosis NCL1 NCL2

Glucocerebrosidase

Aspartylglucosaminidase

2111/726

+/−/−/−

Autosomal recessive Autosomal recessive

Ceramidase

Autosomal recessive Autosomal recessive

117/117,000

−/+/+/−

ERT is the preferred therapy

?/?

+/−/−/−

β-Galactosidase Hexosaminidase A Hexosaminidase A and B

422/310 222/224 422/310

?/−/−/− −/−/+/−

No clear evidence of efficacy for HCT in any of these disorders

α-Glucosidase

201/191

−/+/−/−

ERT: preferred therapy

?/?

+/−/−/−

Palmitoyl protein thioesterase Tripeptidyl peptidase I

Acid lipase

Canavan disease

Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive Autosomal recessive or autosomal dominant

Aspartoacylase

Hypophosphatasia

ERT and/or SD: preferred therapies Long-term favorable outcomes have been seen with HCT, particularly in patients with Norrbottnian form Impressive, favorable results with HCT

−/−/−/−

27-Hydroxylase

Autosomal recessive

Multiple sulfatase deficiency

+/+/+/+ −/+/+/− +/+/+/−

+/−/−/−

Wolman syndrome

Sialidosis

59/119

1056/514

α-Galactosidase

Comments

+/−/−/−

α-Mannosidase

Autosomal recessive

Sialic acid storage disease

Treatment options: HCT/ERT/SD/GT

Autosomal recessive Autosomal recessive Autosomal recessive X-linked

Niemann–Pick disease A B C

Cystinosis

Incidence in thousands/ carrier frequency (estimates)

Acid sphingomyelinase Acid sphingomyelinase NPC1 (cholesterol trafficking)

A and B: 264/249 211/230

−/+/−/− +/+/−/− +/−/−/−

704/363

+/−/−/− −/−/−/−

Very limited clinical use of HCT and rapid disease course make it difficult to reach a conclusion ERT may have a role in type A and B patients. HCT may be effective in type B and C patients; however, experience remains very limited HCT: extremely high morbidity and mortality Increased in Ashkenazi Jews

Cystine transporter

281/219

−/−/+/−

Sialic acid transporter

603/363

?/−/−/−

HCT: role is unknown

Neuraminidase

4222/1027

?/−/−/−

HCT: role is unknown

Multiple sulfatase factor, all sulfatases Tissue nonspecific alkaline phosphatase

1407/593

+/−/−/−

HCT: extremely limited clinical experience HCT: extremely limited clinical experience

+/−/−/−

ERT, enzyme replacement therapy; GT, gene therapy; HCT, hematopoietic cell transplantation; SD, substrate depletion; ?, unknown. Data from [170].

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Fig. 77.1 Transport of a lysosomal enzyme in a normal cell and correction of storage in the cell of a patient with mucopolysaccharidosis. The mannose-6phosphate-recognition signal is added to the lysosomal enzyme precursor in the late Golgi compartments, where enzyme modified by mannose-6-phosphate binds to mannose-6-phosphate receptors. The enzyme–receptor complex is packaged into a transport carrier vesicle and delivered to early endosomes, in which low pH promotes the dissociation of the enzyme from the receptor. The enzyme is then delivered to the mature lysosome, and the mannose-6-phosphate receptor is recycled to the Golgi apparatus. A small amount of the mannose-6-phosphate-modified enzyme escapes capture by the mannose-6-phosphate receptors and is released into the extracellular space. This enzyme can be recaptured by binding to a mannose-6-phosphate receptor in a clathrin-coated pit on the cell surface. In a patient who has undergone hematopoietic stem cell transplantation, enzyme released from a donor-derived cell can be taken up by a mucopolysaccharidosis cell, which corrects aberrant glycosaminoglycan storage. (Reproduced from Muenzer J, Fisher A. Advances in the treatment of mucopolysaccharidosis type I. N Engl J Med 2004; 350: 1960–9, with permission.)

Hematopoietic Cell Transplantation for Storage Diseases

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Table 77.4 Guidelines for the pre-hematopoietic cell transplantation (HCT) evaluation (section A) and post-HCT (section B) follow-up of patients with mucopolysaccharide, mucolipidosis, and glycoprotein metabolic disorders Section A

Pre HCT

Neurology Neuropsychology

Examination including assessment for signs or symptoms of hydrocephalus in patients with Hurler syndrome Test of cognitive ability according to age and developmental level, e.g. Mullen Scales of Early Learning to obtain Early Learning composite or WISC III Language assessment to obtain receptive and expressive language scores Vineland Adaptive Behavior Scales MRI of brain and upper cervical spine with attention to the odontoid process in patients with Hurler syndrome Echocardiogram, EKG Chest X-ray (posteroanterior and lateral) Polysomnogram ENT as needed for placement of tubes Brainstem auditory evoked response if requested by audiology department Routine evaluation and if possible ocular pressures in patients with Hurler syndrome

Neuroradiology Cardiology Pulmonology Audiology Ophthalmology Genetic counseling Neurophysiology Occupational, physical, and speech and language therapy Donor studies Molecular biology studies Lumbar puncture

Electrophysiology studies (EMG) with surface electrodes to evaluate for carpal tunnel syndrome if clinically indicated in patients with Hurler syndrome Assessment and therapies as needed, of particular importance in children with Hurler syndrome Donor enzyme activity level, if possible, in addition to infectious disease markers On admission to hospital, consider obtaining blood samples for DNA (e.g. mutation analysis, pharmacogenomics) If there are clinical concerns about possible, significant increased intracranial pressure, a lumbar puncture with determination of opening and closing pressures may be performed with determinations of cerebrospinal fluid glucose and protein in patients with Hurler syndrome

Section B

Post HCT

Blood and marrow transplant (BMT) team

BMT physician, neurologist, neuropsychologist or developmental pediatrician, cardiologist, pulmonologist/sleep disorder specialist, endocrinologist, audiologist, ophthalmologist, occupational and physical therapist, speech and language therapist, advanced practice nurse, nurse coordinator, social worker; others as needed, including occupational and physical therapists Examination Test of cognitive ability according to age and developmental level, i.e. Mullen Scales of Early Learning (Early Learning Composite), or Stanford Binet Intelligence Scale (Composite Score), or Wechsler Preschool and Primary Scale of Intelligence (Full Scale IQ) Language assessment to obtain receptive and expressive language scores Vineland Adaptive Behavior Scales MRI of brain and upper cervical spine (attention to odontoid process) Echocardiogram EKG Chest X-ray Consider follow-up polysomnogram as clinically indicated ENT as needed for placement of tubes Brainstem auditory evoked response if requested by audiology department Electroretinogram Orthopedic evaluation of spine, hips, and knees, and hand surgery assessment for carpal tunnel syndrome Growth hormone stimulation test Free thyroxine Thyroid-stimulating hormone levels Dual-energy X-ray absorptiometry scan Bone age X-ray Electrophysiology studies (EMG) with surface electrodes to evaluate for carpal tunnel syndrome Assessment and therapies as needed

Neurology Neuropsychology

Neuroradiology Cardiology Pulmonology Audiology Ophthalmology Orthopedic surgery Endocrinology

Neurophysiology Occupational, physical, and speech and language therapy Engraftment and chimerism studies

Variable number of tandem repeat or XY fluoresence in situ hybridization studies and enzyme level (days +21–30, +60, +100, +6 months, +1 year, and yearly post HCT thereafter as needed

EKG, electrocardiogram; EMG, electromyelogram; ENT, ear, nose, and throat; MRI, magnetic resonance imaging.

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Table 77.5 Guidelines for the pre-hematopoietic cell transplantation (HCT) evaluation (section A) and post HCT (section B) follow-up of patients with leukodystrophies Section A

Pre HCT

Neurology Neuropsychology

Examination Age-appropriate battery of neuropsychologic tests, i.e. Wechsler Preschool and Primary Scale of Intelligence (WPPSIrevised) and Wechsler Intelligence Scale for Children III (WISC-III) Wechsler Adult Intelligence Scale III to obtain: • Full Scale IQ Score • Verbal IQ Score • Performance IQ Score Other testing to measure school achievement, language, visual perception, attention, memory, motor function, executive function. and behavior Vineland Adaptive Behavior Scales MRI of brain including contrast agent (gadolinium) with severity score determinations (Loes Score [80]); Magnetic resonance spectroscopy if available and deemed to be potentially informative MRI of brain

Neuroradiology – cerebral X-ALD Neuroradiology – GLD [15] and metachromatic leukodystrophy CSF Pulmonology Ophthalmology Endocrine Neurophysiology Occupational, physical, and speech and language therapy

Examination of CSF (including CSD protein): may be informative in patients with advanced disease at HCT to evaluate status of blood–brain barrier disruptions (i.e. degree of CSF protein elevation; e.g., in infants with GLD, CSF protein levels of 500–600 mg/dL can be seen) Chest X-ray Assessment as needed To assess treatment of adrenal insufficiency (i.e. dosing of hydrocortisone) in boys with X-ALD; routine assessment of all patients for thyroid function, progress toward puberty, growth, and development Electrophysiology studies (EMG): a long-term evaluation in boys transplanted for cerebral X-ALD who are reaching the age at which they are at risk for developing AMN, i.e. approximately ≥20 years of age. The question of whether such men will develop AMN is unanswered Assessment and therapies as needed

Section B

Post HCT

Blood and marrow transplant (BMT) team Neurology Neuropsychology

BMT physician, neurologist, advanced practice nurse, nurse coordinator, social worker; others as needed, including occupational and physical therapists Examination Age appropriate battery of neuropsychologic tests, i.e. Wechsler Preschool and Primary Scale of Intelligence (WPPSIrevised) and Wechsler Intelligence Scale for Children III (WISC-III) Wechsler Adult Intelligence Scale III to obtain: • Full Scale IQ Score • Verbal IQ Score • Performance IQ Score Other testing to measure school achievement, language, visual perception, attention, memory, motor function, executive function, and behavior Vineland Adaptive Behavior Scales MRI of brain including contrast agent (gadolinium) with severity score determinations (MRI severity or Loes Score [80]); recommended minimum number of examinations – approximately 2–3 months post HCT to evaluate for resolution of gadolinium enhancement (hallmark of active demyelination in cerebral X-ALD) and at 1 year post HCT to evaluate severity score at time when stabilization has been achieved [81]. Subsequent brain MRIs as clinically indicated Magnetic resonance spectroscopy if available and deemed to be potentially informative MRI of brain at 1 year post HCT and annually, if clinically indicated, until stable myelination is demonstrated Examination of CSF (including CSD protein): may be informative in patients with advanced disease at HCT to evaluate status of blood–brain barrier disruptions (i.e. degree of CSF protein elevation; e.g. in infants with GLD, CSF protein levels of 500–600 mg/dL can be seen) Chest X-ray Assessment as needed To assess treatment of adrenal insufficiency (i.e. dosing of hydrocortisone) in boys with X-ALD; routine assessment of all patients for thyroid function, progress toward puberty, growth, and development Electrophysiology studies (EMG): a long-term evaluation in boys transplanted for cerebral X-ALD who are reaching the age at which they are at risk for developing AMN, i.e. approximately ≥20 years of age. The question of whether such men will develop AMN is unanswered Patient’s variable number of tandem repeats and appropriate enzyme levels (days +21–30, +60, +100, +6 months, +1 year, and yearly post HCT thereafter); follow-up evaluation of VLCFA has been discontinued as published studies showed a 55% reduction in these levels after HCT but not complete normalization [85]. VLCFA levels do not appear to have any clinical significance or correlation with disease activity or clinical status

Neuroradiology – cerebral X-ALD

Neuroradiology – GLD and MLD CSF Pulmonology Ophthalmology Endocrine Neurophysiology Engraftment and chimerism studies

AMN, adrenomyeloneuropathy; CSF, cerebrospinal fluid; EMG, electromyogram; GLD, globoid cell leukodystrophy; ML, metachromatic leukodystrophy; MRI, magnetic resonance imaging; VLCFA, very long chain fatty acid; X-ALD, X-linked adrenoleukodystrophy.

Hematopoietic Cell Transplantation for Storage Diseases

MPSs and mucolipidoses In this section, MPS disorders (Hurler, Hurler/Scheie, Scheie, Hunter, Sanfilippo, Morquio, Maroteaux-Lamy, and Sly) and mucolipidoses (MLs; I-cell disease and pseudo-Hurler polydystrophy) will be presented and discussed (Table 77.1). Epidemiology and etiology The MPS diseases are a group of lysosomal storage disorders caused by deficiency of degradative enzymes of glycosaminoglycans (GAGs) [26], which include heparan, dermatan, keratan, and chondroitin sulfates, that individually or in combination accumulate intracellularly, leading to cellular dysfunction. These GAGs are usually excreted in urine and can be detected by initial diagnostic screening tests. Genes and their complementary DNAs (cDNAs) encoding most of the enzymes have been cloned, leading to characterization of their primary structures, production of recombinant enzymes, and elucidation of disease-causing mutations. MPS disorders, their genetics and respective enzymes, incidence and carrier frequencies, and treatment options are presented in Table 77.1. All of the MPS and ML disorders are autosomal recessive in their inheritance except for Hunter syndrome (X-linked). ML II (I-cell) and ML III (pseudo-Hurler polydystrophy) are related, rare genetic diseases in which there is abnormal lysosomal enzyme transport in cells of mesenchymal origin. Newly synthesized lysosomal enzymes are secreted into the extracellular medium rather than being targeted correctly to lysosomes. Affected cells show dense storage material-filled inclusions; lysosomal enzymes are present at elevated levels in serum and body fluids of affected patients [27]. Molecular and clinical biology A precise correlation between the numerous genotypes and the phenotypes of these MPS disorders remains elusive. Enzyme activity assays are available for diagnosing most of the MPS disorders, although they are not helpful to definitively identify a carrier. Characterization of the mutation(s) within a family can be helpful. Naturally occurring disease models exist in dogs, cats, rats, mice, and goats; mouse models have been created by targeted gene disruption [26]. Phosphotransferase, deficient in ML, is a low-abundance, membrane-bound enzyme complex of three subunits that are the products of two genes. Biochemical studies suggest that the enzyme contains two domains: catalytic and recognition sites for lysosomal enzymes. In general, ML II and ML III must be distinguished on clinical criteria and on disease progression. Prenatal diagnosis is reliable, and carrier detection is also possible [27]. Clinical description Shared clinical features among the MPS disorders include progressive courses, multiorgan system involvement, organomegaly, dysostosis multiplex, and facial dysmorphia. Hearing, vision, respiratory, and cardiovascular function are often affected. Severe cognitive deficits with developmental delay are observed in Hurler (MPS IH), the severe phenotype of Hunter (MPS IIA), and Sanfilippo syndromes (MPS III) [26]. ML II is characterized by severe psychomotor retardation and shares many clinical and radiologic features with Hurler syndrome. However, clinical differentiation is possible due to earlier onset of signs and symptoms, absence of GAGs in urine, and the more rapidly progressive course, leading to death between 5 and 8 years of age. Neonates with I-cell disease usually show characteristic facial features, craniofacial abnormalities, and restricted joint movement despite generalized hypotonia. Other presenting features may include striking gingival

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hyperplasia, congenital hip dislocation, fractures, hernias, and bilateral talipes equinovarus. The clinical course includes progressive failure to thrive and developmental delay, usually evident by age 6 months; linear growth decelerates during the first year and stops during the second. Joint immobility progresses, with claw hand deformity and kyphoscoliosis ensuing. Hepatomegaly is prominent while splenomegaly is minimal. Respiratory infections are frequent, as is otitis media. Cardiomegaly and murmurs are common, and aortic insufficiency is not infrequent [27]. Nontransplant approaches A wide variety of nontransplant approaches are available for the MPS disorders, including ERT, substrate depletion, and gene transfer. Their applicability, if appropriate, is addressed in Table 77.1. Hematopoietic cell transplantation To date, HCT represents an effective therapy with the longest clinical experience for selected MPS disorders. MPS and ML disorders that can benefit from HCT include Hurler (MPS IH), Maroteaux–Lamy (MPS VI), and Sly (MPS VII) syndromes, as well as I-cell disease (ML II); unfortunately, either limited or lack of effectiveness of HCT in benefiting the CNS and/or the skeletal system has significantly dampened enthusiasm for transplant in patients with Hunter (MPS II), Sanfilippo (MPS III), and Morquio (MPS IV) syndromes (Table 77.1). MPS disorders for which HCT can be beneficial: MPS I, MPS VI, and MPS VII Hurler (MPS IH), Hurler/Scheie (MPS IH/S), and Scheie syndromes (MPS IS). During the last 27 years, since the first allogeneic HCT for MPS IH [12], hundreds of MPS IH patients have been transplanted. Large HCT experiences [28] with both unrelated [17] and related donors [18] have provided guidance for patient selection and timing of HCT, and a realistic assessment of the effects of HCT on various organs and tissues in MPS IH, as well as neurocognitive function (Fig. 77.2). While it is difficult to provide a precise estimate of long-term engrafted survival for Hurler HCT patients, the likelihood is as high as 80–85% using bone marrow or UCB (Fig. 77.3) [22,28]. When performed early in the disease course in conjunction with intensive speech therapy, HCT can preserve intellectual function and prevent manifestations of the classic severe Hurler phenotype [17,18,22,28]. The mechanism by which progressive mental retardation occurs remains unclear; however, increasing atrophy and ventricular size appear to be associated with decreased cognitive ability. Generally, with successful HCT, children with MPS IH can achieve normal neurodevelopment [22] and, in the experience of most HCT centers, do not require ventriculoperitoneal shunting [29,30]. While age and cardiopulmonary and neurodevelopmental status are important determinants of outcome in Hurler patients following HCT, other contributing factors include quality and quantity of developmental services and therapies (e.g. speech and language therapy) as well as the number and severity of specific post-HCT complications such as graft-versushost disease, infections, and organ toxicities [17,18,22,28–36]. On occasions when primary or secondary graft failure has occurred, successful long-term engrafted survival with a favorable quality of life has been achieved following a second HCT [37]. Considerable attention has been paid to issues related to quality of life, including musculoskeletal, motor, and adaptive function in Hurler patients after HCT [38–40], as well as to optimizing patient safety during and after HCT. ERT using laronidase prior to, during, and after HCT has been administered to reduce the total body burden of GAGs, and theoretically to reduce the likelihood of an

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Fig. 77.2 Neurocognitive function as measured by intelligence or developmental quotients (IQ or DQ) in 15 patients with Hurler syndrome before and after haematopoietic cell transplantation. BMT, bone marrow transplantation. (Reproduced from [28], with permission).

Fig. 77.3 Cumulative incidence of neutrophil (a) and platelet (b) engraftment after cord blood transplant, and Kaplan–Meier estimates of the probability of event-free survival (c). In (a), myeloid engraftment was defined as an absolute neutrophil count of at least 500/mm3 on three consecutive days. The one patient who remained aplastic after the first cord blood transplant received a second cord blood transplant on day +63. Myeloid engraftment occurred on day +26 after the second transplant. In (b), platelet engraftment was defined by a platelet count of at least 50,000/mm3 without need for transfusion for at least seven consecutive days. In (c), event-free survival was defined by survival with full (>99%) donor chimerism. Tick marks indicate the most recent follow-up visit for each patient. (Reproduced from [22], with permission.)

MPS-related peritransplant complication [24,25]. Busulfan pharmacokinetics are not altered in children with metabolic storage diseases, including MPS IH, and the rates of donor-derived engraftment are no different in these patients compared with children with other diseases [41]. Discussions continue regarding optimal hematopoietic cell source, preparative regimen, and enzyme activity level following HCT [42,43]. With successful donor-derived engraftment, Hurler patients can experience favorable long-term HCT outcomes. These outcomes include stabilization of the CNS with preservation of neurocognitive function, and motor development within the range of normal and capacity for

independence in activities of daily living. Careful neuropsychologic and neuroradiologic assessments are needed [38–40,44–46]. Hepatosplenomegaly, impaired joint mobility, and upper airway obstruction with sleep apnea [47] resolve within months of HCT; GAG disappears from hepatocytes and Kupffer cells [48]. Corneal clouding stabilizes or slowly resolves in many patients [28], and ocular pressures may normalize; however, for many patients, electroretinogram abnormalities are evident long term [49]. HCT does not reverse the progressive, profound conductive and sensorineural hearing abnormalities observed in many Hurler patients, although nearly 50% of children do

Hematopoietic Cell Transplantation for Storage Diseases

show normalization or improved auditory acuity after HCT [28]. Myocardial muscle function is stabilized or improved, and coronary artery patency has been demonstrated up to 14 years after HCT [50,51]. The long-term outcome of cardiac valvular thickening and insufficiency require continued monitoring [50]. However, some disease features show much poorer responses due to poor penetration of laronidase into the relevant tissue. Principal among these are skeletal abnormalities also known as dysostosis multiplex. Successfully transplanted children often require major orthopedic surgical procedures for genu valgum [52], acetabular hip dysplasia [53], kyphoscoliosis, carpal tunnel syndrome, and trigger digits [54,55] by 6 years of age. Orthopedic aspects of MPS disorders were the subject of a consensus

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conference held in Manchester, United Kingdom, in 2003 (Table 77.6). Interestingly, upper cervical spine stabilization has occurred long term in many donor-engrafted Hurler patients [56]. Nevertheless, severe spinal cord injury has been observed 6 years from successful HCT [57]. The decision of whether to proceed with HCT for patients with Hurler syndrome is a complex one. Factors include the child’s neurocognitive and developmental status with emphasis on the potential for growth, the presence or absence of hydrocephalus with or without a ventriculoperitoneal shunt, and a history of pulmonary and airway complications such as frequent pneumonias, chronic hypoxia, sleep apnea, and cardiac failure. It is imperative that a comprehensive pre-HCT evaluation be performed and that detailed information be provided to families with

Table 77.6 Consensus statement prepared by facilitator Ms Jean Mossman on the orthopaedic management of mucopolysaccharidosis (MPS) patients who have had a hematopoietic cell transplant (HCT). Consensus position statements arising from conference held October 3–4, 2003 in Manchester, UK A. General session Speakers and contributors to this session and their affiliations: Peters, C. currently: The Children’s Mercy Hospital, Kansas City, USA, previously: University of Minnesota, Minneapolis, USA; Wynn, R. Royal Manchester Children’s Hospital, Manchester, UK; Steward, CG. Bone Marrow Transplant Unit, Royal Hospital for Children, Bristol, UK; Wraith, E. Willink Biochemical Genetics Unity, Royal Manchester Children’s Hospital, Manchester, UK; Guffon, N. Centre de Référence des Maladies Héréditaires du Métabolisme, Hôpital Edouard Herriot, Lyon, France. • As a result of HCT and other emerging treatments for MPS diseases, the management of skeletal abnormalities has become increasingly important • HCT has favorably altered the natural history of some MPS diseases and can partially correct the skeletal abnormalities • Multiple lines of accumulating evidence in patients and animals provide compelling evidence that early HCT has a positive impact on skeletal disease, in particular carpal tunnel syndrome and trigger finger • HCT improves the soft tissue abnormalities associated with MPS diseases • HCT is expected to be more effective as prophylaxis than treatment • Skeletal disease varies between types of MPS disease • The etiology of skeletal disease is uncertain: it is not clear how glycosaminoglycan accumulation leads to the skeletal dysplasia • At present, it is not possible accurately to predict which children will develop skeletal abnormalities of sufficient severity to require intervention, and ongoing review of the likely orthopedic problems is essential • The outcome of orthopedic surgery for skeletal and soft tissue problems (genu valgum, hip dislocation, trigger finger, and carpal tunnel syndrome) in the group of patients for whom these symptoms are a problem is encouraging • Most parents of children who have had surgery have been pleased with the results • Assessing the long-term outcome of surgical intervention in children who have received bone marrow transplants is important and requires follow-up into adulthood • An international registry of both treated and untreated patients, including procedures and outcomes, is needed as a matter of urgency • Data fields collected by registries should have input from patients and families to ensure that the data collected are relevant to the decisions future parents will make regarding treatment for their children B. Knee surgery session Speakers and contributors to this session and their affiliations: Ogilvie, JW, currently: Shriners Hospital for Children, Salt Lake City, USA, previously: University of Minnesota, Minneapolis, USA; Meadows, T. Royal Manchester Children’s Hospital, Manchester, UK. • >50% of engrafted patients develop knock knees • Good correction can be achieved with both stapling and osteotomy • Changes with growth can result in failure of both procedures, and more than one procedure may be necessary • Stapling can fail as a result of permanent growth failure of the growth zone on the other side of the knee • Assessment for knee surgery: in children where there is clinical suspicion of genu valgum, a posteroanterior standing X-ray of the entire lower limbs is indicated Stapling • Epiphyseal stapling should be considered when the tibiofemoral angle >~15° and • Epiphysis is ossified enough to hold staple • Age >4–5 years may be optimal and • There is an expectation of a further 1–2 years of growth remaining • The staple should be removed when angle is reduced to 0–5°. Two staples, of a design that provides adequate anchorage but which can be removed, should be used Osteotomy Osteotomy should be considered when: • Degree of deformity >~20° • Stapling has failed to correct the problem • The deformity is not at the knee

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Table 77.6 Continued • The patient is too old and therefore there is not enough growing time left • Acute correction versus graduated correction: Patient preference, in the light of a clear explanation and open discussion of the risks and benefits of both approaches, should contribute to the decision about which approach to use • Patient groups have an important role to play in producing information setting out the risks and benefits Osteotomy

Stapling

More morbidity Several days in hospital Days or weeks to become ambulatory, depending on procedure

Less morbidity Outpatient or short stay if not done alongside other procedures Ambulatory within days

C. Hip surgery session Speakers and contributors to this session and their affiliations: Meadows, T. Royal Manchester Children’s Hospital, Manchester, UK; Ogilvie, JW, currently: Shriners Hospital for Children, Salt Lake City, USA, previously: University of Minnesota, Minneapolis, USA; Garin, C. Hôpital Edouard Herriot, Lyon, France. • The same basic principles for indication for surgery apply as in non-MPS hip dysplasia: • Shallow acetabulum • Shenton’s line broken • Patients for whom longevity is assumed • If the degree of dysplasia is likely to lead to • early or premature arthritis • adverse effect on quality of life is likely The aim of surgery is to: • Reduce the hip • Provide cover • Early rehabilitation Hip surgery should be undertaken before the ability to remodel the acetabulum is lost • Assessment – Anteroposterior X-ray of the pelvis – Consider magnetic resonance imaging Surgery may be to the pelvis, femur or both The operation technique will depend on: • Age • Severity of problem • Surgical preference Both hips may be operated on at the same time, depending on the technique used, the general wellbeing of the patient and the preference of the patient/ family and the surgeon One technique which has been used successfully: 1) Single stage a) bilateral femoral osteotomy b) bilateral iliac osteotomy Femoral varus with or without rotation osteotomy Degas-type iliac osteotomy 2) 6 weeks in a cast a) progressive ambulation b) gentle physical therapy D. Carpal tunnel syndrome session Speakers and contributors to this session and their affiliations: Van Heest, A and Khanna, G. University of Minnesota, Minneapolis, USA. • Screening testing for all children with MPS using nerve conduction velocity is essential. Children do not complain of pain, numbness or weakness until the carpal tunnel syndrome is severe and neurologic damage may be permanent. Nerve conduction velocity testing can diagnose carpal tunnel in its milder form, when median nerve compression is reversible • Nerve conduction velocity with decreased velocity or decreased amplitude or increased latency in median nerve function compared with ulnar nerve function over the same carpal distance is diagnostic of carpal tunnel syndrome • Untreated carpal tunnel syndrome leads to loss of thenar muscle function and loss of sensation in the thumb, index, long, and part of the ring finger • Carpal tunnel syndrome in MPS diseases is due to dysplasia of the carpal bones, leading to a narrower bony carpal tunnel, combined with glycosaminoglycan deposits on the flexor tendons within the carpal tunnel, resulting in increased pressure on the median nerve

Hematopoietic Cell Transplantation for Storage Diseases

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Table 77.6 Continued • Studies have demonstrated that carpal tunnel release surgery improves median nerve function • With improved blood and marrow transplant techniques, treating patient with MPS IH, approximately 50% of children acquire carpal tunnel syndrome. Decreased risk of developing carpal tunnel syndrome exists for patients transplanted at a younger age and in patients with normal or carrier levels of enzyme post transplant E. Trigger finger session Speakers and contributors to this session and their affiliations: Van Heest, A and Khanna, G. University of Minnesota, Minneapolis, USA. • Untreated locked trigger finger can lead to permanent hand joint contractures • Surgical release of the A1 and A3 pulleys with possible resection of half the flexor digitorum superficialis tendon may lead to prevention of contractures if locked digits exist. Stretching exercises for the fingers and night-time splinting do not resolve the underlying cause • Screening for trigger finger includes palpation of the flexor tendons for nodules as well as documentation of active and passive range of motion of each finger joint F. Spinal surgery for kyphosis session Speakers and contributors to this session and their affiliations: Garin, C. Hôpital Edouard Herriot, Lyon, France; Ogilvie, JW, currently: Shriners Hospital for Children, Salt Lake City, USA, previously: University of Minnesota, Minneapolis, USA; Williamson, B. Royal Manchester Children’s Hospital, Manchester, UK. • Purpose of spinal surgery is: • to keep the spine balanced • to protect neural elements • to maximize mobility and function by minimizing the deformity • to retain quality of life • Spinal surgery should only be undertaken after a surgeon with expertise in operating on children with MPS diseases has clinically reviewed the case • Indications for surgery: • these may be different from the indications that would apply to non-MPS children • consistent deterioration on serial X-rays • clinical need as judged by expert opinion • some surgeons operate only when the degree of curvature is in the 60–70° region • There is a role for patient organizations in educating patients and their families on the need to monitor change in the spine and to be alert to neurologic changes that might arise from spinal changes: • Weakness • Pain • Decreasing activity tolerance • Deteriorating bowel or bladder function • Assessment: standing 2 m posteroanterior and lateral X-ray • Surgical approach • Anterior column support • Anterior posterior surgery • The role of braces • There is no evidence that bracing alters the natural history of spinal changes prior to surgery • On this basis, if families can tolerate no bracing, it is an acceptable approach to take • Post-surgical management • A cast should be applied in the operating room at the end of surgery and should remain in place for between 6 weeks and 3 months • Subsequently, the patient should wear a brace until the bones have healed/united/fused • Physiotherapy should only be undertaken with a treatment plan agreed in advance with the surgeon • Normal activity can be resumed at 12 months, although this does not include contact sports and other potentially dangerous activities such as tumbling G. Spinal surgery for scoliosis session Speakers and contributors to this session and their affiliations: Garin, C. Hôpital Edouard Herriot, Lyon, France; Ogilvie, JW, currently: Shriners Hospital for Children, Salt Lake City, USA, previously: University of Minnesota, Minneapolis, USA; Williamson, B. Royal Manchester Children’s Hospital, Manchester, UK. • Purpose of spinal surgery is: • to keep the spine balanced • to protect neural elements • to maximize mobility • to retain quality of life • Indication for surgery: apply same principles as for non-MPS children • Spinal surgery for scoliosis should only be undertaken after a surgeon with expertise in operating on children with MPS diseases has clinically reviewed the case

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particular attention to disease aspects that are typically resistant to the beneficial effects of HCT, such as skeletal abnormalities, some ocular and auditory features, and cardiac aspects. Long-term, coordinated, comprehensive monitoring not only for late effects from HCT (see Chapter 105), but also for disease-specific aspects, should be performed in close collaboration with educators and therapists (e.g. physical, occupational, speech, and language) (Table 77.4). Hurler/Scheie syndrome demonstrates intermediate phenotype between MPS IH and MPS IS, with normal to mildly delayed neurocognitive development, and survival beyond the first decade and often into the third decade [26]. A 10-year-old child with MPS IH/S underwent HCT and demonstrated resolution of hepatosplenomegaly, amelioration of facial features, stabilization of cardiac function, improved joint range of motion, with persistent and progressive skeletal abnormalities, and unchanged corneal haze though visual acuity improved. Neurocognitive function was preserved at a level that was below average [58]. While HCT can successfully treat MPS IH/S or MPS IS as well, such patients are treated typically with α-L-iduronidase ERT [59]. Maroteaux–Lamy syndrome (MPS VI). Maroteaux–Lamy syndrome (MPS VI) is another MPS disorder that can be treated by HCT. The principal clinical features of children with MPS VI are dysostosis multiplex with severe short stature, corneal haze, pulmonary complications related to decreased intrathoracic volume and limited expansion due to severe hepatosplenomegaly, and cardiac valvular abnormalities [26]. HCT has been used successfully to treat MPS VI for nearly 25 years [13,60], with resolution of hepatosplenomegaly, airway obstruction, and sleep apnea, prevention of further cardiopulmonary deterioration, and improved joint mobility. Visual acuity improved in some cases [13,60], although corneal haze does not necessarily resolve. As in other MPS disorders, HCT has not treated effectively skeletal abnormalities (Table 77.6).

shares clinical features with MPS IH, including facial dysmorphia, progressive conductive and sensorineural hearing loss, upper airway obstruction with sleep apnea, cardiopulmonary dysfunction, hepatosplenomegaly, joint stiffness, and short stature [26]. Dysostosis multiplex is less severe and corneal haze is absent in Hunter syndrome. The attenuated form of MPS II is associated with less neurocognitive impairment and, in some cases, normal intelligence; survival can extend into the fifth and sixth decades, in contrast to MPS IIA (severe Hunter syndrome) and its shortened life expectancy of one to two decades [26]. While HCT seems to help patients with MPS IIA and MPS IIB (attenuated Hunter syndrome) with respect to organomegaly, airway obstruction, and cardiac function, boys with MPS IIA still have demonstrated profound neurocognitive disabilities despite early, successful HCT [44,62,64–68]. The failure of HCT to favorably alter the long-term neurocognitive outcome in MPS IIA suggests that iduronate-2-sulfatase is not being effectively delivered to essential components of the CNS. In cases of transplanted MPS IIB patients, there have been somatic benefits, and intellectual function has remained intact as expected based upon the natural history of the disorder. Ethical issues continue to arise in the face of limited somatic benefit and minimal-to-absent neurocognitive gains in patients with MPS IIB undergoing HCT [69]. Sanfilippo syndrome (MPS III). Neurobehavioral manifestations are the hallmarks of all four types of MPS III [26]. Characteristic findings include extreme hyperactivity, aggressive behaviors, attention deficits, and progressive neurocognitive delay often associated with expressive language disabilities. As in MPS II, HCT is able to effectively treat the various somatic aspects of MPS III; however, the uniformly poor neurocognitive and developmental outcomes despite fully donor-engrafted marrow transplants performed early in the disease course (Fig. 77.4)

Sly syndrome (MPS VII). The use of HCT for MPS VII is limited by the rarity of the disorder and its predilection toward hydrops fetalis, although attenuated forms do exist [26]. The neonatal form is one of the few lysosomal storage diseases with clinical manifestations in utero or at birth [26]. MPS VII, in certain circumstances, can be ameliorated by HCT provided the neurodevelopmental and clinical status of the patient is satisfactory at the time of HCT [61]. In this case report, the 12-yearold MPS VII patient experienced major improvements in motor and pulmonary function with diminished upper respiratory tract and middle ear infections, leading to an improved quality of life.

Historical experience with HCT for ML-II (I-cell disease) existed initially only in patients with endstage cardiopulmonary disease [62]. Over the past decade, transplant experience with a small cohort of patients ranging in age from 0.3 to 1.7 years has demonstrated that long-term engrafted survival can be achieved in selected patients who continue to make developmental progress, albeit at slower than normal rates [63]. We have observed that obstructive sleep apnea may require tracheostomy prior to HCT as well as continuous positive airway pressure or bilevel positive airway pressure support. Further, careful long-term monitoring is required as airway complications and pulmonary hypertension can occur. MPS disorders for which HCT is not beneficial: MPS II, MPS III, MPS IV Hunter syndrome (MPS II). There are two distinct clinical phenotypes of MPS II. The more common severe form presents before the age of 3 years with profound neurocognitive and developmental delay, and

Bone marrow transplant

90

80

70

60 DQ or IQ

ML types II and III (I-cell disease and pseudo-Hurler polydystrophy)

100

8

50 7

40

2

4

6

30

20

10 5

1

3

0 0

24

48

72

96

120

144

Age in months

Fig. 77.4 Decline in intellectual function (developmental quotient or intelligence quotient) in eight children with mucopolysaccharidosis III (Sanfilippo syndrome). (Reproduced from [70], with permission.)

168

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The de novo mutation rate in X-ALD is 7.8% of cases, and in 42% of index cases the first recognized mutation occurs in the mother [75]. The ALD gene (located at Xq28) encodes the ALD protein (ALDP, ABCD1), which is a member of the ATP-binding cassette transport protein family (ABC transporters) and is likely involved in transport of fatty acyl coenzyme A substrates or their cofactors into peroxisomes [76]. Impaired ALDP function leads to the accumulation of VLCFAs in body fluids and tissues. However, the contribution to cerebral and adrenal dysfunction is poorly understood, although recent reports reflect significant efforts directed at achieving a better understanding of the underlying pathophysiology [77,78].

resonance imaging [MRI] positive and gadolinium-positive) and the spectrum of clinical manifestations are [80–82]: 1 posterior in approximately 80–85% of cases: graphomotor, spatial perception and visual memory difficulties, and visual agnosia ending in cortical blindness; 2 frontal in 10–15%: “acquired” attention deficit hyperactivity disorderlike presentation, behavioral disinhibition, verbal fluency problems, and memory and new learning difficulties; 3 pyramidal tracts in approximately 5%: corticospinal signs in the extremities. Neurologic deterioration occurs in all boys, with deficits that can include vision/visual processing, hearing/auditory processing, speech, gait, fine motor skills, and activities of daily living. Disability, dementia, and death can occur within months to several years after clinical onset. Adult-onset AMN, typically first manifesting itself in the second or third decades, is characterized by an axonopathy with spinal cord atrophy, initially involving the lower extremities, with progressive long tract signs when progressive to the upper extremities. There is stiffness and clumsiness of the legs which is slowly progressive; mild-to-moderate spastic paraparesis, impaired vibratory sense and graphesthesia of the distal legs; peripheral neuropathy (mild); bowel and bladder control problems; impotence; sparse hair; adrenal insufficiency; and hypogonadism manifested by low testosterone and high luteinizing hormone levels. The MRI of the brain shows characteristic ascending involvement of the pyramidal tracts from the pons to the internal capsules. Of note, men can develop cerebral demyelination that is similar to that seen in childhood cases. A complete evaluation of a boy with cerebral X-ALD includes a thorough neurologic examination; comprehensive neuropsychologic assessment with particular attention to the performance IQ (PIQ), which is a sensitive indicator of deficits in visual perceptual and spatial processing, speed and efficiency of task completion and novel problem solving; and an MRI of the brain. Boys diagnosed with X-ALD due to a family history merit careful, complete serial monitoring beginning at age 3 years and continuing into adolescence, with the objective being to detect cerebral demyelination at a very early stage and to intervene with HCT in a timely and presumably effective manner. To date, MRI scanning of the brain with gadolinium is the most effective clinical tool to perform serial monitoring. It is strongly recommended that scans be performed at least every 6 months, and more often if there is evidence of the earliest stages of dysmyelination. This is particularly relevant for boys under 10 years of age with X-ALD who have the highest risk for developing childhood-onset cerebral demyelination that is rapidly progressive. However, in no circumstances should HCT be performed in the absence of cerebral disease (see below).

Clinical description

Nontransplant approaches

There is no genotype–phenotype correlation in X-ALD; that is, six distinct clinical phenotypes exist independent of VLCFA abnormality or ALDP expression, mutation or family history. Genotypic analysis definitively identifies the 20% of female heterozygous carriers who can have normal levels of fasting plasma VLCFAs. The clinical phenotypes and their relative percentages are as follows [79]: • childhood cerebral (30–40%); • adolescent cerebral (5–10%); • adult cerebral (~5%); • adrenomyeloneuropathy (AMN; 40–50%); • AMN with cerebral involvement (25% of the total AMN cases); • Addison only (variable percentage, dependent on age). Childhood-onset cerebral disease, with its median age of onset of 7 years, encompasses adrenal insufficiency in 90% of affected boys. Locations for the inflammatory demyelinating process (i.e. magnetic

The primary nontransplantation approaches to X-ALD have been Lorenzo’s oil with a special low-fat diet, immunosuppression, and more recently antioxidant agents. There is evidence that, in patients with the biochemical diagnosis of X-ALD who are started on Lorenzo’s oil and the special diet, are compliant, and start at an early age, there is a decreased likelihood of developing childhood-onset cerebral demyelination [83]. However, not all boys are protected, and for those who do develop cerebral manifestations by MRI and/or clinical features, HCT remains the only potentially effective long-term treatment.

[70,71] have been attributed to reduced efficiency of uptake of MPS III-specific hydrolases, leading to diminished clearance of substrate. Morquio syndrome (MPS IV). The two distinct biochemical forms of MPS IV have similar clinical features, including severe dysostosis multiplex, dwarfism, atlantoaxial instability, short trunk, and hyperextensible joints with ligamentous laxity. Corneal haze is mild, hepatosplenomegaly moderate, cardiac abnormalities unusual, and intelligence preserved [26]. HCT is incapable of ameliorating the severe skeletal deformities. Therefore, this shortcoming of HCT and the MPS IV clinical features make HCT a suboptimal intervention for this disease [72].

Leukodystrophies and other white matter diseases In this section, leukodystrophies (X-ALD, GLD, and MLD) will be presented and discussed in detail (see below and Table 77.2); other white matter diseases, such as Alexander disease, Pelizaeus–Merzbacher disease (PMD), vanishing white matter disease (VWMD), and Zellweger syndrome will be presented and discussed in a limited manner (Table 77.2). X-linked adrenoleukodystrophy Epidemiology and etiology Adrenoleukodystrophy is an X-linked peroxisomal disorder of VLCFA metabolism which has a minimum frequency of 1 : 16,800 in the total population and approximately 1 : 20,000 males (Table 77.2) [73,74]. It predominantly affects males, although 40% or more of female heterozygous carriers exhibit mild-to-moderate noncerebral signs of the disease. Molecular and clinical biology

Hematopoietic cell transplantation The first transplant for childhood cerebral X-ALD was performed in 1982 in a boy with advanced-stage disease; despite a successful graft, he died of progressive X-ALD [84]. However, Aubourg and colleagues in Paris successfully transplanted a boy with early stage cerebral X-ALD

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and published their findings in 1990 [14], thus beginning the modern era of successful transplantation for this disorder. Lessons learned and considerations to be given in each case of cerebral X-ALD include: (1) the timing of HCT and disease status; (2) the optimal hematopoietic cell source (marrow, peripheral or UCB, HLA match, cell dose, and carrier status of the donor, if applicable); (3) the conditioning regimen: high dose versus reduced intensity; (4) multispecialty, multidisciplinary evaluation and treatment; and (5) quality of life for the patient and his family. The long-term results (5–10 years) of transplant in 12 boys were reported in 2000 by Shapiro and colleagues [85]. They described the prospects for achieving stability in the extent of MRI demyelination and the changes in neuropsychologic function, with a propensity for deterioration before ultimate stabilization in visual processing as evidenced by PIQ score in boys with a posterior pattern. Interestingly, VLCFA concentrations decreased by 55% but remained elevated in the long term, suggesting that VLCFA levels are not an optimal surrogate marker for this disease. The first extensive report of the international HCT experience for childhood and adolescent cerebral X-ALD was published in 2004 by Peters and colleagues from 43 HCT centers and described 126 subjects, of whom 94 were deemed to have complete data [20]. In addition to providing overall, related, and unrelated donor transplant Kaplan–Meier probabilities of survival, this report described for the first time the extraordinary successes possible in boys transplanted for earlystage cerebral disease (zero or one neurologic deficit and MRI severity score <9, with 92% survival at 5 years; Fig. 77.5). Insight into the probability of neurologic deterioration following HCT was also provided (Table 77.7). Specifically, in boys with no neurologic deficits before HCT, there was still a 44% likelihood that the neurologic deficit score would increase afterwards. The concept of an ALD-disability rating scale (ALD-DRS) was introduced, although not yet validated (Table 77.8). This scale proved informative in examining neurologic deficits, neuropsychologic function, MRI severity, and ALD-DRS levels after HCT in patients with cerebral X-ALD according to baseline PIQ (Table 77.9). Both PIQ and ALD-DRS levels were significantly poorer after HCT in boys whose baseline PIQ was below 80. The ALD-DRS levels before and after HCT were reported and gave insight into the potential for significant worsening of the level of disability depending on the status of the disease at time of HCT (Table 77.10). Finally, the neuropsychologic functional outcomes of boys after HCT was assessed and reported based upon their MRI pattern of demyelination (Table 77.11). Baseline PIQ scores after HCT and mean changes in PIQ scores were significantly lower and greater, respectively, in boys with a parietal–occipital lobe (i.e. posterior) pattern of demyelination.

There has been considerable discussion over the last decade or longer regarding the putative mechanism by which HCT halts the progress of CNS demyelination in cerebral X-ALD. It has been speculated that immunosuppression contained within the conditioning regimen, together with long-term donor-derived engraftment, would halt the inflammatory process and contribute donor-derived microglia, respectively. However, recently, further insight into this process has been provided by the collaborative group from the programs in Dusseldorf and Göttingen led by Schönberger and Gärtner [86,87]. In their study of post mortem tissues in a boy transplanted for advanced-stage cerebral disease, they noted stable development of wild-type X-ALD genotype and peroxisomal ALDP expression in a great variety of extraneural and CNS human tissues by immunohistochemical staining. Interestingly and specifically, there was regular peroxisomal punctate staining for ALDP in marrow and cytoplasmic staining in gray matter neurons. Further studies are needed. Other important HCT experience for childhood and adolescent cerebral X-ALD includes an informal study from selected centers in Brazil,

Fig. 77.5 Kaplan–Meier estimate of survival for cerebral X-linked adrenoleukodystrophy following hematopoietic cell transplantation by number of neurologic deficits (e.g. limitations of vision, hearing, speech, gait, fine motor skills, and/or activities of daily living) and magnetic resonance imaging (MRI) severity score before transplantation. The solid line indicates patients with zero or one neurologic deficit and an MRI severity score less than 9 (n = 25). The dashed line indicates patients with two or more neurologic deficits or an MRI severity score of 9 or greater (n = 37). Ticks on the probability lines indicate dates of censoring at last followup. y, years. (Reproduced from [20], with permission.)

Table 77.7 Neurologic deficit (e.g. impairments of vision, hearing, speech, gait, fine motor skills, and/or activities of daily living) score before and after hematopoietic cell transplantation (HCT) in 94 patients with cerebral X-adrenoleukodystrophy After HCT number (%) Before HCT

Number

0

1

2

More than 2

Unknown or not available

0 1 2 More than 2 Unknown or not available Totals

32 28 21 9 4 94

18 (56) 3 (11) – – – 21

4 (13) 10 (36) 1 (5) – – 15

1 (3) 4 (14) 6 (29) – – 11

9 (28) 9 (32) 14 (67) 9 (100) – 41

– 2 (7) – – 4 (100) 6

–, none. Bold text indicates patients for whom neurologic deficit scores were unchanged.

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Table 77.8 X-linked adrenoleukodystrophy (ALD) Disability Rating Scale (ALD-DRS) Scale level

Description

0 I II III IV

No difficulties Mild learning or coordination difficulties from ALD; patient does not require support or intervention Moderate learning, sensory, and/or neurologic abnormality; patient requires support or intervention in a few areas Severe learning, sensory, and/or neurologic abnormality; patient requires support or intervention in many areas Loss of cognitive ability and disorientation; patient requires constant supervision

Neurologic deficits Median, number (range) Observations, number PIQ Median, number (range) Observations, number MRI Severity Score Median, number (range) Observations, number ALD-DRS level Median, number (range) Observations, number

Baseline PIQ below 80

Baseline PIQ of 80 or more

2 (0–4) 32

1 (0–4) 43

45 (45–63) 6

78.5 (45–122.5) 26

12.8 (8–19) 8

8 (0–23.5) 23

IV (I–IV) 28

II (0–IV) 36

p 0.29

<0.01

0.25

Table 77.9 Neurologic deficits (e.g. impairments of vision, hearing, speech, gait, fine motor skills, and/or activities of daily living), neuropsychologic function, magnetic resonance imaging (MRI) severity, and adrenoleukodystrophy (ALD) Disability Rating Scale (ALD-DRS) levels after hematopoietic cell transplantation in patients with cerebral X-linked ALD according to baseline performance intelligence quotient (PIQ).

<0.01

Table 77.10 X-adrenoleukodystrophy Disability Rating Scale (ALD-DRS) levels before and after hematopoietic cell transplantation (HCT) in 94 patients with cerebral X-ALD Level after HCT, number (%) Level before HCT

Number

0

I

II

III

IV

No data, because of death

0 I II III IV Missing data Totals Deaths

13 21 36 12 1 11 94 35

8 (62)* 1 (4.8) 1 (2.8) – – – 10 1

3 (23) 3 (28)† 1 (2.8) – – – 10 2

1 (7.6) 4 (19)† 10 (2.8)‡ – – 1 (9) 16 5

– 3 (14) 1 (2.8) 1 (8.3) – – 5 0

1 (7.6) 6 (28)† 19 (53)¶ 10 (83)§ 1 (100)* – 37 16

– 1 (4.8) 4 (11) 1 (8.3) – 5 (45) 11 11

*1 death; †2 deaths; ‡3 deaths; §4 deaths; ¶9 deaths.

Table 77.11 Comparison of neuropsychologic functional outcomes according to magnetic resonance imaging pattern of demyelination following hematopoietic cell transplantation (HCT) for cerebral X-linked adrenoleukodystrophy

Parietal-occipital lobes, n = 25

Frontal lobes or pyramidal tracts, n=7

Verbal intelligence quotient* Baseline (range) After HCT Mean change (95% confidence interval)

99 (59–27) 82 (46–123) -13.7 (−20.5 to −6.8)

Performance intelligence quotient* Baseline (range) After HCT Mean change (95% confidence interval)

96 (45–122.5) 64 (45–122.5) -21.6 (−30.6 to −12.6)

NS, not significant. * VIQ and PIQ baseline evaluations and those after HCT: median (range). Bold denotes statistical comparisons and corresponding p values.

Total, n = 32

No neuropsychologic function assessment after HCT because of death, n = 29

p

89 (70–107) 79 (57–113) -6.4 (−17.8 to 4.9)

98 (59–127) 80.5 (46–123) −12.1 (−17.1 to −7.1)

92 (74–124) – –

NS – 0.25

92 (61–100) 93 (54.108) 0.4 (−6.8 to 7.7)

94.5 (45–122.5) 73 (45–122.5) -16.8 (−24.6 to −8.9)

77.5 (45–138) – –

<0.01 – 0.03

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Germany, and the United States in which 20 boys with very early-stage disease have received HCT, with 100% engrafted survival achieved. A single-center report of its experience with UCB HCT for 12 patients with X-ALD, including six with early and very early stage cerebral disease (MRI severity score 3–8, although one patient’s MRI severity score was not reported) revealed significant morbidity and mortality before (one death from adrenal crisis) and after (one death from sinusoidal obstructive syndrome, and one graft failure) HCT [88]. In the Cord Blood Transplantation (COBLT) study, the median age of the UCB recipient was 1.8 years, and it is unclear if X-ALD patients had evidence of cerebral disease at the time of HCT [89]. In conclusion, given the phenotypic variation in X-ALD, the National Marrow Donor Program, International Bone Marrow Transplant Registry, and the Working Party on Inborn Errors of the European Group for Blood and Marrow Transplantation recommend that HCT be reserved for X-ALD patients with early but definite evidence of cerebral disease by brain MRI [90]. Based upon the long-term outcomes of HCT for cerebral X-ALD, the worldwide HCT experience for this disease, and knowledge of the natural history, the following guidelines can be promulgated: MRI severity score and gadolinium enhancement are highly predictive of the likelihood of disease progression; for example, an MRI severity score of at least 1–3 points or more, and gadolinium enhancement in boys under 10 years of age is associated with approximately 90% likelihood of severe, progressive demyelination [81,82,85,91]. Boys with advanced disease (i.e. very late stage) manifested by low PIQ (<80), a very sensitive neuropsychologic parameter for visual processing, neurologic deficits, and high MRI severity score, typically over 13 points, are poor candidates for HCT. Boys with very early-stage and early-stage cerebral disease defined by an MRI severity score between 1 and 8 points who typically are without neurologic and neuropsychologic abnormalities are the best candidates for HCT. Other efforts to improve results related to HCT for cerebral X-ALD have focused on the use of N-acetyl-L-cysteine in patients with very late-stage cerebral disease [92], use of advanced imaging techniques such as magnetic resonance spectroscopy to evaluate the brain after HCT [93], and the use of magnetic resonance spectroscopy as well to evaluate for the earliest evidence of changes associated with demyelination [94]. Despite significant progress in the field of HCT for cerebral X-ALD, a number of limitations and unanswered questions must be acknowledged. These include those related to the following: 1 the fact that, in most instances – about two-thirds of cases – HCTs have been performed in symptomatic boys with their cerebral X-ALD rather than in boys with early stages of disease who had undergone serial MRI monitoring after receiving the biochemical diagnosis of ALD due to a positive family history of this X-linked disease; 2 our continuing inability to predict phenotype in a boy or an adult man; 3 the need for a measure of cerebral disease velocity; 4 the implementation of a more precise system for staging of cerebral disease prior to HCT that builds upon the following: very early stage = MRI score 1–3; early stage = MRI score 4–8; late stage = MRI score 9–13; very late stage = MRI score greater than 13; 5 the pressing need for newborn screening for X-ALD [95]. In the meantime, novel approaches to boys and men with very late-stage cerebral disease are also needed. Further, the field of HCT for men with AMN and cerebral disease is beginning to be orchestrated on an international front through centers in Germany, The Netherlands, and the United States. Clearly, the need for such cooperation and collaboration is essential, together with international databases and registries with attention to longitudinal outcomes.

Globoid cell leukodystrophy Epidemiology and etiology GLD is an autosomal recessive, lysosomal disorder arising from a deficiency of galactocerebrosidase (GALC) enzyme activity (Table 77.2) [96]. Molecular and clinical biology There is white matter degeneration in the CNS and peripheral nervous system, with large macrophages (globoid cells). Clinical description Early-onset GLD, also known as classic Krabbe disease, is the predominant form, while late-onset cases are relatively rare. Early-onset disease is characterized by profound psychomotor retardation, failure to thrive, spasticity, optic atrophy, and cortical blindness, and manifests itself early in infancy with fisting and hypertonicity that progresses rapidly to seizures, tonic spasms, and incapacitation, culminating in death often by age 2 years. Late-onset disease, which can present from later in childhood up to adulthood, demonstrates a much more protracted course and is characterized by loss of vision, progressive spasticity of the lower extremities, neurocognitive decline, albeit in some cases very slowly, and localized neuropsychologic deficits, gait disturbances, long tract signs, extremity weakness, which can be asymmetric, and difficulties with coordination and balance [96]. Nontransplant approaches Nontransplant approaches to patients with GLD primarily fit under the broad category of supportive care measures and may be directed toward the treatment of seizures, spasticity, feeding difficulties, and constipation. Hematopoietic cell transplantation The first definitive report on the use of HCT for GLD was published in 1998 and described five cases (four late onset, and one Krabbe disease) [15]. Hematopoietic cell sources included HLA-identical marrow in four cases and UCB in one. In all cases, high-dose conditioning was administered; total body irradiation was given to one patient. It is generally regarded that this report provided proof of principle for the use of HCT for GLD. The four cases of late-onset GLD received their HCT between the ages of 2.6 and 11 years of age, with follow-up from 3 to 9 years. The patient with Krabbe disease underwent HCT at age 2 months, and follow-up of 14 months was reported. The outcomes may be summarized as follows: (1) GALC normalized in all patients; (2) there was normal, stable neuropsychologic function in the three lateonset disease patients; (3) MRI of the brain showed an “improvement in demyelination” based upon reduced abnormal signal intensity; (4) cerebrospinal fluid (CSF) protein normalized in all cases except for the Krabbe infant who still had a CSF protein level of over 150 mg/dL at follow-up, although this was decreased from greater than 550 mg/dL at baseline. A recent report on the use of UCB HCT in patients with classic Krabbe disease included 25 infants, of whom 11 were “asymptomatic” and 14 were symptomatic prior to transplant [23]. Comparison of “outcomes” between the two groups of patients focused on survival, engraftment/GALC, developmental and neurologic status, and MRI of the brain. The authors found the following: (1) engrafted survival among the “asymptomatic” infants was seen in all 11 of 11, while it occurred in six of the 14 patients who were symptomatic at HCT; (2) none of the patients achieved normalization of CSF protein; (3) among the symptomatic infants, all had major developmental and neurologic deficits after HCT; (4) significant residua were also present among those

Hematopoietic Cell Transplantation for Storage Diseases

transplanted in an “asymptomatic” condition. Of these 12 patients, only four could be described as “normal.” Problems included mild-to-moderate expressive language delays and mildly-to-severely impaired gross motor function. A novel development stemming from this preliminary experience with UCB for “presymptomatic or asymptomatic” infants with Krabbe disease was the implementation of a newborn screening program in New York State for GLD, which commenced in August 2006. The early experience reveals that one of the two infants who were identified with GLD by this newborn screening process is surviving after neonatal UCB HCT (Dr Patti Duffner, Buffalo Children’s Hospital, personal communication). Additional investigations have been conducted in patients transplanted for GLD. Peripheral neuropathy is a significant clinical problem in patients with classic Krabbe disease. The effect of UCB HCT was studied in 12 patients with GLD, of whom nine had classic Krabbe disease and three had late-onset disease. Serial nerve conduction studies were performed in patients after HCT, although two did not have preHCT studies. Further studies are needed to more fully assess the clinical significance of the nerve conduction velocity results and how best to express these results; in the case of this report, values were expressed as age-matched standardized ratios [97]. With the development of diffusion-tensor MRI (DTI), there is the promise of more sensitive means to assess myelination patterns. With DTI, the MR signal is sensitized to microscopic movement of water molecules. Water motion in white matter myelination is anisotropic (i.e. has a tendency to diffuse along one direction rather than in all directions), a feature that can be quantitatively and reproducibly measured with anisotropy maps. Another report on patients with GLD described the serial assessment of DTI anisotropy measurements in seven patients with early-onset Krabbe disease who had received UCB HCT. Fractional anisotropy ratios were reported, and all patients exhibited worsening of the white matter signal intensity by conventional MRI [98]. The current limitations of HCT for GLD and remaining unanswered questions relate to the following areas: (1) late-onset GLD cases are very rare, and consequently the experience with HCT is still quite limited; (2) the long-term neurologic, neurocognitive, neurodevelopmental, and quality of life outcomes of patients with classic Krabbe disease who have received UCB HCT in the first 1–2 months of life; (3) the impact of newborn screening for GLD in New York State, and whether there is justification for its expansion to other states; (4) international discussion, review, and planning for the future, which is is urgently needed.

Metachromatic leukodystrophy Epidemiology and etiology MLD is an autosomal recessive lysosomal disorder arising from deficiency of arylsulfatase A (ARSA) enzyme activity and also characterized by increased urinary sulfatides [99]. Molecular and clinical biology There is defective desulfation of sulfated glycolipids present in myelin sheaths of the CNS and peripheral nervous system, and to a lesser extent in visceral organs (e.g. kidney, gallbladder, and liver). Lysosomal accumulation occurs which manifests itself as metachromatic staining. Of absolutely critical importance is confirmation of the diagnosis by documenting elevation of urinary sulfatides. This is necessary due to the common ARSA pseudodeficiency allele in the general population. MLD caused by saposin B deficiency can present as any form of MLD caused by ARSA deficiency. Multiple sulfatase deficiency associates the signs of MLD with MPS and icthyosis. Saposin B deficiency and multiple

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sulfatase deficiency share with MLD increased CSF protein, slowed nerve conduction velocities, and increased urinary sulfatides. Clinical description The phenotypes of MLD include late-infantile MLD and late-onset forms. Late-infantile (early onset, 0.5–4 years of age) presents generally in the first or second year of life due to loss of motor milestones including gait disturbance, decreased deep tendon reflexes, abnormal speech, loss of neurocognitive skills, optic atrophy, progressive spastic quadriparesis, increased CSF protein, and slowed nerve conduction velocities, culminating in dementia and death within several years. The early juvenile form (late onset, 4–6 years) shows gait and postural abnormalities, emotional and behavioral disturbances, optic atrophy, progressive spastic quadriparesis, increased CSF protein, and slowed nerve conduction velocities. The late juvenile form (late onset, 6–16 years) exhibits behavioral abnormalities, poor school performance, language regression, gait disturbance, slowly progressive tetraparesis, increased CSF protein, and slowed nerve conduction velocities. Finally, the adult form (late onset, >16 years) is characterized by mental regression, psychiatric symptoms, incontinence, slowly progressive spastic tetraparesis, normal or increased CSF protein, and normal or slowed nerve conduction velocities. Nontransplant approaches There is a potential role for ERT for the treatment of MLD and specifically for peripheral nervous system involvement. Further study through clinical trials is needed. Hematopoietic cell transplantation The experience with HCT for MLD extends over 20 years; however, despite advances in the field, HCT for this disease remains very challenging. For example, all patients transplanted for symptomatic lateinfantile MLD have done poorly in all aspects. Even in the few cases of presymptomatic HCT for late-infantile MLD, the results have been disappointing, although cognitive but not motor function has been preserved in some patients. All children who were transplanted prior to the development of symptoms have become wheelchair bound and totally dependent in activities of daily living. Late-onset forms of MLD with disease manifesting during school age, adolescence or adulthood present with progressive motor signs and symptoms, including gait disturbance, clumsiness, tremor, and dysarthria. Gradual decline in neurocognitive function is also observed. Neurocognitive and neurobehavioral symptoms seem to predominate in those patients whose disease first manifests in late teenage years or in early to middle adulthood. In fact, many such cases are misdiagnosed as schizophrenia or major psychosis. New-onset attention deficit hyperactivity disorder in school-age children is typical, and they are often treated for years until mental decline is observed. Attention deficits, impulsive behavior, disinhibition, impulsivity, loss of spatial skills, memory loss, and personality changes, together with features of frontal lobe dysfunction, often progress over years to decades. Peripheral nervous system disease is variably present. This indolent course in adult cases, and the potential for intervening at an appropriate stage of disease for juvenile-onset cases, permits the potentially effective use of HCT for these forms of MLD. HCT can succeed in stabilizing CNS disease and function while having little to no beneficial effect on the peripheral nervous system [100–109]. University of Minnesota experience with HCT for MLD from 1984 to 2005 included over 45 cases, the vast majority of which were juvenile or adult-onset cases. An unpublished review of this experience, performed by Peters and Taskinen, confirmed information presented earlier (see above). Outcomes of HCT for MLD were recorded from the perspective of:

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1 HCT related: survival, engraftment, graft-versus-host disease, and complications; 2 disease-specific measures: neurologic (hearing, speech, gait, bowel and bladder function, and adaptive function/activities of daily living), neuropsychologic function (IQ) and neurodevelopmental status, and neuroradiology – attempts to develop an MRI severity score in conjunction with Dr D. Loes. Background and proof of principle regarding the use of HCT for MLD was provided by the report of the first and successful HCT for MLD, which was performed by Krivit and colleagues at the University of Minnesota in 1984 [100]. Subsequent transplant experience in North America, Europe, and Asia (published and unpublished) has primarily focused on late-onset disease. A child with late-infantile MLD underwent HCT. Prior to HCT at age 0.7 years, the neurologic status was deemed to be normal; however, at age 9 years, this child has no speech, although is able to communicate, is in a wheelchair, and requires constant aid. From a neuropsychologic perspective, the age-equivalent score was approximately 0.6 years with developmental indices of approximately 100. After HCT, it was not technically possible to perform formal testing. The developmental status was felt to be approximately at the 3-year level when the child was 9 years of age. Systematic MRI studies of this patient were not performed. For late-onset or juvenile cases of MLD, follow-up after HCT ranged from 1.8 to 20.3 years. From a neurologic standpoint, after HCT, all patients were requiring a wheelchair except for one patient who was using a cane or walker. Bowel and bladder incontinence issues were prominent; speech was not used or was limited. It is noteworthy that the patient with the longest follow-up had receptive language skills at the level of approximately 10 years with a chronologic age of 21 years. For late-onset or adult patients, the follow-up after HCT ranged from 4.7 to 15.2 years. Neurologic status after HCT showed that they were dependent upon a wheelchair or a walker with a spastic gait or walking; some had frontal lobe dementia; some were incontinent of bowel and bladder; most patients were requiring constant aid. The average neuropsychologic level of function was below the normal range; however, some patients did have preservation of cognitive function in the normal-to-low normal range. In late-onset MLD, cognitive function declined during the 1–2 years after HCT but remained stable as long as 20 years after HCT. HCT “arrested” the course of the central demyelinating process in lateonset MLD if treated early. HCT did not help the demyelinating peripheral neuropathy. This observation led to a phase I clinical trial in six patients who had previously received matched sibling donor HCTs [110]. They received a single infusion of mesenchymal stem cells (MSCs) (2–10 × 106 MSCs/ kg of recipient body weight) from the original donor. There was no infusion-related toxicity. It was reported that four patients with MLD had “significant improvements in nerve conduction velocities after MSC infusion” (e.g. peroneal nerve 5–6 m/s and median nerve 8–9 m/s); however, no clinically apparent changes occurred in patients’ overall health or mental and physical development after MSC infusion. The current limitations of HCT for MLD and unanswered questions relate to: (1) the fact that it is virtually impossible to effectively treat (i.e. transplant) cases of late-infantile MLD due to the rapid rate of disease progression; (2) late-onset cases still show significant peripheral nervous system disease after HCT, and the time to achieve CNS stabilization appears to be longer than that observed in patients with cerebral X-ALD or late-onset GLD; (3) the possible role of enzymes (ERT) for the peripheral nervous system in MLD remains unknown or speculative. Important ERT experience with the mouse model of MLD has been published [111,112], and a clinical trial of ERT in children with lateinfantile MLD has been occurring in Copenhagen, Denmark, sponsored by Zymenex.

Other white matter disorders Alexander disease Alexander disease is a rare, fatal disorder of the CNS that occurs primarily in infants and children. Mutations in the glial fibrillary acidic protein gene have now been shown in a number of biopsy- or autopsy-proven patients with Alexander disease [113–115]. Histologic demonstration of Rosenthal fibers in brain specimens is no longer required for diagnostic purposes. In infants and young children, Alexander disease causes developmental delay, psychomotor retardation, paraparesis, feeding problems, megalencephaly, seizures, and hydrocephalus. Pseudobulbar and bulbar signs predominate in juvenile cases. Characteristic MRI findings have been reported in Alexander disease. Adult cases, which can be more variable, may resemble multiple sclerosis and include palatal clonus. At this time, the only treatment is supportive (Table 77.2). Pelizaeus–Merzbacher disease PMD disease is an X-linked disorder characterized by delayed motor development presenting in the first year of life and progressing to include nystagmus, ataxia, spasticity, and mental retardation [116]. Loss of myelin is evident on MRI or histopathology. Abnormal CNS conduction velocities manifesting in evoked potentials reflect this myelin defect. Mutations occur in the PLP gene, which encodes the major CNS myelin protein proteolipid protein (PLP). PLP is a gene subject to dosage control, as overexpression of PLP is the most common type of mutation in PMD. At the cellular level, the extent of pathophysiology in PMD can be correlated with the type of mutation in the PLP gene. In the most severe mutations (the connatal form of PMD) there is accumulation of material including PLP in oligodendrocytes triggering apoptosis. Less severe mutations can lead to abnormalities of myelin sheath formation. There is no rationale for the use of HCT to treat PMD (Table 77.2). Vanishing white matter disease VWMD, also called childhood ataxia with central hypomyelination, is one of the most prevalent inherited childhood disorders of white matter [117].The disease has extremely wide phenotypic variation and may affect people of all ages. Typical cases have their onset in late infancy or early childhood, with neurologic involvement characterized by progressive spasticity and ataxia, seizures, dysarthria, and optic atrophy. Acute illness, head injury, and stress can exacerbate the course. Brain MRI shows diffuse, symmetric hypomyelination. Mutations in the elF2B translation initiation factor gene have been identified. There are no specific treatments for VWMD aside from avoidance of clinical situations recognized to exacerbate the disease, and there is no rationale for the use of HCT to treat VWMD (Table 77.2). Zellweger syndrome Zellweger syndrome is a rare autosomal recessive severe peroxisomal disorder affecting infants who demonstrate characteristic facial features, profound hypotonia, seizures, and liver and renal dysfunction [118]. It can be diagnosed on the basis of abnormalities of VLCFAs and plasmalogens (phospholipids in which the group at a C1 of glycerol is an ether-linked alcohol rather than an ester-linked fatty acid). Brain MRI shows polymicrogyria. Treatment focuses on addressing feeding difficulties, various therapies, supportive care measures, management of liver dysfunction (e.g. supplemental vitamin K and fat-soluble vitamins) to control bleeding tendencies; ursodeoxycholic acid, adrenal hormone replacement, and decosahexaenoic acid supplementation. There is no rationale for the use of HCT to treat Zellweger syndrome (Table 77.2).

Hematopoietic Cell Transplantation for Storage Diseases

Glycoprotein metabolic and miscellaneous disorders In this section, glycoprotein metabolic diseases (fucosidosis, Gaucher disease, α-mannosidosis, and aspartylglucosaminuria [AGU]) will be presented and discussed (see below) (Table 77.3). Miscellaneous disorders (cerebrotendinous xanthomatosis, Fabry disease, Farber lipogranulomatous disease, gangliosidoses [GM1, GM2, and Tay–Sachs and Sandhoff disease], glycogen storage disease II [Pompe disease], neuronal ceroid lipofuscinosis [CLN1 and CLN2], Niemann–Pick disease, Wolman syndrome, Canavan disease, cystinosis, sialic acid storage disease, sialidosis, multiple sulfatase deficiency, and hypophosphatasia) will be presented and discussed in a limited fashion (Table 77.3). Epidemiology and etiology The glycoprotein metabolic diseases (fucosidosis, Gaucher disease, αmannosidosis, and AGU) are all autosomal recessive inborn errors of metabolism (Table 77.3). Molecular and clinical biology Fucosidosis arises from deficient activity of the lysosomal enzyme fucosidase [119]. The gene (FUCA1) maps to chromosome 1p24. At least 24 disease-causing mutations have been reported. Of these, only four cause an amino acid substitution; the remaining mutations are presumed to result in unstable or defective messenger RNA, for example premature stop codons, frameshifts, defective splicing or large deletions. Although the disease is panethnic, the majority of patients have been from Italy or the south-western region of the United States. A high frequency of consanguinity has been noted in affected families. Gaucher disease arises from deficient activity of the lysosomal enzyme glucocerebrosidase [119a]. Gaucher disease is most common in the Ashkenazi Jewish population, where the frequency of the Gaucher disease-causing alleles is 0.0343. The 1448C mutation exists at polymorphic levels in northern Sweden, causing type 3 disease among homozygotes. The gene is located on chromosome 1; a pseudogene with high homology is approximately 16 kilobases downstream from the active gene. Most of the disease-causing mutations are missense leading to the synthesis of glucocerebrosidase (also called β-glucosidase), with decreased catalytic function and/or stability. There are a variety of other mutations observed. The most common mutation in the Ashkenazi Jewish population is at the cDNA nt 1226 where an A to G transition results in an N370S amino acid substitution. This is associated with non-neuronopathic disease only; clinical manifestations can be relatively mild. Other mutations (e.g. L444P) are highly associated with neuronopathic disease. However, there is much diversity in phenotypic expression within all genotypes. α-Mannosidosis arises from deficient activity of the lysosomal enzyme α-mannosidase [119]. The gene maps to chromosome 19p13.2–q12. AGU arises from deficient activity of the lysosomal enzyme aspartylglucosaminidase [120]. The gene is located at 4q34–q35. A missense mutation (C163S) causing disruption of a disulfide bridge in the polypeptide structure is the predominant founder mutation in the Finnish population, representing 98% of the disease mutations. Several other “private” mutations have been detected in families with different ethnic backgrounds. Clinical description Fucosidosis is characterized by, in severely affected patients, onset of psychomotor retardation in the first year of life, growth retardation, coarse facies, dysostosis multiplex, and increased sweat chloride [119].

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At least two clinical phenotypes have been described, though in actuality there may be a continuum with a wide spectrum of severity. Attenuated phenotypes are characterized by angiokeratoma, longer survival, and more normal levels of sweat sodium chloride. Three types of Gaucher disease have been delineated. Type 1, by far the most common, is distinguished from type 2 and type 3 disease by the lack of primary CNS involvement. Type 2, the acute neuronopathic form of the disease, has an early onset with severe CNS manifestations and death usually by age 2 years. Patients with type 3 (subacute neuronopathic) Gaucher disease have neurologic symptoms with a later onset and a more indolent course than that seen in type 2 disease. Hepatosplenomegaly, bone lesions, and occasional involvement of the lungs and other organs occur in all types of Gaucher disease. The more severe (infantile, type I) phenotype of α-mannosidosis includes rapidly progressive mental retardation, hepatosplenomegaly, severe dysostosis multiplex, and often death by between ages 3 and 12 years. The attenuated (juvenile–adult, type II) phenotype accounts for approximately 10–15% of cases. It is characterized by a slower rate of progression with survival into adulthood. There is likely a continuum of clinical severity between these two forms. Aspartylglucosaminuria is characterized by global developmental delay with onset between 2 and 4 years of age. Delayed speech and motor clumsiness are often preceded by frequent upper respiratory tract infections. Patients reach developmental capacities equivalent to that of a 5–6-year-old around puberty and then slowly deteriorate to a severely delayed level by adulthood. Mild connective tissue changes lead to coarse facial features, thickened calvarium, and osteoporosis. Seizures are common later in the disease course, with abnormalities in the differentiation between gray and white matter as well as delayed myelination apparent on brain MRI. Nontransplant approaches The primary nontransplant approaches for fucosidosis, α-mannosidosis, and AGU are supportive in nature, while ERT is the standard of care for Gaucher type 1 patients and to a more limited extent for type 3 patients. Hematopoietic cell transplantation For fucosidosis, detailed studies of a valid animal model, namely the springer spaniel, include the use of HCT after total lymphoid irradiation to correct the enzymatic deficiency [121]. There is very limited experience with HCT in children with fucosidosis [122,123]. Due to disease variability, a definitive conclusion regarding the benefits of HCT cannot be reached at this time. For Gaucher disease, the first successful HCT was performed at Huddinge Hospital in Stockholm, Sweden, by Ringdén and his team [124,125]. The early experience with HCT for type 1 patients dates to the mid-1980s at Westminster Children’s Hospital in London under the direction of Hobbs and co-workers, who reported on the beneficial effect of pre-HCT splenectomy in patients with type 1 Gaucher disease and their rapid and remarkable improvement [126]. A biochemical marker (plasma chitotriosidase) declined to normal levels in patients with Gaucher disease by 5–12 years after HCT [127]. Reports have focused on the marked improvement in Gaucher bone disease after HCT, including resolution of bone crises and resumption of normal growth [124,128,129]. Due to the rapidly progressive nature of the acute neuronopathic (type 2) form of Gaucher disease, attempts to perform HCT in these patients have been limited and without clinical benefit. Alternative approaches to neurologic disease in Gaucher disease are being investigated, and include substrate depletion and chaperone therapy [125].

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Work performed at Huddinge Hospital led to the characterization of the Norrbottnian type of Gaucher disease. The study focused on the CNS symptomatology and function, correlation of clinical signs with laboratory, neuropathologic, and biochemical findings, the effects of splenectomy on the course and severity of disease, and the effect of HCT on this form of type 3 Gaucher disease [130]. Patients experienced correction of hepatomegaly, disappearance of Gaucher cells, resolution of bone pain, and growth spurts. Further, encouraging results have been reported in the long-term (10-year) follow-up of the neurologic and neuropsychologic function of these patients [131]. Neurocognitive function has stabilized in the normal or above-normal range in most patients. This was a critical observation, thereby underscoring the value of HCT for patients with Norrbottnian type 3 Gaucher disease. Further emphasis on the efficacy of HCT comes from the inability of long-term ERT to stabilize the neurologic and neuropsychologic status of patients with Norrbottnian Gaucher disease (Table 77.3) [132]. Walkley et al. [133] demonstrated that HCT is effective in the feline model of mannosidosis. The first reported case of α-mannosidosis undergoing HCT was reported by Will and colleagues [134]. A subsequent successful case of HCT with long-term survival was reported by Wall et al. [135], with description of the resolution of sinopulmonary infections and organomegaly, improved dysostosis multiplex, and stabilization of neurocognitive function. More recently, three surviving, fully engrafted patients with α-mannosidosis transplanted at the University of Minnesota were demonstrating a very good-to-excellent quality of life with preservation of normal neurocognitive and cardiopulmonary function [136]. α-Mannosidosis patients have experienced pulmonary complications from 10 to 20 weeks after HCT. No infectious etiologies have been identified; it appears that selected storage disease patients are at increased risk for pulmonary complications, including hemorrhage and/ or bronchiolitis obliterans. This may be attributed to release of lysosomal storage material from damaged parenchymal cells following HCT and the resultant inflammatory response. Generally, patients transplanted early in their disease course prior to the onset of significant diseaserelated complications are the best candidates for HCT. The medical center in Helsinki, Finland, has experience with HCT in three patients with follow-up ranging from 1 to over 5 years including serial MRI and biochemical and clinical examinations [137]. MRIs of the brain in transplanted patients with at least 2 years of follow-up showed nearly normal gray–white matter relationships, and improved neuropsychologic function had also occurred [138]. However, more recently, longer-term follow-up has raised questions about the efficacy of HCT in these patients with respect to their ultimate neurodevelopmental outcomes [139].

Miscellaneous disorders Cerebrotendinous xanthomatosis Cerebrotendinous xanthomatosis is a rare autosomal recessive disorder due to defective activity of mitochondrial enzyme sterol 27-hydroxylase (CYP27A) [140]. The gene is found on chromosome 2. More than 300 patients have been reported worldwide and over 50 different mutations in CYP27A identified, most of which result in absences or inactive enzyme. The clinical features include diarrhea, cataracts, tendon xanthomas, and neurologic signs such as dementia, psychiatric disturbances, pyramidal and/or cerebellar issues, and seizures. Strongly suggestive cerebellar findings are noted on brain MRI. Abnormalities of bile acids in urine and serum, and elevated plasma and tissue cholestanol, are the biochemical hallmarks at diagnosis and in subsequent monitoring on treatment with chenodeoxycholic acid. There is no role for HCT in this disorder (Table 77.3).

Fabry disease Fabry disease is an X-linked recessive inborn error of glycosphingolipid catabolism due to deficient activity of lysosomal hydrolase αgalactosidase A in affected hemizygous males [141]. This defect leads to systemic deposition of glycosphingolipids, predominantly globotriaosylceramide in the following tissues: body fluids, endothelium, perithelium, smooth muscle cells of blood vessels, ganglia, heart, kidneys, eyes, etc. Onset is usually in childhood or adolescence with pain and paresthesias of the extremities, vessel ectasia (angiokeratoma) in skin and mucous membranes, and hypohidrosis. Corneal and lenticular opacities are often noted. With aging, proteinuria, hyposthenuria, and lymphedema appear. Severe renal impairment leads to hypertension and uremia. Causes of death are usually renal, cardiac or cerebrovascualar. Heterozygous females may have an attenuated form of the disease. The gene is located at Xq22.1. Over 150 mutations have been identified, including partial gene rearrangements, splice-junction defects, and point mutations, thereby emphasizing the heterogeneity of the molecular defects causing this disease. The primary treatment is ERT with additional supportive care measures as indicated, such as treatment for neuropathic pain, anticoagulants in patients who are prone to strokes, and hemodialysis for patients with endstage renal disease. HCT is not considered an appropriate therapy (Table 77.3). Farber lipogranulomatous disease Farber disease is a rare lysosomal storage disorder caused by deficiency of acidic ceramidase [142]. Formation of subcutaneous and periarticular ceramide-containing nodules causes painful joint swelling, progressive disability, and hoarseness. The pathophysiology whereby the defect in ceramide metabolism leads to chronic granulomatous inflammation remains to be elucidated. Most patients with Farber disease die of progressive neurologic deterioration early in infancy; however, since there is a wide spectrum of clinical severity/CNS involvement, depending on residual lysosomal ceramide, there is a broad spectrum of disease severity. Children with Farber disease without CNS involvement (Farber disease type 2/3) still have a severe disease due to chronic granulomatous inflammation with progressive joint deformities causing pain and immobility, and effects on the lower airways causing chronic respiratory failure with death before age 20 years. HCT performed in patients with Farber disease and CNS involvement has been unsuccessful, with the two patients dying of progressive neurodegeneration while peripheral symptoms including granuloma resolved [143]. Gangliosidoses (GM1, GM2, Tay Sachs, Sandhoff) The GM2 gangliosidoses are a group of inherited disorders caused by excessive accumulation of ganglioside GM2 and related glycolipds in lysosomes, especially in neurons [30,144]. With infantile onset, the rapidly progressive neurodegenerative disease leads to death by 4 years of age (classic Tay-Sachs disease, Sandhoff disease, and GM2 activator deficiency), while later-onset subacute or chronic forms show more slowly progressive neurologic conditions compatible with survival into late childhood, adolescence or even adulthood. HCT does not appear to successfully correct these disorders (Table 77.3) [30,144]. Deficiency of lysosomal β-galactosidase is manifested clinically as two different diseases, GM1 gangliosidosis and Morquio B (MPS IVB, discussed previously) [144a]. GM1 gangliosidosis can have its onset in infancy, childhood/adolescence or adulthood. The clinical course is generally progressive neurologic deterioration. The role of HCT for GM1 gangliosidosis remains unclear since most cases are too advanced at diagnosis or progress too rapidly for benefit to be derived from HCT. HCT may be helpful in later-onset cases at an early stage of the disease process.

Hematopoietic Cell Transplantation for Storage Diseases

Glycogen storage disease type II (Pompe disease) Glycogen storage disease type II is also called acid maltase deficiency or Pompe disease [145]. It is an autosomal recessive disorder of glycogen metabolism arising from deficiency of lysosomal hydolase acid αglucosidase activity. The clinical presentation encompasses a range of phenotypes, all of which include varying degrees of myopathy, but differ with respect to onset, extent of organ involvement, and rate of progression to death. The most severe is the classic infantile-onset form, which was described by Pompe and is characterized by prominent cardiomegaly, hypotonia, hepatomegaly, and death due to cardiorespiratory failure [145]. At the other extreme is a slowly progressive proximal myopathic adult-onset disease with onset as late as the second to sixth decade and involving only skeletal muscle. The gene is located on chromosome 17q25. The primary therapies are ERT and supportive care measures. There is not a role for HCT in this disorder (Table 77.3). Neuronal ceroid lipofuscinosis (NCL1, NCL2) Two forms of neuronal ceroid lipofuscinosis (NCL1, infantile, and NCL2, classic late-infantile) are recognized as true lysosomal enzyme storage disorders. NCL1 is characterized by normal development until age 6–12 months, followed by psychomotor retardation, microcephaly, myoclonus, and visual deterioration. NCL2 presents between 2 and 4 years of age with severe myoclonic seizures and slow progression to blindness. Since both diseases are lysosomal, the implication is that HCT might be effective therapy [146–148]. However, this has not been borne out by presymptomatic patients with NCL2 who have been transplanted, or by the existing animal model studies. Additional clinical data are needed to determine whether the natural history of NCL2 is too rapidly progressive for HCT to be beneficial (Table 77.3). Niemann–Pick disease types A, B, and C Niemann–Pick disease types A and B are lysosomal storage disorders resulting from deficient acid sphingomyelinase enzyme activity [149– 151]. Niemann–Pick type A is a rapidly progressive disorder of infancy characterized by failure to thrive, hepatosplenomegaly, neurodegeneration, and death, often by 2–3 years of age. HCT has not been effective in preventing the inexorable neurodevelopmental decline. Niemann– Pick type B is a phenotypically variable disorder that is usually diagnosed in childhood due to marked hepatosplenomegaly. HCT appears to effectively treat the somatic manifestations of Niemann–Pick type B disease [151]. Niemann–Pick type C is an autosomal recessive lipidosis resulting from a unique error in cellular trafficking of exogenous cholesterol, and is associated with lysosomal accumulation of unesterified cholesterol [152]. A case has been reported of a 3-year-old girl with Niemann–Pick type C disease who underwent allogeneic HLA-identical sibling donor HCT who engrafted [153]. She survived with improvement in somatic disease features including hepatosplenomegaly and marrow and lung infiltration; however, her neurologic status continued to deteriorate [153]. It is therefore unclear whether HCT has a role in the effective treatment of the CNS in Niemann–Pick type C. Wolman syndrome Wolman syndrome is an autosomal recessive disorder due to deficient enzyme activity of acid lipase resulting in massive accumulation of cholesteryl esters and triglycerides in most body tissues [154,155]. The disease occurs in infancy and is typically fatal in the first year of life. HCT has been performed successfully in a very small number of patients [155]. Canavan disease The clinical features of Canavan disease include poor head control and hypotonia at 2–4 months of age, generalized seizures, opisthotonic

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posturing, loss of very early milestones, increased head circumference, white matter changes (leukodystrophy) on brain MRI, hypotonia that progresses to spasticity, and, late in the course, decerebrate or decorticate posturing [156]. This autosomal recessive disorder arises due to deficient aspartoacylase activity. There is no role for HCT in the treatment of Canavan disease; the primary treatments are supportive in nature (Table 77.3). Cystinosis Cystinosis is a rare, autosomal recessive lysosomal storage disorder due to defective carrier-mediated transport of the amino acid cystine across the lysosomal membrane [157]. The major clinical manifestation is renal failure at age 9–10 years. The gene is located on chromosome 17p13 and codes for cystinosin. Therapy includes replacement of renal losses due to Fanconi syndrome, provision of thyroxine, insulin, pancreatic enzymes, and testosterone for deficient patients, and symptomatic care for ophthalmic complaints. There is no clear role for HCT in cystinosis (Table 77.3). Sialic acid storage diseases The lysosomal free sialic acid storage diseases, namely Salla disease and infantile free sialic acid storage disease, are autosomal recessive disorders with various degrees of psychomotor retardation [158]. Dysmyelination is an invariable characteristic as seen on brain MRI. The fundamental problem is accumulation of free sialic acid in lysosomes due to an impaired carrier-mediated transport system in the lysosomal membrane. There is no clear indication that HCT is beneficial (Table 77.3). Sialidosis Sialidosis is an autosomal recessive disorder consisting of two clinical phenotypes. Type I, the milder form, is characterized by development of ocular cherry-red spots and generalized myoclonus in the second or third decade, as well as seizures, hyperreflexia, and ataxia. Type II disease has an early age of onset, and is a severe and rapidly progressive disorder with an MPS-like phenotype with organomegaly, dysostosis multiplex, and mental retardation [119]. The gene which codes for neuraminidase is found on chromosome 6p21. There is no definitive treatment (Table 77.3). Multiple sulfatase deficiency Multiple sulfatase deficiency is a rare (1 : 1.4 million) autosomal recessive disorder characterized by deficiencies in all 12 known sulfatases, and leading to a clinical presentation that generally resembles late-infantile MLD [159]. There has been very limited experience with HCT for this disease, and no definite conclusion can be drawn at this time as to its utility (Table 77.3). Hypophosphatasia Hypophosphatasia is an autosomal dominant or autosomal recessive disorder caused by defective osteoblasts and chondrocytes due to inactivation of tissue-nonspecific alkaline phosphatase. There is limited experience to date with HCT for this disorder [160].

Future directions ERT, substrate depletion, alternative stem cells, complementary and combination therapies [24], gene therapy, alternative approaches to HCT with or without stem cell selection, and newborn screening are all part of this rapidly advancing field of clinical medicine [21,161–165]. Alternative and complementary therapies to HCT, as well as combinations of therapies for these inherited metabolic storage diseases, are either in

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place or being developed. ERT has been available for patients with Gaucher disease type 1 for over 15 years [166]. Recently, ERT has made the transition from clinical trial to proven therapy for Fabry, Pompe and Niemann–Pick type B diseases, and selected MPSs including MPS I, II, and VI. In the case of Fabry disease, ERT reduced severe neuropathic pain, stabilized renal function, and improved cardiac and vascular structure and function [167]. In Pompe disease, a fatal cardiac and skeletal muscle disorder due to acid maltase deficiency, ERT has improved cardiac function and structure and increased overall muscle strength [168]. Important lessons from these experiences include the variability of the clinical response to ERT depending upon the disease, the organ or tissue, the dose of enzyme, and the schedule of administration. Of particular importance is the observation that there is limited-to-no penetration by exogenous intravenous enzyme into the CNS or peripheral nervous system. In the area of substrate depletion or deprivation, a number of disorders have been treated, including Gaucher disease, GM2, Fabry disease, and cystinosis. Drugs that slow the rate of formation of accumulating glycolipids are under development as well. One such agent is N-butyldeoxynorjirimycin (OGS-918), which has shown promise in Gaucher disease. While benefits have been observed in these various

disorders, the extent of amelioration has been limited in scope and degree. Alternative stem cells, including both embryonic and adult stem cells, are being used in transplantation and gene transfer/therapy investigational protocols. This is important since bone marrow-derived MSCs remain host derived despite successful HCT with engraftment in patients with lysosomal and peroxisomal disorders [169]. The scope of gene transfer applications in therapy for human diseases has continued to expand over the past two decades and includes genetic storage diseases. Hematopoietic stem cells have been considered appropriate targets for therapeutic gene transfer due to their ability to selfrenew as well as differentiate into multiple lineages. Diseases such as Gaucher disease, Hurler syndrome, X-ALD, MLD, and other enzymedeficiency states may be amenable to treatment by gene transfer into hematopoietic stem cells, due to enzyme production, release, and uptake leading to metabolic correction. In conclusion, enhanced interactions among the many disciplines and specialists caring for patients with these complex disorders are of paramount importance to the coordinated advancement of this very exciting field of clinical medicine and research.

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138. Autti T, Rapola J, Santavuori P et al. Bone marrow transplantation in aspartylglucosaminuria-histopathological and MRI study. Neuropediatrics 1999; 30: 283–8. 139. Arvio M, Sauna-Aho O, Peippo M. Bone marrow transplantation for aspartylglucosaminuria. Follow-up study of transplanted and non-transplanted patients. J Pediatr 2001; 138: 288– 90. 140. Gallus GN, Dotti MT, Federico A. Clinical and molecular diagnosis of cerebrotendinous xanthomatosis with a review of the mutations in the CYP27A1 gene. Neurol Sci 2006; 27: 143– 9. 141. Desnick RJ, Ioannou YA, Eng CM. α-Galactosidase A deficiency: Fabry disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 3827–76. 142. Moser HW, Linke T, Fensom AH, Levade T, Sandhoff K. Acid ceramidase deficiency: Farber lipogranulomatosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 3827– 76. 143. Vormoor J, Ehlert K, Groll AH et al. Successful hematopoietic stem cell transplantation in Farber disease. J Pediatr 2004; 144: 132–4. 144. Gravel RA, Kaback MM, Proia RL, Sandhoff K, Suzuki K, Suzuki K. The GM2 gangliosidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGrawHill; 2001. pp. 3827–76. 144a. Suzuki Y, Oshima A, Nanba E. B-galactosidase deficiency (b-galactosidosis): GM1 gangliosidosis and Morquio B disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 3775–810. 145. Hirschhorn R, Reuser AJJ. Glycogen storage disease type II: acid α-glucosidase (acid maltase) deficiency. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 3389–420. 146. Hofmann SL, Peltonen L. The neuronal ceroid lipofuscinoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 3877– 96. 147. Santavuori P, Lauronen L, Kirveskari E et al. Neuronal ceroid lipofuscinoses in childhood. Neurol Sci 2000; 21: S35–41. 148. Deeg HJ, Shulman HM, Albrechtsen D et al. Batten’s disease: failure of allogeneic bone marrow transplantation to arrest disease progression in a canine model. Clin Genet 1990; 37: 264–70. 149. Schuchman EH, Desnick RJ, Niemann-Pick disease types A and B: acid sphingomyelinase deficiencies. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 3589–610. 150. Bayever E, Kamani N, Ferreira P et al. Bone marrow transplantation for Niemann-Pick type

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IA disease. J Inherit Metab Dis 1992; 15: 919– 28. Vellodi A, Hobbs JR, O’Donnell NM et al. Treatment of Niemann-Pick disease type B by allogeneic bone marrow transplantation. Br Med J (Clin Res Ed) 1987; 295: 1375–6. Patterson MC, Vanier MT, Suzuki K et al. Niemann-Pick disease type C: a lipid trafficking disorder. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 3611–34. Hsu YS, Hwu WL, Huang SF et al. NiemannPick disease type C (a cellular cholesterol lipidosis) treated by bone marrow transplantation. Bone Marrow Transplant 1999; 24: 103–7. Assmann G, Seedorf U. Acid lipase deficiency: Wolman disease and cholesteryl ester storage disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 3551–72. Krivit W, Peters C, Dusenbery K et al. Wolman disease successfully treated by bone marrow transplantation. Bone Marrow Transplant 2000; 26: 567–70. Beaudet AL. Aspartoacylase deficiency (Canavan disease). In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 5799–806. Gahl WA, Thoene JG, Schneider JA. Cystinosis: a disorder of lysosomal membrane transport. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGrawHill; 2001. pp. 5085–108. Aula P, Gahl WA. Disorders of free sialic acid storage. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGraw-Hill; 2001. pp. 5109–20. Hopwood JJ, Ballabio A. Multiple sulfatase deficiency and the nature of the sulfatase family. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease, 8th edn. New York: McGrawHill; 2001. pp. 3725–32. Whyte MP, Kurtzberg J, McAlister WH et al. Marrow cell transplantation for infantile hypophosphatasia. J Bone Miner Res 2003; 18: 624– 36. Albert MH, Schuster F, Peters C et al. T-celldepleted peripheral blood stem cell transplantation for alpha-mannosidosis. Bone Marrow Transplant 2003; 32: 443–6. Yabe H, Inoue H, Matsumoto M et al. Unmanipulated HLA-haploidentical bone marrow transplantation for the treatment of fatal, nonmalignant diseases in children and adolescents. Int J Hematol 2004; 80: 78–82. Corti P, Peters C, Balduzzi A et al. Reconstitution of lymphocyte subpopulations in children with inherited metabolic storage diseases after haematopoietic cell transplantation. Br J Haematol 2005; 130: 249–55. Gaipa G, Dassi M, Perseghin P et al. Allogeneic bone marrow stem cell transplantation following CD34+ immunomagnetic enrichment in patients with inherited metabolic storage diseases. Bone Marrow Transplant 2003; 31: 857–60.

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165. Resnick IB, Abdul Hai A, Shapira MY et al. Treatment of X-linked childhood cerebral adrenoleukodystrophy by the use of an allogeneic stem cell transplantation with reduced intensity conditioning regimen. Clin Transplant 2005; 19: 840– 7. 166. Barton NW, Brady RO, Dambrosia JM et al. Replacement therapy for inherited enzyme deficiency – macrophage-targeted glucocerebrosi-

dase for Gaucher disease. N Engl J Med 1991; 324: 1464–70. 167. Schiffmann R, Brady RO. New prospects for the treatment of lysosomal storage diseases. Drugs 2002; 62: 733–42. 168. Kaye EM. Lysosomal storage diseases. Curr Treat Options Neurol 2001; 3: 249–56. 169. Koç ON, Peters C, Aubourg P et al. Bone marrow-derived mesenchymal stem cells remain

host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol 1999; 27: 1675– 81. 170. Meikle PJ, Hopwood JJ, Clague AE, Carey WF. Prevalence of lysosomal storage disorders. JAMA 1999; 281: 249– 54.

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Rajni Agarwal

Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders

Introduction The human body is equipped with a complex phagocytic and immune system to defend against a wide variety of microbes ranging from viruses to bacterial and fungal offenders. The first line of defense is a set of two systems, namely the monocyte–phagocyte system and the granulocyte– phagocyte system. The primary cells of these two systems, monocytes and granulocytes, arise from a common committed progenitor in the bone marrow [1–3]. The pattern of the inflammatory response in the monocyte–phagocyte system is similar to that seen in the granulocyte– phagocyte system. Any quantitative or qualitative defect in the elements of these defense systems will result in an inadequate host defense against microbial infections. The monocyte–phagocyte system is defined as a continuum from monoblasts to promonocytes to monocytes with eventual migration of the monocytes into tissues such as lungs, liver, spleen, and the brain to form fixed tissue macrophages which perform specialized functions suited for the specific location. The life span of monocytes varies between 3 and 6 days. Tissue macrophages do not usually undergo cell division, and their life span is estimated to range from weeks to years. However, if the cell proliferation regulatory controls are lost or altered, uncontrolled proliferation and accumulation of macrophages may take place in multiple organs. One such example is the hemophagocytic lymphohistiocytosis (HLH) described later in this chapter. In HLH (familial or secondary), excessive accumulation of tissue macrophages results in organ dysfunction/enlargement. The excessive “cell-eating” behavior of the macrophages seen in HLH results in low blood counts. Abnormal proliferation and accumulation of macrophages at specific anatomic sites may result in signs and symptoms pertinent to those sites. For example, central nervous system (CNS) involvement may result in serious clinical focal neurologic manifestations including hypotonia or hypertonia, convulsions, hemiplegia/tetraplegia or blindness. The functions of the monocyte–macrophage system are manifold [3]. The mononuclear phagocytes play an important role as phagocytic and immunoregulatory cells. As phagocytic cells, macrophages secrete a variety of antimicrobial agents including lysozyme, neutral proteases, acid hydrolases, and cytokines. The macrophages, for example, secrete the cytokine interleukin-1 (IL-1). IL-1, in turn, stimulates vigorous production of hydrogen peroxide and toxic oxygen metabolites not only by

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

the macrophages, but also by the neutrophils. The activation of macrophages in response to insults such as infection is central to their role in host defense. Intact chemotactic mechanisms result in the accumulation of macrophages at the site of insult. Defective mononuclear phagocytic chemotaxis may result in an inadequate host defense against infection, such as is seen in mucocutaneous candidiasis. The immunoregulatory functions of the macrophages are also diverse and complex. Macrophages process and present antigens to lymphocytes and secrete cytokines important for the development of lymphocytes and the regulation of their replication. The immune responses are mediated via secretory products that have pleotropic effects affecting nearly all cell types. Macrophages also participate in removing immune complexes from the circulation. The inflammatory cascade of the granulocyte–phagocyte system has two major components: (1) vasodilatation and increased vascular permeability to phagocyte products, and (2) “leukocytic events.” These leukocytic events include several sequential steps, some of them overlapping in nature (Fig. 78.1): 1 margination; 2 adhesion to the endothelial surface; 3 emigration, which includes “rolling and diapedesis”; 4 phagocytosis and intracellular degranulation and killing; 5 release of leukocyte products. Initially, proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-1, IL-6, and interferon-γ are released by endothelium, T cells, and B cells, resulting in vasodilatation and increased vascular permeability. Immunoglobulin (Ig) and the complement system activate phagocytes and enhance microbial opsonization. A redundancy of chemoattractants such as various cytokines and complement fragments is generated to ensure that the leukocytes will be attracted to the site of insult. The chemoattractant molecules also upregulate certain adhesion receptors on the leukocytes as well as promoting recruitment of the cytoskeletal actin for movement of phagocytes. Following margination or peripheral orientation of leukocytes in the moving blood stream, the white cells adhere in great numbers to the endothelial surface and eventually migrate to the infected or injured site. The initial step in the emigration of white cells, often referred to as “rolling” or “tethering,” is mediated by a family of glycoprotein adhesion molecules called selectins (L-, E-, and P-selectins). Some of the rolling leukocytes become firmly attached to the vascular endothelium via β integrin adhesion receptors on the leukocytes. The integrins are the glycosylated heterodimers of noncovalently linked β and α chains. The type of β integrin is determined by the type of β subunit in the complete molecule. An important type of β integrin is the β2 integrin.

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Fig. 78.1 The acute inflammatory response. Neutrophils are among the first cells to arrive at the scene of an infection and are important contributors to the acute inflammatory response. As the neutrophil rolls along the blood vessel wall, the L-selectin on its surface binds to carbohydrate structures such as sialylLewisx on the adhesion molecules on the vascular endothelium, and its progress is eventually halted. As the neutrophil becomes activated, it replaces L-selectin with other cell surface adhesion molecules such as integrins. These molecules bind E-selectin, which is present on the blood vessel wall as a result of the influence of inflammatory mediators such as bacterial lipopolysaccharides and the cytokines interleukin-1 and tumor necrosis factor-α. The activated neutrophil then enters the tissues, where it is attracted to the infection site by a number of chemoattractants. The neutrophil can then phagocytose and destroy the C3bcoated bacteria. (Reproduced from [134], with permission.)

Three different β2 integrin molecules result when the same β2 integrin subunit (CD18) is linked with different α subunits (CD11a, CD11b or CD11c). Defective adhesion has been noted in an inherited heterogeneous defect called leukocyte adhesion deficiency type I (LAD 1). In this disorder, mutations in the common β2 integrin subunit are the underlying cause of defective chemotaxis. Two patients have been described with the absence of neutrophil receptors for E-selectin resulting in defective adhesion, and the disorder is called LAD type 2. The emigration of the adhered or firmly attached leukocytes at the endothelial surfaces into the site of injury or infection involves the presence of a chemotactic gradient that creates the directional movement, the chemotaxis, of the cells. The phagocytes move by extending pseudopodia, the interior of which consists of a branching network of actin filaments and the contractile protein myosin. Defects in neutrophil motility involving actin have been noted in neutrophil actin deficiency syndromes. In the next step, the accumulated leukocytes recognize the opsonized targets by attaching to the latter via opsonin receptors on the leukocyte surfaces. The major humoral opsonins that coat the microbes include IgM, IgG1, IgG3, C3b, and C3bi. In LAD 1, ingestion of C3biopsonized microbes is also impaired. Once the leukocyte–opsonin interaction has occurred, the process of engulfment is initiated. As the final step, the engulfed material is destroyed through intracellular cytocidal and digestive activity. This process involves the oxygenindependent (in the hypoxic necrotic environment of tissue injury) and oxygen-dependent pathways of microbial killing. In the oxygen-independent pathway, numerous cationic antimicrobial proteins contained within the neutrophil azurophilic granules are utilized. These antimicrobial proteins include proteins such as defensins and serpocidins. In

addition, the azurophilic and specific granules also contain other degradation enzymes such as lysozymes and hydrolases. Deficiencies in neutrophil granule proteins or defects in granule formation or degranulation are associated with recurrent bacterial infections as seen in Chediak– Higashi syndrome (CHS). The oxygen-dependent pathway of intracellular microbial killing, often referred to the “respiratory burst oxidase” or NADPH pathway, utilizes the generation of superoxide radicals through mitochondrially based reactions. Mutations in the oxidase subunit result in the well known disease entity of chronic granulomatous disease (CGD). In addition to the normally functioning granulocytes, adequate numbers of granulocytes are necessary for normal host defenses. Granulocytopenia due to congenital defects, malignancy or after administration of chemotherapeutic agents results in increased susceptibility to bacterial infections. The congenital disorders that affect the monocyte– macrophage and granulocyte system present in early childhood with recurrent, severe, and often life-threatening infections. This chapter will address the various disorders of macrophages and granulocytes and the role of hematopoietic cell transplantation (HCT) as an important treatment modality (Table 78.1).

General considerations for HCT Timing of HCT The timing of intervention in the phagocytic disorders with HCT depends on the nature of the disease, the presence of serious infections, and the status of organ function. Even though the risk attendant to HCT may be

Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders Table 78.1 Hematopoietic cell transplantation for macrophage and granulocyte disorders Macrophage disorders

Hemophagocytic lymphohistiocytosis

Granulocyte disorders

Quantitative disorders Kostmann’s syndrome Reticular dysgenesis Cyclic neutropenia Cartilage–hair dysplasia Dyskeratosis congenita Swachman–Diamond syndrome Qualitative disorders Neutrophil actin deficiency – abnormal chemotaxis Leukocyte adhesion (CD11/CD18) deficiency – abnormal adhesion Chediak–Higashi syndrome – abnormal granulation Chronic granulomatous disease – abnormal oxidation Griscelli’s syndrome

a concern, a “wait-and-see” approach may not be in the patient’s best interest. Timing of HCT should be determined by the prognosis and the anticipated risk of life-threatening infections or irreversible organ damage. Disorders with a variable clinical course need to be approached differently than disorders in which delayed intervention with HCT might result in high mortality despite the provision of optimal supportive care. For example, in cyclic neutropenia, low counts can be managed with granulocyte colony-stimulating factor (G-CSF) and without intervention with HCT. On the other hand, in severe forms of reticular dysgenesis (RD), early intervention with HCT is warranted [4]. The course of CGD can be extremely variable, but certain phenotypes of CGD can be more severe than others [5]. Choice of donor for HCT Since the disorders discussed in this chapter affect young children, genotypically identical donors will likely be available for no more than 20–30% of patients. Therefore, 70–80% of HCT candidates are without an ideally matched related donor for HCT. Since many of these disorders occur in children of consanguineous parents, an extensive family search should be conducted to identify a suitable related donor. The siblings or any other related potential donors should be tested, if technically possible, for the presence of the disease in question before they are considered to serve as donors. Many congenital disorders of the phagocyte system are transmitted as autosomal recessive conditions. The carrier state does not result in serious clinical disease, and heterozygous individuals may qualify as donors. One possible exception would be X-linked CGD, where severely imbalanced “lyonization” may result in clinically significant disease [6]. For HCT in HLH, preference should be given to the sibling donor. However, the sibling donor should be screened to rule out the presence of any occult disease. If all the available tests including low natural killer (NK)-cell activity are negative, the sibling may be used as a donor. The caveat that should be kept in mind is that no definitive tests exist to exclude the carrier state. In the event that the sibling is suspected to be a possible carrier, a search for a matched unrelated donor should be performed [7]. Identification of such a donor may require several months,

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and HCT risks may rise during the search process. In general, T-celldepleted haploidentical grafts from parents have been successfully used as matched unrelated donors to perform HCT in these patients. The difficulties and risks, however, with these HCT procedures remain a major concern. Preparative regimens Most of the HCT procedures for phagocytic disorders have been performed utilizing high-dose conditioning. The two major chemotherapeutic agents in use are busulfan (BU) and cyclophosphamide (CY). CY, in certain circumstances, is used in combination with total body irradiation (TBI). It remains the physician’s choice to use a particular regimen, as both radiation-based and nonradiation-based regimens are relatively well tolerated by children. Since the introduction of hyperfractionated TBI, many of the previously observed long-term effects have diminished. Recent experiences suggest that lower doses of TBI may result in prolonged mixed and eventual complete chimerism, which might be effective and less toxic in HCT procedures for some disorders [8]. More data need to be generated to prove the usefulness of this approach for children with conditions discussed in this chapter. At this time, reducedintensity conditioning (RIC) regimens seem to be attractive and should be explored. The RIC approach can be especially useful where mixed chimerism could restore the cellular function, for example in patients with CGD.

Macrophage disorders HLH HLH is the most frequent disorder of the macrophage system. HLH is characterized by multisystem inflammation, a reactive process resulting from the hyperactivation of antigen-presenting cells (macrophages and histiocytes) and CD8+ T cells. In this disorder, accumulation of activated macrophages along with the hemophagocytosis of hematopoietic cellular elements takes place, resulting in organ enlargement with dysfunction and cytopenia. The pathologic findings in HLH arise from persistently elevated levels of proinflammatory cytokines. It is now believed that increased exposure to the cytokines generated by uncontrolled activation of histiocytes and T cells leads to progressive organ dysfunction that leads to death in affected patients. Ongoing hypercytokinemia results from failure of natural immune downregulation due to defective NK- and cytotoxic T-lymphocyte function [9]. Several genetic defects have been described in HLH. The first genetic defect causing HLH was reported in 1999 involving mutation of the perforin gene (PRF1) [10]. Perforin is stored in the NK, CD8+ T, and NK-T cells, and is required to deliver granzyme B to target cells, which initiates apoptotic programs leading to target cell death [11]. The perforin gene mutation is shown to be involved in 20–40% of all affected families and up to 50% of North American families [12]. In 2003, another gene mutation in UNC 13D on chromosome 17q25 was shown to lead to HLH [13]. The affected protein, Munc13–4, is essential for cytolytic granule secretion preceding vesicle membrane fusion. The mutated Munc protein results in defective cytolytic granule exocytosis. A third gene defect associated with HLH has recently been identified encoding the protein syntaxin 11. This involves the gene STX11 on chromosome 6q24. Syntaxin 11 has been postulated to play a role in intracellular trafficking [14]. Mutations in UNC 13D and STX11 affect 20% and 10% of HLH patients respectively [15]. From a diagnostic standpoint, a rapid screening assay for PRF mutations by flow cytometry has been developed. Genetic sequencing of the PRF and MUNC13– 4 genes is also available on a clinical basis in the United States [16].

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HLH includes two different conditions: (1) primary or familial HLH, an autosomal recessive disorder, and (2) secondary HLH, which includes an infection-associated hemophagocytic syndrome and a malignancyassociated hemophagocytic syndrome. Since HCT procedures have been performed only for autosomally recessive primary HLH, we will discuss this entity here. Primary HLH has been reported from all continents and all ethnic groups. In a retrospective study, the incidence of the disease in Swedish children was estimated to be 0.12 per 100,000 children per year [17]. Since primary HLH is an autosomal recessive disorder, an increased incidence has been reported in families with consanguinity [18]. Most patients develop the disease in early life, with about 70% of children presenting in the first year of life. However, the age of presentation may be up to 8 years. The age of onset is usually similar among the multiple affected siblings. However, on occasion, a difference of more than 1–3 years in age has been noted in affected siblings. The diagnosis of HLH may be difficult. Diagnostic guidelines have been developed based on clinical, laboratory, and histopathologic criteria by the Histiocyte Society in protocol 2004 [12,19]. In the absence of a family history or genetic diagnosis, five or more of the eight diagnostic criteria need to be met for a diagnosis of HLH. The guidelines encompass clinical, laboratory, and histopathologic findings in the disease (Table 78.2). The clinical manifestations may vary widely. Although serious CNS involvement may occur in the disease, the most common early findings include fever, hepatomegaly, and splenomegaly. The laboratory findings include cytopenia, hypertriglyceridemia, elevated ferritin, and hypofibrinogenemia. Markedly reduced or absent NK-cell and T-cell activity have been found to be associated with primary HLH [20]. The association between NK activity and HLH is of great interest due to its potential diagnostic value. It has been found that almost all children with verified familial disease have extremely low or absent NK-cell activity, and this value only normalizes after successful HCT. By contrast, those patients with secondary HLH who have low NK-cell activity at presentation normalize their NK-cell activity later. The differentiation of familial HLH from secondary HLH may be dif-

Table 78.2 Revised diagnostic guidelines for hemophagocytic lymphohistiocytosis (HLH) (HLH-2004): the diagnosis of HLH can be established if either (1) or (2) is fulfilled 1. A molecular diagnosis consistent with HLH 2. Diagnostic criteria for HLH fulfilled (five out of the eight criteria below) A. Initial diagnostic criteria (to be evaluated in all patients with HLH) Fever Splenomegaly Cytopenia (affecting >2 of 3 lineages in the peripheral blood): Hemoglobin <90 g/L (in infants <4 weeks: hemoglobin <100 g/L) 9 Platelets <100 × 10 /L 9 Neutrophils <1.0 × 10 /L Hypertriglyceridemia and/or hypofibrinogenemia: Fasting triglycerides >3.0 mmol/L (i.e. >265 mg/dL) Fibrinogen <105 g/L Hemophagocytosis in bone marrow or spleen or lymph nodes No evidence of malignancy B. New diagnostic criteria Low or absent natural killer cell activity (according to local laboratory reference) Ferritin >500 μ/L Soluble CD25 (i.e. soluble interleukin-2 receptor) >2400 U/mL

ficult as about half of the patients present with infections, and the family history at the time of presentation may or may not be helpful. In this situation, testing of NK-cell activity and its normalization pattern may be helpful in distinguishing primary from secondary disease. This observation makes it mandatory to test for the NK-cell activity prior to the initiation of HCT. The histopathologic criteria of diagnosis include infiltration of the bone marrow, lymph nodes, liver, and spleen with nonmalignant lymphocytes and histiocytes in addition to the evidence for hemophagocytosis in these tissues [21]. HCT procedures have been performed only for autosomally recessive primary HLH. If not treated, the primary HLH is rapidly fatal with a median survival of about 2 months [22]. Several treatment strategies with cytotoxic agents have been tried in the past without much success. The most effective of these moderately effective strategies was the combination of vinblastine and steroids. The treatment of HLH has also included etoposide (VP-16) in combination with other agents [23]. Approximately 60% of patients have also received CNS treatment with intrathecal methotrexate and cranial irradiation [18]. Immunosuppressive drugs such as cyclosporine and antithymocyte globulin (ATG) have been shown to be effective in HLH [24] for shortterm control of disease. Historically, the probability of survival at 5 years had been found to be 10%, with all patients ultimately succumbing to their disease. In 1994, the Histiocyte Society developed a treatment protocol (HLH-94) designed for treatment of primary HLH [25]. The protocol includes initial treatment with CSP, VP-16, and steroids with consideration for HCT when patients attain remission. Fischer et al. achieved the first major breakthrough in the treatment of this fatal disease. An allogeneic HCT procedure was performed successfully that had cured the disease in the patient [26]. Since then, several reports have been published describing the efficacy of HCT using related or unrelated donors. Most of the reports have originated from single HCT centers or from transplant registries [27–30]. The current consensus indicates that HCT should be performed after an initial 8 weeks of treatment with chemotherapy if a suitable donor is available. Patients without a suitable donor should continue to receive standard-dose chemotherapy. It is preferred that the patient should be in a stable or quiescent disease state before an HCT procedure is contemplated. In a follow-up study to the first publication of results with HLH-94, it is clear that the HCT procedure for primary HLH offers an excellent chance of cure. On the HLH-94 protocol, the estimated overall 3-year probability of survival after HCT for patients recruited during the period of 1995–2000 was 64%. The specific survival between different types of graft used, however, varied. The 3-year survival was 71% with matched related donors, 70% with matched unrelated donors, 50% with family haploidentical donors, and 54% with mismatched unrelated donors [31]. The results, however, indicate that the long-term diseasefree outcome was comparable for unrelated donor transplants and matched sibling donors (approximately 70% at 3 years). In contrast, the use of T-cell-depleted grafts from haploidentical donors has met with less success (50% survival at 3 years). A recent study by Ouachee-Chardin et al. published the largest cohort of 48 patients with HLH undergoing HCT [32]. The major determinant for the failure and death in the transplanted patients was found to be active disease at the time of HCT. In fact, the combination of active disease with a T-cell-depleted haploidentical graft resulted in the worst outcome (Fig. 78.2). It may be possible that the active disease hampered donor cell engraftment because of a possible inhibitory effect of cytokines on hematopoiesis. As suggested by this and other reports, it is of great importance to perform HCT as soon as complete remission has been obtained for the disease [16,32].

Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders (a)

(b)

(c)

(d)

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Fig. 78.2 Survival of patients with hemophagocytic lymphohistiocytosis (HLH) after hematopoietic stem cell transplantation (HSCT). (a) Overall survival (n = 48) calculated from the time of the first attempts at HSCT. (b) Survival according to donor origin. Because of low numbers and similar data, patients who received a transplant with a matched sibling donor (MSD) and an unrelated donor (URD) have been grouped together (p = 0.3 log-rank test) (c) Survival related to status of HLH at HSCT (complete remission [CR] versus active disease [AD], p = 0.053 log-rank test). (d) Survival according to a combination of donor compatibility and disease activity (for patients with AD, MSD-URD versus haploidentical HSCT, p = 0.03 log-rank test; for patients in CR, p = 0.39 log rank test. y, years. (Reprinted from [32], with permission.)

Human leukocyte antigen (HLA)-matched sibling donors are the preferred choice for HCT in this disorder. However, one caveat that should be kept in mind while selecting a sibling donor is that a healthy-appearing sibling donor may develop HLH later. If this occurs, it is possible that the disease will be transferred from the donor to the recipient. As mentioned earlier, the presence of extremely low or no NK-cell activity could be used as a guide to select the sibling donor for HCT in primary HLH. In the event that a suitable sibling donor is not available, alternative donors should be sought. Alternative donor options include closely matched relatives, unrelated donors of marrow or human umbilical cord blood. The preparative regimens in most of the published cases have consisted of chemotherapy, containing myeloablative doses of chemotherapeutic drugs. Most of the centers have used high-dose BU, CY, and VP-16 as the conditioning regimen [30]. In some patients, additional immunosuppression has been provided by ATG or the antieukocyte function-associated antigen-1 monoclonal antibody. These regimens have been well tolerated by children with HLH. TBI has not been favored as histiocytes may not be sensitive to this measure. There are limited data available on reduced-dose conditioning in HLH [33]; it is not yet possible to make definitive recommendations for such regimens. Following HCT, patients usually achieve full donor chimerism. However, repeated analyses should be performed at regular intervals. In some cases, a mixed chimeric state can persist with low quantities of

donor cells present in the graft. It is known that mixed chimerism of over 10–20% is associated with stable complete remission for up to 20 years [16,32]. Secondary graft rejections have been seen below the threshold level of 10% donor cells in the chimera. In patients with declining levels of donor cells, follow-up studies are important as a progressive decrease in donor cells may indicate early relapse and the need for further intervention. Overall, the major causes of death following HCT for HLH are transplant-related complications including graft failure, infections, and graft-versus-host disease (GVHD). In a few cases, progression of the CNS disease has occurred within weeks after HCT. Active or poorly controlled CNS disease before HCT initiation has led to deterioration after transplantation. The relapse rates for the underlying disease have been reported in the range of 0–55% [28–30]. Actuarial 5-year diseasefree survival in HLH patients who underwent HCT has been compared with those HLH patients who did not receive the HCT procedure. This disease-free survival was reported to be 66% in HLH patients who were transplanted versus only 10% in those patients who did not receive HCT (Fig. 78.3). Most long-term survivors of HCT for HLH experience good quality of life and are able to discontinue all disease-related medications. Stabilization and improvement of neurologic symptoms is commonly seen. The volume loss of the cerebrum and demyelination often seen during the height of HLH disease activity is reversible within 1–3 years of successful engraftment. Long-term follow-up of survivors of bone

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100

BMT– BMT+

90 80 Survival (%)

70

66.0 (37.8)

60 50 40 30 20

10.1 (9.6)

10

p = 0.00006

0 0

12

24

36

48

60

Months from diagnosis

Fig. 78.3 Kaplan–Meier survival estimates of 122 children with hemophagocytic lymphohistiocytosis treated with or without bone marrow transplantation (BMT). (Reprinted from [135], with permission.)

marrow transplantation for HLH indicates that most children return to normal quality of life. However, children who have severe neurologic problems in the beginning due to HLH may have persistent cognitive problems and learning disabilities. For this reason, neuropsychiatric evaluation is recommended before the transplant and on a periodic basis at regular intervals post HCT. In summary, there have been major strides in the understanding of HLH at the molecular level. HLH protocol 2004 includes changes reflective of increased understanding in the diagnosis and treatment of HLH. The use of HCT in this disease has provided the best results and remains the treatment of choice as chemotherapy alone is not curative. Adequate therapy to achieve remission before HCT should be attempted since the transplant outcome appears to be improved when patients are in remission.

Granulocyte disorders The granulocyte disorders are discussed here as quantitative disorders, in which the number of granulocytes is low, or as qualitative disorders, where the function of granulocytes is altered. Both groups of disorder are very heterogeneous and vary widely in their clinical severity. Quantitative granulocyte disorders Neutropenia is defined as a condition in which the circulating neutrophil count is less than 500/μl. Severe neutropenia means an absolute neutrophil count of less than 200/μl. Severe neutropenia is often associated with life-threatening infections, especially if it is prolonged or is associated with immune deficiency. Neutropenia is common to a heterogeneous group of disorders resulting from decreased production or release of maturing and differentiating myeloid precursor cells from the bone marrow. Neutropenia may be primary congenital or secondary acquired. This part of the chapter will cover the intrinsic maturation and differentiation defects of the early myeloid precursors. Severe congenital neutropenia (Kostmann’s syndrome) Severe congenital neutropenia (SCN), also known as Kostmann’s syndrome, is an inherited disorder with an undefined inheritance pattern. It occurs with an estimated frequency of about 1–2 cases per million, with equal gender distribution. A persistent absolute neutrophil count of 0.2 × 109/L or less is required for making a diagnosis. Recurrent bacterial

infections usually occur in the first year of life. Typically, these patients experience in early infancy recurrent pneumonia, otitis media, gingivitis, and perineal or urinary tract infections. Until recently, 50% of these patients died as a result of infections before 1 year of age, and only 30% survived beyond 5 years of age [34]. The biology of SCN is not completely understood. At the cellular level, a maturation arrest of myelopoiesis at the promyelocyte or myelocyte stage occurs, resulting in neutropenia [35]. Contrary to the earlier suggestions of defective G-CSF production or defective response of the cells to G-CSF, the current data indicate a normal or even increased endogenous G-CSF biological profile and activity in the cells of patients with SCN. In vitro measurements of colony-forming units-granulocyte– macrophage also appear to be normal. Early G-CSF receptor mutations at birth have not been noted in the SCN patients. These mutations have, however, appeared in those SCN patients who have developed leukemia or myelodysplastic syndrome (MDS) later in their lives [36]. This observation suggests that these GCSF receptor mutations are acquired and may not have a causal relationship with the genesis of this disease. The identification of mutations within the ELA-2 (elastase-2) gene as a cause of cyclic neutropenia has led to similar studies in patients with SCN. Heterozygous ELA-2 mutations have been found in 60% of the patients with SCN [37]. Analysis of SCN families with an autosomal dominant inheritance of elastase mutation and a case with mosaic expression of mutant ELA-2 has provided the strongest evidence to implicate ELA-2 mutations in the etiology of SCN. In addition to ELA-2 mutations, a number of genetic aberrations have been found in patients with disorders associated with Kostmann’s syndrome. Genes that have been found to be mutated in a minor subgroup of patients are GF-1, CXCR4, SBDS, WASP, the gene for glucose-6-phosphatase translocase, Taz-1, AP3B1, and some others [38]. It is now well established that patients with SCN are at risk for the development of MDS and acute myeloid leukemia (AML). The acquisition of G-CSF mutations in SCN patients can be used advantageously to predict the risk of subsequent development of leukemia/MDS in these patients. In a recent report from the SCN International Registry, 375 patients with SCN were followed for the development of MDS or AML. The potential risk for the development of MDS/AML increased with the duration of SCN. The cumulative incidence of MDS/AML was 9% after 6 years of therapy. The incidence increased to 23% after 10 years and 33% after 12 years [39,40]. These ominous findings have been linked to point mutations in the G-CSF receptor gene [41,42]. Also, cytogenetic abnormalities such as monosomy 7 have also been noted in these patients [43]. In light of these findings, SCN can be considered a preleukemic condition. Based on the clinical response to G-CSF treatment, SCN patients have been categorized into G-CSF responders and G-CSF nonresponders. This conclusion was initially deduced from data on 304 patients enrolled on the SCN International Registry in 1994 [44]. In this study, 95% of the patients had responded to the G-CSF treatment. The G-CSF responders have been treated with G-CSF therapy in doses of 3–10 μg/kg/day. This response was biologically explained on the basis of normal or increased expression of G-CSF receptors on the cells. Since the myeloid precursor progenitors in SCN patients retain the capacity to differentiate to normal myeloid cells in response to pharmacologic doses of G-CSF [44–46], a continuous treatment strategy with recombinant G-CSF has been adopted for most of the SCN patients. The continuous treatment with G-CSF results in an increase in the number of mature myeloid cells in the circulation and a concomitant decrease in the incidence of serious infections. Those patients who do not respond to G-CSF doses of 120 μg/kg/day, and still suffer from bacterial infections, are classified as the G-CSF

Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders

Reticulocytes (%)

nonresponders or patients refractory to this treatment. This population constitutes about 5% of the patients in the SCINR study group mentioned above. In the nonresponders, the G-CSF gene defect has not been elucidated. The G-CSF nonresponders, and those patients who develop cytogenetic and G-CSF receptor mutations during the course of the disease, are considered candidates for HCT. All SCN patients who develop MDS/AML are also considered candidates for HCT. The monitoring for cytogenetic and G-CSF receptor mutations is, therefore, emphasized for two reasons: first, for timely intervention with HCT, and second, for the prediction of risk for developing MDS/AML in these patients, with a subsequent HCT procedure. A limited number of patients with SCN, including those documented in the International Bone Marrow Transplant Registry, have undergone HCT procedures using HLA-identical sibling donors. One patient received CY as a single preparative agent and attained only partial lymphoid engraftment for 6 months [45]. Another patient received an HLA-identical sibling donor transplant at 20 months of age. This patient is reported to be alive and disease free 20 years after the HCT procedure [46]. The clinical course of this patient is depicted in Fig. 78.4. The patient engrafted around day 15 and achieved full and durable chimerism. In 2001, a successful fully matched unrelated donor transplant procedure was performed in a G-CSF nonresponder [47]. The preparatory regimen contained TBI, VP-16, and CY. ATG was administered to prevent graft rejection. The patient engrafted promptly, and the preexisting pulmonary abscesses resolved. In 2004, Myers et al. reported successful engraftment following unrelated donor transplantation in an alloimmunized patient with SCN [48]. At our own institution, we have successfully transplanted a G-CSF nonresponder with unrelated donor graft using a high-dose conditioning regimen (unpublished data). At the time of writing, this patient is 9 months post HCT with full donor chimerism. The conditioning for matched unrelated donor transplants has been fully myeloablative with either BU, CY, and ATG, or TBI, BU,

10

5

and ATRG. With advances in supportive care, fully matched unrelated donor transplants have an excellent chance of success in patients with SCN. RIC regimens are currently being developed for consideration in patients with a high risk for toxicity [49]. Pretransplant chemotherapy in SCN patients with MDS/AML has important bearing on the outcome of HCT in these patients. In a report by Choi et al., a cohort of six patients has been reported that developed MDS/AML while on therapy with G-CSF. All six patients received HCT. Four of these six patients who died because of transplant-related complications had frank AML and had received pretransplant chemotherapy. Two patients who survived had early MDS and had not received any chemotherapy before the transplant. These findings emphasize the importance of frequent monitoring of all SCN patients in order to promptly recognize the development of clonal chromosomal abnormalities and MDS for early intervention with HCT in these patients [40]. RD RD is a rare congenital disorder characterized by severe combined immunodeficiency and agranulocytosis manifesting with life-threatening fulminant infections. The term “reticular dysgenesis” was introduced in 1959 when male twins were reported with this syndrome and subsequently died of the disease [50]. An autopsy was performed on these patients, the findings showing depletion of lymphocytes and Hassall’s corpuscles in the thymus, absent lymph nodes, tonsils or Peyer’s patches, and lack of myeloid activity in the bone marrow [50]. At the molecular level, the primary defect in RD has not yet been identified. At the cellular level, a maturation arrest in the myeloid lineage beyond the promyelocyte stage has been observed along with global impairment of lymphoid maturation. However, erythropoiesis and megakaryopoiesis are found to be normal in RD, indicating the existence of an intact pluripotent hematopoietic stem cell pool in these patients. These observations are utilized to make a diagnosis of RD using established clinical and laboratory data, namely agranulocytosis,

ATS PC TX TBI

0 10

WBC/mm3 × 103

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6 4 2

Platelets/mm3 × 103

0 500 400 300 200 100 0 –15 –10 –5

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5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Days post-transplantation

Fig. 78.4 Clinical course of a patient receiving transplant for infantile agranulocytosis (Kostmann’s syndrome). Note the presence of granulocytes following transplantation. ATS, antithymocyte serum; PC, procarbazine; TBI, total body irradiation; TX, transplant. (Reproduced from [46],with permission.)

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lymphocytopenia, a bone marrow finding of early myeloid maturation arrest, failure of in vitro assays to produce mature granulocytes from precursors, lack of lymphocyte functions, and thymic aplasia. In contrast to other variants of severe combined immune deficiency with neutropenia, no sustained responses to myeloid growth factors have been observed in RD. So far, the only curative treatment described for RD is allogeneic HCT. Untreated patients with RD die from infections within the first days or weeks of life. Prior to 1983, nine cases with this disorder were reported. Their survival ranged from 3 days to 17 weeks [8,51–54]. In 1983, a cure was reported for RD following an HCT procedure from an HLA-identical sibling [55]. A preparative regimen of BU and CY was used, and the patient achieved full hematopoietic and lymphoid reconstitution. Several other reports have since been published on successful HCT in RD patients using HLA-identical, T-cell-depleted, HLA-haploidentical, and umbilical cord blood unrelated donor grafts [27,37,55–62]. In one report using unrelated umbilical cord grafts, the patient failed to engraft initially but fully engrafted after a second unrelated cord blood transplantation procedure [61]. A challenging feature of HCT for RD is not only the choice of the donor, but also the intensity of the conditioning treatment to obtain both lymphoid and myeloid reconstitution. This observation has been repeatedly substantiated from data on HLA-haploidentical transplants. A Tcell-depleted haploidentical graft had been successfully transplanted following administration of BU, CY, and ATG [53]. In one report, using haploidentical donors, two patients could be retransplanted successfully only when an intensive high-dose conditioning regimen consisting of 700 cGy TBI was utilized prior to the HCT procedure [60]. In another report, 10 patients receiving HLA-haploidentical grafts were studied; half of them were given intensive BU/CY conditioning, while the other half received either low-dose BU with CY, or ATG with CY without BU [62]. Three of the five patients who had received intensive high-dose conditioning survived with lymphoid and myeloid reconstitution. The group that did not receive the intensive preparatory regimen or was treated with the regimen without BU failed to demonstrate stable engraftment of donor hematopoietic stem cells. This finding again indicates the paramount importance of using intensive conditioning prior to the HCT procedure in RD patients. Cyclic neutropenia Cyclic neutropenia is usually a familial disorder in which patients have regular cyclic fluctuations in the number of blood neutrophils. The genetic transmission may be autosomal dominant with variable expression [63]. The gene responsible for cyclic neutropenia has been identified [64]. Clinical signs and symptoms include fever, malaise, periodontitis, mucosal ulcers, sore throat, and lymphadenopathy. Most patients live normal life spans with a gradual amelioration of symptoms. Ten percent of patients experience frequent serious infections. Laboratory investigation reveals periods of severe neutropenia (<200 neutrophils/μl) lasting for 3–10 days alternating with periods of normal neutrophil counts. The cyclic fluctuation occurs usually every 21 days with a range of 14–36 days. For each individual patient, the cycle length is constant. Bone marrow precursors also reveal concomitant cyclic oscillations. Bone marrow myeloid precursor cells are present in highest numbers at the time of the nadir of peripheral neutropenia. Therapy with G-CSF improves the neutrophil counts and reduces the frequency of symptoms. Clinical manifestations of cyclic neutropenia do not justify the use of HCT. However, HCT has contributed towards a better understanding of this disorder. In an autosomal recessive collie dog model, cyclic neutropenia can be corrected by HCT [65]. To date, no human transplants have been reported for cyclic neutropenia. In one patient who was trans-

planted with an allogeneic donor graft with cyclic neutropenia, the disease was found to be transferred to the recipient during an HCT procedure [66]. Cartilage–hair hypoplasia Cartilage–hair hypoplasia, an autosomal recessive disorder, is encountered primarily in the Amish and Finnish populations. The abnormal gene for cartilage–hair hypoplasia is located on the short arm of chromosome 9 [67]. Characteristic findings include short-limbed dwarfism, fine hair, moderate-to-severe neutropenia (absolute neutrophil count 100– 200/μl) and defects of cell-mediated immunity [68,69]. The combination of neutropenia and cellular immunity defects leads to recurrent bacterial and viral infections, especially reactivation of varicella zoster infection, in these patients. Allogeneic HCT has been used to correct the hematologic and immunologic defects of cartilage–hair hypoplasia [70,71]. Expectedly, the phenotypic hair and bony abnormalities have not been corrected by HCT. Six patients with cartilage–hair hypoplasia have been reported to have undergone HCT. One of the six patients received the preparative regimen consisting of CY, cytosine arabinoside, and thioguanine. This patient achieved complete lymphoid engraftment of donor origin and demonstrated otherwise mixed chimeric hematopoiesis. The remaining five patients received BU and CY and achieved full donor chimerism for lymphoid as well as all other hematologic parameters. The experience with allogeneic HCT is still limited, but severely affected patients seem to benefit from the procedure. Dyskeratosis congenita Dyskeratosis congenita, a rare congenital syndrome, was originally described in 1906. The condition is genetically heterogenous, and all three patterns of inheritance have been recognized [72]. However, the majority of the patients are male, and the defective gene has been linked to chromosome Xq28. This genetic defect results in a defective nucleolar protein called dyskarin, which is associated with human telomerase RNA [72]. In dyskeratosis congenita, like Fanconi’s anemia, associated DNA repair defects have been noted. Clinically, dyskeratosis congenita is a multisystem disorder marked by nail dystrophy, oral leukoplakia, increased skin pigmentation, and a variable degree of bone marrow aplasia. Bone marrow failure is the principal cause of death, followed by predisposition to malignancy and pulmonary failure. Immunologic manifestations include defective cell-mediated immunity [73,74]. Allogeneic bone marrow transplantation has been used successfully to correct the hematologic abnormalities [75]. It is noted that patients with this syndrome require less intensive conditioning [74]. Twenty-two patients have been reported in the literature to have undergone HCT with high-dose conditioning. Of the 22 patients, only four patients received grafts from unrelated donors. Ten of the 22 patients survived longer than 3 years post transplant. In addition to developing short-term complications like organ damage and sepsis, long-term complications carried a high mortality rate [76]. Veno-occlusive disease, pulmonary fibrosis, and renal microangiopathy developed months after the HCT. In a published report of five patients with dyskeratosis congenitaassociated severe aplastic anemia who had received HCT using HLAidentical related donor grafts, four patients died 2–8 years post HCT with renal microangiopathy, veno-occlusive disease of the liver, Evans’ syndrome or invasive aspergillosis [76]. The one patient living at the time of publication had developed anemia, polyarthritis, pulmonary fibrosis, and gastrointestinal malabsorption syndrome 7.5 years after the HCT procedure. In view of the occurrence of the late HCT-associated complications, it is important that a complete baseline evaluation of kidneys, liver, and respiratory tract be performed prior to the initiation of the HCT procedure in these patients.

Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders

Since patients with dyskeratosis congenita have shown poor tolerance to high-dose conditioning regimens, reports using RIC approaches have emerged [77,78]. In one study using RIC, the patient received 200 cGY TBI and 90 mg/m2 fludarabine in a matched sibling donor setting. GVHD prophylaxis was with mycophenolate and cyclosporine. This patient achieved 100% donor chimerism with excellent clinical outcome 2 years post transplant [77]. In another report, two children with dyskeratosis congenita underwent unrelated donor transplant with RIC with fludarabine, CY, and ATG. The prophylaxis for GVHD was provided with steroids and cyclosporine. Both of the patients did very well with full donor chimerism [78]. Whether long-term toxicity is also reduced with low-intensity conditioning regimens remains to be seen in longer follow-ups. However, the use of certain conditioning agents such as BU and irradiation should be avoided because of the increased predisposition of dyskeratosis congenita patients to chromosomal instability, tumorigenicity, and pulmonary and vascular complications. Shwachman–Bodian–Diamond syndrome Shwachman–Bodian–Diamond syndrome (SBDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, metaphyseal dysostosis, and bone marrow hypoplasia [34]. Cyclic or persistent neutropenia is a common finding. However, in 10–25% of patients, trilineage pancytopenia may also be seen. Recurrent bacterial infections occur early in childhood and can contribute to early mortality. Progression to myelodysplasia and transformation to acute leukemia are seen in patients who have pancytopenia as the predominant finding. Overall survival without any treatment varies with different subpopulations. In patients without hypoplasia and leukemic transformation, the projected 50% survival is 35 years. However, this expectation is reduced to 24 years in patients with pancytopenia and further decreased to 10 years in those with leukemic transformation [34]. Approximately 90% of patients meeting clinical criteria for the diagnosis for SBDS carry mutations in the SBDS gene. SBDS maps to the 7q11 centromeric region of chromosome 7 [79]. SBDS is a highly conserved gene of unknown function. In humans, the SBDS protein is localized to both the cytoplasm and nucleus, and shuttles in and out of the nucleolus in a cell cycle-dependent manner. Conceivably, HCT would be the only curative treatment option for the bone marrow dysfunction in SBDS. However, mixed results have been observed with allogeneic HCT as a therapeutic approach for SBDS patients with hematologic abnormalities. Fifty-eight patients have been reported as undergoing HCT for bone marrow failure and/or leukemic transformation with SBDS. Most of the published data comes from case reports and small series of cases [79–83]. Patients have received grafts from matched sibling donors, matched unrelated donors, and human umbilical cord blood [79]. The conditioning regimens were either with BU/CY/ATG or TBI/CY. Forty percent of the transplant patients died due to HCT-related complications of sepsis, regimen-related toxicity, and GVHD. Risk of graft failure did not appear to be significantly elevated. As expected, the patients with marrow aplasia had a relatively better survival than the patients with MDS or leukemic transformation. A single report exists for the use of RIC in a patient with SBDS/AML who received an unrelated donor transplant. Full engraftment is reported in this surviving patient [80]. At the moment, follow-up of these patients is relatively short (3 years). Long-term studies will be needed to look at the real impact of HCT in SBDS patients. Qualitative neutrophil disorders The major role of neutrophils is to defend the host against a variety of infectious agents. To accomplish this task, the neutrophils must first sense infection, migrate to the site of infection (chemotaxis), adhere to

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the membranes of the blood vessel wall, and then destroy the offender (phagocytosis) through the release of granules that take part in the activation of oxidative-dependent and oxygen-independent pathways of microbial killing. The presence of these different components of the microbial killing cascade is essential for the appropriate function of neutrophils. In the qualitative disorders discussed below, neutrophilia is usually present in response to infection. However, one or more of the functional components of the neutrophil attack may be faulty. Neutrophil actin abnormality This is a rare inherited autosomal recessive disorder in which defective granulocyte motility leads to inadequate chemotaxis resulting in serious recurrent infections. Granulocyte counts are, however, either normal or even elevated. This disorder was initially described in an infant in 1974 who had failed to form abscesses during recurrent life-threatening infections with Gram-positive and Gram-negative organisms [84]. A Rebuck skin window showed abnormal cellular migration. Although actin levels are normal in the affected neutrophils, the polymerization of actin is defective, leading to poor or absent migration of neutrophils to the chemotactic stimuli. After the initial report, more children have been reported with neutrophil actin deficiency or its variants [85]. Allogeneic HCT has been attempted in a few patients with this disorder [85,86]. According to the published reports, one of three patients survived and was cured. The successfully treated patient received a high-dose regimen with BU, CY, and ATG. The other two patients engrafted but died due to treatment-related complications, mainly infections. It is possible to correct the defect in neutrophil actin abnormality by HCT using a heterozygous histocompatible sibling donor. Since the disease course is very aggressive, supportive care alone is usually not sufficient to allow prolonged survival of these patients. LAD 1 First described in 1974, LAD 1 is a rare heterogeneous autosomal recessive disorder in which neutrophil adhesion, chemotaxis, and ingestion of the C3bi-opsonized microbes is impaired [87]. The neutrophils fail to migrate in the Rebuck skin window and also exhibit defects in neutrophil functions of adhesion and phagocytosis. The condition is usually detected in early childhood as a result of frequent, life-threatening bacterial and fungal infections, delayed umbilical cord separation, and neutrophilia. The infections are mostly localized in the skin, subcutaneous tissues, middle ear, and oropharynx. At the molecular level, the basic defect lies in the β2 integrin adhesion molecule (CD11/CD18). The CD11/CD18 glycoprotein family consists of three heterodimeric proteins each composed of α and β chains. Translocation of α and β chains to the cell surface requires assembly of the αβ heterodimer. The mutations in the CD11/18 deficiency have been found to be in the β2 chain, which prevent the assembly of the αβ heterodimer and thereby translocation of the heterodimer to the cell surface [88]. The cells with the mutated glycoprotein are unable to adhere or migrate in response to chemotactic stimulation. Since the β2 chain is constant for each family and is present on the surface of the leukocytes, flow cytometric analysis with pertinent monoclonal antibodies can help identify those affected by the disorder. The severe clinical phenotype with less than 0.3% of the normal amount of β2 integrins is more commonly seen as compared with the moderate variant in which β2 integrin levels of 2.5–11% of normal are observed. Since most of the children with LAD experience severe infections and die early in the course of their disease, early diagnosis and treatment, possibly with HCT, should be attempted. Several reports describe the course of HCT in patients with LAD 1. So far, 14 patients have been reported to have received HCT. Of these 14, five patients underwent HLA-matched sibling donor HCT, and nine patients had received grafts

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from two antigen-mismatched or haploidentical related donors [89,90]. In this study of 14 patients, the majority achieved either full or mixed chimerism. In most of the patients with mixed chimerism, a variable number of donor cells were noted with no increased incidence of infections, except in one patient with 2–15% donor cells who developed mild gingivitis. Also, two patients with immunologic graft rejection achieved full chimerism after treatment with anti-CD2 or antieukocyte functionassociated antigen-1 monoclonal antibody. More recently, there have been reports of successful unrelated HCT utilizing fully matched donors and one antigen-mismatched donor [91,92]. Although the follow-up is short for these patients, engraftment was prompt and the patients fared well. Beside CD11/CD18 deficiency, there are other leukocyte adhesion molecules called selectins, defects in which may result in significant disease. Two patients have been described with the absence of neutrophil receptors for E-selectin, resulting in defective adhesion. To date, HCT has not yet been attempted in patients with selectin deficiency.

CHS CHS is an autosomal recessive disorder. Clinically, it is characterized by partial ocular and cutaneous albinism, and an increased susceptibility to bacterial infections and compromised platelet function. Partial albinism and photophobia are usually noted in early infancy, and increased susceptibility to respiratory and cutaneous infections soon becomes apparent. Decreased platelet function leads to bleeding tendency. Other organ systems involved include the hair, adrenal glands, pituitary gland, and peripheral nerves [93]. The disease is manifested in two forms, namely an initial stable phase followed by an accelerated phase. Stable-phase disease may or may not be associated with serious infections, while accelerated-phase disease may be complicated by fulminant infections and massive hepatosplenomegaly, lymphadenopathy, anemia, neutropenia, and thrombocytopenia [94]. The accelerated phase is uniformly fatal, and no long-term survivors of the accelerated phase have been reported. In some patients, the diagnosis of CHS is not apparent until the accelerated phase has developed. Although phagocytosis per se occurs at a higher than normal rate in neutrophils, and post-phagocytic metabolic burst is also normal [95], the basic abnormality lies in the production of abnormal lysosomes and granule formation in cells of the myeloid cell series [96]. This abnormality leads to defective microbial killing and clinical manifestations of CHS. The molecular defect has been reported to be an abnormal calcium uptake pump in CHS lysosomes, resulting in an inefficient and incomplete post-phagocytic delivery of lysosomal enzymes into the phagosomes [96]. This defect results in delayed intracellular destruction of bacteria by the Chediak–Higashi leukocytes [97]. In addition, cellular response to chemotactic stimuli also appears to be defective. In the laboratory, light and electron microscopy can be used to diagnose CHS by demonstrating abnormal granules and lysosomes. Neutrophils containing giant azurophilic granules along with abnormal lysosomes can be demonstrated. Monocytes and macrophages show abnormal lysosomes as well. Both lymphocytes and NK cells also have giant cytoplasmic granules with decreased NK function. In platelets, the content of dense granules is decreased, leading to abnormal platelet function. The bone marrow examination of these patients displays ineffective myelopoiesis. The first HCT procedure for CHS using a sibling donor was performed in a patient in the accelerated phase [98]. CY alone was used as a preparative regimen. The patient, however, could not achieve sustained engraftment, probably due to inadequate preparation. The patient

relapsed back into the accelerated phase, and a second transplant procedure from the same donor was attempted. This time, however, a preparative regimen containing CY and TBI was used. This strategy resulted in stable engraftment. Another patient who was in the stable phase of the disease was similarly treated with CY and TBI, resulting in full donor engraftment and correction of the functional defect [99]. A series of 10 other patients has been published [100]. Two patients were in stable disease and eight patients were in the accelerated phase of CHS. Seven patients received HCT from sibling donors, and the other three received one-antigen-mismatched or haploidentical donor grafts. The preparative regimen included BU, CY, and etoposide in eight patients. One patient received additional cytosine arabinoside. Three of the seven long-term survivors achieved full donor chimerism, while four patients demonstrated mixed chimerism. In one patient with a low level of donor chimerism, persistent bacterial infections were seen. As expected, in none of these patients were the cutaneous, ocular, and neurologic manifestations corrected. Few reports describe patients receiving RIC leading to mixed chimerisms with as low as 20% donor cells. These patients have continued to do well without developing the accelerated phase of the disease [101,102]. It is clear that HCT can be used successfully to treat patients with CHS in both the stable as well as the accelerated phase. HCT should be considered in the early stages as soon as the diagnosis is established. Patients with stable-phase disease are expected to tolerate the procedure better than those treated in the advanced phase when serious organ damage might have taken place. If matched sibling donors are not available, unrelated donors can be considered. As evident from the discussion above, a good number of patients develop mixed chimerism in spite of the aggressive preparatory regimen used. Most of these mixed chimeric patients do not progress to the accelerated phase of CHS and continue to do well. There are results from long-term follow-up of patients several years post HCT. Eleven out of 20 patients who underwent HCT for CHS 20 years ago have shown signs of progressive neurologic deterioration consistent with the progression of CHS [103]. Similar findings were also suggested by the earlier follow-up reports on patients with CHS [104]. CGD CGD is a rare clinical syndrome resulting from defective neutrophil function. Both autosomal recessive and X-linked patterns of inheritance have been described. Although the carriers are usually asymptomatic, females with extreme lyonization may be affected. The median age at presentation of the disease is 7 years (range 0.4–15 years). Clinically, the course of CGD is extremely variable. Some patients present within their first year of life, while others may present later. Most patients suffer from recurrent bacterial and fungal infections. Those presenting later in life may have fewer life-threatening infections than those who present early. The most common bacterial infections are from catalase-positive bacteria such as Staphylococcus aureus and enteric bacilli. Aspergillosis is the most common fungal infection. Infections are usually cutaneous, sinupulmonary, and lymph adenitis. The occurrence of hepatic abscesses is characteristic in these patients. Serious and persistent infections may lead to formation of granulomas in vital organs. In CGD, neutrophils are characterized by a defective respiratory burst formation during phagocytosis and an inability to generate superoxide. The impaired superoxide production usually occurs transiently in each phagosome and leads to recurrent bacterial and fungal infections. Those bacteria that have their own mechanism to produce hydrogen peroxide to generate hypochlorous acid via the myeloperoxidase pathway of the phagocytic killing pose little problem. However, microbes such as cata-

Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders

lase-positive bacteria can produce very low amounts of hydrogen peroxide in the phagocytic vacuoles. This defect results in insufficient production of hypochlorous acid to contribute to significant bacterial killing, culminating in recurrent infections with these bacteria [105]. The molecular basis of CGD has now been well defined. The respiratory burst defects have been found to be due to mutations in one of the four subunits of the NADPH oxidase complex. Of the four subunits, two are membrane associated, while the other two are located in the cytosol [106]. The gene for one of the membrane-associated subunits is located on the X chromosome, while the others are encoded by genes on chromosomes 1, 7, and 16. The disease is classified according to the underlying respiratory burst unit defect. In two large studies of 140 pediatric patients, defects in the X-linked gene for the gp91phox subunit of cytochrome B accounted for 62% of the cases. The remainder were found to be due to autosomal recessive defects in p47phox (27%), p67phox (5%), and p22phox (6%). The nitroblue tetrazolium test is a very important laboratory tool to detect the disease and also to detect the carrier state. With the availability of excellent supportive care, the overall survival of patients with CGD has improved, with life expectancy extending into the 30-year range. Although prophylaxis with trimethoprim–sulphamethoxazole has improved the outcome in these patients [107–109], resistant fungal infections still pose significant life-threatening problems with resultant early mortality. Allogeneic HCT may be appropriate in patients with frequent serious infections despite aggressive medical management. The availability of excellent supportive care with improved outcomes and the extreme variability in the disease course makes selection of appropriate patients for HCT very difficult. Pretransplant evaluation is still of critical importance. The older patients are anticipated to have a higher incidence of HCT-related complications including graft failure and/or rejection. Tissue damage due to chronic inflammation should be carefully evaluated. Hepatic abscesses, ileocecal inflammation, and gastrointestinal

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obstruction can influence the outcome of HCT. The patients should also be evaluated for other associated disease entities such as antibodynegative collagen vascular disease, retinitis pigmentosa, Macleod’s red blood cell syndrome, and Duchenne muscular dystrophy. A significantly higher mortality in patients with the X-linked recessive form of the disease has been reported and should be considered while selecting the patients [109]. Also, the possibility of extreme lyonization should be kept in mind while considering related donors. The number of reports of successful HCT in patients with CGD has increased during the last two decades [110–116]. Most of these HCT procedures have been performed using HLA-matched related donors. With such donors, RIC preparative regimens containing CY or CY with antithymocyte serum were used initially. This resulted in expected autologous recovery with later recurrence of the disease. In one case, 1–2% cells from the donor could be identified for about 3 years, after which time these cells also disappeared [117]. This observation of autologous recovery suggested the necessity for using high-dose conditioning. Subsequently, CGD patients were therefore treated with CY and reduced doses of BU (8 mg/kg). This strategy has produced better outcomes, resulting in mixed chimerism (10–23%) with donor cells persisting for periods of up to 6 years [118]. Horwitz et al. published their results on 10 patients with CGD who underwent matched sibling donor transplants with RIC and a T-cell-depleted graft [119]. The results of this study were mixed as six patients achieved 100% donor chimerism and two patients rejected the graft. Three patients died due to serious infections and GVHD. Although the use of RIC appears to be rational, it has not yet proved to be successful. Recently, reports of successful unrelated HLA-matched HCT with high-dose conditioning have appeared in the literature [120]. Figure 78.5 illustrates the course of an 18-year-old patient with CGD who received an unrelated HCT. This patient was diagnosed with CGD at age 1 year and suffered repeatedly severe life-threatening infections despite

Fig. 78.5 Clinical course of a patient with chronic granulomatous disease (CGD) treated with unrelated donor bone marrow transplantation (UBMT). AMPH-B, amphotericin B; AZT, aztreonam; CRP, C-reactive protein; CsA, cyclosporine A; CY, cyclophosphamide; 5FC, flucytosine; FCZ, fluconazole; G-CSF, granulocyte colony-stimulating factor; Hb, hemoglobin; MCZ, miconazole; mPSL, methylprednisolone; MTX, methotrexate; PAPM/BP, panipenem/betamipron; PIPC, piperacillin; plts, platelets; p.o., orally; SBT/ABPC, sodium sulbactam/sodium cefoperazone; TBI, total body irradiation; WBC, white blood cell count. (Reproduced from [12], with permission.)

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prophylactic antibiotics and granulocyte transfusions. The patient had also been treated with interferon-γ. The patient subsequently developed an Aspergillus infection. Eventually, an unrelated donor HCT procedure was performed successfully in this patient. Although the high-dose regimens have led to more success in obtaining sustained hematopoietic reconstitution with long-term survival, the attendant increased risk of worsening infections and regimen-related toxicity cannot be ignored, especially in older patients. Other complications of HCT in CGD patients include a high risk for graft failure and an increased risk for GVHD. As the occurrence of repeated infections over a long period of time in the older patients may result in significant organ damage, it may predispose this patient population to a higher risk for HCT-related complications such as GVHD. Therefore, active infections or inflammation should be attended in a timely and appropriate fashion as described below. The increased risk for GVHD in CGD patients may possibly be due to the occurrence of repeated infections and/or inflammation resulting in a sustained elevation of TNF-α levels in these patients [121–123]. Administration of TNF-α antagonists has been proposed to neutralize the circulating TNF-α [124]. A study of 27 patients with CGD has been reported evaluating complications (including GVHD) and survival according to different risk factors (infection and/or inflammation) present at the time of HCT [125]. Twenty-five patients received the graft from a compatible sibling donor, and the remaining two patients received their HCT from HLA-matched unrelated donors. All patients were treated with high-dose conditioning. In this study, an overall survival of 23 of 27 had been reported, with 22 of 23 patients cured of CGD (median follow-up of 2 years). Moreover, a slightly higher incidence of severe GVHD (grade III–IV) was found in those patients who had pre-existing overt infections and/or active inflammatory conditions prior to the HCT (4 of 11 patients). Of the 16 patients who did not have any overt infection/inflammation, only three developed mild grade II GVHD. Also, the high-dose conditioning used in the study did not pose a problem in the majority of the patients who did not have pre-existing infections. It is, therefore, important to identify the presence and nature of the infected foci clinically, by laboratory means, and by using the appropriate radiological methods such as computed tomography, positron emission tomography and combined positron emission tomography/computed tomography to institute appropriate therapy [126]. The active infections/ inflammation should be treated adequately using antimicrobials with intracellular action in combination with G-CSF-primed granulocyte transfusions [113]. This aggressive treatment should be carried out before and during the HCT procedure. In conclusion, patients with CGD can be cured with sibling donor or unrelated donor HCT. However, overall outcome is superior with sibling donor transplants. Pretransplant evaluation of patients with severe disease and the treatment of active infections and inflammation prior to and during HCT plays an important role in the overall outcome of CGD patients. The optimal conditioning regimen includes complete myeloablation to prevent autologous recovery and immunosuppression to prevent GVHD in order to attain good results. Since CGD results from single-gene defects in proteins expressed in myeloid cells, this disorder can be considered as an excellent candidate for gene replacement therapy targeted to the hematopoietic cells. Gene replacement therapy can be theoretically very useful in patients with as few as 10–20% nitroblue tetrazolium-positive cells. This concept is supported by the fact that the female carriers in the X-linked variant of CGD have as low as 10% nitroblue tetrazolium-positive cells and are asymptomatic. So far, successful gene therapy has been limited to in vitro experiments where reconstitution of the respiratory burst activity has been displayed in Epstein–Barr virus-transformed lymphocytes [127]. The in vivo clinical attempts to mediate gene transfer into long-lived

human hematopoietic cells have met with a lack of availability of appropriate viral vectors to carry out gene transfer. Griscelli’s syndrome Griscelli’s syndrome is an autosomal recessive disorder that affects both lymphoid and myeloid cells. The basic defect is characterized by lymphoid and NK-cell dysfunction with clinical manifestations of partial albinism with immunodeficiency [128,129]. The disorder is uniformly fatal and is also characterized by an abnormal regulation of the immune system resulting in an accelerated lymphohistiocytic phase along with macrophage hyperactivation as seen in HLH. Variable granulocyte dysfunction may coexist. Two different gene loci, RAB27A and MYO5A, have been identified in patients with the syndrome [130]. Unlike MYO5A, the GTP-binding protein RAB27A is involved in the control of immune regulation and appears to be a key effector of cytotoxic degranulation. Also, all patients with Griscelli’s syndrome who have mutations in the RAB27A gene have developed HLH. A systematic diagnostic approach is essential in suspected cases of Griscelli’s syndrome. The diagnostic steps should include clinical, laboratory, and histopathologic assessment as mentioned in diagnostic guidelines for HLH. The NK-cell activity should also be tested. Finally, mutation analysis of the RAB27A gene should also be performed to confirm the diagnosis. This systematic approach is very important to identify those patients who have a worse prognosis and need timely intervention with HCT. Allogeneic HCT is the only potentially curative treatment of choice in Griscelli’s syndrome. Although the data are limited due to the rarity of the disease, there are several reports of HCT performed in patients with Griscelli’s syndrome using sibling, matched related as well as matched unrelated donors [129,131–133]. In one report of three patients who received an HLA-matched sibling graft, two patients died due to transplant-related complications [129,131]. The third patient achieved full chimerism with normal immune and hematopoietic function and had done very well after the HCT procedure. In another published case of a 6-month-old patient with Griscelli’s syndrome, mobilized peripheral blood hematopoietic cells from her phenotypically HLA-identical mother were used [132]. In this patient, the conditioning included BU, CY, and etoposide. The patient received a CD34+ cell dose of 15.4 × 106/kg along with the CD3+ cell dose of 17.6 × 103/kg recipient body weight. Three months after the transplant procedure was performed, a lymphoproliferative syndrome occurred and was successfully treated. Twenty-six months after HCT, the patient has shown mixed chimerism (52% donor cells) and is well. A recent case report has shown successful HCT in a patient with Griscelli’s syndrome using a matched unrelated donor graft [133]. In this patient, high doses of BU were used along with thiotepa and fludarabine as conditioning prior to HCT. The number of nucleated cells infused was 7.8 × 108/kg recipient body weight. Engraftment in this patient was prompt, and the patient is well 30 months post HCT with complete donor chimerism and normal full blood cell counts. In summary, in addition to a systematic diagnostic approach, an early intervention with HCT in patients with the RAB27A gene mutation is recommended. If a sibling donor is not available, an immediate search for an unrelated donor should be performed. If no histocompatible donors are available, strong consideration should be given to an unrelated donor or haploidentical HCT as the condition is rapidly fatal.

Conclusion Allogeneic HCT remains the procedure of choice for most of the diseases discussed in this chapter. An HLA-matched sibling is the preferred donor. However, if such a sibling is unavailable, a search for a histo-

Hematopoietic Cell Transplantation for Macrophage and Granulocyte Disorders

compatible matched unrelated or haploidentical related donor should not be delayed. In specific cases such as X-linked CGD with severely imbalanced lyonization or in primary HLH, the risk–benefit ratio should be assessed before choosing a sibling versus an unrelated donor. Early diagnosis and HCT is very essential in some of the disorders, such as leukocyte adhesion deficiency and CHS, due to the aggressive and fatal nature of these disorders. An efficient pretransplant evaluation is therefore important.

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The outcome of HCT in macrophage–phagocyte disorders has improved during the last decade. In addition to high-quality supportive care after HCT and improved GVHD prevention, the factors that affect a successful outcome with HCT include a careful early diagnosis, nature or course of the disease, pre-existing serious infections, other pre-existing comorbidities, and the status of vital organ functioning at transplantation.

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reticular dysgenesis. Bone Marrow Transplant 1996; 17: 1171–3. Knutsen AP, Wall D. Umbilical cord blood transplantation in severe T-cell immunodeficiency disorders: two-year experience. J Clin Immunol 2000; 20: 466–76. Bertrand Y, Muller SM, Casanova JL et al. Reticular dysgenesis: HLA non-identical bone marrow transplants in a series of 10 patients. Bone Marrow Transplant 2002; 29: 759–62. Dale DC, Graw RG. Transplantation of allogeneic bone marrow in canine cyclic neutropenia. Science 1974; 183: 83–4. Horwitz M, Benson KF, Person RE et al. Mutations in ELA-2, encoding neutrophil elastase, define a 21-day biologic clock in cyclic hematopoeisis. Nat Genet 1999; 23: 433–6. Jones J, Yang TJ, Dale JB et al. Canine cyclic hematopoiesis: marrow transplantation between littermates. Br J Hematol 1975; 30: 215–23. Krance RA, Spruce WE, Forman SJ et al. Human cyclic neutropenia transferred by allogeneic bone marrow grafting. Blood 1982; 60: 1263–6. Sulisalo T, Sistinen P, Hastbacka J et al. Cartilage-hair hypoplasia gene assigned to chromosome 9 by linkage analysis. Nature Genet 1993; 3: 338–41. Makiti O, Kaitila I. Cartilage-hair hypoplasia – clinical manifestations in 108 Finnish patients. Eur J Pediatr 1993; 152: 211–17. Lux SE, Johnston RB, August CS et al. Chronic neutropenia and abnormal cellular immunity in cartilage-hair hypoplasia. N Engl J Med 1970; 282: 231–6. O’Reilly R, Brochstein J, Dinsmore R. Marrow transplantation for congenital disorders. Semin Hematol 1984; 21: 188–221. Berthet F, Siegrist C, Ozsahin H et al. Bone marrow transplantation in cartilage hair hypoplasia: correction of immune deficiency but not of chondroplasia. Eur J Pediatr 1996; 155: 286–90. Connor J, Gatherer D, Gray FC et al. Assignment of the gene for dyskeratosis congenital to Xq 28. Human Genet 1986; 72: 348–51. Ogden G, Connor E, Chisolm DM. Dyskeratosis congenital: report of a case and review of literature. Oral Surg Oral Med Oral Pathol 1988; 65: 586–91. Putterman C, Safadi R, Zlotogora J, Banura, R, Eldor A. Treatment of the hematological manifestations of dyskeratosis congenital. Ann Hematol 1993; 66: 209–12. Mehmoud HK, Shaefer V, Schmidt CG et al. Marrow transplantation for pancytopenia in dyskeratosis congenital. Blut 1985; 51: 57–60. Vanderson R, Agnes D, Gerard S et al. Unusual complications after bone marrow transplantation for dyskeratosis congenita. Br J Hematol 1998; 103: 243–8. Güngör T, Corbacioglu S, Storb R et al. Nonmyeloablative allogeneic hematopoietic stem cell transplantation for treatment of dyskeratosis congenita. Bone Marrow Transplant 2003; 31: 407– 10. Dror Y, Freedman MH, Leaker M et al. Lowintensity hematopoietic stem-cell transplantation across human leucocyte antigen barriers in dyskeratosis congenita. Bone Marrow Transplant 2003; 3: 847–50. Shimamura A. Shwachman–Diamond syndrome. Semin Hematol 2006; 43: 178–88.

80. Mitsui T, Kawakami T, Sendo D et al. Successful unrelated donor bone marrow transplantation for Shwachman–Diamond syndrome with leukemia. Int J Hematol 2004; 79: 189–92. 81. Donadieu J, Micheal G, Merlin E et al. Hematopoietic stem cell transplantation for Shwachman– Diamond syndrome: experience of the French neutropenia registry. Bone Marrow Transplant 2005; 36: 787–92. 82. Cesaro S, Oneto R, Messina C et al. The EBMT Severe Aplastic Anaemia and Paediatric Diseases Working Party. Haematopoietic stem cell transplantation for Shwachman–Diamond disease: a study from the European Group for blood and marrow transplantation. Br J Hematol 2005; 131: 231–6. 83. Vibhakar R, Radhi M, Rumelhart S et al. Successful unrelated umbilical cord blood transplantation in children with Shwachman–Diamond syndrome. Bone Marrow Transplant 2005; 3: 855–61. 84. Boxer LA, Hedly-White T, Stossel TP. Neutrophil actin dysfunction and abnormal neutrophil behavior. N Engl J Med 1974; 297: 1093–9. 85. Coates TD, Torkildson JC, Torres M, Church JA, Howard TH. An inherited defect of neutrophil motility and microfilamentous cytoskeleton associated with abnormalities in 47-KD and 89-KD proteins. Blood 1991; 78: 1338–46. 86. Camitta BM, Queensberry P, Parkman R et al. Bone marrow transplantation for an infant with neutrophil dysfunction. Exp Hematol 1977; 5: 109–11. 87. Skubitz KM. Qualitative disorders of leukocyte. In: Lee GR, Forester J, Lukens J, Paraskevas F, Greer JP, Rodgers GM, editors. Wintrob’s Clinical Hematology, 11th edn. Baltimore: Lippincott Williams & Wilkins; 2003 pp. 1801–1817. 88. Larson R, Springer TA. Structure and function of leukocyte integrins. Immunol Rev 1990; 114: 181–217. 89. Todd RF, Freyer DR. The CD 11/CD 18 leukocyte glycoprotein deficiency. Hematol Oncol Clin North Am 1988; 2: 13–31. 90. Le Deist F, Blanche S, Keable H et al. Successful HLA nonidentical bone marrow transplantation in three patients with the leukocyte adhesion deficiency. Blood 1989; 74: 512–16. 91. Hattori H, Tsuruta S, Horikoshi Y et al. Successful human leukocyte antigen mismatched related bone marrow transplantation in a 6 year old boy with leukocyte adhesion deficiency syndrome. Pediatr Int 2001; 43: 306–9. 92. Mancias C, Infante AJ, Kamani NR. Matched unrelated donor bone marrow transplantation in leukocyte adhesion deficiency. Bone Marrow Transplant 1999; 24: 1261–3. 93. Davis WC, Douglas SD. Defective granule formation and function in the Chediak–Higashi syndrome in man and animals. Semin Hematol 1972; 9: 431–50. 94. Blume R, Bennett JM, Yankee RA et al. Defective granulocyte regulation in the Chediak– Higashi syndrome. N Engl J Med 1968; 279: 1009–15. 95. Root R, Rosenthal AS, Balestra DJ. Abnormal bactericidal, metabolic and lysosomal functions of Chediak–Higashi syndrome leukocytes. J Clin Invest 1972; 51: 649–65. 96. Styrt B, Pollack CR, Klempner MS. An abnormal calcium uptake pump in Chediak–Higashi neutro-

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79

John E. Wagner, Margaret L. MacMillan, & Arleen D. Auerbach

Hematopoietic Cell Transplantation for Fanconi’s Anemia

Introduction Fanconi’s anemia (FA) is a genetically and phenotypically heterogeneous autosomal and X-linked recessive disorder characterized by congenital malformations, progressive marrow failure, and marked predisposition to malignancy. While the basic biochemical defects responsible for the syndrome are beginning to be elucidated, animal models and longitudinal studies evaluating the phenotypic consequences of specific mutations provide clues about the function of FA genes. In this chapter, the pathogenesis and pathology of this congenital disorder as well as the results of various treatment strategies, focusing on allogeneic hematopoietic cell transplantation (HCT), will be reviewed. As of 2008, allogeneic HCT remains the only treatment modality with the potential of correcting the hematologic defect common to most, if not all, patients with FA.

History In 1927, Fanconi described a family in which three male children between the ages of 5 and 7 years had pancytopenia and birth defects [1]. Based on his observations in this family and others, Fanconi’s chief criteria for the diagnosis of FA were pancytopenia, hyperpigmentation, skeletal malformations, small stature, urogenital abnormalities, and familial occurrence. According to Fanconi, Naegeli suggested in 1931 that the term “Fanconi’s anemia” be used to describe such patients [2]. Fanconi’s proposed diagnostic criteria for FA were widely accepted well into the 1980s. Consideration of FA in the differential diagnosis of a patient manifesting clinical features of the syndrome depends on the clinician’s concept of the FA phenotype. Most cases reported in textbooks and in the literature prior to the present decade present a clinical picture similar to that of the original cases described by Fanconi. Diagnosis of FA was usually made when aplastic anemia (AA) or leukemia developed in individuals with the characteristic physical abnormalities. Literature reviews have thus been biased, more likely excluding those without the “classic” phenotype. Glanz and Fraser [3], however, were among the first to report that the phenotype was quite variable. Compared with probands, affected siblings were more likely to have a milder phenotype, being diagnosed only after the diagnosis of FA in another affected family member and not because of their phenotypic presentation. In addition, patients with

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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“Fanconi-like” marrow failure who completely lacked congenital malformations, previously described as having the Estren–Dameshek syndrome [4], were found in the same sibships with “classic” FA. While there is increasing awareness of the immense heterogeneity with regard to malformations and stature, delayed diagnosis is still the standard until the onset of marrow failure regardless of race or presence of congenital malformations [5,6]. This repetitive finding continues to suggest the need for increased awareness among physicians of the wide array of clinical features associated with the syndrome.

Clinical features The FA phenotype is extremely variable. Congenital malformations may range from none to many [7], and may involve any of the major organ systems (Table 79.1). Abnormalities involving the central nervous system, gastrointestinal system, and skeletal system in addition to radialray defects (i.e. radius and thumb abnormalities) have been added to the original FA phenotype [6]. FA patients may present with Vertebral anomalies, Anal atresia, Cardiac abnormalities, Tracheo-Esophageal fistula, Renal anomalies, and radial Limb (VACTERL), and may also have hydrocephalus. These abnormalities comprise the VACTERL syndromes. FA patients may also present with a phenotype characteristic of Holt–Oram syndrome, thrombocytopenia–absent radius syndrome, Baller–Gerold syndrome, Saethre–Chotzen syndrome (TWIST mutation), Dubowitz syndrome, velocardiofacial syndrome, Diamond– Blackfan anemia, dyskeratosis congenita, Seckel syndrome, and Nijmegen breakage syndrome. Thus, the clinician must recognize the considerable overlap of FA phenotype with these other syndromes and not be misled by pre-existing “diagnostic labels.” More than 1000 cases of FA have been reported in varying detail in the world’s literature; an analysis of these cases clearly demonstrates that FA patients not only have morphologic abnormalities in many cases, but are at progressively increasing risk of cancer as they age [8–12]. The International Fanconi Anemia Registry (IFAR) was established at the Rockefeller University in 1982 to collect clinical, genetic, and hematologic information from a large number of FA patients over time to study the full spectrum of clinical features of the disease. The primary source of case material for the IFAR is physician reporting. Diagnosis of FA was confirmed in all cases by study of chromosomal breakage induced by diepoxybutane (DEB). Using the IFAR and its collection of clinical information on 754 DEB-confirmed FA patients from North America ascertained over a 20-year time period, correlations between genotype and phenotype were initiated [10]. Based on the clinical information in the IFAR, congenital malformations associated with FA are even more variable than previously

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Table 79.1 List of congenital malformations in Fanconi’s anemia Congenital malformation

Frequency (%)

Skin pigmentation (café-au-lait spots, hyper- and hypopigmentation) Thumb and radius (thenar hypoplasia, clinodactyly of fifth digit, syndactyly of fingers, hyperextensible thumbs, arachnodactyly, absence or hypoplasia of radius) Skeletal (dysplastic ulna, micrognathia, frontal bossing, spina bifida, Klippel–Feil syndrome, vertebral anomalies, Sprengel’s deformity) Kidney and urinary tract (ectopic, horseshoe, rotated, hypoplastic or absent, dysplastic, hydronephrosis, hydroureter, reflux) Genital (males: micropenis, undescended or atrophic or absent testes, hypospadius, phimosis, azospermia; females: bicornate uterus, aplasia or hypoplasia of vagina and uterus, atresia of vagina, uterus, and ovary) Cardiac (patent ductus arteriosis, ventricular septal defect, pulmonic or aortic stenosis, coarcation of the aorta, double aortic arch, cardiomyopathy, tetrology of Fallot) Ears (deafness [usually conductive], atresia or dysplasia, canal stenosis) Eyes (microophthalmia, short or almond-shaped palpebral fissures, ptosis, epicanthal folds, hyper- and hypotelorism) Nose (flattened nasal bridge, nasal pit) Gastrointestinal (esophageal atresia, duodenal atresia, anal atresia) Central nervous system (microcephaly, hydrocephalus, Bell’s palsy, central nervous system arterial malformations, abnormal pituitary, absent corpus callosum, hyperreflexia) Growth retardation (short stature)

71 59 31 57 50 (males) 29 Unknown 47 1 7 1 67

Fig. 79.1 Nine-month old twins; one healthy and one affected by Fanconi’s anemia (FA). Photographs demonstrate the “classic” physical features associated with FA, specifically growth retardation, bilateral hypoplastic radii and absent thumbs, microophthalmia, and microcephaly.

recognized [6]. From a developmental standpoint, it is interesting that radial ray abnormalities in FA patients can be bilateral or unilateral. Even patients with bilateral abnormalities usually exhibit asymmetry, with limbs having different specific anomalies. Notably, approximately one-third of FA patients do not manifest major congenital malformations. However, most have alterations in growth. Other very common findings are skin pigmentation abnormalities, hypoplastic thenar eminence, and/or microophthalmia. Increased awareness of the facial anomalies as well as the complete spectrum of minor malformations seen in these patients should enable an earlier diagnosis to be made among patients without major congenital anomalies [7]. Short stature is a well-recognized feature of FA and is often secondary to hormonal deficiencies. The syndrome is usually associated with abnormal growth parameters both prenatally and postnatally (Fig. 79.1). The mean stature of FA patients in the IFAR is near the fifth centile, with weight and head circumference often on the fifth centile. A

prospective study of 54 patients with FA, 30 males and 24 females from 47 unrelated families, showed that endocrinopathies are a common feature of FA, primarily manifesting as glucose/insulin abnormalities, growth hormone insufficiency, and hypothyroidism [13]. Although short stature is a feature of FA, it is notable that 23 patients (43%) were within 2 standard deviations of the 50th centile, and 5 (9%) were above the mean height for the general population. Expectedly, patients with endocrine dysfunction are more likely to have short stature. These data indicate that short stature is an integral feature of FA, but also that the addition of endocrinopathies magnifies the growth failure in a significant proportion of patients. The finding of abnormal endogenous growth hormone secretion may demonstrate an underlying hypothalamic–pituitary dysfunction that results in poor growth. Since correction of growth hormone or thyroid hormone deficiency may improve final height outcome and quality of life, endocrine evaluations are recommended for all FA children at an early age well before use of androgens and HCT if possible.

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Chapter 79

Fig. 79.2 Cumulative incidence of hematologic abnormalities and malignancy over time in patients with FA. Of 754 FA patients within the International Fanconi Anemia Registry (IFAR) database, 601 (80%) experienced bone marrow failure (BMF), 120 (60%) hematologic malignancy, and 79 (40%) were nonhematologic malignancy. The risk of developing BMF and hematologic and nonhematologic malignancies increased with advancing age with a 90%, 33%, and 28% cumulative incidence, respectively, by 40 years of age. (Reproduced from Kutler et al. [10], with permission.)

Infertility is also common. Approximately half of all female patients with FA are infertile. Menopause usually starts during the fourth decade. However, 15% of females cited in the literature or reported to the IFAR who reached at least 16 years of age and were not receiving androgen therapy had at least one pregnancy [14]. While pregnancy is possible in some females, it is often associated with significant complications, such as rapid and marked progression of marrow failure, pre-eclampsia, and premature labour. In contrast, males are very rarely fertile [15]. Genital malformations and hypoplastic gonads are common findings in males with FA. Results of semen analyses typically reveal very low or absent sperm counts as well as evidence of abnormal spermatogenesis. Hematologic abnormalities occur in virtually all patients with FA at a median age of 7 (range birth to 41) years (Fig. 79.2) [8,10,16]. Based on clinical data in the IFAR (n = 754 patients), the cumulative incidence of bone marrow failure by age 40 years was 90%. Initial hematologic findings were diverse. Thrombocytopenia was often associated with elevated levels of fetal hemoglobin and macrocytosis, and usually preceded onset of anemia or neutropenia. Notably, some patients presented with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) without prior diagnosis of AA. Of the 754 IFAR FA patients, 120 (16%) patients experienced MDS and/or AML (Plate 79.1). The cumulative incidence of MDS or AML by age 40 years was 33% [10]. Based on a survey of FA patients performed by Rosenberg et al. [9], the median age of onset of leukemia was 11.3 years. Presence of clonal cytogenetic abnormalities becomes increasingly common with age. The actuarial risk of identifying clonal cytogenetic abnormalities at the time of marrow failure is 67% by 30 years of age. Among the most frequent clonal abnormalities observed are duplications and triplications of the long arm of chromosome 1, gains of portions of the long arm of chromosome 3, and monosomy 7 or loss of material from the long arm of chromosome 7. Deletions of 5q or 11q, rearrangements of 6p, and gains of chromosomes 8 and 21 have also been noted by different groups (Plate 79.2) [16,17] (B. Hirsch, unpublished data).

In addition, AML in FA patients rarely involves the chromosomal rearrangements commonly observed in non-FA patients with AML (e.g. t[9;11], t[8;21], t[6;9], t[11;19], and inv 6). Frequently, abnormal karyotypes in FA AML are complex. Thus, many of the early descriptions of these karyotypes included designations of “marker” or “add,” referring to structurally abnormal chromosomes that could not be completely identified by G- or R-banding. Recent advances in single and multicolor fluorescence in situ hybridization, and utilization of these and other molecular/cytogenetic techniques as adjuncts to conventional cytogenetic analysis, now permits more definitive characterization of clones with the possibility of identifying new specific recurrent abnormalities (Plate 79.2). As earlier identification of clonal cytogenetic abnormalities would clearly alter the frequency of medical surveillance and potentially treatment options, annual marrow examinations with cytogenetics is mandatory. Clearly, early and accurate diagnosis of FA is important. Patients diagnosed early in life may have earlier endocrine intervention to optimize growth and development, as well as more frequent monitoring of the hematologic profile. While investigational, harvesting of hematopoietic stem cells (HSCs) at a time when the marrow cellularity approaches normal and before the development of clonal cytogenetic abnormalities, MDS or leukemia may be warranted. Harvested autologous HSCs could be useful for patients undergoing allogeneic HCT as a “back-up” in the event of graft failure or for future applications, such as their use as a source of HSC or multipotent adult stem cells for genetic correction. Until recently, storage of autologous HSCs has rarely been possible since most patients were previously diagnosed at the time of marrow failure.

Diagnostic tests Due to considerable overlap of the FA phenotype with that of a variety of genetic and nongenetic diseases, diagnosis of FA is often difficult and unreliable on the basis of clinical manifestations alone. While FA should be strongly suspected if there are phenotypic features consistent with the diagnosis plus an elevated mean corpuscular volume for age, patients with FA may not have any clinical manifestations. Schroeder et al. [18] first suggested the use of spontaneous chromosomal breakage as a cellular marker for FA, but longitudinal studies of chromosome instability in FA patients showed this finding to be inconsistent. In contrast, hypersensitivity of FA cells to the clastogenic (chromosome-breaking) effect of crosslinking agents provides a reliable cellular marker for the diagnosis of this disorder (Fig. 79.3). DEB and mitomycin C (MMC) are the agents most widely used for FA diagnosis. Extensive experience with MMC and DEB testing has demonstrated the sensitivity, specificity, and reproducibility of the results [19,20]. Crosslinker hypersensitivity can be used to identify the preanemic patient as well as the patient with AA, MDS, or leukemia who may or may not have the physical stigmata associated with FA. It is recommended that all patients exhibiting any congenital malformation known to be associated with FA or AA at any age, or any patient with MDS with complex cytogenetic abnormalities, have a peripheral blood sample tested for crosslinker hypersensitivity [6]. Because of the lack of concordance of FA phenotype among affected siblings, all full siblings of an FA patient should also be screened. Data from DEB testing indicate that there is great variability in the degree of hypersensitivity in FA patients, although there is no overlap with the normal range [19]. In approximately 25% of FA patients, DEB testing will reveal two populations of phytohemagglutinin A (PHA)stimulated peripheral blood lymphocytes: one demonstrating an FA phenotype with chromatid breaks and exchanges, and the other a normal one [21]. The clinical significance of this type of mosaicism is unclear and is under investigation. Thus far, no correlation has been discerned

Hematopoietic Cell Transplantation for Fanconi’s Anemia

lymphocytes exposed to crosslinking agents, are sometimes used as a research tool [24,25]. While it is clear that duration of the G2/M phase is increased in FA cells compared with unaffected cells, clinical diagnostic laboratories do not often use this method. Further, it has been reported that measurement of serum α-fetoprotein levels may be a sensitive and reliable test for FA [26]. Due to the fact that normal levels of α-fetoprotein have been observed in 15.8% of patients (9 of 57 tested) referred to the University of Minnesota (Wagner, Zierhut, and MacMillan, unpublished data), it should be considered supportive evidence for the diagnosis and does not replace the need for chromosomal breakage studies. Of note, all three of three patients evaluated with biallelic mutations in BRCA2 had extremely high levels of α-fetoprotein (>100 μg/L).

between the degree of crosslinker hypersensitivity and the severity of the phenotype in FA patients or individual patient sensitivity to chemoradiotherapy [22] (Wagner and Auerbach, unpublished data). However, data exist suggesting that genes not in the FA pathway can modify the crosslinker hypersensitivity [23]. Importantly, the clinician should know that the chromosomal breakage test could also be applied to the study of fetal cells obtained by chorionic villous sampling (CVS), amniocentesis, or percutaneous umbilical blood sampling. If the specific mutation is known within the family, testing for the presence of the mutation on limited quantities of uncultured cells is possible and rapid. Alternative diagnostic methods for FA, such as by cell-cycle analysis using flow cytometric methods on

11 breaks

Complementation groups and gene cloning

2 radial figures

Thirteen complementation groups representing distinct genes (FA-A through FA-G, including FA-D1 and FA-D2, FA-I, FA-J, FA-L, FA-M, and FA-N) have been described [27–39]. Complementation groups FAA (~62%), FA-C (~15%), and FA-G (~9%) account for the majority of complement group assignments in FA patients. While the genes responsible for the defect have been identified for each of the 13 FA complementation groups (Table 79.2) [30–47], there are patients for whom all known genes have been excluded, suggesting that additional FA genes exist. Inheritance is autosomal recessive except for FA-B which is Xlinked recessive. The FA genes encode unique proteins, many of which do not exhibit any known functional domains. At least eight FA proteins, FANCA/B/ C/E/F/G/L, and M, form a nuclear multiprotein complex termed the FA core complex, which is required for the monoubiquitination of both FANCD2 and FANCI [39]. Additional FA-associated proteins, including FAAP100 and FAAP24, have also been identified as part of the complex, but mutations in these associated proteins have not yet been detected in FA patients. The genes for FANCD2 and FANCI appear to be ancestrally related, both require ataxia telangiectasia mutated (ATM)/ ATM and Rad3-related (ATR) phosphorylation to be activated, and both require an intact FA core complex for monoubiquitination at specific lysine residues. FANCD2 and FANCI together form a complex, and

1 tri-radial figure

Average number of aberrations/cell (DEB 0.1 mg/mL) Patient

10.78

Control

0.08

Normal range

1181

0.00-0.12

Fig. 79.3 Diepoxybutane (DEB) test. Metaphase cell from a patient with Fanconi’s anemia after treatment with DEB in vitro. Arrows designate chromatid gaps and/or breaks; asterisks designate radial figures. This cell has nine gap/breaks and two multiradial figures. (Reproduced courtesy of Betsy Hirsch, PhD, Director of the Cytogenetics Laboratory of the University of Minnesota Medical Center, Fairview, Minneapolis, MN.) Table 79.2 Fanconi’s anemia (FA) genes: description and frequency

FA group

FA gene

MIM #

Location

Exons

Amino acids

Frequency in the International Fanconi Anemia Registry

A

FANCA

607139

16q24.3

43

1455

62

B C D1 D2 E F G I

FANCB (FAAP95) FANCC FANCD1/BRCA2 FANCD2 FANCE FANCF FANCG/XRCC9 FANCI/KIAA1794

300515 227645 605724 227645 600901 603467 602956 609053

Xp22.31 9q22.3 13q12.3 3p25.3 6p21.3 11p15 9p13 15q26.1

10 14 27 44 10 1 14 38

859 558 3481 1451 536 374 622 1268

2 15 3 3 1 2 9 1

J

FANCJBRIP1/BACH1/

605882

17q22

20

1249

2

L M N

FANCL/PHF9/(FAAP43) FANCM/Hef (FAAP250) FANCN/PALB2

608111 609644 610832

2p16.1 14q21.3 16p12

14 22 13

375 2048 1186

<1 <1 <1

Cloning principal investigator (citation) Lo Ten Foe et al., 1996; FA/BC Consortium, 1996 [41a,b] Meetei et al., 2004 [42] Strathdee et al., 1992 [40] Howlertt et al., 2002 [46] Timmers et al., 2001 [30] de Winter et al., 2000 [45] de Winter et al., 2000 [44] de Winter et al., 1998 [43] Smogorzewska et al., 2007; Sims et al., 2007 [31,32] Levran et al., 2005; Levitus et al., 2005 [33, 34] Meetei et al., 2003 [35] Meetei et al., 2005 [36] Reid et al., 2007; Xia et al, 2007 [37,38]

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ubiquitination of each protein is important for the maintenance of ubiquitination of the other. The ubiquitinated FANCI/D2 complex localizes to chromatin in response to DNA damage. FANCC, the first FA gene discovered, encodes multiple transcripts, due to alternative splicing and polyadenylation signals. Each transcript shares the same 1674 nucleotide coding sequence, encoding a 558 amino acid protein (63 kDa) that shows no homology to known proteins. A variety of FANCC mutations has been identified; the most common is c.711 + 4A > T (alias IVS4 + 4A > T in the literature). In North America, this mutation is found only in patients of Ashkenazi Jewish ancestry, and accounts for approximately 85% of FA mutations in this population [48,49]. The carrier frequency for this intron 4 splicing mutation in Ashkenazi Jews is one in 89. Several other relatively common FANCC mutations found in FA patients of northern European ancestry are 322delG, c.808C > T (p.R185X), c.1916C > T (L554P), and c.1897C > T (p.R548X) [22,50,51]. Like FANCC, FANCA also expresses multiple transcripts, with a major species of 5.5 kilobases (kb), encoding a 4368 nucleotide coding sequence. The predicted FANCA protein is a 1455 amino acid peptide (~163 kDa) that, like FANCC, shares no significant homology with any known protein. Sequence analysis has identified two overlapping nuclear localization signals, as well as a partial leucine zipper consensus domain, but neither of these domains has yet been demonstrated to be functional [41,42]. The FANCA gene contains 43 exons spanning approximately 80 kb, ranging from 34 to 188 base-pairs [52]. FANCA mutation analysis reveals a large number of different mutations, indicating that FANCA is highly polymorphic and may be hypermutable [53]. There are many private or semiprivate mutations, as well as ethnic-specific mutations, making large-scale screening for FANCA mutations difficult [53–59]. The gene for the FA-G complementation group (FANCG) was the third FA gene to be cloned, and was found to be identical with human XRCC9, which maps to 9p13 [43]. The complementary DNA (cDNA) is predicted to encode a polypeptide of 622 amino acids, with no sequence similarities to any other known protein or motifs that could point to a molecular function for FANCG/XRCC9. Four pathogenic mutations in FANCG were originally described; three were in patients of German ancestry, and one was in a consanguineous Lebanese family. FANCG has subsequently been shown to be highly polymorphic, and to exhibit ethnic-specific mutations in Korean/Japanese, Portuguese– Brazilian, French–Acadian, and northern European FA families, as well as many private mutations [60–62]. Notably, only a few FA patients have been described in the literature belonging to groups FA-B, -D1, -D2, -E, -F, -I, -J, -L, -M, and -N. Information regarding FA mutations can be obtained from the Fanconi Anemia Mutation Database at http://www.rockefeller.edu/fanconi/mutate. Proposed functions of FA genes Pathophysiology of FA at a cellular level has yet to be completely defined. Defects in DNA repair, cell-cycle checkpoints, oxygen metabolism, and induction of apoptosis have all been described in FA cells. The published data have been marked by contradictory results, which can be attributed partially to inconsistencies in experimental conditions, including variations in administration and dosage of DNA crosslinking agents administered, the complementation group of the FA cells used, and the use of primary cells versus transformed cell lines. Although the first FA gene was cloned more than 15 years ago, the biochemical function of some FA proteins has only recently being elucidated. The FA genes all encode unique proteins, which typically lack recognizable functional domains. Exceptions are FANCJ/BRIP1, which has a DEAH-box helicase domain, FANCM, which contains a DNA helicase

and endonuclease domain, FANCL, which contains a WD40 repeat, a plant homeo domain finger, and a E3 ubiquitin ligase domain, FANCN, which contains an amino-terminal prefoldin-like domain and two Cterminal WD40-like repeats, and FANCG, which has at least seven tetratricopeptide repeat motifs [33–38]. Recent studies of post-translational modifications of a complex containing FANCD2 and FANCI suggest that FA proteins function in regulating discrete cellular signalling pathways. Formation of the FA nuclear core complex by various FA proteins, and FA-associated proteins, including FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL FANCM, FAAP24, and FAAP100 [39], has been shown to be essential for the monoubiquitination of the FANCD2/FANCI complex in response to DNA damage by crosslinking agents. Activation of the FANCD2/FANCI complex causes it to translocate to nuclear foci and associate with chromatin, suggesting a role for the FANCD2/FANCI complex in DNA repair functions. Recent data suggest that the FANCD1 (BRCA2)/FANCN (PALB2) complex is recruited to these foci, to initiate DNA repair. Phosphorylation of FANCD2 and FANCI by ATM/ATR, is an independent posttranslational modification, which is required for activation of FANCD2 [63,64] and FANCI [31]. The FA core complex possesses a putative helicase and an E3 ubiquitin ligase subunit, as well as subunits with molecular motifs and domains which suggest a general role for the complex as molecular scaffolding during DNA repair, in addition to any other functions for individual subunits [39]. The nature of the interaction of the BRCA1-binding protein FANCJ (BRIP1) in the FA pathway has still not been elucidated. FANCJ exhibits a helicase domain, and a domain required for BRCA1 binding. Mutations in either of these domains results in an FA-like phenotype. Grompe and van de Vrugt [65] have proposed a schematic representation of the FA pathway (Plate 79.3). Monoubiquitination of FANCD2 and FANCI can be detected by immunoblotting with FANCD2 or FANCI antibodies, respectively [32]. Protein extracts from normal cells show both unmodified (short) and the monoubiquitinated (long) forms of these proteins, while extracts from cells from FA complementation groups FA-A, -B, -C, -E, -F, -G, -L, and -M show only the unmodified short FANCD2 and FANCI forms. It is postulated that the entire FA nuclear core complex acts as a multisubunit E3 ubiquitin ligase, which binds to the E2-conjugating enzyme UBE2T, and that this entire complex participates in FANCD2/FANCI monoubiquitination [66]. This is a dynamic and reversible process, with evidence that USP1 is the enzyme that deubiquitinates FANCD2 [67]. It has been suggested that western blotting to detect the presence or absence of the FANCD2 long form be used as a diagnostic screen for FA, and as an assay to detect complementation group after transduction by cDNA-containing retroviral vectors [68]. While this may be a useful adjunct to standard tests of hypersensitivity to crosslinking agents, there are insufficient data at this time to determine its sensitivity, specificity, and reproducibility for routine diagnosis, especially for patients who cannot be complemented by any of the cloned genes and those who exhibit mosaicism. Although the FANCC protein participates in the FA nuclear complex responsible for monobiquitination of FANCD2, abundant levels of endogeneous FANCC are also found in the cytoplasm. There is strong evidence that the cytoplasmic form of FANCC interacts with other cytoplasmic proteins in control of apoptosis [69–72]. Cytoplasmic complexes are also found. There is emerging evidence that FA complexes, often acting in concert with heat shock proteins, function to suppress the activation state of intracellular effectors of apoptosis and facilitate survival signaling pathways, including the Janus kinase (JAK)-dependent signal transducers and activators of transcription (STAT) activation pathway, thus promoting cell survival. The exact composition of the cytoplasmic FA complexes has not been fully defined, but included

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1183

among the cytosolic proteins that bind to FANCC in vitro are the mitotic cyclin-dependent kinase cdc2 [73,74], the molecular chaperone GRP94 [75], NADPH cytochrome p450 reductase [76], and STAT1 [77,78]. FANCC is also suggested to have a role as a redox regulator of glutathione S-transferase P1 (GSTP1) [77,79]. Evidence also indicates that the molecular chaperone heat shock protein 70 (Hsp70) interacts with FANCC to protect hematopoietic cells from cytotoxicity and apoptosis on exposure to interferon-gamma (IFN-γ) and tumor necrosis factor-alfa (TNF-α) [80]. Together these interactions protect cells against a variety of environmental factors, including oxidative stress and radiation, and suppress double-stranded RNA-dependent protein kinase (PKR) activity and caspase 3 activation [81,82]. Subsequent studies have shown that FANCA and FANCG also participate in the complex affecting PKR [83]. More recently, Hsp90, which regulates diverse signaling pathways, has been found to associate with FANCA [84]. In addition, other studies suggest that FANCA may have direct interactions with BRCA1 [85] and brahma-related gene-1 (BRG1), a component of the SWItch/sucrose nonfermentable (SWI/SNF) complex involved in chromatin remodeling [86]. While there has been tremendous progress, the exact role of FA proteins in DNA repair, cell-cycle checkpoints, oxygen metabolism, and induction of apoptosis still remain to be elucidated. Complementation group assignment, mutation analysis, and genotype–phenotype correlations Once a patient is diagnosed as affected with FA, identification of the complementation group assignment should be undertaken, as the risk of developing bone marrow failure or a malignancy at a very young age varies with specific complementation groups and mutations. The complementation group can be determined by correction of crosslink hypersensitivity of the patient’s cells by transduction with retroviral vectors containing the normal FA cDNAs for the various groups. The method of crosslink hypersensitivity correction is similar to that established for testing for crosslink hypersensitivity of lymphoblastoid cell lines, fibroblasts, or peripheral blood-derived patient T cells [87,88]. After the complementation group has been determined by retroviral typing, mutation detection can be targeted to a specific gene, using high-throughput sequencing. Rapid mutation screening methods such as denaturing highperformance liquid chromatography can also provide accurate mutation screening for FA [89–91]. Alternative methods such as multiplex ligation-dependent probe amplification or quantitative polymerase chain reaction are presently used to detect large genomic deletions which comprise at least 40% of mutations in FANCA, as these are not detected by routine genomic DNA sequencing. It is forseen that gene array technology will soon be applied for rapid detecton of mutations, including genomic deletions in most FA genes, with complementation group assignment made directly by finding biallelic mutations in one of the FA genes. The heterogeneous nature of FA makes an understanding of the correlation between genotype and phenotype potentially important in the clinical management of FA patients. IFAR data [10] confirm that specific complementation groups play a significant role in both the timing of bone marrow failure and survival. FA-C patients, for example, have a significantly earlier onset of bone marrow failure (Fig. 79.4) and poorer survival compared with FA-A and FA-G patients. Subdividing the FA-C group based on the region of the gene that is mutated reveals that patients with intron 4 or exon 14 mutations have an earlier onset of bone marrow failure and poorer survival compared with patients with exon 1 mutations, confirming the earlier findings of Gillio et al. and Yamashita et al. [22,92]. As reported by Gillio et al. [22], patients with FANCC genotypes can be divided into three clinical groups: (1) patients

Fig. 79.4 Survival and time to marrow failure was compared for patients with Fanconi’s anemia in complementation groups A, G, and C. Survival and incidence of marrow failure was similar groups A and G. Comparison of the combined groups A/G with group C showed that patients in group C had a significantly shorter survival (a) and higher incidence of bone marrow failure (b). (Reproduced from [10], with permission.)

with the c.711 + 4A > T (IVS4) mutation; (2) patients with at least one exon 14 mutation (p.R548X or p.L554P); and (3) patients with at least one exon 1 mutation (c.322delG or p.Q13X) and no known exon 14 mutation. IVS4 and exon 14 subgroups are associated with a severe phenotype manifested by multiple major congenital malformations, early onset of hematologic disease, and poorer survival compared with exon 1 patients and with the non-FA-C IFAR population (Fig. 79.5) [10].

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1

IVS4 exon 14

exon 1 1

p <0.00005 p <0.00005

IVS4

non C

exon 1

p <0.0013

p <0.0458

exon 14 p <0.00005 p <0.00005

p <0.00005 p <0.00005

0.75

Cumulative survival

Cumulative proportion without hematologic onset

non C

0.5

0.25

0.75

0.5

0.25

0

0 0

5

10

15

20 25 Age (years)

30

35

40

0

5

10

15

20 25 Age (years)

30

35

40

45

BRCA2 Not BRCA2

0.0

0.2

Cumulative incidence 0.4 0.6 0.8

It is particularly important to point out a special category of FA patients with biallelic mutations in FANCD1/BRCA2. As detailed previously, many of the known FA proteins organize into a multiprotein FA core complex which in turn functions in a post-translational modification (monoubiquination) of FANCD2. FA proteins downstream to FANCD2 (e.g. helicase BRIP1 [FANCJ], and breast cancer susceptibility proteins PALB2 [FANCN] and BRCA2 [FANCD1]) cooperate with the core complex in recognition and repair of DNA [39]. While there are similarities in the response to clastogenic agents and abnormal skin pigmentation, intrauterine growth retardation, and short stature, biallelic mutations in FANCD1/BRCA2 are associated with an extremely high predilection to leukemia and cancer at a very early age [93]. For all patients with biallelic mutations in FANCD1/BRCA2, the incidence of leukemia is 41% at age 5 years, in contrast to FA in general, where incidence is 1% during the first 5 years of life (Fig. 79.6). In addition, biallelic FANCD1/BRCA2 mutations are also associated with the development of solid tumors, mostly of brain and kidney [94]. Of interest, medulloblastoma is particularly common in children of Ashkenazi Jewish descent who carry the BRCA2 founder mutation c.6174delT. All these children have died, and none has had leukemia or bone marrow failure. With few exceptions, children with biallelic FANCD1/BRCA2 mutations have either solid tumor or leukemia but not both. However, 3 of the 13 reported cases of early leukemia also had a solid tumor [95–97]. One child developed Wilms’ tumor at 7 months, a medulloblastoma at 6 years, and B-lineage acute lymphoblastic leukemia at 10 years of age. The only other FA patients with a similar phenotype to FA-D1/BRCA2 patients are those in complementation group FA-N, with biallelic mutations in the BRCA2-interacting protein partner and localizer of BRCA2 (PALB2). All reported cases had embryonal cancers and early death [37]. As expected, the family history is particularly important in BRCA2 families, as most (but not all) of these kindreds had a significant breast and other cancer history. Family members of the other known genes

1.0

Fig. 79.5 Within complementation group C patients (n = 78 in the International Fanconi Anemia Registry database), marrow failure occurs at an earlier time point in patients with at least one intron 4 (IVS4 + 4A > T) or at least one exon 14 (R548X or L554P) mutation (n = 50) compared with patients with at least one exon 1 mutation (322delG and QX13) and no mutations in exon 14 or intron 4 (n = 27). Left-hand panel: the median age of hematologic disease onset is 2.7 (range 0–11) years in the IVS4 group and 2.1 (range 0.8–5.2) years in the exon 14 group. In comparison, the exon 1 subgroup displays a mild phenotype with few major congenital malformations, and the median age of onset of hematologic disease is 7.6 (range 2.6–21.8) years. Right-hand panel: earlier hematologic disease onset and a greater number of congenital malformations are associated with poorer survival. IVS4 and exon 14 subgroups experienced median survivals of 14.9 and 9.7 years, respectively. In contrast, exon 1 patients had a median survival of 19.7 years (p <0.00005). Reproduced from Seyschab et al. [22], with permission.)

0

50

100 150 200 250 300 350 400 450 500 550 600 Time to hematologic malignancy (months)

Fig. 79.6 Cumulative incidence of hematologic malignancy among BRCA2 mutation carriers (14 patients) versus all other complementation groups in the International Fanconi Anemia Registry (IFAR) (746 patients). Among Fanconi’s anemia (FA) patients with biallelic mutations in BRCA2, the cumulative incidence of hematologic malignancy is 41% (95% confidence interval 20–85%) at 5 years of age. In contrast, the incidence of hematologic malignancy in non-BRCA2 FA patients in the IFAR registry at 5 years is 1% (95% confidence interval 0.5–2.1%). (Reproduced from Wagner et al. [93], with permission.)

downstream of FANCD2 (FANCJ and FANCN) also have some increased risk of breast cancer [98,99]. Surprisingly, in a large study of FA kindreds, there was no overall increased risk of cancer of FA heterozygotes, although there was some evidence that FANCC mutations are possibly breast cancer susceptibility alleles [100]. Clearly, the question of whether FA genes function in a single FA/breast cancer pathway needs further study.

Hematopoietic Cell Transplantation for Fanconi’s Anemia

120 100

Percentage

The ability to correlate genotype with outcome has considerable clinical significance. For the first time, the pace of hematologic abnormalities and survival can be predicted in patients with specific genotypes. This knowledge aids the family and clinician in timing the various treatment options as well as prescribing a tailored surveillance plan. For example, patients with FANCC IVS4 or exon 14 mutations or biallelic mutations in BRCA2 should be followed more closely for early marrow failure and development of clonal cytogenetic abnormalities, in the case of BRCA2, necessitating the consideration of very early allogeneic HCT.

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80 60 40 20

Somatic mosaicism 0

0

5

10

25

50

0

5

10

25

50

100

120 100

Percentage

In addition to genotype, the presence of somatic mosaicism may be important in predicting the clinical course of the patient with FA. It is currently estimated that 25% of patients with FA will exhibit two populations of T cells, one that is sensitive to the clastogenic effects of DNA crosslinking agents and one that is resistant. T-cell mosaicism has been reported for various diseases, such as severe combined immune deficiency, Duchenne muscular dystrophy, tyrosinemia, epidermolysis bullosa, and Bloom’s syndrome [101–105]. In each of these cases, restoration of the normal allele was demonstrated only in the lymphoid lineage. The question now is whether there is any clinical significance to somatic mosaicism in patients with FA. Gregory et al. [106] demonstrated somatic mosaicism in the HSC population. In this study, loss of the maternal FANCA mutation in HSCs was confirmed by the presence of revertant myeloid and erythroid precursors as well as T and B lymphocytes. Although not definitive, the data suggested that back-mutation in a single HSC was the most likely mechanism for the revertant mosaicism in this patient. Other potential mechanisms that account for somatic mosaicism include mitotic gene conversion, intragenic mitotic recombination, and the introduction of compensatory mutations. Regardless of the mechanism, spontaneous genetic reversion would be a rare event. If the event occurs during embryogenesis, it would be predicted that somatic mosaicism would be found in all tissues. If the event occurs at a later time point, somatic mosaicism will be restricted to specific tissues. In the case illustrated above, 100% of skin fibroblasts exhibited the maternal FANCA mutation, as would be predicted based on the subject’s clinical findings of short stature and radial ray defects. While it would be predicted that hematopoiesis would be clonal, this has not been confirmed. However, in a second case, clonal hematopoiesis has been confirmed (Wagner, Davies, and Auerbach, unpublished data). Like the first case, hematopoietic progenitors were insensitive to low doses of MMC, paralleling the dose–response curve of progenitors from an unaffected donor (Fig. 79.7). It is important to recognize that somatic mosaicism originating in a pluripotential stem cell can be viewed as “spontaneous” or “natural” gene therapy. The clinical course of the patients described above may be relevant and instructive with regard to the future potential of HSC gene therapy for this disease. The main premise of gene therapy in FA patients is that corrected FA cells will have a proliferative advantage over noncorrected cells. In both cases described above, hematopoietic function was distinctly better than that in affected siblings. In both cases, these patients were detected on testing as possible bone marrow donors. However, in the first case, the possibility that HSC gene correction alone may not be sufficient is also illustrated. In this case, residual FA HSCs became dysplastic with eventual suppression of normal hematopoiesis. Therefore, the risk of malignant transformation of residual FA HSCs is an open question. The clinical course observed in the first patient suggests that it may be necessary to eradicate residual nonreverted stem cells in those with somatic mosaicism (and uncorrected stem cells after

80 60 40 20 0 100

MMC concentration

Fig. 79.7 Mitomycin C (MMC) dose–response curves in patients with Fanconi’s anemia (FA). Marrow mononuclear cells were plated in methylcellulose containing differing concentrations of MMC (0, 5, 10, 25, 50, and 100 mM). After 2 weeks in culture, colonies were enumerated and compared with the number of colonies in the untreated control. Results demonstrate that colony-forming cell MMC resistance in patients 1 and 2. The upper panel (patient 1) demonstrates partial resistance; the lower panel (patient 2) demonstrates complete MMC resistance comparable to marrow colony-forming cells from a normal healthy volunteer. The dashed line in the lower panel reveals marked MMC sensitivity of marrow progenitors obtained from the patient’s affected sibling.

gene therapy) using low-dose chemotherapy to reduce the risk of developing MDS or AML. The success of this approach has been suggested by Battaile et al. [107]. The hypothesis that normal HSCs will preferentially expand in vivo, particularly in the presence of chemotherapy agents, was tested in a murine model. On mixing of wild-type with FANCC knockout HSCs, the ratio of normal to FA HSC remained stable over months. After a single injection of MMC, selection of normal cells was rapid and robust. Under these conditions, even a very small number of wild-type HSCs was able to significantly repopulate the hematopoietic system. These results suggest that selective pressure must be applied not only to eradicate the risk of malignant transformation of residual FA cells in patients with somatic mosaicism or in recipients of genetically corrected HSCs, but also to promote the clonal expansion of corrected cells to rapidly restore normal hematopoiesis. While it is clear that some FA patients have T-cell and hematopoietic progenitor cell mosaicism, it is also clear that the presence of mosaicism does not prevent marrow failure or MDS in all, or the requirement for allogeneic HCT. As discussed below (University of Minnesota, Initial

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Trials 1995–98), the presence of DEB-resistant T cells is associated with a higher risk of graft rejection after alternative donor HCT. This finding suggested the need for more intensive immunosuppression to overcome the alloreactivity of DEB-resistant cells. Use of a fludarabine (FLU)based preparative therapy has eliminated this association.

Proposed mechanisms of marrow failure and leukemogenesis While patients with FA have an extraordinary predisposition for the development of marrow failure and leukemia, the pathogenesis and pathophysiology of these disease entities are unclear. Because the prognosis of FA patients with AML is exceedingly poor, understanding the pathogenesis of this complication and development of assays to predict its occurrence could improve the timing of HCT and/or the collection of autologous HSC for genetic modification. Murine models have been developed for various FA genes, including Fancc, Fanca, Fancg, and Fancd2, providing insights into the mechanisms of marrow failure [108–120]. The phenotype of the two homozygous Fancc mutant animals is similar in the two murine models. Lymphocytes from these animals demonstrate the classic hypersensitivity to bifunctional crosslinking agents and G2/M cell-cycle abnormalities, although skeletal and urinary system birth defects, pancytopenia, and increased cancer susceptibility have not been observed. While the peripheral blood appears normal, mice with an exon 9 deletion demonstrate age-dependent decreases in progenitor cell frequencies and hypersensitivity to IFN-γ-mediated by fas-induced apoptosis [108]. Whitney et al. [108] suggest that the FANCC protein may directly or indirectly suppress an IFN-γ-mediated mitotic inhibitory pathway. Wang et al. [117] studied the function of constitutively expressed normal human FANCC protein in mouse hematopoietic cells. HSCs from FANCC-overexpressing transgenic mice were resistant to the cytolytic effect of a fas-triggering antibody, suggesting that fas-mediated apoptosis is involved in the pathogenesis of marrow failure in FA. Antisense oligonucleotides complementary to FANCC messenger RNA inhibited the in vitro clonal growth of normal erythroid and granulocyte–macrophage progenitor cells, even in the presence of exogenous growth factors. In contrast, peripheral blood CD34+ cells isolated from an FA-C patient and infected with FANCC cDNA exhibited a 5- to 10fold increase in the number of progenitor colonies in vitro. These results suggest that the FANCC gene plays a direct role in the survival of hematopoietic progenitor cells. It has been proposed that FANCC regulates an apoptotic pathway during hematopoiesis and may respond to an apoptotic pathway induced by DNA damage [118–120]. Bagby et al. [121] have considered the possibility that marrow failure was mediated by insufficient production of hematopoietic growth factors secondary to the FA mutation. While no abnormalities were noted in stromal production of stem cell factor or macrophage colony-stimulating factor, the production of interleukin 6, granulocyte–macrophage colonystimulating factor, and granulocyte colony-stimulating factor was variable. Because these responses were variable between affected siblings, the abnormalities observed are not likely to be the direct effect of the inherited mutation. Abnormal levels and responses to cytokines have also been reported in patients with FA [122,123]. Rathburn et al. [124] have proposed that marrow failure in patients with FA results from the accumulated losses of HSCs caused by repeated episodes of low IFN-γ released in response to recurrent minor inflammatory stimuli. The capacity of IFN-γ to induce TNF-α gene expression is increased in the serum of children with FA [125]. Whitney et al. [108] postulate that the apoptotic phenotype in hematopoietic stem and progenitor cells creates an environment for the selection of IFN-γ-resistant clones. Their findings argue that therapies designed to correct the

abnormal pattern of IFN-γ sensitivity in FA cells may prevent or delay the onset of marrow failure and the development of MDS and leukemia. More recently, data from Pang et al. [80] show that TNF-α inhibits HSC expansion, interferes with HSC self-renewal, and compromises the ability of HSCs to reconstitute hematopoiesis. Their findings argue that therapies designed to correct the abnormal pattern of IFN-γ sensitivity in FA cells or reduce the levels of TNF-α may prevent or delay the onset of marrow failure and the development of MDS and leukemia. Studies are ongoing evaluating the safety and potential efficacy of entanercept (Enbrel) to reduce the levels of TNF-α; no results have been reported to date.

Nontransplant treatment strategies Androgens Primary treatment for bone marrow failure in patients with FA has historically been androgens with or without prednisone. Androgen therapy (most commonly oxymetholone initiated at 2 mg/kg/day) was first evaluated in patients with FA by Shahidi and Diamond [126]. Hematologic improvement occurs in 50% [127,128]. Patients responding to androgens typically do so within several months of drug initiation [129]. In addition prednisone (5–10 mg every other day) was frequently added in an attempt to promote vascular stability and decrease bleeding tendency in those with severe thrombocytopenia [129]. Whether the addition of prednisone plays any role remains unknown and has largely been abandoned. Further, it is unknown whether alternative androgen formulations offer advantages or disadvantages relative to oxymetholone, although there are clinical trials ongoing currently. Based on a review of the literature, Alter and Young [129] observed a 3-year survival advantage in those treated with androgens. Ideally, if used, androgen therapy should be initiated early after the onset of marrow hypoplasia and peripheral blood cytopenia. Although there has not been a formal analysis to determine the response rate in those treated at early versus late stages of marrow aplasia, early treatment may pre-empt the need for red cell or platelet transfusions. Once a response has been documented, the androgen dose should be tapered slowly and rarely discontinued. Except in South African Afrikaaners and at the period of early adolescence, few patients have been successfully tapered off without recurrence of marrow aplasia and peripheral blood cytopenia [129]. Importantly, patients treated with androgens must be monitored for peliosis hepatis, hepatic adenomata, and hepatocellular carcinoma (Plate 79.4) by ultrasound, computed tomography, and/or magnetic resonance imaging, and α-fetoprotein levels. If any of these complications occurs, alternative therapies, such as hematopoietic growth factors or HCT, should be investigated. Reports on the use of antithymocyte globulin (ATG), cyclosporine, high-dose methylprednisolone, and other immunosuppressants have been anecdotal [129]. Individual patients may respond to such therapy transiently with augmentation in peripheral blood counts. Overall, immunosuppressive therapy is not expected to be effective in patients with FA, in contrast to patients with acquired severe AA, but this has never been formally evaluated. Vitamins and antioxidants Shahidi and others have recommended the use of antioxidants to potentially reduce free radical oxygen toxicity that may play a role in the pathogenesis of the progressive marrow failure and epithelioid malignancy observed in patients with FA [130]. Daily intake of beta-carotene, vitamin C, vitamin E, and selenium has been suggested. Whether such therapy alters the natural history of marrow failure or the risk of

Hematopoietic Cell Transplantation for Fanconi’s Anemia

hematologic or nonhematologic malignancy (i.e. cervical carcinoma or head and neck cancers) is unknown. In contrast, there is probably a role for folic acid and iron supplementation in those receiving therapy to increase the red cell mass. Iron levels, however, should be evaluated prior to the initiation of iron supplementation as some patients will already have excess iron stores for unclear reasons.

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demonstrating hypersensitivity to alkylating agents and radiation, Gluckman et al. [137] proposed the use of low-dose CY and limited field irradiation schemata. These modifications ultimately led to markedly reduced risks of RRT, enhanced survival, and improved health quality of life. HCT with HLA-identical sibling donors

Hematopoietic growth factor therapy Two pilot studies [131,132] have been reported on the safety and efficacy of hematopoietic growth factor therapy in the treatment of neutropenia in patients with FA. While granulocyte–macrophage colony-stimulating factor and granulocyte colony-stimulating factor have both been used successfully to increase neutrophil counts with little systemic toxicity, it is unknown whether such therapy will alter the risk of MDS, development of clonal cytogenetic abnormalities, or risk of leukemia, if used long term. Erythropoietin has also been used to increase red cell counts and decrease the need for red cell transfusions, but no formal study has been published. However, due to the fact that many FA patients have highly elevated endogenous erythropoietin levels, it is unclear whether the addition of exogenous erythropoietin will be beneficial in the majority. Supportive care Eventually, FA will result in marrow failure, necessitating blood product support for the treatment of thrombocytopenia and anemia. While epsilon-aminocaproic acid may be helpful in patients with thrombocytopenia and bleeding, hematopoietic growth factor therapy and antibiotics are often needed in patients with neutropenia and fever. For the patient not pursuing allogeneic HCT as a treatment option, such combination therapy can aid in sustaining life for years. Risk of iron overload should be minimized by early chelation therapy. For the patient considering allogeneic HCT, use of red cell and platelet transfusions should be avoided if possible to reduce the risk of alloimmunization. If transfusions are required, an effort should be made to transfuse only irradiated, cytomegalovirus-negative (or filtered) red cell or single-donor platelet products. Whether specific, selected volunteer donors should be identified to limit exposure to human leukocyte antigens (HLAs) has not been proven. It is important that the clinician consider strategies for maximizing hematopoiesis even though a decline is ultimately unavoidable in most FA patients. For example, every effort should be made to avoid marrowsuppressive drugs, such as antibiotics, like Bactrim, often used in the treatment of chronic otitis media or urinary tract infections. In addition, young children with FA should be immunized against varicella-zoster virus and influenza as severe marrow suppression after such viral infection has been observed.

Registry studies HCT from HLA-identical sibling donors is generally associated with excellent outcome if performed early prior to the development of MDS or leukemia, particularly within the first two decades of life. Since the seminal work done by Gluckman [137] in the early 1980s demonstrating the need for a reduced-intensity preparative regimen, most patients have received a limited field irradiation as proposed by Gluckman, or a variant of this. Although reported more than 10 years ago, data from the largest review to date [138] using the database of the International Bone Marrow Transplant Registry (IBMTR), revealed a 2-year probability of survival of 66% (95% confidence interval [CI] 58–73%) for recipients of HLAidentical sibling hematopoietic cells (n = 151), with 91% of survivors having a Karnofsky performance score of over 90% at last follow-up. Graft failure was observed in 8% (95% CI 5–14%), grade II–IV acute GVHD was observed in 42% (34–50%), and chronic GVHD was observed in 44% (95% CI 34–53%) among patients surviving more than 90 days with evidence of engraftment. Interstitial pneumonitis was relatively common and was observed in 12% (95% CI 8–19%) of patients. Importantly, age at transplant, pretransplant platelet count, conditioning regimen, and GVHD prophylaxis were identified in multivariate analysis as factors that correlated with survival after HLA-identical sibling donor HCT. Older age (by decade) and lower pretransplant platelet count were associated with lower rates of survival, mostly as a result of increased graft failure and chronic GVHD. While lower platelet count may reflect longer-standing disease or greater transfusion exposure, the reason for the association between pretransplant platelet count and survival has thus far remained unclear. In addition, use of ATG as part of the conditioning regimen was associated with better survival. More recently, updated results of IBMTR data were reported on the survival of FA patients transplanted with HLA-identical sibling donor HSCs (Fig. 79.8) [139]. Among 209 patients transplanted between 1994 and 1999 from matched siblings, the 3-year survival was 81% (95% CI 72–90%) in patients less than 10 years of age (n = 109) and 69% (95% CI 59–79%) in older patients (n = 100).

100 HLA-identical sibling, ≤10y (n = 109)

Hematopoietic cell transplantation As of 2008, allogeneic HCT remains the only treatment with the potential for correcting the hematologic manifestations of FA. Early experiences with HCT were negative. Poor outcome was primarily the result of excessive regimen-related toxicity (RRT), including severe acute graft-versus-host disease (GVHD) [133,134]. However, early transplants used conditioning regimens devised for patients with acquired AA, administering cyclophosphamide (CY) at doses of 200 mg/kg with or without irradiation. In these patients, cardiomyopathy, severe mucositis, gastrointestinal hemorrhage, hemorrhagic cystitis, and severe skin toxicity were common. This early clinical experience prompted in vitro laboratory studies that confirmed the hypersensitivity of FA cells to CY [135,136] and irradiation [134]. As a result of these in vitro tests

Probability (%)

80

HLA-identical sibling, >10y (n = 100)

60 40

Unrelated, ≤10y (n = 36)

20 P = 0.0001 0

0

1

Unrelated, >10y (n = 58) 2

3

4

5

Years

Fig. 79.8 Probability of survival after human leukocyte antigen (HLA)identical sibling donor hematopoietic cell transplantation in patients with Fanconi’s anemia as reported to the International Bone Marrow Transplant Registry (IBMTR). (Reproduced from [139], with permission.)

6

1188

Chapter 79

Single-center studies Use of limited field irradiation in combination with low-dose CY has been the “conventional” therapeutic plan for most FA patients undergoing HCT from HLA-identical sibling donors in the United States and Europe. Based on the reports from single institutions, recipients of this approach can expect hematopoietic recovery to be in excess of 85% and survival rates in excess of 75% (Table 79.3) [140–147]. However, over the past decade, there has been a growing interest in the use of nonirradiation-conditioning regimens. Results of single institutional trials using such regimens are summarized in Table 79.4 [148–160]. Updated results with HCT for FA using HLA-identical donors and nonirradiation-conditioning regimens from various centers are summarized here. The largest single institutional experience with nonirradiation-containing regimens in patients with HLA-identical sibling donors has been at the Federal University of Parana, Curitiba, Brazil. As part of a CY de-escalation study, Bonfim et al. [161] have recently updated results at the current dose of CY 15 mg/kg × 4 days (60 mg/kg total dose; n = 42). Engraftment occurred in 41 of 42 patients, with a median time to neutrophil and platelet recovery (defined as >20,000/μL) of 19 (range 11– 57) days and 21 (range 14–57) days, respectively. While no patient died of transplant-related mortality, complications included late graft failure in 4 of 41, grade 3–4 mucositis in 27 of 42, grades II–IV acute GVHD in 5 of 41 and chronic GVHD in 4 of 41 patients. At a median follow-up of 2.5 years, 95% are alive. Notably, Flowers et al. [153] and de Mederios et al. [160] reported the results of the study at higher dose levels (CY 140–200 mg/kg and 100 mg/kg, respectively). While sustained engraftment may have been higher with higher doses of CY, overall survival was lower (Table 79.4). As an alternative to higher dose, MacMillan et al. at the University of Minnesota Medical Center and Children’s Hospital, Minneapolis, United States, explored low-dose CY with FLU based on the early promising results reported by Aker et al. [156]. To date, 18 patients (median age 8.0 [range 3.6–22.5] years) have received CY (20 mg/kg), FLU (175 mg/m2), and ATG (150 mg/kg) followed by the infusion of T-cell-depleted bone marrow (n= 12) or umbilical cord blood (UCB) (n = 6) through October 2007. Primary neutrophil recovery and engraftment was observed in 100%. No patient had grade 3–5 RRT or developed acute or chronic GVHD. Of the 18 patients with AA and an HLA genotypic identical donor, 17 of 18 are alive and well at a median of 3 (range 0.1–5.8) years. While promising, longer follow-up is required before determining whether such an approach will reduce the risk of late effects classically associated with radiation, such as infertility/sterility, cataracts, endocrinopathies, or risk of epitheloid cancers which is particularly common in adults with FA even without HCT. Whether such regimens are adequate for FA patients with advanced MDS or leukemia has yet to be evaluated. Summary of HCT from HLA-matched sibling donors in FA patients Together, the clinical data suggest that: (1) the optimum time for HCT is when the patient is young, has had few red cell and/or platelet transfusions, and is free of MDS or leukemia; and (2) the optimal therapy is one that minimizes exposure to DNA crosslinking agents, such as highdose CY or total body irradiation (TBI), at least for patients with FA and bone marrow failure and an HLA-identical sibling donor. In contrast to the very positive results observed in FA patients with bone marrow failure, or those with advanced MDS or leukemia are likely to have a significantly lower probability of survival. Based on a review of the literature, 7 of 14 FA patients with MDS and leukemia and an HLAidentical sibling donor were alive at the time of the report (Table 79.4). For those patients with pre-existing severe organ dysfunction or older

age (e.g. age >35 years), alternative therapies, such as with androgens, transfusions with chelation, and/or hematopoietic growth factors, should be considered, due to a high risk of TRM. For those patients with MDS or leukemia, the effectiveness of pretransplant chemotherapy or a nonTBI-containing preparative regimen is unknown. HCT with alternative donors The majority of FA patients do not have an HLA-identical unaffected sibling donor and therefore require an alternative (i.e., a HLA-mismatched related or unrelated) donor. Historically, HCT for FA using alternative donors has been less successful than that after HLA-identical sibling transplantation due to high rates of graft failure, RRT, GVHD, and opportunistic infections. Published data are summarized in Table 79.5 [138,153,140,162–167]. Registry studies In 1995, Gluckman et al. [138] were the first to report the outcomes of alternative donor HCT in patients with FA (n = 29 with an HLAmismatched related and n = 19 with an unrelated donor) from multiple institutions, using the database of the IBMTR. Overall survival was 29% at 2 years. Of note, graft failure was common (24%), suggesting that the reduced-intensity conditioning used in these patients was insufficient to achieve reliable engraftment. In 2000, Guardiola et al. [165] reported the outcomes of unrelated donor HCT in patients with FA (n = 69) on behalf of the European Group for Blood and Marrow Transplantation and the European Fanconi Anemia Registry. Overall survival was 33% at 3 years. The primary causes of death were acute GVHD (n = 18), primary or secondary graft failure (n = 13), chronic GVHD (n = 4), infections (n = 11), and sinusoidal obstructive syndrome of the liver (n = 1). However, in contrast to prior analyses, risk factors for mortality were identified. Malformations (three or more), use of androgens prior to HCT, positive recipient cytomegalovirus serology, and the use of a female donor were associated with poor survival. In 2007, Wagner et al. [166] reported the outcomes of unrelated donor HCT in patients with FA (n = 98) on behalf of the Center for International Blood and Marrow Transplantation Research. Of 83 patients surviving at least 21 days, the overall incidence of neutrophil recovery was 78% at a median of 11 days after HCT. In multivariate analysis, the use of a FLU-containing regimen was associated with a higher probability of neutrophil engraftment. Among recipients of non-FLU-containing regimens, engraftment tended to be poorer in those with evidence of DEB T-cell mosaicism (52% versus 89%; odds ratio 0.14, 95% CI 0.02–1.23; p = 0.076). The overall incidence of grade III–IV GVHD was 29%, with a higher incidence in recipients of a non-T-cell-depleted marrow (relative risk 4.59; 95% CI 1.82–10.10; p <0.001). The day 100 mortality rate was significantly lower in recipients of a FLU-containing regimen (24% versus 65%, respectively; p <0.001). Corresponding 3year adjusted overall survival rates were 52% versus 13%; (p <0.001). Factors associated with higher survival were younger recipient age (<10 years), CMV seronegativity in the recipient, a history of fewer than 20 blood product transfusions, and use of FLU in the preparative regimen. More recently in 2007, Gluckman et al. [167] reported the outcomes of unrelated donor HCT in patients with FA (n = 93) on behalf of Eurocord-Netcord and European Group for Blood and Marrow Transplantation, restricting the analysis to recipients of a matched (six of six match, n = 12) or partially HLA matched (five of six match, n = 35; three or four of six match, n = 45) unrelated UCB unit. The incidence of neutrophil recovery was 60 ± 5%, with a higher incidence of recovery in recipients of FLU and a unit containing 4.9 × 107 or more nucleated cells/kg recipient body weight. The incidence of acute grade II–IV and

CY 20 mg/kg + TAI 5 Gy

CY 20 mg/kg + TAI 5 Gy (n = 45) CY 40 mg/kg + TAI 5 Gy (n = 5) CY 20 mg/kg, TAI 4 Gy + ATG 160 mg/kg

CY 20 mg/kg + TAI 5–6 Gy (n = 12) CY 20–80 mg/kg, TBI 3–6 Gy (n = 10)‡ CY 100–200 mg/kg (n = 5) CY 20 mg/kg + TAI 4 Gy

Gluckman, 1993 [143]

Socié et al., 1998 [144]

DuFour et al., 2001 [146]

CSP (n = 18) CSP + MTX (n = 8) MTX alone (n = 1) CSP + MP + ATG

8.7 years (3–15 years) 9 years

7.6 years

27‡

30

N/A

N/A

10 years (1–36 years)

7.7 years

8 years (6–14 years) 13 years

Median recipient age (range)

23/25 (2.5– 19.5 years) 27/30

19

46/49

2/45

92%

5/5 (7–14 years) 16/17

7/8

Sustained engraftment

2/30

2/25

0

10/49

N/A

42% (grades II–IV)

0.17

1/5

4/8

Acute GVHD (grades III–IV)

90% alive 2007 at 10 years

81% alive at 3 years

76% alive at at 5 years 74% alive at 4.5 years 14/19 alive (1.7–48 months)

5/5 alive 1992 (18–66 months) 17/17 alive (6–76 months) 65% alive at 5 years

7/8 alive 1989 (4–60 months)

Outcome

ATG, antithymocyte globulin; CSP, cyclosporine A; CY, cyclophosphamide; GVHD, graft-versus-host disease; MP, methylprednisolone; MTX, methotrexate; N/A, not available; TAI, thoracoabdominal irradiation; TBI, total body irradiation; TCD, T-cell depletion. * Includes an unspecified number of patients receiving HCT from sibling donor umbilical cord blood. † Includes one patient who received a one-antigen-mismatched sibling bone marrow. Four patients received HLA-identical sibling donor umbilical cord blood HCT. ‡ Includes two patients who received phenotypically identical parental donor HCT.

Farzia et al., 2007 [147]

45*

19

Varied

Gluckman et al., 1995 [138]

17 (1.5–10.0 years) 151

5

CSP + ATG (n = 13) CSP, MTX + ATG (n = 6)

MTX + other 12% CSP + other 62% MTX + CSP 21% TCD 5% CSP

CY 20 mg/kg + TAI 4 Gy

Ayas et al., 2001 [145]

CSP (n = 1) CSP + MTX (n = 4) CSP + ATG (n = 17) + ATG 120 mg/kg

CY 20 mg/kg + TBI 5 Gy (n = 5)

Di Bartolomeo et al., 1992 [141] Kohli-Kumar et al., 1994 [142]

8

50†

CSP (n = 8) TCD (n = 2)

CY 20 mg/kg + TBI 6 Gy (n = 8)

Hows et al., 1989 [140]

CSP

GVHD prophylaxis

Conditioning regimen

Reference

Number of cases

Table 79.3 Human leukocyte antigen (HLA)-identical sibling donor hematopoietic cell transplantation (HCT): clinical results with radiation-based regimens

Hematopoietic Cell Transplantation for Fanconi’s Anemia

1189

1190

Chapter 79

Table 79.4 Human leukocyte antigen (HLA)-identical sibling donor hematopoietic cell transplantation: clinical results with chemotherapy only-based regimens Reference

Conditioning regimen

Barrett et al., 1977 [148] Kersey et al., 1979 [149] Holl et al., 1981 [150] Flowers et al., 1992 [151] Ratanatharathorn et al., 1993 [152]

CY 200 mg/kg + Procarb + ATG CY 200 mg/kg + 6MP 1 g/m2 CY 200 mg/kg + Procarb 25 mg/kg + ATG CY 100 mg/kg + BU 14 mg/kg BU 8 mg/kg + TLI 5 Gy

Flowers et al., 1996 [153]

CY 200 mg/kg (n = 17) CY 135 – 50 mg/kg (n = 8) CY 140 mg/kg + ATG (n = 9) CY 120 mg/kg (n = 7) BU 8 mg/kg + CY 40 mg/kg CY 100 mg/kg (n = 16) FLU 180 mg/m2 + CY 20 mg/kg + ATG FLU 75–180 mg/m2 + ATG + OKT3 FLU 125 mg/m2 + CY 20 mg/kg + ATG CY 40 mg/kg + BU 6 mg/kg ± ALG CY 60 mg/kg

Maschan et al., 1997 [154] De Mederios et al., 1999 [155] Aker et al., 1999 [156] Ebell, 2002 [158] Tan, 2006 [157] Torjemane et al., 2006 [159] De Medeiros et al., 2006 [160]

Number of cases

Engrafted

Outcome (range)

1/1 1/1 (GVHD) 1/1 1/1 (sepsis) 2/2

41*

92%

Alive >11 months Died, day 35 Alive >4.5 years Died, day 41 1 alive >20 months 1 died, day 45 (HUS, ARDS) 25/41 alive (median 3.1 years)

1 16 1** 7 11 17 35

1/1 16 1/1 5 10 14 35

Alive >17 months 88% alive (mean 1.6 years) Alive >11 months 5/7 alive 100% at 2 years 72% alive (median 16 months) 100% alive at 1 year

1 1 1 1 2

6MP, 6-mercaptopurine; ALG, antilymphocyte globulin; ARDS, adult respiratory distress syndrome; ATG, antithymocyte globulin; BU, busulfan; CY, cyclophosphamide; FLU, fludarabine; GVHD, graft-versus-host disease; HUS, hemolytic–uremic syndrome; Procarb, procarbazine. * Includes those patients who received HLA-matched (n = 1), one-antigen-mismatched (n = 1), or two-antigen-mismatched transplants. ** The patient received an HLA-identical sibling donor umbilical cord blood transplant.

chronic GVHD was 32 ± 5% and 16 ± 4%, respectively. The overall survival was 40 ± 5% with higher survival rates in CMV-seronegative patients and in recipients of FLU and a unit containing 4.9 × 107 or more nucleated cells/kg recipient body weight. Survival by HLA match was 74 ± 13% (six of six match, n = 12), 48 ± 9% (five of six match, n = 35), and 25 ± 7% (three or four of six match, n = 45). Single-center studies In addition to outcomes reported by the United States and European registries, single institutional trials have been reported and are summarized in Table 79.5. Updated results with HCT for FA using alternative donors from various centers are summarized. At the Memorial Sloan-Kettering Cancer Center [168] between March 1999 and December 2006, 21 patients aged 5–35 (median 11.5) years with FA underwent HCT using a mismatched related (n = 8) or unrelated (n = 13) donor. All received a preparative therapy consisting of TBI 450 cGy, FLU 150 mg/m2, CY 40 mg/kg, and rabbit ATG 10 mg/kg followed by infusion of a T-cell-depleted graft using CD34 selection and erythrocyte rosetting (peripheral blood hemtopoietic cells; n = 18). All were engrafted, and chronic GVHD occurred in two. With a median follow-up of 4.6 years and 2.2 years in recipients of mismatched related and unrelated donors, respectively, 14 of 21 patients are alive and well (7 of 10 patients with bone marrow failure, and 7 of 11 with MDS or AML). At the Children’s Hospital in Cincinnati between 2000 and 2006, 13 patients aged 1–15 (median 7) years with FA underwent HCT using an unrelated donor. All received a preparative therapy consisting of TBI 450 cGy, FLU, CY 40 mg/kg, and ATG, similar to that used at the University of Minnesota and Memorial Sloan-Kettering Cancer Center. All received a graft that had been T-cell depleted using CD34 selection (peripheral blood stem cells; n = 8). Sustained engraftment was observed in six of eight, and none developed acute GVHD. As of the date of the report, 8 of 13 are alive, with infection listed as the cause of death in 4 others.

Between 1993 and 2007 at the University of Minnesota Medical Center and Children’s Hospital, in a series of phase II trials, 102 patients aged 0.8–48.5 (median 11.2) years with FA underwent HCT using an HLA-mismatched related or unrelated donor. All received preparative therapy consisting of TBI, CY40 mg/kg, and ATG. All received a graft T-cell depleted using counterflow elutriation (1993–99) or CD34 selection (after 1999). As shown in Fig. 79.9, there has been steady progress in improving survival. The Minnesota trial 1 considered TBI dose escalation. Between June 1993 and March 1999, patients (n = 30) were treated with TBI 450 and 600 cGy, in addition to CY and ATG [169, with updates]. Sustained neutrophil recovery was poor at 61% (95% CI 3–79%) and did not differ with TBI dose. Factors associated with increased graft failure were presence of DEB T-cell mosaicism (p = 0.04) and lower cell dose (<3.4 × 107 nucleated cells/kg; p = 0.05). Of 25 patients evaluable for engraftment, 7 of 12 patients with DEB T-cell mosaicism experienced primary graft failure in contrast to 3 of 13 patients without DEB T-cell mosaicism. The incidence of neutrophil recovery for patients with and without DEB T-cell mosaicism was 42% (95% CI 14–70%) and 83% (95% CI 61–100%), respectively (p = 0.05), suggesting that the presence of DEB-resistant T cells increased the risk of graft rejection, and that a regimen of CY/TBI 450–600 cGy/ATG was insufficient for their complete eradication. The Minnesota trial 2 was on the addition of FLU to CY/TBI 450 cGy. Between April 1999 and December 2003, in an attempt to promote engraftment, 41 patients were treated with FLU 150 mg/m2 in addition to CY, TBI 450 cGy, and ATG. The incidence of neutrophil recovery was 98% (95% CI 93–100%). No factor, including the presence of DEB T-cell mosaicism, was associated with engraftment. The incidence of grade III–IV acute GVHD was 19% (95% CI 7–30%) and that of chronic GVHD was 14% (95% CI 3–24%). While overall survival was 53%, when stratified on the basis of risk group (high risk defined as age ≥18 years, history of systemic fungal or Gram-negative infection, or advanced MDS or leukemia), the probability of survival was 61% (95%

CY 40 0 mg/kg + Ara-C

Dini et al., 1997 [163]

TCD + CSP Varied Varied

CY 40 mg/kg + TBI 4.5–6.0 Gy

Varied

Varied

MacMillan et al., 2000 [165]

Wagner et al., 2007 [166]

Gluckman et al., 2007 [167]

93

98

29

69

6

7

29

2

8.6 (1–45)

12 (0.8–33)

12.1 (3.7–48.5)

10.8 (4.0–37.4)

n.r.

7–28 (n = 2)

9

14, 23

12 (5–13)

(years)

Recipient age

21% (TCD) 70% (no TCD) 32 ± 5%%

69% (no FLU) 60 ± 5%

2/18

34%

n.r.

4 evaluable

1 grade III–IV of

51% grade II–IV

1 grade III–IV

4 evaluable

1 grade III–IV of

Acute GVHD

89% (FLU)

63%

83%†

100%

4 of 5 evaluable

76%

2 of 2

3 of 5

engraftment

Sustained

25 ± 7% (HLA 3–4/6, n = 45)

48 ± 9% (HLA 5/6, n = 35)

74 ± 13% (HLA 6/6, n = 12)

13% (no FLU) at 3 years

52% (FLU) at 3 years

34% at 1 year

33% at 3 years

3/6 surviving 3 months–4 years

3 surviving >10* years

29% surviving 2 years

0/2 alive BU 14 mg/kg

1/5 alive at >4 years

Outcome

thoracoabdominal irradiation; TBI, total body irradiation; TCD, T-cell depletion.

Ara-C, cytosine arabinoside; CSP, cyclosporine; CY, cyclophosphamide; FLU, fludarabine; GVHD, graft-versus-host disease; MTX, methotrexate; n.r., not reported specifically for Fanconi’s anemia patients; Pred, prednisone; TAI,

† Probability of secondary graft failure was 19%.

* Updated survival.

Varied

Varied

Varied

Guardiola et al., 2000 [164]

18 mg/m2 TAI or TBI 5 Gy

MTX + CSP (n = 5)

CY 40 mg/kg + TBI 4.0–4.5 Gy

Davies et al., 1996 [162] MTX + Pred + XomaZyme

MTX + CSP Varied

CY 100–120 mg/kg + TBI 12 Gy

Varied

Flowers et al., 1996 [153]

5

CSP (n = 5) TCD (n = 4)

CY 20 mg/kg TBI 6 Gy

Hows et al., 1989 [140]

Gluckman et al., 1995 [138]

of cases

GVHD prophylaxis

Conditioning regimen

Number

Reference

Table 79.5 Alternative donor hematopoietic cell transplantation: clinical results

Hematopoietic Cell Transplantation for Fanconi’s Anemia

1191

1192

Chapter 79

Cumulative proportion

1.0

II

I

p <0.01

MT0605 (86%) I

I I I II

0.8

I

MT0318 (71%) I I I II I I I I

0.6

I I

I I

I

I

MT9905 (60%)

0.4 MT9510 (23%) I

0.2 0.0 0

1

2

3

4

5

Years Fig. 79.9 Probability of survival after alternative donor hematopoietic cell transplantation or the treatment of patients with standard risk Fanconi’s anemia at the University of Minnesota between 1993 and 2007. All patients received cyclophosphamide and total body irradiation in combination with fludarabine or thymic shielding.

CI 44–78, n = 31) for standard-risk patients and 33% (95% CI 6–60, n = 12; p = 0.01) for high-risk patients at 2 years. The Minnesota trial 3 involved FLU/CY/TBI with thymic shielding. Based on preclinical work suggesting that thymic shielding during TBI might improve immune reconstitution in recipients of allogeneic HCT [170], we evaluated the safety and potential efficacy of thymic shielding in patients with FA. After CT localization of the thymus, 5HVL Cerrobend blocks were fabricated and used to shield the thymus. Between 2003 and 2006, 16 patients were treated with FLU, CY, TBI 450 cGy, and ATG with thymic shielding. The incidence of neutrophil recovery was 94% (95% CI 92–100%). Incidence of grade II–IV acute GVHD was 32% and that of chronic GVHD 0%. Overall survival was 67%, with higher survival (79%) in standard-risk patients. Notably, thymic shielding was associated with a significantly lower risk of opportunistic infections (9 infections in 16 shielded patients versus 126 in 43 nonshielded patients; p < 0.01). The Minnesota trial 4 studied FLU, CY, and TBI dose de-escalation. TBI dose de-escalation strata were TBI 300 cGy (cohort 1), TBI 150 cGy (cohort 2), and no TBI (cohort 3); otherwise, all patients received the same treatment as in trial 3. Between July 2006 and July 2007, 11 FA patients were enrolled in cohort 1, and two in cohort 2. All patients achieved primary engraftment at a median of 10.5 (range 9–30) days. Two of 11 patients developed grade II acute GVHD, with none having chronic GVHD to date. With a median follow-up of 1 year, 10 of the 11 in cohort 1 are alive with good engraftment and without GVHD. Nonirradation-based conditioning regimens prior to alternative donor HCT As reported above for recipients of HLA-identical sibling donor HCT, use of nonirradiation-based conditioning regimens has been explored in the setting of HLA-mismatched, related and unrelated donor HCT with some success. At the Federal University of Parana (Bonfim, personal communication), patients with FA (n = 7, median age 9 years) were treated with CY 60 mg/kg, as described above for recipients with sibling donors. Neutrophil engraftment was achieved in all patients at a median of 16 (range 10–25) days. One patient developed grade II acute GVHD, and survival at 1 year is 100%. The same group added FLU 125 mg/m2 and rabbit ATG 4 mg/kg to CY 60 mg/kg in FA patients (n = 27, median age 8 years) with UCB donors. Of 21 patients evaluable for engraftment,

only 48% had a complete hematologic recovery. Median time to neutrophil recovery was 23 (range 12–35) days, and median time to platelet recovery (>20,000/μL) was 27 (range 15–50) days. Four of 15 evaluable patients developed grade III–IV acute GVHD, and 5 of 11 evaluable patients developed extensive chronic GVHD. Overall survival at 1 year was 41%, with a trend toward higher survival in patients with five or six of six HLA-matched UCB units (56% versus 28%; p = 0.22) [161]. At the Birmingham Children’s Hospital in the United Kingdom, Motwani et al. [171] transplanted seven children with FA from 2000 to 2004 using a nonirradiation-based preparative therapy consisting of FLU 125 mg/m2, CY 20–30 mg/kg, and ATG. Donor sources included HLAmatched unrelated UCB (n = 4) and HLA-matched unrelated (n = 2) and haploidentical (n = 1) peripheral blood grafts. Five of seven patients achieved sustained engraftment, with the two patients who rejected their grafts later achieving engraftment after a second transplant. All patients developed grade II–III acute GVHD. All are alive 1 year after HCT. HCT for the treatment of MDS and leukemia in FA patients Experience in the treatment of FA patients who have developed leukemia is limited. Untreated patients generally die of disease progression within months to a year of diagnosis. Clinical experience with chemotherapy in these patients is scant, although there are anecdotal reports of responses to chemotherapy and HCT. These data indicate that longterm survival is possible in these patients, and they should be considered candidates for HCT. The apparent successful eradication of the malignant clone in these patients with generally reduced doses of chemotherapy suggests that the malignant cells carry a similar hypersensitivity to alkylating agents and irradiation, characteristic of nonmalignant cells in patients with FA. However, there are currently no laboratory studies available addressing this point, which would be of great interest in planning future therapy. Despite successful engraftment after non-TBI-based preparative therapy, patients with MDS and excessive blasts or AML may be at high risk of relapse after HCT. TBI or other novel approaches should be considered. Further, it is unclear whether chemotherapy should be administered prior to transplantation. There are no reports in the literature to suggest that such therapy is helpful; local experience does not support the use of cytoreduction prior to HCT due to the extremely high risk of severe toxicity and peritransplant mortality; however, numbers are small (Wagner, unpublished observations). At the University of Minnesota, we developed a busulfan-based regimen for FA patients with leukemia. Between December 2002 and July 2007, six FA patients with acute leukemia (four with AML and two with acute lymphoblastic leukemia) received busulfan 3.2 mg/kg, FLU 140 mg/m2, CY 40 mg/kg, and ATG. Stem cell sources included five of six HLA-matched, related, T-cell-depleted marrow (n = 1), five of six HLA-matched, unrelated donor, T-cell-depleted marrow (n = 2), and five of six HLA-matched, unrelated UCB (n = 3). All patients achieved neutrophil engraftment at a median of 16 (range 11–20) days. One patient developed grade I acute GVHD, and no patient has developed chronic GVHD. Relapse occurred in one. The overall probability of survival at 1 year is 52%, with a median follow-up of 2 years. Summary of HCT from alternative donors in the treatment of FA To date, there have been a number of observations regarding risk factors for engraftment, GVHD, and survival in FA patients transplanted with alternative donor HSCs (reviewed in Table 79.6). While it is not surprising that age, organ function, and Karnofsky performance status are indeed predictive of survival, it is noteworthy that more recent analyses suggest that the number of malformations (three or more sites), prior exposure to androgens, and absence of FLU in the preparative therapy predict poorer survival. The current challenges include: (1) determining

Hematopoietic Cell Transplantation for Fanconi’s Anemia Table 79.6 Summary of risk factors Outcome

Variable

p-value

Neutrophil

Age at transplant <10 years Serum AST/ALT >2× normal prior to HCT

0.001 <0.001

Engraftment

Male donor Absence of three-lineage cytopenia prior to HCT T-cell somatic mosaicism Stem cell dose HLA mismatch Recipient CMV serostatus T-replete unrelated donor BMT Fludarabine-containing preparative therapy

<0.04 <0.06 0.05 0.05 0.05 0.05 0.003 0.001

Acute GVHD

T-replete stem cells Serum AST/ALT >2× normal prior to HCT Malformation of urogenital tract and/or kidneys Limb malformations HLA mismatch Recipient CMV serostatus T-cell-depleted unrelated marrow

0.04 <0.04 <0.02 <0.04 0.0005 0.02 <0.0001

Survival

Extensive malformations (≥3 sites) Serum AST/ALT >2× normal prior to HCT Positive recipient CMV serostatus Stem cell dose Age (increment in decades) Karnofsky score prior to HCT Androgen therapy prior to HCT Fludarabine in conditioning regimen

0.005 0.005 0.02 0.03 0.002 0.03 0.04 0.003

Risk factors identified in Guardiola et al., 2000 [164], MacMillan et al., 2000 [165], and Wagner et al., 2007 [166]. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMT, bone marrow transplantation; CMV, cytomegalovirus; GVHD, graft-versus-host disease; HCT, hematopoietic cell transplantation; HLA, human leukocyte antigen.

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the optimal time for HCT (proposed process shown in Fig. 79.10); (2) predicting individual patient sensitivity to chemoradiotherapy; and (3) understanding the effect of the mosaic phenotype on the natural history of the disease. With markedly improved rates of survival in the current era of HCT in FA, emphasis is now being placed on improving quality of life by reducing late effects, particularly the risks of malignancy, sterility, and endocrinopathies. Improvements in therapy are likely to proceed more rapidly if patients are treated according to common protocols and data are shared between institutions. Cancer risk after HCT for FA patients Long-term follow-up studies indicate that, although successful HCT may cure the hematologic complications of FA, patients are still at high risk for malignancy after HCT [9,10,172–174]. In an analysis of 700 patients with FA (n = 79) or AA (n = 621) treated with allogeneic HCT in Seattle or Paris, the Kaplan–Meier estimate for developing any malignancy by 20 years after transplantation was 14% [158]. Among patients with FA, a single hazard peak for solid tumors occurred between 8 and 9 years after HCT. The Kaplan–Meier estimate of developing any malignancy by 20 years after HCT was 42% (95% CI 10–74%), with all being solid tumors, while no lymphohematopoietic malignancies were seen as in patients with severe AA. In the multivariate analysis of the 700 patients with marrow failure (FA and non-FA), the diagnosis of FA (relative risk 11.2; p = 0.0001) and treatment with azathioprine (relative risk 11.7; p = 0.0001) were independent predictors of secondary malignancy, while use of irradiation was not. Importantly, for those transplanted, patients were 17–29 years of age at the time of solid tumor diagnosis. The role of HCT in the development of late solid tumors in patients with FA, however, remains speculative. Increased susceptibility to AML and solid tumors has long been recognized in children and adults with FA cured with HCT. However, the incidence and relative risk of these events have been difficult to quantify until recently. Two studies have recently been reported that address cancer risk in this patient population. Rosenberg et al. [9] assembled a survey cohort in the United States and in Canada. Questionnaires

Fanconi’s anemia Moderate pancytopenia

AML/MDS Novel BMT

Sibling donor

No sibling donor

BMT/UCBT

Standard BMT risk

Fig. 79.10 Proposed treatment algorithm for patients with Fanconi’s anemia. AML, acute myeloid leukemia; BM, bone marrow; BMT, bone marrow transplantation; G-CSF, granulocyte colony-stimulating factor; HLA, human leukocyte antigen; HLA-mm, HLA mismatch; MDS, myelodysplastic syndrome; UCB, umbilical cord blood; UCBT, umbilical cord blood transplant.

HLA-matched unrelated BM or 5-6/6 matched UCB donor

BMT/UCBT

Phase I study

h/o infection or organ failure

Poor BMT risk

HLA-mm BM or <5/6 UCB donor Androgens

? Response

or Transfusions/ G-CSF

No response or toxicity

New agents

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included demographics, physical malformations, and age of cancer onset. Cancer diagnoses were confirmed by medical records, pathology reports, and/or death certificates. In order to calculate the cancer risk relative to the general population, results in patients with FA were compared with the observed numbers of cancers by age and type in established cancer registries. In this study, 27 cancers were observed in 23 individuals of the 145 returning the questionnaire [9]. There were 9 cases of leukemia and 18 cases of solid tumor. The median age of subjects at leukemia diagnosis was 11.3 (range 3–24) years in contrast to 28.9 (range 7–45) years at solid tumor diagnosis. Compared with the general population, these data suggest a 785-fold higher risk of leukemia and 48-fold higher risk of solid tumor in patients with FA. Importantly, the observed-to-expected ratio of solid tumors of the head and neck, esophagus, and vulva is 706, 2362, and 4317, respectively. Further, the relative risk of solid tumor was low until age 28 years. By age 28, 34, and 48 years, the risk was 9%, 22%, and 29%, respectively. As shown in Fig. 79.2, the rise in relative risk of solid tumors is nonlinear with no plateau. A report from IFAR further highlights the susceptibility of FA patients to both benign and malignant tumor development [10]. The cumulative incidence of hematologic neoplasms and solid tumors by age 40 is 33% and 28%, respectively. Evaluation of the types of neoplasms that FA patients developed revealed a wide array of different solid neoplasms (Table 79.7), with a marked predisposition toward squamous cell carcinoma, especially of the upper aerodigestive and anogenital regions. The increase in the cumulative incidence of head and neck cancer in FA patients compared with the Surveillance, Epidemiology, and End Results population is 500-fold [10]. A critical question is whether treatment consisting of chemotherapy and irradiation in combination with HCT alters the underlying cancer risk in FA patients. In the study by Rosenberg et al. [9], there appeared to be a trend toward higher risk in HCT recipients. In the IFAR study [10], the statistical analysis of the relationship between HCT and solid neoplasm development could not be performed due to the small number of HCT patients who developed solid tumors. Notably, the percentage

of patients who had undergone HCT and developed solid neoplasms (2.7%) was actually less than the percentage of patients without HCT who developed solid neoplasms (13%) [10]. While not conclusive, these results suggest that the underlying chromosomal instability characteristic of FA appears to play the critical role in the development of solid tumors with an as yet undetermined effect by HCT therapy. Based on these observations, there are four key conclusions: 1. The risk of solid tumor does not appear to be affected by chemoradiotherapy and HCT, although this remains unproven. 2. Solid tumors begin to appear at 5 years of age, with more than a linear rise in risk without a plateau. 3. As survival rates improve with HCT, and death from marrow failure and leukemia are reduced, a greater proportion of FA patients will be “at risk” as they enter the third and fourth decades of life. 4. Rigorous screening and cancer prevention programs are critical to the survival of FA patients, particularly after the age of 20 years.

Future directions Improved understanding of the function of the FA genes has already profoundly affected our understanding of apoptosis, DNA repair, and regulation of normal hematopoiesis. Additionally, there is a concerted effort by numerous investigators to find novel therapies that can potentially alter the natural history of the disease. In contrast to reports in the past, HCT now has an improved capacity to correct the hematologic manifestations of this syndrome. None the less, risk of morbidity and mortality is still present, and new therapeutic strategies should be explored, such as the development of safer preparative therapies and potentially safer methods of gene correction. In addition, recent developments in assisted reproduction technologies can now permit couples at high risk of having a child with FA to conceive a child free of the disease and with a desired HLA type. Although complicated by numerous ethical and legal issues, the use of preimplantation genetic diagnosis (PGD) is growing rapidly. Gene therapy

Table 79.7 Observed cancers (O), ratio of observed to expected cancers (O/E), and 95% confidence intervals (95% CI) among North American respondents with Fanconi’s anemia Type of cancer

O†

O/E‡

95% CI§

Leukemia (AML) Head and neck Esophagus Liver Vulva Cervix Osteosarcoma Soft tissue sarcoma Brain Total cancers Total solid tumors

9 6 2 2 3 2 1 1 1 27 18

785* 706* 2362* 386* 4317* 179* 79 49 17 50* 48*

360–1490 260–1540 265–8530 45–1395 870–12,615 20–645 1–440 0.6–270.0 0.2–95.0 35–80 30–80

AML, acute myeloid leukemia. * p <0.05 that true O/E ratio equals 1.0 (exact two-sided tests). † Twenty-seven cancers observed in 23 patients. Two patients had two solid tumors (cervix and vulva, and vulva and oesophagus), and one had three solid tumors (oesophagus, liver, and cervix). ‡ Expected cancer incidence rates calculated from the Connecticut Tumor Registry. § Limits of the 95% CIs rounded to the nearest 5 for values ≥10. Reproduced with permission from Rosenberg and Greene [9].

The possibility of using gene transfer methods for correcting genetic diseases of HSCs has received much attention. Thirteen FA genes have been cloned, and the sequences for these genes have been placed in the public domain. Currently, gene vector technology is being used in the laboratory to test whether a patient belongs to one of these thirteen complementation groups. Once the complementation group is known, mutation screening for a particular family is targeted. This technology will rapidly expand our ability to predict the pace of disease progression and risk of hematologic malignancy, as well as identify which patients are appropriate for gene transfer studies. The availability of FA genes has spurred considerable interest in the development of gene therapy for this disease for two reasons: (1) few FA patients have HLA genotypically identical HSC donors; and (2) the risks of morbidity and mortality after unrelated donor HCT in specific high-risk subpopulations are substantial. An alternative treatment would be the infusion of genetically corrected HSCs. Retroviral transduction of FANCA, -C, or -G cDNA into primary FA cells has already been shown to correct crosslinking agent hypersensitivity and chromosomal instability in hematopoietic colony-forming cells as well as improve progenitor cell survival compared with uninfected cells. While there have been numerous reports [175–177] demonstrating that recombinant virus transduction of FA cDNA into FA CD34+ hematopoietic progenitor cells improves clonogenic potential with phenotypic correction of colony-forming cells in vitro, as shown by resistance to MMC-induced cell death, loss of susceptibility to chromosomal breakage, and restoration

Hematopoietic Cell Transplantation for Fanconi’s Anemia

of cell-cycle kinetics, clinical trials of gene transfer have been disappointing. Liu et al. [175] reported the clinical results in four FA-C patients treated with autologous hematopoietic progenitor cells transduced with the FANCC gene. In this study, a G1FASvNa.52 retroviral vector containing the FANCC and neomycin resistance coding sequences was used. After infusion, the FANCC transgene was present transiently in the peripheral blood and bone. Function of the FANCC transgene was suggested by a marked increase in hematopoietic colony-forming cells, including colonies in the presence of MMC, after successive infusions of transduced cells. Despite a growth advantage in vitro, there has been only transient evidence of gene-corrected HSC amplification in vivo. A great deal remains to be learned regarding gene therapy in the treatment of FA. Optimization of HSC mobilization, collection, and gene transduction, as well as long-term expression and safety questions, have yet to be elucidated. More recent work suggests that lentiviral vectors might be a strategy for efficient gene correction of quiescent HSCs. Galimi et al. [178] have demonstrated correction of FANCA−/− and FANCC−/− mice. Long-term repopulating hematopoietic progenitor cells were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying normal FANCA or FANCC genes. Notably, no cell purification or cytokine prestimulation was necessary, reducing the risk of nonspecific HSC loss, especially important in this setting where there are few HSCs available. Resistance to clastogenic agents was fully restored by lentiviral transduction, which allowed for subsequent in vivo selection of the corrected cells by exposure to low-dose CY. While gene therapy may not be readily applicable to FA patients today, it is a particularly attractive candidate disease for such a therapeutic approach owing to an inherent selective advantage. In some instances, collection of autologous HSCs may be considered, but only early in the course of the disease, prior to marrow aplasia or MDS when a limited number of “normal” HSCs are still available. Multipotent stem cells and tissue repair Embryonic stem cells are pluripotent cells derived from the inner cell mass of the blastocyst that can be propogated indefinitely in the undifferentiated state or differentiated into all cell lineages, at least in vivo. Recently, pluripotent mesenchymal stem cells have also been isolated from adult marrow [179]. After injection into an early blastocyst, single adult stem cells have contributed to most, if not all, somatic cell types. Irrespective of their origin, embryonic stem cells and adult stem cells hold great promise for the treatment of many degenerative and inherited diseases. For example, adult stem cells from a closely HLA-matched healthy donor could be used alone or in combination with HSCs. Autologous adult stem cells could be genetically modified and then be used in the treatment of diseases such as FA and cancer. In the setting of FA, adult stem cells will likely be used as a strategy to enhance tissue repair after chemoradiotherapy. Alternatively, genetically corrected adult stem cells from the FA patient him- or herself could be used as a source of HSCs. Due to their proliferative capacity, adult stem cells can be genetically corrected with high efficiency. Gene correction of adult stem cells followed by differentiation into HSCs is being explored as a particularly attractive strategy for treating the bone marrow failure common to most patients with FA. Assisted reproduction and PGD For all high-risk couples (typically when at leasat one child is known to have FA), reproductive counseling is mandatory. At a minimum, couples need to be aware of the risks of FA in future children so that they can make an informed decision about birth control, prenatal testing, and use

1195

of various assisted reproduction techniques, including embryo screening with PGD [180]. If the specific couple is interested in the use of in vitro fertilization (IVF) with PGD for the purpose of avoiding the implantation of affected embryos, complementation group assignment is required today. Once known, parental-specific FA gene mutations or polymorphisms are determined for genetic screening of embryos. In addition, couples can also screen for a specific HLA type, for example in the circumstance that an HLA-matched, unaffected child is desired to serve as an allogeneic HSC donor. In these circumstances, it is important to identify a team experienced in IVF that understands the specific goals of the couple and can provide reliable estimates of the chance for success. Importantly, the IVF procedure does not have to be performed at the location of the PGD team or transplant center. As with IVF for infertile couples, the procedure requires daily injections to stimulate follicle development and maturation. After retrieval, each ovum is fertilized. After 2–3 days, at the 8- to 10-cell stage, a single cell is removed and tested for FA and HLA. Embryos that are free of disease and HLA identical (if appropriate) are identified and implanted at that time or at a future time, depending upon the desire of the family (Plate 79.5) [180]. Due to the risk of error, CVS or amniocentesis must be performed to confirm that the fetus is healthy and HLA identical. At the time of this review, three such errors are known where affected children have been born. However, in most circumstances, prior to delivery, arrangements are made for collection and shipment of the UCB from the HLA-identical healthy sibling to the transplant center. Prior to HCT, the newborn donor is evaluated for FA and HLA a third time, due to the critical importance of these tests to the child being transplanted. Procedural steps are as follows [181]: • hold a conference with the transplant physician and genetic counselor; • obtain complementation group assignment and mutation analysis; • HLA type the mother, father, and child affected with FA (if appropriate); • referr to the IVF center; • CVS/amniocentesis to confirm the health and HLA status of the fetus; • UCB collection and harvesting; • confirmatory testing on UCB or the newborn baby; • transplantation of HLA-matched UCB. While the breadth of applicability remains to be identified, and as the debate regarding the use of PGD to select embryos with desirable characteristics continues, it is clear from the number of referrals that there is a significant interest in using this technology. In addition to being aware of such technology, transplant physicians need to be intimately involved from the outset, as the patient’s condition may not allow sufficient time for the delivery of the healthy HLA genotypic-identical sibling donor.

Summary Clearly, all patients with FA should be followed routinely by a hematologist, even prior to the onset of marrow failure. To better understand the natural history of this disease and the significance of cytogenetic clonal abnormalities, patients must have marrow examinations annually and more frequently after the onset of marrow failure and/or development of clonal hematopoiesis. Moreover, the hematologist must be aware of the importance of family genetic counseling, the availability of prenatal diagnosis, and PGD. However, the hematologist must also be aware that the availability of predictive testing has led to complex ethical issues, such as deliberate conception of a fetus and, more recently, embryo selection for the possibility of providing an unaffected HLAidentical sibling HSC donor. These and other ethical issues are discussed in detail elsewhere [182].

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For general purposes, a treatment algorithm is suggested (see Fig. 79.10). Clearly. specific issues for a given patient with FA may necessitate alternative considerations. However, at the time of diagnosis, it is critical that all siblings of the proband be tested for FA regardless of phenotype and HLA type. In those circumstances where a potential donor already exists, transplant should be done earlier prior to severe pancytopenia. In those circumstances where no related donor

exists, alternatives include use of assisted reproduction techniques with PGD and a search of the unrelated volunteer registries and UCB banks. While outcomes with unrelated donor HCT have substantially improved over the past 5 years, this therapeutic approach should still be reserved until the development of a severe cytopenia or any sign of clonal progression. New treatment strategies are on the horizon.

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identical donors: results of a TBI dose escalation trial. Br J Haematol 2000; 108: 1–10. Amagai T, Kina T, Hirokawa K, Nishikawa S, Imanishi J, Katsura Y. Dysfunction of irradiated thymus for the development of helper T cells. J Immunol 1987; 139: 358–64. Motwani J, Lawson SE, Darbyshire PJ. Successful HSCT using non-radiotherapy-based conditioning regimens and alternative donors in patients with Fanconi anaemia – experience in a single UK centre. Bone Marrow Transplant 2005, 36: 405– 10. Deeg HJ, Socié G, Schoch G et al. Malignancies after marrow transplantation for aplastic anemia and Fanconi anemia: a joint Seattle and Paris analysis of results in 700 patients. Blood 1996; 87: 386–92. Socie G, Devergie A, Girinski T et al. Transplantation of Fanconi’s anaemia: long-term follow-up of fifty patients transplanted from a sibling donor after low dose cyclophosphamide and thoracoabdominal irradiation for conditioning. Br J Haematol 1998; 103: 249–55. Reed K, Ravikumar TS, Gifford RR, Grage TB. The association of Fanconi’s anemia and squamous cell carcinoma. Cancer 1983; 52: 926–8. Liu JM, Kim S, Read EJ et al. Engraftment of hematopoietic progenitor cells transduced with the Fanconi anemia group C gene (FANCC). Hum Gene Ther 1999; 10: 2337–46. Walsh CE, Mann MM, Emmons RVB, Wang S, Liu JM. Transduction of CD34-enriched human peripheral and umbilical cord blood progenitors using a retroviral vector with the Fanconi anemia group C gene. J Invest Med 1995; 43: 379– 85. Walsh C, Fu K, Brecher M, Kirby S, DiBartolomeo P, Jacobs P. Retroviral mediated gene transfer for group A Fanconi anemia patients. Fanconi Anemia Research Fund Scientific Symposium 2000: 46a. Galimi F, Noll M, Kanazawa Y et al. Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors. Blood 2002; 100: 2732–6. Jiang Y, Jahagirdar B, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418: 41–9. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. JAMA 2001; 285: 3130–3. Verlinsky Y, Kuliev A, editors. An Atlas of Pre-implantation Genetic Diagnosis. New York: Parthenon, 2000. Robertson JA, Kahn JP, Wagner JE. Conception to obtain hematopoietic stem cells. Hastings Cent Rep 2002; 32: 34–40.

Section 7 Complications of Hematopoietic Cell Transplantation

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Mechanisms and Treatment of Graft Failure

Introduction The ability of hematopoietic stem cells (HSCs) to circulate and migrate from blood to marrow and back is an intrinsic aspect of the hematopoietic system that has been conserved through evolution. The biologic role and physiologic significance of this constitutive HSC circulation remains unclear, yet it is this capacity to traffic that leads to hematopoietic cell engraftment and forms the essential requirement for success of hematopoietic cell transplantation (HCT) in the treatment of hematologic and nonhematologic diseases. The failure to achieve sustained engraftment following HCT is associated with considerable morbidity and mortality, most notably infections and hemorrhagic complications due to marrow hypoplasia, peripheral cytopenias, and/or relapse of disease. This chapter will review the mechanisms and contributing factors that result in graft failure.

Overview of trafficking and homing of HSCs The restoration of adequate blood cell production after transplantation requires a series of balanced interactions between the infused intact HSC and its complex multidimensional supporting marrow microenvironment (see Chapters 6 and 7). As an initial step, the infused HSCs must adhere to the bone marrow (BM) endothelium with sufficient strength to overcome the shear forces of blood flow [1]. The dominant HSC cell surface molecules that mediate adhesion and arrest are the selectin ligands P-selectin glycoprotein ligand-1 and the hematopoietic cell L-/E-selectin ligand that interact principally with endothelial E selectin [2–4]. Other HSC surface adhesion molecules that mediate adherence to the BM endothelium are a subset of the integrin superfamily principally very late antigen-4, α4β7, and lymphocyte function-associated antigen1, that interact with endothelial immunoglobulin superfamily receptors (e.g. vascular cell adhesion molecule-1 [VCAM-1]), and the hyaluronate receptor CD44 [5,6]. HSCs that are null for the β integrins cannot migrate to their BM niche even though they proliferate and differentiate appropriately in the fetal liver [7]. Following firm adherence, the transendothelial movement and intraparenchymal homing to hemopoietic niches located within the inner endosteal surface of the bone is predominantly regulated by a gradient of extracellular matrix-bound stromal cell-derived factor-1 (SDF-1; also known as CXCL12) binding to the CXCR4 receptor on HSCs [8]. The SDF-1/CXCR4 axis is nonredundant, which means this is an exception among the chemokine family as this one receptor, CXCR4, binds this Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

one chemokine, SDF-1, and vice versa. The requirement for CXCR4 expression on HSCs for homing and engraftment is well documented [9,10]. Mice genetically deficient in CXCR4 develop fetal liver hematopoiesis but die prenatally due to the lack of BM hematopoiesis [8]. In keeping with this observation, in vitro exposure of human CD34+ cells to uridine triphosphate, which reduces downregulation of CXCR4 expression, results in superior homing efficiency in nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) mice [11]. Following successful homing, the initial adhesion of the HSC within the hematopoietic niche appears regulated at least in part by annexin II as inhibitors reduce binding to the BM microenvironment [12]. The marrow niches represent complex biologic units composed of different cell types that include primitive and potentially self-renewing mesenchymal stem cells, as well as cells with the defined phenotype of parathyroid hormone receptor-bearing osteoblasts [13,14]. Recent observations suggest that mesenchymal stem cells promote and enhance engraftment when co-transplanted with HSCs [15]. Osteoblasts, possibly in conjunction with sinusoidal endothelial cells, appear to also play a pivotal role in the regulation of HSC engraftment by producing a number of molecules, such as annexin II, VCAM-1, intercellular adhesion molecule, CD44, CD164, and osteopontin, that promote engraftment [12,16–18]. Stimulation of osteoblasts with parathyroid hormone following transplantation results in a cyclic adenosine monophosphatemediated expansion of the HSC pool that enhances engraftment and improves the survival of lethally irradiated animals [13,19]. In addition to the regulators of HSC adhesion and homing, the function of the HSC is further regulated by its intrinsic genetic programs for quiescence, self-renewal, proliferation, differentiation, and apoptosis that are codependent on communication with a network of interacting cells localized in the marrow microenvironment, including various T-cell subpopulations, adipocytes, and fibroblasts. Given the complexity of HSC trafficking and control, it is surprising that clinical HCT in general has a relatively low rate of graft failure.

Definitions Graft failure is defined as the lack of hematopoietic cell engraftment following autologous and allogeneic HCT. Its criteria are predominantly operational, and graft failure is divided into primary (early) and secondary (late) phases. Primary graft failure The day of myeloid engraftment is commonly defined as the first of three consecutive days on which the absolute neutrophil count exceeds 5 × 108/L [20,21]. Myeloid engraftment typically occurs within 28 days

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of the graft infusion, irrespective of whether an autologous, allogeneic matched related or unrelated product of marrow or blood progenitor cells is used. The day of platelet recovery is more loosely defined as the first day of a platelet count of at least 20, 50 or 100 × 109/L, sustained without transfusion for 7 days [22]. Platelet recovery may be substantially delayed compared with myeloid recovery or sometimes not achieved at all, especially if the higher platelet value (100 × 109/L) is used as the threshold [23]. A platelet threshold of 20 × 109/L often requires administration of platelet transfusions for minor procedures or as per individual physician practice, and may less accurately reflect achievement of durable engraftment. A hemoglobin level of at least 8 g/dL without transfusion support is an accepted threshold for red cell engraftment [24]. Primary graft failure is defined as a failure to achieve these threshold counts beyond day 28 post transplantation. An isolated cytopenia does not necessarily invoke graft failure as this may be a transitory phenomenon related to a medication, infection, a lineage-specific immune-mediated cytopenia or graft-versus-host disease (GVHD). Secondary or late graft failure Patients meeting the criteria for initial engraftment but subsequently developing loss of a previously functioning graft defined by at least two cytopenic lines are considered to have late or secondary graft failure [25]. Late graft failure is a phenomenon more often associated with allogeneic HCT than with autologous transplantation [26]. Some possible causes for late graft failure include graft rejection related to residual host immunity, persistent or progressive disease, low donor cell yield, medication side-effect, infection or GVHD. Graft rejection and poor graft function Graft rejection is the immune-mediated rejection of the donor allograft by residual host effector cells that occurs because of the genetic disparity between the recipient and the donor. Graft rejection is a possible cause of graft failure following allogeneic HCT. Primary graft rejection is the absence of evidence of donor hematopoiesis, and late rejection is defined as a loss of donor cells after initial graft function [27]. The determination of graft rejection requires an analysis of the blood or marrow for chimerism. In contrast to graft rejection, poor graft function describes the failure to achieve adequate blood counts following allogeneic HCT in the presence of complete donor hematopoietic cell chimerism [28]. Poor graft function is also divided into primary (early) and secondary (late).

There remains a lack of consensus regarding the use of these terms. Mixed chimerism is not unique to reduced-intensity conditioning. Prior to the addition of antithymocyte globulin (ATG), almost 60% of patients that received a BM allograft following high-dose conditioning for severe aplastic anemia (SAA) had mixed chimerism, of which two-thirds converted to complete donor-type hematopoiesis while the remainder experienced late graft failure [30]. Interestingly, patients with mixed chimerism did not have a statistically significant increased risk of graft failure compared with those who developed complete chimerism by day 28 after transplantation [30].

Incidence of graft failure The incidence of graft failure varies widely in published reports. This variance may reflect, in part, that the consequences associated with graft failure (i.e. morbidity and mortality due to infection, hemorrhage, and disease progression) are reported and not the graft failure itself. To estimate the incidence of graft failure following autologous HCT, it is reasonable to consider that, in most centers, the 4-month nonrelapse mortality is 10% or less, of which only a small subset can be attributed to graft failure. Another surrogate marker for estimating the incidence of graft failure following autologous HCT is the requirement for hematopoietic cell rescue using a back-up product. In a single-center study of 300 consecutive patients that underwent high-dose therapy followed by autologous hematopoietic cell rescue between 1980 and 1997, 14 (4.7%) required their back-up product [31]. Unmanipulated BM, acute myelogenous leukemia, and CD34+ selection of granulocyte colonystimulating factor (G-CSF)-mobilized blood mononuclear cells (MNCs) collected by apheresis were risk factors for graft failure in this study. Thus, it is reasonable to estimate that the incidence of graft failure following autologous HCT is between 1% and 5%. The incidence of graft failure is higher in recipients of allogeneic grafts and is reported to occur in 5–20% of patients [32,33]. Some transplant centers opt to store autologous blood or marrow-derived hematopoietic progenitor cells for salvage purposes, but this is not considered a standard of care in the United States or Canada [31]. Eightyfive of 122 (70%) European centers completed questionnaires regarding their policy to store autologous back-up. Among the centers that responded, autologous back-up products were routinely stored in approximately 10% and 45% of patients receiving unmanipulated grafts from human leukocyte antigen (HLA)-matched family and unrelated donors, respectively [31].

Causes of graft failure Terms for graft failure following reduced-intensity conditioning for allogeneic HCT Reduced-intensity conditioning for allogeneic HCT is associated with incomplete eradication of host hematopoietic elements. As a consequence, a significant percentage of patients have multilineage mixed chimerism for several months after transplantation before converting to complete donor type. Primary engraftment following allogeneic HCT using reduced-intensity conditioning is defined by neutrophil, platelet, and hemoglobin count recovery as outlined above, in addition to achievement of 5% or more donor T cells (CD3+) by day 28 after transplantation [25,29]. A failure to surpass this 5% threshold at any time after transplantation is considered primary graft failure, while secondary graft failure is reserved for cases that initially surpass the 5% threshold but whose T-cell chimerism subsequently falls below this level. Mixed chimerism describes patients enumerating 5–95% donor T cells, and full or complete chimerism is considered the achievement of more than 95% donor T-cell origin [25].

Contributing causes for graft failure are listed in Table 80.1. Graft failure following allogeneic HCT is more complex than graft failure after autologous HCT because of the presence of confounding factors such as histocompatibility and ABO matching, graft-versus-host (GVH) and host-versus-graft (HVG) reactions, and the use of post-transplantation immune suppression. These factors increase the risk of immunemediated graft rejection. Graft rejection It is well established that donor T cells in the allograft promote hematopoietic engraftment as depletion of T lymphocytes from the marrow graft before transplantation is associated with a substantial increase in the occurrence of graft rejection [34–36]. The increased risk of graft rejection following T-cell-depleted marrow grafts is due, in part, to an enhanced susceptibility of the graft to regimen-resistant host natural killer (NK) cell- and lymphocyte-mediated rejection [37,38].

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Mechanisms and Treatment of Graft Failure Table 80.1 Factors associated with graft failure

No Transfusion

Quantitative progenitor issues: Low cell yield (e.g. CD34+) Presence of splenomegaly

Immunologic issues: Donor and recipient human leukocyte antigen disparity Donor and recipient ABO disparity Manipulation of the graft (i.e. T-cell depletion, CD34+ cell selection) Prior transfusion history Adequate post-transplantation immunosuppressive medication Reduced-intensity conditioning regimen with residual host effector cells Presence of acute or chronic graft-versus-host disease Other issues: Patient and donor age (advanced age versus younger age) Viral infections (cytomegalovirus, HHV-6, HHV-8, parvovirus) Storage techniques HHV, human herpesvirus.

The relative contributions of individual host cell subsets to graft rejection differ based on the genetic disparity between the donor and the recipient, and the status of host antidonor reactivity. Murine models of transplantation have confirmed that antidonor cytotoxic T lymphocyte (CTL) populations sensitized to major and minor histocompatibility antigens conferred resistance against allogeneic BM [39]. In human transplantation, the presence of radioresistant antidonor CTL populations sensitized to donor minor histocompatibility antigens in individuals who previously received a transplant, or in patients with SAA who became sensitized through repeated blood transfusions, was associated with high levels of graft rejection [40]. Figure 80.1 shows a lower overall survival rate for transplant recipients who had a pretransplantation history of transfusions compared with those without prior transfusions; the difference was mainly due to death from graft rejection [40]. In this study, both groups of patients received grafts from HLA genotypically matched family donors, and both populations were adequately balanced for transplant preparative regimen, disease duration, and cause of aplasia. In preclinical models of BM transplantation (BMT), radioresistant host NK cells are also capable of efficiently lysing donor hematopoietic cell targets and rejecting BM grafts, especially those that lack expression of major histocompatibility complex (MHC) class I antigens [41,42]. In vivo studies showed that class I-deficient β2-microglobulin−/− BM grafts were more susceptible to NK-mediated rejection by syngeneic hosts, and that resistance could be restored by adding an exogenous source of β2microglobulin [43]. Mice with SCID, which cannot develop T or B cells due to a failure in T- and B-cell receptor rearrangements, but which have normal NK cell function, demonstrated that NK cells alone can mediate BM allograft rejection [44]. The treatment of wild-type recipient mice with the α-NK cell polyclonal antibody, α-ASGM1, known to abolish

0.8

Survival

Qualitative marrow issues/altered marrow microenvironment: Disease category – Inherited diseases of erythropoiesis (i.e. thalassemia) – Acquired marrow failure diseases (i.e. myelodysplastic syndrome, myelofibrosis, severe aplastic anemia) Disease status – Presence of marrow involvement with disease – Longer than 1 or 2 years from the time of initial diagnosis Prior therapy Iron overload

Bone Marrow Transplantation

1

0.6 Transfusion 0.4

0.2

0 0

2

4

6

8 10 Years

12

14

16

18

Fig. 80.1 Effect of transfusion status on actuarial survival. Fifty-three patients with aplastic anemia and no preceding history of blood product transfusions versus 115 previously transfused patients underwent allogeneic hematopoietic cell transplantation using bone marrow from human leukocyte antigen-matched sibling donors. Tick marks denote censoring times of surviving patients. The p-value was 0.04 and was calculated using the logrank and two-sided tests. (Adapted from [40], with permission.)

both NK cell and CTL activity in vivo, resulted in a significant improvement in survival in lethally irradiated mice that underwent transplantation across MHC barriers [45]. The improved survival in resistant strain combinations was the result of successful donor cell engraftment. Evidence that NK cells mediate resistance to engraftment in clinical HCT is lacking, due in part to the difficulty of discerning T-cell- from NK-cell-mediated resistance in humans. Human NK cells are highly sensitive to cyclophosphamide (CY), which is commonly used in transplantation preparative regimens, and the quantity of hematopoietic cells given clinically is probably sufficient to override resistance by host NK cells. It has been demonstrated, in murine models of BMT, that NK cell-mediated graft rejection increases as the cytoreductive conditioning is reduced [46]. Thus, it is tempting to speculate that NK cells may play a role in graft rejection following reduced-intensity conditioning regimens in humans. The molecular basis underlying T-cell-mediated graft rejection remains incompletely defined. In vitro T-cell-mediated cytotoxicity is essentially dependent on the Fas–Fas ligand (FasL) interaction and the perforin-dependent pathways [47]. Disruption of either of these two major cytotoxic pathways did not reduce resistance to allogeneic BM cells [45]. Further, several independent studies using FasL-deficient mice confirmed that these animals maintained strong engraftment barriers [48]. Double (perforin and FasL) and triple (perforin, FasL, and tumor necrosis factor receptor)-deficient mice that underwent HCT across minor histocompatibility barriers failed to show a diminished capacity to resist BM allografts compared with wild-type recipients [49]. The elimination of any one selected cytokine in recipient mice, including tumor necrosis factor-alpha, transforming growth factor-beta, and polarizing T helper type 1 and type 2 cytokines did not alter allogeneic hematopoietic cell resistance [45]. In a murine model of resistance using recipients who lacked perforin, FasL, and other known death ligand receptor pathways, the host CD8+ T memory cells were strong mediators of graft resistance against donor minor histocompatibility mismatched BM progenitor cell allografts [45]. Understanding the pathways of resistance mediated by effector cell populations after high-dose and reducedintensity conditioning regimens will be important for the development of effective strategies that lead to stable engraftment after allogeneic HCT, especially for those patients who suffer graft failure.

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In addition to T-cell- and NK-cell-mediated BM rejection, antibodymediated BM rejection occurring either by antibody-dependent cellmediated cytotoxicity or complement mediated cytotoxicity has been described [50,51]. Preformed antibody present at the time of the BM infusion is unaffected by standard transplantation conditioning regimens or by the immune-suppression medications administered in the peritransplant period. Strategies such as plasmapheresis, high-dose intravenous immunoglobulin, splenectomy, and immunoadsorption that are commonly used in solid organ transplantation for the elimination of antibody-mediated rejection [52] are generally not part of HCT conditioning regimens, and consequently their effectiveness at abrogating graft rejection has not been formally evaluated. In a murine model of transplantation, it was shown that a single priming against an MHC-disparate strain resulted in antibody mediated rejection that was very rapid (<3 hours), whereas T-cell-mediated rejection in nonprimed mice took more than 6 days [53]. The antibody-mediated rejection was not due to a BM homing defect as rejection occurred despite direct intraBM infusions of donor BM cells. Rejection was dependent on host FcR+ cells [53]. Antibody-mediated rejection was overcome and complete chimerism achieved in mice using the combination of a high dose of donor BM cells, high-dose immunoglobulin, and T-cell-depleting antibodies to target T cells that survive total body irradiation (TBI) conditioning [53]. These results suggest that a similar strategy may be effective at preventing antibody-mediated acute BM rejection in sensitized patients known to have antidonor reactive alloantibodies before HCT. Preclinical models indicate that dendritic cells in the transfusion product play an important role in sensitizing hosts to disparate minor histocompatibility antigens [54]. Exposing blood products to gamma irradiation in vitro before transfusion almost eliminated sensitization to minor histocompatibility antigens and prevented rejection of dog leukocyte antigen-identical marrow grafts [55]. Leukocyte depletion of blood products also reduced rejection in the canine model [56], suggesting that patients with SAA who are BMT candidates should be managed with irradiated leukocyte-poor blood products. As already discussed, T-cell-depleted grafts are associated with a higher incidence of graft rejection following HCT. These findings support a role for donor T-cell-mediated function in successful BM engraftment, yet the mechanism by which donor T cells inhibit the host’s ability to reject marrow allografts remains poorly defined. In murine transplantation models, donor CD8+ T cells have been reported to be much more potent facilitators of short- and long-term engraftment than CD4+ T cells [57,58]. It is likely that the donor T cells directly suppress or eliminate host effector cell populations. In a murine model of transplantation, BM engraftment across MHC class I or II barriers required CD8+ donor T cells with intact perforin pathways to achieve sustained engraftment and prevent graft rejection [57]. Another possibility is that donor T cells provide necessary cytokines that may influence donor hematopoietic progenitor cells to home to the marrow niches or augment their proliferation despite the fact that the host effector cells may not have been suppressed [59]. Secretion of granulocyte–macrophage colony-stimulating factor (GM-CSF) by alloreactive T cells augments HSC proliferation [60]. Another possibility is that the donor T cells can produce factors that render stem cells more resistant to the lytic machinery of the host effector cells. In support of this, interferon-gamma has been demonstrated to render some tumor cells resistant to the lytic mechanisms of cytotoxic cells [61]. Efforts to characterize cell populations in the donor graft that facilitate engraftment without causing GVHD remain an area of active research. Unfractionated CD4+ T cells do not possess efficient facilitating potential [57]. Yet transplantation of CD4+CD25+ T cells in a murine model of reduced-intensity conditioning for BMT using complete MHC-

mismatched strains supported engraftment of donor BM cells [62]; these findings suggest that regulatory T cells may provide an approach for the promotion of engraftment following allogeneic HCT. BM CD8+ subpopulations, specifically CD8α+CD8β+TCR β −TCRγδ−CD3+ cells when used in the amounts contained in a BM graft, facilitate engraftment of donor cells without provoking GVHD [63]. CD8α−/− mice lack facilitator cells in murine models of BMT, whereas these cells are replete in mice whose CD8+ and NK killer function was depleted [64]. These findings suggest that the CD8α molecule and not lytic function is important in facilitating engraftment of donor cells after HCT. Understanding how HSCs are recognized and resisted is a complex biologic question. From the preclinical models, it is evident that a variety of host cell populations contribute to resistance and that their roles are influenced by multiple factors, including genetic disparity, prior sensitization, graft content, and recipient preparation. Likewise, there are populations of cells contained within the donor allograft that affect hematopoietic cell engraftment. Unraveling how transplanted HSCs are targeted and suppressed will undoubtedly contribute to developing strategies that ensure successful engraftment possibly without provoking GVHD. Histocompatibility antigens The MHC maps to position p21.3 on the short arm of chromosome 6 and encodes at least 12 genetic sites, named the HLA loci that comprise the genes of the HLA system (for a comprehensive review of this topic see Chapter 12). HLA-A, B, C, DR, DQ, and DP gene products (antigens) define histocompatibility important in allogeneic HCT [65]. Historically, human HLA typing was performed using a panel of antibodies derived from individuals exposed to HLAs by pregnancy or transfusion. These serologically defined antigens (e.g. HLA-A1) are referred to as low resolution. In the last decade, such serologic typing has given way to polymerase chain reaction-based methods that can detect single nucleotide differences between two unique HLA alleles of the same antigen. In this way, a donor–recipient pair who are serologically matched for HLA-A, B, and DR may harbor allelic mismatches at these as well as the other HLA loci. The increased precision in HLA typing has had clinically notable improvements in outcome, as accurate matching at the MHC is considered essential to minimize GVHD and graft failure [66]. Comparing studies that have evaluated the impact of HLA compatibility with the risk of graft failure following allogeneic HCT is challenging. The main reason relates to the difficulty in assessing the true degree of incompatibility in earlier studies; when DNA-based typing methods are applied, allele disparities among phenotypically identical individuals are uncovered, and these allele disparities are reportedly functionally relevant [67,68]. Also, donor selection in many studies was often originally based on matching criteria for HLA-A, B, and DR, and may not reflect matching at HLA-C, DP, and DQ. Among 205 patients who underwent transplantation from HLA-A, B, C, DRB1, and DQB1 molecularly matched unrelated donors, a single-allele HLA-DP mismatch was identified in 90 recipient–donor pairs, and a two-allele mismatch was determined in 60 pairs [69]. Two-allele HLA-DP mismatches conferred a significant risk of severe acute GVHD, indicating an important role in the alloimmune response; its effect on graft failure was not evaluated. Despite the uncertainty of the true degree of mismatch, several important concepts consistently emerge with respect to HLA compatibility and the risk of graft failure. First, despite high-dose conditioning, a small number of patients will reject a marrow graft from an HLA-identical sibling, and this risk increases with increasing number of HLA mismatches on the nonshared haplotype. In one study of BMT from family members, graft failure

Mechanisms and Treatment of Graft Failure

occurred in 7% of recipients of phenotypically HLA-matched grafts, in 9% of those incompatible for one HLA locus, and in 21% of patients receiving grafts from incompatible donors for two HLA loci [21]. In virtually every study reporting on graft failure following unrelated donor HCT, the degree of HLA mismatch negatively influenced the risk of graft failure [20,23,70,71]. In these studies, immune-mediated rejection was assumed to be the cause of graft failure if donor T cells were not identified in the recipient’s blood or marrow. A graft with higher numbers of hematopoietic progenitor cells correlated with a lower incidence of graft rejection following transplantation using an HLA serologically mismatched donor [20]. The reason for this beneficial effect was not understood. It was possible that the higher number of cells with repopulating ability were able to overwhelm residual host effector cells. Previous studies have shown that megadoses of purified HSCs can engraft in allogeneic recipients, an effect hypothesized to occur via a veto mechanism [72]. Alternatively, a more cellular graft may contain a critical number of cells with support function, such as regulatory T cells or other facilitator cells, that may not be present in marginal grafts [62]. The pediatric experience is somewhat different from the adult experience as graft failure demonstrates more tolerability to HLA disparity [73]. Second, the vector (also referred to as the “direction” of the mismatch) after HLA-incompatible T-cell-replete grafts for HVG and GVH alloreactivity can be defined and is important in assessing graft failure risk [74]. The presence of donor antigens or alleles not shared by the recipient determines HVG allorecognition and correlates with an increased risk of graft failure. The presence of recipient antigens or alleles not shared by the donor provides the immunologic basis for GVH alloreactivity and correlates with an increased risk of GVHD. Examples of HVG and GVH vector mismatches are shown in Table 80.2. Third, the risk of graft failure may not be contributed equally by class I and class II mismatching. Some have reported that class I mismatching, and in particular HLA-C mismatching, is associated with an increased risk of graft failure compared with class II mismatching [75]. Fourth, the risk of graft failure was more evident in transplants with low-resolution versus only high-resolution mismatches [70]. This indicated that the immunogenicity of allele and antigen mismatches were different, with fewer detrimental effects associated with two alleles of the same antigen (e.g. an HLA-A*0401 versus HLA-A*0404 mismatch within the A2 antigen family) compared with mismatching between two antigens (e.g. A4 versus A15) [68]. Transplants from unrelated donors with a single class I allele mismatch that was not serologically detectable were used without an apparent increase in the rate of graft failure [68].

Table 80.2 Mismatching according to the vector of human leukocyte antigen disparity Vector

Definition

Donor

Recipient

HVG

Presence of donor alleles or antigens not present in the recipient Presence of recipient alleles or antigens not present in the donor

B*0801, 4402† B*0801, 4402‡

B*0801, 4405 B*0801, 0801

B*0801, 4402† B*0801, 0801‡

B*0801, 4405 B*0801, 4405

GVH

GVH, graft-versus-host; HVG, host-versus-graft. † These combinations contain bidirectional (HVG and GVH) mismatch vectors. ‡ These combinations contain unidirectional mismatches.

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ABO match ABO incompatibility between donor and recipients occurs in approximately 20–30% of HLA-matched allogeneic transplants [76]. Incompatibility is defined as major when the recipient plasma has isohemagglutinins against donor red blood cell antigens, and minor when the donor has isohemagglutinins against recipient red blood cell antigens. Bidirectional ABO incompatibility occurs when combined features of major and minor incompatibility are present. In many studies, ABO incompatibility had no influence on the kinetics of neutrophil and platelet recovery and on the risk of graft failure [77]. In contrast, a single-center study of 224 patients with acute leukemia transplanted from unrelated donors found that major ABO incompatibilities were statistically significantly associated with an increased risk for graft failure [78]. There also remains conflicting data regarding ABO incompatibility and its relevance to other transplant outcomes such as GVHD, relapse, and survival [79].

Additional factors that influence graft failure risk Age It remains unclear whether patient and donor age impact the incidence of graft failure following HCT. In autologous transplantation, increased patient age adversely impacted the mobilization of sufficient numbers of progenitor cells to support successful engraftment in patients with multiple myeloma, Hodgkin’s disease, and non-Hodgkin’s lymphoma [80,81]. In another report, there was no difference in successful mobilization rates and engraftment between older and younger myeloma patients in spite of significantly different median ages (52 versus 68 years) [82]. In this latter study, the evaluation of age as a continuous variable failed to demonstrate any correlation between age and the total number of CD34+ cells collected. In another study, increased donor age was significantly associated with lower circulating CD34+ cell counts and lower CD34+ cell yields following G-CSF mobilization [83]. The lack of prospective studies designed to evaluate whether donor and recipient age impact the risk of graft failure preclude firm conclusions. Animal models have provided insight into age-related events that may eventually result in patient-related investigations. Do “old stem cells transplanted into younger patients” impact the likelihood of achieving sustained engraftment? An emerging hypothesis is that we age through accumulated damage to our stem cells, including HSCs, and that these damaged cells die by apoptosis or are removed by senescence [84]. This accumulated damage can lead to diminished stem cell function and, consequently, a reduced capacity for organ regeneration potential or, as in the case of HCT, a reduced capacity for hematopoietic recovery. Phenotyping and progenitor assays have indicated that the HSC population known as the side population in older mice are still stem cells but compete poorly with the side population cells from younger mice in competitive long-term repopulation assays [85]. It appears that the lower engraftment from older mice is due, at least in part, to a defect in the ability of the side population cells to home to their marrow niche. Direct BM injections of side population cells compared with the intravenous route of administration largely negated the difference between young versus old side population cells in competitive repopulation assays [85]. The tumor suppressor p53 has been implicated as a potential regulator of age-associated HSC function [86]. The p53 hypermorphic (p53+/m) mice had a higher activity of p53 than wild-type mice and displayed phenotypes of premature aging [86]. Organs from aged p53+/m mice exhibited reduced cellularity and atrophy, suggesting defects in stem cell regenerative capacity. Transplantation of 500 HSCs from old p53+/m

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mice into lethally irradiated recipients resulted in reduced engraftment compared with aged, matched, wild-type p53+/+ and p53+/− HSCs [86]. Thus, alteration of p53 activity affects stem cell numbers, proliferation potential, and hematopoiesis. Telomeres are noncoding regions of DNA that cap the ends of eukaryotic chromosomes to protect against the enzymatic breakdown of coding DNA, and prevent fusion or other chromosomal aberrations [87]. Telomeres shorten by 50–150 base pairs with each cell division in cells that do not express telomerase. The maintenance of telomere length by induced expression of telomerase postpones senescence, and its constitutive expression is a general requirement for immortalization of cells [87]. Several studies involving adult and pediatric patients have shown that, following allogeneic HCT, the telomeres in the chimeric blood and marrow cells were 0.4–2 kilobases shorter than those of their respective donors and aged-matched controls [88,89]. This degree of accelerated telomere loss is equivalent to 15–70 years of aging in healthy controls. These observations support the notion that the increased replicative demand on HSCs during the early period of hematopoietic cell reconstitution following transplantation results in measurable accelerated telomere shortening. It could be reasoned that transplantation using a marginal graft with a very limited number of HSCs or transplantation using HSCs from older donors or HSCs that might have short telomeres to start with, for whatever reason, could put high demands on their proliferative potential that could eventually result in exhaustion and subsequent late graft failure. In this respect, cord blood cells might have an advantage that outweighs concerns regarding the limited number of cells available for transplantation. Two cases of late graft failure following high-dose allogeneic HCT have been described that were associated with significant telomere shortening in the chimeric donor hematopoietic cells compared with the donor’s native cells [90]. In these cases, graft failure due to medication, infection, relapse, immune-mediated rejection or GVHD was ruled out. Given the increasing number of long-term survivors of HCT and the increasing age of recipients and donors, it is important to understand age-associated regulators of HSC function. It is conceivable that this knowledge may eventually be used as selection criteria to better maintain the integrity of the repopulating HSCs and reduce the risk of graft failure or other adverse outcomes. Diagnosis The rate of graft failure varies with the underlying disease, yet it is difficult to draw firm conclusions as studies include patients with various stages of diseases, and differ with respect to the conditioning regimen, the graft content, and a variety of other variables considered important in contributing to graft failure. Aplastic anemia In general, patients with SAA who receive BM grafts following highdose conditioning have a graft failure rate that is higher than that associated with acute leukemia [27]. Transplantation early in the disease course and before sensitization to histocompatibility antigens reduces the graft rejection rate in patients with SAA, yet it is likely that only a minority of patients do not receive transfusions prior to transplantation [27,40]. Myeloid disorders The graft failure rate for patients undergoing allogeneic HCT for chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), and myelofibrosis (MF) is increased compared with patients with acute leukemia, and this finding is consistent irrespective of high-dose or reducedintensity conditioning [91–97]. The increased graft failure rate for CML

compared with acute leukemia was also observed in the pediatric population [94]. It is possible that CML, MDS, and MF patients who have not received pretransplantation intensive chemotherapy better resist donor cell engraftment owing to the presence of residual intact host effector cells. In one study, the overwhelming majority of patients who experienced graft failure received only interferon-alpha prior to receiving their reduced-intensity preparative regimen [98]. Thus, more intensive immunosuppression before or immediately after HCT may be required to better support engraftment, particularly if reduced-intensity regimens are being considered in patients with CML, MDS, and MF. Patients with MDS and MF may have a defective supportive microenvironment that further increases resistance to donor cell engraftment [95,96]. Hemoglobinopathies Thalassemia is associated with a relatively high graft failure rate [99,100]. Risk factors for graft failure include hepatomegaly (>2 cm below the costal margin), presence of portal fibrosis in the pretransplant liver biopsy, and quality of iron chelation during the years before transplant (considered good if regular deferoxamine therapy was initiated within 18 months of the first transfusion and administered continuously for 8–10 hours for at least 5 days per week) [100]. The incidence of graft failure following high-dose conditioning and HCT from HLA-matched sibling donors was around 10% for risk class 1 (none of the risk factors) patients, and 20–25% for patients in risk class 3 (all three risk factors present) [100]. These results support the finding that, as in SAA, pretransplantation transfusion history adversely impacts the risk of graft failure. In contrast to thalassemia, data collected from multiple centers from over a 13-year period on patients with sickle cell anemia confirmed that none of the 67 recipients of HLA-matched related BM grafts developed graft failure following high-dose busulfan (BU) plus CY conditioning [101]. Disease status Many studies have demonstrated that a long interval from diagnosis to HCT is associated with an increased risk of mortality among patients with acute myelogenous leukemia, CML, MDS, and SAA [20,23,27,93,97,102]. In these studies, the graft failure rate was increased with high-risk category disease versus low-risk disease. In part, this may be the consequence of previous intensive treatments causing damage to the supportive microenvironment. The difficulty in achieving sustained engraftment in patients with advanced-stage disease may also relate to the rapid expansion of the malignant clone within the BM that results in loss of graft; this can be true for autologous as well as allogeneic HCT. Previous treatments Experimental and clinical data suggest that prior exposure to cytotoxic agents and radiation therapy damages hematopoietic progenitors and may impair graft function. Cytotoxic agents differ considerably in their effects on HSCs. Some agents can affect late pluripotent stem cells and committed progenitor cells in cycle without damaging early stem cells. Animal studies showed that the cytotoxic agents melphalan, 1,3-bis(2chloroethly)-1-nitrosurea (BCNU), and BU significantly reduced the repopulating capacity of BM progenitor cells compared with CY, etoposide, and cytarabine [103]. Salvage regimens containing agents that potentially affect progenitor cell function would be expected to compromise engraftment following autologous HCT, whereas regimens not using progenitor cell cytotoxic drugs would be preferable. In keeping with these preclinical studies, exposure to a salvage regimen that con-

Mechanisms and Treatment of Graft Failure

tained BCNU or melphalan was shown to be a significant factor that adversely affected progenitor cell yield and performance of the graft, and was associated with an increased risk of graft failure following autologous HCT [104]. Dose intensity may also be important as patients who received higher-dose CY, doxorubicin, vincristine, and prednisone (CHOP) had a significantly prolonged time to engraftment following autologous HCT compared with patients that received standard-dose CHOP [105]. Additionally, the cytogenetic evaluation of a BM aspirate for abnormal clones that may have developed from prior conventionaldose therapy in patients undergoing autologous harvesting is important. Infusion of autologous products that harbored abnormal cytogenetic clones led to an increased risk of poor graft function with the subsequent development of post-transplantation overt MDS or secondary leukemia [106]. Prior exposure to chemotherapy and radiation therapy can impair the supportive function of the hematopoietic microenvironment and may negatively impact graft function. In mouse models, irradiation caused profound changes in the expression of the endothelial trafficking molecules VCAM-1 and P-selectin, which are important for hematopoietic cell recruitment to the BM niches [107]. In other preclinical studies, exposure to clinically relevant high- (120 cGy/min) and low- (5 cGy/ min) dose rate irradiation resulted in the appearance of a delayed stromal lesion with a distinct phenotype that disrupted the marrow microenvironment and was associated with a poor recovery of allogeneic bloodforming elements (cobblestone area-forming colonies [CAFCs]) that persisted even when rechallenged several months after radiation exposure [108]. In a clinical study, the frequency of CAFCs was significantly reduced in the marrow of allograft recipients, irrespective of the cell dose and source, compared with the number obtained from marrow using normal controls [109]. CAFC numbers in patients transplanted in early stages of myeloid malignancies (acute myelogenous leukemia in first complete remission) were significantly higher than CAFC numbers in the marrow of patients transplanted in more advanced stages of disease or in those with MDSs [109]. Taken together, these findings suggest that the health of the marrow microenvironment may be a limiting factor in supporting the sustained engraftment of allogeneic hematopoietic donor cells. The clinical relevance of these observations with respect to graft failure is not established but is of interest. Preparative regimen The transplant preparative regimen may influence the graft failure rate. Attempts to overcome the relatively high graft failure rate among patients with SAA who received multiple pretransplantation transfusions have included intensifying the conditioning regimen by combining CY 200 mg/kg with TBI or total lymphoid irradiation (TLI) [27,110,111]. Although rejection rates were reduced with the combinations, overall survival was not improved due to the higher rates of death from GVHD, interstitial pneumonia, and secondary malignancies. The improved graft failure rate with combination therapy was considered a result of more effective host immune effector cell eradication. Animal studies showed a synergistic immunosuppressive effect between CY and ATG for overcoming marrow allograft rejection, thereby obviating the need for TBI [112]. On the basis of these models, a regimen of CY and ATG was used to successfully rescue patients whose first graft was rejected [113]. A subsequent uncontrolled study of patients receiving this regimen for their first transplant reported a reduced graft failure rate and improved overall survival compared with historical data [114]. Yet a prospective clinical trial where patients were randomly assigned to receive CY alone or CY in combination with ATG confirmed that the 5-year probabilities of survival were similar in both groups, as was the incidence of graft failure and GVHD [115]. The improved outcomes of allogeneic BMT

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for SAA were likely related to advances in supportive care, and the addition of immunosuppression with ATG resulted in no significantly improved outcomes. A randomized study that compared CY plus TBI versus BU/CY host conditioning for patients receiving unmanipulated marrow grafts for the treatment of chronic-phase CML failed to identify a difference in graft failure rates or in the time to functional platelet and neutrophil engraftment. Increasing the intensity of the transplant preparative regimen in already fully myeloablative conditioning did not decrease the risk of graft failure [22]. The addition of cytarabine to a standard BU/CY transplantation regimen for pediatric patients did not impact on the risk of graft failure or the time to platelet and neutrophil engraftment in recipients of autologous or allogeneic HCT [116]. In contrast, it is likely that increasing the intensity of host conditioning following reduced-intensity regimens can impact the incidence of graft failure. Nonfatal graft failure occurred in 20% of the 44 patients who received sublethal TBI with post-transplantation mycophenolate mofetil (MMF) and cyclosporine (CSP), and autologous hematopoietic cell recovery occurred in each case [117]. In a similar patient population, the addition of fludarabine to sublethal TBI reduced the graft failure rate to 12% [91]. Graft failure rates can vary widely even when comparing transplant preparative regimens that use drugs within the same class. A randomized study of fludarabine versus cladrabine plus BU and low-dose TBI as conditioning for allogeneic HCT halted enrollment in the cladrabine cohort early when an interim analysis found this arm was associated with a high risk of graft failure [118]. The graft failure in these cases was not associated with endogenous hematopoietic cell reconstitution. Transplantation following reduced-intensity conditioning is associated with multilineage mixed chimerism in many patients that can persist for several months after HCT [29]. It is difficult to assess the impact of mixed chimerism on the risk of graft failure because often interventions such as immunosuppressive drug withdrawal, CD34+-selected donor cell boost or donor leukocyte infusions (DLIs) are employed [29,119]. These interventions are thought to promote conversion to full donor type, yet they are not without risk and may result in the development of GVHD. A significant percentage of patients who received a Campath-containing reduced-intensity regimen required DLI to ensure conversion to complete donor type and protect against immune-mediated graft rejection [120]. A regimen of CY, ATG, and thymic irradiation with postgrafting immunosuppression consisting of a short course of CSP revealed that the median donor T-cell level on day 30 post transplant was 50% (range 1–95%), and almost 30% of the patients below this level developed graft failure by day 100 after transplantation [119]. The graft failure risk following conditioning with 2 Gy TBI ± fludarabine with post-transplantation immunosuppression consisting of MMF and CSP was 50% among patients who had donor T-cell levels below 25% by day 30 post transplantation, compared with a 4% graft failure rate if the donor T-cell level was between 51% and 75% by this time [121]. In some studies, the post-transplant evaluation of the level of donor T- and NK-cell chimerism at days 14 and 28 after transplant were important predictors of graft failure [29,122]. Conditioning with TLI and ATG revealed no predictive results with respect to the risk of graft failure and the level of T-cell chimerism at day 30 or 60 post transplantation [123]. There is an array of transplant conditioning regimens that consider patient- and donor-specific factors including but not limited to disease risk, sensitivity to graft-versus-tumor reactions, patient comorbidity, donor age, degree of HLA and ABO incompatibility, and degree of prior allosensitization. Controlled experimental and clinical studies are required to better understand how an alteration in a preparative regimen impacts the incidence of graft failure.

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Post-transplantation immunosuppression Suppression of HVG reactions with post transplantation immunosuppressive medications promotes donor hematopoietic cell engraftment. In preclinical models of BMT using sublethal TBI conditioning in dog leukocyte antigen-identical littermate dogs, CSP alone was insufficient to suppress HVG reactions, and all recipients experienced nonfatal graft failure [124]. By comparison, methotrexate and CSP led to prolonged hematopoietic chimerism in all cases, although just over half of the animals studied ultimately lost their grafts. The combinations of MMF and CSP or MMF and sirolimus led to stable mixed chimeras in all recipients for at least 4 months [124,125]. In other canine transplantation models, it was shown that extending the length of post-transplantation immunosuppressive medication from 35 to 100 days favorably influenced stable donor engraftment [126]. These preclinical studies have been instrumental in designing optimal combinations of post-transplantation immunosuppressive medications. In humans, it remains unclear whether withdrawal of immunosuppression in the setting of persistent mixed chimerism post transplantation promotes engraftment or increases the likelihood of graft failure.

Graft source The preferred source of hematopoietic cells for autologous transplantation has shifted from BM to blood MNCs collected by apheresis. Chemotherapy, either in combination or as a single agent in addition to G-CSF and GM-CSF, represents the benchmark mobilization protocol (see Chapter 41). The advantages with cells collected with these protocols include a more rapid neutrophil and platelet recovery that leads to a shorter hospital stay, convenience of collection, and potentially a lower chance of tumor contamination [127]. Beyond the more rapid engraftment with the use of mobilized hematopoietic progenitor cells, an impact on graft failure rates following autologous HCT has not been shown. There remains a large variation in practice with respect to the use of BM versus G-CSF-mobilized blood as a source of hematopoietic cells for allogeneic HCT [128]. Randomized studies confirm a significant reduction in the number of days to reach neutrophil and platelet engraftment following HCT with G-CSF-mobilized peripheral blood progenitor cells (PBPCs), yet the incidence of primary and secondary graft failure was similar in both arms [128]. AMD3100 is a direct antagonist of the interaction between the chemokine SDF-1 and its receptor CXCR4, and rapidly mobilizes hematopoietic progenitors for clinical transplantation [129]. Transplantation of human AMD3100-mobilized MNCs compared with G-CSF-mobilized MNCs into NOD/SCID mice confirmed equal repopulating frequencies [130]. Clinical studies of autologous and allogeneic transplantation of AMD3100-mobilized PBPCs demonstrated prompt and stable engraftment in selected patient populations [131]. Whether AMD3100 will improve the kinetics of platelet and neutrophil recovery and impact the incidence of graft failure remains to be determined. The field of umbilical cord blood transplantation is still in its infancy, and investigators are learning how this source of cells compares with and differs from other forms of HCT. Biologic features of cord blood suggest that they are highly proliferative, which is important to overcome the limited number of cells that are obtained for transplantation [132]. It is prudent to anticipate the need for a back-up graft given that umbilical cord blood transplantation is associated with an increased risk of graft failure even when using well-matched, suitably sized cord blood units [133]. The majority of patients who had graft failure were rescued by this type of a planned approach initiated before day 50 after transplantation [133]. Double-unit umbilical cord blood trans-

plantation with two closely and suitably matched cord bloods may provide better engraftment by providing a higher CD34+ and CD3+ cell dose [134]. Effect of cell numbers Most studies have demonstrated, at least to some degree, that a minimum threshold number of progenitor cells were required to ensure reliable neutrophil and platelet engraftment, and reduce the risk of graft failure following autologous and allogeneic HCT. Historically, the nucleated cell or MNC dose was the surrogate to assess the HSC content in the BM graft. Studies that evaluated the predictive value of nucleated cell or MNC dose on the rapidity of hematopoietic recovery following autologous BMT demonstrated no statistically significant correlations between the infused numbers of nucleated cells/kg or MNCs/kg and the time to neutrophil or platelet recovery [135]. For allogeneic HCT, it was reported that 5 × 108 nucleated cells/kg recipient body weight was a reasonable estimate of the number of BM cells required for a successful transplant whereas a dose of fewer than 3 × 108 nucleated cells/kg recipient weight correlated with an increased risk of graft failure following full-dose conditioning [136]. Subsequent studies regarding the predictive value of BM nucleated cell dose and the risk of graft failure following allogeneic HCT failed to yield consistent results [137]. Unlike the nucleated cell dose, an increased number of granulocyte– macrophage colony forming units (CFU-GMs) in the autograft positively correlates with more rapid time to neutrophil and platelet recovery. Threshold doses below 0.1–3.0 × 104 CFU-GM/kg result in significant delays in engraftment [138]. The CFU-GM assay is limited in usefulness as the imprecision of CFU-GM quantification, especially when the progenitor content is low, leads to a subjective nature of data interpretation that results in high interlaboratory variability. An additional limitation includes the requirement for expensive biologic reagents and an approximate 2-week delay before results are obtained. The expression of specific cell surface markers can be rapidly assessed within a few hours by flow cytometry. CD34 is a cell surface glycoprotein expressed on hematopoietic progenitor cell populations that enables short- and long-term hematopoietic reconstitution in preclinical studies. The infusion of more than 2 × 106 CD34+ cells/kg recipient weight was suggested as the minimum target dose for acceptable engraftment kinetics following autologous HCT [139]. A more rapid neutrophil and platelet reconstitution was consistently observed by the infusion of higher CD34+ cell doses [140]. Platelet recovery appeared most affected by grafts with low CD34+ cell numbers. Patients considered as “poor mobilizers,” defined as a collection of less than 1 × 106 CD34+ cell/kg, safely underwent autologous HCT with no difference in overall survival, although 50% of the patients failed to reach a platelet count of over 100,000/μL by day 100 after transplant [141]. An absolute minimum CD34+ cell dose below which autologous HCT would not be recommended has not been defined, yet it is the opinion of the authors that BM harvesting to supplement the PBPC graft be considered for patients with a CD34+ cell dose below 1 × 106 CD34+ cell/kg. There are inconsistent results regarding the impact of the CD34+ cell dose and its relationship to graft failure following full-dose allogeneic HCT. This may reflect the fact that a minimum threshold number of cells are required to promote engraftment, above which there is no correlation. The widespread practice of infusing as many cells as possible to give the recipient the best possible chance for a rapid and complete hematologic recovery has made it difficult to analyze the effect of cell dose on the risk of graft failure. Further, comparison among studies is difficult because patients differ with respect to the preparative regimen, underlying disease, disease status, pretransplantation therapy received, post-transplant immunosuppressive medications, donor–recipient sex

Mechanisms and Treatment of Graft Failure

mismatch, degree of HLA compatibility, and a variety of other patientand donor-specific factors. Overall, it appears that, within the general range of cells collected in an unmanipulated graft, its composition does not appear to impact on the risk of graft failure and the time to platelet and neutrophil recovery following full-dose conditioning for allogeneic HCT [142]. Graft composition appears important at affecting the balance between donor and host cells following transplantation using reduced-intensity conditioning. Several studies have confirmed that, following reducedintensity conditioning, high doses of donor CD34+ cells lead to a more rapid T-cell engraftment with lessened risk of graft failure [143]. It has also been reported that a higher CD8+ T-cell dose in the donor inoculum correlated with a lower risk of graft failure [144]. As our understanding of repopulation capabilities of CD34+ subpopulations and graft anti-tumor reactions of effector cell subsets within the inoculum improves, protocols that manipulate graft content to reduce graft failure and enhance antitumor activity will be developed for autologous and allogeneic HCT. Graft manipulation CD34+ cell selection of BM- or blood-derived autografts obtained from patients with non-Hodgkin’s lymphoma and breast cancer does not adversely affect the time to neutrophil and platelet recovery or the graft failure rate [145]. The CD34+ cell dose required to support engraftment appears similar in patients that receive positively selected G-CSFmobilized PBPC grafts and historical controls who receive unmanipulated products [146]. The in vitro use of CY derivatives was a common method of purging acute myelogenous leukemia cells from autologous cell products. In most studies, mafosfamide purging resulted in an approximate 1-log fold decrease in the CFU-GM number/kg patient weight [147,148]. In one study that compared two purging strategies, dose-adjusted mafosfamide versus constant-dose mafosfamide, 18% and 48%, respectively, of patients had no detectable CFU-GM after purging [147]. Approximately 20% of these patients died from graft failure after autologous HCT even with the use of an unmanipulated back-up BM graft. Unmanipulated grafts are more effective in mediating stable engraftment following allogeneic HCT compared with grafts selectively enriched or depleted for one of a variety of cell populations, and this is true irrespective of the intensity of the transplant conditioning regimen. Pan-T-cell depletion, or selective CD8+ and CD6+ T-cell depletion of the donor inoculum, reduced the risk of acute GVHD, but survival in these studies was not improved because of the increased risk of death from graft failure, tumor relapse, and infection [149–151]. Preclinical studies have confirmed that T-cell receptor-positive (TCR+) and TCR−CD8α+ subpopulations in the BM graft have facilitative potential [63,64]. The TCR−CD8+ facilitators are CD3− and share phenotypic characteristics with CD8αα lymphoid dendritic cells and veto cells. In murine models of BMT, enrichment of the donor inoculum for these subpopulations enhances engraftment without provoking GVHD. The impact on graft antitumor activity has not been determined. This work has not yet been translated to human transplantation.

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to a mismatched related donor [65]. Given the improved outcomes with unrelated donor HCT and the impact of advanced donor age on outcomes, including an increased risk of graft failure, it may be advantageous to use a younger unrelated donor or even umbilical cord blood rather than an older related donor. A detailed evaluation of these options with respect to engraftment and sustained graft function will be required. Optimal preparative regimens Allograft recipients who are heavily transfused prior to HCT develop graft failure at a significantly higher rate; likewise, patients who are refractory to platelet transfusions are also at increased risk for graft failure [27,40]. In general in these patients, the practice is to use regimens that include TBI and/or TLI in combination with ATG so as to reduce HVG reactions [114]. The current practice of transfusing leukocyte-depleted and irradiated blood products for patients being considered for allogeneic HCT remains an important approach for decreasing allosensitization [152]. Allogeneic HCT following reduced-intensity conditioning is associated with increased graft failure rates for patients with high-risk MDS or MF and those with advanced stages of leukemia compared with lowrisk leukemia patients [29,102,153,154]. In these instances, graft failure is often associated with concomitant disease progression. High-dose or reduced-intensity regimens that still provide a moderate level of disease control should be considered in these patients. Appropriate cell numbers The number of transplanted cells is an important determinant in the establishment of a functioning graft [136,138–141]. Cell numbers should be adjusted whenever possible to meet required needs; occasionally, this may require a BM harvest to supplement the low CD34+ cell yield obtained from a patient who is a “poor mobilizer.” In allogeneic HCT, a higher cell inoculum appears to enhance the probability of sustained engraftment in patients transplanted for nonmalignant hematopoietic disorders such as SAA, as well as recipients of grafts from HLA-mismatched donors [155]. The higher CD34+ cell dose has to be balanced against a potential increased risk of chronic GVHD.

Monitoring of graft performance The evaluation of graft function may be performed at different levels including the assessment of adequate production of blood cells, assessment of hematopoietic precursors in the marrow, and evaluation of stromal cell function. Chimerism studies of T, B, and NK cells as well as myeloid cells have become a vital part of graft monitoring, particularly for patients transplanted after reduced-intensity preparations [29]. Studies that evaluate the kinetic change of the proportion of donor cells are mandatory to project the fate of the graft, determine the risk of graft failure, and possibly direct interventions.

Interventions for graft failure Strategies used for risk reduction for graft failure Donor selection Donor and recipient pairs that are HLA matched are associated with a lower rate of graft failure compared with mismatched pairs [65,66]. Recent improvements in the outcome of transplants from unrelated donors suggest that unrelated HLA-matched donors may be preferable

Strategies for the treatment for early and late graft failure following autologous and allogeneic HCT are highlighted in Table 80.3. Use of autologous back-up In circumstances where the autologous product has been manipulated, for example by CD34+ cell enrichment, it is prudent to have stored an

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Table 80.3 Treatment strategies for graft failure Graft failure following autologous HCT

Graft failure following allogeneic HCT

Autologous back-up should be available especially for manipulated products Hematopoietic growth factor support Consideration for allogeneic hematopoietic cell transplantation Autologous back-up Hematopoietic growth factor support Alterations in immunosuppressive medication to convert mixed to full donor chimerism Donor lymphocyte infusion to convert to full donor type CD34+ cell boost for poor graft function Regrafting using same or alternate donor

HCT, hematopoietic cell transplantation.

unmanipulated portion of the graft as a back-up product [31]. Infusion of the back-up product has been shown to restore hematopoiesis [31]. In the event that an autologous back-up product is not available or successful, a search for a donor graft should be initiated. In this instance, the allogeneic rescue should be preceded by a reduced-intensity conditioning regimen. Autologous rescue has been successful in restoring hematopoiesis in patients that have experienced graft failure after allogeneic HCT [31]. The relatively low risk of graft failure following allogeneic HCT and the inherent difficulty in collecting uncontaminated autologous cells in patients with hematologic neoplasms or marrow failure syndromes makes the routine decision to store autologous products problematic.

Changes in immune suppression Preclinical models of BMT have confirmed that suboptimal immune suppression in the immediate post-transplantation period can increase the risk of early graft failure following reduced-intensity conditioning for allogeneic HCT [124]; adequate dosing of immunosuppressive medication supports engraftment by inhibiting HVG reactions [125,126]. It remains unclear whether immunosuppressive drug withdrawal will hasten or abrogate graft loss in instances of persistent mixed chimerism. Detailed studies are required to define how an alteration in post-transplantation immune suppression will affect engraftment and graft failure. Donor lymphocyte infusion Unstimulated DLI was administered pre-emptively to patients with SAA who were considered at high risk for graft failure because of decreasing levels of donor cell chimerism despite full-dose conditioning [162]. In these patients, low-dose DLI promoted the conversion to complete donor type, and none experienced graft failure. Following reduced-intensity conditioning, patients with mixed chimerism are converted to full donor type with DLI, although it is uncertain how many would have become full donor chimeras without an intervention [119]. DLI is associated with the development of severe acute GVHD in a significant proportion of recipients [163]. Selective depletion of cellular subsets within the DLI might promote engraftment yet not precipitate GVHD. In this respect, the removal of naïve and a subset of memory T cells may appear a possible candidate [164]. The timing of when to administer DLI for graft failure prevention is unclear and requires careful consideration of competing risks and benefits. Is conversion from mixed chimerism to complete donor type absolutely necessary? Conversion to full donor chimerism was not required for graft-versus-tumor reactions in some studies [165], whereas others have suggested that sustained graft-versustumor activity requires conversion to full donor chimerism [29].

Use of growth factors after graft dysfunction Myeloid growth factors are reported to significantly shorten the time to neutrophil recovery following autologous PBPC infusion [156,157]. The impact on the time to platelet recovery is less clear; it was reported that post-infusion growth factors significantly delayed platelet recovery, especially in patients who received less than 2.5 × 106 CD34+ cells/kg [156]. Growth factors have been used for the treatment of graft failure following autologous HCT, with variable improvements in blood counts leading to adequate myelopoiesis, and long-term disease-free survival reported in some patients [158]. In contrast, post-transplantation growth factor support for patients with graft failure who received a chemically purged autologous product consistently confirms a lack of response [159,160]. There are no data comparing GM-CSF to G-CSF to GM-CSF followed by G-CSF. A trial of post-transplantation growth factors is a reasonable approach for graft dysfunction following autologous HCT, while consideration of a more definitive therapeutic intervention is being evaluated. The role of commercially available growth factors to abrogate poor graft function or graft failure following allogeneic HCT remains unclear. In a randomized study evaluating patients with graft failure after related or unrelated allogeneic HCT, GM-CSF followed by G-CSF was confirmed to improve 100-day survival compared with GM-CSF alone [161]. As expected, beneficial effects on red blood cell and platelet recovery were not noted. It is reasonable to consider hematopoietic growth factors as an adjunct in the management of graft dysfunction following allogeneic HCT, yet it is unlikely that this therapy alone will be effective to restore adequate blood counts in the majority of patients.

CD34+ infusion Poor graft function after allogeneic HCT is defined by at least two lines of cytopenia beyond 28 days after transplantation with complete or nearcomplete donor chimerism [28]. In patients with poor graft function, a CD34+-selected cell boost following G-CSF mobilization was associated with a high likelihood of hematopoietic cell recovery and a low risk of GVHD [28]. Patients did not receive conditioning prior to the CD34+ cell boost. It is unclear how long to wait before requesting a second donation of cells. Once the diagnosis of poor graft function is established, we consider 2–3 weeks of severe cytopenia a reasonable time, keeping in mind that it may take another 1–4 weeks to actually collect the cells. Poor graft function may result from a viral infection, a medication side-effect, and GVHD, and identifying the underlying cause should be the primary focus as appropriate treatment may establish that the cytopenias are transitory. Regrafting Regrafting with a second allogeneic HCT from the same donor or an alternate can successfully rescue patients with graft failure (Table 80.4). A retrospective multicenter report analyzed 82 consecutive second early allogeneic transplants for primary (n = 28) or secondary (n = 54) graft failure in patients with acute and chronic leukemia and SAA [166]. HLA-matched siblings were used in 64 cases, the same donor was used for both transplants in 56 cases, and the first transplant was T-cell depleted in 30 cases. The median intertransplant interval was 2 months.

Mechanisms and Treatment of Graft Failure

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Table 80.4 Second transplants for graft failure

Graft failure

Author

n

Guardiola et al. [166]

82

28 1°GF 54 2°GF

Min et al. [167]

20

7 1°GF 10 2°GF 3 other

Gaziev et al. [168] Jabbour et al. [169]

32

4 1°GF 28 2°GF 8 1°GF 1 2°GF

Chewning et al. [170]

16

9

11 1°GF 5 2°GF

Median time to second transplant (days)

Regimen before second transplant

Diagnosis

Donor

Source of cells

Same donor

AML CML SAA AML ALL CML SAA Thal

64 MRD 18 other

72 BM 19 PB

56 Yes 26 No

62

82 Yes

73%

30% 3-yr OS

18 MRD 2 other

6 BM 14 PB

20 Yes

49

9 Yes 11 No

75%

71% 3-yr OS

32 URD

32 BM

675

32 Yes

58%

60% 3-yr OS

AML CML MDS ALL AML CML FA MDS

3 MRD 6 other

8 PB 1UCB

28 Yes 4 No 8 Yes 1 No

54

9 Yes

66%

20% 2-yr OS

3 MRD 13 other

2 BM 13 TCD 1 CB

6 Yes 10 No

45

16 Yes

100%

35% 3-yr OS

Response

Outcome

1°GF, primary graft failure; 2°GF, secondary graft failure; ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; BM, bone marrow; CML, chronic myelogenous leukemia; FA, Fanconi’s anemia; MDS, myelodysplastic syndrome; MRD, matched related donor; OS, overall survival; PB, granulocyte-colony stimulating factor-mobilized blood mononuclear cells; SAA, severe aplastic anemia; TCD, T-cell depleted; Thal, thalassemia; UCB, umbilical cord blood.

All patients received conditioning prior to the second transplant; 69 patients received a combination of sublethal irradiation, cytotoxic therapy or anti-T-cell therapy, and 13 patients received anti-T-cell therapy alone. The probability of neutrophil recovery was almost 75%, and the probability of platelet recovery to over 50 × 109/l was almost 30%. The probability of developing clinically significant acute GVHD was 41%. The estimated 3-year overall survival was 30%, a result superior to the 8% that was reported for graft failure in the setting of SAA without a second transplant. The requirement for a conditioning regimen prior to a second transplant was raised in another report [167]. Among 788 consecutive recipients of HLA-matched unmanipulated BM grafts following full-dose conditioning, 20 (2.5%) patients received a second transplant for primary (n = 7) or secondary (n = 10) graft failure or aplasia (n = 3) caused by residual recipient cells producing antierythrocyte antibodies. The same donor was used for the second transplant in all cases. The second graft consisted of BM (n = 6) or G-CSF-mobilized blood MNCs (n = 14) without ex vivo manipulation. No conditioning was given prior to the second transplant in just over half of the recipients, while the remaining patients were prepared with total nodal irradiation with or without antiT-cell antibodies. Fifteen (75%) patients achieved a complete hematologic recovery. and this did not appear dependent on receiving conditioning. Forty percent of patients developed clinically significant acute GVHD. Patients that did not respond to the second transplant died from complications related to graft failure. Chimerism studies before the second transplant were not performed. The lack of prospective randomized trials has made definitive recommendations regarding a second transplant difficult, and many issues remain unresolved (Table 80.5) [26].

Table 80.5 Considerations for a second transplant • Should the same or a different donor be used? • Should bone marrow or granulocyte colony-stimulating factor-mobilized blood be used? • When is a CD34+ selected boost versus an unmanipulated graft preferred? • Should a preparative regimen be used? If so, what? • Is post second transplantation immunosuppressive medication necessary? • What is the role of donor lymphocyte infusion for converting partial chimerism?

Summary Successful HCT is achieved when the infused hematopoietic progenitor cells traffic to their BM niche and restore hematopoiesis. Graft failure refers to an inadequate recovery of blood cells following HCT and remains a relatively uncommon complication, yet its presence can be associated with significant morbidity and mortality. Graft failure is the end-stage result of many contributing factors. A detailed evaluation of the underlying cause of graft dysfunction is important because appropriate treatment may establish that the cytopenias are transitory. Although more study is needed, several good treatment options are available to overcome graft failure and improve the long-term survival in transplant recipients.

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after allogeneic bone marrow transplantation. Acta Haematol 2000; 104: 185–92. 168. Gaziev D, Polchi P, Lucarelli G et al. Second marrow transplants for graft failure in patients with thalassemia. Bone Marrow Transplant 1999; 24: 1299–306.

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170. Chewning JH, Castro-Malaspina H, Jakubowski A et al. Fludarabine-based conditioning secures engraftment of second hematopoietic stem cell allografts (HSCT) in the treatment of initial graft failure. Biol Blood Marrow Transplant 2007; 13: 1313–23.

81

Margaret R. O’Donnell

Blood Group Incompatibilities and Hemolytic Complications of Hematopoietic Cell Transplantation

Introduction The etiologies of hemolytic disorders following hematopoietic cell transplantation (HCT) can be categorized as either immune mediated or complications related to thrombotic microangiopathy (TM) syndromes. The majority of immune-mediated hemolytic reactions are caused by alloantibodies directed at red cell antigens in blood group-incompatible donor–recipient pairs. Autoimmune mediated hemolysis is less common but can be seen following both allogeneic and autologous HCT. In these instances, hemolysis may be related to drugs, infections or recurrent disease (primarily in lymphoproliferative disorders), or be a manifestation of chronic graft-versus-host disease (GVHD). The factors leading to hemolysis in the context of TM following HCT are poorly understood. The supposition has been that injury of the endothelial cells caused by radiation, dose-intensive chemotherapy, immunosuppressive agents such as tacrolimus (FK506), sirolimus or cyclosporine (CSP), or cytokine release associated with GVHD is responsible for thrombus formation in small vessel beds. This chapter addresses the issues of prevention, diagnosis, and treatment of these disorders in the context of HCT.

Hemolytic complications of ABO incompatibility The inheritance of blood group antigens (ABO, Rh, Jk, etc.) is independent of that of the human leukocyte antigen complex present on tissues. In most series of sibling allogeneic HCT, there is discordance of ABO groups in 30–40% of donor–recipient pairs, with similar or slightly higher rates in unrelated donor (URD) HCT. While ABO incompatibility may increase the complexity of HCT, it is not usually viewed as a barrier to successful engraftment of myeloid or megakaryocytic precursors, and does not affect the incidence of GVHD. Recipients of ABOincompatible HCT products do require more red blood cell (RBC) and platelet transfusions than those receiving compatible products, with a subset of patients with ABO major mismatches developing pure red cell aplasia (PRCA), which usually resolves within a few weeks to months. ABO incompatibility is defined as major when the recipient plasma contains isohemagglutinins against donor RBC antigens; it is an ABO minor mismatch if the donor has isohemagglutinins directed against

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

recipient RBC antigens; and it is bidirectional if both features are present. There are two scenarios for the development of hemolysis in the first weeks post HCT (Table 81.1) [1–5]: 1. Pre-existing isohemagglutinins cause immediate and ongoing lysis of RBCs as long as the isohemagglutinins persist (major or bidirectional). 2. Generation of new isohemagglutinins by passenger lymphocytes in the graft as a response to foreign antigens in the recipient causes delayed hemolysis, usually 2–4 weeks following HCT (minor and bidirectional). The time of onset and duration of hemolysis are affected by the titer of isohemagglutinin and its rate of clearance, as well as the quantity of target antigen available. In a proportion of major ABO-incompatible HCT, the isohemagglutinin targets not just circulating RBCs, but also early marrow precursors expressing the antigen at the level of the colony forming unit-erythroid, leading to PRCA [6–9]. Other factors that may have implications for the development of hemolysis are the conditioning regimen (high dose versus reduced-intensity conditioning [RIC]), HCT source (marrow versus peripheral blood) and donor (sibling versus URD versus umbilical cord blood [UCB]), and immunosuppressive agents. The development of RIC regimens to accommodate a wider spectrum of patients creates an environment in which donor and host hematopoiesis coexist for protracted periods of time; this has led to concerns for the possibility of higher rates of chronic hemolysis and PRCA [10–12]. Peripheral blood HCT products carry a 10-fold higher number of T lymphocytes, which are less activated than T cells in marrow products. B-cell recovery also is reported to occur earlier post peripheral blood HCT, with augmented immunoglobulin production early post HCT compared with marrow, which might lead to a higher probability of delayed hemolysis in minor incompatible HCTs. Delayed RBC production and increased transfusion requirements have been documented with ABO incompatibility by several authors in both high-dose and RIC settings. Worel et al. showed that generation of reticulocyte counts of over 3% took a median of 32 days for major ABO mismatches versus 21 and 22 days for matched and ABO minor mismatched recipients, respectively, and the number of RBC transfusions up to day 100 following ablative marrow HCT doubled (12 for major versus six for matched and eight for ABO minor mismatched) (p = 0.045) [13]. In 2001, Bolan et al. reported that RBC production was delayed beyond 100 days in over 60% of patients treated with RIC [14]. Early post-HCT RBC production may appear adequate in RIC recipients due to autologous RBC recovery; however, as the chimeric balance progressively becomes donor in origin, autologous RBC production declines during the second to fourth months post RIC HCT. The agents used in RIC (fludarabine, cyclophosphamide, melphalan, and low-dose

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Table 81.1 Immunohematologic problems of ABO-incompatible hematopoietic cell transplantation Major mismatch (20%) Example: Recipient O, donor A, B, AB or recipient A or B, donor AB Immediate hemolysis of RBCs infused with the graft Delayed hemolysis of donor RBCs by persistent recipient isohemagglutinins Delayed erythrocyte production Pure red cell aplasia Minor mismatch (22–24%) Example: Recipient A, B, or AB, donor O or recipient AB, donor A or B Immediate hemolysis of recipient RBCs by donor-derived isohemagglutinins in the graft Delayed hemolysis of recipient RBCs by newly generated isohemagglutinins from donor lymphocytes (“passage lymphocytes”) Bidirectional mismatch (1–2%) Example: Donor A, recipient B or vice versa Both immediate and delayed hemolysis are possible with isohemagglutinins of the donor against recipient RBCs, and recipient isohemagglutinins directed against donor RBCs RBC, red blood cell.

radiation) allow the persistence of recipient B cells and plasma cells for much longer periods of time than with ablative conditioning [15]. During the period of mixed chimerism, these cells are capable of continuing production of recipient-type isohemagglutinins. Although marrow cultures confirm that engraftment of erythroid precursors at the level of burst-forming units-erythroid occurs in the same ratio as for myeloid and megakaryocytic precursors, later erythroid precursors that express ABH antigens starting at the level of colony forming unit-erythroid often do not appear on marrow biopsy until immunoglobulin G (IgG) and IgM titers of recipient isohemagglutinins fall to 1 : 1 [16]. The reported incidence of PRCA following RIC has varied widely from 6% to 50%, compared with rates of 5–16% post high-dose conditioning [14]. Griffith et al. followed chimerism studies including plasma cells and B cells post fludarabine/cyclophosphamide conditioning in 12 ABO-mismatched recipients with renal cell carcinoma or other malignancy. Six developed PRCA, which persisted for 3–4 months after T-cell and myeloid hematopoiesis had shifted to 100% donor; in all six patients with PRCA, recipient plasma cells were detectable by chimerism studies (5–42%) and persisted longer than B cells, implying that the plasma cells were the source of the isohemagglutinin production [17]. There have also been reports that GVHD regimens of CSP alone or combined with prednisone create a milieu conducive to persistence of isohemagglutinin production, since CSP (and by inference tacrolimus) permits B cells to remain longer than combinations that include methotrexate or mycophenolate mofetil (MMF) along with CSP [18]. The development of acute GVHD appears to accelerate clearance of isohemagglutinins, possibly due to a graft-versus-plasma cell effect [19].

Delayed hemolytic reactions In minor ABO-mismatched HCT, hemolysis, which may be severe and occasionally fatal, can either occur immediately due to existing IgM or IgG isohemagglutinins in the product, or emerge abruptly 5–17 days post transplant as a result of development of new isohemagglutinins generated by “passenger” B lymphocytes transferred from the donor at the time of transplantation [5,20,21]. Significant hemolysis occurs in

10–15% of ABO minor mismatched adult HCT grafts compared with 10% for renal allografts and 70% for heart–lung recipients. The incidence of “passenger lymphocyte syndrome” increases with the B-cell content of the graft; it is therefore reasonable to expect a higher incidence of hemolysis with peripheral blood hematopoietic cell products, which contain a 10-fold increase in CD10+ and CD20+ cells compared with marrow grafts [20,21]. The use of granulocyte colony-stimulating factor to mobilize donor peripheral blood hematopoietic cells can also modulate T-lymphocyte cytokine production in such a way that promotes antibody production. While ABO antigens are the most common targets, hemolysis has also been reported against Rh, Jka, Kidd, and Lewis blood group antigens [21–25]. Several factors influence the severity of the hemolysis, including the rapidity of rise of donor antibody in the recipient post transplant, secretor status, and rapidity of engraftment (i.e. type of graft) [20,21]. Massive hemolysis in which most, if not all, recipient RBCs are lysed over 2–3 days seems to be complement mediated, analogous to posttransfusion purpura. The incidence of hemolysis following ABO minor mismatched HCT has varied from 5–10% for marrow up to 33% for peripheral blood HCT. A recent report evaluated the rate of isohemagglutinin production following 24 peripheral blood cell and 14 UCB minor mismatched products. Fifteen of 24 peripheral blood cell recipients were found to have donor-derived isohemagglutinins between 6 and 88 days post HCT; 50% of those with detectable isohemagglutinins showed some evidence of hemolysis. None of the UCB recipients developed isohemagglutinins, which correlates with the data that the naïve B cells present in the UCB graft lack the exposure to ABH antigens necessary to generate isohemagglutinins [26].

Management of ABO and Rh incompatibility The strategies for managing blood product support are outlined in Table 81.2. As a general rule, group O RBCs are the preferred products in the immediate pre- and post-HCT period, thereby diluting the percentage of RBC targets for donor-generated isohemagglutinins in ABO minor or bidirectional mismatched HCT [27–29]. Prior to HCT, both recipient and donor isohemagglutinin titers should be ascertained in mismatched HCT. If the recipient has high titers against donor RBC antigens, it is important to minimize RBC content infused, and one should consider plasma exchange using plasma lacking that antibody shortly before HCT in patients with titers of 1 : 256 or higher, with the intent of lowering the titer to 1 : 16 or less. Red cell exchange prior to HCT has been used to minimize the late hemolytic reactions seen 5–16 days post infusion in minor ABO-mismatched recipients with high titers against recipient RBCs; these procedures are usually performed 2–3 days before HCT infusion, after immunosuppression has been started, to prevent rebound phenomena. However, pretransplant titers are not always predictive of the severity of delayed hemolysis in the “passenger lymphocyte” syndrome, and therefore exchange transfusions are being used less frequently. RBC and platelet products should be volume-reduced post HCT to minimize infusion of isohemagglutinins in the plasma [30]. Another strategy is infusion of donor-type fresh frozen plasma to provide a noncellular source of A or B antigen to absorb recipient isohemagglutinins [31]. Complications associated with these strategies to deplete isohemagglutinins include citrate toxicity, risk of transmission of infection, and platelet depletion. There are also risks of febrile transfusion reactions, transfusion-related lung injury, and, with fresh frozen plasma infusion, volume overload. Some authors have also reported using small aliquots of donor RBCs 12–24 hours before HCT. This intervention requires forced alkaline diuresis and intensive monitoring for hemolytic reactions [32]. Red cell depletion and reduction of isohemagglutinins are quite

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Table 81.2 Management of blood support for ABO-incompatible transplant recipients

Major mismatch Example: Recipient O, donor A

RBCs

Plasma

Remove RBCs from HCT product

Give donor-type platelets and plasma- or volume-reduce out all of donor group products If recipient anti A titer is >1 : 256, consider: 1 Plasma exchange pre transplant 2 Daily infusion of donor-type FFP to lower titer to 1 : 1, or lower stable titer to <1 : 16

Transfuse O cells post transplant until anti-A titer is undetectable and recipient types as group A Minor mismatch Example: Recipient B, donor O

Bidirectional mismatch Example: Recipient B, donor A

Rh incompatible Major Minor

Transfuse group O cells Red-cell exchange with group O cells pre HCT to dilute recipient-type RBCs down to <30% of RBC volume

1 Remove plasma from HCT graft to prevent immediate hemolysis if titer is ≥1 : 128 2 Give platelets and plasma lacking anti-B isohemagglutinin or volume-reduce the product 3 Monitor the Coombs test, beginning day 5–20, every 2–3 days to monitor for hemolysis caused by “passenger” lymphocyte syndrome

Transfuse O cells Remove RBCs from the HCT product Red-cell exchange with group O cells to deplete the percentage of B cells prior to HCT

Plasma-deplete the donor HCT product before infusions Use group AB plasma and platelet products before and after transplant (or volume-reduce out of group products) Consider plasma exchange for high titer (>1 : 256) IgG isohemagglutinin

Deplete RBCs from graft. Use Rh− product Use Rh− product

Consider prophylactic removal of Rh antibody in patient by red-cell exchange Consider RhoGAM

FFP, fresh frozen plasma; HCT, hematopoietic cell transplantation; RBC, red blood cell.

effective in preventing immediate hemolysis in major ABO mismatches; however, 10% of patients receiving myeloablative and up to 35% of patients receiving RIC HCT will develop delayed hemolysis. Following HCT, patients with ABO incompatibility should be monitored for the emergence of donor-derived erythrocytes, persistence of recipient isohemagglutinins, and evidence of hemolysis using lactic dehydrogenase, reticulocyte counts, and the direct agglutinin test. Patients with major ABO mismatches and high isohemagglutinin titers pre HCT are particularly at risk for delay of RBC engraftment, hemolysis, and PRCA. Isohemagglutinin titers in recipients with major ABO mismatches should be monitored at least twice monthly until their disappearance. Patients with rising titers and increasing transfusion requirements merit marrow evaluation to exclude PRCA. Clearance of isohemagglutinin appears to be affected by both donor source and GVHD. Investigators from Seattle examined the rate of fall of isohemagglutinin titers in 383 major or bidirectional ABO-mismatched HCT recipients following ablative conditioning [33]. The time to undetectable titer was significantly shorter (46 versus 61 days; p = 0.016) in unrelated compared with sibling recipients. Among related donor–recipient pairs, those who developed GVHD had a twofold more rapid decline in antibody titers. In this study, ABO incompatibility did not affect the incidence of GVHD or survival. Remberger and colleagues recently reported that ABO major mismatch was a significant risk factor for graft failure among 224 URD HCT recipients who received fully ablative conditioning and T-cell-depletion as a component of GVHD prophylaxis. The rate of primary and secondary graft failure was 7.5%

for ABO major mismatch and 0.6% for matched donor–recipient pairs [34]. Graft failure has not been reported in high frequency in non-T-celldepleted studies. In two other studies, ABO incompatibility did affect survival at day 100 in subgroups of patients (chronic myelogenous leukemia patients in one, and acute myeloid leukemia and myelodysplasia in the second) [14,35]. Neither report specified the factors influencing the poorer outcomes for ABO-incompatible HCT. One may speculate that more protracted transfusional support requiring indwelling venous access devices, which placed patients at higher risk of infection, might be a factor. Iron overload is a potential deleterious condition for those patients who developed PRCA. The largest study of the impact of ABO incompatibility on HCT outcome was recently compiled by the International Bone Marrow Transplant Registry. They evaluated 3103 sibling ablative HCTs performed between 1990 and 1998 with CSP/methotrexate for GVHD prophylaxis for early-stage leukemia. Two-thirds of the recipients were ABO matched; 14% were major, 14% were minor, and 4% were ABO bidirectional mismatches. There was no difference in survival, relapse, transplant-related mortality or acute and chronic GVHD. The only differences were a higher incidence of grade II–IV GVHD in bidirectional mismatched pairs, and longer RBC transfusion dependence for ABO major mismatches [36]. Despite immunosuppression, patients can also develop alloimmunization to non-ABO RBC antigens post HCT, most commonly involving Rh, Kell or Kidd (Jk) antigens. Development of non-ABO antibodies has been reported in high frequency in patients receiving ABO-incom-

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patible HCT: 9.6% versus 1.6% for those receiving ABO-matched products [22]. De la Rubia et al. [23] reported a 3.7% incidence of detection of new alloimmunization to Jka, Kell, M, Lewis, and Rh antigens, usually within the first 30 days post HCT. In the majority of cases in which the specificity of the antibody was identified by elutriation from RBCs, the antibody was directed at antigens which were not detected in either donor or recipient at the time of HCT. The isohemagglutinins could have been produced by either residual host- or donor-derived lymphocytes in response to new RBC antigens presented post HCT. Clinically, the agglutinins that develop in response to antigens which are different from those expressed on either donor or recipient RBCs have little hemolysis associated with them compared with quite severe hemolytic reactions, which have been seen with Rh− donor lymphocytes infused into Rh+ recipients. However, there have been two reports of severe hemolysis associated with the Jka system. In both instances, the donor had previously been sensitized to Jka, but the method used for the pretransplantation antibody screen did not detect the Jka antibody. It is not uncommon for an individual with known anti-Jka to have no serologically identifiable serum antibody a few months after the antibody was originally identified, making the risk of a clinically significant hemolytic event higher in Jka+ recipients [22,25]. Treatment of PRCA PRCA represents the most extreme form of hemolytic reaction post HCT, with recipient isohemagglutinins targeting medullary precursors at the CFU-E stage, which is the earliest point of ABH antigen presentation. Recent reports do suggest that there is a difference in both the incidence and timing of this complication dependent upon the conditioning regimen. In high-dose conditioning, the incidence is lower and appears as early and prolonged transfusion dependence, while RIC recipients often have early autologous RBC production when the donor graft is just emerging, with a later development of RBC dependence. Red cell production usually returns within 3–4 weeks following disappearance of circulating isohemagglutinins; however, some patients have remained RBC dependent for over 5 years [37]. In the majority of cases, tapering of immunosuppression permits a “graft-versus-plasma or B cell” effect which shuts off isohemagglutinin production by either memory B cells or plasma cells. Other strategies have employed pharmacologic doses of erythropoietin alone or combined with steroids [38–40]. In more refractory cases, plasma exchange or the use of rituximab has been successful; rarely, donor lymphocyte infusion has been used to promote full donor chimerism and to ablate isohemagglutinin production [41–44]. In addition to PRCA associated with ABO incompatibility, aplasia has also been reported in conjunction with parvovirus 19 infection in both HCT and solid organ transplantation [45]. Most of these patients responded to immunoglobulin administration. Two children with hepatic allografts have been reported to develop PRCA associated with tacrolimus, which resolved when the drug was withdrawn; four renal allograft recipients developed PRCA associated with MMF [46,47]. In situations where PRCA develops outside the setting of major ABO incompatibility, a change in calcineurin inhibitor from tacrolimus to CSP, or discontinuation of MMF, may produce red cell recovery. Non-ABO-related Coombs-positive autoimmune hemolytic anemia (AIHA) can also develop late post HCT as an autoimmune manifestation of chronic GVHD [48,49]. Drugs such as antibiotics and fludarabine or infections with Mycoplasma may also produce hemolysis. Impaired Bcell regulation may occur as a consequence of functional or quantitative T-cell depletion of the HCT product with an AIHA incidence of 3% in one large series.

Patients who develop AIHA after HCT usually receive the same battery of immunosuppressive agents as nontransplant patients – steroids, immunoglobulin, rituximab or splenectomy – as well as less conventional approaches such as plasma immunoabsorption, vincristine or reinfusion of aliquots of donor T cells to exert immunoregulation on the autoreactive T cells. Hemolysis and TM syndrome “Thrombotic microangiopathy” describes syndromes of Coombs-negative hemolysis associated with RBC fragmentation, often combined with clinical features of renal or neurologic dysfunction and thrombocytopenia suggestive of thrombotic thrombocytopenia purpura (TTP) or hemolytic–uremic syndrome (HUS). Injury to the endothelial substructure by high-dose chemotherapy, radiation, calcineurin inhibitors such as CSP, tacrolimus or sirolimus, and cytokine production in acute GVDH is the proposed inciting mechanism. Inflammatory cytokines (tumor necrosis factor and interleukin-8) released by tissue injury associated with GVHD have been linked to increases in prothrombotic events such as increases in tissue plasminogen activator and ultralarge von Willebrand factor assembly; interleukin-6 inhibits ultralarge von Willebrand factor cleavage [50–53]. The incidence has varied widely from low levels of less than 1% for autologous HCT to as high as 70% in some small series of allogeneic HCT [54–60]. In contrast to idiopathic sporadic TTP/HUS syndrome, there is no defining laboratory marker such as ADAMTS13 deficiency or decrease in large von Willebrand multimers to separate this entity from coexisting post-transplantation complications [61–63]. In a 2004 literature review of 447 cases of post-transplantation TM culled from more than 5400 transplant procedures in 35 articles, George et al. reported an overall incidence of 8.2%, with the majority of cases occurring within the first 3 months after HCT [64]. However, the criteria used to define the syndrome differed in 28 of these series and included a spectrum of clinical settings from nontransfusion-dependent anemia with schistocytes and mild renal impairment to severe anemia, thrombocytopenia with dialysis-dependent renal failure, and/or neurologic symptoms which usually were manifested as confusion or seizures. In the most severely affected cases, mortality ranged from 50% to 90% within 3 months of diagnosis, usually due to uncontrolled GVHD or infection. Viral infections such as human herpesvirus 6, parvovirus 19, cytomegalovirus, and adenovirus, as well as Aspergillus species, have also been reported to cause TM either directly or by inciting inflammatory cytokines which secondarily lead to endothelial damage. Tarantolo et al. reported a small series of TM caused by a Bartonella-like agent which responded to doxycycline [65]. Escherichia coli strains, which are the most common initiators of HUS in the nontransplantation setting, do not appear to be prevalent pathogens post HCT. The median time of onset of TM in adult allogeneic HSCT is 47 days, although an earlier onset at a median of 25 days was reported by Cutler et al. in patients receiving a combination of sirolimus and tacrolimus [66–68]. The most recent series from St Judes Hospital in Memphis, Tennessee, showed a 9.6% incidence of TM, with a much later onset of 171 days for pediatric patients; however, in this population, T-cell depletion was used as a component of GVHD prophylaxis for all unrelated or mismatched related donors. In this report, a risk factor for developing TM was identified as T-cell depletion, rather than either acute GVHD or URD status, which are the major risk factors for TM in adults [69]. In addition to grade II–IV GVHD, female sex, receipt of an URD product, and diagnosis of lymphoid malignancy were the major predisposing factors for TM in a review of over 1200 patients treated at M.D. Anderson Cancer Center at Houston, Texas, using tacrolimus-based GVHD prophylaxis [70]. The incidence of TM in this group was 5.9%, compared with a 10.8% incidence of TM using tacrolimus and sirolimus

Blood Group Incompatibilities and Hemolytic Complications of Hematopoietic Cell Transplantation Table 81.3 Criteria for thrombotic microangiopathy (TM) (A) Bone Marrow Transplant Clinical Trials Network Toxicity Committee consensus definition for TM 1. RBC fragmentation and ≥2 schistocytes per high-power field on peripheral smear 2. Concurrent increased serum lactate dehydrogenase above institutional baseline 3. Concurrent renal* and/or neurologic dysfunction without other explanations 4. Negative direct and indirect Coombs test results (B) Common toxicity criteria for bone marrow transplantation-associated TM Grade 1 Evidence of RBC destruction (schistocytosis) without clinical consequences Grade 2 Evidence of RBC destruction with increased creatinine Grade 3 Evidence of RBC destruction with creatinine >3 times the upper limit of normal not requiring dialysis Grade 4 Evidence of RBC destruction with renal failure requiring dialysis, and/or encephalopathy RBC, red blood cell. * Doubling of serum creatinine from baseline (baseline = creatinine before hydration and conditioning) or a 50% decrease in creatinine clearance from baseline.

in the series reported from Dana Farber Cancer Institute in Boston. The TM rate for the CSP-based control group in the Dana-Farber series was 4.2% [66]. The use of RIC has not led to a lower incidence of TM, with an incidence of 15–23% in three small series; in several of these cases, however, these patients had a prior myeloablative HCT, often within the previous 6 months [68,71,72]. In the M.D. Anderson report, which includes more than 500 RIC recipients, the risk of TM was equivalent to that for high-dose conditioning recipients. In an effort to provide consistency for reporting transplantationrelated toxicities and to provide a common working definition for future therapeutic interventions, the United States Blood and Marrow Transplant Clinical Trials Network (BMT CTN) proposed criteria for defining and grading TM, shown in Table 81.3, which include red cell fragmentation and a negative Coombs test combined with renal or neurologic dysfunction [73]. While thrombocytopenia is also very common in this entity, the BMT CTN chose not to include this parameter as thrombocytopenia may be caused by several other coexisting conditions in the 3–4 months post HCT, in which TM is most likely to occur. Screening for disseminated intravascular coagulation was also not included in the BMT CTN criteria, but is included along with thrombocytopenia in the more recently proposed European Group for Blood and Marrow Transplantation schema [74]. Trials evaluating automated methods for reproducibly quantifying schistocytes are ongoing and may help standardize criteria for multi-institutional trials [75]. Other markers which have been used to define nontransplant TTP/HUS, such as ADAMTS13 levels or

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size of von Willebrand multimers, have not been useful in the post-HCT setting [76,77]. Therapy for post-HCT TM Patients who exhibit grade I TM by National Cancer Institute common toxicity criteria for TM actually do not meet the current CTN definition of TM. However, these patients may represent a population in which attention to factors that may increase endothelial damage and RBC fragility may lessen the risk of evolution to overt TM. Strategies include optimizing antihypertensive therapy, adjusting CSP, tacrolimus, and sirolimus levels to lower therapeutic levels in patients without active GVHD, and providing appropriate magnesium replacement. In patients with overt TM, treatment strategies have included discontinuation or change of calcineurin inhibitors, and either plasma infusions or plasma exchange. In patients with renal dysfunction or neurologic abnormalities, discontinuing CSP or tacrolimus for a few days may help distinguish TM from drug-related toxicity. However, a significant number of TM patients also have active grade II or more GVHD which requires ongoing therapy. In the M.D. Anderson series, tacrolimus was continued at reduced doses in the majority of patients and discontinued only for severe renal or neurologic impairment. In patients requiring discontinuation of tacrolimus, steroids were either started or the dose increased to provide additional coverage for GVHD. The majority (95%) of patients in the M.D. Anderson trial also received plasma exchange on a daily basis, with tapering of exchanges based on clinical response [70]. Resolution of TM-associated clinical and laboratory findings occurred in 60% of patients at a median of 29 days in this report, in contrast to much lower responses to plasma exchange in (18–30%) earlier series. Mortality, however, remained high, with only 50% of the responders and none of the nonresponders alive at 6 months. Control of GVHD and infection appeared to be crucial for long-term survival post TM. Ultimately, only 11 of 66 plasma exchange recipients were long-term survivors at 3 years post HCT, comparable to the 70–95% mortality in other post-HCT TM series, and in contrast to survival of over 60% in idiopathic non-HCT TM treated with plasma exchange [78–87]. New treatment approaches need to be developed for this entity. Based on recent experience using defibrotide for sinusoidal occlusive syndrome of the liver, a similar problem of endothelial injury and thrombosis, clinical trials are being designed to explore this drug for TM [67,88].

Conclusion As the scope of HCT evolves to encompass wider age ranges, differing sources of hematopoietic cells, newer conditioning regimens, and alternative immunosuppressive agents, we need to be conscious that changes in the kinetics of chimerism or the introduction of new drugs to either conditioning or GVHD prophylaxis and treatment can have a significant impact on the incidence and timing of hemolysis post allogeneic HCT. It is important to maintain a high level of suspicion and to initiate appropriate testing for hemolysis of both immune and thrombotic etiologies in patients with unexplained anemia and increasing transfusion requirements. Better definition of TM syndromes may allow earlier intervention, which should improve outcomes.

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Chapter 81

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Jeffrey McCullough

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation

Introduction Patients who are candidates for or are undergoing hemapatopoietic cell transplantation (HCT) have unique transfusion requirements. These are due to the need to minimize the likelihood of alloimmunization, the severe immunosuppression these patients undergo, their temporary inability to produce blood cells, and the fact that their blood type may change and they may become a temporary or permanent chimera. Thus, an effective blood bank and transfusion medicine program are essential for a successful HCT program. This chapter includes a brief description of the blood components and derivatives used by HCT patients and the relevant transfusion strategies.

Red blood cell components Red cells Red blood cells (RBCs) are the cells that remain after most of the plasma has been removed from whole blood. This blood component is often called “packed red cells” or “packed cells.” The unit of red cells has a volume of about 350 mL, and will contain about 200 mL red cells with an hematocrit of about 60%. The fluid portion of the unit is primarily an additive solution preservative used to enable red cells to be stored for 42 days. About 20 mL plasma remains from the original unit of whole blood. One unit of red cells will increase the hemoglobin and hematocrit in an average-sized (70 kg) adult by about 1 g/dL and 3%, respectively. Red cells are the component of choice for any patient with severe anemia. Most patients who require red cell replacement do not also need intravascular volume replacement because anemia has developed slowly. Thus, almost all transfusions given for red cell replacement are packed red cells, and whole blood is rarely used. Red cell transfusions are usually given to HCT patients because of anemia. While the decision to transfuse is usually based on the hemoglobin level, other clinical factors should also be considered. Most patients with hemoglobins of 10 g/dL or above do not require transfusion, and most patients with a hemoglobin of less than 7 g/dL will benefit from transfusion [1–5]. Because of the heterogeneity of patients and these clinical situations, there is no single standard indication for red cell transfusion. In deciding whether an individual requires transfusion, the clinical condition of the patient is of primary importance, and patients

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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should not be transfused based only on their hemoglobin level. Patients with cardiovascular disease may benefit from transfusion at hemoglobin of 9 g/dL [4,5]. When there is sudden acute blood loss, the major threat to the patient is the loss of intravascular volume and resultant cardiovascular collapse. In most “normal” patients, the loss of approximately 1000 mL blood can be replaced by colloid or crystalloid solutions alone. An otherwise healthy individual can tolerate the loss of up to half of the red cell mass without need for replacement. Because many patients have some degree of cardiovascular compromise, they will require red cell replacement after smaller volumes of blood loss. If blood loss is judged sufficient to require transfusion, it is not necessary to wait until symptoms such as pallor, diaphoresis, tachycardia or hypotension develop. Transfusion is with the standard RBC component from the stock supply of the blood bank. Leukocyte-reduced red cells Red cell and platelet blood components contain contaminating leukocytes that can alloimmunize transfusion recipients, leading to multiple problems (Table 82.1). In order to avoid these problems, leukocytes are removed from the red cells. This is done using filters that remove about 99.9% of the leukocytes leaving fewer than 5 × 106 per unit. In the past, filtration was performed at the bedside, but currently this is done in the laboratory in order to obtain more consistent leukocyte removal and prevent the accumulation of cytokines produced by the contaminating leukocytes. The indications for leukocyte-reduced red cells are: 1 prevention of alloimmunization to leukocytes and platelets; 2 prevention of transmission of cytomegalovirus (CMV) (see below); 3 treatment of patients with multiple febrile transfusion reactions; 4 prevention of immunomodulatory effects of transfusion. The kinds of patient who should receive leukocyte-reduced red cells include any individuals with a disease that can potentially be treated by HCT and those who will receive multiple transfusions during their life, such as patients with hemoglobinopathies (Table 82.2). In addition to alloimmunization to leukocytes, transfusion has other immunologic effects generally referred to as transfusion-related immune modulation [6]. The first observation of transfusion-related immune modulation was the beneficial effect of transfusion on renal graft survival. In the last decade, considerable debate has developed over whether transfusion also leads to increased cancer recurrence and/or postoperative infection (see below) [6–8]. Because the clinical situations in which leukocyte-reduced blood components are valuable continues to increase [7], many blood

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation Table 82.1 Adverse effects of leukocytes in blood components Immunologic effects Alloimmunization Febrile nonhemolytic transfusion reactions Refractoriness to platelet transfusion Rejection of transplanted organs Graft-versus-host disease Transfusion-related acute lung injury Immunomodulation Increased bacterial infections Increased recurrance of malignancy Infectious disease Cytomegalovirus Human T-lymphotrophic virus-I Epstein–Barr virus

Table 82.2 Clinical situations in which leukodepleted red cells are recommended Hematopoietic stem cell recipients Acute leukemia Chronic leukemia Congenital platelet function abnormalities Congenital immune deficiency syndrome Hematologic malignancies potentially treated with stem cell transplantation Solid tumors potentially treated with stem cell transplantation Intrauterine transfusions Exchange transfusion for hemolytic disease of the newborn Hemoglobinopathy or thalassemia

centers now leukocyte-reduce all of their blood components. Some countries with a national blood program have introduced universal leukoreduction, with apparent beneficial results [9,10]. Washed red cells Washed red cells are red cells suspended in an electrolyte solution. Most plasma, platelets, and leukocytes have been removed, although leukocyte removal is not as complete as with frozen deglycerolized red cells. After washing, the red cells can be stored for only 24 hours because of the possibility of bacterial contamination. Since the major advantage is the removal of the plasma by washing, washed red cells are indicated for patients who have severe reactions caused by plasma.

Coagulation factor components

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available – especially factor V – multiple factor deficiencies such as patients with severe liver disease or anticoagulated with warfarin, and massive transfusion in selected patients. FFP is used as the replacement solution for plasma exchange in patients with thrombotic thrombocytopenic purpura, but it is not indicated for use as a volume expander or a nutritional source. Bleeding due to dilution of multiple coagulation factor deficiency can occur in situations where massive transfusions are required. This dilution occurs because red cell components contain little plasma. If coagulation factor replacement is necessary in these patients, FFP is usually used. Patients who are undergoing massive transfusion may also develop disseminated intravascular coagulation (DIC) because of the severity of the underlying problem. Thus, a bleeding diathesis may develop that is a combination of the dilutional coagulopathy of massive transfusion and the “consumption coagulopathy” of DIC. Treatment of the underlying cause of the DIC is essential since, without intervention, transfusion of blood components merely adds more substrate for the coagulation process. In mild forms of DIC, transfusion is usually not necessary. However, in the more extreme forms, there is usually a deficiency of factors V and VIII, fibrinogen, and platelets. Replacement of coagulation factors in the management of DIC should be based on laboratory abnormalities and not on arbitrary formulas. When replacement is necessary, FFP is usually used to replace all factors. Coagulopathy may also develop in patients undergoing HCT. The use of FFP as prophylaxis for patients with coagulopathy who are to undergo invasive procedures is discussed later in this chapter. Plasma Plasma separated from whole blood between 8 and 24 hours after transfusion can be frozen, stored for up to 1 year, and used as a source of coagulation factor replacement but with reduced levels of factors V and VIII. This plasma product can be used in most situations requiring replacement of multiple coagulation factors and is gaining wider use. However, to assure replacement of coagulation factors in a manner equivalent to fresh blood, FFP is recommended. Cryoprecipitate Cryoprecipitate is the cold-insoluble portion of FFP that has been thawed between 1 and 6°C. It is stored at −18°C or below and can be kept for up to 1 year. Cryoprecipitate contains coagulation factor VIII, fibrinogen, and von Willebrand factor. Each bag of cryoprecipitate contains about 250 mg fibrinogen, and this is now its major use. Cryoprecipitate is not a suitable source of coagulation factors II, V, VII, IX, X, XI or XII.

Fresh frozen plasma

Blood group compatibility of components used to replace coagulation factors

Fresh frozen plasma (FFP) is plasma separated from whole blood and placed at −18°C or lower within 8 hours of collection. The unit of FFP has a volume of about 200–250 mL and contains all of the coagulation factors that are present in fresh blood. FFP is not considered to contain red cells and is usually administered without regard to Rhesus (Rh) type. However, occasional rare reports have suggested that units of FFP contain a small amount of red cell stroma that can cause immunization to red cells. Because the plasma contains ABO antibodies, the plasma must be compatible with the recipient’s red cells. The generally accepted indications for FFP [11] are documented coagulation factor deficiencies for which factor concentrates are not

FFP need not be ABO-identical but should be compatible with the recipient’s red cells and can be given without regard to Rh type. Red cell compatibility testing is not necessary. Cryoprecipitate should also be administered as ABO compatible. Although the volume of each unit is small, most therapy involves many units, and thus the total volume of plasma may be large. Cryoprecipitate can be administered without regard to Rh type. Compatibility testing is not necessary. In HCT, patients’ cryoprecipitate is usually used to replace fibrinogen, but the recipient’s fibrinogen may become elevated by the fibrinogen contained in cryoprecipitate if many units are given to patients who are not hypofibrinogenemic.

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Platelet components

Transfusion of platelets

Platelet concentrates prepared from whole blood

The decision as to whether to transfuse platelets depends on the clinical condition of the patient, the cause of the thrombocytopenia, the platelet count, and the functional ability of the patient’s own platelets. Virtually all patients undergoing HCT become thrombocytopenic and require platelet transfusion. Most platelets are transfused to patients with transient thrombocytopenia due to chemotherapy for malignancy or those undergoing HCT [19]. Most platelet transfusions are used for the prevention of bleeding (as prophylaxis) rather than the treatment of active bleeding [19]. Very few controlled studies of prophylactic platelet transfusion were carried out when platelet therapy first became available. Some studies supported the use of prophylactic transfusions [20–23], but other studies were not able to show a benefit of prophylactic platelet transfusion [24,25]. Despite the lack of substantial convincing clinical trial data, it became common practice for physicians to use platelet transfusions to prevent serious bleeding when the platelet count was less than 20,000/μL, because there is little risk of serious spontaneous hemorrhage when the platelet count is more than 20,000/μL, but the risk increases with lower counts [26]. In an effort to evaluate the bleeding risks of thrombocytopenia, occult blood loss in the stools was quantitated using chromium-51-labeled red cells in patients with different degrees of thrombocytopenia. In stable thrombocytopenic patients, no increase occurred in stool blood loss at platelet counts of 10,000/μL, and blood loss increased only when the platelet count reached 5000/μL [25,27]. Since the early days of platelet transfusion, considerable experience has been gained in the management of thrombocytopenic patients, which led to improvements in their outcome while maintaining much lower platelet counts than were previously believed to be safe. Serious bleeding usually occurred only when the platelet count was below 10,000/μL, and fatal bleeding is unlikely to occur at platelet counts above 5000/μL. Gmur et al. [28] found that the threshold for prophylactic platelet transfusion could be 5000/μL in uncomplicated patients, 10,000/μL in individuals with fever or bleeding, and 20,000/μL in patients with coagulopathy or bleeding sites. Heckman et al. [29] found no difference in red cell use, days of fever, hospitalization, thrombocytopenia, remission, death or major bleeding complications when prophylactic transfusion thresholds of 20,000/μL and 10,000/μL were compared. Rebulla et al. [30] reported that, in patients with acute leukemia undergoing first induction of remission, there was no difference in deaths, red cell transfusions or severe hemorrhage, but there was a 21.5% reduction in platelet use using a transfusion threshold of 10,000/ μL compared with 20,000/μL. Wandt et al. [31], summarizing experience in 17 centers in Germany, found no difference in bleeding complications when 10,000/μL or 20,000/μL thresholds were used, and hemorrhagic deaths actually occurred in two patients with platelet counts of 36,000/μL and 50,000/μL. In stable marrow transplant recipients, lowering the transfusion threshold from 20,000/μL to 10,000/μL did not increase severe bleeding or deaths due to bleeding [32], but reduced platelet use by about 25%. Aderka et al. [33] proposed, based on analysis of 196 patients with acute leukemia, that prophylactic platelet transfusion was not necessary when the platelet count was above 10,000/μL. More recently, Zumberg et al. [34] reported no differences in the incidence or severity of bleeding in 159 HCT patients prospectively randomized to receive platelet transfusions at a threshold count of 20,000/μL or 10,000/μL. Establishing the indications for platelet transfusion remains complex because many studies are not powered to show modest differences, most do not use bleeding as the endpoint, and the patient population is heterogeneous. A National Institutes of Health Consensus Conference

Platelet concentrates, often referred to as “random-donor” platelet concentrates, are prepared by centrifugation of whole blood, although the centrifugation technique used in Europe and Canada differs from that used in the United States [12]. At least 75% of the units of random-donor platelets must contain at least 5.5 × 1010 platelets. Most units contain 6–8 × 1010 platelets, and each blood bank should be able to provide an estimate of the platelet content from their quality control data. Although there is no required volume of the random-donor platelet concentrate, the volume is usually about 50 mL. Platelet concentrates prepared by apheresis Several different instruments are available for the collection of platelets by apheresis, all of which separate the platelets using centrifugation. The instrument operation is controlled by a microprocessor that regulates the blood flow rate, the anticoagulant added to the whole blood entering the system, the centrifuge conditions, the component separation, and recombination of the remaining components that are then returned to the donor. The official name of this component is “platelets, pheresis,” but it is usually called single-donor platelet or plateletpheresis concentrate, which is a suspension of platelets in plasma prepared by cytapheresis. For quality control purposes, a unit or bag of plateletpheresis concentrate must contain at least 3 × 1011 platelets in a minimum of 75% of the units tested, but most plateletpheresis concentrates contain approximately 3.5–4 × 1010 platelets. Plateletpheresis usually requires about 1.5 hours and involves processing 4000–5000 mL of the donor’s blood through the blood cell separator. Platelets obtained by plateletpheresis are processed, tested, and labeled in a manner similar to that for whole blood. This includes ABO and Rh typing, and required testing for all transfusion-transmitted diseases. The number of platelets contained in each concentrate is determined, although this information may not necessarily be recorded on the label. Each platelet concentrate has a volume of approximately 200 mL and contains very few (<0.5 mL) red cells so that red cell crossmatching is not necessary. The white blood cell content varies depending upon the instrument and technique used for collection. Apheresis procedures separate leukocytes from platelets, so that platelet concentrates produced by apheresis are usually considered to be leukocyte-reduced and contain fewer than 5 × 106 leukocytes. Platelet storage conditions and duration Platelets are stored at 20–24°C to maintain functional effectiveness for 5 days [13–15]. The variables that affect the quality of platelets during storage include the temperature, anticoagulant preservative solution, storage container, type of agitation, and volume of plasma [15,16]. Citrate phosphate dextrose and citrate phosphate dextrose-adenine solutions are satisfactory platelet preservatives; undisturbed storage is inferior to gentle agitation, and horizontal agitation is preferable to endover-end agitation. The composition, surface area, and size of the storage container influence the ability for carbon dioxide to diffuse out and oxygen to enter the platelet concentrate, and storage containers specifically designed to optimize platelet quality are now used routinely. Maintenance of the pH above 6.0 is the crucial factor indicating satisfactory platelet preservation. This combination of storage container, agitation, preservative solution, temperature, and the use of about 50 mL plasma provides satisfactory preservation of random-donor platelets for up to 7 days. However, several instances of bacterial contamination of platelet concentrates stored for this period were reported [17,18], and the storage time was reduced to the present limit of 5 days.

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation

recommended that the 20,000/μL value traditionally used for prophylactic platelet transfusion could be safely lowered for many patients [35]. Beutler [36] recommended lowering the indications for prophylactic platelet transfusion, and now many physician and hospital guidelines use platelet counts of 10,000/μL or 5000/μL as the indication for prophylactic transfusion to patients with uncomplicated conditions [37]. The guideline of a platelet count of 10,000/μL for prophylactic transfusion applies to the stable, uncomplicated patient. However, many HCT patients are febrile, have severe mucosal bleeding or have other complications that necessitate transfusion at higher platelet counts [19].

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the patient is considered to be refractory. If platelet recovery is used as an indicator, the expected result is about 65% because some of the platelets normally are sequestered in the spleen. It has been suggested that frequent transfusions of a lower dose of platelets will reduce the number of platelets needed [42]. Conversely, when larger numbers are transfused, the time between transfusions is extended [43–45], suggesting that overall fewer platelets or transfusions might be needed. A study of platelet dose is being carried out and results should be available in the next few years. ABO (H) and Rh in platelet transfusion

Treatment of active bleeding Platelet transfusions are not necessary for bleeding patients with a platelet count above 100,000/μL because these patients have a normal bleeding time. With platelet counts below 100,000/μL, the bleeding time is increasingly prolonged [38]. The optimum platelet count to achieve in a bleeding patient is not known. In one study [39] of patients undergoing surgery with platelet counts less than 100,000/μL, prophylactic platelet transfusions were given to those whose platelet count was less than 50,000/μL. Bleeding was similar in patients with levels less than 50,000/ μL who received platelet transfusion compared with those whose platelet count was above 50,000/μL and who did not receive platelet transfusion. The bleeding did not relate to the platelet count but instead to the severity of the surgical procedure. Thus, in actively bleeding patients, platelet transfusion should be considered in those with a platelet count of less than approximately 75,000/μL. An attempt to achieve a level of above 50,000/μL is recommended. If the patient’s platelets are dysfunctional due to causes such as drugs or uremia, the bleeding time may be much longer than would be expected based on the degree of thrombocytopenia. In those situations, the decision to give a platelet transfusion is made on clinical grounds alone. Dose of platelets and outcome of platelet transfusion There is a dose–response effect from platelet transfusion. Within 1 hour after transfusion, the platelet count increases by approximately 10,000/ μL when 1 × 1011 platelets are transfused into a 70 kg patient [40]. Since a transfusion usually contains approximately 3.5–4.0 × 1011 platelets, the platelet count should increase by 35,000–40,000/μL in an average-sized adult. In order to achieve these increases in platelet count, four to six whole-blood-derived platelet concentrates are pooled or one apheresis product is used. For some individuals, the volume of plasma in the final pooled component is too large, and plasma must be removed before transfusion. The loss of platelets during this concentration step is reported to be from 15% to as much as 55% [41]. Thus, concentration or volume reduction of pooled platelets can be carried out successfully, and is often done for small patients or those who are receiving large volumes of blood components or intravenous fluids. The 1-hour post-transfusion platelet count is an excellent predictor of an effective platelet transfusion [40]. If a very accurate determination of the response to platelet transfusion is needed, the 1-hour corrected count increment (CCI) or the percent recovery can be determined. This takes into account the number of platelets transfused and the size of the patient. The CCI is calculated as follows: ⎛ Posttransfusion − pretransfusion⎞ × ( body surface area ) ⎝ platelet count ⎠ CCI = Number of platelets transfused The expected CCI is about 15,000/μL × 1011 platelets transfused per square meter of body surface area. If the CCI is less than 5000–7500,

ABO antigens are intrinsic to the platelet, and some are also adsorbed onto its surface. When 51Cr-labeled group A platelets are transfused to normal group O volunteers, the recovery is reduced [46], and the higher the ABO isoagglutinin titers, the greater the reduction in recovery of transfused platelets. This ABO effect has been substantiated clinically in several studies. There is a reduced recovery when platelets from human leukocyte antigen (HLA)-matched donors are transfused to alloimmunized patients who are ABO incompatible with the donor platelets [47], and blood group O patients may be refractory to platelets transfused from group A donors but not from platelets transfused from group O donors [47]. Two other studies substantiate the concept that if platelets that contain ABH antigens are transfused into patients with circulating antibody directed against those antigens, the intravascular recovery of the transfused platelets is substantially decreased [48,49]. Decreased recovery of transfused platelets also occurs when the incompatibility involves transfusion of ABO antibody directed against the recipient’s ABO antigens (e.g. group O platelets transfused to a group A patient) [49]. This may be due to the formation of circulating immune complexes by soluble ABH substance and ABO antibodies, and can occur with the antigen–antibody complexes from either donor or recipient antibody and donor or recipient ABH substance. Thus, the reduced platelet recovery can occur when the ABO incompatibility is either the “major” or “minor” type. Although ABO incompatibility is associated with reduced post-transfusion platelet recovery, this is not of a magnitude that would contraindicate transfusion of ABO-mismatched but HLAmatched platelets. A separate consideration involving the ABO system and platelet transfusion is the potential administration of large volumes of ABO-incompatible plasma if transfusions involve “minor” incompatibility (e.g. group O platelets transfused to group A patients). This plasma may reduce the survival of the transfused platelets, but another concern is the potential for hemolysis when large amounts of ABO-incompatible antibody are transfused. Most institutions have a policy to avoid or limit the amount of ABO-incompatible plasma transfused in either plasma or platelet products [50]. Limiting the volume of ABO-incompatible plasma that a patient can receive can be in terms of a percentage of the patient’s estimated blood volume or an absolute volume limit. For instance, we limit adults to no more than 1 L of ABO-incompatible plasma per week. It is recognized that a rare platelet unit may have a very high anti-A or anti-B titer, but our approach seems to be satisfactory. As mentioned earlier, the platelet product can be concentrated further by removing some of the plasma. Rh antigens are not present on the platelet surface. However, the few red cells contained in the platelet concentrate can lead to immunization, and thus Rh must be considered in platelet transfusion. In Rh-negative cancer patients, reported rates of immunization to the D antigen range from 0% to 18% [51–54]. Because oncology patients and HCT recipients receive a large number of platelet transfusions, it is usually not possible to provide all Rh-negative platelets for Rh-negative patients. Thus, it is common to provide platelets to these patients without regard

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to Rh type. Rh immune globulin is not administered to prevent alloimmunization. Even when circulating anti-D develops, this antibody does not interfere with the circulation of Rh (D)-positive platelets [55]. If Rh immune globulin is to be administered to prevent alloimmunization, the dose of Rh immune globulin can be determined from the number of units of platelets that the patient receives. For instance, since most therapeutic doses of platelets (e.g. one single-donor unit or four to five pooled random-donor units) contain less than 1 mL red cells, one standard dose of 300 μg of Rh immune globulin is sufficient [52]. Lack of response (refractoriness) to platelet transfusion Many patients do not attain the expected post-transfusion increment in platelet count and are said to be “refractory” to platelet transfusion. In patients who receive large numbers of platelet transfusions, the CCI may decrease for reasons that are not well understood [56]. While these patients may not fit the exact definition of refractoriness, smaller posttransfusion increments in platelet count are not unusual. Refractoriness to platelet transfusion can be caused by factors related to the patient and/or factors related to the platelet concentrate. Factors related to the patient In patients who have HLA or platelet antibodies, such as those with autoimmune thrombocytopenic purpura or patients who are immunized to antigens of the HLA system, survival of circulating platelets is extremely brief, sometimes only a matter of minutes [25]. Splenomegaly also causes sequestration of platelets and a reduced post-transfusion increment [57]. In a careful study of 941 platelet transfusions administered to 133 patients, factors associated with a poor response to platelet transfusion include DIC, amphotericin administration, palpable spleen, presence of HLA antibody, presence of platelet antibody, status post HCT, and fever [58]. It is not clear whether active bleeding is associated with a reduced response to platelet transfusion. Thus, patients can be refractory due to alloimmunization (HLA and/or platelet antibodies) or nonimmune clinical factors. In some patients, refractoriness is clearly associated with the presence of HLA antibodies; however, many of these patients also have one or more clinical factors present which could account at least partially for the refractoriness, and in at least one study [59], nonimmune factors were the major cause of refractoriness. Thus, in practice, it is very difficult to determine whether a patient is refractory due to immune or nonimmune factors, and this affects the strategy used to manage these patients. In the past, the incidence of refractoriness was rather high, but during the 1990s it was only about 15% after the first 8 weeks of therapy [60]. This decrease may be due to changing transfusion practices, described in the section on prevention of alloimmunization, or to more intensive chemotherapy. Factors related to the platelet concentrate ABO incompatibility can reduce the intravascular recovery and survival of transfused platelets. Transfusion of platelets near the end of their storage period can also reduce recovery [61]. Transfusion of similar numbers of apheresis or whole-blood-derived platelets should give similar increments in platelet count. There is no reason to believe that transfusion of whole-blood-derived platelets could give the misleading impression of refractoriness due to poor increments based only on the source of the platelets.

platelets should be attempted. Platelets from HLA-identical siblings, HLA-identical unrelated donors, and even partially matched donors can provide a good post-transfusion response [62–64]. Most blood banks have large files of HLA-typed volunteer donors so that HLA-matched platelets can be provided for many patients. However, most HLAmatched platelets obtained from unrelated donors have some antigens mismatched with the patient. Although the average response to these partially matched transfusions is similar to that from fully matched HLA-identical transfusions, about 30% of HLA-matched transfusions do not provide a satisfactory response [63]. Also, since these patients require frequent platelet transfusions, many donors who are matched with the patient must be available to sustain the patient for days or a few weeks, so that in practice it is often difficult to maintain a patient on HLA-matched platelets. Platelet crossmatching for platelet transfusion. Immune platelet refractoriness is usually due to HLA antibodies, but platelet-specific antibodies may also be involved. Thus, one approach that might be effective in overcoming the 30% failure of HLA-matched transfusions is crossmatching the patient’s serum and potential donor’s cells. The development of this approach was slow for many years because the platelet antibody detection techniques were complex, and because the relative role of platelet-specific versus HLA antibodies has not been clear, the choice of the optimum method for crossmatching was complicated. Several different platelet antibody methods were used, and there was great variability in results, with the positive predictive value ranging from 73% to 100% and the negative predictive value from 52% to 92% in 10 studies summarized by Heal et al. [49]. Although several methods have been reported to be effective, none has been suitable for general use and none has gained wide practical application. During the past several years, a solid phase red cell adherence assay was developed specifically for platelet crossmatching [65,66]. This method has overcome the previous barriers of test complexity and speed of results. In clinical use, the method predicted a successful transfusion outcome in 97% of patients with no clinical factors to cause nonimmune platelet destruction [66]. Successful prediction of transfusion response using the solid-phase red cell adherence assay was confirmed by O’Connell et al. [67] and by Friedberg et al. [68], who, in a separate study of 962 single-donor platelet transfusions to 71 refractory patients, showed that the solid-phase red cell adherence assay was superior to HLA matching. This method is now widely used because of its effectiveness and practicality. In a separate study [69], HLA matching and platelet crossmatching provided similar degrees of successful 1-hour CCIs (40–60%). If only transfusions were used in which four HLA-A,B locus antigens were identical between donor and recipient (A match) or in which only three HLA-A,B locus antigens were identified in the donor, HLA matching was superior to platelet crossmatching. However, Moroff et al. [69] concluded that a similar number of successful transfusions could be obtained by either HLA matching or platelet crossmatching, and this is the general belief currently. Other approaches to the refractory patient. Other strategies that have been attempted but are not usually successful and are not recommended include plasma exchange, treatment of the patient’s plasma with a staphylococcal protein A column, cyclosporine, and intravenous immunoglobulin.

Strategies for managing patients who are refractory to platelet transfusion

A practical approach to the patient who is refractory to platelet transfusion

HLA matching for platelet transfusion. If the use of ABO-matched platelets of approximately 24 hours’ storage fails to produce a satisfactory increment, transfusion of either HLA-matched or crossmatched

The lack of a response to platelet transfusion is often associated with bleeding. These patients are usually quite ill, with problems such as fever, sepsis, DIC or viral infections. Therefore, clinical factors such as

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation

infection that might cause refractoriness should be sought, and, if they are present, appropriate treatment should be initiated. Depending on the patient’s condition and the degree of concern about the platelet count, additional simple steps that can be attempted to improve the response to platelet transfusion include the transfusion of ABO-identical platelets and platelets less than 48 hours old. The techniques for platelet transfusion should be reviewed to be sure the platelets are not being damaged or lost due to improper handling after leaving the blood bank, use of incorrect filters or improper storage conditions. An additional step is to be certain that the platelet concentrates contain an adequate number of platelets, which can be done by checking the blood bank quality control testing results. If questions continue, the platelet count and content of the specific concentrates being used for the patient can be determined. If these measures fail, platelets that are matched to the recipient should be used. This can be accomplished by HLA matching of donor and recipient, or by using platelets that are compatible in a crossmatch. Most HLA-matched platelets obtained from unrelated donors will not be matched for all four of the HLA-A and B locus antigens, and thus there will be some degree of antigen mismatch between the patient and donor. Although the average response to these partially matched transfusions is similar to that from fully matched transfusions, about 30% of HLA-matched transfusions do not provide a satisfactory response [63,64]. Also, since these patients require frequent platelet transfusions, many donors who are matched to the patient must be available to sustain the patient for days or a few weeks. For many patients, this approach may not be practical. An additional or alternative approach is to crossmatch the patient’s serum and potential donor’s cells. As described above, the success rate is similar to that with HLA-matched transfusions. Thus, it seems that either HLA matching or platelet crossmatching can be used to obtain about the same effect. One practical difference is that usually crossmatched platelets can be obtained more rapidly than HLA-matched platelets because crossmatching is done on platelets already collected and available in inventory, whereas HLA-matched donors must be located and scheduled for donation. A suggested practical strategy for dealing with patients who are refractory to platelet transfusion is as follows: 1 Treat any correctable clinical factors present that may cause platelet refractoriness. Until these factors are eliminated, recognize that any of the steps listed below may not be effective. 2 Ensure that the patient is receiving the correct dose of platelets. 3 Give at least one test transfusion of platelets that are not more than 48 hours old. 4 Ensure that platelets being transfused are ABO identical. 5 If these steps have failed and transfusion is urgently needed, give transfusions of either crossmatch-compatible or HLA-matched platelets, whichever is available soonest. Give at least one and preferably two or three such transfusions. 6 Continue to use either crossmatched or HLA-matched platelets until the desired increment is obtained. 7 Determine whether the patient has HLA or platelet-specific antibodies, or both. This precaution may be of value in making future decisions about platelet transfusion if the patient does not respond to HLA-matched or crossmatched platelets. 8 Determine whether the patient is receiving medications such as vancomycin that might cause drug-dependent antiplatelet antibodies. If so, carry out a drug-platelet antibody test. Some patients do not respond to either HLA-matched or crossmatched platelets. Often these are patients in whom engraftment has failed, who have graft-versus-host disease (GVHD), who are septic or who are experiencing other severe complications of cytopenia. One approach is to increase the dose of platelets to two or even three single-donor, or 20

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to 30 random-donor, units per day. While this may enable physicians to feel that they are doing something helpful, usually the patients fail to achieve a substantial increase in platelet count. It is not known whether the transfusions are helpful despite the lack of increase in circulating platelets. Prevention of alloimmunization and platelet refractoriness Because of the difficulty in managing patients who are refractory to platelet transfusion, there is considerable interest in preventing alloimmunization. Immunization may be caused by pregnancy but is also due to the leukocytes contained in the platelet and red cell transfusions [9,70]. Although the exact mechanism of alloimmunization has not been defined, it appears that intact viable leukocytes present HLA class I and II antigens for processing [71]. Thus, strategies to modify the transfusion products have been adopted. In general, these strategies have involved limiting the number of donor exposures, removing the leukocytes or treating the components to render the leukocytes nonimmunogenic. There is a very substantial body of data that establishes that leukocyte reduction reduces the incidence of alloimmunization and delays and/or reduces the onset of refractoriness [72–74]. Clinical trials are difficult to accomplish perfectly, and many of these studies have one or another shortcoming; however, there is little disagreement that leukocyte reduction is effective. In a study of more than 500 newly diagnosed patients with acute leukemia [60], the use of leukocyte-reduced red cells and platelet concentrates reduced the incidence of alloimmunization, and no difference was found between pooled random-donor and single-donor platelet concentrates. This confirms that the important factor is the reduction of the leukocyte exposure rather than the number of different donors. Filtration of the components at the time they are produced in the laboratory is more effective than bedside filtration for prevention of alloimmunization because the leukoreduction can be standardized and controlled better [75]. Ultraviolet (UV) radiation inhibits the ability of lymphocytes to either proliferate or stimulate in a mixed lymphocyte culture, but UVB light does not interfere with platelet function in vitro or in vivo at doses that will abrogate lymphocyte function. In a large clinical trial, UVB irradiated platelets reduced the incidence of alloimmunization to the same extent as leukoreduced platelets [60]. Thus, although UVB light appears to be effective in preventing alloimmunization, it is not presently used, and no systems are licensed by the Food and Drug Administration (FDA) for routine blood bank use. Role of thrombopoietin in platelet transfusion It was hoped that the long-sought platelet growth factor thrombopoietin (TPO), discovered more than a decade ago, would reduce the need for platelet transfusions. TPO could influence transfusion therapy in HCT patients in at least two ways. It could be given to the patient in hope of speeding the recovery of platelet production and shortening the period of thrombocytopenia, thus reducing bleeding complications and the need for platelet transfusions. Alternatively, TPO could be given to platelet donors to elevate the donor’s platelet count, and increase the number of platelets collected and transfused, in hope of increasing the time between transfusions and reducing the number of donor exposures that the patient received. In patients with advanced cancer, TPO increased the platelet count in a dose-dependent manner, elevated the nadir platelet count, shortened the period of thrombocytopenia, and decreased platelet transfusion requirements [76]. However, this observation has had little impact on platelet use because these patients did not receive many platelet transfusions. The response to TPO in HCT patients receiving myeloablative therapy is even less impressive. TPO therapy has not shorted the dura-

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tion of thrombocytopenia or decreased the need for platelet transfusions [76], possibly because endogenous TPO levels are already very high in these patients, TPO receptors may be downgraded or TPO may actually inhibit release of mature platelets from megakaryocytes. Because the many pluripotent functions of TPO have not been well studied, the reasons for its role or lack thereof in the HCT setting are not completely understood. When a TPO-like agent was administered to normal plateletpheresis donors, the level of circulating platelets increased, and thus the yield of platelets following plateletpheresis increased two to four times. When these high-dose concentrates were used as a single transfusion, they provided a larger increase in platelet count than present transfusions, prolonged the interval between transfusions, and reduced the number of donor exposures [43]. However, in separate studies, TPO inhibitors occurred in normal subjects, and development of that agent has been discontinued.

Granulocyte concentrates and granulocyte transfusion A series of studies in the 1970s established that granulocyte transfusions provided improved survival in patients with documented Gram-negative sepsis who remained granulocytopenic for at least 10 days (reviewed in [77]). Most clinical trials of granulocyte transfusion involve patients with Gram-negative bacteremia [77], there is little information regarding other organisms, and patients with fever of unknown origin do not experience improvement as a result of granulocyte transfusion [77]. Other than bacteremia, the site of infection does not seem to correlate with response to granulocyte transfusion [77]. Prophylactic granulocyte transfusions using doses available in the 1970s were not helpful [78]. A major reason for the limited effectiveness of granulocyte transfusions was thought to be the small dose of cells [79]. There is renewed interest in granulocyte transfusion because hematopoietic growth factors such as granulocyte-colony stimulating factor (G-CSF) can be used to stimulate granulocyte production in normal blood donors, increasing the level of circulating granulocytes and the yield of granulocytes [79]. The level of circulating granulocytes can be increased to about 30,000/μL with G-CSF alone, and, by combining the use of corticosteroids and G-CSF, the level of circulating granulocytes can be increased to approximately 40,000/μL [80]. Leukapheresis of these donors results in a dose of up to 7 or 8 × 1010 granulocytes in some cases [79]. G-CSF-mobilized granulocytes have normal in vitro function and migrate to sites of infection [79]. These doses of granulocytes far exceed those used in the studies of the 1970s and may make transfusions clinically effective. There have been several reports of the clinical use of G-CSF-mobilized granulocytes, but studies have been small or uncontrolled, involved different definitions or patient eligiblity or prophylactic transfusions [81], or they were in children where all doses were very high due to the patient’s smaller size (reviewed in [77]). Thus, it is not known whether the large doses of granulocytes that can be provided today can be clinially effective. A large, controlled multicenter clinical study has been initiated by the National Heart, Lung, and Blood Institute-funded Transfusion Medicine/Hemostasis Clinical Trials Network to determine whether granulocyte transfusions are clinically helpful.

Transfusion strategies in HCT Pretransplantation For pretransplant transfusion strategy, patients can be thought of in three groups: immunocompetent but not requiring transfusion, immunocompetent and requiring transfusion, and immunocompromised. An example

of the first group is patients with inborn errors of metabolism, who rarely require transfusion before HCT. The other two groups of patients have a variable degree of immunocompetence and may become alloimmunized to leukocytes or platelets following transfusion. Alloimmunization may cause transfusion reactions but, more importantly, may interfere with engraftment in some patients. The presence of HLA antibodies is associated with marrow graft rejection [82]. Pretransplant transfusions are associated with increased graft failure rates [83,84]. For immunocompetent patients such as those with aplastic anemia, hemoglobinopathies or myelodysplastic syndrome who require pretransplant transfusions, efforts should be made to prevent alloimmunization. Patients with aplastic anemia should be evaluated quickly, and HCT, if indicated, should be performed as soon as possible to minimize the number of pretransplant transfusions. Many of these patients can be managed safely when prophylactic platelets are given only when the platelet count is less than 5,000/μL [85]. For patients such as those with hemoglobinopathies or myelodysplastic syndrome who require red cell or platelet transfusions before HCT, the following are recommended: 1 Red cells should be depleted of leukocytes. 2 Platelet concentrates should be depleted of leukocytes. This can be done by filtration or by certain apheresis collection procedures. 3 The use of single-donor instead of pooled random-donor platelet concentrates should be considered. However, the largest study of this issue did not show a difference in the rate of alloimmunization between single-donor and pooled random-donor platelet concentrates [60]. 4 Family members should not be used as blood or blood component donors (before transplant) because of the risk of alloimmunization causing an unsuccessful marrow graft [85]. Patients with malignancies who will undergo HCT usually have received multiple transfusions during the initial chemotherapy of their underlying disease. However, the effects of this on subsequent marrow engraftment are not as severe as for patients with aplastic anemia because standard chemotherapy of their malignancy is extremely immunosuppressive. Thus, transfusion therapy for these patients can be that necessitated by their chemotherapy. The increasing use of reduced-intensity preparative regimens may make engraftment more problematic in heavily alloimmunized patients. Therefore, red cells and platelets should be leukocyte reduced in order to minimize the possibility of alloimmunization. Transfusions from family members probably should be avoided [85]. Although one study showed no increase in graft failure in patients with leukemia who received transfusions from family donors [86], this is not generally recommended. Post-transplantation Because of the severe immunosuppression caused by the pretransplant preparative regimen, fatal GVHD can occur as a result of transfusion of viable lymphocytes that are present in blood components. Transfusionrelated GVHD can be prevented by irradiating all blood components with at least 2,500 cGy (see below). About 10–25 days elapse between HCT and marrow engraftment, although this period is becoming more variable as different sources of hematopoietic cells are used. The return of production of different blood cell lines varies, so that although the duration of transfusion therapy may range from 2 to 6 weeks, the need for different components varies with different types of transplant. Almost all patients require platelet and red cell transfusions, but since HCT does not usually interfere with the production of coagulation factors, transfusion of FFP and cryoprecipitate is necessary only if coagulopathy develops. Although HCT patients are severely neutropenic, granulocyte transfusions are usually not necessary, although some centers have begun using high-dose granulocyte concentrates obtained from donors stimulated with G-CSF.

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation

Because of their complex situation, HCT patients may place a major demand on the blood bank for blood components, especially platelets. The red cells used can be routine red cell components, although leukocyte-reduced red cells are recommended to minimize the likelihood of inducing alloimmunization. Another unique consideration in the transfusion management of HCT patients is the source of platelets. Either whole blood-derived or single (apheresis)-donor platelets can be utilized. Platelets should be leukocyte reduced to prevent alloimmunization. Results of studies of leukocyte-reduced platelet concentrates in situations other than HCT have indicated that this strategy is effective [72–74]. One large study [60] found that leukocyte reduction decreased platelet refractoriness even in patients who were already alloimmunized, but leukocytereduced platelets may not prevent secondary alloimmunization. Although these studies of alloimmunization have not been carried out in HCT patients, the effectiveness of leukocyte reduction in other situations indicates that leukocyte-reduced platelets should be used in HCT patients. If patients become refractory to platelet transfusion, HLA-matched unrelated or family donor platelets can be used (see above). Platelets can be obtained from the marrow donor if the patient is refractory and experiencing serious bleeding problems, but this decision requires considerable thought because of ethical considerations regarding the donor. Transfusion prophylaxis for invasive procedures Most patients will undergo placement of a central venous access line, and many may undergo other invasive diagnostic procedures such as lumbar puncture, bronchoscopy, thoracentesis, bone marrow aspiration or even liver biopsy. At the time of these procedures, the patient may have abnormal coagulation test results or be thrombocytopenic. Unfortunately, there are insufficient clinical data to determine the value of prophylactic transfusion of FFP to correct coagulopathy [87] or platelet transfusion to increase the platelet count above an arbitrary level [88,89]. However, based on an extensive summary of experience, but in the absence of firm data, the American Society of Clinical Oncology has recommended that a platelet count of 40,000–50,000/μL “is sufficient to perform major invasive procedures” [89]. Bone marrow aspiration and biopsy can be done safely at platelet counts of less than 20,000/μL, but “there are sparse data about the safety of other invasive procedures at much lower platelet count levels” [89].

Prevention of transfusion-transmitted CMV infection CMV disease is an important source of post-transplant mortality (see Chapter 90). Most CMV infections in HCT patients occur in those with CMV antibody, and probably are due to reactivation of virus from a previous infection, not to acquisition of a new strain. However, in CMV antibody-negative patients who receive unscreened blood products, the risk of developing CMV infection is high. The likelihood of these patients acquiring CMV infection is almost eliminated by the use of CMV antibody-negative blood components [90,91]. Therefore, it is now customary to provide these blood components to CMV antibodynegative HCT recipients whose marrow donor is CMV antibody negative. Because a substantial portion of blood donors are CMV antibody positive, providing CMV antibody-negative blood components is sometimes difficult, especially as the indications for CMV-free blood components have increased. Although infectious viral particles cannot be recovered from normal donors previously infected with CMV who have antibodies to CMV, blood from these donors can transmit CMV. Because it is presumed that leukocytes are the reservoir of CMV in these asymptomatic blood donors, prevention of transfusion-transmitted CMV has been attempted using leukocyte-reduced components.

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Two small studies [92,93] and one large controlled trial [91] have established that leukocyte-reduced components are as effective as antibody-negative components in preventing transfusion-transmitted CMV. With both CMV antibody-negative and leukocyte-reduced blood, there is a small residual risk of CMV transmission of about 2–3%. In the large study [91], there was no difference in the CMV seroconversion rate between patients who received leukocyte-reduced components versus those who received CMV antibody-negative components. However, the few cases of clinical CMV infection occurred in the leukocyte-reduced group, compared with only seroconversion in the CMV antibody-negative group. This has caused some transplant physicians to prefer CMV antibody-negative blood components when they can be obtained. In most centers, leukocyte-reduced and antibody-negative components are considered equivalent and used interchangeably depending on the logistical considerations of each transfusion service. The use of CMV antibody-negative blood components in potential HCT patients who are CMV antibody negative has been suggested. This would prevent these patients from becoming infected with CMV as a result of transfusions received earlier in their disease and before entering an HCT program. This practice is gaining in popularity, and the conversion to routine use of leukocyte-reduced blood components makes it more practical. Other approaches that have been attempted for the prevention of CMV infection include the use of CMV intravenous immunoglobulin or ordinary intravenous immunoglobulin, although the increasing use of leukocyte-reduced blood products has obviated these other strategies.

Prevention of transfusion-associated GVHD GVHD from blood transfusion was originally identified in immunodeficient children. It is now clear that GVHD can occur in a wide variety of immunocompromised and even some immunocompetent patients, but the more common situation in which GVHD occurs is HCT. Following HCT, GVHD is caused by the immunocompetent marrow donor cells, but viable lymphocytes contained in blood components can cause transfusion-associated GVHD in susceptible patients. Irradiation of blood components Warning: Hematopoietic stem cells are sensitive to irradiation. Marrow, peripheral blood, or cord blood stem cells that are intended for transplantation should never be exposed to irradiation. To prevent transfusion-associated GVHD, blood components are subjected to irradiation, which interferes with the ability of lymphocytes to proliferate. Gamma irradiation or X-rays damage the lymphocytes by forming electrically charged particles or ions that alter DNA, making lymphocytes unable to proliferate [94]. UV irradiation is not an effective method of damaging lymphocytes to prevent GVHD. Blood banks that provide large numbers of irradiated products usually irradiate with a dedicated instrument. These instruments use caesium-137 or cobalt-60 as the source of the radioactivity. Recently, relatively simple X-ray devices have been produced for use in the blood bank. Gamma irradiation at a dose of only 500 cGy will abolish lymphocyte proliferation in mixed lymphocyte culture [95], and doses up to 5000 cGy reduce the incorporation of carbon-14-thymidine into mitogen-stimulated lymphocytes by 85.0–98.5% [96]. Red cell survival in vivo and certain in vitro assays is normal after up to 10,000 cGy [97]. Granulocyte chemotaxis may be slightly reduced by even 500 cGy, but this defect does not become significant until above 10,000 cGy [96]. Very high doses such as 40,000 cGy are required to interfere with phagocytosis and microbial killing. In vitro platelet function studies have generally been normal following up to a 5000 cGy dose [95]. Studies involving

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lower doses of irradiation such as 2500 or 3000 cGy have shown normal platelet in vivo survival [98] and post-transfusion increments [99]. Thus, it appears that doses of irradiation up to 5000 cGy do not have an adverse effect on red cells, platelets or granulocytes. One difficulty in selecting an irradiation dose is the lack of a definitive in vitro assay to establish a clinically effective dose. Because of the occurrence of transfusion-associated GVHD in several individuals who received blood components supposedly irradiated with at least 1500 cGy [100–102], a dose of 3000 cGy was recommended [100]. During the past few years, the limiting dilution assay has been proposed as a good indicator of the effects of irradiation on lymphocytes because this assay detects a 5 logl0 reduction in viable T cells, compared with a 1–2 log10 reduction in the mixed lymphocyte culture [94]. Studies using the limiting dilution assay showed that 2500 cGy completely eliminated T-lymphocyte growth, and, based on this experience, the FDA has recommended a minimum dose of 2500 cGy. Important practical factors are the configuration of the blood containers in the irradiated field, the distribution of irradiation within the field, and quality control methods to assure that the desired dose is actually being administered [94].

Autologous bone marrow transplantation Although there may be some differences in the preparative regimen for autologous compared with allogeneic (HCT), patients undergoing autologous bone marrow transplantation are severely immunocompromised for several weeks. The use of irradiated blood components has been adopted for autologous HCT without clinical or laboratory study. Noncellular blood components Transfusion-associated GVHD has occurred in patients with congenital immune deficiency following transfusion of fresh liquid plasma [100,105,106] but has not been reported to be caused by previously frozen components such as FFP or cryoprecipitate. Plasma components contain fragments of leukocytes but few, if any, viable lymphocytes. They would not be expected to cause transfusion-associated GVHD. Although irradiation of FFP and cryoprecipitate is probably not necessary, many blood banks do irradiate these components to avoid clerical errors in which a cellular blood component might not be irradiated when necessary.

Transfusion therapy for HCT donors Storage of irradiated components As the use of irradiated blood components has increased, interest has developed in irradiating the components after collection and storing them for use days or weeks later. Doses of 2000 or 3000 cGy to units of red cells result in potassium levels that are two and three times normal after storage for 4–5 days [103]. This leakage suggests damage to the red cell membrane or the sodium–potassium pump. Some blood banks wash red cells that have been stored for several days after irradiation. However, in a thorough review, Strauss concluded that washing is not necessary for most clinical situations [104]. Based on these studies, red cells can be stored for only 28 days after irradiation. Because there is no reduction in platelet recovery and survival when previously irradiated platelets are stored [98], platelets can be irradiated and stored for the usual 5 days.

Quality control of irradiation Quality control of blood irradiators is extremely important to ensure that the components receive the expected dose. Moroff et al. [94] have proposed the following as appropriate quality control measures for blood irradiation: 1 the use of qualitative indicators to confirm that irradiation has been performed as intended. Labels can be placed on the blood container that change when exposed to irradiation, indicating that the unit has been successfully irradiated; 2 periodic measurement over the delivered dose using appropriate dosemetric techniques; 3 periodic surveys to detect isotope leakage; 4 daily confirmation of timer accuracy.

Leukocyte reduction to prevent GVHD Because blood filters are very effective in removing leukocytes, the question has arisen as to whether blood filtration might be an alternative to irradiation for prevention of GVHD. Although, on average, filters produce components that contain fewer than 5 × 106 leukocytes, a small proportion of units may contain considerably more leukocytes. Thus, filtration is not an acceptable approach to prevention of transfusionassociated GVHD.

Patients (autologous donors) or normal allogeneic donors of marrow may require some replacement of red cells. The volume of replacement needed can be predicted from the size of the patient and the donor [107]. It is important to anticipate whether red cell transfusion of the normal allogeneic donor will be necessary and, if so, to take steps to avoid exposure to allogeneic red cells. If red cell replacement is contemplated, the normal donor should provide autologous red cells for transfusion during marrow donation [107]. Usually, two units of red cells suffice. Erythropoietin has been used to increase erythropoiesis in autologous blood donors before elective surgery, and this step may be a helpful strategy for allogeneic marrow donors, although there are no definitive data on this. Various strategies using chemotherapy and/or hematopoietic growth factors are being used to mobilize hematopoietic cells for transplantation from either patients or normal allogeneic donors. G-CSF can be administered to normal allogeneic donors to increase the level of circulating CD34+ cells, and leukapheresis used to obtain an adequate dose of cells for transplantation. Thus, HCT can be accomplished without subjecting these donors to general anesthesia, which is necessary for marrow donation. Donation of autologous peripheral blood hematopoietic cells by apheresis (see Chapters 37, 38, and 41) involves much less red cell loss than marrow donation, and red cell replacement is not necessary for healthy allogeneic blood stem cell donors. Although these donors do not have transfusion requirements, other donor-related issues that must be considered include the effect of the G-CSF on the donors, the methods of vascular access, the optimum apheresis procedures, and the effect of the donation of hematopoietic cells on the donor’s hematologic status. These issues are discussed more fully in Chapters 37, 38, and 41. Red cell transfusion may be necessary for autologous donors if the patient is anemic from the disease. Those patients/donors should receive leukocyte-reduced red cells, and the indications for transfusion are those described earlier in this chapter. Complications of blood transfusion The complications of transfusion can be categorized as immunologic and nonimmunologic. Approximately 1–3% of transfusions result in an adverse effect during or shortly after the transfusion, but the incidence of longer-term adverse effects is difficult to establish. As many as 20% of transfusions may result in some kind of adverse effect, but these

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation

effects are considered serious in only about 0.5% of transfusions [108]. The fatality rate immediately surrounding transfusion is estimated to be about 1 per 100,000 patients transfused, or about 35 deaths annually in the United States [109]. Hemolytic transfusion reactions Hemolysis can occur as a result of HCT that is incompatible for ABO or other red cell antigens (see Chapter 81). While these create complex transfusion situations, they are not typical transfusion complications and will not be considered here. Most hemolytic transfusion reactions are due to ABO incompatibility, and a common cause of transfusion fatalities reported to the FDA is ABO-incompatible transfusion. It is estimated that a hemolytic transfusion reaction occurs following about 1 in 150,000 units of red cells transfused [110]. About half of these are of no clinical consequence, so the rate of clinically apparent reactions is about 1 in 25,000–35,000. Other causes of red cell hemolysis in transfusion recipients, such as improper storage or techniques of administering the blood, are not immunologic. ABO-incompatible hemolytic transfusion reactions are very dangerous because the patient has preformed ABO antibodies that often are immunoglobulin M (IgM) and bind complement, causing activation of the complement system with associated systemic manifestations and leading to red cell lysis. In addition, cytokines cause important biologic effects in hemolytic transfusion reactions. There is substantial production of interleukin-1 (IL-1), IL-8, monocyte chemoattractant protein-1, and tumor necrosis factor in response to red cell incompatibility [110], and these act like a final common pathway for both the IgM and IgG red cell-incompatible systems. The nature and severity of the symptoms do not correlate with the severity or ultimate outcome of a hemolytic transfusion reaction. Some patients may experience a severe reaction after only 20 mL of ABOincompatible red cells, whereas others may tolerate an entire unit without signs or symptoms. The reaction may begin almost immediately upon beginning the transfusion or up to several hours after transfusion. The signs and symptoms that may accompany a hemolytic transfusion reaction are due to complement activation and release of cytokines, and include fever, chills, flushing, low back pain, hypotension, dyspnea, abdominal pain, vomiting, diarrhea, tachycardia, chest pain or unexpected bleeding. Non-ABO antibodies can also cause a hemolytic transfusion reaction, but usually these antibodies are IgG immunoglobulins and cause extravascular rather the intravascular hemolysis. Extravascular hemolysis is usually a slower process with red cells removed by phagocytes and little or no complement activation. Usually, there are few, if any, symptoms. A delayed hemolytic transfusion reaction can occur in a patient in whom no red cell antibody was detected at the time of compatibility testing but who experiences accelerated destruction of the transfused red cells after an interval during which reactivation of a previous immune response to the transfused red cells occurs. The interval after transfusion may be as little as 24 hours or up to about 1 week. A delayed hemolytic transfusion reaction may be symptomatic or asymptomatic. The most common sign is a decrease in hemoglobin after transfusion, and is the way most delayed hemolytic transfusion reactions are identified. Such a reaction can appear to be unexplained blood loss following transfusion. In this situation, repeat red cell antibody detection testing should be done, which will usually reveal a red cell antibody that was not present before transfusion. Febrile nonhemolytic transfusion reactions These reactions occur in association with about 0.5–1.0% of red cell transfusions and up to 20% of platelet transfusions [111]. Traditionally,

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it was believed that they are due to leukocyte antibodies present in the patients that react with leukocytes present in the transfused components. The severity of the reaction is directly related to the number of leukocytes in the blood component. Febrile leukocyte reactions can be prevented by removing leukocytes from the blood components. However, this traditional concept is now out of date, and cytokines probably play a major role (see the later discussion of reactions to transfused platelets). In red cell transfusions, the relative contribution of leukocyte antibody–antigen reactions versus cytokines released from contaminating leukocytes during storage is not known. Both mechanisms are probably important, and both can be prevented by leukocyte reduction of the red cells shortly after they are collected, which is known as prestorage leukocyte reduction. Previously, the use of leukocyte-reduced blood components was not recommended routinely after a first febrile nonhemolytic transfusion reaction because there is only a small likelihood that a patient will experience a subsequent reaction. As the use of leukocytereduced red cells and platelets has become common for HCT patients, the issue of when to convert to leukocyte-reduced blood has become moot. The increased use of leukocyte-reduced blood should decrease febrile nonhemolytic transfusion reactions, and this has been documented with the use of leukocyte-reduced platelets [111]. Patients are often premedicated with antipyretics and antihistamines to prevent or modify febrile nonhemolytic transfusion reactions. Antipyretics do seem to be effective in modifying the febrile response to pyrogens in structured studies. Premedication is often not effective in preventing chills and fever from platelet transfusions. There is no evidence that antihistamine modifies febrile nonhemolytic transfusion reactions in platelet recipients [112]. Thus, while antipyretics and antihistamines can be used, expectations should be modest, and the major benefit is probably from the antipyretic [113]. Allergic and anaphylactic reactions Allergic reactions are thought to be due to proteins in donor plasma to which the recipient was previously sensitized. Recipient mast cells and basophils containing IgE antibody become activated and release complement components, cytokines or other vasoactive substances that cause the reaction. Reactions due to this mechanism may range from mild and involve hives only to severe anaphylaxis with laryngospasm and shock. Allergic reactions that involve hives only with no other symptoms are probably the most frequent kind of reaction, occurring after 1–2% of transfusions. These reactions are the only situation in which the transfusion is stopped and the patient can be given an antihistamine and the transfusion can be restarted after 15–30 minutes. Patients who are IgA deficient and have anti-IgA antibodies may experience an anaphylactic reaction if they receive blood components that contain IgA [114]. The treatment is the same as for any anaphylactic reaction. The reactions can be prevented by using red cells or platelet concentrates, washed to remove plasma IgA, and using plasma components prepared from IgA-deficient donors. Although it is a complex process, IgA-deficient patients can be managed successfully while undergoing HCT [115]. Pulmonary reactions: transfusion-related acute lung injury Following transfusion, a very severe type of transfusion reaction is the acute, sometimes fatal, pulmonary reaction that has been termed transfusion-related acute lung injury (TRALI). TRALI is a clinical syndrome, not a single disease [116,117]. It is defined as new acute lung injury occurring within 6 hours of transfusion and including hypoxemia and chest X-ray indicative of alveolar or interstitial disease but without circulatory overload [116,117].

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Patients develop acute respiratory distress, hypoxemia, diffuse pulmonary infiltrates on X-ray and the general clinical presentation of noncardiogenic pulmonary edema. Fever and hypotension may also occur, and symptoms begin less than 6 hours after the transfusion is initiated. The incidence of TRALI is not known because it is a clinical diagnosis that has not had a clear specific definition in the past. TRALI has been estimated to occur once in 432,000–557,000 transfusions depending on the specific blood component [116]. There are two main hypotheses of the etiology of TRALI. Leukocyte (HLA or granulocyte-specific) antibodies in donor plasma have been thought to be the inciting cause of TRALI, being found in almost 90% of cases. An alternative hypothesis is that a phenomenon such as platelet activating factor or cytokines that accumulate in blood during storage cause general activation of neutrophils. Then a second insult such as a leukocyte antibody antigen reaction leads to pulmonary endothelial adherence and damage. It has been suggested that TRALI is more common than presently believed and that, to avoid it, plasma from multiparous donors should not be used. The American Red Cross now produces plasma products for transfusion only from male donors. Apheresis platelets are still produced from both male and female donors. The prompt recognition that pulmonary symptoms occurring during or shortly after transfusion could be due to TRALI allows prompt initiation of respiratory supportive therapy. This intervention is important because TRALI is a transient phenomenon that clears usually within 72 hours.

the large dose of granulocytes being administered to infected neutropenic patients, or to cytokines contained in the granulocyte concentrate. Since granulocyte concentrates do not undergo a leukocyte crossmatch, leukocyte incompatibility may be present in many granulocyte transfusions and may also account for some reactions. At one time, it was believed that granulocytes transfused in association with amphotericin infusion resulted in severe pulmonary reactions with sequestration of granulocytes in the lungs. Subsequent experience has not supported this. With the large number of intravenous medications these patients receive and the importance of transfusing granulocytes as soon as possible, it is often not practical to space the granulocyte transfusion several hours apart from amphotericin or other similar infusions. Thus, a several hour gap between amphotericin and granulocytes is suggested but not absolutely necessary. Other immunologic complications of transfusion As a result of exposure to blood, patients may form antibodies to red cells, to lymphocyte, granulocyte or platelet surface antigens, or to plasma proteins. The likelihood of antibody formation depends on the immunogenicity of the antigen and the ability of the individual to mount an antibody response. Each kind of antibody can cause a particular clinical problem later if the patient requires subsequent transfusions, organ or tissue grafts, or becomes pregnant. Bacterial contamination of units

Reactions to platelet transfusions Patients with platelet or HLA antibodies may have febrile nonhemolytic reactions, probably due to leukocytes contained in the platelet concentrates. The reactions usually involve chills and fever, but platelets may be trapped in the pulmonary capillaries, causing dyspnea and pulmonary edema. These febrile nonhemolytic reactions occur following 5–30% of platelet transfusions [117]. The reactions have been thought to be due to antibodies to leukocytes and have been considered similar to febrile nonhemolytic reactions from units of red cells. Because these reactions sometimes occur in nontransfused males, are more common when stored platelets are used, and are more related to the plasma than the platelets in the platelet concentrates, it has become clear that bioactive substance other than leukocyte antibodies might be involved [109]. Leukocytes in the platelet concentrates produce cytokines during storage, and platelet transfusion reactions are correlated with the concentration of IL-1 and IL-6 in the plasma. A landmark study [117] established that many (if not most) reactions to platelet transfusion are due to cytokines, not antigen–antibody reactions. Removal of the leukocytes soon after collection of the blood prevents the accumulation of cytokines in the platelet concentrate and avoids platelet transfusion reactions [75,118]. Removal of leukocytes by bedside filtration at the time of transfusion does not decrease the incidence of platelet transfusion reactions [119] because cytokines will have accumulated from contaminating leukocytes during storage. Thus, platelet concentrates should be depleted of leukocytes either as part of the apheresis collection procedure or by filtration at the time of production and not later, at the time of transfusion. Allergic reactions can also occur with platelet transfusions. In one study, the IL-6 levels in platelet concentrates were correlated with allergic reactions [120], suggesting that cytokines might be involved in allergic as well as febrile reactions. Reactions to granulocyte transfusions Transfusion reactions are common following granulocyte transfusions. Many of these reactions are probably due to the physiologic activity of

Bacterial contamination continues to be an important complication of blood transfusion [121]. Bacterial contamination probably occurs most commonly from skin flora at the venepuncture site. Other possibilities are contaminated bags or solutions, contamination of ports during waterbath thawing of frozen products or during platelet pooling, or asymptomatic bacteremia in the donor. The extent of the problem is difficult to ascertain, but a positive bacterial culture has been reported in 0.3– 0.4% of units of red cells and platelets [122], although most of these units do not cause a clinical problem. The two general types of problem are bacterial contamination of platelet concentrates stored at room temperature and transmission of bacteria, especially Yersinia enterocolitica, from red cells stored at refrigerator temperatures [121]. The American Association of Blood Banks now has a standard that requires blood banks to utilize a method of local choice to minimize the possibility of bacterial contamination. The approaches to prevent transfusion of contaminated blood components include Gram staining, routine leukocyte depletion, the Limulus lysate assay for endotoxin, automated culture systems, antigen detection systems, immunologic methods, nucleic acid probes, the visual identification of infected units due to their different color, or diversion of the first 30 mL of blood to prevent skin contaminants from entering the unit of blood. None of these methods is ideal. The most commonly used method is a modification of blood culture systems used for patient blood cultures. One observation from this large-scale culturing of millions of blood units is that bacterial contamination may be less than previously believed [123–125]. Although rates were lower than previous reports, these strategies have decreased the likelihood of transfusing a contaminated unit. Unfortunately, although some bacterial contaminated units were identified and not transfused, other contaminated units were not detected and resulted in septic transfusion reactions [123]. Thus, while the problem of contaminated blood products, especially platelets, has been reduced, this potential serious adverse effect has not been eliminated. Because of the reduction in transmission of viral diseases that has occurred in the last decade, bacterial contamination of blood components is now potentially the most serious complication of transfusion, especially in the immunocompromised recipient.

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation Table 82.3 Strategies to reduce transfusion-transmitted disease Improve donor selection Improve transmissible disease testing Reduce donor exposure Autologous blood Directed-donor programs Limited-donor programs Decrease blood use Changed indications for transfusion Pharmacologic stimulation or substitution Modification of the blood component Viral and bacterial inactivation Blood substitutes

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Table 82.4 Estimated risk of transfusion-transmitted infections in the United States* United States

Human immunodeficiency virus Hepatitis C virus Hepatitis B virus Bacteria

Risk donations

Estimated number of cases annually†

2,135,000 935,000 205,000 75,000

5.6 12.8 58.5 160

* Includes nucleic acid amplification technology. † Based on 12,000,000 units of whole blood collected annually in the United States. Reproduced from Stramer and Chambers [133].

Nonimmunologic complications of blood transfusion Nonimmunologic complications of transfusion include hypothermia from transfusion of large volumes of red cells that have been stored in the refrigerator, citrate toxicity that may manifest itself as hypocalcemia, and a bleeding tendency since red cells suspended in the additive preservative solution do not provide coagulation factor replacement. Proper management of fluid balance can avoid circulatory overload. Iron overload is a complication of patients who receive many transfusions, such as patients with hemoglobinopathies. It is a problem in HCT for patients with thalassemia major and may require an iron removal program following HCT (see Chapter 73) [126,127] . Air or particulate emboli that were a problem in the early days of transfusion no longer occur if proper techniques are used to administer the blood. Transfusion-transmitted diseases The acquired immune deficiency syndrome (AIDS) epidemic has greatly increased the fear of transfusion-transmitted diseases. As a result, there have been great changes in the nature of transfusion practice and the regulation of blood banks by the FDA. The risks of transfusion can be reduced by improving donor selection, improving transmissible disease testing, reducing the number of donor exposures, and reducing blood use (Table 82.3). Physicians have become much more conservative in the use of blood by changing the indications for transfusion and by using pharmacologic substitutes. The number of donor exposures has been reduced by using autologous blood and directed-donor and limiteddonor programs. Donor eligibility criteria have been changed to reflect the understanding of behavior that places potential donors at risk of transmitting disease. The number of donor screening questions has increased by about 30%, and the nature of the questions has changed to become quite specific. These strategies have been remarkably successful. For instance, changes in donor selection resulted in a 90% decrease in the human immunodeficiency virus (HIV) infectivity of the blood supply in the San Francisco Bay area even before the introduction of the anti-HIV test [128]. Presently, a complex process is used to obtain the safest possible donors. Laboratory testing of donated blood has also greatly expanded (see Table 82.5). Starting with the introduction of the test for HIV, the number of routine infectious disease screening tests done on each unit of blood increased from two to nine. These steps were extremely effective in reducing the risks of blood transfusion. Screening tests for HIV and hepatitis C virus detect antibodies to these viruses and, thus, there is a “window phase” between exposure to the virus and the development of a positive screening test. During this window phase, the blood can be infectious. The most recent step to improve blood safety is the adoption

of nucleic acid amplification tests to detect the actual infectious agent rather than antibodies to the agent [129–131]. All blood collected in the United States and most developed coutries is presently being screened for HIV and hepatitis C virus using nucleic acid amplification. This testing strategy has virtually eliminated the window phase of blood donor infectivity. Present estimates of disease transmission [132–133] are shown in Table 82.4. A brief review of transfusion-transmitted diseases is presented below. Syphilis Transmission of syphilis by blood transfusion was common in the early days of blood transfusion but now it is extremely rare, the last case in the United States being reported in 1966 [134]. Syphilis testing is still required of all donated blood despite the fact that the test is almost always negative when circulating spirochetes are present. In the early days of the AIDS epidemic, it was believed that testing for syphilis might be a surrogate to identify sexually promiscuous donors who would be at increased risk for transmitting HIV. This assumption has not proved to be correct, and syphilis testing today probably does not contribute to blood safety, although it has not been eliminated by the FDA [135,136]. Hepatitis Post-transfusion hepatitis is the most common disease transmitted by blood transfusion and is a major public health problem. It can be caused by hepatitis A, B, and C viruses, CMV, Epstein–Barr virus (EBV), and possibly other viruses. The term “non-A, non-B, non-C” is sometimes used to refer to hepatitis due to none of these three agents. It is difficult to provide a single estimate of the frequency of occurrence of posttransfusion hepatitis because the incidence depends on the blood donor population, when the studies were done, and the type of patients involved. Prospective studies in the 1970s showed rates of post-transfusion hepatitis as high as 25%, but that was substantially reduced in the United States and other countries due to the conversion from paid donors to volunteer donors [137]. Hepatitis A usually has a short period of viremia, symptoms occur early during the viremic phase, and there is no carrier state. Because there is no chronic viremia and symptoms coincide with the acute viremic phase, infectious donors are usually identified by the medical history. Thus, post-transfusion hepatitis A is rare [138,139]. Laboratory testing of blood donors for hepatitis A is not done. Hepatitis A can be transmitted by coagulation factor concentrates or rarely by plasma derivatives. In evaluating a patient with suspected post-transfusion hepatitis, hepatitis A should be considered, but only as a remote possibility.

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Most people infected with the hepatitis B virus are asymptomatic, and thus an apparently healthy individual may meet all of the donor medical history criteria and donate a unit of infectious blood. The adoption of routine screening of blood donors for hepatitis B surface antigen reduced the incidence of post-transfusion hepatitis B. Despite testing for transfusion-transmitted hepatitis B, this is now the most common transfusiontransmitted viral infection [132,133], probably because some infectious carriers have a level of viremia below the limit of detection in the antigen screening test. Nucleic acid amplification testing is not done for hepatitis B because, despite the high sensitivity of this test method, levels of circulating virus may be undetectable in some asymptomatic blood donors. In 1989, an RNA virus similar in classification to a togavirus was identified and termed hepatitis C because it appeared to account for most post-transfusion non-A, non-B hepatitis [140]. Acute hepatitis C is usually mild, with up to 80% of patients being asymptomatic. However, the long-term effects are more serious because the virus tends to be persistent and the condition develops into chronic liver disease [141,142]. It has been estimated that the introduction of hepatitis C testing of blood donors has prevented about 40,000 cases of post-transfusion hepatitis C annually in the United States [142]. With the introduction of nucleic acid amplification testing, very little transfusion-transmitted hepatitis C remains (Table 82.4). Despite the discovery of the hepatitis C virus and implementation of screening donated blood for this virus, a few patients with posttransfusion hepatitis test negative for the known hepatitis viruses. A new RNA virus called hepatitis G or GB virus has been identified in some of these patients. From 1% to 4% of normal blood donors are carriers of hepatitis G when polymerase chain reaction techniques are used [143]. The apparent association of GB virus with hepatitis is probably due to similarities between GB virus and hepatitis C. The designation “hepatitis” virus was premature, and GB virus is not considered a cause of transfusion-transmitted hepatitis [143]. There is no practical test and no established role of hepatitis G in disease transmission. Presently, there is no plan to screen blood donors for the virus. There is also the TT virus. Although the designation of this virus suggests that it is “transfusion-transmitted,” the nomenclature signifies the initials of the patient from whom it was isolated. The TT virus was found in a patient with unexplained hepatitis and was thought to be a new viral hepatitis agent. Although TT virus is prevalent in blood donors and can be transmitted to as many as 30% of transfusion recipients, it is not associated with hepatitis [144]. Human immunodeficiency virus Although HIV-1 infection can be transmitted by blood transfusion, only a very small proportion of AIDS cases are due to transfusion. The risk of acquiring HIV infection following transfusion with anti-HIV-1positive blood is as high as 70–91% [145]. It has been estimated that, before the introduction of HIV-1 antibody testing in May 1985, approximately 12,000 patients were infected with HIV-1 by transfusion [146]. Because there is the interval or “window” between infection and the development of antibody to the infecting virus, HIV infection can be transmitted by anti-HIV-negative donors if they are in this window period [147–149]. Screening of blood for the HIV antigen using enzymelinked assay technology to eliminate the window phase was not effective. Nucleic acid amplification testing is now done on all donated blood and has nearly eliminated transfusion-transmitted HIV (Table 82.4) [131,133]. Human T-lymphotrophic virus I/II Human T-lymphotrophic virus-1 (HTLV-I) is the first retrovirus that has been shown to cause malignancy, adult T-cell leukemia, in humans. The

virus is also associated with a form of myelopathy referred to as HTLVassociated myelopathy or tropical spastic paraparesis. Donors with a true positive test for HTLV-I/II have approximately a 1–5% chance of developing adult T-cell leukemia during a 70-year lifespan, and another 2% may develop tropical spastic paraparesis within 5–10 years of initial infection. Although no cases of transfusion-transmitted adult T-cell leukemia or tropical spastic paraparesis have been identified, transmission of the virus by blood transfusion does occur [150]. In the United States, anti-HTLV-I is found almost exclusively in intravenous drug abusers or persons from areas that are endemic for HTLV-I [151]. Because of the potential for disease transmission, although no cases of transfusiontransmitted adult T-cell leukemia have been identified, routine testing of all donated blood for anti-HTLV-I was initiated in the United States in December 1988. This avoided the potential problem of transfusiontransmitted adult T-cell leukemia or tropical spastic paraparesis. Parvovirus The parvovirus B19 has been transmitted by blood transfusion (see Chapter 94) [152,153]. The prevalence of parvovirus in blood donors is estimated to be 1 in 3,300 to 1 in 50,000 [152,153]. The low prevalence, combined with the brief period of viremia, makes transmission of parvovirus by blood transfusion rare. When transmission occurs, it usually involves coagulation factor concentrates [153], although a few cases of transmission by single-donor components have been reported [153–155]. Epstein–Barr Virus Infection with EBV is followed by a lifelong carrier state, and most adults have been infected (see Chapter 93). EBV can be transmitted by transfusion [156], usually as the “postperfusion” syndrome, a virus-like illness that occurs after transfusion of fresh blood during open heart surgery. Transfusion transmission of EBV has not been a major clinical problem in HCT (see Chapter 93), although it can be transmitted to organ recipients [157] and could cause lymphoproliferative syndrome in HCT patients. No strategies are used to avoid transfusion transmission of EBV. Parasitic diseases It has been known for years that some parasites are transmitted by transfusion. All species of Plasmodium can survive in refrigerated blood and have caused transfusion-transmitted malaria, although most cases involve P. malariae. Malaria parasites are still viable and able to induce malaria infection after storage of the blood for a week in a refrigerator. All components, including RBCs, white blood cells, platelet concentrates, and fresh plasma can transmit malaria [158]. The prevention of transfusion-transmitted malaria relies on the exclusion of asymptomatic carriers based on residence or travel to an endemic area. However, in most cases of post-transfusion malaria, the interview fails to exclude the infectious donors. Laboratory tests are not used in the United States for screening donors for malaria. Thus, periodic spiking fever in a patient who has recently received a transfusion could be due to malaria, but this is very unlikely in North America, although donor selection procedures designed to identify potentially infectious donors are not very effective. Trypanosoma cruzi is endemic in many parts of Central and South America. Many patients with chronic T. cruzi infection may be asymptomatic, and thus could pass the blood donor medical questions and donate blood. Trypanosoma cruzi survives in refrigerated blood and can be transmitted by transfusion [159], although this event is rare in the United States [159,160]. As the number of immigrants from endemic areas increases, transfusion-transmitted T. cruzi could become a larger problem. For instance, in the Los Angeles area, which has a large Latin-American population, about 1 in 1000 donors has antibodies to

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation

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Table 82.5 Laboratory testing for transfusion-transmitted diseases Routinely screened for in US Family

Pathogen

Disease

Yes

No

Hepatitis

HBV, HCV HEV, HGV

Hepatitis Hepatitis

X

X

Retroviruses

Human immunodeficiency virus-1 and 2

Acquired immune deficiency syndrome

X

Human T-lymphotrophic virus-1 and II

Malignantly lymphoproliferative disorders, neuropathy

X

Cytomegalovirus

Cytomegalovirus retinitis, hepatitis, pneumonia

Epstein–Barr virus Human herpesvirus-8

Epstein–Barr syndrome Kaposi’s sarcoma

West Nile Denge

Meningitis, encephalitis Hemorrhagic fever

B19 Gram negative, Gram positive Treponema pallidum Borelia burgdorferi Rickettsia rickettsii Ehrlichia chafeensis

Aplastic anemia Sepsis Syphilis Lyme disease Rocky Mountain spotted fever Ehrlichiosis

Trypanosoma cruzi Babesia microti Leishmania donovani Plasmodium sp.

Chagas’ disease Babesiosis Leishmaniasis Malaria

Herpesviruses

Flavivirus

Parvoviruses Bacteria

X X X X X X X X X

Protozoans X X X X

HBV, HCV, HEV, HGV, hepatitis B, C, E, G viruses, respectively.

T. cruzi [159,161]. A screening test for antibody to T. cruzi has been developed and is being implemented by some blood banks. This is not required by FDA, and it is not clear whether screening for T. cruzi will become routine for all blood collection in the United States. Babesia microti and Babesia bovi are protozoa that occasionally infect humans by tick bites. Many infected individuals are asymptomatic, and thus Babesia microti can be transmitted by blood donated by asymptomatic infected donors [162,163]. The ticks are prevalent in the Northeast, Mid-Atlantic, and Upper Midwest. Because no suitable laboratory screening test exists, some blood banks defer individuals from heavily tick-infested areas during the summer months, but there is no generally adopted strategy to prevent transmission by transfusion. Borrelia burgdorferi, the spirochete that causes Lyme disease, is transmitted by ticks to humans. In up to 40% of persons, the infection is asymptomatic, and the spirochetes survive in stored blood for up to 45 days [164]. Thus, transmission of Borrelia burgdorferi by transfusion is theoretically possible, but this has not been reported [163]. Although a serologic test is available, it is not a suitable laboratory test for donor screening, and the widespread prevalence of the tick makes it impractical to defer donors from endemic areas. Creutzfeldt–Jakob disease A variant of Creutzfeldt–Jakob disease (CJD) that appears to be associated with ingestion of beef from cows affected with bovine spon-

giform encephalopathogy or “mad cow disease” [165] raised the question of whether the variant of CJD could be transmitted by blood transfusion. CJD has been transmitted by dura, corneas, and pituitary growth hormone from CJD patients and electroencephalographic electrodes used on CJD patients [166]. Prions, thought to be involved in CJD, are highly resistant to inactivation with methods currently used in the production of plasma derivatives. Thus, even one unit of plasma contaminated with the CJD agent could render a large lot of plasma derivatives infectious. However, it appears that, during the manufacture of plasma derivatives, prions do not segregate with the plasma fractions that result in coagulation factor concentrates or immune globulins. There is no evidence of blood-borne transmission or of a recent “outbreak” of CJD in the United States [167]. There have been two reports from the United Kingdom of transfusion recipients who developed CJD after receiving blood from donors who later developed CJD [168,169]. While this does not definitively establish transmission of variant CJD, the occurrence is statistically highly unlikely, suggesting that transfusion transmission might occur. Tests of the causative agent, presumably a prion, are under development but not available for donor screening. In an effort to decrease the transfusion risk, if one exists, potential blood donors are deferred if they have spent prolonged time in areas of high incidence of variant CJD such as the United Kingdom and parts of Europe.

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West Nile virus This virus, newly arrived in the United States, has characteristics of a transfusion-transmissible disease. Mosquitoes are the vector, mosquito bites are common, there is transient viremia shortly after infection, and most infected persons are asymptomatic, especially during the period of viremia. Thus, an infected individual could donate blood. As the West Nile virus epidemic became more widespread, transfusion transmission was identified [170–172]. The existing nucleic acid amplification test systems for HIV and hepatitis C virus provided a platform for introduction of West Nile virus testing, and currently all donated blood in the United States is tested for West Nile virus, so this risk should be nearly but not entirely eliminated. Summary of transfusion-transmitted diseases The diseases of most concern for transmission by blood transfusion have been discussed here. However, there is a concern that emerging pathogens, the mobility of people, and continued immigration can alter the situation with transfusion-transmitted diseases. Blood safety depends on many strategies, not just the laboratory test. The donor medical history and the use of volunteer donors are excellent examples. When considering a new screening test, many factors about the infectious agent and the disease must be taken into consideration. Examples are the prevalence of the disease, infectivity of the agent, status of the epidemic, and likelihood of a carrier state. Although some of the diseases may be regional in nature, the mobility of the population may make regional screening practices unsuitable. However, when all of the scientific discussion is completed, the issues of social policy and the public expectations of its blood supply remain. The public expects the transfusion medicine community to take steps to achieve the maximum possible safety. In the past, policy-makers and politicians have shown little tolerance for failure to take steps that would decrease risks to their constituents regardless of the cost. For instance, the cost of laboratory tests and additional regulations during the past decade have probably increased the cost of blood by 50%, and it has been estimated that introduction of nucleic acid amplification testing for

HIV and hepatitis C virus may have cost as much as $10,000,000 per quality-adjusted life year saved. A more rational approach to blood safety has been sought by many; however, the decision-making process is still a complex one with no easily discernible structure for these decisions. Two additional strategies being developed to further improve blood safety are the development of blood substitutes that are free of disease, and the development of methods of inactivating viruses and bacteria in blood components. Three decades of work and many millions of dollars have not resulted in a safe and effective blood substitute, and it does not appear that one will be available in the near future. In contrast, there has been great progress in the development of techniques to inactivate pathogens in blood products [173–176]. Pathogen-inactivated platelets are used routinely in many parts of Europe, and pathogen-inactivated plasma is already widely used, and a new even more effective method has recently been licensed there. It is possible that the paradigm for reducing transfusion-transmissible infections will shift from the present reactive to a proactive one involving treatment of blood products to render them noninfectious.

Conclusion The kinds of blood product available for transfusion have become sophisticated, and transfusion strategies are complex. Beginning with the diagnosis and treatment of the underlying disease, transfusion therapy is designed to minimize its interference with successful engraftment. Following transplantation, patient transfusion needs can be met effectively. The blood supply is safer than ever, and although some risks of infection remain, most patients can be supported safely and effectively.

Acknowledgment Portions of this chapter were taken from McCullough, J. Transfusion Medicine, Philadelphia: Elsevier/Churchill Livingstone; 2005.

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of 167 operations in thrombocytopenic patients. Am J Hematol 1987; 26: 147–55. Daly PA, Schiffer CA, Aisner J, Wiernik PH. Platelet transfusion therapy: one hour posttransfusion increments are valuable in predicting the need for HLA matched preparations. JAMA 1980; 243: 435–8. Moroff G, Friedman A, Robkin-Kline L et al. Reduction of the volume of stored platelet concentrates for use in neonatal patients. Transfusion 1984; 24: l44–6. Hersh JK, Hom EG, Brecher ME. Mathematical modeling of platelet survival with implications for optimal transfusion practice in the chronically platelet transfusion-dependent patient. Transfusion 1998; 38: 637–44. Goodnough LT, Kuter DJ, McCullough J et al. Prophylactic platelet transfusions from healthy normal apheresis platelet donors undergoing treatment thrombopoietin. Blood 2001; 98: 1346–51. Klumpp TR, Herman JH, Gaughan JP et al. Clinical consequences of alterations in platelet transfusion dose: a prospective, randomized, double-blind trial. Transfusion 1999; 39: 674– 81. Cid J, Lozano M. Lower or higher doses for prophylactic platelet transfusions: results of a meta-analysis of randomized controlled trials. Transfusion 2007; 47: 464–70. Aster RH. Effect of anticoagulant and ABO incompatibility on recovery of transfused human platelets. Blood 1965; 26: 732–43. Duquesnoy RJ, Anderson AJ, Tomasulo PA, Aster RH. ABO compatibility and platelet transfusions of alloimmunized thrombocytopenic patients. Blood 1979; 54: 595–9. Lee EJ, Schiffer CA. ABO compatibility can influence the results of platelet transfusion: results of a randomized trial. Transfusion 1989; 29: 384– 9. Heal JM, Blumberg N, Masel D. An evaluation of crossmatching, HLA, and ABO matching for platelet transfusions to refractory patients. Blood 1987; 709: 23–30. Fung MK, Downes KA, Shulman IA. Transfusion of platelets containing ABO-incompatible plasma. Arch Pathol Lab Med 2007; 131: 909–16. Goldfinger D, McGinnis MA. Rh incompatible platelet transfusions-risks and consequences of sensitizing immunosuppressed patients. N Engl J Med 1971; 284: 942–4. Menitove JE. Immunoprophylaxis for D– patients receiving platelet transfusions from D– donors? Transfusion 2002; 42: 136–8. Molnar R, Johnson R, Seat LT, Geiger TL. Absence of D alloimmunization in D– pediatric oncology patients receiving D-incompatible single-donor platelets. Transfusion 2002; 42: 177–82. Cid J, Ortin X, Elies E et al. Absence of anti-D alloimmunization in hematologic patients after Dincompatible platelet transfusions. Transfusion 2002; 42: 173–6. Pfisterer H, Thierfelder S, Kottusch H, Stich W. Untersuchung menschlicher thrombocyten auf Rhesus-antigene durch abbaustudien in vivo nach 51Cr-markierung. Klin Wochenschr 1967; 45: 5519–22. Slichter SJ, Davis K, Enright H et al. Factors affecting post-transfusion platelet increments, platelet refractoriness, and platelet transfusion

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intervals in thrombocytopenic patients. Blood 2005; 105: 4106–14. Aster RJ, Jandl JH. Platelet sequestration in man. II. Immunological and conical studies. J Clin Invest 1964; 43: 856–69. Bishop JF, McGrath K, Wolf MM et al. Clincal factors influencing the efficacy of pooled platelet transfusions.. Blood 1988; 71: 383–7. Doughty HA, Murphy MF, Metcalfe P et al. Relative importance of immune and non-immune causes of platelet refractoriness. Vox Sang 1994; 66: 200–5. The Trial to Reduce Alloimmunization to Platelets Study Group. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med 1997; 337: 1861–9. Lazarus HM, Herzig RH, Warm SE, Fishman DJ. Transfusion experience with platelet concentrates stored for 24 to 72 hours at 22°C. Transfusion 1982; 22: 39–43. Yankee RA, Graff KS, Dowling R, Henderson ES. Selection of unrelated compatible platelet donors by lymphocyte HL-A matching. N Engl J Med 1973; 288: 760–4. Lohrman HP, Bull MI, Decter JA et al. Platelet transfusions from HLA compatible unrelated donors to alloimmunized patients. Ann Intern Med 1974; 80: 9–14. Duquesnoy RJ, Filip DJ, Rodey GE et al. Successful transfusion of platelets “mismatched” for HLA antigens to alloimmunized thrombocytopenic patients. Am J Hematol 1977; 2: 219–23. Rachel JM, Sinor LT, Tawfik OW et al. A solidphase red cell adherence test for platelet crossmatching. Med Lab Sci 1985; 42: 194–5. Rachel JM, Summers TC, Sinor LT, Plapp FV. Use of a solid phase red blood cell adherence method for pretransfusion platelet compatibility testing. Am J Clin Pathol 1988; 90: 63–8. O’Connell BA, Lee EJ, Rothko K, Hussein MA, Schiffer CA. Selection of histocompatibility apheresis platelet donors by cross-matching random donor platelet concentrates. Blood 1992; 79: 527–31. Friedberg RC, Donnelly SF, Boyd JC, Gray LS, Mintz PD. Clinical and blood bank factors in the management of platelet refractoriness and alloimmunization. Blood 1993; 81: 3428–34. Moroff G, Garratty G, Heal JM et al. Selection of platelets for refractory patients by HLA matching and prospective crossmatching. Transfusion 1992; 32: 633–40. Class FHJ, Smeenk RJT, Schmidt R et al. Alloimmunization against the MHC antigens after platelet transfusions is due to contaminating leukocytes in the platelet suspension. Exp Hematol 1981; 9: 84–9. Semple JW, Freedman J. Recipient antigen-processing pathways of allogeneic platelet antigens: essential mediators of immunity. Transfusion 2002; 42: 958–61. Sniecinski I, O’Donnell MR, Nowicki B, Hill LR. Prevention of refractoriness and HLA-alloimmunization using filtered blood products. Blood 1988; 71: 1402–7. Saarinen UM, Kekomaki R, Siimes MA, Myllyla G. Effective prophylaxis against platelet refractoriness in multitransfused patients by use of leukocyte-free blood components. Blood 1990; 75: 512–17.

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74. van Marwijk Kooy M, van Prooijen HC, Moes M et al. Use of leukocyte depleted platelet concentrates for the prevention of refractoriness and primary HLA alloimmunization: a prospective, randomized trial. Blood 1991; 77: 201–5. 75. Heddle NM, Blajchman MA. The leukodepletion of cellular blood products in the prevention of HLA-alloimmunization and refractoriness to allogeneic platelet transfusions. Blood 1995; 85: 603–6. 76. Kuter DJ. Whatever happened to thrombopoietin? Transfusion 2002; 42: 279–83. 77. Strauss RG. Therapeutic granulocyte transfusions in 1993. Blood 1993; 81: 1675–8. 78. Strauss RG, Connett JE, Gale RP et al. A controlled trial of prophylactic granulocyte transfusion during initial induction. N Engl J Med 1981; 305: 597–8. 79. Price TH. Granulocyte transfusion therapy: it’s time for an answer. Transfusion 2006; 46: 1–5. 80. Liles WC, Juang JE, Llewellyn C et al. A comparative trial of granulocytecolony-stimulating factor and dexamethasone, separately and in combination, for the mobilization of neutrophils in the peripheral blood of normal volunteers. Transfusion 1997; 37: 182–7. 81. Oza A, Hallemeier C, Goodnough L et al. Granulocyte–colony-stimulating factor–mobilized prophylactic granulocyte transfusions given after allogeneic peripheral blood progenitor cell transplantation result in a modest reduction of febrile days and intravenous antibiotic usage. Transfusion 2005; 46: 14–23. 82. Storb R, Prentice RL, Thomas ED. Marrow transplantation for treatment of aplastic anemia: an analysis of factors associated with graft rejection. N Engl J Med 1977; 296: 61–5. 83. Storb R, Thomas ED, Buckner CD et al. Marrow transplantation in 30 “untransfused” patients with severe aplastic anemia. Ann Intern Med 1980; 92: 30–6. 84. Champlin RE, Horowitz MM, van Bekkum DW et al. Graft failure following bone marrow transplantation for severe aplastic anemia: risk factors and treatment results. Blood 1989; 73: 606–13. 85. Storb R, Weiden PL. Transfusion problems associated with transplantation. Semin Hematol 1981; 18: 163–76. 86. Ho WG, Champlin RE, Winston DJ, Feig SA, Gale RP. Bone marrow transplantation in patients with leukaemia previously transfused with blood products from family members. Br J Haematol 1987; 67: 67–70. 87. Segal JB, Dzik WH. Paucity of studies to support that abnormal coagulation test results predict bleeding in the setting of invasive procedures: an evidence-based review. Transfusion 2005; 45: 1413–25. 88. Howard SC, Gajjar A, Ribeiro RC et al. Safety of lumbar puncture for children with acute lymphoblastic leukemia and thrombocytopenia. JAMA 2000; 284: 2222–4. 89. Schiffer CA, Anderson KC, Bennett CL et al. Platelet transfusion for patients with cancer: clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol 2001; 19: 1519–38. 90. Miller WJ, McCullough J, Balfour HH et al. Prevention of CMV infection following bone marrow transplantation: a randomized trial of blood

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product screening. Bone Marrow Transplant 1991; 7: 227–34. Bowden RA, Slichter SJ, Sayers M et al. A comparison of filtered leukocyte reduced and cytomegalovirus (CMV) seronegative blood products for the prevention of transfusion-associated CMV infection after marrow transplant. Blood 1995; 86: 3598–603. Gilbert GL, Hudson IL, Hayes JJ. Prevention of transfusion-acquired cytomegalovirus infection in infants by blood filtration to remove leucocytes. Lancet 1989; 1: 1228–31. Murphy MF, Grint PCA, Hardiman AE, Lister TA, Waters AH. Use of leukocyte-poor blood components to prevent primary cytomegalovirus (CMV) infection in patients with acute leukemia. Br J Haematol 1988; 70: 253–5. Moroff G, Leitman SF, Luban NLC. Principles of blood irradiation, dose validation and quality control: a practical approach. Transfusion 1997; 37: 1084–92. Leitman SF, Holland PV. Irradiation of blood products: Indications and guidelines. Transfusion 1985; 25: 293–300. Valerius NH, Johansen KS, Nielsen OS et al. Effect of in vitro X-irradiation on lymphocyte and granulocyte function. Scand J Hematol 1981; 27: 9–18. Button LN, DeWolf WC, Newburger PE, Jacobson MS, Kevy SV. The effects of irradiation on blood components. Transfusion 1981; 21: 419– 26. Read EJ, Kodis C, Carter CS, Leitman SF. Viability of platelets following storage in the irradiated state. A pair-controlled study. Transfusion 1988; 23: 446–50. Duguid JKM, Carr R, Jenkins JA et al. Clinical evaluation of the effects of storage time and irradiation on transfused platelets. Vox Sang 1991; 60: 151–4. Park BH, Good RA, Gate J et al. Fatal graftversus-host reaction following transfusion of allogeneic blood and plasma in infants with combined immunodeficiency disease. Transplant Proc 1974; 6: 385–7. Sproul AM, Chalmers EA, Mills KI et al. Third party mediated graft rejection despite irradiation of blood products. Br J Haematol 1992; 80: 251–2. Lowenthal RM, Challis DR, Griffiths AE et al. Transfusion-associated graft-versus-host disease: report of a case following administration of blood. Transfusion 1993; 33: 524–9. Rivet C, Baxter A, Rock G. Potassium levels in irradiated blood [Letter]. Transfusion 1989; 29: 185. Strauss RG. Routine washing of irradiated red cells before transfusion seems unwarranted. Transfusion 1990; 30: 675–7. Douglas SD, Fudenberg HH. Graft versus host reaction in Wiskott–Aldrich syndrome: antemortem diagnosis of human GVH in an immunologic deficiency disease. Vox Sang 1969; 16: 172–8. Hathaway WE, Githens JH, Blackburn WR et al. Aplastic anemia, histiocytosis and erythrodermia in immunologically deficient children: probable human runt disease. N Engl J Med 1965; 271: 953–4. Thompson HW, McCullough J. Use of blood components containing red cells by donors of allogeneic bone marrow. Transfusion 1986; 26: 98–100.

108. Eder AF, Chambers LA. Noninfectious complications of blood transfusion. Arch Pathol Lab Med 2007; 131: 708–18. 109. Sazama K. 355 reports of transfusion-associated deaths. Transfusion 1990; 30: 583. 110. Popovsky MA, editor. Transfusion Reactions, 3rd edn. Bethesda, MD: AABB Press; 2001. 111. Enright H, Davis K, Gernsheimer T, McCullough JJ, Woodson R, Slichter SJ. Factors influencing moderate to severe reactions to PLT transfusions: experience of the TRAP multicenter clinial trial. Transfusion 2003; 43: 1545–52. 112. Wang SE, Lara PN Jr., Lee-Ow A et al. Acetaminophen and diphenhydromine as premedication for platelet transfusions: a prospective randomized double-blind placebo controlled trial. Am J Hematol 2002; 70: 191–4. 113. Tobian AAR, King KE, Ness PM. Transfusion premedications: a growing practice not based on evidence. Transfusion 2007; 47: 1089–96. 114. Sewell LD. IgA deficiency: what we should – or should not – be doing. J Clin Pathol 2001; 54: 337–8. 115. Meena-Leist CE, Fleming DR, Heye M, Herzig RH. The transfusion needs of an autologous bone marrow transplant patient with IgA deficiency. Transfusion 1999; 39: 457. 116. Kleinman S, Caulfield T, Chan P et al. Toward an understanding of transfusion-related acute lung injury: statement of a consensus panel. Transfusion 2004; 44: 1774–89. 117. Heddle NM, Klama L, Singer J et al. The role of the plasma from platelet concentrates in transfusion reactions. N Engl J Med 1994; 331: 625–8. 118. Bordin JO, Heddle NM, Blajchman MA. Biologic effects of leukocytes present in transfused cellular blood products. Blood 1994; 84: 1703–21. 119. Goodnough LT, Riddell J, Lazarus H et al. Prevalence of platelet transfusion reactions before and after implementation of leukocyte-depleted platelet concentrates by filtration. Vox Sang 1993; 65: 103–7. 120. Muylle L, Wouters E, Peetermans ME. Febrile reactions to platelet transfusion: the effect of increased interleukin 6 levels in concentrates prepared by the platelet-rich plasma method. Transfusion 1996; 36: 886–90. 121. Klein HG, Dodd MY, Ness PM, Fratantoni JA, Nemo GJ. Current status of microbial contamination of blood components: summary of a conference. Transfusion 1997; 37: 95–101. 122. Goldman M, Blajchman MA. Blood productassociated bacterial sepsis. Transf Med Rev 1991; 5: 73–83. 123. Ramirez-Arcos S, Jenkins C, Dion J et al. Canadian experience with detection of bacterial contamination in apheresis platelets. Transfusion 2007; 47: 421–9. 124. Eder AF, Herron R, Strupp A et al. Transfusionrelated acute lung injury surveillance (2003–2005) and the potential impact of the selective use of plasma from male donors in the American Red Cross. Transfusion 2007; 47: 599–607. 125. Eder AF, Kennedy JM, Dy BA et al. Bacterial screening of apheresis platelets and the residual risk of septic transfusion reactions: the American Red Cross experience (2004–2006). Transfusion 2007; 47: 1134–42. 126. Lucarelli G, Angelucci E, Giardini C et al. Fate of iron stores in thalassemia after bone marrow transplantation. Lancet 1993; 342: 1388–91.

Principles of Transfusion Support Before and After Hematopoietic Cell Transplantation 127. Angelucci E, Baronciani D, Giardini C et al. Iron removal in ex-thalassemics after BMT: preliminary results from the phlebotomy program. Bone Marrow Transplant 1993; 12(Suppl 1): 105–7. 128. Busch MP, Young MJ, Samson SJ et al. Risk of human immunodeficiency virus (HIV) transmission by blood transfusions before the implementation of HIV-I antibody screening. Transfusion 1991; 31: 4–11. 129. Hewlett IK, Epstein JS. Food and drug administration conference on the feasibility of genetic technology to close the HIV window in donor screening. Transfusion 1997; 37: 346–51. 130. Report of the Interorganizational Task Force on Nucleic Acid Amplification Testing of Blood Donors. Nucleic acid amplification testing of blood donors for transfusion-transmitted infectious diseases. Transfusion 2000; 40: 143–59. 131. Stramer SL, Caglioti S, Strong DM. NAT of the United States and Canadian blood supply. Transfusion 2000; 40: 1165. 132. O’Brien SF, Yi QL, Fan W, Scalia V, Kleinman S, Vamvakas E. Current incidence and estimated residual risk of transfusion-transmitted infections in donations made to Canadian Blood Services. Transfusion 2007; 47: 316–25. 133. Stramer SL. Current risks of transfusion-transmitted agents. Arch Pathol Lab Med 2007; 131: 702–7. 134. Cable R. Evaluation of syphilis testing of blood donors. Transf Med Rev 1996; 10: 296–302. 135. Schmidt PJ. Syphilis, a disease of direct transfusion. Transfusion 2001; 41: 1069–71. 136. Greenwalt TJ, Rios JA. To test or not to test for syphilis: a global problem. Transfusion 2001; 41: 976. 137. Alter HJ, Holland PV, Purcell RH et al. Posttransfusion hepatitis after exclusion of commercial and hepatitis-B antigen-positive donors. Ann Intern Med 1972; 77: 691–9. 138. Hollinger FB, Khan NC, Oefinger PA et al. Posttransfusion hepatitis type A. JAMA 1983; 250: 2313–17. 139. Noble RC, Kane MA, Reeves SA, Roeckel I. Posttransfusion hepatitis A in a neonatal intensive care unit. JAMA 1984; 252: 2711–21. 140. Alter HJ, Purcell RH, Shih JW et al. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989; 321: 1494–500. 141. Tong MJ, El-Farra NS, Reikes AR, Co RL. Clinical outcomes after transfusion-associated hepatitis C. N Engl J Med 1995; 332: 1463–6. 142. Alter HJ. To C or not to C: these are the questions. Blood 1995; 85: 1681–95. 143. Mosley JW, Rakela J. Foundling viruses and transfusion medicine. Transfusion 1999; 39: 1041. 144. Wang JT, Lee CZ, Kao JH, Sheu JC, Wang TH, Chen DC. Incidence and clinical presentation of posttransfusion TT virus infection in prospectively followed transfusion recipients: emphasis

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on its relevance to hepatitis. Transfusion 2000; 40: 596. Menitove JE. Status of recipients of blood from donors subsequently found to have an antibody to HIV. N Engl J Med 1986; 315: 1095–6. Centers for Disease Control (CDC). Human immunodeficiency virus infection in transfusion recipients and their family members. Morbid Mortal Weekly Rep 1987; 36: 137. Cohen ND, Munoz A, Reitz BA et al. Transmission of retroviruses by transfusion of screened blood in patients undergoing cardiac surgery. N Engl J Med 1989; 320: 1172. Roberts CS, Longfield JN, Platte RC et al. Transfusion-associated human immunodeficiency virus type I from screened antibody-negative blood donors. Arch Pathol Lab Med 1994; 118: 1188– 92. Irani MS, Dudley AW, Lucco LJ. Case of HIV-I transmission by antigen-positive, antibody negative blood [Letter]. N Engl J Med 1991; 325: 1174–5. Okochi K, Sato H. Adult T-cell leukemia virus, blood donors and transfusion: experience in Japan. Prog Clin Biol Res 1985; 182: 245–9. Williams AE, Fang CT, Slamon DJ et al. Seroprevalence and epidemiologic correlates of HTLV-I infection in U.S. blood donors. Science 1988; 240: 643–6. Lefrere JJ, Mariotti M, de la Croix I et al. Albumin batches and B19 parvovirus DNA. Transfusion 1995; 35: 389–91. Brown KE, Young NS, Alving BM, Barbosa LH. Parvovirus B19: implications for transfusion medicine. Transfusion 2001; 41: 130. Cohen BJ, Beard S, Knowles WA et al. Chronic anemia due to parvovirus B19 infection in a bone marrow transplant patient after platelet transfusion. Transfusion 1997; 37: 947–52. Zanell A, Rossi F, Cesana C et al. Transfusiontransmitted human parvovirus B19 infection in thalassemic patients. Transfusion 1995; 35: 769– 72. Henle W, Henle G, Scriba M et al. Antibody responses to the Epstein–Barr virus and cytomegalovirus after open-heart and other surgery. N Engl J Med 1970; 282: 1068–74. Alfieri C, Tanner J, Carpentier L et al. Epstein– Barr virus transmission from a blood donor to an organ transplant recipient with recovery of the same virus stain from the recipient’s blood and oropharynx. Blood 1996; 87: 812–17. Mungai M, Tegtmeier G, Chamberland M, Parise M. Transfusion-transmitted malaria in the United States from 1963 through 1999. N Engl J Med 2001; 344: 1973–8. Leiby DA, Herron RM, Read EJ et al. Trypanosoma cruzi in Los Angeles and Miami blood donors: impact of evolving donor demographics on seroprevalence and implications for transfusion transmission. Transfusion 2002; 42: 549. Nichols WG, Price TH, Gooley T, Corey L, Boeckh M. Transfusion-transmitted cytomegalo-

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83

I. Benjamin Paz

Vascular Access and Complications

Introduction The administration of intravenous fluids was first attempted by Drs Thomas Latta and Robert Lewins during the cholera epidemic in England in 1832. After studying the blood and excreta of cholera patients, Dr William Brooke O’Shaughnessy correctly deduced that the changes in composition of the blood were secondary to the fluid and electrolyte losses in the excreta, and proposed that the treatment of this disease should include the replenishment of fluid and salts intravenously. This was first attempted by Dr Latta, who administered a hypotonic solution by direct intravenous injection through a silver needle connected to a syringe by rubber tubing. Dr Lewins reported this case in an 1832 issue of The Lancet [1]. This practice was largely abandoned until the late 1800s, when the administration of intravenous fluids became available thanks to the advent of sterile electrolyte solutions and steel hypodermic needles. In 1945, plastic catheters for continuous vascular access were introduced following the discovery of penicillin and the need for multiple intravenous injections. The development in the 1960s of long nylon catheters that could be placed into the central veins constituted a major technologic development [2]. These catheters were initially inserted into cancer patients via peripheral vein cutdown and later via percutaneous approaches into the subclavian and jugular veins, with the objective of limiting the chemotherapy-associated phlebitis. A major breakthrough occurred when Broviac et al. described the use of a soft, cuffed, silicone catheter for long-term central venous access in children on total parenteral nutrition [3]. This was shortly followed by the development by Hickman et al. of a similar catheter with larger lumen for bone marrow transplant recipients [4]. These catheters could remain in place for an extended period of time, facilitating the outpatient care of these patients, and providing higher flow for chemotherapy, blood transfusions, and parenteral nutrition. These devices were further modified by the addition of a subcutaneous reservoir, which could be easily and intermittently accessed via percutaneous needles. Later, the development of new polymers such as silicone, polyvinyl chloride, Teflon, and polyurethane led to the manufacture of a large variety of intravenous catheters. These catheters had different physical properties designed for specific applications, including chemotherapy, parenteral nutrition, hemodynamic monitoring, and hemodialysis [5].

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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The use of central venous access for treatment and support has improved the quality of life and permitted the delivery of complex chemotherapy regimens to cancer patients. Long-term use of venous access has become so frequent that all health-care personnel now need to understand the indications, selection, and maintenance of catheters, as well as the prevention, diagnosis, and treatment of catheter-related complications.

Indications and patient selection Although there are no established guidelines for the placement of central venous access, the use of complex multidrug chemotherapy and bone marrow transplant in cancer treatment has made the establishment of reliable long-term venous access an essential component of cancer therapy. Important considerations in deciding whether a catheter is indicated are patient preferences, the frequency and duration of the therapy, the frequency of blood draws, the nature of the therapy (vesicating agents are better delivered into a central vein without risk of extravasation), the need for support therapies (total parenteral nutrition), the need for hematopoietic cell collection, plasmapheresis, and bone marrow reinfusion. Long-term venous access needs to be considered an elective procedure. The patient should be recovered from any acute infections or treatment-related complications that could prevent the safe placement of these catheters. In cases in which patients have not fully recovered and there is an absolute need for central venous access, a temporary percutaneous central line or a peripherally inserted central venous catheter (PICC) can provide access until the patient has recovered. History of prior vascular access insertions (number and location), thoracic surgery, radiation, mediastinal, and thoracic disease should alert the surgeon to possible changes in the normal venous drainage. Physical examination documenting the integrity of the skin, normal venous anatomy without evidence of abnormal collaterals, changes secondary to surgical treatment and reconstruction, sites of prior catheter insertions, evidence of venous obstruction, and pulmonary reserve should be completed in every patient. If there is any evidence of venous obstruction or history of bilateral placements of central venous accesses, the examination should be complemented with a venous imaging study. In patients with a previous history of venous thrombosis, the site can be used for access if the vein is shown to be patent by ultrasound or venogram. Duplex Doppler ultrasound can visualize the patency and flow of the neck and arm veins. Intrathoracic veins are poorly visualized with this technique. Computed tomography and magnetic resonance imaging are very good at documenting thrombosis and patency of major intrathoracic veins (Fig. 83.1). Venous angiogram

Vascular Access and Complications

(a)

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(b)

Fig. 83.1 (a) Computed tomography (CT) scan showing a right internal jugular vein thrombosis. (b) CT scan showing a superior vena cava thrombosis above the entrance of the innominate vein.

Table 83.1 Patient and treatment factors that affect catheter selection Catheter related factors: • Duration of therapy (short or long term) • Frequency of treatment (intermittent or continuous) • Number of lumina required Therapy for which the catheter is intended: • Transfusions of blood products • Parenteral nutrition • Chemotherapy administration (types of chemotherapy agent) • Hematopoietic cell collection • Supportive care Patient related factors: • Previous history of chest or neck surgery or radiation (mastectomy, neck biopsy) • Presence of a chest, neck or mediastinal tumor • Pulmonary function (asthma, pleural effusion) • Cardiac function (arrhythmias, pacemakers) Venous access factors: • Current venous access • Previous history of venous access • Previous history of catheter-related complications • Previous or current history of veno-occlusive disease or lymphedema Hematological and coagulation status: • Red cell blood count • Platelet count • History of bleeding disorders • Anticoagulation (coumarin, aspirin, Plavix) Infection risk: • White blood cell count • History of recent infections • History of catheter-related infections Allergies: • Anesthetics • Cleansing solutions • Dressings

is still considered the “gold” standard in the study of venous anatomy and should be performed whenever noninvasive examinations fail to provide a definitive diagnosis. Chest radiography can disclose important information such as the presence of pleural effusions, lung metastases, and mediastinal tumors, which can modify the site selection for the placement of central venous access. Table 83.1 provides a complete list of conditions that should be considered in patient selection. Relative contraindications to the placement of a permanent central venous access are granulocyte counts less than 1500/mm3. These patients have a higher incidence of septic episodes that might prompt early catheter removal. Thrombocytopenia and platelet dysfunction are frequently encountered in cancer patients. Preoperative platelet transfusion to 50,000/mm3 allows for the safe placement of catheters in the majority of patients without bleeding complications. In those patients with refractory thrombocytopenia to platelet transfusions, venous cutdown might be the safest approach for catheter placement. Many cancer patients have abnormalities in their clotting factors secondary to malnutrition or chemotherapy treatments. Vitamin K or fresh frozen plasma is usually necessary for the correction of these abnormalities. Patients who cannot tolerate the recumbent or Trendelenburg position can frequently have their catheters placed through a cutdown approach under local anesthesia. Except for those patients who require a line for long-term antibiotic treatment, the only absolute contraindication for the placement of a long-term indwelling catheter is the presence of an active infection.

Catheter selection Long-term venous catheters are designed for external access, such as the Hickman, Broviac, Groshong or Quinton catheters [6], or for access through a subcutaneous port such as a Port-a-Cath or Infusaport [7]. These catheters are available with different lumen diameters and as single- and multilumen devices [8]. Central venous catheters peripherally placed through an antecubital vein (PICC lines) [9] have become increasingly popular due to their ease of placement and their ability to be placed by trained nursing personnel. Unfortunately, these devices are associated with a higher incidence of catheter-related complications [10]. An important general consideration in the selection of the appropriate catheter is that catheter resistance to infusion depends on the catheter length and diameter of the lumen. During laminar flow, the flow-rate through a catheter is governed by Poiseuille’s law:

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Flow = [13.1416 × (P1 − P2) × R4]/8 × n × L

where P1 and P2 are the fluid pressures at the proximal and distal end of the tube, respectively, R is the radius of the tube, n is the viscosity of the fluid, and L is the length of the tube. Flow is then directly proportional to the fourth power of the radius of the tube and inversely proportional to the length [11]. Therefore, variations in the lumen diameter have a greater effect on the flow rate than do changes in the length of the catheter [11]. Peripherally inserted central catheter PICC lines are inserted via the antecubital veins in the arm and advanced into the central veins, with its tip located in the lower third of the superior vena cava (SVC). It is usually inserted at the patient’s bedside following the application of local anesthetic. The use of hand-held Doppler ultrasound increases the insertion success rate, especially in patients with several previous peripheral access insertion attempts. Ultrasound guidance is the preferred method for PICC placement in patients with no clinically identifiable peripheral vein. PICC lines have several advantages: ease of insertion, low complication rates associated with insertion, low cost of insertion, and the possibility of being placed by trained nurses [12]. As a result, PICC is the device of choice for patients who require a long-term venous access for antibiotic therapy, parenteral nutrition, pain management, and fluid replacement, but do not require frequent blood samples or infusion of blood products [9]. They are made either of silicone or polyurethane and are available in varying sizes, either single- or dual-lumen, and can remain in situ for up to a year [9]. The main disadvantages of these lines are mechanical phlebitis, frequent malfunction due to occlusion, the site of insertion, which might interfere with the patient’s arm activity, and the local care required, which can be difficult to manage for nonhospitalized patients [10]. Implantable subcutaneous ports An implantable port is a totally implanted vascular access device that is inserted on the chest wall or in the antecubital area. It is made up of a subcutaneous port containing a silicone septum, which is connected to a silicone catheter. It is implanted in a subcutaneous pocket, and the catheter is inserted in the vein via cutdown or venous puncture using the Seldinger technique. The port can only be accessed by the use of a

noncoring (Huber) point needle, which is inserted through the skin into the septum. The needle can only remain in situ for up to 7 days at a time. The advantage of this port is that it does not interfere with daily activities, does not require local care when it is not accessed, and has a less significant impact on body image. The port requires monthly flushing and is associated with a lower infection risk. Disadvantages of ports include the requirement of a needle for access, flow rate being limited by needle diameter, low reliability for obtaining blood draws, higher frequency of catheter occlusion, and the possibility of soft tissue chemotherapy extravasations if the needle is misplaced or migrates out of the port after insertion. Subcutaneous ports are primarily indicated in patients who require intermittent therapy and do not need continuous intravenous access. Externally tunneled catheter The Broviac and Hickman catheters were developed in the mid-1970s and constituted the first examples of the skin-tunneled catheter. The tunnel provides a distance between the vein entry site and the skin exit site, and this, along with a Dacron cuff, has been shown to reduce infection risk. The cuff is usually placed about 2 cm from the entry site; the local tissue reaction and granulation tissue entering into the cuff prevent infections and catheter dislodgement. These devices provide reliable long-term access and are made of either silicone or polyurethane. Single-, double-, and triple-lumen catheters are available. These devices are usually placed by surgeons, radiologists or other trained personnel under fluoroscopic guidance either via cutdown or percutaneous venous access. Externally tunneled devices are preferred for those patients who require continuous or frequent catheter access for treatment, blood samples or support treatment (parenteral nutrition or blood product transfusion) or are receiving potentially toxic therapy if the infusion extravasates into the subcutaneous tissue. The choice of number and size of lumina should be determined based on the intensity and complexity of the therapy. Pooled studies vary significantly with respect to patient characteristics, catheter maintenance schedules, and diagnosis criteria for infection. Safdar and Maki report an overall mean infection rate per 100 catheter days of 0.21 for external devices and 0.04 for subcutaneous ports [13]. Randomized studies have failed to show a significant difference in infection incidence between the implantable ports and the external devices. Other important differences between these devices are outlined in Table 83.2.

Table 83.2 Differences between devices for central venous access Peripherally inserted central venous catheter lines

Externally tunneled devices

Subcutaneous ports

Lumen Maintenance Activity Blood sample Uses

Single and dual lumina Daily; training required Some restrictions Low reliability Chemotherapy, TPN, antibiotic therapy, supportive therapy, transfusions

Single and dual lumina Monthly; no training required No restrictions Moderate reliability Chemotherapy, TPN, antibiotic therapy, supportive therapy, transfusions

Cost Access Flow Complications Placement Removal

Low insertion cost Painless Determined by the lumen High Trained nurses; at bedside Easy

Single, dual, and triple lumina Daily; training required Some restrictions High reliability Hematopoietic cell collection and infusion, photophoresis, plasma/red cell exchange, transfusions, supportive therapy, chemotherapy High maintenance cost Painless Determined by the lumen High Physician, in operating room or radiology Easy; in the office

TPN, total parenteral nutrition.

High insertion cost Needle access Determined by the needle Low Physician, in operating room or radiology Second procedure required

Vascular Access and Complications

The hematopoietic cell transplant patient merits special attention. In recent years, the use of peripheral blood progenitor cells has nearly supplanted bone marrow as the preferred autologous hematopoietic progenitor cell source for reconstitution after high-dose chemotherapy. In the past, peripheral blood progenitor cells were collected via a largebore, stiff, temporary, dual-lumen central venous catheter that provided flow rates of at least 50 mL/min. The apheresis procedure is lengthy and time-consuming, since large blood volumes must be processed during multiple sessions until enough progenitor cells have been collected. On the other hand, the transplant process also requires long-term multilumen venous access, but only requires a smaller, soft, and flexible catheter for chemotherapy administration, supportive care management (including frequent blood sampling, intravenous antibiotics, analgesics, antiemetics, blood components, and parenteral nutrition), and reinfusion of hematopoietic cells. Since different catheter properties are required, the transplant procedure traditionally involved the insertion of two venous access devices, one for apheresis and another for the treatment and supportive care. Over the last few years, the advent of soft silicone or polyurethane catheters specially designed for apheresis treatment has enabled the use of a single catheter for the entire transplant process. These catheters are shorter and have large lumina, staggered at the tip to prevent recirculation during the hematopoietic cell collection [14]. The design of these catheters makes them more likely to kink during placement if special precautions are not taken to maintain adequate flow rates. They also have a very short distance from the cuff to the proximal connectors, which make them uncomfortable for the frequent venous access required in this patient population. A compromise between these catheters is to place a large bore (12 Fr) Hickman catheter with two round lumina of 1.3 mm, each of which provides comfortable access while allowing 50 mL/min flow rates for a successful apheresis.

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dence of arterial puncture and other related complications [18]. In women and children, where the space between the first rib and the clavicle is very small, it is the preferred site of insertion for large-bore catheters to avoid catheter pinch-off syndrome, which leads to catheter fracture and embolization [19]. A common problem associated with this insertion site is catheter occlusion if the operator is not attentive while tunneling the catheter. The best approach is to allow for a soft inverted U-curve to prevent kinking within the tunnel, to tunnel the catheter from a lateral entry site in the anterior chest to a medial venous entry site in

Fig. 83.2 X-ray showing kinking of the catheter at the internal jugular vein insertion site.

The main veins used for central venous access are the internal and external jugular and subclavian veins, which are used for all types of central venous access device; occasionally, the femoral veins are selected when the above sites are not available or for short-term access. The cephalic and basilic veins are the preferred sites for PICC venous access. The right side of the patient is usually preferred for access from the internal [15] and external jugular vein, and the left side is preferred for access from the subclavian vein [16] because the vessel anatomy allows easier access to the SVC and provides the easiest route for the practitioner inserting the device. Regardless of the insertion site, it is important to pay special attention to the tunneling of the catheter to avoid kinking, and to the location of the tip of the catheter (Fig. 83.2). An increased risk of venous thrombosis is associated with the tip of the catheter being located in the upper third of the SVC or in the innominate vein. Even though the lower third appears to be the favored location for the SVC tip, there is still some debate over the optimal location for the SVC tip. A good point of reference under fluoroscopy in the patient in the Trendelenburg position is the carina of the main bronchus bifurcation (Fig. 83.3) [17]. Even though right atrial placement is associated with arrhythmias, atrial thrombus, and potential cardiac or valve perforations when the catheter is kept for an extended period of time, it is still performed in some centers. Internal jugular vein This is a good site due to its high success in insertion rate and low incidence of complications. The internal jugular vein also allows for easier insertion by ultrasound guidance, thereby decreasing the inci-

Fig. 83.3 Intraoperative fluoroscopy showing the tip of a dual-lumen silicone catheter at the carina. Also note the soft curve at the internal jugular vein entry site.

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the neck, and to choose a low neck venous access. This is the site of choice for healthy bone marrow donors [20]. External jugular vein Even though this is an easily visible vein, it varies in size, and its junction with the subclavian is angulated, making it more difficult to advance the catheter into the SVC. This vein is usually accessed via cutdown, making it an ideal site for those patients who cannot tolerate the Trendelenburg position or who have a coagulation disorder. Unfortunately, the size of this vein is too small in most patients to place a large-bore multilumen silicone catheter. To avoid kinking within the tunnel, these catheters are best tunneled from a medial insertion site in the anterior chest to a lateral neck incision. Subclavian vein This large vein has been the access of choice for long-term central venous access for many years. The main advantages of using this vein are that it usually requires a short tunnel and can be easily accessed from a lateral approach, which makes the procedure easier, especially in conscious patients. Also, anatomic landmarks for catheter insertion vary less from patient to patient [21]. The disadvantages are that when the vein thromboses, it causes upper extremity swelling, and that the small space between the first rib and the clavicle is often a problem for the placement of large-diameter catheters. Pneumothorax is the most common complication when using this route. Basilic vein Even though this vein is associated with more valves, it is the preferred vein for insertion of peripherally inserted central catheters because it is a shorter, straighter vein, which enables easier advancement of the catheter to a central vein location. Cephalic vein This is an easily palpable vein that is longer than the basilic vein. The angle of entrance into the subclavian vein can result in a more difficult peripherally inserted central catheter insertion. Femoral vein This vein is used more often in children. The tip of the catheter should be located in the inferior vena cava below the renal veins. Maintaining a sterile dressing can be difficult, and there is a high risk of complications such as thrombosis and infection [22].

Insertion technique Long-term intravenous catheters are better placed in a surgery or radiology suite or a similar outfitted procedure room in order to minimize the incidence of infections. Most procedures are performed on an outpatient basis or immediately prior to chemotherapy admission. Local anesthesia with monitored care and short-acting barbiturates, or conscious sedation with short-acting agents, provides safe and excellent patient sedation [23]. The use of prophylactic antibiotics during the insertion of longterm venous access is controversial; a systematic review of several randomized studies failed to show a significant benefit for this practice [24]. The use of intraoperative fluoroscopy to document appropriate catheter placement and to prevent complications is highly recommendable.

In a prospective study done by experienced operators, placement of catheters without fluoroscopic guidance resulted in a 6% incidence of catheter misplacement that required repositioning under fluoroscopy at a later time [25]. The most common technique used in catheter placement is the percutaneous method of Seldinger, which uses the subclavian or internal jugular vein. Special care should be taken during the introduction of the wire. In most cases, the guidewire should not be introduced without fluoroscopic guidance for more than 18 cm, which is the average distance in adults from most puncture sites to the atrial-caval junction in adults [26]. Alternatively, a cutdown over the cephalic, external or internal jugular vein can provide venous access. For percutaneous approach, the patient is placed supine, in the Trendelenburg position. The anterior chest, neck, and shoulders are prepared in a sterile manner. The site for venous access is infiltrated with anesthetic, and the right side is most commonly preferred for the internal jugular vein, and the left for the subclavian vein [15,16,27]. The use of ultrasound can aid the placement of the needle, especially when an internal jugular vein approach is attempted [18,28]. A systematic review of 18 randomized trials showed that ultrasound guidance for internal jugular vein cannulation was more effective and less expensive than the landmark method, resulting in a faster and higher rate of success in catheter placement in both children and adults [29]. The needle is advanced bevel up carefully and slowly into the vein, while aspirating with the attached syringe (without Luer-lock). No more than three attempts to puncture any given vein should be tried, and the bilateral percutaneous approach should not be attempted in the same session without the confirmation of absence of complications. Vein penetration is confirmed by easy blood flow into the syringe, the syringe is disconnected, preventing air penetration with the thumb, and a flexible J-guidewire is advanced through the needle for no more than 18 cm for most insertion sites and 16 cm for the right internal jugular vein; fluoroscopy confirms the placement of the wire at the atrial-caval junction. Placement of the catheter without fluoroscopic guidance should be discouraged. Pulsatile flow indicates an arterial puncture. When in doubt, the needle should be connected to a pressure transducer before proceeding. After an arterial puncture, the needle needs to be withdrawn, and local pressure should be applied for 5–10 minutes. Occasionally, in patients with an existent Port-a-Cath, this can be converted to a tunneled device by wiring the existing catheter. This can usually be accomplished if the previous catheter was placed by venipuncture rather than cutdown. This procedure is contraindicated in patients who might have a catheter-related infection and should include routine culture of the removed catheter [30]. Resistance to the advancement of the wire is usually secondary to either misdirection of the wire into a secondary vein or migration outside the vein. Fluoroscopy confirms the wire position; if the wire is outside the vessel, the needle needs to be removed with the wire to prevent its shearing. If the wire requires to be redirected, the needle should be replaced with a 2.5-inch 16-gauge Angiocath, prior to withdrawing the wire. After the successful placement of the wire, the exit site for the catheter, as well as the subcutaneous tunnel between this site and the vein access, is anaesthetized, and the puncture site is enlarged to admit the catheter and introducer sheath. Placement of the sutures at the catheter exit and vein access site prior to tunneling the catheter prevents potential needle catheter punctures. The catheter is tunneled, measured, and custom-cut to reach the SVC–right atrium junction (approximately the fourth anterior intercostal space) or the tracheal bifurcation under fluoroscopy. The catheter cuff is placed within 2 cm of the exit site. The dilator and the peel-away introducer sheath are advanced slowly over the wire under frequent fluoroscopy control, gently withdrawing and advancing the wire to confirm that the dilator and sheath are threading over the wire and not migrating outside the vessel. The dilator and

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Fig. 83.4 X-ray showing a dual-lumen silicone catheter inserted into the internal jugular vein. Fig. 83.5 X-ray showing placement of a subcutaneous single-lumen venous port into the right internal jugular vein.

wire are withdrawn, and the catheter is advanced through the introducer to the SVC–right atrium junction. Catheter tip placement within the right atrium is associated with a high incidence of right atrial thrombus and occasional supraventicular arrhythmias [31]. Prior to the removal of the introducer, the catheter position should be confirmed by fluoroscopy. Failure to position the beam perpendicular to the patient will provide a false impression of the catheter position. Difficulties in advancing the catheter through the introducer usually imply that the introducer is bent, in which case the introducer should be peeled away slowly while advancing the catheter. This is a frequent occurrence if the catheter is placed through the costoclavicular ligament. The catheter should never be handled with sharp instruments, and only nontoothed forceps should be used if needed (Fig. 83.4). The port placement is similar except for the creation of the subcutaneous pocket. The pocket needs to be placed over a hard surface (chest, arm, thigh or pelvis) and should be easily palpable and big enough to allow a no-tension wound closure. The port is sutured to the muscular fascia to prevent port migration or turning. When the catheter placement has been completed, the catheter position should be examined with fluoroscopy to rule out any kinking that would prevent normal functioning. Catheter infusion (no resistance) and withdrawal (no turbulence) should be tested in the surgical suite prior to heparinization of the lumina. In the recovery room, a chest X ray should be obtained to rule out complications (Fig. 83.5). Catheter care Implanted ports require flushing with heparin solution (2–3 mL 100 U/ mL) every 4 weeks or after each use. During treatment, the noncoring (Huber) needle is replaced every fifth to seventh day. External catheters require daily care with antiseptic solutions; a meta-analysis showed a 50% reduction in catheter-related infections when chlorhexidine gluconate alcohol-based solutions rather than povidone–iodine solutions were used for entry site care [32]. There is considerable controversy regarding the best occlusive dressing. A systematic review of all randomized trials failed to show a difference in the incidence of infection between gauze and tape versus transparent polyurethane film dressings [33].

Hickman catheters need to be flushed daily with heparin solution (2–3 mL 100 U/mL) or after each use, and protective caps need to be replaced biweekly. Several studies, especially in children, have documented that the use of vancomycin (25 μg/mL) in the lock solution reduces the incidence of catheter-related infections without increasing the incidence of vancomycin-resistant infections [24,34]. Recently, studies have shown high levels of catheter patency with the use of nonheparin-containing solutions when a positive pressure catheter cap is used [35]. Catheters with Groshong tips require weekly flushing with 5 mL of saline solution.

Complications Complications related to catheter insertion Complications during catheter placement can be related to the method of insertion or to the experience of the operator. The overall incidence of major complications is approximately 2–5% [36]. The use of imaging during catheter insertion can significantly reduce the incidence of complications, especially during insertion. The incidence of complications with the subclavian or internal jugular approach is similar in prospective randomized studies [37]. In general, the incidence of catheter malposition is more frequent with the subclavian approach, and the incidence of arterial puncture is more frequent with internal jugular insertion. Pneumothorax This is the most frequent major complication when using the percutaneous technique. A higher incidence of up to 17% is seen in nutritionally compromised and emaciated patients. This complication is also related to the number of attempts to access the vessel and the experience of the operator. Several steps can be taken in order to avoid this complication. The use of a cutdown technique should be encouraged in high-risk patients. Using an ultrasound-guided percutaneous internal jugular vein approach can also effectively avoid this complication. The withdrawal

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of air during the insertion of the needle is usually secondary to a loose connection between the syringe and the needle rather than a lung puncture. Pneumothorax can be due to a tear of the pleura or the lung parenchyma. This complication is usually recognized in the postoperative upright expiration chest film, but in some cases is not seen until a few hours or days after insertion. Patients that complain of shortness of breath or pleuritic pain hours or days after the procedure should have a repeat chest X-ray to rule out a delayed pneumothorax or other catheterrelated complication. A pneumothorax of less than 30% in an otherwise asymptomatic patient can be observed as long as the pneumothorax does not increase in subsequent films. Symptomatic patients or those with a larger pneumothorax are best treated with a small anterior chest tube thoracostomy connected to a Heimlich (one-way) valve, water seal or suction [38,39]. Arterial punctures This frequent and underreported complication is seen more commonly with the internal jugular than the subclavian vein approach. This complication can be avoided with the use of ultrasound guidance to access the internal jugular vein [29]. The presence of pulsatile flow from the needle confirms the arterial puncture; removal of the needle and compression of the vessel for 5 minutes is usually all that is necessary. If initially missed, the placement of the guidewire to the left of the thoracic spine should be evidence of an arterial puncture (in patients with persistent left vena cava, the wire will be to the left of the spine as well; an intraoperative venogram will confirm the diagnosis). If unsure, an Angiocath needs to be placed over the wire and connected to a blood pressure transducer. The introducer sheath should not be inserted until the operator is sure that the guidewire is in the vein.

sheath is 20 cm long; usually no more than 16 cm from the right side and 18 cm from the left side is required to reach the desired catheter tip location. Furthermore, the dilator should only be introduced until the peel-away sheath is in the vein. It can then be withdrawn into the sheath to prevent potential perforations with the tip of the dilator. Most of these patients can be treated with a large lateral tube thoracostomy connected to a closed thoracic suction system. Many of these systems have a blood reinfusion collecting system. Thoracotomy is indicated in patients with persistent bleeding (more than 500 mL per hour) or with massive hemothorax (more than 1500 mL). Catheter tip malposition This problem is usually recognized and corrected at the time of surgery with the use of fluoroscopy. Catheters placed in the azygos or internal mammary veins can mimic a SVC placement. These catheters frequently do not withdraw blood easily, and the tip of the catheter does not move with the heart rate; a lateral view of the thorax or a venogram will confirm the position. Placement of the catheter in the proximal vena cava or brachiocephalic trunk usually results in venous thrombosis (Fig. 83.7). The ideal tip location is at the atrial-caval junction, with a free-floating tip. Catheter migration after the placement can usually be corrected with an angiographic guidewire placed through the femoral vein. Catheter occlusion Placement of the catheter medial and close to the clavicle may cause a compression of the catheter in between the clavicle and the first rib. This complication, which may cause fracture and distal embolization of the catheter, can be recognized by the compression of the lumen (pinch-off sign) (Fig. 83.8). It can be prevented by selecting a more lateral and lower puncture site. If recognized during placement, the catheter should be repositioned through a different venous access site.

Hemothorax This complication, usually due to the perforation of one of the major vessels with the introducer, the needle or the wire during the catheter placement, is fortunately rarely seen (Fig. 83.6) [37,40]. Careful attention to the technique and use of fluoroscopy during advancement of the introducer should prevent this complication. Special attention should be given to the length of the introducer sheath. In the majority of trays, this

Catheter entry site bleeding and hematomas

Fig. 83.6 X-ray showing a large extrapleural hemothorax (arrows) following placement of a dual-lumen external silicone catheter in a patient with a mediastinal tumor. Notice the placement of a large chest tube.

Fig. 83.7 X-ray showing right internal jugular vein insertion in the upper third of the superior vena cava. Short catheters usually result in venous thrombosis and catheter dislodgement.

These complications, which can be prevented with the correction of clotting factors, are frequent in thrombocytopenic patients. Compression

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Fig. 83.8 X-ray showing subclavian insertion of subcutaneous port with compression of the catheter between the first rib and the clavicle (“pinch-off” sign).

over the tunnel and the catheter entry site while the coagulopathy is corrected usually prevents the problem. Air embolization This is an infrequently recognized complication during catheter placement that can result in a life-threatening complication. This is especially true in children and patients with spontaneous breathing. The best method to avoid this complication is to ask the patient to do a Valsalva maneuver, or by requesting the anesthesiologist to provide positive ventilation while introducing the guidewire into the needle or the catheter into the peel-away sheath [41]. Cardiac tamponade This insertion complication usually occurs secondary to the guidewire or the dilator. It can be avoided if neither is advanced beyond the atrialcaval junction. The use of fluoroscopy during the procedure helps to prevent this usually fatal complication. This complication, which is sometimes not immediately recognized, can be the result of the migration of the catheter into the pericardium after its placement [42]. Cardiac arrhythmias This complication is usually secondary to the wire placement, and some surgeons use the presence of arrhythmias to confirm the placement of the wire into the right atrium. This can usually be avoided if the wire is not advanced beyond 18 cm. Usually, these arrhythmias are limited to a few extrasystoles or a short run of supraventricular tachycardia that stops after withdrawing the wire. This complication can also occur after placing the catheter if the tip stimulates the atrial node. This can be avoided by not placing the catheter beyond the atrial-caval junction. Occasionally, this is not recognized while the patient is still in the operating room; the arrhythmia might be diagnosed when the patient is in recovery or might be suspected days after the catheter placement. Patients describe a rapid heart rate usually when they adopt certain positions. To establish the diagnosis, they should be connected to a monitor while they change positions. In rare occasions, a 24-hour Holter monitor is necessary. The presence of a new atrial arrhythmia after surgery usually requires repositioning of the catheter. Nerve injury This infrequent complication is due to the needle placement. It is more common with supraclavicular approaches. This can be avoided by using ultrasound during the needle placement. Rarely, it occurs secondary to a hematoma or other local complication. The brachial plexus is the most commonly affected. Even though the most common symptom is pain, occasionally the patient might also present motor symptoms. Ordinarily,

Fig. 83.9 X-ray showing catheter tip placed in the innominate vein resulting in venous thrombosis. The injection of contrast through the catheter in this single frame from a videofluoroscopic study shows no distal flow but contrast reflux around the catheter (fibrin sheath) to small collaterals in the supraclavicular region.

these symptoms are detectable immediately after the procedure. Failure to immediately recognize them is usually due to the effects of the local or general anesthesia. The presence of severe pain that persists days after the procedure, especially if it radiates to the arm or is associated with motor deficits, should be suspicious for a nerve injury. Imaging studies to rule out a hematoma and nerve conduction studies are sometimes needed to confirm the diagnosis. Patients with persistent and out of the ordinary pain days after the procedure sometimes require the removal and repositioning of the catheter into another site. Motor symptoms may require months to recover. Catheter-related complications Catheter occlusion Inability to withdraw or infuse through a catheter can be secondary to catheter tip malposition, kinking, thrombosis of the vessel or the catheter, or intraluminal precipitation of medications. After confirming the correct position of the catheter with a chest X-ray, 5000 U in 2 mL urokinase should be injected into the catheter. After 30 minutes, the urokinase should be withdrawn and the catheter tested again. This can be repeated twice and left for longer periods (24 hours) if not effective [43]. Alternatively, 2 mg recombinant tissue plasminogen activator can be used instead of urokinase; the tissue plasminogen activator should be left for 30–120 minutes and the dose repeated for a second and third time if catheter patency is not established [44]. This protocol restores catheter flow in 87% of patients [45]. When compared with regular heparin flushing, the use of prophylactic urokinase has failed to prevent these complications [46]. Inability to withdraw from a catheter might also be due to the development of a fibrin sheath that excludes the catheter from the lumen of the vein. This complication, which occurs in 50–80% of the catheters, does not always result in catheter occlusion. In these patients, a Valsalva maneuver or repositioning of the patient can sometimes allow a successful blood draw. A venogram can usually confirm this etiology (Fig. 83.9). These patients rarely respond to a urokinase bolus. An infusion

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Catheter malfunction

Complete occlusion of all the catheter lumina

Partial occlusion of one or more catheter lumina

Catheter contrast study to determine if there is some minimal patency

Chest X-ray to rule out catheter kinking or other mechanical factors

If occurs immediately following infusion, suspect chemical

Urokinase or t-PA catheter injection. Repeat up to three times

Correctable catheterrelated problem

Noncorrectable catheter-related problem

Hydrochloric acid or bicarbonate irrigation. Repeat up to three times

Catheter contrast study to rule out fibrin sheath or venous thrombosis

Appropriate intervention

Catheter removal

Pain with infusion

Chest X-ray to rule out catheter fracture or dislodgement

Urokinase or t-PA infusion for 12 hours

Fig. 83.10 Catheter malfunction algorithm. t-PA, tissue plasminogen activator.

of 200 U/kg/hour, not to exceed 100,000 U/hr, for 6–12 hours is usually more effective. Patients with contraindication for thrombolytic therapy should not be treated with this protocol; fibrinogen levels should be checked and kept above 100 mg/dL. Chemical occlusion can be treated with instillation of a mixture of 0.01 N hydrochloric acid and heparin for calcium–phosphorus precipitation or low-pH medications and sodium bicarbonate for high-pH medications [47]. The solution is irrigated in and out for 2 minutes, clamped for 1 hour, and then aspirated. A suggested algorithm for the management of catheter occlusions is outlined in Fig. 83.10. Catheter damage/fracture External catheters can be damaged at the site of the clamp or the suture. The use of needleless connections for infusions and irrigations should prevent needle damage. Most catheters have kits to replace the external portion of the catheter. Repair of the catheter has not been associated with an increased incidence of infection. Intravascular catheter fractures usually occur as a consequence of compression between the first rib and the clavicle (Fig. 83.11). This usually can be recognized on a chest X-ray by the “pinch-off” sign [48]. Patients with this syndrome usually have intermittent and positional occlusion of the catheter flow. A segment of the catheter that embolizes into the heart or the pulmonary circulation can usually be retrieved with a snare through the femoral vein by an interventional radiologist (Fig. 83.12) [49]. Drug extravasation Accidental dislodgement of the needle from the port may lead to extravasation of the chemicals into the subcutaneous tissue. This may lead to chemical cellulitis, tissue damage, necrosis or loss of soft tissue. Clinical signs of extravasation are changes at the infusion site with pain, burning, swelling or erythema. If these are suspected, the infusion should be stopped and the needle withdrawn. Management of extravasation injuries depends on the type of drug infusing and on the amount extravasated. Occasionally, the patient will complain of pain along the tunnel or at the insertion site during infusion. The catheter should not be used until a contrast study is obtained to document the catheter integrity and patency. In rare occasions, the extravasation is due to a fibrin sheath that

Fig. 83.11 X-ray showing catheter fracture at the junction between the first rib and the clavicle with the free catheter segment in the right ventricle.

excludes the catheter from the main lumen of the vein, causing a backflow of the solutions around the catheter to the entry site. Catheter-related venous thrombosis Venograms done in prospective studies 6 weeks after catheter placement have shown evidence of venous thrombosis in up to 36% of the patients, with only 6% of them being symptomatic. The incidence of catheterrelated infections increases the incidence of catheter-related venous thrombosis in hematology patients [50]. Ipsilateral arm swelling, pain,

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and development of collaterals should alert the physician to the possibility of venous thrombosis. Venogram, computed tomography scan, and duplex Doppler venous imaging can establish the diagnosis and the site of the obstruction (Fig. 83.13). The treatment should be directed to the prevention of pulmonary embolisms, avoidance of the clot propagation, prevention of the post-phlebitic syndrome, and preservation of the catheter whenever possible. With these considerations in mind, the catheter should be removed only if it is no longer necessary or after failure of the initial therapy [51]. It is also important to note that up to 20% of patients with catheter-related venous thrombosis may have this hypercoagulable state due to inherited conditions such as a factor V Leiden gene mutation [52]. The 60-day mortality of upper extremity or internal jugular deep vein thrombosis is reported to be up to 30%, usually secondary to the underlying disease rather than the vein thrombosis; pulmonary embolism is seen in 3–5% of the patients [53,54]. Patients should be treated with systemic heparin or subcutaneous low molecular weight heparin (LMWH) for 5–10 days prior to changing to oral anticoagulation. Continuation on LMWH should be considered for patients with a high risk for recurrent thrombosis [55]. Fibrinolytic

agents are also effective in the management of this complication and seem to be associated with less post-phlebitic syndrome. The incidence of catheter-related venous thrombosis is associated with the catheter tip position (the more proximal, the more likely the development of venous thrombosis), the size of the catheter (especially triple lumen), and the access site (percutaneous access having a higher incidence of thrombosis than vein cutdown). The use of low-dose daily coumarin (1 mg/day) or LMWH as a prophylaxis for catheter-related deep vein thrombosis remains controversial. The use of low-dose coumarin prophylaxis has been shown to be safe in patients treated with high-dose chemotherapy and stem cell transplantation as long as the platelet count is above 50,000 /dL [56]. A systematic review of the evidence showed that there is no conclusive evidence to recommend the routine use of venous thrombosis prophylaxis [57,58]. A suggested algorithm for the management of catheter-related venous thrombosis is outlined in Fig. 83.14.

Fig. 83.12 Single frame from a continuous fluoroscopy scan showing retrieval of a catheter fragment from the right ventricle using an endoloop inserted through the femoral vein.

Fig. 83.13 Venous ultrasound scan showing subclavian vein thrombosis on the left. Note the venous thrombus in the lumen. On the right, there is external compression of the vein with minimal change.

Catheter-related infection Catheter-related systemic infections are potentially the most life-threatening complications, especially when they occur in the immunocompromised patient. The incidence of this complication can significantly be diminished by strictly following the catheter care protocols discussed in the previous sections. The diagnosis of a catheter-related infection requires the isolation of a three- to fivefold increase in colony-forming organisms per unit of blood from cultures drawn from the catheter, compared with concomitant peripherally drawn blood cultures, or more than 100 colony-forming units on cultures obtained from the device in the absence of peripheral cultures [59]. Infections are related to the

Neck or extremity swelling or pain

Chest X-ray to rule out catheterrelated problems. Venous duplex ultrasound to rule out thrombosis

Computed tomography scan with contrast to rule out venous thrombosis

Fig. 83.14 Algorithm for venous thrombosis.

Initiate anticoagulation therapy with low molecular weight heparin, followed by coumarin

Remove catheter if no longer needed, symptoms do not improve or anticoagulation is contraindicated

Do not remove catheter if catheter is needed and symptoms improve

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frequency of use, number of lumina, type of dressing, and patient age (with a higher incidence of infections among younger patients). Several randomized studies comparing external tunneled catheters with implantable ports have failed to show differences in the incidence of infections among these devices. In a systematic review of 200 prospective studies, Maki et al. [60] report that the incidence of catheter-related infections with external cuffed tunneled catheters is 1.6% per 1000 catheter days compared with 0.1% per 1000 catheter days for implanted ports, and 1% per 1000 catheter days for PICCs. The usage of a second antimicrobial cuff in the external devices has failed to reduce the incidence of systemic or local infections. Most of the infections are secondary to Gram-positive cocci (coagulase-negative staphylococci) and Gram-negative bacilli (enterobacteriaceae and Pseudomonas aeruginosa); only 3.5% of the infections are fungally related (Candida). Whenever an infection is suspected, appropriate antibiotic coverage should be instituted after the blood cultures are drawn, and antibiotic selection needs to be reassessed after the cultures are back. There are no randomized studies documenting the catheter salvage rates following antibiotic treatment. Several studies report catheter salvage rates of up to 50% following antibiotic treatment, at the expense of higher infection recurrence [61,62]. Indications for catheter removal are the persistence of symptoms or positive blood cultures after 3 days of appropriate antibiotic coverage, hypotension, severe systemic

compromise, infections secondary to fungi or Staphylococcus aureus (these organisms being rarely eradicated with antibiotic treatment), and recurrence of infections secondary to the same organism after successful treatment. Tunnel or pocket infections are less common. The presence of swelling, tenderness, and warmth over the area is usually an indication of a local infection. In leukopenic patients, pain and fever are frequently the only symptoms. These infections are usually secondary to Gram-positive cocci. Culture of the exit site or the skin around the Huber needle will normally isolate the microorganism. Local care, removal of the Huber needle, and systemic antibiotics are measures needed in the treatment of most of these infections. Staphylococcus aureus and fungal infections usually require removal of the catheter. A recent prospective randomized study using new catheter technology with silicone impregnated catheters with minocycline and rifampin showed that these noncuffed, nontunneled catheters were as likely to be colonized as standard cuffed, tunneled silicone catheters [63]. In the same study, bloodstream infection was four times less likely to originate from antimicrobially impregnated than from tunneled catheters (0.36 versus 1.43 per 1000 catheter–days), but the antimicrobially impregnated catheters remained in place for a shorter period of time (mean 30.2 versus 43.8 days). A suggested algorithm for the management of catheter-related infections is outlined in Fig. 83.15.

Fever or other systemic symptoms of infection. Local changes, drainage or pain over the tunnel

Culture peripheral blood and from each catheter lumen; urine and sputum culture; remove unused catheters

Non-leukopenic. May continue observation as long as there is no systemic infection

Skin changes, tunnel drainage or pain over the catheter tunnel or insertion sites

Leukopenic. Broad antibiotic coverage

Local cultures from the tunnel and entry sites. Rule out venous thrombosis

Threefold increase in colonies when compare catheter with peripheral blood cultures

Broad Gram-positive bacteria antibiotic coverage

Antibiotic coverage according to blood and other culture results

Good systemic response and negative repeat catheter cultures

Keep catheter

New catheter-related infection due to the same organism

No response after 3 days of treatment or positive cultures due to Staphylococcus aureus or fungal elements

Remove catheter

Fig. 83.15 Algorithm for catheter-related infection.

Vascular Access and Complications

Catheter removal There are several methods for removal of a long-term intravenous cuffed catheter. In general, removal of the catheter and the cuff is preferred and recommended by the catheter manufacturer. This can be accomplished by catheter traction when the cuff is close to the insertion site [64]. Anesthetic infiltration around the cuff and gentle freeing of the cuff through the tunnel with a curved hemostat clamp usually helps with cuff removal. When the cuff is high in the tunnel, a cutdown technique is

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usually necessary to remove the cuff [65]. Alternatively, a traction technique can be used, which often results in leaving the cuff behind [66]. The incidence of retained cuff infections is low and requires cuff removal [67,68]. The traction technique for catheter removal can result in the fracture of the catheter at the cuff, requiring a cutdown to remove the cuff and the remainder of the catheter. The length of the fractured segment helps to determine the location of the cuff. This complication is more frequent with small-lumen and non-silicone catheters. Compression of the tunnel prevents bleeding or air embolism during the procedure.

References 1. Cosnett JE. The origins of intravenous fluid therapy. Lancet 1989; 333: 768–71. 2. Roy R, Wilkinson R, Bayliss C. The utilization of long nylon catheters for prolonged intravenous infusions. Can Med Assoc J 1967; 96: 94–7. 3. Broviac JW, Cole JJ, Scribner BH. A silicone rubber atrial catheter for prolonged parenteral alimentation. Surg Gynecol Obstet 1973; 136: 602– 6. 4. Hickman RO, Buckner CD, Clift RA, Sanders JE, Stewart P, Thomas ED. A modified right atrial catheter for access to the venous system in marrow transplant recipients. Surg Gynecol Obstet 1979; 148: 871–5. 5. Wenzel RP, Edmond MB. The evolving technology of venous access. N Engl J Med 1999; 340: 48–50. 6. Kumagai T, Sakamaki H, Tanikawa S et al. Utility and safety of Hickman catheters for venous access after bone marrow transplantation. Intern Med 1998; 37: 286–91. 7. Biffi R, Pozzi S, Agazzi A et al. Use of totally implantable central venous access ports for highdose chemotherapy and peripheral blood stem cell transplantation: results of a monocentre series of 376 patients. Ann Oncol 2004; 15: 296–300. 8. Lazarus HM, Trehan S, Miller R, Fox RM, Creger RJ, Raaf JH. Multi-purpose silastic dual-lumen central venous catheters for both collection and transplantation of hematopoietic progenitor cells. Bone Marrow Transplant 2000; 25: 779–85. 9. Harter C, Ostendorf T, Bach A, Egerer G, Goldschmidt H, Ho AD. Peripherally inserted central venous catheters for autologous blood progenitor cell transplantation in patients with haematological malignancies. Support Care Cancer 2003; 11: 790–4. 10. Cheong K, Perry D, Karapetis C, Koczwara B. High rate of complications associated with peripherally inserted central venous catheters in patients with solid tumours. Intern Med J 2004; 34: 234– 8. 11. Beebe DS, Beck D, Belani KG. Comparison of the flow rates of central venous catheters designed for rapid transfusion in infants and small children. Paediatr Anaesthes 1995; 5: 35–9. 12. Horattas MC, Trupiano J, Hopkins S, Pasini D, Martino C, Murty A. Changing concepts in longterm central venous access: catheter selection and cost savings. Am J Infect Control 2001; 29: 32– 40. 13. Safdar N, Maki DG. Risk of catheter-related bloodstream infection with peripherally inserted central venous catheters used in hospitalized patients. Chest 2005; 128: 489–95.

14. Restrepo A, Devore P, Encarnacion CE et al. Performance of a hybrid central venous catheter utilized for both peripheral blood stem cell harvest and transplant support of patients undergoing autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 2002; 30: 389–95. 15. Muralidhar K. Left internal versus right internal jugular vein access to central venous circulation using the Seldinger technique. J Cardiothorac Vasc Anesth 1995; 9: 115–16. 16. Unal AE, Bayar S, Arat M, Ilhan O. Malpositioning of Hickman catheters, left versus right sided attempts. Transfus Apher Sci 2003; 28: 9–12. 17. Petersen J, Delaney JH, Brakstad MT, Rowbotham RK, Bagley CM, Jr. Silicone venous access devices positioned with their tips high in the superior vena cava are more likely to malfunction. Am J Surg 1999; 178: 38–41. 18. Karakitsos D, Labropoulos N, De Groot E et al. Real-time ultrasound-guided catheterisation of the internal jugular vein: a prospective comparison with the landmark technique in critical care patients. Crit Care 2006; 10: R162. 19. Hinke DH, Zandt-Stastny DA, Goodman LR, Quebbeman EJ, Krzywda EA, Andris DA. Pinchoff syndrome: a complication of implantable subclavian venous access devices. Radiology 1990; 177: 353–6. 20. Sadler DJ, McCarthy M, Saliken JC et al. Imageguided central venous catheter placement for apheresis in allogeneic stem cell donors. J Clin Apher 2000; 15: 173–5. 21. von Goedecke A, Keller C, Moriggl B et al. An anatomic landmark to simplify subclavian vein cannulation: the “deltoid tuberosity”. Anesth Analg 2005; 100: 623–8. 22. Lorente L, Henry C, Martin MM, Jimenez A, Mora ML. Central venous catheter-related infection in a prospective and observational study of 2,595 catheters. Crit Care 2005; 9: R631–5. 23. Pratila MG, Fischer ME, Alagesan R, Alagesan R, Reinsel RA, Pratilas D. Propofol versus midazolam for monitored sedation: a comparison of intraoperative and recovery parameters. J Clin Anesth 1993; 5: 268–74. 24. van de Wetering MD, van Woensel JB. Prophylactic antibiotics for preventing early central venous catheter Gram positive infections in oncology patients. Cochrane Database Syst Rev 2007(1): CD003295. 25. Muhm M, Kalhs P, Sunder-Plassmann G, Apsner R, Brugger S, Druml W. Percutaneous nonangiographic insertion of Hickman catheters in marrow transplant recipients by anesthesiologists and intensivists. Anesth Analg 1997; 84: 80–4.

26. Andrews RT, Bova DA, Venbrux AC. How much guidewire is too much? Direct measurement of the distance from subclavian and internal jugular vein access sites to the superior vena cava-atrial junction during central venous catheter placement. Crit Care Med 2000; 28: 138–42. 27. Madden B, Paruchuru P, Kunst H. Sucking noise and collapse after central venous catheter removal. J R Soc Med 2000; 93: 592–3. 28. Muhm M. Ultrasound guided central venous access. BMJ 2002; 325: 1373–4. 29. Hind D, Calvert N, McWilliams R et al. Ultrasonic locating devices for central venous cannulation: meta-analysis. BMJ 2003; 327: 361. 30. Brodwater BK, Silber JS, Smith TP et al. Conversion of indwelling chest port catheters to tunneled central venous catheters. J Vasc Interv Radiol 2000; 11: 1137–42. 31. Gilon D, Schechter D, Rein AJ et al. Right atrial thrombi are related to indwelling central venous catheter position: insights into time course and possible mechanism of formation. Am Heart J 1998; 135: 457–62. 32. Chaiyakunapruk N, Veenstra DL, Lipsky BA, Saint S. Chlorhexidine compared with povidone-iodine solution for vascular catheter-site care: a metaanalysis. Ann Intern Med 2002; 136: 792–801. 33. Gillies D, O’Riordan L, Carr D, Frost J, Gunning R, O’Brien I. Gauze and tape and transparent polyurethane dressings for central venous catheters. Cochrane Database Syst Rev 2003(4): CD003827. 34. Safdar N, Maki DG. Use of vancomycin-containing lock or flush solutions for prevention of bloodstream infection associated with central venous access devices: a meta-analysis of prospective, randomized trials. Clin Infect Dis 2006; 43: 474–84. 35. Schilling S, Doellman D, Hutchinson N, Jacobs BR. The impact of needleless connector device design on central venous catheter occlusion in children: a prospective, controlled trial. JPEN J Parenter Enteral Nutr 2006; 30: 85–90. 36. Whitman ED. Complications associated with the use of central venous access devices. Curr Probl Surg 1996; 33: 309–78. 37. Ruesch S, Walder B, Tramer MR. Complications of central venous catheters: internal jugular versus subclavian access – a systematic review. Crit Care Med 2002; 30: 454–60. 38. Reed MF, Lyons JM, Luchette FA, Neu JA, Howington JA. Preliminary report of a prospective, randomized trial of underwater seal for spontaneous and iatrogenic pneumothorax. J Am Coll Surg 2007; 204: 84–90. 39. Roggla M, Wagner A, Brunner C, Roggla G. The management of pneumothorax with the thoracic

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41.

42.

43.

44.

45.

46.

47.

48.

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vent versus conventional intercostal tube drainage. Wien Klin Wochenschr 1996; 108: 330–3. Meisenberg BR, Callaghan M, Sloan C, Sampson L, Miller WE, McMillan R. Complications associated with central venous catheters used for the collection of peripheral blood progenitor cells to support high-dose chemotherapy and autologous stem cell rescue. Support Care Cancer 1997; 5: 223–7. Wysoki MG, Covey A, Pollak J, Rosenblatt M, Aruny J, Denbow N. Evaluation of various maneuvers for prevention of air embolism during central venous catheter placement. J Vasc Interv Radiol 2001; 12: 764–6. Fletcher S, Bodenham A. Safe placement of central venous catheters: where should the tip lie? Br J Anaesth 2000; 85: 188–92. Deitcher SR, Fraschini G, Himmelfarb J et al. Dose-ranging trial with a recombinant urokinase (urokinase alfa) for occluded central venous catheters in oncology patients. J Vasc Interv Radiol 2004; 15: 575–80. Gorski LA. Central venous access device occlusions. Part 1. Thrombotic causes and treatment. Home Healthc Nurse 2003; 21: 115–23. Deitcher SR, Fesen MR, Kiproff PM et al. Safety and efficacy of alteplase for restoring function in occluded central venous catheters: results of the cardiovascular thrombolytic to open occluded lines trial. J Clin Oncol 2002; 20: 317–24. Solomon B, Moore J, Arthur C, Prince HM. Lack of efficacy of twice-weekly urokinase in the prevention of complications associated with Hickman catheters: a multicentre randomised comparison of urokinase versus heparin. Eur J Cancer 2001; 37: 2379–84. Gorski LA. Central venous access device occlusions. Part 2. Nonthrombotic causes and treatment. Home Healthc Nurse 2003; 21: 168–73. Mirza B, Vanek VW, Kupensky DT. Pinch-off syndrome: case report and collective review of the literature. Am Surg 2004; 70: 635–44. Dinkel HP, Muhm M, Exadaktylos AK, Hoppe H, Triller J. Emergency percutaneous retrieval of a

50.

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silicone port catheter fragment in pinch-off syndrome by means of an Amplatz gooseneck snare. Emerg Radiol 2002; 9: 165–8. van Rooden CJ, Schippers EF, Barge RM et al. Infectious complications of central venous catheters increase the risk of catheter-related thrombosis in hematology patients: a prospective study. J Clin Oncol 2005; 23: 2655–60. Kovacs MJ, Kahn SR, Rodger M et al. A pilot study of central venous catheter survival in cancer patients using low molecular weight heparin (dalteparin) and warfarin without catheter removal for the treatment of upper extremity deep vein thrombosis (the catheter study). J Thromb Haemost 2007; 5: 1650–3. Mandala M, Curigliano G, Bucciarelli P et al. Factor V Leiden and G20210A prothrombin mutation and the risk of subclavian vein thrombosis in patients with breast cancer and a central venous catheter. Ann Oncol 2004; 15: 590–3. Hingorani A, Ascher E, Markevich N et al. Risk factors for mortality in patients with upper extremity and internal jugular deep venous thrombosis. J Vasc Surg 2005; 41: 476–8. Joffe HV, Kucher N, Tapson VF, Goldhaber SZ. Upper-extremity deep vein thrombosis: a prospective registry of 592 patients. Circulation 2004; 110: 1605–11. Lee AY, Levine MN, Baker RI et al. Lowmolecular-weight heparin versus a coumarin for the prevention of recurrent venous thromboembolism in patients with cancer. N Engl J Med 2003; 349: 146–53. Magagnoli M, Masci G, Castagna L et al. Prophylaxis of central venous catheter-related thrombosis with minidose warfarin in patients treated with high-dose chemotherapy and peripheral-blood stem-cell transplantation: retrospective analysis of 228 cancer patients. Am J Hematol 2006; 81: 1–4. Cunningham MS, White B, Hollywood D, O’Donnell J. Primary thromboprophylaxis for cancer patients with central venous catheters – a reappraisal of the evidence. Br J Cancer 2006; 94: 189–94.

58. Levine M, Kakkar AK. Catheter-associated thrombosis: thromboprophylaxis or not? J Clin Oncol 2005; 23: 4006–8. 59. Safdar N, Fine JP, Maki DG. Meta-analysis: methods for diagnosing intravascular devicerelated bloodstream infection. Ann Intern Med 2005; 142: 451–66. 60. Maki DG, Kluger DM, Crnich CJ. The risk of bloodstream infection in adults with different intravascular devices: a systematic review of 200 published prospective studies. Mayo Clin Proc 2006; 81: 1159–71. 61. Oppenheim BA. Optimal management of central venous catheter-related infections – what is the evidence? J Infect 2000; 40: 26–30. 62. Coyle VM, McMullan R, Morris TC, Rooney PJ, Hedderwick S. Catheter-related bloodstream infection in adult haematology patients: catheter removal practice and outcome. J Hosp Infect 2004; 57: 325–31. 63. Darouiche RO, Berger DH, Khardori N et al. Comparison of antimicrobial impregnation with tunneling of long-term central venous catheters: a randomized controlled trial. Ann Surg 2005; 242: 193–200. 64. Galloway S, Bodenham A. Safe removal of longterm cuffed Hickman-type catheters. Hosp Med 2003; 64: 20–3. 65. Reed WP, Newman KA, Tenney JH. An improved technique for the removal of long term implantable central venous lines. Surg Gynecol Obstet 1985; 161: 479–80. 66. Kohli MD, Trerotola SO, Namyslowski J et al. Outcome of polyester cuff retention following traction removal of tunneled central venous catheters. Radiology 2001; 219: 651–4. 67. Ruppel LJ, Brown RA, Borson RA, Whitman ED. Retained Hickman catheter cuff as an infection source following allogeneic bone marrow transplant. Bone Marrow Transplant 1994; 14: 169–71. 68. al-Wali WI, Wilcox MH, Thickett KJ, Simmons KM, Spencer RC. Retained Hickman catheter cuff as a source of infection. J Infect 1993; 26: 199– 201.

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Nelson J. Chao & Keith M. Sullivan

Pharmacologic Prevention of Acute Graft-Versus-Host Disease

Historical development In 1955, a description of graft-versus-host disease (GVHD) in animals, then known as “secondary disease,” was reported by Barnes and Loutit [1]. Named to differentiate it from the primary disease of radiation sickness, fatal secondary disease was observed in irradiated mice given allogeneic spleen cells but not in recipients of syngeneic cells. By the late 1950s, it was apparent that the skin abnormalities and diarrhea of secondary disease and runt disease (a wasting syndrome in unirradiated newborn mice given allogeneic spleen cells) were the result of immunologically competent cells introduced into an immunoincompetent host. The term “graft-versus-host” was introduced to describe the vector of this immunologic assault [2,3]. Early human transplants of allogeneic marrow were often complicated by GVHD [4–8]. Features were remarkably similar to those seen in animal studies of GVHD [9] and to reports of GVHD developing in immunodeficient children who had received blood transfusions [10]. Because it has been difficult to separate the illness caused by immunologic attack from the consequences of this assault (including immunodeficiency, organ dysfunction, and infection), both aspects have been considered a part of human GVHD. Historically, the term acute GVHD describes a distinctive syndrome of dermatitis, hepatitis and enteritis developing within 100 days of allogeneic hematopoietic cell transplantation (HCT). However, with the use of reducedintensity conditioning (RIC) regimens, the clinical picture of acute GVHD can occur after 100 days, thus making the day 100 cut-off less relevant. This distinction is not merely semantic because there is a need to clearly define these syndromes in clinical trials.

Pathogenesis In a remarkably perceptive summary in 1966, Billingham [11] defined the criteria for the development of GVHD as follows: 1 The graft must contain immunologically competent cells. 2 The host must possess important transplantation alloantigens that are lacking in the donor graft, so that the host appears foreign to the graft, and is, therefore, capable of stimulating it antigenically. 3 The host itself must be incapable of mounting an effective immunologic reaction against the graft, at least for sufficient time for the latter to manifest its immunologic capabilities; that is, it must have the security of tenure.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

These criteria now require some modification to incorporate current understanding of the biology of GVHD (see Chapters 16). The occurrence of autologous GVHD suggests that inappropriate recognition of host self-antigens may occur, and transfusion-associated GVHD may develop in certain immunocompetent individuals. A multistep model of antigen expression, cytokine production, T-cell activation, and tissue injury has been described and Fig. 84.1 depicts this interactive construct [12,13].

Alloreactivity Abundant animal data demonstrate that T lymphocytes contained in the donor inoculum proliferate and differentiate in vivo in response to disparate histocompatibility antigens on host tissues, and directly, or through secondary mechanisms, attack recipient cells, thus producing the signs and symptoms of acute GVHD (see Chapters 16 and 86) [9,11,14]. The afferent arm of acute GVHD consists of antigen presentation, activation of T cells, clonal proliferation, and differentiation [15,16]. Cell death of host targets results from alloreactivity of donorderived T cells. In the efferent phase, release of cytokines from activated lymphocytes contributes to cell death, either directly or through recruitment of secondary effectors, such as natural killer cells [17]. Based on mechanistic roles postulated for interleukin-1 (IL-1), IL-2, lipopolysaccharide, and proinflammatory cytokines such as IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor (TNF), immunomodulatory agents have been administered in vivo to control GVHD. This alloreactivity is enhanced by the microbial environment, especially of the gastrointestinal tract. Compared with conventionally housed irradiated mice, enteric GVHD was significantly reduced in germ-free mice given incompatible marrow [18]. Human studies confirm this gnotobiotic effect [19,20]. Microorganisms likely act as triggers of GVHD, perhaps by sharing antigenic epitopes with gut epithelial cells or by activating latent virusinducing antigens on cell surfaces to become targets of alloreactivity [21]. Gut damage could also lead to leakage of lipopolysaccharide into the circulation, thereby releasing cytokine effectors of GVHD [22].

Predictive factors Prior to a discussion regarding prevention of acute GVHD, it is important to review the incidence and predictive factors. Some of these factors have been utilized as targets for agents in the prevention of GVHD. Human leukocyte antigen (HLA) disparity between the hematopoietic cell donor and recipient is the most powerful factor governing the severity and kinetics of GVHD. The importance of class I and II allele matching in reducing the risk for GVHD has been widely demonstrated in

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Tissue injury/inflammation

Allogeneic recognition

Tregs

Upregulation of histocompatibility antigens

Effector T cells

Production of cytokines (TNF, IL-1, IL-2, γ-IFN...)

GVHD Fig. 84.1 Interactive events leading to graft-versus-host disease (GVHD). T-cell activation and tissue injury release cytokines that produce the clinical manifestations of GVHD. IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; Tregs, regulatory T cells.

unrelated donor HCT cohorts [23–26]. In a National Marrow Donor Program analysis of individuals undergoing HCT from HLA-A- and B-matched unrelated donors, HLA-DRB1 allele disparity was independently associated with an increased risk of severe acute GVHD [27]. Further studies have determined the effects of HLA matching (low or high resolution or both) on engraftment, GVHD, and mortality in 1874 donor–recipient pairs retrospectively typed at high resolution for HLAA, B, C, DRB1, DQ, and DP. Mismatches at HLA-A, B, C, and DRB1 each had similar adverse effects on mortality. Only HLA-A mismatches demonstrated significant adverse effects on GVHD. These adverse effects on outcome were more evident in transplants with low-resolution versus only high-resolution mismatches. Mismatches for HLA-DQ or DP did not significantly affect outcome. When high-resolution mismatches at HLA-A, B, C, and DRB1 were considered together, adverse effects on survival and GVHD were observed [28]. Minor HLA peptides have been identified and sequenced, and minor histocompatibility antigen (mHA) disparities appear correlated with the risk for GVHD in adults given HLA-identical donor grafts [29,30]. The importance of mHA matching is further supported by the observed low rate of GVHD in populations that feature genetic homogeneity [31]. Sex mismatching and donor parity have been associated with an increased risk of acute GVHD [32–34]. Female donor T cells could recognize H-Y minor antigens on host Y chromosome-containing cells. Moreover, previously parous donors could have experienced alloimmunization as a result of unshared minor antigens of the fetus [35]. Age is another key factor associated with the development of acute GVHD [36–38]. Among patients under 20 years of age given the original 102-day course of intermittent methotrexate (MTX) as GVHD prophylaxis, the incidence of significant (grade II–IV) acute GVHD was approximately 20% [39]. In contrast, the incidence of acute GVHD was 30% in patients 45–50 years old, and 79% in those 51–62 years old [40]. Moreover, using older matched unrelated donors has led to a higher incidence of GVHD in the recipients. The source of allogeneic hematopoietic cells may also influence the development of GVHD. For more than two decades, experience was derived from marrow transplant cohorts describing rates and risk factors for GVHD [41–44]. Virtually all marrow grafts were infused as fresh products, although one group reported apparent reduction in rates of GVHD with cryopreserved marrow [45]. Of considerable interest to stem cell biologists is that umbilical cord blood cells appear less immunologically reactive than bone marrow cells

[46]. When applied in human HCT, there is an apparent reduction in rates of GVHD in recipients, who are usually of younger age [47–49]. Allogeneic peripheral blood progenitor cells (PBPCs) is becoming a more common source for HCT. Initial reports suggested no significant increase in the rates of acute and chronic GVHD when compared with the historical bone marrow transplant (BMT) experience [50,51]. However, with longer follow-up, the increased risk for chronic GVHD appears established [52], and a number of controlled trials comparing outcomes in related marrow or PBPC transplantations have been reported. Faster engraftment, fewer hospitalizations, less donor morbidity, and economic advantages are noted with the use of peripheral blood. A meta-analysis conducted of 16 studies (five randomized and 11 cohort) found that the relative risk (RR) for acute GVHD after PBPC transplantation was 1.16-fold greater (p = 0.006) when compared with BMT [53]. There was also a trend toward lessened rates of relapse. In a cohort study of PBPC compared with BMT from unrelated donors, GVHD rates were similar, but nonrelapse mortality (NRM) was lower and survival was higher after PBPC transplantation (see Chapter 43) [54]. Studies of CD34+ cell dose have shown advantages in resource utilization and cost in autologous peripheral blood HCT. In allogeneic PBPC transplantation, a higher (6.3–10.0 × 106 CD34+ cells/kg) hematopoietic cell dose was associated with a sharp increase in GVHD risk among cyclosporine (CSP) recipients [55]. In contrast, CD34+ cell dose did not appear to influence the occurrence or severity of acute or chronic GVHD in allogeneic BMT [56]. Of interest, a low PBPC dose (<3 × 106 CD34+ cells/kg) was associated with slower hematologic recovery, increased rates of fungal infections and NRM, and decreased overall survival. As shown in Fig. 84.1, activated donor T cells release proinflammatory cytokines which upregulate HLA expression (see Chapter 16). Accordingly, investigators have asked if cytokine gene polymorphisms that alter function and regulation might be predictive of GVHD. An initial study in blood and tissue samples failed to show a clear correlation or prediction of GVHD [57]. Another report found that the genotype in the donor of IL-1 receptor antagonist (allele 2) was protective against severe GVHD developing in the recipient [58]. Single nucleotide polymorphisms were determined in sibling transplants, and recipient genotypes for IFN-γ intron 1, IL-10−1064, and TNF d3 were found to be associated with acute GVHD, whereas the IL-6174 genotype was associated with chronic GVHD [59]. In a similar study of genetic and clinical factors, polymorphisms of IL-10 in both donor (RR 3.5) and recipient (RR 7.9) predicted for acute GVHD along with donor cytomegalovirus (CMV) seropositivity and mHA mismatching [60]. More recently, polymorphisms in the innate immunity gene (NOD2/Card15) have also been associated with chronic GVHD. The incidence of acute GVHD after matched sibling HCT varies inversely with the efficacy of post-transplant immunosuppressive prophylaxis and with the intensity of the preparatory regimen. Multivariate analysis revealed that an increased dose of pretransplant total body irradiation (TBI) was associated with an increased risk of GVHD. Among HLA-identical HCT recipients who received GVHD prophylaxis with MTX and CSP, acute GVHD developed in 48% of patients given 1575 cGy TBI, compared with 21% of patients given 1200 cGy TBI (p = 0.02) [61]. One hypothesis is that more intensive myeloablative conditioning might eliminate persisting host cells and restrict the development of mixed donor–host chimerism, which has been associated with a decreased probability of GVHD [62,63]. Alternatively, more intensive conditioning could increase gastrointestinal damage, modify the microbial environment, release cytokine mediators, and/or attenuate the dose of administered GVHD prophylaxis. Some studies indicate that donor or host infection (as denoted by seropositivity for the herpesviruses) predicts the subsequent development of GVHD [64], whereas others find no predictive association [65,66].

Pharmacologic Prevention of Acute Graft-Versus-Host Disease

Prophylaxis of GVHD had consisted of the most commonly utilized immune-suppressive drugs. These drugs have initially been used as single agents and then in combination. These drugs will be discussed individually below; however, the efficacy for prophylaxis of GVHD relies on combination of these agents. Table 84.1 summarizes the available randomized studies using these drugs. Understanding the relative contributions of these drugs has led to better regimens and ultimately to better outcomes. It is important to point out that the diagnosis of GVHD is a clinical one, with the pathology helping sometimes in confirming the diagnosis. Therefore, the overall grading based on clinical findings is a potential methodologic problem with GVHD prevention trials. There can be significant interobserver differences at a single institution. Moreover, there are significant differences between institutions as to what is determined to be, for example, grade II acute GVHD.

Nonspecific immunosuppressive drugs Corticosteroids This class of drug has been combined with other immunosuppressants in the prophylaxis against GVHD, but is most frequently used as frontline therapy in the treatment of established GVHD. However, we still do not fully understand their mechanism of action. Pharmacology The most commonly utilized corticosteroid is methylprednisolone, which differs from prednisolone and prednisone only by the addition of 6-α-methyl group (Fig. 84.2). The 6-α-methyl group blocks the specific binding of this corticosteroid to transcortin, the plasma protein that carries steroids. Instead, methylprednisolone is primarily bound to albumin. The frequent side-effects from methylprednisolone may be dependent on the albumin level in the host. The lack of transcortin binding leads to a larger partition coefficient, and the result is a significantly greater penetration into bronchial alveolar fluids, which may provide a therapeutic advantage for treatment of pulmonary inflammatory states. Signaling by the steroid molecules is achieved through binding to a specific cytosolic receptor, thereby initiating its pharmacologic effects (Fig. 84.3). This binding of steroid to receptor activates the complex, enabling it to translocate rapidly into the nucleus. There it is associated with specific sequences known as the glucocorticoid response elements. These response elements regulate the messenger RNA expression of certain proteins, leading to the known biologic effects. Generally, larger doses produce a greater intensity and duration of effects. The utility of corticosteroids in GVHD is thought to be related to their lympholytic activity [67]. The presumed activity is that corticosteroids destroy the lymphoid cells, which they certainly do in vitro and in vivo. However, there are cells that survive in both murine and human studies that can mount an immune response [67]. Thus, other mechanisms, such as downmodulation of cytokines, are likely to be responsible as well [68]. There are data to suggest that the major effect of corticosteroids is the suppression of proinflammatory cytokines rather than direct cellular cytotoxicity of lymphocytes [69]. In clinical trials that measured cytokines, the levels of TNF-α decreased after the initiation of corticosteroids used for the therapy of GVHD [70].

1259

chosis or neurotoxicity, as well as myopathy. Other chronic effects such as osteoporosis, cataract formation, and aseptic necrosis also occur. Corticosteroids are sensitive to a variety of drug interactions, specifically anticonvulsants, which may increase the total clearance of unbound corticosteroids. Clinical use Corticosteroids are given intravenously when used as prophylaxis or therapy for acute GVHD. The drug of choice has been methylprednisolone followed by oral prednisone when patients are able to eat sufficiently or when a patient’s diarrhea has resolved so that one can be confident that they will absorb the medication without difficulty. One regimen, which combines CSP, MTX, and prednisone (beginning after all MTX doses have been administered), is among the most potent combination for the prevention of acute GVHD, resulting in only 9% grade II–IV acute GVHD in the best transplant candidates (patients in first remission of acute leukemia or first chronic phase of chronic myeloid leukemia) for allogeneic BMT [71]. The doses of methylprednisolone given for prophylaxis have varied, and most GVHD prevention regimens do not include corticosteroids. In support of the delayed use of corticosteroids are the results noted in a prospective randomized study comparing CSP, MTX, and methylprednisolone versus CSP and MTX in patients receiving BMT from an HLA-identical sibling [72]. MTX was given in four doses (days 1, 3, 6, and 11), and methylprednisolone was not begun until day 14. Acute GVHD was more common in the patients treated without methylprednisolone. This study also noted no difference in relapse or in chronic GVHD. However, a second randomized study found no difference between these two regimens in preventing either acute or chronic GVHD [73]. Based on these data, our current recommendation is to save the use of corticosteroids for the treatment of acute GVHD. Most prophylactic regimens taper the steroid dose to zero by 180 days after HCT. When steroids have been used for the treatment of acute GVHD, the usual methylprednisolone dose is 1–2 mg/kg administered in divided doses with a tapering schedule after about 2 weeks of 10% every 3–5 days. Occasionally, higher pulse doses up to 10 mg/kg have been used. Chronic GVHD treatment dose is started in the range of 1 mg/kg on a daily basis. The goal thereafter is to switch patients to an alternating-day regimen to spare side-effects of corticosteroids [74]. For those patients in the older age range, a small dose of prednisone, similar to those used in autoimmune diseases, may be necessary to suppress reactivation of GVHD. Corticosteroids have also been used as local treatment for GVHD. Beclomethasone 17, 21-dipropionate (BDP) is a synthetic diester of beclomethasone, a corticosteroid analog that has appeal in the treatment of gastrointestinal GVHD by virtue of its ability to direct therapy to inflamed gastrointestinal mucosa. After an initial promising result, a randomized, placebo-controlled, double-blind study evaluating BDP in the treatment of grade II GVHD with gastrointestinal symptoms has been performed [75]. Patients were randomized to receive prednisone for 10 days plus either BDP (8 mg/day) or placebo for 50 days. The results of this study demonstrated that oral BDP was more effective than placebo in restoring oral intake in patients with grade II GVHD with gastrointestinal symptoms, and allows prednisone to be rapidly tapered without a flare of intestinal symptoms. Oral BDP was well tolerated, with discontinuation of drug being higher in the placebo arm rather than the treatment arm. Importantly, there was a survival advantage for those on BDP that lasted past the first year of randomization.

Toxicity There are many adverse effects associated with corticosteroids. In addition to their immunosuppressive effects, which set the stage for infectious complications, most of the adverse effects are related to Cushingoid features, hyperglycemia, fluid retention, hypertension, occasional psy-

Methotrexate MTX is an analog of aminopterin, the folic acid antagonist introduced in 1948 for the treatment of acute leukemia. As one of the

AML in CR1 and CML in CP Acute leukemia and CML

Nonmalignant and malignant diseases Advance-stage malignant diseases

Nonmalignant and malignant diseases Malignant diseases

Hematologic malignant diseases Hematologic malignant diseases Hematologic malignant diseases Hematologic malignant diseases Severe aplastic anemia Severe aplastic anemia

Storb et al. (1986, 1989 [82,150])

Forman et al. (1987 [151])

Santos et al. (1987 [93])

Sullivan et al. (1989 [39])

Storb et al. (1990 [152])

Chao et al. (1993 [71])

Deeg et al. (1997 [153)

Ratanatharathorn et al. (1998 [100])

Chao et al. (2000 [73])

Ruutu et al. (2000 [154])

Locatelli et al. (2000 [155])

Severe aplastic anemia

Nonmalignant and malignant diseases

Disease groups

Storb et al. (1989 [19])

HLA-identical sibling donors Ramsay et al. (1982 [149])

Investigator (year, reference)

34

53 37

90 55

165 96

62 164

75 60

63 74

44 59

40

40 25

54 42

50 53

24 43

35 22

32

Patients (n)

MTX + ATG + PSE versus MTX MTX + CSP versus MTX MTX + CSP versus CSP MTX + PSE versus CSP + PSE CSP + MP versus CY + MP Long MTX + BC versus Short MTX versus Long MTX MTX + CSP + PSE versus MTX + CP CSP + PSE versus MTX + CSP + PSE CSP versus CSP + MP MTX + CSP versus MTX + TACR MTX + CSP versus MTX + CSP + PSE MTX + CSP versus MTX + CSP + PSE MTX + CSP versus CSP

Regimens compared

18

42 20

34 41

40 34

39 40

28 36

28 32

19 32

18

24 19

26 23

30 26

23 30

16 23

16

Median age (year)

21 (p = 0.01) 48 18 (p = 0.01) 53 33 (p = 0.01) 54 47 (p = 0.05) 28 32 (p = 0.05) 68 82 (p = 0.016) 59 (p = 0.0016) 25 46 (p = 0.02) 25 23 (p = 0.02) 9 73 (p = 0.01) 60 44 (p = 0.01) 32 20 (n.s.) 18 56 (p = 0.001) 19 30 (n.s.) 38

Acute GVHD (%)

Table 84.1 Randomized trials of single-agent and combination immunomodulation for prevention of acute graft-versus-host disease (GVHD)

6 (n.s.) 9 58 (n.s.) 36 26 (n.s.) 24 ? ? ? 40 (n.s.) 18 44 (n.s.) 51 (n.s.) 33 62 (p = 0.01) 40 60 (n.s.) 57 21 (n.s.) 44 49 (n.s.) 56 52 (n.s.) 46 48 (p = 0.06) 36 44 (n.s.) 30

Chronic GVHD (%)

52 (n.s.) 44 73 (n.s.) 58 65 (n.s.) 54 53 (n.s.) 57 38 (p = 0.03) 20 24 (n.s.) 30 (n.s.) 41 52 (n.s.) 46 59 (n.s.) 46 26 (n.s.) 23 57 (p = 0.02) 47 51 (n.s.) 60 72 (n.s.) 65 94 (p = 0.05) 78

Overall survival (%)

1260 Chapter 84

Hematologic malignant diseases

Hematologic malignant diseases Hematologic malignant diseases Hematologic malignant diseases

Antin et al. (2002 [160])

Antin et al. (2002 [161])

Ruutu et al. (2002 [162]) 123

91 119

10 90

120 3

121

191

191

27

29 28

90 25

90

69

21 31

19

Control vs. Short IVIg Placebo versus rIL-11 Placebo versus IL-1RA Control versus ursodeoxycholic acid

Control versus IVIg

MTX + CSP versus MTX + TACR No ATG versus ATG 7.5 mg/kg No ATG versus ATG 15 mg/kg

CSP + MTX versus CSP + MMF Placebo versus Palifermin

38

41 40

11 44

NA 3

NA

NA

NA

32

28 28

34 29

35

46

49 46

46

51 (p = 0.0051) 34 (if ≥20 years) 73 (n.s.) 65 33 (n.s.) 25 59 (n.s.) 61 10 (p = 0.006) 2 (grade III–IV)

74 (p = 0.001) 56 36 (n.s.) 41 50 (p = 0.001) 11

37 (n.s.) 48 40 (n.s.) 38

70 (n.s.) 76 NA (n.s.) NA NA (n.s.) NA NA (n.s.) NA

NA

NA

70 (n.s.) 76 65 (n.s.) 38 59 (n.s.) 41

NA

64 (n.s.) 63 NA

50 (n.s.) 42 66 (n.s.) 44 45 (n.s.) 35 55 (p = 0.01) 71

35 (n.s.) 33

50 (n.s.) 54 52 (n.s.) 55 46 (n.s.) 41

68 (n.s.) 52 84 (n.s.) 82

AML, acute myeloid leukemia; ATG, antithymocyte globulin; BC, donor buffy coat cells; CML, chronic myeloid leukemia; CP, chronic phase; CR, complete remission; CSP, cyclosporine; CY, cyclophosphamide; HLA, human leukocyte antigen; IVIg, intravenous immunoglobulin; MP, methylprednisolone; MTX, methotrexate; n.s., not significant; PSE, prednisone; rIL-11, recombinant IL-11; TACR, tacrolimus.

Nonmalignant and malignant diseases

Feinstein et al. (1999 [159])

HLA-identical and nonidentical donors Nonmalignant and Sullivan et al. (1990 [158]) malignant diseases

Bacigalupo et al. (2001 [132])

Nonmalignant and malignant diseases

Hematologic malignant diseases

Blazar et al. (2006 [157])

HLA-nonidentical donors Nash et al. (2000 [105])

Hematologic malignant diseases

Bolwell et al. (2004 [156])

Pharmacologic Prevention of Acute Graft-Versus-Host Disease

1261

1262

Chapter 84 OH

OH O

HO

O O

OH

OH

H

MTX can induce tolerance following marrow transplantation. In canine transplantation, MTX given after the graft was effective in controlling GVHD and in inducing tolerance when donor and recipient were matched for the dog leukocyte antigen system [76].

H

Pharmacology H

H

H

O

H

O Cortisol

Prednisone

OH

OH O

HO

O HO

OH

OH

H H

H H

H

O

H

O Prednisolone

Methylprednisolone

Fig. 84.2 Structure of corticosteroids.

S

S R R

Cytoplasm

Nucleus

S R

DNA (GRE) mRNA

Proteins

MTX was one of the first chemotherapy agents introduced for the therapy of malignancies. Studies have detailed its pharmacokinetics and mechanisms of action at the intracellular level. How these mechanisms translate to its in vivo effects and how it functions in the prevention of GVHD, facilitation of engraftment, and induction of tolerance are not well understood. At the cellular level, MTX exerts its cytotoxic effect by inhibiting dihydrofolate reductase (DHFR). DHFR is the intracellular enzyme responsible for converting folic acid to reduced folate cofactors. The reduced state of the folates, namely the tetrahydrofolates, are responsible for the transport of single carbon groups that are required for purine and thymidylate synthesis [77]. Once the single carbon group is delivered, the oxidized folates must be converted back to tetrahydrofolates by DHFR. MTX binds to DHFR, thereby preventing its ability to reduce the oxidized folates to tetrahydrofolate, and blocking further purine or thymidylate synthesis [78]. Following the administration of MTX, approximately 50% is bound to plasma proteins, especially albumin, with the highest tissue–plasma equilibrium reported in the kidney, liver, gastrointestinal, tract and muscle [79]. The gastrointestinal tract is an important site of distribution and metabolism of MTX, perhaps accounting for one of the primary sites of toxicity. One concern in using MTX is its clearance in those patients who have pleural or ascitic fluid since these reservoir spaces can have a substantial impact on the disposition of MTX. These spaces can be a reservoir from which MTX can be continuously released, leading to severe toxicity. At the low intravenous doses, MTX clearance correlates with glomerular filtration, and renal excretion of unmetabolized drug is the major route for elimination. Polyglutamate formation is also important to MTX metabolism [80]. The formation of MTX polyglutamates intracellularly may account for its retention within the cell and its cytotoxic effects. The enzyme folyl polyglutamate synthetase adds glutamate residues to folates or antifolates such as MTX. These polyglutamate moieties are then retained inside the cell, leading to prolonged antifolate activity. The polyglutamate derivatives of MTX appear to be as toxic to DHFR as the native compound. If the retained MTX polyglutamates are produced differentially in lymphocytes compared with other normal cells, the effective concentrations of the drug would differ. For example, these differences in intracellular concentrations may account for selective toxicities, and may selectively inhibit certain subpopulations of cells [81]. Toxicity

Fig. 84.3 Diagram of the mechanisms of action of steroids and the intracellular receptor. The complex of the drug and receptor translocates efficiently into the nucleus where it interacts with glucocorticoid response elements (GREs), leading to the generation of messenger RNA and protein synthesis.

antimetabolites, it functions as a decoy substrate for critical biochemical reactions leading to cell death. The amino group substitution found in MTX results in a less potent antifolate but a drug with more predictable toxicity profile and equivalent clinical results. The precise mechanism by which MTX prevents GVHD is not understood; however, it is likely related to MTX’s ability to inhibit cellular growth and division. Therefore, antigen-activated T cells that are rapidly proliferating would be particularly sensitive to this antimetabolite.

The most common adverse effects of MTX when used for GVHD prophylaxis are hematopoietic, renal, hepatic, and gastrointestinal mucosal toxicity. Patients may have an elevation in creatinine and elevation of bilirubin levels following the administration of MTX. As many of these are also the side-effects of CSP or tacrolimus, dose attenuation of MTX and CSP or tacrolimus may be necessary. Full target doses may not be achievable in patients with grade III–IV mucositis resulting from the preparatory regimen because of airway obstruction and severe oropharyngeal bleeding. Clinical use The use of MTX following allogeneic BMT has been exclusively with the intravenous formulation. The drug doses administered have been relatively small given the inability to dose escalate MTX significantly (Table 84.2). Initial studies in Seattle tested MTX as a single agent on

Pharmacologic Prevention of Acute Graft-Versus-Host Disease

1263

Table 84.2 Outline of some regimens used for prophylaxis of graft-versus-host disease (GVHD) Cyclosporine

Methotrexate*

Day

Dose

Route

Day

Dose

Route

−2 to +3 +4 to +14 +15 to +35 +36 to +83 +84 to +97 +98 to +119 +120 to +180 +181

5 mg/kg 3 mg/kg 3.75 mg/kg 5 mg/kg 4 mg/kg 3 mg/kg 2 mg/kg Off

IV QD over 20 hours IV QD over 20 hours IV QD over 20 hours po BID po BID po BID po BID

+1 +3 +6 +11

15 mg/m2 10 mg/m2 10 mg/m2 10 mg/m2

IV IV IV IV

Tacrolimus/methotrexate† Day

Dose

Route

−2 to +30 (or until good oral intake) (maintain level of 10–30 ng/ml) +30 to 120 (maintain level of 10–30 ng/ml) +120 to +180 (maintain levels around 10 ng/ml) +181 Off

0.03 mg/kg convert IV to oral at a ratio of 1 : 4

IV QD over 24 hours po BID

Tacrolimus

Sirolimus

Day

Dose

Route

Day

Dose

Route

−2 to +30 (or until good oral intake) (maintain level of 10–30 ng/ml)

0.02 mg/kg

IV QD over 24 hours

12 mg 4 mg

po po

+30 to 120 (maintain level of 10–30 ng/ml) +120 to +180 (maintain levels around 10 ng/ml) +181

convert IV to oral at a ratio of 1 : 4

po BID

−3 −2 to +100 (maintain level of 3–12 ng/ml) +101 to +180

taper to off by +180

Off

BID, twice a day; IV, intravenous; po, taken orally; QD, four times daily. * If “mini”-dose methotrexate is used, the doses are 10 mg/m2 on day +1, and 5 mg/m2 on days +3 and +6. † Methotrexate dosing is similar to the regimen with cyclosporine in the first section of the table.

a weekly basis up to day 100 after HCT, but, because of the toxicity of weekly administration of MTX, the dose was changed to four doses and combined with CSP [82]. MTX, when used with CSP or tacrolimus, generally is injected intravenously on days 1, 3, 6, and 11 following BMT. This schema is currently the most commonly utilized prophylaxis regimen today. Another schema that is commonly used following unrelated stem cell transplantation has been named “mini-dose MTX,” usually used in conjunction with tacrolimus [83]. In this regimen, MTX is given on days 1, 3, 6, and 11 at 5 mg/m2. The overall prevention of GVHD was similar to that of standard-dose MTX with lower systemic toxicities. In general, patients should be very well hydrated, have their urine alkalinized with sodium bicarbonate, and MTX should not be administered until the urine pH is 8 or above. Moreover, dose attenuation should be considered for elevations of creatinine or bilirubin, and for grade III–IV mucositis. One suggested schema for dose attenuation of MTX is shown in Table 84.3. There has been one report of leucovorin rescue after MTX [84]. In this small series, use of leucovorin was not associated with an adverse outcome, although this practice is not commonly used. Leucovorin circumvents the blocked tetrahydrofolate synthesis by supplying reduced folates directly, therefore bypassing the blocked DHFR.

Table 84.3 Suggested dose adjustments for methotrexate Dose (%) Bilirubin (mg/Dl) <2.0 2.1–3.0 3.1–5.0 >5.0

100 50 25 Hold dose

Creatinine (mg/dL) <1.5 1.5–1.7 1.8–2.0 >2.0

100 75 50 Hold dose

Specific T-cell immunosuppressive drugs Cyclosporine CSP, also known as cyclosporine A, has led to a considerable expansion of the field of allogeneic transplantation given its potent immunosup-

1264

Chapter 84

The cytosolic binding protein for CSP was isolated in 1984 from lymphocytes and named cyclophilin for its high affinity for CSP [87]. The analogous major isoform for the binding protein for tacrolimus has been termed tacrolimus-binding protein (FKBP). Both cyclophilin and FKBP are highly basic and very abundant in the cytoplasm. Although these two proteins do not have sequence homology, they both function as the enzyme prolyl cis-trans isomerase (or rotamase) [88]. Rotamase accelerates the conversion of cis and trans rotamers of proline-containing peptides or proteins. This conversion step is thought to be the ratelimiting step during normal protein folding. Both of these molecules are potent inhibitors of rotamase activity. The binding of CSP or tacrolimus to these rotamases suggests that inhibition of this enzyme may be an important step leading to immunosuppression. The mechanism of action of CSP is exerted through binding to cyclophilin (as tacrolimus is bound to FKBP). This complex of drug and binding protein interrupts the activation cascade at the level of NFAT dephosphorylation, and therefore NFAT is not able to enter the nuclear domain and activate IL-2 (Fig. 84.5). CSP is a highly lipophilic drug that is extensively metabolized with subsequent biliary and, to a lesser extent, urinary secretion. Over 15 metabolites have been isolated and identified. Some metabolites also have immunosuppressive activity, while others are nephrotoxic. Thus, the clinical efficacy and interactions between CSP and the other drugs may be confounded by its metabolites. However, the most commonly reported inhibition or induction of CSP metabolism is by cytochrome p450 enzymes, specifically HLp and PCN1 enzymes that are members of the cytochrome P450 IIIA gene family [89].

pressive activity. CSP is a cyclic peptide that was extracted in 1969 from two strains of fungi isolated from soil samples. CSP was tested as an antifungal agent and demonstrated only limited effects, but it was highly effective as an immunosuppressive agent. These studies led to it being administered to patients in 1978 [85,86]. Pharmacology CSP is a neutral hydrophobic cyclic peptide composed of 11 amino acids (Fig. 84.4). Over the past decade, much work has been devoted to understanding the mechanism of CSP’s activity. Moreover, CSP, as well as tacrolimus, has been used as a chemical probe to dissect the pathways of T-cell activation. This understanding will lead to novel immunosuppressants that may be more specific and potentially less toxic. While CSP and tacrolimus are distinct in their molecular structure (Fig. 84.4), their mechanism of action will be discussed together since they are almost indistinguishable in their cellular functions. What is known thus far is that CSP (and tacrolimus) block the calciumdependent signal transduction pathways distal to engagement of the Tcell receptor (TCR) (Fig. 84.5). This block interrupts the activation of T cells since the signaling from the TCR is prevented. In dissecting the signaling pathway, several critical intracellular molecules have been identified; these include calmodulin, calcineurin, and the nuclear factor of activated T cells (NFAT). The activation cascade includes calcium binding to calmodulin, which leads to binding calcineurin. The activated calcineurin may dephosphorylate the cytoplasmic unit of NFAT, which allows translocation of the NFAT from the cytoplasm into the nucleus to form a competent transcriptional activator of the IL-2 gene [87].

O N

N

HO H N

N

O

O N O

O

N O O N O

N H

O

H N

N

H N

O

O

Cyclosporine HO

O

O

O O O

N

H O

OH O

OH

N H

O O

O O

O O

O O Tacrolimus

H O

OH

O

O

Sirolimus

OH

Fig. 84.4 Structure of cyclosporine, tacrolimus, and sirolimus.

Pharmacologic Prevention of Acute Graft-Versus-Host Disease

Ag TCR

PTK/PLCy1

Cytoplasm

Calmodulin

Ca2+

DAG IP3

PKC

Cyclosporinecyclophilin

P

Calmodulin

P

Calcineurin

Nucleus

NF-ATc

NF-ATn

IL-2 gene transcription

Fig. 84.5 A simplified diagram of the signal transduction found in the activation of a T cell through the T-cell receptor (TCR) and the point where cyclosporine (CSP) and tacrolimus interact with its binding proteins to block the generation of nuclear factor of activated T cells (NF-AT) and its subsequent generation of interleukin-2 (IL-2). Sirolimus, while similar to tacrolimus in structure, does not inhibit the signaling through the TCR but rather inhibits signaling through the CD28/B7 family of molecules and prevents the egress of cells from the G1 phase. DAG, diacylglycerol; IP3, inositol triphosphate; PKC, protein kinase C; PLC, phospholipase C; PTK, protein tyrosine kinase.

Toxicity The major clinical toxicities that need to be monitored with CSP administration are renal insufficiency and elevation of bilirubin levels. Both of these complications appear to be dose related [90]. The acute nephrotoxicity is due to vasoconstriction and ischemia of the afferent arterioles in the kidney. These acute changes are reversible but, if present for too long, can lead to an irreversible interstitial injury with glomerular thrombosis resulting in permanent azotemia. A major difficulty clinically is to differentiate specific side-effects of CSP from those of other drugs such as amphotericin, MTX, and aminoglycoside, or from an underlying disease process such as GVHD. Other side-effects that are commonly encountered are hypertension, hyperglycemia, headaches, and hirsutism. Rarer events include gum hypertrophy, brittle nails, acne, nausea, and vomiting. One specific clinical concern is the apparent association of hypomagnesemia and seizures in patients on CSP. Many patients who have a seizure while on CSP may also have concurrent hypomagnesemia and hypertension. Correction of these two abnormal findings are important in the management of patients. Hypertension alone can occasionally become a significant problem. Usually, nifedipine is the drug of choice since it is an effective antihypertensive, and it has not been associated with changes in CSP levels.

1265

A rare but potentially reversible toxicity associated with CSP is posterior reversible encephalopathy syndrome [91]. The clinical syndrome is characterized by headaches, confusion or decreased consciousness, visual complaints, and seizures. There are commonly associated findings of posterior cerebral white matter vasogenic edema in studies such as magnetic resonance imaging. Prompt recognition is important, so that the findings can be reversed. CSP should be stopped, and treatment of the hypertension and seizures (if present) instituted. A different immunosuppressant should be used in lieu of restarting CSP. Another rare but important toxicity of CSP (and tacrolimus) is the induction of a hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura syndrome. Patients present with schistocytes and an elevated creatinine with or without mental status changes. In some circumstances, this toxicity will abate if one calcineurin inhibitor is substituted for the other. Clinical use CSP can be administered intravenously or orally. It is initially administered intravenously since, in the early phase following transplantation, many patients develop mucositis and gastrointestinal damage from the preparatory regimen as well as from MTX given for GVHD prophylaxis. Once the patient is eating and drinking without significant difficulties, CSP may be switched to an oral preparation. CSP is available as an olive oil solution and as soft gelatin capsules, as well as in the microencapsulated form. CSP is erratically and incompletely absorbed after oral administration, with many factors reported to influence oral CSP bioavailability. For example, food intake may impact its absorption, especially if the meal is high in fat content. Absorption kinetics of CSP have been described as either zero order or a series of first-order processes. CSP dosage usually is governed by research protocols, beginning the drug at a higher dose as a “loading dose,” with a gradual taper over time. However, there are several variations of dosing schedules. An example of a CSP regimen in given in Table 84.2. There have been several studies attempting to correlate the efficacy of prophylactic CSP with its level in either whole blood, plasma or serum. The only assay that is completely specific for the parent compound is high-pressure liquid chromatography. Other assays such as polyclonal or monoclonal antibody immunoassays measure varying amounts of CSP metabolites. The target level that one uses will vary depending on which assay the diagnostic laboratory utilizes for measuring CSP levels. While the specific analytic methods are clearly more precise and preferable to the nonspecific methods for therapeutic drug monitoring, the technical difficulties in current high-pressure liquid chromatography methods make it less practical to utilize it widely. Moreover, there are limited data to suggest that there is a correlation between CSP levels and clinical outcome. The results from these studies suggest that there may be a complex relationship between dose, blood level, and occurrence of GVHD [92–94]. These studies failed to demonstrate a clear direct correlation between drug level and occurrence of GVHD. From a practical standpoint, CSP is administered to achieve a “therapeutic level,” but, beyond that, it is important to deliver the planned dose per protocol. It is also important to be certain that the level drawn is a trough level in steady state, otherwise the values will not be interpretable. CSP levels are used to increase the dose if it is below the therapeutic threshold. In general practice, the dose is not decreased for elevated levels if no toxicity is present, but if the level is above two times the upper limit of the therapeutic range, it is reasonable to decrease the dose to avoid potential neurotoxicity (i.e. seizures). Therefore, there is a range (up to two times normal) where CSP levels do not result in any change in clinical management. There are also concerns about the possible interactions between CSP and other medications. Many different drugs have been implicated in increasing or decreasing CSP levels

1266

Chapter 84

Table 84.4 Cyclosporine (CSP) drug interactions Decrease CSP levels

Increase CSP levels

Phenytoin Phenobarbital Carbamazepine Primidone Trimethoprim Rifampicin Nafcillin Octreotide Sulfonamides

Erythromycin* Ketoconazole Itraconazole Fluconazole Voriconazole Diltiazem Verapamil Nicardipine Acetazolamide Alcohol Colchicine Fluoroquinolones Imipenem

* Not all macrolides are identical in effects because some do not form stable complexes with cytochrome P450 enzymes.

in patients. Such medications and their effects are listed in Table 84.4 [95,96]. Tacrolimus (FK506) Tacrolimus is a macrolide antibiotic extracted from the soil fungus Streptomyces tsukubaensis [97]. It is completely different from the cyclic peptide CSP, but it does exhibit a very similar selective immunosuppressive activity (Fig. 84.4). Its mechanism of action is also through the inhibition of signaling through the TCR (Fig. 84.5). Pharmacology Tacrolimus is highly lipophilic, and its method of administration is similar to that of CSP. Its mechanism of action has been reviewed above in conjunction with CSP. It is generally given intravenously in the early phases following allogeneic transplantation, and then switched to the oral formulation. There is rapid distribution of the drug to the central compartment after a short intravenous infusion. Following oral administration, tacrolimus is erratically and incompletely absorbed [98, 99]. The major difference compared with CSP is that the absorption of tacrolimus does not seem to depend on the presence of bile salts. Tacrolimus undergoes extensive distribution, with highest levels found in the lung, kidney, heart, and spleen. While in the blood, it is distributed into red blood cells. Less than 1% of the intravenous or oral dose appears unchanged in the urine, indicating that the drug is almost completely metabolized in the liver prior to its elimination. It is eliminated primarily through monodemethylation, dimethylation, and hydroxylation. The half-life of tacrolimus is approximately 9 hours, and longer in those patients who have hepatic dysfunction. Tacrolimus is monitored in a similar fashion to CSP. Appropriate doses and levels of tacrolimus have not yet been clearly defined in patients undergoing allogeneic BMT, but the general blood levels have been 10–20 ng/dL. Toxicities Tacrolimus is moderately well tolerated in humans. Most of the data on adverse events can be categorized into neurotoxicity, renal toxicity, and hyperglycemia, and generally have been reported in solid organ transplantation recipients. Tacrolimus can cause an increase in serum creatinine concentration similar to that of CSP, probably secondary to decrease in glomerular filtration. Hypertension in previously normotensive

patients has also been described. Occasionally, hypertension can be quite refractory to antihypertensive medication. The most commonly utilized antihypertensives have included the calcium channel blockers. Central nervous system effects have also been found, including headaches, tremors, parathesias, photophobia, and mental status changes. Seizure and coma have also been reported. Other findings include pulmonary symptoms such as dyspnea, musculoskeletal pain, itching, gastrointestinal complaints (including anorexia, nausea, vomiting, and abdominal pains), and fatigue. While the adverse effects of CSP and tacrolimus appear to be similar, there may be some advantage of tacrolimus in hypertensive patients. Hyperkalemia has also been observed following tacrolimus administration. Clinical use Tacrolimus has been evaluated in combination with MTX for the prevention of GVHD. In a multicenter trial, 329 recipients of HLAidentical marrow transplants were randomized to receive a short course of MTX plus either tacrolimus or CSP [100]. The incidence of grade II–IV acute GVHD was significantly lower in patients who received tacrolimus (32% versus 44% with CSP), even though more unfavorable patients were enrolled in the tacrolimus group. This difference was largely due to a reduction in grade II disease. The incidence of chronic GVHD was similar in the two groups (56% and 49%, respectively), but severe chronic disease was more likely with CSP. There were, however, surprising adverse findings in the tacrolimus group. First, there was inferior 2-year survival (41% versus 50% with CSP) and overall survival (47% versus 57% with CSP) that was largely due to poorer survival of patients with advanced disease (25% versus 42%). Second, there was a higher incidence of regimen-related adverse events, which predominantly occurred in patients with advanced disease. The conclusion of the study was that, in patients without advanced disease, tacrolimus plus MTX was more effective than CSP plus MTX in the prevention of acute GVHD, with no difference in disease-free or overall survival. The survival disadvantage in patients with advanced disease warrants further study. Another randomized trial also compared tacrolimus and MTX against CSP and MTX [101]. Tacrolimus was more effective than CSP for the prophylaxis of acute GVHD, but there was no difference in patient survival or relapse. Although these trials were performed in patients with HLA-identical marrow transplants, uncontrolled [102–104] and controlled [105] observations indicate that tacrolimus also has activity against acute GVHD in recipients of matched unrelated donor transplants. The doses used in GVHD prophylaxis trials begin at 0.03– 0.04 mg/kg/day as a continuous infusion. When patients are able to sustain near-normal oral food and fluid intake, the drug is given orally at a dose of 0.15 mg/kg/day administered in two divided doses. Sirolimus (rapamycin) Sirolimus is a lipophilic macrolide that was identified more than 20 years ago during an antibiotic screening [106]. This molecule is produced by a strain of Streptomyces hygroscopicus isolated from a soil sample from Easter Island (Rapa Nui). Although lacking antibacterial activity, it inhibited yeast growth and growth of filamentous fungi. The first demonstration of the immunosuppressive activity of sirolimus was obtained from studies demonstrating the inhibition of immunoglobulin E production, as well as efficacy in experimental allergic encephalomyelitis and adjuvant arthritis [106]. The interest in sirolimus as an immunosuppressive agent coincided with the discovery of the immunosuppressive activity of tacrolimus, since the structure of both of these molecules contained a distinctive hemiketal-masked αβ-diketopipecolic acid amidic component (Fig. 84.4) [107].

Pharmacologic Prevention of Acute Graft-Versus-Host Disease

Pharmacology Sirolimus binds to the same family of intracellular receptors as tacrolimus, termed tacrolimus-binding proteins or FKBPs. In a similar fashion to tacrolimus, sirolimus has two domains, a domain bound by FKBP, and an effector domain forming a composite surface that interacts with the mammalian target of sirolimus [108]. The intracellular target of sirolimus (termed the mammalian target of rapamycin) is ubiquitously expressed in human tissue, with the highest level found in the testes and skeletal muscle. While sirolimus and tacrolimus bind to the same family of intracellular binding proteins, the mechanisms of immunosuppression are clearly distinct. Sirolimus inhibits the growth of hematopoietic and lymphoid cells in vitro. The most striking observation is that it suppresses cytokine-driven growth of these cells. The data demonstrate a significant increase in the proportion of G1-phase cells when sirolimus is added [109]. The mechanism of action of sirolimus is to inhibit specifically the progression of cells from G1 into the S phase, suggesting that it may interfere with the signaling that is critical for the cyclin/ cyclin-dependent kinase complex required for cellular proliferation. Data using human peripheral blood T cells demonstrate that the block of G1 may be related to an increase in a species of titratable inhibitor of G1 cyclin/cyclin-dependent kinase [110]. In addition to regulation of the cell cycle, sirolimus may also exert an effect on signal transduction pathways that mediate specific cytokine responses. For example, sirolimus has been shown to inhibit IL-1-driven IFN-γ production [111]. Moreover, the secondary signals required for T-cell activation and proliferation are dependent on ligation of the B7 family with CD28. Lack of the secondary signal has been shown to result in anergy in the responding T cell. While signaling through the TCR is not affected by sirolimus, signaling through CD28 is inhibited by sirolimus [112]. CD28 signaling leads to a sustained downregulation of the inhibitor I kappa B. CD28-mediated downregulation of I kappa B-α is prevented by sirolimus. These data suggest that sirolimus affects the CD28 signaling pathway, which may be a significant mechanism of its action and distinct from that of either CSP or tacrolimus. Moreover, in murine experimental models, sirolimus has been shown to selectively expand naturally occurring CD4+CD25+Foxp3+ regulatory T cells in vivo [113]. Sirolimus has also been used to expand these cells ex vivo for cellular therapies directed against activated T cells. This approach is being tested in patients as a method to prevent GVHD. Toxicity Sirolimus appears toxic to the gastrointestinal system, including elevation of liver function tests and diarrhea. In contrast to the renal sideeffects that are common with CSP and tacrolimus, nephrotoxicity is rarely encountered with sirolimus. However, sirolimus may potentiate the nephrotoxicity of CSP [114]. Other toxicities of sirolimus include hypertriglyceridemia, a decrease in platelets and leukocytes, epistaxis, blood pressure changes, headaches, nausea, mucous membrane irritation, and infections. The use of sirolimus at high doses may also increase the risk of hemolytic-uremic syndrome in patients with known GVHD [115]. Adverse reactions previously observed with other macrolide antibiotics are also of concern. Testicular atrophy has been observed in both mice and non-human primates. These early observations suggest that liver function abnormalities, thrombocytopenia, and neutropenia may be its dose-limiting toxicities. Clinical use Sirolimus has been used as the front-line regimen for the prevention of GVHD [116]. One study tested the feasibility and activity of adding sirolimus to tacrolimus and low-dose MTX as GVHD prophylaxis in recipients of alternative donor transplants following a myeloablative

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regimen. All evaluable patients engrafted. An absolute neutrophil count of 500/μL was achieved on day 18 (range 11–32 days) after HCT. Sustained platelet counts of more than 20,000/μL were attained on day 29 (range 14–98 days) after HCT. Grade 0–I acute GVHD occurred in 75% of patients. Grades II, III, and IV acute GVHD occurred in 13%, 8%, and 5%, respectively (total grade II–IV GVHD 26%). The authors concluded that sirolimus was tolerable, adequate blood levels were achievable, and there was a low rate of acute GVHD compared with historical data in this high-risk population. A second study explored sirolimus and tacrolimus without MTX following matched related donor peripheral blood transplantation. A total of 30 patients were included in a phase II study. Grade II GVHD occurred in three patients (10%), and no patient developed grade III or IV GVHD. Neutrophil and platelet engraftment were prompt, occurring on days 14 and 13 after HCT, respectively. All patients survived to hospital discharge (median 18 days), and peritransplantation toxicity was mild. Four patients developed thrombotic microangiopathy, and three patients developed hepatic sinusoidal obstructive syndrome (venoocclusive disease). Mucositis was modest, engraftment was prompt, and transplant-related toxicity was modest. Chronic GVHD occurred in 11 patients. Relapse-free and overall survival at 100 days were 93% and 97%, respectively, and were 71% and 67% at 1 year. These data suggest that sirolimus in combination with tacrolimus is a promising alternative to MTX-based regimens for GVHD prophylaxis after matched related donor peripheral blood stem cell transplantation. Mycophenolate mofetil Mycophenolate mofetil (MMF) is a morpholinoethyl ester of mycophenolic acid (MPA). MPA is produced by several species of Penicillium molds, and this agent possesses antibacterial, antifungal, antiviral, antitumor, and immunosuppressive properties [117]. The immunosuppressive activity of MMF occurs after hydrolysis to MPA. Therefore, MPA is the active moiety, but it is formulated as MMF to enhance its bioavailability. MMF inhibits the proliferation of T and B cells and the production of antibodies [118,119]. Most of the clinical data are in the solid organ literature. The developmental work on MMF has utilized the dog model in BMT. Studies in the dog model of allogeneic transplantation by Storb et al. [120] demonstrated that the combination of MMF and CSP was highly effective in preventing host-versus-graft rejection and graft-versus-host reaction following a RIC regimen. These studies have led to the marked increase in this form of transplantation worldwide. The contribution of MMF to the development of this approach was significant. Pharmacology Purines are essential for the growth and survival of cells. Cells have two pathways to produce purines: de novo and salvage pathways. Lymphocytes are highly dependent on the de novo synthesis, while other cells can utilize both (Fig. 84.6). In the de novo pathway, the ribose-phosphate portion of the purine nucleotide is derived from 5-phosphoribosyl1-pyrophosphate, which is derived from adenosine triphosphate and the sugar ribose-5-phosphate. Therefore, 5-phosphoribosyl-1pyrophosphate is an essential intermediary in the synthesis of purines. Following T-cell stimulation, there is a brisk and sustained increase in 5-phosphoribosyl-1-pyrophosphate, and guanosines and deoxyguanosines activate 5-phosphoribosyl-1-pyrophosphate synthetase [121]. Therefore, depletion of guanosines would lead to a decrease in 5phosphoribosyl-1-pyrophosphate synthetase and inhibition of purine synthesis, which is required for T-cell activation. MPA action is through the inhibition of inosine monophosphate dehydrogenase. This enzyme catalyzes oxidation of inosine monophosphate

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Ribose-5P + ATP

de novo pathway

PRPP

significant improvement in the prevention of GVHD was not suggested, compared with CSP and MTX, MMF in combination with CSP could be considered in cases in which MTX is contraindicated.

5-Phosphoribosyl-1-pyrophosphate MMF Guanosine MP

Inosine MP

Adenosine MP

HGPRT

APRT

salvage pathway Guanine

Hypoxanthine Xanthine

Adenosine Uric Acid

Fig. 84.6 The salvage and de novo pathway of purine synthesis. Inositol monophosphate (IMP) is at a central position, and mycophenolate mofetil (MMF) inhibits IMP dehydrogenase, necessary for the conversion of inosine monophosphate (MP) to guanosine MP. APRT, adenine phosphoribosyltransferase; ATP, adenosine triphosphate; HGPRTase, hypoxanthine–guanine phosphoribosyltransferase; PRPP, 5-phosphoriboxyl1-pyrophosphate.

to xanthine monophosphate. Xanthine monophosphate is a required intermediate metabolite in the synthesis of guanosine triphosphate. The enzyme inosine monophosphate dehydrogenase is a critical enzyme for the de novo biosynthesis of purine nucleotides, specifically guanosine monophosphate. Blockade of guanosine monophosphate synthesis leads to a negative feedback inhibition of 5-phosphoribosyl-1-pyrophosphate and prevention of T-cell activation. The therapeutic index of MMF depends on the lymphocytes’ reliance on de novo synthesis of purines, allowing for greater immunosuppressive activity with less toxicity. Toxicity The major effect seem with MMF has been suppression of the hematopoietic system, with neutropenia as an important side-effect. Gastrointestinal side-effects have also been reported occasionally. The optimal dose of MMF has not been determined, but doses as high as 15 mg/kg four times daily have been reported to be tolerable. Of note, MMF does not seem to deplete guanosine triphosphate in neutrophils in contrast to lymphocytes and monocytes. Its selective action on lymphocytes seems to spare the higher risk of acute infections. Doses up to 1.5 g twice daily had no nephrotoxicity, myelosuppression or other serious side-effects in patients with rheumatoid arthritis [122]. Methods to monitor drug levels of MMF are being developed. Clinical use MMF has been used as part of the front-line regimen for the prevention of GVHD, especially within the setting of reduced-intensity preparatory regimens. This drug is commonly used as part of post-transplant immune suppression as there are data in the dog model that it supports donor cell engraftment. It is possible that the prolonged administration of MMF helps to delay the onset of GVHD since the use of MMF instead of MTX as immunosuppressive therapy led to delay in the onset of GVHD after reduced-intensity HCT. However, preliminary results of a prospective trial in which MMF was given twice daily for 27 days in combination with CSP suggest that this combination is not superior to the standard regimen of MTX plus CSP for preventing GVHD after ablative HCT from related and unrelated donors [123]. In this study, the median time to engraftment after HCT was 15 (range 10–20) days. The incidence of acute GVHD was 62%, which was comparable to a group of historical controls receiving CSP and MTX for GVHD prophylaxis. Although a

Antibodies Many antibodies have been used for the prevention and treatment of GVHD. Most of the promising data have come from studies using selected monoclonal antibodies directed at specific epitopes. However, these antibodies are not generally available, and most of the studies have involved small groups of patients. Several antibodies will be discussed, namely intravenous immunoglobulin (IVIg), anti-CD3, antithymocyte globulin (ATG), daclizumab or basiliximab (anti-IL-2 receptor), infliximab (anti-TNFα receptor), etanercept (soluble TNF receptor), and alemtuzumab (Campath-1H). Anti-CD3 OKT3 is an anti-CD3 monoclonal antibody that is commercially available. This antibody has been used to treat GVHD, but because it is a stimulatory antibody to the T cell, the results have not been uniformly successful. Carpenter and colleagues have shown that soluble antihuman CD3 monoclonal antibodies induce apoptosis of activated T cells [124]. They evaluated different anti-CD3 antibodies, and demonstrated that the ability to induce apoptosis varied among them. Compared with OKT3, one monoclonal termed HuM291 induced more sustained phosphorylation of extracellular signal-related kinase-2, greater release of IFN-γ, and more activation-induced T-cell death. The use of HuM291 (visilizumab) in a phase I study in patients with steroid refractory acute GVHD was highly encouraging. This antibody is not capable of crosslinking with Fc receptors on accessory cells, and therefore does not activate proliferation of T cells. In one study, of the 17 enrolled patients, five achieved a complete resolution of GVHD, eight had partial improvement, two showed no change, and two progressed. Eight of the 13 responders had sustained responses, but the estimated 1-year survival was only 24%. Epstein–Barr virus reactivation was also a problem, and therefore monitoring for Epstein–Barr virus is important with early intervention with rituximab if necessary. Intravenous immunoglobulin Polyclonal human immunoglobulins purified and sterilized from serum pooled from many donors have been used to manipulate the immune system [125]. There are many preparations of IVIg with different titres of anti-CMW activity. To date, there are no known clinical differences with the use of any of these commercial preparations. Occasionally, one preparation may be preferable to another based on the need to conserve on the volume that a patient receives. The initial studies focused on its use to prevent infectious complications following BMT [126,127]. However, in a large randomized trial of IVIg, this product was also associated with a decrease in the incidence of GVHD, especially in BMT recipients under the age of 20 years. Moreover, there was a decrease in the overall mortality in favor of those who had received IVIg [127]. While this agent is effective in the early phase following BMT, its prolonged use, in the absence of hypogammaglobulinemia after day +100, does not reduce late complications or chronic GVHD, and may be associated with impairment of humoral immune recovery [127]. Moreover, serious issues such as the extremely high costs of these products and occasional limited availability have led to a decrease in their use. Antithymocyte globulin ATG is a polyclonal immunoglobulin prepared by injecting horses or rabbits with human thymocytes or with a T-cell line. These antibodies

Pharmacologic Prevention of Acute Graft-Versus-Host Disease

are capable of destroying human leukocytes. The half-life of these antibodies differs significantly between horse compared with rabbit ATG, and the functional half-life of these antibodies is not well defined, although rabbit ATG is more potent than horse ATG. ATG has been used as part of the preparatory regimen prior to infusion of donor cells to decrease the incidence of graft rejection, or following BMT to prevent GVHD. In two prospective studies using ATG as part of GVHD prophylaxis, ATG was not effective [128,129]. Its use as therapy of GVHD has also been tested. The response rates are encouraging, but the overall survival remains poor [130]. The doses varied widely but were generally in the range of 10–30 mg/kg/day (for horse ATG). Since ATG is a foreign xenogeneic protein and an antibody, serum sickness can occur. Corticosteroids, acetaminophen, and histamine-1 and histamine-2 blockers are frequently used to prevent or treat the symptoms associated with the infusion of ATG. The use of ATG after BMT has been associated with an increase in lymphoproliferative disorders [131]. More recent data using rabbit ATG suggest that this agent may have a beneficial effect on the incidence of chronic GVHD [132]. Anticytokine therapy Several cytokines have been implicated in GVHD. IL-2 is a critical molecule in T-cell survival and stimulation. There are two monoclonal antibodies in clinical use directed against the IL-2α receptor (daclizumab), as well as a chimeric anti-IL-2 monoclonal antibody (basiliximab) [133]. The bulk of the data have been obtained from solid organ allograft rejection studies. The use of anticytokine agents in stem cell transplantation is limited. The initial results of the use of dacluzimab are encouraging, although these are phase II data, and further studies are necessary to confirm these encouraging results. As with all antibodies, toxicities associated with such proteins are common and include myalgia, fevers, rash, pruritus, arthritis, and headaches. Further caution is warranted in targeting the IL-2 receptor since CD25 is also expressed in regulatory T cells. In a prospective study with contemporaneous controls, the use of an immunotoxin directed against this epitope resulted in a higher incidence of acute GVHD [134]. The data suggested that CSP blocked the induction of CD25 expression on alloreactive T cells but had no detectable effect on CD25 expression on regulatory T cells, thus enhancing the loss of regulatory T cells. TNF is another cytokine that has been implicated in acute GVHD. Use of a polyclonal neutralizing antibody against TNF-α resulted in a 70% reduction in GVHD-associated mortality, and also in diminished lesions in the skin and the intestines in an experimental model [135]. Similar findings were observed using a neutralizing antibody against murine TNF with reduction of splenomegaly in GVHD models [136]. Higher TNF levels have also been described in those patients who develop GVHD, although this finding has not been universally observed [137]. There are two anti-TNF molecules. Infliximab is a chimeric monoclonal antibody that binds to TNF. There is also a fusion molecule, etanercept, that consists of the soluble TNF receptor (p75) linked to the Fc portion of human IgG1 [138]. These molecules are successful in the therapy of Crohn’s disease and rheumatoid arthritis, and could be equally useful following allogeneic BMT, but well-designed studies will be necessary. There are preliminary data for the use of infliximab in the therapy of acute GVHD, but these data have not been reported in the peer-reviewed literature. Given the known importance of cytokines such as TNF in the genesis of GVHD, the use of such agents will be explored further both for therapy and prevention. Alemtuzumab (Campath-1H) Alemtuzumab is a humanized monoclonal antibody directed against CD52. This antibody was formerly known as Campath-1H and is

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active against early- and late-stage chronic lymphocytic leukemia as well as T-cell prolymphocytic leukemia. Alemtuzumab targets the 21–28 kD cell surface glycoprotein CD52, a protein present on the surface of mature lymphocytes but not on lymphoid progenitors. The function of CD52 is unknown, but it is found on more than 95% of peripheral blood lymphocytes and monocytes. Upon binding to CD52, alemtuzumab initiates its cytotoxic effect by complement fixation and antibody-dependent cell-mediated cytotoxicity mechanisms [139]. Alemtuzumab has been associated with frequent infusion-related events including hypotension, rigors, fever, shortness of breath, bronchospasm, chills, and/or rash. These side-effects necessitate close follow-up of patients, especially during their first dose infusion. Other serious infusion-related events were reported: syncope, pulmonary infiltrates, acute respiratory distress syndrome, respiratory arrest, cardiac arrhythmias, myocardial infarction, and cardiac arrest. An important and potentially fatal complication of therapy with CMV. Several groups have utilized this monoclonal antibody as a backbone to a preparatory regimen in RIC to decrease GVHD. The first report of using Campath-1H in an RIC regimen was investigated in 44 patients with hematologic malignancies [140]. Forty-two of the 43 evaluable patients had sustained engraftment. There were no cases of grade III–IV acute GVHD. Only two patients developed grade II acute GVHD, and only one had chronic GVHD. The estimated probability of NRM was low at 11%. These data suggested that inclusion of Campath-1H in a reduced-intensity preparative regimen is associated with durable engraftment, minimal toxicity, and low incidence of GVHD. The use of this monoclonal antibody has been extended for use in haploidentical HCT [141]. In a standard-risk group of 19 patients with aplasia or in remission at transplantation, there was a 63% 1-year survival rate (95% confidence interval 38–80%) and 2.9-year median overall survival time (95% confidence interval 6.2–48 months). Thus, inclusion of Campath-1H in a reduced-intensity preparative regimen is associated with durable engraftment, minimal toxicity, and a low incidence of GVHD. While some groups have used this monoclonal antibody as a backbone of their preparatory regimen, it is not used universally because of concerns regarding infections and relapses.

Other agents Induction of anergy An exciting to the prevention of GVHD is to induce tolerance by inhibition of co-stimulatory molecules. To this end, the use of a chimeric molecule cytotoxic T lymphocyte antigen 4 (CTLA-4)–Ig has been investigated. Data generated from studies of T-cell activation have clearly demonstrated that there is a critical need for a second costimulatory signal for a positive immune response. Lack of secondary signals results in specific T-cell anergy. CTLA-4–Ig is a fusion protein designed in an attempt to prevent co-stimulation signals. Variable results have been observed using the CTLA-4–Ig fusion protein to block GVHD across major histocompatibility barriers [142]. These results may vary according to the model of GVHD selected (i.e. major histocompatibility differences) or the activation state of T cells. Methods to block costimulation hold significant promise in achieving antigen-specific unresponsiveness or anergy, and therefore a state of tolerance [143,144]. In a clinical trial of tolerance induction, haploidentical bone marrow was treated ex vivo with CTLA-4–Ig in the presence of recipient peripheral blood [143]. Laboratory parameters such as T-cell precursor frequency against allogeneic targets were markedly reduced. The preliminary clinical data were encouraging, with a low incidence of GVHD and

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Chapter 84

Table 84.5 Immunosuppressive drugs, targets, and mechanisms of action discussed in this chapter Drug

Target

Mechanism(s) of action

Corticosteroids Methotrexate Cyclosporine Tacrolimus Sirolimus Mycophenolic acid Dacluzimab/basiliximab Intravenous immunoglobulin Antithymocyte globulin Infliximab Etanercept Thalidomide Clofazimine Hydroxychloroquine CD4-D1 peptide CTLA-4-Ig

Steroid receptor DHFR Calcineurin Calcineurin mTOR IMPD IL-2 receptor Multiple Multiple TNF receptor TNF Unknown Unknown TAP proteins CD4 CD28

Multiple, including cytokine expression, lymphocyte activation, and cell depletion Blocks DNA synthesis Blocks lymphocyte activation Blocks lymphocyte activation Blocks co-stimulatory signal and cell division Blocks DNA synthesis Blocks lymphocyte activation Unknown Blocks lymphocyte activation Blocks lymphocyte activation Blocks lymphocyte activation Unknown Unknown Blocks lymphocyte activation Blocks lymphocyte activation Blocks lymphocyte activation

DHFR, dihydrofolate reductase; IL, interleukin; IMPD, inosine monophosphate dehydrogenase; mTOR, mammalian target of rapamycin; TNF, tumor necrosis factor.

encouraging overall survival. Such new approaches may circumvent the need for prolonged immunosuppression after BMT, and may be clinically beneficial for patients who require an allogeneic BMT. Photopheresis/pentostatin The use of extracorporeal photopheresis or pentostatin is a choice for patients with chronic GVHD. This combination approach has been explored as means to induce tolerance in a reduced-intensity preparatory regimen. Patients received a preparative regimen of photopheresis, pentostatin, and TBI followed by allogeneic stem cell infusion from six of six or five of six HLA-matched related donors, or six of six HLAmatched unrelated donors. GVHD prophylaxis consisted of CSP and a short course of MTX. Sixteen of 18 patients developed full donor chimerism with no transplant-related mortality at day 100 after HCT. Grade II–IV acute GVHD and extensive chronic GVHD developed in 19% and 18% of patients, respectively. At a median follow-up of 14 (range 1–35) months, the 1-year failure-free and overall survival were 64% and 65%, respectively [145].

Conclusion This chapter has focused primarily on the most frequently used drugs for immunosuppression or immunomodulation for GVHD (Table 84.5). Other drugs or nonpharmacologic treatment options that appear to be promising are psoralen and ultraviolet light A, radiation therapy for chronic GVHD, and other novel molecules such as a fusion immunotoxin directed against CD3 [146]. As we learn more about effector cell trafficking to target organs of GVHD, more directed therapy to adhesion molecules and homing receptors may be developed [147]. Because GVHD and solid organ rejection remain such a difficult clinical problem, there is a need for continued efforts to develop novel immunosuppressive or immunomodulatory agents. As shown in Table 84.1, the number of randomized, controlled trials for prevention of acute GVHD has been modest over the past 25 years. Moreover, there is only one agent approved by the US Food and Drug Administration for GVHD prevention (Gamimune) and one pending consideration for GHVD (oral beclomethasone). Well-designed randomized trials in this complex disorder are critical for advancements in the control of GVHD [149].

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adults given related donor marrow allografts. Biol Blood Marrow Transplant 1999; 6: 369–78. 160. Antin JH, Lee SJ, Neuberg D et al. A phase I/II double-blind, placebo-controlled study of recombinant human interleukin-11 for mucositis and acute GVHD prevention in allogeneic stem cell transplantation. Bone Marrow Transplant 2002; 29: 373–7. 161. Antin JH, Weisdorf D, Neuberg D et al. Interleukin-1 blockade does not prevent acute graftversus-host disease: results of a randomized, double-blind, placebo-controlled trial of interleukin-1 receptor antagonist in allogeneic bone marrow transplantation. Blood 2002; 100: 3479– 83. 162. Ruutu T, Eriksson B, Remes K et al. Ursodeoxycholic acid for the prevention of hepatic complications in allogeneic stem cell transplantation. Blood 2002; 100: 1977–83.

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T-Cell Depletion to Prevent Graft-versus-Host Disease

Graft-versus-host disease (GVHD) accounts for a significant fraction of the morbidity and mortality associated with allogeneic hematopoietic cell transplantation (HCT) [1,2]. GVHD negatively impacts survival directly as a result of organ damage, and indirectly as a consequence of infectious complications prompted by GVHD prophylaxis, therapy and immune organ damage. Donor T-cell depletion (TCD), when it was initially introduced, offered the potential for prevention of GVHD without the toxicity associated with immunosuppressive drugs [3]. Numerous TCD methods have been utilized over the past three decades. These have included ex vivo negative selection approaches using antibodies, physical separation or now photodepletion techniques, ex vivo positive selection through CD34+ columns, or in vivo administration of commercially available anti-T-cell antibody preparations [4–11]. Most early trials documented that TCD could substantially limit acute and chronic GVHD. However, this reduction in GVHD did not translate into improved overall survival because of unexpected high rates of graft failure [12–14], Epstein–Barr virus (EBV)-associated lymphoproliferative disorders [15–17], and disease recurrence [18–20] following TCD HCT. There have been very few randomized studies evaluating TCD, but those that have been performed have not convincingly demonstrated a survival advantage to this approach [21]. Nonetheless, there still remains substantial interest in perfecting the technique, particularly for recipients of human leukocyte antigen (HLA)-mismatched grafts. If advances in graft engineering can accomplish the goal of GVHD prevention without adversely affecting engraftment, immune competence, and antileukemic activity, substantial improvements in overall transplant outcome can become a reality.

this GVHD did not develop after infusion of fetal liver/spleen tissue, which lacks mature T lymphocytes [23]. In the 1960s, Bortin and Saltzstein extended Uphoff’s findings and further demonstrated that irradiated mice given allogeneic embryonic liver and thymus cells became true chimeras that did not reject donor skin grafts [24]. These pivotal observations pointed to the critical connection between mature donor T lymphocytes and GVHD, and suggested that selective in vitro removal of these cells from the allograft could prevent this transplant complication. In 1968, Dicke et al. reported on the use of differential centrifugation on a discontinuous albumin gradient to separate lymphocytes from hematopoietic precursors, and demonstrated that irradiated mice given spleen cell fractions devoid of small lymphocytes resulted in 80–100% survival without evidence of GVHD. In contrast, mice inoculated with fractions that contained increasing numbers of small lymphocytes all died from severe GVHD [25]. Subsequently, Reisner et al. demonstrated that mouse bone marrow and spleen cell suspensions, when treated with soy bean and/or peanut agglutinin, could generate an agglutinated fraction that is enriched in hematopoietic activity but depleted of T cells. Treatment of irradiated mice with this fraction conferred hematopoietic recovery with a low incidence of GVHD [26]. In the 1970s, absorbed antisera with retained activity against mature lymphocytes were developed and successfully used in animal models as the method for ex vivo TCD. Mice, rats, and dogs were successfully transplanted with minimal to no GVHD, across both major and minor histocompatibility barriers [27–30]. In the late 1970s, the development of monoclonal antibody technology helped to advance efforts to eliminate T cells from the donor graft and markedly decrease the incidence of GVHD in animal models [31,32].

Early preclinical models of TCD

T-cell dose

In the 1950s, Barnes and Loutit noted that, following irradiation of mice, infusion of isologous marrow and spleen resulted in long-lasting survival, while inoculation of allogeneic cells afforded only a temporary recovery, with most animals later succumbing to a wasting condition called “secondary phase of irradiation syndrome” [22]. They subsequently proposed that this “secondary disease” was the result of a graftversus-host (GVH) immunologic response. Uphoff demonstrated that

Bone marrow used for human transplantation contains approximately 1–2 × 1010 nucleated cells, of which up to 10–20% (~109 cells) are mature lymphocytes. An average patient transplanted with unmodified marrow would thus receive a T-cell dose of the order of 1–4 × 107 cells/kg recipient body weight. Initially, it was not known what degree of TCD would be necessary to prevent GVHD, as immunofluorescence and erythrocyte rosette assays for quantifying T lymphocytes were not sufficiently sensitive to evaluate marrow that had been manipulated with antibodies. Limiting dilution assays suggest that at least a 2-log depletion of T cells to 1–3 × 105 cells/kg is necessary to prevent GVHD in the HLA-identical related donor marrow transplant setting without exogenous immune suppression [33–36]. The target for most current TCD

Introduction

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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studies is a T-cell inoculum below 0.5–1.0 × 105 cells/kg recipient body weight for HLA-matched related stem cells. Granulocyte colonystimulating factor (G-CSF)-mobilized blood stem cell products contain approximately 10-fold more T cells than does unstimulated bone marrow grafts. Therefore, methods removing only 2 log of T cells would likely be insufficient for GVHD prevention. CD34+ selection of peripheral blood progenitor cells (PBPCs) with a 3–4-log reduction in CD3+ count to about 1 × 105 cells/kg is associated with lower rates of GVHD [37,38]. Although there appears to be a threshold effect at about 1 × 105 CD3+ cells/kg for PBPC transplantation, this T-cell number must be considered in the context of the stem cell dose as well, since CD34+ count is also an important determinant of engraftment, GVHD, and survival after PBPC transplantation [39–42]. The T-cell threshold for patients receiving unrelated or haplotype-mismatched stem cell products is likely lower. While these data give a clue as to the relationship between T-cell dose and GVHD, there is likely to be significant variability between donor– recipient pairs. The extent of TCD needed to prevent GVHD likely depends not only on T-cell dose, but also on other factors including donor source, stem cell source, degree of major and minor HLA disparity, and other potential polymorphisms between donor and recipient pairs [43–47]. While ex vivo methods of TCD allow a precise determination of T-cell dose in the graft, in vivo administration of T-cell antibodies yields a level of T-cell reduction that is much less predictable, owing to the variable half-lives of these antibody preparations and the dose of antibodies used. Certainly, there is evidence that the dose of antithymocyte globulin (ATG) used impacts both GVHD incidence and immune reconstitution [48].

Specificity of TCD TCD techniques may vary not only in the extent of T-cell removal, but also in the selectivity of depletion. Some strategies have removed all lymphoid fractions, including natural killer (NK) cells and B cells. These other cellular elements may indeed play an important role in immune surveillance, engraftment, and elimination of residual disease post transplantation. Other approaches have exclusively removed T cells, while still others have focused on specific phenotypically defined T-cell subsets. More recent studies have attempted to target donor T lymphocytes expressing activation antigens after in vitro exposure to recipient antigen-presenting cells in order to eliminate only those cells likely to cause GVHD. A retrospective analysis of alternative donor transplantation from the Centre for International Blood and Marrow Transplant Research (CIBMTR) compared TCD techniques with “narrow” and “broad” spectra of reactivity. Examples of “narrow”-specificity techniques included antibodies targeting mature T cells only, such as anti-T-cell receptor (TCR), anti-CD6, and anti-CD5, or T cells with specific functional roles (e.g. anti-CD8). Broad-specificity techniques include antibodies such as Campath, multiple antibody combinations, and physical separation techniques such as lectin agglutination. Patients who received TCD using narrow-specificity antibodies had a superior 5-year leukemia-free survival compared with those receiving broad-specificity TCD, due to a combination of increased relapse, graft failure, and transplantrelated mortality in the broad-specificity TCD cohort [49]. The incidence of acute GVHD was not significantly different between the groups. Unfortunately, after 30 years of research, it still is uncertain what anatomic T-cell subset mediates GVHD in humans. The recognition that certain T cells (Tregs) have regulatory function that can suppress GVH reactions may play a role in the design of future TCD strategies [50–52]. Several groups are exploring approaches that enrich for Tregs in hopes of limiting GVHD.

Acute GVHD HLA-identical related donor high-dose transplantation Early studies of TCD using murine monoclonal antibodies alone reported GVHD rates of approximately 50% even in HLA-identical sibling transplant recipients [53,54]. These results suggested that opsonization of T lymphocytes alone might be insufficient to prevent acute GVHD in humans, and that additional lymphocyte lysis, facilitated by addition of complement to the antibody, was necessary. Small pilot studies in the mid 1980s confirmed that TCD using monoclonal antibodies plus rabbit complement which removed 2–3 log of T lymphocytes uniformly decreased the incidence of clinically significant GVHD to 10–20% after matched sibling transplantation [55–57]. In the late 1980s and early 1990s, similar results were reported in larger phase II trials of TCD using anti-CD52 (Campath-1), anti-CD5, anti-CD6, and anti-CD8 antibodies plus complement, or immunotoxins [58–63]. In some of these studies, the low incidence of GVHD was achieved even in the absence of immunosuppression post transplantation. [59,60]. Addition of anti-CD52 antibody directly to the hematopoietic cell product as it is being infused (Campath “in the bag”) has also resulted in a low incidence of grade II–IV acute GVHD in recipients of marrow and mobilized peripheral blood cells [9]. A low frequency of acute GVHD (<20%) has also been noted with ex vivo physical separation techniques, such as soy bean lectin agglutination and counterflow centrifugal elutriation [35,64]. Studies of HLA-identical sibling PBPC transplantation using ex vivo CD34+ positive selection initially reported a wide variation in acute GVHD incidence from 8% to 100% [65–68]. This variability was likely influenced by factors such as the number of CD3+ cells infused, immunosuppressive agents given post transplant, and the manner in which these devices were used. A study comparing three devices (CEPRATE, ISOLEX 300i, and CliniMACS) found no apparent differences in the allograft composition or clinical outcomes, although ease of processing did differ [69]. Initial studies using CD34 selection columns showed that a 2-log reduction in T cells from matched related donor PBPCs was insufficient to control GVHD despite the use of cyclosporine ± methotrexate as post-transplant immune suppression [65,66]. However, a subsequent study demonstrated that when the T-cell content in the sibling PBPC graft was reduced by 3.0–3.5 log using the CEPRATE or ISOLEX 300i system, grade II–IV acute GVHD was only 10% [67]. Unrelated donor high-dose transplantation Since an HLA-identical sibling is available in only 25–30% of patients requiring transplant, most transplant candidates will need an alternative graft source, such as an unrelated donor or a partially HLA-mismatched family member. Transplantation from these “alternative” donors is generally associated with a higher risk of GVHD and inferior overall outcome. Several single-institution studies in the 1990s suggested that the incidence of acute GVHD after unrelated TCD bone marrow transplantation (BMT) was lower than that associated with transplantation of unmanipulated unrelated marrow [70–72]. In a report from the National Marrow Donor Program in 1993, the use of TCD was the most significant factor predicting freedom from severe (grade III–IV) acute GVHD [73]. A subsequent analysis from the CIBMTR involving 1868 leukemia patients receiving marrow transplants from donors other than HLAidentical siblings revealed that the incidence of grade II–IV acute GVHD was 34% and 38% for the TCD groups compared with 57% for the nonTCD cohort (p < 0.0001) [49]. Despite 25 years of TCD studies, only one large prospective randomized trial has been performed. This study was conducted in 410 recipients of HLA-identical unrelated bone marrow. Patients were randomized

T-Cell Depletion to Prevent Graft-versus-Host Disease Table 85.1. Impact of T-cell depletion (TCD) of outcome for unrelated bone marrow transplantation adapted from unrelated donor marrow transplantation Effect of GVHD prophylaxis method

Outcome

TCD superior c/w cyclosporine/methotrexate

Grade II–IV acute GVHD Grade III–IV acute GVHD Speed of neutrophil recovery Regimen-related toxicity Need for parenteral nutrition Length of hospital stay

TCD inferior c/w cyclosporine/methotrexate

CMV infection Fatal Aspergillus infection EBV PTLD CML relapse

TCD not significantly different c/w cyclosporine/methotrexate

Overall survival Disease-free survival Transplant-related mortality Relapse other than CML Graft failure Platelet recovery Chronic GVHD

CML, chronic myelogenous leukemia; EBV, Epstein–Barr virus; GVHD, graft-versushost disease; PTLD, post-transplantation lymphoproliferative disorder. Adapted with permission from Wagner et al. [21].

to receive either T-cell-depleted bone marrow (depleted by the monoclonal antibody T10B9 or elutriation) with post-transplant cyclosporine alone versus T-cell-replete bone marrow with post-transplant cyclosporine and methotrexate as GVHD prophylaxis [21]. Both groups received cyclophosphamide and total body irradiation for conditioning. Patients in the TCD arm received additional treatment to promote engraftment: ATG in the elutriation group and cytarabine in the T10B9 antibody group. Mean T-cell dose infused for the TCD arm was 1 log lower than in the non-TCD arm. There was no difference in incidence of primary graft failure in the two groups. The incidence of grade II–IV acute GVHD was significantly lower in the TCD arm compared with the nonTCD arm (39% versus 63%; p < 0.0001), and the cumulative incidence of grade III–IV acute GVHD was 18% versus 37%, respectively (p < 0.0001). Regimen-related toxicity was lower in the TCD arm. With the exception of chronic myelogenous leukemia (CML) in first stable phase, there was no difference in relapse rates. Unfortunately, infections rates, particularly from cytomegalovirus (CMV) and other herpesviruses, were higher in the TCD group. Overall disease-free survival at 3 years was 30%, and there was no difference between the TCD and nonTCD arms (27% versus 34%, respectively; p = 0.16) (Table 85.1). It is uncertain whether this trial represented a true test of the potential impact of TCD as the methods utilized only removed 1–2 log of T cells. Two smaller randomized studies evaluating in vivo antibody therapy with ATG in patients undergoing myeloablative unrelated transplantation were reported by investigators from Italy. In these successive studies, cyclosporine plus methotrexate was compared with cyclosporine plus ATG (15 mg/kg in one study, 7.5 mg/kg in the other) in recipients of matched unrelated donor BMT. Only the higher dose of ATG was associated with a significant reduction in grade III–IV acute GVHD. However, this reduction in severe acute GVHD did not improve outcome because 30% of the patients on the high-dose ATG arm died of severe infections. Overall survival was not affected by the use of either dose of ATG [48].

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Haploidentical donor transplantation Overcoming major HLA barriers to enable transplantation of mismatched grafts remains a major goal of allogeneic transplantation. It has long been hoped that TCD could make that goal a reality. Although early studies of TCD in HLA-mismatched related marrow transplants were plagued by high incidences of graft failure and GVHD [74], later series have demonstrated better results [75,76]. In single-institution studies, the incidence of grade II–IV GVHD has ranged from 18% to 40% in recipients of HLA-mismatched BMT after TCD using monoclonal antibody and complement alone. A report of 201 patients receiving T-cell-depleted high-dose conditioning and HCT from partially HLAmismatched family donors reported a rate of grade II–IV acute GVHD of 13% when partial TCD was achieved with monoclonal antibodies in addition to steroids, cyclosporine, and ATG [77]. However, a majority of these patients also received steroids, ATG, and cyclosporine as additional prophylaxis. Outcome was compromised by increases in infection and relapse. Improved engraftment of T-cell-depleted haploidentical transplantation has been achieved through infusion of high doses of CD34+mobilized peripheral blood cells depleted of T cells after CD34+ selection [78,79]. In these initial studies, patients received aggressive conditioning and ATG to prevent donor graft rejection. It is not clear how intensive conditioning must be to achieve hematopoietic recovery after TCD transplantation. Successful engraftment of haploidentical family donor PBPCs using a reduced-intensity conditioning regimen plus alemtuzumab has been reported with a 16% incidence of grade II–IV acute GVHD [80]. Immunologic analysis of fully haploidentical extensively T-celldepleted transplantation has led to an appreciation of the role of NK cells and their seemingly paradoxical role in allorecognition and GVHD. Donor–recipient disparity in HLA class I alleles, particularly at HLA-C, often is associated with mismatches in killer immunoglobulin-like receptors (KIRs) which form the basis for NK-cell allorecognition and activation [81,82]. The biology of KIR mismatch and NK-cell reactivity is discussed in detail in Chapter 13. The concept of NK-cell reactivity in clinical transplantation was first brought to light by studies from Perugia, Italy [78,83,84]. In that study, patients with acute leukemia underwent haploidentical transplantation with an intensive high-dose conditioning regimen coupled with extensive TCD using sheep red blood cell rosetting and CD34 selection of the PBPC product. No posttransplant immune suppression was given. Among 60 patients transplanted, only four cases of GVHD were observed. Notably, patients in this group whose donor–recipient HLA disparity predicted for KIR mismatch and NK-cell reactivity in the GVH direction experienced no graft failure, acute GVHD or myeloid leukemia relapse. High frequencies of alloreactive donor NK cells were detected in recipient blood post transplant that could specifically lyse leukemia targets missing KIR ligands, and not control target cells that expressed the “self” KIR ligand. These results generated speculation that HLA class I mismatching in the unrelated donor setting would also lead to leukemia relapse if the mismatch predicted for a KIR mismatch in the GVH direction. One retrospective study in mismatched unrelated donor transplantation, in which cyclosporine, methotrexate, and pretransplant ATG were used as GVHD prophylaxis, has demonstrated that KIR ligand mismatch in the GVH direction improved treatment-related mortality, disease-free survival, and overall survival [85]. Specific KIR and HLA genotypes associated with NK activation have been demonstrated to have prognostic importance also in the setting of HLA-identical T-cell-depleted transplantation for acute myeloid leukemia (AML) [86]. However, other studies in conventional non-TCD mismatched unrelated donor transplantation, including an analysis from the combined CIBMTR,

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European Group for Blood and Marrow Transplantation, and Dutch registry, have failed to demonstrate any benefit in outcomes for recipients with KIR mismatch [87–89]. It is possible that, in the non-TCD setting, the beneficial effects of NK cells were offset by the deleterious effects of alloreactive T cells in the absence of TCD. Extensive TCD may be necessary to observe the effect. In one study, NK cell counts at day 30 after transplant were associated with reduced GVHD, an improved graft-versus-leukemia (GVL) effect, and better survival in 52 recipients of TCD transplants [90].

TCD and reduced-intensity transplantation Over the past decade, there has been a dramatic upswing in the use of reduced-intensity allogeneic transplantation (RIT) [91,92]. The primary antitumor activity in RIT procedures stems not from the cytotoxic effects of low-dose chemotherapy, but from the alloimmune effect of the donor cells. Thus, there would appear to be little rationale for TCD in RIT since TCD could blunt the GVL effect, and defeat the purpose of the transplant itself. Further, the nonablative preparative regimens in RIT might not be sufficiently immunosuppressive to support and sustain the engraftment of T-cell-depleted stem cells or marrow. Nonetheless, both ATG preparations and alemtuzumab have been used to achieve simultaneous depletion of donor and host T cells to ensure engraftment and reduce GVHD in RIT. As with high-dose transplantation, these antibodies lead to delayed immune reconstitution and a higher incidence of disease relapse and virus infections, especially from CMV and adenovirus [93–98]. As a consequence, many studies of TCD RIT incorporate T-cell add-backs after transplant to improve immune function, facilitate full donor chimerism, and restore the GVL effect [99–101] There is no consensus on which antibody preparation yields the best results. In a comparative analysis of two prospective studies using either alemtuzumab plus cyclosporine or cyclosporine plus methotrexate as GVHD prophylaxis after reduced-intensity conditioning, there was a significantly lower incidence of acute and chronic GVHD in the patients receiving alemtuzumab, but a higher incidence of CMV reactivation and disease relapse/progression [93]. However, long-term disease and overall survival status were similar between the two cohorts when responses were considered after donor lymphocyte infusion (DLI) in the patients on the alemtuzumab arm. These data suggest that TCD can be used to induce a state of mixed chimerism with minimal GVHD, and provide a platform for additional adoptive immunotherapy interventions such as DLI or vaccinations to bolster immune function and the GVL effect. Unfortunately, there has yet to be a prospective randomized trial evaluating the consequence of in vivo anti-T-cell antibody administration in RIT. Ex vivo TCD has also been studied in RIT. CD34+ selection of donor PBPCs after reduced-intensity conditioning has led to a low rate of GVHD [102]. However, both engraftment and disease relapse have been problems in the absence of additional DLI.

syndrome patients from Seattle [104]. Similarly, prophylaxis with alemtuzumab in RIT suggests a lower rate of chronic GVHD [91]. However, the large prospective multicenter randomized trial of unrelated donor marrow transplantation failed to demonstrate any difference in the incidence and time to development of chronic GVHD between patients receiving TCD versus standard GVHD prophylaxis of cyclosporine and methotrexate, although the overall incidence reported in this trial was low and may be underrepresentative [21]. A follow-up report from the study suggested that survival 3 years after the diagnosis of chronic GVHD was similar [105]. It is possible that the 1-log reduction in T cells achieved by the methods used in this study was insufficient to protect against chronic GVHD. However, the lack of protection against chronic GVHD despite lower risks of acute GVHD may also reflect intrinsic differences in the pathogenesis of these two conditions. The retrospective study from the CIBMTR looking at the effect of broad- and narrow-specificity TCD demonstrated no protection by narrowspecificity antibody TCD against chronic GVHD despite a reduction in acute GVHD [49]. However, exhaustive TCD with broad-based approaches did reduce the incidence of chronic GVHD in this analysis. The increasing utilization of PBPC transplantation and its attendant increase in chronic GVHD makes TCD a potentially attractive modality in this setting. One report comparing TCD of bone marrow and peripheral blood grafts found a higher incidence of chronic GVHD in the TCD peripheral blood group, although no overall difference in survival [106].

Organ dysfunction In single-institution TCD series, low rates of hepatic, renal, and pulmonary complications have been reported [107–110]. In an analysis of 199 allogeneic transplants, the incidence of severe pulmonary complications was 8% among those who received CD6 TCD as the sole form of GVHD prophylaxis, but 33% among those who received cyclosporine and methotrexate [107]. The protective effect of TCD against pulmonary complications was independent of acute GVHD. The incidence of sinusoidal obstructive syndrome (hepatic veno-occlusive disease) has been reported to be quite low after TCD BMT [108,109]. This reduction in hepatotoxicity may be due to the fact that the TCD patients are spared from the injurious effects of methotrexate and/or cyclosporine/tacrolimus. Alternatively, decreases in alloreactivity may lead to lower levels of circulating cytokines that could damage hepatic endothelium. In a prospective randomized study of 410 patients, recipients of T-cell-depleted marrow experienced less regimen-related toxicity within the first 28 days of transplant, including stomatitis (p < 0.0001) and hepatic (p = 0.0003), pulmonary (p = 0.012), and central nervous system complications (p = 0.024) [21]. An assessment of quality of life 3–6 months after transplantation from a single institution showed no difference between TCD and non-TCD patients [111].

Chronic GVHD Most single-institution studies of ex vivo T-cell-depleted BMT have reported a low incidence of chronic GVHD. A retrospective analysis from the National Marrow Donor Program had suggested that TCD reduced the risk of both acute and chronic GVHD after unrelated BMT [73]. Ex vivo anti-CD6 antibody-based TCD has been associated with a very low incidence of chronic GVHD in related marrow recipients [60]. Data from the randomized ATG trials in Italy suggested a reduction in chronic GVHD for those receiving ATG, with a reduction in long-term pulmonary complications and late deaths [103]. Protection against chronic GVHD by ATG has been suggested by data on myelodysplastic

Engraftment Graft failure or rejection has been reported to occur more frequently in patients receiving TCD marrow than in recipients of T-cell-replete grafts. Whereas graft failure usually occurs in 1% or less of patients with leukemia receiving HLA-identical products, studies from the 1980s and early 1990s reported graft failures rates of 3–40% depending on the methodology used [12–14,112–114]. Registry analysis from the CIBMTR in that era identified a significantly increased risk for graft failure compared with unmanipulated marrow transplantation (relative risk 9.29; p < 0.0001) [18].

T-Cell Depletion to Prevent Graft-versus-Host Disease

Graft failure after TCD marrow transplantation may occur in two patterns: (1) failure of initial engraftment or graft rejection within 2 weeks of BMT, and (2) delayed graft failure, which can occur months following transplant [12,115]. Early graft failure after TCD transplantation likely represents primary immunologic rejection of donor hematopoietic elements by host lymphoid cells that have survived the conditioning regimen. Host T lymphocytes with donor-specific activity have been isolated from the blood of patients at the time of graft rejection [116–125]. It is likely that TCD or marrow manipulation removes cells that are important for promoting engraftment. However, it remains uncertain what roles specific donor cells play in establishing and maintaining a graft in human transplantation. In one trial of HLA-mismatched unrelated BMT in which patients were given grafts engineered to contain graded doses of CD4+ cells and CD8+ cells, it appeared that the CD8+ cell dose, but not CD4+ dose, influenced graft rejection [126]. The mechanism behind late graft failure remains unknown. Unlike primary rejection, donor-specific host T lymphocytes have not been isolated from the blood of recipients at the time of late graft failure. Since mixed hematopoietic and lymphoid chimerism is common after TCD transplantation, late graft rejection may result when this state of immune tolerance is broken by some event. Viral infections with CMV or human herpes virus-6 may also contribute to late graft failure after BMT [127–130]. Attempts to thwart immunologic rejection after BMT have included increasing host immunosuppression, intensification of the ablative regimen to create space or additional immunosuppressive agents post transplant [9,75,131–133]. Other approaches have included narrowing the spectrum of TCD, infusion of increasing doses of CD34+ cells in the graft, and administration of donor lymphocytes after transplantation. Graft failure can also be a major complication in allogeneic PBPC transplantation when extensive TCD is employed. In this setting, the risk of graft failure appears to be inversely correlated with the CD3+ content in the PBPC graft. In one analysis of 257 patients undergoing matched sibling PBPC transplantation with CD34 selection, the incidence of graft failure was 18% among recipients whose grafts contained 2.0 × 105 CD3+ cells/kg or less, compared with 1% among those who received more than 2 × 105 CD3+ cells/kg (p = 0.001). Low CD3+ dose was a significant predictor of graft failure in the multivariate analysis [38]. Similarly, another study demonstrated that a CD3+ dose of less than 0.83 × 105 cells/kg was an independent factor associated with poor survival, primarily from graft failure and disease relapse after PBPC HCT [134]. However, in a report from 2007, all 52 patients transplanted with CD34+-selected TCD matched-sibling mobilized PBPCs engrafted after conditioning with fractionated total body irradiation, thiotepa, and fludarabine without ATG, despite receiving a median of only 0.99 × 103 cells/kg [135]. Even in patients in whom hematopoietic recovery is observed, there is an increased frequency of mixed hematopoietic chimerism [136,137]. This has been particularly noted in the T-cell compartment. It is this mixed chimerism which may promote tolerance to subsequent organ allograft transplantation [138]. Mixed immunologic chimerism may predispose to the relapse of certain underlying malignancies, such as CML and myelodysplastic syndrome, but not others [139].

Immune reconstitution and infectious complications Immune reconstitution is delayed after TCD [140–147]. In general, the total lymphocyte count is lower in recipients of TCD grafts. Prolonged inversion of CD4+ : CD8+ ratios is observed with delays in CD4+ T cell recovery [140]. The number of T cells with memory phenotypes is decreased, and delays in functional recovery of T cells after TCD HCT have also been described. The proliferative responses of PBMCs to

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mitogenic stimuli are impaired far longer after TCD transplants than after conventional procedures [142,144]. T lymphocytes from recipients of TCD BMT have restricted variability in their TCR repertoires, limiting the breadth of potential cellular responses [145]. In addition, the capacity to generate new T cells is impaired post TCD, as noted by decreased numbers of thymic emigrants measured by TCR excision circle analysis [146,147]. Delayed immune reconstitution and loss of TCR repertoire diversity may place TCD HCT recipients at increased risk for opportunistic infections. CMV reactivation and adenovirus infections occur more frequently after TCD transplants and with increased frequency in recipients of in vivo anti-T-cell antibodies, particularly alemtuzumab [93–98]. One study suggested that partial TCD did not abrogate the transfer of antiCMV T-cell immunity, although protection against CMV appears lost with more extensive depletion [148,149]. In the large randomized study of TCD, the overall rate of fatal infections did not differ between the TCD and non-TCD arms. Bacterial infections were not increased. However, a higher incidence of severe CMV (28% versus 17%; p = 0.02) and life-threatening Aspergillus (16% versus 7%; p = 0.01) infections was observed in the patients receiving TCD [21,150]. A number of studies have demonstrated a higher probability of EBV reactivation in recipients of TCD grafts [15–17,151]. The reported incidence of post-transplantation lymphoproliferative disorder (PTLD) after TCD BMT ranges from 5% to 30%. Recipients of TCD transplants using HLA-mismatched or unrelated donor marrow appear to be at particularly high risk, as are patients with severe GVHD and those treated with specific anti-T-cell monoclonal antibodies [151]. Recipients of cord blood transplantation are also at high risk for EBV-PTLD, especially if they are conditioned with a nonmyeloablative regimen that includes ATG [152]. DLI [153] has been successful in treating PTLD, as has rituximab [154]. Rituximab has also been utilized in the pre-emptive mode in the setting of rising EBV viral loads in the absence of documented disease [155]. Strategies to accelerate recovery of T cell immunity after TCD BMT are in the process of being developed. Improvement in thymic function through administration of interleukin-7 (IL-7), luteinizing hormonereleasing hormone antagonists, and other compounds are being investigated [156–158]. Infusions of donor lymphocytes rendered less alloresponsive through a variety of techniques, including co-stimulatory blockade, photodepletion, and/or CD25 depletion, are under study, particularly after CD34+-selected haploidentical transplantation [159–161]. The production of virus-specific T lymphocytes with the capacity to recognize CMV, EBV, and adenovirus but without alloreactive potential is being studied in both the therapeutic and the prophylactic setting [162].

Disease relapse The increased risk of disease relapse after T-cell-depleted BMT was initially recognized in patients with CML [18–20,163–166]. In patients receiving matched sibling BMT for first chronic-phase CML, the risk of relapse with TCD has been estimated to be five- to sixfold higher than for conventional BMT. Interestingly, the incremental risk of relapse is less apparent after TCD transplantation using matched unrelated marrow [167,168]. In one retrospective single-institution series, the 3-year probability of relapse for CML in chronic phase was only 8% for recipients of unrelated TCD allografts, compared with 47% for those who received TCD marrow from HLA-identical siblings [168]. A study from the European Group for Blood and Marrow Transplantation demonstrated that, in patients with CML who received unrelated donor BMT, TCD was not associated with a higher incidence of relapse in the multivariate analysis [169]. However, in the large multicenter randomized trial of

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TCD, the 3-year relapse incidence was higher in the TCD patients transplanted for CML (20% versus 7%; p = 0.017) [21]. The increased incidence of CML relapse after TCD HCT has been linked, at least in part, to the reduction in GVHD. It is well established that allogeneic HCT is superior to autologous or syngeneic transplant because donor T cells can mediate alloimmune responses, and patients who develop clinically significant GVHD have a better leukemia-free survival after BMT [1,170,171], Direct evidence of the importance of donor T cells in the GVL effect has been derived from DLI studies for patients with CML who have relapsed after allogeneic BMT, where complete remission rates of 70–80% have been achieved [172,173]. The diseases for which DLI appears efficacious are the same ones in which TCD has been linked to increased relapse rates. These include, in addition to CML, both multiple myeloma and chronic lymphocytic leukemia [174,175]. In contrast to CML, TCD appears to have minimal effect on the relapse rates of patients transplanted for acute leukemia [176–178]. Retrospective data from the CIBMTR suggested that TCD is associated with a 1.7–2.0-fold increased risk for recurrence in patients with acute lymphoblastic leukemia in any phase, and in patients with AML who are transplanted in relapse or in first complete remission [18]. In this same analysis, AML patients transplanted in second complete remission actually had a lower risk of relapse with TCD. In two separate randomized trials comparing TCD with methotrexate and cyclosporine as GVHD prophylaxis for patients undergoing HLA-matched related or unrelated BMT, a higher relapse rate was observed after TCD BMT only in patients with CML, and not in patients with acute leukemia [21,179]. The risk of leukemia relapse may also vary depending on the extent and specificity of TCD employed. “Narrow-specificity” antibodies appear to be associated with a lower relapse rate compared with “broadspecificity” TCD methods. The 5-year probability of leukemia relapse was 28% for recipients of narrow-specificity TCD BMT versus 51% for recipients T-cell depleted by other techniques (p < 0.001) [49]. The 5year relapse rate for recipients of “narrow-specificity” TCD was similar to that observed in patients who received unmanipulated BMT. The intriguing data on the role of NK-cell reactivity after TCD mismatched transplantation give further support to the notion that TCD does not compromise the GVL effect in AML [83–85].

using CD34 selection, ATG or Campath, where the extensive depletion leads to profound and long-lasting impairment of immunity [95,101– 103]. Unfortunately, GVHD is common after these DLIs. While the incidence of chronic GVHD after prophylactic DLI is similar to that after conventional transplantation, a report suggests that this chronic GVHD after T-cell add-back is associated with less mortality, but retains its protective effect on relapse [101]. Retrospective studies in patients with CML and multiple myeloma undergoing high-dose transplantation have suggested that TCD plus DLI is a reasonable alternative to conventional GVH prophylaxis, and may in some circumstances be associated with superior outcome [181,184,185]. An update of the Spanish and British RIT experience in Hodgkin’s lymphoma suggested that, with the addition of DLI, current disease-free survival was superior for patients treated with cyclosporine and alemtuzumab compared with those receiving cyclosporine and methotrexate for GVHD prophylaxis [186]. Mounting evidence points to an important role for NK cells in limiting disease relapse, particularly in the setting of TCD. In haploidentical human transplantation with KIR mismatches in the GVH direction, donor-derived alloreactive NK-cell clones capable of lysing recipient target leukemia cells have been isolated at a high frequency early post transplant in the absence of clinical GVHD [85]. It is presumed these NK cells are also targeting host antigen-presenting cells and thereby prevent GVH induction. At least in certain circumstances, HLAmismatched transplantation combined with intensive conditioning and extensive TCD could provide a platform for the induction of NKcell-mediated antitumor activity without GVHD. Trials of NK-cell add-back to boost antitumor activity are underway. Whether such NK-cell-mediated antileukemic activity exists in mismatched transplantation in the absence of extensive TCD remains to be seen. Low doses of IL-2 administration in vivo may also enhance GVL activity through NK-cell stimulation without inducing GVHD. Prolonged infusion of low-dose recombinant IL-2 following autologous or TCD allogeneic BMT is well tolerated, and results in a marked increase in NK-cell numbers [187]. In a pilot clinical trial, infusion of IL-2 for at least 4 weeks after TCD BMT appeared to result in a lower incidence of disease relapse relative to historical controls [188]. In a recent report of a randomized clinical trial of subcutaneous IL-2 administration in patients with AML after autologous transplantation, the cohort randomized to IL-2 experienced lower relapse rates than the cohort randomized to observation [189].

Efforts to reduce relapse after T-cell-depleted HCT Efforts aimed at reducing leukemia relapse after TCD have primarily focused on immune-based strategies to enhance GVL activity without compromising GVHD prevention. These approaches have included: selective purging of T-lymphocyte subsets (i.e. CD8+ T cells), prophylactic DLI, and induction of NK-cell-mediated antitumor activity. Selective CD8+ TCD has attracted particular interest because preclinical data suggest that CD8+ T cells preferentially mediate GVHD. Early recovery of CD8+ T cells after transplantation has been associated with subsequent development of GVHD [180]. Initial trials in HLA-identical sibling BMT in the early 1990s suggested that CD8+ depletion with post-transplant cyclosporine alone could reduce the incidence and severity of GVHD without compromising the GVL effect [32,33]. Similar decreases in GVH without loss of the GVL effect have been documented in CD8 depletion of donor lymphocytes [181,182]. However, results of CD8 depletion of mobilized peripheral blood cells achieving a 2-log reduction have been disappointing, particularly in the unrelated transplant setting [183]. DLI or T-cell add-back strategies have been used in conjunction with TCD as a strategy to restore GVL activity and prevent disease relapse. Prophylactic DLI strategies may be particularly useful after TCD HCT

Novel approaches to graft manipulation Elimination of alloreactive T cells Efforts to limit the risk of GVHD associated with either stem cell or DLIs are under investigation, and include photoinactivation, anergy induction, and elimination of cells expressing activation antigens (e.g. CD25 or CD69). These approaches usually involve a brief co-incubation of donor cells with recipient-derived antigen-presenting cells. Theoretically, this approach would preferentially purge alloreactive lymphocytes responsible for GVHD, but retain “nonalloreactive” T cells, which might improve post-transplant immune reconstitution against infectious pathogens [190–193]. If minor histocompatibility antigens responsible for the GVH, but not the GVL effect, can be identified, targeted depletion of those cells recognizing those minor antigens could represent a major advance [194]. Adoptive therapy with CD4+CD25+Foxp3+Tregs CD4+CD25+Foxp3+ regulatory T cells (Tregs) have been identified as important mediators of self-tolerance and auto/alloimmunity, and immune

T-Cell Depletion to Prevent Graft-versus-Host Disease

reconstitution after hematopoietic cell transplantation. Murine transplant models have demonstrated that Tregs are potent suppressors of alloimmune responses and can protect against GVHD [50,52,195]. Lower levels of Tregs have been associated with the development of acute and chronic GVHD in some, but not all, human series [196–198]. Efforts to expand Tregs ex vivo for subsequent administration are underway [199]. However, the optimal dosing and timing of Treg administration requires further study. In vivo expansion of Tregs with low doses of recombinant IL-2 are being investigated in patients with established chronic GVHD based upon the observations from clinical trials that this cytokine preferentially expands Tregs in cancer and transplant patients [200]. Enrichment of CD8+ NK/T cells CD8+ NK/T cells are another lymphoid subset, which, like CD4+CD25+Foxp3+ T cells, appears to have regulatory properties that can modulate alloreactivity. Preclinical models have shown that adoptive transfer of CD8+ NK/T cells expanded in culture exhibit potent cytotoxic activity against allogeneic tumor targets without causing GVHD [156]. NK/Tregs of either donor or host type appear to prevent acute GVHD in mice by secreting IL-4 that prompts donor T cells to acquire an antiinflammatory, IL-4-secreting T helper cell type 2 phenotype [201–203]. Enrichment of these NK/T cells has been promoted by a reducedintensity conditioning regimen including total lymphoid irradiation and ATG. In this study, a very low rate of acute GVHD was observed without apparent compromise of the GVL effect [204]. Herpes simplex–thymidine kinase suicide gene insertion Herpes simplex–thymidine kinase (HSV-TK) gene insertion into donor T cells represents another approach for controlling GVHD after

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allogeneic transplantation or DLI. These cells have been rendered susceptible to destruction after exposure to ganciclovir [205]. Infusion of HSV-TK+ T lymphocytes with the TCD marrow graft did not hamper engraftment, and resulted in stable establishment of circulating TK+ cells in the recipient post transplant [206]. A case of chronic cutaneous GVHD responsive to ganciclovir has been reported in a patient who had received T cells bearing the HSV-TK gene at the time of BMT [207]. HSV-TK gene insertion has also been studied in the DLI setting without compromise of antitumor activity [208,209]. In one study, a direct correlation between antitumor response with peak levels of TK+ cells in the circulation was seen. Moreover, circulating TK+ cells could be disrupted by the administration of ganciclovir. In some patients, an immune response against the TK protein has been observed and has been linked to a decrease in circulating TK+ cells, although the development of HSVTK-specific CD8+ T cells did not interfere with the TK DLI response [210].

Summary Although it is clear that TCD reduces the incidence of acute GVHD, and, in most cases, transplant-related organ toxicity, there is as yet no definitive evidence that TCD improves overall survival after transplantation. The optimal extent and specificity of TCD remains uncertain. Nonetheless, many investigators remain optimistic that graft engineering will one day fulfill its promise of separating the GVH and GVL effects. Further delineation of the cellular subsets which promote engraftment and control alloimmune, antitumor, and antiviral responses is needed to optimize graft engineering.

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in first remission (CR1): preliminary results from Cancer and Leukemia Group B (CALGB) 19808. American Society of Hematology Annual Meeting 2007; Abstract 157. Chen BJ, Cui X, Liu C, Chao NJ. Prevention of graft-versus-host disease while preserving graftversus-leukemia effect after selective depletion of host-reactive T cells by photodynamic cell purging process. Blood 2002; 99: 3083–8. Koh MB, Prentice HG, Lowdell MW. Selective removal of alloreactive cells from haematopoietic stem cell grafts: graft engineering for GVHD prophylaxis. Bone Marrow Transplant 1999; 23: 1071–9. Solomon SR, Mielke S, Savani BN et al. Selective depletion of alloreactive donor lymphocytes: a novel method to reduce the severity of graftversus-host disease in older patients undergoing matched sibling donor stem cell transplantation. Blood 2005; 106: 1123–9. Vaclavkova P, Cao Y, Wu LK, Michalek J, Vitetta ES. A comparison of an anti-CD25 immunotoxin, Ontak and anti-CD25 microbeads for their ability to deplete alloreactive T cells in vitro. Bone Marrow Transplant 2006; 37: 559–67. De Witte MA, Toebes M, Song JY et al. Effective graft depletion of MiHAg T-cell specificities and consequences for graft-versus-host disease. Blood 2007; 109: 3830–8. Hanash AM, Levy RB. Donor CD4+CD25+ T cells promote engraftment and tolerance following MHC-mismatched hematopoietic cell transplantation. Blood 2005; 105: 1828–36. Miura Y, Thoburn CJ, Bright EC et al. Association of Foxp3 regulatory gene expression with

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graft-versus-host disease. Blood 2004; 104: 2187–93. Zorn E, Kim HT, Lee SJ et al. Reduced frequency of FOXP3+ CD4+CD25+ regulatory T cells in patients with chronic graft-versus-host disease. Blood 2005; 106: 2903–11. Clark FJ, Gregg R, Piper K et al. Chronic graftversus-host disease is associated with increased numbers of peripheral blood CD4+CD25high regulatory T cells. Blood 2004; 103: 2410– 16. Hoffmann P, Eder R, Kunz-Schughart LA et al. Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells. Blood 2004; 104: 895–903. Zorn E, Nelson EA, Mohseni M et al. IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood 2006; 108: 1571–9. Zeng D, Lewis D, Dejbakhsh-Jones S et al. Bone marrow NK1.1(−) and NK1.1(+) T cells reciprocally regulate acute graft versus host disease. J Exp Med 1999; 189: 1073–81. Lan F, Zeng D, Higuchi M, Huie P, Higgins JP, Strober S. Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: “natural suppressor” cells. J Immunol 2001; 167: 2087–96. Lan F, Zeng D, Higuchi M, Higgins JP, Strober S. Host conditioning with total lymphoid irradiation and antithymocyte globulin prevents graftversus-host disease: the role of CD1-reactive

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natural killer T cells. Biol Blood Marrow Transplant 2003; 9: 355–63. Lowsky R, Takahashi T, Liu YP et al. Protective conditioning for acute graft-versus-host disease. N Engl J Med 2005; 353: 1321–31. Munshi NC, Govindarajan R, Drake R et al. Thymidine kinase (TK) gene-transduced human lymphocytes can be highly purified, remain fully functional, and are killed efficiently with ganciclovir. Blood 1997; 89: 1334–40. Tiberghien P, Ferrand C, Lioure B et al. Administration of herpes simplex-thymidine kinase-expressing donor T cells with a Tcell-depleted allogeneic marrow graft. Blood 2001; 97: 63–72. Aubin F, Cahn JY, Ferrand C et al. Extensive vitiligo after ganciclovir treatment of GvHD in a patient who had received donor T cells expressing herpes simplex virus thymidine kinase. Lancet 2000; 355: 626–7. Link CJ, Burt RK, Traynor AE et al. Adoptive immunotherapy for leukemia: donor lymphocytes transduced with the herpes simplex thymidine kinase gene for remission induction. HGTRI 0103. Hum Gene Ther 1998; 9: 115–34. Ciceri F, Bonini C, Marktel S et al. Anti-tumor effects of HSV-TK engineered donor lymphocytes after allogeneic stem cell transplantation. Blood 2007; 109: 4698–707. Traversari C, Marktel S, Magnani Z et al. The potential immunogenicity of the TK suicide gene does not prevent full clinical benefit associated with the use of TK-transduced donor lymphocytes in HSCT for hematologic malignancies. Blood 2007; 109: 4708–15.

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Manifestations and Treatment of Acute Graft-Versus-Host Disease

Background Historical development The first description of graft-vs.-host disease (GVHD) is ascribed to Barnes and Loutit in 1955, who reported on ‘secondary disease’ in animals undergoing transplantation [1]. this disorder was named to differentiate it from the primary disease of radiation sickness, as fatal secondary disease was observed in irradiated mice given allogeneic spleen cells but not in recipients of syngeneic cells. By the late 1950s, it was apparent that the skin abnormalities and diarrhea of secondary disease and runt disease (a wasting syndrome in unirradiated newborn mice given allogeneic spleen cells) were the result of immunologically competent cells introduced into an immunoincompetent host. Only then was the term graft-vs.-host introduced to describe the contribution of the donor in this immunologic assault [2,3]. Early human transplants of allogeneic bone marrow were often complicated by GVHD [4–7]. The clinical features of human GVHD were very similar to those reported in the animal literature [8], as well as to reports of GVHD developing in immunodeficient children who received blood transfusions [9]. The term acute GVHD traditionally describes a distinctive syndrome of cutaneous, hepatic and gastrointestinal inflammation developing within 100 days of allogeneic hematopoietic cell transplantation (HCT). The term chronic GVHD describes a more pleiotropic syndrome that develops after day 100 (see Chapter 87). However, it is now recognized that acute GVHD can occur after day 100, and chronic GVHD may occur before then. Late-onset acute GVHD predominantly occurs after reduced-intensity conditioning regimen-based allogeneic transplantation [10]. Pathogenesis The criteria for the development of GVHD were summarized by Billingham in 1966 as follows [11]. 1 The graft must contain immunologically competent cells. 2 The host must possess important transplantation alloantigens that are lacking in the donor graft, so that the host appears foreign to the graft, and is, therefore, capable of stimulating it antigenically.

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

3 The host itself must be incapable of mounting an effective immunologic reaction against the graft, at least for sufficient time for the latter to manifest its immunologic capabilities; that is, it must have the security of tenure. While adequate for a number of years, these criteria require some modification to better incorporate our current understanding of the biology of GVHD. The occurrence of autologous GVHD suggests that inappropriate recognition of host self-antigens may occur, and transfusion-associated GVHD may develop in certain immunocompetent individuals. A multistep model of antigen expression, cytokine production, T-cell activation, and tissue injury has been described (reviewed in Chapter 16) [12,13]. Alloreactivity Abundant animal data demonstrate that T lymphocytes contained in the donor inoculum proliferate and differentiate in vivo in response to disparate major and minor histocompatibility antigens expressed on host tissues, and directly, or through secondary mechanisms, attack recipient cells, thus producing the signs and symptoms of acute GVHD (see Chapter 16) [8,11–14]. The afferent arm of acute GVHD consists of antigen presentation, activation of T cells, clonal proliferation, and differentiation [15,16]. In the efferent phase, direct cytotoxicity and the release of cytokines from activated lymphocytes contributes to cell death, either directly or through recruitment of secondary effectors, such as natural killer (NK) cells [17]. Based on mechanistic roles postulated for interleukin-1 (IL-1), IL-2, lipopolysaccharide (LPS), and proinflammatory cytokines such as IL-6, interferon-gamma, and tumor necrosis factor (TNF), immunomodulatory agents have been administered in vivo to control GVHD. A review of methods to prevent acute GVHD through pharmacologic and graft manipulation can be found in Chapters 84 and 85. Microbial environment The microbial milieu of the host may also influence the development of GVHD. Compared with conventionally housed irradiated mice, enteric GVHD was significantly reduced in germ-free mice given incompatible marrow [18]. Human studies confirm this gnotobiotic effect [19,20]; however, this approach may be associated with an increased susceptibility to fungal overgrowth in the gastrointestinal tract [21]. Microorganisms could act as triggers of GVHD, perhaps by sharing antigenic epitopes with gut epithelial cells or by activating latent virus-inducing antigens on cell surfaces to become targets of alloreactivity [22]. Gut damage also leads to translocation of bacteria across the mucosal barrier into the blood stream, and elaboration of LPS in the circulation, thereby releasing cytokine effectors of GVHD [23,24]. Laminar airflow protec-

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tive isolation with gut decontamination has been shown to decrease the incidence of acute GVHD and improve survival in patients with aplastic anemia prepared with cyclophosphamide alone [19], but improved outcomes have not been generalizable to all transplant scenarios [25,26]. Updated analyses of two previously reported randomized clinical trials of antimicrobial prophylaxis show significant lessening of enteric GVHD after administration of either antifungal prophylaxis with fluconazole compared with placebo [27], or anerobic intestinal decontamination with metronidazole plus ciprofloxacin, compared with ciprofloxacin alone [20]. Preclinical models demonstrate that administration of an LPS antagonist reduces TNF levels and intestinal GVHD histopathology after HCT [23]. Even newer approaches aimed at preservation of enteric epithelial integrity are currently under study. Keratinocyte growth factor has specificity for gut epithelial cells and can prevent epithelial injury from chemoradiotherapy, reduce levels of LPS and TNF, and decrease enteric GVHD in animal models [28]. However, the use of keratinocyte growth factor after transplantation has not been shown to reduce acute GVHD in a multicenter study [29]. A randomized study is ongoing. Tolerance Chapter 15 reviews the mechanisms of graft–host tolerance after allogeneic HCT. Tolerance can be achieved by elimination (clonal deletion) in the thymus of host reactive cells; accordingly, thymic damage could abrogate self-tolerance [30,31]. Alternatively, functional suppressor CD4+CD25+FoxP3+ regulatory T cells may be important in transplantation tolerance [32], and strategies to selectively expand or transfer these T cells to abrogate experimental acute GVHD have been developed [33]. Tolerance can also be attained via the transplacental bidirectional passage of maternal and fetal antigens during pregnancy. In this fashion, tolerance to noninherited maternal antigens (NIMAs) in offspring and to inherited paternal antigens in the mother can occur [34]. In experimental models, transplantation from NIMA-exposed offspring to mothers results in less acute GVHD than transplantation from inherited paternal antigens-exposed mothers to offspring, suggesting that NIMAinduced tolerance exists. This tolerance may be dependent on regulatory T cells [35]. Consistent with this finding is that, among haploidentical sibling transplants, mismatches for noninherited paternal antigens appear associated with higher rates of GVHD, suggesting that exposure to maternal human leukocyte antigens (HLAs) in utero could have a lifelong effect on subsequent immune responses [36,37].

Incidence and risk factors Incidence Clinically relevant, grade II–IV acute GVHD occurs in 35–40% of patients transplanted from a matched sibling donor [38], and in 40% to greater than 50% of recipients of unrelated donor grafts [39,40]. Severe acute GVHD (grade III–IV) occurs in up to 20% of recipients of related donors [38], and up to 35% of unrelated donors [39,40]. The median time to the diagnosis of acute GVHD varies with conditioning, with recipients of high-dose therapy and transplantation being diagnosed at a median of 17 days [41], in comparison with recipients of reducedintensity conditioning transplantation being diagnosed at a median of 3 months [10], and commonly is associated with the tapering of immunosuppressive agents. Donor–recipient risk factors HLA disparity between the hematopoietic cell donor and recipient is the most powerful factor governing the severity and kinetics of acute GVHD.

The incidence of acute GVHD is much increased with HLA-nonidentical donors when compared with HLA-identical donors [42]. A higher incidence of acute GVHD has also been observed with HLA-matched unrelated donors when compared with related donors, necessarily implicating minor HLA antigens in the pathogenesis of GVHD [43]. Although some studies have identified certain HLA antigens associated with increased rates of GVHD [44], these correlations have not been observed consistently [45,46]. Chapter 12 details the evolving precision of molecular HLA typing. The importance of class I and II allele matching in reducing the risk for GVHD has been widely demonstrated in unrelated donor HCT cohorts [47–49]. A large National Marrow Donor Program analysis of individuals undergoing HCT from HLA-A- and B-matched unrelated donors demonstrated that HLA-DRB1 allele disparity was independently associated with an increased risk of severe acute GVHD [50]. Since then, the importance of HLA-C has been demonstrated, with a clear association between mismatch at this locus and acute GVHD and adverse transplantation outcomes [51–53]. Similarly, mismatch at the extended class II antigen, HLA-DP, has also been associated with an increased risk of acute GVHD [54,55]. Minor HLA peptides have been identified and sequenced, and these minor histocompatibility antigen disparities appear correlated with the risk for GVHD in adults given HLA-identical donor grafts [56–58]. The importance of minor histocompatibility antigen matching is further supported by the observed lower rate of GVHD in populations that feature genetic homogeneity [59]. In more genetically heterogeneous racial groups, data are less consistent as to increased rates of GVHD [60–62]. Disparities in antigens with presentation restricted to certain class I and class II alleles have been associated with GVHD, demonstrating that altered peptide ligand binding may have profound effects on alloreactivity [63,64]. Sex mismatching and donor parity have been associated with an increased risk of acute GVHD, with male recipients of female grafts having the highest rates of GVHD [44–46]. These findings have been confirmed in the pediatric setting as well [65]. The pathogenesis of this phenomenon is likely due to the recognition of Y chromosome-encoded neoantigens by female donor T cells. Previously parous donors may have experienced maternal alloimmunization from unshared minor antigens of the fetus [66]. These presensitized cells would then mount a vigorous secondary type response when transplanted. Age is another key factor associated with the development of acute GVHD. In a large analysis of National Marrow Donor Program donor– recipient pairs, increasing donor age was associated with an increased incidence of severe acute GVHD (relative risk 1.08 per decade; p = 0.002) [67]. It is possible that the increased proportion of mature T cells in older donors and the relative paucity of naïve T cells could account for these increased rates of GVHD. Cord blood transplantation, associated with the lowest rates of acute and chronic GVHD, fits well with this model, but the absolute size of the T-cell inoculum may be responsible for differences here. Since sibling ages are often closely linked, it has been difficult to demonstrate the effect of age on GVHD incidence in matched donor transplantation. In one retrospective analysis, when stratified by age (less than 18 versus 18 or more years), the incidence of acute GVHD was 27% in the younger group and 63% in the older group [68]. In a single-center review of risk factors for acute GVHD, recipient age greater than 40 years was associated with an increased risk of severe acute GVHD [69]. The association of increasing donor age and acute GVHD is also noted among pediatric transplant recipients, where rates of acute GVHD are universally lower [70]. The influence of ABO blood type on acute GVHD remains controversial. ABO glycotransferase peptides may act as minor histocompatibility antigens and stimulate allogeneic T-cell responses [71]. A large

Manifestations and Treatment of Acute Graft-Versus-Host Disease

Center for International Blood and Marrow Transplant Research (CIBMTR) analysis of 3103 donor–recipient pairs demonstrated an increased risk of severe acute GVHD among bidirectional ABOmismatched pairs (hazard ratio 1.869; p = 0.006) [72]; however, several smaller studies were not able to replicate this finding [73–76]. Since ABO incompatibility has other consequences on graft processing and transfusion requirements after transplantation, it is often considered in the search strategy in unrelated donor transplantation. Donor and recipient cytomegalovirus (CMV) serostatus may also influence the rates of acute GVHD. For CMV-seronegative bone marrow transplantation (BMT) recipients, matching with seronegative donors also appears to reduce the risk of both CMV infection and GVHD [44].

Source of hematopoietic cells The source of allogeneic hematopoietic cells may also influence the development of GVHD. For more than two decades, experience was derived from BMT recipient cohorts describing rates and risk factors for GVHD [44–46,77]. Recently, peripheral blood progenitor cells (PBPCs) have become the most utilized source of hematopoietic cells for both related and unrelated donor transplantation [78]. PBPC products are associated with more rapid engraftment, fewer hospitalizations, less donor morbidity, and economic advantages when compared with bone marrow products [79]. Initial phase II studies of matched, related donor PBPC transplantation demonstrated an increased incidence of acute GVHD [80–83]. To address this question further, there have been eight randomized controlled trials designed to compare GVHD outcomes between matched sibling bone marrow and PBPC transplantation [84–91]. In the largest randomized trial involving 350 patients enrolled between 1995 and 1999, acute GVHD grade II–IV and severe acute GVHD grade III–IV were significantly increased in the PBPC group (52% versus 39%, p = 0.014; and 28% versus 16%, p = 0.01; respectively). In this trial, however, an abbreviated three-dose course of methotrexate was used in both arms as GVHD prophylaxis [90]. The Seattle group demonstrated a nonsignificant increase in the hazard ratio for both acute GVHD (hazard ratio 1.21, 95% confidence interval 0.81–1.81) and severe acute GVHD (hazard ratio 1.27, 95% confidence interval 0.55–2.89) in the PBPC arms [84]. Other trials have reported nonsignificant increases [87,91] or similar risks in the PBPC and bone marrow groups [85,86,89], with only one small trial demonstrating a statistically significant increase in acute GVHD in the bone marrow arm [88]. A meta-analysis of 16 studies (five randomized and 11 cohort) found that the relative risk for acute GVHD after PBPC transplantation was 1.16-fold greater (p = 0.006) when compared with BMT [92]. A more recent meta-analysis examining individual patient data from randomized trials only noted a similar effect size for acute GVHD (odds ratio 1.14; p = 0.2) but a statistically significant increase in severe acute GVHD after PBPC transplantation (odds ratio 1.13; p = 0.03) [93]. In addition, this meta-analysis was able to demonstrate a survival advantage for patients with advanced malignancies who received PBPC transplants, possibly linking the higher rates of acute and chronic GVHD with an increase in graft-versus-tumor effects. In the unrelated donor setting, initial small phase II studies of PBPC transplantation with retrospective comparisons to bone marrow did not demonstrate differences in the rates of acute GVHD [94,95], but a large retrospective review of over 1300 patients from the CIBMTR database has demonstrated that acute GVHD may be more prevalent (56 versus 46%; p < 0.001) after PBPC transplantation than BMT for leukemia and myelodysplasia [96].

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Umbilical cord blood transplantation appears to be associated with the lowest rates of acute GVHD. Umbilical cord blood units also have the lowest numbers of T cells. At least two large retrospective analyses have demonstrated equivalent or lower rates of GVHD when compared with mismatched bone marrow grafts [97,98]. Many of the differences in the rates of acute GVHD noted between different stem cell sources can be attributed to the graft composition of these hematopoietic stem cell sources. Graft composition and hematopoietic stem cell dose The clinical yields of granulocyte colony-stimulating factor-mobilized PBPC collections are generally superior to bone marrow harvests when CD34+ progenitor numbers are compared. The differential yield after collection of both bone marrow and PBPCs was examined in 40 healthy donors who underwent bone marrow harvesting followed by granulocyte colony-stimulating factor-stimulated PBPC collection 1 week later for their HLA-identical siblings. Total nucleated cells, CD34+ cell yield, and colony-forming unit-granulocyte–macrophage activity were 2.3, 3.7, and 3.7 times higher, respectively, after PBPC harvesting when compared with bone marrow harvesting [99]. The dose of CD34+ progenitors has been clearly shown to affect the development of chronic GVHD [100,101], particularly after PBPC transplantation [102], but this association is less clear for acute GVHD. In a study of risk factors for acute GVHD, a CD34+ cell dose of greater than 8 × 106/kg was associated with an increased risk of acute GVHD, but this remains the largest study demonstrating an association [103]. More recently, an analysis of 100 recipients of matched sibling PBPC transplantation after high-dose conditioning reported that the dose of CD34+ stem and progenitor cells did not influence the rate of acute GVHD [100]. Similarly, in a study of 214 patients who received matched related bone marrow grafts, there was no relationship between CD34+ cell dose and acute GVHD [104]; these results were similar to the largest analysis, which examined both bone marrow and PBPC transplantation [102]. Finally, after reduced-intensity conditioning and transplantation of unrelated donor progenitor cells, no effect of CD34+ cell dose was noted [105]. The other cellular component that is vastly different when peripheral blood and bone marrow hematopoietic cell products are compared is the number and phenotype of T cells. Since T cells are found in much higher concentrations in PBPC grafts, it has been assumed that this accounts for the differences in GVHD outcome between bone marrow and PBPC grafts, but this too has yet to be proven [100,103,106,107]. T-cell subsets may be responsible for the differences in acute GVHD incidence. CD8+ T cells may be crucial for the development of acute GVHD, and CD8+ cell dose was the only cellular fraction that correlated with the development of acute GVHD in a reduced-intensity transplantation model [108], although this has not been confirmed by all [107,109]. Depletion of CD8+ cells can influence the rates of acute GVHD, which also supports the notion that these cells are important in the pathophysiology of acute GVHD [110]. Regulatory T cells, which are a subset of CD4+CD25+ T cells that constitutively express the transcription factor FoxP3 (forkhead boxP3), are suppressors of effector T cells and can control GVHD in murine models [111]. These cells have also been postulated to influence the development of human acute GVHD, with higher concentrations of transplanted regulatory T cells associated with a lower incidence of acute GVHD [112–114]. However, this finding has not been observed by all [115,116]. The pace of regulatory T-cell reconstitution and the occurrence of acute GVHD has also been correlated [113]. The impact of other T-cell subsets as well as the dose of NK cells administered on acute GVHD remains to be determined.

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Cytokine levels and gene polymorphisms Polymorphisms in cytokine genes (such as IL-6, IL-10, and others) that lead to altered expression and levels of cytokines have been linked to differences in acute [117–129] and chronic [130–132] GVHD incidence, and also to differences in host response to infectious agents after transplantation [133–134]. An additional group of distinct polymorphisms that occur in the family of the NK cell receptor and ligand group, collectively termed the killer immunoglobulin-like receptor family, have been linked to differences in both GVHD and relapse rates after transplantation [135–139]. There are additional genes involved in drug metabolism that have been linked to toxicity and GVHD after allogeneic transplantation [140,141]. Genes with only loose associations with the immune system can also influence rates of GVHD [142–146]. These polymorphisms may be critical in both host and recipient-derived tissues, as demonstrated by polymorphisms of IL-10 (an inhibitor of TNF-α), where both donor (relative risk 3.5) and recipient (relative risk 7.9) polymorphisms predict the development of acute GVHD [147]. Currently, the presence of cytokine polymorphisms is not ascertained prior to allogeneic transplantation, but several investigators have suggested that search strategies that include donor genotype of cytokines and other relevant genes be incorporated into search strategies [148– 150]. Conditioning regimen intensity and immunomodulation The pathophysiology of acute GVHD, reviewed in Chapter 16, describes an afferent loop caused by tissue injury related to conditioning therapy. As such, it has been postulated that lower-intensity preparative regimens, by virtue of causing less tissue injury and lower concentrations of elucidated cytokines, would be associated with a reduced incidence of acute GVHD. In a retrospective single-center review of 238 patients who underwent reduced-intensity compared with traditional high-dose transplantation, the hazard rate for grade II–IV acute GVHD was 1.73 (p = 0.015) despite a similar GVHD prophylaxis strategy in both arms [151]. Similarly, the European Group for Blood and Marrow Transplantation noted a 40% reduction in the incidence of acute GVHD among 315 reduced-intensity regimen patients when compared with over 400 high-dose transplant patients (p = 0.01) [152]. Even among patients undergoing high-dose transplantation, the intensity of the conditioning regimen may influence acute GVHD outcomes. Among HLA-identical transplant recipients who received GVHD prophylaxis with methotrexate and cyclosporine, acute GVHD developed in 48% of patients given 1575 cGy total body irradiation, compared with 21% of patients given 1200 cGy total body irradiation (p = 0.02) [153]. An alternative hypothesis to the conditioning-related injury theory is that more intensive conditioning might eliminate persistent host cells and prevent the development of mixed donor–host chimerism, which has been associated with a decreased probability of GVHD [154–155].

Predictive assays The role of in vitro assays to predict the occurrence of acute GVHD is largely historical, except in some situations of highly transfused recipients, such as patients with advanced aplastic anemia. The frequencies of alloreactive cytotoxic T-lymphocyte precursors and helper Tlymphocyte precursors may have pretransplant predictive value in selecting donor–recipient pairs with a reduced risk of GVHD [156,157]. Skin-explant models have been developed in which donor lymphocytes sensitized in vitro against recipient cells are cultured with recipient skin [158,159]. In other models, a mixed epidermal cell lymphocyte reaction is used to detect incompatibility [160]. Although correlations with

GVHD appear to be statistically significant, individual variations have been observed [161]. One report suggests a tighter correlation by combining cytokine gene polymorphisms and explant results [162].

Clinical features of acute GVHD Cutaneous acute GVHD The skin is the most commonly affected organ in acute GVHD, with over 75% of acute GVHD patients having some cutaneous involvement, and 44% of patients having skin involvement as their only manifestation of acute GVHD [163]. The initial manifestation of acute GVHD is most commonly a maculopapular exanthem [164]. Lesions may be pruritic or painful and red to violaceous in color, and may involve the palms and soles, which helps to differentiate the rash of acute GVHD from a drug eruption, which generally spares these areas (see Plate 23.16). The characteristic predilection for these sites appears related to concentration of hematopoietic stem cells in the rete ridges (see Plate 23.18) [165]. As the rash intensifies, confluent involvement of the cheeks, ears, neck, and trunk is noted, often associated with papule formation (see Plate 23.22). In its most severe form, bulla formation ensues and epidermal necrosis with desquamation occurs, mimicking toxic epidermal necrolysis (see Plate 23.17). The bullae contain sterile serous fluid, and unless the acute alloreactive inflammatory reaction has abated, the regenerating skin exposed by the bullae may be erythematous as well. Gastrointestinal acute GVHD The gastrointestinal tract is the second most commonly involved organ in acute GVHD, with up to half of individuals being affected [163]. In general, signs of enteric GVHD develop as the chemoradiotherapy effects resolve following the first several weeks after transplantation, but these syndromes may overlap, making an early diagnosis difficult. Symptoms of acute GVHD of the distal small bowel and colon include profuse watery diarrhea, intestinal bleeding, crampy abdominal pain, and, in its severest form, ileus. The diarrhea is often green, mucoid, watery, and mixed with exfoliated cells, forming fecal casts. A variant of gastrointestinal GVHD involving the upper gastrointestinal tract associated with anorexia, nausea, and dyspepsia is known to occur, and patients with upper gastrointestinal disease may not manifest lower tract involvement. Imaging of the gastrointestinal tract using computed tomography often reveals diffuse edema of the intestinal tract. These findings are nonspecific and should be followed with endoscopy and biopsy as indicated. Endoscopic findings of enteric GVHD range from normal to extensive edema (see Plates 23.24, 23.26, and 23.27) and mucosal sloughing (see Plate 23.30). Lesions may be most prominent in the cecum, ileum, and ascending colon, but may also involve the stomach, duodenum, and rectum. Gastrointestinal endoscopy and biopsy are mandatory for the diagnosis of upper tract disease [166,167], since anorexia and nausea are common symptoms of conditioning-related injury. Endoscopy and biopsy are recommended for the diagnosis of lower gastrointestinal tract in order to exclude other diagnoses. Hepatic effects The liver is the least commonly affected organ in acute GVHD, with less than 20% of acute GVHD patients having some degree of hepatic involvement [163]. Cholestatic jaundice is the most common manifestation, but a concomitant or isolated transaminitis is not uncommon [168– 172]. When severe, hepatic failure with encephalopathy may occur. Percutaneous or transjugular liver biopsy is recommended for the diag-

Manifestations and Treatment of Acute Graft-Versus-Host Disease

nosis of hepatic GVHD, when feasible, as there are no imaging studies that reliably can differentiate hepatic GVHD from other causes of liver dysfunction. Other findings Ocular symptoms have been described in patients with acute GVHD [173–176]. Manifestations may include photophobia, hemorrhagic conjunctivitis, pseudomembrane formation, corneal ulceration, and lagophthalmos. Involvement of the central nervous system with acute GVHD has been postulated to occur [177–178], although in most cases the definite etiology of the central nervous system pathology cannot reliably be ascribed to an alloimmune effect. Infections and leukoencephalopathy related to calcineurin inhibitors remain the primary diagnostic alternatives. The syndromes of diffuse alveolar hemorrhage and idiopathic pneumonia syndrome are more common among individuals with acute GVHD [179], but whether this in fact represents a true pulmonary manifestation of acute GVHD remains unclear. The kidney appears to be susceptible to alloimmune attack in the context of chronic GVHD, but acute GVHD involving the kidney has not been reported. However, there appears to be an association between thrombotic microangiopathy after transplantation and acute GVHD [180,181]. Thrombocytopenia and anemia have been reported in patients with GVHD, and failure of platelet recovery was shown to be 3.5-fold more common in patients with acute GVHD [182,183]. In addition, persistent thrombocytopenia may carry negative prognostic value [184].

Diagnosis and differential diagnosis Acute GVHD is a clinicopathologic syndrome involving the skin, liver, and gut. The clinical features of acute GVHD may be difficult to distinguish from other events early after transplantation. For example, the skin rash of acute GVHD is often confused with eruptions related to allergic reactions. Similarly, the pattern of hepatic enzyme perturbation may be difficult to distinguish from sinusoidal obstructive syndrome of the liver or viral hepatitis. Indeed, these complications may coexist, further complicating the diagnosis and management of hepatic GVHD. Finally, the nausea and anorexia of upper gastrointestinal GVHD and diarrhea of lower gastrointestinal GVHD are often indistinguishable from conditioning-related injury and intestinal infection with agents such as Clostridium difficile, Candida and CMV. Occurring around the time of neutrophil engraftment, an engraftment syndrome, consisting of fever in

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the absence of infection, diffuse erythroderma, and noncardiogenic pulmonary edema, may also be difficult to clinically distinguish from acute GVHD [194]. Biopsy of the target organs of GVHD (reviewed in Chapter 27) is often obtained for confirmation of the diagnosis of acute GVHD; however, the diagnosis remains largely a clinical one, and therapy often must be initiated before the results of biopsies are known. Early after transplantation, cutaneous acute GVHD may be difficult to distinguish from the effects of chemoradiotherapeutic conditioning, allergic reactions to medications or viral exanthems, and thus serial biopsies and observations may be required. However, the utility of skin biopsy in the diagnosis of acute GVHD remains questionable. Several retrospective reviews have demonstrated the general lack of utility of the skin biopsy in the diagnosis and prognostication of cutaneous GVHD [195–197]. Similarly, biopsy of the lower and upper gastrointestinal tract may be difficult to interpret early after transplantation, since the apoptotic changes associated with acute GVHD can often be confused with conditioning-related injury. Viral or other infectious etiologies of diarrhea after transplantation are more readily distinguished, however. Biopsy of the liver is often indicated, as the histologic findings of acute hepatic GVHD, including portal triad inflammation, are often easily distinguishable from the findings of sinusoidal obstructive syndrome of the liver, viral hepatitis or the cytotoxic effects of medications, unless massive necrosis obscures the diagnostic picture. Several biological markers of acute GVHD have been postulated to predict the occurrence of acute GVHD better than clinical acumen. Assays for single inflammatory molecules such as TNF-α [198] and IL-13 [199], or whole-blood proteomic assays [200,201], may, in the future, be the best markers of acute GVHD, but at the moment are experimental in nature. Grading and severity scores Table 86.1 presents the commonly used modified Seattle Glucksberg criteria (also termed the Consensus criteria) for the staging and grading of acute GVHD [193,194]. A modification of grade II disease includes symptoms of nausea, anorexia, food intolerance or vomiting confirmed to be enteric GVHD on upper intestinal biopsy [167,194]. A more recent staging system proposed by the International Bone Marrow Transplant Registry (IBMTR) uses the same individual organ staging but modifies the overall grading scheme slightly (Table 86.2) [195]. While both systems predict overall survival equally well, the Consensus system

Table 86.1 Modified Glucksberg grading of acute graft-versus-host disease Organ stage

Skin*

Liver

Gut

1 2 3 4

Rash <25% Rash 25–50% Rash >50% Generalized erythroderma with bullae

Bilirubin 2–2.9 mg/dL Bilirubin 3–6 mg/dL Bilirubin 6.1–15 mg/dL Bilirubin >15 mg/dL

Diarrhea 500–1000 cm3/d or biopsy-proven upper gastrointesinal involvement Diarrhea 1000–1500 cm3/d Diarrhea 1500–2000 cm3/d Diarrhea >2000 cm3/d or severe abdominal pain with or without ileus

Organ stage

Skin*

Liver

Gut

I II III IV

Stage 1–2 Stage 3 or – Stage 4 or

None Stage 1 or Stage 2–3 or Stage 4

None Stage 1 Stage 2–4 –

* Use the “rule of nines” to determine the body surface area. Adapted with permission from [203].

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Table 86.2 International Bone Marrow Transplant Registry grading of acute graft-versus-host disease Organ stage

Skin*

Liver

Gut

A B C D

Stage 1 Stage 2 Stage 3 Stage 4

None Stage 1 or 2 Stage 3 Stage 4

None Stage 1 or 2 Stage 3 Stage 4

Adapted with permission from [204].

predicted earlier survival better in a comparison of 607 patients. The IBMTR index, however, was easier to use and was associated with less physician bias or error in stage assignment [196]. Reliable incidence figures for acute GVHD are limited by interobserver and intercenter variation. This variability largely derives from uncertainty in establishing grade II GVHD [197,198]. Other factors may influence acute GVHD outcome, independent of severity scores. For example, in a logistic regression training and validation model to assess nonrelapse mortality in patients with chronic myeloid leukemia who had undergone unrelated donor BMT, mortality upon the diagnosis of acute GVHD was influenced not only by bilirubin elevation and the requirement for first- and second-line immunosuppressive therapy, but by protean factors such as caloric intake and performance status [199]. Using this scoring system, algorithms to predict mortality at 200 days from transplantation could be constructed, which can be used in real time by clinicians for prognostic purposes. Similarly, a retrospective analysis demonstrated that up to 20% of patients will develop serious acute (or chronic) GVHD by 1 year from transplantation. In this analysis, serious GVHD was defined by death or major disability related to GVHD or its treatment, three or more major infections in a single year during persistent GVHD, hospitalization for greater than 60 days in a single year because of GVHD or any hospitalization for suicidality because of GVHD or its treatment [200].

Therapy of acute GVHD Primary therapy The mainstay of therapy for acute GVHD is corticosteroids, with the route of administration determined by the severity of acute GVHD and the degree of organ involvement. Corticosteroids are lympholytic and decrease the inflammatory cytokine cascades that propagate acute GVHD. Other agents have been utilized as front-line therapy, but none is superior to corticosteroids [41,201]. There is substantial variability between investigators and centers regarding the stage of acute GVHD that should be treated, and the dose of corticosteroids to be used, particularly in early-stage GVHD. Cutaneous involvement with less than 50% body surface area involvement in the absence of hepatic or gastrointestinal involvement (stage I or II skin disease, or overall grade I acute GVHD) can be treated with the topical administration of medium- or high-potency glucocorticoids alone; however, individuals with multiorgan involvement, as well as some individuals with near-50% body surface cutaneous involvement, require the systemic administration of corticosteroids. The recommended initial dose of corticosteroids for moderate-to-severe (grade II–IV) acute GVHD is 2 mg/kg/day of methylprednisolone, or an equivalent steroid [202,203]. Doses higher than this have not been associated with improvement in response rates. In a prospective trial comparing methylprednisolone at 2 mg/kg/day with 10 mg/kg/day in 94 patients with grade II–IV acute GVHD, response rates, progression to grade III–IV disease,

Fig. 86.1 Nonrelapse mortality following primary therapy for acute graftversus-host disease (GVHD). CR, complete response; MR, mixed response; NC, no change; P, progression; PR, partial response. (Reproduced from [41], with permission.)

nonrelapse mortality, and overall survival were similar in both treatment groups [204]. For steroid-responsive acute GVHD, treatment is generally continued until the complete resolution of the signs and symptoms of acute GVHD and for at least 1–2 weeks thereafter. At this point, a gradual tapering of the corticosteroid dose is initiated, generally at no greater than a 10% reduction per week, but modified by factors such as the durability of the response to steroids and the toxicity of high-dose steroid administration. In a prospective trial, patients with acute GVHD who were responding by day 14 of corticosteroid treatment were randomized to a fast taper of corticosteroids over 86 days (n = 14) or a slow taper over 147 days (n = 16). The median time to resolution of acute GVHD was 42 (range 12–74) days in the short-treatment group, compared with 30 (range 6– 30) days for the long taper schedule (p = 0.01), without significant differences in the number of patients with acute GVHD flares, chronic GVHD, median dose of corticosteroid administered, infection or death, suggesting that lower total cumulative doses of steroids appear equally effective and might minimize steroid-related complications [205]. The response rate to corticosteroid therapy, when analyzed in large retrospective reviews, is approximately 50% [41,206], although, in smaller clinical trials, the response rate has been reported to be higher [207]. It is known that the response to steroids and long-term outcome of acute GVHD is correlated with the initial stage at presentation [193]. Long-term survival varies, and for patients with grade 0–I disease approaches 50%, while for those with grade IV disease, the long-term survival rate has been reported to be as low as 11% [208]. Response to treatment is a key predictor of outcome, as mortality in patients with grade II–IV acute GVHD is lowest in those who achieve a complete response to initial treatment (Fig. 86.1) [41,206,208]. A very early response, as evidenced by the ability to begin a steroid taper on day 5 of therapy, has also been shown to be associated with a favorable prognosis, but this study included patients with grade I disease [204]. It is likely that early responders do better at least in part because the steroid taper begins sooner and reduces the risk of complicating opportunistic infections, which are commonly a cause of death, even among responders. In general, dermal disease responds promptly, but lower gastrointestinal involvement responds poorly to therapy [206]. This may be a

Manifestations and Treatment of Acute Graft-Versus-Host Disease

reflection of the fact that the regeneration of a denuded intestinal mucosa often requires days to weeks to occur, and thus caution must be utilized in establishing failure of therapy in lower gastrointestinal GVHD, since the escalation of immunosuppression to treat refractory or incompletely responding GVHD often can result in fatal infections. Other risks for failure of initial therapy include early onset of GVHD, HLA disparity, and age [41,206]. The complete response rate to corticosteroids is low enough to have prompted numerous investigators to examine the role of additional immunosuppressive agents in the initial therapy of acute GVHD. The most widely studied agent in this setting is antithymocyte globulin (ATG). Dugan et al. reported a response rate of 67% in patients treated with ATG and prednisone as initial therapy of acute GVHD [209], and Graziani et al. reported a response rate of 80% in patients given ATG early in the course of acute GVHD, in conjunction with steroids [210]. Cragg et al. randomly assigned 100 patients to receive prednisolone or prednisolone with equine ATG as initial therapy of grade II–IV acute GVHD, and failed to demonstrate a difference in response rates (76% in both arms) or survival at 1 and 2 years between groups. However, infectious morbidity was higher in the combined immunosuppression group [207]. In order to minimize the systemic effects of long-term high-dose steroids, Hockenbery et al. administered oral beclomethasone and systemic steroids to patients with upper gastrointestinal GVHD, and compared outcomes with patients treated with systemic steroids alone. In patients receiving oral beclomethasone, the relapse rate was significantly lower and mortality was significantly reduced at day 200 when compared with patients receiving steroids alone [211]. A number of other biologic agents have been attempted as part of initial therapy for acute GVHD. Lee et al. randomized 102 patients to receive the monoclonal anti-CD25 (the IL-2 receptor α chain) antibody daclizumab (1 mg/kg) on days 1 and 4 and weekly thereafter or placebo in conjunction with corticosteroids. While response rates were similar in both groups (53% versus 51%), survival at 100 days and 1 year was inferior in the daclizumab group [212]. A previous randomized study evaluating another antibody against the IL-2 receptor (BT563, inolimomab) also did not demonstrate an advantage over placebo when combined with prednisone and cyclosporine [213]. In another randomized trial of 143 patients, methylprednisolone and placebo were found to yield similar clinical results to methylprednisolone when combined with a pan-T-cell ricin-A-chain immunotoxin [214]. Manifestations of acute GVHD responded more quickly to the immunoconjugate, but benefits were not durable. Finally, Uberti et al. reported a 75% response rate to the TNF-α inhibitor etanercept [215]. While none of these experiences has changed the standard of care for the initial therapy of acute

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GVHD, the Bone Marrow Transplant Clinical Trials Network is now evaluating four promising newer agents (denileukin diftitox, etanercept, mycophenolate mofetil, and pentostatin), each in combination with corticosteroids, for the initial therapy of acute GVHD. Secondary therapy Patients in whom initial therapy fails (commonly defined as progression of acute GVHD after 3 days, no change after 7 days or incomplete response after 14 days of methylprednisolone treatment) have been given a variety of salvage regimens. To date, there is no clear standard for salvage treatment, and therapy of steroid-resistant acute GVHD remains unrewarding despite seemingly high response rates to certain second-line therapies, due to the risks of infection during intense immunosuppression. Numerous agents have been tested in this setting. (Table 86.3). The agent used most commonly in the setting of failure of response to corticosteroid therapy is ATG [216,217]. ATG use has been associated with response rates between 30% and 54%, but this translates to long-term survivorship in only 5–20% of treated patients [218–221]. Earlier administration of ATG has been associated with improved survival [221]. The monoclonal murine anti-CD3 antibody OKT3 may be effective as second-line therapy for GVHD [222], but its usefulness may be offset by toxicity and a cytokine release syndrome [223], as well as a higher than anticipated risk of Epstein–Barr virus-associated lymphoproliferative disease [224]. In a trial of OKT3 with high-dose methylprednisolone (10 mg/kg) in comparison with high-dose methylprednisolone alone, the response rate to the combination was higher (53% versus 33%), but this improvement in initial response did not result in superior overall survival at 1 year (45% versus 36%; p = 0.41) [225]. A nonmitogenic anti-CD3 antibody that does not bind the human Fc receptor, visilizumab, has also been tested. Clinical results are similar to OKT3, with a response rate of 32%, and an Epstein–Barr virus reactivation rate of 43% [226]. A pilot study of anti-CD3 and anti-CD7 murine monoclonal antibodies conjugated with the ricin A immunotoxin has also been performed [227]. In an attempt to target activated T cells, antibody therapy directed against the α- subunit of the IL-2 receptor (CD25) has been analyzed. The first antibody (the anti-Tac antibody) was reported in 1981 [228,229]; however, the humanized form (daclizumab), first reported upon in 1989 [230], is used more commonly today. The original trials of anti-Tac therapy as well as therapy with similar anti-IL-2 monoclonal antibodies yielded similar results, with complete responses noted in 10–65% of treated patients [231–234]. The largest of these trials, which evaluated

Table 86.3 Agents used as therapy for steroid-refractory acute graft-versus-host disease (GVHD) Biologic therapy Polycolonal antibodies Monoclonal antibodies Immunotoxin/conjugate Tumor necrosis factor-alpha blockade Chemotherapy Phototherapy Cellular therapy Topical/directed therapy

Antithymocyte globulin (ATGAM, Thymoglobulin) OKT3, visilizumab, ABX-CBL, daclizumab, inolimomab, basiliximab, alemtuzumab, alefacept Denileukin diftitox Infliximab, etanercept Mycophenolate mofetil, pentostatin, calcineurin inhibitors, sirolimus Psoralens + ultraviolet A light, extracorporeal photopheresis Mesenchymal stem cells Oral beclomethasone (as dipropionate) or budesonide for intestinal GVHD, intra-arterial steroid or methotrexate infusion

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85 patients treated with the murine antibody BT563 (inolimomab), demonstrated a complete response in 29% and a partial response in an additional 34%, for an overall response rate of 63% [235]. Three phase II trials of the humanized IL-2 receptor antibody have been published. In the original dose-escalation trial, there was a response rate of 40%, with a predilection for improvement in cutaneous and gastrointestinal disease [236]. Subsequently, a response rate of 50% was shown in a small cohort of patients with grade III–IV acute GVHD [237], and the largest series of patients reported demonstrated a complete response rate of 29% for patients treated with daclizumab weekly for five consecutive weeks, and a complete response rate of 47% for patients treated on a more accelerated schedule (1 mg/kg on days 1, 4, 8, 15, and 22) [238]. A chimeric murine–human monoclonal IL-2 receptor antibody, basiliximab, has also been used in steroid-refractory acute GVHD. In phase II studies using different doses and schedules of basiliximab, the overall response rate was uniformly high (71–82.5%), with the complete response rate varying between 17.5% and 53% [239–241]. Similarly, targeting activated T cells through the IL-2 receptor denileukin diftitox (Ontak) is recombinant fusion protein comprised of amino acids 1 through 133 of IL-2 fused to the membrane translocation and catalytic domains of diphtheria toxin (amino acids 1 through 387). In a phase I study of Ontak in steroid-refractory acute GVHD, 71% of patients had a clinical response to therapy, with 46% of patients achieving a complete response at the maximum tolerated dose level of 9 μg/kg administered on days 1, 3, 5, 15, 17, and 19 [242]. A similar dose-escalation trial that administered 4.5 μg/kg daily for 5 days followed by weekly therapy for 1 month showed a complete response rate of 41% at 36 days [243]. Another approach at targeting activated T cells is with the murine monoclonal antibody ABX-CBL, which targets CD147. CD147, a member of the immunoglobulin superfamily, is found on activated T cells, as well as B cells, monocytes, and dendritic cells [244]. While active, this agent demonstrated inferior survival when compared with ATG in a multicenter, randomized phase III study [245]. In an effort to target T cells as well as antigen-presenting cells, the monoclonal antibody alemtuzumab (Campath) has also been reported to be effective as therapy of steroid-refractory acute GVHD [246–249]. A different approach to therapy of steroid-refractory acute GVHD is to target the inflammatory cytokines that lead to T-cell activation and proliferation, with the soluble factor TNF-α having been targeted the most. Etanercept is a genetically engineered molecule comprising two p75 extracellular domains of the human TNF-α receptor fused with human immunoglobulin G1, which has been used to treat steroidrefractory GVHD. Busca et al. treated 13 patients with steroid-refractory acute GVHD with 25 mg of etanercept administered subcutaneously twice weekly for 4 weeks. Six of the 13 patients responded (46%), with five of six responses being complete [250]. Combination therapy with daclizumab and etanercept has been reported, with a response rate of 66% [251], and there is a report of ATG therapy in combination with etanercept [258,259]. The chimeric antibody infliximab has been used more extensively than etanercept. The antibody both neutralizes TNF-α and lyses cells that produce it. Initial case series and dose-escalation trials documented the potential effectiveness of this agent [253–256], and larger series confirmed that this drug may be effective, with response rates of 59–67% noted, with a predilection for improvement in gastrointestinal GVHD [257,258]. Also noted was a high incidence of fungal infection following therapy [258,259]. The T-cell cytotoxic agent pentostatin has been attempted as therapy in steroid-refractory acute GVHD. The accumulation of 2′deoxyadenosine 5′-triphosphate as a result of pentostatin administration leads to impaired lymphocyte growth and apoptosis of dividing lymphocytes. In a phase I dose-escalation study, the maximum tolerated dose

was determined to be 1.5 mg/m2, and the overall response rate was 77% [269]. Mycophenolate mofetil similarly suppresses T-cell proliferation by reversibly inhibiting purine synthesis. Basara et al. initially reported a 65% response rate in 17 patients with steroid-refractory acute GVHD and updated the series to include 36 patients [261,262]. Similar response rates were noted in other small studies [263–265]. Sirolimus, an inhibitor of the mammalian target of rapamycin, is a T-cell immunosuppressant that acts via a reduction in DNA transcription, DNA translation, protein synthesis, and cell cycling [266]. Sirolimus may also be immunosuppressive through the maintenance of regulatory T-cell populations [267–269] and via inhibition of dendritic cell activity through a reduction in antigen uptake [270,271], cellular maturation [272], intracellular signaling [273], and apoptosis induction [274,275]. Benito et al. reported that 12 of 21 steroid-refractory patients (57%) had an objective response to sirolimus (five complete response and seven partial response), but toxicity was limiting in this series [276]. An alternative approach employs use of ultraviolet A with a psoralen sensitizer to control refractory cutaneous GVHD, where high response rates have been noted [277–279]. More interest has focused on extracorporeal photopheresis (ECP) in management of refractory acute GVHD. Postulated mechanisms of action of ECP, in addition to apoptosis of treated lymphocytes, include a decrease in the production of proinflammatory cytokines and an increase in production of antiinflammatory cytokines, both of which lead to a lower ability to stimulate T-cell responses [280]. In addition, ECP may help eliminate CD8+ T-effector cells while inducing regulatory T cells [280,281]. In the largest reported series of ECP as therapy of acute GVHD, Greinix et al. reported a 60% complete response rate, although there were no responders with gastrointestinal GVHD [282]. Other series have reported similar results [283,284]. The use of cellular therapy to treat resistant acute GVHD has now been explored. Mesenchymal stem cells (MSCs) are immunosuppressive, and transplantation of these cells from haploidentical or third-party donors has been shown to improve GVHD in experimental models [285]. The first report of haploidentical MSC infusion for therapy of resistant acute GVHD was by Le Blanc et al., who reported a single successful outcome [287]. Since then, other case series reporting the successful use of MSCs in resistant visceral acute GVHD have been reported, the largest being a report where six of eight treated patients responded to a single infusion of MSCs [287]. There is even a notion that these cells may help repair the damaged organ, thus speeding up recovery from GVHD even further. There are other experimental approaches to the management of refractory acute GVHD, including intra-arterial steroid [288–290] or methotrexate [291] injection for gastrointestinal and hepatic GVHD, and modulation of T-cell responses using alefacept [292] and IL-1 receptor blocking antibodies [293], but all of these approaches remain experimental. Supportive care In addition to immunosuppressive therapy, supportive care is critical for the favorable outcome of acute GVHD. Organ-specific supportive measures for the skin include the use of topical emollient therapy and meticulous wound care, up to and including care in a burn unit. For the gastrointestinal tract, gut rest and hyperalimentation are required. Antimotility agents such as loperamide are often employed, and there is evidence that topical therapy with nonabsorbable corticosteroids may be of value [211]. Octreotide or other somatostatin analogs may be of benefit in controlling the secretory component of gastrointestinal GVHD [294,295]. Bloody diarrhea often requires frequent transfusion support.

Manifestations and Treatment of Acute Graft-Versus-Host Disease

The use of ursodeoxycholic acid has been advocated as a supportive measure in hepatic acute GVHD, and hepatotoxic agents should be avoided. Systemic supportive measures include the judicious use of antibiotics for proven or suspected infections, the routine use of antiviral prophylaxis against herpesviruses, and routine monitoring for CMV reactivation. Finally, routine antifungal prophylaxis with extended spectrum azoles or echinocandins with activity against Aspergillus species and other molds is recommended.

Special scenarios Hyperacute GVHD Hyperacute GVHD is a very rare complication of allogeneic HCT. Although clinically similar to traditional acute GVHD, hyperacute GVHD occurs earlier, often several days prior to engraftment, as early as 7 days from transplantation, although no standard definition exists. The pathophysiology of hyperacute GVHD remains unclear. It has been postulated to be caused by mature, immunocompetent cells transferred with the stem cell product, including NK-T cells [296]. In one analysis, the incidence of hyperacute GVHD was lower in patients receiving the lowest number of T cells transferred with the hematopoietic cell graft, but this has not been confirmed [297]. Risk factors for hyperacute GVHD include mismatched or unrelated donor transplantation, highdose conditioning, donor–recipient sex mismatching, second transplantation, and the lack of post-transplant immunosuppression [297–300]. Therapy for hyperacute GVHD is similar to that for acute GVHD, although therapy is often less successful when compared with traditional acute GVHD [297], and the long-term outlook for hyperacute GVHD appears no different than for patients with traditional acute GVHD [297,298]. Autologous GVHD Histocompatibility differences between donor and recipient may not always be needed to produce GVHD; instead, inappropriate recognition of self-antigens may produce a GVHD-like syndrome after autologous HCT [301,302]. In a thymus damaged by conditioning therapy after autologous HCT, HLA class II-bearing cells may be absent, and clonal deletion of reactive antiself cells may not occur. Administration of cyclosporine in the context of thymic damage may further prevent the development of immune regulatory cells and permit the development of autoreactive CD8+ and CD4+ T cells that recognize class II determinants [303]. This syndrome has even been purposefully induced with the administration of cyclosporine after autologous transplantation to invoke graft-versus-malignancy effects [304–306]. Clinical involvement of the skin, liver, and gastrointestinal tract have all been reported in autologous GVHD [307–309], although the incidence of each of these complications is low. A recent series of patients conditioned with alemtuzumab prior to transplantation, however, reported a high incidence of autologous GVHD, pointing to the dysregulation of the immune system as the pathophysiology of autologous GVHD [310]. Therapy for autologous GVHD is similar to that for classic acute GVHD, with very favorable results with corticosteroids alone. Transfusion-associated GVHD Risk factors, incidence and etiology Transfusion-associated GVHD (TA-GVHD) is a rare complication of blood component transfusion therapy. Since the original description of

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TA-GVHD over 40 years ago, over 200 patients have been reported worldwide [311]. In most reported cases, patients had received whole blood, packed red cells, platelet or granulocyte transfusions, or fresh plasma [312,313]. No cases have been observed in patients receiving transfusion of cryoprecipitate or fresh frozen plasma [314,315]. The pathobiology of TA-GVHD appears similar to the sequence of GVHD events following allogeneic HCT [316,317]. While normal hosts may be affected by TA-GVHD, several groups are at a distinctly higher risk. Individuals with congenital immune deficiency disorders, cancer patients receiving immunosuppressive chemotherapy, and surgical patients undergoing major surgical procedures or suffering from severe trauma represent the most commonly affected [318]. Among normal individuals, genetic factors appear to be the most important risk factors [319,320]. Particularly in populations with common HLA haplotypes, blood from a donor homozygous for an extended HLA haplotype could be transfused into a recipient who is heterozygous for the same haplotype. In this setting, donor cells would not be rejected as foreign by the recipient and could precipitate TAGVHD [321,322]. As such, in Japan, the incidence is estimated to be approximately 1 in 500 after major cardiothoracic procedures [323]. The incidence of TA-GVHD overall remains elusive, as the disorder is often not recognized and is underreported. Diagnosis and clinical features The clinical features of TA-GVHD are very similar to those of posttransplant GVHD, with an erythematous rash, elevation of liver function tests, and profuse watery diarrhea as the main presenting clinical features. Histologic examination of affected tissues confirms the characteristic lesions of acute GVHD. The onset of TA-GVHD is generally earlier than for traditional GVHD, often occurring 7–10 days after transfusion therapy. The one distinguishing feature of TA-GVHD is the development of pancytopenia, which indicates that host marrow and hematopoietic progenitors are an immunologic target of TA-GVHD. The ensuing pancytopenia and resultant infectious complications of TA-GVHD is the most common cause of death in TA-GVHD, which often occurs 3–4 weeks after the diagnosis is made. Chimerism analysis of circulating peripheral blood cells demonstrates donor origin and represents the sine qua non of TA-GVHD. Prevention The efficacy of blood product irradiation has long been recognized, and initial BMT practice routinely employed irradiation of blood products with 1500 cGy [324]. This dose of gamma irradiation from cobalt-60 or caesium-137 (137Cs) sources effectively prevents lymphocyte proliferation without adverse effects on blood cell morphology or function [325]. Although there has been variation across individual centers, 97% of institutions surveyed by the American Association of Blood Banks employ doses of 1500–3500 cGy [326]. This practice appeared to be highly effective, and only a single case of TA-GVHD has been reported in a patient given blood components irradiated with 2000 cGy from a 137 Cs source [327]. Based upon more sensitive limiting dilution assays of T-cell inactivation, more recent studies have called for higher doses of blood production irradiation, and since July 1993, the Food and Drug Administration has required a dose of 2500 cGy to the internal midplane of the canister with no area receiving less than 15 Gy, with well-defined procedures for quality assurance, dose mapping, and storage times [328– 331]. A Medline search of articles on TA-GVHD published in English since 1960 indicates that most were published between 1985 and 1996, reflecting perhaps more effective current prevention [332]. Newer methods to prevent TA-GVHD are in development. The use of photosensitizing psoralens and ultraviolet A light to inactivate infectious organisms and prevent proliferation of T cells has been explored,

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and in preclinical models this approach appears to be an effective means to prevent TA-GVHD [333]. Another approach that has been attempted is lymphocyte depletion of transfused blood products; however, since the minimum threshold of competent T cells to induce TA-GVHD is yet unknown, this method remains experimental. There are case reports of individuals developing TA-GVHD despite lymphocyte depletion [334,345]. Thus, gamma irradiation remains the standard. Although debate continues as to which patients should receive irradiated blood products, it is universally recommended for transplant recipients. This practice continues for at least 6 months post transplantation, but may be needed for longer periods in patients with active chronic GVHD and severe immunodeficiency. Therapy In the nontransplant setting, therapy with agents used to prevent or treat traditional GVHD after allogeneic transplantation has rarely been successful, since the development of irreversible pancytopenia is often established. This is the main contributor to the near-90% fatality rate associated with TA-GVHD. Despite this, an attempt at immunosuppression using corticosteroids and other immunosuppressive agents is often attempted, and there are some favorable outcomes reported [336,337].

Often, however, allogeneic HCT is required to restore hematopoiesis, since an insufficient number of stem cells are transferred with the blood product that is responsible for the TA-GVHD.

Conclusion Despite a seemingly improved understanding of the biologic mechanisms of acute GVHD, this syndrome is often the first hurdle to challenge the HCT patient. The diagnosis of acute GVHD remains largely a clinical one, with biopsy of affected organs an important adjunct. After decades of research, the standard therapy for this problem remains simple corticosteroids, although novel approaches to the initial management of acute GVHD are being developed. In addition, there remains no standard for secondary therapy of acute GVHD, as numerous agents have modest activity in this setting. Unfortunately, most of these approaches are associated with high mortality related to infectious morbidity. Future endeavors in acute GVHD include novel serologic assays for the diagnosis of acute GVHD, pre-emptive therapy for incipient acute GVHD, and the standardization of therapeutic measures when primary therapy fails.

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in patients with severe hepatic graft-versus-host disease: an open-label, uncontrolled trial. Clin Ther 2004; 26: 407–14. Shapira MY, Resnick IB, Bitan M et al. Rapid response to alefacept given to patients with steroid resistant or steroid dependent acute graft-versushost disease: a preliminary report. Bone Marrow Transplant 2005; 36: 1097–101. Antin JH, Weinstein HJ, Guinan EC et al. Recombinant human interleukin-1 receptor antagonist in the treatment of steroid-resistant graft-versus-host disease. Blood 1994; 84: 1342–8. Bianco JA, Higano C, Singer J, Appelbaum FR, McDonald GB. The somatostatin analog octreotide in the management of the secretory diarrhea of the acute intestinal graft-versus-host disease in a patient after bone marrow transplantation. Transplantation 1990; 49: 1194–5. Ippoliti C, Champlin R, Bugazia N et al. Use of octreotide in the symptomatic management of diarrhea induced by graft-versus-host disease in patients with hematologic malignancies. J Clin Oncol 1997; 15: 3350–4. Tanaka Y, Kami M, Ogawa S et al. Hyperacute graft-versus-host disease and NKT cells. Am J Hematol 2000; 63: 60–1. Saliba RM, de Lima M, Giralt S et al. Hyperacute GVHD: risk factors, outcomes, and clinical implications. Blood 2007; 109: 2751–8. Kim DH, Sohn SK, Kim JG et al. Clinical impact of hyperacute graft-versus-host disease on results of allogeneic stem cell transplantation. Bone Marrow Transplant 2004; 33: 1025–30. Davies SM, Weisdorf DJ, Haake RJ et al. Second infusion of bone marrow for treatment of graft failure after allogeneic bone marrow transplantation. Bone Marrow Transplant 1994; 14: 73–7. Sullivan KM, Deeg HJ, Sanders J et al. Hyperacute graft-v-host disease in patients not given immunosuppression after allogeneic marrow transplantation. Blood 1986; 67: 1172–5. Hess AD. Autologous graft-versus-host disease. J Hematother Stem Cell Res 2000; 9: 297. Santos GW. Autologous graft-versus-host disease. Leukemia. 1992;6 Suppl 4:110–111. Miura Y, Thoburn CJ, Bright EC, Hess AD. Cytolytic effector mechanisms and gene expression in autologous graft-versus-host disease: distinct roles of perforin and Fas ligand. Biol Blood Marrow Transplant 2004; 10: 156–70. Giralt S, Weber D, Colome M et al. Phase I trial of cyclosporine-induced autologous graft-versushost disease in patients with multiple myeloma undergoing high-dose chemotherapy with autologous stem-cell rescue. J Clin Oncol 1997; 15: 667–73. Pati AR, Godder KT, Abhyankar SH, Gee AP, Henslee-Downey PJ. Cyclosporine-induced autologous graft-versus-host disease following autologous blood stem cell transplantation for lymphoma. Bone Marrow Transplant 1996; 17: 1081–3. Yeager AM, Vogelsang GB, Jones RJ et al. Cyclosporine-induced graft-versus-host disease after autologous bone marrow transplantation for acute myeloid leukemia. Leuk Lymphoma 1993; 11: 215–20.

307. Holmberg L, Kikuchi K, Gooley TA et al. Gastrointestinal graft-versus-host disease in recipients of autologous hematopoietic stem cells: incidence, risk factors, and outcome. Biol Blood Marrow Transplant 2006; 12: 226–34. 308. Saunders MD, Shulman HM, Murakami CS et al. Bile duct apoptosis and cholestasis resembling acute graft-versus-host disease after autologous hematopoietic cell transplantation. Am J Surg Pathol 2000; 24: 1004–8. 309. Yeager AM, Vogelsang GB, Jones RJ et al. Induction of cutaneous graft-versus-host disease by administration of cyclosporine to patients undergoing autologous bone marrow transplantation for acute myeloid leukemia. Blood 1992; 79: 3031–5. 310. Zenz T, Ritgen M, Dreger P et al. Autologous graft-versus-host disease-like syndrome after an alemtuzumab-containing conditioning regimen and autologous stem cell transplantation for chronic lymphocytic leukemia. Blood 2006; 108: 2127–30. 311. Greenbaum BH. Transfusion-associated graftversus-host disease: historical perspectives, incidence, and current use of irradiated blood products. J Clin Oncol 1991; 9: 1889–902. 312. Parkman R, Mosier D, Umansky I et al. Graftversus-host disease after intrauterine and exchange transfusions for hemolytic disease of the newborn. N Engl J Med 1974; 290: 359–63. 313. Weiden PL, Zuckerman N, Hansen JA et al. Fatal graft-versus-host disease in a patient with lymphoblastic leukemia following normal granulocyte transfusion. Blood 1981; 57: 328–32. 314. Greenbaum BH. Transfusion-associated graftversus-host disease: historical perspectives, incidence, and current use of irradiated blood products. J Clin Oncol 1991; 9: 1889–902. 315. Schroeder ML. Transfusion-associated graftversus-host disease. Br J Haematol 2002; 117: 275–87. 316. Nishimura M, Uchida S, Mitsunaga S et al. Identification of HLA class II antigens as the targets of effector clones which may cause transfusionassociated graft-versus-host disease. Transfus Med 1997; 7: 89–94. 317. Nishimura M, Uchida S, Mitsunaga S et al. Characterization of T-cell clones derived from peripheral blood lymphocytes of a patient with transfusion-associated graft-versus-host disease: Fas-mediated killing by CD4+ and CD8+ cytotoxic T-cell clones and tumor necrosis factor beta production by CD4+ T-cell clones. Blood 1997; 89: 1440–45. 318. Schroeder ML. Transfusion-associated graftversus-host disease. Br J Haematol 2002; 117: 275–87. 319. Juji T, Takahashi K, Shibata Y et al. Posttransfusion graft-versus-host disease in immunocompetent patients after cardiac surgery in Japan. N Engl J Med 1989; 321: 56. 320. Capon SM, DePond WD, Tyan DB et al. Transfusion-associated graft-versus-host disease in an immunocompetent patient. Ann Intern Med 1991; 114: 1025–6. 321. Kruskall M, Alpher C, Awdeh Z. HLAhomozygous donors and transfusion-associated

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graft-versus-host disease. N Engl J Med 1990; 322: 1004–7. Shivdasani RA, Haluska FG, Dock NL et al. Brief report: graft-versus-host disease associated with transfusion of blood from unrelated HLA-homozygous donors. N Engl J Med 1993; 328: 766–70. Juji T, Takahashi K, Shibata Y et al. Posttransfusion graft-versus-host disease in immunocompetent patients after cardiac surgery in Japan. N Engl J Med 1989; 321: 56. Thomas ED, Herman EC, Jr., Greenough WB, III et al. Irradiation and marrow infusion in leukemia. Observations in five patients with acute leukemia treated by whole-body exposures of 1,400 to 2,000 roentgens and infusions of marrow. Arch Intern Med 1961; 107: 829–45. Button LN, DeWolf WC, Newburger PE, Jacobson MS, Kevy SV. The effects of irradiation on blood components. Transfusion 1981; 21: 419–26. Anderson KC, Goodnough LT, Sayers M et al. Variation in blood component irradiation practice: implications for prevention of transfusionassociated graft-versus-host disease. Blood 1991; 77: 2096–102. Drobyski W, Thibodeau S, Truitt RL et al. Thirdparty-mediated graft rejection and graft-versushost disease after T-cell-depleted bone marrow transplantation, as demonstrated by hypervariable DNA probes and HLA-DR polymorphism. Blood 1989; 74: 2285–94. Moroff G, Luban NL. The irradiation of blood and blood components to prevent graft-versus-host disease: technical issues and guidelines. Transfus Med Rev 1997; 11: 15–26. AABB Technical Manual. 15 ed. AABB; 2005. Standards for Blood Banks and Transfusion Services. 24 ed. AABB; 2006. Pelszynski MM, Moroff G, Luban NL, Taylor BJ, Quinones RR. Effect of gamma irradiation of red blood cell units on T-cell inactivation as assessed by limiting dilution analysis: implications for preventing transfusion-associated graft-versus-host disease. Blood 1994; 83: 1683–9. Schroeder ML. Transfusion-associated graftversus-host disease. Br J Haematol 2002; 117: 275–87. Grass JA, Wafa T, Reames A et al. Prevention of transfusion-associated graft-versus-host disease by photochemical treatment. Blood 1999; 93: 3140–7. Akahoshi M, Takanashi M, Masuda M et al. A case of transfusion-associated graft-versus-host disease not prevented by white cell-reduction filters. Transfusion 1992; 32: 169–72. Hayashi H, Nishiuchi T, Tamura H, Takeda K. Transfusion-associated graft-versus-host disease caused by leukocyte-filtered stored blood. Anesthesiology 1993; 79: 1419–21. Prince M, Szer J, van der Weyden MB et al. Transfusion associated graft-versus-host disease after cardiac surgery: response to antithymocyteglobulin and corticosteroid therapy. Aust N Z J Med 1991; 21: 43–6. Cohen D, Weinstein H, Mihm M, Yankee R. Nonfatal graft-versus-host disease occurring after transfusion with leukocytes and platelets obtained from normal donors. Blood 1979; 53: 1053–7.

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Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy

Introduction Chronic graft-versus-host disease (GVHD) is a multisystem chronic alloimmune and autoimmune disorder that occurs later after allogeneic hematopoietic cell transplantation (HCT) [1,2]. It is characterized by immunosuppression, immune dysregulation, decreased organ function, significant morbidity, and impaired survival. Like acute GVHD, there are two opposing aspects: first, chronic GVHD is the main contributor to late post-transplant morbidity, and mortality; and second, in patients with malignant disease, chronic GVHD is associated with important therapeutic graft-versus-tumor (GVT) effects. Overly aggressive immunosuppressive therapy of chronic GVHD may attenuate important GVT reactions and increase the risk of infections, while ineffective (or no) therapy increases chronic GVHD-related transplant morbidity and mortality. Due to insufficient understanding of chronic GVHD biology, our current treatments are based on global immunosuppression and are nonspecific. How to separate beneficial (GVT) effects from harmful effects (GVHD) and use them optimally to the patient’s advantage is a major challenge for HCT research. Depending on a number of factors, chronic GVHD occurs in about half (ranging from 10% to 80%) of 3-month survivors after allogeneic HCT. It is estimated that about 3500 new cases of chronic GVHD are diagnosed each year in North America. Due to its protracted clinical course, an unknown but much larger number of patients live with this diagnosis. There are no contemporary epidemiologic data describing trends in incidence and severity of chronic GVHD. It seems that, due to the changing patterns of utilization of HCT and decreasing early transplant-related mortality, the proportion of patients who develop chronic GVHD is increasing [3]. Reliable incidence estimates are compromised by lack of standardized diagnostic criteria, variability in observer experience, limited expert follow-up, differences in the statistical methods applied, and the sometimes protean nature of chronic GVHD symptoms, which can mimic alternative diagnoses. For many years, chronic GVHD has been a very difficult problem to address. Clinical research in this area lags behind other innovations in HCT. For clinicians, HCT researchers, and patients, chronic GVHD is a major management challenge. The reasons are multiple, and include polymorphic chronic clinical course, disease onset after patients leave transplant centers, poor understanding of disease biology, lack of therapeutic advances, logistical challenges to providing continuous multidis-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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ciplinary care, and difficulties in obtaining research funding from private or public sources. Consequently, there is no United States Food and Drug Administration-approved medication for chronic GVHD. One of the major obstacles to faster advances in chronic GVHD treatment has been the lack of standardized criteria and definitions for diagnosis and clinical trials. Most recently the international transplant community has focused more on chronic GVHD, as demonstrated by the National Institutes of Health-sponsored chronic GVHD consensus project. A series of consensus documents has been published addressing the areas of diagnosis and staging, histopathology, strategies for the development and validation of biomarkers, response criteria, ancillary therapy and supportive care, and the design of clinical trials (Table 87.1) [4–9]. Although many of these newer criteria still need to be validated and refined, they will also advance the standards and uniformity of chronic GVHD clinical research. This is an opportunity for the transplant community to unite and make a significant impact in our understanding and treatment of chronic GVHD.

Risk factors for onset of chronic GVHD A number of factors have been reported consistently associated with a higher risk of developing chronic GVHD (Table 87.2) [3,10]. Among them, the prior history of acute GVHD seems the most powerful predictor. Therefore, it is not surprising that many risk factors which increase risk of acute GVHD, such as age of the patient or the level of human leukocyte antigen (HLA) and other genetic disparity, also predict chronic GVHD. In interpreting these reports, it is important to remember that all these studies used the traditional definition of chronic GVHD (i.e. any GVHD after day 100). Therefore, the recently described late-onset acute GVHD that occurs after day 100, or persistent acute GVHD after day 100, would have been classified as chronic GVHD. There has been a great deal of discussion on the impact of hematopoietic cell source on the risk of chronic GVHD. A recent individualpatient meta-analysis using data from nine randomized trials enrolling 1111 adult patients convincingly demonstrated the increased risk of overall and extensive chronic GVHD in matched sibling transplant patients who received peripheral blood hematopoietic cells instead of bone marrow. Three-year incidences of extensive and overall chronic GVHD after peripheral blood progenitor cells versus bone marrow were 47% versus 31% (p < 0.000001) and 68% versus 52% (p < 0.000001) respectively [11–13]. An ongoing national randomized clinical trial will address this question in the unrelated donor–recipient setting. The reason for the increased incidence of chronic GVHD after peripheral blood HCT is not yet mechanistically explained. It does not seem to correlate with the number of infused CD3+ cells. The increased chronic GVHD risk may be connected to the infused CD34+ cell fraction or associated

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Table 87.1 Documents published by the National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease (GVHD) Document

Key definitions and recommendations included

Diagnosis and staging [4]

Minimal clinical diagnostic criteria Clinical distinction between acute and chronic GVHD New organ severity scoring system New global scoring system Indications for systemic therapy Strategies for developing better predictors of NRM

Histopathology [5]

Minimal diagnostic criteria for active GVHD Biopsy features suggestive of chronic GVHD Standardized terminology for reporting results List of clinical data that should accompany biopsy Organ-specific criteria for adequate tissue sampling Standardized research forms for histology reporting

Biomarkers [6]

Rationale for developing biomarkers in chronic GVHD Definition of biomarkers and potential applications Proposal for a pathophysiology-based classification Current data in search for biomarkers in chronic GVHD Methodologic considerations in identification and validation of biomarkers in chronic GVHD

Response criteria [7]

Proposal of quantifiable set of chronic GVHD measures suitable for outpatient use by transplant and nontransplant providers and in the adult and pediatric setting Introduction of ancillary objective and patient-reported measures of function, performance, and quality of life Provisional criteria and guidelines for determining partial response and progression Guidelines for use of response assessment in trials

Ancillary and supportive care [8]

Evidence-based rated organ-specific guidelines for ancillary and supportive care including skin, mouth, eyes, vulvar vaginal, gastrointestinal/liver, lungs, hematopoietic system, neurologic system, immunologic system/infection prevention, musculoskeletal system, and psychosocial factors Clinical monitoring guidelines in patients with chronic GVHD Need for multidisciplinary team approach

Design of clinical trials [9]

Approaches and definitions to ensure consistent and interpretable results of clinical studies designed to assess interventions for treatment of chronic GVHD

NRM, nonrelapse mortality

Table 87.2 Factors associated with increased risk for the development of chronic graft-versus-host disease (GVHD) Risk factors consistently demonstrated in published studies as being associated with higher risk of chronic GVHD

Less commonly reported risk factors for the development of chronic GVHD

Prior acute GVHD

Higher infused CD34+ cell dose in peripheral blood stem cell transplantation Lower infused CD34 dose in bone marrow transplantation Faster achievement of complete hematopoietic chimerism Cytomegalovirus seropositivity or reactivation Transplant for chronic myelogenous leukemia or aplastic anemia Corticosteroids in GVHD prophylaxis Lower incidence with umbilical cord transplantations

Increasing recipient age Greater human leukocyte disparity Transplant from a female donor to male recipient Peripheral blood stem cell source Donor leukocyte infusions

cell populations. It does not seem to be related to donor exposure to granulocyte colony-stimulating factor [12]. In some studies, the incidence of extensive chronic GVHD seems to be decreased after umbilical cord blood transplantation [13]. Although a number of studies consistently demonstrated a decreased incidence of acute GVHD with ex vivo T-cell depletion, the effect of these procedures on chronic GVHD is less clear [14].

In spite of increased utilization of reduced-intensity conditioning (RIC) for allogeneic HCT, the impact of RIC on chronic GVHD remains an active question and has been difficult to evaluate systematically. Factors confounding such analyses include: short follow-up of studies; absence of prospective comparative trials due to fundamental differences in the groups receiving RIC versus high-dose conditioning regimens in terms of baseline risk factors for GVHD; use of a variety of

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Fig. 87.1 Duration of immunosuppressive treatment for chronic graft-versus-host disease (GVHD). (a) Cumulative incidence of discontinued (D/C) immunosuppressive treatment without recurrent malignancy (lower curve and left-hand scale), and the competing risks of death or recurrent malignancy during continued immunosuppressive treatment (upper curve and right-hand scale) among all patients (n = 751). Time to permanent discontinuation of immunosuppression is prolonged among patients who received mobilized peripheral blood (b), among male recipients with female donors (c), and among patients with human leukocyte antigen disparity (d). Other panels show the same results for patients with (– – –) or without (—) the indicated risk factor. In (c), results for female recipients with either male or female donors were similar to those for male recipients with male donors. (Reproduced from [21], with permission.)

RIC regimens; lack of uniform GVHD prophylaxis; and lack of consistent criteria for the diagnosis and staging of chronic GVHD. In some studies, RIC regimens have reduced the incidence of acute GVHD as assessed at day 100 post transplant, but this has not necessarily translated into a demonstrated reduction in the incidence of chronic GVHD [10]. Acute GVHD after RIC occurs often in a delayed fashion, leading to an overlap syndrome of acute and chronic GVHD, which may potentially increase the estimated incidence of chronic GVHD, emphasizing the need for better standardization in future clinical trials. Early achievement of complete donor chimerism, which seems important for the development of chronic GVHD, is frequently delayed after RIC, and may affect the incidence and severity of chronic GVHD [15]. Chronic GVHD after RIC appears to be frequently limited rather than extensive, and patients may be able to be withdrawn from immunosuppressive therapies at an earlier time period [10]. In summary, our ability to predict chronic GVHD remains insufficiently reliable, and there is a need for developing better prognostic criteria, which will likely also include biologic markers [6].

Prognostic factors for outcomes in patients with chronic GVHD Chronic GVHD is major cause of late nonrelapse mortality (NRM) after allogeneic HCT. Across studies, the characteristics most consistently associated with an increased risk of late NRM among patients with chronic GVHD are thrombocytopenia (<100 × 109/L) and progressive onset of chronic GVHD from acute GVHD. A number of other factors associated with increased NRM in patients with chronic GVHD have been commonly reported and include elevated bilirubin, generalized or lichen planus-like skin involvement, poor Karnofsky performance status, steroid therapy at the time of onset, diarrhea, weight loss, gastrointestinal involvement, lack of oral involvement, history of prior acute GVHD grade II–IV, HLA mismatch, increased age, and lack of therapeutic response to chronic GVHD treatment [16–22]. It is important to emphasize that most studies evaluating prognostic factors for NRM have been retrospective, included patients from various treatment eras, and had patient populations heterogeneous in regard of the hematopoietic cell source (bone marrow versus peripheral blood), donor type (related versus unrelated) or age (inclusion of pediatric patients). These heterogeneities could potentially affect the reproducibility of such prognostic

factors in different patient populations [20]. No studies at this time have described factors prognostic for NRM in the setting of RIC transplantation [10]. Due to a common lack of details in available datasets, little to nothing is known about the impact of organ-specific clinical presentations on major outcomes such as NRM. The recently proposed scoring system for chronic GVHD may help address this deficiency. Prospective validation and refinement of risk factors for NRM in patients with chronic GVHD in the modern transplant era should be a major goal of future research, so that patients receive appropriate therapy for their risk group. Patients judged at low risk for NRM might be preferentially enrolled in studies of new topical or organ-specific therapies, while high-risk patients might receive more aggressive systemic therapy. Very few studies have addressed prognostic factors for major endpoints other than NRM. In an important study, Stewart et al. identified prognostic factors for time to permanent discontinuation of systemic immunosuppression, which is considered a surrogate endpoint for cure in chronic GVHD (Fig. 87.1). Factors which were found to identify poor prognosis included peripheral blood hematopoietic cell source, female donor to a male recipient, HLA mismatch, elevated bilirubin, and involvement of multiple organ sites at onset of chronic GVHD [21]. Arora et al. identified the achievement of complete therapeutic response as an important predictive factor for permanent withdrawal of immunosuppression [19].

Pathogenesis in humans Despite the condition being one of the main complications after allogeneic HCT, biologic mechanisms leading to chronic GVHD are not well understood. The fact that chronic GVHD usually does not occur after autologous or identical twin transplantation suggests that alloreactivity is a key requirement. The delayed onset of the disease indicates that processes leading to chronic GVHD have a long evolution or latency. Since older age is one of the major clinical predictors of chronic GVHD, it is likely that thymic dysfunction plays some role in chronic GVHD. This concept suggests that therapeutic approaches may be different in children versus adults, which is supported by available data in trials that have included pediatric patients, where response rates are often different from those in adult patients. Although useful information has emerged from murine experiments regarding possible mechanisms of chronic

Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy

Acute GVHD: rash, GI, liver

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Chronic GVHD: skin, eyes, mouth, GI liver, musculoskeletal, lungs, GU

Alloreactivity Autoimmunity Immunodeficiency - Classic acute - Late acute - Chronic overlap

Day

0

Activity (inflammation)

50

100

180

injur y

- Classic chronic

1 year

repair

2 years

3 years

5 years

Damage (fibrosis)

Fig. 87.2 Key events in pathogenesis of chronic graft-versus-host disease (GVHD). Chronic GVHD is a biologically and clinically unique process with slow evolution, and is distinct from acute GVHD. Alloreactivity mediated by donor T cells is considered a key component for initiation of the processes. Immune dysregulation ensues through the disruption of thymic and peripheral mechanisms of tolerance. The resulting inflammatory process is both alloreactive and autoreactive, and leads to target organ tissue injury, immunodeficiency, secretion of profibrotic cytokines, and repair or irreversible organ damage. Chronic GVHD is today diagnosed by the recognition of typical clinical symptoms and signs, and not arbitrarily by using the day 100 cut-off time point. Some patients may present with both typical acute GVHD symptoms (late, persistent or recurrent acute GVHD) and classical chronic GVHD symptoms (overlap chronic GVHD). Late acute GVHD presentation seems to convey worse prognosis. Average time of onset for chronic GVHD is about 6 months post transplant, and most cases present within the first 2 years after transplantation. Average duration of chronic GVHD is 2–3 years, but 10–15% of patients may have active disease beyond 5 years after diagnosis. A better understanding of the biologic events which initiate and promote the development of chronic GVHD, and of its most severe clinical presentations, is a major issue for researchers. Similarly, the nature of mechanisms which convey the beneficial chronic GVHD mediated graft-versus-tumor effects should be elucidated. GI, gastrointestinal; GU, genitourinary.

GVHD, the animal models do not duplicate the complete clinical syndrome seen in humans [23]. It is likely that no single biologic mechanism accounts for the diverse features of chronic GVHD, and that chronic GVHD will be dependent on a unique combination of immune factors in each patient–donor pair. Investigations of these factors in humans have been further complicated by the lack of standard clinical definitions of chronic GVHD. Although acute GVHD is a major predictor of chronic GVHD, it is well accepted that chronic GVHD is a biologically unique process. This fact is emphasized by the observations that chronic GVHD can occur without prior acute GVHD (de novo onset), and that interventions that are successful in preventing or treating acute GVHD most commonly fail to decrease chronic GVHD [14,24–26]. Most investigators now consider chronic GVHD as a disease of immune dysregulation that involves donor-derived immune cells and host cell populations and tissues. This process is likely initiated by donor-derived T cells and is both alloreactive (directed against the recipient’s histocompatibility antigens) and autoreactive (directed against antigens present on both the donor and the recipient) [23]. The activated immune response then proceeds unchecked by the thymic or peripheral mechanisms of deletion and immunoregulation [27,28]. Critical donor or recipient tolerancepromoting mechanisms may be absent [29,30]. This pathologic immune response can then attack target tissues directly through cytolytic mechanisms, secretion of inflammatory and fibrosing cytokines or promotion of B-cell activation and autoantibody production [23,31–35]. Inflammatory processes lead to tissue damage, counterregulation, and repair with fibrosis or irreversible organ damage.

Due to the diversity and unpredictability of this process, contemporary chronic GVHD treatment utilizes global suppression of all or many of the key components of this inflammatory cascade. The current understanding of the evolution of the chronic GVHD syndrome from the time of transplantation until resolution is presented in Fig. 87.2. Biologic events that occur between peak times of acute and chronic GVHD prevalence and which lead to clinical chronic GVHD are a major focus of researcher interest. Although an integrative interpretation and understanding of chronic GVHD biology is still lacking, a number of elements of this mosaic are becoming more evident.

Clinical manifestations and diagnosis of chronic GVHD Symptoms of chronic GVHD usually present between 3 months and 2 years after allogeneic HCT, about two-thirds of cases develop within the first 12 months. In about 80% of patients, chronic GVHD is preceded by a history of acute GVHD. Manifestations of chronic GVHD may be restricted to a single organ or tissue, but typically two or three organs are involved. The organs most commonly affected are skin, mouth, and eyes, with more than 50% of patients demonstrating these manifestations. Other common disease sites include the liver, lung, gastrointestinal tract, musculoskeletal system, and female genital organs. Publications rarely report more detailed descriptions of chronic GVHD presentations in specific organs (e.g. skin sclerotic changes versus lichenoid versus deep fascial involvement) or systematically characterize the severity of manifestations. Frequency estimates of classic chronic GVHD

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presentations are therefore lacking. The average time of onset of different manifestations varies; for example, skin sclerotic lesions usually present beyond 1 year after transplantation. Therefore, salvage therapy trials may have many patients with sclerotic type skin, while these patients are rare in trials of initial presentation. Chronic GVHD can lead to debilitating consequences such as joint contractures, loss of sight, endstage lung disease, and mortality from infection. Many other factors add to the complexity of evaluating signs and symptoms in patients with chronic GVHD. These include late effects of prior cancer therapies or transplant conditioning, side-effects of steroids, immunosuppressive agents or other medications (photosensitivity to voriconazole, for example), metabolic and growth-related defects, symptoms of underlying or recurrent malignancy, and secondary cancers [36,37]. Diagnosis The diagnosis of chronic GVHD requires at least one diagnostic sign (i.e. sign that is found only in chronic GVHD, such as poikiloderma, sclerotic skin features or oral lichen planus-like changes) or at least one distinctive sign that is highly suggestive of chronic GVHD (e.g. nail dystrophy, vitiligo-like depigmentation or bronchiolitis obliterans diagnosis based only on pulmonary function tests and computerized tomography findings) but requires laboratory or biopsy confirmation in the same or another organ (Table 87.3) [4]. A detailed color atlas and classification of the skin and oral manifestations of chronic GVHD is available at http://www.asbmt.org/GVHD/index.htm. Appropriate studies should be performed to rule out other potential diagnoses such as infection, drug toxicity, second cancers, etc. A biopsy read as “consistent with” or “unequivocal” GVHD is considered sufficient to support the diagnosis of chronic GVHD if accompanied by at least one distinctive clinical manifestation [5]. In addition to setting diagnostic criteria for chronic GVHD, the recent National Institutes of Health (NIH) consensus conference also provided a definition of acute GVHD to help differentiate the two syndromes. In the past, any manifestation of GVHD that was present beyond 100 days after HCT was called chronic GVHD, even if the manifestation was indistinguishable from acute GVHD. Changes in HCT practice in the past two decades have altered our understanding of the natural history of both acute and chronic GVHD and brought previous definitions into question. For instance, acute GVHD may present beyond 3 months after transplantation in patients who have received RIC, in patients with persistent GVHD or in those that have GVHD flares during tapering of immunosuppression. Manifestations of acute and chronic GVHD can also be present simultaneously, for example in patients treated with donor lymphocyte infusion. Therefore, the current consensus is that clinical manifestations and not the time after transplantation determines whether the clinical syndrome of GVHD is considered acute or chronic [4]. Some signs and symptoms are common to both chronic and acute GVHD (erythema, maculopapular rash, nausea, vomiting or diarrhea, and elevated liver function tests), and thus cannot be used to distinguish the two. Instead, two main categories of GVHD are now recognized, each with two subcategories (Table 87.4). The broad category of acute GVHD includes classic acute GVHD (maculopapular erythematous rash, gastrointestinal symptoms or cholestatic hepatitis) occurring within 100 days after transplant or donor lymphocyte infusion, while persistent, recurrent or late acute GVHD (usually seen after withdrawal of immunosuppression) occurs beyond 100 days of transplantation or donor lymphocyte infusion. The arbitrary day 100 distinction is retained in separating these two acute GVHD categories to facilitate reporting in clinical trials. Both acute GVHD subentities should occur without the presence of diagnostic or distinctive chronic GVHD manifestations.

A second broad GVHD category encompasses classic chronic GVHD, consisting only of manifestations that can be ascribed to chronic GVHD, and acute and chronic overlap syndrome, in which features of both acute and chronic GVHD appear together. In the absence of histologic or clinical signs or symptoms characteristic of chronic GVHD, the persistence, recurrence or new onset of characteristic skin, gastrointestinal tract or liver abnormalities should be classified as acute GVHD regardless of the time after transplantation. With appropriate stratifications, patients with persistent, recurrent or late acute GVHD or overlap syndrome can be included in clinical trials with patients who have chronic GVHD. The newly defined entity “late onset” of acute GVHD has been shown in preliminary studies to be highly associated with poor survival when chronic GVHD patients were reclassified according to the new definition (Fig. 87.3) [38,39]. Couriel et al. [38] have found that patients with late acute GVHD manifestations had significantly higher 2-year NRM (75% versus 35%) than those with classic chronic GVHD. A higher proportion of patients with late acute GVHD had platelet counts less than 100 × 109/L, progressive presentation, and higher serum lactate dehydrogenase levels than patients with classic chronic GVHD manifestations. To ensure adequate risk stratification and interpretation, future studies of chronic GVHD should include distinction of “late-onset” acute GVHD from “classic” chronic GVHD in the study protocols. Organ-specific manifestations Skin Diagnostic manifestations include: 1 poikiloderma (e.g. atrophic and pigmentary changes; Plates 87.1, 27.52); 2 lichen planus-like eruption (e.g. erythematous/violaceous flat-topped papules or plaques with or without surface reticulations or a silvery or shiny appearance on direct light; see Plates 27.42 and 27.53); 3 deep sclerotic features (e.g. smooth, waxy, indurated skin – “thickened or tight skin,” caused by deep and diffuse sclerosis over a wide area; Plates 87.2–87.4); 4 morphea-like superficial sclerotic features (e.g. localized patchy areas of moveable smooth or shiny skin with a leathery-like consistency, often with dyspigmentation; Plate 87.5); 5 lichen sclerosus-like lesions (e.g. discrete-to-coalescent gray-to-white moveable papules or plaques, often with follicular plugs, with a shiny appearance and leathery consistency; Plate 87.6). Severe sclerotic features characterized by thickened, tight, and fragile skin are often associated with poor wound healing, inadequate lymphatic drainage, and skin ulcers from minor trauma. A distinctive feature for chronic GVHD (not seen in acute GVHD, but not sufficiently unique to be considered diagnostic of chronic GVHD) is depigmentation. However, depigmentation would contribute to the diagnosis of chronic GVHD in combination with biopsy or laboratory confirmation of GVHD in skin or another organ. Sweat impairment and intolerance to temperature change from loss of sweat glands are seen in chronic GVHD. Other common, nondistinctive skin manifestations found with both acute and chronic GVHD include erythema, maculopapular rash, and pruritus. Nails Dystrophy consisting of longitudinal ridging, nail splitting or brittleness, onycholysis, pterygium unguis, and nail loss (usually symmetric and affecting most nails) are distinctive signs of chronic GVHD but are not sufficient for diagnosis (Plate 87.7). Hair Distinctive features of chronic GVHD include new scarring and nonscarring scalp alopecia (after recovery from chemotherapy or radiotherapy)

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Table 87.3 Signs and symptoms of chronic graft-versus-host disease (GVHD) and related diagnostic interventions

Organ or site Skin

Diagnostic (sufficient to establish the diagnosis of chronic GVHD)

Distinctive (seen in chronic GVHD, but insufficient alone to establish a diagnosis of chronic GVHD)

Poikiloderma Lichen planus-like features Sclerotic features Morphea-like features Lichen sclerosus-like features

Depigmentation

Nails

Dystrophy Longitudinal ridging, splitting or brittle features Onycholysis Pterygium unguis Nail loss (usually symmetric; affects most nails)† New onset of scarring or nonscarring scalp alopecia (after recovery from chemoradiotherapy)

Scalp and body hair

Mouth

Lichen planus-like features Hyperkeratotic plaques Restriction of mouth opening from sclerosis

Eyes

Genitalia

Gastrointestinal tract

Lichen planus-like features Vaginal scarring or stenosis Esophageal web Strictures or stenosis in the upper to mid third of the esophagus†

Scaling, papulosquamous lesions Xerostomia Mucocele Mucosal atrophy Pseudomembranes† Ulcers† New-onset dry, gritty or painful eyes‡ Cicatricial conjunctivitis Keratoconjunctivitis sicca‡ Confluent areas of punctate keratopathy Erosions† Fissures† Ulcers†

Other features*

Diagnostic intervention

Sweat impairment Ichthyosis Keratosis pilaris Hypopigmentation Hyperpigmentation

Clinical examination and skin biopsy

Clinical examination

Thinning scalp hair, typically patchy, coarse or dull (not explained by endocrine or other causes) Premature gray hair

Dental and oral evaluation, mucosal or salivary gland biopsy

Photophobia Periorbital hyperpigmentation Blepharitis (erythema of the eyelids with edema)

Ophthalmology evaluation, Schirmer tear test, corneal staining and conjunctival examination, rarely conjunctival or lacrimal gland biopsy Gynecologic examination, biopsy

Exocrine pancreatic insufficiency

Clinical history and physical, swallowing studies, lower and/or upper gastrointestinal endoscopy, biopsy Bilirubin, alkaline phosphatase >2 × ULN†; ALT or AST >2 × ULN† Liver biopsy Pulmonary function tests, computed tomography scan with inspiratory and expiratory films, lung biopsy if indicated Physical therapy evaluation to evaluate range of movement, muscle strength, and stiffness, creatine kinase, aldolase, EMG, muscle biopsy, magnetic resonance imaging Complete blood count, differential, quantitative immunoglobulins, Coombs’ test, bone marrow biopsy

Liver

Lung

Bronchiolitis obliterans diagnosed with lung biopsy

Bronchiolitis obliterans diagnosed with pulmonary function tests and radiology‡

Muscles, fascia, joints

Fasciitis Joint stiffness or contractures secondary to sclerosis/fasciitis

Myositis or polymyositis‡

Hematopoietic and immune system

Clinical examination

Edema Muscle cramps Arthralgia or arthritis

Thrombocytopenia Eosinophilia Lymphopenia Hypo- or hypergammaglobulinemia AIHA and ITP

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Table 87.3 Continued

Organ or site

Diagnostic (sufficient to establish the diagnosis of chronic GVHD)

Distinctive (seen in chronic GVHD, but insufficient alone to establish a diagnosis of chronic GVHD)

Other

Other features*

Diagnostic intervention

Pericardial or pleural effusions, Diagnostic ultrasound, computed ascites tomography scans, paracentesis Peripheral Neurology examination, nerve conduction studies and EMG Neuropathy Nephrotic syndrome 24-hour urine collection for urinary protein excretion, protein electrophoresis, kidney biopsy if indicated Myasthenia gravis Neurology examination, EMG, antiacetylcholine receptor antibodies Cardiac conduction Electrocardiogram, Holter monitor, abnormality or creatine kinase, lactate cardiomyopathy dehydrogenase and isoenzymes, troponin

AIHA, autoimmune hemolytic anemia; ALT, alanine aminotransferase; AST, aspartate aminotransferase; EMG, electromyogram; ITP, idiopathic thrombocytopenic purpura; ULN, upper limit of normal. * Can be acknowledged as part of the chronic GVHD symptomatology if the diagnosis is confirmed. † In all cases, infection, drug effects, malignancy, and other causes must be excluded. ‡ Diagnosis of chronic GVHD requires biopsy or radiographic confirmation (or Schirmer test for eyes).

Table 87.4 Categories of acute and chronic graft-versus-host disease (GVHD)

Category

Time of symptoms after hematopoietic cell transplantation or donor lymphocyte infusion

Presence of acute GVHD features*

Presence of chronic GVHD features†

Acute GVHD Classic acute GVHD Persistent, recurrent or late-onset acute GVHD

≤100 days >100 days

Yes Yes

No No

Chronic GVHD Classic chronic GVHD Overlap syndrome

No time limit No time limit

No Yes

Yes Yes

* Maculopapular erythema, gastrointestinal symptoms, and elevated liver function tests. † See Table 87.3.

and loss of body hair. Other characteristics seen with chronic GVHD include premature graying, thinning or brittleness, but these findings are not diagnostic (Plate 87.8).

pain, dry mouth, and sometimes decreased oral range of motion due to fibrosis, which all can interfere with critical oral functioning. Eyes

Mouth Diagnostic features of oral chronic GVHD include: 1 lichen planus-like changes (white lines and lacy-appearing lesions of the buccal mucosa, tongue, palate or lips; Plate 87.9); 2 hyperkeratotic plaques (leukoplakia); 3 decreased oral range of motion in patients with sclerotic features of skin GVHD. Distinctive features of chronic GVHD include xerostomia (dryness), mucoceles, mucosal atrophy, pseudomembranes, and ulcers (infectious pathogens such as yeast or herpesvirus; secondary malignancy must be excluded) (Plates 87.10 and 87.11; see also Plate 27.49). Usual oral symptoms that patients experience include oral sensitivity to food, oral

Distinctive manifestations of chronic GVHD include: 1 new onset of dry, gritty or painful eyes; 2 cicatricial conjunctivitis; 3 keratoconjunctivitis sicca; 4 confluent areas of punctate keratopathy (Plate 87.12). Other features include photophobia, periorbital hyperpigmentation, difficulty in opening the eyes in the morning because of mucoid secretions, and blepharitis (erythema of the eye lids with edema). New ocular sicca documented by low Schirmer test values with a mean value of both eyes 5 mm or less at 5 minutes, or a new onset of keratoconjunctivitis sicca by slit-lamp examination with mean values of 6–10 mm on the Schirmer test, is sufficient for the diagnosis of chronic GVHD if accompanied by

Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy

Lungs

1.0

P= 0.0005 at 2 years

0.9 0.8 0.7

Chronic GVHD

0.6 0.5 0.4 Late acute GVHD

0.3 0.2 0.1 0.0

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0

5

10

20 15 25 Months post rapamycin

30

35

40

*Unadjusted estimate Fig. 87.3 Survival of patients with late acute graft-versus-host disease (GVHD) versus classic chronic GVHD. The y-axis indicates probability of survival. (Reproduced from [38], with permission.)

distinctive manifestations in at least one other organ. Ocular symptoms include eye dryness, irritation, photophobia, and pain. Genitalia Diagnostic features for the genitalia include lichen planus-like features and vaginal scarring or stenosis (often associated with oral GVHD). Vulvar vaginal symptoms may include itching, pain, dyspareunia, and dysuria (Plate 87.13). Gastrointestinal tract Diagnostic features for the gastrointestinal tract include esophageal web, stricture, or concentric rings documented by endoscopy or barium contrast radiograph. Chronic GVHD may be associated with pancreatic exocrine insufficiency, which may require enzyme supplementation to ameliorate the diarrhea caused by the malabsorption. Manifestations common to both acute and chronic GVHD (as well as other causes, such as drug side-effects, motility disorders, infections or malabsorption) include anorexia, nausea, vomiting, diarrhea, weight loss, and failure to thrive. Wasting syndrome may be a manifestation of chronic GVHD but is often multifactorial (e.g. decreased caloric intake, poor absorption, increased resting energy expenditures, and hypercatabolism). Endoscopic findings of mucosal edema and erythema or focal erosions with histologic changes of apoptotic epithelial cells and crypt cell drop-out may be seen but are not considered diagnostic of chronic GVHD unless the patient also has distinctive features in a non-gastrointestinal system. Patients with unresolved acute GVHD may have more severe intestinal mucosal lesions, including ulcers and mucosal sloughing.

The only diagnostic manifestation of pulmonary chronic GVHD is biopsy-proven bronchiolitis obliterans (BO) (Plate 87.15). BO diagnosed via pulmonary function and radiologic testing requires at least one other distinctive manifestation in a separate organ system to establish the diagnosis of chronic GVHD. BO is characterized by the new onset of an obstructive lung defect. Clinical manifestations may include dyspnea on exertion, cough or wheezing. Some patients may be asymptomatic early in the disease process. Pneumothorax, pneumomediastinum, and subcutaneous emphysema are rare and often represent advanced disease. Restrictive pulmonary function abnormalities secondary to advanced sclerosis of the chest wall are attributable to skin GVHD. BO is clinically diagnosed when all of the following criteria are met: 1 Forced expiratory volume in 1 second (FEV1)/forced vital capacity ratio less than 0.7, and FEV1 less than 75% of predicted. 2 Evidence of air trapping or small airway thickening or bronchiectasis on high-resolution chest computed tomography (with inspiratory and expiratory images), residual volume over 120% or pathologic confirmation of constrictive bronchiolitis. 3 Absence of infection in the respiratory tract, documented with investigations directed by clinical symptoms, such as radiologic studies (radiographs or computed tomographic scans) or microbiologic cultures (sinus aspiration, upper respiratory tract viral screen, sputum culture or bronchoalveolar lavage); 4 BO organizing pneumonia not due to infections may represent a manifestation of either acute or chronic GVHD and is considered a common feature (see also Chapter 96). Musculoskeletal system Diagnostic features include fascial involvement often affecting the forearms or legs and often associated with sclerosis of the overlying skin and subcutaneous tissue (Plate 87.16). Fascial involvement may develop without overlying sclerotic changes of the skin and can result in joint stiffness or contractures when present near joints. Fasciitis is detected on examination by stiffness, a restricted range of motion (e.g. often decreased dorsal wrist flexion or inability to assume a Buddha prayer posture), edema of the extremities with or without erythema (early sign), peau d’orange (edematous skin with prominent pores resembling the surface of an orange) or joint contractures (late complications) (Plate 87.17). Clinical myositis with tender muscles and increased muscle enzymes is a distinctive but nondiagnostic manifestation of chronic GVHD (see Plate 27.54). Myositis may present as proximal myopathy, but this complication is rare and does not explain the frequent complaints of severe cramps. Evaluation of myositis involves electromyography and measurement of creatine phosphokinase or aldolase; imaging by magnetic resonance imaging may also prove useful. Arthralgia and arthritis are uncommon and are occasionally associated with the presence of autoantibodies. Hematopoietic and immune systems

Liver Hepatic acute and chronic GVHD typically presents as cholestasis, with increased bilirubin and/or alkaline phosphatase levels, but it may also present as acute hepatitis [40]. Because of many possible alternative diagnoses, liver biopsy is helpful to confirm GVHD involvement of the liver, but this is often difficult to do because of other medical conditions. Note that because of the histologic similarity between acute and chronic liver GVHD, the diagnosis of chronic GVHD cannot be made on the basis of liver biopsy alone but requires a distinctive manifestation in at least one other organ system (Plate 87.14) (see also Chapter 95).

Although immune deficiency is ubiquitous in chronic GVHD, it is not used to establish the diagnosis. Hematopoietic abnormalities are common in chronic GVHD, but also are not specific and are not used to establish the diagnosis. Cytopenias may result from stromal damage or autoimmune processes. Lymphopenia (≤500/μL), eosinophilia (≥500/μL), hypogammaglobulinemia or hypergammaglobulinemia may be present. Autoantibodies may develop with autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura. Thrombocytopenia (<100,000/ μL) at the time of chronic GVHD diagnosis has been associated with a poor prognosis (see Plate 27.51).

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Other findings Serositis (pericardial/pleural effusions or ascites), peripheral neuropathy, myasthenia gravis, and cardiac function complications have been attributed to chronic GVHD, but these manifestations are rare. For these manifestations, chronic GVHD is often a diagnosis of exclusion unless the patient has additional features of chronic GVHD. Recently, two large retrospective studies have reported cumulative incidences of GVHDassociated nephrotic syndrome of 6.1% and 8%, suggesting this manifestation may be more frequently encountered than previously thought. The first study found that all patients with nephrotic syndrome had received RIC, whereas patients receiving high-dose conditioning at the same institute during the same period had no reported cases of nephrotic syndrome. Patients who developed nephritic syndrome suffered significant morbidity, including thromboembolic complications and progressive renal failure, and were unlikely to respond to immunosuppression [41]. The second study found no differences between conditioning regimens, but rather observed an association with hematopoietic cell source, with increased probability of nephrotic syndrome in patients receiving peripheral blood progenitor cells versus bone marrow. All patients in this study responded to immunosuppressive therapy [42] (see also Chapter 97).

Histopathology and diagnosis of chronic GVHD Diagnosis of chronic GVHD is a clinical diagnosis, but histologic confirmation has an important role in the final assessment. Biopsies are recommended to confirm chronic GVHD in situations where distinctive

but nondiagnostic clinical features of chronic GVHD are present, alternative diagnoses are entertained, clinical signs are confined to internal organs, or clinical assessment is made difficult by concomitant medical conditions [4]. In these instances, biopsy confirmation should be viewed as essential for establishing a diagnosis of chronic GVHD. Failure to obtain biopsies can result in erroneous treatment. Jacobsohn and colleagues found that 7% of patients referred to Johns Hopkins University for consultation regarding chronic GVHD did not have biopsies before starting treatment, and had been incorrectly diagnosed and treated for active chronic GVHD prior to referral [43]. Many practical issues in the histopathology of GVHD are unresolved [5]. It is often not possible to distinguish persistent, recurrent or late acute GVHD from chronic GVHD by histology (Table 87.5). Further, uniform minimal diagnostic criteria for chronic GVHD have not been established for affected organs, and histologic grading systems have not been validated in a prospective fashion. A number of factors can influence or cause difficulty in histologic interpretation. Immunosuppressive treatment blunts the inflammatory response, infections and drug reactions can mimic chronic GVHD, the histology changes over time, and there is residual destruction of epithelia or glandular structures and irreversible fibrosis, which all pose problems in separating old damage from ongoing or new chronic GVHD activity. Sampling and technical factors can also cause a false-negative histologic assessment of chronic GVHD. Further progress in this field may be accomplished by introduction of diagnostic tools such as immunohistochemistry and other molecular approaches to aid diagnosis, classification, and potentially direct therapy of chronic GVHD. Development

Table 87.5 Histopathology and diagnosis of chronic graft-versus-host disease (GVHD) by organ system Organ or system

Minimal criteria for active GVHD

Skin, any stage

Apoptoses in epidermal basal layer or lower malphigian layer or outer root sheath of hair follicle or acrosyringium ± lichenoid inflammation ± vacuolar change ± lymphocytic satellitosis

Skin lichen planus-like

Skin sclerotic Skin morpheic

Skin fasciitis Liver Gastro-intestinal Oral mucosa and conjunctiva Minor salivary or lacrimal gland Lung

Global assessment of dysmorphic or destroyed small bile ducts ± cholestasis, lobular and portal inflammation Variable apoptotic criteria (≥1/piece) in crypts

Specific criteria for chronic GVHD

Combination of epidermal orthorkeratosis, hypergranulosis, and acanthosis with lichenoid changes ± syringitis of eccrine units ± panniculitis Collagenous deposition with thickening throughout papillary dermis, or pandermal collagenosis ± panniculitis Clinically focal or localized lesion predominated by sclerosis in lower reticular dermis or along dermal hypodermal border ± epidermal and appendigeal involvement Fibrous thickening of fascial septa with adjacent inflammation ± panniculitis Ductopenia, portal fibrosis, chronic cholestasis reflect chronicity but are not specific for chronic GVHD Destruction of glands, ulceration or submucosal fibrosis reflect long-standing disease but are not specific for chronic GVHD

Lymphocytic infiltration of mucosa with variable apoptosis Infiltration and damaged intralobular ducts, fibroplasia in periductal stroma, inflammation with destruction of acinar tissue Obliterative bronchiolitis: dense eosinophilic scarring beneath the respiratory epithelium, resulting in complete fibrous obliteration or some degree of luminal narrowing*

* Obliterative bronchiolitis should be distinguished from bronchiolitis obliterans organizing pneumonia, which is also associated with GVHD but has a different clinicopathologic presentation and a more favorable outcome. Reprinted with permission from Shulman et al. [5].

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100% 90% 80% 70%

Fig. 87.4 Percent distribution of the National Institutes for Health (NIH) staging organ severity scores 0–3 in a chronic graft-versus-host disease (GVHD) consultation clinic (n = 52). Patients were enrolled in to the National Cancer Institute (NCI) protocol 04-C-0281 natural history study of chronic GVHD. GI, gastrointestinal; KPS, Karnofsky performance score; V/V, vulvar vaginal (n = 23 female patients). Twenty-two patients had at least one organ with an NIH score of 3, indicating seriously disabling manifestations. (Courtesy of I. Pusic, MD.)

60%

NIH 3 NIH 2 NIH 1 NIH 0

50% 40% 30% 20% 10% 0% KPS

V/V

of these tools in chronic GVHD is lagging compared with the progress in other fields of oncology.

Classification and staging of chronic GVHD There are several ways to classify chronic GVHD: according to the type of onset, need for systemic immunosuppressive therapy or mortality risk. The disease may evolve directly from acute GVHD (“progressive”) or after resolution of acute GVHD (“quiescent”), and also may develop without prior clinically manifest acute GVHD (“de novo” onset). De novo onset occurs in about 15–30% of cases and seems to be less common in recipients of unrelated donor HCT. Progressive onset has been most consistently associated with impaired survival. Historically, chronic GVHD was classified as limited (localized skin involvement and/or liver dysfunction) or extensive (generalized skin involvement, liver histology showing aggressive hepatitis or involvement of any other target organ) [44]. This classification, developed by investigators in Seattle, was formulated on the basis of the results of a retrospective analysis in a cohort of 20 patients dated in the precyclosporine era. The aim of this system was to identify patients who needed systemic immunosuppressive therapy. Although widely utilized by the transplant community, the Seattle classification has been also shown to be nonreproducible among individual investigators and with limited value in predicting late NRM [18]. Several investigators have developed prognostic scales for NRM based on studying a number of clinical factors in chronic GVHD patients drawn from single institutions or from large registry databases [16–19]. All major prognostic schemes were efficient in predicting NRM. The survival of the most favorable chronic GVHD group in each scheme was consistently around 80%, while the survival in poorest prognostic groups varied around 10–50%. On average, about 15% of patients with chronic GVHD have severe disease with poorest prognosis, and about 50% have mild disease presentations associated with best survival [18]. None of these classifications provides information about the number and extent of the organs involved or severity of organ function impairments, and therefore do not allow for investigation of the details of the disease which may have prognostic or therapeutic decision-making value. A new chronic GVHD clinical staging system (0–3) is now recommended for scoring of individual organs that describes the severity for each affected organ/site at any given time, and which includes functional impact [4]. This system is applied only after the diagnosis of chronic GVHD is established. Eight organs are assessed for scoring: skin, mouth, eyes, gastrointestinal tract, liver, lungs, joints and fascia, and genital

Joint

Lung

Liver

GI

Eye

Mouth

Skin

tract. In general, a score of 0 means no manifestations/symptoms, a score of 1 means no significant impairment of function or activities of daily living, a score of 2 means significant impairment of activities of daily living but no major disability, and a score of 3 indicates significant impairment of activities of daily living with major disability (Fig. 87.4). The scoring can be easily conducted in the clinic, and the only mandated laboratory tests for its completion are liver function tests. Utilization of pulmonary function tests for FEV1 and diffusing capacity of the lung for carbon monoxide assessments is strongly encouraged for lung scoring, although evaluation can be done also by assessing the symptoms of shortness of breath and need for supplemental oxygen. A global staging of severity (none, mild, moderate, and severe) is derived by combining organ-specific scores, and is intended to replace the current “limited-extensive” scoring system [4,44]. Staging mild chronic GVHD involves only one or two organs with maximum score of 1, whereas moderate chronic GVHD involves at least one organ with score 2 or 3 or more organs with scores of 1. A lung score of 1 is also considered as moderate-stage chronic GVHD. These staging criteria and a simple scoring form developed for this purpose are intended for baseline or cross-sectional use at sentinel time points in the course of the disease (e.g. diagnosis and yearly follow up), both in clinical practice or in trials [4]. The scoring forms can be easily performed in the office by general practitioners. This staging system is not intended to be used for monitoring of therapeutic response in intervention trials, which is addressed by separate response criteria [14].

Measuring response in therapeutic clinical trials To date there has been no focused attention to experimental therapeutics in chronic GVHD. This is a gap that must be addressed urgently, since current therapies for chronic GVHD are of limited efficacy and there is no satisfactory regimen available to offer patients who fail front-line steroid-based therapy [45]. There is no United States Food and Drug Administration-approved medication for use in chronic GVHD. The lack of standardized response criteria to measure therapeutic efficacy has been identified as one of the major obstacles for pursuing therapeutic trials in chronic GVHD [14]. A wide variety of endpoints has been considered in therapeutic trials for chronic GVHD, and the characteristics and potential limitations are summarized in Table 87.6 [9]. Overall survival and survival to discontinuation of systemic immunosuppression are accepted as major endpoints reflecting benefit in therapeutic trials of chronic GVHD, but to reach these long-term out-

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Table 87.6 Some potential endpoints for use in chronic graft-versus-host disease (GVHD) treatment trials Endpoint

Time to endpoint

Characteristics

GVHD response

Short

Patient-reported outcomes

Short

Biomarker of GVHD response

Very short

Time to treatment failure* Transplant-related mortality

Intermediate Long

Overall survival Relapse-free survival to permanent discontinuation of immunosuppressive treatment

Long Long

Scales for objective measurement of response have not yet been validated. Results may be affected by changes in ancillary treatment and supportive care. Endpoint is subject to bias if trials are not blinded Missing data are a frequent problem. Few sensitive instruments are available. Results may be affected by changes in ancillary treatment and supportive care. Measures patients’ experience rather than chronic GVHD; subject to bias if trials not blinded; best used as secondary endpoints Area of very high interest but no validated biologic markers of therapeutic response exist. Very few markers – candidates for testing have been identified. Research hampered by the absence of studies and the multiplicity of interfering transplant and chronic GVHD therapy-related factors Endpoint measures failure rather than success. Not validated in trials Endpoint measures failure rather than success. Also known as “chronic GVHD-specific mortality” in the literature Objective endpoint, ultimate gold standard, but may not be specific to chronic GVHD Best used as a primary endpoint in phase III studies.

* Secondary or salvage treatment is considered to be an indication of failure.

comes may take several years, making these endpoints unsuitable for early-phase drug development studies. Therefore, shorter-term measures of therapeutic efficacy such as partial or complete responses which are reliably predictive of long-term major outcomes need to be developed for allowing faster drug development in chronic GVHD. There are suggestions from some studies that the clinician’s estimate of therapeutic response can predict for survival or permanent withdrawal of immunosuppression, but no standardized and validated response criteria exist to predict such major endpoints [19,46]. Current definitions of response are usually based on subjective assessments of global complete (disappearance of all symptoms) or partial response (50% reduction in symptoms) based on nonreproducible physician assessments. There is a considerable variability in tools used from one study to the next (see the review by G. Akpek available at: http://www.asbmt.org/ GVHDForms.htm). There has been a trend towards introducing “surrogate” measures of benefit in chronic GVHD trials, such as the ability to taper steroids. However, this endpoint is controlled by the physician and of uncertain value unless a double-blinded study is employed, or the taper is conducted according to a fixed pre-defined algorithm [47]. The NIH-sponsored consensus project provided for the first time a set of standardized measures and provisional definitions to be used as quantitative response criteria in chronic GVHD. The tools were based on existing instruments or strategies used in chronic GVHD and in other fields of medicine. The proposed response measures are broadly divided in two main groups: clinician-assessed and patient-reported measures (Table 87.7). These are further divided into chronic GVHD-specific core measures that are always recommended for use in trials, and chronic GVHD-nonspecific ancillary measures. In other patient populations with chronic diseases, such outcomes have been extensively applied [48–50]. Quality of life measures (QOL) such as Short Form 36 Health Survey (SF-36) or Functional Assessment of Cancer Therapy – BMT Module (FACT-BMT) reflect patient experiences with the disease process rather than the disease status. The response measures proposed by the NIH consensus project should enhance uniformity of data collection and improve standardization of chronic GVHD clinical trials. For routine clinical care, the 0–3 organ-scoring system allows for a quick assessment of chronic GVHD for planning therapy (topical versus

systemic). A new skin score and a global chronic GVHD score for response assessments have been proposed recently [51,52]. It is imperative for these new measures to be prospectively validated for their ability to reliably predict major clinical outcomes in chronic GVHD studies. Further development and refinement of the response measures should be one of the most active areas in the future chronic GVHD clinical research.

Prevention As in acute GVHD, the most attractive approach for controlling chronic GVHD would be prevention of the most severe clinical manifestations while maintaining the crucial GVT effect. Clinical studies have identified many recipient, donor, and transplant characteristics which are associated with chronic GVHD. Some of these factors (such as recipient’s age or underlying diagnosis) are not modifiable, while others (such as choosing a better matched donor, donor gender or using bone marrow instead of peripheral blood as a hematopoietic cell source) may offer some limited flexibility. However, all of these approaches have very limited impact on chronic GVHD, and more effective methods of prevention are needed. Although acute GVHD is the best predictor for the development of chronic GVHD, a number of strategies which successfully decreased acute GVHD have not resulted in decreased rates of chronic GVHD [14,24–26,53–55]. Other unsuccessful attempts to prevent chronic GVHD include addition of thalidomide, intravenous immunoglobulin (IVIg) or pre-emptive treatment of subclinical chronic GVHD found in skin and lip biopsies [56–58]. Graft manipulation by ex vivo T-cell depletion is successful in reducing acute GVHD, but the effects on chronic GVHD are less evident and may depend on the methods for graft manipulation [14]. There has been a resurgence of interest in using antibodies in preparative regimens for in vivo prevention of GVHD. The addition of antithymocte globulin or anti-CD52 monoclonal antibody to preparative regimens seems to provide protection against extensive chronic GVHD, but is associated with other unwanted effects of indiscriminant lymphodepletion such as infections or attenuation of GVT effects [59,60].

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Table 87.7 Conceptual framework of chronic graft-versus-host disease (GVHD) response measures as proposed by the National Institutes of Health consensus project for clinical trials in chronic GVHD Measure

Clinician assessed

Patient reported

I. Chronic GVHD-specific core measures Signs

Organ-specific measures*

Not applicable

Symptoms

Clinician-assessed symptoms†

Patient-reported symptoms†

Global rating

Mild, moderate or severe 0–10 severity scale 7-point change scale

Mild, moderate or severe 0–10 severity scale 7-point change scale

II. Chronic GVHD nonspecific ancillary measures Function Grip strength 2-minute walk time

Patient-reported function‡

Quality of life

Patient-reported health-related quality of life‡

–

* Clinician-assessed organ-specific measures include assessments of skin, eyes, mouth, platelet and eosinophil counts, gastrointestinal symptoms, liver function tests, pulmonary function tests, and optional evaluations by the range of motion of joints affected by skin sclerosis, and by gynecologic examination for vulvar vaginal symptoms. Karnofsky performance status and patient weight are also recorded. † Patient-reported symptoms include 0–10 intensity scale reporting symptoms of itching, chief ocular complaint, oral pain, dryness, and sensitivity, and the symptom bother scale published by Lee et al. [92]. ‡ Patient self-reports: for function HAP (adults) and ASK (children); for quality of life, SF-36 and FACT-BMT(adults), and CHRIs (children) [7].

The major obstacle for developing effective and optimal prevention approaches for chronic GVHD is still our rudimentary understanding of basic chronic GVHD biology. The recent 2007 state of the art Bone Marrow Transplant Clinical Trials Network conference identified studies of GVHD biology as a major priority to generate critically needed information for advances in controlling both acute and chronic GVHD, improved graft manipulations, cellular interventions, and the development of more selective targeted therapies.

Treatment The typical course of chronic GVHD is protracted, lasting on average 2–3 years. Ultimately, about 85% of patients who survive beyond 5 years after diagnosis are able to discontinue systemic therapy [21]. The goal of therapy of chronic GVHD is to stop the destructive immunologic process, alleviate symptoms, and prevent disease progression which may lead to irreversible disability or death. The ultimate goal is to establish immunologic tolerance and withdraw immunosuppressive therapy. It is also important to recognize that patients with chronic GVHD, even after permanent withdrawal of immunosuppression, remain at increased risk for a number of late complications of allogeneic transplantation [36,37]. These patients require continued monitoring by providers familiar with the late effects of transplantation and cancer therapy. Therapy of chronic GVHD depends heavily on a well-coordinated multidisciplinary team which among others includes transplantation specialists, a primary health-care provider, organ-specific consultants, nurses, and a number of ancillary services including social services, vocational specialists, and patient and family support groups and systems. Principal components of chronic GVHD therapy include systemic treatment with immunosuppressants or immune modulators integrated with ancillary therapy and supportive care. Systemic therapy Symptomatic mild chronic GVHD is often effectively treated with local therapies alone (e.g. topical steroids to the skin or cyclosporine

Table 87.8 Indications for starting systemic therapy in chronic graft-versushost disease (GVHD) patients Global severity of chronic GVHD

High risk for mortality*

Systemic treatment

Mild Mild Moderate Severe

No Yes No/yes No/yes

No Yes† Yes Yes

Systemic therapy should be considered for patients who meet criteria for moderate or severe global severity (any involvement of three or more organs or with a National Institutes of Health score of 2 or greater in any single organ, or any lung involvement). Global staging system of severity (none, mild, moderate or severe) is derived by combining organ-specific 0–3 scores [4] and is intended also to replace the current “limited-extensive” scoring system [44]. Mild chronic GVHD stage involves only one or two organs with a maximum score of 1. Moderate chronic GVHD involves at least one organ with score 2 or 3, or more organs with scores of 1. A lung score of 1 is also considered as moderate-stage chronic GVHD. * Platelets <100 × 109/L or receiving steroids at time of diagnosis of chronic GVHD, or progressive onset, or >50% of body surface area skin involvement. † The potential benefits of the graft-versus-tumor effect and the risk of chronic GVHD may need to be weighted.

eyedrops). However, systemic therapy should be considered for patients who meet criteria for moderate-to-severe global severity (involvement of three or more organs, or with an NIH score of 2 or greater in any single organ, or any lung involvement) (Table 87.8) [4]. Good medical practice and judgment dictate flexibility in this recommendation. Some experts incorporate the presence or absence of published high-risk features (e.g. thrombocytopenia or progressive onset) and the underlying reason for transplantation (e.g. malignant versus nonmalignant disease) or current comorbid conditions (e.g. infection) into decisions of whether or not to treat with systemic immunosuppression. Appropriate early intervention with effective systemic therapy may prevent progression to severe chronic GVHD.

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Primary therapy When possible, patients found to have chronic GVHD should be entered on investigational treatment protocols. If such studies are not available, the most widely used initial systemic treatment of chronic GVHD relies on prednisone alone in conjunction with cyclosporine or tacrolimus [3,61–64]. In contrast to the treatment of acute GVHD, where more rapid disease course and high frequency of gastrointestinal symptoms dictate initially intravenous administration, in chronic GVHD these drugs are most commonly administered by the oral route. Patients start treatment with prednisone usually at 1 mg/kg/day in a single oral dose. Cyclosporine or tacrolimus is dosed twice a day to keep levels therapeutic. If a patient is responding or showing signs of stable disease, a prednisone taper starts by reducing the dose by 25% per week over 4 weeks, to a target dosage of 1 mg/kg every other day. Responses are evaluated every 3 months after alternate-day dosing has been achieved. The 3month timeframe for evaluation of response to a given therapy is based on the observation that 90% of patients who ultimately respond to therapy will show signs of response at that point [22]. If the disease has completely resolved, patients are gradually weaned from medication, with dose reductions made approximately at 2-week intervals, usually starting with a steroid taper. Patients who continue to respond are maintained on the same therapy and are re-evaluated in another 3 months. Once patients reach their maximal response, therapy is continued for another 3 months and then weaned. For those who have not responded by the 3-month timepoint or who progress, alternative salvage regimens should be instituted. This front line standard of practice evolved based on two reports by Sullivan et al. from the mid 1980s. In a randomized placebo-controlled, trial they reported that prednisone alone was superior to prednisone plus azathioprine for primary treatment of patients with standard-risk extensive chronic GVHD (5-year survival 61% and 47%, respectively). However, in patients classified as high risk on the basis of platelet counts lower than 100 × 109/L who were directly assigned to the treatment with prednisone, survival was only 26% at 5 years [61]. When a group of 40 similar high-risk patients was treated in a subsequent phase II study with alternating-day cyclosporine and prednisone, the long-term survival rate was 51% [62]. After this encouraging report, most centers adopted cyclosporine and prednisone for initial treatment of all patients, not just those deemed at high risk. Koc et al. reported the results of a randomized nonblinded trial comparing prednisone alone with prednisone plus cyclosporine in patients with extensive chronic GVHD without thrombocytopenia. In this trial (n = 287 evaluable patients), the cumulative incidence of NRM, overall survival, relapse, need for secondary chronic GVHD therapy, and discontinuation of immunosuppressive medications were not significantly different between the two arms. However, survival without recurrent malignancy was better in the prednisone-only arm (71% versus 61%; p = 0.03), although the incidence of avascular necrosis was also higher (22% versus 13%; p = 0.04) [63]. Thus, there is no current evidence that initial combination therapy of cyclosporine with prednisone improves control of chronic GVHD in patients with platelet counts over 100 × 109/L. Because cyclosporine exerted superior survival in the subset of patients with thrombocytopenia and has demonstrated steroid toxicitysparing effects, transplant clinicians commonly favor the combination of a calcineurin inhibitor with prednisone for initial systemic therapy of chronic GVHD. Attempts to improve outcomes of primary therapy in chronic GVHD continue, although very few studies have addressed this issue. Two randomized trials of thalidomide as part of initial therapy yielded no clinical benefit of addition of this agent to standard cyclosporine and prednisone [65,66]. Currently, two multicentered randomized phase III

trials are examining whether the addition of mycophenolate mofetil (United States trial; see also #NCT00089141 at http://www. ClinicalTrials.gov) or enteric-coated mycophenolic acid (European trial; #NCT00298324 at http://www.ClinicalTrials.gov) to prednisone plus cyclosporine/tacrolimus improves the success rate of primary therapy. Secondary therapy About 50% of patients with chronic GVHD fail to achieve a complete or partial response to therapy by 1 year after diagnosis [19]. Therefore, there is a frequent need for initiation of secondary treatments. Indications for initiation of secondary treatment include failure of primary steroid-based therapy or of any subsequent line of therapy, as manifested by progression of chronic GVHD, or stable persistence of chronic GVHD despite 4–12 weeks of sustained therapy, or inability to taper immunosuppression without recurrence of clinical manifestations. Earlier initiation of secondary therapy would be appropriate in patients with more severe chronic GVHD, while a longer trial of initial therapy would be appropriate in patients with sclerotic skin changes or other slowly reversible manifestations of chronic GVHD. Due to the recalcitrant nature of the disease, BO represents a special situation where the duration of full steroid doses of 1 mg/kg/day typically lasts for months, and any disease stability is considered a success. A delay in initiating secondary therapy would also be appropriate when the next agent to be used has a high risk of toxicity. Inability to tolerate therapy (e.g. steroid myopathy or calcineurin- and/or sirolimus-induced thrombotic microangopathy) may also be considered an indication for secondary treatment. There is no current standard of care for chronic GVHD patients who fail front-line steroid-based therapy. A number of mostly smaller phase II studies of secondary or salvage regimens have been published, most of which report response rates of 25–75%. Responses are frequently incomplete and not durable [38,46,52,67–88]. Agents used for secondary therapy of chronic GVHD are shown in Table 87.9. Most of these agents have been used with marginal success and with poor clinician satisfaction [89]. Since there is no therapeutic standard, choices in salvage therapy for chronic GVHD are therefore made based on the patient’s history of previous treatments, using agents with nonoverlapping toxicities, and with sensitivity to the patient’s personal preferences. In patients with advanced chronic GVHD, it is important to maintain awareness of the possibility that some symptoms may be due to irreversible organ damage and not to active disease. It is also very difficult to interpret responses among different trials since each trial uses its own definition of response. Some studies also include the ability to taper or discontinue steroids as an endpoint. As the decision to taper steroids is highly subjective, inclusion of such endpoints in an open-label phase II trial is very difficult to interpret. Finally, the relation of these responses to major endpoints such as survival or permanent disability is unknown. For all these reasons, the most appropriate secondary therapy for a patient with chronic GVHD is a clinical trial. There is an urgent need for developing better treatments and better standardized clinical trials of new agents for chronic GVHD. Ancillary therapy and supportive care Chronic GVHD is characterized by polymorphic clinical manifestations. Prolonged systemic immunosuppressive treatment including corticosteroids is necessary to control the disease. Treatment, delayed immunological reconstitution, and the immunodeficiency associated with chronic GVHD increase the risk of infection. Clinical manifestations of chronic GVHD can persist for prolonged periods of time, causing significant morbidity. Some of these changes, such as contractures, may be

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Table 87.9 Summary of secondary systemic therapies for chronic graft-versus-host disease (GVHD) Agents

Class

Common side-effects*

Serious side-effects*

Comment

Commonly used Mycophenolate mofetil [71,72] Tacrolimus [73]

De novo pathway purine synthesis inhibitor Calcineurin inhibitor

Gastrointestinal

Gastrointestinal bleeding, leukopenia, infections Thrombotic microangiopathy, CNS Central catheter-related (infections, clots)

Most popular first-choice salvage

Caution when in combination with calcineurin inhibitors, serious interaction with voriconazole Requires intravenous hydration before and after each dose Skin and musculoskeletal system respond best Annual eye examinations needed, well tolerated, mostly used as adjuvant Option when rapid response needed

Renal, nausea

Used instead of cyclosporine, limited success Consistently high rate of response (40–80%) in salvage trials, increasingly popular

Need for central venous access, iron deficiency, logistics

Sirolimus [38]

Photoactivation of blood mononuclear cells by ex vivo 8-methoxypsoralen, immunomodulation mTOR inhibitor, antifibrotic

Hyperlipidemia, infections, renal dysfunction, cytopenia

Thrombotic microangiopathy

Pentostatin [52]

Nucleoside analog

Nausea, vomiting

Infections, renal, CNS

Rituximab [70,74]

Infusional reactions

Infections

Hydroxychloroquine [75]

Chimeric anti-CD20 monoclonal antibody Antimalarial agent

Gastrointestinal symptoms

Retinal damage, myositis, polyneuropathy

High-dose pulse steroids [76]

Corticosteroid

Numerous

Myopathy, infections, psychosis

8-methoxypsolaren and ultraviolet A irradiation Antileprotic, antiinflammatory, immunomodulatory

Nausea, skin phototoxicity

Nonmelanoma skin cancer

Neuropathy, somnolence, constipation

Neutropenia, teratogenic, deep venous thrombosis, skin hypersensitivity

Ursodeoxycholic acid [79]

Bile acid

–

Clofazimine [80]

Antimycobacterial

Diarrhea, abdominal pain, headache Gastrointestinal, hyperpigmentation

Rare use or new reports Acitretin [81]

Retinoid

Skin flaking, dryness, cheilitis, alopecia, night blindness Gastrointestinal symptoms, marrow suppression Infections

Pancreatitis, hyperlipidemia

Patients with skin sclerosis, alone or as adjuvant

Second cancers

No formal reports in salvage

Anaphylaxis, serum sickness Infections, second cancers

No formal reports in salvage

Gastrointestinal symptoms, dizziness, headache, insomnia, fatigue Hypersensitivity

Infections

Few case reports, combined with other agents

Infections

Infections

Infections

Rare reports, usually in combination 5 of 8 patients responded

Myelosuppression, liver toxicity Profound myelosuppression

Accumulation in effusions

Cannot be used in renal failure

Hemorrhagic cystitis, cardiac

2 cases reported, both responded, sclerotic skin

Extracorporeal photopheresis [46]

Less frequently used Psoralen and ultraviolet A [77] Thalidomide [66,69,78]

Azathioprine Antithymocyte globulin Total lymphoid irradiation [82] Daclizumab [68,83]

Infliximab [83] Etanercept [84]

Low-dose methotrexate [67] Cyclophosphamide 200 mg/ kg and stem cell rescue [85,106]

Competitive inhibitor of purine synthesis Polyclonal antibody Low dose 100 cGy ionizing irradiation Humanized anti-IL-2 receptor monoclonal antibody Chimeric anti-TNF-α monoclonal antibody Recombinant human soluble TNF receptor fusion protein Antimetabolite, antiinflammatory Alkylating agent

Leukopenia

–

Best for skin lichen planus lesions, no systemic effect Disappointing in primary therapy, promising as salvage agent, toxicity a serious limitation Liver GVHD as adjuvant, or for very mild cases Best in sclerotic skin and oral disease

Rarely used today

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Table 87.9 Continued Agents

Class

Common side-effects*

Serious side-effects*

Comment

Cyclophosphamide pulses ~1 g/m2 [86] Imatinib [87]

Alkylating agent

Myelosuppression

Hemorrhagic cystitis

Tyrosine kinase inhibitor, PDGF inhibitor

Myelosuppression, weight gain

Drug-induced hepatitis

Montelukast [88]

Leukotriene antagonist

Headache, nausea

–

3 cases reported, all responded, liver and oral GVHD One case reported, effective in a patient with bronchiolitis obliterans 15 of 19 patients responded, 3 of 5 with lung disease

CNS, central nervous system; mTOR, mammalian target of rapamycin; PDGF, platelet-derived growth factor; TNF-α, tumor necrosis factor-alpha. * Only most relevant common or serious side-effects that have been described with these agents in hematopoietic cell transplantation and/or other patient populations are selected here. This is not an exhaustive list of all possible common or serious side-effects.

irreversible. Thus, ancillary therapy and supportive care to prevent infections, optimize nutrition, ameliorate morbidity, and optimize functional performance and capacity are critical components of management [8]. As used in the NIH consensus recommendations, the term “ancillary therapy” refers to any intervention such as topical corticosteroids, cyclosporine eyedrops or any other nonsystemic therapeutic intervention directed at control of symptoms. The term “supportive care” includes a broad spectrum of interventions that are directed at control of organspecific or systemic symptoms. Supportive care includes antibiotics for prevention of infections, management of osteoporosis, metabolic problems, physical therapy, as well as a number of educational, preventive, and psychosocial measures. Ancillary and supportive care therapies are commonly added to systemic immunosuppression treatment. Occasionally, their use may circumvent the need for systemic treatment, or allow doses of systemic agents to be reduced. A summary of ancillary therapy and supportive care interventions guidelines is presented in Table 87.10 [8]. Specific dispensary information is also available from the website: http://www.asbmt.org/GvHDForms. These recommendations were largely generated for use in adult patients. In pediatric patients, there are many areas of uncertainty. For example, because of body surface area differences, topical therapies may actually result in significant systemic absorption in pediatric patients. In general, close serial monitoring of all organ systems is recommended to promote early detection and intervention [8]. Periodic monitoring recommendations include interval medical history, physical examination and review of medications, blood work (complete blood count and differential, chemistry panel, therapeutic drug levels, IgG level, lipid profile, 25-OH vitamin D status, thyroid function, and ovarian and testicular function), pulmonary function tests, and bone dual-energy X-ray absorptiometry densitometry. Periodic specialist evaluations should include ophthalmology (Schirmer test, corneal evaluation, and tests for glaucoma), dental and oral examination, and dermatology, gynecology, rehabilitation medicine, and pain and supportive care team assessments. The scope and frequency of monitoring should be individualized as clinically indicated, and ranges from monthly to at least yearly. More frequent monitoring is advised for those with active chronic GVHD, especially during the high-risk periods such as during tapering of medications. Especially challenging cases should be considered for consultation referral at one of the major national centers with multidisciplinary expertise in studying chronic GVHD. Infection is the most common cause of mortality in patients with chronic GVHD, and prophylaxis of infections requires special emphasis. The immune defects in chronic GVHD are broad, encompassing macrophage function, antibody production, and T-cell function.

Recommendations supported by published evidence are usually derived from studies with patients who had conditions other than chronic GVHD. All patients with chronic GVHD are considered at risk for infection with encapsulated bacteria, particularly Streptococcus pneumoniae, but also Haemophilus influenzae and Neisseria meningitides. Prophylactic antibiotics should be given to all patients with chronic GVHD as long as systemic immunosuppressive treatment is being administered. Penicillin V K is the prophylactic agent of choice when the frequency of penicillin-resistant Streptococcus pneumoniae is low. Alternatives include azithromycin or other macrolides and newer-generation quinolones, although drug interactions can cause problems. Daily use of trimethoprim–sulfamethoxazole has also been used for this indication. Although no studies have evaluated the degree of protection provided by pneumococcal (polyvalent polysaccharide or heptavalent), H. influenzae b conjugate or influenza vaccination in patients with chronic GVHD, most experts advocate their use, since the risk of adverse outcomes with vaccination is low. No live viruses, including the new live attenuated influenza vaccine and measles, mumps, and rubella, should be given. Household contacts should not be given oral polio vaccine. Universal administration of IVIg after HCT has not been shown to confer clinical benefit and should be avoided. IVIg supplementation may be considered for patients more than 90 days after HCT who have recurrent sinopulmonary infections and serum IgG levels less than 400 mg/ dl. Invasive mold infections are a significant concern in patients receiving immunosuppressive treatment for chronic GVHD. Some centers prescribe prophylactic mold-active agents for patients with chronic GVHD who are on higher doses of steroids (>0.5–1.0 mg/kg/day), but this approach remains investigational since the benefits and risks are unknown. All patients receiving immunosuppression should receive Pneumocystis carinii prophylaxis.. It is unknown how long prophylaxis should be continued after stopping immunosuppression, and practices vary widely among centers. Agents used for Pneumocystis prophylaxis include trimethoprim–sulfamethoxazole, pentamidine, dapsone, and atovaquone. Trimethoprim–sulfamethoxazole also provides prophylaxis against Toxoplasma and Nocardia. Some experts use long-term antiviral prophylaxis to prevent recurrent herpes simplex virus and varicella zoster virus (VZV) infection among HCT recipients with severe, longterm immunodeficiency. If VZV-seronegative patients with chronic GVHD are exposed to varicella (either primary or post-vaccination illness), VZV IG should be given within 96 hours. Cytomegalovirus (CMV) disease after day 100 has become more common. Patients with active GVHD, history of CMV reactivation

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Table 87.10 Summary of ancillary therapy and supportive care interventions Organ system

Organ-specific intervention

Skin and appendages

Prevention • Photoprotection. Surveillance for malignancy Treatment • For intact skin – Topical emollients, corticosteroids, antipruritic agents, and others (e.g. PUVA, calcineurin inhibitors) • For erosions/ulcerations – Microbiologic cultures, topical antimicrobials, protective films or other dressings, debridement, hyperbaric oxygen, wound care specialist consultation

Mouth and oral cavity

Prevention • Maintain good oral/dental hygiene. Consider routine dental cleaning and endocarditis prophylaxis. Surveillance for infection and malignancy Treatment • Topical high and ultrahigh potency corticosteroids and analgesics. Therapy for oral dryness

Eyes

Prevention • Photoprotection. Surveillance for infection, cataract formation, and increased intraocular pressure Treatment • Artificial tears, ocular ointments, topical corticosteroids or cyclosporine, punctal occlusion, humidified environment, occlusive eye wear, moisture chamber eyeglasses, cevimeline, pilocarpine, tarsorraphy, gas-permeable scleral contact lens, autologous serum, microbiologic cultures, topical antimicrobials, doxycycline

Vulva and vagina

Prevention • Surveillance for estrogen deficiency, infection (herpes simplex virus, human papilloma virus, yeast, bacteria), and malignancy Treatment • Water-based lubricants, topical estrogens, topical corticosteroids or calcineurin inhibitors, dilators, surgery for extensive synechiae/obliteration, early gynecology consultation

Gastrointestinal tract and liver

Prevention • Surveillance for infection (viral, fungal) Treatment • Eliminate other potential etiologies. Dietary modification, enzyme supplementation for malabsorption, gastroesophageal reflux management, esophageal dilatation, ursodeoxycholic acid

Lungs

Prevention • Surveillance for infection (Pneumocystis carinii, viral, fungal, bacterial) Treatment • Eliminate other potential etiologies (e.g. infection, gastroesophageal reflux). Inhaled corticosteroids, bronchodilators, supplementary oxygen, pulmonary rehabilitation. Consideration of lung transplantation

Hematopoietic

Prevention • Surveillance for infection (cytomegalovirus, parvovirus) Treatment • Eliminate other potential etiologies (e.g. drug toxicity, infection). Hematopoietic growth factors, immunoglobulin for immune cytopenias

Neurologic

Prevention • Calcineurin drug level monitoring. Seizure prophylaxis including blood pressure control, electrolyte replacement, anticonvulsants Treatment • Occupational and physical therapy, treatment of neuropathic syndromes with tricyclic antidepressants, selective serotonin reuptake inhibitors or anticonvulsants

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Table 87.10 Continued Organ system

Organ-specific intervention

Immunologic and infectious diseases

Prevention • Immunizations and prophylaxis against Pneumocystis carinii, varicella zoster virus, and encapsulated bacteria based on Centers for Disease Control guidelines. Consider immunoglobulin replacement based on levels and recurrent infections. No current evidence to support the use of mold-active agents. Surveillance for infection (viral, bacterial, fungal, atypical) Treatment • Organism-specific antimicrobial agents. Empiric parenteral broad-spectrum antibacterial coverage for fever

Musculoskeletal

Prevention • Surveillance for decreased range of motion, bone densitometry, calcium levels and 25-OH vitamin D. Physical therapy, calcium, vitamin D, bisphosphonates Treatment • Physical therapy, bisphosphonates for osteopenia and osteoporosis

PUVA, psoralen and ultraviolet irradiation. Modified with permission from Couriel et al. [8].

during the first 3 months, and lymphopenia are at higher risk of CMV reactivation and death [90]. Some centers continue to monitor for CMV infection after day 100 by pp65 antigenemia or polymerase chain reaction tests, followed by pre-emptive therapy, based on the individual risk as determined by donor and recipient serology. Some investigators advocate early empirical treatment of influenza with neuraminidase inhibitors during influenza outbreaks, using prediction rules based on symptoms and signs, although there is no evidence to support this practice [8].

QOL and other health-related outcomes in chronic GVHD Health related QOL is a multidimensional construct (physical, functional, emotional, social, and spiritual), and in clinical medicine is typically elicited through self-reported assessment that provides a measure of overall satisfaction with life and sense of wellbeing. Chronic GVHD is associated with substantial QOL deficits, particularly in the areas of physical and functional status [3]. Chronic GVHD is also found to be associated with loss of employment in long-term survivors. The need for protracted treatment, particularly with corticosteroids, may result in decreased QOL and functional losses secondary to significant morbidity. It is important to emphasize that QOL questionnaires are measures of the patient’s experiences of the disease, and they do not necessarily accurately represent the severity and activity of the disease. In some studies, it has been found that patients who had chronic GVHD paradoxically reported their QOL as being better than transplant survivors without chronic GVHD [91]. A 30-item survey capturing patient selfreport of chronic GVHD symptoms has been developed and validated for the purpose of use in clinical care and studies in chronic GVHD [92]. It is complementary to the frequently used generic QOL instruments such as SF-36 or FACT-BMT. Prior studies have not thoroughly characterized the impact of chronic GVHD diagnosis on overall health status. Fraser et al. reported recently

a study in 584 individuals who had undergone allogeneic HCT between 1976 and 1999 and survived 2 or more years [93]. These patients completed a 255-item health questionnaire. Global assessment of health status was performed by measurement of six health status domains. In multivariable analyses, subjects with active chronic GVHD were more likely to report adverse general health and mental health, functional impairments, activity limitation, and pain, but not anxiety or fear, when compared with those with no history of chronic GVHD. Importantly, health status did not differ between those with resolved chronic GVHD and those who never had chronic GVHD (Fig. 87.5). This study emphasizes the reversible nature of chronic GVHD, and the need for timely and assertive implementation of effective therapies. A number of valuable web-based resources and organizations exist which can be recommended to patients, including National Bone Marrow Transplant Link (NBMTLink; http://www.nbmtlink.org/), Blood and Marrow Transplant Information Network (BMTInfoNet; http://www.bmtinfonet.org/), and the National Marrow Donor Program (http://www.marrow.org/). Transplantation survivors have an increased long-term risk for new solid cancers. Chronic GVHD and its therapy are independently associated with nearly a threefold higher risk for later developing of squamous cell carcinoma (SCC), particularly of skin and oral mucosa, while there is no additive increased risk for non-SCC cancers [37]. Curtis et al. identified that major risk factors for the development of SCC are long duration of chronic GVHD therapy (p<0.001), use of azathioprine, particularly when combined with cyclosporine and steroids (p<0.001), and severe chronic GVHD (p = 0.004). The risk was further heightened to more than 50-fold when other drugs, therapy with psoralen and ultraviolet A, or limited field irradiation was used in addition to azathioprine, cyclosporine, and steroid therapy. These data provide further impetus for the development of more effective and less carcinogenic treatment regimens for patients with moderate or severe chronic GVHD. Clinical surveillance for SCC seems appropriate among long-term transplant survivors patients exposed to persistent chronic GVHD or prolonged immunosuppressive therapy.

Chronic Graft-versus-Host Disease: Clinical Manifestations and Therapy

90

Prevalence (percent)

80

none

resolved

P<.001

active

70

1321

P<.001

60

P<.001

50

P=.002

40

P=.02

P<.001

30

P=.98

20 10 0 general health

mental health

functional impairment

activity limitation

pain

anxiety

any domain

Fig. 87.5 Prevalence of survivors of hematopoietic cell transplantation with adverse health outcomes by chronic graft-versus-host disease (GVHD) activity status (none, resolved or active). Subjects with active chronic GVHD were more likely to report adverse general health, but health status did not differ between those with resolved chronic GVHD and those who never had chronic GVHD. (Reproduced from [93], with permission.)

Graft-versus-leukemia effect A number of observational studies demonstrate that chronic GVHD is associated with lower relapse rates in both early and advanced-stage leukemia [18,94–101]. However, the biologic mechanisms of this important graft-versus-malignancy effect are poorly understood. As mentioned earlier, these studies used the day 100 definition of chronic GVHD and are hampered by lack of specificity of their GVHD designation. Study of this question is further hampered by limitations of the biostatistical methods utilized in the setting of multiple time-dependent competing events. Refinement and development of best statistical approaches to adequately study the magnitude of post-transplant events such as relapse and chronic GVHD is an active area of research [102,103]. In a landmark registry-based study, Horowitz et al. studied 2254 patients with acute myeloid and lymphoblastic leukemia in first remission or chronic myeloid leukemia in first chronic phase, most of whom received HLA-identical sibling bone marrow after high-dose conditioning. Patients who had only chronic GVHD or both acute and chronic GVHD had lower relative risks of relapse as compared with patients without GVHD (relative risk [RR] 0.43, p = 0.01, and RR 0.33, p = 0.0001, respectively). In the same analysis, relapse rates in patients who received identical twin grafts or T-cell-depleted grafts were at increased risk of disease relapse (RR 2.09, p = 0.005, and RR 1.76, p = 0.002, respectively) [96]. Of interest, Bierman et al. used similar methodology to study GVT effects in a large group of patients after high-dose conditioning and allogeneic HCT for advanced non-Hodgkin’s lymphoma, and did not find any evidence of an impact of acute or chronic GVHD, T-cell depletion or identical twin grafts on relapse risk, indicating that the clinical expression of the allogeneic graft-versus-malignancy effects may depend on the underlying disease and/or the number or nature of prior therapies [104]. Lee et al. analyzed effects of chronic GVHD severity in three cohorts including a total of 3472 patients who received high-dose conditioning and allogeneic HCT from related or unrelated donors for various stages of acute or chronic myeloid leukemia [18]. Mild manifestations of chronic GVHD did not increase NRM as compared with patients without chronic GVHD. In all patients, NRM increased significantly with severe manifestations of chronic GVHD. Significantly lower relapse rates were seen in all patients with chronic GVHD, RR being 0.5 (p < 0.001), 0.6 (p < 0.01), and 0.6 (p < 0.01), respectively in each of the three patient cohorts. This large study did not show evidence of decreasing relapse rates with increasing chronic GVHD severity. Leukemia relapses were uncommon (8–9%) after the development of chronic GVHD. However,

increasing severity of chronic GVHD was not associated with a longer time to relapse, and patients with mild chronic GVHD had the best disease-free survival. Patients with no chronic GVHD had a disease-free survival similar to patients with moderate chronic GVHD, and patients with the most severe chronic manifestations had the worst disease-free survival. Other studies in leukemia patients demonstrated that limitedstage chronic GVHD provided survival benefit by reducing risk of relapse without increasing risk of death from chronic GVHD [99,100]. These important findings establish the concept that only moderate or severe cases of chronic GVHD adversely impact disease-free survival, and identify such patients as a primary target of future therapeutic or preventive interventions. There are surprisingly few data on the GVT effects of chronic GHVD in RIC HCT, a setting where curative effects of transplantation are almost entirely dependent on immunologic effects. Baron et al. analyzed GVT effects in 322 patients given HLA-matched related or unrelated donor grafts after RIC transplantation, and noted higher probability of achieving complete remission in patients who developed clinical extensive chronic GVHD (hazard ratio 1.7; p = 0.07). Grade II–IV acute GVHD had no significant impact on the risk of disease relapse/progression. Acute GVHD was associated with an increased risk of NRM and decreased probability of progression-free survival. In contrast, extensive chronic GVHD was associated with reduced risk of disease relapse/progression (hazard ratio 0.4; p = 0.006) and increased probability of progression-free survival (p = 0.003) [105]. These findings suggest that chronic GVHD may contribute substantially to the success of RIC transplants for malignant disease.

Future directions As this chapter has pointed out repeatedly, it is staggering to realize how little we know or understand about chronic GVHD. The last few years have shown a significant increase in interest in chronic GVHD at a clinical level, but the basic research needed to support the clinical advances remains scant. Is there a pathway out of this dilemma? Yes, but it requires collaboration among investigators. The protracted and varied natural history of chronic GVHD makes the individual-center approach (which has dominated research into acute GVHD) difficult at best. The failure of this approach is evident by the few published studies on chronic GVHD. To be able to conduct timely clinical studies, including studies which use patient blood and tissue samples, investigators must work together, regardless of their physical location. Development of pre-emptive treatment approaches which

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would interfere with the processes of evolving chronic GVHD is of highest interest. The recent NIH Consensus Conference, the two multicenter initial therapy trials, the trials sponsored by the Pediatric Oncology Group, and the trials being planned by the Bone Marrow Transplant Clinical Trials Network are the first steps in this direction [107]. Patients must be enrolled in natural history studies, treatment studies, and studies characterizing the immunologic defects common in this disease. As importantly, the transplant community needs to pressure the NIH and private funding organizations to support studies in chronic GVHD. With steadily increasing numbers of transplant survivors, chronic GVHD has become as large an, or a larger, issue than acute GVHD. It deserves the same intensive, aggressive research that acute GVHD has received.

Only with that level of dedication will progress be made in this disorder.

Acknowledgment The authors would like to thank Drs Edward Cowen and Maria Turner of the National Cancer Institute Dermatology Branch, Janine Smith of the National Eye Institute, and Mark Schubert of the Fred Hutchinson Cancer Research Center, who kindly allowed us to use photographs from their archive. Our gratitude also goes to Sandra Mitchell, PhD, for her invaluable expert assistance with selecting and composing the photographs for colour plates relating to this chapter.

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66. Arora M, Wagner JE, Davies SM et al. Randomized clinical trial of thalidomide, cyclosporine, and prednisone versus cyclosporine and prednisone as initial therapy for chronic graft-versushost disease. Biol Blood Marrow Transplant 2001; 7: 265–73. 67. Giaccone L, Martin P, Carpenter P et al. Safety and potential efficacy of low-dose methotrexate for treatment of chronic graft-versus-host disease. Bone Marrow Transplant 2005; 36: 337–41. 68. Teachey DT, Bickert B, Bunin N. Daclizumab for children with corticosteroid refractory graftversus-host disease. Bone Marrow Transplant 2006; 37: 95–9. 69. Vogelsang GB, Farmer ER, Hess AD et al. Thalidomide for the treatment of chronic graftversus-host disease. N Engl J Med 1992; 326: 1055–8. 70. Cutler C, Miklos D, Kim HT et al. Rituximab for steroid-refractory chronic graft-versus-host disease. Blood 2006; 108: 756–62. 71. Mookerjee B, Altomonte V, Vogelsang G. Salvage therapy for refractory chronic graftversus-host disease with mycophenolate mofetil and tacrolimus. Bone Marrow Transplant 1999; 24: 517–20. 72. Lopez F, Parker P, Nademanee A et al. Efficacy of mycophenolate mofetil in the treatment of chronic graft-versus-host disease. Biol Blood Marrow Transplant 2005; 11: 307–13. 73. Carnevale-Schianca F, Martin P, Sullivan K et al. Changing from cyclosporine to tacrolimus as salvage therapy for chronic graft-versus-host disease. Biol Blood Marrow Transplant 2000; 6: 613–20. 74. Zaja F, Bacigalupo A, Patriarca F et al. Treatment of refractory chronic GVHD with rituximab: a GITMO study. Bone Marrow Transplant 2007; 40: 273–7. 75. Gilman AL, Chan KW, Mogul A et al. Hydroxychloroquine for the treatment of chronic graftversus-host disease. Biol Blood Marrow Transplant 2000; 6: 327–34. 76. Akpek G, Lee SM, Anders V, Vogelsang GB. A high-dose pulse steroid regimen for controlling active chronic graft-versus-host disease. Biol Blood Marrow Transplant 2001; 7: 495–502. 77. Vogelsang GB, Wolff D, Altomonte V et al. Treatment of chronic graft-versus-host disease with ultraviolet irradiation and psoralen (PUVA). Bone Marrow Transplant 1996; 17: 1061–7. 78. Parker PM, Chao N, Nademanee A et al. Thalidomide as salvage therapy for chronic graftversus-host disease. Blood 1995; 86: 3604–9. 79. Fried RH, Murakami CS, Fisher LD, Willson RA, Sullivan KM, McDonald GB. Ursodeoxycholic acid treatment of refractory chronic graft-versushost disease of the liver. Ann Intern Med 1992; 116: 624–9. 80. Lee SJ, Wegner SA, McGarigle CJ, Bierer BE, Antin JH. Treatment of chronic graft-versus-host disease with clofazimine. Blood 1997; 89: 2298– 302. 81. Marcellus DC, Altomonte VL, Farmer ER et al. Etretinate therapy for refractory sclerodermatous chronic graft-versus-host disease. Blood 1999; 93: 66–70. 82. Robin M, Guardiola P, Girinsky T et al. Low-dose thoracoabdominal irradiation for the treatment of refractory chronic graft-versus-host disease. Transplantation 2005; 80: 634–42.

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83. Rodriguez V, Anderson PM, Trotz BA, Arndt CA, Allen JA, Khan SP. Use of infliximab–daclizumab combination for the treatment of acute and chronic graft-versus-host disease of the liver and gut. Pediatr Blood Cancer 2007; 49: 212–15. 84. Busca A, Locatelli F, Marmont F, Ceretto C, Falda M. Recombinant human soluble tumor necrosis factor receptor fusion protein as treatment for steroid refractory graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. Am J Hematol 2007; 82: 45–52. 85. Arat M, Ilhan O, Iayan EA, Celebi H, Koc H, Akan H. Treatment of extensive chronic sclerodermatous graft-versus-host disease with highdose immunosuppressive therapy and CD34+ autologous stem cell rescue. Blood 2001; 98: 892–3. 86. Mayer J, Krejci M, Doubek M et al. Pulse cyclophosphamide for corticosteroid-refractory graftversus-host disease. Bone Marrow Transplant 2005; 35: 699–705. 87. Majhail NS, Schiffer CA, Weisdorf DJ. Improvement of pulmonary function with imatinib mesylate in bronchiolitis obliterans following allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2006; 12: 789–91. 88. Or R, Gesundheit B, Resnick I et al. Sparing effect by montelukast treatment for chronic graft versus host disease: a pilot study. Transplantation 2007; 83: 577–81. 89. Lee SJ, Vogelsang G, Gilman A et al. A survey of diagnosis, management, and grading of chronic GVHD. Biol Blood Marrow Transplant 2002; 8: 32–9. 90. Boeckh M, Leisenring W, Riddell SR et al. Late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants: importance of viral load and T-cell immunity. Blood 2003; 101: 407–14. 91. Bush NE, Haberman M, Donaldson G, Sullivan KM. Quality of life of 125 adults surviving 6–18 years after bone marrow transplantation. Soc Sci Med 1995; 40: 479–90.

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88

Helen L. Leather & John R. Wingard

Bacterial Infections

Introduction Susceptibility to infection has posed one of the most formidable challenges in the clinical management of patients undergoing hematopoietic cell transplantation (HCT) from the earliest days of this treatment. A variety of advances in infection control have permitted major strides in the supportive care of transplant recipients, and these have translated into improved outcomes. Increased understanding of the pathogenesis of infectious syndromes, introduction of new antimicrobial agents, adoption of empirical antibiotics during aplasia before engraftment, development of novel strategies to prevent and treat infections, and recognition of the contributory role of infectious pathogens to the morbidity of other transplant complications, especially graft-versus-host disease (GVHD), have all been responsible for improved survival rates. Advances including peripheral blood hematopoietic cells as alternative sources of bone marrow cells have resulted in faster engraftment of both platelets and neutrophils, leading to shorter length of stay, fewer days of fever, and less antibiotic use. The increasing use of reduced-intensity transplants, which are associated with less gastrointestinal and overall chemotherapy-induced toxicity, are also associated with reduced infectious complications. However, shifts in the patterns of opportunistic pathogens, changing antimicrobial susceptibility, differences in host immunodeficiency with implementation of new immunosuppressive regimens for GVHD, introduction of new conditioning regimens for use in the treatment of solid tumors, and the increasing use of alternative donors continue to pose new and different challenges and opportunities for the management of infectious complications.

Biology of bacterial pathogens Bacteria are found in a great variety of environments. They are classified as prokaryotic microorganisms distinguished by absence of a membrane enclosing their DNA and absence of other membrane-bound organelles. More than 2500 species of bacteria are known. Classification consists of the broad grouping of medically significant bacteria based on their staining properties using the Gram stain (positive or negative), morphology (cocci and bacilli) and oxygen tolerance (aerobic or anaerobic). Other properties, including DNA and RNA homology, ribosomal RNA homology, biochemical properties, specialized growth

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

requirements, and antigenic attributes, have also been used for further characterization. In general, bacteria have cell walls made of peptidoglycan, a molecule unique to bacteria and thus a target for many antimicrobial agents. The cell wall provides the basic shape of the organism and confers rigidity. The Gram stain detects differences in the cell wall structure of bacteria. These differences are useful in distinguishing both antibiotic susceptibility and the potential for interacting with host defenses. Gram-positive bacteria have walls that consist of thick layers of peptidoglycan, teichoic acids, teichuronic acids, and other polysaccharides. Inhibition of peptidoglycan synthesis is the mechanism of action of a variety of antibiotics, including penicillins, cephalosporins, and glycopeptides. The cell wall of Gram-negative organisms consists of an outer membrane composed of protein, lipopolysaccharide (LPS), and phospholipid, and a thin inner layer of peptidoglycan. Protein components of the membrane, known as porins, serve as permeability conduits for hydrophilic molecules. The size and number of porins determine permeability to various molecules, including antibiotics. Several properties are important in the ability of a given bacterial species to colonize or invade the human host (Table 88.1). These include adherence to host cells, the capacity to acquire iron from the host, elaboration of toxins, and successful competition with other microbial colonizers for nutrients. Adhesins on the surface of bacteria interact with specific receptors on host tissue targets. Differences in either the adhesin molecules or host cell receptor binding determine tissue tropism. Various structures exist on the surface of medically important bacterial pathogens, which are important virulence factors. A capsule of polysaccharide can afford protection against phagocytosis. Immunologic diversity of such capsules within species is common (the basis for serotyping), and can permit evasion of the normal host defenses since opsonizing antibodies directed against the specific antigens of the capsule are important for phagocytosis. In Gram-negative bacteria, pili are important means by which bacteria adhere to epithelial cells. Gram-positive bacteria may have surface proteins (e.g. the M protein of group A streptococci or staphylococcal protein A) that serve as virulence factors. Bacterial products such as superantigens may provoke host responses that are deleterious to maintenance of host physiology. Exotoxins produced during exponential growth and released by both Gram-positive and Gram-negative bacteria interfere with normal host cell metabolism and contribute to host toxicity. Certain toxins, known as bacteriocins, are elaborated to enhance an organism’s competition with other microorganisms within the normal flora to establish an ecologic niche. The production of certain extracellular enzymes, such as hyaluronidase, neuraminidase, elastase, and collagenase, facilitates overcoming

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Chapter 88 Table 88.1 Bacterial virulence factors

Molecule

Effects on host

Exotoxins Endotoxins Bacteriocins Exoenzymes Proteases Polysaccharide capsules Plasmids, phages, transposons

Interfere with cellular physiology Elicit sepsis syndrome Establish ecologic niche in host flora Disrupt anatomic barriers, facilitate tissue invasion Degrade humoral immune responses and complement Resist humoral immunity and phagocytosis Confer antibiotic resistance, multiple virulence properties, rapid adaptation to environment Allow rapid adaptation to environmental changes Form barrier to exclude antibiotics

Regulatory factors Biofilms

Table 88.2 Targets for various antibiotics

Bacterial targets

Antibiotics

Peptidoglycan synthesis Lipopolysaccharide Penicillin-binding proteins β-Lactamases Ribosomes

Penicillins, cephalosporins, glycopeptides Antibodies to endotoxins or cytokines mediating sepsis syndrome Penicillins, cephalosporins, carbapenems Clavulanic acid, sulbactam, tazobactam Aminoglycosides, macrolides, tetracyclines, chloramphenicol, oxazolidinones, streptogramins Rifamycins Sulfonamides, trimethoprim Fluoroquinolones Daptomycin Oxazolidinones, linezolid; streptogramins, quinupristin/dalfopristin

RNA polymerases Folic acid synthesis DNA gyrase Bacterial membrane Depolarization

anatomic barriers and promotes spread past tissue barriers. Proteins may be produced that facilitate entry into host cells. Disruption of host humoral responses by proteases permits traversing of mucosal barriers and survival in extracellular tissue as well as serum. Avoidance of phagocytosis or killing of microorganisms once ingested is the role of a variety of virulence factors. The polysaccharide capsule of many organisms is designed both to resist humoral immunity and impede phagocytosis. Bacterial LPS (endotoxin) serves as an important barrier against bile salts, lytic enzymes, DNA, and heavy metals. LPS also contains a carbohydrate moiety (the O antigen) that resists complement binding and differs antigenically from strain to strain. The generation of such antigenic diversity is a mechanism that bacteria use to evade host humoral immune responses. LPS induces the sepsis syndrome by causing the release of a variety of proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin 1, interferon-gamma, and various colony-stimulating factors by monocytes and macrophages, and by triggering a variety of host enzymatic mechanisms, including complement, clotting, fibrinolytic, and kinin pathways. A number of gene products on extrachromosomal elements, such as plasmids, phages, insertion elements and transposons, provide bacteria with mechanisms to rapidly adapt to a competitive environment, conferring antibiotic resistance and multiple virulence properties, which can be shared between a wide variety of unrelated microbes. A variety of regulatory proteins provide bacteria with the capacity to respond favorably to rapid shifts in the environment, such as temperature, iron, and pH. These proteins facilitate adherence and invasiveness and modify the expression of other proteins on the cell surface to optimize the organism’s competitive stance. Antigenic variation of these proteins is the mechanism used by bacteria to evade host immune responses. These

characteristics of bacteria offer a range of bacterial targets for antimicrobial agents (Table 88.2). The host microbial flora itself may act as a barrier against opportunistic pathogens. Ordinarily, complex bacterial communities reside on the body’s surfaces and within the gastrointestinal tract without deleterious effects. However, when the interactions are altered by the use of antimicrobial agents or other infection control measures, this natural barrier may be compromised, and overgrowth of pathogens that might otherwise not be capable of establishing a niche can occur. The normal commensal flora prevent the establishment of opportunistic pathogens by competition for the same nutrients or receptors on host cells through the production of toxic products, fatty acids or other metabolites that inhibit growth of potential competitors and stimulation of the host immune system. Some bacteria routinely cause disease in susceptible hosts who have normal defenses, and these certainly can cause morbidity in the HCT recipient. However, more commonly, infectious morbidity in the HCT recipient is caused by bacterial organisms (such as many Gram-negative bacteria, α-streptococci, and Staphylococcus epidermidis) that ordinarily are not pathogens. These organisms, known as opportunistic pathogens, exploit deficits in host defenses that occur as a result of the transplant procedure or nosocomial procedures, or they exploit microbial shifts caused by infection control measures. Whether or not an opportunistic pathogen causes disease is a reflection of an interplay between its own inherent virulence, whether it has access to the host in sufficient numbers, and the state of the host defenses. Thus, effective infection control measures must address issues related to minimizing exposure to potential pathogens, the strategic use of antimicrobial agents to suppress microbial burden, and, where possible, bolstering host defenses.

Bacterial Infections

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Table 88.3 Mechanisms of antibiotic resistance Mechanism

Examples

Genetic basis

Types of bacteria

Antibiotic affected

Inhibition of antibiotic by enzymes

β-Lactamases

Chromosomal, plasmids, transporons

Staphylococci, many Gramnegative bacteria

Penicillins, cephalosporins

Acetyl transferases Phosphotransferases Nucleotidyltransferases Mutations involving porins

Plasmids Plasmids Plasmids Chromosomal

Many Many Many Many

New proteins

Chromosomal, plasmids, transporons Chromosomal, plasmids

Tetracyclines, macrolides, aminoglycosides, lincosamides Vancomycin, other glycopeptides Penicillins, cephalosporins

Plasmids

Many Gram-negative bacteria and staphylococci Gram-positive and Gramnegative bacteria Gram-positive bacteria Gram-positive and some Gram-negative bacteria Many Gram-negative

Aminoglycosides, chloramphenicol Chloramphenicol, aminoglycosides Aminoglycosides β-Lactams, aminoglycosides, carbapenems, nalidixic acid, fluoroquinolones Tetracyclines, macrolides, fluoroquinolones

Chromosomal, plasmids

Gram-negative bacteria

Trimethoprim

Chromosomal, plasmids Chromosomal

Gram-negative bacteria Staphylococcus epidermidis, Pseudomonas aeruginosa

Nalidixic acid, fluoroquinolones Multiple

Reduced membrane permeability Increased efflux of antibiotics Altered targets

Exclusion barrier

Changes in ribosomal binding sites Changes in cell wall Changes in penicillinbinding proteins Changes in dihydropteroate synthetase Changes in dihydrofolate reductase Changes in DNA gyrases Biofilm formation

Chromosomal, plasmids Chromosomal, plasmids

Antibiotic resistance A number of pathogens are problematic by virtue of documented resistance, including Acinetobacter baumannii, extended-spectrum βlactamases (ESBLs), Pseudomonas aeruginosa, vancomycin-resistant Enterococus faecium, vancomycin-resistant Stalphylococcus aureus, and community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The mechanisms through which drug resistance is most commonly conferred are described below. One of the most successful attributes bacteria have to enhance survival is the capacity for genetic variability. This capacity is frequently used by bacterial organisms to foil clinicians’ attempts to control infection by antibiotics. Through point mutations, rearrangement of entire sequences of DNA to different locations within the bacterial genome (e.g. transposons) or the transfer of DNA from one bacterial organism to another (e.g. via plasmids), development of antibiotic resistance can occur in a variety of ways (Table 88.3). A common mechanism is inactivation of antibiotics by enzymes. For example, staphylococci and many Gram-negative bacteria produce a class of enzymes known as β-lactamases that render inactive β-lactam antibiotics, the group of molecules that includes the penicillins, the cephalosporins (e.g. cephalothin, cefuroxime, ceftazidime, cefepime, cefoxitin, etc.), carbapenems (e.g. imipenem and meropenem), and monobactams (e.g. aztreonam). β-Lactamase genes found on transposons or plasmids permit spread of high-level resistance widely to other classes of drugs such as the fluoroquinolones and trimethoprim– sulfamethoxazole (TMP–SMX). Unfortunately, prevalent use of the βlactam class of antibiotics, particularly third-generation cephalosporins, has resulted in selection pressure to give advantage to resistant organisms and further encourage spread. ESBLs are an increasing cause of treatment failure. The ESBLs are most often noted in Klebsiella pneumonia and Escherichia coli species. Immunocompromised patients infected with these organisms should be placed on Gram-negative

Gram-negative Gram-negative Gram-negative Gram-negative

bacteria bacteria bacteria bacteria

Sulfonamides

isolation precautions and treated with carbapenems. Carbapenem-resistant pathogens have been reported, including the Klebsiella pneumoniae carbapenemase, OXA carbapenemase that is most commonly reported in Acinetobacter baumannii isolates, and metallo-β-lactamases that are found in Stenotrophomonas maltophilia, Acinetobacter spp., Pseudomonas aeruginosa, and Flavobacterium species. Carbapenem-resistant pathogens have been most commonly reported in Europe or the eastern part of the United States [1]. Reduced entry of the antibiotic into bacterial organisms by alteration of permeability, thereby restricting drug access to target sites, can be caused by mutations involving different porins. Such changes are potential mechanisms for resistance to nalidixic acid, fluoroquinolones (such as norfloxacin, ciprofloxacin, levofloxacin, and ofloxacin), aminoglycosides, and β-lactams, among others. Alternatively, resistance to some antibiotics may occur by an increase in efflux of antibiotics from the bacterial organism. This can take place by mutations in chromosomal genes or the acquisition of new genes from plasmids that lead to new membrane proteins, which reduce the retention of certain antibiotics, such as tetracyclines in Gram-negative bacteria and macrolides (such as erythromycin, azithromycin, and clarithromycin) in Gram-positive bacteria. Alteration of the target of the antibiotic is another strategy. Multiple examples are notable. Changes in ribosomal binding sites may lead to resistance to tetracyclines, aminoglycosides, or macrolides, while changes in the amino acid residues on the constituents of cell wall building blocks may confer resistance to antibiotics such as vancomycin. Mutations in the genes that encode for penicillin-binding proteins alter susceptibility to β-lactams, such as the penicillins and cephalosporins. A variety of target enzymes (as shown in Table 88.3) that can be changed by mutations can potentially lead to resistance to sulfonamides, trimethoprim, nalidixic acid, and fluoroquinolones, among other antibiotics. The emergence of widespread antibiotic resistance can impede the best efforts of clinicians to control infections by various bacterial patho-

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gens. Clearly, a cornerstone of any infection control program is the wise use of antibiotics. Repeated pleas by the Centers for Disease Control and Prevention (CDC) and other authoritative bodies for clinicians to practice antimicrobial stewardship have regrettably failed to slow the continuing emergence of drug resistance. However, several strategies have been offered [2–4]. Patients colonized or infected with resistant organisms should be isolated [3,4]. Each institution should maintain a program of surveillance for resistance to detect shifts in susceptibility and trends of problem organisms. Selected antimicrobial agents may need to be restricted to reduce the prevalence of specific resistant organisms. Certain antibiotics initiated empirically for suspected organisms (e.g. vancomycin) should be discontinued once it has been determined that they are not needed. Implementation of an antibiotic cycling program where a particular class of antibacterial is withdrawn from use for a defined period of time as a strategy to limit bacterial resistance has also been evaluated. Results with this approach are inconsistent. Other initiatives include limiting susceptibility reporting such that the more powerful antibacterials are not listed, and as such are less likely to be selected for treatment. Such efforts will reduce selection pressures and spread of antibiotic resistance among microbial populations [5].

Compromised host defenses Host defenses are compromised by a variety of factors following HCT. The degree and type of compromise of host defenses change over time [6]. Three periods have been described: (1) early recovery, corresponding to the first several weeks after transplantation, the pre-engraftment phase; (2) mid-recovery, corresponding to the second and third months after transplant, the early post-engraftment phase; and (3) late recovery, corresponding to the interval beyond 3 months. Table 88.4 lists the predominant deficits that are present at various intervals. Early recovery (phase I) The cytoreductive agents used in conventional high-dose conditioning regimens damage rapidly dividing cell populations, particularly bone marrow progenitors and mucosal epithelial cells. For several weeks after

Table 88.4 Host defenses compromised by hematopoietic cell transplantation (HCT) that make patients vulnerable to bacterial infections Early recovery Neutropenia Oral and gastrointestinal mucosal damage due to cytoreductive therapy Skin barrier compromised by CVCs Mid-recovery Skin and gastrointestinal mucosal damage due to GVHD Decreased cellular immunity due to GVHD and immunosuppressive therapy Skin barrier compromised by CVCs Decreased cellular immunity due to viral infections, especially CMV Late recovery Decreased cellular immunity; persistent with chronic GVHD Nonspecific suppressor cells with chronic GVHD Reduced opsonization Decreased reticuloendothelial function IgG subclass deficiencies CMV, cytomegalovirus; CVCs, central venous catheters; GVHD, graft-versus-host disease; IgG, immunoglobulin G.

HCT, pancytopenia and damage to the mucosal barriers are predominant deficits in the host defenses against infectious pathogens. The duration of neutropenia varies according to the number of stem cells used to effect reconstitution, the stem cell source, the occurrence of certain viral infections, especially cytomegalovirus (CMV), the use of immunosuppressive agents after transplantation as part of the GVHD prophylaxis, the use of agents to purge the marrow stem cells ex vivo from contaminating tumor cells, and the use of cytokines to stimulate recovery. In the autologous transplant setting, the number of prior treatment regimens, the prior use of radiotherapy, and the use of certain chemotherapeutic agents that are known to damage hematopoietic progenitors, such as nitrosoureas and melphalan, negatively influence the ability to obtain stem cells, leading to delays in engraftment. In the allogeneic transplant setting, human leukocyte antigen (HLA) disparity between donor and recipient, T-lymphocyte depletion of the graft, the use of umbilical cord as a stem cell source, and inadequate conditioning of the recipient to eliminate host immune cells can impede robust engraftment as well. The duration and the depth of neutropenia independently influence the risk for infection [7]. Although the risk for bacterial infection increases incrementally as the circulating neutrophil count falls below 1000/mm3, the risk becomes substantial below 500/mm3. Bacteremia and life-threatening bacterial infection occur mostly at neutrophil counts below 100/mm3. Indeed, in HCT recipients, 90% of the bacterial infections occur at neutrophil counts below 100/mm3. The degree of mucosal damage varies according to the preparative regimen. Busulfan, etoposide, melphalan, cytarabine, cyclophosphamide, and total body irradiation are associated with varying degrees of mucositis. Although stomatitis is readily observable, damage to the mucosa of the entire gastrointestinal tract occurs. Attempts to reliably quantify the degree of mucosal damage are primitive, and have hampered efforts to correlate the degree of mucosal damage with infection risk along the same lines that the neutrophil count and the risk for infection have been quantified. However, the use of the d-xylose absorption test, a measure of mucosal functional integrity, has been evaluated in leukemic patients. An association between the degree of d-xylose absorption and the risk for infection has been noted [8]. Reactivation of herpes simplex virus (HSV) type 1 (which occurs in approximately 70% of seropositive patients, usually during the first or second week after HCT unless acyclovir prophylaxis is given) can lead to diffuse or localized ulceration of the mucosa of the oral cavity and the lower esophagus. This change has been associated with bacteremia from organisms that reside on the buccal mucosa. Reactivation of HSV type II occurs in seropositive patients and can result in urethral, labial, perineal, and perianal skin and mucosal breakdown. The use of cytotoxic agents after HCT, such as methotrexate or cyclophosphamide to prevent GVHD, can also result in exacerbation of mucositis and delays in healing, as can the corticosteroids. The nearly universal use of indwelling central venous catheters (CVCs) today poses a risk for infection, both early and late after HCT. Such catheters compromise the integrity of the integument as a physical barrier to potential pathogens residing on the skin. Marrow puncture sites and peripheral venous catheters can also compromise the skin barrier. The use of reduced-intensity conditioning (RIC) HCT is increasing, utilizing the immune system rather than chemotherapy to achieve disease control. The RIC regimens that are used limit the development of neutropenia, and mucositis is also significantly diminished. As a consequence, this new type of HCT has demonstrated differences in infection patterns compared with high-dose allogeneic HCT [9–11]. The incidence of early bacteremias (day 0–30) is low, and these account for a low rate of bacteremia during the first 100 days.

Bacterial Infections

Mid-recovery (phase II) Following engraftment, patients enter a period of profound deficiency of cellular and humoral immunity. The degree and duration of immunodeficiency are influenced by the type of HCT, the degree of donor and recipient histocompatibility, whether T lymphocytes have been eliminated from the allogeneic marrow graft, the use of immunologic or pharmacologic purging of autologous marrow, the post-transplant immunosuppressive treatment given as GVHD prophylaxis, the occurrence of certain viral infections (especially CMV), and the occurrence and severity of GVHD. The integrity of mucosal barriers may continue to be compromised by recurrent HSV infections, the use of cytotoxic agents, and intestinal involvement by GVHD. Indwelling venous catheters typically remain in place during this period, adding to the patient’s vulnerability for infection from skin bacterial flora. Late recovery (phase III) With time, there is gradual recovery of both cellular and humoral immunity (see Chapter 17). Generally, immune recovery is more rapid after autologous than allogeneic HCT. Among autograft recipients, those given high-dose preparative regimens (as typically used for acute leukemia) are more immunodeficient, and are more immunodeficient for longer intervals, than those given less intensive, reduced-intensity regimens, such as those used for solid tumor therapy. Immune recovery following peripheral blood autotransplantation may be more rapid than after autologous marrow transplantation. Recipients of CD34-selected grafts have a slower pace of T-cell immune reconsitution [12]. Immune recovery after matched-related HLA-identical transplantation appears to be more rapid than after HLA-disparate or HLA-matched unrelated donor transplantation. The occurrence of GVHD also influences the tempo of immune recovery and is associated with dysregulated immune responses (see Chapter 86). If chronic GVHD occurs, cellular and humoral immunodeficiency may persist for months and even years [13]. Reticuloendothelial function can also be severely impaired, especially in patients with chronic GVHD. Immunoglobulin deficiencies can occur. Even in the face of normal levels of the isotypes, IgG subclass deficiencies, especially of subclass II, can be present, rendering patients vulnerable to severe infection by encapsulated bacteria. By 1 year, immune recovery is nearly complete in recipients of matched-related transplants without GVHD and off immunosuppressive medications. However, in matched unrelated donor and mismatched family-related donor transplants, immune recovery may lag substantially. Even in the absence of overt GVHD, vulnerability for recurrent sinopulmonary infections can persist for much longer periods than for those seen after HCT from HLA-matched siblings [14]. Responses to immunizations may be impaired up to 1 year or longer.

Spectrum of bacterial infections Just as the defects in host immunity vary over time, so does the spectrum of infections. In Fig. 88.1, the various infectious syndromes that occur at different times after HCT are portrayed [6]. As noted previously, relative to viral and fungal infections, bacterial infections predominate during the early recovery phase, whereas viral and fungal infections predominate during the mid- and late recovery phases. However, there are important differences that vary over time, as noted in Tables 88.5 and 88.6. Early recovery (phase I) Among neutropenic patients with fever, approximately 50% will have an infection that can be documented, and in the remainder no cause can be definitively identified. Because signs and symptoms of infection are

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attenuated during neutropenia, and untreated bacterial infections may be rapidly life threatening, neutropenic fevers should operationally be regarded as infectious until proven otherwise. Bacterial pathogens account for more than 90% of the first infections during neutropenia. Gram-negative bacteria are the most virulent bacterial pathogens during neutropenia, and historically have been major causes of morbidity and mortality. Over the past 20 years, the percentage of infections caused by Gram-negative organisms has steadily decreased from 70% to 30%. However, several centers have again started to notice an increase in the proportion of Gram-negative bacterial infections [15]. The most common Gram-negative bacteria have been Escherichia coli, Klebsiella spp., and Pseudomonas aeruginosa. In recent years, several other organisms such as Acinetobacter spp., Stenotrophomonas spp., Alcaligenes xylosoxidans, Serratia spp., Legionella, and Burkholderia cepacia have been increasingly identified [16]. The portal of entry for many organisms is the damaged mucosa of gastrointestinal tract. Perianal fissures or skin breakdown are other potential sources, especially for Pseudomonas aeruginosa. Occasionally, venous catheters can also serve as an entry site for Gram-negative bacteria. Gram-positive bacteria have emerged as major pathogens, and bacteremia rates for Gram-positive organisms now exceed those for Gramnegative bacteria [15,16]. Gram-positive bacteria now account for 70% of bacterial infections, compared with 30% 25 years ago. This increase is in large part attributable to the nearly universal use of indwelling CVCs. The widespread use of antibiotics with Gram-negative coverage to decontaminate the gut and reduce Gram-negative infections has also contributed to the proliferation of Gram-positive infections. Occasionally, Gram-positive organisms may invade the host via the gastrointestinal tract as well. Staphylococcus epidermidis, Staphylococcus aureus, α-hemolytic streptococci and enterococci are the most common organisms [17]. The portal of entry for α-hemolytic streptococci is frequently the oral mucosa. Thus, patients with stomatitis due to chemotherapy or radiotherapy, or with HSV-induced mucosal ulcerations, are particularly at risk. Several large series have reported bacteremia rates of 15–25% in HCT populations [18–21]. Where speciated, Streptococcus mitis, an organism that normally resides on the buccal mucosa, is the most common α-hemolytic Streptococcus. Although Gram-positive bacteria are less virulent than Gram-negative ones, approximately 10% of αhemolytic streptococcal bacteremias are associated with a toxic shocklike syndrome that can be fatal even with prompt antimicrobial therapy [19,20,22]. The use of fluoroquinolones in several series has been associated with an increased risk for streptococcal infection [23,24]. Corynebacterium jeikeium infections are associated with infected marrow needle puncture sites or infected peripheral venous catheters, often with associated thrombophlebitis. Typically, they are resistant to multiple antibiotics and frequently require catheter removal [25]. After institution of antibiotics during first fever, the microbial flora changes. Superinfections, as manifest by recurrent or persistent fever during the second or subsequent week of neutropenia, are more heterogeneous in etiology (Table 88.5). Gram-negative bacteria, especially those that are resistant to the antibiotic regimen used to treat the first febrile episode, are of paramount concern [26]. These bacteria account for only roughly 10% of superinfections, but are highly virulent and have rapid life-threatening potential if appropriate antibiotic modification is not made. The most common etiologic pathogen for blood stream infections is Staphylococcus epidermidis, which accounts for roughly half of superinfections. These organisms are less virulent than Gram-negative bacteria and are quickly isolated from blood cultures of bacteremic patients. Thus, clinicians can wait until multiple blood cultures are positive to ensure that the isolate is a true pathogen rather than a harmless

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Host immune system defect

Devise risk

Phase I, pre-engraftment < 30 days

Phase II, post-engraftment 30–100 days

Neutropenia, mitosis and acute graft-versus-host disease

Impaired cellular immunity and acute chronic graft-versushost disease

Phase III, late phase > 100 days Impaired cellular and humoral immunity and chronic graft-versushost disease

Central line Respiratory and enteric viruses

Allogeneic patients

Herpes simplex virus*†

Cytomegalovirus*† Varicella zoster virus Epstein–Barr virus lymphoproliferative disease*

Facultative Gram-negative bacilli Staphylococcus epidermidis

Encapsulated bacteria (e.g. pneumococcus)

Gastrointestinal tract streptococci species All Candida species Aspergillus species

Aspergillus species Pneumocystis jiroveci Toxoplasma gondii Strongyloides stercoralis 30

0

100

360

Days after transplant High incidence (≥10%)

Low incidence (<10%)

Episodic and endemic

Continuous risk

Fig. 88.1 Phases of opportunistic infections among allogeneic hematopoietic stem cell transplante recipients.

* Without standard prophylaxis † Primarily among persons who are seropositive before transplant

Syndrome

Relative frequency*

Relative life-threatening potential

First fever Staphylocococci Staphylococcus epidermidis Staphylococcus aureus α-Hemolytic streptococci Gram-negative bacilli

+++ + + +

+ +++ ++ +++

Subsequent fevers Antibiotic-resistant Gram-negative bacilli

+

+++

Staphylococci Staphylococcus epidermidis Staphylococcus aureus Fungi

+++ + ++

+ +++ +++

Table 88.5 Bacterial and fungal infectious syndromes encountered early after hematopoietic cell transplantation (HCT), during the preengraftment phase (early recovery or phase I)

* Increasing frequency and life-threatening potential depicted by increased number of “+” signs.

contaminant. Less common is Staphylococcus aureus; however, Staphylococcus aureus is highly virulent, and requires prompt and vigorous antibiotic treatment. Other potential causes of superinfection include fungal pathogens and Clostridium difficile-associated diarrhea (CDAD).

Mid-recovery (phase II) With recovery of the neutrophil count, most bacterial infections resolve and antimicrobial agents can be discontinued. Fever of obscure etiology is occasionally noted in the mid-recovery period (Table 88.7). Sinusitis

Bacterial Infections

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Table 88.6 Bacterial infections encountered after engraftment (mid- and late recovery, or phases II and III) Relative frequency* Infection Mid-recovery (phase II) Staphylococci Fungi Gram-negative bacilli Late recovery (phase III) Encapsulated bacteria Fungi

Allogeneic HCT with no GVHD

Allogeneic HCT with GVHD

Autologous HCT

++ ++ −

++ +++ +

++ + −

− −

++ +

− −

* Increasing frequency depicted by increased number of “+” signs; “−” indicates uncommon infections. GVHD, graft-versus-host disease; HCT, hematopoietic cell transplantation.

Table 88.7 Causes of fevers of obscure origin after engraftment during midand late recovery (phases II and III) Cytomegalovirus Central venous catheter infections Occult sinusitis Hepatosplenic candidiasis Pulmonary or disseminated Aspergillus infection

is a common cause of fever of uncertain etiology during this period [27]. Frequently, it occurs without specific focal signs or symptoms, but can readily be detected by either radiographs or computed tomographic scans of the sinuses [28], and further investigated by nasal endoscopy. Another potential cause of fever is an occult infection of the CVC. Blood cultures are helpful but may be unrevealing initially. Removal of the catheter is occasionally required to exclude this possibility in a patient with persistent obscure fever, and is discussed under treatment of intravascular catheter-related infections. Patients who have undergone allogeneic HCT are more susceptible to infections during the mid-recovery period than are autograft recipients [29], particularly if acute GVHD occurs and more intensive immunosuppression is necessary (Table 88.6). Although viral and fungal infections predominate during this period, occasionally Gram-negative bacteremias can occur as a result of disruption of the gastrointestinal mucosa barrier from acute GVHD. Also, Gram-positive bacteremia frequently occurs related to infections associated with the CVCs [17,30]. Late recovery (phase III) Over time, with gradual immune recovery, the risk for infection progressively declines. Autograft recipients have a very low risk for bacterial infection late after the procedure. If the indwelling CVC remains in place, however, infections associated with the catheter can continue to occur, especially if the catheter continues to be used or if fibrin sheaths develop. In the absence of chronic GVHD, allogeneic transplant recipients have a progressively declining risk for bacterial infection. However, patients with chronic GVHD are highly susceptible to recurrent bacterial infections, especially from encapsulated bacteria, including Streptococcus pneumoniae, Hemophilus influenzae, and Neisseria meningitidis, due to low CD4 lymphocyte counts, poor reticuloendothelial function, and low levels of opsonizing antibodies [13,14,31,32]. The continuing use of immunosuppressive therapy, especially corticosteroids, with their deleterious effects on phagocytosis, renders the patient susceptible for

recurrent infection. As noted earlier, patients who are the recipients of mismatched or matched unrelated donor transplants are, even in the absence of chronic GVHD, vulnerable to recurrent infections [14], especially sinopulmonary infections.

Treatment strategies First fever during neutropenia With the recognition that most febrile episodes during neutropenia are infectious in origin, awareness of their life-threatening potential, and knowledge that most first infections are due to bacteria, evaluation should be prompt and thorough. Special attention should be directed to the oral cavity, skin, lung, catheter sites, and perianal area. Cultures of suspected sites of infection should be obtained, and in all patients at least two sets of blood samples should be submitted for bacterial and fungal cultures. Each set consists of an aerobic and anaerobic blood culture bottle. Patients with respiratory signs or symptoms should have a chest X-ray in addition to physical examination and routine blood cultures. Patients with signs and symptoms of a urinary tract infection or an abnormal urinalysis should have a clean catch urine specimen sent for analysis and culture. Two or three blood cultures should be obtained either simultaneously or separated by 30–60 minutes [33]. According to the Infectious Disease Society of America (IDSA) guidelines for the management of febrile neutropenia, at least one of these cultures should be from a peripheral venous puncture, and the other can be from an intravenous line [34]. Many centers do not draw peripheral blood cultures, preferring to draw two sets of cultures from the indwelling catheter. There appears to be no value in obtaining more than a single set of cultures per 24-hour period for patients with neutropenic fever. Preferably 20–30 mL per culture set for an adult is recommended [35], with proportionately smaller volumes for children: 1–2 mL blood per culture for neonates; 2–3 mL for infants aged 1 month to 2 years; 3–5 mL for older children; and 10–20 mL for adolescents is recommended. Numerous satisfactory manual or instrumental culture systems are commercially available (reviewed in [33]). Most systems perform well, but no one medium or system is capable of detecting all microorganisms. Several systems are suitable for special considerations. There are products to minimize the inhibitory influence of antibiotics for patients who are already receiving antibiotics, and systems that are useful for detecting filamentous fungi or mycobacteria (such as the lysis centrifugation system). Each has its strengths and shortcomings (reviewed in [33]). In general, it is advisable that each transplant team meets periodically with the director of the hospital microbiology laboratory to review the blood

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culture system in place at that institution and the infections encountered in the HCT patients, in order to accommodate specific situations where special media would be advisable. After evaluation, antibacterial agents should be instituted promptly on an empirical basis. This strategy has reduced the morbidity and mortality associated with infection from more than 50% to less than 10% in the last 25 years, and has become universally adopted as the standard of care for management of the initial fever during neutropenia. A variety of antibiotic regimens have been evaluated in controlled clinical trials. It remains debatable as to whether one regimen is superior to another. What is generally agreed upon, however, is that antibiotics should be begun promptly at the first sign of fever, without waiting for isolation of an organism in blood or other cultures, even in the absence of signs or symptoms of infection, since the host is unable to mount an effective inflammatory response. When choosing an antibiotic, a number of factors must be considered, such as the local susceptibility patterns, as well as the susceptibility of organisms to available antibiotics. Other important considerations include toxicity of the individual agents (particularly in individuals with impaired organ function), allergy history (severity of allergy and class of agent that caused the allergy previously), the likelihood of the emergence of drug resistance, the risk for superinfection (both the frequency and the type of superinfection), and cost. A consensus committee of the IDSA first provided guidelines for the management of fever during neutropenia in 1990; these were updated in 1997 and 2002 [34], and are currently being updated again. Although developed primarily for hospitalized patients with neutropenia following conventional chemotherapy, they have been updated to include outpatient management of febrile neutropenia and are suitable for HCT patients as well. These guidelines are evidence based, ranking both the strength and the quality of evidence of each recommendation. The IDSA’s suggested strategies for first fever are indicated in Fig. 88.2 [34]. The first decision concerns where the patient will be managed – inpatient or outpatient – which is dependent upon the risk status of the patient. With the increasing role of RIC regimens followed by HCT and rapid recovery seen with autologous peripheral blood cell transplants,

some HCT patients are managed exclusively as outpatients. Management of neutropenic fever should be no exception provided the patient is stable and low risk. Indications and candidates for the outpatient management of febrile neutropenia will be discussed under patient management of neutropenia. In hospitalized individuals with bacteremia, there are three potential management options. The first strategy is treatment with a single agent, or monotherapy [34]. Successfully studied agents include the thirdgeneration cephalosporin ceftazidime [36–40], the fourth-generation cephalosporin cefepime [38,39,41–46], the antipseudomonal penicillin piperacillin–tazobactam [40,44,47,48], and carbapenems including imipenem [37,41,45,46] and meropenem [49,50]. A number of studies have compared the outcomes of monotherapy with those achieved with combination therapy in the management of neutropenic fever. The majority of studies, including several meta-analyses, have not demonstrated any difference in outcomes between the approaches [51–54]. A recent metaanalysis of monotherapy versus combination therapy demonstrated lower rates of bacterial superinfection and fewer treatment failures among the monotherapy recipients [53]. Disadvantages of monotherapy include limited Gram-positive activity, particularly with ceftazidime, and lack of efficacy against viridans streptococci. Cefepime and the carbapenems have excellent activity against viridans streptococci and pneumococci. None, however, has activity against MRSA, which must be considered in hospitals with high MRSA rates. Other organisms that are poorly covered by monotherapy include vancomycin-resistant enterococci (VRE), penicillin-resistant Streptococcus pneumoniae and coagulase-negative Staphylococcus aureus, ESBL-producing organisms, and carbapenemase-producing Pseudomonas aeruginosa bacteria. In the era of increasing bacterial resistance to antimicrobial agents, particularly VRE, clinicians need to implement strategies to reduce unnecessary antibiotic consumption. Cefepime in particular has reduced the need for the addition of vancomycin to the empirical regimen when compared with ceftazidime [55]. In a recent study, vancomycin was added to the empirical regimen in 51% and 80% of cases, respectively [56]. When appropriateness of vancomycin use was evaluated against

Fever (temperature ≥38.3°C) + neutropenia (<500 neutrophils/mm3)

Low risk

Oral

High risk

IV

Vancomycin not needed

Two drugs

Monotherapy Ciprofloxacin + Amoxicillin–clavulanate (adults only)

• Cefepime, • Ceftazidime, or • Carbapenem

Vancomycin needed

• • • •

Aminoglycoside + Antipseudomonal penicillin, Cefepime, Ceftazidime, or Carbapenem

Reassess after 3–5 days

Vancomycin + Vancomycin + Cefepime, ceftazidime, or carbapenem ± aminoglycoside

Fig. 88.2 Algorithm for initial management of febrile neutropenic patients. IV, intravenous. (Reproduced from [34], with permission from the University of Chicago Press.)

Bacterial Infections

IDSA guidelines, patients receiving cefepime had less inappropriate use compared with ceftazidime patients (28% versus 58%; p < 0.001) [56]. One of the main concerns of monotherapy is the emergence of antimicrobial resistance. ESBLs have been shown to reduce the utility of ceftazidime monotherapy [57,58]. Fourth-generation cephalosporins have not been subject to the same problem, and in fact ceftazidimeresistant strains of Enterobacter cloacae remain susceptible to cefepime [58]. Institutions that have adopted an “antibiotic cycling” program, or have changed their preference of cephalosporin from ceftazidime to cefepime, have seen a reversal in the resistance patterns to ceftazidime of inducible Enterobacteriaceae [59]. Imipenem and meropenem have also been extensively evaluated in the management of febrile neutropenia [37,41,45,46,49,50]. In trials comparing carbapenems with third- and fourth-generation cephalosporins, there were no appreciable differences in time to recrudescence of fever, days of therapy or breakthrough infections. One potential concern with the routine use of carbapenems is the lack of coverage against Stenotrophomonas maltophilia, an organism that is increasingly isolated in HCT patients. Depending upon individual institutions’ susceptibility patterns, carbapenems are a suitable choice for monotherapy. Piperacillin–tazobactam has also been evaluated in the empiric treatment of neutropenic fever. Several studies have demonstrated efficacy compared with other broad-spectrum antibacterials such as cefepime [40,44,47,48]. The most recent large randomized trial demonstrated noninferiority compared with cefepime among patients with hematologic malignancies or undergoing autologous HCT. While not in the current version of the IDSA guidelines for the management of neutropenic fever, it is anticipated that, based on the body of data, it will be included in the next update. Among patients with documented penicillin or cephalosporin allergies, suitable alternatives include aztreonam or a carbapenem. The other class of antibiotics that has been evaluated less extensively includes fluoroquinolones such as ciprofloxacin [60–62] and levofloxacin. Ciprofloxacin has been studied, with varying results. One potential concern is development of resistance, based on widespread prophylactic use as part of “gut decontamination.” If a patient develops a fever while on an oral fluoroquinolone, changing to an intravenous fluoroquinolone is a poor choice since the cause of fever may be an organism not susceptible to fluoroquinolones. A second option for high-risk febrile neutropenia patients is a combination of two agents, such as an aminoglycoside in combination with an antipseudomonal penicillin, cefepime, ceftazidime, fluoroquinolone or a carbapenem. In general, the evidence suggests that two-drug combinations have similar response rates to monotherapy regimens. An advantage of combination therapy is a broad coverage of pathogens, including organisms that might be resistant to individual agents. A second advantage is potential synergy against Gram-negative bacteria and coverage against anaerobes. Combination regimens are attractive for patients at high risk for Pseudomonas aeruginosa infections, although the role of combination therapy for the prevention and treatment of such infections has recently been challenged [63–65]. Disadvantages include nephrotoxicity, ototoxicity, hypokalemia, the need for monitoring of aminoglycoside levels to assure therapeutic levels and to minimize toxicity, and a lack of coverage against most Gram-positive bacteria. A regimen of two β-lactams, such as a cephalosporin plus an ureidopenicillin, has been evaluated at some centers and appears effective. Such combinations offer broad-spectrum activity with little toxicity and good anaerobic coverage, but there is little coverage against Gram-positive organisms, it is more costly and antagonism is possible in certain bacterial infections. The final option is to combine an agent with Gram-positive activity, such as a glycopeptide, for example vancomycin, or other novel agents with activity against Gram-positive bacteria, for example linezolid with

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either option one or two above [66–69]. These combinations offer the advantage of excellent coverage against Gram-positive bacteria in addition to the advantages and disadvantages noted in the first option. Opinion remains divided as to the need for empiric vancomycin because many Gram-positive infections are easily controlled once they are identified, and because of concerns as to the emergence of resistance if the agent is overused [70]. Certainly, the emergence of VRE in many centers has led to calls for restricted use of this drug from the CDC as well as multiple infectious disease committees [71]. VRE infections will be discussed below. The role of glycopeptides as part of the empiric antibacterial strategy has recently been evaluated in three separate metaanalyses [67,68,72]. In these evaluations, the addition of a glycopeptide to the initial antibacterial regimen for febrile neutropenia did not result in differences in treatment failure or all-cause mortality, but was associated with an increase in adverse events. Inclusion of vancomycin in first-line therapy could be considered in seriously ill patients with suspected staphylococcal infections or with infected catheters, or in centers where fulminant Gram-positive bacterial infections are common. If cultures prove negative, its use should be curtailed. The role of linezolid as part of the initial treatment for neutropenic fever is unclear. Linezolid has demonstrated comparable efficacy to vancomycin when added to the initial empiric treatment of patients with febrile neutropenia with proven or suspected infection due to a Grampositive pathogen [69]. Broad use of agents that are traditionally restricted to severe infections such as VRE is not wise, particularly as the development pipeline for new antibacterials is so limited. Further, linezolid is known to cause bone marrow suppression, a toxicity that is best avoided where possible in HCT recipients. Subsequent fever during neutropenia The response to the initial antibiotic regimen dictates subsequent management decisions. If the patient defervesces, the initial regimen can be continued without modification until resolution of neutropenia (absolute neutrophil count [ANC] ≥500/mm3 for 2 consecutive days), regardless of whether an infection was documented. Occasionally, if the patient has defervesced and no infection has been documented, antibiotics can be stopped before resolution of neutropenia. However, this step should be undertaken only with caution, preferably in patients who have been afebrile for 5–7 days. The patient should be monitored closely and therapy resumed if fever should recur (Fig. 88.3) [34]. Generally, it is advisable to avoid stopping antibiotics if ongoing active oral or gastrointestinal mucositis is present. In the past, it was customary to operationally define neutropenic resolution at 500/mm3. However, studies in children undergoing cancer chemotherapy have suggested that stopping antibiotics at lower levels, such as 250/mm3, is also satisfactory as long as the counts are rising. If fever persists, patients require continued careful monitoring. If no infection has been documented and no signs of progressive infection are seen, the initial regimen can be continued without modification. If an infection has been documented, therapy targeted against the pathogens should be instituted. If signs of progressive infection occur, modification of the initial regimen should be instituted to cover suspected pathogens that were not covered by the initial regimen (Fig. 88.4) [34]. If fever persists or recurs 5 or more days after initiation of antibiotics and the patient is likely to remain profoundly neutropenic, the patient is at high risk for fungal infection. Consideration should be made to initiation of antifungal therapy, as discussed in Chapter 89, and the patient should be evaluated clinically and radiologically. Other causes of persistent or recurrent fever include Clostridium difficile, another bacterial infection or a noninfectious source. Provided the patient is

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Chapter 88

Duration of antibiotic therapy

Afebrile by days 3–5

ANC ≥500 cells/mm3 for 2 consecutive days

Stop antibiotics 48 h after afebrile + ANC ≥500 cells/mm3

Persistent fever

ANC <500 cells/mm3 by day 7

Initial low risk Clinically well

Stop when afebrile for 5–7 days

Initial high risk • ANC <100 cells/mm3 • Mucositis • Unstable signs Continue antibiotics

ANC ≥500 cells/mm3

ANC <500 cells/mm3

Stop 4–5 days after ANC >500 cells/mm3

Continue for 2 weeks

Reassess

Reassess Stop if no disease and condition is stable

Fig. 88.3 Suggested scheme for estimating the duration of antibiotic administration under various conditions. See Table 88.8 for rating system for patients at low risk. ANC, absolute neutrophil count. (Reproduced from [34], with permission from the University of Chicago Press.)

Persistent fever during first 3–5 days of treatment: no etiology Reassess patient on days 3–5

Continue initial antibiotics

If no change in patient’s condition (consider stopping vancomycin)

Change antibiotics

• If progressive disease • If criteria for vancomycin are met

Antifungal drug, with or without antibiotic change If febrile through days 5–7 and resolution of neutropenia is not imminent

Fig. 88.4 Guide to treatment of patients who have persistent fever after 3–5 days of treatment and for whom the cause of fever is not found. (Reproduced from [34], with permission from the University of Chicago Press.)

hemodynamically stable, continued fever alone should not prompt significant antibacterial modifications in the absence of positive culture data. Fever following resolution of neutropenia In the HCT population, patients occasionally remain febrile following neutrophil recovery. In this febrile, non-neutropenic population, where reassessment for undiagnosed infection is negative, antibiotics can be stopped after 4–5 days of an ANC over 500/mm3 (Fig. 88.3) [34], while evaluation to determine the cause of the fever is pursued. Patient management of neutropenia In recent years, a variety of considerations have led to the development of outpatient antibiotic regimens including the development of programmable infusion pumps, the widespread availability of home-care services, the desire for patients to receive more of their care in the outpatient

setting, the introduction of highly effective and nontoxic antibiotic regimens, and an emphasis on cost containment. A variety of studies have identified factors that distinguish patients who are at low risk for serious morbidity from those who are at risk for septic shock and other complications of neutropenic fever [73–76]. Factors such as the presence of comorbid diseases, the development of fever as an inpatient, underlying hematologic malignancy, prolonged neutropenia (>10 days), and receipt of allogeneic HCT identify those at high risk. Low-risk patients include those with underlying solid tumors receiving conventional chemotherapy, those with a short anticipated duration of neutropenia (<7 days), and those with no comorbid conditions. Following the stratification of the different clinical risk groups, further research has been centered on developing an internationally validated scoring system to identify low-risk patients at the onset of a febrile illness [75]. In a multivariate model, several characteristics have been identified that are considered independent predictive factors of favorable outcome. Integer weights were applied to these characteristics to develop a risk-index score, which was subsequently validated (Table 88.8) [75]. A risk-index score of 21 or more identified low-risk patients with a positive predictive value of 91%, a specificity of 68%, and a sensitivity of 71% [75]. This scoring system demonstrated lower misclassification rates and better sensitivity compared with earlier models. One of the limitations of the model is the applicability to the HCT setting. A small percentage of the patients studied were HCT recipients, but this model requires further validation in a larger cohort of transplant recipients. If applicable to this population, it could be possible to select low-risk patients for early intravenous-to-oral switch programs while remaining neutropenic. Outpatient antibiotic therapy or early discharge of patients at low risk for serious sequelae has been evaluated in non-HCT patient groups [73,76,77]. A meta-analysis of randomized trials evaluating the efficacy of oral versus intravenous antibacterials for the treatment of febrile neutropenia in low-risk cancer patients has demonstrated the safety and efficacy of oral antibiotics in this setting. Neutropenic patients that are low risk for mortality can safely receive oral antibiotics in lieu of hospitalization and intravenous administration [78]. Careful selection of suitable low-risk patients is imperative. Outpatient antibiotic regimens have also been evaluated in autologous HCT patients (neither of these

Bacterial Infections Table 88.8 Scoring index for identification of low-risk febrile neutropenic patients at time of presentation with fever Characteristic

Score

Extent of illness* No symptoms Mild symptoms Moderate symptoms No hypertension No chronic obstructive pulmonary disease Solid tumor or no fungal infection No dehydration Outpatient at onset of fever Age <60 years†

5 5 3 5 4 4 3 3 2

Note: highest theoretical score is 26. A risk index score of ≥21 indicates that the patient is likely to be at low risk for complications and morbidity. The scoring system is derived from the Multinational Association of Supportive Care in Cancer [75]. * Choose one item only. † Does not apply to ≤16 years of age. Reproduced with permission from Klastersky et al. [75], courtesy of the University of Chicago Press.

is truly treatment, rather prophylaxis) and appear to be quite promising for some HCT patients at low risk for serious sequelae. Stable febrile and neutropenic patients who are undergoing RIC regimens are also candidates for outpatient therapy. Vancomycin resistant enterococci Enterococcal infections are the third most common cause of hospitalacquired bacteremia in hospitalized patients [79]. Enterococcus faecalis and E. faecium are the most frequently identified species, with E. faecium being the most common VRE infection, and a smaller number being caused by E. faecalis and E. avium and other species. Of great concern is the development of resistance of Enterococcus to multiple antimicrobial agents. Enterococci are intrinsically resistant to certain β-lactams (particularly cephalosporins and penicillinase-resistant penicillin), lowconcentration aminoglycosides, clindamycin, fluoroquinolones, and TMP–SMX. Acquired resistance to high concentration of β-lactams and aminoglycosides, glycopeptides (vancomycin, teicoplanin), tetracycline, erythromycin, fluoroquinolones, rifampin, chloramphenicol, fusidic acid, and nitrofurantoin is also well documented [80]. There are six vancomycin-resistant enterococcal phenotypes, namely VanA, VanB, VanC, VanD, VanE, and Van G [80,81], of which VanA and VanB are the most common and clinically relevant. VanA resistance, characterized by acquired inducible resistance to high concentrations of vancomycin (minimum inhibitory concentration [MIC] 64–1000 μg/mL) and teicoplanin (MIC 16–512 μg/mL), can be induced by the presence of either drug. VanA resistance is mediated by transposon Tn1546. Of concern, high-level VanA vancomycin resistance has been transferred from E. faecalis to Streptococcus aureus via the Tn1546 transposon [82]. VanB-type resistance is characterized by acquired inducible resistance to low-to-high concentrations of vancomycin (MIC from 4 to over 1064 μg/mL), but sensitive to teicoplanin (MIC 0.25– 2 μg/mL). Both VanA and VanB VRE phenotypes are most common amongst E. faecium and E. faecalis, and less common among other Enterococcus spp. such as E. gallinarum and E. casseliflavus, which are typically associated with the VanC phenotype [80–82]. Colonization and infection with VRE is a concern in high-risk neutropenic patients [83–86]. Colonization rates in patients undergoing

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HCT have been reported to be between 5.5% and 40% [83,84]. Approximately one-third of these patients develop VRE bacteremia, which is associated with high mortality rates [87,88]. Several risk factors have been identified that are associated with colonization or infection with VRE [80,81,89,90]. Antibiotic exposure, particularly to vancomycin, cephalosporins, and antianaerobic agents such as metronidazole and imipenem, are reported to increase risk [81,91]. Other variables that increase risk are a longer duration of hospitalization, greater severity of underlying illness, hemodialysis, receipt of corticosteroids, parenteral nutrition, neutropenia, mucositis, presence of feeding tubes, and a close proximity to colonized or infected patients. Enterococci are resilient organisms and are able to be easily transmitted via health-care workers and contaminated medical equipment, as well as inadequately cleaned rooms following occupation with a VRE-positive patient. It is essential that standard infection control measures, as suggested by the CDC guidelines for preventing opportunistic infections in HCT recipients [6], be strictly adhered to [92]. These measures include hand washing with antibacterial soap before and after entering HCT recipients’ rooms, and disinfecting patients’ rooms and equipment, including structural surfaces (e.g. walls, floors, and bed frames) with an approved Food and Drug Administration or Environmental Protection Authorityregistered disinfectant [6,92,93]. Patients must also be placed under contact precautions until all antibiotics are discontinued and three separate repeat cultures, each 1 week apart, are negative. More recently, the Healthcare Infection Control Practices Advisory Committee has published recommendations for the management of multidrug-resistant organisms in health-care settings, which includes VRE [94]. These guidelines are strict and recommend development and implementation of protocols to obtain active surveillance cultures from high-risk patients, including HCT candidates. Implementation of contact precautions from the time the surveillance cultures are sent pending the results is also recommended. Implementation of surveillance programs for MRSA and VRE appears necessary based on increasing infection rates across a number of institutions. Whether these guidelines can be easily implemented remains to be seen, with likely significant increases in resource consumption [93,94]. Initial treatment of VRE-infected patients includes draining any collections if present, which is less likely in severely neutropenic patients with an absence of neutrophils. All indwelling vascular access devices should be removed immediately. There are a number of drugs that have been used in the management of VRE infection, with traditional approaches including ampicillin, gentamicin, chloramphenicol [95], tetracycline, and nitrofurantoin [80]. Newer agents include quinupristin–dalfopristin [96,97], a combination of streptogrammins; linezolid, an oxazolidinone [98,99]; and daptomycin [100,101], a cyclic lipopeptide, all of which have shown good response rates in VRE bacteremias, although reports of resistance to these agents are unfortunately on the rise. Choice of agent should be guided by species of Enterococcus, particularly as quinupristin–dalfopristin has limited activity against E. faecalis. An alternative approach currently under investigation is the routine screening of patients undergoing HCT for VRE stool colonization to identify those that are at high risk for VRE blood stream infections post transplant. Implementation of empiric antibiotics with activity against VRE at the time of first fever in patients colonized with VRE will provide broad coverage against VRE and may reduce mortality associated with documented VRE infections. This approach is currently under investigation and should not be implemented widely [84]. Clostridium difficile-associated diarrhea Clostridium difficile, a Gram-positive spore-forming, anaerobic bacillus, of endogenous or exogenous origin, can establish itself in the colon and

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proliferate. Clostridium difficile infection results in a wide range of clinical conditions ranging from asymptomatic colonization to colitis to toxic megacolon. Approximately 1–5% of patients with C. difficile have severe disease resulting in colectomy, intensive care admission, or death. Clostridium difficile usually produces two toxins, namely toxin A and toxin B. Patients are classified as C. difficile positive when either toxin A or B is detected in the stool. There are several methods of detecting C. difficile, of which the toxin-specific enzyme-linked immunoassay (ELISA) method is the most common. The ELISA test can detect both toxin A and B, or only toxin A, depending on the reagent used. Reagents that detect both toxin A and toxin B are preferred as a small number of cases (1–2%) of C. difficile involve strains of C. difficile that only produce toxin B. The enzyme immunoassay is capable of detecting 100–1000 pg of toxin, which results in a relatively high false-negative rate [102]. As a consequence, between 10% and 20% of patients need greater than one stool sample to detect toxin. Good clinical practice involves sending multiple stool specimens (up to three), to increase the diagnostic yield by 5–10% [102,103]. The ELISA test is sensitive (71– 94%) and has a high specificity (92–98%) [102] and also has the advantage of a quick turnaround for results. A number of other tests have been used, but many of these tests are less sensitive and take considerably longer to generate a result, delaying implementation of treatment. Recently, a hypervirulent epidemic strain of C. difficile has been associated with a number of CDAD outbreaks in Canada, the United States, the United Kingdom, and Europe. This strain was associated with an increase in incidence of CDAD as well as severity of the disease. The hypervirulent strain was identified as the North American Pulsed Field type 1 (NAP1), restriction enzyme analysis type “B1” and polymerase chain reaction isotype 027 [104–107]. NAP1/B1/027 produces a binary toxin that is associated with more severe CDAD cases. The NAP1/ B1/027 strain has an altered repressor gene, tcdC, which regulates toxin A and B production, resulting in 16–23-fold more toxin A and B than wild-type strains [104]. Presence of the NAP1/B1/027 strain is associated with a more severe disease course [107,108], increased relapse rates following conventional treatment [109,110], disease in uncommon populations [111,112], and a higher attributable mortality [107]. Most laboratories do not have capabilities to determine whether the C. difficile is the NAP1/B1/027 strain. Specimens can be sent to the CDC for testing. Clostridium difficile has emerged as a prominent cause of hospitalacquired infection. Clostridium difficile is thought to be the cause of 15–25% of all cases of antibiotic-associated diarrhea, and a higher percentage of colitis associated with antibiotic treatment [102,103]. Among HCT recipients, CDAD is reported to occur in 5–15% of patients with diarrhea [113–116]. The most widely recognized risk factor for the development of CDAD is antimicrobial therapy, an increased duration of therapy, the use of broad-spectrum antibacterials, and the use of multiple agents. Almost all antimicrobial agents have been associated with the development of CDAD, including clindamycin, penicillins, and cephalosporins. The fluoroquinolones emerged as a predominant risk factor for CDAD during the recent outbreak of NAP1/B1/027 CDAD in Canada [108]. Where possible, treatment of CDAD involves discontinuation or streamlining of antibiotic therapy. Among HCT recipients, this is often not feasible due to underlying infection. First-line treatment options for CDAD include metronidazole and vancomycin. Two older comparative trials demonstrated similar response rates with these agents [117,118]. A more recent randomized trial demonstrated equivalent response rates in mild CDAD. In severe cases of CDAD, vancomycin was associated with an improved response rate [119]. Failure rates associated with metronidazole therapy have increased from approximately 7% in the

1990s to 16–38% in more recent series [110,120]. No specific cases of metronidazole resistance have been documented in North America. With most cases of CDAD being mild in nature, metronidazole 250 mg orally four times a day, or 500 mg orally three times a day, remains the preferred treatment. It is significantly cheaper and is not associated with the VRE-resistance issues that plague vancomycin therapy. If alternative treatment is necessary due to intolerance or failure to respond to initial therapy, vancomycin 125 mg orally four times daily should be initiated. Treatment failure in immunocompetent patients is often defined as failure to stop diarrhea after 4–5 days of therapy. This definition may not be applicable in immunocompromised patients, since neutropenic patients require a longer time to respond [121]. If clinical symptoms progress, alternative treatment options should be considered. Vancomycin must be administered orally, not intravenously. Oral therapy results in high concentrations in the stool, whereas intravenous therapy does not achieve therapeutic concentrations in the stool. If intravenous therapy is indicated due to mucositis, metronidazole can be administered intravenously, as bactericidal concentrations are achieved in the colon when administered via this route [102]. Other new treatment options that have been investigated for CDAD include tolevamer [122], rifaximin [123], and nitazoxanide [124,125]. Nitazoxanide has been compared to metronidazole for the treatment of CDAD, resulting in similar time to resolution of symptoms of colitis, similar complete response rate at the end of 7 days of treatment, and similar sustained clinical responses 31 days after starting treatment [124]. Tolevamer at two different dosage levels has been compared to oral vancomycin, resulting in similar time to resolution of diarrhea [122]. Patients with CDAD should be treated with appropriate therapy for 10–14 days. Relapses are reported to occur following successful treatment in up to 25% of cases [103,110], although relapse rates among neutropenic patients are reported to be lower, i.e. 3.3% [121]. Relapses can be treated with another 10–14 days of therapy. Relapses are primarily due to reinfection with a new strain and are not associated with bacterial resistance [102]; therefore, the drug used for the initial infection can be used for the relapse. In patients who have multiple relapses, addition of cholestyramine, a nonabsorbable anion exchange resin, which acts to bind the toxin, can be considered. Other treatment options that have been investigated include a slow tapering schedule of oral vancomycin over a period of 6 weeks or, in appropriate candidates, intracolonic vancomycin. The use of probiotic therapy to restore normal colonic fecal flora has also been investigated. Administration of Lactobacillus or Saccharomyces boulardii is reported to protect against C. difficile-induced colitis. These approaches should be avoided in neutropenic patients due to risk of active infection with the probiotic organism as opposed to colonic colonization. Finally intravenous immunoglobulin (IVIg) therapy has been used with some success. The basis of decreasing transmission of C. difficile is good infection control. This is discussed later under preventive strategies.

MRSA and CA-MRSA Infections caused by MRSA are increasing, not only nosocomially acquired infections, but also CA-MRSA. The MRSA strains found in the community are genetically different from strains typically found in health-care facilities. The types of infection caused by the two different types of MRSA are also different. CA-MRSA typically causes skin and soft tissue infections and less frequently pneumonia, whereas healthcare-associated MRSA is more commonly associated with respiratory tract and urinary tract infections. There are also differences in antibacterial susceptibilities between health-care-associated MRSA and

Bacterial Infections

CA-MRSA. Health-care-associated MRSA is not only resistant to oxacillin, penicillins, and other β-lactams, but is usually also resistant to other classes of drug, including the fluoroquinolones, macrolides, and clindamycin. CA-MRSA, however, maintains susceptibility to fluoroquinolones, macrolides, clindamycin, tetracyclines, and TMP–SMX [126]. These issues need to be considered in HCT patients who move routinely between the inpatient and outpatient units. Historically, CAMRSA has not been a concern, although it now should be part of the differential when patients are readmitted. Vancomycin has been the reliable cornerstone of MRSA treatment for the past 20 years. Over the past few years, a number of vancomycinintermediate Staphylococcus aureus infections and vancomycin-resistant Staphylococcus aureus infections have been reported. Likewise, vancomycin failures in the absence of vancomycin-intermediate Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus are increasingly reported. The Clinical and Laboratory Standards Institute has recently revised the vancomycin MIC interpretative criteria for Staphylococcus aureus in light of reports of decreasing effectiveness [127]. Pathogens are considered sensitive according to the new standard if the MIC breakpoint is 2 μg/mL or less, intermediate if the MIC breakpoint is 4–8 μg /mL, and resistant if the MIC breakpoint is 8 μg/mL or greater. Lowering of the breakpoints will enable increased detection of resistant isolates. Based on Surveillance Network data for 2005, approximately 0.2% of Staphylococcus aureus isolates and 0.8% of coagulasenegative Staphylococcus aureus isolates have a vancomycin MIC of 4 μg/mL or greater [127]. Despite the low percentage of isolates with a higher MIC, it may be necessary to aim for higher vancomycin trough concentrations in documented MRSA infections. Historically, a vancomycin trough of 5–10 mg/L has been sufficient for most MRSA infections. With the potential for “MIC creep,” it would be prudent in cases of documented MRSA bacteremia to target a trough vancomycin concentration of 10–15 mg/L in certain infections. Similarly, in cases where the MIC is known and is greater than 2 μg/mL, even higher trough concentrations of vancomycin of 15–20 mg/L have been suggested as being necessary [128]. The changing spectrum of MRSA infections is concerning. CAMRSA infections are being reported in health-care facilities, and strains of MRSA typically associated with health-care facilities are being reported in the community. Acute awareness of the change in infection patterns is necessary when selecting empiric treatment for HCT candidates. Likewise, the reduced efficacy of “workhorse” antibacterials must be considered among patients failing to clear infections.

Neutropenic enterocolitis/typhilitis Neutropenic enterocolitis (also known as typhilitis) is a syndrome characterized by neutropenic fever (ANC < 100/mm3), abdominal pain (especially right lower quadrant pain), diarrhea, and radiographically observed bowel wall thickening [129,130]. Characteristic radiologic findings include one or more of the following: thickened bowel wall (greater than 4 mm) with a predilection for the cecum, fluid-filled dilated bowel loops, right lower quadrant inflammatory mass, pericecal fluid or inflammatory changes in the pericecal soft tissues. Rebound tenderness may or may not be present. Neutropenic enterocolitis occurs in approximately 6% of adult patients receiving treatment for acute leukemia [129]. Pathologically, the structural integrity of the bowel wall is compromised, with microbial invasion, bowel wall necrosis, and edema; hemorrhage and perforation sometimes occur. Neutropenic enterocolitis has a high mortality rate. Recognition of clinical signs and symptoms, prompt evaluation, and institution of broad-spectrum antibacterial treatment (to cover Gram-negative and anaerobic bacteria) is imperative.

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Treatment of late bacterial infections Recurrent sinopulmonary infections are frequently associated with chronic GVHD or immunoglobulin deficiency [13,14,31]. They can be treated successfully by a variety of antibiotics, including penicillin, TMP–SMX, cefaclor, cefuroxime, and the new-generation macrolide antibiotics. If imunoglobulin deficiency is present (i.e. IgG < 400 mg/ dL), IVIg can be helpful [6], but it is very costly. An alternative strategy that can be considered is chronic antibiotic prophylaxis, but emergence of antibiotic-resistance may limit the utility of such an approach. Treatment of intravascular catheter-related infections Intravascular catheters are used almost universally in HCT recipients. Tunneled CVCs such as Hickman, Broviac, and Groshong catheters, as well as implantable devices such as the PortACath, are most commonly used. Peripherally inserted central catheter lines are also gaining greater use. The majority of infections are caused by Gram-positive organisms, particularly coagulase-negative Staphylococcus, and, less commonly, Staphylococcus aureus, although Gram-negative infections with Pseudomonas spp., Acinetobacter baumannii, and Stenotrophomonas spp. are increasingly documented. The IDSA has, as previously mentioned, developed guidelines for the management of intravascular catheter-related infections [30], and these are useful in therapeutic decision-making processes. For those patients with a suspected CVC-related infection, the guidelines recommend that two sets of blood cultures should be drawn, with one set drawn percutaneously. This approach is most helpful if the blood sample drawn through the CVC is negative, as it excludes CVC-related infection. Once results are available identifying the organism, choice of antibiotic therapy must be based on susceptibilities. Guidelines for managing tunneled CVC infections are outlined in Fig. 88.5 [34] and specifically address when to remove the catheter and the duration of antibiotic therapy. Patients should be educated on how to best access their lines if they are performing catheter care at home, to minimize the risk of catheter-associated infection. See Chapter 83 for further discussion of this topic.

Preventive strategies General measures The foremost principle of infection prophylaxis is minimization of the possibility that encounters with the health-care team and exposure to the hospital environment place patients at greater risk for acquiring infection. Accordingly, clinical procedures, especially the placement of venous and urinary catheters, should be avoided when possible. General consensus guidelines for prevention of infection in HCT patients have been published [6]. Hand washing by care providers is of utmost importance to avoid transmission of infectious agents from one patient to another or from staff to patients. Opinion is divided as to the need for decontaminating regimens to reduce the endogenous flora and the use of pathogen-free diets. However, abstinence from fresh fruits and vegetables is frequently advised. Particular attention has been placed on the need for minimizing exposure to airborne organisms. Opinion again is divided as to the value of various isolation strategies. The consensus guidelines for the prevention of opportunistic infections in HCT recipients have recommended that all allogeneic HCT patients be placed in rooms with greater than 12 air exchanges per hour and point-of-use high-efficiency particulate air (HEPA) filters capable of removing particles 0.3 mm or more in diameter be used [6]. The relative merits of laminar air flow versus HEPAfiltered rooms have not been established.

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Tunneled central venous catheter (CVC)—or implantable device (ID)—related bacteremia

Uncomplicated

Complicated

Tunnel infection, port abscess

• Remove CVC/ID and treat with antibiotics for 10–14 days

Septic thrombosis, endocarditis, osteomyelitis • Remove CVC/ID and treat with antibiotics for 4–6 weeks; 6–8 weeks for osteomyelitis

Coagulase negative staphylococcus

• May retain CVC/ID and take systemic antibiotic for 7 days plus antibiotic lock therapy for 10–14 days • Remove CVC/ID if there is clinical deterioration, persisting or relapsing bacteremia

S. aureus

• Remove CVC/ID and use systemic antibiotic for 14 days if TEE (−) • For CVC/ID salvage therapy if TEE (−), use systemic and antibiotic lock therapy for 14 days • Remove CVC/ID if there is clinical deterioration, persisting or relapsing bacteremia

Gram-negative bacilli

• Remove CVC/ID and treat for 10–14 days • For CVC /ID salvage, use systemic and antibiotic lock therapy for 14 days • If no response, remove CVC/ID and treat with antibiotic therapy for 10–14 days

Candida Spp.

• Remove CVC/ID and treat with antifungal therapy for 14 days after last positive blood culture

Fig. 88.5 Approach to the management of a patient with a tunneled central venous catheter or a surgically implanted device-related blood stream infection. TEE, transesophageal echcardiogram. (Reproduced from [30], with permission from the University of Chicago Press.)

Knowledge that most infectious pathogens originate from the endogenous flora, as well as the observations that in many instances these organisms are acquired after entry to the hospital environment, have led to investigation of the utility of bacterial and fungal surveillance cultures. Bacterial surveillance cultures have been found to be useful in detecting antibiotic-resistant bacteria [131]. Such surveillance culture programs have been useful primarily in the setting of prolonged neutropenia for detecting causes of superinfection. The disadvantages of routine use of surveillance cultures are its high cost, the enormous workload for the microbiology laboratories, and frequent low predictive value [132]. Accordingly, routine surveillance of individual patients has not been used widely. Nevertheless, there is consensus that ongoing hospital-wide and unit-specific infection surveillance programs need to be in place to detect shifts in infectious pathogens as well as patterns of antibiotic resistance [131]. Clostridium difficile is a major cause of diarrhea in HCT patients. Contaminated environmental surfaces and the hands of health-care personnel are major sources of organism transmission to susceptible patients. Minimization of antibiotic exposure and attention to infection control measures to reduce person-to-person contact are of paramount importance in preventing outbreaks [132,133]. Most institutions utilize alcohol-based hand sanitizers for health-care workers. Alcohol-based solutions do not effectively kill Clostridium difficile spores; therefore, personnel should use soap and water to wash their hands in units where there are infected patients. A variety of factors influence the risk for infection associated with CVCs: the type of catheter, site of insertion, catheter size, duration of use, degree of host immunodeficiency, type of precautions used during catheter insertion, and skill with which the catheter is inserted. The Hospital Infection Control Practices Advisory Committee has published guidelines for the prevention of infections related to intravascular devices [134]; these include training of the health-care team that will place and maintain the lines, monitoring infection rates with different kinds of catheter over time, hand washing before and after handling catheters or their dressings, the use of barrier precautions during catheter insertion and care, the use of skin antiseptics before catheter

insertion, the use of either sterile gauze or transparent dressings for covering the catheter site, change of dressings when they are soiled or wet, avoiding touching of the catheter exit site when the dressing is changed, the use of a single-lumen rather than a multilumen catheter when possible, the use of a tunneled catheter if long-term use is anticipated, and the use of a subclavian rather than a jugular or femoral site unless necessary. Several controlled trials have evaluated the use of silver and antibiotic-impregnated catheters to reduce the risks for infection in non-HCT recipients [135–137]. This promising strategy was successful in the context of these clinical trials; however, the long-term effects in terms of antibiotic resistance are not known. Further, since the studies were not conducted with tunneled catheters, how applicable the results are to the HCT setting is yet to be tested. Antibiotic prophylaxis during neutropenia The frequency and severity of Gram-negative bacterial infections during neutropenia and concomitant gastrointestinal mucosal damage led to an early emphasis on suppressing the intestinal flora to prevent invasive infection. Early efforts to prevent bacterial infections employed the use of oral nonabsorbable agents. Agents such as vancomycin, gentamicin, neomycin, colistin, and polymyxin B have been advocated in various combinations. A variety of controlled trials have been conducted, and some have shown a beneficial effect. Unfortunately, difficulties with patient compliance, concerns about the emergence of resistance, the high cost of several of these regimens, and lack of evidence for a treatment effect on all-cause mortality and attributable mortality have led to poor acceptance. Interest in such regimens today remains, but more due to the potential for decreasing the elaboration of proinflammatory cytokines involved in the pathogenesis of GVHD than for infection control. TMP–SMX has also been found to reduce both Gram-positive and Gram-negative bacterial infections in patients with hematologic malignancies and undergoing HCT [138,139]. Its broad-spectrum activity against a number of Gram-negative organisms is offset by its lack of activity against Pseudomonas aeruginosa, a major pathogen in HCT patients. Moreover, concerns about delays in engraftment, patient

Bacterial Infections

tolerance, and selection of resistant organisms have limited its use. Because of these concerns and the paucity of data in HCT patients, this regimen has not been adopted widely. The fluoroquinolones have been shown in controlled trials to significantly reduce Gram-negative bacterial infections, appear to be as or more effective than other oral antibiotic regimens, and have been evaluated in HCT patients [140,141]. Norfloxacin [142–145], ciprofloxacin [145–151], ofloxacin [152–154], perfloxacin [155,156], levofloxacin [157], and moxifloxacin [158] have been most widely studied in HCT patients. Ciprofloxacin provides an advantage over norfloxacin in that oral administration achieves systemic blood levels; however, it is not active against most methicillin-resistant staphylococci (the majority of staphylococcal isolates). Levofloxacin offers greater activity against Gram-positive organisms, including Staphylococcus and Streptococcus spp. Concerns about the emergence of fluoroquinolone resistance have also been raised. The effect of fluoroquinolone prophylaxis on microbial resistance was recently addressed in a systematic review and metaanalysis of 56 randomized trials of antibiotic prophylaxis in neutropenic hosts [159]. Within the analysis, the trials were separated into two cohorts, namely those that compared fluoroquinolones with placebo/no intervention, and those that compared fluoroquinolones with other antibiotics. Many trials did not report colonization data, microbiological surveillance endpoints, or routine susceptibility data. Of the fluoroquinolone versus placebo/no treatment trials reporting these endpoints, there were no increases in colonization with organisms resistant to fluoroquinolones, and no difference in the proportion of patients developing infections caused by bacteria resistant to fluoroquinolones. Several microbiologically documented infections were observed in these trials, of which more were caused by fluoroquinolone-resistant organisms in the fluoroquinolone arm compared with placebo. A number of trials compared a fluoroquinolone with TMP–SMX. In these trials, fluoroquinolone use was associated with fewer fluoroquinolone-resistant bacteria in the fluoroquinolone arm than TMP–SMX-resistant bacteria in the TMP–SMX arm. Fewer fluoroquinolone-resistant infections were noted in the fluoroquinolone arm. This analysis demonstrated that there was no difference in the infection rate of fluoroquinolone-resistant pathogens when prophylactic fluoroquinolones were used. Limitations of the analysis are the small number of trials that reported colonization rates and microbiological surveillance results. Continued surveillance and analysis is necessary to evaluate local concerns. The risk for streptococcal infections may also be greater among recipients of fluoroquinolone prophylaxis [24]. The addition of penicillin or rifampin to the fluoroquinolone can reduce the risk for streptococcal infection [149,160,161]. However, resistance to penicillin is also high in the community and may not confer additional protection. Historically, preservation of the anaerobic flora has generally been believed to offer protection against more virulent pathogens. Little activity against anaerobes is a feature of the older quinolone, and this may be desirable in protecting against the more virulent pathogens. There are several groups that have evaluated the role of antibiotic prophylaxis in neutropenic patients over the past decade [140,141,162– 166]. Early meta-analyses demonstrated that fluoroquinolones, while effective in preventing Gram-negative bacteremia, had no effect on days of fever or infection-related mortality [162,163]. Other analyses have similarly shown a reduction in the number of documented infections compared with placebo, particularly for Gram-negative pathogens [164,165]. Reduction in infection-related mortality due to bacterial causes has also been demonstrated [166]. More recently, a meta-analysis of 95 randomized clinical trials in 9283 afebrile neutropenic patients concluded that prophylactic antibiotics significantly reduced the risk for death compared with placebo or no treatment (relative risk [RR] 0.67,

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95% confidence interval [CI] 0.55–0.81) [140,141]. Within the analysis, there were 55 trials that specifically addressed fluoroquinolone prophylaxis. Similar results were observed, with fluoroquinolone prophylaxis leading to decreases in all-case mortality (RR 0.52, 95% CI 0.35–0.77) and infection-related mortality. Other important endpoints including fever, and clinically and microbiologically documented infection rates, were also reduced. The CDC/IDSA/American Society for Blood and Marrow Transplantation consensus panel and the IDSA practice guidelines for the use of antimicrobial agents in neutropenic patients with cancer do not currently recommend the use of routine prophylactic antibacterials in this patient population [6,34]. Unfortunately, both guidelines are out of date and do not encompass recent literature. Data published since the development of guidelines suggest that antibiotic prophylaxis is indicated among high-risk patients who are expected to have prolonged (>7 days) and profound (ANC < 0.1 × 109/L) neutropenia. Bacterial prophylaxis after engraftment Morbidity and mortality from chronic GVHD are frequently due to infection. Antibiotic prophylaxis, while not providing universal protection against encapsulated organisms, substantially reduces the mortality from chronic GVHD [167,168]. Accordingly, all patients with chronic GVHD should be placed on daily antibiotic prophylaxis with penicillin, TMP–SMX or a suitable alternative guided by local susceptibility patterns for at least as long as active GVHD therapy is prescribed [6]. The host deficits that mediate vulnerability for infection persist even after successful completion of GVHD therapy and some centers continue antibiotic coverage longer, but duration of need has not been adequately studied. If penicillin is used, additional prophylaxis for Pneumocystis jirovecii (formerly Pneumocystis carinii) should also be administered. Even in the absence of chronic GVHD, patients with recurrent sinopulmonary infections and autografted recipients undergoing HCT with total body irradiation-containing regimens [168] should be considered for prophylaxis to prevent infection from encapsulated organisms. Agents such as penicillin, cefaclor, cefuroxime, TMP–SMX, and the newgeneration macrolides are particularly useful. Moreover, patients who have previously undergone splenectomy should be considered for antibiotic prophylaxis against encapsulated bacteria.

Adjunctive measures Growth factors Growth factors such as granulocyte colony-stimulating factor (G-CSF) or granulocyte–macrophage colony-stimulating factor (GM-CSF) to stimulate more rapid hematopoietic recovery, as well as to obtain an enriched hematopoietic cell product, have become widespread in the field of HCT, and are especially useful in autologous HCT (see Chapter 45). Unfortunately, although neutrophil recovery is hastened in most prospective randomized trials, the effects on preventing bacterial infections have either been nonexistent or marginal. A meta-analysis of 34 randomized controlled trials of prophylactic GCSF and GM-CSF demonstrated that colony-stimulating factors were associated with a small reduction in the risk of documented infections (RR 0.87, 95% CI 0.76 – 1.00; p = 0.05), with an absolute decrease in the risk of documented infection of 8%, which did not translate into improved infection rates or treatment-related mortality [169]. The meta-analysis included a number of subgroup analyses including differences between colony-stimulating factors, that is G-CSF versus GM-CSF as well as differences between transplant types, that is autologous versus allogeneic HCT. In these analyses, there was no difference in

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Chapter 88

documented infections in trials that utilized G-CSF, whereas a benefit was demonstrated for GM-CSF. There was no advantage to colony-stimulating factor use in the autologous HCT setting, whereas there was a significant reduction in documented infections among allogeneic recipients receiving colony-stimulating factors. Other outcomes that were evaluated included microbiologically and clinically documented infection, days of fever, days of parenteral antibiotics, and infection-related mortality. There was no difference in microbiologically documented infection regardless of analysis type. Clinically documented infection was no different among the groups analyzed. Days of fever were not statistically different in the overall analysis and in the G-CSF recipients, whereas GM-CSF recipients had fewer days of fever. There were fewer days of parenteral antibacterials administered in all analyzed groups with the exception of allogeneic HCT recipients [169]. Finally infection-related mortality did not differ in any of the groups analyzed. Therefore, the benefit to colony-stimulating factor on documented infection and associated complications is small and should be weighed against the cost of these agents. Current consensus guidelines from the American Society of Clinical Oncology recommend colony-stimulating factors following autologous HCT and not following allogeneic HCT [170]. Of interest is the use of hematopoietic growth factors as an adjunct to antibiotic therapy for established infection or for patients with persistent fever. However, controlled trials have failed to show improved outcomes in either HCT or non-HCT neutropenic patients other than shortening of neutropenia duration, antibiotic utilization, and hospitalization. The current American Society of Clinical Oncology guidelines do not recommend the routine use of colony-stimulating factors as adjunctive treatment with antibiotic therapy in patients with febrile neutropenia. The exception to this recommendation is in patients who are at high risk for infectionassociated complications or those patients who are likely to have a poor clinical outcome based on known prognostic factors such as prolonged and profound neutropenia (>10 days and ANC < 0.1 × 109/L), being elderly (>65 years), uncontrolled primary disease, pneumonia, hypotension, multiorgan dysfunction, invasive fungal infection or being hospitalized at the time of fever [170]. The advantages, disadvantages and indications for the use of these cytokines are discussed in more detail elsewhere in the book (see Chapter 45). Several other cytokines, including transforming growth factor-beta [171] and keratinocyte growth factor [172], have been shown in preclinical studies to enhance mucosal stem cell regrowth. Clinical trials have shown that both recombinant human keratinocyte growth factor (palifermin) and repifermin are able to reduce the severity of mucositis [173,174]. Since mucositis is associated with infection [8,175], whether or not these molecules reduce the risk for infection was an important consideration. Palifermin treatment is associated with a reduction in the incidence of febrile neutropenia (75% palifermin versus 92% placebo; p < 0.001), and a nonsignificant trend towards a lower rate of bacteremia [173]. Repifermin was associated with a lower rate of fever; however, no difference in rates of febrile neutropenia was observed [174]. Other agents that have been evaluated for their ability to decrease mucosal toxicity and subsequent infections in cancer patients include GM-CSF mouthwash, glutamine, and low-level laser therapy. None of these agents has consistently demonstrated reductions in infection rates, antibacterial consumption or length of stay of HCT recipients. Of note, the use of GM-CSF mouthwash in HCT recipients is discouraged in the new mucositis guidelines published by the Multinational Association of Supportive Care in Cancer. Granulocyte transfusions During the 1970s, granulocyte transfusions were frequently used to treat or prevent infections in severely neutropenic patients [176,177].

Unfortunately, they were found to have only marginal benefit in most individuals, perhaps in part due to the low number of granulocytes mobilized. Moreover, they often transmitted CMV from donors and morbidity frequently developed from the acquired CMV infection. Today, with modern antibiotic regimens, the need for granulocyte transfusions has diminished. However, they can be useful where bacterial infection from an organism resistant to multiple antibiotics is present or the infection progresses despite the administration of optimal antimicrobial agents [178,179]. The use of granulocyte donors who are serologically negative for CMV eliminates the risk of transmitting CMV. The use of growth factors such as G-CSF or GM-CSF, alone or in combination with dexamethasone, to stimulate granulocyte production and mobilization into the peripheral blood has led to the potential for collecting large numbers of granulocytes from normal donors [178], and the use of these “enhanced” granulocyte transfusion products for the treatment of severe infection. It has yet to be demonstrated whether this approach offers significant clinical benefit. A recent Cochrane review concluded that there is inconclusive evidence from available trials to support or reject routine use of granulocyte transfusions [179]. Randomized controlled trials are needed to definitively answer the role of granulocyte transfusions in the treatment of infections in patients with neutropenia or neutrophil dysfunction; one randomized trial is currently underway. An alternative approach is to collect G-CSF-mobilized granulocytes and administer them to patients prophylactically post transplant, during the expected neutropenic window, to facilitate accelerated neutrophil recovery [180]. This approach has been shown to reduce the number of patients who develop fever while hospitalized, the number of febrile days, and the duration of antibacterial therapy in a small series. Additional research on the validity of such an approach is needed, and may be suitable only for patients at high risk for serious or antibioticresistant infections. Immunobiologic agents Several studies have shown that the use of IVIg reduces bacterial, fungal, and viral infections after allogeneic HCT [181–184], but others have not [185,186], including a controlled trial in autologous HCT patients [186]. The relative merits of IVIg as a general antimicrobial measure versus the specific antimicrobial agents mentioned above remain unclear. Generally speaking, IVIg is well tolerated. Its disadvantages include its high cost, large fluid volumes, need for parenteral administration, and occasional side-effects. Beyond day 90, IVIg given as a monthly infusion was of no benefit in reducing bacterial infections, and routine use is not recommended in the CDC guidelines [6,182]. Moreover, there was no reduction in the incidence of bronchiolitis obliterans, overall survival or survival from chronic GVHD, and serum IgG and IgA levels in patients who received IVIg were lower than those of control subjects, suggesting that the use of IVIg might retard humoral immune recovery. Although there may be other reasons to consider the use of immune globulin (e.g. to reduce GVHD or interstitial pneumonia), its high cost and cheaper antibiotic alternatives do not make this approach attractive for prevention of bacterial infections. Activated protein C has been shown to be effective in the treatment of sepsis syndrome, which frequently complicates HCT and is associated with significant morbidity and mortality. This syndrome is the end result of the activation of inflammatory and coagulation cascades in response to infection [187]. Activated protein C is an endogenous protein that is important in maintaining circulatory homeostasis. In sepsis, concentrations are depleted, a factor that has been associated with poor outcomes in septic patients. Activated protein C has several actions; those of most importance in sepsis are the anti-inflammatory, profibrinolytic, and anticoagulant

Bacterial Infections

activities. Drotecogin alfa, a recombinant human activated protein C, has been shown to reduce the relative and absolute risks of death in septic individuals [187,188]. In the pivotal Food and Drug Administration licensing trial, HCT recipients were excluded due to increased risk from bleeding complications in an already thrombocytopenic population. Since Food and Drug Administration approval, a number of centers have used this agent in the high-risk thrombocytopenic HCT recipients with success [189], although the benefits of administration should be weighed against the potential risks of treatment.

Conclusion The morbidity and mortality from infection have dramatically improved in recent years as insights into the deficits in host bacterial pathogenesis and epidemiology of infections, the introduction of more effective and less toxic antimicrobial regimens, and improved strategies of using various antimicrobial agents have translated into effective therapeutic approaches. Such advances have facilitated enormous strides in reducing transplant-associated mortality, thereby improving the acceptability of

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HCT as a treatment option for an ever-widening array of malignant and nonmalignant diseases. Variations in the epidemiology of opportunistic pathogens, the evercontinuing emergence of antimicrobial resistance among microorganisms, changes in host characteristics due to new preparative and immunosuppressive regimens, the application of HCT to new patient populations (older ages, different hematopoietic cell sources, and different underlying diseases) and the adoption of post-transplant immunoadjuvant therapies to reduce the risk for relapse in the autologous transplant setting require continuing vigilance, and will pose new challenges for the control of bacterial infections. Better diagnostic tools to detect pathogens more accurately and rapidly, and to detect them earlier in the course of infection, are needed. Newer antimicrobial agents that are safer and more effective, with a broader spectrum of activity, must be sought. The expanding array of biologic agents to modify pathogen virulence and host responses to infection offer the opportunity to reduce host susceptibility to infection, and will likely prove important adjuncts to antimicrobial agents directed against the growing repertoire of opportunistic pathogens.

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172. Housley RM, Morris CF, Boyle W et al. Keratinocyte growth factor induced proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract. J Clin Invest 1994; 94: 1764–77. 173. Spielberger R, Stiff P, Bensinger W et al. Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 2004; 351: 2590–8. 174. Freytes CO, Ranatanatharathorn V, Taylor C et al. Phase I/II randomized trial evaluating the safety and clinical effects of repifermin administered to reduce mucositis in patients undergoing autologous hematopoietic stem cell transplantation. Clin Cancer Res 2004; 10: 8318–24. 175. Vera-Llonch M, Oster G, Ford CM, Lu J, Sonis S. Oral mucositis and outcomes of allogeneic hematopoietic stem-cell transplantation in patients with hematologic malignancies. Support Care Cancer 2007; 15: 491–6. 176. Clift RA, Sanders JE, Thomas ED et al. Granulocyte transfusions for the prevention of infection in patients receiving bone marrow transplants. N Engl J Med 1978; 298: 1052–7. 177. Winston DJ, Ho WG, Young LS et al. Prophylactic granulocyte transfusions during human bone marrow transplantation. Am J Med 1980; 68: 893–7. 178. Price TH, Bowden RA, Boeckh M et al. Phase I/II trial of neutrophil transfusions from donors stimulated with G-CSF and dexamethasone for treatment of patients with infections in hematopoietic stem cell transplantation. Blood 2000; 95: 3302–9. 179. Stanworth SJ, Massey E, Hyde C et al. Granulocyte transfusions for treating infections in patients with neutropenia or neutrophil dysfunction. Cochrane Database Syst Rev 2005(3): CD005339. 180. Oza A, Hallemeier C, Goodnough L et al. Granulocyte-colony-stimulating factor-mobilized prophylactic granulocyte transfusions given after allogeneic peripheral blood progenitor cell transplantation result in a modest reduction of febrile days and intravenous antibiotic usage. Transfusion 2006; 46: 14–23. 181. Sullivan KM, Kopecky J, Jocom J et al. Immunomodulatory and antimicrobial efficacy of

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intravenous immunoglobulin in bone marrow transplantation. N Engl J Med 1990; 323: 705– 12. Sullivan KM, Storek J, Kopecky J et al. A controlled trial of long-term administration of intravenous immunoglobulin to prevent late infection and chronic graft-vs.-host disease after marrow transplantation: clinical outcome and effect on subsequent immune recovery. Biol Blood Marrow Transplant 1996; 2: 44–53. Graham-Pole J, Camitta B, Casper CJ et al. Intravenous immunoglobulin may lessen all forms of infection in patients receiving allogeneic bone marrow transplantation for acute lymphoblastic leukemia: a pediatric oncology group study. Bone Marrow Transplant 1988; 3: 559–66. Petersen FB, Bowden RA, Thornquist M et al. The effect of prophylactic intravenous immune globulin on the incidence of septicemia in marrow transplant recipients. Bone Marrow Transplant 1987; 2: 141–7. Winston D, Antin JH, Wolff SN et al. Multicenter, randomized, double-blind doses of intravenous immunoglobulin for the prevention of graft versus host disease and infection after allogeneic bone marrow transplantation. Bone Marrow Transplant 2001; 28: 187–91. Wolff SN, Fay JW, Herzig RH et al. High-dose weekly intravenous immunoglobulin to prevent infections in patients undergoing autologous bone marrow transplantation or severe myelosuppressive therapy. A study of the American Bone Marrow Transplant Group. Ann Intern Med 1993; 118: 937–42. Bernard GR, Vincent JL, Laterre PF et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699–709. Abraham E, Laterre PF, Garg R et al. Drotecogin alfa (activated) for adults with severe sepsis and low risk for death. N Engl J Med 2005; 353: 1332–41. Pastores SM, Shaw A, Williams MD et al. A safety evaluation of drotecogin alfa (activated) in hematopoietic stem cell transplant patients with severe sepsis: lessons learned in clinica research. Bone Marrow Transplant 2005; 36: 721– 4.

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Janice (Wes) M.Y. Brown

Fungal Infections after Hematopoietic Cell Transplantation

Introduction Invasive fungal infections (IFIs) are increasingly recognized as the leading infectious causes of mortality following allogeneic hematopoietic cell transplantation (HCT) in adults and children [1–3]. The rising incidence of these infections, the emergence of noncandidal and nonAspergillus infections, the refinement of new diagnostic modalities, and the introduction of the broadest array of antifungal drugs in the history of medicine have resulted in a sustained renaissance in the study of IFIs. The substantially higher mortality rate associated with IFIs in HCT patients when compared with other immunocompromised patients, even in this era of effective antifungal therapies, reflects the unique degree and nature of immunosuppression resulting from allogeneic HCT [4]. There are three major classes of clinically important fungus – yeasts, molds, and dimorphic fungi. One genus of yeast, Candida spp., and one genus of mold, Aspergillus spp., cause more than 80% of all IFIs following HCT. Candida spp. are distinguished by their lack of true hyphae or branching forms, and exist primarily as unicellular forms known as blastoconidia or blastospores. Molds, such as Aspergillus spp., are most commonly transmitted via inhalation of the conidial (spore) forms and have true hyphae. The dimorphic fungi, such as Coccidioidomycosis imitis or Histoplasma capsulatum, have both yeast forms and true hyphae, and are still relatively uncommon in nonendemic areas following transplantation. This chapter will focus on infections due to Aspergillus and Candida spp., the two clinically most important fungi following HCT. Since the epidemiology and pathophysiology of Candida spp. and Aspergillus spp. are unique, they will be discussed separately. The European Organization for Research and Treatment of Cancer (EORTC) and the Mycoses Study Group of the National Institute of Allergy and Infectious Diseases (MSG-NIAID) in the United States have devised classifications of IFIs based on the degree of certainty of the diagnosis. Infections are classified as definite/proven, probable or possible based on the underlying clinical condition and the results of histopathology, culture, and radiologic studies. The classifications differ slightly with respect to HCT recipients as the likelihood that invasion has occurred is great, specific radiologic findings may not be present, and invasive diagnostic procedures are not always feasible. Definite infection requires evidence of infection on histopathologic specimens or the isolation of fungus from typically sterile sites. Although

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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the culture of fungus from nonsterile specimens, for example sputum, and radiographic findings consistent with IFIs define probable infection in other populations, following HCT an X-ray consistent with IFIs is generally considered sufficient even in the absence of supporting microbiologic data. Therefore, the classification of possible fungal infections, formally defined as an X-ray consistent with IFIs but without positive cultures, has little clinical significance following HCT [5]. While these classifications are of importance for clinical studies of antifungal therapy, there is no difference in the outcome/survival of patients classified as having definite versus probable IFIs, especially aspergillosis. Following HCT, even patients who meet the formal criteria for probable and possible IFIs should be treated aggressively with appropriate antifungal medications. Every reasonable attempt should be made to obtain samples necessary to properly identify the pathogen to ensure appropriate treatment. This principle takes on renewed significance in light of the increasing number of reports of a variety of infections due to fungi other than Aspergillus spp. or Candida spp.

Epidemiology of IFIs In newer analyses, the published overall incidence of IFIs is substantially lower than the previously reported incidence of between 8% and 15% of all patients undergoing HCT [3,6,7]. Autopsy studies continue to demonstrate the inadequacy of our traditional diagnostic modalities. In one recent report, 83% of cases of IA diagnosed post mortem did not have positive cultures pre mortem [8]. IFIs are most commonly due to Candida or Aspergillus spp. (invasive aspergillosis [IA]) and occur following autologous or allogeneic HCT, although the incidence and outcome differ in these two groups. Practices and demographics at individual centers will influence the rate and type of IFIs; however, many centers report that the incidence of IA is 2–6% following autologous and 5–9% following allogeneic transplantation. Although the incidence of invasive candidal infections has generally decreased at institutions where prophylactic fluconazole therapy is used, this practice has been associated with an increase in invasive Aspergillus infections [9]. In an interim analysis of their prospective, the EORTC and the MSGNIAID multicenter surveillance program reported that the aggregate cumulative incidence of aspergillosis at 12 months was 0.5% after autologous HCT, 2.3% after allogeneic HCT from a human leukocyte antigen (HLA)-matched related donor, 3.2% after transplantation from an HLAmismatched related donor, and 3.9% after transplantation from an unrelated donor. Moreover, there was no difference in the incidence at 12 months following high-dose versus reduced-intensity conditioning (RIC)

Fungal Infections after Hematopoietic Cell Transplantation

regimens [10]. Published incidences of IFIs in pediatric centers are equally variable, generally ranging from 4.5% to 14% [2,11]. In a systematic review of the literature, the case fatality rate in patients with IA following allogeneic HCT was 80–90% compared with a case fatality rate of 8–60% in hematology and solid organ transplant patients despite prompt institution of aggressive antifungal therapy [12–14]. There is a bimodal pattern to the occurrence of IFIs. This pattern was recognized early in the history of HCT, and the specific associated risk factors were identified. Interestingly, the pattern persists, although a shift in the pathogens associated with early and late infections has occurred, reflecting changes that include the shortening of time to neutrophil engraftment, the use of prophylactic antifungal agents, and issues associated with graft-versus-host disease (GVHD). Currently, although the incidence of infections due to candidal species is lower than was reported in the earlier years of HCT, the mean time to diagnosis of candidal infections remains at 2 weeks post transplantation, during or immediately following the period of neutropenia and disruption of the mucocutaneous barriers. IAs also occur during this period and are generally associated with either colonization of the airways or protracted neutropenia peri transplantation. Thus, although neutropenia remains a risk for IA, the majority (58–100%) of patients currently are diagnosed following the resolution of neutropenia. In most large studies, the first peak of invasive Aspergillus infection occurs at approximately day 15 after transplantation (day +15), and the second, larger, peak occurs at between day +60 and day +136 in association with GVHD and systemic corticosteroid use [1,3,5,7,10]. The timing of IFIs in relation to specific risks is represented schematically in Fig. 89.1. Specific risk factors for the development of invasive Aspergillus infection differ based on when patients are diagnosed in temporal relation to HCT. For patients diagnosed prior to day +40, risk factors include increases in genetic disparities between donor and recipient, certain conditioning and GVHD prophylactic regimens, and undergoing HCT in a room lacking laminar airflow. Mortality was higher if IFIs were associated with bacteremia or viral infections [6,15]. For patients diagnosed after day +40, risk factors included systemic steroid administration, late-onset cytomegalovirus (CMV) infection, certain underlying malignancies and hematopoietic disorders, genetic disparities between donor and recipient, prolonged neutropenia, acute grade III–IV GVHD or extensive chronic GVHD. One study reported that 21 or more days of steroid use of 1 mg/kg/day or greater was specifically associated with increased risk [1]. Recipients of bone marrow transplants from matched unrelated donors have been reported to be at a high risk, especially for late fungal disease generally attributed to the increased risk for GVHD [1,6,16]. Other risk factors include the history of IFIs prior to transplantation and the underlying disease that serves as the indication for transplantation. Examples of underlying conditions that increase the risk for fungal infection, particularly due to Aspergillus spp., include

Aspergillus

Candida Risk factor

Neutropenia 10

Marrow infusion

20

30

Chronic GVHD

Acute GVHD and therapy 40

50

60

70

80

Days after marrow transplant

90

12

100 Months

Fig. 89.1 Risk for fungal infection after hematopoietic cell transplantation. GVHD, graft-versus-host disease.

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chronic granulomatous disease, aplastic anemia, and myelodysplastic syndrome. IFIs are not limited to adult HCT recipients. A recent retrospective analysis reports a 16% incidence of IFIs after allogeneic HCT and 8% after autologous HCT in children with a mortality rate comparable to that of adults. A separate retrospective cohort study using the 2000 Kids Inpatient Database, a national database of hospital inpatient stays during the year 2000, revealed that the highest incidence of IA was seen in children who had undergone allogeneic bone marrow transplantation (4.5%) and those with acute myelogenous leukemia (4%). Other risk factors cited include prolonged neutropenia, GVHD, age above 10 years, and steroid administration. As is seen with adults, children who received a total of 0.25–1.00 g/day of methylprednisolone for longer than 5–10 days, or greater than 2 mg/kg/day of prednisone for longer than 10 days, had a higher incidence of IFIs [11]. IFIs have also been described as one of the most common causes of pneumonia death in pediatric HCT recipients, and the overall in-hospital mortality of immunocompromised children with IA in one study was 18% [2,11,17]. IA was associated with a significantly higher mortality rate, longer median length of hospital stay, and substantially higher median total hospital charges than for immunocompromised children without IA [2].

Biology and pathophysiology The most important principle in understanding the pathophysiology of IFIs is to understand the nature of the immunodeficiencies that render HCT recipients so susceptible to lethal infection. Armed with this knowledge, rational immunologic strategies may be formulated to improve antifungal immune reconstitution. In the case of candidal infections, animal models demonstrate that the crucial determinant of fungal clearance is specific host immune reactivity and not the administration of antifungal agents. Candida Candidal organisms are commensurals, residing on the skin and in the airways and gastrointestinal (GI) tract. HCT-associated risk factors that predispose individuals to candidal invasion include mucositis, neutropenia, GVHD, co-infections of the urogenital tract (such as herpes simplex virus), administration of broad-spectrum antibiotics, total parenteral nutrition, and central venous catheters. Antibiotics including penicillins, quinolones and cephalosporins alter GI colonization [18–20]. Sulfonamides, aminoglycosides, and doxycycline may decrease antifungal neutrophil activity [21,22]. It is generally believed that the GI tract is the most common source of the introduction. Thus, while invasive infections must be distinguished from colonization, identification of the colonizing organism may be helpful in the selection of the appropriate antifungal agent in the setting of presumed or proven invasive disease. Of the numerous species of Candida, only C. albicans, C. glabrata, C. guillermondii, C. krusei, C. lusitaniae, C. dubliniensis, C. tropicalis, C. pseudotropicalis, C. parapsilosis and C. inconspicua are associated with fungal disease [23,24]. Certain species exhibit intrinsic resistance to specific antifungals. Candida krusei is intrinsically resistant to fluconazole, and C. lusitaniae is resistant to amphotericin. Candida glabrata exhibits variable resistance to fluconazole and amphotericin [21,25]. Prior to the widespread use of fluconazole prophylaxis, candidal organisms represented 10–20% of all blood stream isolates and 5–50% of nosocomial sepsis [26]. Since the administration of prophylactic fluconazole became a more widely used practice, an increase in the relative incidence of nonalbicans Candida spp. has been inconsistently reported. Currently, following HCT for adult patients, C. albicans is the most common isolated species (52–64%), followed by C. tropicalis (11– 25%), C. parapsilosis (7–24%), C. glabrata (8–20%) and C. krusei

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Chapter 89

(1–5%). Pediatric programs often report a relatively increased incidence of infections due to C. parapsilosis [21,27–29]. Syndromes of candidal infection Multiple clinical syndromes associated with Candida spp. have been described. A high degree of clinical suspicion is required in order to make a pre mortem diagnosis. Mucocutaneous infection Colonization of the skin or GI tract may pose greater risk for invasion. Esophagitis or involvement of other areas of the GI tract may occur in the absence of thrush, and the associated complaints of dysphagia, substernal chest pain, nausea, vomiting or upper GI bleeding are indistinguishable from esophagitis due to other etiologies. The stomach is the second most common GI site of candidal infection, followed by the large and small bowel. Findings at endoscopy include white plaques, single or multiple ulcers, erosions or pseudomembrane formation. Rarely, patients may present with perforation. Patients who experience visceral perforation from other causes should be considered to have a substantial risk for candidemia. Candidemia Methods used to culture blood are insensitive in the isolation of Candida spp., and, as a result, fewer than 50% of patients with proven dissemination have positive blood cultures. Despite the insensitivity of this test, Candida spp. are some of the most common organisms isolated from blood cultures at large medical centers. Techniques such as the lysis centrifugation (Isolator) system have not significantly improved the sensitivity of blood cultures. Following HCT, there is a higher incidence of dissemination associated with candidemia. Candidal species are capable of producing glycocalyx, potentially contributing to a biofilm on intravenous catheters. Thus, candidemia is highly associated with infection of a central venous catheter. Most data indicate that the prompt removal of intravenous catheters significantly shortens the period of candidemia and reduces the risk for recurrent candidemia. Changing catheters over a wire does not appear to reduce the risk. It would be ideal to delay replacement of a central venous catheter until blood cultures are negative. Resolution of symptoms (primarily fever) following the removal of the central venous catheter does not obviate the need for antifungal therapy [30–32].

retrospective analysis of autopsy-proven cases of hepatic candidiasis, CT scans or ultrasound examinations revealed evidence of tissue infection in only 18% of patients prior to their death. Other imaging modalities may be more useful. One prospective study examined the role of magnetic resonance imaging (MRI) scans in patients with suspected hepatosplenic candidiasis. Patients were divided into groups based on whether they received acute (<2 weeks), subacute (>2 weeks but <3 months) or chronic (>3 months or completed) antifungal treatment. MRI scanning offers high positive and negative predictive values (85% and 100%, respectively, with a sensitivity of 100%) and a specificity of 96% in the diagnosis of hepatosplenic candidiasis in the acute setting. In 11 of 13 patients with hepatosplenic abnormalities, the lesions were less than 1 cm in diameter and were described as welldefined, high-signal intensity foci on T1-weighted images. Additionally, MRI may be able to distinguish active from chronic lesions that are presumed to be healed and which tend to be larger (1–3 cm) with irregular, more angular margins [34]. The lungs are often involved in disseminated candidiasis representing delivery of organisms by nonportal perfusion. As with hepatosplenic candidiasis, patients may have fever but infrequently complain of symptoms referable to the respiratory tract. Unfortunately, cultures of sputum or bronchoalveolar lavage fluid have limited sensitivity and specificity. CT scans of the chest are recommended in the setting of candidemia following HCT. In a retrospective analysis, the most common thinsection CT findings in HCT recipients with histopathologically proven pulmonary candidiasis were multiple nodules, which were seen in 15 of the 17 (88%) patients studied. In nearly half of the patients, the nodules were described as centrilobular with a tree-in-bud pattern, and in 75% of these scans the nodules were bilateral. The majority of patients (71%) had associated pulmonary lesions including areas of air space consolidation in 11 (65%) patients, areas of ground-glass opacity (35%), pleural effusion, thickening of the bronchial walls, and cavitation [35]. The early institution of antimycotic therapy improves survival. In one series, therapy started within 10 days of the diagnosis of pulmonary candidiasis resulted in 41% mortality compared with over 90% mortality if therapy was started after 10 days of symptoms [36]. An endovascular source of infection should be considered in patients believed to have disseminated candidiasis who persistently exhibit symptoms or develop new lesions despite appropriate antifungal therapy. Cardiovascular system

Dissemination with deep-tissue infection Despite the high risk for visceral dissemination in HCT patients with a history of documented candidemia, it is frequently not diagnosed until post mortem examination. Although the liver, spleen, and lungs are most frequently affected, virtually all other organs are at risk for seeding, including the kidneys, brain, heart (myocarditis or endocarditis), central nervous system (CNS), vascular endothelium, bone, endocrine glands, and eye (endophthalmitis). Microabscesses are the hallmark of candidal tissue invasion and are not necessarily accompanied by a marked inflammatory response, especially following HCT [33]. Histologic examination may reveal a granulomatous response with epithelioid or histiocyte response. Special stains (periodic acid–Schiff or methenamine silver stains) may increase the identification of fungal organisms present in the microabscesses. Hepatosplenic candidiasis is probably the most common syndrome of disseminated infection and is believed to require candidal entry via the portal system. Patients do not usually have signs and symptoms associated with organ involvement; therefore, radiologic imaging is recommended. With a reported sensitivity of over 90%, computed tomography (CT) scans of the abdomen performed with intravenous contrast are more sensitive than ultrasound examinations. However, in one

Candida spp. are capable of attaching to the endothelium and to catheters, which may explain the presence of endovascular infections, such as endocarditis and thrombophlebitis, with or without concomitant deep venous thrombosis. Thrombophlebitis is most commonly a post mortem diagnosis; however, the classic, albeit rarely seen, presentation includes a patient with localizing extremity edema and a history of protracted broad-spectrum antibiotic and/or total parenteral nutrition administration via a central venous catheter. Although rare, Candida spp. are the most common cause of fungal endocarditis. Most of the symptoms are indistinguishable from bacterial endocarditis, and it is notable that patients may have minimal symptoms. One clue that a patient may have fungal endocarditis may be the occurrence of large peripheral embolic lesions as fungal vegetations tend to be large and friable. Blood cultures are frequently positive, and endophthalmitis is a common finding. Endovascular candidal infections have high mortality rates, but significantly improved outcomes have been historically reported if surgical intervention is part of the therapy of thrombophlebitis or endocarditis. The availability of better tolerated antifungal agents has been accompanied by reports of successful medical therapy of candidal endocarditis; however, the optimal approach to candidal endocarditis has not been determined. A recent review of 879 cases of endocarditis due to Candida

Fungal Infections after Hematopoietic Cell Transplantation

spp. reported in the peer-reviewed literature from 1966 to 2002 included 31 cases treated with antifungal monotherapy, 25 cases treated with medical antifungal combination therapy, and 107 cases treated with adjunctive surgical plus medical antifungal therapy. Adjunctive surgery was associated with a lower mortality rate, and a higher mortality was associated with infection prior to 1980, antifungal monotherapy, infection due to C. parapsilosis, and left-sided endocarditis [37–39]. Eye and CNS disease Successful therapy of fungal keratitis or endophthalmitis has been reported with either topical or oral voriconazole treatment, and aqueous humor drug concentrations exceeding the minimal inhibitory concentration [40–42]. Formal ophthalmologic evaluation is recommended in HCT recipients with visual complaints and is mandatory in the setting of disseminated candidal infection. Vitreous biopsy may be necessary to diagnose candidal endophthalmitis. In retrospective analyses, Candida was identified as one of the most common causes of CNS infection following HCT. In two large retrospective analyses performed prior to the widespread use of fluconazole prophylaxis in allogeneic HCT, Candida spp. were identified as the responsible pathogen in 18% and 33% of CNS lesions. Candida meningitis or brain abscess often occurred in association with fungemia (63% of cases) or neutropenia (63%) and widespread dissemination. The high incidence of CNS involvement following HCT mandates that the treatment or prevention of IFIs should take into account the CNS compartment [43]. Skin lesions The early post-transplant period is the time of highest risk for disseminated candidal infection, a time period that overlaps with the period of broad-spectrum antibiotic administration (febrile neutropenia) and acute GVHD. The cutaneous lesions of disseminated candidiasis may be subtle or dramatic, and include macronodular lesions (0.5–1.0 cm in diameter, pink or red), lesions resembling ecthyma gangrenosum or purpura fulminans. Diagnosis requires biopsy and culture of the lesions. Aspergillus Aspergillus spores are thermotolerant and resistant to desiccation. Although molds are not commensurals, exposure to spores likely occurs frequently as they are found throughout nature, particularly associated with decaying vegetation and soil, barns, compost piles, indoor potted plants, peppers, spices, and smoking marijuana [44]. Filimentous fungi, including Aspergillus, have also been isolated from water in a pediatric HCT unit; however, there have not yet been large studies confirming nosocomial transmission via the water supply, even in high-risk populations [45]. Virtually all IA described has been caused by five species – A. fumigatus, A. flavus, A. niger, A. terreus and A. nidulans – probably due to the fact that these pathogenic species can grow at 37° C, in contrast to nonpathogenic species that lack this trait. The marked predominance of A. fumigatus may be explained in part by its ubiquity in nature and its very rapid growth rate, with hyphal extension occurring at a rate as high as 1–2 cm/hour. The small size and hydrophobic coating of the spores facilitate efficient aerosolization and possibly facilitate immune evasion. Invasive infections are rarely seen in immunocompetent patients. The inoculum size necessary to cause disease is suspected to be minimal in severely immunocompromised individuals because of (1) the occurrence of nosocomial outbreaks of Aspergillus spp. infections with highest incidence in the most immunocompromised patients, even in the absence of

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documentation of high ambient spore counts; and (2) the lower frequency of positive cultures of the respiratory tracts of HCT recipients compared with patients with other risk factors. Additionally, patients may have been colonized for an extended period and develop disease after subsequent immunocompromise. Conidia that escape mucociliary defenses are rapidly ingested by the alveolar macrophages. Soon after tissue invasion, the hyphae are damaged and destroyed by neutrophils and, to some degree, by platelets and monocytes. A variety of different antigens are expressed during the morphologic changes as soon as the conidia begins germination as the initial stages of hyphal formation. The hallmark of Aspergillus disease is angioinvasive dissemination often resulting in the hemorrhage, infarction, and cavitation that are the classic hallmarks of infection. Similarly, cavitation may appear as a consequence of vascular compromise. As Aspergillus spp. can thrive in an acidic environment with low oxygen tension, vascular disruption establishes these growth conditions and impairs the delivery of antifungal agents and immune effectors. These findings may help explain the observation that complete eradication is very difficult with medical therapy alone. Steroids cause a 30–40% acceleration in the growth rate in vitro of some species of Aspergillus [46], and inhibit the control of fungal infection by a variety of mechanisms, including (1) impaired ingestion and/or killing of conidia and hyphae by alveolar macrophages, monocytes, and neutrophils, (2) delayed cytokine production and cell recruitment, and (3) reduced tumor necrosis factor (TNF) production, resulting in an attenuation of monocyte activation [47,48]. Pathogen-specific virulence determinants and the cellular pathogenesis have not yet been defined for Aspergillus spp. In addition to the ability to grow at body temperature, pathogenic species of Aspergillus demonstrate efficient binding to laminin and fibrinogen, and they produce elastases, phospholipases, several superoxide dismutases, and catalases; however, the specific role of each of these biochemical characteristics in the pathogenesis of invasive infection has yet to defined [49]. Elucidation of pathogen-related virulence factors has been greatly facilitated by the recently completed sequencing of the 29.4-megabase genome sequence of the clinical isolate Af293, which included the identification of eight chromosomes containing 9926 predicted genes. Microarray analysis revealed temperaturedependent expression of a distinct set of genes, as well as 700 A. fumigatus genes [50]. Aspergillosis: clinical syndromes The term “aspergillosis” is currently used to refer to invasive disease, in contrast to “aspergilloma” which refers to a focal lesion, typically in the lung. In settings other than following HCT, each of these terms has a distinct histopathologic and clinical significance. Following HCT, even the patient with a single lesion identified on chest CT must be considered to have microscopic invasion or to be at imminent risk for invasion even at the time of first diagnosis. This risk for and likelihood of invasive disease has important therapeutic ramifications. As the biology of Aspergillus would predict, the most common sites of inoculation are the bronchial tree and paranasal sinuses. Therefore, concomitant pneumonia and sinusitis should suggest fungal disease. Pulmonary aspergillosis following HCT is most commonly accompanied only by fever, but many patients may be completely asymptomatic. The paucity and nonspecific nature of the symptoms likely account for the fact that as many as two-thirds of cases are diagnosed at autopsy [51,52]. Necrotic skin or mucosal lesions, invasive sinusitis, hemoptysis, and pneumonia with pain are findings that should raise suspicion of an invasive infection due to Aspergillus spp. The changing risk factors for IA have been paralleled by a change in the spectrum of radiologic findings. Likely as a result of a limited

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immune response, parenchymal lesions due to Aspergillosis spp. may appear in a multitude of forms including nodules, with or without a circumferential hemorrhage (often described as a “halo”) or central cavitation, infiltrates, or a combination of both. Radiographic findings may be highly variable following HCT. Proposed criteria requiring the presence of “classic” lesions, such as halos or crescents, will ensure missing the diagnosis in many cases, and, further, these are not specific findings. In a recent systematic review of the high-resolution CT findings in patients with pulmonary infection after bone marrow transplantation, the presence of large nodules and visualization of the halo sign were most suggestive of fungal infection but were not sufficiently specific to infections due to fungi. For example, nodules that were 1 cm or more in diameter were seen in 13 (62%) of 21 patients with fungal pneumonia but also in five (19%) of 26 patients with bacterial pneumonia, three (10%) of 30 with respiratory syncytial virus pneumonia, and three (14%) of 22 with CMV pneumonia. The halo sign was present in 10 of 21 patients with fungal pneumonia, two of 26 with bacterial pneumonia, three of 30 with respiratory syncytial virus pneumonia, and one of 22 with CMV pneumonia [53]. An analysis of common diagnostic modalities in patients with infections more than 100 days post HCT revealed a significantly higher sensitivity, negative predictive value, and accuracy for CT than for chest X-ray (89% versus 58%, p < 0.0001; 78% versus 47%, p < 0.0001; 90% versus 68%, p < 0.0001, respectively) [54]. IA appearing more like bronchopneumonia may have a poorer prognosis than IA with a more angioinvasive appearance [55]. Molecular (polymerase chain reaction [PCR]) detection methods have shown some promise to increase diagnostic yield when compared with classic microbiologic studies of fluid from bronchoalveolar lavage following HCT; however, studies with sufficient power to define the sensitivity and specificity of PCR are still in progress [56]. Although the substantial benefit of confirming the diagnosis of an IFI are well recognized, empiric therapy is frequently instituted to the perceived risk and low yield of an invasive diagnostic procedure. The limited published data includes a case series in which the first attempt at a radiologically guided fine-needle biopsy was suggestive of IFI in five of 21 patients, 14 of whom were ultimately shown to have IA. However, 13% of patients had a clinically insignificant pneumothorax or self-limiting hemoptysis despite the fact that all patients were given platelet transfusions, and the median time of the procedure was 131 days post transplantation [57]. Sinusitis is usually symptomatic but is usually not distinguishable from sinusitis due to other pathogens. Due to the pigments produced by Aspergillus and the invasive nature of the fungus, drainage may be green, black or bloody. Signs and symptoms suggestive of an invasive process include erosive lesions of the nasal septum, cavernous sinus thrombosis, pain with eye movement, extraocular muscle plegias, diplopia, and periorbital swelling. Infections limited to the lower airways include laryngitis, tracheitis, and tracheobronchitis. The invasive potential for tracheobronchitis is documented by the detection of Aspergillus antigen in the blood prior to initial clinical presentation, and the subsequent development of parenchymal lesions [58]. A high degree of clinical suspicion must exist in order to diagnose tracheobronchitis as airway thickening may be absent or not appreciated. Sputum samples may persistently yield colonies of mold – often more than a few colonies – but negative cultures do not rule out this diagnosis. Bronchoscopy should be considered in patients with unexplained or chronic cough which may or may not be productive and is refractory to antibacterial therapy. A variety of bronchoscopic and post mortem findings have been described, including pseudomembranes, plaques, and balls of fungus. Sites of skin trauma including catheter insertion sites are also portals of fungal entry; however, these infections are relatively uncommon.

Cutaneous aspergillosis may invade and become fatal [59]. Skin lesions typically start as focal lesions, and necrosis is characteristic. If the lesion is not at a site of known trauma or there is more than one lesion, the possibility of hematogenous spread from an intravascular focus of infection including endocarditis should be considered. Histopathologic examination and culture of biopsies of the lesion should be performed to confirm the diagnosis. CNS involvement was reported in 10 of 18 HCT patients diagnosed with IA. Manifestations of CNS disease include microabscesses, macroabscesses, meningitis, and extension from sinus disease. In retrospective analyses of brain abscesses following bone marrow transplantation, fungal organisms were isolated in 60–92% of all cases, and Aspergillus spp. was the most common organism [59,60]. It is important to note that although pulmonary disease was present in the vast majority (87%) of cases of Aspergillus brain abscess, this finding was not invariably present. Prior to the current era of antifungal agents, CNS disease had been associated with a nearly 100% mortality rate [61,62]. However, successful treatment of CNS fungal infection has been reported by a number of groups, most commonly following treatment with voriconazole, which can achieve fungicidal drug concentrations within the CNS. In one retrospective, analysis of 81 patients with CNS aspergillosis, neurosurgical intervention was associated with improved survival compared with medical therapy alone (p = 0.02). Although HCT recipients had a poorer survival, seven (22%) of 32 survived for a median of 203 days [63,64]. Dissemination to virtually every tissue has been described, including the myocardium, thyroid, kidneys, eyes, and GI tract. With the exception of fungal endophthalmitis, tissue biopsy is necessary in order to establish the diagnosis. In contrast to candidal endophthalmitis, biopsy of vitreal tissue often fails to identify the organism in Aspergillus infection. Thus, empiric broad-spectrum antifungal therapy is mandated in this infection. Treatment may include vitrectomy and/or intravitreal instillation of antifungal agents even in the absence of a confirmed diagnosis. Although still investigational, molecular diagnostic tests may be helpful in the future evaluation of this infection. GI involvement may result from direct invasion or angioinvasive spread and should be a diagnostic consideration for persistent symptomatology in the setting of protracted therapy for GI GVHD but may occur in any immunocompromised host, presenting a diagnostic challenge. Any region of the GI tract may be involved, and a variety of findings, including diffuse infiltration, necrotizing lesions, and pseudomembrane formation, have been described [65]. To further complicate matters, there is a particularly high association with CMV in this setting. Emerging fungal infections Reports of serious invasive infections caused by less commonly encountered fungi continue with an apparently increasing frequency. These fungi are often referred to collectively as “emerging fungal infections,” and the most commonly reported non-Aspergillus molds include Zygomycetes spp., Fusarium spp., Scedosporium spp., and dematiaceous (dark-walled) fungi [66–68]. Although the increase may in part be due to improving fungal identification and speciation, concern remains that this trend may in part be due to the growing use of antifungal agents. Little is known about the epidemiology and treatment of infections due to these fungi following HCT. As these fungi often exhibit intrinsic resistance to many antifungal agents, this trend underscores the need for a detailed history of prior antifungal therapy, definitive identification of the pathogen, and appropriate use of antifungal susceptibility testing. Zygomycetes Infections by molds in this class include zygomycosis, mucormycosis, and cunninghamellosis, and may occur in healthy hosts, although

Fungal Infections after Hematopoietic Cell Transplantation

diabetes, iron overload, neutropenia, and GVHD are the leading risk factors. Patients present most commonly with invasive rhinocerebral or pulmonary disease. Amphotericin preparations are considered to be primary therapy, but recent reports underscore the potential efficacy of posaconazole. Although controversial, an increase in the incidence of zygomycoses has been reported by centers after expanding the use of voriconazole following HCT. Aggressive surgical debridement is also a mainstay of successful therapy. Scedosporium spp. Infections with Scedosporium spp. (Scedosporium apiospermum and S. prolificans) may mimic aspergillosis in their predilection for sinopulmonary disease; however, clues to these organisms include a higher risk of embolic skin lesions, ophthalmologic infections, and CNS disease. Given a substantial frequency of resistance to amphotericin preparations, primary therapy with voriconazole, itraconazole or posaconazole is recommended. Fusarium spp. Fusarium is the most common mold to be cultured from the blood, and the hematogenous spread may be manifested by a stuttering appearance of skin lesions which are frequently hemorrhagic, tender nodules. Infection may be introduced from a site of identified skin injury, but frequently the entry site is occult. Disseminated disease is not uncommon. Intrinsic resistance to multiple antifungal agents is common, and successful therapy with combinations of agents, including terbinafine, has been reported. Dematiaceous molds (phaeohyphomycosis) A multitude of species comprises this class of mold, which are identified in culture by the pigment in their cell walls. These organisms can cause a range of infections including dermal, sinopulmonary, and CNS disease. Historically, itraconazole has been considered primary therapy, but response to voriconazole and posaconazole has also been reported. Diagnosis not based on culture Blood cultures are rarely positive, even in IA, and they have an unacceptably low sensitivity in candidemia. Decades of investigation have led to the development of nonculture-based diagnostic tests. Currently, the most promising for clinical use include tests to detect fungal components and tests based on molecular techniques to detect fungal nucleic acids. Galactomannans (GMs) are cell wall components of fungi, and measurement of this antigen in the blood and urine has been studied for over two decades as a potential diagnostic test for IFIs. The two most studied GM antigenemia tests are the latex agglutination test and enzyme-linked immunosorbent assay (ELISA). A number of recent studies have reported that the latex agglutination test failed to distinguish between infection and exposure to noninfectious Aspergillus spp. antigens, and was less sensitive and persistently less positive than ELISA [69]. Multiple factors impact the utility of the ELISA test to screen blood, including the risk for IA, the age of the patient, and the administration of antifungal therapy. Further, false-positive results have been consistently reported with the administration of piperacillin/tazobactam and the ingestion of certain food. Higher levels of false positives in pediatric patients have been reported, but a recent prospective study in 64 pediatric patients did not demonstrate that there were more false-positive results than reported in adult populations [70,71]. Inter- and intraspecimen variability of the GM assay has been reported. The overall performance of the test improves by testing samples in duplicate and requiring positivity in two consecutive tests. Decreasing the cut-off point of the optical density

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reading to 0.5 improves the sensitivity (60–100%) and positive predictive value of the test, but also decreases the specificity (89–99%) [72– 74]. In multiple analyses, ELISA either did not consistently detect IA prior to the development of signs or symptoms, or did so within the week prior to conventional diagnosis. Serial quantitative serum GM measurements by ELISA were found to have some utility in determining the response to therapy [47,75]. A variety of PCR-based assays, including species-specific probes, have been developed and appear to hold more promise in the early diagnosis of IFIs [76,77]. Retrospective and prospective studies of the utility of PCR to screen the blood include a report of accurate identification of the genus and species of pathogenic fungi with a reported 100% sensitivity and 98% specificity. In one large study, colonization was infrequently associated with a positive PCR signal in the blood. However, the signal was positive only 4–7 days prior to a radiologic diagnosis and 5.7 (range 0–14) days prior to the institution of antifungal therapy. Additionally, the sensitivity decreases and the specificity increases depending on whether two rather than one test is considered to be the threshold for a diagnosis. Reports of less than optimal specificity and positive predictive value continue to be a concern [78,79]. The utility of a PCR assay was determined for 197 bronchoalveolar lavage samples from 102 immunocompromised patients. The sensitivity, specificity, and negative predictive value were 94.0%, 94.0%, and 98.1%, respectively, when a combination of culture, radiographic, and histologic studies were used to confirm the diagnosis [80]. The GM assay (optical density cut-off of 0.5) was compared with a quantitative PCR assay on bronchoalveolar fluid in 49 patients with proven or probable IA, and in uninfected control patients. The GM assay was found to have a sensitivity of 76% and a specificity of 94%, while the quantitative PCR assay had a sensitivity and specificity of 67% and 100%, respectively. The sensitivity of the GM assay and quantitative PCR in culturenegative cases was 59% and 36%, respectively. Thus, in some cases, these tests may serve as adjuncts in the evaluation of high-risk patients [81]. Molecular-based assays also offer the potential for a more rapid diagnosis. When compared with blood cultures, a multiplex PCR, designed to amplify unique sequences of C. albicans, provided results in 8 hours and had a sensitivity of 96.9% [36]. These important data and recent comparative studies confirm that no single test is yet able to serve as a reliable screening tool for blood or bronchoalveolar fluid but complements conventional diagnostic approaches. In addition to aiding in the diagnosis of IFIs, nonculturebased diagnostic modalities offer great potential in the development of strategies to stratify risk and/or develop pre-emptive treat strategies such as those utilized for CMV. Immune response to Candida spp. The association between neutropenia and IFIs has long been recognized; however, the significant increase in IFIs in immunocompromised hosts has brought into focus the role of other immune effectors in defense against infection. The morphologic changes the fungi undergo in the course of invasion, with the attendant changes in surface molecule expression, add to the complexity of host–pathogen interaction. The high incidence of invasive candidal infection associated with human immunodeficiency virus infection first highlighted the clinically significant role of the CD4+ cell in anticandidal defense. Studies demonstrate that invasive candidal infection follows adhesion of the organism to the cell surfaces, which induces proinflammatory cytokine production [82]. Various immune effectors are involved in the response to candidal infections. Neutrophils damage candidal pseudohyphae and phagocytose the blastospores [83]. Both dendritic cells (DCs) and macrophages attach to

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mannose–fucose receptors and ingest candidal blastospores, presenting fungal antigens to T cells. Unlike macrophages, DCs do not require opsonization of the candida, and serum does not enhance ingestion. DCs that ingest heat-killed Candida induced lymphocyte proliferation [84]. In vitro and mouse studies demonstrate that, following DC or macrophage presentation of candidal antigens, recovery from infection is dependent on CD4+ T helper type 1 (Th1)-type cytokine enhancement of monocyte and neutrophil function. In adoptive transfer studies, naïve CD4+ but not CD8+ cells were demonstrated to protect against death due to Candida. Studies using antibodies and/or knockout mice have demonstrated that interferon-gamma (IFN-γ), TNF, and interleukin-18 (IL18) are important in the defense against invasive candidal disease. Additionally, neutralization of IL-18, IL-1 or IL-12 results in a decrease in candida-induced IFN production. Therefore, the production and secretion of epithelial cytokines of the Th1 type appear to recruit activated lymphocytes in response to infection with Candida spp. [85]. The cellular immune response to Candida spp. is complex, and evidence suggests that specific effector populations may be linked to the route of invasion. Gamma/delta and alpha/beta T-cell knockout mice were more resistant than wild-type mice to disseminated candidiasis following intravenous injection. However, these mice are highly susceptible to candidal dissemination following high-dose oral challenge, as are transgenic mice with combined natural killer and T-cell defects [86,87]. The immune response against Candida spp. is not purely beneficial to the host. The role of antibodies in infection with Candida spp. is complex. Patients with disseminated disease may have high titers of immunoglobulin G (IgG); however, serum is not protective against infection. Studies in mice reveal that the administration of subsets of antibodies may actually potentiate disease progression, whereas others agglutinate blastospores, assisting with clearance of fungus. However, antibody-mediated agglutination of candidal organisms is not sufficiently protective. To complicate matters, Candida spp. produce proteases that digest IgG and IgA. The role of alveolar macrophages is equally complex. The ability of alveolar macrophages to ingest and destroy candidal organisms has been well established in vitro. Alveolar macrophage-deficient mice demonstrated reduced candidal clearing, and fewer recruited neutrophils due to a reduction in the level of the neutrophil chemoattractant macrophage inflammatory protein-2 and decreased myeloperoxidase activities. However, this attenuated immune response was associated with an improved survival. Fungal determinants of pathogenicity are not well understood but are, in part, due to varying degrees of immunogenicity and must be analyzed in the context of immunodeficiency. Species of Candida differ in their association with invasive disease. These differences may be explained by fungal determinants of virulence as well as variations in the intricacies of immunomodulation induced by the organism. Illustrative of these principles is the fact that C. glabrata is considered to be intrinsically less virulent. However, studies in mice demonstrate that the improved survival following C. glabrata infection may in part be due to an associated vigorous IFN-γ, TNF, and IL-12 response, and a minimal production of the anti-inflammatory cytokine IL-10. Similarly, death due to infection with C. albicans is associated with a delayed and severely reduced IFN-γ, TNF, and IL-12 response [88]. The immune response to different candidal forms is also specific. DCs appear to differentiate yeast from hyphae as the ingestion of early hyphal forms inhibits IL-12 production to a degree far greater than the quiescent yeast cells [89]. A few studies have begun to elucidate the dysregulation of Th-type cytokine production in mice following T-cell-depleted allogeneic HCT. Early post transplant, production of the Th2 type cytokines IL-4 and IL-10 was substantial, in contrast to a significantly impaired production

of Th1-type cytokines IL-12, IFN-γ, and TNF. Th1-type antifungal resistance could be restored following treatment with Th2 cytokine antagonists. In the haploidentical model, this shift to a Th2-type immunoreactivity was seen despite the establishment of full, donor-type chimerism by the second week post transplantation, and the mice were profoundly susceptible to lethal fungal infections. By 5 weeks post transplantation, the susceptibility to infection had resolved, and was temporally correlated with an improved Th1-type response [90]. Studies in immunosuppressed mice confirm that antifungal therapy did not result in optimal antifungal clearance unless a favorable cytokine response was restored. In this model, the concomitant administration of soluble IL-4 receptor, IL-12 or antibodies against IL-10 was synergistic with the administration of antifungal agents. In summary, although Th1-type cytokines profoundly influence the outcome of infection, recent observations in transgenic mice parallel clinical observations which suggest that the innate immune response is paramount, and that the key, but secondary, role of the adaptive immune response involves the development of resistance to reinfection [91]. Immune response to Aspergillus spp. A growing body of work is beginning to elucidate the immune response to Aspergillus spp. As observed with Candida spp., different morphologic forms of the fungus elicit different immune responses. The current understanding of the immune response to the conidia and hyphal forms of Aspergillus spp. is represented schematically in Fig. 89.2. Following inhalation of conidia, the alveolar macrophages and DCs are the first line of defense. Any conidial forms that escape are subject to clearance by tissue macrophages. IA is associated with hyphal germination and subsequent tissue destruction. Neutrophils are crucial in the clearance of the earliest hyphal (germlings) as well as the full hyphal forms [50]. The effect of polymorphonuclear cells on conidial growth is still unresolved, although recent evidence supports the role for mechanisms that are both dependent on independent of reactive oxygen species. Cell-free supernatants of deregulated polymorphonuclear cells that were able to suppress fungal growth were found to be rich in lactoferrin. Abrogation of this fungal suppression resulted from maneuvers to reduce lactoferrin or its ability to sequester iron [92]. Antigenic and/or metabolic differences between the morphologic forms may also influence adaptive immune responses, as studies have demonstrated that robust Th1 lymphocyte and humoral responses are seen only after inhalation of fungal spores that still retain the ability to germinate and invade [93]. A limited number of early animal studies demonstrated the importance of the acquired immune response. These reports demonstrated the prevention of aspergillosis following vaccination of turkey poults and the adoptive transfer of immune splenic macrophages in immunoincompetent mice [94,95]. Since these early studies, a number of preclinical studies have convincingly confirmed that immunomodulation with cytokines can enhance the antifungal activity of neutrophils and monocytes/ macrophages, as well as upregulate protective Th1-type adaptive immune responses. Further, the elements of this response are being defined. For example, DCs have been shown to have a pivotal role in the defense against aspergillosis in mice and human cells [96]. In addition to phagocytosing both conidia and hyphae, DCs migrate to lymph nodes and spleen, influencing T-cell reactivity [97]. Multiple recent animal and clinical studies have consistently demonstrated that the specific nature of the resultant T-cell reactivity influences the host’s ability to resist IA. As documented in the immune response to Candida spp., a Th1-type immunoreactivity, as indicated by production of TNF-α, IL-12, IFN-γ, and IL-2, is protective against intravenous or inhaled challenge with A. fumigatus. In contrast, Th2-type immunoreactivity is deleterious, and a high production

Fungal Infections after Hematopoietic Cell Transplantation

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Th1 response IL-12 IFN-γ TNF-α

Ab

DC B cell

IL-10 IL-12 IL-18

CD8+ TCR MHC II

Th2 response IL-4 IL-5 AB-mediated Direct complement opsonization

CD4+

MΦ MHC I

Germinating conidia Conidia Hyphae

Fig. 89.2 Immune response to conidia and hyphal forms of Aspergillus spp. AB, antibody; MΦ, macrophage; TCR, T-cell receptor. See text for other abbreviations.

of IL-4 and IL-10 correlates with an increased susceptibility to infection. These observations have been confirmed using appropriate knockout mice and cytokine neutralization [98,99]. Immunocompetent or immunized mice produce significant quantities of IFN-γ and IL-12 in the lungs and spleen, in contrast to susceptible cortisone-treated mice, which had an increase in IL-10 production in these organs. Rapid Toll-like receptor-dependent priming of A. fumigatus-specific T cells has been demonstrated in lymph nodes draining the lung, resulting in differentiation into IFN-γ-producing Th1 CD4(+) T cells. In contrast, T-cell proliferation, trafficking, and Th1-type differentiation in the airways were independent of Toll-like receptors and myeloid differentiation primary response gene [100,101]. Evidence of similar response pathways has been found in humans. Lymphoproliferative responses to A. fumigatus antigens were more robust in healthy individuals and patients with aspergillosis who were felt to have a favorable response to antifungal agents when compared with patients with progressive infection. Responses by a healthy individual included (1) a lymphoproliferative response to cellular extracts of A fumigatus, the 88 kDa dipeptidylpeptidase, and/or the 90 kDa catalase, and (2) the release of IFN-γ in culture supernatants on stimulation with A. fumigatus antigens. A higher IFN-γ:IL-10 ratio in culture supernatants correlated with a favorable clinical response to antifungal therapy. In contrast, administration of steroids suppressed Aspergillusspecific lymphoproliferation and release of IFN-γ. Evidence to support the therapeutic use of cytokines in immunocompromised hosts with IFI is, however, still scant and inconclusive. Highly purified human Th1type cells with activity against A. fumigatus in vitro and reduced alloreactivity have been generated from healthy donors [102]. Adoptive transfer of pathogen-specific, nonrecipient reactive lymphocyte clones resulted in a sustained high-level IFN-γ, low levels of IL-10, and a reduced mortality due to IFI in 35 recipients of a haploidentical HCT [103].

Despite these intriguing observations, it may be that nonmyeloid responses are only pivotal in situations such as profound T-cell depletion, delayed reconstitution or profound immune suppression that are characteristic of haploidentical and/or umbilical cord transplantation and GVHD. Indeed, the administration of anti-T-cell antibodies such as antithymocyte globulin or alemtuzumab (Campath-1H) has not been associated with increases in IFI in small studies of RIC or high-dose preparative regimens. Reducing the incidence of GVHD and its attendant immunosuppressive therapy would likely have the most profound effect on the incidence of and mortality associated with IFIs under most conditions of HCT [104,105]. Alternatively, inadequate or aberrant lymphoid reconstitution may result in attenuation of innate immunity. An effective antimicrobial response is typically characterized by a complex cytokine response, and the response to A. fumigatus is no exception. Increased levels of IFN-γ, granulocyte–macrophage colony-stimulating factor (GM-CSF), TNF-γ, and IL-2, but not IL-10 or IL-4, were detected in supernatants of co-cultured A. fumigatus and human peripheral blood mononuclear cells. TNF-α appears to be required for the initiation of the innate fungal response, and CXC chemokine subsets and their receptor CXCR2 affect myeloid recruitment. CCL3/macrophage inflammatory protein-1 alpha and the chemokine ligand monocyte chemoattractant protein/CCL2 (MCP-1/CCL2) are critical to recruitment of monocyte lineage leukocytes and NK cells, respectively. The role of natural killer (NK) cells has not been extensively studied, but some intriguing data suggest a potentially important role. Neutralization of MCP-1/ CCL2 resulted in impaired NK recruitment and an increase in fungal invasion and mortality, as did antibody depletion of NK cells [106]. Other host factors that predispose individuals to IA are being investigated. To date, groups have reported that polymorphisms in Toll-like receptor genes 1 and 6 or Fc-γ aRIIa R-131 may be associated with increased susceptibility to IA. Other polymorphisms were found to be

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associated with risks for acute GHVD, chronic GVHD, severe bacterial infections, and time to engraftment. Ultimately, donor and recipient gene polymorphisms may be used to analyze genetic risk factors that influence the selection of donor–recipient pairs and could help in the understanding of mechanisms involved in the host defenses of the bone marrow transplant recipient [107]. Effect of immunosuppressive agents Virtually all clinical studies have identified systemic corticosteroid use as a key risk factor for the development of IFIs. Hydrocortisone in vitro causes 30–40% acceleration in the growth rate of some species of Aspergillus [50]. Steroids have also been shown to impair alveolar macrophages, monocytes, and the neutrophil killing of fungi [52]. Steroid-treated mice had a delayed initial clearance of the conidia, increased splenic production of IL-10, poorly regulated IL-6 and IL-2b production, and an inflammatory response with few lymphocytes [100]. Data from murine models suggest that GM-CSF could promote resistance to conidia by maintaining proinflammatory responses including IL-1α, TNF-α, and macrophage inflammatory protein-1α secretion even in the setting of corticosteroid administration [108,109]. The immunologic sequelae of GVHD, however, further confound the clear understanding of the precise additional risk of IFIs resulting from the administration of corticosteroids. Calcineurin inhibitors have not been reported to be independent risk factors for IFIs, and it is interesting to note that cyclosporine has antifungal activity. In contrast, treatment of GVHD with the TNF-α blocker infliximab was associated with proven or probable IFIs in 19% of patients with severe GVHD. In a time-dependent Cox regression model among patients with severe GVHD, the adjusted IFI hazard ratio of infliximab exposure was 13.6 (p = 0.004; 95% confidence interval [CI] 2.29–80.2) [110]. Preparative regimen, graft manipulation, progenitor cells The preparative regimen for HCT may influence the incidence of IFIs. Protocols that result in significant mucositis were associated with an increase in invasive candidal infections. Protracted neutropenia increased the risk for invasive infections with both Candida and Aspergillus spp. Grafts composed of mobilized peripheral blood stem cells result in a substantially shorter period of neutropenia, and in vivo selection of CD34+ cells from an autologous graft did not result in an increase in IFIs [111]. A multicenter study of 1188 recipients of autologous transplantation confirmed a low incidence of IFI including proven or probable IA of 0.8% and candidemia of 0.3%. IFIs were diagnosed at a median of 35 days post transplantation [112]. The reported incidences of IFIs following RIC regimens compared with high-dose preparative regimens for are highly variable. This variability is not surprising given the diversity of these protocols with respect to drugs utilized, patient selection, GVHD prophylaxis, and care protocols [113–115]. It is notable that patients with a history of IFIs, including bilateral pulmonary infiltrates due to IA, have survived and even cleared infection following RIC regimens, a fact that suggests the potential benefit for rapid immune reconstitution with a minimal period of neutropenia. However, this observation has not been universal. The reported incidence of late IA following RIC transplant would be predicted to be dependent on the incidence of grade III or IV GVHD and the dose of steroid administration, as with all other HCT. Higher rates of IFI have been reported following haploidentical HCT and umbilical cord HCT, which have been attributed to profound T-cell depletion and protracted neutropenia, respectively. A variety of approaches to reduce transplant-related mortality are being actively investigated and include nonmyeloablative preparative regimens, ex vivo culture of cells, and multiple cord transplantation. These variables,

combined with other important factors such as T-cell depletion, the status of the underlying disease, and recipient selection, make direct comparison with recipients of a peripheral blood hematopoietic cell graft from a HLA-matched donor difficult [116,117].

Prevention and treatment It is very difficult to sterilize tissue that has been invaded with fungus, especially in the setting of immune compromise associated with allogeneic HCT; thus, relapse can occur in the event of subsequent immunosuppression. Given the morbidity and mortality associated with IFIs, preventative measures as well as therapeutic maneuvers are essential. Prevention of infection Transplantation strategies that reduce (1) the duration and degree of mucosal injury, (2) fungal colonization, (3) the duration and nature of the resultant immunodeficiency, (4) the severity of GVHD, and (5) the need for corticosteroids, parenteral nutrition or intravenous catheters would all contribute to a decrease in IFIs. Candida spp. is a mucocutaneous commensural organism, and violating the integrity of these surfaces is directly related to the risk for infection. In contrast, the incidence of Aspergillus has been shown to be related to environmental exposure, which may have occurred prior to the diagnosed infection. Environmental considerations Although not a common occurrence, transmission of candidal species has been documented from infections or colonization of the skin and nails of health-care workers. Simple maneuvers, such as strict handwashing protocols, prohibition of direct patient contact by health-care personnel who have fungal skin or nail infections, and staff education are successful. Interestingly, false or acrylic nails and dermatitis are associated with increased risk for superficial fungal infection and, therefore, nosocomial transmission. Central to the prevention of aspergillosis is the avoidance of inhalation of spores. Other nonendemic molds and emerging fungi are also most commonly found in the soil as they are saprobes and, by definition, feed or grow upon decaying animal or vegetable matter. Given published associations, avoidance of contact with soil and plants, including marijuana smoking, gardening, and maintenance of compost piles, would be prudent. The optimal duration of this prohibition is not clear. The efficacy of wearing high-efficiency particulate air (HEPA)-filtered air masks has also not been proven in controlled studies but is a reasonable consideration. Behavioral modifications that result from wearing the mask may theoretically decrease the transmission of other infections such as those due to respiratory viruses, especially as an outpatient. In the inpatient setting, a number of studies have demonstrated the benefit of laminar airflow rooms and HEPA-filtered air, which have been incorporated into most large transplant centers. Although it is noteworthy that Aspergillus has been isolated from water, prohibition of showers or bathing has not been demonstrated to reduce infection, but surveillance may be helpful to reduce risk [118]. The Centers for Disease Control publish recommendations regarding the reduction of environmental risks for reducing IFIs [119]. Administration of antifungal agents The efficacy of antifungal agents in the prevention of IFIs is an area of active investigation. Strategies include (1) primary prophylaxis, defined as the administration of antifungal drugs in the absence of any evidence or history of infection, (2) secondary prophylaxis, defined as the administration of agents to patients with a history of prior IFIs, and (3) empiric

Fungal Infections after Hematopoietic Cell Transplantation

treatment, defined as the administration of agents based on a defined but nondiagnostic clinical indication, but without proven IFIs. The prototypic empiric therapy is the use of antifungals in patients with fever during neutropenia. Primary prophylaxis The concept of primary chemoprophylaxis of IFIs has been supported by in vitro data and by clinical studies with respect to candidiasis. Studies in animal models of HCT demonstrated that administration of antifungals prior to infection was successful in improving mortality even when compared with therapy given early post infection [120]. Numerous clinical studies have been reported and are included in Table 89.1; however, few had a study design that would provide statistically significant results. Administration of fluconazole during the early posttransplant period through engraftment or day +75 post transplantation [121] has been shown to reduce the incidence of all invasive candidal infections (27% versus 8%) and, in particular, hepatic candidal infection (16% versus 3%) [9]. Additionally, after 8 years of follow-up, 68 of 152 patients who received fluconazole at a dose of 400 mg/day survived following allogeneic HCT, compared with 41 of 148 patients who received placebo from day 0 through day +75 (p = 0.001). In this same study, the incidence and mortality rate of severe GVHD were significantly higher in the placebo group; however, these observations could not be attributed specifically to a decrease in IFIs [121]. Despite the efficacy of fluconazole prophylaxis in allogeneic HCT, questions remain. The optimal dose and duration of prophylactic administration of fluconazole has not yet been determined. In one randomized study of 253 pediatric and adult HCT patients comparing 400 mg/day versus 200 mg/day of fluconazole during the neutropenic period, no significant difference was observed in IFIs by day +50 [122]. However, other studies have failed to demonstrate any protective benefit of 100 mg/day. Similarly, the role of fluconazole prophylaxis following autologous transplantation has not been thoroughly studied; however, it would be prudent to consider fluconazole prophylaxis if significant mucositis or prolonged neutropenia is expected to result. One major concern is that widespread implementation of fluconazole prophylaxis may result in the emergence of resistance in previously susceptible species of Candida or an increase in the incidence of infection with species intrinsically resistant to fluconazole. The development of azole resistance is a well-documented occurrence in the setting of therapy. Of potentially greater significance is that crossresistance to other azole antifungal agents may be engendered by exposure to fluconazole. Administration of antifungal agents may also have unexpected effects on the spectrum of fungal diseases. An interesting observation in one large retrospective analysis of patient outcomes and autopsy data is that, although there was no increase in infections due to resistant candidal species, there was an increase in the incidence of IA following the institution of routine fluconazole prophylaxis [9]. The investigators postulated that the reduction in death due to invasive candidal infections prolonged survival so patients eventually succumbed to mold infection. Finally, the administration of fluconazole may decrease the sensitivity of blood cultures especially with respect to C. albicans [123]. The major limitation of fluconazole is its lack of activity against molds. Thus, the availability of well-tolerated, broad-spectrum agents renewed the search for a regimen that would prevent the more lethal IFIs. Lipid formulations of amphotericin B, all three broad-spectrum triazoles, caspofungin, and micafungin have all been or are being studied for their utility as a prophylactic antifungal in various high-risk populations. All studies to date have confirmed the ability to prevent invasive candidiasis; however, the data regarding preventing proven mold infections and reducing fungal-related mortality have been limited [124].

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Prior to the introduction of lipid formulations of amphotericin and itraconazole, the only licensed, broad-spectrum antifungal agent was amphotericin B deoxycholate (D-AmB, Fungizone). The high incidence of nephrotoxicity associated with D-Amb limited the tolerated dose in high-risk patients (e.g. high-dose allogeneic HCT recipients) as the majority of these patients were receiving at least one other nephrotoxic agent. Lipid formulations of amphotericin accumulate in alveolar macrophages and may enhance killing of conidia when ingested by macrophages, but the published studies of the efficacy of aerosolized D-Amb or amphotericin B lipid complex (ABLC) lack a design that would provide statistically significant results [125–127]. Randomized trials to date have included investigations of prophylactic administration of lipid-associated amphotericin and itraconazole. Two randomized trials of lower-dose lipid-associated amphotericin have been published. Although underpowered to demonstrate a significant difference, a trend toward a decrease in IFIs was noted in which patients received 1–2 mg/kg three times per week of lipid-associated amphotericin during neutropenia. No difference in mortality was observed during a short follow-up period between the groups. Risk-stratified chemoprophylactic regimens and novel dosing schedules have also been studied, such as twice per week ABLC in patients on greater than 30 mg prednisone or once per week high-dose therapy (10 mg/kg AmBisome). These regimens were well tolerated, although efficacy could not be determined [128–130]. Two meta-analyses of randomized controlled trials of itraconazole as antifungal prophylaxis are worth noting as they arrive at different conclusions regarding the statistical significance of differences regarding documented IFIs and tolerability of itraconazole [131,132]. Both analyses, however, do conclude that itraconazole is more effective than fluconazole at preventing documented and suspected IFI [132,133]. One center described a successful strategy for prophylaxis against IFIs in high-risk post-transplant patients utilizing oral itraconazole or intravenous AmBisome (for those intolerant) until the resolution of neutropenia, the patient was taking less than 10 mg of prednisone per day or the disappearance of Aspergillus colonization. Although this is a descriptive trial, results from studies of a variety of antifungal agents and treatment strategies have recently been published or are in progress. The results of one such large, multicenter, randomized trial comparing micafungin with fluconazole in the neutropenic HCT recipient demonstrated that micafungin was as well tolerated and as efficacious as fluconazole in the prophylaxis of candidal infections, and superior with respect to the reduction in the incidence of mold infections [134]. However, this study only addressed prophylaxis during the early post-transplant period, when a minority of mold infections following HCT is diagnosed. In a smaller trial focused on pharmacokinetic analysis of the coadministration of micafungin with fluconazole, HCT patients who received both drugs had a trend toward a lower incidence of IFI than patients who had received fluconazole alone [135]. Most recently, two randomized, multicenter trials of posaconazole prophylaxis have been published. In the first, 304 patients with acute myelogenous leukemia or myelodysplastic syndrome who were neutropenic following chemotherapy were assigned to receive posaconazole for up to 12 weeks and were compared with patients who received either fluconazole (n = 240) or itraconazole (n = 58). The incidence of proven or probable IFI in the posaconazole group was 2% compared with 8% in the combined other groups. Although survival was significantly longer in the posaconazole group, serious adverse events were more frequently reported in the posaconazole group (6%) than in the combined group (2%) [136]. A second randomized, double-blind trial demonstrated that the overall incidence of IFI and fungal-associated mortality at 112 days following the initiation of antifungal drug in 301 patients receiving posaconazole was comparable to the incidence in 299

600

600

112

140

53 300

355

253

210

103

355

331 35

76

161

Clinical Trials Network

Ullmann (2007) [137]

Alangaden (1994) [173]

Winston (2003) [133]

Ehninger (1996) [174] Slavin (1995) and Marr (2000) [121]

van Burik (1998) [9]

MacMillan (2002) [122]

Nucci (2000) [175]

Foot (1999) [176]

Wolff (2000) [177]

O’Donnell (1994) [125] Riley (1994) [178]

Tollemar (1993) [179]

Kelsey (1999) [180]

2–3 months Until PMN engraftment

Until PMN engraftment Until PMN engraftment

1 mg/kg/day 2 mg/kg/day tiw

Until PMN engraftment

Until PMN engraftment

Until PMN engraftment then 100 mg/day through day +100 Until PMN engraftment

At least 5 days

Day +393 Day +75

Day +100

Until PMN engraftment

Up to 112 days

75–180 days

Duration of treatment

5–10 mg/day 0.1 mg/kg/day

400 mg/day vs. 0.2 mg/kg/day

5 mg/kg/day

100 mg bid

400 mg/day vs. 200 mg/day

400 mg/day

200–400 mg/day (Itra) vs. 400 mg/day (Flu) 200 mg/day 400 mg/day

100–200 mg/day

600 vs. 400 mg/day

400 vs. 400 mg/day

Dose

No significant difference in IFIs ↓ Rate of colonization

↓ Colonization

26 received Ampho B for persistent fever during neutropenia Trend toward decrease in IFIs in fluconazole group ↓ IFIs ↓ IFIs ↓ High-dose Ampho B

↓ IFIs

No infections in either group ↓ Early candidal death ↓ GI GVHD →Survival ↓ Hepatic candidiasis →Mold infections No significant difference in IFIs

↓ Proven or probable aspergillosis No difference in overall survival ↓ Proven or probable aspergillosis ↓ Death due to fungus ↓ IFIs and Candida albicans colonization →Candida glabrata colonization IFIs in Itra group

Effect of study drug on fungally related conditions

Included chemotherapy

All allogeneic, retrospective Fewer days of antibiotic therapy Trend toward decreased hospitalization and improved survival

Ampho B associated with greater toxicity

Included chemotherapy ↓ Ampho B use if protracted neutropenia Pediatric, majority (90%) HCT

Randomized, HCT Pediatric and adult patients

Autopsy

Included 10 pediatric patients (100 mg/day) 8-year follow-up

Patients with GVHD; Included galactomannan assay in diagnostic criteria Historical controls ↓ Ampho B use

Included galactomannan assay in diagnostic criteria

Comments

Ampho B, amphotericin B; bid, twice daily; Flu, fluconazole; GI, gastrointestinal; GVHD, graft-versus-host disease; IFIs, invasive fungal infections; Itra, itraconazole; PMN, polymorphonuclear leukocyte; tiw, three times a week.

AmBisome vs. placebo AmBisome vs. placebo

Fluconazole vs. ampho B Ampho B Ampho B vs. placebo

Itraconazole vs. placebo Itraconazole solution

Fluconazole

Fluconazole

Itraconazole or fluconazole Fluconazole Fluconazole

Voriconazole vs. fluconazole Posaconazole vs. fluconazole Fluconazole

Number Drug

Study

Table 89.1 Studies of antifungal prophylaxis following hematopoietic cell transplantation (HCT)

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Fungal Infections after Hematopoietic Cell Transplantation

receiving oral fluconazole as prophylaxis in the setting of GVHD following HCT, despite the fact that proven or probable IA was diagnosed in significantly fewer patients in the posaconazole group (2.3% versus 7.0%; odds ratio 0.31, 95% CI 0.13–0.75; p = 0.006) [137]. Interestingly, a large, multicenter randomized, double blind trial comparing fluconazole with voriconazole as fungal prophylaxis following high-dose conditioning and allogeneic HCT completed by the Clinical Trials Network and presented at the American Society of Hematology meeting in December 2007 reported that there was no significant difference in the proven, probable, and presumptive IFIs. Moreover, fungalfree, event-free, and overall survival rates at 6 and 12 months were similar in both groups. In summary, the data to date reinforce the limitations of universal prophylaxis. An ideal strategy would parallel the pre-emptive strategies used for CMV infection and involve serially screening patients with a sensitive test, such as the molecular diagnostic methods. The results of such an analysis would further refine the risk for IFIs, delineate treatment-related toxicity and risk for engendering antifungal resistance, and determine the cost of antifungal prophylaxis.

Fever during neutropenia A variety of multicenter studies using amphotericin B, lipid preparations of amphotericin, fluconazole, itraconazole, and voriconazole have not identified a superior drug or regimen as empiric therapy for fever during neutropenia. The use of weekly screening chest X-rays is recommended, although CT scans of the chest are more likely to detect lesions earlier and may be associated with an improved outcome in neutropenic patients, even in the absence of fever. However, the cost–benefit analysis of using CT to screen has not been definitively determined. Antifungal agents The armamentarium of available antifungal agents with a variety of mechanisms of action and routes of administration continues to grow. All licensed agents offer improved tolerability when compared with amphotericin B. The mechanism of action of the currently licensed agents is represented schematically in Fig. 89.3. Available formulations of licensed agents, dosing schedules, commonly encountered toxicities, and drug interactions are summarized in Table 89.2.

Secondary prophylaxis

Polyenes

It is difficult to determine the precise degree to which mortality is increased by a pretransplant history of IFIs. Outcome depends on a variety of factors, most notably the pathogenic organism, whether or not the infection has been cleared, or at least, stabilized, and the preparative regimen. Representative of several case reports is a series of 15 patients with a history of stable hepatosplenic candidiasis that underwent transplantation while receiving 0.5 mg/kg/day of amphotericin B. In 11 of the 15 patients, the abnormalities seen on CT scans were either improved or resolved, and in one patient the lesions were stable. The remaining three patients died with evidence of fungal disease; however, the pathogen identified was not the same as that identified prior to transplantation [138]. It is generally accepted that a history of Aspergillus likely represents greater risk than one of invasive candidiasis and adds to the mortality risk of HCT, particularly following the transplantation of grafts from HLA matched unrelated donors. Most reports have confirmed a higher mortality rate following HCT after a high-dose preparative regimen in patients with a history of IA; however, the cause of death may not be directly attributable to the fungus. Successful transplantation with low rates or IFI reactivation has been demonstrated following a variety of approaches to the stabilization of disease, including: aggressive primary therapy of the initial infections including surgical debridement; therapeutic dosing of antifungal agents during the peritransplant period, attempts at immunotherapy including IFN-γ, or the transfusion of granulocyte colony-stimulating factor (G-CSF)-mobilized white blood cells prior to engraftment [138–140]. Successful allogeneic transplantation following an RIC regimen has been reported in patients with either a history of or evidence of refractory IA. In one report, three of four patients with active Aspergillus demonstrated regression of fungal lesions and one with slight progression on chest CT [141]. One group has established a risk model for progression of infection based on 129 patients with a history of IA prior to undergoing an allogeneic transplantation following conventional high-dose conditioning or RIC. Prolonged neutropenia, advanced stage of underlying disease, and less than 6 weeks of antifungal administration prior to transplantation were associated with increased risk of progression in both groups. Progression of disease after day 30 post transplantation was associated with CMV disease, bone marrow or cord blood as source of graft, or grade II–IV GVHD. Using these risk factors, patients could be stratified with respect to incidence of progression of IFI following HCT [142].

Amphotericins: amphotericin B deoxycholate and lipid-associated amphotericin preparations. The binding of these drugs to ergosterol, a key element of the fungal cell membrane, results in the physical disruption of that membrane. The efficacy of amphotericin B, the first drug in this class, is limited by significant toxicities. Most common adverse effects include potentially severe infusion-related symptoms (fevers, rigors, cardiovascular instability, and hypoxemia), nephrotoxicity, hypokalemia, and anemia. The risk of nephrotoxicity increases with concomitant administration of other nephrotoxic agents, and full-dose amphotericin B is frequently not tolerated following HCT. Two lipidassociated preparations of amphotericin – ABLC and liposomal amphotericin – are currently available. These preparations differ in many respects due to the lipid with which they are complexed. Although neither of these formulations has been shown to provide superior efficacy in the treatment of IFIs, both have significantly fewer infusionrelated toxicities and are less nephrotoxic than D-Amb. There are no randomized dose–response studies, and doses as high as 15 mg/kg/day

Cytoplasm

Plasma membrane

Cell wall

Azoles [14C]a Demethylase

Polyenes

Hydrophobins

Ergosterol

Lanosterol

Galactomannan

Mannoproteins

Chitin

β1-3 glucan (α; β1–6)

β glucan

Echinocandins

Fig. 89.3 Site of action of licensed antifungal drugs.

Amphotec

Abelcet AmBisome

Diflucan

Sporonox

Vfend

Noxafil

Cancidas Mycamine Eraxis

ABCD

ABLC Liposomal amphotericin

Azoles Fluconazole

Itraconazole

Voriconazole

Posaconazole

Candins Caspofungin Micafungin Anidulafungin Broad Broad Broad

Broad

Broad

Broad

Candida spp.

Broad Broad

Broad

Broad

Spectrum

200–600 mg 6 mg/kg2 4 mg/kg bid 200–300 mg bid 100 mg bid – 200 mg tid

PO solution IV

PO PO

70 mg/50 mg 50–100 mg 100 mg/50 mg

Renal, hepatic

200–600 mg

PO capsules

IV IV IV

Renal, hepatic

200–800 mg 100–400 mg

PO IV

Hepatic ?Severe hepatic

Hepatic ?Severe renal, ?Severe hepatic

Renal, hepatic

Renal Renal, hepatic

200–400 mg

Renal

Renal Renal

Renal Renal

Renal

Dose adjustment

IV

2.5–7.5 mg/kg 2.5–15.0 mg/kg

2.5–5.0 mg/kg

PO topical IV IV IV

0.5–1.0 mg/kg

Dose

IV

Route

Hepatic GI, phlebitis, headache Alcohol is diluent

GI, hypokalemia, edema (hepatic) Visual disturbances, hepatic, mental status, rash, neutropenia As above Constitutional, GI, hepatic

GI, hypokalemia, edema (hepatic)

Hepatic, hypokalemia, edema

Hepatic

Infusion-related, nephrotoxicity, hypokalemia, hemolysis GI Infusion-related, nephrotoxicity Nephrotoxicity Nephrotoxicity

Most common toxicities

Increases caspofungin levels: CSA Increases levels of sirolimus, nifedipine, itraconazole CSA modestly increases anidulafungin levels

As above Increases levels of CSA, tacrolimus, phenytoin

As above for itraconazole except potentially more pronounced and more numerous

Decreases itraconazole levels: increased gastric pH, rifampin, isoniazid, phenytoin, carbamazepine, cisapride Itraconazole increases levels of: CSA, tacrolimus, steroids, digoxin, astemizole, cisapride, warfarin, vinca alkaloids, busulfan

Decreases fluconazole levels: rifampin Fluconazole increases levels of: CSA, phenytoin, glipizide, glyburide, warfarin

Less potential for nephrotoxicity than Ampho B Less potential for nephrotoxicity than Ampho B

Less potential for nephrotoxicity than Ampho B

Concomitant administration of other nephrotoxic agents increases risk for Ampho-induced renal failure

Drug interactions

ABCD, amphotericin B colloidal dispersion; ABLC, amphotericin B lipid complex; Ampho B, amphotericin B; bid, twice daily; CSA, cyclosporine; GI, gastrointestinal; IV, intravenous; PO, oral administration.

Fungizone

Polyenes Amphotericin B

Trade name

Table 89.2 Currently licensed antifungal agents

1358 Chapter 89

Fungal Infections after Hematopoietic Cell Transplantation

of liposomal amphotericin for infections involving the CNS have been reported. Echinocandins Caspofungin. Caspofungin (Cancidas) is an echinocandin lipopeptide that interferes with the synthesis of the major component of the cell wall of many clinically important fungi, including Candida and Aspergillus spp. Sites of Aspergillus hyphal growth appear to be particularly affected. Echinocandins inhibit the synthesis of (1,3)-β-D-glucan, and do not demonstrate crossresistance with other licensed antifungal agents in studies of in vitro susceptibility. The response efficacy of caspofungin treatment is equivalent to amphotericin B and fluconazole in the treatment of candidal esophagitis [143]. However, only 4–7% of patients on caspofungin, compared with 24% of patients on amphotericin B, discontinued the drug as a result of toxicity. Cyclosporine is known to increase levels of caspofungin; however, small case series have not revealed clinically significant toxicity resulting from co-administration of these two agents [144]. Tacrolimus does not appear to affect levels of caspofungin. Although licensed for the treatment of IA refractory to other licensed therapy, a very limited number of patients with IA have been reported, and further study is required to determine the true safety and efficacy of caspofungin in the treatment of this IFI following HCT. Hope remains for the use of this agent in combination therapy of IFIs. The utility of caspofungin has also been studied as empiric therapy in the setting of fever during neutropenia. In a randomized, multinational trial of 1055 patients with fever during neutropenia, noninferiority of caspofungin was established when compared with liposomal amphotericin and caspofungin in the incidence of breakthrough fungal infection, although caspofungin was better tolerated [145]. Anidulafungin and micafungin. Anidulafungin (LY303366, Eraxis) and micafungin (FK463, Mycamine) are recently approved enchinocandins. Anidulafungin has excellent activity against both Candida spp. and many Aspergillus spp. In a recent randomized, double-blind, noninferiority trial, anidulafungin was found to be noninferior to fluconazole in the treatment of invasive candidiasis [146]. Further, in vitro additive effects have been reported for anidulafungin in combination with amphotericin B against Aspergillus species and Fusarium species isolates, and synergistic activity when combined with itraconazole or voriconazole against Aspergillus species. It is distinguished by its degradation via a biotransformation process that does not involve the cytochrome P450 system with products excreted via the biliary system. Thus, it appears to be well tolerated even in hepatic and renal dysfunction [147]. Similarly, no difference in efficacy was seen between micafungin 100 mg/day and liposomal amphotericin 3 mg/kg in a comparative noninferiority study of 531 adult patients with candidemia or invasive candidiasis. Micafungin, however, was associated with fewer adverse events [148]. Findings of a multicenter, randomized trial of micafungin as prophylaxis during the early post-transplant period are discussed above. Azoles Azoles inhibit production of ergosterol, an essential component of the fungal cytoplasmic membrane. Resistance may result from increased drug efflux, or altered C14a-demethylase. Currently, the four most important drugs of this class for use in HCT recipients are fluconazole, itraconazole, voriconazole, and posaconazole. Unlike earlier drugs in this class, the newer triazoles do not significantly affect hormone production, but these have significant drug interactions with commonly used immunosuppressant agents. The degree of these interactions is influenced by individual factors, routes of administration, and

1359

differences in effects of the cytochrome P450 system. Differences in metabolism and in the inhibitory potency of cytochrome P450 3A4 and P-glycoprotein influence the onset, magnitude, and resolution of drug interactions and their potential effect on clinical outcomes [149]. Crossresistance between the triazoles is variable and may become a clinically more important issue following widespread use of these agents. The potential for antagonism between azoles and amphotericin has been described [141]. Fluconazole. After well-designed multicenter trials demonstrated that fluconazole (Diflucan), 400 mg daily, was efficacious in preventing invasive candidiasis and in reducing mortality due to fungal infection following allogeneic HCT [121], its prophylactic use in this setting has become widespread. Despite its excellent activity against most Candida spp., it is not useful in the treatment of infections due to Aspergillus spp. or most other emerging fungal pathogens. Candida glabrata and C. krusei demonstrate innate resistance to fluconazole. Fluconazole is available for parenteral and oral administration. Oral fluconazole is well absorbed, even in the setting of some GI disruption. The distribution of drug includes blood, tissue, bladder, and cerebral spinal fluid. Dose reduction in renal failure is recommended. Blood levels of cyclosporine, warfarin, phenytoin, and oral hypoglycemic agents may increase when administered with fluconazole. Adverse effects are distinctly uncommon, the most important of which is that elevations in aminotransferase levels may occur in as many as 10% of patients. Itraconazole. Itraconazole (Sporonox) is a broad-spectrum agent available in two oral formulations. The absorption of the original capsular formulation is so erratic that it is generally considered to be inadequate following HCT. The solubilization of itraconazole in cyclodextrin solution results in excellent oral bioavailability; however, the most common toxicity of both oral formulations is dose-related nausea and GI distress. Dose adjustment should also be considered in significant hepatic insufficiency. Great inter- and intrapatient variability of plasma levels has been observed, and monitoring of levels, although not readily available at all centers, is ideal. Antagonism with amphotericin has also been observed in in vitro testing [150]. Other toxicities include hypokalemia and edema, also primarily associated with higher doses. Rash is infrequent, but a variety of dermatologic eruptions has been described, including pustular lesions. Itraconazole has been demonstrated to increase the blood levels of several drugs including cyclosporine, tacrolimus, methylprednisolone, vinca alkaloids, benzodiazepines, and busulfan. Therefore, the potential for toxicity associated with itraconazole administration may result from its effect on other agents. Voriconazole. Voriconazole (Vfend) is a recently licensed triazole that is structurally related to fluconazole but has activity against both Candida and Aspergillus spp. Like itraconazole, it also has activity against Histoplasma spp., Blastomyces spp., and Cryptococcus neoformans. Voriconazole and posaconazole have potential activity against scedosporiosis and fusariosis. A great deal of attention has been paid to two recent randomized, multicenter trials involving voriconazole [151,152]. The first trial compared voriconazole with liposomal amphotericin B for empiric antifungal therapy in patients with fever and neutropenia [152]. Of the 837 patients included in the analysis, 415 had undergone HCT, including 76 (18.3%) and 79 (18.7%) recipients of allogeneic HCT who were assigned to the voriconazole and liposomal amphotericin arms, respectively. Voriconazole was administered intravenously at a dose of 4 mg/kg twice daily after two doses of 6 mg/kg. Oral administration of voriconazole (200 mg twice daily) was permitted after 2 days of intravenous administration. The target dose of intravenous liposomal amphotericin was

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Chapter 89

3 mg/kg/day; however, dose reduction to 1.5 mg/kg/day was permitted. The voriconazole group (median duration of therapy 7 [range 1–113] days) had fewer cases of severe infusion-related reactions (p < 0.01) and of nephrotoxicity (p < 0.001). However, a significantly higher incidence of visual disturbances (22% versus 1%; p < 0.001) and hallucinations (4.3% versus 0.5%; p < 0.001) than in those receiving liposomal amphotericin B (median duration of 7 [range 1–81] days) was also reported. It is also notable that there were fewer documented breakthrough IFIs in patients treated with voriconazole than in those treated with liposomal amphotericin B (1.9% versus 5.0%; p = 0.02). The significantly higher 30-day mortality in patients with breakthrough fungal infections demonstrated the limitations of empiric therapy. Thus, although voriconazole did not fulfill the protocol-defined criteria for noninferiority to liposomal amphotericin B with respect to overall response to empiric therapy, these results suggest a role in the empiric treatment of patients with persistent fever during neutropenia [151]. In a multicenter, randomized trial, voriconazole was compared to DAmb for the treatment of IA [152]. A total of 144 patients in the voriconazole group and 133 patients in the D-Amb group with definite or probable aspergillosis received at least one dose of treatment. Thirtyseven (25.7%) and 30 (22.6%) recipients of allogeneic HCT were assigned to the voriconazole and D-Amb arms, respectively. A detailed discussion of this study is beyond the scope of this chapter; however, a few key points are worth highlighting. First, a total of 102 patients were excluded from the modified intent-to-treat population. The most common reason for the lack of confirmation was the inability of the data review committee to confirm the presence of a halo or air crescent sign in patients without supporting mycologic or pathologic evidence (35 in the voriconazole group and 25 in the amphotericin B group). Following HCT, halo and air crescent signs are frequently not visualized, and, as described previously, these findings may represent an immune response. One of the most important observations of the study was that the mean duration of therapy was significantly different between the groups. The median duration of voriconazole treatment was 77 days, and other licensed antifungal therapy was given to 52 patients in the voriconazole group. The median duration of D-Amb treatment was 10 days, and, during the first 14 days of therapy, administration of D-Amb was suspended for more than 1 day in 13 patients. Other licensed antifungal therapy was given to 107 patients in the amphotericin B group. The survival rate at 12 weeks was 70.8% in the voriconazole group and 57.9% in the amphotericin B group, although the percentage of patients with a complete response was similar. Adverse effects of voriconazole included visual disturbances (44%) and hallucinations or confusion (10.4%), skin reactions including rash, pruritus or photosensitivity (8.2%), and hepatic inflammation (3.6%). In contrast, a significant percentage of patients receiving amphotericin B therapy experienced infusion-related (24.9%) and renal toxicity. Thus, the inability to administer full-dose amphotericin B, especially early after the identification of IA, may explain the difference in outcome between the two groups. Perhaps the most notable observation of the study was that 24% of patients with CNS aspergillosis survived, which may be due to the measurable brain tissue levels achieved during voriconazole treatment [152]. Voriconazole is available in an intravenous form and a highly bioavailable oral form. In healthy subjects, anaphylactoid type reactions have been described. The visual disturbances require some mention as they occur in as many of 44% of patients and are often infusion related. A variety of disturbances have been described, including altered or enhanced visual perception, blurred vision, changes in color vision, and photophobia. They may persist throughout the course of therapy; however, no long-term visual sequelae have been noted in animals or in clinical trials. Rashes, with or without photosensitivity, occurred in 6%

of patients. Hepatotoxicity may occur more frequently following HCT due to drug interactions with other potentially hepatotoxic medications. The list of known drug interactions is lengthy, and concomitant administration may result in increased levels of cyclosporine, tacrolimus, benzodiazepines, vinca alkaloids, ergot alkaloids, and omeprazole. Voriconazole is considered to be contraindicated when used concomitantly with sirolimus; however, several case reports confirm safe concomitant use if an empiric initial dose reduction of sirolimus is combined with monitoring of sirolimus levels [153]. Like itraconazole, the metabolism of the drug has been shown to be highly variable, although further study is necessary to demonstrate correlation between plasma or tissue levels and clinical outcome. Measurable plasma levels should be confirmed during therapy as very low plasma levels have been reported even in the setting of parenteral administration. Because of potential accumulation of the intravenous vehicle, patients with moderate-to-severe renal insufficiency (creatinine clearance <50 mL/min) or receiving hemodialysis should receive the drug orally, or be closely monitored if parenteral therapy is more appropriate. Dose reduction in these circumstances also results in a reduction in effective drug exposure, which thus may be subtherapeutic. In the case of significant hepatic insufficiency, the maintenance dose should be halved. Posaconazole. Posaconazole (Noxafil) is a newly licensed, orally bioavailable, extended-spectrum triazole antifungal agent with a favorable toxicity profile. Studies of the efficacy of posaconazole in the prophylaxis of IFI are discussed above. In case reports and small series, posaconazole has been shown to have potential as a salvage therapy for a variety of fungi, including refractory aspergillosis, scedosporiosis, mucormycosis, cryptococcosis, fusariosis, and coccidioidomycosis. Successful treatment of disseminated fusariosis with posaconazole during neutropenia and subsequent allogeneic HCT has been described [154]. The mean total exposure as defined by overall area under the curve or peak concentration at steady state was comparable in patients with and without mucositis. Despite this, it is possible that increases in dose or frequency may be necessary if absorption cannot be assured [155]. As is seen with other azoles, dose reductions for cyclosporine and tacrolimus are recommended. Posaconazole increased the maximum blood concentration and the area under the curve–time curve for tacrolimus by 121% and 358%, respectively, on day 14 compared with day 1 (both p = 0.001), but there was no effect on posaconazole pharmacokinetics [156].

Combination therapy Despite the vastly improved armamentarium of well-tolerated antifungal drugs, the mortality rate associated with IFI remains unacceptably high. The fact that the mortality rates remain higher following HCT than in any other at-risk population indicates that an improved understanding of the immune deficits will likely result in the most significant improvement in outcome. A parallel avenue of research into the efficacy of combinations of antifungals is also being driven by the high IFI-associated mortality rate. There is no paucity of case reports, limited case series or analyses using historical controls. Frequently, these reports involve therapy of infections due to emerging fungal pathogens such as Fusarium spp. or Scedosporium spp. given the high morbidity and mortality rates associated with these infections [157,158]. However, there has not been, to date, an adequately powered, randomized, protective analysis of a single agent compared with treatment with a combination of agents for these or any other IFI. In such a trial, accurate stratification of immunologic risk factors will be crucial in order to interpret the results.

Fungal Infections after Hematopoietic Cell Transplantation

Other agents In addition to the investigational echinocandins and triazoles referred to above, other agents being evaluated include liposomal formulations of nystatin, inhibitors of fungal elongation factor-2 or chitin synthesis, and immunomodulators. Immunotherapy Although the availability of more antifungal agents offers the possibility of improved survival from IFIs, all clinical and laboratory data support the need to improve reconstitution of an effective immune response to ensure fungal clearance. Adjunctive immunotherapy may take the form of administration of antibodies against well-defined antigens, transfer of adoptive immune effects, modification of graft composition, active immunization or the administration of immunomodulators. Hematopoietic cell growth factors A number of in vitro studies confirm the salutatory effect of macrophage colony-stimulating factor (M-CSF), G-CSF, and GM-CSF on cellular response to fungus and the shortening of the neutropenic period following chemotherapy and transplantation of bone marrow cells. GM-CSF has been shown to enhance the killing of Aspergillus hyphae and Candida spp., as well as prevent the suppression of neutrophil killing of A. fumigatus hyphae resulting from steroid administration [159]. M-CSF has also been demonstrated to enhance the ingestion and killing of fungi, but this agent is no longer being developed. Despite these findings, there has yet to be a definitive study documenting the reduction in incidence of or mortality due to IFIs. Changes in the graft composition have shortened the time to neutrophil engraftment, which has resulted in a significant reduction in the incidence of IFIs during the early post-transplant period. Therefore, it may be difficult to statistically appreciate any further reduction in the risk of IFIs resulting from the shortening of the median duration of neutropenia from 14 days to 10.5 days due to G-CSF after a high-dose regimen. In fact, post-transplantation administration of hematopoietic growth factors may have deleterious effects. Recent studies demonstrate that G-CSF treatment influences post-transplantation lymphocyte recovery, resulting in a potentially detrimental shift towards a lymphocyte profile associated with increased susceptibility to fungal infection. In a study of patients following T-cell-depleted haploidentical HCT, G-CSF administration post transplant impaired T-cell reconstitution without influencing the rate of engraftment or the incidence of GVHD. The most important observation with respect to intracellular pathogens and fungi was that the administration of G-CSF resulted in a profound Th2-type immunoreactivity, as measured by increased production of IL-4 and IL-10, concomitant decreased IL-12 production, and the absence of IL-12 receptor β2 chain expression by CD4+ cells. The appearance of IL-12-producing cells took longer than 12 months following G-CSF administration, in contrast to 1–3 months post transplant in patients who did not receive G-CSF. CD4+ and antifungal reactivity in vitro recovered more rapidly in the absence of G-CSF. Thus, in recipients of T-cell-depleted grafts, functional lymphoid immune response to IFIs may be negatively influenced by the administration of hematopoietic growth factors [160]. Adjunctive therapy with IFN-γ is actively being studied, with a particular focus on the treatment of IA and the influence on the incidence of GVHD. Antifungal vaccine strategies The antibody response to invasive candidal infection is complex and has been demonstrated to be both protective as well as deleterious. The role of antibodies in aspergillosis is not well understood. Therefore, a passive antibody approach for either fungus is not yet near development. A natural offshoot of the demonstration of the potentially significant nonmyeloid

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response and the ability to adoptively transfer resistance to disease in mouse models is the study of the efficacy of vaccination. In fact, killed vaccines against aspergillosis were reported decades ago when such fungal infections were clinical rarities. Differences in fungal strain tropism and virulence may influence the specific nature of the immunoreactivity, but progress has been made in the elucidation of antifungal immunity. Vaccination prior to challenge appears more effective in fungal clearance than vaccination or antibody administration following infection [161]. The study of possible vaccine strategies for aspergillosis continues. Vaccination of mice with a crude extract induces a Th1-type response comparable to that resulting from nasal instillation of conidia. Protection against invasive pulmonary aspergillosis was documented following adoptive transfer of antigen-specific CD4+ lymphocytes from these vaccinated mice. In contrast, vaccination with a recombinant antigen, Asp f 2, led to an increase in IL-4 production without an increase in IL-12, consistent with a Th2-type response, which would be predicted to increase susceptibility to fungus. In a more recent study, vaccination with either recombinant Asp f 3 or truncated versions resulted in apparent attenuation of illness and histologic changes associated with inhaled conidia. Further, histopathologic analysis suggested that vaccinated individuals had a preservation of enhancement of macrophage response in contrast to the predominantly neutrophilic tissue response in unvaccinated mice [162]. These complexities must be further understood prior to the development of an effective vaccine. Other investigational immunotherapy: pulsed DCs and graft modification In mouse models, splenic or bone marrow-derived DCs pulsed with candidal yeasts, or yeast RNA, enhanced reconstitution of IFN-producing CD4+ Candida-specific cells and protected against IFI following allogeneic HCT. These cells also expressed fungal mannoproteins, upregulated major histocompatibility complex (MHC) class II antigens and co-stimulatory molecules, and produced IL-12 [163]. Similar findings were reported using ex vivo-generated human DCs derived from CD34+ progenitors or monocytes harvested prior to autologous or allogeneic HCT. Following exposure of these DCs to Aspergillus antigens, these cells partially restored the fungal immunoreactivity of lymphocytes collected from patients 1 month post transplantation. These findings suggest that antigen-pulsed DCs, including those generated ex vivo, have the potential to effectively enhance immunity after HCT when the antibody response may be inadequate [96,164]. Enhancement of immune reconstitution may also be achieved via modification of graft composition. In studies of murine HCT, co-transplantation of lineage-restricted progenitors known as common myeloid progenitors (CMPs) and granulocyte–macrophage progenitors (GMPs) resulted in a significant increase in the absolute number of myeloid cells, the majority of which were CMP/GMP-derived. Co-transplantation of GMP/GMP with hematopoietic stem cells protected against death following lethal challenge with either of two lethal pathogens associated with neutropenia: A. fumigatus and Pseudomonas aeruginosa. Survival correlated with the measurable appearance of progenitor-derived myeloid cells in the spleen despite persistent peripheral neutropenia [165]. Of potentially greatest clinical utility is the demonstration that the protection resulting from myeloid progenitor infusion was not MHC restricted [166]. Examples of other areas of active investigation include attempts to attenuate the risk of profound neutropenia by in vitro expansion of autologous myeloid populations, and cotransplantation of CD34+ cells from an HLA-haploidentical donor [167]. Granulocyte transfusions Although granulocyte transfusions may be a logical consideration to attenuate the risk of neutropenia, profound technical challenges impede

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their implementation. Historically, small case series have resulted in equivocal data. In more recent studies, the use of G-CSF for leukocyte mobilization has been associated with an improved outcome and the need to transfuse less frequently when compared with corticosteroidmobilized granulocytes. G-CSF has been shown to improve the yield of leukocytes, and, of potentially greater significance, G-CSF-mobilized apheresis products contain a high percentage of myeloid progenitor cells (Brown, unpublished data). Recent nonrandomized studies and numerous case reports support the safety and potential benefit of this adjunctive therapy to either control infection or reduce risk of recurrent infection peri transplantation or during intensive chemotherapy. Repeated infusions from the same and different donors are typically necessary. In fact, in one elegant analysis, the increase in the absolute neutrophil count was higher or comparable following a transfusion from a community donor versus a family member. Other strategies to enhance and/or accelerate myeloid reconstitution that may prove to be therapeutically feasible include ex vivo expansion of CD34+ cells and activation of myeloid cell lines [168–170]. Surgical approaches to IFIs The role of surgery in invasive candidal infections is limited to intravascular lesions such as endocarditis and thrombophlebitis. However, there is a growing body of data to support combination medical and surgical therapy for invasive mold infections in patients with hematologic malignancies, most of whom were neutropenic at the time of surgery, but the potential risk versus benefit is typically more difficult following HCT [171]. Small series have been published describing survival following both open and thoracoscopic approaches in the excision following HCT, even in the setting of GVHD, but this success was not universally observed. Resection should be considered in patients who are deemed acceptable surgical candidates with (1) a single lesion, (2) one predominant, large lesion, or (3) lesions abutting vascular structures given the high risk for hemorrhage. Even in the setting of disseminated or CNS disease, there may be a role for surgical intervention, which may include drainage, excision, and/or instillation of antifungal agents. Successful surgical approaches often include the intraoperative frozen section demonstration that the margins of the excision are free of disease. This approach is perhaps of greatest importance in surgery involving the sinus or periorbital regions. Intralesional administration of antifungal agents (typically amphotericin B) has also been described in cavitary pulmonary and CNS lesions, with and without an attempt at excision, but the data are limited to individual case reports. Surgical debridement is almost invariably necessary for the successful treatment of fungal sinusitis, skin lesions, and intravascular lesions following HCT; however, survival benefit has not been definitively demonstrated. Fungal eye infections deserve special mention given their high risk for dissemination and the diagnostic and therapeutic challenges. These infections should not be treated without the active participation of an experienced ophthalmologist to determine the accurate diagnosis and response to treatment. Fluid and/or tissue sampling or intravitreal instillation of antifungal agents is frequently necessary. Conflicting data exist regarding the optimal antifungal agent.

Approach to the patient with suspected invasive fungal disease following HCT Outcomes are better for patients who are diagnosed and treated as early and as aggressively as can be tolerated. Patients with persistent, unexplained fever should be treated empirically while being evaluated for IFIs. Although diagnostic blood tests have not been validated, they may, if they are available, provide adjunctive data; however, further evaluation should proceed immediately (see Plate 27.45).

In the setting of documented candidemia, immediate central venous catheter removal is strongly recommended. A CT scan with intravenous contrast or MRI of the chest and abdomen should be performed to determine the extent of disease in order to select the appropriate antifungal agent as well as the necessary duration of therapy. There are no randomized trials to guide the duration of therapy in either disseminated candidal infections or IA. However, symptomatic and/or radiologic exacerbations are not uncommon if therapy is stopped while patients remain on immunosuppressive agents or have persistent radiologic abnormalities. Two to 6 months of therapy are the rule, rather than the exception, during which time serial CT scans should be performed. In vitro susceptibility testing should be performed on any nonalbicans Candida spp. or C. albicans isolates obtained in the setting of fluconazole therapy. However, the correlation between nonazole susceptibility and clinical outcome has not been determined. The vast majority of C. albicans remains susceptible to all agents; however, the intrinsic resistance patterns of certain species, including C. krusei (intrinsically resistant to fluconazole), C. glabrata (variably resistant to fluconazole and amphotericin), and C. lusitanea (resistant to amphotericin), should be considered, and appropriate agents chosen pending the results of in vitro susceptibility testing [172]. As patients with IA may be asymptomatic, abnormal findings on CT scans should prompt attempts to isolate the organism, including induction of sputum, bronchoalveolar lavage, and/or biopsy for accessible lesions. Culture of the specimens is crucial to confirm identification of fungus and determination of in vitro antifungal susceptibility. Given the potential of CNS involvement in IA following HCT, it would be prudent to consider an MRI in invasive disease. Symptoms of sinus involvement should prompt a CT scan of the sinuses, and surgical evaluation is warranted if invasive sinusitis is documented. For all IFIs, empiric therapy should not be delayed during clinical evaluation. Antifungal CNS penetration should also be considered if CNS disease is documented. First choice should be given to drugs that can be safely administered at full dose. Even with agents with excellent oral availablity, it is prudent to administer agents parenterally until stabilization or improvement of infection can be demonstrated, especially in the setting of GI pathology such as GVHD. Increasing reports of noncandidal, non-Aspergillus infection or fungi resistant to various antimicrobials with increasing frequency underscore the importance of aggressive attempts to isolate the organism. Although the correlation between in vitro fungal susceptibility testing and clinical outcome has not yet been proven, it is prudent to avoid agents against which the fungus demonstrates in vitro resistance. In vitro fungal susceptibility testing will likely gain increasing importance. As discussed, there is potentially a role for surgical excision or debridement in selected patients. Reducing steroid dosage as rapidly as possible is also crucial to clearance of fungus.

Conclusion IFIs remain the leading cause of infectious death following allogeneic HCT at most large centers. Strategies to reduce the incidence of GVHD, to lower the dose of corticosteroids, and to enhance the immune response to fungi following HCT, coupled with a growing armamentarium of better-tolerated antifungal agents, offer hope for a reduction in the morbidity and mortality of these infections. Surgery in selected cases may also result in improved survival. Prophylactic and empiric strategies based on improved diagnostic assays are currently under study. Modification of the graft composition designed to accelerate the recapitulation of fungal immune response without increasing GHVD would be ideal. The specter of emergent fungal infections cannot be overlooked, especially following protracted administration of broad-spectrum antifungal drugs and as the use of these drugs becomes more widespread.

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John A. Zaia

Cytomegalovirus Infection

Introduction Since the era of hematopoietic cell transplantation (HCT) began, human cytomegalovirus (CMV) has been a problem that has limited the success of the procedure [1]. This chapter describes CMV infection as seen from two perspectives: the natural history of CMV infection defined when there were no effective antiviral agents, and the clinical aspects of CMV infection today when antiviral therapy and a new understanding of host immunity to CMV have become available [2]. The chapter brings a description of these aspects of CMV to the student, the transplant physician, and other interested medical care providers who deal with the continued problems of prevention and management of this infection.

Molecular and clinical biology Structure and biology of human CMV CMV, also known taxonomically as human herpesvirus-5, is classified as a β herpesvirus and is a DNA virus with typical herpes virion structure (Figs 90.1 and 90.2). The genomic size of the DNA is approximately 240 kilobase pairs, making it the largest of the known herpesviruses [3], and this large genome encodes nearly 200 proteins based upon an analysis of open reading frames [4]. A more detailed virologic description of CMV is beyond the scope of this chapter, and the interested reader is referred to reviews of this subject [5]. From a clinical standpoint, a significant aspect of viral replication is the ability of the CMV genome to exist in cells in either an active or a quiescent state of transcription. In the active state of transcription, there is a sequential expression of three categories of genomic elements termed immediate early (IE), early, and late viral proteins. The importance of this phenomenon to the clinician is that the inhibition of viral DNA replication with antiviral drugs such as ganciclovir does not stop IE gene transcription and, although it can restrict new virus production, it would not affect potentially pathologic processes mediated by IE proteins. Interestingly, virus-induced cytopathology is present in vitro at times when only IE gene expression has occurred. For example, immediately after infection, basic housekeeping proteins are downregulated [6], and a family of proteins encoded in the unique short region of the

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

genome interferes with peptide processing in the cytosol and endoplasmic reticulum [5,7]. Although the actual role of these IE–early virus proteins in the pathogenesis of CMV disease is not well understood, it is possible that a component of CMV pathology is contributed by these gene products. The ineffectiveness of the CMV inhibitors, which act by blocking viral DNA replication, to effectively treat advanced CMV infection suggests that this concept might have some clinical relevance. CMV latency The term “latency” refers to the presence of the viral genome in the absence of immediate and active production of infectious virus. After primary CMV infection, as indicated by seroconversion using antibody assays for CMV, the virus can become inactive, or latent, and then subsequently reactivate. Antibody-positive persons, whether they are HCT donors or recipients, carry CMV and are at risk for future reactivation of infection. Active infection is detected by markers for CMV, such as rising CMV DNA load or antigenemia in the blood using polymerase chain reaction (PCR)-based or antigen-based assays, or actual detection of virus in tissue culture assays. If a patient is seronegative for CMV antibody and has no detectable markers for infectious virus, it is likely that there is no latent infection present. Although false-negative serologic results occur, for practical purposes, CMV-seronegative hematopoietic progenitor cell transplant donors (D−) virtually never transmit CMV to seronegative recipients (R−). The donor who is seropositive (D+) for CMV antibody and has no detectable evidence for infectious virus is assumed to have latent CMV infection, and the blood products from this person are infectious for the marrow recipient whether CMV seropositive (R+) or R−. Studies have demonstrated the ability to isolate infectious virus from peripheral blood mononuclear cells that were placed in long-term culture with allogeneic stimulation [8]. Thus, it appears that a site of latency of CMV is the blood monocyte, and following certain signal transduction events leading to cytokine stimulation, the virus can be activated from latency. Clinical observations have indicated that transmission of virus by blood is relatively inefficient compared with transmission by solid organs such as kidney and heart [9]. The actual activation of the virus within these transplanted cells appears to be dependent on cellular activation signals [8], and such cellular activation might explain the peak period of CMV infection as the new graft becomes established 4–8 weeks post HCT. It is possible that other cells of the blood can serve as a vehicle for transmission of CMV with transfusions, and CMV has been demonstrated in circulating endothelial cells after HCT [10].

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Fig. 90.1 Electron micrograph of cytomegalovirus (CMV) virions and defective particles. CMV-infected human foreskin fibroblasts were subjected to glutaraldehyde and osmium tetroxide fixation and electron microscopy, revealing intact virions (solid arrows), defective virions (open arrows), and dense bodies (arrowheads). Magnification: ×38,750; inset magnification: ×182,500. (Photomicrograph prepared by John Hardy, City of Hope National Medical Center.)

Envelope proteins gB (gpUL55) gM/gN (gpUL100/73) gH/gL/gO (gpUL75/115/74)

Internal proteins Tegument pp65 (ppUL83) pp71 (ppUL82) pp150 (ppUL32) pp28 (ppUL99)

CMV

Capsid MCP (pUL86) p34 (pUL46) p37 (pUL80a)

Non-structural IE1 (pUL123) IE2 (pUL122) DNApol (pUL54) US 2-11 (pUS2-11)

Fig. 90.2 Significant proteins of human cytomegalovirus (CMV). A schematic cross-sectional representation of the CMV virion and infected cells is shown indicating the components of the viral envelope, the internal proteins, and the nonstructural proteins. The alphanumeric labels refer to the usual names of these proteins with the official nomenclature in parenthesis.

Epidemiology and etiology of CMV-associated disease Changing pattern of CMV epidemiology The epidemiologic description of CMV infection in the HCT population is well described and has established that CMV infection can originate from both endogenous and exogenous sources [11]. The curtailment of blood-borne CMV exposure, by cessation of routine granulocyte transfusion therapy after HCT and by the use of filtered blood product support, demonstrated that CMV can be transmitted in transfused blood cells to

a susceptible recipient. However, the major effect on CMV epidemiology was the introduction of effective antiviral agents during the 1980s. The incidence of unmodified CMV infection and disease is best described by the control groups from the blinded studies that attempted to alter the occurrence of CMV complications after HCT. Thus, the incidence of CMV infection in D−R− HCT dropped from approximately 20% to nearly 0% with the introduction of CMV-seronegative blood support [12]. In D+R+ and D−R+ transplant recipients, the incidence of CMV disease was fairly stable until the introduction of effective antiviral chemotherapy in the 1990s. Pre-emptive antiviral treatment strategies dramatically reduced the early occurrence of CMV disease but moved disease onset to late times after HCT. As shown in Table 90.1, in the 1980s, the disease occurred at a median time of 50–60 days post HCT, but with the introduction of pre-emptive ganciclovir therapy, the median time of onset has shifted to 160–176 days post HCT. Thus, our current therapeutic maneuvers used to prevent early CMV disease have altered the natural history of infection, leading to so-called “late-onset” disease [13]. CMV-associated disease in HCT allograft recipients in the pre-antiviral era The first descriptions of CMV-associated disease in marrow allograft recipients appeared in the 1970s and described a relatively new disease, CMV-associated interstitial pneumonia (CMV-IP) [14,15]. CMV infection occurred in 42–69% if the HCT recipients were CMV seropositive pre HCT (reactivation of CMV), and in 13–36% if they were CMV seronegative (primary CMV infection). The rate of CMV disease also followed this pattern and occurred in 15–25% of the CMV seropositive recipients. The most detailed early analysis of CMV infection in marrow allograft recipients was made by Meyers et al. [16] in a prospective assessment of infection in 545 HCT recipients studied between 1979 and 1982. Because this study so accurately reflects the epidemiology of CMV in the period prior to effective intervention, the details of this study are worth reviewing. Evidence of CMV infection, based on positive culture and/or seroconversion, was present in 51% of patients. Of these, infectious virus was cultured from 43%, and CMV seroconversion was noted in 31% of patients. Among the infected patients, approximately 40% had virus recovered without undergoing antibody seroconversion, indicating the poor reliability of antibody determinations in this population, and an additional 16% underwent seroconversion without isolation of virus. The median time to infection in the throat and urine was 54 and 59 days, respectively, after HCT. The excretion of virus and seroconversion were significantly associated with the serologic status of the recipient and donor prior to HCT. The incidence of CMV infection in D+R− transplants was significantly increased over D−R− transplants (53% versus 26%; p < 0.001). This risk factor for CMV infection persisted even when other seropositive blood support was used in the seronegative HCT recipient. This observation underlines the importance of the donor hematopoietic cell source in contributing to an exogenous source of CMV infection [16]. This important aspect of CMV epidemiology was shown in the pretreatment era and continues today for the CMV infection rates analyzed according to donor and recipient pre-HCT CMV antibody (Table 90.2). At that time, the rate of CMV infection was greatest in the CMVseropositive recipient groups, regardless of negative or positive donor serology (i.e. both D+R+ and D−R+). In the seronegative recipient group in the 1980s, when there was minimal protection from exposure to CMV in blood products, both the D+R− and D−R− groups had relatively high infection rates [12,17]. But with the protection of the seronegative recipient from CMV by filtration of blood products, the D−R− group now has minimum risk for CMV infection [18].

Cytomegalovirus Infection

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Table 90.1 Changing epidemiology of cytomegalovirus (CMV) disease after allogeneic hematopoietic cell transplantation Reference

Year

Pre-emptive treatment

CMV disease (%)

Median day of disease onset

Winston et al. [19] Bowden et al. [118] Schmidt et al. [25] Zaia et al. [28]

1987 1991 1991 1995

Einsele et al. [29]

1995

Boeckh et al. [30] Zaia et al. [119] Boeckh et al. [13]

1996 1997 2003

No No No Yes – BAL No† Yes – BC Yes – PCR Yes – AG Yes – BAL/BC Yes – AG

17/37 (46) 12/60 (20) 30/84 (36) 11/103 (1)* 4/60 (7)† 13/34 (38)‡ 5/37 (14%)§ 23/114 (20) 10/117 (8.5)** 26/146 (18)¶

52 60 51 60* 125† <100‡ >100§ 160 176** 169¶

Results include only control groups from clinical studies in which there was no pre-emptive treatment strategy, except for Schmidt et al., in which the pre-emptive treatment control group is used. AG, antigenemia assay; BAL, bronchoalveolar lavage; BC, blood culture; PCR, polymerase chain reaction. * Data are from subjects undergoing routine bronchoalveolar lavage (BAL) who received early ganciclovir treatment for positive CMV culture in BAL specimens. † Data are from subjects undergoing routine BAL who did not receive ganciclovir because of negative CMV culture in a BAL specimen. ‡ Data are from subjects undergoing routine CMV BC surveillance who received early ganciclovir treatment for positive CMV culture. § Data are from subjects undergoing routine CMV leukocyte PCR assay who received early ganciclovir treatment for positive CMV PCR. ¶ Data are from subjects undergoing routine CMV leukocyte AG assay who received early ganciclovir treatment for positive CMV AG. ** Data are from subjects undergoing routine CMV culture of BAL and blood who received early ganciclovir treatment for positive CMV BAL or BC.

Table 90.2 Incidence of cytomegalovirus (CMV) infection in marrow allograft recipients by donor/recipient CMV serology CMV antibody in recipient R+ (%)*

R− (%)*

Source

CMV antibody in donor

Meyers et al. [16]

D− D+

81/118 (69) 97/140 (69)

58/208 (28) 44/77 (57)

Miller et al. [17]

D− D+

15/21 (71) 13/23 (57)

20/107 (19) 6/30 (20)

220/393 (56) 268/467 (57)

13/628 (2) 31/262 (12)

Nichols et al. [18]

−

D D+

CMV antibody determination prior to hematopoietic cell transplantation (HCT). D, donor; R, recipient. * Percent with CMV infection documented post HCT.

The incidence of CMV infection and disease during this pretreatment era is recorded in the control arms of clinical trials of the various candidate agents evaluated for anti-CMV effect in allograft populations (Table 90.3). The data from five centers are shown separately for marrow recipients with and without pretransplantion CMV antibody, and compared with results obtained when more current pre-emptive anti-CMV strategies were in place. Again, the high rates of infection and disease in the studies by Winston et al. and Bowden et al. [19,20] fell dramatically with the introduction of CMV-negative blood support (Table 90.3) [21,22]. Note that, even in the latter part of the 1980s, the control groups that did not receive selected blood support had high infection rates, suggesting that other anticancer or anti-graft-versus-host disease (GVHD) management regimen changes did not account for the decrease in CMV attack rates [12,17,23,24]. In the current era, for the CMV-seronegative recipient, approximately 10–15% will develop CMV blood infection from the donor or from blood product support, and up to one-half of these will still get disease [18].

For CMV-seropositive recipients in the pre-antiviral era, infection rates ranged from 45% to 86%, and prevalence rates for CMV disease ranged from 21% to 43% (Table 90.3) [21–26]. Of note, the availability of antivirals does not alter the CMV reactivation rate, unless they are used prophylactically (see below), and CMV infection continues to occur in up to two-thirds of all CMV-seropositive recipients [18,27]. It was thought that if this reactivation of CMV could be controlled by anti-CMV chemotherapy, the problem of CMV infection should be mitigated. The historic data in Table 90.3 indicate that CMV disease has been decreased, but antiviral agents have not solved the problem. CMV infection and disease rates in HCT allograft recipients in the post-antiviral era As indicated in the studies of pre-emptive ganciclovir therapy, the incidence of CMV reactivation has remained 60–70% despite the fact that the diagnostic methods that confirm this infection rate have improved considerably in sensitivity [27,28]. The prevalence of CMV infection from past observations using blood culture or antigenemia as a virus detection method [23,25,29,30] remain the same as with use of modern PCR methods [29,31]. On the other hand, the use of antiviral agents at the time of engraftment or when CMV infection first occurs reduces the incidence of CMV disease in the first 100 days post HCT to 1–2%. However, when treatment ends, the eventual incidence of CMV disease in a cohort can be expected to be as high as 16% [24,30]. In the largest analysis of outcomes since the onset of pre-emptive anti-CMV treatment of HCT recipients, Nichols et al. [18] have reported that, in 1750 HCT recipients treated between 1992 and 1998, CMV disease occurred in 10.6% overall and with 15.8% of those transplants in which either the recipient or donor or both were CMV seropositive. A recent report from Einsele et al. [27] confirms the continued high rate of CMV blood infection after HCT. Importantly, the advent of reduced-intensity conditioning for allogeneic HCT has not changed the incidence of CMV reactivation [32]. Infection rates in autologous/syngeneic recipients Wingard et al. [33] have reported the incidence of CMV infection in a cohort of 143 autologous marrow recipients to be 45%. However, in

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Table 90.3 Historic incidence of cytomegalovirus (CMV) infection and disease

Bowden et al. [20] Winston et al. [19] Sullivan et al. [21]* De Witte et al. [22]† Bowden et al. [118] Bowden et al. [12] Miller et al. [17] Nichols et al. [120] Meyers et al. [26] Sullivan et al. [21] De Witte et al. [22] Schmidt et al. [25] Goodrich et al. [23] Goodrich et al. [24] Nichols et al. [120]

CMV antibody in recipient pretransplantation

Infection (%)

Time

Disease (%)

Time

No No No No No No No No Yes Yes Yes Yes Yes Yes Yes

8/20 (40) 21/37 (57) 1/58 (2) 0/28 (0) 26/60 (43) 7/30 (23) 21/61 (34) 44/890 (5) 49/65 (75) NP (50) 42/48 (86) 56/104 (54)‡ 107/194 (55) 14/31 (45) 488/860 (57)

d 56 d 53 NA NA d 54 d 59 NP NP d 40 NP NP d 35 d 40 NP NP

3/20 (15) 17/37 (46) 0/61 (0) 0/28 (0) 12/60 (20) 2/30 (7) 3/55 (5) 21/890 (2) 20/65 (31) 67/308 (22) 10/48 (21) 84 (36) 15/35 (43) 9/31 (29) 164/860 (19)

d 58 d 52 NA NA d 60 NP NP NP NP NP NP d 51 NP NP NP

Results include only control groups from these clinical trials, except for [22], which was a prospective but uncontrolled study, and [25] and [23], in which CMV infection data were collected prior to therapy and hence include all patients. NA, does not apply; NP, data not provided in source document. * Data from the subgroup of subjects who were CMV-seronegative recipients of a CMV-seronegative donor marrow and who received CMV-seronegative blood support. † Study includes only CMV-seronegative recipients receiving leukocyte-poor blood support. ‡ Study includes only the CMV infection rate for bronchoalveolar lavage specimens.

these autologous HCT recipients, there was only a 2% rate of CMV disease, suggesting that pathogenesis is a function of factors linked to allogeneic transplantation. In that study, the incidence of CMV-IP was no different than that of the allograft recipients having no acute GVHD. Reusser et al. [34] have also studied the epidemiology of CMV in autologous marrow recipients and noted a rate of approximately 10% CMV disease in this same population. Boeckh et al. [35] reported that, in a group of 67 autologous marrow or blood progenitor cell recipients, CMV reactivated in 39%, as determined by antigenemia assay, and CMV disease occurred in 6% of 67. In a comparable group analyzed by leukocyte PCR, Hebart et al. [36] described a similar rate of CMV reactivation, with a 7.5% incidence of disease. Other recent studies of autologous HCT, including those in which selected CD34+ progenitor cells were used, indicate a variable incidence of associated CMV disease in this population. The risk for CMV disease in this population has been linked to heavy pre-HCT chemotherapy, concomitant anti-T-cell therapy, and use of CD34-selected peripheral blood progenitor cells. In syngeneic transplants, on the other hand, Appelbaum et al. [37] reported that, despite a similar occurrence of CMV infection, there was no CMV disease in 100 recipients of syngeneic marrow. Rare occurrence of CMV disease among this group would likely be due to the same risk factors as noted for autologous HCT recipients.

Immune response to CMV infection Cellular immunity to CMV As noted almost three decades ago, cytotoxic T-lymphocyte function is most critical for protection from severe disease and mortality in the setting of HCT [38], and HLA-restricted cytotoxic T-lymphocyte function was associated with survival from CMV-IP [39,40]. Subsequently, using new methods of quantifying CMV-specific cell-mediated immunity, studies demonstrated that patients lacking CD4-specific CMV recognition were at greater risk for CMV complications [41,42].

Immunologic recovery of CMV-specific cellular immunity by 3 months post HCT is a critical factor in protection from CMV infection, and the factors indicating that a recipient is high risk for CMV due to impaired cellular immunity include use of high-dose corticosteroids, defined as more than 1 mg/kg/day, CD4+ cell count less than 100 × 109/L, and CD8+ cell count less than 50 × 109/L [43]. The two predominant immunologic targets of CMV-specific cellmediated immunity are CMVpp65 and CMV-immediate-early (reviewed in [44,45]), yet there is a wide range of responses to CMV proteins [46]. Analysis of T-cell receptor usage among such peptide-specific responses permits quantitative measure of cell-mediated immunity, and Cwynarski et al. [47] first applied this technology to HCT recipients, demonstrating, in the early expansion of CD8+ lymphocytes, that those patients developing at least 107 CMV-specific CD8+ cells appeared to be protected from CMV disease. Gratama et al. [48] have reported that, after T-cell-depleted HCT, the absence of CMV-specific CD8+ lymphocytes correlated with the need for ganciclovir and with the occurrence of CMV disease. Of note, it appears that CMV reactivation occurs in most at-risk patients, inducing an adaptive immune response in some but not others [49]. Lacey et al. [50] demonstrated that the CMV-specific CD8+ cell expansions can occur prior to day 40 post HCT, and can reach levels as high as 25% of total CD8+ cells. Importantly, these cells were clonal expansions and were derived from the donor [50]. This donor origin of CMV-specific T cells underlies the increased risk for CMV infection in T-cell-depleted HCT and the delay in recovery of T-cell immunity in CMV-seropositive recipients of seronegative donors [42]. More recently, the role of the innate immune system in protection from CMV infection in HCT recipients has been explored. The natural killer (NK) cells of the innate immune system are controlled by activating and inhibiting killer immunoglobulin-like receptors (KIRs) that are responsive to the surface proteins, usually HLA proteins, on the target cell surface [51]. There are at least 37 NK-related genotypes, currently divided into a haplotype A, which carries a single activating KIR gene,

Cytomegalovirus Infection

In the pre-ganciclovir era, the analyses of risk for CMV disease after HCT demonstrated the following risk factors: (1) host factors, such as previous CMV infection, age, diagnosis/remission disease status, and delayed immune reconstitution post HCT; (2) treatment factors such as total body irradiation, preparative chemotherapy, and GVHD prophylaxis; and (3) the degree of allogeneic mismatch between the recipient and donor [1,16,55]. These risk factors continue to be important, but the ability to intervene with pre-emptive anti-CMV treatment has focused attention on the importance of recognizing which HCT recipients are at highest risk for CMV disease. Studies have shown that the important factors present at 3 months after HCT, which predict risk for late occurrence of CMV disease, are CMV virus load, any GVHD, postengraftment lymphocytopenia of less than 100/mm3, CD4 counts of less than 50/mm3, and CMV-specific T-cell immunodeficiency [13,42]. Any CMV antigenemia before day 95 post HCT, a CD4 count less than 50/mm3 or any GVHD before day 95 post HCT significantly increases the probability of late CMV disease (Fig. 90.3). Specific assessments of innate and adaptive immunity relative to host protection from CMV infection post HCT are described below. Once the risk for CMV infection is present, several other significant risk factors deserve attention. The use of corticosteroids for management of GVHD is the single most important risk factor. Nichols et al. [18] have shown that 1–2 mg/kg corticosteroid was associated with significant rise in CMV DNA in the blood during ganciclovir therapy. At doses of 2 mg/kg or higher, there was a 10-fold likelihood of a rising CMV DNA load while on therapy. Einsele et al. [27] have reported that risk factors for late-onset CMV disease are chronic GVHD and the need to treat with anti-CMV therapy for 4 weeks or more prior to day 100 when treatment is based on clearance of blood infection. As in the pre-antiviral era, CMV disease prevalence and overall mortality in current studies are higher in CMV-seropositive recipients than in seronegative recipients (e.g. CMV disease was seen in 17.6% of D+R+, in 20.9% of D−R+, in 5.3% of D+R−, and in 1.1% of D−R− transplants [18]). The D+R+ and D+R− groups had the highest risk for overall mortality, and CMV-associated morbidity, including ganciclovir-related neutropenia, explained the increase in the D+R+ group. However, the increased mortality in the D+R− group was explained not by overt CMV infection but by an increased occurrence of bacterial and fungal infections, which was mitigated in those receiving ganciclovir therapy. The implication of this is that it is likely that even subclinical CMV infection in CMV D+R− settings appears to contribute to poor outcome, presumably by the influence of occult CMV infection on host immunity. Thus, HCT allograft recipients must be managed based on their various risks for CMV disease. Yet, even those in the lowest-risk groups face some

KIR2DS4, and haplotype B, with both activating and inactivating KIR genes (see Chapter 13). In sibling donor HCT, donor haplotype B was associated with a significant reduction in CMV reactivation [52]. The presence of additional activating KIR genes or identity between donor and recipient activating KIR genotypes has also been associated not only with decreased CMV reactivation, but also with improved survival [53]. It appears that the early events of CMV reactivation are controlled by NK cells, and then the progression of infection is controlled by the adaptive immune system interacting with this innate protective mechanism.

Clinical description of CMV-associated disease Definitions of CMV disease CMV infection per se in HCT recipients, which can be primary or reactivated infection, is usually defined as the isolation of CMV in tissue culture or the identification of markers for CMV in tissue specimens or blood by histologic and histochemical means, or by specific antigen staining, by direct CMV DNA/RNA or CMV antigen detection, or by a fourfold or higher rise in CMV antibody titer [54]. CMV-associated interstitial pneumonia is defined as a progressive interstitial pulmonary process, as evidenced by chest X-ray findings and increased hypoxia, with evidence of CMV infection in the lung and without evidence for other causes of pneumonitis. CMV enteritis is defined as an enteropathic syndrome with pain, nausea and vomiting, or diarrhea and evidence of CMV infection at the site of an inflammatory or ulcerative mucosal lesion. In general, other CMV-associated organ-related syndromes, such as hepatitis and encephalitis, are defined as syndromes with specific organ dysfunction and the concomitant presence of active CMV infection. With the exception of CMV retinitis, the diagnosis of CMV disease cannot be made with confidence without histologic evidence of CMV infection in the involved organ. Analysis of risk for disease Although it would appear to be obvious, the most significant risk factor for the occurrence of CMV-associated disease after allogeneic HCT is the development of CMV infection. It is important to note CMV infection as a risk factor because it confirms that the heterogeneous syndromes that can present during CMV infection are, in fact, due to this infection, and that this ubiquitous agent is not merely present and masking another pathologic process. In addition, it allows us to focus on management of patients by recognizing the factors that predict for CMV infection and disease.

Pp65 antigenemia
Fig. 90.3 Cumulative incidence of late cytomegalovirus (CMV) disease in patients with the presence and absence of risk factors present at 3 months. Panel (a) compares patients with any pp65 antigenemia before day 95 or CD4 count less than 50/mm3 with patients having no CMV antigenemia or CD4 count less than 50/mm3. Panel (b) compares patients with any graft-versus-host disease (GVHD) (grade II–IV or clinical chronic) with patients having no GVHD before day 95. (Reproduced from [13], with permission.)

Probability of late CMV disease

0.5

GVHD

p = 0.02

0.4

1371

p = 0.03

0.3 AG pos or T4 <50 (n = 92)

0.23

AG neg and T4 >50 (n = 39)

0.08

Any GVHD before day 95 (n = 119) 0.21

0.2

0.1

No GVHD before day 95 (n = 27)

0.04

0.0 0 (a)

12

24

Months since transplant

36

0 (b)

12

24

Months since transplant

36

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chance of CMV-related disease after HCT, and therefore it is important for the transplant team to consider all HCT recipients when considering anti-CMV strategies and to base treatment on relative risks of CMV disease. CMV-associated interstitial pneumonia Prior to the development of methods for prevention of CMV infection, CMV-IP occurred in 15–30% of HCT allograft recipients [1,56]. Bronchoalveolar lavage (BAL) is the preferred method of diagnosis of CMVIP, and the specimen should be analyzed for infectious virus and for cytologic evidence of CMV. As shown in Fig. 90.4, inclusion bodies typical for CMV can often be seen in the CMV-positive BAL samples. No matter when CMV-IP occurs after HCT, hypoxia is the major physiologic abnormality observed, and radiologic abnormalities suggestive of interstitial pneumonitis are the frequent pattern on X-ray films

(Fig. 90.5). Although infiltrates usually become diffuse and are basilar, there is considerable variation in the X-ray pattern, which can reveal segmental, lobar, diffuse interstitial or nodular infiltrates [57]. The differential diagnosis includes radiation-induced pulmonary disease, cytotoxic chemotherapy-induced pulmonary damage, pulmonary hemorrhage, pulmonary edema, metastatic neoplasia, and other infections of fungal, viral or bacterial etiology. As illustrated in Fig. 90.4, the histopathology of CMV-IP involves thickening of the interalveolar membranes with cellular infiltrates and edema [14,57]. Of interest, CMV infections occur in an erratic distribution and are not clearly related to sites of typical CMV pathology [58,59]. Zaia [60] analyzed clinical and virologic aspects of 32 consecutive autopsied cases of CMV-IP. When the group was arbitrarily subdivided into those who survived 14 days or less and those who lived longer than 14 days from the time of initial clinical disease (hypoxia), the mean time to death was 8.4 days versus 28.5 days, indicating the variable duration of CMV-IP. The amount of CMV per gram of lung, as measured by infectious titer or by the amount of CMV DNA, did not significantly correlate with the duration of disease. Similarly, it has been reported that quantitative assessment of infectious CMV in the BAL specimens, as a measure of virus burden in infected lungs of allograft recipients, does not distinguish patients in terms of disease severity or predictive outcome [61]. However, as noted earlier, it is recognized that CMV DNA level in blood is predictive of risk for late-onset CMV disease [13]. CMV-associated enteritis Gastrointestinal syndromes associated with CMV are an important problem in the allogeneic HCT recipient. Ulcerations associated with CMV infection can be identified in the esophagus, the stomach, the small bowel, and the large intestine [62,63]. The diagnosis is made by the association of CMV infection with mucosal pathology and appropriate symptoms. Van Burik et al. [64] have reported a median onset of CMV enteritis of 91 (range 17–527) days post HCT, and a 2-year overall survival rate in this population of 35%.

Fig. 90.4 Histopathology of cytomegalovirus (CMV) interstitial pneumonia (IP). (a) Photomicrograph of a thin section of lung from a patient with CMVIP stained with hematoxylin and eosin. Note the cell in the alveolar space containing a Cowdry type A intranuclear inclusion typical of CMV. Magnification: ×640. (b) Photomicrograph of a Papanicolaou-stained bronchoalveolar lavage specimen from a patient with CMV-IP. Note similar CMV-induced “owl’s eye” intranuclear inclusions. Magnification: ×1000.

Other CMV-associated syndromes Since at least two-thirds of all at-risk transplant recipients develop CMV infection, the accurate association of other syndromes with this infection can be difficult. Nevertheless, in addition to mononucleosis-like syndrome with fever, arthralgia, and malaise, both hepatitis and suppressed marrow function, including neutropenia and thrombocytopenia, have

Fig. 90.5 Clinical course of treated cytomegalovirus (CMV) interstitial pneumonia. This is a series of X-ray films is from two patients (UPN475 and UPN478) treated with ganciclovir as described in Table 90.5; radiographs are from days 0, 21, and 42 of treatment. Note the resolution of the right lower lobe pneumonitis in both patients.

Cytomegalovirus Infection

been associated with acute CMV infections [65]. The course of asymptomatic CMV infection is not well described in the allogeneic HCT recipient, but it appears that febrile episodes are a significant part of this infection. In order to understand the clinical effects of asymptomatic CMV infection, Zaia [60] evaluated consecutive patients with CMV infection during HCT and found a significant association of fever between days 42 and 56 in those with otherwise asymptomatic infection. There was no increased rate of neutropenia in this group. However, neutropenia is associated with CMV infection during this same time period; for example, in 66 patients examined by Meyers and Thomas [66], there was a fall in leukocyte and platelet count compared with the control group (59% versus 36%; p = nonsignificant). Treatment CMV-associated interstitial pneumonia The treatment of CMV-IP has had mixed success in immunocompromised persons, probably due to variations in patient groups, that is, different diagnoses, different levels of immunosuppression, and varying contribution from host factors, known and unknown, that modify CMV infection. Overall, the outcome for the untreated disease has historically been very poor, with a mortality reported in the 1970s of 65% [15] and in the 1980s of approximately 85% (Table 90.4). As shown in this table, until the availability of ganciclovir, attempts to treat CMV disease with antiviral agents were unsuccessful despite the fact that many of these agents had produced excellent in vitro antiviral effects. But even ganciclovir used alone, at a total dose ranging from 7.5 to 15.0 mg/kg/day in 10 persons, had a uniformly bad outcome for CMV-IP, with only one surviving patient [67]. In a study reported by Winston et al. [68], ganciclovir used at a dose of 10 mg/kg/day resulted in a survival rate of 22% in nine marrow recipients with CMV-IP. Erice et al. [69] reported

Table 90.4 Historic treatment of cytomegalovirus (CMV) interstitial pneumonia with antiviral agents

Source

Agent

Survival rate at 3 months (%)

Kraemer et al. [121] Meyers et al. [122] Meyers et al. [123] Wade et al. [124] Wade et al. [125] Meyers et al. [126] Winston et al. [127] Shepp et al. [128] Shepp et al. [67] Winston et al. [68] Erice et al. [69] Reed et al. [71] Blacklock et al. [129] Reed et al. [130] Ringden et al. [131] Reed et al. [74] Emanuel et al. [75] Schmidt et al. [76] Ljungman et al. [132] Aschan et al. [133]

Vidarabine Leukocyte IFN IFN and vidarabine Acyclovir IFN and acyclovir rDNA IFN rDNA IFN IFN and acyclovir Ganciclovir Ganciclovir Ganciclovir Ganciclovir and steroid CMVIg CMVIg Foscarnet Ganciclovir/IVIg Ganciclovir/IVIg Ganciclovir/IVIg Ganciclovir/IVIg Foscarnet

16.6 0 14.2 12.5 23 0 60 12.5 10 22.2 45 16.6 50 21.4 0 48 65 85 31 0

CMVIg, CMV immune globulin; IFN, interferon; IVIg, intravenous immune globulin; rDNA IFN, recombinant interferon.

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that ganciclovir used at 7.5 mg/kg/day was associated with a 45% survival rate, in a study which included non-HCT patients. Thus, unlike the initial experience in the use of ganciclovir for the treatment of CMV diseases in patients with acquired immune deficiency syndrome (AIDS), in which prompt clinical response was the rule [70], ganciclovir alone was not able to improve the outcome of CMV-IP in the HCT recipient. The inability of agents that act only to suppress virus replication to treat CMV-IP successfully has suggested that there are additional factors contributing to pathogenesis besides virus cytopathology. Because CMV-IP could be caused by both CMV infection and the host response to infection, it was suggested that antiviral therapy be combined with immune-response modification [59]. The initial use of combined treatment, reported by Reed et al. [71], involved ganciclovir (7.5 mg/kg/day) plus methylprednisolone (16 mg/kg/day) in six HCT recipients. Only one patient survived, and severe marrow and renal toxicities were observed in five patients. CMV immune globulin (CMVIg), used alone, was evaluated for the treatment of CMV-IP, with mixed results, but the combination with ganciclovir by Reed et al. [72] and by Bratanow et al. [73] provided the first improvement in outcome of CMV-IP. Subsequently, several centers published results using this regimen. In the study by Reed et al. [74], two intravenous immunoglobulin (IVIg) products were used in conjunction with ganciclovir. One product contained high-titer CMV-specific antibody and was given at a dose of 400 mg/kg on days 1, 2, and 7, and then at half this dose on days 14 and 21. Of 50 patients in this group, 25 (50%) had at least a 6-week survival time, and 16 patients (32%) were alive at 6 months, at a median followup time of 9 months. In an additional group, patients were treated with a standard, commercially available immune globulin containing a lowertiter CMV-specific antibody given at 500 mg/kg every other day for nine doses. Thirteen patients were entered into this group; five (38%) were alive at 6 months. In the study reported by Emanuel et al., 10 patients received a regimen of IVIg 500 mg/kg every other day for 10 doses plus ganciclovir 7.5 mg/ kg/day in three divided doses for 20 days followed by IVIg 500 mg/kg once weekly for 8 weeks and ganciclovir maintenance therapy 5 mg/kg/day three times weekly for 20 doses [75]. Seven of the 10 patients were alive at a median follow-up time of 10 months. In the study by Schmidt et al., 40 patients were treated with therapy that included antiviral induction treatment lasting 3 weeks, or until there was documented clearing of pulmonary CMV infection by BAL analysis, followed by a maintenance treatment lasting until significant immunosuppressive medications were stopped [76]. In this regimen, ganciclovir was given at 10 mg/kg daily and IVIg 500 mg/kg every other day for 21 days, followed by ganciclovir at 5 mg/kg daily 5 days per week and IVIg at 500 mg/kg weekly until day 180 after HCT. With this treatment of 40 cases of CMV-IP after HCT, 80% were alive at 6 weeks, and 40% were alive at a median follow-up time of 18 months. Ganciclovir plus IVIg treatment of CMV-IP has met with varying success in some HCT centers, probably due to variations in patient populations. For example, Ljungman noted that patients who did not receive total body irradiation compared with those who did benefited more from ganciclovir plus IVIg treatment of CMV-IP (75% versus 27% survival rates at 30 days, respectively; p = 0.009), and, in general, patients with multiple organ dysfunction or respiratory failure were less likely to respond to antiviral therapy [77]. Thus, although these results were not derived from randomized and placebo-controlled studies, ganciclovir plus IVIg has become the recommended treatment for CMV-IP in the HCT recipient (a method of treatment being outlined in Table 90.5). Repeat BAL is suggested after the initial 21 days of treatment if there is any question regarding response to the initial antiviral therapy. Reactivation of CMV is the usual course when ganciclovir is stopped, and therefore it is recommended that maintenance therapy be continued for the duration of major immunosuppressive treatment. The expected course of resolution of CMV-IP on treatment

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is shown in Fig. 90.5. Fever and hypoxia usually resolve within the first week of treatment, but X-ray changes persist for many weeks. The patient with a slow response to treatment prompts the consideration of ganciclovir-resistant CMV, which is rare in this setting, or the presence of secondary infections (Fig. 90.6). Both of these questions can be addressed by repeat BAL. CMV infection is usually eliminated from the lavage specimen by 21 days after the start of treatment, and deterioration of pulmonary function with continued CMV lung infection at this time suggests ganciclovir resistance, although this is rare in the setting of HCT. If ganciclovir resistance occurs, or if ganciclovir cannot be used because of marrow toxicity, foscarnet is indicated [2]. As derived from experience with treatment of retinitis in persons with AIDS, foscarnet is given at a dose of either 90 mg/kg twice daily or 60 mg/kg three times daily with saline infusion over 2 hours during an initial 7-day induction period. Maintenance therapy follows this at a dose of 90 mg/kg/day for an extended period of time, determined by the immunosuppression of the patient. Foscarnet is nephrotoxic, and it is recommended that the calculation of the dosage be determined by creatinine clearance. There has been no systematic evaluation of the efficacy of foscarnet plus immune globulin for the treatment of CMV-IP. CMV-associated enteritis and other syndromes A controlled study using ganciclovir has shown that, despite antiviral effect, there was no significant improvement of CMV gastroenteritis [78]. Nevertheless, because of this antiviral effect, it is common practice

Table 90.5 Treatment of cytomegalovirus interstitial pneumonia Induction phase – 21 days GCV IVIg

5 mg/kg IV every 12 hours 500 mg/kg IV every other day

Maintenance phase – during continued immunosuppression GCV 5 mg/kg IV daily on 5 or 6 days per week IVIg 500 mg/kg once weekly If the absolute neutrophil count is less than 1000/μl for 2 consecutive days, stop GCV until count recovers; consider the use of the growth factors granulocyte colony-simulating factor or granulocyte–macrophage colony-simulating factor. GCV, ganciclovir; IV, intravenously; IVIg, intravenous immune globulin.

to use ganciclovir without IVIg for CMV enteritis according to the dosing schedule shown for CMV-IP (Table 90.5). For other CMVassociated syndromes such as hepatitis and retinitis, this same regimen shown in Table 90.5 is used. Boeckh et al. [65] documented a case of CMV-associated marrow suppression with response to therapy using a growth factor and an antiviral agent. In addition, ganciclovir has been used for treatment of marrow failure after HCT [79]. Complications of CMV therapy The major complications associated with ganciclovir treatment are neutropenia and creatinine elevation. Neutropenia occurs in approximately 30% of patients and, based on the experience of Goodrich et al. [24], it lasts for a median of 12 days with an upper range of 74 days. Salzberger et al. [80] analyzed ganciclovir-associated neutropenia in 278 HCT recipients and noted that neutropenia occurred significantly more frequently when there was an elevated bilirubin level (≥6 mg/dL) during the first 20 days after HCT, or a serum creatinine level of 2 mg/dL or higher after day 21 of transplantation, or low marrow cellularity between days 21 and 28 post HCT. Persons with one risk factor had a 21% incidence of neutropenia, but those with more factors had a 57% occurrence of neutropenia. Of importance, this treatment-related neutropenia was an independent risk factor for poor overall survival (relative risk [RR] 3.8; p = 0.001), for poor event-free survival (RR 2.1; p < 0.0001), and for relapse (RR 1.7; p = 0.03) [80]. Therefore, in the event of ganciclovir-induced neutropenia, the use of granulocyte colony-stimulating factor or granulocyte–macrophage colony-stimulating factor is recommended, and if this fails, therapy should be switched to foscarnet. To minimize this problem, a stopping rule for ganciclovir use must be employed, such as cessation of drug when the absolute neutrophil count is lower than 1000/μL on 2 consecutive days.

Prevention of CMV infection after HCT From a practical standpoint, the central risks that define how the patient should be managed relative to probability of CMV disease are defined by the chances that the individual will develop CMV infection, and the likelihood that inadequate immune function exists for resolution of the infection. When considered in this way, the important risk factors for development of severe CMV disease are patient age, pretransplantation CMV antibody seropositivity of donor or recipient, HLA-mismatch recipient–donor status, occurrence of acute GVHD, and ongoing immunologic function post HCT.

Fig. 90.6 Late complication of treated cytomegalovirus interstitial pneumonia. Patient UPN454 was treated on day 0 (d 0) with ganciclovir according to the regimen in Table 90.5, with resolution of pneumonitis (d 21 and d 42). Subsequently, the patient developed a right lower lobe nodule (d 189), progressing to right lower lobe pneumonia (d 194 and d 204) and then death from Aspergillus infection and chronic graftversus-host disease.

Cytomegalovirus Infection

Recognizing those at high risk for CMV infection, there are two general approaches to the prevention of CMV disease using either ganciclovir or foscarnet: (1) treatment of early blood-borne CMV infection prior to onset of disease; and (2) treatment of all at-risk patients for the defined period of risk. The first approach is a “pre-emptive” strategy and was first shown to be effective in the HCT setting where the period of risk is well defined [23,25]. With this method the requirement for accurate detection of CMV infection is the limiting feature, but the benefit is that fewer patients are exposed to the antiviral chemotherapy. The second approach, termed “general prophylaxis,” is used when selected patients can be identified as being at particularly high risk for CMV infection, and all such patients are treated for the period of risk. For both strategies, the risk of CMV disease governs the management, and this is determined by analysis for host-specific risk factors as discussed above. Pre-emptive treatment strategy for prevention of CMV after HCT Patients should be monitored for CMV infection, usually using the test for CMV DNA in the blood, although other methods for detection of CMV can be used [81]. Surveillance is started based on medical history, such that patients with a history of CMV infection in the past 6 months should have a preconditioning test, and all others should begin at approximately 21 days post HCT. When less sensitive assays such as blood cultures are used, twice-weekly monitoring can be effective [28], but, in general, once weekly surveillance is preferred using the more sensitive antigenemia assay or a DNA-based assay [82]. Documented CMV blood infection should be pre-emptively treated starting with ganciclovir for at least 2–3 weeks [29,83–85], and continued during the period of maximum immunosuppression [2]. With the availability of methods to measure CMV blood levels, the antiviral treatment is monitored to document the effect of treatment [2]. Management of pre-emptively treated CMV infection As noted in Table 90.6, ganciclovir is the drug of choice for pre-emptive treatment and is administered at an “induction” dose of 5 mg/kg twice daily for at least 7 days or until evidence of falling CMV load. CMV infection will clear in most patients within 2 weeks [29,86], but for those with stable or increasing CMV levels, the induction period should continue. However, if secondary treatment for GVHD is required, and corticosteroid use is 1 mg/kg/day or more, the pre-emptive surveillance program should be reactivated. Maintenance therapy consists of ganciclovir 5 mg/kg/day for 5–7 days per week for an additional period of time based on the patient’s risk factors. For example, if the CMV blood levels are negative on two occasions, and the subject is on a nonsteroid, non-T-specific regimen, maintenance can stop at 3–6 weeks. For patients on steroid or other secondary therapy for GVHD, treatment should continue for up to day 100 or longer post HCT. Monitoring should continue after treatment stops to observe for relapsed infection, which then requires additional treatment using the induction/maintenance regimen as before until virus clears. During maintenance therapy, sporadic CMV-positive assays can occur, and these do not require additional treatment unless consecutive positives occur. However, for select risk groups, such as those receiving antithymocyte globulin or Campath, prophylactic ganciclovir should be considered (see below). A major question concerns what to do for an HCT recipient who is at high risk in the period after 3 months post HCT. After 3 months post HCT, it is expected that not all patients will be reconstituting immunity to CMV, and those in the high-risk category are defined as those who have had a treated CMV blood infection already, those receiving corti-

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costeroid therapy greater than 1 mg/kg or other secondary therapy for GVHD, and those with lymphopenia [13,42,43]. An interventional strategy has been tested for preventing late CMV infections based on documented T-cell immunity. Using this approach, Avetisyan et al. withheld ganciclovir treatment of CMV infection occurring at longer than 3 months post allogeneic HCT if the recipient had evidence of cellular immunity to CMV as determined by functional interferon-gamma assays [87]. In this prospective study, most CMV infections occurred before month 3 post HCT and were treated preemptively. Of the 37% of infections occurring after month 3, therapy could be deferred in only 25% of subjects, but these patients did not develop subsequent CMV disease. Boeckh et al. have studied the use of prophylactic valganciclovir in subjects at high risk for CMV after day 100 post HCT. Valganciclovir reduced the need for pre-emptive treatment with ganciclovir but was no better than placebo in reducing incidence of late CMV disease. The optimal management of the high-risk patient between days 100 and 270 post HCT is still not clear, but pre-emptive ganciclovir remains the best approach, in which careful monitoring of CMV blood infection should continue with treatment as necessary. Management situations commonly present the following questions. Should a pre-emptive strategy be used for autologous HCT recipients? All CMV-seropositive recipients of HCT are at risk for CMV reactivation, and in the autologous HCT population, the rate of reactivation ranges from 25% to 45%, with CMV disease occurring in 2–10% [30,33,34,36]. Risk for disease in this group appears to be related to immune reconstitution post HCT [88]. Nevertheless, CMV disease occurs in recipients of autologous HCT, and the risk factors in this population include the prior amount of pre-HCT chemotherapy, treatment with corticosteroids after HCT, and use of CD34+-selected peripheral blood progenitor cells. Autologous HCT recipients with these risk factors should be managed with pre-emptive monitoring and early treatment with ganciclovir [2]. Should a pre-emptive strategy be used for reduced-intensity conditioning of HCT recipients? The risk for CMV-associated disease in patients after reduced-intensity HCT conditioning does not appear to be any less than for high-dose allogeneic recipients [32]. In addition, if there is cell selection of the hematopoietic cell product, there is increased risk for CMV infection [89,90]. Hence, recipients of reduced-intensity HCT should receive preemptive monitoring for and early treatment of CMV infection [2]. What should be done for antiviral drug resistance? Breakthrough CMV infection is not uncommon while on seemingly adequate antiviral therapy, and can be a difficult clinical problem. Boeckh et al. [91] documented that ganciclovir therapy used at engraftment results in breakthrough CMV-positive blood in 50% of patients monitored by antigenemia, and in 66% of those followed by PCR. Studies have shown that approximately 15–25% of patients treated preemptively with ganciclovir or foscarnet will require re-treatment [29]. Yet ganciclovir resistance is uncommon, and rising CMV DNA levels are probably the best laboratory data for assessing potential antiviral drug resistance. Decisions usually have to be made in the absence of definitive evidence of drug resistance, and foscarnet should be substituted in place of ganciclovir in the following situations: (1) whenever there is marrow toxicity and the CMV assays remain persistently positive or show rising CMV blood load; (2) when persistent infection is present at the time of increased treatment of GVHD with corticosteroid or other secondary anti-GVHD drugs; and (3) when there are changing clinical signs suggestive of CMV disease.

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Table 90.6 Cytomegalovirus (CMV) prophylaxis in hematopoietic stem cell transplant (HCT) recipients Pre-emptive treatment Treat all recipients with documented CMV infection in blood or lung; for PCR assays, require two positive results within a 3–7-day period, unless a highly sensitive assay (e.g. quantitative PCR) is used Induction therapy Ganciclovir, 5 mg/kg IV twice daily for 7–14 days* Foscarnet, 60 mg/kg IV twice daily for 7–14 days*† Maintenance therapy Ganciclovir, 5 mg/kg IV daily on 5 or 6 days per week, or 1 g orally three times daily (adults) or 30 mg/kg orally three times daily (children), or Valganciclovir‡, 900 mg by mouth daily on 5 or 6 days per week (pediatric dose is 500 mg/m2 to maximum of 900 mg), or Foscarnet, 60 mg/kg twice daily or 90 mg/kg daily*† Duration of maintenance therapy is 2–5 weeks on the basis of (a) time to significant reduction in immunosuppression (long course), or (b) time to return of CMV infection indicator to negative (short course) General prophylaxis Treat all HCT recipients at highest risk for CMV on the basis of immunosuppressive regimen using ganciclovir or foscarnet§ Dosage Ganciclovir, 5 mg/kg twice daily for 7 days, then daily on 5 or 6 days per week§, or Valganciclovir‡, 900 mg by mouth daily (pediatric dose is 500 mg/m2 to maximum of 900 mg) Foscarnet, 60 mg/kg twice daily for the period of prophylaxis*† Note: Doses must be adjusted for renal insufficiency. * If no maintenance therapy is used, the induction period continues until the CMV detection assay result becomes negative. Note that the CMV load can initially increase, and treatment should continue until the CMV load is negative. † Patients should be prehydrated with normal saline before foscarnet is provided, and the magnesium ion and calcium ion levels in serum should be maintained with appropriate supplements. ‡ Valganciclovir is not approved for prevention of CMV infection in HCT recipients. § Start therapy when the absolute neutrophil count is 750 × 109 neutrophils/L, and stop therapy when the immunosuppression is significantly reduced or the absolute neutrophil count is less than 1000 × 109 neutrophils/L for 2 consecutive days.

When should general prophylactic anti-CMV treatment be used? Not all HCT recipients will do well with pre-emptive anti-CMV management, especially those at the highest risk for CMV infection [92]. The alternative approach to prevention of CMV in high-risk patients is to treat all patients with ganciclovir, usually at the time of engraftment [2,24]. In the first randomized, placebo-controlled trial of this approach, Goodrich et al. [24] studied the effect of ganciclovir given at a dose of 5 mg/kg intravenously twice daily for 5 days and then every day until day 100 after HCT in patients who were CMV seropositive at the time of engraftment. This method significantly reduced CMV excretion (3% versus 45%; p = 0.0001) and CMV-associated disease (10% versus 29%; p = 0.0008). Subsequent CMV disease occurred in 10% of the treated group after treatment was stopped at day 100. However, there was no difference between control and treated groups in terms of mortality, either during treatment (19% versus 12%, respectively) or at day 180 (26% versus 30%, respectively), and this lack of effect on mortality is different from the effect observed with pre-emptive use of ganciclovir [23,29]. Patients on ganciclovir who became neutropenic had a significantly increased risk of bacterial infection [80]. Furthermore, the cost of treating all allogeneic marrow transplant recipients in this way is expensive. Therefore, because of the toxicity of these agents and their expense, the general use of ganciclovir in all CMV-seropositive persons is currently not recommended [2]. Verdonck et al. [93] developed a risk-adapted strategy in which patients with GVHD requiring increased steroid therapy were given a 2-week course of ganciclovir. Other patients in this study were followed with antigenemia assays and treated pre-emptively. This approach was

used in 41 patients, and half of the patients never received ganciclovir; of those treated, 26% required a second 2-week course. Using this approach, there was no CMV disease and no neutropenia [93]. In general, HCT recipients of matched unrelated donor hematopoietic cells do well with pre-emptive anti-CMV management. However, if T-cell depletion or any in vivo anti-T-cell therapy is used for matched unrelated or haploidentical HCT, anti-CMV chemotherapy should be initiated early after HCT. Similarly, if the risk for CMV disease increases during the course of any allogeneic HCT due to significantly increased immunosuppression, the method of risk-adapted general prophylaxis can be adopted. In such patients, subsequent routine surveillance for CMV is not necessary, and CMV diagnostic assays should be based on clinical indications [2]. What clinical specimen should be used for CMV monitoring? CMV detection in blood is considered the preferred specimen for detection of CMV because the predictive value of a positive result is high [16]. BAL reaches a site of early reactivation of CMV, and was used in the initial demonstration of the effectiveness of pre-emptive therapy [25]. However, it has been demonstrated that the positive predictive value of results from BAL is no better than from blood specimens [94], and that monitoring of blood for CMV is better than using BAL [95]. In the study by Goodrich et al. [23], allogeneic HCT recipients were treated with ganciclovir at the time of first CMV infection, as monitored in weekly cultures of throat, urine, and blood. In this study, throat infection was less reliable than blood, and urine was nonpredictive of subsequent disease [23].

Cytomegalovirus Infection Table 90.7 Comparison of cytomegalovirus (CMV) diagnostic methods used in pre-emptive ganciclovir therapy Median days to first positive* (range)

Median days until clearance* (range)

Antigenemia PCRPL PCRPBL

42 (27–75)† 45 (18–56)† 32 (18–46)† 35 (2–90)‡

13 (7–41)† 12 (7–41)† 23 (14–53)† 12 (7–49)‡

Hybrid capture NASBA

34 (16–84)‡ 21 (4–82)§

10 (7–49)‡ 19 (2–32)§

Blood culture

51 (37–93)† 59 (48–81)‡

0 (0–7)† NA‡

Assay

NA, not applicable; NASBA, nucleic acid sequence-based assay; PCR, polymerase chain reaction; PCRPBL, leukocyte PCR; PCRPL, plasma PCR. * Median day to initial positive based on weekly testing and days to negative test after start of pre-emptive antiviral therapy. † Adapted from Boeckh et al. [91]. ‡ Adapted from Hebart et al. [134]. § Adapted from Hebart et al. [98].

What is the optimal assay for detecting CMV? Pre-emptive management strategy places a burden on the transplant team to detect CMV infection before progression to overt disease, and clearly the window of opportunity for such early treatment varies with the sensitivity of the assay. There are three general types of assay for the detection of CMV: culture-based assays, antigen assays, and DNA/RNA assays. The tissue culture assay for CMV, called the “shell vial” assay [96], and the detection of the CMV antigen CMVpp65 rely on CMV replication and become positive at a median time of 42 days after HCT [91]. Since CMV-IP occurs at a median time of 50–60 days post HCT, the use of these assays presents only a small margin of time for treatment prior to disease onset. Using DNA/RNA-based methods, detection of CMV in blood can be made at approximately 35 days post HCT [29,97,98]. Thus, the DNA/RNA-based assays widen the opportunity for early treatment by 1 week. Although this seems small, Einsele et al. [29] demonstrated that this was sufficient to significantly reduce CMV disease and overall mortality. As important, the quantitative aspects of these tests allow them to be useful for monitoring the response to treatment. Table 90.7 compares the various assays in terms of time to CMV detection and time to clearance of infection after start of antiviral treatment. Ultimately, the choice of assay must be made based on the resources at each medical center, and the type of HCT patients treated [82]. What is the role of acyclovir and related agents in pre-emptive strategies? Acyclovir is not a first-line agent for prevention of CMV infection, but because of its safety profile, it can have an important role in the management of the HCT patient. Acyclovir and the acyclovir prodrug valacyclovir have a 50% inhibitory concentration for CMV strains of 63.1 ± 30.2 mM [99], and since peak acyclovir levels in the plasma are above this level, it was not surprising that acyclovir could modify CMV infection. However, it was surprising that prophylactic administration of acyclovir in allogeneic HCT recipients and in renal allograft recipients significantly lowered patient mortality [26,100,101]. Of note, when acyclovir was used in the same dose and at the same institution, but in

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autologous marrow recipients, there was no significant effect on CMV infection or disease compared with a control group [102], suggesting that any improvement in survival might be to do with the effect of acyclovir on other viral infections. A most interesting result of acyclovir prophylaxis was reported by the European HCT group [103,104]. In a blinded, controlled study, recipients of matched related or unrelated HCT were randomly assigned to receive either (1) high-dose acyclovir for 6 months (500 mg/m2 intravenously three times daily) from day 5 before to day 30 after HCT, and then oral acyclovir (800 mg four times daily), (2) the same high–dose acyclovir for 1 month, or (3) a standard herpes simplex prevention regimen for 30 days (400 mg orally four times a day). The use of highdose acyclovir for both 30 days and 6 months was associated with reduced CMV infection compared with the standard dose. The occurrence of procedure-related morbidity, including CMV disease, was the same for all groups, but there was a 19% survival advantage at 3 months and at 1 year in the group with high-dose, long-term acyclovir use [103,104]. This result suggests that either CMV infection or some other virus or viruses influence fatal complications without recognizable overt viral disease. The use of short-term high-dose acyclovir as a supplement to ganciclovir preventive treatment had no such outcome advantage [105]. Valacyclovir has been used for primary CMV prophylaxis or for suppressive anti-CMV therapy after ganciclovir induction therapy in recent studies [106,107]. In the randomized, double-blinded study of Ljungman et al. [107], in which 748 patients received intravenous acyclovir 500 mg/m2 three times daily for the initial 28 days post HCT, and then either oral valacyclovir 2 g four times daily or acyclovir 800 mg four times daily until 18 weeks post HCT, valacyclovir was significantly more effective in suppressing CMV reactivation (28% versus 40%, respectively; p < 0.0001). In this study, pre-emptive ganciclovir or foscarnet was used for CMV blood infection, and there were fewer patients with infections that initiated anti-CMV treatment in the valacyclovir group compared with the acyclovir group (23% versus 37%, respectively) and significantly more time to onset of indicator infection in the valacyclovir group. If cost-effectiveness studies and other studies of outcome can confirm the benefit of supplementing pre-emptive strategies with valacyclovir, it is possible that valacyclovir will eventually have a role in the prevention of CMV infection in the HCT recipient.

How is foscarnet used in the HCT patient? Foscarnet is a phosphonate that requires no metabolism for activity and, because of its excellent antiviral profile, has become an important agent in the treatment of CMV infection in the immunosuppressed population. Initial experiences with this agent indicated that nephrotoxicity, hypocalcemia, and hypophosphatemia were significant problems [108]. However, with the recognition of the importance of hydration, the safe use of foscarnet has been demonstrated in HCT populations [85]. Foscarnet is used at a dose shown in Table 90.6 over 1–2 hours with concomitant administration of saline solution for hydration. It has also been shown that, for recipients at high risk for CMV disease, foscarnet and ganciclovir could be used in combination [84] at the same induction doses as before and with maintenance therapy for an additional 2 weeks if needed by alternating each drug every other day. In addition, foscarnet effectively prevents CMV disease when used at 60 mg/m2 twice daily for 14 days in a pre-emptive strategy with PCR surveillance [85]. Thus, foscarnet has a role in the pre-emptive management of the transplant recipient. However, the choice of foscarnet is often difficult in patients receiving other nephrotoxic agents after HCT, and, for this reason, ganciclovir is usually started when CMV infection occurs.

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What is the role of oral formulations of ganciclovir? With the availability of valganciclovir, orally administered ganciclovir is no longer used because of its poor absorption [109,110]. Valganciclovir has much greater bioavailability, and an oral dose of 900 mg can result in a blood level of drug exposure (area under the curve) similar to an intravenous dose of 5 mg/kg ganciclovir in adults. Valganciclovir is approved for use as a preventive measure in solid organ transplantation but, despite wide use, does not have an approved indication in HCT recipients.

but without improved survival [114]. In the HCT recipient, utilizing either conventional IVIg or CMVIg [115], there was a decrease in the incidence of CMV infection compared with controls, but the effect on CMV-associated disease was variable, with no effect seen in some studies. A controlled use of passive immunization with a monoclonal antibody to CMV glycoprotein H failed to protect CMV-seropositive recipients from CMV reactivation, from total ganciclovir exposure during pre-emptive treatment, from GVHD or from bacterial and fungal infections [116]. In summary, immunoglobulins are not recommended for the prevention of CMV infection after HCT [117].

When should cidofovir be used in the HCT recipient? Cidofovir has been used for the pre-emptive management of the HCT patient [111]. The dosage for cidofovir is 5 mg/kg per dose in two doses given 1 week apart, followed by maintenance therapy provided every other week. The drug is given with probenecid and adequate preinfusion hydration. In the United States, cidofovir is not approved for the prevention of CMV infection in the HCT recipient. It is nephrotoxic, and its use in the HCT setting remains a third-line agent when pre-emptive use of ganciclovir or foscarnet has failed. Is there a role for IVIg for prevention of CMV in HCT? One of the most controversial areas in the management of the transplant patient in regard to CMV infection is the use of passive immunization with IVIg [112,113]. In solid organ transplantation, in which CMVIg is an approved product, there is convincing evidence for a protective effect,

Conclusion The availability of antiviral agents has greatly altered the management of the allogeneic HCT recipient, and yet control of CMV remains less than optimal because of the toxicity of available agents. Even when used successfully to prevent early disease, the patient often remains at risk for late-onset disease. An immune deficiency underlies prolonged susceptibility to CMV in this population, and this continued inability of the host to develop protective immunity in some patients remains the ultimate challenge for improved treatment. It is possible that antiviral agents alone will never completely solve the problem of CMV infection after HCT. It remains to be determined whether less toxic antiviral agents will provide a more effective method for preventing CMV disease in this population.

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Herpes Simplex Virus Infections

Introduction Members of the herpesvirus family are the cause of significant morbidity and mortality in the post-hematopoietic cell transplantation (HCT) setting. They all possess the unique characteristics of latency and reactivation, and each virus emerges during a specific time period after transplantation. Herpes simplex virus (HSV) infections occur during the early, neutropenic or pre-engraftment period soon after the initiation of immunosuppressive therapy [1,2]. In the days prior to the use of antiviral prophylaxis, approximately 70% of HSV-seropositive HCT recipients would shed the virus and, of these, 70% would develop HSV disease [3]. With the advent of acyclovir prophylaxis, the incidence of HSV disease has dropped below 5% [3–5]. But while the problem of HSV infection after HCT has substantially diminished in the early posttransplant period, HSV infection has shifted to the period after prophylaxis has ended, and acyclovir-, foscarnet-, and cidofovir-resistant HSV strains have started to emerge.

Virology HSVs, HSV-1 and HSV-2, belong to the group of human herpesviruses designated alphaherpesviruses. Varicella zoster virus (VZV) rounds out this group. All herpesviruses are morphologically similar and consist of four components: (1) an electron-dense core containing viral DNA; (2) an icosadeltahedral capsid consisting of 162 capsomeres; (3) an amorphous layer of proteins surrounding the capsid called tegument; and (4) a lipid envelope [6]. The HSV genome is a linear, double-stranded DNA molecule containing at least 152 kilobase pairs encoding at least 84 different polypeptides. For viral replication to occur, infection of the human host cell must take place. HSV attaches to cell surface receptors, fuses its envelope to the plasma membrane, the capsid is de-enveloped, and the DNA is transported to the nucleus. Viral replication then proceeds in the nucleus with transcription, DNA synthesis, capsid assembly, DNA packaging, and envelopment. Synthesis of viral proteins takes place in three sequential periods designated a (immediate–early), b (early), and g (late). The a proteins shut off host protein synthesis and initiate synthesis of b proteins. The b proteins are responsible for viral nucleic acid metabolism and are the main target of current antiviral therapy. These include viral DNA polymerase and viral thymidine kinase. Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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The antiviral drug acyclovir, for example, is a nucleoside analog that is a substrate for the HSV thymidine kinase. Acyclovir is selectively phosphorylated by HSV-infected cells to acyclovir monophosphate. It is then phosphorylated by host cellular enzymes to acyclovirtriphosphate, a competitive inhibitor of the viral DNA polymerase. Acyclovir triphosphate is incorporated into the growing viral DNA chain causing chain termination. Resistance to acyclovir occurs because of spontaneously arising mutations (at a frequency of 10−4) in the viral thymidine kinase or DNA polymerase genes. It is also this thymidine kinase of HSV that has recently been the instrument and focus of gene therapy research. A number of groups [7–9] have transferred the HSVTK “suicide gene” into donor lymphocytes, which confers selective sensitivity to the antiviral drug ganciclovir. Thus, after engraftment, if graft-versus-host disease (GVHD) occurs, ganciclovir can be administered to eliminate donor T cells, among which are the mediators of GVHD, thereby terminating GVHD. Finally, the g proteins are the structural components of the virion. A more detailed description of the virology of HSV can be found in recent reviews [6].

Pathogenesis and immunology HSV possesses two unique properties that it shares with the other alphaherpesvirus, VZV: neurotropism and latency. These two related characteristics explain a great deal of the pathogenesis and pathology of HSV infection and disease. HSV infection begins after close contact of a susceptible individual with someone excreting virus. This usually involves exposure to HSV at mucosal surfaces or abraded skin. For HSV-1, it is usually the oral mucosa, and for HSV-2, it is usually the genital mucosa. However, either virus type can infect at the other site. Viral replication takes place at these mucosal sites and progresses to the sensory or autonomic nerve endings [10]. Virus is then transported intra-axonally to the nerve cell bodies in the dorsal root ganglia [11]. For oral HSV-1 infection, this usually results in trigeminal ganglion infection, and for HSV-2 genital infection, this usually produces sacral nerve root ganglion (S2–S5) infection. Virus continues to replicate in these ganglia and in contiguous neural tissues, and then can spread to other mucosal or skin surfaces through centrifugal migration via peripheral sensory nerves. After primary infection resolves, a state of latency is established. During latency, infectious HSV can no longer be recovered from the ganglia, but viral DNA can be recovered from some ganglion cells. The viral genome remains in an episomal state for the life of the infected human host. The mechanism(s) whereby latency is established and

Herpes Simplex Virus Infections

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maintained is not understood. It is likely that latent infection of neurons involves intrinsic non-permissive properties of sensory neurons and viral functions, and maintaining it likely involves the CD8+ T cell [6,12,13]. Latency allows the virus to survive in a sequestered state not vulnerable to the inhibitory effects of antiviral agents or the host’s immune system. What the property of latency allows is subsequent reactivation of virus. Again, the mechanism whereby reactivation occurs is not known, but a number of stimuli have been closely associated with reactivation: physical or emotional stress, fever, exposure to ultraviolet light, tissue damage, and immune suppression. These are all (if one substitutes radiation therapy for ultraviolet light) present during the early phase of HCT. It is likely that tissue damage and the repair process may have a greater influence on reactivation than immune suppression. The immunologic response to primary and reactivation infection involves both humoral and cell-mediated immune responses. It is assumed that the more crucial immune response is the cell-mediated response since agammaglobulinemic patients handle HSV infection well, and the presence of neutralizing antibody does not prevent reactivation. Also, patients with T-cell immune deficiencies (transplant recipients, those infected with human immunodeficiency virus, and congenitally T-cell-deficient patients) tend to have more widespread, invasive, and disseminated disease. In animal models, it appears that T cells play the major role in preventing dissemination of HSV [14], although antibodies can reduce the viral titers. Monoclonal antibodies have been shown to protect animals against HSV challenge and reduced subsequent disease and latency [15,16]. Other cell populations (CD4 and CD8 T lymphocytes, natural killer cells, macrophages, dendritic cells, and T regulatory cells) and various cytokines also play a role in limiting and terminating infection [17], but it is becoming clear that the key to controlling lytic infection is the CD8+ T cell [6]. HSV is capable of modulating and subverting the host immune response. Programmed death (apoptosis) of a virus-infected cell is one host response intended to prevent the spread of virus to uninfected cells. HSV has been shown to prevent apoptosis in differentiated cells. HSV can also prevent the immune activation of CD8+ cytotoxic T lymphocytes. It does this via the a47-gene product (ICP-47), which induces retention of the major histocompatibility complex class I molecules in the cytoplasm, resulting in a lack of peptide presentation on the cell surface [18]. A more detailed description of the pathogenesis and immunology of HSV infection can be found in recent reviews [6,19].

index profiles, although the mean lymphocyte stimulation index of seropositive patients is lower, that is, 4.5. Immediately after HCT, the lymphocyte response to HSV drops in these seropositive recipients. However, after day 40, the lymphocyte response returns to pretransplant levels. This return of response occurs in those who develop HSV reactivation and is not related to the immune status of the donor. Seronegative recipients continue to show no HSV-specific lymphocyte response. Thus, it appears that the return of specific lymphocyte-mediated immunity to HSV requires exposure to antigen in the form of reactivation infection, and that passively transferred donor immune lymphocytes are insufficient to establish immune reactivity in the recipient. It was also observed that acyclovir treatment of HSV infection after HCT was associated with a delay in restoration of the lymphocyte response [21], and that this delay in HSV-specific immune reconstitution was manifested clinically as more frequent HSV recurrences as well as a shorter interval between recurrences in those treated with acyclovir. In this regard, in an oral acyclovir prophylaxis study, seropositive patients on acyclovir had a significantly lower lymphocyte response than those on placebo within the first 7 weeks of HCT [5]. This response slowly improved until it matched the placebo group at 3 months after transplantation. This was reflected in a 68% incidence of HSV reactivation in the placebo group compared with a 21% incidence in the acyclovir prophylaxis group during the 4-week prophylaxis period. Ultimately, however, after acyclovir was stopped, 58% of the prophylaxis group had HSV infection. Thus, in recipients undergoing prophylaxis, there may be a delay in HSV-specific immune reconstitution.

HSV immunology in the HCT recipient

HSV-1

Almost all of the components of both the innate immune system and the adaptive immune system are deficient after the conditioning regimen for HCT has been administered [20]. It is probably the combination of the stimuli of mucosal damage, induced by the cytotoxic agents of the conditioning regimen, and global, but primarily T-cell-mediated immune deficiency that is responsible for the high reactivation rate seen after HCT. Certainly, the more severe and invasive HSV infections that occur in the early transplantation period are related to a cellular immunodeficiency. Virtually all HSV infection in HCT is due to reactivation and not primary infection. In one early study, 62 (82%) of 76 seropositive patients but only one (1.5%) of 65 seronegative patients developed HSV infection [1]. Approximately 80% of seropositive, immunocompetent persons have a positive lymphocyte proliferation response to HSV antigens, with a mean lymphocyte stimulation index (mean counts per minute of cells exposed to HSV antigen/mean counts per minute of cells exposed to antigen from uninfected cells) of 10.4. This compares with only 7% of seronegative persons, with a mean stimulation index of 1.5× control. Of note, patients pre-HCT have similar lymphocyte stimulation

Primary HSV-1 infections usually occur in young children under the age of 5 years and are usually asymptomatic. The seroprevalence of HSV-1 slowly increases with age until a 90% rate is achieved by the fifth decade of life [22]. A recent balanced household survey from 1999 to 2004 estimated the overall prevalence of HSV-1 in United States adults to be 57% [23]. The prevalence of HSV-1 antibody correlates with socioeconomic status, with higher prevalence in the lower socioeconomic groups [24]. In the United States, African-Americans appear to acquire HSV-1 earlier in life, but the prevalence rates in the white population become similar to African-American rates (90%) by the fifth decade of life [22].

Epidemiology As virtually all HSV disease occurring in the HCT setting is due to reactivation, the serologic status of the recipient determines risk for disease, and therefore the requirement for prophylaxis. The likelihood of any one patient being HSV-1 or HSV-2 seropositive is proportionate to a number of factors, including age, geographic origin, socioeconomic status, race, and, in the case of HSV-2, past sexual activity. However, once the transplantation process is initiated, these factors have no bearing on the risk of reactivation, although other factors do, such as the type and severity of the cytotoxic conditioning regimen. HSVs have a worldwide distribution, and there are no animal vectors or reservoirs. HSV-1 infection is more prevalent and acquired earlier in life than HSV-2.

HSV-2 Antibodies to HSV-2 appear later in life than HSV-1 antibodies. They begin to appear in puberty and correlate with past sexual activity. The HSV-2 seroprevalence of adults in the United States is approximately 17% [23]. In the United States, there is an ethnic difference in sero-

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prevalence of HSV-2 antibody, with 25% in white women, 20% in white men, 80% in African-American women, and 60% in African-American men. Thus, it is highly likely that an adult candidate for HCT will be seropositive for HSV-1 and fairly likely to be seropositive for HSV-2.

Epidemiology of HSV infection in the HCT recipient Prior to the use of antiviral prophylaxis, 82% of seropositive patients reactivated infection after HCT [1]. The median time to infection was during the third week post-HCT. Only 1.5% of seronegative patients had HSV infection. Approximately 90% of infections involved the oral mucous membranes, while 6% involved the genital area. Since the advent of antiviral prophylaxis, the incidence of HSV infection has diminished and shifted to a later time period. While on prophylaxis, few patients – 0% and 21%, respectively, in two separate studies – reactivated HSV infection compared with those on placebo (70% and 68%, respectively) [3,5]. However, in one study, when prophylaxis was discontinued after 4 weeks, 38% of treated seropositive patients reactivated at a median time of 8 weeks after stopping the drug [5]. In another study, where prophylaxis was administered for 18 days, 70% (7 of 10) of seropositive patients on prophylaxis reactivated at a median time of 25 days after cessation of the drug [3]. Five (71%) of these patients developed symptomatic skin lesions, while two (29%) had asymptomatic shedding.

Clinical manifestations Prior to the era of antiviral prophylaxis, the majority (92%) of HSV reactivations presented as oropharyngeal or orofacial lesions, and only 6% presented as genital eruption [1]. Two (3%) had esophageal involvement and two (3%) had lung involvement following oropharyngeal infection. In the prophylaxis era, it appears that the distribution is 85% oropharyngeal and 15% genital presentation [25], possibly reflecting the relatively higher acyclovir resistance of HSV-2 compared with HSV-1. However, HSV infection now occurs predominantly after prophylaxis cessation. In addition, the infections are probably less severe [3,5]. Oropharyngeal HSV infection can be confused with the mucositis that frequently occurs after conditioning therapy for HCT (Plates 27.6 and 27.7). The painful mucosal ulcerations appear similar. The pain suffered by the patient can become a major management problem, but the damage done to the oral mucosa can lead to portals of entry and sites of infection for bacteria (viridans streptococci) and fungi (Candida). Oropharyngeal HSV infection can also lead to extension of infection into the esophagus and lung, but again, with prophylaxis, the severity of oropharyngeal reactivation is much less. Oral labial and facial reactivation is less likely to be confused with regimen-induced mucositis. Also, although the cutaneous lesions are usually classically appearing herpetic lesions, presenting as clusters of vesicular lesions, they can be quite severe and extensive, especially in someone who is not receiving or has not received antiviral prophylaxis. Genital HSV infection usually presents as patches of vesicular lesions in or around the genital area. It can appear in the rectum, perianal area, and buttocks and hips. If it occurs unilaterally, it can be confused with VZV infection. This differentiation becomes important as these two infections are treated differently. HSV-2 can also present as a cystitis or urethritis. Oropharyngeal HSV infection can spread contiguously to the esophagus and the lungs. HSV esophagitis can be severe and, clinically, it cannot be distinguished from cytomegalovirus (CMV) (Plate 27.35) or Candida esophagitis. Morphologically, HSV infection can be distinguished from Candida esophagitis upon endoscopic inspection, but it requires a biopsy to distinguish it from CMV infection. HSV can also cause disease further down the gastrointestinal tract, including the small

intestine [26] and colon [27]. It is not clear, however, whether lower gastrointestinal tract involvement is due to the extension of higher tract infection or blood-borne dissemination, or whether it is even the primary etiology of disease at these sites as other etiologies (CMV, Clostridium difficile or GVHD) are often coexistent. HSV pneumonia probably results as an extension of oropharyngeal infection and usually presents as an interstitial pneumonitis. HSV interstitial pneumonitis has a high fatality rate [28] which does not appear to have changed in the era of prophylaxis [29]. Also, it does not appear that the acyclovir-resistant strains of HSV are any less virulent in the lung. One also has to be careful with regard to attributing pneumonia to HSV when the patient may have an ongoing oropharyngeal reactivation and the bronchoscopically obtained specimen is contaminated by virus in the upper airway or aspirated virus in the lung (see the section on diagnosis, below). HSV hepatitis is fairly rare. In one series, eight cases were found over a 14-year period in a transplantation population base of 3000 [30]. HSV hepatitis probably represents true HSV dissemination as multiple organ involvement was found in the majority of cases. Also, HSV-2 has been reported to cause fulminant hepatitis in early HCT despite antiviral prophylaxis [31]. The mortality rate was 100% in this small series [30]. Cases from the preprophylaxis era occurred before day 20, while those who received prophylaxis did not develop disease until after day 40. Common presenting signs and symptoms included fever, abdominal pain, and elevated serum transaminase levels. HSV encephalitis is a rare complication after HCT. It occurs in the general population with a frequency of 1 in 250,000–500,000 persons per year, and does not appear to be more common in HCT [32]. In the “normal” host, the clinical presentation consists of acute onset of fevers and focal, primarily temporal lobe, neurologic signs and symptoms. However, in the immunocompromised host, the presentation is atypical, with a subacute but progressively deteriorating course.

Diagnosis Diagnosis of HSV infection relies upon both clinical and laboratory criteria. A clinical diagnosis of cutaneous HSV infection can be made when clumps of vesicular lesions on an erythematous base near or around the perioral or genital areas are present. However, there may be difficulty in differentiating HSV infection from VZV infection if these lesions appear to be unilateral and in a limited dermatomal distribution, for example a cluster of lesions on the hip or on one side of the face. In the past, rapid diagnosis of cutaneous lesions was made with adequate scrapings of fresh, early lesions examined microscopically after Giemsa (Tzanck preparation), Wright or Papanicolaou staining. The demonstration of intranuclear inclusions and/or giant cells confirmed a diagnosis of herpesvirus infection. However, these staining techniques do not differentiate between HSV or VZV, and therefore the preferred test is for HSV (and VZV) antigens or DNA. These scrapings can be submitted for antigen detection by direct fluorescent antibody, peroxidase or enzyme-linked methods. These are highly specific and sensitive tests if the specimens are obtained early in newly erupted lesions. Polymerase chain reaction (PCR) can be used to detect HSV DNA in swabs or scrapings from mucocutaneous lesions, is more sensitive than culture, and may be the standard of the future [33]. The standard of diagnosis of HSV infection is virus isolation. The positive culture will also provide the laboratory with an isolate that can subsequently be tested for antiviral resistance. Culture for HSV is recommended when acyclovir resistance is suspected, for example in emergence of infection during acyclovir prophylaxis. Clinical specimens submitted for culture should be transported on ice and immediately inoculated into a cell culture system. A cytopathic effect may develop

Herpes Simplex Virus Infections

within 24–48 hours, but definitive identification may take from 48 to 96 hours. A more rapid cultural technique is the spin-amplified culture, or shell vial, technique with subsequent staining for specific HSV antigen. The turnaround time for this method can be less than 24 hours. A tissue biopsy is usually required to make a diagnosis of visceral HSV infection (Plate 23.36 – see above). The tissue should be submitted for culture and histologic examination. The demonstration of tissue pathology is important in differentiating between HSV disease and HSV excretion and contamination. A positive culture from bronchioalveolar lavage fluid can result from contamination of the fluid from oropharyngeal excretion rather than from the pneumonia. Likewise, a positive culture from tissue or fluid obtained from the esophagus or stomach during an endoscopic procedure might also have come from oropharyngeal excretion of HSV. The standard of diagnosis for HSV encephalitis is isolation of the virus from brain tissue. However, the current method of choice is the much less invasive test of HSV DNA determination by PCR of cerebrospinal fluid [34]. In the post-HCT setting, serologic testing for HSV specific antibody is of very little use. What defines reactivation and infection is detection of HSV in specimens from mucous membranes and tissue. Antibody testing in the HCT candidate is useful in determining past HSV exposure. It is only the seropositive patient who is likely to reactivate HSV infection after HCT, defining those at risk for reactivation during HCT, and therefore the optimal candidate for anti-HSV prophylaxis (see below). Moreover, it has been reported that the risk of HSV reactivation is proportional to the pretransplant HSV immunoglobulin G titer [35].

Treatment The drug of choice for the treatment of HSV mucocutaneous infection in the HCT recipient is acyclovir (Table 91.1). It is usually given intravenously but can be given orally if the patient is capable of taking and absorbing the oral formulation. An early study demonstrated that intravenous acyclovir shortened the time to resolution of pain, crusting of lesions, total healing, and excretion of virus [36]. Unfortunately, recurrent infection was more common in the acyclovir-treated group. This study is the basis for the current standard of therapy: acyclovir 250 mg/ m2 (or 5 mg/kg) intravenously every 8 hours for at least 7 days. A study of oral acyclovir therapy for mucocutaneous HSV infections in HCT recipients demonstrated similar benefits [37]. Acyclovir was given as 400 mg orally five times a day for 10 days. The choice of oral versus intravenous therapy is based on the ability to take oral medications and on the severity of disease. Newer oral drugs that result in higher serum levels can probably also be used, although they have not been studied extensively in HCT patients. These include valacyclovir, a prodrug of acyclovir, and famciclovir, a prodrug of penciclovir. Valacyclovir is administered at 500–1000 mg orally three times a day for 7 days, while famciclovir is administered at 500 mg orally three times a day for 7 days. If the patient has more severe, visceral, disseminated or central nervous system disease, acyclovir at 10 mg/kg (or 500 mg/m2) intravenously every 8 hours is recommended. If infection with acyclovir-resistant HSV is suspected or proven, the drug of choice is foscarnet, a nonthymidine kinase-dependent agent. It is given at 40 mg/kg intravenously every 8 hours. For more severe or visceral involvement, 60 mg/kg every 8 hours is recommended. The major and limiting toxicity of foscarnet is nephrotoxicity, making it very difficult to use in the HCT setting. There have also been reports of HSV strains resistant to both acyclovir and foscarnet [38–40]. In such cases, cidofovir has shown some benefit and is recommended at 5 mg/kg intravenously once a week for 2 weeks, and then once every 2 weeks. However, this drug is also nephrotoxic. A less nephrotoxic dosing sched-

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ule may be 1 mg/kg/day five times a week. Recently, cidofovir resistance developing in vivo was reported in HCT recipients being treated for HSV disease [41]. Fortunately, these isolates remained sensitive to both acyclovir and foscarnet.

Prophylaxis Acyclovir prophylaxis should be offered to all HSV-seropositive allogeneic recipients to prevent HSV reactivation during the early posttransplant period. This is the recommendation of the Centers for Disease Control and Prevention, the Infectious Disease Society of America, and the American Society for Blood and Marrow Transplantation [42]. The recommended regimen is either acyclovir 200 mg orally three times a day or acyclovir 250 mg/m2 intravenously every 12 hours. It is also recommended that acyclovir be started at the beginning of conditioning therapy and continue until engraftment or until mucositis resolves, that is, approximately 30 days after HCT for allogeneic HCT recipients. The duration of acyclovir prophylaxis can be extended beyond 30 days for persons with “frequent recurrent HSV.” It should also be extended for those HCT recipients with active GVHD. A recommended alternative to acyclovir is valacyclovir. If a patient is already receiving ganciclovir, valgancyclovir, valacyclovir, foscarnet, cidofovir or maribavir, acyclovir is not necessary. If a patient is placed on long-term (i.e. 1 year or longer) prophylaxis for VZV reactivation prophylaxis, there should be no break between the end of HSV and the end of VZV prophylaxis. Despite this strong recommendation and the fact that most HCT centers use HSV prophylaxis, the decision to use universal prophylaxis is not without controversy. Furthermore, the duration of prophylaxis varies among centers. Although antiviral prophylaxis has clearly been shown to dramatically reduce the incidence of viral shedding and disease during the period of prophylaxis, reactivation and disease still occur after Table 91.1 Prophylaxis and treatment guidelines for herpes simplex virus (HSV) infections in hematopoietic cell transplantation (HCT) Drug

Route

Dose

Interval

Prophylaxis: Acyclovir Acyclovir Valacyclovir

IV p.o. p.o.

250 mg/m2 or 5 mg/kg 200 mg 500–1000 mg

Every 12 hours t.i.d. b.i.d.

Treatment: Acyclovir Acyclovir Valacyclovir Famciclovir

IV p.o. p.o. p.o.

250 mg/m2 or 5 mg/kg 400 mg 500–1000 mg 500 mg

Every 8 hours 5 times a day b.i.d. b.i.d.

For visceral or disseminated disease: Acyclovir IV 500 mg/m2 or 10 mg/kg

Every 8 hours

For acyclovir-resistant HSV: Foscarnet IV

Every 8 hours

40–60 mg/kg

For acyclovir/foscarnet-resistant HSV: Cidofovir IV 5 mg/kg

Or

IV

1 mg/kg

b.i.d., twice daily; IV, intravenous; p.o., orally; t.i.d., three times daily.

weekly ×2, then every 2 weeks every other day or three times a week

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prophylaxis has ended [3–5]. If the duration of prophylaxis is extended much beyond the recommended 30 days to either 75 days or 6 months, there appear to be fewer reactivations and less severe disease [35,43,44]. Also, prophylaxis not only delays reactivation but also delays immunologic reactivity to HSV antigens and specific immune reconstitution to this virus [5]. Whether this delay is detrimental or beneficial to the HCT recipient is unknown. Another argument for prophylaxis is the observation that resistant virus often emerges in the treatment setting but rarely in the prophylactic setting [5,45]. In a recent study [46] to determine the efficacy of long-term prophylaxis for VZV infection in HCT [47], three cohorts of HSV- and VZVseropositive patients received prophylaxis for either 1 month, 1 year, or longer than 1 year after transplantation. The 2-year probability of HSV disease was 31.6%, 3.9%, and 0% respectively. Thus, long-term prophylaxis resulted in less disease/reactivation and prevented the emergence of drug-resistant HSV. In addition, although higher doses of prophylactic agents (acyclovir 800 mg orally twice daily or valacyclovir 500 mg orally twice daily after engraftment) were used, no hematologic toxicity was noted. If long-term (at least 1 year) prophylaxis becomes standard for VZV prevention in HCT, certainly there should be fewer HSV reactivations and fewer acyclovir-resistant HSV emerging. There may be other benefits of using HSV prophylaxis in the HCT setting. Meyers et al. [48] demonstrated a significant decrease in CMV infection and disease and significantly improved survival in those HCT recipients given high doses (500 mg/m2 every 8 hours) of intravenous acyclovir prophylaxis until 30 days after HCT. However, others using lower doses (250 mg/m2) of acyclovir could demonstrate no effect on the frequency of CMV infections [37]. A more recent study demonstrated that valacyclovir was even more effective as a CMVprophylactic agent when given after the initial intravenous/high-dose acyclovir prophylaxis [49]. There were also fewer HSV infections in the valacyclovir group versus the acyclovir group (7% and 10%, respectively), but the difference was not significant. There have also been reports of earlier engraftment in patients receiving acyclovir prophylaxis [5], but this has not been confirmed by others [50,51]. If a new, as yet unapproved, antiviral drug, maribavir [52], proves to be a nonmarrow toxic, effective prophylactic agent against CMV infection, it will obviate

the need for anti-HSV prophylaxis, at least from the time at which maribavir is initiated (probably at engraftment). The ultimate preventive measure is the development of a vaccine that will prevent primary HSV infection in the general population. Preferably it would be a killed, subunit, replication-defective viral or naked DNA vaccine, as a live vaccine would have the unknown potential of reactivating during HCT. Work is in progress [6,19,53], but there will be no effective vaccine in the near future, and it will not be of help to those who are already seropositive. It is unlikely that a vaccine could ever prevent HSV reactivation in the profoundly immunocompromised seropositive population of HCT recipients.

Conclusion Since the inception of acyclovir prophylaxis, the incidence of HSV reactivation and disease has dramatically decreased during the early period of HCT. However, prophylaxis as now administered (until engraftment or approximately 30 days) under current recommendations has resulted in a delay of HSV-specific immune reconstitution, and reactivation now occurs during a later period in HCT. However, severe, visceral, and disseminated HSV infection still occurs and demands rapid and specific diagnosis in order to initiate effective therapy, and although resistant virus is less likely to occur under prophylactic conditions, it still remains a significant problem in treating patients with HSV infection after HCT. A possible solution to the late reactivation or “rebound” phenomenon after early cessation of prophylaxis and the problem of the emergence of acyclovir-resistant HSV may be to extend the duration of HSV prophylaxis (in addition to using higher doses than currently recommended) while at the same time protecting the HCT recipient against VZV reactivation, which may be an even more devastating herpesvirus infection. However, further examination of this approach will have to be undertaken before a recommendation for longer duration of prophylaxis can be made. If a recommendation for long-term, highdose VZV prophylaxis is made, the secondary benefits for the HCT recipient would be fewer HSV reactivations/disease and a lower incidence of resistant HSV, and the issue of duration of prophylaxis becomes a moot question.

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12. Divito S, Cheerpes TL, Hendricks RL. A triple entente: virus, neurons, and CD8+ T cells maintain HSV-1 latency. Immunol Res 2006; 36: 119–26. 13. Sheridan BS, Knickellbein JE, Hendricks RL. CD8 T cells and latent herpes simplex virus type 1: keeping the peace in sensory ganglia. Expert Opin Biol Ther 2007; 7: 1323–31. 14. Kapoor AK, Nash AA, Wildy P et al. Pathogenesis of herpes simplex virus in congenitally athymic mice: the relative roles of cell-mediated and humoral immunity. J General Virol 1982; 60: 225–33. 15. Balachandran N, Bacchetti S, Rawls WE. Protection against lethal challenge of BALB/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type-2. Infect Immun 1982; 37: 1132–7. 16. Eisenberg R, Cerini CP, Heilman CJ et al. Synthetic glycoprotein d-related peptides protect mice against herpes simplex virus challenge. J Virol 1985; 55: 1014–17. 17. Manickan E, Rouse BT. Role of different T-cell subsets in control of herpes simplex virus infection determined by using T-cell-deficient mouse models. J Virol 1995; 69: 8178–9.

Herpes Simplex Virus Infections 18. York IA, Roop C, Andrews DW et al. A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 1994; 77: 525–35. 19. Koelle DM, Corey L. Herpes simplex: insights on pathogenesis and possible vaccines. Ann Rev Med 2008; 59: 381–95. 20. Storek J, Witherspoon RP. Immunologic reconstitution after hematopoietic stem cell transplantation. In: Atkinson K, editor. Clinical Bone Marrow and Blood Stem Cell Transplantation. Cambridge, UK: Cambridge University Press; 2000. pp. 111– 46. 21. Wade JC, Day LM, Crowley JJ, Meyers JD. Recurrent infection with herpes simplex virus after marrow transplantation. Role of the specific immune response and acyclovir treatment. J Infect Dis 1984; 149: 750–6. 22. Nahmias AJ, Lee FK, Bechman-Nahmias S. Seroepidemiological and sociological patterns of herpes simplex virus infection in the world. Scand J Infect Dis 1990; 69: 19–36. 23. Xu F, Sternberg MR, Kottiri BJ et al. Trends in herpes simplex virus type 1 and type 2 seroprevalence in the United States. JAMA 2006; 296: 964– 73. 24. Rawls WE, Iwamoto K, Adam E, Melnick JL. Measurement of antibodies to herpesvirus type 1 and 2 in human sera. J Immunol 1970; 104: 599– 606. 25. Sable CA, Donowitz GR. Infections in bone marrow transplant recipients. Clin Infect Dis 1994; 18: 273–84. 26. Kingreen D, Nitsche A, Beyer J, Siegert W. Herpes simplex infection of the jejunum occurring in the early post-transplantation period. Bone Marrow Transplant 1997; 20: 989–91. 27. Naik HR, Chandrasekar PH. Herpes simplex virus (HSV) colitis in a bone marrow transplant recipient. Bone Marrow Transplant 1996; 17: 285–6. 28. Ramsey PG, Fife KH, Hackman RC, Meyers JD, Corey L. Herpes simplex virus pneumonia. Ann Intern Med 1982; 97: 813–20. 29. Ljungman P, Ellis MN, Hackman RC, Shepp DH, Meyers JD. Acyclovir-resistant herpes simplex virus causing pneumonia after marrow transplantation. J Infect Dis 1990; 162: 244–8. 30. Johnson JR, Egaas S, Gleaves CA, Hackman R, Bowden RA. Hepatitis due to herpes simplex virus in marrow-transplant recipients. Clin Infect Dis 1992; 14: 38–45. 31. Gruson D, Hilbert G, Le Bail B, Portel L, Boiron JM, Reiffers J, Gbikpi-Benissan G. Fulminant hepatitis due to herpes simplex virus-type 2 in early

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phase of bone marrow transplantation. Hematol Cell Ther 1998; 40: 41–4. Whitley RJ. Herpes simplex encephalitis: adolescents and adults. Antiviral Res 2006; 71: 141–8. Strick LB, Wald A. Diagnostics for herpes simplex virus: is PCR the new gold standard? Mol Diagn Ther 2006; 10: 17–28. Lakerman FD, Whitley RJ, the National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group. Diagnosis of herpes simplex encephalitis: application of polymerase chain reaction 50 cerebrospinal fluid from brain biopsied patients and correlation with disease. J Infect Dis 1995; 171: 857–63. Lundgren G, Wilczek H, Lonnqvist B, Lindholm A, Wahren B, Ringden O. Acyclovir prophylaxis in bone marrow transplant recipients. Scand J Infect Dis 1985; 47(Suppl): 137–44. Wade JC, Newton B, McLaren C, Flournoy N, Keeney RE, Meyers JD. Intravenous acyclovir to treat mucocutaneous herpes simplex virus infection after marrow transplantation: a double-blind trial. Ann Intern Med 1982; 96: 265–9. Shepp DH, Newton BA, Dandliker PS, Flournoy N, Meyers JO. Oral acyclovir mucocutaneous herpes simplex virus infections in immunocompromised marrow transplant recipients. Ann Intern Med 1985; 102: 783–5. Bryant P, Sasadeusz J, Carapetis J, Waters K, Curtis N. Successful treatment of foscarnetresistant herpes simplex stomatitis with intravenous cidofovir in a child. Pediatr Infect Dis J 2001; 20: 1083–6. Blot N, Schneider P, Young P, Janvresse C, Dehesdin D, Tron P, Vannier JP. Treatment of an acyclovir and foscarnet-resistant herpes simplex virus infection with cidofovir in a child after an unrelated bone marrow transplant. Bone Marrow Transplant 2000; 26: 903–5. Darville JM, Ley BE, Roome AP, Foot AB. Acyclovir-resistant herpes simplex virus infections in a bone marrow transplant population. Bone Marrow Transplant 1998; 22: 587–9. Wyles DL, Patel A, Madinger N, Bessesen M, Krause PR, Weinberg A. Development of herpes simplex virus disease in patients who are receiving cidofovir. Clin Infect Dis 2005; 41: 676–80. Center for Disease Control and Prevention. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients: recommendations of the CDC, the Infectious Disease Society of America, and the American Society of Blood and Marrow Transplantation. MMWR 2000; 49 (RR-10): 15–16.

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43. Shepp DH, Dandliker PS, Fournoy N, Meyers JD. Sequential intravenous and twice-daily oral acyclovir for extended prophylaxis of herpes simplex virus infection in marrow transplant patients. Transplantation 1987; 43: 654–8. 44. Ljungman P, Wilczek H, Gahrton G et al. Longterm acyclovir prophylaxis in bone marrow transplant recipients and lymphocyte proliferation responses to herpesvirus antigens in vitro. Bone Marrow Transplant 1986; 1: 185–92. 45. Wade JC, McLaren C, Meyers JD. Frequency and significance of acyclovir-resistant herpes simplex virus isolated from marrow transplant patients receiving multiple courses of treatment with acyclovir. J Infect Dis 1983; 48: 1077–82. 46. Erard V, Wald A, Corey L, Leisenring WM, Boeckh M. Use of long-term suppressive acyclovir after hematopoietic stem-cell transplantation: impact on herpes simplex virus (HSV) disease and drug-resistant HSV disease. J Infect Dis 2007; 196: 266–70. 47. Boeckh M, Kim HW, Flowers MED, Meyers JD, Bowden RA. Long-term acyclovir for prevention of varicella zoster virus disease after allogeneic hematopoietic cell transplantation – a randomized double-blind placebo-controlled study. Blood 2006; 107: 1800–5. 48. Meyers JD, Reed EC, Shepp DH et al. Acyclovir for prevention of cytomegalovirus infection and disease after allogeneic marrow transplantation. N Engl J Med 1988; 318: 70–5. 49. Ljungman P, de la Camara R, Milpied N et al. Randomized study of valacyclovir as prophylaxis against cytomegalovirus reactivation in recipients of allogeneic bone marrow transplants. Transplantation 2002; 99: 3050–6. 50. Perren TJ, Powles RL, Easton D, Stolle K, Selby PS. Prevention of herpes zoster in patients by longer-term oral acyclovir after allogeneic bone marrow transplantation. Am J Medical 1988; 85(Suppl. 2A): 99–101. 51. Lundgren G, Wilczek H, Lonnqvist B, Lindholm A, Wahren B, Ringden O. Acyclovir prophylaxis in bone marrow transplant recipients. Scan J Infect Dis Suppl 1985; 47: 137–44. 52. Winston DJ, Young JA, Pullarkat V et al. Maribavir prophylaxis for prevention of cytomegalovirus infection in allogeneic stem-cell transplant recipients: a multicenter, randomized, double-blind, placebo-controlled, dose-ranging study. Blood. Epub 2008 February 19, PMID 2006228. 53. Koelle DM. Vaccines for herpes simplex virus infections. Curr Opin Investig Drugs 2006; 7: 136–41.

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Dora Y. Ho & Ann M. Arvin

Varicella-zoster Virus Infections

Introduction Varicella-zoster virus (VZV), like other pathogens of the herpesvirus family, can cause severe infections in hematopoietic cell transplant (HCT) recipients [1,2]. As in other immunodeficient patients, serious VZV disease after transplantation is related to the compromise of Tlymphocyte function [3,4]. VZV infections are encountered in HCT patients who are experiencing their initial contact with the virus or who have recurrent disease due to the reactivation of latent virus. Primary VZV infection manifests clinically as varicella or “chickenpox,” in which the exposure of a susceptible individual, who has not had previous VZV infection, to the virus results in systemic symptoms of fever and malaise, and a characteristic vesicular rash. After primary infection, VZV establishes latency in cells of the dorsal root ganglia. Serum immunoglobulin G (IgG) antibodies to VZV provide evidence of a past primary infection and indicate that the individual is latently infected with the virus. The reactivation of endogenous latent VZV usually causes herpes zoster, in which the vesicular eruption appears in a localized, dermatomal distribution. Recurrent VZV infection also presents as atypical, nonlocalized herpes zoster in HCT recipients that cannot be distinguished from varicella by its clinical manifestations. Both primary and recurrent infections of VZV can cause significant morbidity and, in some cases, even mortality in HCT recipients. In addition to describing the clinical patterns of VZV infection after HCT, significant progress has been made toward understanding viral pathogenesis and the host response to VZV among HCT recipients. Fortunately, most diseases caused by either primary or recurrent VZV infection, when recognized promptly, can now be treated effectively with antiviral therapy. While live attenuated VZV vaccines are available for the prevention of varicella in the pediatric population and zoster in the elderly population, these vaccines are not recommended for use in immunocompromised patients. Instead, an experimental inactivated varicella vaccine has been tested in HCT recipients. Prophylactic approaches using antiviral drugs have also been evaluated.

The virus VZV is a member of the alfaherpesvirus subgroup of the herpesvirus genus. It is an enveloped virus that has a double-stranded DNA genome surrounded by an icosahedral capsid [5]. The VZV genome contains

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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approximately 125,000 base-pairs, arranged as long unique and short unique segments, each of which contains terminal repeat sequences. The VZV genome has coding regions for at least 70 distinct genes. Information about most of the VZV gene products is limited, but VZV proteins regulate viral gene transcription during replication and form the viral capsid, tegument, and virion envelope structures. Probable functions for some VZV proteins have been deduced from homologies with herpes simplex virus type 1 (HSV-1), which is the prototype of the alfaherpesvirus subgroup. Like HSV, replication of VZV usually involves synthesis and activation of a viral thymidine kinase, which makes the virus susceptible to inhibition by the antiviral agent acyclovir. Since the thymidine kinase is not required for VZV replication, mutant strains that do not express this antiviral target can be selected by exposure to the drug. The viral glycoproteins gB, gC, gE, gH, and gI, as well as viral proteins with structural and regulatory functions, including the IE4, IE62, and IE63 proteins, are known to be targets of the host response following VZV infection. VZV is spread as cell-free virus to susceptible hosts, but is transmitted by cell-to-cell spread in the infected host. VZV gE and gI are important for cell-to-cell spread in vitro [6,7] and recent evidence suggests that gE interacts with the insulin-degrading enzyme of the host cell [8].

Epidemiology Primary VZV infection Primary VZV infection, or varicella, is much less common than disease caused by VZV reactivation during the first year after HCT, accounting for only about 5% of VZV infections in this population. The lower incidence of varicella is due to the fact that more than 85% of individuals in the United States have had primary VZV infection by 8 years of age as a result of the annual varicella epidemics that occur in this and other temperate regions of the world. Nevertheless, if an HCT recipient has never had VZV infection before, the attack rate for varicella after close exposure to an index case will reflect the risk of transmission to any susceptible individual, defined as an individual who has not been infected previously. Attack rates range from about 30% with classroom exposure, to 90% with household contact. Direct contact with lesions is not required since, in contrast to other herpesviruses, VZV is transmissible by the respiratory route. The incidence of primary VZV infection is higher in pediatric HCT recipients, and, as expected, the risk correlates inversely with the age of the child. In one series, 10 of 54 children (18%) with VZV infections after HCT had primary VZV infection demonstrated serologically [5]. Although 25 adult patients presented with a disseminated cutaneous VZV exanthem, only 2 patients had serologic

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Table 92.1 Incidence of varicella-zoster virus (VZV) infections following hematopoietic cell transplantation (HCT) Clinical presentation

Year

Underlying disease

Transplant type

VZV infection* n (%)

Localized zoster (%)

Atypical zoster† (%)

Varicella (%)

1980 [9] 1982 [10] 1985 [1] 1986 [11] 1989 [12]‡ 1989 [13] 1991 [14] 1994 [15] 2000 [16] 2000 [17] 2000 [18]‡ 2001 [19] 2003 [20] 2007 [21]§ 2007 [21]§

Leukemia Leukemia/aplastic anemia Leukemia/aplastic anemia Hematologic malignancy Leukemia/solid tumors Leukemia/lymphoma Hodgkin’s disease Leukemia/lymphoma/other Leukemia/other Leukemia/other Malignancies/other Leukemia/lymphoma/other Hematologic malignancy Leukemia/lymphoma/other Leukemia/lymphoma/other

Allogeneic/syngeneic Allogeneic/syngeneic Allogeneic Allogeneic Autologous Autologous Autologous Autologous/allogeneic Allogeneic Allogeneic Autologous/allogeneic Autologous Cord blood Allogeneic Autologous

33 98 1394 73 236 153 28 1186 100 151 109 164 40 720 212

85 91 75 77 100 62 100 100 42 92 88 80 87

15 7 13 20 0 32 0 0 40 4 12 20 13

0 2 18 2 0

21 52 17 36 23 28 32 18 41 38 30 16 63 25 21

18 4

* Percentage of patients who developed any VZV infection. † Including central nervous system, disseminated, or visceral disease. ‡ Pediatric patients only. § Including only patients that did not receive acyclovir for VZV prophylaxis, but received acyclovir from day 7 before HCT to engraftment, if herpes simplex virus positive.

evidence of primary VZV infection [1]. While the possibility of reinfection cannot be eliminated in some cases, the epidemiologic evidence suggests that patients who have been infected previously are usually protected against infection following a new VZV exposure, presumably because VZV IgG titers are maintained despite immunosuppressive disease or therapies. The addition of the varicella vaccine to the routine childhood immunization schedule has reduced the extent of the annual epidemics, making it less likely that HCT recipients who have no preexisting immunity to VZV will be exposed to varicella, and more likely that pediatric HCT recipients will have vaccine-induced immunity to VZV. Recurrent VZV infection The reported incidence of recurrent VZV infection after HCT ranges from 16% to 63% (Table 92.1) [1,9–21]. The incidence of recurrent VZV infection after HCT is higher than that observed among solid organ transplant recipients, which is about 7%, but it is comparable to rates of VZV reactivation in patients with Hodgkin’s disease receiving combined modality therapy. For purposes of comparison, the estimated annual incidence of herpes zoster in adults without underlying disease is 0.5% [16]. The reactivation of herpesviruses follows a predictable temporal pattern after HCT [2]. HSV-1 causes clinically apparent disease at about 2–3 weeks, and cytomegalovirus (CMV) disease usually occurs during the second to third months after transplantation. Epstein–Barr virus may also reactivate in the third month, whereas VZV recurrences present at a median of 5 months after HCT. In general, the risk of recurrent VZV infection is highest between 2 and 10 months after transplantation, although cases have been reported within the first week and continue to occur after the first year (Fig. 92.1). Locksley et al. found that 80% of patients who developed VZV infection following HCT had disease

within 9 months [1]. When options for antiviral therapy were limited, 21% of these patients had visceral dissemination of the virus, and 12% died from complications of recurrent VZV. In a review of 100 consecutive adult patients who received allogeneic HCT between 1992 and 1997, 41% developed VZV reactivation at a median of 227 (range 45– 346) days [16]. Of these episodes, 12% occurred in the first 100 days; the attack rate was 59% among patients who survived for at least 2 years. In another retrospective analysis of VZV infection after HCT in children [18], 33 (30%) of the total 109 children who were transplanted during a 7-year period developed post-transplant VZV infection, including 6 cases of varicella and 27 cases of zoster. Twenty-four of these 33 (73%) children had VZV infection within 1 year following HCT. The cumulative incidences of post-transplant VZV infection at 1 and 5 years were 26% and 45%, respectively. Factors that are associated with higher rates of VZV reactivation following HCT include genotypic nonidentity for human leukocyte antigen (HLA) between donor and recipient, and acute or chronic graft-versushost disease (GVHD), which are linked variables [17]. In one series, 64% of patients whose donor was HLA nonidentical had herpes zoster, compared with 44% of HCT recipients with matched donors and no GVHD. The risk of late VZV infection was increased fourfold (Table 92.2) [22]. In a second series, patients with a limited chronic GHVD had a lower incidence of VZV reactivation compared with those with extensive chronic GVHD [16]. The presence of nonspecific suppressor cells associated with chronic GVHD correlated with a higher risk of recurrent VZV (Table 92.2) [10]. A CD4 T-cell count of less than 200 cells/μL and a CD8 T-cell count of less than 800 cells/μL at 30 days after transplantation correlated with an increased risk of zoster within the first year [19]. The cumulative evidence indicates that patients undergoing allogeneic or autologous HCT have about the same overall risk of recurrent VZV disease, although cord blood HCT seems to pose the highest risk for VZV

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reactivation [20] (Table 92.1). In one study, 28% of autologous transplant recipients developed recurrent VZV infection [13]. The initial manifestation of recurrence was localized disease in 77% of these patients, while 23% had atypical nonlocalized zoster, which is comparable to the distribution of these clinical syndromes among allogeneic HCT recipients. Thirty-two percent of patients who received an autologous HCT for Hodgkin’s lymphoma had VZV infection [14], which is consistent with experience in Hodgkin’s and non-Hodgkin’s lymphoma patients [23]. The somewhat higher incidence of zoster in lymphoma patients may account for the fact that patients who receive autologous HCT have rates of VZV reactivation that are similar to those in allogeneic

35

25

Re-infection with VZV

20

15

10

D D D D D D D D D D D D D D V V

D D D D D D D D V V

D D D D V V

D D D D D D DDD VVVD

D D D D D D V D V

1 2 3 4 5 6 7 8 9 10 11 12

D D D 5 D DD DD DD VD

D D D D D D D V V V V V V

D D D D D D D D V V V V V V V V V V V

V V VDD

V

VD

V

V

13–15 16–18 19–21 22–24 25–27 28–30 31–33 34–36 37–39 40–42 43–45 46–48 49–51 52–54 55–57 58–60 61–63 64–66 67–69

Number of patients with VZV infection

30

HCT recipients, even though GVHD is less common. Morbidity and mortality caused by VZV infection after autologous HCT cannot be compared with the original analyses of VZV-related disease after allogeneic HCT because antiviral therapy was available when the studies of autologous HCT patients were done. In the series of Schuchter et al., 15% of autologous HCT recipients had cutaneous dissemination, 5% had visceral dissemination, and 25% had morbidity, including post-herpetic neuralgia and neurologic dysfunction [13]. In a more recent series of pediatric HCT patients, 61 of 201 (35%) allogeneic HCT recipients versus 10 of 109 (9%) autologus HCT recipients developed zoster. Risk factors for development of zoster included age over 10 years, allogeneic HCT, and total body irradiation in allogeneic recipients [24]. The underlying disease which provided the indication for HCT probably does not influence VZV reactivation. However, a history of symptomatic herpes zoster before marrow transplantation was associated with a higher risk of early post-transplant VZV disease among Hodgkin’s lymphoma patients undergoing autologous HCT [14]. VZV reactivation has not been found to predict relapse of the underlying disease in HCT recipients.

Month after transplantation

Fig. 92.1 Varicella-zoster virus (VZV) infections by month after marrow transplantation: ⵧ, uncomplicated herpes zoster; V, varicella; D, herpes zoster with subsequent dissemination. Reprinted with permission of the Chicago University Press from Locksley et al. [1].

Natural immunity following varicella usually protects against systemic reinfection. For this reason, symptomatic reinfection with VZV is very rare among immunocompetent individuals. Surveillance of 9947 cases of varicella suggested that 4.5–13.5% of children presenting with varicella had a documented previous episode of varicella, suggesting that clinical reinfection may be more common than previously thought [25]. However, most of these cases of varicella were not laboratory confirmed, and other childhood viral illness can also present with vesicular rashes. Further, a study in adults showed that a history of previous varicella infection in adults was not reliable based on absence of VZV IgG antibodies in pre-illness serum specimens [26]. Reinfection probably occurs occasionally among severely immunocompromised patients who have had primary VZV infection previously. However, this hypothesis is difficult to confirm because the distinction between atypical nonlocalized herpes zoster (which is much more common in immunocompromised patients) and reinfection requires a comparison of the VZV isolates causing the initial infection and the new episode by DNA sequence analysis. Based on clinical criteria only, children with leukemia have been reported to have second episodes of varicella following close contact despite a past history of varicella and serologic evidence of pre-existing immunity. Nevertheless, anecdotal experience suggests that exposure of HCT patients who have had past primary VZV infection rarely causes any clinically apparent signs of reinfection [2].

Table 92.2 Risk of recurrent varicella-zoster, virus (VZV) infection after hematopoietic cell transplantation (HCT) in relation to human lcukocyte antigen (HLA) matching of recipient and donor and the occurrence of graft-versus-host disease (GVHD) in the recipient. Adapted from Atkinson et al. [22] HLA identical With GHVD

No. of patients Late VZV infections VZV at any time

No GHVD

No suppressor cells

With suppressor cells

HLA nonidentical

Syngeneic

25 24% 44%

21 38% 57%

19 47% 79%

14 36% 64%

19 11% 21%

Nonspecific suppressor cells were detected in cocultures with donor lymphocytes, assessing their effect on the proliferation responses to alloantigens or concanavalin-A.

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Fig. 92.2 The pathogenesis of varicella-zoster virus (VZV) infection in the human host. VZV infection is acquired by inoculation of mucosal epithelial cells. Since VZV is a T-cell tropic virus, infection of T cells in lymphoid tissues of Waldeyer’s ring can amplify the infection, and infected T cells can then transport VZV to the skin via a cell-associated viremia. Infection of skin produces the vesicular rash associated with chickenpox. During skin infection, VZ virions gain access to the sensory nerve cell body by retrograde axonal transport from fine nerve endings or via cell-associated viremia, to establish a lifelong latent infection within the sensory ganglia. Herpes zoster results from reactivation of latent VZV. During reactivation, VZ virions gain access to skin via anterograde transport along neuronal axons, causing a localized dermatomal rash. In immunocompromised patients, reactivation may result in T-cell infection and dissemination, with or without a dermatomal rash.

Mechanisms of pathogenesis and viral immune evasion Primary VZV infection Events during the 10- to 21-day incubation period of primary VZV infection have been difficult to document with laboratory methods. The epidemiologic evidence suggests that infection is initiated by the inoculation of respiratory mucosal sites (Fig. 92.2) [5]. Infectious virus is then presumed to be transported to regional lymphoid tissue, possibly undergoing a phase of local replication at these sites. VZV causes a cell-associated viremia, which appears to target T lymphocytes, although the possibility that other mononuclear cells become infected has not been eliminated definitively. Tonsillar T cells are highly permissive for VZV infection, suggesting that VZV could be transferred efficiently from respiratory epithelial cells into these local T-cell-rich tissues as a first stage of spread within the naïve host [27]. Cell-associated viremia has been demonstrated late in the incubation period, just before and after the appearance of varicella skin lesions.

VZV was isolated by viral culture from 24% of samples of peripheral blood mononuclear cells (PBMCs) taken within 24 hours after the onset of rash [28]. Cell-associated VZV viremia was detected by in situ hybridization with a probe for VZV DNA, which showed viral DNA in lymphocytic cells, and by polymerase chain reaction (PCR) testing for viral DNA in PBMC specimens from otherwise healthy subjects tested immediately after the appearance of skin lesions [29]. VZV infection of tonsillar T lymphocytes was enhanced in activated, memory CD4 T lymphocytes that expressed skin homing markers, which suggests that cell-associated viremia may be particularly associated with T-lymphocyte infection [27]. When the cutaneous exanthem develops, VZV replication in epithelial cells may amplify viremia by transfer of infectious virus into migrating T-lymphocyte populations, leading to secondary “crops” of skin lesions. These observations about the pathogenesis of primary VZV infection suggest that the efficiency with which cell-associated viremia and replication at skin sites is terminated, either by the host response or by antiviral therapy, is likely to influence the extent of the cutaneous exanthem and the risk of visceral dissemination (Fig. 92.3).

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Fig. 92.3 The pathogenesis of primary and recurrent varicella-zoster virus (VZV) infection is modulated by innate immune responses of cutaneous epithelial cells. This diagram depicts new concepts about events in the pathogenesis of VZV infection of skin. During primary VZV infection, T cells within the tonsil lymphoid tissues become infected by VZV. Infected T cells transport the virus to the skin shortly thereafter, exiting through capillary endothelium by the usual mechanisms for trafficking of migratory T cells. The infected T cells then release infectious VZV at skin sites of replication. The remainder of the 10- to 21-day incubation period is the interval required for VZV to overcome the innate interferon-alfa (IFN-α) and other antiviral responses in enough epidermal cells to create the typical vesicular lesions containing VZV at the skin surface. Signaling of innate responses, such as signal transducers and activators of transcription-1 (Stat1), interleukin-1 alfa (IL-1α), and nuclear factor kappa B (NF-κB), in adjacent skin cells prevents a rapid, uncontrolled, cell-to-cell spread of VZV. When uninfected T cells that are trafficking through skin in normal immune surveillance encounter early stage cutaneous lesions, they may become infected and amplify viremia. When VZV reactivates from latency in sensory neurons, it is delivered to skin by axonal transport. VZV replication in the involved dermatome encounters innate barriers as the initial host response, followed by the appearance of VZV-specific T cells that clear active infection. If these responses are delayed, the virus may enter migrating T cells to cause disseminated VZV infection.

The study of VZV pathogenesis in animal models has been hampered by the restricted replication of the virus in nonhuman species. Mice that have inherited severe combined immunodeficiency (SCID) can be used to support grafts of human tissues that differentiate to contain the usual human cell populations. These animals, which are referred to as SCIDhu mice, provide a unique opportunity to examine VZV–cell interactions in intact human tissues independently of the effects of the host immune response on viral replication (Plate 92.1) [30,31] (reviewed in [32]). Conditions in the model are similar to those in the period immediately after bone marrow transplantation, since VZV-specific immunity is absent. Experiments in the SCID-hu mouse model demonstrate that VZV infects CD4+ and CD8+ human T lymphocytes, proving that VZV shares pathogenic mechanisms that are characteristic of the lymphotropic as well as the neurotropic herpesviruses [32]. The tropism of VZV for human T cells and its infectivity for skin are both essential elements of its pathogenicity in human disease, but comparative analyses of VZV strains demonstrate that these tropisms are mediated by different virulence determinants. For example, the VZV strain used to make the live attenuated varicella vaccine, the vaccine Oka strain, was indistinguishable from low-passage VZV in its infectivity for CD4+ and CD8+ T cells, whereas its pathogenic potential in human skin was reduced substantially compared with the parent Oka strain, as assessed by the extent of the cutaneous lesions, viral protein synthesis, infectious virus yields, and release of infectious virus. Transfer of VZV from infected T cells into skin, followed by the appearance of VZV skin lesions, was demonstrated in the SCID-hu model [33]. These experiments also revealed the substantial upregulation of interferon-alfa (IFN-α) production by epidermal cells adjacent to VZV-infected cells and blocking experiments with IFN-α/β receptor antibodies showed the biologic importance of these responses. Investigations of VZV and HSV-1 replication in the SCID-hu model also revealed significant differences in cell tropisms that correlate with

clinical observations about their pathologic effects in intact and immunocompromised individuals. In SCID-hu mice, VZV caused extensive necrosis in deeper dermal layers of skin implants, but HSV-1 was confined to the epidermis. In contrast to VZV, HSV-1 was not infectious for human CD4+ or CD8+ T cells, which is consistent with the clinical differences, since primary HSV infection is not associated with viremia in the intact host. HCT recipients and other immunocompromised patients are also susceptible to cell-associated viremia when VZV reactivates, whereas HSV viremia is a rare occurrence during recurrent HSV infection even with intensive immunosuppression. Recurrent VZV infection The hypothesis that VZV becomes latent in sensory ganglia following primary infection and that herpes zoster is caused by its reactivation from latency was proved by using restriction enzyme analysis to demonstrate that a single VZV strain caused both varicella and a subsequent episode of herpes zoster in a child with Wiskott–Aldrich syndrome [33]. During the primary attack, VZV is postulated to pass centripetally from the skin to the corresponding ganglia; hematologic seeding of the ganglia secondary to cell-associated viremia is also likely. Once in the ganglia, VZV establishes latency, apparently without viral replication and without causing cell damage. In contrast to HSV, infectious VZV has not been recovered from ganglion tissue by explant culture or co-cultivation techniques. However, the presence of VZV nucleic acid sequences has been documented in human ganglia taken at autopsy from individuals with no evidence of recent VZV infection [5,35–42]. Multiple VZV RNA sequences have been detected by in situ hybridization and PCR, in contrast to HSV, in which only limited gene transcription has been identified in latently infected neurons. Transcription of VZV genes corresponding to open reading frames (ORFs) 4, 18, 21, 29, 40, 62, 63, and 66 have been reported in human dorsal root ganglion (DRG) from autopsies, although observations about specific VZV genes

Varicella-zoster Virus Infections

have varied between studies [41,43,44]. ORF63 transcripts are the most abundant and the most commonly found [45]. Latent infection appears to be more common in the trigeminal ganglion than in any of the thoracic ganglia [39]. In addition to gene transcription, expression of VZV proteins in the cytoplasm of neurons has been described in autopsy specimens from subjects without zoster. IE63-expressing cells were reported in 4 of 21 [46], 3 of 3 [47], and 10 of 10 [37] adult ganglia, but not in infant/fetal ganglia controls. Two of these reports also describe IE62, IE4, ORF21, and ORF29 proteins in some specimens [37,47]. However, these studies are difficult because adult neurons often contain cytoplasmic neuronal cell pigments, and postmortem changes may influence expression of viral as well as host cell proteins. The most extensive analysis by Mahalingam et al. identified very few IE63+ neurons in only a few subjects [46], suggesting that VZV transcripts are translated occasionally, perhaps during abortive replication. Selective gene transcription, especially of ORF63, appears to be a more consistent signature [45]. However, basic questions about viral and host mechanisms involved in VZV neuropathogenesis and persistence in human sensory ganglion cells remain unanswered. Investigations using molecular probes for VZV DNA sequences indicate that the virus persists in both neuronal and satellite cells, although neuronal infection appears to predominate [5,38,42]. More recently, the study of VZV neuropathogenesis has been facilitated by exgrafting human DRG under the kidney capsules of SCID mice [48]. DRG xenografts contained neurons and satellite cells within a typical DRG architecture. VZV clinical isolates predominantly infected the neurons within the DRG, while there are recent data showing that some satellite cells are also infected [5]. An initial phase of viral replication was followed within 4–8 weeks by a transition to VZV latency, characterized by the absence of infectious virus release, the cessation of virion assembly, and a reduction in VZV genome copies. Furthermore, VZV-infected T cells transferred virus from the circulation into the DRG, suggesting that VZV lymphotropism facilitates its neurotropism. The maintenance of VZV latency, as defined by the absence of symptomatic VZV reactivation, is also influenced directly by the host response to VZV, as is evident from the high incidence of VZV reactivation after HCT when VZV immunity is impaired. Although HSV reactivation causes little or no damage to ganglion cells, VZV reactivation is associated with extensive viral replication in the affected ganglia, producing pathologic changes including necrosis, inflammation, and destruction of neural cell bodies. In contrast to HSV, CMV, and Epstein–Barr virus, which can be recovered from asymptomatic patients by viral culture methods, technical problems have interfered with the detection of subclinical VZV reactivation. Episodes of subclinical VZV reactivation, consisting of cell-associated viremia in HCT recipients detected by VZV PCR, were documented in 19% of patients tested at a mean of 94 days after transplantation [43]. Disseminated VZV infection has been diagnosed at autopsy in HCT patients who had no cutaneous lesions [1,44,49]. However, testing PBMCs with the VZV PCR assay provided the first virologic evidence that these severely immunocompromised patients can experience VZV reactivation in the absence of clinically apparent cutaneous infection, and resolve the infection without developing signs of visceral dissemination. Herpes zoster, like recurrent HSV, has been attributed to the spread of the reactivated virus along neural pathways from the site of latency in DRGs. Some immunodeficient patients with localized herpes zoster develop VZV viremia, presumably because T lymphocytes become infected at the site of local cutaneous replication. Subclinical, cell-associated viremia in HCT patients without cutaneous disease suggests that the virus may also be taken up directly by circulating lymphocytes at the neuronal site of viral reactivation. This mechanism for causing

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viremia could account for the clinical observation of atypical, generalized herpes zoster in some HCT recipients. Since the activation of T lymphocytes makes this cell population more permissive for VZV infection in vitro [27], cell-associated VZV viremia in HCT patients may be potentiated by the characteristic persistence of activated T lymphocytes in circulation for a prolonged period after transplant [50]. Viral immune evasion Like many viruses, VZV has mechanisms to inhibit recognition by the host immune system. We have found that VZV causes a significant reduction of the cell surface expression of major histocompatibility complex (MHC) class I protein on infected fibroblasts and on infected T cells, as demonstrated in the SCID-hu model [51]. Although their synthesis was not affected, MHC class I molecules were retained in the Golgi apparatus in VZV-infected cells. This effect may enable the virus to evade CD8+ T-cell immune recognition during VZV pathogenesis, including the critical phase of T-lymphocyte-associated viremia. In related experiments, we showed that VZV has the capacity to block the upregulation of MHC class II protein that is usually induced when cells are exposed to IFN-γ [52]. Induction of MHC class II expression is required for recognition of virus-infected cells by antiviral CD4 T cells. When skin biopsies of varicella lesions were analyzed by in situ hybridization, MHC class II RNA transcripts were detected in cells near the lesion but not in cells that were infected with VZV. VZV infection inhibited transcription of IFN regulatory factor-1 in the IFN-γ signaling pathway and expression of signal transducers and activators of transcription-1a (Stat1a) and Janus kinase-2 (Jak2) proteins. The inhibition of MHC class II expression on VZV-infected cells in vivo may transiently protect cells from CD4 T-cell immune surveillance, facilitating local virus replication during the first few days of cutaneous lesion formation during primary or recurrent VZV infection, and could allow a period of viral replication in skin cells even when the host has VZV-specific CD4 T cells.

The host response Primary VZV infection is associated with the development of virusspecific IgG and IgM antibodies and the acquisition of cellular immunity, which can be demonstrated by proliferation of T cells that are stimulated with VZV antigens in vitro. Although VZV IgG antibodies can neutralize virus infectivity and function in antibody-mediated cellular cytotoxicity, humoral immunity seems to be less important in the host response to VZV than cell-mediated immunity. Progressive varicella occurs in immunocompromised children despite the production of VZV IgG and IgM antibodies, whereas these children fail to develop VZV-specific T-lymphocyte proliferation [53]. Immunocompromised patients, including HCT recipients, develop VZV reactivation in spite of high concentrations of circulating antibodies to VZV. No quantitative relationship between VZV antibody titers in the donor or the recipient and the subsequent development of herpes zoster has been established in HCT patients [54]. A recent retrospective study examined anti-VZV IgG titers after HCT, and also measured VZV DNA copy numbers by real-time PCR during clinical VZV reactivation using cryopreserved serum samples [55]. No significant difference was found between anti-VZV titers in patients with VZV infection and in those without VZV infection at each time point after HCT. However, patients with disseminated zoster tended to have lower pre-existing anti-VZV titers and higher serum VZV DNA copy numbers than those with localized zoster cases. A strong inverse correlation was found between pre-existing anti-VZV titer and serum VZV DNA at onset. Therefore, it was concluded that pre-existing antibody

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does not prevent the development of VZV reactivation in HCT recipients, but may contribute to decreased viral load at onset. Periods of diminished VZV-specific T-lymphocyte proliferation have been correlated with an increase in susceptibility to herpes zoster among immunocompromised patient populations, including HCT recipients. In the individual patient, the loss of T-cell proliferation to VZV is a necessary, but not a sufficient, condition for symptomatic reactivation. Conversely, VZV-specific T-lymphocyte proliferation has been correlated with a decreased risk of herpes zoster. Analyses of VZV-specific cell-mediated immunity have demonstrated a gradual recovery of T-cell proliferation to VZV antigens, with a larger percentage of HCT patients having detectable responses as the interval following transplant increases [4,11,43,56,57]. The interval to recovery may be somewhat shorter in autologous HCT recipients. This reconstitution of cell-mediated immunity to VZV antigens is generally observed by 9–12 months after HCT, which correlates with the time when VZV reactivation and susceptibility to severe VZV disease become less common. In HCT patients, IFN-γ production was associated with the recovery of T-cell proliferation to VZV antigen and increased with time following transplantation. Interleukin-10 production by PBMCs stimulated with VZV antigen was also observed consistently after HCT, and was highest in patients who recovered T-cell proliferation to VZV antigen [56]. When VZV T-cell proliferation responses were followed prospectively in unvaccinated patients in a study of inactivated varicella vaccine, autologous transplant patients had a mean VZV stimulation index of 9.9 (standard error [SE] 2.32) within 30 days before transplantation, responses were undetectable at 30 days, and then increased to 8.0 (SE 1.63) by 90 days; mean responses continued to increase over the first year (Fig. 92.4) [23]. Evaluation of VZV immunity at 6 months after transplantation using a quantitative flow cytometry method showed that the frequencies of CD4 T cells that made tumor necrosis factor-alpha in

80

Vacc mean Nonvacc mean

50

70

40

TNF-α 30 20 10 0 pre

30 days

90 days

120 days

6 months 12 months

Sample time

Fig. 92.4 CD4 T-cell proliferation in response to varicella-zoster virus (VZV) antigen. T-cell proliferation in response to VZV antigen, measured as the stimulation index, is shown as the mean stimulation index (y-axis) for vaccinated (ⵧ) and unvaccinated (䉬) participants; the time of immunologic evaluation is shown on the x-axis in relation to time of hematopoietic cell transplantation. Inactivated varicella vaccine was given pretransplant and at 30, 60, and 90 days; the stimulation index for vaccinees at 30 days represents responses after the pretransplant dose; at 90 days, three doses had been given, and at 120 days, 6 months, and 12 months, vaccinees had received four doses. The error bars represent standard errors. The numbers of patients tested at each time point were: pretransplantation, 58 unvaccinated/53 vaccinated; 30 days, 50 unvaccinated/49 vaccinated; 90 days, 43 unvaccinated/42 vaccinated; 120 days, 34 unvaccinated/39 vaccinated; 6 months, 30 unvaccinated/34 vaccinated; 12 months, 27 unvaccinated/33 vaccinated. (Reproduced from Hata et al. [23], with permission.)

Proportion of CD4+ T cells (%)

Stimulation index means

response to VZV stimulation was 0.19%, compared with 0.30% in VZV immune adults without underlying disease; the mean percentage of CD4 T cells that made IFN-γ was 0.11%, which was equivalent to frequencies in healthy adults (Fig. 92.5) [23]. These analyses excluded patients who developed herpes zoster, indicating that VZV-specific T-cell responses can recover either as a result of subclinical VZV reactivation, as documented by VZV PCR testing of HCT recipients who had no signs of infection, or through re-expansion of memory T-cell populations that persisted through the preparative regimen for transplantation [43]. In this prospective study, the risk of zoster was reduced by 19% per unit increase in stimulation index over 1.6; a stimulation index of over 5.0 correlated with greater than 93% protection [23]. Pretransplant immunity in both the donor and the recipient may facilitate the reconstitution of VZV-specific cell-mediated immunity [58]. Nevertheless, it is apparent that the recovery of virus-specific cellular immunity is delayed for months, and recovery of VZV-specific T-cell immunity often does not occur until after the patient has had an episode of herpes zoster, which provides in vivo re-exposure to VZV antigens. Meyers et al. detected T-lymphocyte proliferation to VZV in 16 of 18 patients (89%) following symptomatic recurrences of VZV, compared with 15 of 29 patients (51%) who did not develop herpes zoster [4]. When herpes zoster occurs, HCT recipients show an increase in VZVspecific CD4 T-cell proliferation. We found that the mean stimulation index response before onset of herpes zoster was 2.2 (SE 0.4), compared with 28.1 (SE 6.68) when the episode of VZV reactivation had resolved [23], which is likely to explain why HCT recipients are usually not susceptible to repeated episodes of herpes zoster. During the first year, HCT patients recover cytotoxic T cells that recognize and lyse autologous target cells expressing VZV proteins [43]. Fifty percent of HCT patients showed recovery of VZV-specific cytotoxic T lymphocyte (CTL) function when tested at a mean of 155 days

60 50 Interferon-γ 40 30 20 10 0 Vaccine

No vaccine

Vaccine

No vaccine

Fig. 92.5 Intracellular production of interferon-gamma (IFN-γ) or tumor necrosis factor-alfa (TNF-α) in T cells incubated with varicella-zoster virus (VZV). The percentage of CD4+ T cells that expressed CD69 and intracellular cytokine, IFN-γ ( ) or TNF-α ( ), was determined at 6 months after hematopoietic cell transplantation in 19 vaccinated and 19 unvaccinated patients. Results are shown as mean percent responder cells ± standard error. Differences in VZV-specific responder cell frequencies in vaccinated and unvaccinated subjects were analyzed by unpaired t test: for CD4+, CD69+, and IFN-γ, p = 0.07; for TNF-α, p = 0.03. (Reproduced from Hata et al. [23], with permission.)

Responder cell frequency

Varicella-zoster Virus Infections

≥ 1:400,000 1:300,000

1:200,000

1:100,000

BMT

NI

IE62 protein

NI

BMT gp I protein

Fig. 92.6 Precursor frequencies of cytotoxic T-lymphocytes (CTLs) specific for the immediate early protein (E62) and glycoprotein I (gp I) of varicellazoster virus (VZV) in bone marrow transplant recipients (BMT) and healthy subjects (NI). The mean + standard deviation for precursor frequency estimates are indicated next to the individual data points (●) generated by testing individual hematopoietic cell transplant recipients and healthy immune subjects. (Reproduced from Wilson et al. [47], with permission.)

after transplant (Fig. 92.6). However, the mean precursor frequency of T lymphocytes that recognized the VZV IE62 protein or glycoprotein E (formerly designated gp I) was more than twofold lower among HCT recipients than the frequency of CTLs that recognized these viral proteins in PBMCs from healthy immune subjects. In these experiments, cultures from HCT recipients showed a significant reduction in the proliferation of CD4+ T cells when compared with the pattern of cell phenotypes in cultures from healthy subjects. CD8+ T cells predominated in VZV-stimulated cultures, reflecting the relative increase in circulating CD8+ T cells that is common after HCT. Although CD8+ T lymphocytes have been defined as the “classic” cytotoxic effecter cell, human CD4+ cells also function effectively as antiviral CTLs against many viruses, including VZV [59]. The diminished CD4+ T-cell response to VZV antigen may explain why the overall frequencies of CTL precursors specific for the IE62 or gE (gp I) proteins remained significantly lower after HCT than in healthy, VZV-immune individuals. Helper and cytotoxic T-cell responses specific for the IE63 protein as well as proteins encoded by ORFs 4 and 10 are maintained for decades after primary VZV infection in the intact host [60,61], and their absence could be related to the high risk of VZV reactivation causing herpes zoster. In contrast to virus-specific T-cell immunity, natural killer (NK) cell activity comparable to that of healthy subjects is recovered during the first few months after HCT [62]. Some HCT recipients had a predominance of NK cells, expressing CD16 surface antigen, in assays for cytotoxicity against VZV-infected targets [43]. The importance of NK cells in the host defense against VZV has been exemplified by cases of severe or fatal disseminated VZV infection in patients with an absence of circulating NK cells during the VZV infection or with innate NK cell deficiencies [63–66]. The capacity of lymphocytes from some HCT patients to lyse targets expressing VZV proteins by a mechanism that is not antigen specific may help to limit VZV replication prior to the recovery of virus-specific T-cells.

Clinical manifestations Primary VZV infection The diagnosis of varicella is usually suspected clinically in high-risk as well as healthy children when the characteristic vesicular exanthem appears [3,18,67,68]. The interval from exposure of the nonimmune

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child to the appearance of the rash is about 14 days, with a range of 10–21 days; the incubation period may be somewhat shorter in immunocompromised children. Because of its low incidence, specific descriptions of the clinical course of varicella after HCT are limited, but it is reasonable to generalize from the literature about varicella in other high-risk populations [67,68]. The initial manifestations of varicella in immunocompromised children are similar to those observed in healthy children. Prodromal symptoms may precede the rash by 24–48 hours, usually consisting of headache, irritability, malaise, and fever. Cutaneous lesions most often appear first on the scalp, face or trunk, and are usually pruritic. Each lesion begins as a small erythematous macule which evolves into a vesicle of 1–4 mm diameter, on an irregular erythematous base, the classic “dewdrop on a rose petal.” In high-risk children, including HCT recipients, the vesicles may be unusually large and can involve deeper skin layers. Vesicles on mucous membranes, including the conjunctiva, the oropharynx, the rectum or the vagina, are common even among otherwise healthy children. Varicella is typically accompanied by lowgrade fever, but temperature elevations may be as high as 40.5°C in high-risk patients, and fever often persists beyond the usual 3–4 days. New vesicle formation typically continues for about 3 days, with a range of 1–7 days, among healthy children. This phase is often prolonged in immunocompromised children, with more than half of patients developing new lesions for more than 7 days [68]. Although the majority of immunocompetent children with varicella have fewer than 500 lesions, it is not unusual for patients on immunosuppressive therapy to develop more than 1500 lesions. Successive crops of varicella vesicles develop in a centrifugal pattern, appearing last on the extremities. These later crops of lesions tend to be more extensive in immunocompromised patients, and may involve the palms and soles as well as arms and legs. In lesions that are resolving normally, the vesicle fluid rapidly becomes cloudy, and the lesion may develop an umbilicated appearance as it becomes crusted. Among healthy children, the lesions that erupt later in the disease sometimes resolve after a maculopapular phase, but many of the late lesions also progress through the vesicular stage in immunocompromised patients. In general, the time to complete crusting of lesions is prolonged. Extensive hypopigmentation and scarring may be seen as the crusts resolve, presumably because of the involvement of deeper layers of the dermis in immunocompromised patients. Patients who acquire the infection during the first 9–12 months after transplantation appear to be at highest risk of developing severe varicella [1]. The potential complications of varicella after HCT can be anticipated from the clinical experience with primary VZV infection in other immunocompromised patient populations. HCT recipients can be compared with patients whose immunosuppression is due to treatment of lymphoproliferative malignancies or solid tumors. In one large series, 32% of these patients developed visceral dissemination in the course of varicella, with VZV infection of the lungs, liver, and central nervous system (CNS) [67]. The mortality rate was 7% overall, with an increase to 25% in patients who developed varicella pneumonia. The risk for visceral involvement was significantly higher among patients whose absolute lymphocyte counts were less than 500/μL, and the rate of visceral dissemination increased to 71% when the lymphocyte count declined to less than 100 cells/μL. Visceral dissemination was associated with new lesion formation for a median of 10 days in more than half of patients who were evaluated before effective antiviral therapy for VZV was available. Based on the analysis of placebo recipients in the original studies of antiviral treatment of varicella in high-risk children, the mortality rate was 17%; 27% of the patients developed varicella pneumonia, 19% had hepatitis, and 6% had CNS disease [68]. Among the complications of primary VZV infection, varicella pneumonia is the most common cause of life-threatening illness. Respiratory

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symptoms, except for mild rhinitis and cough, are unusual in uncomplicated varicella and require urgent evaluation in the immunocompromised patient. Pneumonia most often develops within 3–7 days after the appearance of the rash and progresses rapidly to respiratory failure. Clinical signs of pneumonia may be limited to tachypnea, cough, and dyspnea with no accompanying abnormal findings by auscultation. The degree of hypoxemia may be marked even when abnormalities on the chest X-ray are minimal. Varicella can be associated with abnormal liver function tests in the immunocompetent host, but hepatic involvement is usually subclinical. In contrast, immunocompromised children are at risk of fulminant varicella hepatitis, with progression to hepatic failure. Severe varicella is usually accompanied by signs of disseminated intravascular coagulopathy, including epistaxis, hematuria, and gastrointestinal bleeding. Hemorrhage into the cutaneous lesions is recognized as a poor prognostic sign in high-risk patients. Bleeding is most often due to disseminated intravascular coagulopathy and hepatic failure, but isolated thrombocytopenia can occur. Vomiting and abdominal pain are uncommon in children with uncomplicated varicella. Severe abdominal or back pain should be considered a sign of life-threatening infection, and may be associated with inappropriate antidiuretic hormone secretion in the absence of any obvious CNS disease. This atypical presentation of VZV infection has been reported after HCT [69,70]. While the pathogenic mechanism is not understood, disseminated VZV infection can cause pancreatitis and disease of the gastrointestinal tract, such as esophagitis, which may account for these symptoms, or pre-eruptive infection of sensory ganglia may occur. Varicella meningoencephalitis is unusual in immunocompromised children, but, when it occurs, it is usually associated with other signs of visceral involvement. Some patients develop a rapidly deteriorating course progressing to death. Symptoms of CNS disease most often occur between 2 and 6 days after the eruption of the rash and can progress rapidly to coma. Encephalitis may precede the appearance of the rash [71]. The clinical signs include vomiting, headache, altered sensorium, and seizures. The cerebrospinal fluid (CSF) usually shows a mild-tomoderate pleocytosis and elevated protein. Inappropriate antidiuretic hormone secretion may occur in patients who have meningoencephalitis [72]. Severe hypertension is a poor prognostic sign in immunocompromised patients with varicella. The mechanism is uncertain except in cases that are associated with nephritis. Adrenal cortical necrosis is noted at autopsy in patients with disseminated VZV infection. In addition to complications directly related to viral replication, secondary bacterial infections are a risk in immunocompromised patients with primary VZV infection, as they are in otherwise healthy children. Staphylococcus aureus and Streptococcus pyogenes (group A streptococcus) are the most common pathogens, causing cellulitis, subcutaneous abscesses, and lymphadenitis. Varicella gangrenosa, a very rare syndrome of necrotizing fasciitis caused by Streptococcus pyogenes, is life threatening even in the intact host. Secondary bacterial infection of sites other than skin, including bacterial pneumonia, septic arthritis, and osteomyelitis, may follow varicella. Staphylococcal sepsis can occur, and children with indwelling catheters can develop line-related infections. Although information is limited, varicella occurring late after HCT is usually not associated with complications. Four children who had varicella 2 years after transplantation had clinical disease that resembled primary VZV infection in children with no immunodeficiency, and four children who developed varicella from 105 to 350 days after HCT had no complications [1,58]. However, progressive varicella with severe pneumonia was reported in one child who acquired varicella 6 years after successful HCT [73].

Table 92.3 Distribution of dematomal involyement with herpes zoster in bone marrow transplant recipients and healthy individuals with recurrent varicella-zoster virus (VZV) infections HCT recipients*

Healthy individuals†

195

116

Dermatomes involved (%) Cranial Cervical Thoracic Lumbar Sacral

16 17 47 20 12

15 22 53 18 8

Dissemination (%) Cutaneous Visceral

23 13

0 0

7

0

No. of patients

Mortality (%) * Adapted from Locksley et al. [1]. † Adapted from Brunell [77].

Recurrent VZV infection Localized herpes zoster Localized herpes zoster is the most common clinical presentation of VZV infection in HCT recipients who are seropositive at the time of transplantation, accounting for 85% of cases in 231 HCT recipients in the study by Locksley et al. [1]. The rash of localized herpes zoster is usually preceded by pain and paresthesias in the involved dermatome [5,68,74]. These symptoms may begin as long as 5 days before the eruption and vary from mild discomfort to very severe debilitating pain. The etiology of the initial pain is sometimes misdiagnosed as pleurisy, myocardial infarct, cholecystitis, or renal colic. In some instances, the prodrome is not followed by any skin eruption, but a typical antibody response is observed; this syndrome is referred to as “zoster sine herpete” [75,76]. In healthy subjects, herpes zoster involves the thoracic dermatomes in about half the cases, and cranial nerve disease occurs in 13– 20% of cases [68]. Recurrent VZV affects similar dermatomes in HCT patients, with thoracic dermatomal disease in 41–47% of cases and cranial nerve involvement in 16% (Table 92.3) [1,16,77]. The cutaneous lesions of localized herpes zoster appear as clusters of varicella-like vesicles in one or several sites anteriorly and posteriorly along the dermatome; typically, the lesions do not cross the midline, but some cases of bilateral dermatomal involvement have been described. Discrete vesicles often enlarge to form confluent lesions. In immunocompromised patients, including HCT recipients, the local vesicular eruption may be very extensive, evolving to occupy the whole dermatome, the lesions may become hemorrhagic, and the duration of cutaneous disease is prolonged [68]. In the normal host, new lesion formation usually continues for 3–7 days, followed by a phase of pustulation and crust formation; complete healing is expected by 2–3 weeks. In high-risk patients, the average time for cessation of new lesion formation was 8 days, and crusting was not complete until an average of 18 days. Immunosuppressed patients may occasionally develop a chronic cutaneous reactivation of VZV that persists for months [74,78]. As clinical experience with severely immunocompromised patients due to human immunodeficiency virus infection increases, knowledge about the spectrum of disease caused by VZV continues to expand [79]. Many of these complications are now recognized in HCT recipients with VZV reactivation.

Varicella-zoster Virus Infections

When VZV reactivation involves cranial nerves, complications are more likely in both immunocompromised and immunocompetent patients [3]. In one series, corneal damage, facial scarring, cranial nerve VII palsy or hearing loss occurred in 35% of patients [1]. The distribution of the ophthalmic branch of the trigeminal nerve is a common site of herpes zoster. The development of vesicular lesions on the nose is a sign of involvement of the ophthalmic branch and indicates a risk for ocular complications. The clinical findings of ocular herpes zoster include conjunctivitis, keratitis, anterior uveitis, or, rarely, panophthalmitis (reviewed in [80,81]). Although corneal lesions are common, blindness is an unusual complication of herpes zoster ophthalmicus. Loss of vision associated with herpes zoster is usually secondary to retrobulbar neuritis. Although it is less common than CMV retinitis, chorioretinopathy caused by VZV is observed after HCT. Progressive outer retinal necrosis (PORN), a distinct form of VZV necrotizing chorioretinitis, is found almost exclusively in patients with acquired immune deficiency symdrome. However, two cases of VZV-related PORN after allogeneic HCT have been reported recently [82,83]. VZV reactivation may produce facial palsy when it involves cranial nerve VII. Herpes zoster oticus is associated with VZV reactivation from the geniculate ganglion, producing the Ramsay Hunt syndrome with unilateral deafness and vestibular symptoms. Oral lesions of the palate may develop without any cutaneous lesions when the second branch of cranial nerve V is affected. Cerebral angiitis is a syndrome of cerebral vascular inflammation with thrombosis and microinfarcts associated with VZV reactivation in cranial nerve ganglia (reviewed in [84]). In severe cases, massive thrombosis with contralateral hemiparesis may result [85]. Infarct in the distribution of the middle cerebral artery can be demonstrated by computed tomography scan. Pathologic findings include a granulomatous inflammatory process in the arterial wall, mononuclear cell infiltrates, vascular necrosis, and viral particles within the smooth muscle cells of the affected arteries. The syndrome is rare, and there is no evidence that HCT or other immunocompromised patients are at increased risk for cerebral angiitis. However, it is important to recognize this complication in patients who develop hemiparesis about 6–8 weeks after the symptoms of herpes zoster. When the anterior horn cells are involved in VZV reactivation, inflammation and necrosis may follow, with resulting motor deficits in about 10% of otherwise healthy individuals with cervical or lumbosacral zoster [76]. Full recovery of the motor deficit is expected in 85–90% of the cases. Transverse myelitis and ascending paralysis are rare neurologic syndromes caused by recurrent VZV infection with or without immunosuppression. Immunocompromised patients are at increased risk of postherpetic neuralgia, which is the most common complication of herpes zoster in all patient populations. The definition of postherpetic pain varies from any pain lasting after the crusts of the cutaneous lesions have disappeared to pain that persists more than 2 months. Estimates of the incidence of postherpetic neuralgia vary depending upon the definition used. In one study of healthy individuals, 9% of patients with herpes zoster developed postherpetic neuralgia, whereas Locksley et al. observed postherpetic neuralgia in 25% of patients with herpes zoster after HCT [1], and Offidani et al. reported an incidence of 32% [19]. Koc et al. found that postherpetic neuralgia and peripheral neuropathy were the most common complications of VZV reactivation after HCT, occurring in 68% of 100 consecutively identified cases [16]. Postherpetic neuralgia is uncommon in children, even in those who are immunocompromised [74,86,87]; only 2–3% of pediatric HCT recipients who developed herpes zoster had persistent pain [18]. Secondary bacterial infection of skin lesions and local scarring are common in HCT recipients with herpes zoster, occurring with an incidence of 17% and 19%, respectively.

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Cutaneous and visceral dissemination In contrast to otherwise healthy subjects, HCT patients often develop cutaneous dissemination, defined as more than five vesicular lesions beyond the primary dermatome. Cutaneous dissemination has been reported in 17–24% of HCT recipients, and was equally common among those who had recurrent VZV during the early or late period after transplant [1,16]. HCT recipients are also at risk of visceral dissemination during episodes of VZV reactivation (see Fig. 92.1). Since it is a sign of VZV infection of circulating lymphocytes, cutaneous dissemination provides a marker for the risk for visceral dissemination. However, visceral dissemination also occurs in patients whose cutaneous lesions are localized to the primary dermatome. Without antiviral therapy, visceral dissemination was observed in 13% of HCT patients with herpes zoster [16]. The potential sites of visceral dissemination with recurrent VZV are the same as those with disseminated primary VZV infection. The clinical complications that result include pneumonia, hepatitis, disseminated intravascular coagulopathy, and encephalitis. The mortality that accompanies VZV reactivation is almost always due to viral pneumonia, but fatal fulminant hepatitis and disseminated intravascular coagulopathy without VZV pneumonia have been reported [88]. Acute GVHD is the only risk factor associated significantly with VZV dissemination. VZV has also been identified as the causative agent of late interstitial pneumonia in HCT patients with chronic GVHD [89]. Occasionally, HCT recipients present with signs of visceral dissemination 24–96 hours before the localized cutaneous eruption of recurrent VZV becomes evident, resulting in delayed diagnosis, or being misdiagnosed as GVHD or other complications [90–92]. Among these patients, the clinical presentation is characterized by abdominal pain, often mid-epigastric or periumbilical, with or without associated nausea, vomiting or fever. Pancreatitis, hepatitis, gastrointestinal hemorrhage, intestinal necrosis, disseminated intravascular coagulopathy, and inappropriate secretion of antidiuretic hormone have been described in patients with abdominal symptoms preceding the rash [49,69,90–93]. In a series of 10 cases, patients had abdominal pain and elevated aminotransferases and pancreatic enzymes, associated with nausea (60%), fever over 38°C (60%), and vomiting (50%) [94]. Four patients had skin rash, which appeared at an interval of 4–14 days after the onset of abdominal pain. Five patients had pneumonitis, and VZV infection was fatal in five cases. Visceral dissemination can also occur without any signs of cutaneous disease [95]. Three HCT patients had fatal VZV infection and no skin lesions during the course of their illness despite evidence of widespread organ infection at autopsy [1]. In a recent retrospective review of 310 children undergoing HCT in a single institution over a 7-year period, 61 of 201 (32%) allogeneic and 10 of 109 (9%) autologous HCT recipients developed zoster. Of 37 with available data on alanine transaminase values 4 weeks preceding the clinical diagnosis of zoster, 22 (59%) patients experienced a rising ALT level, unassociated with GVHD. Thus, while there are many causes of elevation of transaminases post HCT, it was concluded that the possibility of VZV reaction should be considered, specially among those with increased risk factors for zoster, to allow for earlier diagnosis and therapeutic intervention [24]. Immunosuppression also predisposes to CNS infection with recurrent VZV, although symptomatic neurologic complications of herpes zoster are rare [96,97]. Two of 100 consecutively identified patients with recurrent VZV after HCT developed VZV encephalitis [16]. From the recent series of 310 children with HCT [24], 3 of 73 patients for whom sufficient information was available had CNS involvement. Despite its rare occurrence, cases of fatal VZV encephalitis have been reported [98]. In some cases, neurologic symptoms preceded eruption of skin lesions [71]. In evaluating these patients, it is important to note that abnormal CSF

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findings are common even in immunocompetent patients with herpes zoster. Lymphocytic pleocytosis and elevated protein were observed in about 40% of healthy individuals who had no abnormalities by neurologic examination, whereas the incidence of herpes zoster-associated encephalitis is estimated to be only 0.2–0.5% in this population [14,99,100]. The clinical symptoms and signs of CNS disease are headache, photophobia, meningismus, and altered state of consciousness [99,100]. The neurologic symptoms usually appear within a few days after the exanthem. The temperature is usually normal or mildly elevated. Seizures are rare, but the electroencephalogram may show diffuse slowing or epileptiform activity. As would be expected, the occurrence of visceral dissemination increases the mortality of recurrent VZV infection substantially from an overall rate of 7% to 55%. The mortality rate is higher in patients who develop both pneumonia and encephalitis than in those patients who have encephalitis only. Atypical nonlocalized herpes zoster Atypical nonlocalized herpes zoster after HCT is sometimes categorized as varicella because the HCT recipient is presumed to lack VZV immunity, regardless of prior immune status. However, identifying the syndrome as a distinct clinical presentation is useful because the pathogenesis of infection and the timing of the host response may be affected by differences between the endogenous and exogenous routes of infection, and residual immunity may modify the clinical course. The incidence of atypical nonlocalized herpes zoster is variable, with reports of its occurrence ranging from none to as many as 25% of episodes of VZV reactivation after HCT. Clinically, patients with atypical nonlocalized herpes zoster have cutaneous vesicles that are identical to those of varicella. The number of lesions is quite variable, but the eruption is often extensive, involving face, trunk, extremities, palms, and soles. This syndrome occurs in autologous as well as allogeneic HCT recipients, accounting for 21% of episodes of recurrent VZV in one series [14]. The morbidity of untreated infection is high, with 45% of patients developing visceral dissemination, resulting in a mortality rate of 28% during the period before antiviral therapy was available [1]. Second episodes of recurrent VZV Second episodes of VZV reactivation after HCT are uncommon. About 2% of patients had two episodes of herpes zoster, occurring at an average of 25 months after transplantation, with a range of 4–41 months. Most of the reported second episodes after HCT have involved the same dermatome, but reactivation from another ganglia may occur [1]. Subclinical reactivation of latent VZV The occurrence of subclinical VZV reactivation in HCT recipients has been shown by using a PCR method to demonstrate cell-associated VZV viremia [43]. Subclinical VZV reactivation was detected in 19% of 37 HCT patients who were screened randomly and had no signs of infection. Two of the seven patients who had subclinical VZV viremia developed clinical signs of herpes zoster subsequently, at intervals of 60 days and 130 days later. Three patients who had subclinical VZV viremia between 17 and 85 days after HCT had cleared the cell-associated viremia when they were retested by VZV PCR; two patients were not re-evaluated by VZV PCR but did not develop herpes zoster. Thus, five of seven patients (71%) who had subclinical viremia did not progress to clinical infection.

Laboratory diagnosis The diagnosis of VZV infection can be made by a variety of techniques, including viral culture, viral antigen detection, and viral DNA detection.

Serologic testing is useful to demonstrate that the patient has had prior primary VZV infection and is therefore harboring the virus in sensory ganglia, but serologic tests are not useful for diagnosis of active VZV infection. PCR technology has revolutionized the diagnosis of many infectious diseases, including that of VZV, because it is sensitive and rapid. The caveat is that clinical specimens contain inhibitors that may cause false-negative results in PCR assays. Viral isolation in tissue culture cells VZV can be detected in clinical samples using standard tissue culture methods for viral isolation [101]. The highest yield for viral culture depends upon obtaining vesicular fluid along with infected cells from the base of the cutaneous lesion. Vesicular fluid can be collected in a tuberculin syringe or by using a cotton or a Dacron swab. Swabs should be put in viral transport medium immediately, agitated, and pressed against the side of the vial to remove absorbed fluid; the swab should be taken out of the vial before it is sent to the laboratory. If storage for more than a few hours is required, the specimen should be kept on dry ice or frozen at −70°C; storing the specimen at −20°C, in a standard refrigerator freezer, for 24 hours usually inactivates the virus. The optimal recovery of infectious virus in tissue culture requires the use of diploid cell lines or human embryonic lung fibroblasts. The cytopathic effect appears within 2–4 days after inoculation. Since the average time to detection of the virus in tissue culture is 7 days, diagnosis by viral culture is not rapid enough to influence clinical decisions in most circumstances. The sensitivity of tissue culture for detection of VZV is also substantially less than for identification of HSV and CMV. Procedures such as centrifugation enhancement may shorten the time required to detect the cytopathic effect. There are some differences in the morphology of plaques produced by VZV compared with these other herpesviruses, but the identity of the virus isolate must be proved by immunofluorescence staining with virus-specific antisera. As in the case of CMV, the shell vial culture method improves the sensitivity of VZV isolation and permits earlier identification of positive specimens. This method combines centrifugation and staining with fluoresceinconjugated monoclonal antibodies to VZV; positive results may be available within 1–3 days of inoculation. VZV detection by tissue culture methods is less sensitive than the direct flourescence antigen (DFA) test. The likelihood of recovery of VZV from cutaneous lesions is directly related to the stage of the lesion, with clear vesicles being much more likely to be positive than specimens from lesions that have become pustular or crusted. Varicella lesions are usually positive for 3 days, while virus can be recovered for a week or longer in herpes zoster. VZV can be isolated from PBMCs by tissue culture inoculation of specimens from immunocompromised patients with recurrent as well as primary VZV infections. In contrast to meningoencephalitis associated with primary VZV infection, the virus has been isolated from the CSF of patients with herpes zoster. Bronchial washings may yield VZV in patients with pneumonia. The lungs are the most common autopsy organ from which VZV has been isolated, but the virus has been recovered from many sites, including heart, liver, pancreas, gastrointestinal tract, brain, and eyes. Direct detection of VZV antigens in infected cells by immunofluorescence The optimal method for the rapid diagnosis of cutaneous VZV infection is to obtain epithelial cells from a fresh lesion and to stain the specimen using fluorescein-conjugated monoclonal antibodies to VZV antigens [101,102]. The VZV-specific monoclonal antibodies bind to viral

Varicella-zoster Virus Infections

proteins that are synthesized within infected cells; fixing the cells prior to staining makes the cell membrane permeable, allowing the detection of VZV proteins in the cytoplasm and the nucleus, as well as on the cell surface. This method is referred to as the DFA technique. The most important step in obtaining the specimen is to disrupt the roof of the vesicle in order to collect intact cells from the base of the lesion. Cells can be recovered efficiently by rotating the blunt end of a wooden applicator stick in the unroofed lesion, or by scraping with a scapel and transferring the material to a glass slide. Unless at least five intact cells are visible on the slide, the specimen should not be considered adequate for processing by the DFA method. It is important to include parallel staining of a portion of the specimen with reagents to detect HSV since VZV and HSV lesions are often indistinguishable clinically, and to provide a control for the specificity of the assay. The proper interpretation of DFA slides requires experience; the most common error is to identify false-positive results because of lack of expertise at distinguishing background nonspecific fluorescence. If the clinical course of the patient is not consistent with a diagnosis of VZV infection that was based on the DFA method, the laboratory result should be questioned. DFA and indirect immunofluorescence or immunoperoxidase methods can also be used to demonstrate the presence of VZV in properly prepared tissue sections of lung, liver, and other organs from patients with disseminated VZV infections. Enzyme immunoassay methods are also useful for detecting VZV antigens in specimens from skin lesions [101]. When laboratory facilities for DFA testing are not available, the Tzanck stain and other cytologic methods can be used to detect multinucleated giant cells in a lesion scraping; however, it is essential to realize that false negatives are common, and that these methods do not differentiate VZV from HSV infection. Herpes viral particles can be detected by electron microscopy, but few diagnostic laboratories are equipped to perform this procedure. The method is not rapid and the morphology of herpesviruses is too similar to distinguish these viruses by electron microscopy. Viral DNA detection VZV DNA sequences can be detected using radiolabeled or biotinylated nucleic acid probes for in situ hybridization or Southern blot procedures [29,103,104]. However, in recent years, PCR has become the diagnostic test of choice for detecting VZV in clinical samples from patients with varicella or zoster. PCR assays for VZV are commercially available for routine diagnosis and can be applied to blood, CSF, skin biopsies, and various other kinds of tissue [105]). PCR-based techniques have been used for VZV genotyping and to differentiate wild-type VZV strains from Oka vaccine strains in the investigation of varicella outbreaks following immunization [106–108]. Further, this technology is particularly useful for the early diagnosis of VZV infection in the CNS. Since CNS infection by different herpesviruses may lead to indistinguishable clinical manifestations, PCR assays to enable parallel detection of more than one of these herpesviruses are also being developed [109–111]. Other molecular methods in development to detect VZV DNA include loopmediated isothermal amplification [112,113] as well as microarrays [114–116]. Serologic diagnosis Serologic screening of prospective HCT recipients for VZV antibodies is a valuable tool to establish immune status before transplantation. Many serologic methods are available for measuring IgG antibodies to VZV [3,100]. The most sensitive serologic assays for detection of VZV antibodies are fluorescent-antibody staining of membrane antigen (FAMA), but this is a research method only. Other methods that are

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relatively reliable for establishing immune status include enzyme-linked immunosorbent assay (ELISA) and anticomplement immunofluorescence. While the commercially available ELISA kits for detecting VZV antibodies have a high degree of specificity, these methods are not as sensitive as FAMA. While these methods do not usually yield falsepositive results, from 10% to 15% of immune individuals may be wrongly identified as not having had previous VZV infection. Complement fixation methods are not satisfactory for determining immune status. Latex agglutination is a relatively sensitive and specific method for establishing VZV immune status [117,118]. Although seroconversion can be documented with primary VZV infection, and boosts in antibody titers accompany recurrent VZV, the serologic diagnosis of acute infection requires paired sera and is rarely helpful for clinical purposes. VZV IgM can be detected in patients with varicella, but the majority of patients with recurrent infection also produce VZV IgM, and VZV IgM can be detected intermittently in healthy asymptomatic individuals [119]. Therefore, testing for VZV IgM antibodies is not useful in clinical practice, and should not be done. The test is commercially available through some reference laboratories, but testing is rarely done with the proper control of a known VZV IgMpositive specimen. Quantitative antibody detection has been used to measure the immune response to VZV after vaccination in clinical trials of varicella vaccines. The immune response following natural infection is usually more robust, with VZV antibody titers up to more than 10-fold higher than those following vaccination. Glycoprotein (gp) ELISA assays that allow quantitative VZV antibody measurement have been developed to evaluate VZV-specific immune status [120,121]. A titer of 5 gpELISA units/mL of gpELISA VZV antibodies at 6 weeks post vaccination in initially seronegative children has been proposed as an approximate correlation of protection from infection [120,122]. However, this method is not commercially available, and is subject to false-positive results [123]. At present, there is no reliable serologic method to assess VZV immunity after varicella vaccination. Further, the detection of VZV IgG antibodies after vaccination using commercially available methods does not provide evidence of protection against breakthrough varicella, which can be expected to occur in some vaccinated individuals with close exposure.

Antiviral therapy for VZV infection Acyclovir At the present time, acyclovir (9-[(2-hydroxyethoxy)methyl]-9Hguanine) is the drug of choice for the treatment of primary and recurrent VZV infections. The antiviral activity of acyclovir against VZV follows the same pathway that mediates its interference with the replication of HSV. The metabolism of the drug to the triphosphate form by the viral thymidine kinase produces a compound that functions as a competitive inhibitor and chain terminator of viral DNA polymerase. However, while HSV-1 and HSV-2 isolates are usually inhibited in vitro by 0.125 μg/mL and 0.215 μg/mL of acyclovir, respectively, the mean concentrations required to inhibit VZV isolates are often 0.82–4.64 μg/mL, with a range from as low as 0.3 to as high as 10.8 μg/mL [124]. The plasma concentrations achieved by the intravenous administration of acyclovir at doses of 10 mg/kg or 250–500 mg/m2 range from 15 to 25 μg/mL [68]. These concentrations are several-fold above the in vitro inhibitory concentrations for most VZV isolates. In contrast, only about 20% of the oral dose of acyclovir is absorbed. Oral administration to pediatric patients at doses of 600 mg/m2 given four times produced peak plasma concentrations of approximately 1.0–1.5 μg/mL [125]. The oral dose of 200 mg acyclovir given five times a day to adults (approximately 115 mg/m2 for an adult male) produces plasma concentrations

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of approximately 0.5 μg/mL; increasing the unit dose to 600 mg resulted in plasma concentrations of 1.3 μg/mL [126]. Thus, acyclovir concentrations required to inhibit some VZV isolates can be expected to be significantly above the mean peak plasma concentration achieved by oral dosing.

The administration of intravenous acyclovir to immunocompromised children with varicella has the potential to terminate the cell-associated viremia that produces malignant progressive varicella in these patients, and to prevent the onset of varicella pneumonitis, each of which is correlated with a high risk of fatal infection [3,5,67,127]. The dosage of intravenous acyclovir for varicella in high-risk patients is 500 mg/m2 per dose every 8 hours, with administration continuing for 7 days. When this dosage was tested in placebo-controlled trials, the effect of the drug on the number of days to defervescence and resolution of cutaneous lesions was not significant, but varicella pneumonitis was prevented in the acyclovir recipients [128,129]. Primary VZV infection in HCT recipients occurring within the first year after transplant should be considered to require intravenous acyclovir therapy. The goal of antiviral therapy for varicella in high-risk patients is to initiate drug treatment within 72 hours after the appearance of the cutaneous rash. Parents and patients need to be educated about the typical appearance of varicella lesions because many cases occur without any known exposure, and prompt diagnosis during the early phase of infection is important to the success of antiviral therapy. Immunocompromised children who have been vaccinated are at risk for breakthrough vesicles, and parents should also be educated about the signs and symptoms, even though vaccination is expected to provide some protection from severe disease. Since there are other causes of vesicular rashes in childhood and in immunocompromised patients, the clinical diagnosis should be confirmed by the laboratory using the DFA technique. While most children do not have clinical signs of dissemination immediately, the interval during which preventive therapy can be initiated is very short. The decision to initiate antiviral therapy must be made before progression of the rash becomes obvious because visceral dissemination takes place at the same time. The average period to onset of varicella pneumonitis is 6 days, with most cases occurring within 4–8 days among untreated high-risk patients. In addition to preventing life-threatening dissemination, acyclovir therapy can also be expected to minimize the extent of the cutaneous disease, and shorten the time to complete healing significantly [67]. More rapid resolution of the cutaneous lesions may reduce the risk of secondary bacterial infections as well. Immunocompromised children or adults who present with signs of disseminated VZV infection should receive immediate treatment with intravenous acyclovir. The efficacy of acyclovir for the treatment of established varicella pneumonia or other visceral sites of infection has not been determined in a controlled trial. Five recipients of placebo in the original acyclovir trial who developed pneumonitis were placed on the drug approximately 6–8 days after the appearance of the varicella rash, and all of these patients improved after initiation of the drug [129]. However, in another series, three of four high-risk patients who were not treated until at least 5 days after the onset of the cutaneous lesions had evidence of visceral dissemination at the initiation of treatment; all three patients had progressive varicella, and two patients died [130]. Beyond 9–12 months after HCT, it may be acceptable to monitor the clinical course of varicella without giving acyclovir, assuming that the patient has no evidence of GVHD and is not receiving immunosuppressive therapy. However, given the predictable benefits of a short course of acyclovir for varicella, even in healthy children, its administration is justified.

Burning Pain Healing

0.90 0.80 0.70 Probability

Antiviral treatment of varicella

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0.60 0.50 0.40 0.30 0.20 0.10 0.0 1

2

3

4

5

6

7

10

14

21

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Fig. 92.7 Probability of lesion burning (23 patients; 䊉——䊉), of lesion pain (31 patients; 䊊- - - -䊊), and of not having total healing (31 patients; 䉭- - - -䉭) by treatment day. Reproduced from Balfour et al. [131], with permission.)

Antiviral treatment of herpes zoster Acyclovir has been shown to be effective for the treatment of recurrent VZV infection in immunocompromised patients in placebo-controlled trials and through extensive clinical experience with the drug. The dose of acyclovir is 500 mg/m2 or 10 mg/kg given every 8 hours intravenously. Therapy should be continued for 7 days, or for 2 days after cessation of new lesion formation, whichever provides the longer treatment course. In a placebo-controlled trial, local progression and progression of cutaneous dissemination were terminated with acyclovir therapy [131]. One of 52 treated patients had progressive VZV disease, compared with 11 of 42 placebo recipients. Treatment resulted in a shorter time to cessation of new lesion formation, more rapid crusting and healing, and prevention of cutaneous and visceral dissemination in patients who presented with localized herpes zoster (Fig. 92.7). Acyclovir decreased the duration of local viral replication, with viral cultures remaining positive for VZV for only 4 days after the initiation of treatment. Based on this experience, acyclovir therapy initiated within 72 hours after the onset of VZV reactivation can be expected to reduce the duration of new lesion formation in HCT patients to approximately 3 days. On average, early antiviral treatment should cause the cessation of acute pain within 4 days, crusting of lesions by 7 days, and complete healing by 2–3 weeks (Fig. 92.7). Although early acyclovir treatment is likely to produce the best results, clinical benefit can still occur when therapy is delayed for more than 3 days [131]. None of 29 immunocompromised patients whose therapy was initiated more than 3 days after the onset of the rash had progressive herpes zoster, compared with 3 of 17 patients in the placebo group. Although the drug eliminates the life-threatening complications of VZV reactivation in most patients, relapse of herpes zoster is a clinical problem in some HCT patients who are treated with acyclovir. In one series, 5 of 40 patients (12%) developed new lesions, with relapse occurring less than 4 days after treatment was stopped in three of the five patients [132]. Early acyclovir therapy may delay the recovery of VZV specific immunity in some patients. Nevertheless, most patients respond to treatment with a second course of acyclovir. Although acyclovir is clearly beneficial for the treatment of acute herpes zoster, its effect on the incidence of postherpetic neuralgia has been more difficult to establish. Varying definitions of postherpetic

Varicella-zoster Virus Infections

neuralgia have complicated assessments of antiviral and adjunctive therapies. A meta-analysis of several placebo-controlled trials of oral acyclovir therapy for herpes zoster in otherwise healthy individuals indicates that long-term pain is diminished [133,134]. However, HCT recipients may have a higher rate of recurrence of pain after cessation of therapy. Based upon experience in immunocompetent patients, extending the duration of acyclovir therapy from 7 to 21 days does not improve outcome [135]. Evaluations of the administration of steroids as adjunctive therapy in patients without malignancy have yielded conflicting results about clinical benefit [135,136]. Whether the use of steroids along with antiviral therapy reduces the severity of zoster-associated pain in HCT recipients has not been assessed. The fact that acyclovir ameliorates the acute pain associated with herpes zoster more effectively than long-term pain suggests that a different mechanism is responsible for postherpetic neuralgia. Continued VZV replication is not likely to account for postherpetic pain. Instead, it has been suggested that the initial neuronal infection and associated inflammation, which can persist for months, results in deafferentation, ectopic activity of damaged afferents nerves, and enhanced excitability of neurons relaying central pain signals. Current approaches to the management of postherpetic neuralgia (reviewed in [137,138]) include tricyclic antidepressants [137–1442], antiepileptics such as gabapentin [143], pregabalin [144,145], and divalproex sodium [146], as well as opioids [147]. Topical application of lidocaine patches or capsaicin can also provide some relief. Medical interventions are often used in combination, or sequentially, because no single approach is beneficial in most patients. Patients with intractable pain despite the approaches mentioned above may consider intrathecal corticosteroids [148]. The benefits of some other approaches, such as sympathetic blockade, intravenous lidocaine or cryotherapy, have not been convincingly demonstrated. The efficacy of oral acyclovir for herpes zoster in HCT patients has not been firmly established in prospective studies, but this route of administration is appropriate for selected patients who have localized herpes zoster occurring in the late period after HCT. Since the bioavailability of oral acyclovir is low, this route of administration requires giving 800 mg per dose, five times a day. Patients receiving oral acyclovir should be monitored for signs of progressive VZV infection, and should be treated with the intravenous drug if complications arise. When patients are treated with the standard intravenous regimen, there is no need to provide further treatment with oral acyclovir after discharge. However, in some cases, the full course of therapy may be achieved by a combination of intravenous and oral acyclovir. Acyclovir toxicity The safety of acyclovir has been established in numerous clinical trials. While HCT patients also tolerate the drug well, the incidence of sideeffects is higher. In one series, gastrointestinal symptoms of nausea and vomiting occurred in 40% of treated patients [132]. Nephrotoxicity, defined as a 50% rise in serum creatinine, was also more common than in other patient populations treated with acyclovir; 10–25% of HCT patients receiving acyclovir are reported to have an abnormal serum creatinine level, but these elevations may be caused by other medications given concurrently [132]. In any case, since acyclovir is excreted by glomerular filtration, other drugs that affect renal function, such as cyclosporine, can interact to cause elevated plasma concentrations of the drug. The dosage and dose interval for acyclovir administration should be adjusted based on the relative impairment of creatinine clearance. The dosage interval should be lengthened to every 12 hours for clearances of 25–50 mL/min and to 24 hours for clearances of 10–25 mL/min; if the clearance is 0–10 mL/min, the dosage should be reduced to 250 mg/

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m2, given every 24–48 hours. It is also important to maintain adequate hydration in patients receiving acyclovir to avoid precipitation of the drug in renal tubules. A few cases of acute neurotoxicity have been reported in patients with deficient renal clearance who were receiving acyclovir. Abnormal liver function tests should be considered possible evidence of VZV hepatitis rather than drug toxicity. Acyclovir does not have hematologic toxicity and does not interfere with engraftment in HCT recipients. Other antiviral compounds Famciclovir and valaciclovir are nucleoside analogues and are prodrugs of penciclovir and acyclovir, respectively. Both have been licensed for treatment of herpes zoster in the immunocompetent host [149–152]. Similar to acyclovir, penciclovir (9-[4-hydroxy-3-hydroxymethylbut-1yl] guanine) is phosphorylated by the viral thymidine kinase and acts as a competitive inhibitor of the viral DNA polymerase, but unlike acyclovir, it is not an obligate chain terminator. The advantage of these prodrugs is a much better absorption after oral administration than acyclovir, coupled with a safety profile that resembles that of acyclovir. Although controlled trials to establish the clinical efficacy of these antiviral agents in HCT patients have not been done, valaciclovir or famciclovir may be useful in selected patients who are considered to be at low risk of visceral dissemination during recurrent VZV reactivation. Attention to the dosage of these very efficiently absorbed drugs is essential, since toxicity of valaciclovir, manifest as thrombocytopenic purpura and hemolytic–uremic syndrome, has been reported in HCT recipients [153]. Plasma concentrations approach those achieved by intravenous administration of acyclovir unless the patient has altered gastrointestinal function, which may be an obstacle to their use in some HCT recipients. Famciclovir and valaciclovir require less frequent administration and are more convenient for patients, but these drugs are more expensive than oral acyclovir. Although VZV resistance to acyclovir has not been common in HCT recipients, it has been reported in these patients and patients with the acquired immunedeficiency syndrome [154–156]. Among HCT recipients, the failure of VZV infections to resolve, or their recurrence shortly after antiviral therapy is discontinued, is usually a function of the limited host response, and should not be attributed to acyclovir resistance in most cases. When it occurs, antiviral resistance is usually mediated by thymidine kinase mutations. As a result, valaciclovir and famciclovir are not useful for treating VZV infections caused by acyclovir-resistant strains [154–156]. Foscarnet, a pyrophosphate analogue, has antiviral activity against VZV through inhibition of the viral DNA polymerase, and has activity against isolates that are not inhibited by acyclovir or famciclovir [78,154]. Its clinical use is complicated by potential nephrotoxicity and the emergence of resistance [157,158]. Cidofovir is a nucleotide agent that may be useful for resistant strains, but clinical experience is limited. Serious side-effects of this drug include severe nephrotoxicity, metabolic acidosis, neutropenia, and ophthalmic complications. There is no evidence that combinations of these antiviral drugs improve the treatment of VZV infection, and adverse drug interactions may occur. Vidarabine, the first antiviral agent shown to have clinical benefit for the treatment of VZV infections in immunocompromised patients, inhibits VZV by a different pathway, but was less effective than aciclovir and is no longer available [132]. Although sorivudine (BVaraU) might have been useful for managing resistant VZV, its development has been terminated in the United States because of its lethal interactions with 5-fluorouracil, and concern that it might be administered inadvertently to patients who were receiving this drug [159,160]. Brivudine is a

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Chapter 92

nucleoside analogue of thymidine, which also has potent antiviral activity against VZV and HSV. A single daily dose of brivudine at 125 mg is found to be effective in the treatment of herpes zoster, compared with acyclovir 800 mg five times a day or famciclovir 250 mg three times a day, all given orally [161,162]. However, similar to sorivudine, brivudine, when co-administered with 5-fluorouracil or capecitabine (Xeloda, which is metabolized to 5-fluorouracil) can lead to lethal complications. This drug is approved for sale in some European countries including Germany and Italy, but is not licensed for use in the United States. Human and recombinant leukocyte IFN were shown to have significant clinical efficacy in immunocompromised patients with herpes zoster in early studies and provide an alternative to the nucleoside analogue drugs for the treatment of resistant VZV infection [163]. Although VZV strains resistant to acyclovir may emerge during therapy, particularly when the drug is given for prolonged courses and at low doses, VZV strains isolated from later recurrences in patients whose resistant infections have resolved are usually again susceptible to acyclovir. Ganciclovir (9-[1,3-dihydroxy-2-propoxymethyl] guanine; DHPG) has in vitro activity against VZV that is equivalent to acyclovir, but clinical studies of its efficacy for the treatment of VZV infections have not been done because of its greater toxicity. However, it is possible that the administration of ganciclovir to HCT recipients who have CMV infection could alter the course of concurrent VZV infection.

Prophylaxis for VZV infection Varicella-zoster immune globulin Varicella-zoster immune globulin (VZIG) is a passive antibody preparation containing VZV IgG antibodies that is prepared from high-titer immune human serum. VZIG is no longer commercially available, since its production was discontinued by its only United States-licensed manufacturer (Massachusetts Public Health Biologic Laboratories, Boston, MA). An investigational product of VZIG (VariZIG) is available under an Investigational New Drug Application Expanded Access protocol (Cangene Corporation, Winnipeg, Canada) [164]. However, Investigational Review Board approval is required before accessing this product from the sole authorized United States distributor, FFF Enterprises (Temecula, CA). The product is lyophilized, which, when reconstituted properly, is approximately a 5% solution of IgG. The dosage is one vial (125 units)/10 kg body weight given intramuscularly. The minimum dose is 125 units and the maximum dose 625 units. The human IgG content is 60–200 mg per 125 units. Since VariZIG is not readily available, an alternative to VariZIG would be intravenous immune globulin (IVIG), which can be administered at 400 mg/kg [165]. Passive antibody prophylaxis with VariZIG or IVIG is indicated following exposure of VZV-seronegative immunocompromised children or adults who have not had previous VZV infection, including HCT recipients [153,166]. HCT recipients are in general considered nonimmune, regardless of previous history of varicella disease or varicella vaccination in themselves or in their donors. However, if varicella or zoster develops after transplantation, they should then be considered immune [166], and passive antibody prophylaxis would not be indicated after exposure. The effect of passive antibody prophylaxis depends upon administration within 96 hours, and preferably within 48 hours of exposure. Varicella exposure is defined as household contact, shared hospital room or indoor play for at least 1 hour with a child who is in the contagious phase of varicella, which is the interval from 2 days before to 5 days after the onset of the rash. If a patient has received any of the commercial preparations of high-dose IVIG (≥400 mg/kg) for other indications within 3 weeks before the exposure, it is not necessary to administer VZIG [134,166]. The duration of protection after

administration of passive antibody prophylaxis is unclear, but it is thought to last at least one half-life of the immunoglobulin, which is about 3 weeks. Therefore, a second dose of passive antibody prophylaxis should be given if a new exposure occurs more than 3 weeks after a dose of VZIG has been given [166]. While the risk of VZV transmission from an individual with herpes zoster is low, close contact between an HCT patient who has never been infected with VZV and a patient with recurrent VZV lesions also justifies the administration of VZIG. Clinicians must be aware that severe varicella develops in some immunocompromised patients despite the timely administration of VZIG. The incidence of varicella despite VZIG prophylaxis is significantly higher for children with household exposures than for those who have less prolonged contact with the index case, and the attack rate is affected by the VZV IgG titer of the preparation [167]. In the placebocontrolled trial of intravenous acyclovir, one of six (17%) placebo recipients who had received zoster immune globulin or zoster immune plasma before entry required reassignment to open drug because of progressive varicella [129]. In the St Jude experience, the risk of varicella pneumonitis was reduced significantly by passive antibody prophylaxis, but 11% of children developed pneumonia despite receiving zoster immune globulin, zoster immune plasma or VZIG [67]. Because of these risks, HCT recipients who develop varicella should be treated with intravenous acyclovir even if passive antibody prophylaxis was given at the time of exposure. Since the live attenuated varicella vaccine is recommended for universal administration to children in the United States at age 12–15 months, some children will have had vaccine-induced immunity before HCT [168]. These children should be given passive antibody prophylaxis for exposure to wild-type varicella because the protective efficacy of varicella vaccine is not defined in these circumstances. High-risk children may also have close exposure to an immunized child or adult who has breakthrough varicella caused by the wild-type virus or, rarely, to a healthy contact with a varicella vaccine-related rash. Immunocompromised children who have such an exposure should receive passive antibody prophylaxis. However, depending on the timing of administration post vaccination, contacts treated with passive antibody prophylaxis can still develop subclinical or clinical varicella. In an early study that evaluated the efficacy of VZIG in immunosuppressed children after household exposure to varicella, up to 60% developed clinical disease despite detectable VZV antibodies [167]. Based upon reports of clinical experience but without controlled trials, some experts have recommended using acyclovir (or valaciclovir) in combination with passive antibody prophylaxis or alone as postexposure prophylaxis for VZV exposure in susceptible high-risk children [169,170]. There is no indication that passive antibody prophylaxis will reduce the risk of VZV reactivation after HCT in patients who have serologic evidence of prior VZV infection. Passive antibody administration is not effective for the treatment of herpes zoster. Acyclovir prophylaxis The efficacy of acyclovir prophylaxis for preventing recurrent VZV infection in HCT recipients was evaluated in several studies [17,171– 174], but only three of them are prospective, randomized, double-blind trials, as summarized in Table 92.4. For these three studies [171,175– 177], the dose of acyclovir employed ranges from 1600 mg/day to 3200 mg/day, and the duration of prophylaxis varies from 6 months to 12 months. Among all the retrospective or prospective studies, only one study reported a single case of breakthrough zoster infection among 247 patients, and there was no information concerning compliance of the patient with the prophylactic regimen [174]. Otherwise, none of the other studies reported any breakthrough VZV infection during the period

Varicella-zoster Virus Infections

1403

Table 92.4 Randomized, double-blind, placebo-controlled studies on acyclovir prophylaxis for prevention of varicella-zoster virus (VZV) infection after hematopoietic cell transplantation (HCT)

Total duration (months)

Reactivation during prophylaxis

Reactivation after cessation of prophylaxis

Number of patients who received acyclovir prophylaxis

Author

HCT type

Oral dose of acyclovir per day (mg)

Ljungman, 1986 [175] Perren, 1988 [176]; Selby, 1989 [177] Boeckh, 2006 [171]

Allogeneic Allogeneic

1200 3200

6 6

No No

31% at 6 months 42% at 6 months

n = 20 n = 42

Allogeneic

1600

12

No*

21% by 12 months

n = 38

* Two cases of VZV in the acyclovir group were in the modified intent-to-treat analysis, but both cases occurred in patients who were off acyclovir.

of therapy, compared with 30–60% incidence without prophylaxis [1,16,18]. However, regardless of the acyclovir dose or duration employed, prophylaxis with acyclovir in general delays but does not prevent VZV reactivation. Once the administration of acyclovir was stopped, VZV infection recurred, with no overall reduction in infection rate compared with control groups. While most studies used higher doses of acyclovir (1200–3600 mg/ day) for prophylaxis [173,175–177], one study [172] tested long-term prophylaxis with acyclovir at 400 mg/day and observed reactivation after cessation of acyclovir only in patients who resumed taking immunosuppressants. It was thus theorized that administration of very-lowdose acyclovir might permit subclinical VZV reaction to allow the recovery of VZV-specific T-cell immunity. However, a subsequent larger study by Thomson et al. using such low-dose acyclovir for prophylaxis [174] showed a cumulative [172] VZV reactivation incidence of 39% at 12 months after cessation of acyclovir, similar to that of the earlier studies with a higher dose/shorter duration of prophylaxis. Therefore, the use of a low-dose acyclovir regimen does not confer any advantage, but may instead induce the emergence of resistance against acyclovir. Boeckh et al. studied the efficacy of long-term acyclovir prophylaxis in a double-blind controlled trial [171]. Patients who underwent allogeneic HCT were randomized to receive acyclovir 800 mg twice daily or placebo given from 1–2 months until 1 year after transplantation. Again, acyclovir significantly reduced VZV infection at 1 year after transplantation, with a hazard ratio of 0.16. However, the postintervention observation at 2, 3, and 5 years showed no statistically difference in VZV infection between the two groups. There was also no statistically significant difference in reconstitution of VZV specific T-helper-cell responses, HSV infection, CMV disease, chronic GVHD, and overall survival between the groups. The suppressive effect of antiviral drugs has been confirmed in clinical experience in allogeneic HCT patients who received acyclovir and/or ganciclovir, but, similar to the observation from clinical trials, rapid occurrence of herpes zoster was observed when the drugs were discontinued [16,17]. Erard et al. [21] recently examined three sequential cohorts from 1996 to 2003 receiving acyclovir from day of transplantation until engraftment, acyclovir or valaciclovir for 1 year, or acyclovir or valaciclovir for at least 1 year or longer if patients remained on immunosuppressive drugs. They reported that prophylaxis given for 1 year significantly reduced VZV disease with no evidence of rebound VZV disease. Despite the favorable outcomes observed with this study, such findings have not been validated by randomized prospective studies. While it may reduce episodes of VZV reactivation, giving acyclovir as prophylaxis to prevent herpes zoster in HCT recipients is not recommended as routine practice, as stated in the guidelines developed jointly by the Centers for Disease Control and Prevention, the Infectious Dis-

eases Society of America, the American Society for Blood and Marrow Transplantation, and other agencies for preventing opportunistic infections after HCT [178]. Although herpes zoster early after HCT has a risk of causing disseminated disease, prompt initiation of acyclovir for the treatment of recurrent VZV infections has proved to be very effective. None of the placebo recipients in the acyclovir prophylaxis studies had fatal dissemination when given intravenous acyclovir at the onset of recurrent VZV infection. Nevertheless, clinical practice varies, and some institutions elect to use acyclovir or related antiviral drugs for suppression of recurrent VZV infections after transplantation [179]. Antiviral prophylaxis may be indicated for selected patients at high risk, such as those with GVHD who remain severely immunodeficient [17]. A similar approach is endorsed by the Infectious Diseases Working Party of the German Society of Hematology and Oncology, which recommended VZV prophylaxis in HCT recipients with risk factors, such as those with T-cell depletion, ATg therapy, anti-CD3 antibody therapy, or unrelated or HLA-mismatched transplantation [180]. The rationale for avoiding routine use is that prolonged administration of antiviral prophylaxis enhances the emergence of VZV strains that are resistant to the drug. While the newer guanine analogues such as valaciclovir or famciclovir have the benefit of improved oral bioavailability compared with acyclovir, there are no data from controlled trials using the newer drugs for VZV prophylaxis. Varicella vaccine In 1995, a live attenuated varicella vaccine (Oka strain) was approved for administration to healthy children in the United States. Since then, more than 40 million doses have been distributed in this country. Experience from the last 10–20 years, over the pre- and post-licensure periods, has demonstrated the effectiveness and safety of this vaccine in immunocompetent populations. From various investigations, the overall vaccine effectiveness ranges from 71% to 100% against varicella, and 95% to 100% against moderately severe and severe disease [181–183]. However, one study of an outbreak reported an effectiveness of 44% against varicella of any severity and 86% against moderate and severe disease, which is significantly lower than the other reports [184]. After the implementation of universal childhood vaccination, the incidence of varicella declined by 90% and related mortality declined by 66% [185]. Two live, attenuated, VZV-containing vaccines are now available in the United States for prevention of varicella: a single-antigen varicella vaccine (Varivax; Merck & Co, Inc.) and a combination vaccine for measles, mumps, rubella, and varicella (ProQuad; Merck & Co, Inc.). In 1996, the Advisory Committee on Immunization Practices (ACIP) recommended one dose of varicella vaccine for children aged 12 months to 12 years, but two doses, 4–8 weeks apart, for those aged 13 or more years [186]. However, despite the success of the one-dose regimen, with

Chapter 92

a vaccine effectiveness of 85%, varicella outbreaks continue to occur in highly vaccinated school populations [187]. In a randomized trial that compared the efficacy of one dose with that of two doses administered 3 months apart, the two-dose regimen was significantly more effective than a single injection with a 10-year observation period [188]. Based on these findings, in June 2006, ACIP approved a routine two-dose recommendation for children, as well as for all susceptible adolescents and adults, with the goal of further reducing varicella disease and its complications in the United States [166]. A second dose is also recommended for all persons who received one dose previously. In May of 2006, a live attenuated vaccine (Zostavax; Merck & Co, Inc.) was also approved in the United States for prevention of zoster and postherpetic neuralgia in adults of 60 years or older. This zoster vaccine is also based on the Oka–Merck strain, but each dose contains 19,400 plaque-forming units, compared with 1350 plaqueforming units in the varicella vaccine. In a large prelicensure study that enrolled 38,546 adults, the vaccine reduced the incidence of zoster by 51.3%. The burden of illness due to zoster and due to postherpetic neuralgia were also reduced by 61.1% and 66.5%, respectively [189]. The live attenuated varicella vaccine was originally investigated in children with leukemia, in an effort to prevent the development of severe or fatal VZV infection in these patients [190]. While the varicella and zoster vaccines have significantly changed the impact of VZV infection on healthy pediatric and adult populations, these live virus vaccines are in general not recommended for immunocompromised hosts. Some recent studies suggested that live VZV vaccine is immunogenic and safe for patients with solid organ transplantation [191–193]. However, this practice remains controversial. A recent case of a heart transplant recipient who received vaccination and developed cutaneous Oka strain vaccine lesions requiring hospitalization was reported [194]. Extensive post-vaccination rash and disseminated disease have also been observed in children with acute lymphoblastic leukemia vaccinated while on steroid therapy [195]. A case of fatal disseminated infection from the vaccine strain in a child with acute lymphoblastic leukemia was reported, even though the vaccination was done 5 months after complete remission had been achieved and chemotherapy was interrupted for 1 week before and after vaccination [196]. Severe complications, including hepatitis, pneumonitis, and chronic zoster, have also been reported in children with underlying immunodeficiency not apparent at the time of immunization [64,197–199]. Immunizing healthy household contacts who are not immune to varicella with the varicella vaccine can reduce the risk of household exposure of the HCT recipient to varicella in siblings [200]. The risk of transmission of the vaccine strain is low, and illness caused by the vaccine strain in immunocompromised individuals is treatable with acyclovir, whereas infection associated with wild-type VZV can be severe. Further, transmission of vaccine virus to immunocompromised persons has not been documented in the post-licensure period in the United States, despite a distribution of more than 40 million vaccine doses [166]. Since the benefits of vaccination outweigh the potential risk of transmission of vaccine-type virus to immunocompromised persons, the Centers for Disease Control and Prevention ACIP recommends routine vaccination of susceptible household contacts of immunocompromised persons [166]. Health-care workers in contact with HCT patients should also be evaluated for susceptibility to VZV and immunized. There are also no contraindications for eligible household members of immunocompromised persons or health-care workers in contact with HCT patients to receive the zoster vaccine for prevention of zoster and post-herpetic neuralgia. Any vaccine recipients, particularly health-care workers and household contacts, should avoid contact with susceptible immunocompromised person if a vaccine-related rash occurs [166]. Since some vaccine recipients become candidates for HCT, post-transplant reactiva-

tion of vaccine virus has been demonstrated in a few instances. However, the vaccine virus appears to reactivate less commonly than wild-type VZV [201]. If symptomatic reactivation occurs, the vaccine virus is susceptible to inhibition by acyclovir, and episodes of vaccine-related herpes zoster can be treated with this drug [202]. Whether immunization with an inactivated preparation of the live attenuated varicella vaccine could substitute for the “natural” resensitization caused by VZV reactivation after HCT, and whether the early restoration of immunity modified the clinical course of recurrent VZV disease, has been evaluated in two prospective studies [23,203]. The administration of the live attenuated varicella vaccine to elderly individuals without underlying disease boosts VZV-specific immunity and immunization has been evaluated as a strategy to reverse the decline of T-cell responses that is associated with the increased risk of herpes zoster in this population [189,204]. Although live attenuated varicella vaccine cannot be given to immunocompromised patients, the vaccine can be heat-inactivated without loss of immunogenicity [205]. HCT recipients recover VZV immunity, but reconstitution is delayed for months and often does not occur until after the patient has experienced herpes zoster [4,23,47,57]. A single-dose regimen of inactivated varicella vaccine induced short-term immunologic enhancement but did not result in clinical benefit. A three-dose regimen in which vaccine was given to allogeneic or autologous transplant recipients at 30, 60, and 90 days after HCT boosted cell-mediated immunity and modified the clinical severity of herpes zoster, but did not reduce the overall incidence of herpes zoster after HCT [203]. When inactivated varicella vaccine was given to autologous HCT recipients in a regimen that included a pretransplantation dose, as well as doses given at 30, 60, and 90 days after HCT, the risk of herpes zoster was reduced, and protection correlated with reconstitution of VZV CD4 T-cell immunity [23]. In this study, 19 of 58 unvaccinated patients (33%) developed herpes zoster, compared with 7 of 53 (13%) vaccine recipients (p = 0.01) (Fig. 92.8). The difference was 30%, compared with 13% (p = 0.02) when two patients who developed zoster before transplant were excluded. VZV-specific CD4 T-cell proliferation was significantly higher in vaccinees by 90 days, following three doses of vaccine (p = 0.04), and the differences remained significant at 120 days, after four doses (p = 0.0001), and at 6 months (p = 0.004) and 12 months (p = 0.02) (Fig. 92.4).

Probability of VZV

1404

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

No vaccine Vaccine

0

1

2

3

4

5

6

7

8

9

10

11

12

13

Time in months

Fig. 92.8 The occurrence of zoster in autologous hematopoietic cell transplantation (HCT) recipients randomized to receive inactivated varicella vaccine or no vaccine. The Kaplan–Meier plot shows the probability of zoster in study participants who were randomized to receive inactivated vaccine (- - - -) and those who were unvaccinated (——), from enrollment until 12.5 months after HCT. The incidence of zoster was significantly higher in the unvaccinated group (p = 0.01). When the two cases of zoster that occurred before transplant were excluded, the difference remained significant (p = 0.02). Reproduced from Hata et al. [23], with permission.)

Varicella-zoster Virus Infections

As noted in the section on host response, this prospective study demonstrated that VZV-specific CD4 T-cell proliferation was a correlate of protection against VZV reactivation [23]. The risk of zoster was reduced by 19% per unit increase in stimulation index over 1.6, and a stimulation index of greater than 5.0 correlated with over 93% protection. The relationship between a high stimulation index and reduced risk of herpes zoster could reflect the direct contribution of VZV-specific CD4 T cells to controlling VZV replication, since these cells make IFN-γ and other cytokines, and have cytotoxic function. Reconstitution of VZV-specific CD4 T cells by vaccination may also provide helper functions necessary to expand the VZV-specific CD8 T-cell populations that are essential for preserving VZV latency. In terms of basic immunobiology, the reduced frequency of VZV reactivation observed after active immunization of HCT recipients establishes the critical role of virus-specific T-cell responses in preserving the balance between VZV and the host. Sustaining these host responses by repeated doses is likely to be necessary to preserve clinical efficacy against herpesvirus reactivation while immunosuppressive

1405

therapy continues. Immunization of HCT patients against herpesviruses represents a particular challenge because disease is usually due to the reactivation of endogenous viruses during the first several months after transplantation. However, this experience with inactivated varicella vaccine indicates that immunotherapy with vaccines against herpesviruses has the potential to reduce the morbidity caused by these common pathogens after HCT. Inactivated or protein-based VZV vaccines that will be safe for immunocompromised patients are being developed for clinical use.

Conclusion Primary and recurrent VZV infections remain a serious threat to HCT recipients. Nevertheless, early recognition of the clinical signs of varicella and herpes zoster permits effective intervention with antiviral drugs. Immunization with inactivated varicella vaccine has the potential to reduce the morbidity of VZV reactivation after HCT.

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12. Wacker P, Hartmann O, Benhamou E et al. Varicella-zoster virus infections after autologous bone marrow transplantation in children. Bone Marrow Transplant 1989; 4: 191–4. 13. Schuchter LM, Wingard JR, Piantadosi S et al. Herpes zoster infection after autologous bone marrow transplantation. Blood 1989; 74: 1424– 7. 14. Christiansen NP, Haake RJ, Hurd DD. Early herpes zoster infection in adult patients with Hodgkin’s disease undergoing autologous bone marrow transplant. Bone Marrow Transplant 1991; 7: 435–7. 15. Han CS, Miller W, Haake R, Weisdorf D. Varicella zoster infection after bone marrow transplantation: incidence, risk factors and complications. Bone Marrow Transplant 1994; 13: 277–83. 16. Koc Y, Miller KB, Schenkein DP et al. Varicella zoster virus infections following allogeneic bone marrow transplantation: frequency, risk factors, and clinical outcome. Biol Blood Marrow Transplant 2000; 6: 44–9. 17. Steer CB, Szer J, Sasadeusz J et al. Varicellazoster infection after allogeneic bone marrow transplantation: incidence, risk factors and prevention with low-dose aciclovir and ganciclovir. Bone Marrow Transplant 2000; 25: 657–64. 18. Leung TF, Chik KW, Li CK et al. Incidence, risk factors and outcome of varicella-zoster virus infection in children after haematopoietic stem cell transplantation. Bone Marrow Transplant 2000; 25: 167–72. 19. Offidani M, Corvatta L, Olivieri A et al. A predictive model of varicella-zoster virus infection after autologous peripheral blood progenitor cell transplantation. Clin Infect Dis 2001; 32: 1414–22. 20. Tomonari A, Iseki T, Takahashi S et al. Varicellazoster virus infection in adult patients after unrelated cord blood transplantation: a single institute experience in Japan. Br J Haematol 2003; 122: 802–5. 21. Erard V, Guthrie KA, Varley C et al. One-year acyclovir prophylaxis for preventing varicellazoster virus (VZV) disease following hematopoi-

22.

23.

24.

25.

26.

27.

28.

29.

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results of a randomized, double-blind, multicentered study. Antiviral Res 2003; 59: 49– 56. Merigan TC, Rand KH, Pollard RB et al. Human leukocyte interferon for the treatment of herpes zoster in patients with cancer. N Engl J Med 1978; 298: 981–7. Cangene Corporation. VariZIG package insert, 2005. Winnipeg: Cangene Corporation. Available at: http://www.cangene.com/pdf/VariZIG-Monograph.English.pdf. Paryani SG, Arvin AM, Koropchak CM et al. Comparison of varicella zoster antibody titers in patients given intravenous immune serum globulin or varicella zoster immune globulin. J Pediatr 1984; 105: 200–5. Centers for Disease Control. Prevention of varicella: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2007; 56(RR-4): 1–40. Zaia JA, Levin MJ, Preblud SR et al. Evaluation of varicella-zoster immune globulin: protection of immunosuppressed children after household exposure to varicella. J Infect Dis 1983; 147: 737–43. Arvin AM, Gershon AA. Live attenuated varicella vaccine. Annu Rev Microbiol 1996; 50: 59–100. Weinstock DM, Boeckh M, Boulad F et al. Postexposure prophylaxis against varicella-zoster virus infection among recipients of hematopoietic stem cell transplant: unresolved issues. Infect Control Hosp Epidemiol 2004; 25: 603–8. Weinstock DM, Boeckh M, Sepkowitz KA. Postexposure prophylaxis against varicella zoster virus infection among hematopoietic stem cell transplant recipients. Biol Blood Marrow Transplant 2006; 12: 1096–7. Boeckh M, Kim HW, Flowers ME et al. Longterm acyclovir for prevention of varicella zoster virus disease after allogeneic hematopoietic cell transplantation – a randomized double-blind placebo-controlled study. Blood 2006; 107: 1800– 5. Kanda Y, Mineishi S, Saito T et al. Long-term low-dose acyclovir against varicella-zoster virus reactivation after allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2001; 28: 689–92. Sempere A, Sanz GF, Senent L et al. Long-term acyclovir prophylaxis for prevention of varicella zoster virus infection after autologous blood stem cell transplantation in patients with acute leukemia. Bone Marrow Transplant 1992; 10: 495–8. Thomson KJ, Hart DP, Banerjee L et al. The effect of low-dose aciclovir on reactivation of varicella zoster virus after allogeneic haemopoietic stem cell transplantation. Bone Marrow Transplant 2005; 35: 1065–9. Ljungman P, Wilczek H, Gahrton G et al. Longterm acyclovir prophylaxis in bone marrow transplant recipients and lymphocyte proliferation responses to herpes virus antigens in vitro. Bone Marrow Transplant 1986; 1: 185–92. Perren TJ, Powles RL, Easton D et al. Prevention of herpes zoster in patients by long-term oral acyclovir after allogeneic bone marrow transplantation. Am J Med 1988; 85(2A): 99–101. Selby PJ, Powles RL, Easton D et al. The prophylactic role of intravenous and long-term oral acyclovir after allogeneic bone marrow transplantation. Br J Cancer 1989; 59: 434–8.

Varicella-zoster Virus Infections 178. Sullivan KM, Dykewicz CA, Longworth DL et al. Preventing opportunistic infections after hematopoietic stem cell transplantation: the Centers for Disease Control and Prevention, Infectious Diseases Society of America, and American Society for Blood and Marrow Transplantation Practice Guidelines and beyond. Hematology Am Soc Hematol Educ Program 2001: 392–421. 179. Boeckh M. Prevention of VZV infection in immunosuppressed patients using antiviral agents. Herpes 2006; 13: 60–5. 180. Sandherr M, Einsele H, Hebart H et al. Antiviral prophylaxis in patients with haematological malignancies and solid tumours: gidelines of the Infectious Diseases Working Party (AGIHO) of the German Society for Hematology and Oncology (DGHO). Ann Oncol 2006; 17: 1051–9. 181. Dworkin MS, Jennings CE, Roth-Thomas J et al. An outbreak of varicella among children attending preschool and elementary school in Illinois. Clin Infect Dis 2002; 35: 102–4. 182. Izurieta HS, Strebel PM, Blake PA. Postlicensure effectiveness of varicella vaccine during an outbreak in a child care center. JAMA 1997; 278: 1495–9. 183. Vazquez M, LaRussa PS, Gershon AA et al. The effectiveness of the varicella vaccine in clinical practice. N Engl J Med 2001; 344: 955–60. 184. Galil K, Lee B, Strine T et al. Outbreak of varicella at a day-care center despite vaccination. N Engl J Med 2002; 347: 1909–15. 185. Nguyen HQ, Jumaan AO, Seward JF. Decline in mortality due to varicella after implementation of varicella vaccination in the United States. N Engl J Med 2005; 352: 450–8. 186. Centers for Disease Control and Prevention. Prevention of varicella: recommendations of the Advisory Committee on Immunization Practices (ACIP). Centers for Disease Control and Prevention. MMWR Recomm Rep 1996; 45(RR-11): 1– 36.

187. Tugwell BD, Lee LE, Gillette H et al. Chickenpox outbreak in a highly vaccinated school population. Pediatrics 2004; 113(3 Pt 1): 455–9. 188. Kuter B, Matthews H, Shinefield H et al. Ten year follow-up of healthy children who received one or two injections of varicella vaccine. Pediatr Infect Dis J 2004; 23: 132–7. 189. Oxman MN, Levin MJ, Johnson GR et al. A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N Engl J Med 2005; 352: 2271–84. 190. Gershon AA, Steinberg SP, Gelb L. Live attenuated varicella vaccine use in immunocompromised children and adults. Pediatrics 1986; 78(4 Pt 2): 757–62. 191. Chaves Tdo S, Lopes MH, de Souza VA et al. Seroprevalence of antibodies against varicellazoster virus and response to the varicella vaccine in pediatric renal transplant patients. Pediatr Transplant 2005; 9: 192–6. 192. Khan S, Erlichman J, Rand EB. Live virus immunization after orthotopic liver transplantation. Pediatr Transplant 2006; 10: 78–82. 193. Weinberg A, Horslen SP, Kaufman SS et al. Safety and immunogenicity of varicella-zoster virus vaccine in pediatric liver and intestine transplant recipients. Am J Transplant 2006; 6: 565– 8. 194. Kraft JN, Shaw JC. Varicella infection caused by Oka strain vaccine in a heart transplant recipient. Arch Dermatol 2006; 142: 943–5. 195. Lydick E, Kuter BJ, Zajac BA, Guess HA. Association of steroid therapy with vaccine-associated rashes in children with acute lymphocytic leukaemia who received Oka/Merck varicella vaccine. NIAID Varicella Vaccine Collaborative Study Group. Vaccine 1989; 7: 549–53. 196. Schrauder A, Henke-Gendo C, Seidemann K et al. Varicella vaccination in a child with acute lymphoblastic leukaemia. Lancet 2007; 369: 1232.

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197. Ghaffar F, Carrick K, Rogers BB et al. Disseminated infection with varicella-zoster virus vaccine strain presenting as hepatitis in a child with adenosine deaminase deficiency. Pediatr Infect Dis J 2000; 19: 764–6. 198. Kramer JM, LaRussa P, Tsai WC et al. Disseminated vaccine strain varicella as the acquired immunodeficiency syndrome-defining illness in a previously undiagnosed child. Pediatrics 2001; 108: E39. 199. Levin MJ, Dahl KM, Weinberg A et al. Development of resistance to acyclovir during chronic infection with the Oka vaccine strain of varicellazoster virus, in an immunosuppressed child. J Infect Dis 2003; 188: 954–9. 200. Diaz PS, Au D, Smith S et al. Lack of transmission of the live attenuated varicella vaccine virus to immunocompromised children after immunization of their siblings. Pediatrics 1991; 87: 166–70. 201. Hardy I, Gershon AA, Steinberg SP, LaRussa P. The incidence of zoster after immunization with live attenuated varicella vaccine. A study in children with leukemia. Varicella Vaccine Collaborative Study Group. N Engl J Med 1991; 325: 1545–50. 202. Shiraki K, Matsui S, Aiba N. Susceptibility of Oka varicella vaccine strain to antiviral drugs. Vaccine 1993; 11: 1380–2. 203. Redman RL, Nader S, Zerboni L et al. Early reconstitution of immunity and decreased severity of herpes zoster in bone marrow transplant recipients immunized with inactivated varicella vaccine. J Infect Dis 1997; 176: 578–85. 204. Oxman MN. Vaccination to prevent herpes zoster and postherpetic neuralgia. Hum Vaccin 2007; 3: 64–8. 205. Sperber SJ, Smith BV, Hayden FG. Serologic response and reactogenicity to booster immunization of healthy seropositive adults with live or inactivated varicella vaccine. Antiviral Res 1992; 17: 213–22.

93

Wen-son Hsieh & Richard F. Ambinder

Epstein–Barr Virus Infection

Introduction Epstein–Barr virus-associated lymphoproliferative disorder (EBV-LPD) is an occasionally fatal complication of hematopoietic cell transplantation (HCT). Its incidence is highly dependent on the specific approach to HCT. Innovations in transplantation have often been accompanied by an increased risk of EBV-LPD. In some settings, the risk rises to above 25%. The antiviral agents used to treat other herpesvirus-associated disorders occurring in the transplant setting such as herpes simplex mucositis or cytomegalovirus pneumonia are ineffective in the prevention or treatment of EBV-LPD. However, EBV-LPD can often be prevented or treated. With a focus on minimizing EBV-LPD as new HCT strategies are developed and implemented, and treating EBV-LPD when it occurs, this chapter reviews aspects of EBV biology, EBV-LPD pathogenesis, diagnosis and treatment. In addition, aspects of HCT therapies that specifically target EBV-associated malignancies are considered.

Aspects of EBV biology and pathogenesis EBV infection is ubiquitous in all adult human populations [1–3]. Like other herpesviruses, EBV infection may be either latent or lytic (Fig. 93.1). In contrast to most other human herpesviruses, latency predominates in vitro and in vivo. The virus is transmitted in saliva. The virus infects B cells, establishes latency, and drives these cells to proliferate (Fig. 93.2). The proliferation of latently infected B cells leads to expansion of the pool of latently infected cells. Several percent or more of B lymphocytes may harbor virus. As the cellular immune response to viral antigens is established, the proliferating lymphocytes expressing many antigens are cleared. However, viral DNA persists in a tiny percentage of resting memory B cells. These cells express few if any viral antigens and thus escape immune surveillance. They provide a lifelong reservoir of infection. Reactivation from the latent state with virion production, infection of B cells, and further infected B-cell proliferation is believed to occur intermittently. This reactivation and proliferation is held in check by the enormous T-cell response to EBV maintained in healthy seropositive individuals that often accounts for more than 1% of T cells. The virion is enveloped and carries a double-stranded linear viral genome approximately 172,000 base pairs in length [1]. Following

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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infection, the linear DNA is transported to the nucleus. There, the left and right termini of the linear viral genomes fuse to form closed circular episomes. In cells that proceed to lytic infection, these episomes give rise to linear multigenome-length concatemers that are then cleaved to generate genome-length linear molecules. Generation of these linear molecules requires the viral DNA polymerase and other virus-encoded lytic cycle enzymes (Fig. 93.1). In cells that maintain the genome in a latent state, viral episomes persist. Viral episomes replicate once per cell cycle and maintain stable copy numbers over many generations. The only viral protein required for this process is Epstein-Barr nuclear antigen (EBNA)-1, a sequence-specific DNA-binding protein expressed in all forms of EBV latency. The genome encodes more than 80 open reading frames. Most of these are expressed in lytic infection, some are expressed in latent infection, while others are expressed in both. EBV is a predominantly B-lymphotropic virus, although infection of a variety of other cell types is well recognized. In vitro the virus infects resting naïve or memory B cells and drives the infected cells to continuous proliferation. The process is often referred to as immortalization and the resulting cell lines as lymphoblastoid cell lines (LCLs). Injected into immunodeficient mice, LCLs grow as human B-cell lymphomas. These cells are predominantly latently infected. Neither LCL proliferation nor maintenance of EBV episomes is sensitive to inhibitors of the viral DNA polymerase. Integration of the viral genome into the cellular genome is not a regular feature of EBV persistence or virus-associated immortalization. LCLs express six latency nuclear antigens (EBNAs), three latency membrane proteins (LMPs), and perhaps proteins encoded by the Bam HI A rightward transcripts. In addition, two small but abundant noncoding polymerase III transcripts are expressed (the EBVencoded RNAs). Immortalization is the result of the coordinated expression of five viral genes [1]. During primary EBV infection in the normal host, a marked expansion of T cells occurs [4–7]. CD4+ and CD8+ T-cell responses are mainly directed against lytic antigens in the early stages of infection. Ultimately, a robust cellular immune response to latency antigens emerges as well. However, the B cells that harbor the virus in healthy seropositive individuals express few if any viral proteins and thus escape cellular immune surveillance. T-cell responses to both lytic and latency viral antigens are maintained at high levels indefinitely, presumably reflecting intermittent viral reactivation. Primary infection is usually asymptomatic but may be associated with the syndrome of infectious mononucleosis (IM) characterized by fever, pharyngitis, lymphadenopathy, lymphocytosis, and the presence of heterophile antibodies [8]. The determinants of symptomatology are poorly understood, but older age of the host seems likely to be an important

Epstein–Barr Virus Infection

predisposing factor. Symptomatic infection leaves an “immune scar” [9,10]. Expression of interleukin-15R-alpha is lost in acute IM and remains undetectable thereafter in the whole peripheral T- and natural killer (NK)-cell pool. This deficit correlates with defective interleukin15 responsiveness in vitro and appears to persist indefinitely (up to 14 years) after IM has been documented [9,10]. The physiologic consequences of this functional deficit remain to be defined.

Latent (1–9)

Lytic (~80)

• ZTA • EBER RNAs

• RTA

• EBNA-1

• Early antigens

• LMP-2A, B

• Thymidine kinase

• LMP-1

• Protein kinase

• EBNA-2, 3A, 3B, 3C

• DNA polymerase

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As described above, an important part of the viral lifecycle is to expand the numbers of infected B cells so as to maintain a reservoir of infection for the lifetime of the individual. A rare consequence of this persistence program is transformation to malignancy. In general, the term “EBV associated” is used to refer to tumors in which viral DNA, RNA, and protein are detected in tumor cells. EBV is consistently associated with endemic Burkitt’s lymphoma, nasopharyngeal carcinoma and nasal type NK/T-cell lymphoma, AIDS, primary central nervous system lymphoma, and LPD in transplant recipients [2]. There is also an association with Hodgkin’s lymphoma (HL), with 30–50% of HL in North America and Western Europe harboring virus. Symptomatic primary infection with EBV (IM) appears to predispose specifically to EBV(+) HL developing 6 months to 20 years after primary infection [11,12]. The virus is also associated with a variety of other T- and B-cell lymphoproliferative diseases and with about 10% of gastric carcinomas. Although viral associations with breast cancer and hepatocellular cancer among others have been reported, these findings have not been consistently reproduced and should not be regarded as established associations [13,14].

EBV-LPD in HCT

• Capsid proteins

Fig. 93.1 Latent versus lytic infection. In latent infection, no new virions are being produced. Viral DNA is present as a closed circular episome. Latent gene expression is limited to noncoding Epstein–Barr virus-encoded RNA (EBER) transcripts, Epstein-Barr nuclear antigens (EBNAs) 1–6, and latency membrane proteins (LMPs)-1, -2A, and -2B. In lytic infection, new virions are being produced. The viral DNA is present as a linear form. Approximately viral 80 proteins are expressed. These include transcriptional regulatory proteins (ZTA, RTA), viral kinases responsible for phosphorylating antiviral nucleoside analogues such as aciclovir and ganciclovir (thymidine kinase and protein kinase), the viral DNA polymerase which is necessary for the production of new virion DNA and is inhibited by these agents, and the structural proteins of the viral capsid.

Patients undergoing unmanipulated allogeneic bone marrow transplantation (BMT) have a risk of developing EBV-LPD of approximately 1%, while, at the other extreme, T-cell depletion using antibodies specific for CD2 and CD3 was associated with EBV-LPD in five out of seven patients (71%) [15]. Risk factors in the Center for International Blood and Marrow Transplant Research (CIBMTR) study of more than 18,000 allogeneic BMT recipients showed the cumulative incidence of LPD to be 1% at 10 years [16]. More than 80% of cases occurred in the first year after HCT, with the incidence of LPD highest at 2–3 months. A multivariate analysis identified the risk factors for early disease as shown in Fig. 93.3. The factor that conferred the greatest relative risk was the use of anti-CD3 antibody to treat graft-versus-host disease (GVHD). T-cell depletion was also a risk factor, but the risk associated with various approaches to depletion varied substantially. The use of sheep red blood cell rosetting, anti-T or anti-T and anti-NK monoclonal antibodies was associated with relative risks of greater than 10-fold. The use of methods that resulted in balanced loss of B cells and T cells was not associated with significantly increased risk. The use of lectins, Campath-1H monoclonal antibody or elutriation was not associated with

T cell response anti-CD3 ab TCD antithymocyte globulin unrelated donor >1 HLA mismatch related donor acute GVHD

(Follicular center reaction?)

Resting B cell

Proliferating infected B cells

Resting infected memory B cells

Fig. 93.2 B-cell infection in vivo. Virus infection of resting B cells leads to expression of viral latency genes and proliferation of infected cells. These infected cells may account for several percent of total B cells. Following the development of an adaptive cellular immune response, proliferating infected B cells are eliminated. However, virus persists in resting memory B cells in peripheral blood indefinitely. Viral gene expression is highly restricted in these cells, but viral DNA is present in approximately 1–1000 per million peripheral blood mononuclear cells.

Balanced lymphocyte depletion (Campath, elutration)

Fig. 93.3 Risk factors for Epstein–Barr virus lymphoproliferative disease in the first year after allogeneic marrow transplant. ab, antibody; GVHD, graftversus-host disease; HLA, human leukocyte antigen; TCD, T-cell depletion. (Data from [16].)

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a statistically significant increased relative risk. Smaller studies reinforce these conclusions [15,17,18]. It seems likely that the protective effects of B-cell depletion reflects both decreased numbers of virus-carrying donor lymphocytes and the elimination of the target cell for transformation. Other risk factors identified include the use of unrelated and mismatched donors. In contrast to EBV-LPD in the solid-organ transplant setting, the recipient’s age, EBV seronegativity, and underlying disease are not risk factors for LPD in allogeneic BMT [16]. Cases that occur late after HCT differ in their risk factors, pathology, and EBV association. The only significant risk factor in the IBMTR study was extensive chronic GVHD [16]. These tumors are sometimes of T-cell rather than B-cell origin and are sometimes not EBV associated [19,20]. Late cases of LPD not associated with EBV have also been described in solid-organ transplant recipients [21]. HL occurs at increased frequency following allogeneic BMT. In an IBMTR series, HL occurred about 10-fold less frequently than LPD. This nonetheless indicates a six-fold increased risk of HL in BMT recipients in comparison with the general population, and is similar to the increase in risk in human immunodeficiency virus (HIV)-positive patients [22]. HL is almost always a late event. The median time to developing a tumor is 4.2 years, and it is always more than 2.5 years. It is also virtually always EBV associated, just as it is when it occurs in association with congenital immunodeficiency or HIV infection. Histologically, the mixed-cellularity subtype predominates. As with other late LPDs, T-cell depletion and human leukocyte antigen disparity are not risk factors for HL. The occurrence of severe acute or chronic GVHD was, however, a risk factor. Recipients of allogeneic peripheral blood progenitor cell (PBPC) transplants also are at risk for LPD [23]. The median time to the diagnosis of LPD is similar to that for bone marrow recipients. T-cell depletion of the PBPC product and underlying diagnosis of immune deficiency in the recipient are both identified as risk factors in multivariate analysis. Reduced-intensity transplants have also been associated with EBV-LPD [24,25]. Fludarabine alone may be associated with the development of EBV-LPD [26,27], so the contribution of transplantation to the problem is not yet clear. Since EBV generally does not cross the blood–placenta barrier, cord blood hematopoietic cell (CBHC) transplantation might be anticipated to be associated with a lower incidence of EBV-LPD than transplantation from other donor sources. Alternatively, CBHC transplant recipients might be anticipated to have a higher incidence of lymphoma because they resemble T-cell-depleted bone marrow and blood hematopoietic cell products insofar as they lack EBV-specific cytotoxic T cells. As the incidence of EBV-LPD in CBHC recipients appears not to differ from that in recipients of unmanipulated bone marrow grafts, it may be that these factors balance out [28,29]. An especially high incidence was reported in reduced-intensity unrelated CBHC transplantation recipients that involved antithymocyte globulin in the conditioning regimen [30]. Autologous BMT or PBPC transplantation has also been associated with EBV-LPD, although much less frequently than allogeneic transplantation. T-cell depletion (for the removal of T-cell tumor cells) appears to be the major risk factor, but EBV-LPD has also occurred in association with CD34 cell selection [31–33]. The major determinants of the risk period for LPD are presumed to be immunologic, and several investigators have presented evidence that reconstitution of CD8+ T-cell immunity to EBV generally occurs during the 6-month period following allogeneic BMT [18]. It may occur even more rapidly following autologous PBPC transplantation [34,35]. Hierarchies of cellular responses to classes of EBV antigen (latent versus lytic) and to particular antigens are well recognized [36–38]. Thus different peptide epitopes elicit different magnitudes of T-cell response. Magnitude and patterns of response differ between individuals. In allo-

geneic PBPC transplantation, evidence has been presented that suggests similarities in pattern between patients and their respective donors, suggesting that homeostatic mechanisms in the donor have been recapitulated in the matched sibling recipient [39]. Evidence has been presented suggesting a relationship between EBV CD8+ T-cell frequencies and viral load in peripheral blood mononuclear cells (PBMCs). Whereas EBV reactivation as evidenced by elevated EBV viral loads was not highly predictive of the development of EBV-LPD, impaired EBVspecific T-cell recovery in conjunction with elevated EBV viral load was associated with development of EBV-LPD in five out of five patients [40]. Progressive loss and functional impairment of EBV-specific CD4+ and CD8+ T cells has also been associated with high-risk for developing EBV-associated lymphomas in acquired immune deficiency syndrome patients [41,42]. However, the relative importance of CD8+ and CD4+ responses, responses to latent versus lytic viral antigens expressed on tumor cells, or responses to specific individual viral antigens remains to be determined. Several investigators have presented evidence for a critical role of CD4+ T cells. These may exert direct cytotoxic effects or suppress the outgrowth of EBV-transformed B-cell lines [24,43,44].

Clinical and pathologic aspects LPD most commonly presents early after transplantation and as disseminated disease [16]. Fever, generalized lymphadenopathy, respiratory compromise, and rising liver transaminase levels are typical and have usually been associated with a rapidly progressive multiorgan failure and death. Lesions are nodal and extranodal, frequently involving Waldeyer’s ring, the gastrointestinal tract, the liver, and the central nervous system. Tumors that arise later after transplantation (>1 year) are more commonly localized and often have an indolent course. The definitive diagnosis of EBV-LPD requires biopsy with in situ hybridization or immunohistochemistry to define the viral association and immunohistochemistry or flow cytometry [2]. EBV-encoded RNA in situ hybridization is the most sensitive tool for detecting virus in tumor insofar as these small viral RNAs which do not code for protein are expressed in all EBV-associated tumors. LMP-1 staining is also available in most pathology laboratories but misses the presence of virus in the subset of tumors that do not express this viral antigen. Immunohistochemistry for EBNA-1 would in theory be broadly applicable, but the antibodies available are technically less satisfactory. A variety of morphologic classifications have been suggested [45–49]. Four categories of post-transplant LPD are recognized in the World Health Organization classification: early lesions, polymorphic lesions, monomorphic lesions, and HL (Table 93.1). Monoclonal, oligoclonal, and polyclonal lesions as determined by immunoglobulin gene rearrangements are all recognized. There is no consensus on the prognostic predictive value of either morphology or clonality. However, it is clear that, on occasions, patients with monoclonal disease will have spontaneous remissions and that patients with polyclonal disease may progress despite aggressive therapy including donor lymphocyte infusion (DLI) [23]. Whereas in solid-organ transplant recipients, EBV-LPD most commonly arises in host B cells, in allogeneic HCT recipients EBV-LPD usually arises in donor B lymphocytes (although exceptions associated with autologous recovery are well documented). Lesions are typically CD19+ and CD20+, and often show restricted light chain expression [47,50]. EBV is usually associated with B-cell lymphoproliferative disease in the post-transplant setting, with the exception of lesions arising several years after transplantation. T-cell lymphoma in the post-transplant setting is often EBV associated. EBV detection requires in situ hybridization for the EBV-encoded RNA transcripts or the detection of viral antigens [49]. Because of variability of

Epstein–Barr Virus Infection

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Table 93.1 Epstein–Barr virus lymphoproliferative disorder Classification

Description

Early lesions

Plasmacytic hyperplasia which resembles infectious mononucleosis. Architecture is preserved. Little or no cytologic atypia.

Polymorphic lesions

Polymorphic lesions show loss of architecture. These lesions are heterogeneous in their cell population. Cells may or may not show plasmacytic differentiation. There is a spectrum of cytologic atypia.

Monomorphic lesions

The monomorphic category includes diffuse large B cell lymphoma, Burkitt or Burkitt-like lymphoma, plasma cell myeloma, plasmacytoma and some peripheral T-cell lymphoma. The histologic features are those seen in immunocompetent individuals.

Data from [49].

antigen expression in EBV-LPD, the inability to detect particular viral antigens, such as LMP-1 or EBNA-2, should not be regarded as evidence that the lesion is not virus associated. Some EBV-associated tumors, such as Burkitt’s lymphoma, and a subset of EBV-LPD will not express any viral antigens other than EBNA-1, and reagents to detect EBNA-1 are not widely used because of lack of commercial availability and issues related to cross-reactivity. With the availability of effective interventions for the prevention or treatment of LPD in BMT recipients, early diagnosis has become clinically more important. Whereas serology is useful in the diagnosis of primary EBV infection in the general population, in the post-transplant setting it is difficult to interpret because blood products passively transfer antibodies to the viral capsid antigen and EBNA. Thus, most patients who are seronegative prior to transplant and have seronegative hematopoietic cell donors show antibodies to viral capsid antigen and EBNA within a few days after transplantation if they have been transfused (since most blood products are from EBV-seropositive donors). In addition, the typical serologic changes of IM, i.e. a rise in the immunoglobulin M anti-viral capsid antigen antibody or the appearance of heterophile antibodies, do not generally occur in the BMT setting.

Virologic aspects High-dose therapy destroys the viral B-cell reservoir and, at least in some cases, seems to eliminate the host virus [51]. EBV-seropositive BMT recipients receiving allografts from seronegative donors may become seronegative and, in EBV-seropositive BMT recipients receiving allografts from seropositive donors, the host strain of virus may be replaced by the donor strain. These observations are consistent with the observation that B cells are required for long-term maintenance of infection, as exemplified by the absence of viral infection in patients with X-linked agammaglobulinemia [52]. Viral strain or “biotype” has been studied. The A strain, which is also known as the type 1 virus, is most common in LPD in the transplant setting [53,54]. In vitro and in murine xenograft studies, this strain is more efficient in immortalization and transformation than the less common B strain. Many variations in viral genes have been described, including variants in EBNA-1 and LMP-1. However, there is little evidence for a high-risk donor strain. Expression of the full spectrum of latency viral antigens is often detected in LPD following BMT as it is in LPD following organ transplantation [55]. T-cell products expanded using LCL stimulators are generally expected to target LPD insofar as the viral antigens expressed parallel those expressed in LCLs. Antigen escape by mutation in EBVLPD following transplantation has been documented in some instances [54,56].

Viral copy number and monitoring Polymerase chain reaction quantitation of viral DNA shows promise in facilitating diagnosis and guiding early interventions to prevent or treat EBV-LPD. However, there are, as of yet, no simple universally accepted standards with regards to what should be measured, in whom the measurements should be made, and how the results should be interpreted. Aspects of monitoring are discussed below and in Fig. 93.4. EBV DNA in PBMC Results are variously reported as EBV copies (or genome equivalents) per million PBMCs or per microgram PBMC DNA. There is striking variability in the absolute values in healthy seropositive individuals across studies but general agreement that the number of EBV genomes in PBMCs is increased in association with EBV-LPD [57–62]. Measurement of EBV DNA in donors is not a standard part of donor evaluation. There is no evidence to suggest that a healthy donor with high copy number is at greater risk than a healthy donor with an undetectable copy number. Transplant recipients who have normal or undetectable EBV copy number in PBMCs almost never develop EBV-LPD. High copy number following solid organ or HCT identifies a group of patients at risk for EBV LPD with high sensitivity but limited specificity [63]. It should be noted that copy number will generally be low or undetectable in patients treated with rituximab in the months prior to assay (reflecting depletion of B cells). Copy number is high in HIV patients independent of the presence of EBV-associated or other tumor, independent of CD4 count, and independent of HIV RNA load. EBV DNA in plasma This is reported as EBV copies (or genome equivalents) per millilitre of plasma. EBV DNA is only occasionally detectable in healthy EBVseropositive individuals. It is detected in plasma from patients with nasopharyngeal carcinoma, EBV-associated HL, EBV-associated NK/ T-cell lymphoma, EBV-associated gastric carcinoma, IM, HIV, and chronic active EBV. It is also detected in patients with EBV-LPD (outside the central nervous system) [64–66]. In very high-risk HCT recipients, weekly monitoring has been used to trigger “pre-emptive” therapy with rituximab. Monitoring and pre-emptive therapy were credited with reducing the incidence of EBV-LPD from 49% to 18%. PBMC versus plasma versus whole blood It might be presumed that changes in PBMC parallel changes in cell-free blood (plasma or serum). However, this supposition is clearly wrong with regard to nasopharyngeal carcinoma patients. Monitoring EBV copy number in PBMCs does not distinguish patients from healthy

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T est EBV DNA in PBMC

Comments

viral episomes in lymphocytes EBV DNA in plasma

Values >10,000,000 copies/million PBMC are useful in prompting investigation of a diagnosis of EBV LPD in highrisk settings In patients treated with rituximab in previous several months low or undetectable values should be regarded as of no diagnostic or prognostic value Routine monitoring is only advised in conjunction with HCT procedures with a risk of EBV LPD >5% When PBMC and plasma monitoring are both available, elevated PBMC values without elevated plasma values should be interpreted with great caution Values >1000 copies/mL are useful in prompting investigation of a diagnosis of EBV LPD in high-risk settings

virion DNA

free DNA

Other sources of EBV DNA in plasma include EBVassociated cancers, primary EBV infection (IM), immunocompromise (including HIV) Monitoring may be valuable even in rituximab-treated patients Routine monitoring is only advised in conjunction with HCT procedures with a risk of EBV LPD >5%

controls, whereas monitoring EBV copy number in cell-free blood not only distinguishes patients from healthy controls, but is also arguably more useful than anatomic staging for prognosis [67–70]. The reason for the difference lies in what is being measured. Measurements of EBV in PBMCs generally reflect the presence of EBV in resting B cells. Increased numbers of B cells harboring virus or an increase in the tendency of infected cells to produce virus both manifest as increased copy number in PBMCs. In contrast, measurements in plasma or serum indicate either the presence of virion DNA or DNA released from cells. In immunocompetent patients, except in the setting of a primary EBV infection, free virion DNA is rarely detected. In nasopharyngeal carcinoma patients, the EBV DNA detected in plasma or serum is not virion DNA but is derived from tumor cells that have undergone apoptosis, releasing tumor DNA including viral DNA into the circulation. Thus, local radiation therapy or surgical excision of nasopharyngeal tumors results in no change in DNA in PBMCs (a reservoir of EBV-infected cells unrelated to the tumor) but a very rapid fall in cell-free EBV DNA (EBV tumor DNA). In HCT recipients, EBV DNA in cell-free blood likely reflects the presence of virions and the presence of viral DNA released by dying cells. Measurements in whole blood ignore these distinctions but are often convenient [71,72].

Suggested guidelines The authors’ suggested guidelines are presented in Fig. 93.4. At the authors’ institution (Johns Hopkins Hospital), there is no routine monitoring of EBV copy number in PBMCs or plasma in the absence of clinical signs of EBV LPD. High viral copy number in PBMCs or plasma increases suspicion of EBV LPD but never triggers preemptive treatment.

Treatment A variety of treatments for LPD have been explored [73]. Discontinuation of immunosuppression is sometimes associated with regression of LPD in solid-organ transplant recipients, but it is very rarely effective in BMT recipients. The use of antivirals, such as aciclovir, ganciclovir,

Fig. 93.4 Viral copy number measurements and guidelines for interpretation. Commonly used assays measure Epstein–Barr virus (EBV) DNA in mononuclear cells (top) or in plasma (bottom). In plasma, viral DNA may be packaged as a virion or free (released from cells, commonly tumor cells). Virion and free DNA are not distinguished in routine clinical testing. HCT, hematopoietic cell transplantation; HIV, human immunodeficiency virus; IM, infectious mononucleosis; LPD, lymphoproliferative disorder; PBMC, peripheral blood mononuclear cells.

and their congeners, has been advocated, but there is little evidence to suggest therapeutic efficacy against EBV in any setting. These drugs do not inhibit the proliferation of lymphocytes latently infected by EBV either in vitro or in vivo. Furthermore, high rates of EBV-LPD have been noted in recipients of T-cell-depleted grafts despite the use of these drugs [23,74]. Interferon-alpha has also been used with some success [75]. Cytotoxic chemotherapy has been curative in organ transplant recipients with LPD, but there was no similar track record of success in a series of BMT recipients [76]. In contrast, therapies with antibodies, DLI, or adoptive T-cell transfer of EBV-specific cytotoxic T cells have all yielded good results. Experience with DLI to treat EBV-LPD was first reported from the Memorial Sloan Kettering Cancer Center, where patients with EBVLPD following T-cell depletion, allogeneic marrow transplantation were treated with donor leukocytes (1 × 106 CD3+ cells/kg) [77]. Durable remissions were rapidly achieved (14–30 days after infusion) with only modest GVHD. Since the LPD was of donor origin, the effectiveness of the therapy is presumed to reflect EBV-specific reactivity rather than alloreactivity. DLI has also been used in other settings, but not always successfully. In recipients of mismatched-related transplants, DLI given to prevent primary disease did not prevent late LPD (>15 months) [78]. Similarly, in the allogeneic peripheral blood HCT setting, DLI has been used with mixed results [23]. This use of DLI in the treatment of EBV-LPD after BMT was followed by the demonstration that infusion of EBV-specific T cells can also induce tumor regression or prevent the development of tumors [79]. Expanded EBV-specific donor T-cell lines were prepared by stimulating donor lymphocytes with irradiated donor-derived LCLs. The resultant cell lines were mixes of CD8+ and CD4+ T cells as well as NK cells. Patients responded to 4-weekly T-cell infusions with a return of viral copy numbers to normal values and resolution of fever, lymphadenopathy, and pulmonary infiltrates. High-risk patients were treated prophylactically. These infusions prevented the development of EBV-LPD, restored EBV cellular immune responses, and established populations of cytotoxic T-cell precursors that could respond to in vivo or ex vivo challenge with the virus for as long as 18 months [79,80]. Anti-B-cell antibodies have also been used to treat EBV-LPD [57,81]. These include antibodies with specificity for CD21, CD24, and CD20

Epstein–Barr Virus Infection Table 93.2 Treatment for Epstein–Barr virus (EBV) lymphoproliferative disorder (LPD) and Hodgkin’s lymphoma (HL) Early LPD Recommended Rituximab Donor lymphocyte infusion or EBV T cells from donor if available Other sources of EBV-specific T cells Standard lymphoma chemotherapy Not recommended Ganciclovir

HL

Standard HL chemotherapy

Ganciclovir

(rituximab). Each of the monoclonals has met with substantial success and been associated with very modest or no toxicity. They are effective in many instances, particularly when used early. The complete response rate in solid organ transplant recipients was 44% in a multicenter trial [81]. Rituximab has also proven effective in HCT patients with EBVLPD [82,83], although the number of patients studied remains small. Recommended treatment approaches are summarized in Table 93.2. The ready availability of rituximab and its lack of toxicity suggest that it should be considered appropriate as first-line therapy, or as a part of first-line therapy, in most instances of EBV B-cell LPD. It may also have a role as pre-emptive treatment in high-risk patients, although no consensus has yet emerged in this regard [63,66,84]. EBV-specific T cells, when available, are an attractive alternative or adjunct. Similarly, DLI from allogeneic HCT donors is often effective, even at lymphocyte doses not usually associated with GVHD. For monomorphic lymphoma occurring late after transplant, conventional chemotherapy may be effective. EBV-associated adoptive targeted immunotherapy, when available, is appropriate. For post-transplant HL, the response to conventional chemotherapy is excellent, and most patients in the CIBMTR report were alive and well after therapy [85].

T-cell therapy for EBV-associated tumors The successes in the treatment of EBV-LPD in BMT recipients have stimulated interest in the treatment of other EBV-associated lymphoid tumors. Two issues need to be considered in regard to such immunotherapeutic approaches. The first relates to the ability of tumor cells to present antigens and to be killed by cognate T cells. The second relates to the pattern of viral antigen expression associated with the tumor. Defects in antigen processing, downregulation of major histocompatibility complex (MHC) class I molecules, and downregulation of adhesion molecules are well recognized in Burkitt’s lymphoma. At least some HL tumors are not likely to be susceptible to CD8+-mediated killing because Reed–Sternberg cells often fail to express MHC class I antigens [86]. However, downregulation of MHC class I antigens is almost exclusively a phenomenon of HL not associated with EBV. EBVassociated HL very consistently expresses MHC class I and high levels of transporter-associated proteins (TAP-1 and -2), irrespective of the presence of latent EBV infection. Studies of the HL cell lines support susceptibility to lysis by antigen-specific cytotoxic T-lymphocytes in a class I-restricted manner [87]. In HL, cytokines and chemokines elaborated by tumor cells may lead to dysregulation of cellular immune responses. Much of the T-cell infiltrate in HL consists of regulatory cells [88–90]. These cells suppress interferon-γ4 production by lymphocytes including the CD8+ cells specific for EBV antigens expressed in HL.

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Note that the presence of T-regulatory cells is characteristic of HL and not specifically associated with EBV(+) HL. The spectrum of viral antigen expression is also an obstacle. Tumor types have characteristic patterns of viral gene expression. Several patterns of expression are recognized. Burkitt’s lymphoma expresses only a single viral antigen (EBNA-1) in most tumor cells. HL expresses EBNA-1 and the latency membrane antigens (LMP-1 and -2). Nasopharyngeal carcinoma less consistently expresses the membrane antigens. EBV-LPD in organ transplant recipients sometimes expresses the complete spectrum of latency antigens. The patterns of expression are of key importance because the viral latency antigens (EBNA-3A, -3B, and -3C) most commonly targeted by EBV-specific T cells expanded in vitro with LCLs are not expressed in HL, nasopharyngeal carcinoma, and many other EBV-associated malignancies. Thus, adoptive T-cell therapy using LCL stimulators for T-cell expansion has been explored in a number of small studies of patients with LPD following organ transplantation, nasopharyngeal carcinoma, and HL [91]. In LPD, the tumors are generally sensitive to T-cell killing, and there is likely to be a match between antigens expressed by tumor cells and antigens targeted by expanded T cells. EBV-specific T cells are readily expanded in vitro, an increase in EBV-specific T-cell frequency is detected in vivo, and tumor regression often results [91]. However, the approach is limited by the time required (several weeks) to expand autologous EBV-specific T-cell products. An alternative approach has been to use partially matched products expanded from allogeneic donors [92]. In a phase II study, 21 of 33 patients showed a clinical response as measured by tumor regression (12 complete responders) despite evidence that infused T cells survived for only a few days or weeks. In HL and nasopharyngeal carcinoma, the results with LCL-expanded T-cell products are intriguing but less certain [93–95]. In patients with EBV-associated HL, expansion of LMP-2-specific T cells in vivo and trafficking to tumor sites were seen in some patients, although measurable tumor responses were seen in only three of 11 patients with measurable disease [93]. Similar results were seen in small numbers of patients with metastatic nasopharyngeal carcinoma [94,95]. Promising new approaches to expanding antigen-specific T cells, which will more specifically target viral antigens expressed in a given tumor type, are emerging. These include improved methods in expanding T cells directed toward LMP-1 or LMP-2 [96] and in vitro modification of T cells via transduction with recombinant T-cell receptors [97,98]. Expansion of T cells from allogeneic HCT donors may in some instances allow expansion of CD8 T cells with specificities for EBV-antigens expressed in HL that are absent from expansions of autologous products [96].

Summary EBV infection is ubiquitous. Following primary infection, latency is established in B cells and infection is spread throughout the B-cell compartment by virus-“driven” proliferation of infected B cells. The cellular immune response helps to limit the proliferation of infected cells. A reservoir of resting infected B cells with very limited viral antigen expression eludes immune surveillance. With high-dose conditioning regimens and marrow transplantation, the host reservoir of latently infected B cells may be replaced by infected donor B cells. In the 6-month period following HCT, patients are at risk for developing EBV-LPD. This tumor usually arises in the donor B cells. The major risk factors are T-cell depletion and HCT from unrelated or mismatched donors. Treatment with adoptive cellular immunotherapy, either DLI or EBV-specific T-cell infusion, has been associated with impressive clinical results in a small number of patients, as has treatment with monoclonal antibodies. Adoptive immunotherapy in the setting of HCT may also have a role to play in the management of EBV-associated malignancies such as HL.

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latent membrane antigen-specific CD8+ T-cell function in Hodgkin lymphoma patients. Blood 2006; 108: 2280–9. 91. Savoldo B, Goss JA, Hammer MM et al. Treatment of solid organ transplant recipients with autologous Epstein Barr virus-specific cytotoxic T lymphocytes (CTLs). Blood 2006; 108: 2942–9. 92. Haque T, Wilkie GM, Jones MM et al. Allogeneic cytotoxic T-cell therapy for EBV-positive post transplant lymphoproliferative disease: results of a phase II multicentre clinical trial. Blood 2007; 93: 1123–31. 93. Bollard CM, Aguilar L, Straathof KC et al. Cytotoxic T lymphocyte therapy for Epstein–Barr

virus+ Hodgkin’s disease. J Exp Med 2004; 200: 1623–33. 94. Comoli P, Pedrazzoli P, Maccario R et al. Cell therapy of stage IV nasopharyngeal carcinoma with autologous Epstein–Barr virus-targeted cytotoxic T lymphocytes. J Clin Oncol 2005; 23: 8942– 9. 95. Straathof KC, Bollard CM, Popat U et al. Treatment of nasopharyngeal carcinoma with Epstein– Barr virus–specific T lymphocytes. Blood 2005; 105: 1898–904. 96. Bollard CM, Gottschalk S, Huls MH et al. In vivo expansion of LMP 1- and 2-specific T-cells in a patient who received donor-derived EBV-specific

T-cells after allogeneic stem cell transplantation. Leuk Lymphoma 2006; 47: 837–42. 97. Savoldo B, Rooney CM, Di Stasi A et al. Epstein Barr virus specific cytotoxic T lymphocytes expressing the anti-CD30zeta artificial chimeric Tcell receptor for immunotherapy of Hodgkin disease. Blood 2007; 110: 2620–30. 98. Jurgens LA, Khanna R, Weber J, Orentas RJ. Transduction of primary lymphocytes with EpsteinBarr virus (EBV) latent membrane protein-specific T-cell receptor induces lysis of virus-infected cells: a novel strategy for the treatment of Hodgkin’s disease and nasopharyngeal carcinoma. J Clin Immunol 2006; 26: 22–32.

94

Michael Boeckh

Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation

Introduction Viral infections are major cause of morbidity and mortality after hematopoietic cell transplantation (HCT). There has been significant progress in the management of herpes simplex virus, varicella zoster virus, and cytomegalovirus (CMV) after transplantation, but, at the same time, other viruses have gained importance. Herpesviruses 1–5 (herpes simplex virus-1 and -2, varicella zoster virus, CMV, and EBV) are reviewed in Chapters 90–93. This chapter will review the significance of adenoviruses, herpesviruses 6–8, community respiratory viruses, papovaviruses (papillomavirus and polyomaviruses), parvovirus B19, enteroviruses, rotavirus, and Norovirus in the HCT setting. The term “infection” generally refers to asymptomatic shedding or viremia, while “disease” refers to end-organ symptomatic manifestations of the infection, which require detection of the virus in the particular organ. Adenovirus Virology Adenovirus is a nonenveloped DNA virus that replicates and assembles in the nucleus. Virions are released when the cell is lysed. Adenovirus causes both lytic and latent infection [1]. Infection may persist from days to years, and may occur in host tissue (e.g. adenoidal) without causing apparent symptoms in immunocompetent hosts. All adenoviruses share a common group-specific complementfixing antigen. Adenoviruses are categorized in subgroups A to F, and there are presently 51 human serotypes known, although only approximately half have been implicated in human disease. Serotypes from all six subgroups can cause disease in both immunocompetent and immunocompromised subjects, although the disease spectrum associated with strains may differ [2,3]. After primary infection, adenovirus establishes latency in adenoidal tissues [3], with lifelong persistence of specific antibodies. Pathogenesis and immunity Transmission of adenovirus occurs by either respiratory droplet or the oral–fecal route. Adenovirus enters the mucosa and infects epithelial cells, resulting in inflammation and necrosis. Subsequent viremia may lead to infection of kidney, bladder, liver, and lungs in HCT recipients;

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

central nervous system (CNS) disease is rare. After HCT, reactivation from latency is probably more important than exogenous infection [2]. Epidemiology and clinical manifestations Primary infection is usually established in early life with either asymptomatic or symptomatic infection. By age 5, most people have been infected by one or more serotypes of adenovirus. Adenovirus infections are common after HCT [2,4,5], and some recent reports suggest that they may be increasing. This increase may be related to transplantation practices (e.g. T-cell depletion) [6]. However, the incidence of end-organ adenovirus disease is dependent on the degree of immunosuppression and seems to be increasing only in recipients of T-cell-depleted grafts [6]. Clinical manifestations include pneumonia, hepatitis, gastrointestinal disease, nephritis, cystitis, and eye infections [6,7]. Risk factors for adenovirus infection after HCT include graft-versushost diseases, HCT from unrelated donors, use of total body irradiation, T-cell depletion, and younger age [4,6–8]. The degree of T-cell depletion and post-transplant immunosuppression directed at T-cell function seems to be important. For example, transplant protocols employed with haploidentical transplantation are associated with a particularly high incidence of adenovirus infection and invasive disease [9]. Almost all studies report a higher incidence in children [10–12]. In recipients of non-T-cell-depleted grafts, adenovirus disease is rare and seems to be restricted to allogeneic transplant recipients [2]. However, cases of disease in reduced-intensity regimens that include T-cell depletion have been reported [8]. Risk factors for adenovirus disease include younger age [12], viremia and shedding from more than two sites [12–14], total body irradiation [15], GVHD [6], and transplantation from an unrelated donor [13]. High-level plasma viremia (>1000 copies/mL) is a marker for invasive infection in recipients of non-T-celldepleted grafts [16]. Adenovirus disease is associated with a fatality rate of 30–50% [2,6]. Response to treatment seems to be particularly poor in patients with pneumonia or disseminated disease [17]. A recent multivariable analysis in recipients of T-cell-replete grafts indicated that adenovirus infection is independently associated with mortality [2]. Diagnostic techniques Direct detection methods are necessary to establish the diagnosis of adenovirus infection and disease in HCT. As with other viral infections in HCT recipients, serology is not recommended. Adenovirus can be grown in cell culture. Shell vial assays and direct antigen-detection methods are used routinely in clinical practice. For detection of adeno-

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virus in stool, enzyme immunoassays or the polymerase chain reaction (PCR) is required because enteropathogenic serotypes 40 and 41 usually do not grow in culture. Quantitative PCR assays are now routinely used to diagnose adenovirus viremia [14,18–20]. For diagnosis of adenovirus disease, detection of the virus in bronchoalveolar lavage (BAL) or tissue samples is required, using rapid culture techniques or direct fluorescent antibody testing on BAL, and immunohistochemistry or in situ hybridization techniques for tissue samples. The role of quantitative PCR for BAL and tissue is currently not defined. Prevention and treatment There are no controlled treatment studies for adenovirus infection in the immunocompromised host. Intravenous ribavirin has been used, but results are conflicting [21–24]. Recently, cidofovir has been shown to have in vitro activity, and several uncontrolled case series have shown promising results [19,23,25–30]. Ganciclovir has in vitro activity against adenovirus, and has a moderate effect in prevention of adenovirus infection in non-T-cell-depleted patients [10]. However, for treatment of adenovirus disease, ganciclovir is not recommended. The antiretroviral drugs zalcitabine (DDC), alovudine, and stavudine have activity in vitro [31,32]. Differences in the results of treatment studies in terms of in vitro susceptibility and outcome may be due to strain and serotype differences, which often have not been reported in these studies. Specific and non-specific T-cell therapy have been reported in small series [33–35]. In high-risk settings such as haploidentical transplantation or cord blood transplantation [18,36], weekly PCR surveillance for adenovirus viremia and pre-emptive treatment with cidofovir is now used at several centers [9,29,30]. However, this strategy does not completely eliminate adenovirus disease in the highest-risk patients [29]. It has also been suggested that adenovirus DNA in stool may be predictive for disease in highest-risk patients [37], which might represent an earlier opportunity to intervene. Adenovirus-specific T cells therapy is an area of active research [35]. Prospective trials are needed to evaluate prevention strategies.

Respiratory viruses Community-acquired respiratory virus infections are an important cause of morbidity and mortality after HCT [38,39]. The best studied viruses include respiratory syncytial virus (RSV), parainfluenza viruses, and influenza viruses. Less information is available on rhinoviruses and coronaviruses, human metapneumovirus (HMPV), and the recently described human bocavirus (HBoV). The infection epidemiology in HCT recipients usually parallels that observed in the community. This

phenomenon is due to the fact that these viruses circulate in immunocompetent individuals (including health-care personnel and family members). A seasonal distribution with a peak during the winter and spring months is well described for RSV, influenza viruses, and MPV; the other respiratory viruses may show different patterns of seasonality. The most significant impact on morbidity and mortality after HCT has been reported from RSV, parainfluenza viruses, and influenza viruses [39–42]. The significance of the emerging respiratory viruses has not been fully examined. Molecular detection techniques are rapidly replacing traditional methods [43–45]. One feature of respiratory virus infections is that the clinical signs and symptoms at presentation are not specific. This syndromic nature of illness requires multiplex testing platforms [46–48]. Specimen handling is important for maximal diagnostic yield. Nasal wash or swab specimens should be placed on ice or in the refrigerator immediately and transported to the laboratory without delay [49]. For nonmolecular methods, specimen set-up in the laboratory should occur within 2–4 hours. Nonmolecular methods available for testing include standard viral cultures (results available in several days), shell vial centrifugation cultures using specific monoclonal antibodies (results after 1–3 days), direct fluorescent antibody tests (2 hours), and enzyme immunoassays (2 hours). On tissue sections from lung biopsy or autopsy specimens, virus-specific monoclonal antibody staining, viral cultures, or PCR can be used. Respiratory syncytial virus Clinical significance RSV is an RNA virus (paramyxovirus) that causes a wide spectrum of respiratory diseases. These diseases range from life-threatening bronchiolitis in infants and potentially fatal pneumonia in transplant recipients to a mild upper respiratory infection (URI) in immunocompetent adults and older children. The virus is increasingly implicated in a number of respiratory illnesses in immunocompetent or mildly immunosuppressed individuals, such as otitis media, exacerbation of chronic obstructive lung disease, and community-acquired pneumonia. After HCT, and also in severely immunosuppressed nontransplant patients with hematologic malignancies [50], RSV causes upper respiratory tract infection which may progress to fatal pneumonia [51]. Recently, RSV has been linked to late airflow obstruction, a debilitating condition of accelerated loss of lung function after HCT [52]. During the respiratory virus season, the incidence may be as high as 10%, and both allogeneic and autologous transplant recipients can be infected (Table 94.1 and Fig. 94.1) [39,53]. Most of the infections occur during the first 2 months after HCT, but late cases have also been reported [54].

Table 94.1 Respiratory virus infections after hematopoietic cell transplantation: comparison of respiratory syncytial virus, parainfluenza virus-3, and influenza viruses

Virus

Incidence of infection

Progression from URI to pneumonia

Time from URI to pneumonia (median)

Proportion of pneumonia without URI

Pulmonary copathogens in cases with pneumonia

Overall mortality at 1 month after diagnosis of pneumonia

Respiratory syncytial virus Parainfluenza virus 3 Influenza viruses A and B

1.8–6%* 4–7% 1.3–2.6%†

40% 18–44% 18%

7 days 7 days 11 days

20–50% 31% 18%

2.5–33% 53% 50%

45% 35–37% 25–28%

URI, upper respiratory infection. * During the winter season, the incidence may be as high as 10%. † May be significantly higher during outbreaks [217]. Data from [38,39,53,75,76,84].

Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation

Risk factors In a large study, winter season, male gender, and use of bone marrow as stem cell source were identified for the acquisition of RSV in HCT recipients [39]. URI precedes pneumonia in 80% of patients, and approximately 30–50% of patients with RSV URI progress to pneumonia after a median of 7 days. The strongest risk factors for progression to pneumonia are older age and lymphopenia [39]. Patients receiving nonmyeloablative conditioning regimens seem to be largely protected

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from progression to lower respiratory tract disease during the first 3 months after HCT [55]. Following reduced-intensity conditioning regimens, progression to lower tract disease has been described, but fatal disease may be less common [56]; however, larger studies are needed to better define the risk of progression and outcome of disease. One study in pediatric HCT recipients indicates that asymptomatic shedding of RSV can occur [57]. Two studies in adult HCT recipients, one of which used weekly PCR-based surveillance, did not detect asymptomatic shedding [45,58]. Treatment and prevention

Fig. 94.1 Time to first respiratory virus infection after hematopoietic cell transplantation. MPV, metapneumonia virus; PIV, parainfluenza virus; RSV, respiratory syncytial virus. (Reproduced from [45], with permission.)

Without treatment, RSV pneumonia is almost uniformly fatal in highly immunosuppressed HCT recipients [51]. However, recovery from RSV lower respiratory tract disease without specific treatment has been reported [59]. These differences are likely due to less intense immunosuppressive regimens. Pulmonary co-pathogens are detected in up to one-third of the patients with RSV pneumonia and require aggressive treatment. No adequately powered controlled trials exist for the treatment of RSV infection and pneumonia in the HCT setting. Available evidence comes from small uncontrolled cohort studies and one small randomized trial [60]. The data suggest that treatment of early pneumonia (i.e. prior to mechanical ventilation) is associated with improved outcome (Table 94.2). Intermittent short duration (2 g over 2 hours three times per day) or continuous aerosolized ribavirin is considered the treatment of choice for RSV pneumonia. With this regimen, the 30-day all-cause mortality is approximately 40% (Table 94.2). In a large multivariable analysis, RSV associated pneumonia (but not URI) was independently associated with mortality [39]. Systemic ribavirin alone does not seem to be effective for the treatment of pneumonia [61,62]; better response rates are reported for treatment of RSV URI, but small sample sizes limit the

Table 94.2 Outcome of radiographically and virologically documented respiratory syncytial virus (RSV) pneumonia with antiviral therapy (only series with five or more patients considered)

Strategy, authors

Reference

Number of patients

Aerosolized ribavirin alone Harrington et al., 1992 Ljungman, 2001 Nichols et al., 2001

[51] [42] [39]

13 9 5

Survival

Respiratory failure at start of treatment

22% 66% 60%

NR NR NR

Aerosolized ribavirin + pooled intravenous immunoglobulin [218] 12 58% Whimbey et al., 1995 [219] 6 66% Ghosh et al., 2000 Ljungman, 2001 [42] 5 60% [39] 19 68% Nichols et al., 2002

Survival 78% if therapy was started before respiratory failure; 0% if after respiratory failure Survival 80% if therapy was started before respiratory failure; 0% if after respiratory failure NR NR

Aerosolized ribavirin + palivizumab [64] Boeckh et al., 2001

None of the patients had respiratory failure at the start of treatment

12

83%

Aerosolized ribavarin + RSV-specific immunoglobulin 7* 86% De Vincenzo et al., 2000 [65] [53] 11 91% Small et al., 2002

None of the patients had respiratory failure at the start of treatment Including two patients with respiratory failure at the start of treatment; both survived†

Intravenous ribavirin alone Lewinsohn et al., 1996

[61]

10

20%

Survival 40% if therapy was started before respiratory failure; 0% if after respiratory failure

Intravenous ribavirin + aerosolized ribavirin Ljungman, 2001 [42] 5

40%

NR

NR, not reported. * Only patients with radiologic signs of lower tract involvement considered. † T. Small, personal communication, 2003.

Chapter 94

strength of the data [63]. It has not been studied whether combined oral and aerosolized ribavirin is more effective than using aerosolized ribavirin alone. The role of concomitant intravenous immunoglobulin or RSV-specific immunoglobulin or palivizumab, an RSV-specific monoclonal antibody directed at the F protein of RSV, remains poorly defined. Uncontrolled data suggest that high-titer antibody preparations or palivizumab may be required if such adjunctive therapy is given [53,64,65]. However, this issue has not been studied in a controlled fashion. Outcome results of recent series are depicted in Table 94.2. There are several factors that may account for differences in outcome in the available cohort studies. Perhaps the most important factor seems to be the timing of initiation of therapy. Several studies suggest that treatment that is started after respiratory failure has occurred is almost uniformly unsuccessful (Table 94.2). Other factors that may explain the differences in outcome between studies include the presence of lymphopenia at the time of diagnosis, the presence of co-pathogens (e.g. invasive molds and CMV), and the use of immunosuppressive agents. Recent data suggest that lymphocyte, rather than neutrophil, engraftment is an important risk factor for progression and outcome in HCT recipients [38,42,66]. Available studies are too small to control for these parameters, making the interpretation of results of pharmacologic interventions difficult. Most centers now agree that aerosolized ribavirin should be given to HCT patients with RSV lower respiratory tract disease. There is no consensus on the role of antibody preparations. Due to the high mortality of RSV pneumonia, much interest has focused on prevention. Possible strategies are similar to those employed for prevention of CMV. While it is well established that RSV-associated URI precedes pneumonia in the majority of cases, limited information is available on the extent of asymptomatic shedding of respiratory viruses in HCT recipients [57,58]. A recent small controlled study of pre-emptive antiviral therapy based on RSV-associated URI showed a decline in viral load but no significant difference in RSV pneumonia; the study was stopped prematurely due to slow accrual [60]. A retrospective analysis of this strategy carried out at the M.D. Anderson Cancer Center, suggests a reduced risk of progression to lower tract disease [38]. Whether aerosolized ribavirin is indicated for RSV-associated URI remains controversial. Some centers administer it in the presence of lymphopenia (less than 300 per mm3) when the risk of progression is particularly high [38,66]. A nonrandomized study suggests that palivizumab given pre-emptively to patients with RSV-associated URI does not prevent progression to pneumonia [67]. Prophylactic measures recommended throughout the respiratory virus season include isolation of infected patients, hand washing prior to every patient contact, educational efforts targeted at health-care personnel and family members, restricting patient contact with health-care personnel and family members with URI symptoms [66], as well as influenza vaccination of health-care personnel and family members [68,69]. Whether pharmacologic prophylaxis (e.g. palivizumab or RSV immunoglobulin) throughout the respiratory virus season is effective in preventing infection and disease in HCT recipients has not been studied. For pretransplant infections with RSV, parainfluenza virus or influenza virus, most transplant centers postpone transplantation until resolution and cessation of viral shedding occurs, especially when a high-dose allogeneic HCT is planned. Indeed, Centers for Disease Control (CDC) guidelines suggest postponing transplantation in all patients with URI symptoms. This approach is supported by a recent study of pretransplant RSV infection, which showed rapid progression to RSV pneumonia in patients where the transplant procedure was not postponed [70]. However, there is some evidence that low-risk autologous transplant patients may be transplanted without adverse outcome [71]. In addition to this, the outcome of respiratory virus infections appears to be less

severe with nonmyeloablative conditioning regimens [55]. Thus, delaying the transplant procedures based on URI symptoms is recommended if patients are to undergo transplants after high-dose conditioning, but less is known in the setting of reduced-intensity conditioning. Use of prophylaxis (e.g. RSV immunoglobulin or palivizumab) during transplantation in patients with pretransplant respiratory virus infections has not been studied. Parainfluenza viruses Clinical significance Parainfluenza, an enveloped paramyxovirus containing single-stranded RNA, is classified into four serotypes. Of the four types of parainfluenza virus, parainfluenza virus-3 is most common (approximately 90%), followed by serotypes 1 and 2. The incubation time is 1–4 days. Parainfluenza virus infections remain detectable throughout the summer season (Fig. 94.2). The incidence of 7% in two studies (using nonmolecular detection methods) is higher than that reported for RSV (4%) [72,73].

60 RSV 50 40 30 20 10 0 60 Influenzavirus A and B 50 Number of cases

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40 30 20 10 0 60 Parainfluenzavirus 3 50 40 30 20 10 0 J

F

M

A

M

J

J

A

S

O

N

D

Month

Fig. 94.2 Monthly distribution of respiratory syncytial virus (RSV), parainfluenza virus-3, and influenza infections after hematopoietic stem cell transplant in the 1990s at the Fred Hutchinson Cancer Research Center. (Data from [39].)

Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation

Risk factors Only HCT from an unrelated donor has been identified as a risk factor for the acquisition of the parainfluenza virus [73]. In Tcell-depleted patients, CD4 lymphopenia has been reported to increase the risk of all respiratory virus infections, including parainfluenza virus infections [56]. Similar to RSV, URI is the predominant clinical presentation. Progression to pneumonia seems to be less common than with RSV (Table 94.1) [73]. Late airflow obstruction has been strongly linked to parainfluenza virus pneumonia (adjusted odds ratio 18), and to a lesser degree to URI [52]. The most important risk factors for the progression from URI to pneumonia are use of systemic corticosteroids and lymphopenia [56,73]. Recipients of nonmyeloablative conditioning regimens (defined as lowdose fludarabine and low-dose total body irradiation) appear to have a small risk of progression during the first 3 months after HCT [55]; progression may still occur with reduced-intensity regimens containing anti-T-cell antibodies, but larger studies are needed to confirm this [56]. Although the overall progression rate to pneumonia is only 18%, in allograft recipients receiving more than 1 mg/kg of prednisone, the risk is 40%, and 65% with 2 mg/kg [73]. Parainfluenza virus 3 pneumonia may also occur after autologous HCT, but it mainly arises in the setting of CD34 selection or use of high-dose steroids [73]. Parainfluenza virus 3 pneumonia is often associated with serious pulmonary co-pathogens (53%), such as Aspergillus fumigatus. Factors associated with poor outcome after pneumonia include the presence of co-pathogens and mechanical ventilation [73]. Thus, aggressive diagnostic intervention (i.e. BAL) and therapy are indicated in patients with suspected parainfluenza virus pneumonia. In a large retrospective analysis, both URI and pneumonia due to parainfluenza virus 3 were associated with overall mortality in multivariable models [73]. Treatment and prevention The mortality of pneumonia is approximately 35% in recipients of allografts following high-dose conditioning [40,74,75]. Outcome may be improved in reduced-intensity conditioning regimens [56]. In a retrospective analysis, neither aerosolized ribavirin nor intravenous immunoglobulin led to an improved outcome of pneumonia or a reduction in viral shedding following pneumonia [74]. Systemic ribavirin has only been reported in case reports [76]. Randomized treatment studies have not been performed. Whether earlier antiviral treatment (i.e. pre-emptive treatment for URI) is effective in the prevention of pneumonia or late airflow obstruction is unknown [76]. The association of high-dose steroid treatment with progression to disease might suggest that a reduction of immunosuppression may be useful [73]. However, such an approach has not been tested. The role of immunoglobulin for treatment is also poorly defined. One retrospective analysis did not find a benefit of pooled immunoglobulin given for parainfluenza virus pneumonia [73]. However, the antibody content of pooled preparations is highly variable. The highest titers can be obtained from RSV-specific immunoglobulin, which also contains a high titer of antibodies directed against parainfluenza virus [77]; RSVspecific immunoglobulin is not available in the United States. Infection control is the mainstay of prevention strategies. Unfortunately, current infection control practices seem to be far from perfect in keeping parainfluenza virus out of HCT units, as indicated by the high incidence figures (Table 94.2). HCT units have been prone to persistent parainfluenza virus outbreaks [44,78–80]. Possible explanations for the difficulty in preventing parainfluenza virus from entering HCT units include lack of a vaccine for health-care workers and close contacts, very mild or a lack of symptoms in immunocompetent individuals,

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prolonged asymptomatic shedding in infected patients, and persistence of the virus on environmental surfaces [45]. Influenza viruses Clinical significance Influenza viruses belong to the family of orthomyxoviruses and are enveloped, single-stranded, pleomorphic RNA viruses. Influenza is classified into three major types, of which type A is most common, followed by type B [41,81,82]. Influenza type C is very uncommon, even in the immunocompetent population; there are no reports of influenza C in HCT recipients. Influenza virus infections seem to be less common than RSV and parainfluenza virus infections. Both subtypes can cause infection, although type A appears to be more common. Clinical characteristics are listed in Table 94.1. Risk factors Acquisition of influenza is increased in patients with high-risk underlying disease [83,84]. Progression to severe pneumonia can occur similar to RSV and parainfluenza virus [82], but progression appears to be less common and risk factors for progression differ (Table 94.2). In contrast to parainfluenza virus infection, where corticosteroids are an important risk factor for the development of pneumonia, with influenza virus, lower tract infection appears to be less common in patients treated with corticosteroids [83,84,85]. Also, recipients of low-dose conditioning appear to be less likely to develop pneumonia during the first 3 months after HCT [55]. Interestingly, the clinical presentation in HCT often lacks myalgia and high fever, which is commonly seen in immunocompetent individuals (Fig. 94.3) [45]. Treatment and prevention Effective prevention is available for influenza, which may explain the lower incidence. Health-care personnel, family members, and visitors are advised to be vaccinated against influenza early in the season. Antiviral therapy is available for influenza virus infection; however, available agents have not been studied prospectively in HCT recipients. Due to the recent emergence of resistance against the M2-inhibitors amantadine and rimantidine, with a more favorable side-effect profile, these agents should not be used to treat influenza A [86]. Neuraminidase inhibitors (i.e. zanamavir and oseltamivir) are now available [87,88] and are active against most strains of influenza A and B. Uncontrolled studies suggest that pre-emptive therapy with neuraminidase inhibitors (i.e. oseltamivir and zanamivir) may be effective in preventing progression to lower tract disease [38,83,84,89]. Widespread chemoprophylaxis for susceptible immunosuppressed patients in outbreak situations has been recommended [90], and a case-control study of oseltamivir suggests that this approach is safe [91].

Other community respiratory viruses Human rhinovirus Human rhinovirus (HRhV) is classified as a picornavirus characterized by small, naked, single-stranded RNA. There are approximately 100 serotypes, which usually cause mild upper respiratory tract symptoms in immunocompetent hosts. Using culture techniques, rhinovirus is infrequently reported as a cause of respiratory infection in HCT recipients. However, several reports found the organism in patients with pneumonia. Ghosh et al. reported lower tract disease in seven of 22 patients with HRhV infection [92], while Bowden documented lower tract disease in only one of 29 infected patients [82]. HRhV appears to occasionally cause lower respiratory tract disease [93], although a clear

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Chapter 94

RSV

PIV

MPV

Influenza

Negative samples

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 Runny nose

Cough

Watery eyes

Sore throat

Shortness Sputum of breath

Wheezing

Sinus Stuffy Sinus congestion headache nose

Muscle aches

Fever >38ºC

Fig. 94.3 Symptoms associated with respiratory virus infection after hematopoietic cell transplantation. The y-axis shows the fraction of patients showing each symptom. MPV, metapneumonia virus; PIV, parainfluenza virus; RSV, respiratory syncytial virus. (Reproduced from [45], with permission.)

causative role is sometimes difficult to ascertain because of the presence of co-pathogens [82,92,94]. Surveillance studies using reverse transcriptase PCR are ongoing to more precisely define the role of HRhV in HCT recipients. There are presently no drugs or vaccines available for the treatment or prevention of rhinovirus infections. Pleconaril, a capsid-binding compound, was the first anti-HRhV antiviral to be submitted to the United States Food and Drug Administration for regulatory approval, but was not approved for licensure for the treatment of colds in adults, primarily based on drug interactions and modest treatment effects. Other antiviral agents, including protease inhibitors, are now being studied. Human coronaviruses Human coronaviruses (HCoVs) are RNA viruses that can cause URI, croup, wheezing, and pneumonia in immunocompetent individuals. Recently, several new strains have been described, including NL63 (identified in 2004) and HKU1 (identified in 2005), in addition to the previously described OC43 and 229E strains [95]. In a recent study, 5.7% of 823 patients admitted to the hospital had HCoV detected. HCoV infection occurred in 8.8% of immunocompromised patients compared with 4.5% of immunocompetent patients. Fourteen (30%) of the 47 HCoV-infected patients, including three HCT recipients, were also infected with other pathogens (RSV and Aspergillus; influenza A; RSV and HRhV, respectively), and these patients had more severe diseases [96]. The four HCoVs have also been detected in solid organ transplant recipients, patients with underlying malignancies, and HIV-infected individuals [97]. HCoV infections have also been associated with longterm decline in forced expiratory volume in 1 second in lung transplant recipients [97]. Surveillance studies are ongoing to better define the incidence and pathogenesis of HCoV in HCT recipients. Currently, there is no specific treatment. Measles virus Measles virus (rubeola), a paramyxovirus, is a recognized cause of giant cell pneumonia, developing with and without rash, and severe CNS disease in immunocompromised hosts, including HCT recipients [98,99]. The incidence of measles is extremely low at most centers due to the widespread use of measles immunization in the general population. However, recently an outbreak among HCT recipients has been reported [100]. During that outbreak, among 122 susceptible patients, clinically

severe disease occurred in one patient, and seven had mild symptoms. Exanthema was present in all of these patients, although it was often atypical. Koplik spots were observed in five patients. All but one patient had fever and nonproductive cough [100]. The use of ribavirin has been reported for measles in two critically ill immunocompromised patients, but no systematic assessment of this intervention can be made [101]. Human metapneumovirus HMPV is a newly discovered negative-sense nonsegmented RNA paramyxovirus [102]. By age 5, virtually all children are seropositive. The virus can cause upper and lower tract infection during the winter season [103]. Serious lower respiratory tract disease associated with HMPV has been reported in immunocompromised patients. HMPV infection occurs in up to 5% of HCT recipients [45]. Whether asymptomatic shedding occurs is controversial [45,104]. The progression rate to lower respiratory tract disease is presently unknown. Among HCT recipients undergoing BAL for clinical and radiographic pneumonia, 3–4% had HMPV detected [105]. Most of these patients were clinically classified as having idiopathic pneumonia syndrome [105]. HMPV disease in HCT recipients immediately post transplant typically presents with upper respiratory symptoms, including fever, nasal congestion, and cough. Once pneumonia develops, rapidly progressive pulmonary infiltrates can occur. This is frequently accompanied by hypotension, septic shock or both [105]. Radiographic findings range from diffuse bilateral alveolar and interstitial infiltrates to emphysema without infiltrate. Histologic assessment has shown that diffuse alveolar damage with hyaline membrane formation, foci of bronchiolitis obliterans and organizing pneumonia, and diffuse alveolar hemorrhage are common [105]. There is no proven treatment for HMPV disease, which may be fatal in HCT recipients [105]. Although intravenous immunoglobulin has been shown to effectively neutralize HMPV in vitro, its effect in vivo is unknown [106]. The role of steroids is also not defined. Ribavirin has been shown to have in vitro antiviral activity against HMPV [106], but there are no reports in regards to treatment with ribavirin. Human bocavirus HBoV is a newly identified human parvovirus originally identified by random PCR amplification/cloning technique from hospitalized children

Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation

in Sweden [107]. HBoV has been detected worldwide [108–110], and appears to be common in young children. Symptoms associated with HBoV infections include rhinorrhea, cough, fever, wheezing, hypoxia, and diarrhea. Viremia is common [111]. To date there is only limited information on the clinical significance of HBoV infection in immunocompromised patients [112]. Infection control practices Recommendations have been summarized by the CDC/American Society for Blood and Marrow Transplantation [90]. Presently, the guidelines are undergoing revision. Hand washing is the single most effective way of preventing the spread of respiratory viruses and should be performed after each patient contact by all health-care personnel and visitors. Respiratory isolation is used for HCT recipients with respiratory virus infections (masks, gowns, gloves, and eye protection) [113]. The use of masks among health-care workers, family members, and asymptomatic patients remains controversial. One nonrandomized study suggests a benefit of masks [68]. However, questions remain on the type of mask, the frequency of changing the mask, and who should wear masks. Not all transplant centers have a universal mask policy. Recent CDC guidelines recommend using masks during patient transport [90]. Another approach that is likely to reduce transmission is to restrict health-care workers and family members from patient contact if they have a URI and systemic symptoms such as rhinorrhea, watery eyes, sneezing, fever, and myalgia [39,70]. While this is not uniformly done in adult patients, most centers restrict small children from direct patient contact during the respiratory virus season due to their predisposition to URIs and prolonged high-titer shedding [90]. A strategy that restricts access of personnel and other close contacts will only be effective for infections that present with significant drainage and symptoms, such as RSV, rhinovirus, HMPV, and influenza infections. Infections which may present with only mild symptoms (e.g. parainfluenza virus infections) may be missed by this approach [44,45].

Human herpesviruses-6, -7, and -8 Human herpesvirus-6

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Whether monitoring for HHV-6 viremia is useful in predicting these clinical syndromes is not known. In solid organ transplant recipients, there seems to be an interaction between HHV-6 and CMV [132–137]. One study showed a trend toward more CMV infection among patients with over 100 copies of HHV-6 DNA/5 × 104 lymphocytes in peripheral blood lymphocytes [117], and another study suggests interaction of HHV-6 and CMV in BAL fluid of HCT recipients [127]. Data in HCT recipients are limited and results are inconclusive. HHV-6 reactivation typically occurs 2–4 weeks after HCT [116,122–124], preceding CMV reactivation, which occurs a median of 40–42 days following transplant [138]. An interaction between HHV-6 and CMV may be explained by a generalized immunosuppressive effect of HHV-6 [139–142]. Early HHV-6 reactivation leads to a delay in CMV-specific T helper immune responses in HCT recipients [143], but the exact mechanism of the interaction remains unclear. Treatment and prevention Currently, there is no widely accepted clinical approach to HHV-6 screening or prevention following HCT. In vitro studies suggest that HHV-6 is susceptible to foscarnet, ganciclovir, and cidofovir, and moderately susceptible to acyclovir [144–146]. Foscarnet and ganciclovir have been used to treat HHV-6-associated disease, and a decline in viral load has been documented with therapy [147,148]. The high risk of HHV-6 disease in cord blood transplant recipients may justify PCR surveillance and pre-emptive therapy [131]; however, no randomized trials or systematic evaluations have been reported for prophylaxis or pre-emptive therapy strategies. Human herpesvirus-7 HHV-7 is a β herpesvirus (similar to HHV-6 and CMV) and can be detected by PCR [149]. One study found both HHV-6 and HHV-7 in peripheral blood and marrow of healthy subjects [150]. The impact of HHV-7 after HCT has been examined in longitudinal studies [114,125]. So far, it has not been possible to assign a clinical syndrome to HHV-7 – with the exception of one recent report of a CNS infection with myelitis in an unrelated donor transplant recipient [151].

Clinical significance Human herpesvirus-6 (HHV-6) is a β herpesvirus with similarities to CMV and HHV-7. Several longitudinal studies have examined the role of HHV-6 after HCT [114–117]. Similar to other HHVs, it is ubiquitous, infecting most people by 2 years of age [118]. After primary infection, the virus persists in lymphocytes and salivary glands, with 95% of adults having detectable viral DNA in their peripheral blood mononuclear cells and saliva [119]. During times of immunosuppression, the virus may reactivate and cause disease. Type B is far more common than type A. Rarely, HHV-6 DNA is integrated into the human DNA [120]. This may lead to continued detection of the virus in the blood after HCT [120,121]. It is estimated that this occurs in approximately 1% of patients [120]; detection of integrated DNA does not require treatment. Active HHV-6 infection has been documented in 38–46% of patients following HCT [116,122–125]. Clinical signs and symptoms that have been associated with HHV-6 include rash, a CNS syndrome consisting of encephalitis and impaired memory [126], interstitial lung disease [119,127], and delayed platelet engraftment [117]. Secondary graft failure has been documented in case reports [128]. Early nonrelapse mortality has also been associated with HHV-6 in one small series [129]. The best evidence exists for encephalitis. HHV-6 encephalitis appears to be particularly common in cord blood transplant recipients [130,131]. HHV-6-associated encephalitis is diagnosed by detection of HHV-6 DNA in the cerebrospinal fluid and compatible clinical symptoms [126].

Human herpesvirus-8 HHV-8 is a γ herpesvirus (similar to EBV). HHV-8 is associated with Kaposi’s sarcoma in HIV-infected individuals. Two recent case series from areas with high seroprevalence suggest that it can cause febrile syndrome and marrow failure in HCT recipients [152,153]. HHV-8 can be detected by PCR [146]. While transmission from donor to recipient has been documented in the kidney transplant setting [154], there have been no reports of transmission via stem cells or cord blood to date [155]. A case of HHV-8-associated Kaposi’s sarcoma has been reported in an HCT recipient [156].

Popovaviruses BK virus BK and JC virus are human polyomaviruses, and both can cause disease in HCT recipients [157]. BK virus is a nonenveloped DNA virus which multiplies in the nucleus. Seroprevalence in the population is between 60% and 80%. Primary infection usually occurs during childhood, possibly by the respiratory route. The virus can be detected by PCR [158,159]. BK virus is associated with postengraftment hemorrhagic cystitis after HCT [160,161]. BK-associated hemorrhagic cystitis appears to be more

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common after high-dose conditioning than after reduced-intensity conditioning [162]; unrelated donor status is an additional risk factor [162]. The statistical and epidemiologic evidence that links BK virus to hemorrhagic cystitis is quite strong. However, tissue-invasive BK hemorrhagic cystitis has only recently been demonstrated in a few cases [163,164]. Whether BK virus causes hemorrhagic cystitis through lytic viral infection remains controversial. An alternative hypothesis is that BK virusassociated hemorrhagic cystitis in HCT recipients is an immune reconstitution phenomenon [165]. According to this theory, hemorrhagic cystitis is the result of an inflammatory process that occurs when antigenspecific T cells recognize BK virus in the bladder. Immune reconstitution disease has been described for JC virus infection in HIV infected individuals treated with antiretroviral combination therapy. The immune reconstitution model is probably not sufficient to explain BK virusassociated hemorrhagic cystitis in the HCT setting since BK virus has been shown in bladder biopsies, and patients can develop postengraftment hemorrhagic cystitis during profound lymphocytopenia and in the presence of high-dose steroids [163,166]. Several cases of BK virus-associated pneumonia have been reported [167]. After kidney transplantation, BK virus is an important cause of late nephritis [168]. No comprehensive data exist on BK nephropathy in HCT recipients, although individual cases have been reported [169]. Although BK virus is often detected in urine by PCR, an association with clinical symptoms may be difficult to prove [170]. High viral load in urine was associated with a higher risk of BK-associated cystitis, but no threshold was defined [158]. BK plasma viremia is associated with high risk of nephropathy in renal transplant recipients [159,168]. Three recent studies indicate that BK viremia is also a marker of BK virusassociated hemorrhagic cystitis in HCT recipients [161,163,166]. Erard et al. showed that BK viremia occurs in about one-third of HCT recipients at a median of 41 days after transplantation, and is associated with post-engraftment hemorrhagic cystitis [163]. In a case-control study of hemorrhagic cystitis, BK plasma levels greater than 10,000 copies/mL were highly associated with post-engraftment BK-associated hemorrhagic cystitis [166]. However, detection in blood was not associated with hemorrhagic cystitis in two earlier studies [158,171]. In a study of pediatric HCT recipients, BK virus-associated hemorrhagic cystitis occurred between day 24 and 50 after transplant in nine of 117 patients. Infection was characterized by a long duration, correlated with use of busulfan, and resulted in bladder tamponade in two of nine patients [172]. There is no established treatment other than supportive care. Cidofovir has activity in vitro against murine polyomavirus, but there are only anecdotal treatment results in humans to date [173]. Pharmacokinetic studies have demonstrated that cidofovir accumulates in renal tissue and urine [165]. Treatment of infections localized to the urinary tract, with doses lower than those used in systemic CMV infection, might thus be effective. Initial data from renal transplant patients with BK nephropathy suggest that lower doses of cidofovir may be sufficient to treat BK disease [174]. Studies are ongoing to define the optimal dose of cidofovir for treatment of BK virus-associated disease. Leflunomide, an immunosuppressive agent used for treatment of rheumatoid arthritis, also appears to have antiviral activity and has been used in uncontrolled case series [175]. Gyrase inhibitors such as fluoroquinolones appear to have moderate preventative activity [157]. Prospective randomized trials are needed to assess the treatment options.

HCT, and in patients with hematologic malignancies [176–180]. Overall, this complication seems to be rare. PCR detection of JC virus in the cerebrospinal fluid is used for diagnosis [181]; however, its sensitivity is not 100%. Therefore, brain biopsies may be required. Additionally, plasma PCR may also be useful [180]. The optimal treatment has not been defined. Cidofovir has activity in vitro, but little is known about its in vivo efficacy in HCT recipients [182]. A study in HIV-infected individuals failed to show a therapeutic effect of cidofovir. Other treatment options include interleukin-2, cytarabine, chlorpromazine or the antipsychotic drugs ziprasidone, risperidone, mirtazipine and olanzapine [180,183]. However, only individual patients have been treated with these drugs, which makes an assessment of efficacy difficult [184]. Human papillomavirus Human papillomavirus (HPV) is a nonenveloped DNA virus. HPV infections occur rarely after transplantation [185,186]. HPV causes cutaneous infections (warts), and an association with cervical carcinoma has been identified. In one series, three of 238 allogeneic HCT recipients developed anogenital condylomata associated with HPV [185]. Rapid progression of pre-existing warts has been reported [187]. One retrospective study found a higher rate of cervical cytologic abnormalities, both before and after marrow transplantation, when compared with the general population [188]. Severe HPV disease has been reported in HCT recipients with severe combined immunodeficiency caused by common cytokine receptor subunit of janus kinase-3 (JAK-3) deficiency [189]. The clinical significance of these findings is unknown. There are no recommendations about the appropriate use of the recently licensed HPV vaccine in HCT recipients.

Parvovirus B19 Parvoviruses are made of small, naked, single-stranded DNA. Parvovirus infects a variety of animals, including cats and dogs. In 1975, human parvovirus B19 was isolated from asymptomatic blood donors. Seroepidemiologic studies showed evidence of past infection in approximately 60% of young adults. It is most likely spread by respiratory transmission or by blood. Parvovirus B19 can infect erythroid progenitor cells. Parvovirus B19 commonly is associated with aplastic crisis in patients with hemolytic anemia. It is also the cause of erythema infectiosum, a self-limited disease of childhood characterized by fever, fatigue, myalgias, a lace-like rash, and a “slapped cheek” appearance. Parvovirus B19 also is associated with some cases of rheumatoid arthritis. Immunity to parvovirus B19 is thought to be primarily humoral; neutralizing antibodies to capsid protein have a major role in host defense. IgM antibody detection may be useful in the diagnosis of acute infection in immunocompetent persons. The reliability of serologic testing in HCT recipients is questionable. In addition to transient marrow failure, parvovirus B19 can cause chronic anemia, and rarely, pancytopenia in HCT recipients [190–192]. Blood PCR is useful as a diagnostic tool [191]. Intravenous immunoglobulin is effective in treating parvovirus B19 symptomatic infection [193]. Prophylactic intravenous immunoglobulin seems to have a protective effect against parvovirus B19, although this has not been established in a randomized study [194].

JC virus JC virus is a nonenveloped DNA virus classified as a polyomavirus. JC virus is the cause of progressive multifocal leukoencephalopathy in immunosuppressed patients. Several cases have been reported after

Enteroviruses Enteroviruses belong to the picornavirus family. They include polioviruses, Coxsackie viruses, echoviruses, and other serotypes simply

Adenoviruses, Respiratory Viruses, HHV-6, HHV-7, HHV-8, Papovaviruses and Other Viruses After Hematopoietic Cell Transplantation

referred to as enteroviruses. They are naked virions containing singlestranded RNA that replicate in the cellular cytoplasm. Both tissue culture and molecular methods are used for diagnosis. The virus can be detected in throat washes, stool, cerebrospinal fluid, and other body fluids. Asymptomatic infection is common in normal hosts, and these viruses are most prevalent during the summer and fall months. Infection is usually by the fecal–oral route; shedding of the virus from the oropharynx may persist for 1–4 weeks, and from the gastrointestinal tract, for up to 18 weeks. Viremic spread in the host is common and can affect a variety of organs. Infection by enteroviruses in HCT recipients has been reported [11,195,196]. One outbreak of Coxsackie A1-associated diarrhea affected patients in a 13-bed unit over a 3-week period, resulting in death in six of seven infected patients [197]. Coxsackie virus was also identified in stools of four of 78 patients with gastrointestinal infections [198]. All four patients with Coxsackie virus died; two had co-infections with adenovirus and rotavirus [198]. Additional cases of disseminated and life-threatening infection have been reported [199–203]. Severe cases of echovirus infections have been reported, including a disseminated infection with pneumonitis and pericarditis [204,205]. Treatment of enterovirus infections is supportive. The antiviral drug pleconaril is active against enteroviruses, but the drug is not licensed by the Food and Drug Administration.

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Rotaviruses Rotaviruses consist of double-stranded RNA and have a double-shelled capsid. They infect the duodenum and the jejunum, causing vomiting and diarrhea in normal hosts, occasionally accompanied by low-grade fever. Infection usually occurs in the winter, and transmission is by the fecal–oral route. During one outbreak, contaminated toys in a cancer center play room were implicated as the mode of transmission [206]; hospital personnel can transmit the virus as well [207]. Diagnosis is by electron microscopy, enzyme-linked immunoabsorbent assay or PCR. Repeated testing does not appear to increase the diagnostic yield [208]. Rotavirus is a common cause of diarrhea in HCT recipients presenting with gastroenteritis [195,198,209–212], and fatalities have been reported [195]. There is no proven specific treatment other than supportive care. Oral immunoglobulin has been used in individual cases [213], but this approach has not been studied in larger series or randomized trials.

Caliciviruses Caliciviruses (Norovirus [prototype Norwalk virus] and Sapovirus [prototype Sapporo virus]) are made of small, nonenveloped, single-stranded RNA-containing virions that have been associated with outbreaks of

Table 94.3 Overview of antibody preparations and antiviral agents with in vitro activity against other viral infections after hematopoietic cell transplantation (HCT). For details about indications, doses and limitations, see text Virus

Drugs

Clinical use in HCT recipients*

Comment

Adenovirus

Ribavirin (intravenous) Cidofovir

Adenovirus disease Adenovirus disease, pre-emptive therapy

RSV

Ribavirin

RSV pneumonia, pre-emptive therapy for upper respiratory infection Combination therapy for RSV pneumonia Combination therapy for RSV pneumonia

High rate of failures, possibly due to selection bias Response to therapy reported, no direct comparison with ribavirin reported Moderate-to-strong evidence for pneumonia,

Human metapneumonia virus Parainfluenza viruses

RSV-specific monoclonal antibodies (palivizumab) Pooled immunoglobulin Ribavirin

Combination with immunoglobulin preparations appear to be more effective when RSV-specific preparations are used (Table 94.2)

No data

Ribavirin Pooled immunoglobulin

Parainfluenza virus pneumonia Combination therapy for parainfluenza pneumonia

Influenza viruses

M2-Inhibitors (amantidine, rimantidine) Neuraminidase inhibitors (oseltemavir, zanamivir)

Influenza infection and disease, prophylaxis; combination therapy Influenza infection and disease, prophylaxis; combination therapy

Rhinovirus HHV-6

Pleconaril Ganciclovir, valganciclovir Foscarnet Cidofovir Foscarnet Cidofovir Ganciclovir, valganciclovir Cidofovir Leflunomide JC virus Cidofovir, risperidone, Mirtazipine interleukin-2, cytarabine

No data HHV-6 CNS disease HHV-6 CNS disease No data No data No data No data Hemorrhagic cystitis

Small case series

Multifocal leukoencephalopathy

Case studies only

HHV-7 HHV-8 BK virus

CNS, central nervous system; HHV, human herpes virus; RSV, respiratory syncytial virus. * All clinical results in HCT recipients are from non-randomized studies.

No apparent effect for pneumonia of either ribavirin or the combination with immunoglobulin; pre-emptive therapy not systematically tested; reduction of immunosuppression may be effective (see text) M2-inhibitors only effective against influenza A, most isolates now resistant; both groups of drugs extensively studied in immunocompetent subjects; neuraminidase inhibitors appear to have improved toxicity profile, although experience in HCT is limited Drug is not Food and Drug Administration approved Both foscarnet and ganciclovir have been used for the treatment of CNS disease; anti-HHV-6 activity does not appear to be HHV-6 variant-dependent [144]

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diarrhea [214]. There have been no reports of calicivirus outbreaks in HCT recipients, but the potential is high, especially for Noroviruses. Diagnosis is by PCR or electron microscopy. Treatment is supportive.

Conclusion and future perspective Major progress has been made in the diagnosis and prevention of viral infections after HCT over the last decade. Perhaps the most impressive examples are the prevention of CMV disease by ganciclovir and the recognition of the host defense mechanisms that control CMV. During the same time period, the significance and our knowledge of other viral infections has increased, especially that of respiratory viruses, HHV-6, BK, and adenoviruses. The increasing availability of sensitive and specific multiplex diagnostic platforms that detect a large number of viruses will soon lead to better understanding of the infection epidemiology, improved infection control, and the development of more therapeutic options [215]. A systematic and comprehensive evaluation of the disease burden of viral infections is needed. Historically, direct organ involvement, such as pneumonia or gastrointestinal disease, and death were reported as endpoints; however, data on airflow obstruction (for respiratory viruses), health-care utilization, including duration of hospitalization, intensive care, and mechanical ventilation are important as well. For most of the infections discussed in this chapter, very little information is available on disease pathogenesis. Although several studies point towards lymphopenia as an important risk factor for severe manifestations of disease, the exact host defense mechanisms that lead to the often devastating consequences of viral infections in HCT recipients are

poorly understood. Studies of the importance of virus load, T-cell and humoral immunity, as well as cytokine and chemokine expression and genetics, are needed. Genetic studies may be useful to better understand the risk of virus acquisition and progression to end-organ and fatal disease. Most epidemiologic and outcome studies to date have focused on the early period after transplantation. More information is needed on the risk of progression and outcome late after HCT (i.e. after the first 3 months), when lymphopenia is rare. For instance, there may be bidirectional relationships between respiratory viruses and lung function late after transplantation: viral infections may lead to prolonged airflow obstruction, and pre-existing airflow obstruction may predispose individuals to progression from upper to lower respiratory tract disease and a poor outcome. Major progress is needed with regards to prevention and treatment options for the viral infections discussed in this chapter. With the exception of treatment for influenza virus, there is a very limited armamentarium of potent and easy to administer drug treatments (Table 94.3) [215]. Drug development, as well as research of the host defense mechanisms, may ultimately lead to novel drug or immune-based interventions. Immune augmentation strategies could consist of vaccination, T-cell or antibody therapy, or nonspecific immune reconstitution strategies (e.g. keratinocyte growth factor). Finally, the explosion of technical capabilities has led to the discovery of several new viruses in recent years, with novel coronaviruses, HBoV, polyomaviruses, and arenaviruses [216] being the latest additions. It is likely that additional viruses will be discovered. Well-designed disease association studies will be needed in order to determine the significance of these viruses in HCT recipients.

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174. Limaye AP. Treatment of refractory BK virusassociated nephropathy with cidofovir. Am J Transplant 2003; 3: 186–91. 175. Faguer S, Hirsch HH, Kamar N et al. Leflunomide treatment for polyomavirus BK-associated nephropathy after kidney transplantation. Transpl Int 2007; 20: 962–9. 176. Coppo P, Laporte JP, Aoudjhane M et al. Progressive multifocal leucoencephalopathy with peripheral demyelinating neuropathy after autologous bone marrow transplantation for acute myeloblastic leukemia (FAB5). Bone Marrow Transplant 1999; 23: 401–3. 177. Re D, Bamborschke S, Feiden W et al. Progressive multifocal leukoencephalopathy after autologous bone marrow transplantation and alpha-interferon immunotherapy. Bone Marrow Transplant 1999; 23: 295–8. 178. Holzapfel C, Kellinghaus C, Luttmann R et al. [Progressive multifocal leukoencephalopathy (PML) in chronic lymphatic leukemia (CLL). Review of the literature and case report]. Nervenarzt 2002; 73: 543–7. 179. Seong D, Bruner JM, Lee KH et al. Progressive multifocal leukoencephalopathy after autologous bone marrow transplantation in a patient with chronic myelogenous leukemia. Clin Infect Dis 1996; 23: 402–3. 180. Kharfan-Dabaja MA, Ayala E, Greene J, Rojiani A, Murtagh FR, Anasetti C. Two cases of progressive multifocal leukoencephalopathy after allogeneic hematopoietic cell transplantation and a review of the literature. Bone Marrow Transplant 2007; 39: 101–7. 181. Taoufik Y, Gasnault J, Karaterki A et al. Prognostic value of JC virus load in cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy. J Infect Dis 1998; 178: 1816–20. 182. Houston S, Roberts N, Mashinter L. Failure of cidofovir therapy in progressive multifocal leukoencephalopathy unrelated to human immunodeficiency virus. Clin Infect Dis 2001; 32: 150–2. 183. Focosi D, Fazzi R, Montanaro D, Emdin M, Petrini M. Progressive multifocal leukoencephalopathy in a haploidentical stem cell transplant recipient: a clinical, neuroradiological and virological response after treatment with risperidone. Antiviral Res 2007; 74: 156–8. 184. Przepiorka D, Jaeckle KA, Birdwell RR et al. Successful treatment of progressive multifocal leukoencephalopathy with low-dose interleukin2. Bone Marrow Transplant 1997; 20: 983–7. 185. Daneshpouy M, Socie G, Clavel C et al. Human papillomavirus infection and anogenital condyloma in bone marrow transplant recipients. Transplantation 2001; 71: 167–9. 186. Barasch A, Eisenberg E, D’Ambrosio JA, Nuki K, Peterson DE. Oral verruca vulgaris in a bone marrow transplant patient: a case report and review of literature. Eur J Cancer B Oral Oncol 1996; 32B: 137–9. 187. Maruyama F, Miyazaki H, Matsui T et al. Rapid progression of flat warts in a patient with malignant lymphoma after PBSCT. Bone Marrow Transplant 1996; 18: 1009–11. 188. Sasadeusz J, Kelly H, Szer J, Schwarer AP, Mitchell H, Grigg A. Abnormal cervical cytology in bone marrow transplant recipients. Bone Marrow Transplant 2001; 28: 393–7. 189. Laffort C, Le Deist F, Favre M et al. Severe cutaneous papillomavirus disease after haemopoietic

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stem-cell transplantation in patients with severe combined immune deficiency caused by common gamma cytokine receptor subunit or JAK-3 deficiency. Lancet 2004; 363: 2051–4. Azzi A, Fanci R, Ciappi S, Zakrzewska K, Bosi A. Human parvovirus B19 infection in bone marrow transplantation patients. Am J Hematol 1993; 44: 207–9. Schleuning M, Jager G, Holler E et al. Human parvovirus B19-associated disease in bone marrow transplantation. Infection 1999; 27: 114–17. Florea AV, Ionescu DN, Melhem MF. Parvovirus B19 infection in the immunocompromised host. Arch Pathol Lab Med 2007; 131: 799–804. Kurtzman G, Frickhofen N, Kimball J, Jenkins DW, Nienhuis AW, Young NS. Pure red-cell aplasia of 10 years’ duration due to persistent parvovirus B19 infection and its cure with immunoglobulin therapy [see comments]. N Engl J Med 1989; 321: 519–23. Frickhofen N, Arnold R, Hertenstein B, Wiesneth M, Young NS. Parvovirus B19 infection and bone marrow transplantation. Ann Hematol 1992; 64(Suppl): A121–4. Yolken RH, Bishop CA, Townsend TR et al. Infectious gastroenteritis in bone-marrowtransplant recipients. N Engl J Med 1982; 306: 1010–12. Chakrabarti S, Collingham KE, Stevens RH, Pillay D, Fegan CD, Milligan DW. Isolation of viruses from stools in stem cell transplant recipients: a prospective surveillance study. Bone Marrow Transplant 2000; 25: 277–82. Townsend TR, Bolyard EA, Yolken RH et al. Outbreak of Coxsackie A1 gastroenteritis: a complication of bone-marrow transplantation. Lancet 1982; 1: 820–3. Cox GJ, Matsui SM, Lo RS et al. Etiology and outcome of diarrhea after marrow transplantation: a prospective study. Gastroenterology 1994; 107: 1398–407. Aquino VM, Farah RA, Lee MC, Sandler ES. Disseminated coxsackie A9 infection complicating bone marrow transplantation. Pediatr Infect Dis J 1996; 15: 1053–4. Galama JM, de Leeuw N, Wittebol S, Peters H, Melchers WJ. Prolonged enteroviral infection in a patient who developed pericarditis and heart failure after bone marrow transplantation. Clin Infect Dis 1996; 22: 1004–8. Gonzalez Y, Martino R, Badell I et al. Pulmonary enterovirus infections in stem cell transplant recipients. Bone Marrow Transplant 1999; 23: 511–13. Fischmeister G, Wiesbauer P, Holzmann HM, Peters C, Eibl M, Gadner H. Enteroviral meningoencephalitis in immunocompromised children after matched unrelated donor-bone marrow transplantation. Pediatr Hematol Oncol 2000; 17: 393–9. Tan PL, Verneris MR, Charnas LR, Reck SJ, van Burik JA, Blazar BR. Outcome of CNS and pulmonary enteroviral infections after hematopoietic cell transplantation. Pediatr Blood Cancer 2005; 45: 74–5. Biggs DD, Toorkey BC, Carrigan DR, Hanson GA, Ash RC. Disseminated echovirus infection complicating bone marrow transplantation. Am J Med 1990; 88: 421–5. Schwarer AP, Opat SS, Watson AM, Spelman D, Firkin F, Lee N. Disseminated echovirus infection

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after allogeneic bone marrow transplantation. Pathology 1997; 29: 424–5. Rogers M, Weinstock DM, Eagan J, Kiehn T, Armstrong D, Sepkowitz KA. Rotavirus outbreak on a pediatric oncology floor: possible association with toys. Am J Infect Control 2000; 28: 378– 80. Kruger W, Stockschlader M, Zander AR. Transmission of rotavirus diarrhea in a bone marrow transplantation unit by a hospital worker. Bone Marrow Transplant 1991; 8: 507–8. Kamboj M, Mihu CN, Sepkowitz K, Kernan NA, Papanicolaou GA. Work-up for infectious diarrhea after allogeneic hematopoietic stem cell transplantation: single specimen testing results in cost savings without compromising diagnostic yield. Transpl Infect Dis 2007; 9: 265–9. van Kraaij MG, Dekker AW, Verdonck LF et al. Infectious gastro-enteritis: an uncommon cause of diarrhoea in adult allogeneic and autologous stem cell transplant recipients. [In process citation.] Bone Marrow Transplant 2000; 26: 299–303.

210. Troussard X, Bauduer F, Gallet E et al. Virus recovery from stools of patients undergoing bone marrow transplantation. Bone Marrow Transplant 1993; 12: 573–6. 211. Yeager AM, Kanof ME, Kramer SS et al. Pneumatosis intestinalis in children after allogeneic bone marrow transplantation. Pediatr Radiol 1987; 17: 18–22. 212. Liakopoulou E, Mutton K, Carrington D et al. Rotavirus as a significant cause of prolonged diarrhoeal illness and morbidity following allogeneic bone marrow transplantation. Bone Marrow Transplant 2005; 36: 691–4. 213. Kanfer EJ, Abrahamson G, Taylor J, Coleman JC, Samson DM. Severe rotavirus-associated diarrhoea following bone marrow transplantation: treatment with oral immunoglobulin. Bone Marrow Transplant 1994; 14: 651–2. 214. Green KY. The role of human caliciviruses in epidemic gastroenteritis. Arch Virol Suppl 1997; 13: 153–65. 215. Nichols WG, Peck-Campbell AJ, Boeckh M. Respiratory viruses other than influenza virus:

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impact and therapeutic advances. Clin Microbiol Rev 2008; 21: 274–90. Palacios G, Druce J, Du L et al. A new arenavirus in a cluster of fatal transplantassociated diseases. N Engl J Med 2008; 358: 991–8. Martino R, Ramila E, Rabella N et al. Respiratory virus infections in adults with hematologic malignancies: a prospective study. Clin Infect Dis 2003; 36: 1–8. Whimbey E, Champlin RE, Englund JA et al. Combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients. Bone Marrow Transplant 1995; 16: 393–9. Ghosh S, Champlin RE, Englund J et al. Respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin. Bone Marrow Transplant 2000; 25: 751–5.

95

Simone I. Strasser & George B. McDonald

Gastrointestinal and Hepatic Complications

Introduction The frequency and severity of intestinal and liver complications of hematopoietic cell transplantation (HCT) have changed in the last few years. Fewer patients have severe gut and liver damage from acute graftversus-host disease (GVHD), as the development of more effective strategies to prevent GVHD has had a marked effect on its clinical presentation. Protracted jaundice is now far less common, as the frequency of sinusoidal liver injury caused by myeloablative regimens has fallen, and prophylaxis with ursodiol has been effective in mitigating cholestatic liver injury. Antiviral agents have almost completely eliminated intestinal and hepatic infections caused by herpes simplex virus (HSV) and cytomegalovirus (CMV), but sporadic cases of adenovirus, rotavirus, astrovirus, hepatitis B virus (HBV), and norovirus infection may be seen. Fungal infections of the intestine and liver have become uncommon since the advent of more effective prophylaxis. Clinicians have become adept at recognizing unusual presentations of infectious diseases, for example abdominal pain as a manifestation of varicella zoster virus (VZV) infection, and diarrhea with multiorgan failure with adenovirus infection. New challenges, however, have come from the greater use of human leukocyte antigen (HLA)-mismatched and unrelated donors, the increase in certain diseases as indications for HCT (e.g. sickle cell disease, renal carcinoma, myeloma, and autoimmune diseases), the older age of patients being considered for HCT, and the growth of reduced-intensity allogeneic HCT. This chapter is organized by the problems a clinician might encounter in caring for an HCT recipient, in the chronologic order in which these problems are likely to appear. Differential diagnoses of specific problems provide a framework for addressing clinical issues.

Evaluation of intestinal and liver problems before transplant Ulcers, tumors, and infections in the gastrointestinal tract Mucosal ulcerations may bleed profusely when platelet counts drop after HCT, and in immunocompromised patients, ulcers may have an infectious etiology (e.g. CMV, HSV or fungal infection) that requires specific antimicrobial treatment. Thus, symptoms of esophageal pain, heartburn, dysphagia, abdominal pain, nausea, and vomiting should be investigated

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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with endoscopy before HCT, and all intestinal ulcerations healed before the start of conditioning therapy. The goal of pre-HCT therapy in patients with ulcerative colitis or Crohn’s disease is to minimize the extent of gross intestinal ulceration to lower the risk of bleeding after HCT. One must also rule out infections as a cause of colonic ulceration. CMV, Entamoeba histolytica, and clostridial infections are causes of colonic ulceration that may mimic inflammatory bowel disease. CMV disease before transplant is associated with a high risk for early CMV disease and death after HCT [1]. Selected patients with ulcerative colitis and Crohn’s disease have undergone both allogeneic and autologous HCT without complications of bleeding, perforation or dissemination of microorganisms [2,3]. Long-term resolution of Crohn’s disease has been observed following myeloablative allogeneic HCT, and autologous HCT has resulted in improvement as well [2,3]. Patients who are to undergo HCT for lymphoma, leukemia in relapse, myeloma or metastatic breast cancer may have intestinal involvement with malignant cells. Cytoreductive therapy results in necrosis of these cells. Both large intestinal ulcers and perforation due to tumor lysis have been seen, but the low frequency of this complication (estimated at less than one case in 1000 HCTs) does not warrant screening in asymptomatic patients. Intra-abdominal abscesses and fistulae should be managed surgically before HCT. Patients over the age of 50 are at risk for the development of colorectal polyps and cancer. These are seldom considerations in younger patients, but the presence of occult blood in stool specimens from patients over 50 years should prompt endoscopic evaluation, including colonoscopy. Diarrhea Patients with diarrhea should be investigated for organisms that may cause morbidity during the period of immunosuppression after HCT (E. histolytica, Strongyloides, Giardia lamblia, Cryptosporidium, clostridial infections, CMV, rotavirus, and adenovirus) [1,4–6]. Persistent eosinophilia in a patient from an area where parasites are endemic is also an indication for screening for parasitic disease, including coccidial and microsporidial organisms if the patient is immunosuppressed. Specific therapy is available for amebiasis, strongyloidiasis, giardiasis, other helminths, and most coccidia and microsporidia. Cryptosporidiosis may be resistant to therapy in an immunosuppressed patient [5], but restoration of normal immunity after allogeneic HCT can result in clearance of cryptosporidia [7]. Similarly, protracted diarrhea related to immune dysregulation can be treated with allogeneic HCT, for example by restoration of T regulatory cells in children with IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome [8].

Gastrointestinal and Hepatic Complications

Other treatable causes of diarrhea in patients with hematologic malignancies include clostridial infections (C. difficile, C. perfringens, and C. septicum), CMV enterocolitis, adenovirus infections, and overgrowth with Candida albicans. The patient who has had previous typhlitis during neutropenia can present problems if there is persistent fever or tenderness over the cecum. Typhlitis is a syndrome of cecal edema, mucosal friability and ulceration, with fever often associated with polymicrobial sepsis; its cause is usually intestinal clostridial infection, particularly C. septicum [9]. After treatment, the risk of post-HCT typhlitis is no different than that of other patients. Pericolic phlegmons, abscesses, and persistent colitis caused by clostridial infections must be treated before the start of conditioning therapy. Perianal pain Pain near the anal canal in a granulocytopenic patient is due to bacterial infection until proven otherwise. Most anorectal infections begin in the anal glands deep to the crypts at the dentate line. Inflammation of anal crypts may cause pain in the absence of an abscess. Abscesses tend to originate in the intersphincteric space and may extend into the perianal, ischiorectal, or supralevator space [10]. Extensive supralevator and intersphincteric abscesses may be present without being apparent on external examination. Perianal infections must be dealt with before HCT, as extensive tissue necrosis and septicemia may result from uncontrolled infection [11,12]. Antibiotic treatment should cover anaerobic as well as aerobic bacteria [13]. Perineal HSV infection and rarely fungal infections may also lead to painful ulcerations. Fungal liver infections A search for hepatic fungal infection should be undertaken in patients with tender hepatomegaly, fevers, and abnormal liver enzymes. Diagnosis may be established by sensitive liver imaging (high-resolution computed tomography [CT] or magnetic resonance imaging [MRI]) [14] in conjunction with fungal biomarkers such as serology for fungal antigens (galactomannan and glucan assays) [15,16], the polymerase chain reaction (PCR) or culture of liver biopsy material. Fungal liver infection should be controlled with systemic antifungal agents, with newer agents offering reduced toxicity and a broader range of coverage [17,18]. Therapy should be continued through HCT until engraftment is established, which can effect resolution of intractable fungal liver abscesses [19]. Viral hepatitis in allogeneic HCT donors Hepatitis B and hepatitis C will be transmitted from viremic donors to transplant recipients [20]. When equally HLA-matched donors are available, a donor who is not infected by hepatitis viruses should be selected. If the most suitable donor has chronic hepatitis B or C, however, preventing passage of virus and mitigating the effects of infection in the recipient become priorities. Oral nucleos(t)ide analogs, such as lamivudine, adefovir, entecavir, telbivudine, and tenofovir result in rapid HBV suppression and, where possible, should be used to reduce viral load prior to stem cell collection [21,22]. However, HBV may persist in donor peripheral blood stem cells despite clearance from serum [23]. If donor hematopoietic cells are rendered negative for HBV DNA before harvest, passage can be prevented [22]. Regardless of whether a donor has been treated with antiviral therapy, a recipient transplanted from a hepatitis B surface antigen (HBsAg)-positive donor should receive prophylaxis with oral antiviral drugs. In addition, hepatitis B vaccination administered to anti-HBs-negative recipients prior to the start of chemotherapy may reduce the development of post-HCT hepa-

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titis B. Antiviral treatment after HCT should be continued for at least 1 year. HBsAg negative-, anti-hepatitis B core antigen (HBc)-positive donors are viremic in fewer than 5% of cases and can be used as donors if their serum and peripheral blood stem cells are HBV DNA negative using a sensitive quantitative PCR assay. A donor who is naturally antiHBs positive may be the preferred donor if the recipient is HBsAg positive or anti-HBc positive, as adoptive transfer of immunity can effect clearance of virus [24]. If a preferred donor is hepatitis C virus (HCV) antibody and RNA positive, HCV will usually be transmitted, and the rate of spontaneous viral clearance is likely to be low. If time permits, treatment of the donor with pegylated interferon plus ribavirin prior to harvest of donor cells may render them nonviremic and much less likely to transmit infection [25,26]. New small-molecule antiviral drugs in current development may offer clinical benefit in rendering donors nonviremic. If virus is transmitted, the acute phase of HCV infection may cause elevated liver enzymes at 2–3 months post HCT, after recovery of T-cell function; however, severe hepatitis is rare, and the outcome in 10 years of followup is no different than in transplant recipients without hepatitis C infection [27]. In the long-term, antiviral treatment with pegylated interferon plus ribavirin should be offered to the recipient who remains with active hepatitis, as such a patient is at risk for development of cirrhosis and hepatocellular carcinoma [28]. Chronic liver disease in candidates for HCT Patients with active liver disease, particularly those with severe hepatic fibrosis or cirrhosis, are at increased risk for fatal sinusoidal obstructive syndrome (SOS; formerly known as veno-occlusive disease of the liver) following hepatotoxic myeloablative regimens, and the presence of cirrhosis may provide a contraindication to any high-dose conditioning regimen. Patients with cirrhosis are at risk for fatal hepatic decompensation after HCT even if given a reduced-intensity conditioning regimen [29]. Liver biopsy should be considered if there is a clinical suspicion of cirrhosis or extensive fibrosis resulting from chronic viral infection, ethanol or nonalcoholic steatohepatitis. This caution also extends to patients with myelofibrosis, in whom extramedullary hematopoiesis can lead to extensive sinusoidal fibrosis. A reduced-intensity conditioning regimen may allow congenitally immunodeficient children with liver disease to be successfully transplanted without SOS, with subsequent normalization of liver abnormalities [30]. Hepatitis B-infected HCT recipients are at risk for severe hepatitis flares and fulminant liver failure. In the absence of antiviral prophylaxis, HBV reactivation is common, and fatal fulminant hepatitis develops in approximately 15% [20]. Prophylaxis with oral nucleoside therapy starting prior to chemotherapy is therefore recommended for all HBsAgpositive recipients. Even in patients with isolated anti-HBc antibodies, there is a 35% risk of post-HCT HBV reactivation, usually during treatment for acute GVHD; the pathogenesis of infection in these cases is related to a reservoir of virus within the hepatocytes [31]. Severe hepatitis B reactivation has also been seen in anti-HBc/anti-HBs-positive patients and in a patient with occult hepatitis B [32]. Patients from areas where hepatitis B is endemic need close scrutiny after HCT. Recent liver dysfunction in candidates for HCT Patients who come to HCT following recent chemotherapy or radiation therapy may be at risk for unexpected toxicities following myeloablative conditioning regimens, particularly if these therapies have resulted in liver dysfunction. Many chemotherapy drugs cause liver dysfunction either directly (cholestatic injury, hepatocyte necrosis or sinusoidal damage) or indirectly (cholestasis caused by sepsis or endotoxemia, i.e.

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Chapter 95

cholangitis lenta) (reviewed in [33,34]). Although recent exposure to standard chemotherapy has not been associated with an increased risk of fatal SOS after cyclophosphamide (CY)-based regimens [35], patients with persistent jaundice and elevations of aminotransferase enzymes are likely at risk. Two newer drugs, Imatinib (Gleevec) and gemtuzumab ozogamicin (Mylotarg) deserve special mention in this context. Imatinib mesylate may cause acute hepatocellular necrosis and multiacinar collapse, with eventual healing by focal fibrosis [36,37]; we have successfully given CY-based myeloablative conditioning to patients who recovered from the acute injury but who had patchy fibrosis on biopsy. Gemtuzumab ozogamicin causes sinusoidal liver injury in 3–15% of patients [38]; if a patient is transplanted following a hepatotoxic myeloablative conditioning regimen within 3 months of exposure to high doses of gemtuzumab ozogamicin, SOS develops in 15–40% of cases [38,39].

Table 95.1 Differential diagnosis of anorexia, nausea and vomiting after transplant

Gall bladder and bile duct stones

Infectious causes in bold are now so rare that they should be considered only when patients are at risk.

HCT candidates with asymptomatic gallstones (incidentally discovered during a CT scan or ultrasound) do not require operative intervention [40]. Patients with symptomatic gall bladder or common duct stones are at significant risk of sepsis after HCT, and consideration should be given to pretransplant cholecystectomy or an endoscopic biliary procedure. Iron overload HCT candidates with diseases such as thalassemia, aplastic anemia, and chronic leukemia or lymphoma may come to HCT with marked hepatic iron overload, best documented by either measurement of iron in liver biopsy tissue or iron-specific MRI imaging (FerriScan) [41]. Excess iron may interfere with Kupffer cell function and predispose to mold infections [42–44]. In patients with extreme iron overload, effective pre-HCT chelation therapy improves post-HCT survival [45]. While some studies suggest an association between excess tissue iron stores and regimenrelated toxicity, others have failed to demonstrate this. Severe iron overload has been associated with nonspecific liver dysfunction post HCT, which may respond to erythropoietin-assisted phlebotomy or chelation [46,47]. In most patients, the quantitation of tissue iron stores and consideration of iron removal can be deferred until after recovery from HCT.

Problems from transplant through day 200 Nausea, vomiting, and anorexia (Table 95.1) Myeloablative conditioning therapy makes most patients nauseated and anorexic, leading to delayed gastric emptying [48] and poor oral intake, with a nadir at day 10–12 post transplant but often extending to day 20 [49]. Serotonin-antagonist drugs are very effective in relieving symptoms during conditioning therapy but have little effect afterwards. Protracted anorexia may be related to circulating [interleukin(IL)-1, IL-6,] and tumor necrosis factor-alpha, cytokines known to affect appetite centers [49]. Mucositis caused by conditioning therapy leads to oral mucosal swelling, pain, and, in severe cases, sloughing of pharyngeal and esophageal epithelium, intense gagging, an inability to swallow, vomiting, retrosternal pain, and airway obstruction. Opioid therapy is effective in relieving pain but can lead to gastric stasis and intestinal pseudo-obstruction. Some regimens, notably those that contain cytarabine, etoposide, high-dose melphalan or multiple alkylating agents, may cause unusually severe intestinal mucosal necrosis and further delay the return of eating.

Infectious causes

Noninfectious causes

Cytomegalovirus Herpes simplex virus Varicella zoster virus Helicobacter pylori Giardia lamblia Cryptosporidia Rotavirus Oropharyngeal bacteria (phlegmonous gastritis)

Residual effects of conditioning therapy Acute graft-versus-host disease Medication intolerance Intestinal obstruction or pseudo-obstruction Gastroparesis Uremia/dialysis Acute hepatitis Acute pancreatitis Cholecystitis Increased intracranial pressure (central nervous system lesions)

An early sign of acute GVHD is loss of appetite, followed by nausea and vomiting. When the onset of GVHD is before day 15, following a peripheral blood allograft for example, these symptoms are indistinguishable from those resulting from conditioning therapy. Some patients never experience improvement in appetite before the onset of GVHD, but rather have a seamless persistence of anorexia and nausea. After day 20, over 80% of patients with intractable anorexia, nausea or vomiting will have gastric and duodenal GVHD as the sole explanation [50,51]. Endoscopy shows edema of the gastric antral and duodenal mucosa, patchy erythema, and bilious gastric fluid (Plates 27.26, 27.27, and 27.28), and histology may show epithelial cell apoptosis and drop-out, often with localized lymphocytic infiltrates (Fig. 27.5, and Plates 27.20, 27.27, 27.28, and 27.29) [52,53]. However, the predictive value of negative histology for GVHD is not high, as what the endoscopist sees as mucosal edema (Plate 27.26) is not what the pathologist relies upon to make a diagnosis of GVHD (apoptosis of crypt cells; Plates 27.20, 27.27, 27.28, and 27.29). Thus, the gamut of clinical presentation, endoscopic appearance, absence of infection, and histology needs to be integrated to arrive at a diagnosis when histology is equivocal. Immunosuppressive therapy using a 10-day induction course of prednisone 1 mg/kg/day plus oral beclomethasone dipropionate 8 mg/day is an effective therapy that avoids prolonged prednisone exposure [54]. Recipients of autologous grafts may also develop a syndrome of anorexia, nausea, and vomiting that is associated with diffuse gastric edema and erythema [55]. Gastric histology shows typical GVHD (Plate 27.37), and symptoms respond to a 10-day course of prednisone 1 mg/kg/day. Endoscopy also serves to rule out intestinal infection with herpesviruses, bacteria, and fungi. CMV infection of the esophagus and upper intestine accounted for a third of patients with unexplained nausea and vomiting during the pre-ganciclovir era, but CMV infection is now an uncommon cause of these symptoms. CMV infections in the gastrointestinal tract (Plates 27.35 and 27.36) are usually diagnosed between 50 and 150 days after HCT [56], but often appear earlier if patients have CMV infection before HCT [1]. CMV infections can be detected in the gastrointestinal tract in the absence of CMV antigen or DNA in peripheral blood. HSV esophagitis may present similarly in patients not receiving prophylactic acyclovir. Fungal esophagitis may cause anorexia but not the incessant vomiting often seen with GVHD or CMV infection. Studies of gastric emptying and myeloelectric activity in HCT patients have shown that symptoms of nausea and vomiting are frequently accompanied by retention of radionuclide meals and disordered

Gastrointestinal and Hepatic Complications

electrical activity [57]. Promotility agents such as metoaclopramide, domperidone, and low-dose erythromycin are occasionally useful, but in patients with persistent symptoms, endoscopic diagnosis of the underlying cause of altered motility should come before empiric promotility therapy. Anorexia and vomiting may also be manifestations of increased intracranial pressure; other neurologic signs and symptoms usually dominate the clinical picture with these disorders. Oral nonabsorbable antibiotics (particularly nystatin), cyclosporine, mycophenolate mofetil, trimethoprim–sulfamethoxazole, voriconazole, posaconazole, itraconazole, intravenous amphotericin, and high-dose opioids also cause nausea and occasionally vomiting that can be confused with symptoms of GVHD [17,18]. Anorexia and nausea are also common, sometimes intolerable, side-effects of treatment with maribavir. Parenteral infusions of fat, glucose, and amino acids reduce food intake, slow gastric emptying, and cause nausea. Even after total parenteral nutrition has been stopped, appetite suppression may linger for 1–3 weeks [58].

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lary hematopoiesis) [27,35]. At our center, the overall incidence of SOS among patients with hematologic malignancy conditioned with CY 120 mg/kg plus TBI 12–13.2 Gy is 38% (7% severe), and among patients with myelodysplastic syndrome conditioned with targeted busulfan (BU)/CY 120 mg/kg it is 12% (2% severe) [64,66]. Overall, the frequency and severity of SOS have fallen dramatically over the last few years, for several reasons: (1) the strategy of dose escalation of conditioning regimens has been abandoned; (2) fludarabine is replacing CY in many centers; and (3) patients at risk for SOS (with underlying fibroinflammatory liver disease) are being directed to regimens that do not contain liver toxins. Evidence has been presented that prophylaxis with ursodiol prevents SOS [67], but the largest randomized trial of ursodiol that specifically tracked SOS as an endpoint found no such effect [60]. The effect of ursodiol is primarily on cholestatic liver disease, and thus it seems likely that many past patients diagnosed as having SOS on clinical grounds had instead a combination of sinusoidal and cholestatic liver injury. Clinical presentation and diagnosis

Jaundice, hepatomegaly, and abnormal liver tests Development of jaundice following HCT is an ominous prognostic sign, with increased nonrelapse mortality in patients whose total serum bilirubin exceeds 4 mg/dL [59]. Because treatment of severe liver dysfunction is often futile, transplant physicians must recognize risk factors for hepatobiliary problems before the transplant process starts, implement measures to prevent liver damage, and identify post-transplant liver disorders that have specific treatments. Over the last decade, this approach has resulted in a marked reduction in the incidence of liver infections and clinical jaundice, resulting from prevention of hepatic sinusoidal injury, fungal and viral prophylaxis, and the introduction of prophylactic ursodiol for all patients undergoing transplant [17,20,60,61]. Nonetheless, there remain multiple hepatic causes of jaundice and elevations of serum alanine aminotransferase (ALT) and alkaline phosphatase (Table 95.2). Additional processes can contribute to jaundice following HCT while not usually being important causes of jaundice in isolation, for example hemolysis and renal insufficiency. It is imperative to search for causes of progressive jaundice as early intervention to prevent severe hyperbilirubinemia may reduce the risk of death, although this is unproven. Sinusoidal obstructive syndrome Definition SOS is a clinical syndrome of tender hepatomegaly, fluid retention and weight gain, and elevated serum bilirubin that follows high-dose myeloablative therapy [62]. This syndrome is sometimes called veno-occlusive disease, but this is an inaccurate descriptor of the underlying pathobiology. This liver injury is initiated by changes in hepatic sinusoids, and occlusion of hepatic venules is not essential to the development of clinical signs and symptoms [63]. Incidence As SOS is caused by toxins in certain conditioning regimens, and not by the transplant process per se, the reported incidence varies with the composition and intensity of the conditioning regimen, from zero after most reduced-intensity regimens [29] to 50% after CY plus total body irradiation (TBI) of more than 14 Gy [35]. Other important contributors to variability in the incidence of SOS are large variations in the metabolism of CY from patient to patient [64,65] and underlying fibroinflammatory liver diseases (for example, chronic viral hepatitis, nonalcoholic steatohepatitis, cirrhosis, and sinusoidal fibrosis related to extramedul-

The diagnosis of SOS rests on the findings of tender hepatomegaly, weight gain, and jaundice following conditioning therapy, in the absence of other explanations for these signs and symptoms (Table 95.3) [35]. Cholestatic liver injury related to cytokine effects on bilirubin transport by hepatocytes may occur during episodes of neutropenic fever before engraftment, and thus be confused with SOS. The most common combinations of illnesses that mimic SOS are (1) sepsis syndrome requiring large volumes of crystalloid, followed by renal insufficiency and sepsisrelated cholestasis; (2) cholestatic liver disease, hemolysis, and weight gain related to total parenteral nutrition; and (3) hyperacute GVHD and sepsis syndrome. SOS may also coexist with these disease processes. The onset of SOS is heralded by an increase in liver size, right upper quadrant tenderness, renal sodium retention, and weight gain, occurring 10–20 days after the start of CY-based cytoreductive therapy [35] and later after other myeloablative regimens [68–70]. Patients then develop hyperbilirubinemia, usually before day 20 [35]. Other clinical manifestations of portal hypertension may strongly suggest SOS (Table 95.3). Some authors have described a syndrome of “late veno-occlusive disease” following conditioning with BU-containing regimens, where signs of liver disease are first recognized after day 30 after transplantation [70]. After HCT, treatment of relapsed acute myeloid leukemia (AML) with gemtuzumab ozogamicin may also result in SOS [38,71]. Measurement of total serum bilirubin is a sensitive test for SOS but not a specific one, as there are many causes of jaundice after HCT (Table 95.2). Elevations of serum aspartate aminotransferase (AST) and ALT can occur late in the course of SOS, reflecting ischemic hepatocyte necrosis from sinusoidal fibrosis (Plate 27.12), as peak AST/ALT levels (sometimes over 1500 U/L) are seen weeks after toxin exposure. Several plasma proteins have been reported to be abnormally high in patients with SOS, including endothelial cell markers (hyaluronic acid, von Willebrand factor, plasminogen activator inhibitor-1, and tissue plasminogen activator), thrombopoietin, proinflammatory cytokines, vascular endothelial growth factor, and procollagen peptides; some laboratory tests are abnormally low in patients with SOS, including the anticoagulant proteins protein C and antithrombin III and platelet counts (reviewed in [62]). Maximum levels of plasminogen activator inhibitor-1 less than 120 ng/mL have a strong negative predictive value for the diagnosis of SOS and may be useful in excluding this diagnosis [72]. It is not clear whether any other laboratory tests have diagnostic or prognostic utility beyond the clinical criteria of weight gain, jaundice, and hepatomegaly. Imaging studies of the liver are useful for demonstrating hepatomegaly, ascites, periportal edema, attenuated hepatic venous flow, and gall bladder wall edema consistent with SOS [73–75], as well as

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Table 95.2 Liver diseases after hematopoietic cell transplant Disease

Frequency

Timing

Diagnosis

Sinusoidal obstructive syndrome

10–35% (regimen dependent)

Onset before day 20

Cholestasis of sepsis (cholangitis lenta)

Common in neutropenic patients

Acute GVHD

10–20% of allograft recipients Rarely after autograft Uncommon when prophylaxis is used against herpes viruses or hepatitis B

Following sepsis or neutropenic fever (usually before day 30) Day 15–50

Typical clinical features plus exclusion of other causes of jaundice and weight gain Imaging (Doppler ultrasound or CT) Transvenous measurement of wedged hepatic venous pressure gradient and liver biopsy Note atypical presentations (acute hepatitis, anasarca) Exclude other causes of cholestasis Inferential diagnosis in a patient with cholestatic jaundice Confirm GVHD in skin and gut Exclude other causes of cholestasis Liver biopsy Pretransplant blood tests (antigen, antibodies, PCR results) Isolation of virus from other sites (stool and urine for adenovirus) PCR of serum for specific viruses Liver biopsy, histology/PCR/immunostatins Hepatic pain, fever Liver imaging (MRI > CT) Serum fungal antigen(s) Hepatic pain, fever Liver imaging Liver biopsy, culture Clinical evidence linking elevations of serum ALT or alkaline phosphatase to drugs known to cause liver injury Clinical evidence linking shock to subsequent rises in serum ALT

Acute viral hepatitis

HSV, day 20–50 Adenovirus, day 30–80 VZV, day 80–250 HBV and HCV, during immune reconstitution

Fungal abscess

Rare when prophylaxis is used

Day 10–60

Bacterial infection

Rare (pyogenic abscess, mycobacterial infection) Common

Day 10–80

Confined to patients with septic or hemorrhagic shock Transient biliary sludge, common Stones or chloromas, rare Rare

Day 0–30

Formerly common

After day 80

Iron overload

Very common

Pre transplant Long-term follow-up after transplant

Chronic GVHD

Common after allografts

After day 80

Nodular regenerative hyperplasia

Rare complication of toxic liver injury

Years after transplant

Drug liver injury

Ischemic liver disease Biliary obstruction

Idiopathic hyperammonemia Chronic hepatitis C

Day 0–100

Day 15–60

History, examination Biliary ultrasound ERCP > magnetic resonance cholangiography

Day 10–50

Unexplained confusion, coma Venous blood ammonia HCV RNA in serum Elevation of serum ALT after immune reconstitution Transferrin saturation Marrow iron quantitation Liver iron quantitation (FerriScan MRI, liver biopsy quantitation) Prior acute GVHD history Chronic GVHD in other organs Consistent elevations of serum ALT and alkaline phosphatase Liver biopsy Signs of portal hypertension but preserved liver function Liver biopsy histology (reticulin stain), laparoscopic appearance of the liver

ERCP, endoscopic retrograde cholangiopancreatography. See text for other abbreviations.

Gastrointestinal and Hepatic Complications Table 95.3 Clinical, laboratory, and imaging manifestations of SOS Usually present

May be present

Hepatomegaly (change from baseline) Weight gain (usually abrupt) Jaundice

Low urine sodium concentration or fractional excretion of sodium Peripheral edema, ascites, anasarca Elevation of serum alanine aminotransferase Gall bladder wall edema on ultrasound, pain over the gall bladder fossa Isolated weight gain Isolated jaundice Thrombocytopenia Appearance of esophageal varices Pleural effusions, pulmonary vascular congestion, hypoxia Acute renal failure

excluding biliary dilation or infiltrative lesions in the liver and hepatic veins that might also explain hepatomegaly and jaundice. Other abnormal findings may include an enlarged portal vein diameter, slow or reversed flow in the portal vein or its segmental branches [76], high congestion index, portal vein thrombosis, and increased resistive index to hepatic artery flow. Unfortunately, ultrasound findings very early in the course of SOS do not appear to add to the information provided by clinical criteria [77]; however, Doppler evaluation of flow in segmental branches of the portal vein can be useful [76]. Later in the course of SOS, especially in patients with severe disease, ultrasound evidence of altered liver blood flow, particularly reversal of portal flow and portal vein thrombosis, is more common [77]. There may be value in following vascular parameters as indices of improvement in sinusoidal blood flow. Liver biopsy and measurement of the hepatic venous pressure gradient In cases where the cause of liver dysfunction is unclear, a transvenous approach that allows both biopsy and hepatic venous pressure measurements is the most accurate diagnostic test [78,79]. Laparoscopic needle biopsy is an alternative method of obtaining liver tissue [80]. Percutaneous biopsy poses a high risk of bleeding in the thrombocytopenic patient, whereas transvenous biopsy methods can be done safely with platelet counts as low as 30,000/mm3. In HCT patients, a hepatic venous pressure gradient (use of an occlusive balloon technique [81] is essential here) above 10 mmHg is highly specific for SOS [78,79]. Initial histologic changes of SOS are dilation of sinusoids, extravasation of red cells through the space of Disse, necrosis of perivenular hepatocytes, and widening of the subendothelial zone in central veins (Plates 27.11, 27.13, 27.14, and 27.15) [62,63]. A finding of “hemorrhage” in zone 3 of the liver acinus is the result of destruction of sinusoidal endothelium (Plate 27.14). In severe SOS, fragmented hepatocyte cords can be seen, with dislodgement of clusters of hepatocytes (Plate 27.15) into portal veins through disrupted pores into lumina of damaged central veins. The later stages of SOS are characterized by extensive collagenization of sinusoids with a variable degree of obstruction of venular lumens by collagenized walls, leading to an obliteration of sinusoidal blood flow (Plates 27.10, 27.12, and 27.16). In severe SOS – if patients survive beyond day 50 post transplant – a pattern of reverse cirrhosis may develop, with extensive linkage between obliterated central venules by fibrous bridges, collapse, and acinar extinction.

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Clinical course and prognosis The severity of SOS has been classified as mild (SOS that is clinically obvious, requires no treatment, and resolves completely), moderate (SOS that causes signs and symptoms requiring treatment such as diuretics or pain medications, but that resolves completely), or severe (SOS that requires treatment but that does not resolve before death or day 100). There is a range of clinical and laboratory findings that correspond to these operational definitions of disease severity [35]. Some patients have subclinical liver damage, evinced by histologic signs of liver toxicity in the absence of clinical signs and symptoms. Published case fatality rates for SOS vary widely, depending on whether all cases of jaundice or only biopsy-proven cases are classified as SOS [82]. Recovery from SOS was seen in over 70% of patients whose SOS followed CY-containing regimens, and in 84% when SOS was caused by other alkylating agents [35,69]. Despite deep jaundice, patients with severe SOS seldom die of liver failure, but rather from renal and cardiopulmonary failure [35,83,84]. A clinically useful model has been developed that predicts the outcome of SOS after CY-based regimens, derived from rates of increase of both bilirubin and weight in the first 2 weeks following HCT [85]. In some patients, there is a bimodal presentation of SOS; that is, clinical signs of SOS appear in the first 2 weeks post HCT, then wane, and then reappear later, probably related to stellate cell proliferation and collagenization of sinusoids. This clinical pattern is associated with a worse prognosis. In some cases, signs of SOS resolve, but ascites later recurs following development of inflammatory liver disease (e.g. GVHD). A poor prognosis correlates with overt signs of portal hypertension, hepatocyte necrosis, and renal and pulmonary failure. Pathogenesis of SOS: insights from animal models The use of animal and in vitro models has clarified the cellular mechanisms of toxic sinusoidal injury (reviewed in [62]). Sinusoidal endothelial cells are more susceptible than hepatocytes to drugs that cause SOS in patients, notably CY. In animals exposed to toxin, the first morphologic change is loss of sinusoidal endothelial cell fenestration, appearance of gaps in the sinusoidal endothelial cell barrier, rounding up of sinusoidal endothelial cells, and penetration of red cells into the space of Disse [86]. Sloughed sinusoidal lining cells, that is, Kupffer cells, sinusoidal endothelial cells, and stellate cells, embolize downstream and obstruct sinusoidal flow. By the time hepatocyte necrosis is observed, there is extensive denudation of the sinusoidal lining. Drugs and toxins that cause SOS profoundly deplete sinusoidal endothelial cell glutathione (GSH) prior to cell death, and support of sinusoidal endothelial cell GSH prevents cell death [87]. One possible explanation for the rounding up of sinusoidal endothelial cells may be increased activity of matrix metalloproteinases (MMPs), as inhibition of MMP activity completely prevents SOS in animal models [88]. Although SOS is defined as a nonthrombotic obstruction of blood flow, the issue of thrombosis has been a recurring topic of research interest, based largely on plasma studies in patients with SOS and an immunohistologic study [89]. Sequential observations during the development of SOS in the animal model have not demonstrated any evidence of thrombosis, perhaps because of obliteration of sinusoidal endothelial cells. Pathogenesis of SOS: clinical studies The proximate cause of SOS is the conditioning regimen, specifically chemotherapy drugs or irradiation that damage sinusoidal endothelium. Two observations about SOS in patients provide clues as to the mechanism of disease. First, unlike other intrinsic liver diseases, the signs and symptoms of portal hypertension precede evidence of parenchymal damage. In SOS, disruption of the liver circulation (Plate 27.14) is the cause and not the consequence of the parenchymal disease. Second,

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involvement of the hepatic veins is not essential to the development of the clinical picture: 45% of patients with mild or moderate disease and 25% of patients with severe SOS did not have occluded hepatic venules at autopsy [63]. Occlusion of central veins of the liver lobule (Plates 27.10 and 27.12) is associated with more severe disease and the development of ascites. This finding suggests that occlusive lesions involving the central veins, a later development, may exacerbate the acute circulatory impairment that occurs at the level of the sinusoid. Chemotherapy drugs CY is common to the conditioning regimens with the highest incidence of fatal SOS: CY/TBI, BU/CY, and BCV (BCNU, CY, and etoposide). The metabolism of CY is highly variable; patients who generate a greater quantity of toxic metabolites are more likely to develop severe SOS following conditioning with CY and TBI [64]. The liver toxin generated by CY metabolism is acrolein (a metabolite formed simultaneously along with the desired metabolite, phosphoramide mustard), via mechanisms dependent upon GSH. Exposure to one CY metabolite (carboxyethyl phosphoramide mustard [CEPM]) was significantly related to SOS, bilirubin elevation, nonrelapse mortality, and survival in patients receiving conditioning with CY/TBI, but there was no relationship to either engraftment or tumor relapse [64]. These data suggest that a strategy that targets the dose of CY on the basis of a patient’s metabolism will substantially reduce the risk of fatal SOS, without jeopardizing engraftment. Accurate methods to target the dose of CY to a metabolic endpoint, thereby eliminating variable exposure to liver toxins, have been developed [65,90]. BU is another component of regimens with a high frequency of SOS, but BU itself is not hepatotoxic [91,92]. A relationship between BU exposure (measured by area under the curve or average steady-state – concentration, C ss, Bu) following oral dosing in the BU/CY regimen has been reported; however, in adults with chronic myeloid leukemia in chronic phase and children with acute leukemia, there is no correlation between BU exposure and SOS [93,94]. BU may contribute to liver injury by inducing oxidative stress, reducing GSH levels in hepatocytes and sinusoidal endothelial cells [91] and altering CY metabolism [66]. Total body irradiation The doses of TBI given in the setting of HCT are in the range of 10–16 Gy, far less than the dose that causes radiationinduced liver disease. In combination with CY, however, there is a clear relationship between the total dose of TBI and the frequency of severe SOS [64]. The frequency of severe SOS is approximately 1% after CY/ TBI 10 Gy [95], 4–7% after CY/TBI 12–14 Gy [64,96], and 20% after CY/TBI greater than 14 Gy [35]. The synergism between CY and TBI in causing sinusoidal injury may be due to the fact that both agents cause depletion of hepatic reduced GSH as well as sublethal damage to sinusoidal endothelial cells. The order of administration (CY/TBI versus TBI/ CY) does not appear to affect the frequency of liver injury. Gemtuzumab ozogamicin Gemtuzumab ozogamicin specifically targets leukemia cells expressing the CD33 receptor by means of a humanized antibody conjugated to a modified cytotoxic agent, calicheamicin. Gemtuzumab ozogamicin may cause sinusoidal liver injury when used to treat patients with AML [38]. Pretransplant sinusoidal liver injury is a risk factor for SOS; thus, the risk of SOS is 15–40% when high-dose gemtuzumab ozogamicin is given in proximity to a CY-containing myeloablative conditioning regimen [38,97]. Lower doses of gemtuzumab ozogamicin appear to eliminate this risk. The drug may also cause SOS when given after transplant for relapsed AML [38,71]. The clinical presentation is one of acute portal hypertension, ascites, weight gain, elevations of serum AST/ALT, and moderate jaundice, with histology showing intense sinusoidal fibrosis and centrilobular hepatocyte necrosis (Plate 27.1) [70].

Intrahepatic coagulation Some see SOS as a disease of disordered coagulation, in which damage to endothelium in the sinusoids and central veins leads to exposure of tissue factor and initiation of the coagulation cascade. However, sinusoidal endothelial cells embolize downstream in SOS, heparin and antithrombin III infusions are ineffective in preventing fatal SOS, and thrombolytic therapy effects improvement in only a minority of patients. Genetic disorders predisposing to coagulation (factor V Leiden and prothrombin gene 20210 G-A) have no associations with SOS after HCT. Current evidence suggests that disordered coagulation in SOS is an epiphenomenon secondary to widespread centrilobular damage (Plate 27.11), and not a cause of sinusoidal damage. However, thrombosis in the portal vein may result from a hypercoagulable state in patients with severe SOS [98]. Stellate cells and sinusoidal fibrosis Several series have documented the early appearance of a procollagen peptide in serum of patients who develop more severe SOS [99], along with inhibitors of fibrolysis, consistent with the intense fibrosis in centrilobular sinusoids and venular walls that is common in fatal SOS [63]. Immunohistology of liver specimens from patients with SOS for alpha-actin (a marker for stellate cell activation) shows intense staining in sinusoids (Plate 27.16) [62]. The stimuli for stellate cell activation and proliferation in the HCT setting have not been identified; candidates include centrilobular hypoxia, endotoxemia, Kupffer cell damage, loss of sinusoidal endothelial cells, and a melange of circulating growth factors and cytokines [100]. Genetic factors Genetically determined differences in drug metabolism or susceptibility to toxic injury might explain some of the variability in the frequency of SOS. Case-control studies using single-nucleotide polymorphisms have reported associations between SOS and carbamyl phosphate synthetase-1 c.4340C>A (CPS1), factor V c.1691G>A (FV Leiden), HFE C282Y, and glutathione S-transferase (GSTM1 and GSTT1) genes [101–103]. These associations could not be confirmed in a cohort of Seattle patients receiving a uniform conditioning regimen (CY/TBI). Patients with Gilbert’s syndrome, a common polygenic disorder that leads to unconjugated hyperbilirubinemia, have defective hepatic glucuronidation; drugs that are normally excreted in bile as glucuronides may cause increased toxicity in patients with Gilbert’s syndrome [104]. Recently, the genetic cause of a naturally occurring form of hepatic vascular damage that is indistinguishable from SOS at the histologic level has been described in kindreds with SOS with immunodeficiency syndrome that results from truncating mutations in SP110 [105]. When patients conditioned with CY/TBI were studied, six singlenucleotide polymorphisms, as well as one haplotype within the SP110 locus, reached nominal significance, but none was significant after correction for multiple testing. Detection of genetic polymorphisms that lead to SOS will require more highly powered studies. Prevention of SOS in patients receiving myeloablative therapy Prevention of severe sinusoidal liver injury begins with an assessment of the risk of hepatotoxicity from a given myeloablative conditioning regimen. Table 95.4 lists underlying liver disorders that increase the risk of severe sinusoidal liver injury following CY-based conditioning regimens. Patients at risk have several options: (1) conventional therapy that does not involve HCT; (2) a reduced-intensity conditioning regimen [29]; (3) a myeloablative regimen that does not contain CY, for example targeted BU and fludarabine for allogeneic [106,107] or BCNU, etoposide, cytosine arabinoside, and melphan (BEAM) [108] for autologous HCT; (4) modification of CY-based regimens [65,90]; and (5) use of pharmacologic approaches to prevent sinusoidal liver injury. The transplant oncologist must achieve a balance between the optimal

Gastrointestinal and Hepatic Complications Table 95.4 Liver disorders that increase the risk of severe sinusoidal liver injury following cyclophosphamide-based regimens Necroinflammatory disorders Chronic hepatitis B or C Nonalcoholic steatohepatitis Alcoholic hepatitis Fibrotic disorders Cirrhosis Lobular fibrosis Extramedullary hematopoiesis with sinusoidal fibrosis Recent exposure to gemtuzumab ozogamicin Prior liver irradiation Cholestatic disorders Jaundice caused by intrahepatic cholestasis Dubin-Johnson syndrome

conditioning regimen for a given malignancy and its potential for fatal liver toxicity in a given patient. Patients with cirrhosis, for example, may decompensate even after a reduced-intensity allograft [29]. If a CY/TBI regimen must be used for a patient at risk for fatal SOS, modifications should be considered for both CY and TBI dosing, with the understanding that clinical trials to prove efficacy and safety have not been done. The total dose of CY should be in the range 75–100 mg/ kg [65], and TBI doses should not exceed 12 Gy [64,68]. Shielding the liver during TBI will lessen liver injury but leads to relapse of underlying hematologic disease [109]. Accurate methods are available to target CY doses to a metabolic endpoint, based on exposure to the CY metabolites 4-HCY and CEPM [65,90]. If a BU/CY regimen must be used for a patient at risk for fatal SOS, liver toxicity may be less frequent if CY is given before targeted BU [110] (this requires intravenous BU, as oral BU is poorly tolerated after CY infusions), or if dosing of CY is delayed for 1–2 days after completion of BU [111]. BU (and the use of phenytoin to prevent seizures) has marked effects on CY metabolism when CY is given second in order; that is, there is increased exposure to 4-HCY, phosphoramide mustard, and acrolein compared with giving CY first in order [66]. BU itself is not a significant sinusoidal liver toxin [91,92], but appears to contribute to SOS by causing depletion of reduced GSH in the liver, thus making sinusoidal endothelial cells more susceptible to injury from metabolites of CY or TBI [87,112]. It is not clear whether intravenous BU offers any advantage over targeted oral BU with regard to liver toxicity from a BU/CY regimen. A lower incidence of SOS has been reported following intravenous BU/CY, compared with oral BU/CY, when neither BU formulation was adjusted for metabolism [113]. There are no studies that examine the frequency of severe SOS following intravenous versus oral BU/CY when both are dosed to the same steady-state concentration (e.g. 900 ng/mL) [93]. The metabolism of intravenous BU is variable, with a several-fold range in area under the curve for BU, a problem that can be addressed only with therapeutic drug monitoring [114]. Liver toxicity has remained a complication of conditioning with both targeted oral BU/CY [66,115,116] and weight-based dosing of intravenous BU/CY [113,117,118]. Lower hepatic exposure to BU may indeed reduce the frequency of fatal SOS following BU/CY regimens by causing less extreme reductions in hepatic GSH, and not because BU is a liver toxin. In patients with no identifiable risk factors for severe SOS who receive a CY-based myeloablative regimen, fatal SOS develops in around 7% of cases following CY/TBI 12–14 Gy [64] and in less than 5% of cases

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following targeted oral BU/CY [66]. The unpredictability of liver toxicity after CY-based regimens is related to aberrant CY metabolism [64], in turn likely related to polymorphisms in hepatic enzymes or transporters. The only certain way to prevent fatal SOS is to avoid giving conditioning therapy that damages hepatic sinusoidal cells, that is, CY and TBI. Substitution of fludarabine for CY (e.g. a BU plus fludarabine regimen) appears to avoid sinusoidal liver damage [106,107]. Use of drugs to prevent SOS has been achieved in animal models of liver injury (reviewed in [62]), but these strategies have not been proved in the clinical setting. There may be value in prophylaxis of SOS with repletion of intracellular GSH [87], inhibition of MMP enzymes [88] or infusion of defibrotide [119,120]. Adequately powered clinical trials of these modalities with proper stratification for risk of fatal outcomes have not been reported. Prospective studies have shown no benefit from use of heparin [121] or antithrombin III [122] for prevention of fatal SOS. A meta-analysis suggests that ursodiol may prevent SOS [67], but SOS was not differentiated from cholestatic liver disease in these studies [82], and the largest randomized trial showed no effect of ursodiol on the frequency of SOS [60]. Treatment of patients with SOS For the 70–85% of patients with SOS who will recover spontaneously, treatment involves management of sodium and water balance with diuretics, preservation of renal blood flow, and repeated paracenteses for ascites that is associated with discomfort or pulmonary compromise. Patients with a poor prognosis can be recognized soon after disease onset by steep rises in total serum bilirubin and body weight, serum ALT values greater than 750 U/L, portal pressures over 20 mmHg, development of portal vein thrombosis, and multiorgan failure requiring dialysis or mechanical ventilation [59,83–85]. There are no satisfactory therapies for severe SOS; the best current results (45% response) are with intravenous defibrotide (25 mg/kg/day) [123]. Defibrotide, a polydisperse mixture of single-stranded phosphodiester oligodeoxyribonucleotides obtained from controlled depolymerization of porcine intestinal mucosal genomic DNA, induces antithrombotic and profibrinolytic effects in vitro and in vivo. However, its mechanism of action in the treatment of SOS is not known [124]. Clinical responses to defibrotide infusions may take weeks to be seen. Although randomized, placebo-controlled trials of defibrotide for therapy of severe SOS have not been carried out, the complete recovery of some patients with severe SOS and multiorgan failure suggests that the drug has biologic effects in man [123,125]. Numerous other approaches to treatment of severe SOS have been reported over the last 20 years, but none can be currently recommended. Thrombolytic therapy with tissue plasminogen activator was ineffective in patients with SOS and organ failure, and resulted in fatal bleeding. Other reported therapies have included intravenous N-acetylcysteine, human antithrombin III concentrate, activated protein C, prostaglandin E1, prednisone, topical nitrate, vitamin E plus glutamine, and use of a liver-assist device. Transhepatic shunts have been placed in patients with SOS to reduce portal pressure and mobilize ascites, but neither serum bilirubin levels nor patient outcomes were improved [126]. In one case, a transjugular intrahepatic portosytemic shunt led to acute respiratory distress syndrome that was fatal [127]. Patients with SOS have undergone successful portosystemic shunts for persistent ascites, but liver dysfunction had resolved long before these shunts were placed. Peritoneovenous shunts for intractable ascites have been unsuccessful. Successful liver transplants for severe SOS have been reported [128]. When severe SOS develops in a patient with a benign condition (a rare event) or in a patient with a favorable outcome post HCT (e.g. chronic myelogenous leukemia in chronic phase), liver transplantation should be considered. Prevention of sinusoidal injury is likely to be a more effective strategy for improving transplant outcomes than treatment.

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Cholestatic disorders Cyclosporine inhibits canalicular bile transport and commonly causes mild increases in serum bilirubin without an effect on serum ALT or alkaline phosphatase [129]. Tacrolimus less commonly causes cholestasis, except in the setting of toxic blood levels. Sepsis-associated cholestasis is an important contributor to hyperbilirubinemia in the weeks after HCT, mediated by endotoxins, IL6, and tumor necrosis factor-alpha [130,131]. Jaundice may be progressive and marked in patients with ongoing sepsis, particularly when other cholestatic disorders are present. Many drugs used after HCT have been associated with liver dysfunction (e.g. trimethoprim–sulfamethoxazole, itraconazole, voriconazole, fluconazole, posaconazole, and ribavirin), although drugs are usually not responsible for severe liver injury in this setting. Prolonged parenteral nutrition may also contribute to cholestasis. Acute GVHD is the most common cause of severe cholestatic injury, as alloreactive T-cells recognize foreign major and minor histocompatability antigens as well as adhesion molecules expressed on biliary epithelial cells. Hepatic GVHD usually follows cutaneous and/or intestinal GVHD, and is heralded by a gradual rise in serum bilirubin, alkaline phosphatase, and aminotransferase enzymes. However, a blinded histologic study could identify no features characteristic of GVHD when biopsies were done within several weeks of the onset of GVHD [132], suggesting that jaundice occurring early after the onset of GHVD is related to cholestasis caused by exposure to cytokines such as IL-6 [49]. In allograft recipients on minimal immunosuppression or after donor lymphocyte infusion, GVHD may also present as an acute hepatitis (Plate 27.39) with marked elevation of serum ALT [133–135]. A cholestatic condition identical to GVHD occurs rarely in autologous HCT recipients. Characteristic liver biopsy findings in GVHD include lymphocytic infiltration of small bile ducts with nuclear pleomorphism and epithelial cell drop-out (Fig. 27.6 and Plate 27.38). Because these patients are frequently receiving immunosuppressive therapy, inflammatory infiltrates may be minimal, and bile duct epithelial changes may be the only abnormality. Severe, ongoing hepatic GVHD and worsening jaundice are associated with progressive destruction of small bile ducts (Fig. 27.7 and Plate 27.41). Only 30% of patients with liver GVHD have resolution of liver abnormalities after initial immunosuppressive treatment. Prophylactic ursodeoxycholic acid reduces the frequency of cholestasis in general and GVHD-related cholestasis specifically, compared with placebo, and it is recommended that all allograft recipients be routinely treated through day 80 post transplantation [60]. Over 50% of patients with acute liver GVHD will develop chronic GVHD.

Acute hepatitis In most cases, a sudden rise in the serum aminotransferase enzymes (AST and ALT) following HCT is due to a noninfective cause such as zone 3 hepatocyte necrosis in SOS, ischemic hepatopathy (septic shock), acute biliary obstruction (choledocholithiasis), drug-induced liver injury, or the acute hepatitic presentation of GVHD [133–135]. If a likely cause is not apparent, acute viral hepatitis should be suspected, as early diagnosis and treatment may prevent a fatal outcome. Serum ALT levels in patients with severe SOS peak at day 23 on average, with extreme elevations indicating a poor prognosis. Acute hepatitis caused by HSV, VZV (Plate 27.58), adenovirus (Plate 27.59), and HBV can lead to fatal fulminant hepatic failure after HCT [20,136–140], whereas hepatic infections caused by CMV and HCV are seldom severe [27]. With routine use of prophylactic acyclovir/valacyclovir, acute hepatitis due to HSV and VZV is now rare; however human herpesvirus-6 (HHV-6) and HHV-8 reactivation causing hepatitis despite

prophylaxis has been reported [141]. When there is uncertainty about the cause of rising serum ALT and AST levels, DNA blood tests for herpesviruses, adenovirus, and HBV should be performed. Transvenous measurement of the wedged hepatic venous pressure gradient can exclude acute portal hypertension due to SOS, and transvenous liver biopsy (via the transjugular or via femoral vein) may demonstrate a specific diagnosis (Plates 27.58 and 27.59). If acyclovir is not being given, it should be started empirically, particularly if the patient presents with abdominal complaints typical of VZV infection [142]. Adenovirus hepatitis should be suspected if the patient has concomitant pulmonary, renal, bladder or intestinal symptoms; the most effective treatment is cidofovir when given early in the course of infection [137,143,144]. Fulminant hepatitis B may develop during immune reconstitution in patients at risk, but can be prevented with prophylactic antiviral agents [20,145]. If severe hepatitis B reactivation does occur, usually because a diagnosis of HBV was not made prior to HCT [32], antiviral therapy with the most potent antiviral drug available (lamivudine, entecavir or telbivudine or tenofovir) should be initiated immediately; however progression to fatal liver failure is not uncommon [146]. Fulminant hepatitis B has also been reported following discontinuation of prophylactic antiviral therapy, and all patients, particularly those with high pretransplant HBV DNA levels, should be monitored following antiviral drug withdrawal [147,148]. Chronic hepatitis C in HCT recipients usually results in asymptomatic elevation of ALT from days 60 to 120, coinciding with the tapering of immunosuppressive drugs [27]. Severe hepatitis due to HCV has only rarely been reported, and specific antiviral therapy for HCV is not indicated early after HCT. Therapy directed at chronic HCV infection should be considered once the patient has ceased all immunosuppressive drugs and has no evidence of active GVHD [149]. Fungal and bacterial infections Antifungal prophylaxis with fluconazole, itraconazole, and more recently posaconazole has had a significant impact on the incidence of invasive fungal disease in HCT recipients, particularly in those requiring ongoing immunosuppression for treatment of GVHD [17,150–152]. If invasive fungal disease does occur, infection with resistant Candida species or molds may involve the liver [150]. Hepatic infection should be suspected in the presence of fever, tender hepatomegaly, and increased serum alkaline phosphatase levels. High-resolution CT scan or MRI may demonstrate multiple fungal abscesses, and serologic tests for fungal antigens may be useful for diagnosis. Treatment courses may need to be protracted in immunosuppressed patients with visceral fungal infection, but return of neutrophil function after HCT can effect resolution of previously treatment-refractory mold infection [19]. Bacterial liver abscesses are rare in HCT recipients, probably because of the high use of systemic antibiotics; however, latent mycobacterial infection may reactivate within the liver with prolonged immunosuppressive therapy. Disseminated Bacille Calmette-Guerin infection with liver involvement has been reported. Disseminated clostridial infection and gall bladder infection with gas-producing organisms may lead to air in the liver and biliary system. Gall bladder and biliary disease Biliary sludge (calcium bilirubinate and crystals of calcineurin inhibitors) is a common finding in HCT recipients, being identified by ultrasound in approximately 70% and at autopsy in 100% of patients [153]. Biliary sludge is usually asymptomatic; however, passage down the bile duct may cause epigastric pain, nausea, and abnormal serum liver enzymes. Biliary sludge may be a cause of acute “acalculous” cholecystitis, acute

Gastrointestinal and Hepatic Complications

pancreatitis, and bacterial cholangitis [154–156]. Acute cholecystitis is uncommonly seen in HCT recipients and is frequently acalculous [157]. Cholecystitis in this setting may also be due to leukemic relapse with gall bladder involvement or infection by CMV or fungi. Diagnosis is difficult because of the high frequency of gall bladder abnormalities on ultrasound following HCT. Pericholecystic fluid, gall bladder wall necrosis or localized tenderness suggest cholecystitis. A radionuclide bile excretion study, with morphine infusion to enhance gall bladder filling, can be useful: nonvisualization of the gall bladder suggests cholecystitis. Biliary obstruction is a rare event, caused by a variety of disorders (e.g. common bile duct calculi or inspissated biliary sludge, GVHD of the ampullary mucosa; lymphoblastic infiltration of the common bile duct, gall bladder, and ampulla of Vater in Epstein–Barr virus (EBV) lymphoproliferative disease (Plate 27.3); CMV-related biliary disease; dissecting duodenal hematoma complicating endoscopic biopsy; and leukemic relapse [chloroma] in the head of the pancreas) [155,156]. Noninvasive imaging (ultrasound, magnetic resonance cholangiopancreatography or CT cholangiography) is usually adequate to demonstrate biliary pathology, and therapeutic endoscopic retrograde cholangiopancreatography should be required only in patients with clinical evidence of cholangitis following HCT and radiologic evidence of extrahepatic biliary obstruction [156]. Malignant disorders EBV lymphoproliferative disease is now an infrequent complication of HCT, largely because of EBV DNA surveillance and pre-emptive treatment. The highest incidence is in recipients of HLA-mismatched, T-celldepleted grafts and those receiving potent anti-T-cell therapies for GVHD. Symptoms include fever, sweats, generalized malaise, enlarged tonsils, and cervical lymphadenopathy with liver involvement (Plate 27.4) occurring in over 50%, manifest by abnormal serum alkaline phosphatase and massive hepatosplenomegaly. Recurrent cancer in patients transplanted for hematologic malignancy or solid tumors may present with abnormal liver enzymes, hepatomegaly or abnormal imaging studies within the first year after HCT. In the absence of disease elsewhere, fine-needle aspiration or needle biopsy will usually be required for definitive diagnosis. Idiopathic hyperammonemia and coma A syndrome of hyperammonemia and coma has been described in patients who received high-dose chemotherapy, including conditioning for HCT [158]. Patients present with progressive lethargy, confusion, weakness, incoordination, vomiting, and hyperventilation with respiratory alkalosis. The diagnosis is confirmed when the plasma ammonia exceeds 200 μmol/L and there is no evidence of liver failure. This syndrome is rare, but is associated with a high mortality. The pathogenesis of idiopathic hyperammonemia likely involves the unmasking of a latent genetic disorder similar to ornithine transcarbamylase deficiency in children [159,160]. Gastrointestinal bleeding Bleeding that does not require transfusion is very common, particularly when platelet counts are low. Causes include retching trauma to esophageal or gastric mucosa, mucosal injury from conditioning therapy, peptic esophagitis, C. difficile colitis, anal fissures, hemorrhoids, and acute GVHD. The incidence of severe gastrointestinal bleeding after HCT is 1–2% – lower than in the past because of effective prophylaxis against viruses, fungi, and acute GVHD [161]. Mortality from severe intestinal bleeding, however, remains at 40% [161,162].

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Table 95.5 Differential diagnosis of gastrointestinal bleeding after transplant Infectious causes

Noninfectious causes

Cytomegalovirus ulcers Fungal infection (usually molds) Adenovirus infection Clostridial infection (C. difficile, C. septicum) Varicella zoster virus ulcers (esophagus, stomach) Helicobacter pylori-related ulcers Epstein–Barr virus lymphoproliferative disease Herpes simplex virus esophagitis

Mucosal necrosis from conditioning therapy Retching trauma (Mallory–Weiss tear) Acute/chronic graft-versus-host disease Lysis of intramucosal tumor Peptic esophagitis Gastric antral vascular ectasia (may also involve other sites) Mucosal biopsy sites Mycophenolate mofetil ulcerations Amyloidosis (mucosal ischemia)

Infectious causes in bold are now so rare that they should be considered only when patients are at risk.

The most common cause of severe bleeding is refractory acute GVHD, with bleeding from extensive ulceration in the small intestine and cecum (Table 95.5 and Plate 27.32). In some patients with GVHD, bleeding may appear to be coming from specific areas of the mucosa, but when such patients are operated on or come to autopsy, diffuse rather than focal mucosal ulceration is the rule [163,164]. Ulcers in the stomach or duodenum that develop after HCT are usually caused by acute GVHD or CMV infection, but with ganciclovir prophylaxis, bleeding CMV ulcers have become rare [56,161]. Gastric ulcerations may also be caused by infection by VZV, bacteria (phlegmonous gastritis) or EBV (lymphoproliferative disease; see Plate 27.3). Gastric antral vascular ectasia is also a cause of severe upper intestinal bleeding in HCT recipients, particularly those who have received oral BU [165]. Diffuse areas of hemorrhage are seen in the gastric antrum and proximal duodenum, but the underlying mucosa is intact. Histology is diagnostic, revealing abnormal dilated capillaries, thromboses, and fibromuscular hyperplasia in the lamina propria. Patients who are transplanted for advanced systemic sclerosis may bleed from similar vascular lesions. Endoscopic laser therapy is the treatment of choice to control bleeding from vascular ectasia, but multiple laser treatments may be required to obliterate ectatic lesions [165]. Similar vascular ectasias can rarely be found in the small intestine and colon [166]. Other rare causes of bleeding post HCT include ulcers caused by molds [167,168], Dieulafoy lesions, Curling’s (stress) ulcers, duodenal biopsy sites, adenovirus colitis, and C. septicum infection (typhlitis) [161]. Patients with severe amyloidosis who undergo autologous HCT may bleed from multiple ischemic ulcers throughout the intestine. There is no effective therapy for mucosa that is diffusely oozing blood other than raising the platelet count and treating the underlying condition. Continuous infusion of octreotide at 25 μg/hour has resulted in cessation of bleeding from GVHD, but recurrent bleeding developed when infusion was decreased [169]. In GVHD, re-epithelialization of ulcerated intestinal mucosa is very slow. Focal lesions, especially mucosal infection, can be treated with endoscopic cautery, heater probe or epinephrine injection provided platelet counts are adequate, but these therapies are futile in mucosa that is oozing blood diffusely. Unless the underlying disease process is eliminated, endoscopic methods will not cure the bleeding problem. Attempts to resect large segments of diffusely bleeding intestine involved with GVHD have not been successful [163].

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Chapter 95

Table 95.6 Differential diagnosis of dysphagia, painful swallowing, and esophageal pain after transplant Infectious causes

Noninfectious causes

Cytomegalovirus Candida albicans Other fungal species (e.g. Candida glabrata, molds) Oropharyngeal bacteria (bacterial esophagitis) Herpes simplex virus Mycobacterium tuberculosis Varicella zoster virus

Acid–peptic reflux Intramural hematoma Pill esophagitis Postinflammatory esophageal stricture Acute GVHD of the esophagus Esophageal perforation Chronic GVHD of the esophagus (see section on problems in long-term transplant survivors)

Infectious causes in bold are now so rare that they should be considered only when patients are at risk. GVHD, graft-versus-host disease.

Dysphagia, painful swallowing, and esophageal pain Mucositis, acid–peptic esophagitis, and pill esophagitis are currently the leading causes of dysphagia and esophageal pain (Table 95.6). Infections of the esophagus (Plates 27.35 and 27.36) have largely disappeared because of antiviral and antifungal prophylaxis; when fungal esophagitis is discovered in a patient receiving fluconazole, the organism is likely to be a resistant candidal species or a mold. Rarely, fungal esophagitis can lead to perforation [170]. Mucositis caused by conditioning therapy may lead to pain on initiating a swallow and inability to move a bolus past the cricopharyngeus, symptoms that can also be seen with oropharyngeal GVHD and HSV infection. Rarely, nonhealing esophageal ulcerations, strictures, and dysphagia result from conditioning therapy [171]. In patients with gastric stasis related to acute GVHD, painful esophagitis is common, caused by reflux of both gastric acid and bilious fluid. The use of proton pump inhibitor therapy, while effective in treating acid–peptic reflux, may lead to colonization of the aerodigestive system with bacteria and fungi [172]. Symptom relief can be obtained by treating the cause of gastric stasis and placing the patient in the reverse Trendelenburg position when in bed. The abrupt onset of severe retrosternal pain, hematemesis, and painful swallowing suggests a hematoma in the wall of the esophagus, a result of retching when platelet counts are very low [173]. Endoscopy is relatively contraindicated, as many intramural hematomas represent contained perforations. The course of intramural hematomas is one of slow resolution over 1–2 weeks. In patients with severe GVHD, esophageal edema, erythema, and a peeling epithelium lead to ulcerations [174]. Pill esophagitis occurs after ingestion of some medications that might be used after HCT, for example phenytoin (dilantin), foscarnet, captopril, oral bisphosphonates, ascorbic acid, ciprofloxacin, clindamycin, and oral potassium chloride. Diarrhea Diarrhea caused by mucosal damage from conditioning therapy is seldom severe, usually resolving by day 12–15 (Plates 27.8 and 27.9). Cytarabine-containing regimens, high-dose melphalan (200 mg/m2), and regimens containing multiple alkylating agents may cause more severe, protracted diarrhea. Intravenous infusion of octreotide and oral loperamide (at 4 mg orally every 6 hours) may be effective for severe diarrhea associated with conditioning therapy [175]. There is a correlation between the amount of diarrhea in the week following completion of conditioning therapy and the risk of subsequent GVHD [176].

Table 95.7 Differential diagnosis of diarrhea after transplant Infectious causes

Noninfectious causes

Clostridium difficile Cytomegalovirus Adenovirus Rotavirus Clostridium septicum and other clostridial species Astrovirus, norovirus, other small round viruses Mycobacterial infection Fungal infection Giardia lamblia Cryptosporidia Microsporidia Strongyloides stercoralis Epstein–Barr virus lymphoproliferative disease Herpes simplex virus Bacterial enteric pathogens (e.g. Salmonella, Shigella, Campylobacter spp., Yersinia)

Residual effects of conditioning therapy Acute graft-versus-host disease Antibiotic-associated diarrhea Mycophenolate mofetil toxicity Promotility drugs Magnesium salts Carbohydrate malabsorption (e.g. genetic or acquired lactase, sucrase/isomaltase deficiency) Medication side-effect Pancreatic insufficiency Intestinal thrombotic microangiography

Infectious causes in bold are now so rare that they should be considered only when patients are at risk.

Acute GVHD is the most common cause of diarrhea after day 15 [162,177]. The onset of diarrhea can be sudden, with volumes in excess of 2 L daily in severe cases. GVHD in patients who were grafted after reduced-intensity conditioning regimens may have its presentation delayed, occurring after day 100 in many patients [178]. The diarrheal fluid of GVHD is watery, green in color, with ropy strands of mucoid material that reflect transmucosal protein loss [179]. In an allografted patient with skin and liver abnormalities typical of acute GVHD, this diarrheal syndrome is almost diagnostic of intestinal GVHD, particularly when there is falling serum albumin and negative stool studies for infection. In GVHD, abdominal imaging with CT or ultrasound may reveal intestinal edema, particularly in the ileum and right colon [75,180,181], but does not differentiate between CMV infection and acute GVHD. Pneumatosis intestinalis, which may be associated with GVHD or CMV enteritis, may be seen by plain X-ray, CT or MRI. A definitive diagnosis of GVHD in problematic cases requires mucosal biopsy. In mild cases, gastroduodenal and rectosigmoid mucosa are grossly normal, but moderately severe GVHD causes diffusely edematous and erythematous mucosa throughout the gastrointestinal tract [52]. Severe GVHD may lead to ulcerations and large areas of mucosal sloughing in the stomach, small intestine and colon [52,163]. Even when the appearance is normal, biopsies from the gastric antrum or rectosigmoid colon often reveal intestinal crypt cell necrosis and apoptotic bodies diagnostic of acute GVHD (Figs 27.2–27.4 and Plates 27.20 and 27.27–27.30) [117,182]. The diagnostic yield of mucosal biopsy is best when biopsies are obtained either from the stomach and distal colon or from the colon and ileum [182]. Reports by endoscopist and pathologist can be discordant, as mucosal edema and erythema are not criteria that are used for the histologic diagnosis of GVHD; visual inspection of the mucosa and histology should be viewed as complementary [52]. Other histologic findings that support the diagnosis of GVHD include pericapillary hemorrhage [183], infiltrating neutrophils [184] or eosinophils [185]. The use of capsule endoscopy for diagnosis of GVHD has been

Gastrointestinal and Hepatic Complications

described; this method does not allow biopsy, but can provide visual inspection of the small intestine that cannot be seen with routine endoscopy [186]. The predictive value of a negative capsule endoscopy examination of the small intestine appears to be high, and thus useful for excluding more severe acute GVHD [186]. In severe cases of GVHD, whole crypts are destroyed, then adjacent crypts, and finally whole segments of intestinal mucosa (Figs 27.3 and 27.4, and Plates 27.31 and 27.32). Bleeding often accompanies diarrhea in patients with mucosal ulceration [161]. Successful treatment of acute GVHD with immunosuppressive therapy results in a dramatic reduction in stool volume, with resolution of accompanying symptoms of abdominal pain, nausea, and vomiting. The management of patients whose diarrhea and other symptoms of intestinal GVHD persist after 7–14 days of immunosuppressive therapy is unsatisfactory, as the rate of failure of secondary therapy is high [187]. In allograft recipients, infectious causes of diarrhea are far less common than GVHD, accounting for only 10–15% of diarrheal episodes [162,177]. In countries where intestinal parasitism and bacterial contamination of water are endemic, the spectrum of infections may be wider [4]. Clostridium difficile colitis is usually a relatively mild, treatable disease when diagnosed at the onset of diarrhea [177]. The recent emergence of more virulent strains of C. difficile has changed the natural history of this infection, and thus prevention of nosocomial C. difficile infection in both hospital and outpatient settings has become even more important (Plate 27.34). One often overlooked factor is the inappropriate use of proton-pump inhibitors for marginal indications [172], which increases the risk of C. difficile colitis twofold [188]. The most common cause of infectious diarrhea in one prospective study was astrovirus (a small round virus similar to norovirus); diagnosis can be made by PCR of stool specimens or by typical histologic findings in small intestinal biopsies [177,189]. Some serotypes of adenovirus cause necrotizing enteritis and rapidly fatal multiorgan failure involving the gut, liver, lungs, and kidneys [138–140]. There should be a sense of urgency in identifying adenovirus as a cause of enteritis, as early treatment with cidofovir appears to be effective [143,144]. CMV is the only common infectious cause of enteritis after HCT that requires an intestinal biopsy for diagnosis [177]. CMV can be found in mucosal biopsies in patients whose blood is negative for CMV antigen or DNA [1,190]. Otherwise the predictive value of a negative stool examination for other viruses, bacteria, fungi, and parasites is high,

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particularly if molecular methods are used [5,177,191]. After HCT, watery diarrhea secondary to intestinal parasite infection (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica) is rare among patients to come to transplant without diarrhea, but sporadic cases are seen that can be confused with GVHD [4,6]. Strongyloides infection and hyperinfection syndrome have been described after HCT; patients from endemic areas should be screened before HCT. There are several disorders that can closely mimic intestinal GVHD or, more commonly, co-exist with GVHD, including infection with nonculturable enteric viruses, mycobacteria, fungi, and parasites (Table 95.8). Brush border disaccharidase deficiency, bile salt malabsorption, pancreatic insufficiency, mucosal toxicity from mycophenolate mofetil (MMF), and intestinal thrombotic microangiopathy may contribute to diarrhea that might otherwise be attributed to GVHD. That is, persistent diarrhea in an allograft recipient may not be caused directly by the necroinflammatory process of GVHD, but by related disorders. For example, intestinal inflammation often results in downregulation of brush border disaccharidases such as lactase and sucrase/isomaltase, leading to diarrhea if lactose or sucrose is ingested. Failure of bile salt absorption in the small intestine leads to inefficient water transport in the colon. Transient pancreatic insufficiency has been described as a consequence of mucosal edema at the ampulla of Vater [197]. Japanese investigators have described intestinal microangiopathy related to calcineurin inhibitors as a disorder that mimics GVHD [194]. MMF causes intestinal ulcerations and apoptotic crypt cells indistinguishable from the histology of acute GVHD [192,193]. In a patient with intestinal symptoms whose immunosuppressive drug regimen includes MMF, it may be difficult to make a diagnosis of GVHD until MMF has been discontinued. Diarrhea may also result from carbohydrate malabsorption (particularly in patients on antibiotics that affect the colon flora’s ability to salvage carbohydrate), oral magnesium salts, tacrolimus (a motilin agonist), and metoclopramide. The multiple causes of diarrhea not directly caused by GVHD in an allograft recipient may explain why diarrhea is not a component of the acute GVHD Activity Index that predicts day 200 nonrelapse mortality [198]. Abdominal pain It is extremely important to distinguish abdominal pain as an indicator of a rapidly progressive, fatal illness from illnesses with a benign natural

Table 95.8 Difficult-to-diagnose disorders that can mimic or complicate gastrointestinal acute GVHD Disorders

Comments

References

Mycophenolate mofetil toxicity

A wide range of gastrointestinal pathology has been described in mycophenolate mofetil-treated organ transplant patients. Very difficult to differentiate from GVHD in the setting of hematopoietic cell transplantation Diagnosis may be difficult when cytomegalovirus in present only in gastrointestinal mucosa (blood is virus free) Rare and treatable only by withdrawing immunosuppression. Molecular diagnosis is far more accurate than microscopic methods of diagnosis The most common infection(s) in a prospective study, but seldom diagnosed because of lack of commercial diagnostic tests (polymerase chain reaction) The hypothesis is that tacrolimus-related vascular injury leads to ischemic damage to mucosa. Withdrawal of tacrolimus has been followed by improvement in some cases Granulomatous mucosal involvement is part of the spectrum of mycobacterial infections in transplant patients Case report of a mold leading to granulomatous enteritis

[192,193]

Cytomegalovirus infection Cryptosporidial infection Nonculturable virus infections (astrovirus, norovirus) Tacrolimus-related thrombotic microangiography Nontuberculous mycobacterial infections Diffuse fungal infection

[1,56] [6,7] [177,189] [194]

[191,195] [196]

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Chapter 95

Table 95.9 Differential diagnosis of abdominal pain after transplant Infectious causes

Noninfectious causes

Cytomegalovirus enteritis Clostridial colitis (C. difficile, C. septicum) Intestinal perforation (abscess/ peritonitis) Varicella zoster infection Adenovirus enteritis Acute cholecystitis Cystitis (JC/BK virus, adenovirus) Viral pancreatitis Aspergillus vasculitis/intestinal infarction Epstein–Barr virus lymphoproliferative disease Phlegmonous gastritis Helicobacter pylori-related ulcers

Colonic pseudo-obstruction Narcotic bowel syndrome Intestinal mucosal necrosis from myeloablative conditioning Sinusoidal obstructive syndrome Acute pancreatitis/pseudocyst Hemorrhagic cystitis (cyclophosphamide) Intramural hematoma of intestine/rectus sheath Acute graft-versus-host disease Biliary sludge syndrome

Infectious causes in bold are now so rare that they should be considered only when patients are at risk.

history that require only conservative management. The causes of abdominal pain after HCT are listed in Table 95.9. Dilation of the bowel in the absence of a mechanical obstruction (i.e. intestinal pseudo-obstruction) is the most common cause of moderateto-severe abdominal pain. The illnesses that may progress rapidly include intestinal perforation, some infections (e.g. typhlitis caused by C. septicum, adenovirus enteritis, and visceral VZV infection), gall bladder necrosis, and bacterial liver abscess. Fortunately, these disorders are far less common than intestinal pseudo-obstruction, liver pain related to SOS, acute GVHD, and hemorrhagic cystitis – causes of severe pain to be sure, but not imminent death. A systematic approach in evaluating abdominal pain in these difficult patients is important in excluding rapidly fatal diseases.

Does the patient need urgent surgery? Surgery is indicated for intestinal perforation, acute cholecystitis, drainage of abscesses, appendicitis, and in some patients with intestinal or biliary obstruction, typhlitis, and dissecting hematomas [199]. The majority of patients with abdominal pain do not require surgery [199], and in patients who have SOS, general anesthesia may jeopardize liver blood flow and lead to progressive liver failure. SOS presenting as severe abdominal pain can usually be recognized by its timing post HCT, by the finding of liver tenderness in either the epigastrium or the right lobe, away from the gall bladder fossa, and by consistent findings on Doppler ultrasound, which also serves to examine the right upper quadrant for abscess or gall bladder disease. Intestinal perforation may develop in the setting of lysis of a transmural lymphoma or metastatic carcinoma shortly after conditioning therapy, or later from CMV ulcers or diverticular perforation. Perforation may present with only mild-tomoderate abdominal pain and pneumoperitoneum on abdominal CT. Pneumoperitoneum on upright X-ray or CT scan was present in all Seattle patients with perforation, but this finding can be a manifestation of pneumatosis intestinalis, a more benign process than free perforation [200]. Recognition of acute cholecystitis is more difficult, as right upper quadrant pain (usually from SOS) and fever are common in the early post-HCT period, and imaging studies frequently show gall bladder wall

thickening and luminal sludge in completely asymptomatic patients [201]. A radionuclide study with morphine that shows filling of the gall bladder suggests that surgical cholecystitis is not present, but falsepositive results (i.e. lack of gall bladder filling) do occur in severely ill patients on total parenteral nutrition [202]. Dilation of the bowel in the absence of a mechanical obstruction is the most common cause of moderate-to-severe abdominal pain. Most patients with pseudo-obstruction have an underlying intestinal disease, such as enteritis from conditioning therapy, GVHD or infection, but frequently the acute presentation is related to increasing use of μ-opioid and anticholinergic medications. Pseudo-obstruction is more frequent among patients transplanted for lymphoma or myeloma, as a result of intestinal neuropathy from repeated use of vincristine. In visceral VZV infection, abdominal distention, severe pain, fever, and rising serum ALT levels may precede cutaneous manifestations by up to 10 days [136,142]. In rare instances, a skin rash never develops. Acyclovir should be started on clinical suspicion while serum is analyzed by PCR for VZV DNA. Pancreatitis is an uncommon cause of abdominal pain in HCT patients, but in a study of autopsied patients, the prevalence of acute pancreatitis was 28% [154]. Symptoms of pancreatitis were absent in many patients with florid pancreatitis at autopsy, suggesting that symptoms were masked by immunosuppressive drugs. Patients with low platelet counts or prolongation of blood clotting may rarely bleed into the retroperitoneum, abdominal wall or intra-abdominal viscera, particularly after duodenal biopsy, causing significant pain. Intestinal infections presenting with significant pain are listed in Table 95.9. Clostridium difficile colitis is generally mild, but severe colitis can be seen with more virulent strains (Plate 27.34). Typhlitis occurs in granulocytopenic patients infected with C. septicum but is not common after HCT. Symptoms include fever, right lower quadrant pain, nausea and vomiting, diarrhea, occult blood in the stool, and shock [9]. Diagnosis of typhlitis is usually made clinically by imaging studies [203]; laparotomy is rarely necessary provided that imipenem and oral vancomycin therapy for C. septicum are started along with systemic coverage for luminal bacteria and fungi in febrile patients [204]. Modern imaging tests (ultrasound and CT scan) and careful examination should allow the surgeon to be highly selective in choosing candidates for operation, as there is little to be gained by diagnosis of GVHD at operation [163,199]. Clinical judgment may lead to surgical exploration or laparoscopy in problematic cases where there is pneumoperitoneum of unknown cause or possible gall bladder necrosis.

Is the pain a manifestation of acute GVHD? Acute gastrointestinal GVHD usually presents with nausea, vomiting, anorexia, abdominal pain, and diarrhea [179]. The sudden onset of intestinal edema can cause a rigid abdomen with rebound tenderness preceding the development of a skin rash or diarrhea [163]; pain is usually crampy and periumbilical, but can be localized to the epigastrium. The decision to treat a patient empirically when definitive evidence of GVHD is not at hand is difficult, but when the pretest probability of GVHD is high and that of perforation or infection low, prednisone therapy at 2 mg/kg/day should be started [187]. CT abdominal imaging shows intestinal wall thickening in more severe acute GVHD, a nonspecific but highly suggestive finding, particularly when CMV infection is unlikely [75,180]. CT is preferred to ultrasound because of better sensitivity for detecting perforation or intra-abdominal abscess. Although one might withhold prednisone therapy until a definitive biopsy diagnosis has been made, delays in therapy may lead to more extensive mucosal necrosis and morbidity. If the pain later proves to be due to causes other than GVHD, prednisone can be discontinued.

Gastrointestinal and Hepatic Complications

Is visceral VZV infection a possibility? Disseminated VZV may present with difficult-to-explain abdominal pain 2–6 months after HCT. This presentation is most likely in immunosuppressed patients who had positive pretransplant VZV serology and who are not currently receiving acyclovir or ganciclovir therapy. Importantly, the abdominal pain may precede typical VZV skin lesions by several days [142,205,206]. Marked hyponatremia due to the syndrome of inappropriate antidiuretic hormone secretion has also been observed in this setting [207]. Patients should be started on intravenous acyclovir while a definitive diagnosis is sought (VZV DNA in serum, gastric mucosal or liver biopsy; Plate 27.58) [206,208]. Pain from visceral VZV infection may be poorly localized, and the patient may not look particularly unwell at presentation, but if not treated promptly, the disease can follow a rapidly fatal course. Is the pain due to a treatable infection? Recent advances in antiviral and antifungal prophylaxis [17,152,209] have made intestinal and liver infections unusual causes of abdominal pain. Nonetheless, infections of the liver and intestinal tract must be considered in patients with pain, particularly infection caused by CMV, adenovirus, C. difficile, C. septicum (typhlitis) or mold (either blood borne or mucosally invasive) [9,56,138–140,168,210,211]. Is the pain caused by intestinal pseudo-obstruction? In patients with intestinal pseudo-obstruction and gaseous distention, medical management is almost always effective and perforation rare. While there are some infections (VZV [212] and CMV [213]) that lead to pseudo-obstruction, opioid and anticholinergic medications are the usual cause. There are only two sources of intestinal gas: swallowed air (or, in the case of mechanical ventilation, leakage around the endotracheal tube) and fermentation of carbohydrate by colon bacteria. A nasoesophageal sump tube connected to suction will prevent additional swallowed air from accumulating in the gut. Switching from a μ-opioid to a κ-opioid agonist (e.g. butorphanol) may allow pain relief while allowing gut motility to recover. Peripheral μ-opioid blockade with methylnaltrexone can also be effective [214]. Neostigmine (2 mg intravenously) has been successfully used in patients with acute colonic pseudo-obstruction after HCT [215]. If distention is caused by fluid, as in severe acute GVHD, or by true obstruction, a rare event, the approach is to place a nasogastric or nasoenteric sump tube to prevent further accumulation, and then address the underlying cause. Identification of other causes of abdominal pain Once the diseases that need urgent surgical or medical care have been ruled out, one can perform directed diagnostic tests to identify specific causes of pain. Hemorrhagic cystitis is usually seen after CY exposure but also occurs with adenovirus or polyomavirus infection; it is always accompanied by hematuria. Hematomas of the abdominal wall or intestine should be suspected in patients with a rapidly expanding anterior abdominal wall mass or obstructive symptoms after intestinal biopsy in the setting of profound thrombocytopenia. Perianal pain Perianal pain after HCT can be caused by an anal fissure, a thrombosed external hemorrhoid, cellulitis related to tissue maceration, and infections. In patients with granulocytopenia, infections in the perineum or perianal spaces are usually polymicrobial, arising either from anal glands or from tears in the anal canal. After HCT, these infections can be difficult to recognize because they may not produce abscesses but rather a spreading cellulitis. Extensive supralevator and intersphincteric abscesses

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may be present without being apparent on external examination [10,12]. CT or MRI scans or endoscopic ultrasound can give a clear view of the anatomy involved, particularly if there is pus present [216]. When antibiotics covering both anaerobic and aerobic bacteria are given to patients with “cryptitis” (incipient perianal infection), far fewer patients require surgical drainage than in the past [11]. Rarely, surgical drainage will be needed for patients with abscesses and rapidly progressive tissue necrosis.

Problems in long-term transplant survivors Liver disorders Chronic GVHD Patients with chronic hepatic GVHD usually have features of chronic GVHD elsewhere in the body. Evidence of cholestasis (elevated alkaline phosphatase and gamma glutamyl transpeptidase, usually without pruritus) is present in approximately 80% of patients with extensive chronic GVHD. Patients with isolated elevations of alkaline phosphatase in the absence of jaundice should be followed closely, but may not require extended doses of high-dose immunosuppressive therapy. Jaundice is a late feature of the disease process, and by the time that jaundice develops, liver biopsy shows extensive damage to, and loss of, small bile ducts (Plates 27.40 and 27.41) [132,133]. In patients receiving no, or tapering doses of, immunosuppression, chronic liver GVHD may present acutely with abrupt elevations of aminotransferase levels to over 2000 U/ L [133,135]. Serologic and nucleic acid testing and liver biopsy are essential to exclude acute viral hepatitis due to a herpesvirus (HSV or VZV) or a hepatitis virus (hepatitis A–E), and to make a definitive diagnosis of chronic GVHD (Plate 27.39) [217]. A serum autoantibody test for CYP1A2 may prove diagnostically useful in the diagnosis of hepatitic GVHD, as this enzyme appears to be a target antigen in GVHD [218]. Acyclovir should be started pending results of viral tests, and, if negative, treatment with a calcineurin inhibitor and prednisone 1–2 mg/ kg/day should be begun. Immunosuppressive drug treatment of chronic GVHD is successful in 50–80% of patients with extensive multiorgan disease. The use of long-term ursodeoxycholic acid (15 mg/kg/day) is safe and well tolerated, and may result in normalization or improvement of liver enzymes [219]. In longstanding chronic GVHD of the liver, small bile ducts may be absent (Plate 27.41), analogous to ductopenic rejection in liver allografts. Ductopenic GVHD is potentially reversible if ongoing immunologic destruction of epithelium ceases, but this process may take months before resolution of jaundice [133]. Liver transplantation, including living-donor transplantation from the original stem cell donor [220], has been performed for patients with liver failure due to chronic hepatic GVHD, although frequently there are contraindications to this approach [221]. Chronic viral hepatitis and cirrhosis HCV infection in HCT survivors almost always results in chronic hepatitis [27,28]. In the first 10 years of HCV infection after HCT, there is little liver-related morbidity. However, cirrhosis of the liver related to chronic HCV infection is rising in frequency among patients transplanted before the 1990s [28,222]. The rate of progression of HCV is higher in HCT recipients than in matched controls, with reports of cirrhosis in 24% by 20 years follow-up [28]. The reasons for more rapid fibrosis progression after HCT are unclear, but may be related to concomitant liver involvement with GVHD, iron overload [223], and immunosuppression. Clues to the presence of cirrhosis include a switch in the normal ratio of serum aminotransferases so that AST is higher than ALT, thrombocytopenia, and hepatic nodularity and splenomegaly on imaging,

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Chapter 95

although thrombocytopenia may be unrelated to liver disease in HCT survivors. It is important to establish a diagnosis of cirrhosis in patients with chronic hepatitis C, so that monitoring for complications can be undertaken. Hepatocellular carcinoma develops in 2–8% per annum of patients with HCV cirrhosis, and screening (usually with 6-monthly ultrasound and serum alfafetoprotein levels) is recommended [224]. Chronic hepatitis C may also be a risk factor for development of lymphoma [225] and other lymphoproliferative disorders [226] after transplant. All patients with chronic HCV, including those with compensated cirrhosis, should be considered for therapy with combination pegylated interferon-α plus ribavirin, unless contraindications exist [227,228]. Pegylated interferon is currently the standard of care for treatment of chronic hepatitis C; however, the long half-life may be associated with rapid falls in platelet and granulocyte counts. Patients with renal impairment may experience increased hemolysis and anemia with ribavirin therapy. Growth factors (e.g. filgastrim and erythropoietin) may be needed to support patients through therapy. New direct antivirals for hepatitis C are under development and may offer enhanced viral responses in the future [229]. Interferon-based therapy may activate chronic GVHD, and patients should be closely monitored. Liver transplantation should be considered in any HCT survivor with hepatic decompensation or early hepatocellular carcinoma. Living-donor transplantation from the original stem cell donor is an attractive consideration in this situation, as minimal immunosuppression would likely be required in the long term [230]. The prevalence of chronic HBV infection among HCT survivors varies widely depending on the country. The serologic pattern of HBV infection may be atypical in HCT survivors, probably as a consequence of immunosuppression. Clearance of surface antigenemia may be observed, and is particularly likely if the donor was anti-HBs positive because of prior HBV infection [20]. Patients who remain HBsAg positive after HCT are at risk of flares of hepatitis activity, particularly at times of reduction of immunosuppression, such as during taper or cessation of treatment for chronic GVHD. All long-term survivors with chronic hepatitis B should be regularly monitored to assess virologic and disease status, and the need for antiviral therapy. Hepatitis B e antigen and antibody status should be determined in all patients, and liver enzymes monitored every 6–12 months. The HBV DNA level should be determined in those with abnormal ALT levels, and liver biopsy considered to assess the severity of hepatic inflammation and fibrosis. The need for antiviral treatment with an oral nucleoside or nucleotide analogue (e.g. entecavir, telbivudine adefovir, or tenofovir) is based upon the ALT and HBV DNA levels, and the severity of hepatic fibrosis [231], and may change over time, hence the need to continually reassess patients who are not on treatment. Long-term survivors with chronic hepatitis B do not seem to have an increased rate of progression to cirrhosis compared with non-HCT patients. As HCT survivors are at increased risk of second malignancy or recurrent malignancy, care should be taken to reassess HBV viral status prior to reintroduction of chemotherapy. Newer agents, such as rituximab used in the treatment of B-cell malignancy, have a particularly high risk of reactivation of latent hepatitis B (HBsAg negative and anti-HBc positive), and careful monitoring of HBV DNA levels on therapy and early introduction of an antiviral drug (lamivudine, entecavir adefovir, or tenofovir) is strongly recommended [232]. As in the peritransplant period, patients with positive HBsAg should receive an antiviral agent whenever they receive immunosuppressive or cytotoxic therapy [233]. Fungal abscess Fungal abscesses can recur after apparently successful antifungal therapy, when high-dose immunosuppressive drugs are started for GVHD. Non-

sterile herbal remedies contaminated by molds may lead to liver abscesses in immunosuppressed HCT survivors [234]. Acute hepatocellular injury The differential diagnosis of extreme elevations of serum ALT in an HCT survivor includes VZV infection, a hepatitic presentation of chronic GVHD [133,134], flares of chronic hepatitis B or C, and drug-induced liver injury. Drug-induced liver injury may be related to antihypertensive drugs, lipid-lowering agents, hypoglycemic agents, nonsteroidal antiinflammatory drugs, antidepressants, antibiotics or herbal preparations. Some drug reactions may result in chronic liver disease [235]. Iron overload Iron overload may be an important cofactor in liver disease in long-term survivors of HCT, and should be part of a screening panel [236,237]. Iron overload is particularly severe in thalassemic patients who have undergone HCT [238]. Iron overload is caused by a combination of multiple red cell transfusions and dyserythropoiesis leading to aberrant hepcidin regulation and increased iron transport by the intestine. In patients where a marked degree of iron overload was unexpected, HFE gene testing (on buccal mucosa swab or stored DNA sample) should be performed. After HCT, iron accumulation stops and body iron stores fall slowly over time [239]. Clinically significant iron overload is usually present only when serum ferritin levels are over 1000 μg/dL [240]. An elevated serum ferritin level, particularly in patients with chronic GVHD, chronic viral hepatitis or other causes of liver disease, may not reflect tissue iron stores, and it may be important to document the degree of tissue iron overload. In the past, liver biopsy with liver iron determination was required; however, increasingly noninvasive methods (e.g. MRI and FerriScan [41]) are being utilized to provide assessments of liver iron concentration and distribution. The consequences of extreme iron overload in HCT survivors are primarily those of cardiac, pituitary, and pancreatic endocrine dysfunction. Patients with a liver iron content greater than 15,000 μg/g dry weight (or equivalent) should be treated aggressively with both phlebotomy and chelation; when the liver iron content is 7000–15,000 μg/g dry weight, phlebotomy is indicated; but when the liver iron content is under 7000 μg/g dry weight, treatment is indicated only if there is evidence of liver disease [241]. Mobilization of iron from heavily overloaded patients improves cardiac function, normalizes serum ALT levels, and results in improved liver histology [46,241,242]. Iron overload may also be a cause of persistent hepatic dysfunction after HCT that responds to iron mobilization [46,47]. Hepatocellular carcinoma Compared with the general population, patients who survive over 10 years post HCT have an eightfold risk of developing a new solid malignancy. Because of the increased rate of chronic viral hepatitis, particularly hepatitis C, the risk of hepatocellular carcinoma is particularly elevated [243]. Transplant survivors with risk factors for hepatocellular carcinoma (HCV or HBV infection, obesity, diabetes or low platelet count) should be screened at yearly intervals [244]. Nodular regenerative hyperplasia Rarely, patients who have experienced toxic liver injury (SOS) from either chemotherapy or a myeloablative conditioning regimen will develop hepatic nodularity caused by atrophy of zone 3 and hypertrophy of zone 1 hepatocytes, without fibrosis [245]. This process is usually clinically silent unless portal hypertension develops, manifest by variceal bleeding, ascites, splenomegaly, and thrombocytopenia, but with preserved liver function.

Gastrointestinal and Hepatic Complications

Gall bladder and biliary tract disease There appears to be a higher-than-expected incidence of gallstones and stone-related biliary problems after HCT than in an age-matched population, probably related to earlier formation of nucleating microliths (biliary sludge). Chronic cyclosporine or tacrolimus dosing may also lead to gallstones, biliary symptoms, and acute pancreatitis [246–248]. Esophageal diseases Chronic GVHD Some patients with extensive chronic GVHD have esophageal desquamation, webs, submucosal fibrous rings, bullae, and long, narrow strictures in the upper and mid-esophagus [249–251]. Development of severe esophageal GVHD is most frequent in patients whose chronic GVHD has been either neglected or inadequately treated. The most common symptom is dysphagia; some patients present with insidious weight loss, retrosternal pain, and aspiration of gastric contents, leading to pulmonary disease that can be mistaken for bronchiolitis obliterans. In children, a common presentation is weight loss and failure to thrive. The diagnosis is made by barium contrast X-ray and endoscopy, which should be done with caution, as perforations have been reported [249]. Tight strictures are difficult to dilate safely, but failure to dilate strictures may lead to progressive esophageal narrowing. Although strictures that have developed recently can be successfully dilated under cover of immunosuppression, chronic strictures that involve most of the upper esophagus may prove intractable. Esophageal involvement can be prevented by prompt treatment of chronic GVHD at its early stages. Therapy with proton pump inhibitors should be considered because patients with esophageal chronic GVHD usually have uncontrolled acid reflux as a result of poor salivary bicarbonate secretion and lack of esophageal peristalsis. Myasthenia gravis may also complicate chronic GVHD, with dysphagia as its presenting complaint [252].

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usually in patients with concomitant chronic GVHD of the oropharynx [253]. Gastrointestinal symptoms: diarrhea, anorexia, nausea, and weight loss The incidence of diarrhea falls sharply after day 100 except in some patients who have developed acute GVHD after reduced-intensity conditioning regimens [178] and in patients whose severe acute GVHD has never resolved. Some long-term survivors have intestinal symptoms for years following HCT, caused by protracted acute GVHD whose symptoms wax and wane with the intensity of immunosuppressive therapy, with each exacerbation similar to the presenting signs of acute GVHD (satiety, poor appetite, nausea, episodic diarrhea, and weight loss) [254,255]. The endoscopic and histologic appearance of the intestinal mucosa is identical to that seen in acute GVHD. This form of GVHD is not considered typical of chronic GVHD, but frequently occurs in patients with chronic GVHD in other sites [256]. Use of oral beclomethasone dipropionate can be effective in treating patients with protracted acute GVHD involving the gastrointestinal tract [257]. Before the introduction of more effective immunosuppressive drugs, chronic GVHD resulted in extensive collagen deposition in submucosal and subserosal areas of the intestinal tract (Plate 27.33), resulting in refractory malabsorption [258]; this process has not been seen in recent years. There are sporadic cases of C. difficile, CMV [56], and rarely Giardia and Cryptosporidium infection in long-term survivors. Rarely, chronic intestinal viral infection can be seen in patients who remain on immunosuppressive drugs, for example rotavirus, norovirus, and adenovirus. Rare cases of “transmission” of intestinal diseases have been reported, such as inflammatory bowel disease and celiac sprue [259]. Pancreatic insufficiency may also cause diarrhea after transplant [197,260,261]. Pancreatic disease

Strictures Esophageal strictures may also be sequelae of earlier herpesvirus infection, severe mucositis involving the esophagus or chronic reflux of gastric contents. Sporadic cases of fungal and rarely viral esophagitis may occur in patients with chronic GVHD on immunosuppressive and antibiotic therapy. Esophageal cancer In series of secondary cancers developing late after transplant, squamous cell carcinoma of the esophagus has been reported,

Sporadic cases of acute pancreatitis are seen in transplant survivors, usually related to passage of gallstones or sludge; cyclosporine and tacrolimus have also been implicated [246–248]. Diarrhea, steatorrhea, and weight loss secondary to pancreatic insufficiency have developed in some long-term HCT survivors [260,261]. The most likely cause is pancreatic acinar atrophy from previous pancreatic necrosis or prolonged corticosteroid exposure [154]. Transient pancreatic insufficiency has been noted in patients with gut and liver GVHD [197]. Chronic GVHD and extreme iron overload may also contribute to pancreatic damage.

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191. Nicholson O, Feja K, LaRussa P et al. Nontuberculous mycobacterial infections in pediatric hematopoietic stem cell transplant recipients: case report and review of the literature. Pediatr Infect Dis J 2006; 25: 263–7. 192. Papadimitriou JC, Drachenberg CB, Beskow CO et al. Graft-versus-host disease-like features in mycophenolate mofetil-related colitis. Transplant Proc 2001; 33: 2237–8. 193. Maes BD, Dalle I, Geboes K et al. Erosive enterocolitis in mycophenolate mofetil-treated renaltransplant recipients with persistent afebrile diarrhea. Transplantation 2003; 75: 665–72. 194. Nishida T, Hamaguchi M, Hirabayashi N et al. Intestinal thrombotic microangiopathy after allogeneic bone marrow transplantation: a clinical imitator of acute enteric graft-versus-host disease. Bone Marrow Transplant 2004; 33: 1143–50. 195. Cordonnier C, Martino R, Trabasso P et al. Mycobacterial infection: a difficult and late diagnosis in stem cell transplant recipients. Clin Infect Dis 2004; 38: 1229–36. 196. Scott RS, Sutton DA, Jagirdar J. Lung infection due to opportunistic fungus, Phialemonium obovatum, in a bone marrow transplant recipient: an emerging infection with fungemia and Crohn disease-like involvement of the gastrointestinal tract. Ann Diagn Pathol 2005; 9: 227–30. 197. Grigg AP, Angus PW, Hoyt R, Szer J. The incidence, pathogenesis and natural history of steatorrhea after bone marrow transplantation. Bone Marrow Transplant 2003; 31: 701–3. 198. Leisenring W, Martin P, Petersdorf E et al. An acute graft-versus-host disease activity index to predict survival after hematopoietic cell transplantation with myeloablative conditioning regimens. Blood 2006; 108: 749–55. 199. Jones AD, Maziarz R, Gilster J, Domreis J, Deveney CW, Sheppard BC. Surgical complications of bone marrow transplantation. Am J Surg 2003; 185: 481–4. 200. de Magalhaes-Silverman M, Simpson J, Ball E. Pneumoperitoneum without peritonitis after allogeneic peripheral blood stem cell transplantation. Bone Marrow Transplant 1998; 21: 1153–4. 201. Teefey SA, Hollister MS, Lee SP et al. Gallbladder sludge formation after bone marrow transplant: sonographic observations. Abdom Imaging 1994; 19: 57–60. 202. Cabana MD, Alavi A, Berlin JA, Shea JA, Kim CK, Williams SV. Morphine-augmented hepatobiliary scintigraphy: a meta-analysis. Nucl Med Commun 1995; 16: 1068–71. 203. Cartoni C, Dragoni F, Micozzi A et al. Neutropenic enterocolitis in patients with acute leukemia: prognostic significance of bowel wall thickening detected by ultrasonography. J Clin Oncol 2001; 19: 756–61. 204. Schlatter M, Snyder K, Freyer D. Successful nonoperative management of typhlitis in pediatric oncology patients. J Pediatr Surg 2002; 37: 1151– 5. 205. David DS, Tegtmeier BR, O’Donnell MR, Paz IB, McCarty TM. Visceral varicella-zoster after bone marrow transplantation: report of a case series and review of the literature. Am J Gastroenterol 1998; 93: 810–13. 206. Grant RM, Weitzman SS, Sherman CG, Sirkin WL, Petric M, Tellier R. Fulminant disseminated varicella zoster virus infection without skin involvement. J Clin Virol 2002; 24: 7–12.

207. McIlwaine LM, Fitzsimons EJ, Soutar RL. Inappropriate antidiuretic hormone secretion, abdominal pain and disseminated varicella-zoster virus infection: an unusual and fatal triad in a patient 13 months post rituximab and autologous stem cell transplantation. Clin Lab Haematol 2001; 23: 253–4. 208. de Jong MD, Weel JF, van Oers MH, Boom R, Wertheim-van Dillen PM. Molecular diagnosis of visceral herpes zoster. Lancet 2001; 357: 2101– 2. 209. Grigg AP, Brown M, Roberts AW, Szer J, Slavin MA. A pilot study of targeted itraconazole prophylaxis in patients with graft-versus-host disease at high risk of invasive mould infections following allogeneic stem cell transplantation. Bone Marrow Transplant 2004; 34: 447–53. 210. Keates J, Lagahee S, Crilley P, Haber M, Kowalski T. CMV enteritis causing segmental ischemia and massive intestinal hemorrhage. Gastrointest Endosc 2001; 53: 355–9. 211. Cohen R, Heffner JE. Bowel infarction as the initial manifestation of disseminated aspergillosis. Chest 1992; 101: 877–9. 212. Nomdedeu JF, Nomdedeu J, Martino R et al. Ogilvie’s syndrome from disseminated varicellazoster infection and infarcted celiac ganglia. J Clin Gastroenterol 1995; 20: 157–9. 213. Sonsino E, Mouy R, Foucaud P et al. Intestinal pseudoobstruction related to cytomegalovirus infection of myenteric plexus. N Engl J Med 1984; 311: 196–7. 214. Yuan C-S, Israel RJ. Methylnaltrexone, a novel peripheral opioid receptor antagonist for the treatment of opioid side effects. Expert Opin Investig Drugs 2006; 15: 541–52. 215. Ponec RJ, Saunders MD, Kimmey MB. Neostigmine for the treatment of acute colonic pseudoobstruction. New Engl J Med 1999; 341: 137–41. 216. Schwartz DA, Harewood GC, Wiersema MJ. EUS for rectal disease. Gastrointest Endosc 2002; 56: 100–9. 217. Shulman HM, Kleiner D, Lee SJ et al. Histopathologic diagnosis of chronic graft-versus-host disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: II. Pathology Working Group Report. Biol Blood Marrow Transplant 2006; 12: 31–47. 218. Mullighan CG, Bogdanos DP, Vergani D, Bardy PG. Cytochrome P450 1A2 is a target antigen in hepatitic graft-versus-host disease. Bone Marrow Transplant 2006; 38: 703–5. 219. Arat M, Idilman R, Soydan EA et al. Ursodeoxycholic acid treatment in isolated chronic graft-vs.host disease of the liver. Clin Transplant 2005; 19: 798–803. 220. Shimizu T, Kasahara M, Tanaka K. Living-donor liver transplantation for chronic hepatic graftversus-host disease. New Engl J Med 2006; 354: 1536–7. 221. Orlando G, Ferrant A, Schots R et al. Liver transplantation for chronic graft-versus-host disease: case report with 10-year follow-up. Transplant Int 2005; 18: 125–9. 222. Strasser SI, Sullivan KM, Myerson D et al. Cirrhosis of the liver in long-term marrow transplant survivors. Blood 1999; 93: 3259–66. 223. Angelucci E, Muretto P, Nicolucci A et al. Effects of iron overload and hepatitis C virus positivity in determining progression of liver fibrosis in thalas-

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Gastrointestinal and Hepatic Complications 240. Jensen PD, Jensen FT, Christensen T, Nielsen JL, Ellegaard J. Relationship between hepatocellular injury and transfusional iron overload prior to and during iron chelation with desferrioxamine: a study in adult patients with acquired anemias. Blood 2003; 101: 91–6. 241. Angelucci E, Muretto P, Lucarelli G et al. Phlebotomy to reduce iron overload in patients cured of thalassemia by bone marrow transplantation. Italian Cooperative Group for Phlebotomy Treatment of Transplanted Thalassemia Patients. Blood 1997; 90: 994–8. 242. Muretto P, Angelucci E, Lucarelli G. Reversibility of cirrhosis in patients cured of thalassemia by bone marrow transplantation. Ann Intern Med 2002; 136: 667–72. 243. Bhatia S, Louie AD, Bhatia R et al. Solid cancers after bone marrow transplantation. J Clin Oncol 2001; 19: 464–71. 244. Ioannou GN, Splan MF, Weiss NS, McDonald GB, Beretta L, Lee SP. Incidence and predictors of hepatocellular carcinoma in cirrhotic patients. Clin Gastroenterol Hepatol 2007; 5: 938– 45. 245. Wanless IR. Micronodular transformation (nodular regenerative hyperplasia) of the liver: A report of 64 cases among 2,500 autopsies and a new classification of benign hepatocellular nodules. Hepatology 1990; 11: 787–97. 246. Lorber MI, Van Buren CT, Flechner SM, Williams C, Kahan BD. Hepatobiliary and pancreatic complications of cyclosporine therapy in 466 renal transplant recipients. Transplantation 1987; 43: 35–40.

247. Sastry J, Young S, Shaw PJ. Acute pancreatitis due to tacrolimus in a case of allogeneic bone marrow transplantation. Bone Marrow Transplant 2004; 33: 867–8. 248. Ogunseinde BA, Wimmers E, Washington B, Iyob M, Cropper T, Callender CO. A case of tacrolimus (FK506)-induced pancreatitis and fatality 2 years postcadaveric renal transplant. Transplantation 2003; 76: 448. 249. McDonald GB, Sullivan KM, Schuffler MD, Shulman HM, Thomas ED. Esophageal abnormalities in chronic graft-versus-host disease in humans. Gastroenterology 1981; 80: 914–21. 250. Minocha A, Mandanas RA, Kida M, Jazzar A. Bullous esophagitis due to chronic graft-versushost disease. Am J Gastroenterol 1997; 92: 529– 30. 251. McDonald GB, Sullivan KM, Plumley TF. Radiographic features of esophageal involvement in chronic graft-versus-host disease. Am J Roentgenol 1984; 142: 501–6. 252. Mackey JR, Desai S, Larratt L, Cwik V, Nabholtz JM. Myasthenia gravis in association with allogeneic bone marrow transplantation: clinical observations, therapeutic implications and review of literature. Bone Marrow Transplant 1997; 19: 939–42. 253. Shimada K, Yokozawa T, Atsuta Y et al. Solid tumors after hematopoietic stem cell transplantation in Japan: incidence, risk factors and prognosis. Bone Marrow Transplant 2005; 36: 115–21. 254. Patey-Mariaud de Serre N, Reijasse D, Verkarre V et al. Chronic intestinal graft-versus-host

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96

Kenneth R. Cooke & Gregory A. Yanik

Lung Injury Following Hematopoietic Cell Transplantation

Introduction Allogeneic hematopoietic cell transplantation (HCT) is the only curative therapy for a number of malignant and nonmalignant conditions, but successful outcomes are limited by several side-effects, including pulmonary toxicity. Diffuse lung injury remains a significant problem following allogeneic HCT both in the immediate post-transplant period and in the months to years that follow. Lung injury occurs in 25–55% of HCT recipients and accounts for approximately 50% of transplantrelated mortality [1–6]. Historically, approximately one-half of all pneumonias seen after HCT have been secondary to infection, but the judicious use of broad-spectrum antimicrobial agents has tipped the balance toward noninfectious causes [7]. Despite advances in preventing and treating opportunistic organisms, infectious lung injury remains a significant problem, particularly in patients with acute or chronic graftversus-host disease (GVHD) or in individuals who have poor or delayed immune reconstitution. Noninfectious lung injury can be either acute (idiopathic pneumonia syndrome [IPS]) or chronic, depending upon the onset after HCT and the tempo of disease progression. Chronic lung injury is further subdivided into two types: obstructive and restrictive [8–14]. Although noninfectious lung injury occasionally occurs following autologous transplants, the allogeneic setting significantly exacerbates the severity of disease in both the acute and chronic timeframes; in each scenario, pulmonary toxicity is associated with significant morbidity and mortality, and responds poorly to standard therapy. This chapter will review the definitions, risk factors, and pathogeneses of lung injury occurring both early and late after allogeneic HCT.

Infectious interstitial pneumonias Infections contribute to interstitial pulmonary infiltrates in a significant number of HCT recipients. The most common pathogens include community acquired respiratory viruses (e.g. parainfluenza, respiratory syncytial virus [RSV], influenza, and metapneumonia), Mycoplasma, and opportunistic pathogens such as Pneumocystis jiroveci (previously Pneumocystis carinii) (Table 96.1). Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) or surgical lung biopsy is required to distinguish infectious from noninfectious causes (Table 96.2). Besides bacterial, fungal, and cytologic stains, quantitative cultures should be performed on BAL fluid for diagnostic purposes. In addition, direct fluorescent antibody stains, centrifugation cultures (shell vial), and

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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polymerase chain reaction (PCR) assays may be very useful in isolating and/or identifying various viral pathogens. Pneumocystis jiroveci pneumonia may be identified through a number of techniques, including cytologic studies, special stains, and PCR-based assays. Cytomegalovirus (CMV) pneumonitis remains a significant cause of morbidity and mortality following allogeneic HCT. In the absence of a CMV prevention strategy (e.g. pre-emptive monitoring of CMV by plasma PCR or antigenemia, or universal prophylaxis), CMV pneumonitis may develop during the first 100 days following HCT with a peak incidence at approximately 8 weeks. In the current era, most CMV infections occur after the monitoring or prophylaxis period ends. CMV pneumonitis in patients with chronic GVHD has also been well documented [15,16]. With the availability of improved antiviral therapy, the mortality rate associated with CMV pneumonitis has declined significantly in recent years [17]. Risk factors for the development of CMV disease include older patient age, the presence of acute GVHD, recipient CMV seropositivity, transplantation for a hematologic malignancy, and the use of antithymocyte globulin during the transplant process [16,18]. Radiologic manifestations of CMV range from diffuse reticular opacities to widespread air space consolidation [19]. Histopathology remains the gold standard for identification of CMV pulmonary disease. Although molecular techniques have an excellent sensitivity for detection of infection, they may be less specific in regard to identifying CMV pneumonitis. RSV is a single-stranded, enveloped RNA virus that presents as a self-limiting upper respiratory tract infection in immunocompetent individuals, or a potentially fatal pneumonitis in immunocompromised patients [20]. Outbreaks in patients undergoing allogeneic HCT have been associated with mortality rates as high as 78% [21,22]. In the United States, the onset of RSV infections typically begins in November and continues for approximately 24 weeks. The organism is highly contagious, with transmission occurring primarily through surfaces contaminated with virus-laden nasal or oral secretions. Even in immunocompetent patients, native memory responses are incomplete, allowing for repeated infections. The overall virulence of this agent places the immunocompromised patient at particular risk for fatal lower respiratory tract infections during the seasonal period [20]. Radiographically, patchy alveolar or diffuse interstitial infiltrates may be seen. Clinically, affected patients may exhibit a profound dyspnea and hypoxemia, with or without concurrent upper respiratory tract symptoms. Less than 50% of patients with lower tract involvement have preceding or concurrent nasopharyngeal symptoms [20,22]. The use of aerosolized ribavirin (6 g/day) using small-particle generators has resulted in a decrease in RSV shedding, but clinical efficacy has not been proven [20,23]. The clinical impact of recently described viruses, including human metapneumovirus, and non-severe acute respiratory syndrome (SARS)

Lung Injury Following Hematopoietic Cell Transplantation

human coronaviruses is not yet clear. Human metapneumovirus is a paramyxovirus recently recognized as the first human pathogen within the genus Metapneumovirus [24]. The virus was first identified in the Netherlands in 2001, and was recently reported as a potential pathogen in allogeneic HCT recipients [25]. Infected patients typically present within the first 50 days post transplant, and exhibit clinical and radiographic findings similar in appearance to those of IPS. Mortality rates as high as 80% have been reported. Rapid progression of respiratory symptoms may occur, with a median of 4 days between initiation of oxygen support and death [25]. The clinical spectrum of human metapneumovirus as a cause of interstitial pneumonia post transplant remains ill-defined, with issues such as the frequency of asymptomatic shedding, improved detection methods, and treatment strategies all under investigation. The reactivation of latent viruses, especially herpes family and adenovirus, may also clinically mimic IPS (discussed below) [26]. However,

Table 96.1 Common infectious pathogens of diffuse interstitial pneumonia Viral

Cytomegalovirus Adenovirus Human herpes virus-6 Varicella virus Respiratory syncytial virus Parainfluenza – types 1 and 2 Influenza A, influenza B Rhinovirus Metapneumovirus

Protozoan

Pneumocystis jiroveci pneumonia Toxoplasma gondii Mycoplasma Chlamydia

Mycobacterial infections

Miliary tuberculosis

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in the vast majority of cases, clinical symptoms related to systemic involvement are manifested by signs of disease in other organs, including elevation of serum hepatic transaminases with CMV, varicella zoster virus or adenovirus, oral mucosal involvement with herpes simplex viruses, or cutaneous involvement secondary to varicella. Abdominal pain with diarrhea may represent concurrent viral enteritis, as commonly seen in association with CMV, herpes simplex or adenovirus.

Noninfectious, acute lung injury: IPS Definition, clinical course, and spectrum of disease In 1993, IPS was defined by a panel of experts as widespread alveolar injury following hematopoietic HCT that occurs in the absence of an active lower respiratory tract infection and cardiogenic causes [27]. As shown in Table 96.2, diagnostic criteria of IPS include signs and symptoms of pneumonia, nonlobar radiographic infiltrates, abnormal pulmonary function, and the absence of infectious organisms as determined by BAL or lung biopsy [2,27]. A variety of histopathologic findings have been associated with IPS, including diffuse alveolar damage with hyaline membranes, lymphocytic bronchitis, bronchiolitis obliterans organizing pneumonia (BOOP) [28], and interstitial pneumonitis (IP). The term IP has historically been used interchangeably with IPS, and it is the most frequently reported histologic pattern associated with this syndrome [3]. IP is seen in association with diffuse alveolar damage and hemorrhage early after HCT and is accompanied by bronchiolar inflammation and epithelial damage at later time points [28]. The incidence of IPS in the first 120 days after allogeneic HCT following high-dose conditioning ranges from 3% to 15% [5–7,27]. The median time of onset for IPS was initially reported to be 6–7 weeks (range 14–90 days) after HCT [27]. Mortality rates ranged from 50% to 80% overall, and to greater than 95% for patients requiring mechanical ventilation [1,3,5–7,27]. A retrospective study from Seattle showed a lower incidence and earlier onset of IPS than previously reported, but the typical clinical course involving the rapid onset of respiratory failure leading to death remained unchanged [6]. In a more recent publication, the frequency of IPS after allogeneic HCT ranged from 5% to 25%

Table 96.2 Definition of idiopathic pneumonia syndrome I: Evidence of widespread alveolar injury: a. Multilobar infiltrates on routine chest radiographs or computed tomography b. Symptoms and signs of pneumonia (cough, dyspnea, tachypnea, rales) c. Evidence of abnormal pulmonary physiology 1. Increased alveolar to arterial oxygen difference 2. New or increased restrictive pulmonary function test abnormality II: Absence of active lower respiratory tract infection based upon: a. Bronchoalveolar lavage negative for significant bacterial pathogens including: acid-fast bacilli, Nocardia and Legionella species b. Bronchoalveolar lavage negative for pathogenic nonbacterial microorganisms: 1. Routine culture for viruses and fungi 2. Shell vial culture for CMV and RSV 3. Cytology for CMV inclusions, fungi and Pneumocystis jiroveci 4. Direct fluorescence staining with antibodies against CMV, RSV, HSV, VZV, influenza virus, parainfluenza virus, adenovirus, and other organisms c. Other organisms/tests to also consider: 1. PCR for HMP, rhinovirus, coronavirus, and HHV-6 2. PCR for Chlamydia, Mycoplasma, and Aspergillus species 3. Serum galactomannan ELISA for Aspergillus species d. Transbronchial biopsy if the patient’s condition permits III: Absence of cardiac dysfunction, acute renal failure or iatrogenic fluid overload as etiology for pulmonary symptomatology CMV, cytomegalovirus; ELISA, immune-linked immunosorbent assay; HHV, human herpes virus; HMP, human metapneumovirus; HSV, herpes simplex virus; PRC, polymerase chain reaction; RSV, respiratory syncytial virus; VZV, varicella zoster virus.

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Chemoradiation toxicity

TRALI

IP CLS Infection

DAH

Cardiogenic edema

IPS PERDS

ARDS

RLD: BOOP / IP

OLD: BrOb/BOS

Pulmonary fibrosis

depending upon donor source and the degree of antigenic mismatch between donor and recipient [29]. Day 100 mortality was 80%, and the median time from diagnosis to death was 13 days despite aggressive treatment with high-dose steroids and broad-spectrum antimicrobial therapy. The clinical spectrum of IPS encompasses several forms of pulmonary toxicity (Fig. 96.1 and Table 96.3). In one small subset of patients with IPS, acute pulmonary hemorrhage or hemorrhagic alveolitis occurs. Diffuse alveolar hemorrhage (DAH) generally develops in the immediate post-HCT period, and is characterized by progressive shortness of breath, cough, and hypoxemia with or without fever [7,30–32]. Classically, DAH is defined by the demonstration of progressively bloodier aliquots of BAL fluid, but frank hemoptysis is rare [30]. Mortality from DAH is as high as 75% despite aggressive treatment with high dose (2 mg/kg to 1 g/m2) steroids, and death usually occurs within weeks of diagnosis [31]. Some patients with DAH can have microorganisms isolated from blood, BAL fluid or tracheal aspirate within 1 week of alveolar hemorrhage. Majhail and colleagues recently compared patients with DAH and infection-associated alveolar hemorrhage who presented with similar clinical and radiographic findings in the setting of progressively bloodier BAL fluid following allogeneic HCT. Alveolar hemorrhage from infectious and noninfectious causes was found to be related but distinct entities with extremely poor outcomes following therapy with conventional agents, including steroids [33]. Peri-engraftment respiratory distress syndrome (PERDS) also falls within the definition of IPS [7]. PERDS is characterized by fever, dyspnea, and hypoxemia that, by definition, occur within 5–7 days of neutrophil engraftment [34–36]. Although PERDS after autologous HCT appears similar to IPS after allogeneic HCT with respect to clinical presentation and time of onset, the two entities differ sharply with respect to overall outcome: inflammation in the autologous setting responds promptly to corticosteroids and is associated with a favorable

Fig. 96.1. Clinical spectrum of idiopathic pneumonia syndrome (IPS) after hematopoietic cell transplantation. The clinical spectrum of IPS includes a variety of descriptive forms of lung injury that may share clinical features with toxicity incurred by chemoradiotherapy, cardiogenic edema, pulmonary fibrosis, infection, and transfusionassociated lung injury (TRALI). ARDS, acute respiratory distress syndrome; BOOP, bronchiolitis obliterans organizing pneumonia; BOS, bronchiolitis obliterans syndrome; BrOb, bronchiolitis obliterans; CLS, capillary leak syndrome; DAH, diffuse alveolar hemorrhage; IP, interstitial pneumonitis; OLD, obstructive lung disease; PERDS, peri-engraftment respiratory distress syndrome; RLD, restrictive lung disease.

prognosis [34], whereas PERDS occurring in an allogeneic environment responds poorly to standard therapy and commonly results in rapid respiratory failure and death in the majority of patients [6,29,37]. Lung inflammation following the administration of 1,3-bis(2choloroethyl)-1-nitrosurea (BCNU; carmustine) and transfusionassociated lung injury (TRALI) are two forms of noninfectious pulmonary toxicity that can be mistaken for IPS but have distinct etiologies. Initially described in the 1970s, chemotherapy-associated pulmonary toxicity has been reported in association with multiple chemotherapeutic agents, including BCNU, busulfan, and melphalan. In particular, BCNU-related lung injury is acute in onset and develops within the first 3 months following HCT in 10–40% of patients receiving this therapy [38]. Clinically, BCNU-related lung injury is typically associated with a nonproductive cough with increasing dyspnea in the context rapidly progressing, bilateral, interstitial infiltrates on both chest radiograph and computed tomography. Pulmonary function tests (PFTs) reveal a restrictive pattern of lung injury, with diminished forced vital capacity (FVC) and total lung capacity (TLC) noted. Risk factors for BCNU pneumonitis include prior pulmonary irradiation, cigarette smoking, and a dosage greater than 450 mg/m2 [39]. The pathogenesis of BCNU-related lung injury has been ill-defined, although increased production of fibrogenic factors such as platelet-derived growth factor-β, insulin-like growth factor I and transforming growth factor-β1 have been implicated [40]. Treatment with pulsed doses of corticosteroids early in the clinical course significantly decreases the morbidity and mortality associated with this condition and is key for successful outcomes; if left untreated, or recognized late in the clinical course, severe pulmonary fibrosis may develop [40]. TRALI is one of the leading causes of mortality following infusions of plasma containing blood products, estimated to occur in 1 in 1000 to 1 in 5000 transfusions [41,42]. Symptoms present acutely, with the onset of dyspnea and respiratory distress typically 6–8 hours following

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Table 96.3 The spectrum of noncardiogenic, pulmonary toxicity defined by idiopathic pneumonia syndrome Interstitial pneumonitis: • Clinical symptoms: fever, cough, dyspnea, hypoxemia • Onset: within first 100 days post transplant • Etiology: infectious (i.e. CMV, Pneumocystis jiroveci pneumonia) or noninfectious factors (chemotoxicity: BCNU, bleomycin, busulfan, methotrexate) • Radiographic findings: bilateral interstitial infiltrates Diffuse alveolar hemorrhage: • Clinical symptoms: progressive dyspnea, cough, rare hemoptysis • Key finding: progressively bloodier aliquots of lavage fluid • Onset: early, within first 100 days post transplant. • Radiographic findings: diffuse infiltrates, central appearance initially noted • Histology: diffuse alveolar damage with alveolar hemorrhage Peri-engraftment respiratory distress syndrome: • Clinical symptoms: fever, dyspnea, hypoxemia • Onset: very early, within 5–7 days of engraftment, classically after autologous stem cell transplantation • Radiographic findings: bilateral interstitial infiltrates Noncardiogenic capillary leak syndrome: • Clinical symptoms: dyspnea, cough, weight gain, edema • Onset: early, within first 30 days post transplant • Radiographic findings: bilateral perihilar infiltrates, pulmonary edema, pleural effusions Bronchiolitis obliterans organizing pneumonia: • Clinical symptoms: fever, dry cough, dyspnea • Onset: 2–12 months post transplant • Radiographic findings: patchy airspace disease, ground glass appearance, nodular opacities • Histology: peribronchiolar infiltration and fibrosis and the presence of intraluminal granulation tissue Bronchiolitis obliterans syndrome: • Clinical symptoms: cough, dyspnea, wheezing, lack of fever • Pulmonary function testing: obstructive findings (diminished FEV1 or FEV1:FVC) • Onset: 3–24 months post transplant • Radiographic findings: hyperinflation. Otherwise routinely normal • Computed tomography: bronchiectasis, centrilobular nodules, septal lines, ground glass appearance • Histology: lymphocytic bronchitis; bronchiolar inflammation with luminal obliteration CMV, cytomegalovirus; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity.

transfusion. Chest radiographs reveal diffuse pulmonary infiltrates reflecting edema from increased pulmonary vascular permeability. Treatment is generally supportive. Discontinuation of the blood product, corticosteroid administration, forced diuresis, and respiratory support results in recovery within 3–4 days in the majority of patients. In over 70% of cases, antibodies directed against human leukocyte antigen class I or II epitopes on recipient hematopoietic cells have been identified as the primary cause of the TRALI event, but in rare cases, the antibody may be present in the recipient’s plasma, and may be directed against transfused donor leukocytes [43,44].

Table 96.4 Risk factors for idiopathic pneumonia syndrome Graft-versus-host disease prophylaxis (methotrexate) Acute graft-versus-host disease Increasing recipient age Total body irradiation (≥1200 cGy) Myeloablative conditioning Decreased pretransplant performance status Longer duration from diagnosis to transplant Transplantation for malignancy other than leukemia Human leukocyte antigen disparity (donor: recipient)

Risk factors for IPS As shown in Table 96.4, risk factors for IPS include conditioning with high-dose, total body irradiation, acute GVHD, older recipient age, initial diagnosis of malignancy other than leukemia, and the use of methotrexate for GVHD prophylaxis [5,45]. Although recipient age and the use of methotrexate are not always risk factors, total body irradiation and the development of acute GVHD have been identified as factors in multiple reports [2,5,6,46,47]. Recently, the cumulative incidence of IPS within 120 days of HCT was found to be significantly lower after reduced-intensity conditioning than observed following conventional

conditioning, despite greater patient age and a similar incidence of acute GVHD in the reduced-intensity group [48]. Once established, however, pulmonary toxicity was severe in each group and resulted in respiratory failure in the majority of patients. These findings suggest that the intensity of HCT conditioning plays an important role in the development of IPS, and are consistent with data generated from mouse HCT models showing that the lung is sensitive to the combined effects of radiation and alloreactive T cells [49,50].

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Etiology of IPS Potential etiologies for IPS include direct toxic effects of HCT conditioning regimens, occult pulmonary infections, and the release of inflammatory cytokines that have been implicated in other forms of pulmonary injury. The association between IPS and severe GVHD reported in several large series [2,3,5–7] suggests that immunologic factors may also be operative in the development of lung injury. Acute GVHD often precedes IPS, suggesting a possible causal relationship between the two disorders [5,51,52]. Although IPS can also occur when signs and symptoms of GVHD are limited or absent, the consistent association between lung injury and GVHD in experimental models also supports such an etiology [2,3,5,6,50,53–56]. Despite the aforementioned clinical association, the lung has not been traditionally recognized as a classic GVHD target organ, and the specific role of alloreactive donor T lymphocytes in the pathogenesis of IPS remains a topic of considerable debate. Epithelial apoptosis is usually attributed to T-cell-mediated injury and is considered pathognomonic for acute GVHD. Although identified in the lungs of some patients with IPS [28,52], epithelial apoptosis has not been consistently observed in allogeneic HCT recipients with pulmonary dysfunction. A histologic spectrum of pulmonary GVHD has recently been described that ranges from diffuse alveolar injury early after HCT to cicatrical bronchiolitis obliterans, a late and irreversible form of lung injury [28]. Bronchitis/ bronchiolitis with IP was the most common finding and included a lymphocytic infiltration around bronchial structures along with a mononuclear inflammation in the perivascular zones and alveolar septa. The heterogeneity of pulmonary histopathology after allogeneic HCT is complicated further by the nonspecific changes that occur after mechanical ventilation and by the limited quality and quantity of lung biopsy tissue. Animal models of human disease The relationship between alloreactivity and IPS has been explored by several laboratories using a variety of murine models (Table 96.5). Rodent HCT models have consistently shown that animals with systemic GVHD develop lung injury, whereas syngeneic, non-GVHD controls do not [50,53,55,57]. Several patterns of lung injury have been identified including acute alveolitis, late-onset IP, and lymphocytic bronchiolitis [53]. In models where the graft-versus-host reaction is induced to (1) minor histocompatibility antigens, (2) class I or class II major histocompatibility complex (MHC) antigens only or (3) both major and minor histocompatibility antigens, two major abnormalities are apparent after allogeneic HCT: a dense mononuclear cell infiltrate around both pulmonary vessels and bronchioles, and an acute pneumonitis involving the

interstitium and alveolar spaces [55,58,59]. These patterns of inflammation closely resemble those reported in allogeneic HCT recipients [27,28,52,60]. Significant alterations in pulmonary function are associated with lung histopathology, demonstrating that the observed lung inflammation is physiologically relevant [56,57]. Furthermore, lung injury correlates with the presence but not the severity of GVHD, consistent with clinical reports of IPS in allogeneic HCT recipients whose signs and symptoms of GVHD were mild [8,9,29,61–63]. Thus, mouse models of IPS reproduce many of the histologic and functional changes observed during human disease. The pathogenesis of IPS Soluble inflammatory effectors: tumor necrosis factor-alpha and lipopolysaccharide The mixed inflammatory alveolar infiltrates found in mice with IPS are accompanied by significant increases in the total number of lymphocytes, macrophages, and neutrophils in the bronchoalveolar space [55], and by increased tumor necrosis factor-alpha (TNF-α) levels in both lung tissue and BAL fluid [50,54,55,64,65]. The presence of neutrophils and TNF-α in the absence of infection suggests that endogenous endotoxin/lipopolysaccharide (LPS) may contribute to the pathophysiology of IPS. LPS levels are elevated in the BAL fluid of mice with IPS, and the intravenous administration of LPS to mice with advanced GVHD significantly amplifies lung injury [55]. The enhanced inflammation is associated with large increases in TNF-α and LPS in the BAL fluid, and the development of alveolar hemorrhage [55,65]. Further, direct antagonism of LPS early in the time course of HCT reduces systemic levels of TNF-α and significantly decreases the severity of GVHD and IPS compared with control treated animals [66]. A causal role for TNF-α in the development of IPS has been established using strategies that either neutralize its effects [65,67] or use TNF-α-deficient mice as HCT donors [68,69]. Administration of recombinant human TNF-α receptor fusion protein (rhTNFR:Fc), a soluble, dimeric, TNF-binding protein, at the time of LPS challenge effectively prevents enhanced pulmonary inflammation, confirming the linkage between LPS and TNF-α in this setting [65]. Neutralization of TNF-α during the development of IPS also reduces the severity of lung injury during the natural course of disease [65]. Recent studies using genetically altered mice have shown that IPS is dependent upon donor-, rather than host-, derived TNF-α. While TNF-α from both donor accessory cells (macrophage/monocytes) and T cells significantly contributes to lung injury, the T-cell component predominates [69]. The actions of TNF-α are mediated by two receptors: a 55–60 kDa type I receptor (TNFRI; p55/60; CD120a) and a 75–80 kDa type II

Table 96.5 Animal models of idiopathic pneumonia syndrome Hematopoietic cell transplant donors

Stem cell transplant recipients

Mismatch

Conditioning

Reference

B10.BR C57BL/6 C57BL/6 C57BL/6 B6C3F1 hybrid C57BL/6 C57BL/6 LP/J

CBA B10.BR B6.C-H2bm1/By B6.C-H2bm12/KhEg B6C3F1 hybrid B6D2F1 hybrid B6.C-H2bm1/By C57BL/6

Multiple minor antigens Complete mismatch MHC class I MHC class II None (syngeneic) Haploidentical MHC class I Multiple minor antigens

TBI: 1100 cGy TBI: 750 cGy ± cyclophosphamide TBI: 675 cGy TBI: 675 cGy Cytoxan, cisplatin, BCNU TBI: 1100–1300 cGy TBI: 1100 cGy TBI: 1300 cGy

[55,56,65,92] [57,74,97,101] [100] [100] [79] [69,85,96] [99,72] [59,72]

MHC, major histocompatability complex; TBI, total body irradiation.

Lung Injury Following Hematopoietic Cell Transplantation

receptor (TNFRII; p75/80; CD120b) [70]. TNFRI is constitutively expressed, while the expression of TNFRII is strongly modulated by various cytokines and other inflammatory stimuli, including LPS. In an animal model, the absence of TNFRI was associated with decreased pulmonary edema and improved lung compliance on day 7 of allogeneic HCT [71]. However, cellular infiltration into the lung and BAL fluid levels of proinflammatory cytokines were actually higher in TNFRI−/− mice than controls. These findings are consistent with a recent report showing that compared with allogeneic controls, TNFRII-deficient HCT recipients develop significantly less severe IPS, which is associated with reductions in expression of pulmonary intercellular adhesion molecule-1 (ICAM-1) and in leukocyte infiltration into the lungs. This protective effect is comparable to that observed when wild-type mice are transplanted with allogeneic TNF-α-deficient donor cells, thereby strengthening the view of the role of TNF-α–TNFRII interactions in the pathophysiology of IPS [72]. TNF-α likely contributes to the development of IPS through both direct and indirect mechanisms. In addition to being directly cytotoxic, TNF-α increases expression of inflammatory chemokines [69] and MHC antigens, modulates leukocyte migration, and facilitates cell-mediated cytotoxicity. TNF-α may also contribute to lung injury by increasing the severity of GVHD in other target organs such as the gut and liver, thus promoting the release of other inflammatory mediators and their ultimate passage to the pulmonary vascular bed. From this perspective, the structural and functional integrity of the liver is critical; the liver is pivotally located between the intestinal reservoir of Gram-negative bacteria and their toxic byproducts, and the rich capillary network in the lung. In the setting of acute GVHD, an endotoxin surge into the systemic blood stream can arise from increased leakage of LPS across damaged intestinal mucosa, and underlying damage to the liver could decrease its capacity for LPS uptake and clearance. Consistent with this scenario, animals with mild or no GVHD can effectively detoxify endotoxin and protect their lungs from further damage, whereas mice with severe GVHD are unable to do so, and develop severe extensive lung injury including alveolar hemorrhage [55]. These data suggest that the inflammatory mediators TNF-α and LPS both contribute to experimental IPS and may do so along a “gut– liver–lung” axis of inflammation (Fig. 96.2). A clinical linkage of hepatic dysfunction to lung injury after HCT is also suggested by associations between sinusoidal obstructive syndrome and IPS, and between hepatic failure and death from IPS [2,29]. Further, evidence for cytokine activation and LPS amplification has been demonstrated in patients with IPS after allogeneic HCT; increased pulmonary vascular permeability and increases in BAL fluid levels of several cytokines, including LPSbinding protein and soluble CD14, were also observed in these patients [1]. Other soluble mediators Strategies that neutralize TNF-α in experimental models do not completely abrogate lung injury [53,65,67,69,73], suggesting that other inflammatory and cellular mechanisms may also contribute to the development of IPS. For example, IL-1β, nitric oxide, and reactive oxygen species have also been implicated in the development of lung injury after HCT, particularly when cyclophosphamide is included in the conditioning regimen [57,74,75]. From a clinical perspective, a recent analysis of plasma and BAL fluid protein profiles in patients with IPS showed that, in addition to increases in the levels of TNF-α and its soluble receptors, significant elevations in other molecules that participate in the cellular activating effects of LPS (soluble CD14, LPS-binding protein), along with three inflammatory chemokines (interleukin-8 [IL-8], monocyte chemoattractant protein-1 [MCP-1], and monokine induced by interferon-gamma [IFN-γ] [MIG]) that regulate leukocyte recruitment to sites of inflammation, were also evident [76].

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The proinflammatory environment observed following allogeneic HCT is frequently associated with the generation of oxidative stress and increased production of reactive oxygen species [77,78]. Exposure to radiochemotherapy further increases oxidant stress by depleting antioxidants, including reduced glutathione, the major antioxidant in the epithelial lining fluid of the lung, findings which have also been observed in murine IPS models after both allogeneic and autologous HCT [79,80]. While direct evidence that oxidative stress plays a distinct role in the development of IPS injury in human is lacking, experimental models demonstrate that the administration of n-acetylcysteine to HCT recipients substantially reduces lung injury compared with mice receiving high-dose chemotherapy without n-acetylcysteine [79]. Taken together, these data suggest that amelioration of oxidative stress may be an effective strategy to reduce the severity of pulmonary toxicity during IPS.

Cellular effectors and the development of IPS Donor-derived T-cell effectors The role of alloreactive donor T cells in the pathogenesis of IPS remains a topic of considerable debate. The importance of lymphocytes to lung injury after experimental HCT has also been shown by several groups [54–57,69]. Donor T cells are critical to the early proinflammatory events associated with lung injury that develops within the first week of HCT across MHC antigens, whereas in minor histocompatibility antigen-mismatch systems, donor lymphocytes continue to contribute to physiologically significant lung injury at later time points [56,57]. Donor T-cell clones that recognize CD45 polymorphisms result in a rapidly progressive pulmonary vasculitis within 3 days of their injection into nonirradiated recipients [54]. The origin and functional capacity of T cells infiltrating the lung have been examined by using differences in the T-cell Vβ repertoire between donor and recipient [56]. Flow cytometry demonstrated that the TCRαβ+ T cells found in the lung 6 weeks after allogeneic HCT were of donor origin, and when these T cells were recultured with irradiated host antigen-presenting cells, they proliferated vigorously and produced significant amounts of IFN-γ [56]. Donor cytotoxic lymphocytes can contribute to lung injury via three primary cytolytic mechanisms: the perforin–granzyme, Fas/Fas ligand (FasL), and TNF-α pathways. Each pathway has been shown to contribute to lung injury in non-HCT models [81,82]. Further, cytolytic T cells expressing granzyme B are present in the lungs of mice after allogeneic HCT, and they colocalize with macrophages expressing the costimulatory molecules B7.1/CD80 and B7.2/CD86 [57]. Alloantigenspecific killing by donor T cells using both perforin and Fas/FasL pathways has also been identified in the lung after HCT. Pulmonary cytotoxic lymphocyte activity is present as early as week 2 after transplant, and cytotoxicity mediated by Fas/FasL contributes to the progression of lung inflammation [58]. Despite these compelling data supporting a role for alloreactive donor lymphocytes in the development of noninfectious lung injury after HCT, IPS has been reported in patients in whom systemic GVHD is mild or absent, making a causal relationship between the two entities difficult to establish. The relationship between lung injury and GVHD severity has been examined in a HCT model across minor histocompatibility antigens. T-cell depletion at the time of allogeneic HCT reduced the number of T cells by greater than 99% and eliminated evidence of clinical or histologic GVHD. Nevertheless, significant lung injury was noted after allogeneic T-cell-depleted HCT, and donor lymphocytes reactive to host antigens were present in the BAL fluid, but not the spleens, of these animals [56]. The precise mechanisms and locations in which T cells interact with host antigens and ultimately cause injury remain unresolved, but this process is likely to involve interactions with dendritic cells (DCs) either

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BMT Conditioning HOST TISSUE INFa

GUT

IL-1 IL-6

LPS LIVER

Host APC

Stage two

Donor T Cell

Mf

IL-2 CTL Stage one

LPS

IFNg

NK

TNFa

Stage three PMNs

LUNG

Apoptosis Lymphocyte axis

Cytokine axis

Fig. 96.2. Pathophysiology of idiopathic pneumonia syndrome (IPS) after allogeneic hematopoietic cell transplantation (HCT). Data generated using murine HCT models have been incorporated into a working hypothesis of IPS physiology. This schema postulates that the lung is susceptible to two distinct but interrelated pathways of immune-mediated injury that occur along a T-lymphocyte activation axis and a “gut–liver–lung” axis of inflammation. Chemoradiotherapy of HCT conditioning causes cytokine release that enhances the ability of host antigen-presenting cells (APCs) to present alloantigens to mature donor T cells and upregulates chemokine expression in the lung. Once engaged, donor T cells become activated and secrete IFNγ and interleukin-2 (IL-2). Interferon-gamma (IFNγ) primes donor macrophages (Mφ) and monocytes, whereas IL-2 facilitates T-cell activation and the generation of CXCR3expressing type 1 T helper lymphocyte effectors that migrate to the lung early after HCT (stage one) in response to inflammatory chemokine gradients, and contribute to pulmonary toxicity via Fas–FasL-mediated cell killing. The inflammatory axis focuses on the relationship between the cellular activating effects of lipopolysaccharide (LPS) and the downstream production of tumor necrosis factor-alpha (TNFα) as it occurs along a gut–liver–lung axis of inflammation. LPS enters the systemic circulation through gaps in the intestinal mucosa. The ability of systemic endotoxin to reach the alveolar space is related to the consequences of graft-versus-host disease in other target organs, particularly the liver, which is pivotally located immediately downstream (via the splanchnic circulation) of the intestinal reservoir of Gram-negative bacteria and their toxic byproducts. CCR2-expressing donor macrophages primed by IFNγ are recruited to the lung (stage two), where they are triggered by LPS to secrete inflammatory cytokines like TNFα, resulting in enhanced chemokine expression, the recruitment of neutrophils to the lung, and increased tissue damage (stage three). NK, natural killer; PMN, polymorphonucleocyte.

resident in the respiratory system or in peripheral, secondary lymphoid organs. Pulmonary DCs are located in the interstitium and in the bronchial epithelium and submucosa, where DC tissue density diminishes with decreasing airway diameter. During steady-state conditions, pulmonary DCs constitute the sole source of MHC class II expression within the epithelial lining of the airway, and they have been shown to play a critical role in the initiation of both acute and chronic rejection of lung allografts [83]. It is possible that radioresistant host DCs persist longer in the lung than in other organs and allow for sustained presentation of host antigens [84]. Activated donor T cells might therefore remain within the pulmonary microvascular circulation because hosttype DCs function as a persistent site of alloantigen presentation. This scenario could account for the apparent “sanctuary” status of the lung with respect to alloreactive donor T cells, and may have important implications with regard to the evaluation and treatment of IPS after allogeneic HCT even when clinical GVHD is absent. Donor accessory cells Experimental data suggest that synergistic interactions between cells from the lymphoid and myeloid lineage are critical to the development of IPS. Specifically, the production of IFN-γ from activated donor T cells primes mononuclear cells and macrophages to secrete inflammatory cytokines when stimulated with LPS. The contribution of donor accessory cells (monocytes/macrophages/neutrophils)

to IPS has been investigated using several models. Kinetic studies of macrophage recruitment to the lung after allogeneic HCT show that the percentage of donor macrophages in the BAL fluid increases from approximately 40% at week 1 to over 90% by week 4 [85]. Additional experiments have shown that these donor-derived macrophages are a significant, albeit not the primary, source of TNF-α after HCT [69]. Studies completed using HCT donors that differ in their response to LPS (by virtue of a genetic mutation in the Toll-like receptor-4 [Tlr4] gene or the absence of CD14, a key cell surface receptor for the LPS– LPS-binding protein complex) have shown that recipients of LPS-resistant donor cells develop significantly less lung injury compared with recipients of wild-type, LPS-sensitive donors [68,86]. The results obtained using CD14-deficient donors are consistent with the report that monocytes recruited to an inflamed lung upregulate CD14 expression and show enhanced sensitivity to LPS stimulation [87], and with the clinical observations made in the BAL fluid of patients with IPS [1]. Collectively, these data demonstrate that donor macrophages/monocytes cells are recruited to the lungs after allogeneic HCT, and their ability to secrete TNF-α in response to LPS stimulation directly correlates with IPS severity. Polymorphonuclear cells are a major component of the BAL fluid of animals with IPS [55]. In mouse IPS models, the BAL fluid neutrophilia is prominent between weeks 4 and 6 after HCT, and is associated with increases in BAL fluid levels of TNF-α and LPS [55,65]. Neutralization

Lung Injury Following Hematopoietic Cell Transplantation

of TNF-α with rhTNFR:Fc during this time interval prevents the influx of neutrophils and reduces the progression of lung injury and dysfunction [65]. Administration of rhTNFR:Fc following LPS challenge completely abrogates the recruitment of neutrophils into the lungs and prevents further damage (including hemorrhage), underscoring the relationship between neutrophils, TNF-α, and LPS in this setting. Neutrophil products such as elastase, myeloperoxidase, metalloproteinases, and oxidants are abundant in the BAL fluid of patients with acute respiratory distress syndrome, and are believed to contribute to the endothelial and epithelial damage that occurs in this setting [88,89]. Neutrophils are likely to play a role in patients with IPS as well; their appearance in the blood stream is often temporarily associated with the onset of lung inflammation [29,34]. Mechanisms of leukocyte recruitment to the lung during IPS Cellular effectors play a significant role in the development of IPS, but the molecular mechanisms by which leukocytes traffic to the lung and cause damage have yet to be fully elucidated. White blood cell migration to sites of inflammation involves interactions between leukocytes and endothelial cells that are mediated by adhesion molecules, chemokines, and their receptors [90,91]. The recruitment of leukocytes from the vascular space and into target tissue can be divided into four steps: (1) weak adhesion of leukocytes to the vascular endothelial cells; (2) firm adhesion to endothelial cells; (3) transmigration of leukocytes through the vascular wall; and (4) migration of cells through the extracellular matrix along a chemotactic gradient. The role of adhesion molecules in the development of IPS has been examined using mouse models. Various adhesion molecules are upregulated by proinflammatory cytokines such as TNF-α and the messenger RNA (mRNA) expression of ICAM-1, vascular cell adhesion molecule-1, and E-selectin is increased in the lungs of mice with IPS after allogeneic HCT [92]. Further, the severity of IPS is dramatically reduced when ICAM-1-deficient (ICAM-1−/−) mice are used as HCT recipients of either MHC matched or mismatched allogeneic donor cells [59,93]. Surprisingly, in each scenario, ICAM-1 deficiency selectively protects the lung from injury even though ICAM-1 expression is elevated in the liver, colon, and spleen of animals with acute GVHD [94]. Chemokine receptor–ligand interactions facilitate the recruitment of leukocytes to the lung in a variety of inflammatory states [95], and investigators have recently begun to explore their role in IPS. The mixed pulmonary infiltrate observed after experimental allogeneic HCT therefore suggests that those chemokines responsible for the recruitment of lymphocytes, monocytes, and neutrophils will be upregulated during the development of IPS. The pulmonary expression of inflammatory chemokine receptors for T-cell effectors (CCR1 and CXCR3), monocytes and macrophages (CCR2), and neutrophils (CXCR2), and their respective ligands, have been analyzed in mouse IPS models. Compared with syngeneic controls, mRNA expression of each chemokine receptor and ligand is increased in allogeneic HCT recipients [96,97]. Elevations in CCR1 and CXCR3 expression peak early after allogeneic HCT, whereas expression of CCR2 and CXCR2 continue to rise over time. Importantly, the kinetics of chemokine ligand expression correlated with the corresponding receptors; increases in RANTES, macrophage inflammatory protein-1 (MIP-1α; CCR1), and IFN-γ-inducible protein-10 (IP-10; CXCR3) peaked at week 1 and then tapered off, whereas increases in MCP-1 (CCR2) and MIP-2 (CXCR2) peaked at weeks 2 and 4, respectively. These data are consistent with prominent increases in the expression of IFNγ-inducible chemokines observed when lung injury is induced by alloreactive type 1 T helper (Th1) cells [98]. Significant increases in chemokine ligand expression in the lung therefore precede the development of IPS and herald the influx of leukocyte subsets bearing the corresponding chemokine receptors. Enhanced chemokine expression and leukocyte infiltration during IPS can be conceptu-

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alized in three distinct stages. In stage 1 of the process, HCT conditioning and the initial allogeneic donor T-cell response result in systemic inflammation during the first week after transplantation. This proinflammatory environment is characterized by the release of IFN-γ, TNF-α, and LPS, and causes increased chemokine expression in the lung. Production of RANTES, MIP-1α, and IP-10 recruits donor-derived, Th1/type 1 cytotoxic lymphocyte (Tc1) lymphocyte effectors (CXCR3+) within the first 2 weeks of HCT [96–99]. In stage 2, T cells cause local tissue injury and activate endothelial and epithelial cells to secrete additional chemokines, including MCP-1. Enhanced expression of MCP-1 in turn recruits CCR2+ donor monocytes, macrophages, and additional T cells [96,97]. In stage 3, recruited macrophages secrete TNF-α in response to LPS stimulation, which results in additional tissue injury, the upregulation of the chemokine ligands cytokine-induced neutrophil chemoattractant (KC) and MIP-2, and the recruitment of donor, CXCR2+ neutrophils. These neutrophils amplify progressive lung injury and dysfunction. This hypothesis stipulates that the migration of each leukocyte subset controls the recruitment of the next wave of effectors. Thus, the recruitment of Th1/Tc1 effectors initiates the cascade, and interruption of this first stage should have profound effects on the development of IPS. Data in support of this hypothesis have recently been generated by using congenic HCT donors that express allelic differences of CD45 on all leukocytes. Using this system, greater than 95% of CD4+ and CD8+ lymphocytes in the bronchoalveolar space are of donor origin by the first week, and turnover is complete by week 2 after HCT [69]. These findings were complemented by a subsequent study using an irradiated murine MHC class I mismatched model wherein IPS and GVHD are mediated by CD8+ T cells. Elevated levels of MIG (CXCL9) and IP-10 (CXCL10) correlated with the recruitment of CXCR3-expressing CD8+ T cells to the lung as early as day 7. In vivo neutralization of CXCL9 or CXCL10, or HCT using CXCR3−/− donor leukocytes, resulted in a near-complete abrogation of infiltrating CD8+ T cells and IPS severity [99]. Donor T cells initially recruited to the lung may play an active role in regulating the inflammation engendered during the evolution of IPS. For example, experiments using genetically altered mice have shown that TNF-α secreted by donor lymphocytes regulates the chemokine milieu in the lung within the first 2 weeks after HCT, which directly contributes to the subsequent recruitment of monocytes and macrophages as lung injury progresses [69]. Similarly, enhanced pulmonary expression of CCL5 (RANTES) correlates with the initial influx of donor T cells into the lungs after allogeneic HCT, and the autocrine production of CCL5 by these infiltrating cells significantly contributes to the subsequent recruitment of additional T cells and accessory cells over time [85]. By contrast, the role of donor-derived CCL3 (MIP-1α), a chemokine that shares common receptors with CCL5, has been less clear. CCL3 contributes to the recruitment of leukocytes during IPS following nonmyeloablative conditions [100]. However using Mip-1 α−/− donor mice in a myeloablative model exacerbated rather than reduced early lung injury [101], providing further support that receptor–ligand interactions of CCL5, likely via its co-receptor CCR1, may be dominant in the evolution of IPS [102]. Targets of inflammation and injury during IPS Pulmonary endothelial and epithelial cells can express MHC class I, MHC class II, and minor histocompatibility antigens, and the expression of these molecules is enhanced by TNF-α and IFN-γ. It is therefore conceivable that pulmonary parenchymal cells can serve as targets for direct cell-mediated damage. Endothelial cell injury has been observed after allogeneic HCT, and has been implicated as a direct contributor to the development of several complications, including GVHD, sinusoidal obstructive syndrome, and thrombotic microangiopathy [103]. Clinical and experimental IPS is associated with evidence for endothelial cell

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injury and leak, as demonstrated by pulmonary edema, enhanced total protein levels in BAL fluid, and increased wet-to-dry lung weight ratios [57,99]. Moreover, leukocyte infiltration during IPS is accompanied by significant apoptosis of the pulmonary vascular endothelium [92]. Endothelial cell apoptosis coincides with the onset of pulmonary pathology, is associated with elevations in BAL fluid TNF-α levels, and is accompanied by evidence for endothelial cell activation as measured by enhanced mRNA expression of adhesion molecules [92]. The administration of a soluble TNF-α-binding protein (rhTNFR:Fc) from week 4 to week 6 after allogeneic HCT significantly reduces the endothelial cell apoptosis and lung histopathology observed in mice [92]. In this light, TNF-α may therefore function as both an effector and a facilitator of lung injury by contributing directly to endothelial cell injury and death, and regulating the chemokine milieu in the lung during the early stages of IPS. By contrast, epithelial apoptosis, generally ascribed to T-cell-mediated injury and considered pathognomonic for acute GVHD in other target tissues, has not been consistently observed in allogeneic HCT recipients with lung injury. The unique aspects of epithelial anatomy in the lung may help explain this discrepancy. Since there is no stratification or layering of pulmonary epithelial cells as in the skin or intestine, identification of epithelial cell apoptosis by histologic criteria can be more challenging. Experimental studies have, however, provided evidence for epithelial injury during IPS. Panoskaltsis-Mortari and coworkers demonstrated that IPS was associated with injured alveolar type II cells and increased frequencies of cytotoxic T lymphocytes in a model of early-onset IPS [57]. The same group later showed that keratinocyte growth factor, a mediator of epithelial cell proliferation and a growth factor for type II pneumocytes, diminished IPS injury by dampening the immune response to chemoradiotherapy and by accelerating repair of the damaged tissue, specifically alveolar type II epithelial cells [104]. The approach to HCT patients with acute respiratory dysfunction The approach to HCT patients with acute pulmonary dysfunction is complex and requires a variety of diagnostic tests and possible consultation with experts in the fields of pulmonology, cardiology, nephrology, radiology, and critical care medicine. Because symptoms of respiratory distress can progress rapidly once established, the timely coordination of care is essential to optimizing outcomes. In addition, there are several diagnostic challenges to address in this setting, as both pulmonary and nonpulmonary causes of respiratory compromise are possible. Determination of the severity of respiratory dysfunction, including an assessment of the need for supplemental oxygen support, overall fluid balance, renal function, and cardiac output should be followed by radiographic imaging. In general, an initial chest X-ray or computed tomography scan will identify the presence of lobar, multilobar or diffuse pulmonary infiltrates. While such findings may impact the decision-making process (Fig. 96.3), they are nondiagnostic in and of themselves. In the absence of obvious left-sided heart failure or iatrogenic fluid overload, bronchoscopy with BAL should be strongly considered when multilobar or diffuse infiltrates are present. As outlined in Table 96.2, BAL samples should be sent for a variety of diagnostic tests to determine the presence of community-acquired, hospitalacquired, and otherwise opportunistic infections. While accepted at many large transplant centers, the need to complete a BAL in HCT recipients with significant respiratory compromise remains a matter of debate, particularly in those patients who are critically ill. Recently, Yanik and colleagues reported on the results of 444 bronchoscopy procedures completed on 300 (20% of all patients) who received HCT at the University of Michigan from 2001 to 2007 [105]. Thirteen percent of BAL specimens collected in the first 30 days of HCT were positive for infection, and this number increased to 33% between

days 31 and 100. Hence, while the majority of HCT patients requiring BAL within the first 100 days may have IPS, a significant number of individuals will have evidence for infection. To this end, BAL data resulted in changes in medical management in approximately 60% of cases, and modifications in antimicrobial therapy in just under half. Since the medical management of IPS (immunosuppression) and infection (antimicrobial therapy) are rather divergent, making the appropriate diagnosis has significant merit. As shown in Table 96.6, current standard treatment regimens for IPS include supportive care measures in conjunction with broad-spectrum antimicrobial agents and intravenous corticosteroids [6,29]. Unfortunately, responses to standard therapy are limited, and the mortality of patients diagnosed with IPS remains unacceptably high [7]. Moreover, high-dose corticosteroid therapy (>2 mg/kg/day of methylprednisolone equivalent) has not been shown to improve outcome when compared with lower doses of corticosteroids (≤2 mg/kg/day) [48]. Advances in supportive care including the early institution of continuous veno-venous hemofiltration may help to improve survival in some patients, but prospective studies addressing the treatment of IPS, including the specific use of steroids, are lacking in the literature. Translational research studies suggest that etanercept may be a useful therapeutic option for IPS [29]. Etanercept was administered in combination with systemic steroids and empiric antimicrobial therapy to a total of 18 patients with IPS [106]. Etanercept was given subcutaneously at a dose of 0.4 mg/kg twice weekly for a maximum of eight doses. Therapy was well tolerated overall. Thirteen of 18 patients were able to completely withdraw from supplemental oxygen support within 28 days of therapy. Survival at day 28 and day 56 (from the first etanercept dose) was 73% and 60%, respectively. Based upon these encouraging results, larger phase II (pediatric) and phase III (adult) trials are ongoing within the Bone Marrow Transplant Clinical Trials Network (phase III) and the Children’s Oncology Group (phase II). Summary IPS remains a frequent and severe complication of allogeneic HCT, and mortality rates remain unacceptably high with standard therapy. Preclinical and clinical data suggest that both inflammatory and cellular effectors participate in the development of IPS after allogeneic HCT. TNF-α and LPS are significant, albeit not exclusive, contributors to IPS, and cells of both lymphoid and myeloid origin play a direct role in lung injury that occurs in this setting. The contribution of donor, nonlymphoid, accessory cells may be linked to cellular activation by LPS and the ultimate secretion of TNF-α within a “gut–liver–lung” axis of inflammation, whereas donor T-cell effectors can home to and damage the lung even when systemic GVHD is mild or absent. Hence, significant research efforts have resulted in a paradigm shift away from identifying lung injury after HCT solely as an idiopathic clinical syndrome and toward understanding IPS as a process involving two distinct, but interrelated, pathways of immune-mediated injury involving aspects of both the adaptive and the innate immune response. Importantly, new laboratory insights are currently being translated to the clinic and will likely prove important to the development of future strategies to prevent or treat this frequently fatal complication.

Chronic pulmonary dysfunction after HCT: obstructive and restrictive lung disease Definition, risk factors, and clinical course Decline in lung function has long been identified as a significant complication in the months to years that follow allogeneic HCT. Two forms

Lung Injury Following Hematopoietic Cell Transplantation

1465

S/S of respiratory distress Cough, rales Supplemental oxygen requirement

Obtain CXR or chest CT scan Close observation Assess fluid status

Normal Abnormal

Diffuse infiltrates

Lobar infiltrate

Yes Yes

S/S of heart failure or fluid overload? Empiric antibiotic treatment

Yes

Treat

Yes

Treat

No

S/S of sepsis? Response

No

No response

Bronchoscopy/BAL Continue antibiotics

Bronchoscopy

Normal

Abnormal

Treat identified infection

Diagnosis: IPS

Modify antimicrobial regimen

Corticosteroids 2 mg/kg/day Consider

Investigational trials

TNF inhibitors

CVVH

Other

Fig. 96.3. Approach to hematopoietic cell transplantation (HCT) patients with acute respiratory dysfunction. The approach to HCT patients with acute pulmonary dysfunction is complex. The timely determination of the severity of respiratory dysfunction, including an assessment of the need for supplemental oxygen support, overall fluid balance, renal function, and cardiac output should be followed by radiographic imaging and consideration for bronchoscopy and bronchoalveolar lavage (BAL). CT, computed tomography; CVVH, continuous veno-venous hemofiltration; CXR, chest X-ray; IPS, idiopathic pneumonia syndrome; S/S, signs and symptoms; TNF, tumor necrosis factor. Table 96.6 Treatment options for idiopathic pneumonia syndrome Supportive therapy

Immunosuppressive therapy

Supplemental oxygen, mechanical ventilation Empiric broad-spectrum antimicrobial agents pending culture results Management of iatrogenic fluid overload Continuous veno-venous hemofiltration Corticosteroids (2 mg/kg/day) Investigational: cytokine inhibitors, including anti-tumor necrosis factor agents

of chronic pulmonary dysfunction are common in patients surviving longer than 100 days after allogeneic HCT: obstructive lung disease (OLD) and restrictive lung disease (RLD). The incidence of both patterns of lung toxicity ranges from 20% to 50% depending upon donor

source and the time interval after HCT [8–14,63]. In each scenario, collagen deposition and the development of fibrosis either in the interstitial (RLD) or peribronchiolar space (OLD) are believed to contribute to the patterns of lung dysfunction displayed on pulmonary function testing. While both forms of pulmonary dysfunction exist as late-onset, noninfectious lung complication following allogeneic HCT, RLD and OLD can be distinguished by a number of clinical parameters as described below (Table 96.7). Restrictive lung disease RLD is defined by reductions in forced vital capacity (FVC), total lung capacity (TLC), and diffusion capacity of the lung for carbon monoxide (DLCO) as measured by standard PFTs. In restrictive disease, the ratio of the forced expiratory volume in 1 second (FEV1) to FVC (FEV1/FVC) is maintained near 100% [7,10,13,62]. RLD is common after HCT; significant decreases in FVC or TLC have been reported in as many as 25–45%

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Table 96.7 Clinical factors present in obstructive versus restrictive lung disease Clinical factor

Obstructive lung disease

Restrictive lung disease

Onset Symptoms Physical examination Pulmonary function tests FEV1:FVC Total lung capacity DLCO Computed tomography findings

Late (3–12 months) Dyspnea, nonproductive cough Wheezing Obstructive physiology Decreased Normal Decreased Air trapping Bronchial wall thickening Centrilobular nodules Strong association

Early (within 3 months) Dyspnea, nonproductive cough Rales Restrictive physiology Normal Decreased Decreased Patchy consolidation: BOOP “Ground glass” opacities

Chronic graft-versus-host disease

Variable, association with BOOP

BOOP, bronchiolitis obliterans organizing pneumonia; DLCO, diffusing capacity of the lung for carbon dioxide; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity.

of allogeneic HCT recipients at day 100 [10,13,14]. A decline in TLC or FVC at 100 days and 1 year after HCT compared to pretransplant values, even if the absolute values for each measurement remained within the normal range, has been associated with an increase in nonrelapse mortality [10,13]. Total body irradiation-containing conditioning regimens and the presence of acute GVHD have been associated with RLD [10,13,14], but the impact of age on the development of RLD is less clear. Early reports suggested that the incidence of RLD is lower in children compared with adults, and that the incidence increases with advancing recipient age [14], but more recent studies have revealed significant RLD in children receiving HCT [107]. In contrast to OLD, RLD after HCT has not been clearly associated with the presence of chronic GVHD. Histologic features of RLD after HCT are rarely described in the clinical literature, although varying degrees of interstitial and alveolar inflammation and fibrosis, as seen in patients with other forms of interstitial pulmonary fibrosis, would be expected. One exception is BOOP. Although reported in less than 2% of HCT recipients, BOOP is associated with restrictive (rather than obstructive) changes on PFTs [7,108]. Clinical features resemble idiopathic BOOP and include dry cough, shortness of breath, and fever, and radiographic findings show diffuse, peripheral, fluffy infiltrates consistent with air space consolidation. In the context of allogeneic HCT, the development of BOOP is strongly associated with prior acute and chronic GVHD [109]. The diagnosis of BOOP requires histologic evidence on lung biopsy of several signature features: patchy fibrosis, granulation tissue within alveolar spaces, alveolar ducts, and respiratory bronchioles, and the absence of infectious organisms. The term BOOP should not be used interchangeably with bronchiolitis obliterans to describe a patient with chronic lung dysfunction after HCT, although such usage is unfortunately widespread. The two disorders differ with respect to histopathology, pulmonary function characteristics, and, most importantly, response to therapy: BOOP after HCT is quite responsive to corticosteroids and in other settings may resolve spontaneously; bronchiolitis obliterans is not [7,108].

Obstructive lung disease OLD was first recognized as a complication of allogeneic HCT in the mid 1980s and is now a well-documented cause of morbidity and mortality [8,62,110–113]. OLD is characterized by enhanced resistance to airflow on expiration and reflects conditions in the smaller airways and bronchioles. The diagnosis of OLD is based primarily on spirometric

measurements as demonstrated by decreases in FEV1 and FEV1:FVC [110]. Obstructive defects as defined by an FEV1:FVC ratio of less than 70% have been observed in approximately 15–25% of allogeneic HCT recipients by day 100 and can persist for years [10,62,63]. OLD results from extensive narrowing and/or destruction of the small airways. Lung biopsies from patients with OLD have shown lymphocytic bronchitis, acute and chronic IP, and bronchiolar inflammation, including bronchiolitis obliterans [8,63,111,114]. This variation in histopathology is complicated further by the methods used to procure lung tissue: transbronchial biopsies rarely include an adequate sampling of distal bronchial structures, and therefore such specimens can reveal cellular infiltrates involving larger airways and the interstitium but may not detect bronchiolar inflammation. Bronchiolitis obliterans depicts small airway inflammation with fibrinous obliteration of the bronchiolar lumen, and remains the most common form of histopathology associated with OLD, and has been used historically to describe “chronic GVHD of the lung” [8,63,111,114]. Airflow obstruction may, however, occasionally exist without bronchiolitis obliterans and vice versa [115]. Moreover, in the vast majority of cases, OLD is diagnosed by PFT findings without histopathologic confirmation, and in this context two phrases that identify affected patients are found in the literature. “Airflow obstruction” has recently been defined as a more than 5% per year decline in percent predicted FEV1 with the lowest post-transplant FEV1:FVC ratio less than 0.8 [112], and “bronchiolitis obliterans syndrome” describes the deterioration of graft function that accompanies chronic lung allograft rejection [116]. The lack of consistent terminology and the variability in diagnostic criteria have contributed to the wide variation in the reported incidence of OLD after HCT. Afessa and colleagues found that OLD was reported in 8.3% of over 2000 allogeneic HCT patients in nine studies and was identified in 6–20% of long-term survivors with chronic GVHD [7]. By contrast, using the definition described above for airflow obstruction, Chien and coworkers found that new OLD following HCT with highdose conditioning was more frequent (26% overall and 32% among patients with chronic GVHD) than in previous estimates [112]. The onset of OLD is later than IPS (ranging from 3 to 18 months after HCT), and more insidious [110]. Respiratory symptoms include cough, dyspnea, and wheezing, but many patients remain asymptomatic despite showing signs of moderate-to-severe airway obstruction on PFTs [8,62]. Chest radiographs are most often normal except for signs of hyperinflation, but patchy, diffuse infiltrates can be present [8,61,63]. Likewise, chest

Lung Injury Following Hematopoietic Cell Transplantation

computed tomography findings range from normal early in the course of disease to extensive peribronchial inflammation and bronchiectasis with significant air trapping and diffuse parenchymal hypoattenuation at later time points [9,117]. The clinical course of OLD also varies from mild to severe with necrotizing bronchiolitis, a rapid decline in FEV1, and a mortality rate of 25–50%, and, unfortunately, clear predictors of outcome have not been identified [8,9,11,12,61,110]. Since airflow obstruction tends to be fixed rather than reversible, response to bronchodilator therapy is usually marginal. Similarly, responses to immunosuppressive therapy (including various combinations of steroids, calcineurin inhibitors, and azathioprine) are limited, and are typically associated with preservation of remaining lung function rather than in significant improvement. Since enhanced immunosuppression significantly increases the risk of infection, the utility of such therapy is questionable when a clinical response is not seen within the first several months of treatment or when pulmonary dysfunction is longstanding. The partial response to immunsuppressive therapy suggests that early detection of disease may be important [8,9,12,110]. In this light, two reports have suggested that a decrease of maximum mid-expiratory flow rates may be an earlier indicator of OLD than changes in FEV1 [12,61]. Further, faster rates of airflow decline between day 100 and 1 year after HCT correlate with higher mortality risk [113]. More importantly, investigators have found that pretransplantation risk scores based solely or in part on pre-HCT lung function tests can be used to predict early respiratory failure and death in patients undergoing allogeneic HCT [118,119]. Risk factors for OLD are several and include lower pretransplant FEV1:FVC, concomitant infections, chronic aspiration, acute and chronic GVHD, older recipient age, and the use of mismatched donors and high-dose (versus reduced-intensity) conditioning regimens [8,9,62,110,112,120]. Recently, the impact of age and intensity of HCT conditioning was examined by Chien and colleagues, who found that the risk for experiencing a yearly decline in FEV1 in excess of 20% was significantly lower for recipients of reduced-intensity conditioning compared with patients receiving high-dose therapy who were greater than 50 years of age [120]. The CMV status of donor and recipient prior to HCT does not correlate with the development of OLD, but previous RSV and adenoviral infections appear to be possible risk factors for the higher incidence of OLD in the pediatric population [9]. The development of OLD is strongly associated with chronic GVHD [121], particularly in patients with low serum IgG levels [8,110] and with chronic hepatic GVHD [9]. Pathogenesis of chronic pulmonary toxicity The pathophysiology of chronic lung injury after HCT is poorly defined, in part because of the paucity of correlative clinical data and the lack of suitable animal models for either OLD or RLD. The development of chronic pulmonary toxicity likely involves an initial insult to the lung parenchyma, followed by an ongoing inflammatory process involving the interplay between recruited immune effector cells and the resident cells of the pulmonary vascular endothelium and interstitium. Most of what is known about the pathogenesis of OLD is based upon observations made in lung allograft recipients and from data generated in murine heterotopic tracheal transplant models. The absence of an initial inflammatory response from HCT conditioning regimens and the presence of a “host-versus-graft” rather than a “graft-versus-host” reaction are just two of the issues that limit the extrapolation of such data to OLD after HCT. Similarly, our understanding of the mechanisms responsible for RLD is inferred from patients with various forms of interstitial fibrosis and from animal models of these diseases. Despite these limitations, lung allograft rejection and pulmonary fibrosis are characterized by

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epithelial cell injury in the terminal bronchioles or alveoli, respectively, and by a profound defect in re-epithelialization and normal repair. Each response also involves T-cell activation, leukocyte recruitment, and enhanced expression of inflammatory mediators, all of which are likely operative in chronic lung injury after HCT. Murine models have begun to elucidate the mechanisms that contribute to leukocyte migration and rejection of lung allograft epithelium, and may shed light on mechanisms contributing to OLD after HCT. Boehler and colleagues found that rat heterotopic lung allografts undergoing rejection showed a strong Th1 immune response even after fibrosis and airway obliteration was complete [122]. This response was also associated with upregulation of MCP-1/CCL2 and RANTES/CCL5. Subsequent studies by several groups have shown enhanced expression of TNF-α, IL-8, transforming growth factor-beta and IL-1β during clinical allograft rejection [123–126], and ultimately revealed critical roles for both RANTES and MCP-1 in the development of experimental bronchiolitis obliterans [127,128]. Although direct cytotoxicity by cellular or cytokine effectors may be responsible for damage and loss of airway epithelium in the transplanted lung, the inability to regenerate and heal injured epithelium is believed to be an equally important factor in the development of bronchiolitis obliterans. In this context, interactions between activated epithelial cells and lung fibroblasts may be critical [129,130]. The impact that innate immune dysregulation may have on these interactions has been recently highlighted by two studies demonstrating that genetic variations in bactericidal/permeabilityincreasing (BPI) protein and oligomerization domain containing-2/ caspase recruitment domain family member 15 (NOD2/CARD15) influence the risk of airflow obstruction and bronchiolitis obliterans after allogeneic HCT [131,132]. Such genetic approaches, along with the development of a new animal model for bronchiolitis obliterans [133], may ultimately prove useful for deciphering mechanisms of disease that are more relevant to OLD that occurs in the context of HCT. The mechanisms responsible for RLD after HCT are also poorly characterized. The prevailing hypothesis holds that most forms of interstitial fibrosis are initiated by an acute inflammatory event that injures the lung, results in the secretion of inflammatory mediators, and subsequently modulates pulmonary fibrogenesis. The etiology of the initial parenchymal insult may; (1) include direct toxicity from drugs, toxins or ionizing irradiation, (2) represent a dysregulated immune response to an aberrantly expressed environmental or self-antigen, and (3) may or may not involve a role for activated T cells. Like OLD, several proteins with profibrotic activity, including TNF-α, IL-1β, IL-6, transforming growth factor-beta, IL-8, and MCP-1/CCL2 have been identified in the BAL fluid of patients and animals with interstitial fibrosis. A growing body of scientific evidence suggests that the cytokine profile present during an evolving immune response determines its pathologic outcome. In this context, a Th1/Th2 explanation has emerged to help predict whether a specific pulmonary response will ultimately resolve or progress toward end-stage fibrosis; IFN-γ has profound regulatory activity on collagen synthesis and possesses potent antifibrotic effects, whereas Th2 cytokines like IL-4 and IL-13 activate fibroblasts and stimulate the production of extracellular matrix proteins [129,130]. This working hypothesis has been recently supported in dramatic fashion by Burman and colleagues, who showed that complete abrogation of IFN-γ signaling using IFN-γ-deficient donors and IFN-γ receptor-deficient recipients resulted in robust pulmonary inflammation and death early after HCT [134]. The evolution of chronic lung injury after HCT A triphasic model of chronic, noninfectious lung injury after HCT is proposed wherein alloantigen recognition represents the inciting stimulus of the immune response (Plate 96.1). Phase one of the disease is

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Chapter 96

characterized by the development of a mixed leukocytic infiltrate and acute interstitial and peribronchial inflammation similar to that observed in IPS (Fig. 96.1(a)). This injury is initiated early after allogeneic HCT by a systemic proinflammatory environment that leads to chemokine upregulation, the promotion of leukocyte recruitment, and the secretion of inflammatory cytokines in the lung. In phase two, the expression of MHC antigens on the pulmonary epithelial and vascular endothelial cells results in a sustained inflammatory signal and a dysregulated reparative response that promotes the transition from acute to chronic lung injury. This transition is accompanied by a change in the character of the leukocytic infiltrate (to one that is predominantly lymphocytic in nature) along with a shift to a profibrotic environment and a proactive role of the lung fibroblast. If the inciting injurious stimuli involve the bronchiolar epithelium, phase two is associated with a progressive, concentric lymphocytic infiltration, collagen deposition, and early fibrosis in the peribronchial areas, resulting in a chronic bronchiolitis (Plate 96.1(b)). Activated lymphocytes then migrate through the basement membrane of the respiratory epithelium and into the airway mucosa and result in epithelial cell apoptosis and necrosis. Continued epithelial injury leads to areas of denudation and ulceration. As chronic inflammation proceeds into phase three, lung fibroblasts increase dramatically in number and contribute to (1) the proliferation of endothelial cells, (2) enhanced collagen deposition, and (3) the development of intraluminal granulation tissue and dense, concentric, periluminal fibrous bands (Plate 96.1(c)). Ultimately, this process results in complete obliteration of the small airways (cicatricial bronchiolitis obliterans) and significant OLD. By contrast, if epithelial cells in the alveolar septae are the principal targets of injury, persistent antigenic stimulation results in recruitment of lymphocytes and monocytes into the interstitial space, eventually resulting in RLD (Plate 96.1(d)). The resultant inflammation is associated with apoptosis and loss of septal epithelial cells, exudation of proteinaceous material, the recruitment and proliferation of fibroblasts, and the deposition of intraseptal granulation tissue. If this extracellular matrix is not resorbed, chronic inflammation progresses to phase three, wherein collagen is deposited within the alveolar septae, and the chronic leukocytic infiltrates are less evident. This results in interstitial thickening, septal fibrosis, and loss of alveolar architecture leading to dilated cystic air spaces or “honeycombing” (Plate 96.1(e)). Such end-stage histopathology is associated with the significant volume reduction and severely impaired gas exchange that is characteristic of severe RLD. Precisely what determines the anatomic specificity (peribronchiolar versus interstitial) of chronic lung injury remains unclear, and the development of either pattern of chronic lung injury (OLD or RLD) does not necessarily exclude the other [135]. Further, the role of acute inflammation and specifically of alloreactive effector cells in the initial damage to the alveolar or bronchiolar epithelium and the subsequent progression to chronic pulmonary injury are not well established. Moreover, an early, robust inflammatory phase may not be a prerequisite for subsequent fibrosis; persistent epithelial damage and subsequent aberrant “cross talk” between epithelial cells and fibroblasts may be sufficient to cause an exuberant reparative response characterized by proliferation, excess matrix deposition, and the development of fibrotic lung disease [136,137]. This mechanism could explain why some patients with chronic lung dysfunction do not have a clear antecedent history of acute lung inflammation. In the setting of imbalanced immune regulation, a subclinical injury, such as an allogeneic response to lung epithelial cells, could initiate a dysregulated reparative response resulting in scarring of either the terminal airways (OLD) or the interstitial space (RLD). In this context, pulmonary fibroblasts may represent an important link in the development of both RLD and OLD after allogeneic HCT.

TNF-α may be a central factor in the triphasic model proposed above. TNF-α is an important mediator of acute lung injury after HCT, it directly contributes to the development of interstitial fibrosis in several non-HCT models [129,138,139], and the abrogation of TNF-α signaling, either by antibody neutralization or by using mutant mice deficient in both TNF-α receptors, significantly reduces lung fibrosis [140,141]. Strong evidence for a role of TNF-α in the transition from acute to chronic lung injury comes from studies using transgenic rodents with targeted overexpression of TNF-α in the lungs [138,139]. Early lung histopathology includes a lymphocytic infiltrate similar to that seen in experimental IPS models, whereas the histologic changes associated with more prolonged exposure to TNF-α closely resemble those seen at later time points after HCT. The linkage between TNF-α and OLD after HCT is more indirect: increased levels of TNF-α have been shown to increase the expression of several proteins that may contribute to this process. For example, increased levels of TNF-α are associated with enhanced MCP-1 expression in a murine lung allograft rejection model and correlate with upregulation of CCR2 and the recruitment of mononuclear phagocytes into the allograft [127]. Interruption of MCP-1/CCR2 signaling using CCR2deficient recipients results in a reduction of macrophage infiltration and less rejection [127], and modulates the induction of profibrotic cytokine cascades following the intratracheal administration of bleomycin and other irritants such as fluorescein isothiocyanate [142]. Finally, clinical studies have shown that enhanced MCP-1 is associated with the progression from acute to chronic allograft rejection and with the evolution of pulmonary fibrosis [127,143]. Treatment of chronic lung injury after HCT The association of OLD and chronic GVHD has resulted in a general consensus that this form of lung damage is immunologically mediated. Thus, “standard” therapy of OLD combines enhanced immunosuppression in conjunction with supportive care including supplemental oxygen therapy and broad-spectrum antimicrobial prophylaxis. Unfortunately, the response to multiple agents including steroids, cyclosporine, tacrolimus, and azathioprine is limited and tends to occur only early in the course of treatment [8,12,61,63,110]. Patients with more severe disease at the start of treatment have poor prognoses and high mortality rates, suggesting that early recognition of OLD may be important [12,61,110]. The poor response to standard therapy and the unacceptable morbidity and mortality associated with chronic lung injury after HCT are underscored by the need for lung transplant in some HCT recipients with severe OLD [144]. The published literature contains a paucity of therapeutic trials for chronic lung injury after HCT, and no agent or combination of agents has been shown to be particularly efficacious. Whereas a clinical study using inhaled steroids in addition to standard systemic immunosuppression to prevent bronchiolitis obliterans syndrome after lung allografting showed no benefit compared with controls [145], a recent randomized, placebo controlled trial of 58 patients demonstrated that while inhaled cyclosporine did not improve the rate of acute rejection, it did improve survival and extend periods of chronic rejection-free survival in lung allograft recipients [146]. Three recently published case series have exploited the anti-inflammatory effects of the antibiotic azithromycin to treat OLD in both allogeneic HCT and lung allograft recipients. Each study suggested a beneficial effect of this drug on pulmonary function when administered for 12 or more weeks [147–149]. The potential role for TNF-α in the pathogenesis of both OLD and RLD suggests that agents such as etanercept, which neutralize this protein, may hold promise, and several studies have demonstrated a potential benefit of this drug in some HCT patients with chronic lung

Lung Injury Following Hematopoietic Cell Transplantation

injury [150,151]. A phase I–II clinical trial using etanercept in this context has recently been completed at the University of Michigan. Preliminary results demonstrate that etanercept can be safely administered in this patient population, and six of 15 patients showed at least a 10% improvement from baseline in either FEV1, FVC or DLCO within the first 2 months of finishing therapy [151]. Based upon these findings, a larger phase II trial has been developed and is currently accruing patients.

Conclusion Diffuse lung injury remains a significant problem following allogeneic HCT both in the immediate post-transplant period and in the months to years that follow. Although lung injury occasionally occurs following autologous transplantation, the allogeneic setting significantly exacerbates toxicity in both the acute and chronic conditions. Historically, much of this injury was assumed to be due to occult and unidentifiable infections, but a large preponderance of experimental data now demonstrates that IPS has a major immunologic component. Despite these findings, establishing the lung as a true target of GVHD remains a hotly debated topic. Like the gut and skin, the lung is a critical immunologic interface between the sterile body sanctuary and the outside environment. As such, the lung is a rich source of histocompatibility antigens and professional antigen-presenting cells, and is the site of complex immunologic

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networks that involve cytokine production and lymphocyte activation. Inflammatory mediators such as TNF-α and LPS, along with donorderived effector cells, which contribute to GVHD, are associated with acute lung injury in the experimental and clinical settings. Clinically, evidence supporting the concept that the lung is a target organ of acute GVHD is limited, and the major obstacle has been the lack of apoptotic epithelial injury. However, other GVHD target organs such as the thymus do not express this particular form of injury, and recent experimental data demonstrate that direct recognition of alloantigen on host epithelium by cytotoxic effectors may not be required for GVHD induction or target organ injury. In the chronic setting, the lung is widely accepted as a target of GVHD. The striking similarities between the histopathologic features of bronchiolitis obliterans seen in association with OLD after allogeneic HCT and those observed during lung allograft rejection, along with reports of improvement in lung function with immunosuppressive agents, strongly support this concept. The case for immunologic mechanisms contributing to RLD is less strong, and the data from experimental allogeneic models are still very scant. Nonetheless, TNF-α may be viewed as a common thread between acute IPS and chronic lung disease of either the obstructive or restrictive type. It is hoped that as animal models of lung injury after HCT yield further insights, our understanding of these disease processes will improve and ultimately lead to new therapeutic strategies to diagnose, treat, and prevent pulmonary toxicity in our allogeneic HCT recipients.

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proinflammatory role of bronchial epithelial cells. Munich Lung Transplant Group. Transplantation 2000; 70: 362–7. Fattal-German M, Le Roy Ladurie F, Cerrina J, Lecerf F, Berrih-Aknin S. Expression and modulation of ICAM-1, TNF-alpha and RANTES in human alveolar macrophages from lungtransplant recipients in vitro. Transpl Immunol 1998; 6: 183–92. El-Gamel A, Sim E, Hasleton P et al. Transforming growth factor beta (TGF-beta) and obliterative bronchiolitis following pulmonary transplantation. J Heart Lung Transplant 1999; 18: 828–37. Belperio JA, Keane MP, Burdick MD et al. Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome. J Clin Invest 2001; 108: 547–56. Belperio JA, Burdick MD, Keane MP et al. The role of the CC chemokine, RANTES, in acute lung allograft rejection. J Immunol 2000; 165: 461–72. Coker RK, Laurent GJ. Pulmonary fibrosis: cytokines in the balance. Eur Respir J 1998; 11: 1218–21. Hogaboam CM, Smith RE, Kunkel SL. Dynamic interactions between lung fibroblasts and leukocytes: implications for fibrotic lung disease. Proc Assoc Am Physicians 1998; 110: 313–20. Chien JW, Zhao LP, Hansen JA, Fan WH, Parimon T, Clark JG. Genetic variation in bactericidal/permeability-increasing protein influences the risk of developing rapid airflow decline after hematopoietic cell transplantation. Blood 2006; 107: 2200–7. Hildebrandt GC, Granell M, Urbano-Ispizua A et al. Recipient NOD2/CARD15 variants: a novel independent risk factor for the development of bronchiolitis obliterans after allogeneic stem cell transplantation. Biol Blood Marrow Transplant 2008; 14: 67–74. Panoskaltsis-Mortari A, Tram KV, Price AP, Wendt CH, Blazar BR. A new murine model for bronchiolitis obliterans post-bone marrow transplant. Am J Respir Crit Care Med 2007; 176: 713–23. Burman AC, Banovic T, Kuns RD et al. IFNgamma differentially controls the development of idiopathic pneumonia syndrome and GVHD of the gastrointestinal tract. Blood 2007; 110: 1064– 72. Trisolini R, Bandini G, Stanzani M et al. Morphologic changes leading to bronchiolitis obliterans in a patient with delayed non-infectious lung disease after allogeneic bone marrow transplantation. Bone Marrow Transplant 2001; 28: 1167– 70. Pardo A, Selman M. Idiopathic pulmonary fibrosis: new insights in its pathogenesis. Int J Biochem Cell Biol 2002; 34: 1534–8. Selman M, King TE, Pardo A. Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 2001; 134: 136–51.

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Sangeeta Hingorani

Kidney and Bladder Complications of Hematopoietic Cell Transplantation

Introduction Renal dysfunction is a common complication of hematopoietic cell transplantation (HCT) and a major cause of morbidity and mortality. There are many definitions used to describe both the acute kidney injury (AKI) and chronic kidney disease (CKD) that occur after HCT (Table 97.1). The heterogeneous nature of the definitions, types of HCT, and diverse patient populations often make it difficult to draw comparisons across studies. The bladder is also commonly involved after HCT, and processes that affect the bladder can also lead to renal injury. This chapter will focus on the epidemiology, pathogenesis, and treatment of renal and urologic problems. The time course of renal injury varies from days after transplant to months later. The difference between acute and chronic is arbitrarily defined by timing as CKD usually refers to manifestations of renal disease that occur after day 100, and “acute” covers the early injury up to day 100. Similarly, bladder injury, most commonly hemorrhagic cystitis (HC), is divided into early onset and late onset, and the distinction between the two is also one of timing, lending itself to different etiologic factors. In this chapter, the earlyonset or acute injuries will be discussed first, followed by the later or chronic injuries. The disease processes can overlap: AKI that persists is later termed CKD. Although this chapter will discuss the injury that occurs after HCT, baseline or pretransplant renal function can impact outcome [1]. Therefore, baseline assessments not only of serum creatinine, but also urinalyses and more formal estimations of glomerular filtration rate (GFR) are warranted. Accurate assessment of baseline renal function can help guide later medication dosing.

Acute kidney injury Epidemiology AKI is defined as a doubling of baseline serum creatinine within the first 60–100 days after HCT. The incidence of AKI varies from a low of 6.5% in autologous transplants to as high as 79% in patients admitted to the intensive care unit (ICU) after transplant. The incidence also varies based on the type of HCT and the conditioning regimen. AKI is more common in allogeneic transplant recipients compared with autologous transplant recipients, and high-dose regimens compared with reduced-intensity conditioning (RIC). The less severe, or grade 2, AKI is defined as less than a doubling of baseline serum creatinine with a

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

greater than 25% decrease in GFR. Grade 2 is a doubling of baseline serum creatinine without the need for dialysis, and grade 3 is dialysisrequiring AKF (Table 97.1). Grade 3 AKF carries with it a higher mortality. Table 97.1 Definition of terms Term

Definition

Acute kidney injury

Doubling of baseline serum creatinine or fall in GFR to >50% Tripling of baseline serum creatinine, fall in GFR to 75% or creatinine >4.0 mg/dL GFR <60 mL/min/1.73 m2

Acute kidney failure Chronic kidney disease GFR, glomerular filtration rate.

Risk factors for AKI by type of conditioning regimen and transplant High-dose conditioning regimens and allogeneic HCT The incidence of AKI in these patients is as high as 69%. AKI in this group of patients occurs early after transplant, often before day 28. Risk factors for AKI in this group of patients include lung toxicity, hepatic toxicity, sinusoidal obstruction syndrome (SOS), amphotericin use, and sepsis. There are conflicting results regarding the importance of preHCT serum creatinine and the later development of AKI [2,3]. In a smaller study from Turkey, cyclosporine (CSP) use was associated with an increased risk of AKI after high-dose regimens, whereas sepsis played a major role in autologous recipients. They also found that a low serum albumin at baseline increased the risk of later renal dysfunction [4]. Those patients who develop AKI after high-dose conditioning require dialysis more frequently and have a higher mortality than patients without AKI. In the patients who require dialysis, the mortality approaches 83%. In a meta-analysis of patients with AKI after HCT, AKI was an independent predictor of mortality [5]. RIC regimens and allogeneic HCT In a retrospective cohort study comparing 140 high-dose versus 129 RIC transplant recipients, the authors found that despite the older age of the RIC patients and the greater percentage of patients with a GFR of less than 89 mL/min/1.73 m2 at baseline, only 47% of patients developed AKI compared with 73% in the high-dose treatment group within the first 3 months post transplant. AKI occurred slightly later in the RIC group, occurring at a median of 26–60 days post transplant. Fewer

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patients required dialysis in the RIC group (3% versus 12%) and mortality was significantly lower. However, the need for artificial ventilation increased the risk of AKI 10-fold in the RIC group of patients. Other risk factors included graft-versus-host disease (GVHD) and the source of the donor cells, with AKI being more common in patients who received marrow compared with peripheral blood. High-dose conditioning regimen and autologous HCT There are two case series of AKI in autologous transplant recipients. Compared with the allogeneic setting, the incidence is lower – approximately 21% – in patients after autologous HCT. In a study of patients with AL amyloidosis, risk factors for AKI identified at baseline included decreased creatinine clearance, proteinuria, and cardiac involvement. In addition, post-transplant risk factors included melphalan use and bacteremia. A second study done in 232 women who underwent HCT for breast cancer found a similar incidence of AKI but identified liver and lung toxicity as well as sepsis as risk factors for acute injury, similar to risk factors for AKI after high-dose conditioning followed by allogeneic transplantation. It has been suggested that the lower incidence after autologous HCT is related to the avoidance of calcineurin inhibitors. Pathogenesis of AKI after HCT Although many risk factors have been associated with the development of AKI after HCT (Table 97.2), only the most common ones will be

discussed in detail below. Thrombotic microangiopathies (TMAs), including thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), can occur early after HCT, but changes in serum creatinine may not be present at the time of diagnosis. TMAs will be discussed in the section on CKD. Tumor lysis syndrome is a rare cause of AKI in patients after HCT, seen more commonly in patients with residual disease or an underlying lymphoproliferative disorder at time of HCT. The renal injury is related to tubular obstruction and dysfunction secondary to hyperuricemia, hyperphosphatemia, and deposition of uric acid and phosphate within the kidney. Management involves use of uric acid inhibitors, aggressive hydration, and, when needed, dialysis. Tumor lysis syndrome is relatively uncommon in the HCT population as most patients are in remission at the time of transplant. Sepsis Sepsis leads to a combination of insults, both hemodynamic and inflammatory, that coalesce to cause renal failure. The pathophysiology of AKI in this setting is complex and not well understood. There is an initial inflammatory response leading to cytokine-induced systemic arteriolar vasodilatation and endothelial injury [14]. The resultant capillary leak that occurs leads to renal hypoperfusion. In addition, early after sepsis there is a constriction of the renal vessels, further decreasing renal perfusion. Injury to the tubules themselves causes a local release of cytokines and chemokines that cause local inflammation and further

Table 97.2 Summary of the literature on acute kidney failure (AKF) after hematopoietic cell transplantation (HCT) Number of patients

Type of transplant

Incidence: autologous/allogeneic

Definition of AKF

Postulated risk factors

Onset time (median)

Reference

272

89% allogeneic, 11% autologous

39%/54%

Grades 2 and 3

14 days

[2]

275

33% autologous, 67% allogeneic 100% autologous

6.5%/36%

Grade 2

30 days

[6]

21%

Grades 2 and 3

Weight gain, hyperbilirubinemia, amphotericin B use, sepsis, serum creatinine >0.7 mg/dL pre transplant Nephrotoxic drugs, SOS, older age Liver and lung toxicity, sepsis

28 days

[7]

329 180 57

100% allogeneic 100% allogeneic 26% autologous, 73% allogeneic

76% 80% 60%/79%

Grades 2 and 3 Grades 2 and 3 Grades 2 and 3

Within 56 days Within 100 days 27 days

[8] [9] [10]

269

100% allogeneic

60%

Grades 2 and 3

22 days

[11]

147 88

100% allogeneic 100% allogeneic

36% 69%

Grade 2 Grades 2 and 3

33 days 16 days

[3] [12]

47

53% autologous, 47% allogeneic 100% allogeneic, nonmyeloablative

32%/68%

Grades 2 and 3

44 days

[4]

40%

Grades 2 and 3

60 days

[13]

232

253

GVHD, graft-versus host disease; SOS, sinusoidal obstruction syndrome.

Liver failure, low serum albumin, APACHE II score Myeloablative conditioning, female gender, high-risk malignancy, comorbidity SOS, amphotericin use SOS, sepsis, lung and liver toxicity SOS, sepsis, cyclosporine Ventilator use, GVHD, product source (marrow versus peripheral blood)

Kidney and Bladder Complications of Hematopoietic Cell Transplantation

intrarenal injury [15]. In addition to the effects of sepsis, the medications used to treat the infections are often nephrotoxic. Several reports have identified amphotericin, in the conventional or liposomal formulation, as a risk factor for AKI. Hepatic SOS There is a well-known association between sinusoidal liver injury and renal insufficiency. It has been postulated that portal hypertension resulting from hepatic sinusoidal injury leads to both decreased renal perfusion and tubular injury, the former probably being the more important in the genesis of AKI. Clinically, SOS is associated with marked sodium avidity, low urinary sodium resulting in volume overload, edema, and weight gain, as seen in hepatorenal syndromes [16]. Weight gain likely serves as a marker of impending renal failure rather than being a result of the renal injury; in addition, the association between weight gain and the development of AKI described in the literature is conflicting [2,3,17]. Although some have advocated keeping weight gain per se to a minimum (<10% fluid overload) to improve outcome in this patient population, it is unclear if the weight gain is causal or the result of other insults to these patients. Thus, prevention of SOS may be the more important strategy. Nephrotoxic medications Although there are many potential nephrotoxic medications that are given to patients after HCT, the one that is most commonly associated with development of AKI is amphotericin. The finding by many of an increased risk of AKI among patients receiving amphotericin is not surprising because the nephrotoxicity of conventional amphotericin is well known, as is its association with renal failure in the HCT population [18–21]. However, in a study by Hingorani et al. [3], liposomal preparations of amphotericin were also associated with an increased risk of AKI. In this same study, neither the administration of vancomycin nor gentamicin in the 2 weeks prior to development of AKI increased the risk of AKI. Studies comparing conventional amphotericin to liposomal preparations in febrile neutropenic patients found that significantly fewer patients on liposomal amphotericin experienced nephrotoxicity (defined as doubling of baseline serum creatinine or creatinine >3.0 mg/dL) compared with those patients on conventional amphotericin [22,23]. In a subset analysis of the allogeneic HCT recipients, Cagnoni et al. found that patients receiving liposomal amphotericin were less likely to develop nephrotoxicity compared with those who received conventional amphotericin: 32% versus 66%, respectively. Fewer patients in the liposomal group required dialysis. In a prospective, randomized controlled trial comparing conventional amphotericin with liposomal amphotericin in ICU patients with candidal infections, Sorkine et al. found that 66.7% of patients treated with conventional amphotericin experienced a significant increase in their serum creatinine compared with 4% of patients in the group treated with liposomal amphotericin [24]. Use of amphotericin for fever of unclear etiology carries significantly increased risks of AKI in the transplant population. Given the marked increase in risk of AKI in patients who have received either conventional or liposomal amphotericin, we recommend that patients be treated with amphotericin only if clear indications exist, such as documented fungal infection or prolonged use of prophylactic triazoles. Fortunately, newer antifungal drugs such as fluconazole, itraconazole, voriconazole, and caspofungin are now available for indications formerly covered by amphotericin. Depending on the endpoints chosen, each of these medications has been shown to be equally efficacious to amphotericin but with better safety profiles [25]. The new definition proposed by nephrologists for acute kidney failure (AKF) is based on the so-called “RIFLE – Risk, Injury, Failure, Loss,

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and End-stage – criteria. As one progresses through the stages, the criteria are based on changes in GFR and serum creatinine and/or decreases in urine output. Risk is an increase in serum creatinine of 1.5× baseline or a decrease in GFR of over 25%. Injury is a doubling of baseline serum creatinine or a 50% decrease in GFR. Failure is either a tripling of baseline serum creatinine, a serum creatinine of over 4 mg/dL or a 75% decrease in GFR. Loss and end-stage refer to duration of renal failure of 1 and 3 months, respectively [26]. These criteria were established because the degree of renal failure affects outcome in adult patients admitted to ICU. They are also applicable to the HCT population and should be used in future studies of AKI after HCT. Management of AKI In the majority of cases, the management of AKI is supportive. Studies comparing intermittent hemodialysis with continuous renal replacement therapy in patients admitted to ICU are equivocal (reviewed in [15]). The choice of dialysis modality often depends on hemodynamic stability of the patient, degree of volume overload, and blood pressure, which makes it difficult to interpret outcomes as sicker patients are more likely to be placed on continuous renal replacement therapy. What is apparent is that early intervention, regardless of modality choice, can improve outcome.

Chronic kidney disease Epidemiology The cumulative incidence of CKD varies from 13–60% in adult studies [27–29] to as high as 62% in children [30]. CKD usually becomes apparent 6–12 months after HCT, although it has been described as early as 2 months and as late as 10 years post transplant. As in AKI, there are numerous definitions used to define CKD in these patients. Most definitions are based on serum creatinine or an estimated or calculated measurement of GFR based on the Modification in Diet and Renal Disease or the Schwartz formula in children. The National Kidney Foundation has divided GFR into stages 1–5 based on an estimated GFR (eGFR) to help guide clinical treatment plans. The range of GFR is ≥90 mL/min/ 1.73 m2 with some renal damage for stage 1, to a GFR of <15 mL/min/ 1.73 m2 for stage 5 and end-stage renal disease (Table 97.3). Many terms have also been suggested to describe the chronic forms of renal dysfunction occurring after HCT, including “bone marrow transplantation nephropathy” [27], “radiation nephritis” [31–33], and “conditioning-associated HUS” [34]. None of these terms defines the degree of renal dysfunction, anemia or hypertension. The renal pathologies of bone marrow transplantation nephropathy, conditioningassociated HUS, and radiation nephritis are indistinguishable and may reflect a common injury. Total body irradiation (TBI) may play a role

Table 97.3 National Kidney Foundation Kidney Disease Outcomes Quality Initiative: chronic kidney disease by stage Stage

Description

eGFR (mL/min/1.73 m2)

1 2 3 4 5

Kidney damage with normal or ↑ GFR Kidney damage with mild ↓ GFR Moderate ↓ GFR Severe ↓ GFR Kidney failure

≥90 60–89 30–59 15–29 <15 or dialysis

eGFR, estimated glomerular filtration rate.

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in the endothelial injury that leads to the development of a TMA and its subsequent renal injury. In addition to a TMA, there are other manifestations of CKD in this patient population, including nephrotic syndrome and GVHD-related CKD. The latter form of CKD after HCT is often defined as abnormalities in serum creatinine or estimated and calculated GFR at 6–12 months after transplant. The lack of a consistent definition of post-HCT CKD has made it difficult to draw comparisons across studies, to identify risk factors for its development, and to devise prevention and treatment strategies. Clinical syndromes of CKD after HCT Acute kidney injury AKI has been shown to be a risk factor for CKD in various adult and pediatric studies [3,29,35], suggesting that some patients do not recover fully from their AKI and progress to CKD. TMAs: TTP and HUS Definitions and clinical diagnosis TMA syndromes represent a spectrum of clinical diseases characterized by systemic or intrarenal platelet aggregation, thrombocytopenia, and microvascular fragmentation of erythrocytes. Platelet aggregation can result in ischemia and organ injury. When renal injury is predominant, a diagnosis of HUS is usually rendered, while the presence of extensive extrarenal manifestations leads to a diagnosis of thrombotic TTP (reviewed in [36]). TMA syndromes are not uncommon in the setting of HCT. In a series of both retrospective and prospective studies, the incidence of microangiopathic disease after HCT ranged between 2% and 21% [37–39]. The incidence seems to be lower in patients receiving high-dose conditioning regimens compared with those patients receiving RIC regimens [40,41]. TTP tends to occur early after transplant, whereas HUS seems to occur much later after transplant. When the presentation is fulminant, TMA syndromes are often associated with severe AKF and death. More commonly, however, they follow an indolent course, resulting in the eventual development of CKD. For the diagnosis of TMA after HCT, the following clinical characteristics must be met: microangiopathic hemolytic anemia demonstrated by red blood cell fragmentation with two or more schistocytes per highpower field, elevated lactate dehydrogenase levels, renal dysfunction (defined as a greater than 50% increase in baseline serum creatinine or a 50% decrease in creatinine clearance from baseline), and/or neurologic involvement without other identifiable causes, and a negative direct and indirect Coombs test [42]. The new grading system proposed is based on the common toxicity criteria for HCT TMA, and severity is based primarily on serum creatinine level, need for dialysis, and/or encephalopathy [42]. Those patients with more severe forms of TMA have a higher risk of mortality [43]. Risk factors for TMA after HCT Although no clear relationships have been found to date for the development of TMA after HCT, a number of risk factors have been examined. In earlier studies, where HUS was the primary diagnosis, risk factors identified were TBI [27,30,44,45] and calcineurin inhibitor use [27,34,46–50]. However, in later studies of TMA, acute GVHD grades II–IV, older age, and transplant from an unrelated donor were the primary risk factors identified [37,39]. Other investigators have also identified SOS, grafts from matched unrelated donors or haploidentical donors, and lymphoid malignancy as significant predictors of TMA after HCT in addition to the above risk factors [51–54]. No decrease in the incidence of TMA has been found in patients undergoing RIC regimens, and it is thought that fludarabine may directly contribute to the endothelial injury in these patients [41,54]. In many of

these cases, the renal injury is not present at the time of diagnosis of the TMA but occurs later in the course of the disease. TMA can also occur in the HCT population as a result of viral infections [55,56]. Total body irradiation The entity known as “radiation nephritis” has been described as early as the 1920s. The clinical course is variable and manifests as nephritis, hypertension, and proteinuria and/or anemia [57]. The timeframe for each of these clinical presentations is also variable, with the acute nephritis presenting 6–12 months after irradiation, and the subacute, chronic, and late forms occurring 2–5 years later. Although radiation can damage the vascular endothelium, tubular epithelium, and glomerulus, the primary site of injury has not been identified [57,58]. The doses of TBI traditionally associated with radiation nephritis are usually greater than 2000 cGy [59]. To test the theory that radiation causes endothelial injury, Rubin and Cassarett performed renal scans at several time points after irradiating rats unilaterally with 1000–6000 R [57]. A baseline renal scan outlined both kidneys and remained essentially unchanged for 3 months post exposure. Between 3 and 6 months, subtle changes were noted, and at 9 months, the irradiated left kidney showed clearly diminished uptake of radiolabel. The changes on renal scan correlated with microangiopathic pathology showing poor vascularization and disruption of glomerular capillaries. Given the variable time to presentation and degree of renal injury, a secondary insult(s) to the kidney may potentiate the radiation injury. For example, it is known that irradiated vessels are more sensitive to the effects of hypertension. It is therefore possible that hypertension in these patients may cause further vascular injury or impair the kidney’s ability to repair such injury. As with other dividing cells, radiation inhibits endothelial cell proliferation in culture [60]. The normal turnover time of endothelial cells is approximately 2 months [61], which may explain the delayed development of renal injury in some patients after HCT. Based on the dose of radiation received, Miralbell et al. determined the risk of having an abnormal creatinine level at 18 months after HCT [62]. There appeared to be a dose-dependent relationship between radiation and renal injury. At a dose of 12 Gy, the relative risk was 2.9, and at a dose of 13.5 Gy, the relative risk was 8.4 compared with the group of patients who received 10 Gy. In a prospective study using renal shielding, a trend toward a decreased incidence of CKD was observed in the shielded versus the unshielded groups [45]. In this study, all patients received TBI in addition to cytarabine and cyclophosphamide as part of their conditioning regimen. Twenty of the 157 adults (13%) surviving beyond 100 days developed renal insufficiency; 18% were from the unshielded group and 10% were from the single shielded group. No patients who received double-shielding (diminishing the total radiation dose to the kidneys to 9.8 Gy) developed renal insufficiency throughout the follow-up period of 2.5 years. Chemotherapeutic high-dose regimens Numerous studies and case reports have described renal injuries associated with various chemotherapeutic regimens with or without TBI. Certain agents may potentiate the effects of radiation. In a study by Lonnerholm et al., carmustine, etoposide, cytarabine, and cyclophosphamide (BEAC) in conjunction with TBI uniformly resulted in renal insufficiency (eight of eight patients receiving this regimen developing renal dysfunction) compared with none of 14 adults with diagnoses other than lymphoma who received TBI without BEAC [63]. Hebert et al. described the case of a 40-yearold woman with Hodgkin’s lymphoma who received two cycles of chemotherapy and then further conditioning chemotherapy prior to HCT, but did not receive TBI [64]. Her regimen was otherwise similar to that described above and included etoposide, cytarabine, cisplatin, and BEAC. Her serum creatinine prior to transplantation was 0.9 mg/mL and was increased to 4.6 mg/mL 15 months later. Renal biopsy revealed mesangiolysis, glomerular capillary aneurysms, and widening of the

Kidney and Bladder Complications of Hematopoietic Cell Transplantation

subendothelial space with fibrin deposition, findings consistent with a TMA process. In the absence of TBI, these authors reasonably concluded that the cisplatin and other neoplastic agents caused the CKD.

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plexes. Similar findings are seen in patients with HUS and so-called “radiation nephritis” [27,30,31,33,44,72]. Pathophysiology of TMA after HCT

Immunosuppressive agents Immunosuppressive agents have also been associated with the development of CKD. Agents such as CSP and tacrolimus can initiate HUS-like syndromes after HCT [27,38,46–50,54]. The manifestations of CSP toxicity range from asymptomatic azotemia and proteinuria to fulminant multiorgan failure [47]. On histologic exam of kidneys from three HCT patients on CSP who died with renal failure, thrombus formation was observed in capillary tufts and arterioles with subendothelial and mesangial widening and sclerosis. Conversely, none of 55 patients not on CSP had renal capillary or arteriolar thromboses [65]. CSP is also a potent vasoconstrictor of the afferent arteriole of the glomerulus. In a small study of seven renal transplant recipients on CSP, decreases in GFR and renal plasma flow were found and correlated with CSP dose. The decreases seen here were transient and reversed as drug levels returned to trough levels [66]. The nephrotoxic effects of CSP correlate with serum levels of the drug and duration of therapy. CSP is also known to cause arteriolar injury, glomerular sclerosis, and interstitial fibrosis, as well as diffuse expansion of the mesangial matrix [67]. Studies of pediatric cardiac transplant patients also found an association between CSP use within the first 2 months after transplant and decreases in GFR years after transplant [68]. High CSP levels (>500 μg/L) in the first 6 months after cardiac transplant were associated with the development of end-stage renal disease at any time after cardiac transplant [69]. However, in a study of approximately 69,000 patients receiving a nonrenal transplant including heart, lung, liver, intestine, and combined heart–lung, CSP use at initial hospitalization was associated with only a slightly increased risk of developing CKD after transplant (hazard ratio [HR] = 1.24, 95% confidence interval [CI] 1.17–1.30) [70]. A recent study also found that sirolimus in conjunction with a calcineurin inhibitor increased the risk of TMA compared with calcineurin inhibitors alone [71]. Histopathology of TMA after HCT Microscopic examination of kidney biopsy specimens from patients with TMA-associated CKD demonstrates mesangiolysis and loss of endothelial cells with expansion of the subendothelium and occlusion of capillary loops (Fig. 97.1). On electron micrographs, there is extensive widening of the space between the glomerular basement membrane and the subendothelium with amorphous deposits that are not immune com-

Regardless of the offending agent, endothelial damage is thought to represent the inciting factor in the development of TMA syndromes (Fig. 97.2). Endothelial injury activates the coagulation system, leading to thrombin formation and fibrin deposition. The ability to repair such injury depends on the balance between fibrinolytic and coagulant activity [73]. Various studies support this hypothesis (reviewed in [34]). In a mouse model of HCT, aspirin decreased the rate of development of renal injury and diminished the severity of the observed histopathologic lesions, suggesting a role of platelet aggregation after endothelial injury in the pathogenesis of renal disease [74]. Plasminogen activator inhibitor-1 (PAI-1), a glycoprotein produced by endothelial cells, hepatocytes, and platelets, is normally present at low concentrations in plasma. It may also play a significant role in the development of TMA disease. PAI-1 levels are normal in patients without complications post HCT [75]; however, there is some evidence that these levels are elevated in postHCT patients at the time of diagnosis of TTP [76] and may also be elevated in patients with SOS and sepsis post HCT. Tissue plasminogen activator antigen remained normal in these patients [77]. In a rat model of radiation nephropathy, PAI-1 expression was increased in glomeruli with evidence of sclerosis, mesangiolysis, and thrombosis. In situ hybridization demonstrated significant association of PAI-1 expression with sites of glomerular injury [78]. In children with HUS caused by Escherichia coli 0157:H7, levels of fragment 1 + 2 (a peptide produced when prothrombin is cleaved to generate thrombin) and PAI-1 activity were elevated prior to the development of HUS [79], and the long-term prognosis in patients with HUS that leads to progressive renal scarring is worse when associated with higher levels of PAI-1 [80]. Thus, PAI-1 levels appear to correlate with injury in kidneys. A prospective study of plasma levels of these markers in patients with HCT-related AKF and CKD is currently underway. More recent studies have identified both acute GVHD grades II–IV and chronic GVHD as risk factors for the development of TMA. Endothelial injury has been described in patients with chronic GVHD, and it is thought that endothelial cells are direct targets of cytotoxic donor T lymphocytes in these patients [81]. An association between inflammatory cytokines, interleukin-6, interleukin-8, and tumor necrosis factor-alpha (TNF-α), and the balance

Soluble fibrin

PA

I-1

ts

is

+ THROMBIN

ibi

rin

ol

ys

Fragment 1+2

D-dimer

inh

fib

Prothrombin Coag activation

Fibrin Accumulates

Injured endothelium

Fig. 97.1 Potential risk factors and pathophysiology of thrombotic microangiopathy after hematopoietic cell transplantation. PAI-1, plasminogen activator-1 (Reproduced with permission).

Potential primary mediators of endothelial injury Radiation Cyclosporine Chemotherapeutic agents Acute graft-versus-host disease

Potential secondary modifiers of endothelial injury Ischemia Hypertension Medications Baseline renal function Age Acute renal failure

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Fig. 97.2 Thrombotic microangiopathy. The glomerular mesangium is fibrillary and blends with unapparent capillary lumina that are obscured by markedly swollen endothelial cells that impart a “bloodless” appearance. Fibrin thrombi fill some of the capillaries in the left glomerulus (arrowheads), which has erythrocyte fragments in the mesangium (arrow). (Hematoxylin & eosin; magnification: ×200).

thelial deposits on biopsy; it is postulated that these deposits are antigen– antibody complexes representing GVHD in the kidney (Fig. 97.3). However, cases of minimal-change disease (MCD), which is thought to be a T-cell-mediated process, have been described [87]. Comparisons between case reports of membranous nephropathy and MCD after HCT have found that membranous nephropathy occurs in 61% of cases compared with 22% of cases with MCD [88]. The majority of reported patients with membranous nephropathy were male, slightly older, and had a history of acute and chronic GVHD. Both MCD and membranous nephropathy occur later after transplant at 8 and 14 months, respectively, and tend to occur within 1–5 months of the development of GVHD and/or the tapering of immunosuppression for chronic GVHD. Membranous nephropathy is more difficult to treat, with only 27% of patients reported achieving remission compared with 90% of patients with MCD. Others have reported cases of diffuse proliferative glomerulonephritis, antinuclear cytoplasmic antibody-related glomerulonephritis, focal segmental glomerulosclerosis, and IgA nephropathy [89–93] occurring after HCT. The development of each of these diseases seems to be associated with chronic GVHD and/or tapering of immunosuppression. Treatment with high-dose prednisone and/or reinstitution of calcineurin inhibitors usually results in resolution of nephrotic syndrome. Some physicians have used rituximab successfully in patients with nephrotic syndrome after HCT, typically in cases of membranous nephropathy [94]. GVHD-related CKD

between ultralarge von Willebrand factor release from endothelial cells and the rate of breakdown by ADAMTS13 resulting in thrombosis, has recently been described [82]. Other investigators have found systemic release of TNF-α correlated with levels of PAI-1 and tissue plasminogen activator release, supporting the idea of cytokine-induced endothelial damage [83]. Natural history of TMA after HCT Several studies have been done to describe the clinical course of TMA related-CKD following HCT. Again, most have involved small numbers of patients and have had a relatively short follow-up (1–5 years) [27,30,44,84,85]. What is apparent from the studies reported is that often an acute deterioration of renal function is followed by a period of stabilization, but rarely is full renal function restored. Patients with CKD after HCT can progress to end-stage renal disease and have decreased survival compared with other patients with end-stage renal disease [86]. In the study by Cohen et al. of 149 patients who survived 100 days or more after HCT during 1985–90, 19 (13%) developed HUS within 6–12 months after transplantation [27]. Four patients progressed rapidly to end-stage renal disease with a clinical picture resembling HUS; three of the four died. The one surviving patient had a markedly diminished GFR of 17 mL/min (normal 80–100 mL/min). Seven patients suffered a more gradual but continual decline, and the remaining eight patients had stabilization of their renal function after an initial period of decline. Four of the seven patients eventually progressed to dialysis. Elevated lactate dehydrogenase levels and lower platelet levels seemed to predict a poor prognosis. Nephrotic syndrome Chronic GVHD may manifest itself in the kidney as nephrotic syndrome with or without renal insufficiency (reviewed in [87]). Patients usually present with proteinuria, edema, and hypoalbuminemia. The majority of these case reports demonstrated membranous nephropathy with subepi-

Many survivors with CKD after HCT will not present with nephrotic syndrome, meet the definition of a TMA or have a documented viral infection, and therefore are labeled idiopathic CKD. The incidence of idiopathic CKD varies from 13% in patients receiving high-dose conditioning to 16–66% in patients who have been treated with RIC. Risk factors identified in the high-dose group include TBI, CSP use, AKI, and acute and chronic GVHD. In the RIC transplant group, long-term calcineurin inhibitor use and prior autologous transplant, as well as AKI and GVHD, were associated with an increased risk of CKD. However, in other recent studies, TBI was not associated with an increased risk of CKD. Miralbell et al. found that those patients receiving a lower dose of TBI because of renal shielding actually had greater decreases in GFR at 4 months compared with patients who received higher doses of TBI. The effects were no longer apparent after 12 months following HCT [95]. In a cohort study of 142 children undergoing HCT over a period of 5 years in the Netherlands, Kist-van Holthe et al. [96] found no correlation between radiation dose used and renal insufficiency at 1 year. In a large, retrospective study of 1635 HCT patients, TBI, irrespective of dose (range 200–1600 cGy), was not associated with the development of CKD after adjusting for demographic data and baseline characteristics, including original diagnosis, donor type, transplant type, and transplant year [28]. In this same study, TBI (HR = 1.0, 95% CI 0.7–1.4) was not associated with an increased risk of CKD among patients who received an allogeneic graft (n = 1228). The finding that GVHD is a risk factor for the development of CKD in patients after HCT who may not present with nephrotic syndrome or evidence of TMA [28,29,62] suggest that either the kidney is a direct target organ of GVHD via T-cell-mediated renal damage or the chronic systemic inflammatory state of GVHD leads to CKD (Fig. 97.4). A third explanation is that chronic exposure to calcineurin inhibitors, such as CSP, leads to CKD. These are not mutually exclusive hypotheses as T-cell-mediated injury in GVHD is intertwined with cytokine effects [97] and the effects of CSP may be potentiated in the presence of a chronic inflammatory state. In an autopsy study of renal pathology in six autologous and 20 allogeneic HCT patients, renal tubulitis identical to that seen in renal allograft rejection was present in 67% of patients

Kidney and Bladder Complications of Hematopoietic Cell Transplantation

(a)

(b)

Fig. 97.3 Membranous nephropathy. (a) The normocellular glomeruli in this nephrotic bone marrow transplantation patient have uniformly thickened glomerular basement membranes. (Methenamine–silver–hematoxylin; magnification: ×200) (b) Eosinophilic subepithelial deposits are visible between membrane spikes. (Methenamine–silver–hematoxylin; magnification: ×400) (c) Variably sized immune complex deposits stud the subepithelial glomerular basement membrane. (Uranyl acetate and lead citrate; magnification: ×6000)

(c)

Cytotoxic T-cells Cellular mediators HCT patient

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aGVHD cGVHD

Renal injury

Inflammatory cytokines Modulating factors Diabetes Hypertension Proteinuria Calcineurin inhibitors

Renal injury

Local release of inflammatory cytokines and profibrotic cytokines in kidney

Progression of CKD

Fig. 97.4 Proposed conceptual representation of pathogenesis of graftversus-host disease (GVHD)-related chronic kidney disease (CKD) in recipients of hematopoietic cell transplantation (HCT). aGVHD, acute graftversus-host disease; cGVHD, chronic graft-versus-host disease.

[98]. In a case report of minimal-change nephrotic syndrome after HCT, large numbers of CD8+ donor T cells were found infiltrating the interstitium and periglomerular areas of the kidney [99]. In a mouse model of GVHD in which the kidneys were examined, venulitis, endothelialitis, and tubulitis were present as early as 2 weeks post transplant. This lymphocytic inflammatory process then progressed, and by 6 months more severe endothelialitis and venulitis were present along with an increased number of peritubular infiltrates [100]. The authors suggest that these findings represent a continuous process of tissue injury that is different from the early injury that is seen in other target organs of GVHD [100]. This observation may explain why CKD often occurs beyond the first 3 months after transplant. The inflammatory and cytokine cascade that accompanies GVHD may also affect the kidney without invoking the T-cell-mediated injury that is present in GVHD involving the skin, gut, and liver [101,102]. In support of this hypothesis, several studies have demonstrated that elevations in plasma cytokine levels correlated with the development of post-transplant complications and organ dysfunction in the HCT population [103,104]. In animal models of GVHD, tissue destruction of acute GVHD does not require alloantigen expression on the target epithelium for cellular cytotoxicity but can be mediated by inflammatory cytokines [105]. In a case report of minimal change nephrotic syndrome post HCT,

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increased production by donor T-cells of TNF-α and interferon-gamma were related to the development of nephrotic syndrome; the lack of cellular infiltrates on biopsy suggested that the glomerular injury was secondary to cytokine production stimulated by alloantigens at extrarenal sites [104]. In addition to the T-cell activation and cytokine release in acute GVHD, chronic GVHD also involves the activation of B cells and the production of cytokines including transforming growth factor-beta (TGF-β1) [106]. TGF-β1 is important for collagen synthesis and matrix deposition in the kidney and other organs [107,108] and may also be important in the development of CSP-associated nephropathy in nonrenal transplant patients [109]. The effects of CSP on the kidney can be potentiated by increases in the production of TGF-β [110]. All CKD is not secondary to TBI or CSP use but rather a combination of factors including GVHD and the chronic inflammatory state that accompanies it. A renal biopsy may benefit these patients as we can better understand the pathophysiology of CKD and the role of GVHD and chronic inflammation in the development and progression of CKD, and tailor therapy accordingly. Albuminuria and CKD Albuminuria, defined as a urine albumin-to-urine creatinine ratio (ACR) of 30–299 mg/g creatinine, is thought to be a marker of endothelial dysfunction and inflammation, reflecting a systemic endothelial injury that affects multiple organs including the kidney. Newer work postulates that albuminuria results from tubular dysfunction in the trafficking and degradation of albumin [111,112]. In a prospective study of 142 patients with normal baseline serum creatinine undergoing their first HCT, ACRs were measured as an early marker of renal injury. Urine samples were collected weekly from baseline to day 100 after transplant. Total (intact) monomeric albumin was determined in untreated urine samples by liquid chromatography. Albuminuria was defined as an ACR (mg/g creatinine) over 30 and overt proteinuria as an ACR of 300 or more. The prevalence of albuminuria at baseline, day 100, and 1 year was 37%, 64% and 50%, respectively. Overt proteinuria occurred in 4% of patients at baseline, 15% at day 100, and 4% at 1 year. Characteristics associated with albuminuria include age, gender, donor type, hypertension, and acute liver injury with portal hypertension, but not exposure to TBI. Early albuminuria increased the risk of acute GVHD and sepsis, but not AKI. Albuminuria at day 100 was associated with CKD at 1 year (odds ratio = 4.0; 95% CI 1.1–14.6). Nonrelapse mortality risk was elevated (HR = 8.8, 95% CI 1.5–50.6) among patients with overt proteinuria at day 100, and overall survival was decreased [113]. There is evidence in patients with diabetes and albuminuria that treatment with an angiotensin-converting enzyme inhibitor (ACEI) or an angiotensin receptor blocker (ARB) slows the progression of CKD [114,115]. Extrapolating from the studies in the diabetic population, we speculate that ACEIs and ARBs would be useful in patients with albuminuria after HCT. However, clinical trials using an ACEI or ARB to treat HCT patients with albuminuria at day 100 are needed to determine whether or not interventions to reduce albuminuria and proteinuria will impact outcomes in the HCT population. Treatment of TMA and GVHD-related CKD Although the described pathology of the kidney in selected patients with CKD after HCT has demonstrated microangiopathic changes or changes associated with nephrotic syndrome, the pathophysiology of idiopathic chronic renal injury after HCT remains obscure. Histopathology is often not obtainable because patients with CKD after HCT are often not considered candidates for biopsy because of the perceived risks of the procedure in this patient population.

Therapeutic options for the treatment of TMA after HCT are limited. Case series of plasma exchange have been described, with mixed results [54,116,117]. Potential offending agents such as CSP and tacrolimus have been discontinued with some improvement clinically, but again the response depends on the severity of disease. And in some cases, where acute GVHD is contributing to disease, resolution of TMA has not occurred until GVHD was treated [54]. Given the finding of GVHD as a risk factor for the development of TMA, some authors have hypothesized that an immune etiology may be a contributing factor and attempted to treat TMA with rituximab. In a case report, four of five patients who developed TMA after HCT were successfully treated with rituximab [118]. In a report of 12 patients with TTP early after transplant, 50% of patients responded to treatment with defibrotide [119]. Based on treatments used in animal models, primarily of radiationinduced HUS, there are other potential interventions for patients with HUS after HCT. ACEIs have been used in rodent models of HCT-related renal injury, with intriguing results. The use of captopril or enalapril at the time of TBI in these animals resulted in less azotemia, lower blood pressures, and long-term preservation of renal function [59]. In addition, when it did occur, the onset of renal dysfunction was delayed and there was a decrease in proteinuria. Studies that have used ACEIs in combination with dexamethasone [120] or have used them with hydrochlorothiazide [121] or an ARB have found there was greater preservation of renal function with an ACEI and dexamethasone in combination, supporting the contributory role of inflammation in the process. Moreover, diuretics did not confer the same degree of protection, suggesting that it is not merely control of blood pressure that improves outcome. In a rat model of radiation nephritis, ACEIs markedly reduced glomerular lesions and sclerosis, and attenuated the increased levels of PAI-1 mRNA expression seen in irradiated kidneys compared with the untreated group [78]. Thus, a potential mechanism through which ACEIs can potentially benefit patients who develop HUS after HCT is by preventing thrombus formation and sclerosis by inhibiting PAI-1 activity. However, these mouse models were of radiation-induced injury, and since it is likely that TBI does not play a role in GVHD-related CKD, it may be that the potential beneficial effects of ACEIs and ARBs in these patients are their potential to reduce blood pressure as well as inflammation and inflammatory markers in patients. Randomized controlled trials using ACEIs or ARBs have not yet been published in patients undergoing HCT, although a trial is currently underway. There have been case series of patients undergoing renal transplantation successfully after HCT, and it is a viable option for patients with end-stage renal disease after HCT [122–124]. In patients who received their kidney from the same donor as their hematopoietic cells, little or no immunosuppression is required.

Bladder complications after HCT Hemorrhagic cystitis Epidemiology Hemorrhagic cystitis (HC) is a common complication after HCT and a significant cause of morbidity. Clinical syndromes vary from microscopic hematuria to macroscopic hematuria, dysuria, and flank pain with or without renal insufficiency. The grading system for HC based on the National Cancer Institute Common Toxicity Criteria defines grade 1 as the presence of microscopic hematuria, grade 2 as the presence of gross hematuria without clots managed with hydration and diuresis, grade 3 as persistent gross hematuria or clots that requires cauterization or transfusions, and grade 4 as gross hematuria that requires surgical intervention. In some studies, HC has been divided into early onset (within 48 hours of the conditioning regimen) and late onset (occurring any time

Kidney and Bladder Complications of Hematopoietic Cell Transplantation

after 48 hours). Early onset is often attributed to the toxic effects of conditioning regimen, including the use of cyclophosphamide, whereas late onset is thought to be related primarily to viral infections [125,126]. Early-onset HC Early-onset HC occurs during conditioning therapy with cyclophosphamide with or without TBI. Acrolein, a breakdown product of cyclophosphamide, is directly toxic to the bladder mucosa and is one of the primary causes of early-onset HC. The risk of HC from cyclophosphamide is dose dependent [127]. In a retrospective analysis of 81 patients who developed HC at one institution, Tsuboi et al. found that 2-mercaptoethane sodium sulfonate (mesna), often used as prophylaxis against HC, increased the risk of developing early-onset HC fivefold, and bladder irrigation increased the risk ninefold [126]. Additionally, the use of busulfan in the conditioning regimen is associated with an increased risk of early-onset HC [126]. These findings contradict others in the literature (described in the section on treatment) regarding the use of mesna. Late-onset HC Both BK polyoma virus and adenovirus are known to cause HC after HCT; however, it is not clear whether viremia and viruria lead to renal insufficiency or HC in all patients [128,129]. Pretransplant anti-BK virus immunoglobulin G titers of 1 : 20 or more increased the risk of BK viruria in patients, but donor BK viral serology was not associated with a risk of reactivation of BK infection in patients who were seronegative at the time of transplant [130]. In addition, peak BK viral load (≥3 log increase from baseline) in the urine has been shown to increase the risk of HC in patients after HCT [130]. In a study of 130 HCT patients, 51% had antibodies to adenovirus prior to transplant. In this study, risk factors for clinical adenovirus infection were positive antibody status of the donor and grade IV acute GVHD [131]. In a report of 21 patients, adenovirus was implicated in the development of acute renal failure after HCT in 90% of the patients [132]. Additional case reports of acute renal failure associated with adenovirus have been described [133]. Other risk factors that have been identified for late-onset HC include older age, pretransplant conditioning with busulfan, allogeneic HCT, and acute GVHD grades II–IV [126,130,134,135]. Risk factors in children include male gender and unrelated donor graft recipients [136]. Treatment of HC post HCT First-line management of HC often involves supportive care, increased hydration and diuresis, and occasionally continuous bladder irrigation. In a randomized prospective study by Bedi et al., no difference was found between the use of mesna versus forced diuresis during the conditioning regimen to prevent the development of early-onset HC secondary to the uroepithelial toxicity of conditioning [137]. One hundred patients were randomized to receive mesna or hyperhydration to prevent HC during conditioning therapy. Patients in the mesna arm received 1.5 mL/m2/day of fluids compared with the 3 mL/m2/day of fluids given to the patients in the hyperhydration arm. Again, no difference was found in the incidence of early-onset HC [138]. Ballen et al. found that hyperhydration with 5% dextrose normal saline with potassium at 250 mL/hour was more cost-effective and a well-tolerated alternative to the use of mesna to prevent early-onset HC attributed to cyclophosphamide used for conditioning. The nursing time involved to carefully monitor these patients and avoid volume overload was not factored into the cost [139]. A third randomized study evaluated hyperhydration with continuous bladder irrigation or continuous mesna infusion. The incidence of earlyonset HC was similar in the two groups, but, as expected, the continuous

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bladder irrigation group had more hematuria. There were a significantly higher number of urinary tract infections in the bladder irrigation group, and these patients reported more discomfort, restriction of movement, and bladder spasms than the mesna group [140]. Based on these studies, it is clear that hyperhydration and maintenance of adequate diuresis is important. Most centers use mesna when cyclosphosphamide-based conditioning is used, but it may not offer additional benefit if hydration and diuresis are adequate. Continuous bladder irrigation should not be used to prevent HC unless patients have markedly decreased to no urine output. It may have a role in dissolution of clots in more severe forms of HC. There are numerous case reports of improvement in symptoms and stabilization of renal function in patients with HC secondary to BK or adenovirus HC treated with cidofovir [141–146]. Ciprofloxacin has also been shown to reduce BK viral replication, decrease the risk of BK reactivation, and quantitatively decrease BK viruria [147], and the authors suggest it may be beneficial to prevent BK-associated HC [148]. There are isolated case reports of the successful use of intravesicular sodium hyaluronate [149], prostaglandin E2 [150], and cidofovir [143] to treat virally induced HC in patients after HCT. Previous intravesicular therapies have been tried (including formalin, alum, and silver nitrate), with varying results and many complications secondary to therapy (reviewed in [151]). Intravenous ribavarin for adenovirusassociated HC and factor VIIa have also been used successfully to treat HC in patients after HCT [152,153]. Other therapies that have been reported to manage the bleeding associated with severe HC include selective embolization of vesical arteries [154] and cystectomy in a patient who developed renal failure and pulmonary edema secondary to ongoing HC after HCT [155]. There is one case report of the use of hyperbaric oxygen therapy to successfully treat intractable HC [156]. In cases of HC where obstruction of urine flow occurs, percutaneous nephrostomy tubes or ureteral stent placements may be necessary to relieve the obstruction and allow drainage of the urine. There have been case reports of patients with biopsy-proven BK nephropathy (Fig. 97.5) after transplant resulting in CKD and in some cases end-stage renal disease requiring dialysis [157]. Thus, in patients with BK viruria after transplant, it is important to measure serum BK levels by the polymerase chain reaction as these have been associated with the development of CKD and renal failure [157]. Serum BK levels are more reflective of renal involvement and, in renal transplant patients, are associated with BK nephropathy in the graft. In cases of BK nephropathy, first-line therapy is often reduction of immunosuppression when possible prior to institution of cidofovir and other antiviral agents. Complications and imaging findings with HC Various complications have been described in patients who develop HC after HCT; these include hydronephrosis, obstructive uropathy [127,136], ureteral obstruction secondary to clot formation and/or ureteral stenosis [158], necrotizing tubulointerstitial nephritis [133], and ARF [127,132,133,136]. Bladder perforation has also been described [136], as has intractable hemorrhage requiring cystectomy [155]. Imaging findings of HC will vary based on severity of disease and imaging modality. On renal and bladder ultrasonography, focal or diffuse bladder wall thickening can be seen [159]. Clots may be visible within the bladder, and if the clot is causing an obstruction, unilateral or bilateral hydronephrosis may be visualized as well. Sloughed debris secondary to obstruction, clot, inflammation or infection can also cause obstruction and is visualized on ultrasound. In the presence of hydronephrosis, additional imaging studies such as a voiding cystourethrogram and a lasix renogram may be necessary to differentiate obstruction versus reflux. Additionally, the upper tracts may be involved, and thickening of the suburothelial lining of the renal pelvis has been described.

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(a)

(b)

Fig. 97.5 BK nephropathy. (a) The nuclei of tubular epithelial cells infected with BK virus have basophilic inclusions and peripheral chromatin rimming (arrowheads). (Methenamine–silver–hematoxylin; magnification: ×400) (b) Infected nuclei stain positively with antibodies directed against the large T antigen of SV40. (Diaminobenzedine; magnification: ×400)

Perinephric fluid collections secondary to hemorrhage or rupture of a fornix can also be found. With the addition of Doppler studies to the ultrasound, decreased flow to areas of the bladder as well as hypervascularity and areas of active bleeding can be identified. Direct visualization with cystoscopy and cauterization of areas of active bleeding may be necessary. Computed tomography and magnetic resonance imaging can enhance the images and may offer more definition of the bladder wall than ultrasound. However, first-line imaging in cases of HC should be done with ultrasound and Doppler studies.

Summary There are clearly distinct clinical entities involving the kidney that occur after HCT: ARF, TMA, nephrotic syndrome, and GVHD-related CKD. HC is the most common urologic complication reported in patients after HCT. There are those patients who will develop CKD that is not related to TBI or the conditioning regimen, but rather to complications and/or therapy that occurs after HCT, specifically acute and chronic GVHD and prolonged calcineurin use. The burden of management will fall not only on the nephrologists, but also on the transplant physician to ensure close monitoring of renal function, blood pressure, and urinalyses post transplant. It may be that our energies have been misdirected in

trying to reduce exposure to TBI and that we should rather try to decrease the inflammatory and cytokine effects of GVHD and reduce exposure to calcineurin inhibitors to prevent CKD in this population of patients. Although physicians are well attuned to HC after HCT, increasing awareness regarding the potential for both BK and adenovirus to lead to nephropathy, both acute and chronic, is needed. Careful monitoring of patients after the acute hemorrhage is controlled may be needed to prevent ongoing injury. Imaging with renal ultrasound is an important aspect of managing the acute complications such as obstruction but may also be helpful in monitoring ongoing injury and late complications. Renal injury after HCT is a relatively common occurrence. As the indications for and number of transplants performed worldwide increases, so will the burden of kidney disease. Identifying those patients at risk for the development of both AKI and CKD will be important for potential intervention and prevention of kidney injury and progression to end-stage renal disease in this patient population.

Acknowledgments Pathology photographs were kindly provided by Dr Laura Finn, Associate Professor of Pathology at the University of Washington and Children’s Hospital and Regional Medical Center, Seattle, WA, USA.

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76. Anthony M, Zeigler Z, Lister J et al. Plasminogen activator inhibitor (PAI-1) antigen levels in primary TTP and secondary TTP post-bone marrow transplantation. Am J Hematol 1998; 59: 9–14. 77. Salat C, Holler E, Reinhardt B et al. Parameters of the fibrinolytic system in patients undergoing BMT: elevation of PAI-1 in veno-occlusive disease. Bone Marrow Transplant 1994; 14: 747– 50. 78. Oikawa T, Freeman M, Lo W, Vaughan D, Fogo A. Modulation of plasminogen activator inhibitor1 in vivo: a new mechanism for the anti-fibrotic effect of renin-angiotensin inhibition. Kidney Int 1997; 51: 164–72. 79. Chandler W, Jelacic S, Boster D et al. Prothrombotic coagulation abnormalities during Escherichia coli 0157:H7 infections. N Engl J Med 2002; 346: 23–32. 80. Chant I, Milford D, Rose P. Plasminogen activator inhibitor activity in diarrhea-associated hemolytic uraemic syndrome. Q J Med 1994; 87: 737–40. 81. Biedermann BC, Sahner S, Gregor M et al. Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease. Lancet 2002; 359: 2078– 83. 82. Bernardo A, Ball C, Nolasco L, Moake JF, Dong JF. Effects of inflammatory cytokines on the release and cleavage of the endothelial cellderived ultralarge von Willebrand factor multimers under flow. Blood 2004; 104: 100–6. 83. Seeber C, Hiller E, Holler E, Kolb HJ. Increased levels of tissue plasminogen activator (t-PA) and tissue plasminogen activator inhibitor (PAI) correlate with tumor necrosis factor alpha (TNF alpha)-release in patients suffering from microangiopathy following allogeneic bone marrow transplantation (BMT). Thromb Res 1992; 66: 373–83. 84. Berg U, Bolme P. Renal function in children following bone marrow transplantation. Transplant Proc 1989; 21: 3092–4. 85. Van Why S, Friedman A, Wei L, Hong R. Renal insufficiency after bone marrow transplantation in children. Bone Marrow Transplant 1991; 7: 383– 8. 86. Cohen E, Piering W, Kabler-Babbitt C, Moulder J. End-stage renal disease (ESRD) after bone marrow transplantation: poor survival compared to other causes of ESRD. Nephron 1998; 79: 408–12. 87. Rao P. Nephrotic syndrome in patients with peripheral blood stem cell transplant. Am J Kidney Dis 2005; 45: 780–5. 88. Brukamp K, Doyle A, Bloom R, Bunin N, Tomaszewski J, Cizman B. Nephrotic syndrome after hematopoietic cell transplantation: do glomerular lesions represent renal graft-versus-host disease? Clin J Am Soc Nephrol 2006; 1: 685– 94. 89. Chan GS-W, Lam M, Au W et al. IgA nephropathy complicating graft-vs-host disease, another nephropathy causing nephrotic syndrome after bone marrow transplantation. Histopathology 2004; 45: 642–56. 90. Oliveira J, Bahia D, Franco M, Balda C, Stella S, Kerbauy J. Nephrotic syndrome as a clinical manifestation of graft-versus-host disease (GVHD) in a marrow transplant recipient after

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genic bone marrow transplantation. J Thromb Haemost 2004; 2: 1853–5. 154. Gine E, Rovira M, Real I et al. Successful treatment of severe hemorrhagic cystitis after hemopoietic cell transplantation by selective embolization of the vesical arteries. Bone Marrow Transplant 2003; 31: 923–5. 155. Koc S, Hagglund H, Ireton RC, Perez-Simon JA, Collins SJ, Appelbaum FR. Successful treatment of severe hemorrhagic cystitis with cystectomy following matched donor allogeneic hematopoietic cell transplantation. Bone Marrow Transplant 2000; 26: 899–901. 156. Hattori K, Yabe M, Matsumoto M et al. Successful hyperbaric oxygen treatment of life-

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Fouad R. Kandeel

Endocrine Complications Following Hematopoietic Cell Transplantation

Introduction The advent of hematopoietic cell transplantation (HCT) has resulted in the significant extension of life for many children and adults affected with hematologic, immunologic, genetic, and metabolic diseases; however, the conditioning regimens for HCT, as well as the process of HCT itself, may originate some new complications for patients. As continued improvement in post-HCT survival rates now often span decades, the manifestation of long-term endocrine complications in terms of growth, skeletal development, metabolic function, and gonadal and sexual function has become increasingly recognized, and the goal of maintaining an extended, superior quality of life post HCT has likewise attained even greater importance. The present chapter will review the endocrine abnormalities that may result from HCT, and will outline suggested approaches to the investigation and management of these disorders.

Hypothalamic-pituitary dysfunction The hypothalamus produces a number of factors that are directly involved in the regulation of hormone secretion from the anterior pituitary: thyrotropin-releasing hormone (TRH) regulates the secretion of thyroid-stimulating hormone (TSH); gonadotropin-releasing hormone (GnRH) regulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH); growth hormone-releasing hormone (GHRH) and somatostatin regulate the secretion of growth hormone (GH); corticotropin-releasing hormone (CRH) regulates the secretion of adrenocorticotropic hormone (ACTH); and dopamine (also known as primary prolactin-inhibiting hormone) regulates the secretion of prolactin. The anterior pituitary arises from Rathke’s pouch, an ectodermal evagination of the oropharynx. Hormones produced by the anterior pituitary mainly regulate hormone secretion from most other endocrine glands, as well as life processes such as growth and lactation; in some instances, anterior pituitary hormones may also have direct metabolic effects. TSH, LH, and FSH are glycoproteins that share a common α subunit, but differ in the composition of β subunits. Binding of the α and β subunits establishes the active hormone and confers biologic activity at the target organs (the thyroid for TSH, and the gonads for LH

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

and FSH). ACTH is derived from splicing a very large prohormone known as pro-opiomelanocorticotropin, and regulates steroid hormone secretion from the adrenal cortex. GH regulates the production of insulin-like growth factor-1 (IGF-1) by the liver, which mediates GH effects on bone growth. In addition, GH has direct metabolic effects. Prolactin is involved in the regulation of lactation, and has a variety of effects on tissues throughout the body. The hormones produced by the thyroid, gonads, and adrenal glands, as well as IGF-1, exert feedback control on the hypothalamic-pituitary axis. (For a further discussion of normal gland physiology, please refer to the respective sections below.) The posterior lobe of the pituitary gland is neural in origin, and arises embryologically as an evagination of the hypothalamus. The posterior pituitary produces arginine vasopressin and oxytocin, which are involved in the regulation of water metabolism and puerperium, respectively. Radiation exposure can result in hypothalamic-pituitary dysfunction in the following three clinical situations: (1) radiotherapy of brain or extracranial tumors (doses ≥30 Gy), (2) cranial radiotherapy for acute lymphoblastic leukemia (ALL) (doses 18–24 Gy), and (3) total body irradiation (TBI) prior to HCT for hematologic malignancy or solid tumor (doses 10–14 Gy) [1,2]. Since the functions of the hypothalamus and the pituitary are intimately related, it is not always easy to discriminate between damage at these two levels. In general, however, the hypothalamus is more radiosensitive than the pituitary, and more often the site of damage [3]. Hyperprolactinemia, for example, often occurs following external radiotherapy to the hypothalamic-pituitary area [4], suggesting a reduction in dopamine secretion by the hypothalamus. The degree of pituitary dysfunction is typically related to the dose of irradiation received [3]; hypopituitarism has been observed with cumulative total doses of 20 Gy or greater to the brain [4]. The threshold radiation dose may be lower in patients treated simultaneously with high doses of methotrexate or intrathecal chemotherapy; however, Kauppila et al. [4] noted that hypopituitarism was not seen after TBI doses of 12 Gy. In fact, it is generally thought that, in most cases, the hypothalamicpituitary axis remains intact after HCT, and that hormonal disturbances usually occur as a result of injury at the target endocrine organ [4]. Nevertheless, it is possible that lower doses of cranial irradiation prior to HCT in patients with hematologic malignancies may cause hypopituitarism, and/or long-term hypothalamic-pituitary hormone insufficiency [2]. In contrast, end-organ hormone disturbances are common post HCT, even with chemotherapy-only regimens [4]. Further discussion of the pathophysiology of specific hormone disorders, as well as methods for their investigation and treatment, is found in their respective sections below.

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Growth disorders Pituitary GH is secreted in a pulsatile manner that is superimposed on a schedule of diurnal variation. GH secretion is stimulated by GHRH, and suppressed by hypothalamic somatostatin. GH stimulates the production of IGF-1 protein by the liver, which, in turn, exerts negative feedback control on the secretion of hypothalamic GHRH and pituitary GH itself (Fig. 98.1) [5]. The immediate neonatal period generally represents the highest level of GH secretion. GH levels then decrease throughout childhood, but rise during puberty as a result of increased pulse amplitude (but not frequency). GH secretion falls once again with increasing age. The anabolic effects of GH on the linear growth of bones are mainly mediated by IGFs and their binding proteins (IGFBPs), which extend the serum half-life of the IGFs by aiding in their transport to target cells and modulating their interactions with membrane surface receptors [6]. Given that IGF-1 concentrations are more dependent on GH than IGF-2, the measurement of IGF-1 levels is more likely to provide useful clinical correlations in the evaluation of GH deficiency. Serum levels of IGF-1 rise throughout childhood, attaining adult levels at puberty. During puberty, IGF-1 levels rise to two to three times that of the normal adult range. After the age of 20 or 30 years, IGF-1 levels gradually decline. Serum levels of IGF-1, therefore, may be greatly influenced by chronologic age, degree of sexual maturation, and nutritional status. GH also has direct metabolic effects, such as lipolysis, increased amino acid transport into tissues, and increased liver protein synthesis.

Further, GH excess can produce insulin resistance via altered postreceptor insulin action, resulting in decreased carbohydrate utilization and impaired glucose uptake into cells. GH additionally increases hepatic glucose output, which further worsens hyperglycemia [7,8]. Growth is an extremely complex process that occurs at vastly differing rates from infancy through early and mid-childhood and adolescence. The rise in serum gonadal steroids during adolescence plays an important role in triggering the pubertal growth spurt, as the presence of androgens and estrogens increase skeletal maturation. After sexual maturation, the fusion of the long bones and vertebral epiphyseal growth plates signals the cessation of growth and the attainment of final adult height [9]. Reduced growth rates in children are thought to be due to numerous factors, but impaired GH secretion is likely the primary cause. GH deficiency that occurs before the fusion of the epiphyseal plates of the long bones will result in short stature, while GH excess leads to gigantism. GH deficiency in the adult may be associated with reduced lean body mass and musculature, increased body fat, reduced bone mineral density (BMD), reduced exercise performance, and increased plasma cholesterol, comprising an overall increased mortality risk from cardiovascular disease [10–13]. Further, adults with GH deficiency may suffer from depression, anxiety, reduced energy, and social isolation [14]. Despite these observations, there are no clinical syndromes of adult GH deficiency that have been widely recognized. It is believed that the type of conditioning regimen prior to HCT has a significant influence on the development of potential growth dysfunction (Table 98.1). A large body of data suggests that radiation (particularly cranial and TBI) is most responsible for diminished growth in children who have undergone HCT [15]. Potential pathogenic mechanisms include the direct impact of combination chemotherapy and radiation on the skeleton (i.e. skeletal dysplasia, particularly of the spine), radiation-induced hypothalamic/pituitary dysfunction, and hypothyroidism [15]. Radiation may also prompt the development of precocious puberty, which can result in premature epiphyseal fusion and subsequent reduced final height. Children who have received preparation regimens consisting of cranial irradiation followed by high-dose chemotherapy and single-dose TBI are most likely to demonstrate growth defects 2–5 years after HCT, with pubertal growth most affected [3]. With no previous cranial irradiation and fractionated TBI, however, growth was not compromised 3 years after HCT [3]. Because GH deficiency is strongly related to irradiation, growth rates may be adversely affected by HCT; in fact, GH deficiency is found in 32% of children after HCT with TBI [16]. Inadvertent exposure of the liver and other tissues during TBI via secondary radiation may additionally contribute to the defective production of essential growth factors such as IGF-1 [4]. Nevertheless, the reports of final short stature after HCT range from as low as 4% [17] and 14.3% [15] to 25% [18] and 60% [19], while other sources report final stature that generally falls within the normal adult range [3]. This variability in final height outcome may be attributable to the different patient ages at HCT, as the risk of impaired growth is highest in the youngest children transplanted prior to the onset of puberty [20,21]; conversely, some reports have noted improved growth after HCT with younger patient age [22]. Table 98.1 Risk factors for growth dysfunction following hematopoietic cell transplantation

Fig. 98.1 Neuroendocrine control of growth hormone and insulin-like growth factor-1 (IGF-1) secretion. +, positive feedback; −, negative feedback; GH, growth hormone; GHRH, growth hormone releasing hormone. (Adapted from [5], with permission.)

Total body irradiation, particularly single-dose irradiation Cranial irradiation High-dose chemotherapy Younger age at transplantation

Endocrine Complications Following Hematopoietic Cell Transplantation

In the absence of cranial irradiation, there is actually a poor correlation between GH production and concentrations of IGF-1 or IGFBP-3 and growth in children after HCT, suggesting the influence of other factors on growth dysfunction in addition to primary GH deficiency. Endogenous or exogenous glucocorticoid excess, for example, may blunt GH secretion [15]. Some reports postulate that chemotherapy alone, particularly busulfan plus cyclophosphamide (BU/CY), may cause growth deficiencies [18] due to toxicities at the epiphyseal growth plate, even after corticosteroid administration has ceased. Additional considerations include nutritional or vitamin deficiencies, genetics, the nature of the underlying disease (e.g. red cell aplasia, Fanconi’s anemia or thalassemia), and the presence of general illness or graft-versus-host disease (GVHD) [15]. The manifestation of other endocrine hormonal dysfunction may further contribute to impaired growth dynamics. As another example, bone age advancement can be severely delayed in hypothyroidism – usually more so than with GH deficiency – and epiphyseal dysgenesis may be seen when calcification of the epiphyses progresses [23]. The administration of gonadal sex steroids for the treatment of hypogonadism may cause premature growth cessation by accelerating the fusion of long bones and the closure of the growth plates, but the absence of these factors during the prepubertal period appears to have no major significance [23]. As with sex steroids, glucocorticoid deficiency seems to have little effect if the individual is otherwise healthy (i.e. if hypotension and hypoglycemia are absent). Interestingly, children with new-onset diabetes mellitus are frequently taller than their peers, likely due to both increased GH and insulin levels during the preclinical stages of the disease. For more detailed information on this subject, please refer to Chapter 104. Evaluation of growth disorders post HCT Children should be closely monitored and serially measured for appropriate growth (height velocity) after HCT in the context of normal standards, such as the growth charts provided by the National Center for Health Statistics, part of the Centers for Disease Control. Height velocity standards remarkably encompass the range of normal growth patterns between 2 years of age and puberty with great predictability; thus, any deviation of the patient’s plotted growth from the normal curve should be immediately suspect for an aberrant growth process [6,9]. Growth potential may be estimated by the measurement of “bone age,” or the level of ossification of the tubular bones, via radiographs of the left hand and wrist [9]. Final adult height may be predicted with greater accuracy with increasing bone age. In the assessment of GH levels, the possibility for hypothalamic or pituitary dysfunction must always be considered, particularly with known radiation exposure and the presence of other endocrine hormone abnormalities (TSH, ACTH, gonadotropin, etc.) and potential worsening of the underlying disease process (e.g. local extension of Hodgkin’s lymphoma of the nasopharynx to the hypothalamus). The identification of actual GH deficiency, however, is not often easily established, due to low levels of basal GH secretion in both normal and truly GH-deficient patients. Further, the sequential measurement of spontaneous GH secretion is cumbersome and expensive. The development and availability of sensitive radioimmunoassay for IGF-related proteins has greatly simplified the diagnostic process (Fig. 98.2). Thus, the laboratory work-up for GH deficiency should primarily involve the measurement of IGF-1 and IGFBP-3 levels, as well as the results of stimulated GH secretion. GH secretion can be stimulated with either physiologic (fasting, sleep or exercise) or pharmacologic stimuli (levodopa, clonidine, glucagon, propanolol, arginine or insulin). GH deficiency may generally be suspected in cases of subnormal growth velocity combined with GH levels below 10 ng/mL after each of two

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pharmacologic stimulations carried out on two separate occasions. Testing should be performed after an overnight fast in order to prevent carbohydrate or fat ingestion from suppressing a GH response. Nevertheless, pulsatile secretion of GH may cause varying results. Further, different radioimmunoassay systems may return dissimilar measurements on the same blood sample, so the physician must be familiar with the laboratory standards being used. Another complicating factor is that prepubertal children secrete less GH than pubertal subjects, and the former may otherwise be mistakenly diagnosed with GH deficiency. This situation may be addressed by “priming” with a dose of estrogen prior to testing, as estrogen administration has been shown to increase GH responsiveness to provocative stimuli in both prepubertal girls and boys, but this practice remains controversial [24]. Since growth failure is likely to be multifactorial, consideration must be given to causes other than inadequate GH. Management of growth disorders post HCT It is suggested that GH is administered to children with decreased clinical growth rates, and/or decreased IGF-1 and IGFBP-3 levels, and/or low GH values on stimulation tests, while giving due consideration to the potential necessity of replacing other deficient hormones necessary in order to achieve optimal growth. While no randomized trial has yet been reported that clearly delineates the benefits of GH treatment in children [20], some available long-term outcome data are promising in regards to positive growth response to treatment [25]. Supplementation is commonly given with a starting dose of recombinant human GH (rhGH) 0.18–0.35 mg/kg body weight per week, administered in seven daily doses. GH treatment after HCT is typically reported to result in an increase in growth velocity, albeit a smaller one than in children with idiopathic GH deficiency [15]. Age at the initiation of treatment is inversely correlated with expected growth responses, possibly indicating the need for early treatment. First-year height velocity is the most predictive factor in regards to later growth that may occur in years 2 through 4 [26]; GH therapy in children typically is given for 2 years, but can be extended until the closure of long bone epiphyses if there are no contraindications. Suboptimal response to GH therapy may be attributed to poor compliance, improper administration, subclinical (undiagnosed) hypothyroidism (see below), underlying illness, concurrent glucocorticoid therapy, prior irradiation of the spine, epiphyseal fusion, the presence of anti-GH antibodies (which develop in 10–20% of patients receiving rhGH), or an incorrect diagnosis of GH deficiency. It has not yet been possible to determine the link, if any, between GH therapy and the new development of leukemia; if present, the risk of leukemia subsequent to GH therapy appears to be limited to children already at high risk for that disease (e.g. prior malignancies, radiation exposure or the presence of syndromes associated with leukemia) [27], but further studies are needed. Other side-effects of GH treatment may include prepubertal gynecomastia, pancreatitis, benign nevus growth, behavioral changes, kyphosis, scoliosis, tonsillar atrophy, and sleep apnea [9]. In the past, the use of pituitary-derived human GH had the potential for causing Creutzfeldt– Jakob disease, but this risk no longer exists with the development of rhGH. GH replacement should not be initiated, however, until patients have remained free of their underlying hematologic disease for at least 2 years post HCT. The treatment of prepubescent HCT recipients with GH deficiency as well as hypogonadism can be challenging; generally, administration of exogenous sex steroids is low in dosage or is postponed altogether until the desired patient height is reached in order to reduce the likelihood of premature closure of the growth plates. Hormone therapy in GH-deficient adolescents and young adults may encourage the development and maintenance of peak bone and muscle

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Fig. 98.2 Algorithm for the evaluation of growth dysfunction following hematopoietic stem cell transplantation. FT4, free thyroxine; GH, growth hormone; IGF-1, insulin-like growth factor-1; IGFBP-3, insulin-like growth factor binding protein-3; MRI, magnetic resonance imaging; SD, standard deviation; SDS, standard deviation score; TBI, total body irradiation; TSH, thyroid-stimulating hormone. (Adapted from [9], with permission.)

Endocrine Complications Following Hematopoietic Cell Transplantation

mass through the second and third decades of life, possibly staving off the future development of osteopenia, as well as cardiovascular disease. GH therapy may also markedly improve psychological wellbeing in deficient adults [12], even after the conclusion of linear bone growth. GH replacement therapy in adults remains controversial due to the potentially increased risk of cardiovascular complications and malignancies.

Table 98.2 Diagnostic categories for osteoporosis based on T-score of bone mineral density Normal Low bone mass (osteopenia) Osteoporosis

Dysfunctional bone metabolism and osteoporosis The skeleton serves a dual purpose: it is both a rigid, structural foundation for the other organs of the body, as well as a vast mineral reserve, particularly in regards to the metabolism of calcium. Extracellular and intracellular calcium levels are tightly regulated. The amount of calcium in the extracellular fluid represents approximately 1% of total body calcium; the rest is sequestered in bone. About 50% of total serum calcium is in the ionized form, while the remainder is either bound to albumin (40%) or complexed with anions such as phosphate and citrate (10%) [28]. The bound fractions of calcium are metabolically inert and not regulated by hormones, but the calcium ion serves a regulatory role, and is itself principally regulated by parathyroid hormone (PTH) and 1,25-hydroxyvitamin D3. Osteoblasts and osteoclasts are mainly influenced by IGF-1, transforming growth factor-beta, and bone morphogenic proteins. GH, glucocorticoids, thyroid hormones, insulin, and gonadal hormones are other systemic hormones that also exert profound effects on bone remodeling. The main goal of calcium homeostasis is to maintain constant levels of calcium ions in the extracellular fluid while still providing adequate amounts of calcium to cells and bone, and for renal excretion. Compensation occurs for changes of daily calcium intake, bone metabolism, and renal function. Massive blood transfusions (in which citrate is used as a coagulant), however, can reduce the levels of calcium ion enough to cause tetany; further, a substantial rise in serum concentrations of calcium can occur in patients with certain types of solid tumor as a result of production of a PTH-related protein. Certain hematologic malignancies (lymphomas) may also be associated with hypercalcemia and high levels of 1,25-hydroxyvitamin D3 (leading to increased calcium absorption). Increased bone resorption and impaired renal excretion contribute to hypercalcemia [29]. Osteoporosis, defined as a condition of low bone mass and microarchitectural disruption leading to enhanced bone fragility and a consequent increase in fracture risk [30], is the most common metabolic bone disease worldwide, affecting approximately 50% of white and Asian postmenopausal women. Further, at least one in eight older women of other (i.e. non-white and non-Asian) racial backgrounds, as well as one in eight older men, are likely to experience an osteoporotic fracture at some point during their lifetime [31]. Diagnostic criteria for postmenopausal women are based on measurements of BMD and/or bone mineral content; although arbitrary, these categories do offer some indication of fracture risk, which markedly increases with age (Table 98.2). Patient T-scores are reported as standard deviations below the average, optimal peak bone density of a healthy 30-year-old of the same sex and ethnicity. Osteopenia represents lower than normal BMD that is not sufficient to qualify as osteoporosis. The loss of bone mass, or compromised bone strength, has been well documented as a sequel to HCT, preferentially within the first 6 months post transplant. Typically, a 5–15% loss of BMD can be expected in the lumbar spine and femoral neck within 1 year after HCT [32–34]. Osteopenia is observed in as many as 50–60% of all HCT recipients, and osteoporosis develops in 20% [35]. Studies in adult survivors of childhood cancer demonstrate that BMD is reduced at multiple skeletal sites, supporting the premise that inadequate bone mineralization during child-

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Severe osteoporosis (established osteoporosis)

BMD or BMC within ± 1 SD of young adult reference mean BMD or BMC between 1 and 2.5 SD less than young adult mean BMD or BMC more than 2.5 SD less than young adult mean BMD or BMC more than 2.5 SD less than young adult mean in the presence of fragility fractures

BMC, bone mineral content; BMD, bone mineral density; SD, standard deviation. Reproduced with permission from Lorenzo et al. [31].

hood may have long-term adverse effects on BMD in adulthood [36–39]. The only study to have reported BMD in children after HCT using dualenergy X-ray absorptiometry (DEXA) technology showed that BMD was significantly lower than in adults [40]. Fracture rates in both male and female pediatric HCT recipients have been estimated at 172 per 10,000 person–years by one center (Sanders, unpublished data, referenced in [41]); when compared with the peak childhood fracture rates of 133 per 10,000 person–years in a large, population-based cohort study of boys and girls [42], this suggests that reduced BMD in children as the result of HCT may be clinically significant. In transplanted adults, bone loss may occur even prior to HCT, due in part to the direct effects of underlying hematologic cancers on the integrity of bone, such as malignant changes in bone structure, particularly with acute leukemia or multiple myeloma. One study compared evidence of osteopenia or osteoporosis prior to HCT and after HCT, with 39% of patients demonstrating evidence of osteopenia or osteoporosis in the lumbar spine, and 25% in the femoral neck, prior to transplant, increasing to 50% and 45% 1 year after transplant, respectively [33]. Other studies have confirmed similar results [43]. In general, the pathogenesis of metabolic bone dysfunction is multifactorial and not completely understood, particularly in regards to the effects of high-dose treatment followed by HCT on bone mineral metabolism [44]. The three major pathogenic mechanisms thought to be responsible for reduced BMD are: (1) failure to achieve peak bone mass (due to altered calcium or vitamin D homeostasis, or deficiencies in growth or gonadal hormone secretion), (2) bone loss caused by increased bone resorption (disturbance in osteoclast function); and (3) inadequate replacement of lost bone due to decreased bone formation (disturbance in osteoblast function) [31]. HCT and post-transplant therapies may specifically affect bone homeostasis by inducing premature menopause and/or hypogonadism, directly poisoning bone cells, and altering calcium and magnesium metabolism, causing elevated PTH levels. The leading cause of reduced BMD after HCT is prolonged glucocorticoid therapy for GVHD [32,45,46], likely due to the production of cytokines that trigger inappropriate osteoclastic activation, decreased bone formation, and decreased levels of 1,25-hydroxyvitamin D3. Glucocorticocoids affect trabecular bone more than cortical bone, resulting in fractures of the vertebrae, ribs, and ends of long bones. Bone loss is most rapid within 6 months of initiating glucocorticoid therapy, and may occur with doses as low as prednisone 5 mg once daily. The use of chemotherapy and, to a lesser extent, radiation therapy as part of the pretransplantation regimen exacerbates bone loss, due to dose-dependent toxicity of high-dose chemotherapy to bone marrow osteoprogenitors, TBI-related radiation damage, and the suppressive effects of both these modalities on the gonads. Cyclosporine, for example, potentiates hypomagnesemia and

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hypocalcemia. Physical inactivity due to long hospitalization or illness and nutritional deficits further compound these effects [32,47]. Additional general risk factors include older age, thin body habitus, family history of osteoporosis or low-trauma fractures, smoking, and a diet low in calcium or vitamin D [43]. Interestingly, at the end of the first year post transplantation, either bone mass begins to recover (spine and total body) or bone loss is attenuated (femoral sites). This improvement is likely due to multiple factors, including a reduction in immunosuppressant dosage, resolution of immobilization or malnutrition, and convalescence from the transplant procedure itself. Long-term bone loss is mainly determined by the intensity of steroid use. Osteonecrosis, or avascular necrosis, is an infrequent, but significant sequela of HCT, involving bone infarction and the rapid and focal deterioration of bone quality that primarily affects the femoral head, although the ankles and shoulders may also be involved. Osteonecrosis is painful, and may lead to the ultimate collapse of the bone, often necessitating a hip replacement. Osteonecrosis can occur in all age groups, but is usually related to the use of corticosteroids, as glucocorticoids inhibit osteoblast function; relatively low doses, or even brief courses of high-dose therapy, have been implicated. With up to 1% of Western populations receiving long-term glucocorticoid therapy for any reason, osteonecrosis is a significant general concern. Notably, osteonecrosis may occur in up to 5% of HCT patients, often long after transplantation [48,49]. Other risk factors for osteonecrosis include the presence of sickle cell disease and other hemoglobinopathies, polycythemia vera and other lymphoproliferative disorders, pregnancy, septic emboli, alcoholism, pancreatitis, Gaucher’s disease, systemic lupus erythematosus, rheumatoid arthritis, trauma (fracture/dislocation or direct tissue damage), radiation (direct injury to the vascular supply, leading to occlusion), and Cushing’s disease. Osteonecrosis of the jaw is also a rare complication of bisphosphonate therapy. Evaluation of metabolic bone dysfunction post HCT Bone loss and fractures may present with pain and loss of function, negatively impacting quality of life. The strongest risk factors for metabolic bone dysfunction post HCT are female sex and older age. Among the other factors, corticosteroid use is most significant; however, the dose–effect curve for steroid use in the development of osteoporosis is still not completely clear, and prospective data are sparse, hindering the ability to establish general recommendations for the prophylaxis and treatment of bone mineral loss post HCT. The significance of the changes in BMD after HCT in regard to fracture risk has not been established [32]. Long-term fracture risk is not only determined by BMD; vertebral fracture itself is a potent risk for future fracture, as well as a history of falls and attributes of bone geometry [32]. In children, growth charts, radiographic assessment of bone age, serum levels of IGF-1 and IGFBP3, and GH provocation tests may be used to monitor for the occurrence of growth dysfunction, and possible metabolic bone disease (see the previous section for a further discussion). In patients with demonstrated osteoporosis, it is reasonable to check serum calcium, magnesium, and 1,25-hydroxyvitamin D levels. In adults, DEXA is currently the best and most well-referenced method to diagnose transplant-related bone loss [50]. One study [43] recommends that bone densitometry be performed as part of the pretransplantation evaluation in order to assess the degree of bone loss before transplantation. After transplantation, this group recommended that bone densitometry be repeated yearly for the first 2 years; if decreased BMD is seen, then densitometry is continued annually; otherwise, densitometry can be repeated every 3–5 years. DEXA is also the most common method for measuring BMD in children, now that age-

related normative reference data are currently available [51,52]. Bone turnover can also be measured by markers such as urinary hydroxyproline and serum osteocalcin, but biochemical markers are not widely used outside of research settings. Once osteonecrosis is established, almost any imaging modality will reveal its presence, although magnetic resonance imaging (MRI) has the best sensitivity and specificity. Management of metabolic bone dysfunction post HCT The prevention and treatment of osteoporosis focus on therapeutic modalities that act either to decrease the rate of bone resorption or increase bone formation. The aggressiveness of disease intervention depends upon the degree of osteoporosis and the severity of bone loss, including T-scores and a history of prior fractures. Management should be tailored to each patient’s specific response to therapy, their level of tolerance for adverse effects, and the complicating presence of other concurrent illnesses. For adult patients with osteopenia, calcium and vitamin D supplementation is recommended. Patients should be counseled in regard to lifestyle management (exercise, smoking cessation, and limiting alcohol intake) and instructed on body mechanics and posture in order to minimize the risk of falls. Treatment of osteopenic adults with chronic GVHD or osteoporotic patients should additionally include oral or intravenous bisphosphonates, which have been shown to reduce bone loss in patients of both sexes, including those on glucocorticoid therapy, and/or sex hormone replacement therapy (HRT), as appropriate [53], particularly in menopausal female patients. Patients with Hodgkin’s disease and a history of mantle radiation therapy, however, already have a high risk (35%) for the development of breast cancer [54]; in these cases, selective estrogen response modulators such as raloxifene may be utilized, although venothrombosis is a risk. Osteonecrosis of the jaw is a serious but rare complication of amino bisphosphonate therapy in adults [55]; however, the incidence of osteonecrosis of the jaw may approach 1% when bisphosphonates are administered intravenously and monthly to adults for the management of bone malignancy, and may occur more frequently in this setting after recent dentoalveolar trauma [55,56]. Oral bisphosphonates have been implicated less frequently as a cause of osteonecrosis of the jaw among patients without malignant bone disease, but have the potentially severe side-effect of esophagitis. Intranasal or injectable calcitonin may be employed to alleviate pain related to bone fractures, but it has not proved useful in preventing or treating osteoporosis itself [43]. Intermittent administration of injectable, low-dose, synthetic (recombinant human) PTH or a peptide analogue as anabolic therapy has been shown to produce substantial increases in trabecular bone mass with little loss of (and even gain in) cortical femoral bone, and has reduced the risk of fractures. PTH may be reserved for patients in whom antiresorptive therapy is insufficient in preventing fractures, and may be particularly useful in steroid-induced osteporosis. Patients must be carefully monitored for the occurrence of hypercalcemia and hypercalciuria, however [31]. Calcilytic agents are calcium ion receptor antagonists that may enhance the secretion of PTH, and may be able to replace the use of exogenous PTH; these drugs currently remain in clinical development [57,58]. BMD should be evaluated in children at 3 months after allogeneic HCT, with an initial follow-up DEXA scan at 1 year [41]. Supplementation with calcium, vitamin D, and weight-bearing exercise is recommended, although these measures have not proved effective in preventing osteopenia, osteoporosis or fractures [59,60] when administered individually. As noted above, sex hormone replacement therapy has increased bone mass in both adult women and men, and is recom-

Endocrine Complications Following Hematopoietic Cell Transplantation

mended for hypogonadal adolescents if age-appropriate [61,62]. Bisphosphonate therapy may also be pursued in pediatric patients, as recent evidence seems to suggest that it may improve mean annualized BMD in children and adolescents after HCT by 33%, which is threefold higher than in children receiving calcium and vitamin D alone [41]; however, the effects of various pretreatment regimens were not sufficiently randomized in this study, and the group receiving bisphosphonates also had a higher proportion of patients receiving GH therapy and sex hormone replacement. The long-term effects of bisphosphonate treatment in children remain to be established.

Thyroid dysfunction The thyroid gland is the largest endocrine organ. Through the uptake of iodine and the subsequent production and secretion of triiodothyronine (T3) and thyroxine (T4) hormones, the thyroid normally regulates a number of critical homeostatic activities, including bodily growth and development and temperature, weight, and metabolism; the thyroid also influences gonadal and sexual function. Hypothalamic TRH promotes the synthesis and release of TSH from the anterior pituitary, which in turn regulates thyroid growth and its hormone secretion (Fig. 98.3). Other factors that influence thyroid function are peripheral deiodinases, autoregulation of the gland in relation to iodine supply, T3 receptor activation/suppression, nonthyroidal T3 agonists or antagonists, and inhibitory effects from TSH receptor autoantibodies [64]. Activity of the thyroid is increased in response to cold, glucocorticoids, and TSH, and decreased in response to heat, dopamine, interleukin-1 beta, somatostatin, estrogens, T4, and T3. Glucocorticoid excess, however, impairs the TSH response to TRH, while estrogens augment the TSH response to TRH. Overall, thyroid dysfunction is a common disorder, thought to affect between 1% and 2% of the general population in geographic areas without iodine deficiency [65]. Similarly, thyroid dysfunction of any

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type is the most common endocrine complication after allogeneic and autologous bone marrow transplantation (HCT), occurring in 2–56% of patients [66]. There is existing controversy over which aspects of HCT are most responsible for thyroid dysfunction: the process of HCT itself, the conditioning regimens used prior to HCT or patient-related parameters (Table 98.3 and Fig. 98.4). Radiation therapy involving the head and neck can lead to the occurrence of thyroid dysfunction, via direct damaging effects to the thyroid tissue (primary dysfunction), and to the hypothalamus or pituitary gland, as evidenced by detailed evaluations of the hypothalamic-pituitarythyroid axis [67]. Further, the use of pretransplant cranial or neck irradiation (e.g. for Hodgkin’s lymphoma) is also implicated in the significant increase in rates (37–69%) of thyroid dysfunction post HCT, including frank hypothyroidism, decreased thyroid reserve, hyperthyroidism, thyroiditis, the occurrence of thyroid nodules, and thyroid malignancy [68]. Similarly, the use of TBI or total lymphoid irradiation Table 98.3 Risk factors for thyroid dysfunction following hematopoietic stem cell transplantation (HCT) Total body irradiation Single-dose irradiation Pretransplant cranial or neck irradiation Occurrence of significant euthyroid sick syndrome Pediatric patient age at transplantation, particularly <10 years old Presence of or predisposing factors for thyroid autoantibodies in donor and/ or recipients of HCT

Cold Dopamine, IL-1β, Somatostatin

TRH

hypothalamus

Circulation

Pituitary gland

glucocorticoids g

“free” T4+T3

estrogens T4

T3 TSH

T4+T3 (bound + free)

Fig. 98.3 Neuroendocrine control of thyroid gland function. +, positive feedback; −, negative feedback; IL-1β, interleukin-1β; T3, triiodothyronine; T4, thyroxine; TRH, thyroid-releasing hormone; TSH, thyroid-stimulating hormone. (Adapted from [63], with permission.)

Fig. 98.4 Actuarial risk of thyroid dysfunction in 150 patients after bone marrow transplantation (BMT) and the effect of total body irradiation (TBI). (a) Actuarial risk for the overall cohort. (b) Actuarial risk according to type and dose of radiation administered. RT, radiotherapy; TLI, total lymphoid radiation; TSH, thyroid-stimulating hormone. Tick marks indicate follow-up. (Reproduced from [67], with permission.)

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Fig. 98.5 (a) Life-table estimate of the proportion of patients with normal thyroid stimulating hormone (TSH) levels following bone marrow transplantation (BMT), according to disease. (b) Life-table estimate of the proportion of patients with normal TSH levels following BMT, according to disease and graft-versus-host disease (GVHD) prophylaxis. ALL, acute lymphoblastic leukemia; ANLL, acute nonlymphoblastic leukemia; MAP, methotrexate, antithymocyte globulin, and prednisone; MTX, methotrexate; SAA; severe aplastic anemia. (Reproduced from [77], with permission.)

(TLI) during the preparation regimen of HCT is associated with a greatly increased risk of some types of thyroid dysfunction occurring in the months to years after transplantation [66,69,70], with one study noting an incidence as high as 57% at 3 months after transplant, compared with the 14% incidence of thyroid dysfunction observed at the same time point in patients who did not receive TBI or TLI prior to HCT [71]. The incidence of thyroid dysfunction is also higher in patients given singledose irradiation (23–73%), rather than fractionated doses (10–28%) [67,68,72–76]. In one study, the prevalence of hypothyroidism appeared to be higher among children receiving HCT for severe aplastic anemia than for leukemia, although this difference appeared to be the result of the type of GVHD prophylaxis used, rather than the disease itself (Fig. 98.5). Patients with severe aplastic anemia who received methotrexate alone appeared to be at increased risk for developing thyroid dysfunction, but the reasons for this are unclear. It is possible that additional immunosuppression with methotrexate, antithymocyte globulin, and prednisone suppressed immunologic injury of the thyroid gland, while methotrexate alone following TLI may be comparatively inadequate to prevent thyroid injury. On the other hand, patients with acute leukemia, having received greater immunosuppression from TBI and previous long-term chemotherapy, may not require additional immunosuppression to avoid this type of radiation-induced “immune” thyroid injury [77]. Overall, the most common thyroid-related complications post HCT are either functional in type (hypothyroidism due to destruction of thyroid tissue or autoimmune thyroid disease [AITD]), or present as mass lesions of the thyroid (benign nodular goiter or malignant carcinoma) (Table 98.4). The prevalence of thyroid abnormalities is higher in allogeneic HCT recipients than in autologous HCT recipients [79], even in the absence of TBI [71,79–81]. In fact, Tourbert et al. [71] noted a remarkably high incidence of both frank hypothyroidism (10–15%) and euthyroid sick syndrome (ETS) (48% at 3 months after treatment) in a patient group who had received only a BU- or CY-based conditioning regimen without TBI, and did not have a history of previous radiotherapy (Table 98.5). ETS is actually the most commonly observed endocrine dysfunction in the 3-month period after HCT, and is typically characterized by low free triiodothyronine (FT3), free thyroxine (FT4) or both, along with normal or low TSH, without pre-existing hypothalamic-pituitary and thyroid gland dysfunction (Table 98.6). A short-term fall in thyroid hormone levels after HCT generally reflects ETS, with increased conversion of T4 to reverse T3 (which is metabolically inactive), rather than intrinsic change in the hypothalamic-pituitary-thyroid axis [82]. Thus, patients with ETS-related thyroid function abnormalities are not considered to have hypothyroidism. Notably, however, the occurrence of significant post-HCT ETS, as defined by a precipitous drop in serum total

Table 98.4 Late consequences in children treated more than 10 years before with two different doses and types of fractionated total body irradiation Fractionated total body irradiation

Late effects

990 cGY (n = 25) n (%)

1200 cGy (n = 17) n (%)

Total (n = 42)

p

Hypothyroidism Thyroid nodules Thyroid carcinoma

4 (16) 20 (80) 5 (20)

1 (6) 4* (27) 1 (6)

5 (12) 24 (57) 6 (14)

0.63 0.002 0.37

* Two patients were excluded because they were never evaluated by ultrasound. Adapted with permission from Faraci et al. [78].

T4 levels, is predictive of lower survival rates, compared with patients who do not develop ETS (34.5% versus 96.2%; p < 0.0001) (Fig. 98.6) [68,71]. If total T4 levels fall below 4 μg/dL, the probability of death is about 50%; with serum T4 levels below 2 μg/dL, the probability of death reaches about 80% [65]. Reasons for poor prognosis associated with ETS remain unclear, but it has been suggested that ETS may be a reflection of global hormonal dysregulation within the context of severely deranged homeostasis, as well as the fact that the occurrence of ETS may be an independent prognosticator in itself [71]. While there is existing controversy as to whether or not the age at transplantation is associated with an increased risk of thyroid dysfunction, several compelling studies cite an age of less than 10 years as being a significant risk factor, while other studies have found no association [68,74,76,83]. Functional derangement of the thyroid post HCT Hypothyroidism of any type is the most frequent type of clinical or laboratory thyroid dysfunction seen in long-term survivors of HCT [76]; however, the incidence and time of onset for hypothyroidism requiring therapy is highly variable, depending on the type of pretransplant conditioning therapy applied [20,84]. The median time to the diagnosis of hypothyroidism after HCT is nearly 4 years [20], while the cumulative incidence of overt hypothyroidism in 308 patients treated with locoregional radiotherapy for nonthyroid head and neck cancer was 20% at 5 years and 27% at 10 years [85]. Yet, when hypothyroidism is defined as an elevation in serum TSH, the cumulative incidence is even higher, with 48% at 5 years and 67% at 8 years [86]. Other reports more modestly estimate the occurrence of subclinical hypothyroidism after allogeneic HCT as ranging from 7% to 15% [20,74].

Endocrine Complications Following Hematopoietic Cell Transplantation

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Table 98.5 Post-bone marrow transplantation hypothyroidism following a cytoreductive therapy regimen without the use of irradiation Reference

No. of patients

Median follow-up (years)

Chemotherapy

No. of hypothyroidisms

Sklar et al. (1982) Urban et al. (1988) Sanders (1991) Galimberti et al. (1991) Liesner et al. (1994) Boulad et al. (1995) Tourbert et al. (1997)

4 10 11 58 26 7 77

2 3.1 1 3.2 4 6.2 2.6

CY or CY + 6-mercaptopurine or CY + procarbazine CY or CY + BU or melphalan or BACT CY + BU CY + BU CY + BU CY or CY + BU CY + BU or BU + Alkeran or CY + ATG

0 0 0 0 0 1 11

ATG, antithymocyte globulin; BACT, BCNU, cytarabine, cyclophosphamide, and 6-thioguanine; BU, busulfan; CY, cyclophosphamide. Reprinted with permission from Tourbert et al. [71].

Table 98.6 Thyroid function before and after bone marrow transplantation (BMT) (3 and 14 months)

Before BMT1

After BMT (3 mos)2

After BMT (14 mos)3

Normal PTI ETS Normal PTI ETS Normal PTI ETS

n

FT3 (pmol/L) [Normal 3.6–9.4]

FT4 (pmol/L) [Normal 8.5–22]

TSH (mIU/L) [Normal 0.2–4]

59 10 6 23 6 29 34 5 9

6.7 [3.7–10.9] 6.6 [3.5–8.9] 2.6 [1.2–3.1] 5.7 [4.0–8.7] 5.2 [1.2–7.5] 2.5 [1.2–3.5] 6.5 [4.1–10] 5.8 [4.3–7.4] 2.6 [1.2–3.5]

15.5 [9.7–21] 13.8 [9.0–19.7] 11.1 [7.4–13] 12.7 [8.9–20.0] 10.5 [4.2–13.9] 8.8 [3.7–14.6] 14.2 [10.0–19.2] 13.0 [10.3–16.4] 8.6 [4.2–14.1]

1.88 [0.3–3.9] 5.9 [4.0–11.7] 1.15 [0.1–2.0] 2.00 [0.5–3.9] 8.73 [4.4–17.0] 1.23 [0.03–4.0] 1.81 [0.55–3.3] 6.12 [4.2–11.0] 1.78 [0.1–3.2]

Biochemical thyroid hormone levels are given: mean (range). To convert SI units to gravimetric units, divide by following conversion factors: gravimetric conversion factor for TSH, 1.0 = mU/L; Gravimetric conversion factor for FT3, 0.0154 = pg/dL; gravimetric conversion factor for FT4, 12.87 = ng/dl. ETS, euthyroid sick syndrome, defined as FT3 <3.6 pmol/L and/or FT4 <8.5 pmol/L and TSH <4 mIU/L; FT3, free serum triiodothyronine; FT4, free serum thyroxine; PTI, peripheral thyroid insufficiency, defined if TSH >4 mIU/L; TSH, ultrasensitive serum thyrotropin level. 1 Total = 77 (not done = 2); death (0–2 months) = 8. 2 Total = 69 (not done = 8); death (3–14 months) = 18. The mechanisms by which hypothyroidism may develop post HCT 3 Total = 51 (not done = 3); death (14–24 months) = 6. are unclear [80]. As with thyroid dysfunction in general, a history of Reprinted with permission from Tourbert et al. [71].

Fig. 98.6 Actuarial survival curves at 3 months: normal thyroid profile compared to peripheral thyroid insufficiency and euthyroid sick syndrome. (Reproduced from [71], with permission.)

radiation exposure – particularly TBI – seems to be a significant risk factor [76]. Single-dose ablative TBI is associated with a 50% incidence of overt hypothyroidism, whereas fractionated TBI is associated with about 15%, compared with pretreatment regimens with BU and CY treatment alone, which represent approximately 11% of cases of hypothyroidism [20]. The incidence of primary, symptomatic hypothyroidism after HCT without the use of TBI during the pretransplant conditioning regimen has been reported to be around 10–15% (Table 98.5) [71,80]. Because hypothyroidism can occur with such relative frequency in HCT recipients who have not received radiation prior to transplant, nonradiotherapy-related mechanisms for post-HCT hypothyroidism have been proposed. Several cytokines regulate thyroid function, and these may be involved in the pathogenesis of thyroid disorders, including ETS [87]. Additionally, the effect of conditioning chemotherapy and the graft-versus-host effect of infused lymphocytes may trigger human leukocyte antigen (HLA)-related AITD. The de novo development of autoimmune thyroid disorders, such as Hashimoto’s thyroiditis or Graves’ disease, has been reported in the presence of chronic GVHD or other pre-existing autoimmune dysfunction [88–90], although antithyroid autoantibodies occur more rarely than other autoantibodies with chronic GVHD [91]. While a limited number of case reports note the transfer of AITD from donor to recipient without any autoimmune phenomena by the mechanism of adoptive autoimmunity (i.e. the adoptive transfer of even a small number of pathogenic lymphoid clones) following allogeneic HCT [92], this type of adoptive transfer is likely a minor cause of autoimmune

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hypothyroidism, as most cases of post-HCT autoimmunity more likely result from an immunologic dysregulation (or “immunologic chaos”) [93]. Although it is also possible for the remission of pre-existing autoimmune disease to occur after transplantation by the reverse mechanism, this has not been reported with respect to autoimmune thyroiditis, except for one case of Graves’ disease [94]. It has been suggested that up to 3% of the post-HCT population may suffer from autoimmune thyroiditis after 5 years of follow-up, meaning that the 5-year actuarial rates for autoimmune thyroiditis were 2.9% and 4% for allogeneic and autologous transplantation, respectively, which is very similar to the 2–4% lifetime risk of de novo autoimmune thyroiditis, which favors a femaleto-male ratio of 3 : 1 [88], although the true incidence of clinically significant autoimmune thyroiditis after HCT remains unclear [95]. Presentation of functional thyroid derangement post HCT The diagnosis of functional thyroid disease after HCT depends on obtaining a careful history, a thorough search for the physical signs of hypothyroidism or thyrotoxicosis, and a detailed evaluation of laboratory results. While the onset of hypothyroidism may be insidious in the adult general population, after HCT disease development may be rapid, with frank manifestations of hypothyroidism present by 6 weeks, and myxedema by 3 months [96]. The signs and symptoms of mild hypothyroidism may be quite subtle, variable, and nonspecific, such as dry/coarse skin, cold intolerance, periorbital swelling, and a delayed ankle reflex relaxation phase. Unusual symptoms at presentation may include: neurasthenia with muscle cramps, paresthesias, and weaknesses; refractory anemia; disturbances in reproductive function, including infertility, delayed puberty or menorrhagia; idiopathic edema or pleuropericardial effusions; retarded growth; obstipation; chronic rhinitis or hoarseness due to edema of the nasal mucosa or vocal cords; and severe depression progressing to emotional instability and even frank paranoid psychosis. Bone disorders related to hypothyroidism in children may include impairment of linear growth and waddling gait due to maldevelopment of the femoral epiphyses. Radiologic evidence of epiphyseal dysgenesis is virtually pathognomonic for hypothyroidism in infancy and childhood [96]. Although the clinical picture of myxedema is usually clear in nonHCT recipients, it can often be difficult to observe in patients with GVHD of the skin post HCT. Hyperthyroidism of Graves’ disease may present with one or more of the following features: (1) thyrotoxicosis, (2) goiter, (3) ophthalmopathy (exophthalmos), and (4) dermopathy (pretibial myxedema) [64]. The clinical signs of hyperthyroidism in younger individuals include palpitations, nervousness, easy fatigability, hyperkinesias, diarrhea, excessive sweating, intolerance of heat, and preference for cold temperatures, often with marked weight loss without a loss of appetite. Commonly seen manifestations are thyroid enlargement, thyrotoxic eye signs (e.g. ophthalmopathy), and mild tachycardia. Muscle weakness and loss of muscle mass may be severe. Rarely, thyroid dermopathy (thickening of the skin) and osteopathy (subperiosteal inflammation), particularly of the metacarpal and metatarsal bones, may be seen. In children, rapid growth with accelerated bone maturation occurs. In patients over 60 years, cardiovascular and myopathic signs predominate, with the most common complaints being palpitation, dyspnea on exertion, tremor, nervousness, and weight loss [64]. Evaluation of functional thyroid derangement post HCT The diagnostic laboratory workup of hypo- or hyperthyroidism usually requires one or more of the following values: FT3, FT4, FT4I (a calculation that can distinguish the level of T4 present compared with thyroid binding globulin), and TSH (Fig. 98.7). The combination of a low serum FT4 or FT4I and an elevated serum TSH is diagnostic of primary hypo-

thyroidism, whereas the combination of elevated T4 and suppressed TSH confirms the diagnosis of hyperthyroidism. Suppression of both FT4 and TSH, in the absence of ETS, suggests secondary hypothyroidism related to pituitary or hypothalamic deficiencies. While both thyroglobulin and thyroid peroxidase autoantibodies are present in Graves’ disease and Hashimoto’s thyroiditis, TSH receptor antibody is specific for Graves’ disease. A soft, enlarged thyroid gland with high blood flow (bruit) is typical of Graves’ disease, and is usually accompanied by exophthalmos and signs of hypermetabolism. A palpable or enlarged thyroid gland that is firm in consistency and coarse in texture, and a positive test for thyroglobulin and thyroid peroxidase autoantibodies would suggest underlying Hashimoto’s thyroiditis. Radionuclide uptake may also help differentiate Graves’ disease from Hashimoto’s thyroiditis, as increased radioiodine uptake at 6 and 24 hours is the hallmark of Graves’ disease, whereas patchy uptake and/or low uptake is associated with Hashimoto’s thyroiditis. Management of functional thyroid derangement post HCT The management of hypothyroidism focuses on ensuring that patients promptly receive appropriate thyroid hormone replacement therapy once diagnosed, and that their responses are monitored at least annually for the long term. The timely replacement of thyroid hormones in children with even subclinical hypothyroidism is especially important, as a number of recent animal studies suggest that lowering a previously raised TSH level into the normal range in a child who has received thyroid irradiation may reduce the risk of future thyroid malignancy [76]. Replacement doses of levothyroxine in adults range from 0.05 to 0.2 mg/day, with a mean of 0.125 mg/day, or 1.7 μg/kg/day [64]. Dosage varies according to patient’s age and body weight. In the elderly and in patients prone to cardiac problems, hormone replacement should be initiated at a low dosage (0.025 mg/day), and then should be increased at 4–6-week intervals based on FT4 and TSH measurements up to a mean of approximately 1.6 μg/kg/day. Young children require a surprisingly high dose of levothyroxine, compared with adults (e.g. 4–5 μg/kg/day in children aged 6–10 years, versus 1–2 μg/kg/day in adults), and initial dosage may be give at one-half the estimated final dose requirement for 4–6 weeks, and then adjusted based on FT4 and TSH per the adult schedule [64]. The major toxic symptoms of levothyroxine overdosage are those of hyperthyroidism, particularly cardiac complications (arrhythmia) and osteoporosis, in postmenopausal women. After full replacement of T4 using levothyroxine, the addition of T3 in a low dosage may be beneficial in some patients who continue to have mood or memory problems [97]. Once a stable dosage is achieved, annual monitoring of the TSH level is usually sufficient. More frequent levels may be required in older patients. Because of some evidence of spontaneous recovery, however, intermittent trials off HRT may be considered [68]. Hyperthyroidism/Graves’ disease can be treated with radioactive iodine, antithyroid drugs (propylthiouracil or methimazole) or surgery (subtotal thyroidectomy), but in the United States, radioactive sodium iodide (iodine-131 [131I]) in a dosage of 80–150 μCi/g of thyroid weight estimated on the basis of physical examination, and 123I scan is the treatment of choice in patients without contraindications. The major complication of radioactive iodine is hypothyroidism, which ultimately develops in approximately 80% of patients who are adequately treated, and hormone replacement should begin as soon as hypothyroidism is confirmed [64]. Patients with small, asymptomatic goiters can be monitored with clinical examination and periodic ultrasound evaluations. Mass lesions of the thyroid post HCT In general, benign nodular goiter (thyroid nodules) is very common, with an estimated incidence of approximately 4% of the adult population in

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Fig. 98.7 Algorithm for the evaluation of the patient suspected of hypothyroidism or hyperthyroidism following hematopoietic cell transplantation. FT3, free triiodothyronine; FT4, free thyroxine; I-123, radioactive isotope of iodine (gamma emitter); I-131, radioactive isotope of iodine (beta and gamma emitter); N, normal; reverse T3, isomer of T3, which binds to T3 receptors and blocks them; Tc-99, Technetium-99; TPO, thyroperoxidase; TSH, thyroid-stimulating hormone; TSHr antibody, thyroid-stimulating hormone receptor antibody.

the United States. Females are more affected than males in a ratio of 4 : 1, but these numbers vary widely according to regional factors such as iodine intake, and individual factors such as smoking habits, age and sex distribution, and genetics. Iodine deficiency increases the prevalence of goiter. In children, the approximate incidence is less than 1%, but this increases with age, reaching 1.5% between 11 and 18 years, and about 5% in persons over the age of 60 years. Elderly women are the most likely candidates for thyroid nodules. In comparison to the general population, the incidence of thyroid nodules in survivors of HCT is quite high. In one study, 26 of 95 (27.3%) patients who had received high-dose radiotherapy for childhood malignancies had palpable thyroid nodules 5–34 years after therapy [98]. Further, the incidence of nodules found in survivors of Hodgkin’s lymphoma varies from 2% to 65%, depending on the length of follow-up and method of evaluation [99]. Because these populations are screened more often and more carefully, however, the true relative risk cannot be ascertained easily. In contrast to thyroid nodules, primary thyroid cancer is a relatively uncommon occurrence in the general adult population, with a prevalence

of 0.004% (four cases per 10,000 in the Third National Cancer Survey) [64]. Primary thyroid cancer in children is also considered to be a rare event (three to five cases per 1,000,000 per year), constituting 2.9% of all childhood cancers and 3–5% of all thyroid cancers [100]. Although the percentages may seem small, these numbers are still significant, as there are approximately 20,000 new cases of thyroid cancer in the United States each year, and differentiated thyroid cancer claims the lives of approximately 1500 individuals annually [101]. Because thyroid carcinomas often require decades to manifest, the incidence of this disease increases with age, peaking in both males and females late in life – 65–99 years and 50–54 years, respectively; there are just three to five childhood cases of thyroid malignancy per 1,000,000 per year, comprising 2.9% of all pediatric malignancies [100], and 7.5– 10% of cancer in 15–24-year-olds. Thyroid carcinoma also tends to be more aggressive in older individuals, and, as with thyroid nodules, females tend to be affected three times as frequently as males. In fact, thyroid cancer is the eighth most common cancer in women. There are several types of thyroid cancer, but only two of these, known as well-differentiated thyroid cancers, comprise 80–90% of all

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cases: papillary carcinoma and follicular carcinoma. Papillary carcinoma is the most common type of thyroid malignancy, being responsible for approximately 70% of all thyroid cancer cases, and about 85% of all cancers that result from radiation exposure. Follicular carcinomas are the second most common thyroid cancers, comprising approximately 15% of all cases. Follicular carcinoma is considered more aggressive than the papillary type, and is also less common in children. In contrast to papillary cancer, the follicular type is rarely associated with radiation therapy. Although the majority of thyroid nodules are asymptomatic, the main objective in their evaluation is to exclude malignancy. Fortunately, only 5–10% of all thyroid nodules are cancerous, and these tend to be of a well-differentiated type (as noted above). As with thyroid nodules, patients who underwent HCT during childhood harbor an increased risk of developing secondary thyroid carcinoma compared with the general healthy population (0–29 years) [83,100], with an overall reported incidence of 0.2% of children after HCT [83]. Factors that further increase the likelihood of malignancy are: older age (about 45%), male sex (because nodules are less common in men, any given nodule in a male patient has a higher probability of being malignant), and family history of thyroid cancer (Table 98.7). A history of radiation exposure to the thyroid is significant, as irradiation causes subsequent hyperplasia, and thus a greater likelihood for the development of nodules and malignancies [66]. The relative risk of inducing thyroid cancer is estimated at 0.6–14.9 after irradiation, but varies depending on several parameters [102]; in particular, the use of TBI increases the relative risk to 3.6, versus 1.0 without TBI [83]. The irradiation regimens used in HCT range from as low as 2–6 Gy up to 8–14 Gy in high-dose therapy. Table 98.7 Clinical presentation of solitary thyroid nodule (>1 cm diameter) suspicious for malignancy Factors increasing suspicion of thyroid carcinoma: ❏ Age <15 years or >45 years ❏ Male sex ❏ Nodule >4 cm in diameter ❏ History of radiation exposure ❏ History of diseases associated with thyroid cancer – Pheochromocytoma – Hyperparathyroidism – Gardner’s syndrome – Familial adenomatous polyposis – Carney complex – Cowden’s syndrome ❏ Suspicious criteria by ultrasound – Central hypervascularity – Irregular border – Microcalcification ❏ Incidental focal positron-emission tomography-positive lesion in the thyroid Highly suspicious for thyroid carcinoma if present: ❏ Rapid nodule growth (over weeks to months) ❏ Very firm nodule ❏ Fixed nodule (to adjacent structures) ❏ Family history of thyroid cancer ❏ Vocal cord paralysis ❏ Enlarged regional lymph nodes (particularly low anterior cervical) ❏ Symptoms of invasion into neck structures (architectural distortion, e.g. tracheal deviation, muscle involvement, tumor fixation, difficulty swallowing, blockage of the internal carotid artery)

In comparison, as little as 6.5 cGy (1 cGy = 10−2 Gy = 1 rad) to the thyroid gland (e.g. during radiation treatment of tinea capitis) reportedly caused cancer in 0.11% of exposed children, versus 0.02% in sibling controls [103]. Increased doses of radiation exposure appear to correlate with a higher incidence of thyroid cancer [64], while exposure to extremely high doses of radiation (as much as 10,000 cGy) was rarely associated with the development of thyroid malignancy. This latter phenomenon is likely due to the fact that the thyroid gland is largely destroyed by such high doses of radiation, which usually results in hypothyroidism [64]. Notably, the presence or absence of GVHD does not appear to have a significant effect on the development of thyroid carcinoma post HCT [83], although some studies have noted a higher relative risk in the presence of chronic GVHD [100]. Another risk factor for the development of secondary thyroid cancer after HCT is a younger age at the time of transplantation, with children aged 0–9 years having a relative risk of 12.2 compared with 1.0 for those between the ages of 10 and 16 years [83]. Evaluation of mass lesions of the thyroid post HCT Thyroid cancer initially presents as a nodule or lump in the thyroid in 95% of cases [64]. Occasionally, and particularly in children, enlarged cervical lymph nodes are the first sign, although careful examination will often reveal a palpable thyroid nodule as a small primary focus. Rarely, distant metastasis in lung or bone is the first sign of thyroid cancer. The main difficulty in the screening of a patient for thyroid neoplasia is that nodules are often not clinically evident. In geographic areas where dietary iodine is sufficient, thyroid nodules are palpable in only 1% of men and 5% of women. Even high-resolution ultrasound of the neck identifies just 19–67% of all people with thyroid nodules. Ultrasound examination, however, can provide accurate size measurement of nodules, help determine if a nodule is solid/complex versus a fluid-filled cyst, and may predict the possibility of malignancy based on nodule tissue characteristics (blood flow patterns, margins, calcifications, echogenicity, etc.). All patients who have received irradiation, including TBI, should be followed for life with physical evaluations 6 months after HCT, and then annually thereafter [64,81], including careful examination of the neck for goiter or nodules, and thyroid function studies (e.g. FT4 with or without TSH to rule out hypothyroidism) 6 months after HCT and annually thereafter [81]. Notably, serum levels of thyroid hormones do not distinguish benign from malignant mass lesions of the thyroid [66], but may help to establish the presence of a functional thyroid lesion (single hyperfunctioning thyroid nodule, also known as Plummer’s disease), or may reflect hypothyroidism in association with multinodular thyroid goiter or Hashimoto’s thyroiditis. If a thyroid nodule more than 1.0 cm in diameter is found, the work-up to rule out malignancy should include a TSH level, and fine-needle aspiration of the nodule for cytolologic assessment and thyroglobulin measurement in the needle washout (Fig. 98.8). Fine-needle aspiration of thyroid nodules or suspicious neck lymph nodes can be done blindly or under the guidance of ultrasound. In the case of nodules with indeterminate cytology results, however, a radionuclide thyroid scan can prove useful, as hyperfunctional nodules are almost always benign and can be managed medically with radioactive iodine or surgery. Thus, a radionuclide thyroid scan can assist in evaluating regional uptake or function within the thyroid gland, and is capable of localizing the presence of ectopic thyroid tissue. The radioactive tracers used for thyroid scans are 131I or 123I (isotopes of iodine), or technetium-99m (99m Tc). Technetium scanning may be preferred at some diagnostic centers because it can be administered just 2 hours

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Fig. 98.8 Algorithm for the evaluation (eval) of the patient presenting with thyroid nodule following hematopoietic cell transplantation. FNA, fine-needle aspiration; FT4I, free thyroxine index; TFTs, thyroid function tests; TSH, thyroid-stimulating hormone; US, ultrasound.

before the study. 123I tends to be used for thyroid uptake tests and routine thyroid scans, while 131I is reserved for follow-up evaluations of known thyroid cancer patients, due to its higher radiation burden. A normal scan indicates that the iodine uptake is similar in both lobes of the thyroid gland. Nodules may be classified as “cold” (decreased uptake), “warm” (uptake similar to that of surrounding tissue) or “hot” (increased uptake). While a large proportion of thyroid nodules may be cold on radionuclide scan, only 5–15% of these are malignant in the

general population [104]; however, data from several large series [64] suggest that the incidence of cancer in a patient with a history of therapeutic radiation of the head, neck or chest who presents with a solitary cold thyroid nodule is as great as 50%. Although MRI is not a primary diagnostic modality in the assessment of patients with thyroid nodules, nodules are often found incidentally during MRI of the neck for nonendocrine indications. The same is true of computed tomography.

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Metabolic and electrolyte abnormalities Patients undergoing HCT are prone to the development of a wide variety of metabolic abnormalities, most of which occur during the early post-transplant period [105] and may vary in severity from mild to life-threatening [106]. One study of 311 patients [106] reviewed the incidence of several metabolic/electrolyte abnormalities following HCT, demonstrating that a high number (269, 86.5%) of patients developed at least one severe metabolic abnormality within 100 days after allogeneic HCT. In all, 196 patients (63.0%) experienced one to three different metabolic abnormalities, whereas 73 (23.5%) had four to seven abnormalities. The most common abnormalities experienced within 100 days after HCT were hyponatremia (185, 59.5%), hypokalemia (149, 47.9%), hypophosphatemia (105, 33.8%), and hypomagnesemia (52, 16.7%). Less frequently encountered abnormalities were hyperkalemia (25, 8.0%), hyperuricemia (22, 7.1%), hypernatremia (13, 4.2%), and hypermagnesemia (9, 2.9%). Calcium abnormalities were found to be relatively rare, with hypercalcemia observed in one patient (0.3%) and hypocalcemia in one other patient (0.3%). In addition to these electrolyte abnormalities, derangements in glucose and lipid homeostasis were observed, with hyperglycemia being most common (138, 44.4%), followed by hypoglycemia (12, 3.9%), and hypercholesterolemia (3, 1.0%). Induction regimens, immunosuppressive drugs, GVHD, and/or total parenteral nutrition supportive therapy of patients undergoing or following HCT are among the factors contributing to the development of metabolic and electrolyte disturbances seen. For example, the use of cyclosporine and tacrolimus in the prophylaxis and treatment of GVHD has been associated with hyperkalemia [107], hyperglycemia [108], hypomagnesemia [109], and hypertriglyceridemia [110] in HCT recipients. As mentioned elsewhere in this chapter, glucocorticoid therapy may also cause hyperglycemia. GVHD-related gastrointestinal complications may also further the development of metabolic abnormalities. The subsequent occurrence of severe inflammatory or infective processes may lead to insulin resistance by increasing levels of tumor necrosis factor-alpha [111]. Long-term treatment with aminoglycoside antibiotics and amphotericin B with concomitant hypomagnesemia and vomiting can cause renal potassium wasting and hypokalemia [106]. Pentamidine, used for the prevention or treatment of pneumocystis pneumonitis, may cause acute release of insulin due to pancreatic β-cell damage, resulting in severe hypoglycemia followed by hyperglycemia [112]. There was also a case report of hypoglycemia induced by insulin receptor antibodies following HCT [113]. Increased phosphate uptake by replicating neutrophils during the periengraftment period may additionally lead to hypophosphatemia in HCT recipients [114]. The occurrence of severe metabolic abnormalities following HCT tends to be associated with inferior clinical outcomes, and the impact of these metabolic disorders may be independent of other complications, including GVHD and hepatic sinusoidal obstructive syndrome (formerly known as veno-occlusive disease) [106], suggesting that metabolic abnormalities that develop during the early post-transplant period may serve as prognostic indicators for patients undergoing allogeneic HCT. Thus, metabolic parameters should be closely monitored in patients treated with HCT. In the following section, only HCT-associated hyponatremia will be discussed in detail. The diagnosis and treatment of other electrolyte abnormalities are usually more straightforward and thus will not be addressed further. Abnormal water metabolism Serum osmolality is regulated primarily by hypothalamic osmoreceptors that govern secretion of antidiuretic hormone (ADH, also known as

arginine vasopressin [AVP] or vasopressin) by cells of the paraventricular and supraoptic nuclei. The hypothalamic thirst center helps to regulate the intake of free water by stimulating drinking behavior when serum osmolarity increases above the normal range (280–300 mOsm/ kg), and also increases secretion of ADH. ADH increases free water reabsorption from the urine, yielding urine of low volume and relatively high osmolarity in order to return serum osmolarity to normal levels. ADH is also secreted in response to hypovolemia, pain, fear, nausea, and hypoxia. Aldosterone, synthesized by the adrenal cortex, is regulated primarily by serum potassium, but also is released in response to hypovolemia (e.g. hemorrhage or dehydration) through the renin–angiotensin– aldosterone axis. Aldosterone causes absorption of sodium at the distal renal tubule, obligating free water retention and assisting in the correction of hypovolemia. The healthy kidney regulates sodium balance independently of ADH or aldosterone by varying the degree of sodium absorption at the distal tubule; however, hypovolemic states prompt increases in sodium absorption in the proximal tubule. Increases in vascular volume suppress tubular sodium reabsorption, resulting in natriuresis, and help to restore normal vascular volume [115]. Hyponatremia, defined as a decrease in serum sodium concentration of 135 mmol/L or less, is a common electrolyte disorder in hospitalized patients, and is among the metabolic disturbances frequently encountered in oncology. Hyponatremia may be classified as being isotonic (factitious), hypertonic or hypotonic. Isotonic hyponatremia is encountered in the setting of hyperlipidemia and/or hyperproteinemia, which decreases the proportion of plasma volume occupied by water. Although the sodium concentration in the total plasma is low, the sodium concentration relative to plasma water will be normal. Hypertonic hyponatremia results from the presence of abnormally high amounts of osmotically effective solutes other than sodium (e.g. glucose), and hyponatremia results from the movement of water from the intracellular to the extracellular fluid compartments. Hypotonic hyponatremia results from depletion of total body sodium in excess of concurrent body water losses, or from dilution of total body sodium by increases in total body water. Hypotonic hyponatremia may be classified into three types: hypovolemic, hypervolemic, and euvolemic. Hypovolemic hyponatremia is associated with volume contraction and nonosmotic stimulation of ADH release, and may be seen with disorders of renal sodium handling (e.g. diuretic use, mineralocorticoid deficiency or other salt-wasting syndromes; see below). Hypervolemic hyponatremia is characterized by paradoxical retention of sodium ions and water despite excess of total body sodium and water, likely due to increased ADH as the result of perceived hypoperfusion by arterial baroreceptors, and associated disorders include congestive heart failure, cirrhosis of the liver with ascites, and nephrotic syndrome. Euvolemic hyponatremia comprises the most heterogeneous group of disorders, which are difficult to unify pathologically, but includes the syndrome of inappropriate ADH (SIADH) secretion, hypothyroidism, adrenal insufficiency, psychogenic polydipsia, postoperative hyponatremia, and hyponatremia following transurethral resection of the prostate. Current data concerning the incidence of hyponatremia following HCT are limited [116], although one study reported the incidence to be as high as 40% [117]. Risk factors for hyponatremia following HCT include the development of SIADH, hypothyroidism, adrenal insufficiency subsequent to discontinuation of glucocorticoid use, chemotherapy, treatment-induced nausea and vomiting, overhydration, pain, narcotic drugs, diarrhea, infection, renal failure, and physical and emotional stress. Diarrhea, in particular, is a relatively frequent complication of HCT because of mucosal damage, infection, and acute GVHD, and hyponatremia due to diarrhea is therefore frequently observed following HCT [106].

Endocrine Complications Following Hematopoietic Cell Transplantation

SIADH is another important, common cause of hyponatremia following HCT, and may result from cytotoxicity to paraventricular and supraoptic neurons (see below) as a side-effect of conditioning therapy, especially with CY [106]. The presence of abnormal secretory stimuli, such as intrathoracic infection or the use of positive pressure ventilation, may also cause SIADH [118]. Inappropriate release of ADH leads to retention of free water and hyponatremia. Thus, SIADH is characterized by hyponatremia, low serum osmolality, and an inappropriately high urine osmolality in the absence of diuretics, heart failure, cirrhosis, adrenal insufficiency, and hypothyroidism. The rate of occurrence of SIADH, which was found in 16 (11.4%) of 140 HCT patients in one study, was unexpectedly high [117]. SIADH has also been reported to be associated with several hematologic diseases, such as non-Hodgkin’s lymphoma and Hodgkin’s lymphoma [119], although the most common cause of SIADH in non-Hodgkin’s lymphoma is thought to be the use of either vinca alkaloid-containing chemotherapy (see below) or direct invasion into the pituitary gland [117]. The use of an HLA-mismatched donor may be a risk factor for SIADH [117]. Younger age at transplantation may be an additional risk factor for the development of SIADH [117]; because the water content of young children is relatively higher than that of adults, electrolyte imbalances – and, correspondingly SIADH – may occur more readily in a pediatric patient population. Further, the consequences of acute hyponatremia are more serious in young children. SIADH-associated hyponatremia has been observed with the use of various antineoplastic agents due to chemotherapy-induced hypothalamic or posterior pituitary damage. Vinca alkaloids (vincristine and vinblastine) are thought to induce SIADH due to paraventricular or supraoptic cell microtubular damage [120]. Vincristine additionally causes gastrointestinal sodium and water loss, resulting in appropriate ADH secretion [121]. When vincristine is used in combination with CY, doxorubicin, methotrexate, and prednisolone (CHOP-M), the incidence of hyponatremia is about 17% [122]. In another series in which highdose vinblastine, cisplatin, and bleomycin therapy were combined, eight of 12 patients developed hyponatremia and hypo-osmolality [123]. High-dose CY therapy causes SIADH via a primarily direct effect on the hypothalamus [124]. Post mortem examination of a case of fatal CY-induced (1800 mg/m2) hyponatremia revealed infundibular necrosis, decreased intraaxonal secretory granules, and depletion of posterior pituitary ADH [125]. Other mechanisms have also been invoked: in a small series of 19 chemotherapy courses with lower dosages of CY, there was development of hyponatremia, plasma hypotonicity, and urinary hypertonicity without an increase in plasma ADH levels [126]. This suggests that damage to the renal tubules and resultant defects of salt and water transport is the major cause of hyponatremia associated with low-dose therapy. The mechanism of cisplatin-induced hyponatremia is unclear, but it has been suggested that renal toxic effects of cisplatin, rather than a direct effect on ADH secretion, are the major factor [127]. This drug likely induces renal salt wasting, leading to hyponatremia, hypoosmolality, and elevated urinary sodium and urinary osmolality. Such an SIADH-like syndrome is difficult to distinguish from true SIADH when signs and symptoms of fluid volume depletion are subtle or absent. It is not clear whether melphalan has a direct effect on the development of hyponatremia or merely potentiates the effects of other agents. Melphalan-induced hyponatremia and urinary sodium loss, thought to be SIADH, has been reported in two patients who received high-dose (2 mg/kg) melphalan in combination with vincristine and CY [128]. Melphalan was implicated in the causation because challenge with the same regimen but a lower dose of melphalan (0.5 mg/kg) in one patient several weeks later did not cause hyponatremia.

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Although not directly related to HCT, the presence of solid tumors may also be responsible for hyponatremia, either through ectopic secretion of ADH by the tumor itself [118], or through secretion of atrial natriuretic peptide, which causes renal salt wasting [129]. Evaluation of abnormal water metabolism post HCT The rate of development of hyponatremia plays a critical role in its pathophysiology and may impact the approach to management [130]. When serum sodium concentration falls slowly, over a period of several days or weeks, the brain is capable of compensating by extrusion of solutes and fluid to the extracellular space. Compensatory extrusion of solutes reduces the flow of free water into the intracellular space, and symptoms will be milder. When serum sodium concentration falls rapidly over a period of 24–48 hours, however, this compensatory mechanism is overwhelmed. Patients with acutely developing hyponatremia are typically symptomatic with sodium levels approximately 120 mEq/L or less, although patients with chronic hyponatremia may present with much lower levels. The clinical manifestations of significant hyponatremia are related primarily to cerebral edema; headache, nausea, vomiting, muscle cramps, lethargy, restlessness, disorientation, and depressed reflexes may be observed. In rapidly evolving hyponatremia, severe neurologic symptoms may ensue, resulting in seizures, coma, permanent brain damage, respiratory arrest, brainstem herniation, and death. Young, menstruating women seem to be more susceptible to the deleterious effects of this cerebral edema [131]. As the clinical presentation may be highly variable and dependent on the cause, degree, and chronicity of hyponatremia, the evaluation of these patients should be directed toward identifying the particular cause of hyponatremia (Fig. 98.9) [132]. Establishing the hydration status of the patient is a significant first step. A history of fluid loss (e.g. vomiting, diarrhea or diuretic therapy) or one that is consistent with the development of SIADH, such as intake of an offending drug (see above), should be elucidated. On physical examination, pulmonary rales, S3 gallop, jugular venous distention, peripheral edema or ascites suggests hypervolemic hyponatremia. Dry mucous membranes, tachycardia, diminished skin turgor, and orthostasis suggest hypovolemic hyponatremia. Patients who lack findings of hypovolemia or hypervolemia are considered to have euvolemic hyponatremia, although identification of subtle degrees of volume depletion or edema may be difficult [130]. As a result, laboratory testing is almost always required to establish the diagnosis of hyponatremia. Nevertheless a history of corticosteroid use, or signs and symptoms suggestive of adrenal insufficiency or hypothyroidism, as well as other metabolic abnormalities commonly observed post HCT, such as hyperglycemia, hyperlipidemia, and hyperuricemia, should not be neglected. Other nonspecific signs include muscle weakness and cramping; rhabdomyolysis is an occasional consequence of hyponatremia, and should be considered in patients with muscle pain or tenderness. Standard laboratory testing for the evaluation of hyponatremia should include a chemistry panel, plasma osmolality, urine osmolality, and urine sodium concentration. Most cases of hyponatremia are associated with a reduced plasma osmolality, but plasma osmolality may be normal, or even high, in hyponatremia that occurs in association with hyperlipidemia or hyperproteinemia, renal failure, hyperglycemia or after the infusion of sucrose- and maltose-containing immunoglobulin G formulations. A urine osmolality of greater than 100 mosmol/kg is consistent with an inability to excrete free water normally, which is generally due to continued secretion of ADH. Urine osmolality less than 100 mosmol/kg may be due to primary polydipsia, malnutrition or a reset osmostat. The urine sodium concentration is used to help distinguish between effective volume depletion and other causes in which euvolemia or renal salt

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Fig. 98.9 Algorithm for the evaluation of the patient with suspected hyponatremia following hematopoietic cell transplantation. CHF, congestive heart failure; CNS, central nervous system; ECFV, extracellular fluid volume. GI, gastrointestinal; Serum Na = serum sodium concentration; SIADH, syndrome of inappropriate antidiuretic hormone; Urinary Na, urine sodium concentration. (Adapted from [132], with permission.)

Endocrine Complications Following Hematopoietic Cell Transplantation

wasting is present, and should be less than 20 mEq/L with hypovolemia, unless salt wasting is occurring. Urine sodium concentrations are generally above 40 mEq/L in SIADH. In patients with intermediate values, serial monitoring of the urine sodium concentration in response to isotonic saline should be performed [130]. The diagnosis of SIADH is usually made from a combination of results from the history, physical examination (no edema), and laboratory tests, where the following results are generally observed: low plasma osmolality, an inappropriately elevated urine osmolality (above 100 mosmol/kg and usually above 300 mosmol/kg), a urine sodium concentration usually above 40 mEq/L, low blood urea nitrogen and serum uric acid concentration, a relatively normal plasma creatinine concentration, normal acid–base and potassium balance, and normal adrenal and thyroid function. Patients with SIADH may have a low urine sodium concentration if they are also volume depleted, or if their sodium intake is extremely low. In such patients, the diagnosis of SIADH is made by observing the response to a saline load: the urine sodium rises but the urine osmolality remains high [130]. In patients for whom the diagnosis is not apparent after the initial evaluation, measurement of the plasma uric acid and urea concentrations, the fractional excretion of sodium, and adrenal and thyroid function tests may be helpful [130]. Management of abnormal water metabolism post HCT The choice of therapy is primarily governed by the cause and severity of the hyponatremia. Treatment should be directed first towards correcting any underlying causes of hyponatremia, such as cessation of a drug that induces SIADH, glucocorticoid or thyroid hormone replacement in patients with adrenal insufficiency or hypothyroidism, and control of hyperlipidemia and/or hyperglycemia. In these circumstances, the rate of correction of the underlying condition results in a relatively slow correction of the hyponatremia. Rapid correction of hyponatremia is generally not indicated in asymptomatic hospitalized patients, particularly if the baseline plasma sodium is above 120 mEq/L. Such patients are treated conservatively with free water restriction (e.g. restriction to 50–60% of daily fluid requirements to induce a negative water balance) [130]. If correction of volume depletion is indicated, intravenous isotonic saline and/or increased dietary salt (NaCl; 2–3 g/day) is administered [115,132]. Salt therapy is usually contraindicated in edematous patients (e.g. heart failure, cirrhosis, and renal failure) since it will exacerbate the presence of cerebral edema. Notably, the response to isotonic saline differs in states of volume depletion and SIADH. Isotonic saline is typically sufficient in true volume depletion, but ineffective in SIADH. Hypertonic saline is recommended only for patients with symptomatic or severe hyponatremia. To avoid the possibility of overcorrection, the therapeutic goal over the first 24 hours should be to increase serum sodium by 8 mEq/L or less, with a maximum rate of correction of up to and including 10–12 mEq/L in the first 24 hours, and 18 mEq/L or less over 48 hours [115,130,133]. When saline is given to treat hyponatremia, the quantity of sodium required to achieve the desired elevation in the plasma sodium concentration can be estimated from the product of the plasma sodium deficit per liter and the total body water (TBW), which represents the osmotic space of distribution of the plasma sodium: Sodium deficit = TBW × (desired sodium – actual sodium) Aggressive initial correction is warranted in patients with acute hyponatremia who present with seizures or other severe neurologic abnormalities [133]. As the primary problem in these patients is cerebral edema, the risk of delayed therapy is greater than the potential risk of too rapid correction. Aggressive initial correction with hypertonic saline at a rate of 1.5–2 meq/L per hour is indicated for the first 3–4 hours, or

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until symptoms resolve. Further correction should proceed at an overall rate that is no greater than 0.5 mEq/L/hour or 12 mEq/L/day [133]. Studies in experimental animals suggest that the rate of correction over the first 24 hours is more important than the maximum rate in any given hour or several hour period [134], and careful monitoring is essential to avoid exceeding these goals. Rapid correction of chronic hyponatremia has been associated with the development of central pontine myelinolysis, although the pathophysiology of the latter is controversial. More recently, investigators have noted that central pontine myelinolysis often develops when chronic hyponatremia is complicated by hypoxia. Thus, central pontine myelinolysis may be a form of hypoxic encephalopathy associated with hyponatremia and not a complication of therapy [135]. Until further data are available, however, management of hyponatremia should include attention to adequate oxygenation. If the cause of SIADH cannot be corrected, and if water restriction is poorly tolerated or ineffective, demeclocycline (a tetracycline antibiotic that increases electrolyte-free water excretion by inhibiting ADHmediated water reabsorption in the collecting duct) 300–600 mg in divided doses can be used [131,132]. If the thirst sensation is intact, patients treated with demeclocycline rarely become hypernatremic. This drug is contraindicated, however, in patients with renal disease, hepatic cirrhosis or congestive heart failure. The ADH receptor antagonists (AVP receptor antagonists), a new class of agents, may correct hyponatremia by directly blocking the binding of ADH with its receptors. In clinical trials, conivaptan, lixivaptan, tolvaptan, and satavaptan have increased serum osmolality and normalized the serum sodium ion concentration in hyponatremia associated with SIADH, cirrhosis, and congestive heart failure [130]. These drugs may have a potential in cancer-related hyponatremia as well. At present, only intravenous conivaptan is available, and is approved for the treatment of euvolemic hyponatremia (i.e. SIADH). The data are currently insufficient to determine the role of these agents in the emergency treatment of symptomatic hyponatremia. Notably, the use of ADH receptor antagonists is accompanied by an increase in thirst, and, as such, does not always eliminate the need for water restriction during chronic therapy [136]. Initial careful monitoring is required, since there is a potential risk of overly rapid correction of the hyponatremia if the serum sodium is very low and the ADH effect is completely eliminated. Further, conivaptan should not be used in patients with hyponatremia caused by hypovolemia, as blocking the V1a receptor sites on vascular smooth muscle cells may cause or worsen the hypotension associated with volume depletion. Carbohydrate and lipid abnormalities Clinical diabetes mellitus is a syndrome of disordered metabolism with inappropriate hyperglycemia, which may be due to an absolute deficiency of insulin secretion, a reduction in the biologic effectiveness of insulin or both [137]. In 2000, there were approximately 171 million total cases of diabetes worldwide, and this number is expected to swell to 366 million by the year 2030 [138]. Autoimmune type 1 diabetes mellitus affects approximately 9% of North American diabetics, and 20% of diabetics in Scandinavian countries [137]. Noninsulin-dependent type 2 diabetes mellitus, typically associated with insulin resistance and hypertriglyceridemia, is the most common form of diabetes, accounting for 90% of cases globally, and 7% of the entire United States population in 2005 [139]. Metabolic syndrome is a constellation of disorders related to insulin resistance, and is characterized clinically by central (abdominal) obesity, elevated plasma glucose, dyslipidemia, hypertension, and a prothrombotic and proinflammatory state. The development of metabolic syndrome itself is an important risk factor for cardiovascular disease and type 2 diabetes mellitus [140].

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The development of insulin-related metabolic disorders in HCT recipients represents a significant health risk for these patients, due to the potential for microvascular and macrovascular complications in longstanding diabetes. While the link between insulin resistance and atherosclerotic disease has increasingly been the focus of investigation, particularly in regard to dyslipidemia, there are few currently available data on long-term cardiovascular outcomes in cancer survivors. Similarly, although dyslipidemia, hyperglycemia, and impaired glucose tolerance are prominent results of corticosteroid treatment, there are minimal data regarding the long-term risk of diabetes development in steroid-treated patients. A previous study reported that the frequency of steroid-induced diabetes mellitus in renal transplant recipients was 46% [141]. Further, dyslipidemia and hypertension are well-known sideeffects of therapy with the calcineurin inhibitors (e.g. tacrolimus), but it is unknown whether this translates into a higher cardiovascular risk years after the discontinuation of these medications. Notably, one study has suggested that survivors of allogeneic HCT were 3.65 times (95% confidence interval [CI] 1.82–7.32) more likely to report any type of diabetes than siblings, and 2.06 times (95% CI 1.39–3.04) more likely to report hypertension compared with siblings, but did not report other cardiovascular outcomes with any greater frequency, after adjusting for age, gender, race, and body mass index. Allogeneic HCT survivors were also more likely to develop hypertension (odds ratio 2.31, 95% CI 1.45–3.04) than autologous recipients. In fact, autologous recipients were no more likely than nontransplanted siblings to report any of the outcomes studied [140]. The differing results between allogeneic and autologous transplant recipients may be attributable to the greater treatment with TBI in the allogeneic group; this study found a direct association between TBI exposure and an increased risk of diabetes (odds ratio 3.42, 95% CI 1.55–7.52) [140]. Interestingly, hypertension was not found to be influenced by TBI, and the risk of hypertension was not increased in subjects who had a history of chronic GVHD, where exposure to calcineurin inhibitors and steroids was likely more prolonged. Radiation and certain chemotherapeutic agents are also known to be cardiotoxic, including CY and the anthracyclines [140]. Other mechanisms related to the inflammatory and immunologic effects of allogeneic transplantation itself may play a role in the development of diabetes and hypertension, possibly by exacerbating the atherosclerotic process [140]. The prevalence of frank type 2 diabetes in HCT survivors appears to vary from 3.3% to 17% [15,142,143]. Impaired glucose tolerance was seen in 3.3–26% of adult patients treated with both TBI and non-TBI regimens prior to HCT. Hyperinsulinemia has also occurred post HCT, with the highest insulin levels noted in patients receiving preparations of both cytotoxic drugs and TBI [144], but the connections between irradiation, BU, and insulin resistance are not clear. Dyslipidemia of any type has been observed in approximately 27.9% of HCT recipients [15,142,143]. In general, of the several abnormalities of lipoprotein metabolism associated with insulin resistance, hypertriglyceridemia is the most common [145]. A number of immunosuppressive medications have been documented to cause dyslipidemias, including cyclosporine, tacrolimus, sirolimus, mycophenolate, and highdose administration of glucocorticoids. Glucocorticoids stimulate hepatic gluconeogenesis, diminish glucose utilization, oppose insulin action, increase protein breakdown, and activate lipolysis in the periphery; if the patient’s insulin levels are inadequate to overcome insulin resistance and increased hepatic glucose output, steroid-induced diabetes mellitus will develop. Older age was noted to be a risk factor for steroid diabetes [141], as well as for diabetes mellitus in general [146–148]. Iwamoto et al. did not find evidence for increased risk of longlasting steroid-induced diabetes with cumulative doses of glucocorticoids [149], although longterm therapy has been associated with hypercholesterolemia [150].

It has also been shown that prolonged (4 days) ingestion and infusion of a hypercaloric carbohydrate diet (hyperalimentation) produced chronic elevations in both plasma insulin and glucose concentration, with plasma triglyceride concentration elevated fourfold, compared with the values in the basal state [145,151]. The hyperalimentation formulas used post HCT are often high in glucose and lipids, which may contribute to hyperglycemia, insulin resistance, and hyperlipidemia. GVHD commonly affects the liver, first presenting with elevated liver transaminase levels, and then with intrahepatic cholestasis and dyslipidemia. This condition frequently manifests between 2 months and 2 years post transplantation, and may occur in up to 40% of recipients from HLA-matched related donors [152]. Although few cases of severe hypercholesterolemia associated with GVHD of the liver in HCT patients have been reported in the literature [153,154], this clinical presentation may occur more frequently than previously perceived [155]. Consequently, there may be insufficient awareness of this important complication. The patient’s diagnosis at transplant was found to be the most significant risk factor for the development of type 2 diabetes post HCT, with leukemic patients at greater risk than patients with other hematologic diseases, possibly due to greater exposure to pretransplantation chemotherapy agents, as well as to islet cell damage as the result of leukostasis or leukemic infiltration of the pancreas [144]. In this study, the prevalence of type 2 diabetes was found to be 9% among those with leukemia, versus 2% with aplastic anemia, compared with the prevalence of physician-diagnosed diabetes in the general population based on the 1988–1994 National Health and Nutrition Examination Survey III survey: 1.1% among all 20–39-year-olds, and 3.9% among all 40–49year-olds [144]. Additional evidence suggests that cancer survivors in general, and survivors of childhood ALL, are also at increased risk for metabolic syndrome [156]. Other significant risk factors for type 2 diabetes in HCT survivors have included Native American/Hispanic/African-American race or ethnicity and a family history of diabetes, followed by older age, obesity, physical inactivity, and diet, similar to what has been reported in the general population [144]. It has also been postulated that increased time since HCT and the presence of chronic GVHD promote diabetes risk (see above) [144]. While there are case reports of patients developing diabetes following abdominal radiation, the association is unclear, due to limited evidence; one very large study that followed children with abdominal cancer who had received abdominal radiation found that their risk of developing diabetes was no greater than for the population as a whole [140,144]. Autoimmune type 1 diabetes may develop post HCT due to the transfer of type 1 diabetes in a bone marrow allograft donor to a transplanted recipient, or because of recipient HLA susceptibility [144,157]. Further, as with type 2 diabetes, the prevalence of type 1 diabetes was approximately three times higher in HCT survivors than in the general population, at 0.52% versus 0.17% [144]. One case report, however, describes a successful allograft for aplastic anemia in which the donor had type 1 diabetes at the time of transplant, but, after a 21-year follow-up period, the recipient has not developed diabetes, although persistent antibodies have developed against pancreatic islet cells [158]. Pediatric HCT survivors seem more likely to develop either type 1 or type 2 diabetes than the general population [144], with one study of 748 survivors of pediatric HCT demonstrating a higher than expected prevalence of both type 1 and type 2 diabetes in these survivors at a median of 11 years of follow-up [140]. Development of type 2 diabetes can also be causally related to the administration of GH therapy. In one study, GH therapy has been associated with a significant sixfold increase in new cases of type 2 diabetes, including increases in insulin concentrations and other evidence of insulin resistance [159]. The occurrence of

Endocrine Complications Following Hematopoietic Cell Transplantation

GH deficiency itself may be a consequence of metabolic syndrome (see the section on growth dysfunction) [156]. Evaluation of carbohydrate and lipid abnormalities post HCT Clinical signs and symptoms of diabetes mellitus are related to the degree of hyperglycemia and the presence or absence of ketoacidosis or other hyperosmolar states, and may include polyuria, polydipsia, polyphagia, weight loss, and blurred vision, and may be associated with fluid and electrolyte disturbances, including elevated blood urea nitrogen and creatinine, low-normal or mildly reduced sodium levels, elevated serum potassium, low serum bicarbonate (<10 mg/dL), and elevated serum triglycerides [160]. Uncomplicated presentations may reveal normal fluid and electrolyte measures, but if diabetic ketoacidosis is present, laboratory values will represent acidosis and more severe dehydration. The American Diabetic Association has defined the following criteria for the diagnosis of diabetes in the setting of clinical hyperglycemic symptoms: fasting plasma glucose of 126 mg/dL or greater on more than two occasions (preferred test), casual plasma glucose concentration of 200 mg/dL or more, or 2-hour post-glucose load of 200 mg/dL or greater during an oral glucose tolerance test. Metabolic syndrome should be suspected if three of the following five criteria are present: elevated fasting blood glucose over 100 mg/dL, obesity (especially large waist circumference – more than 40 inches [102 cm] in men and more than 35 inches [89 cm] in women), hypertension (>130/85 mmHg), hypertriglyceridemia (>150 mg/dL), and low levels of high-density lipoprotein cholesterol (<40 mg/dL in men and <50 mg/dL in women) [161]. In type 1 diabetes, the onset may be rapid. Antibodies to glutamic acid decarboxylase 65 (GAD65), one of the best characterized islet cell autoantigens, is one of the most sensitive and specific autoantibodies associated with type 1 diabetes: approximately 70–80% of diabetics have GAD65 antibodies prior to or at the onset of disease, compared with 3% of control subjects [162]. Islet cell antibodies, the classical autoimmunity marker for insulin-dependent diabetes mellitus, are also detected in the majority of newly diagnosed type 1 diabetics [163]. When the islet cell antibody assay is combined with that for GAD65, autoimmune disease detection is increased to greater than 90% [164]. Levels of insulin autoantibodies may further increase sensitivity. Measurement of insulin autoantibodies must be done before the exogenous administration of insulin, and levels tend to be higher in individuals who develop dia-betes at a younger age or have a more rapid progression towards clinical disease, and are higher in female patients. The presence of endogenous insulin production may be ascertained via measurement of fasting C-peptide, the connecting amino acid chain that is cleaved from proinsulin during the process of insulin synthesis and is secreted by the β cell on an equimolar basis with the insulin molecule. C-peptide levels thus assist in differentiating type 1 from type 2 diabetes: if C-peptide is not present at the time of diagnosis, total β-cell failure has occurred, suggesting type 1 diabetes; if C-peptide is present, it could indicate a type 1 case early in the course of autoimmune β-cell destruction with some residual insulin secretion or, more commonly, type 2 diabetes. Commonly, patients with type 2 diabetes have elevated C-peptide levels due to the presence of insulin resistance. Management of carbohydrate and lipid abnormalities post HCT Regardless of whether or not the patient has a known history of diabetes, it should be emphasized that management of the hyperglycemic hospitalized patient must be intensive, particularly in the presence of severe illness. Acute metabolic derangements related to diabetic ketoacidosis or hyperosmolar coma require urgent management in order to stabilize the patient and restore normal acid–base balance and volume status, and abrogate electrolyte abnormalities. Although hyperglycemia in itself

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may often be given secondary consideration in this setting relative to the development of other acute post-HCT complications, the onset and persistence of suboptimal glycemic control post HCT could significantly prolong the length of hospital stay, as well as increased risk for morbidity and mortality. Several studies that examined the impact of hyperglycemia on cardiac outcomes in hospitalized patients [165] and other acute care settings have shown that aggressive monitoring and treatment of hyperglycemia can result in both fewer complications and improved clinical outcomes. Further, hyperglycemia is more prevalent in hospital patients than may be recognized. With hyperglycemia defined at a level of over 125 mg/dL, one study noted that 38% of hospital patients were hyperglycemic, of which 26% had previously recognized diabetes and 12% did not [166]. If the threshold for hyperglycemia is lowered to 100 mg/dL, however, it is possible that a far greater percentage of patients may be affected [165]. Thus, efficient management of hyperglycemia in the hospitalized patient may improve patient wellbeing and reduce the cost of care. The currently available classes of insulin preparations may be divided into (1) those intended for prandial use – regular insulin, rapid-acting insulin (lispro, aspart, and glulisine, with an onset of action within 1 hour or less), and (2) those for control of fasting blood glucose and background hyperglycemia – intermediate-acting insulin (NPH, with an onset of action within 1–3 hours, a duration of action up to 24 hours, and peak action at 6–8 hours) and long-acting insulin (glargine and detemir, with a peak action within 2–6 hours and a duration up to 24 hours). In the acute care setting, continuous intravenous rapid-acting or regular insulin with frequent monitoring of plasma or serum glucose may be required in order to control hyperglycemia. Once stabilized, typical treatment may include multiple daily injections of premixed intermediate and rapid-acting products, or freshly mixed or separately injected short- and intermediate-acting or long-acting preparations. The most serious complication of insulin replacement is that of hypoglycemia, the risk of which can be reduced in the inpatient setting by the careful monitoring and adjustment of insulin doses, and in the outpatient setting by teaching patients to recognize the signs of hypoglycemia, as well as the measures that should be taken to reverse hyperglycemia once these symptoms are experienced. Hyperalimentation with high lipid content is often prescribed in HCT patients, but in view of the potential metabolic complications from hyperlipidemia, particularly when combined with the presence of GVHD, hyperalimentation should be used for the shortest period possible, and oral or enteral feeding should be initiated as soon as is clinically feasible. Hyperlipidemia may also be seen as the result of GVHD of the liver. If such a patient with GVHD and hepatic damage is also receiving hyperalimentation, lipid content should be reduced, and insulin may be given. Caution should be exercised when using the antihyperlipidemic medications in patients with hepatic damage, which can limit management options. Low doses of fibrates (i.e. amphipathic carboxylic acids, such as gemfibrozil, for the treatment of hypercholesterolemia), niacin (vitamin B3), and omega-3 fatty acids may be used, particularly when statins are contraindicated. Co-enzyme Q10 may also be prescribed, but may have little to no therapeutic effects. Regardless of the treatment options utilized, liver enzymes should be closely monitored to avoid added hepatocellular toxicity related to the therapeutic agents themselves. Plasmapheresis may be employed in severe cases of hypertriglyceridemia to avoid pancreatitis. Steroid-induced diabetes is treated primarily with insulin administration. The course of treatment depends on the severity of the hyperglycemia and the estimated duration of the steroid therapy. Generally, moderate-to-severe hyperglycemia carries an increased risk of infection (especially fungal), particularly in patients with other risk factors, including immunocompromised states and patent central venous lines,

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as is commonly the case post HCT. Steroid-induced dyslipidemia can range from mild to severe, but, if there is no hepatic damage, this condition responds to therapy as with the nonglucocorticoid-induced lipid disorders. The primary goal of long-term clinical management in individuals with type 2 diabetes or metabolic syndrome is to reduce the risk for clinical atherosclerotic disease, and to prevent the development of other diabetic complications. Patients are initially counseled regarding diet and exercise, and the importance of daily blood glucose self-monitoring. If more intensive treatment is required, sulfonylureas (which increase insulin secretion in response to meals), biguanides (which inhibit hepatic glucose production), thiazolidinediones (which improve the insulin responsiveness of muscle and adipose tissue), incretin hormone-based therapies (e.g. mimetics of exendin-4 and glucagon-like peptide-1 that stimulate β-cell responsiveness to glucose and improve β-cell health, and enhancers that increase physiologic levels of incretin hormones), α-glucosidase inhibitors (which blunt glucose absorption from gut), and/or insulin may be tried as initial therapy, depending on the level of metabolic control that exists in the patient at the time of treatment initiation. The overriding goal of treatment is to achieve and maintain blood glucose concentrations that are as close to the normal range as possible. Lipid abnormalities and/or hypertension, if present, should also be adequately treated.

Adrenal dysfunction The adrenal cortex is a source of many steroid hormones, of which the most important are the glucocorticoids (namely cortisol), the mineralocorticoid aldosterone, and the adrenal androgens. Anterior pituitary ACTH secretion or exogenous ACTH administration results in the rapid synthesis and secretion of steroids; plasma levels of these hormones can rise within minutes, and chronic stimulation leads to adrenocortical hyperplasia and hypertrophy. Conversely, chronic ACTH deficiency will decrease steroidogenesis, and will be accompanied by adrenocortical atrophy. ACTH secretion from the pituitary is under stimulatory control of the hypothalamic factor CRH and the inhibitory effects of cortisol or related glucocorticoids (Fig. 98.10). Cortisol secretion is tightly regulated by ACTH, and plasma levels of cortisol will parallel those of ACTH. The neuroendocrine mechanisms of the HPA axis normally permit: (1) episodic secretion as related to circadian rhythms, and (2) cortisol secretion in large quantities in response to stressful stimuli. Endogenous glucorticoids cause insulin resistance, increasing blood glucose concentrations via their actions on glycogen, protein, and lipid metabolism. Additionally, glucocorticoids can promote catabolic changes in muscle, skin, and connective tissue, inhibit osteoblast function, increase blood pressure, suppress immunologic responses, affect mood by increasing the manifestations of depression, euphoria, psychosis, apathy, and lethargy, increase the risk of peptic ulcer disease, inhibit linear skeletal growth, and suppress the thyroid axis. Primary hypoadrenalism (Addison’s disease) is rare in the general population, with an estimated incidence of 0.8 cases per 100,000 [167]. As with the general population, transplant recipients have a low incidence of primary adrenal failure after HCT. Secondary hypoadrenalism, however, is quite common. Although the adrenal gland is known to be relatively radioresistant, TBI has been implicated in adrenal suppression post HCT. Low-dose cranial irradiation, however, has not demonstrated disruption of spontaneous ACTH or cortisol secretion in several studies [4]. In general, however, secondary adrenal insufficiency is mainly the result of long-term, exogenous corticosteroid therapy, such as in HCT patients being treated for GVHD. Chronic administration of glucocorticoids suppresses the function of the HPA axis through a process that is dependent both on the dose and

Fig. 98.10 Neuroendocrine control of adrenal glucocorticoid secretion. Adrenocorticotropic hormone (ACTH) is secreted from the anterior pituitary under the influence of two principal secretagogues, corticotropin-releasing hormone (CRH) and arginine vasopressin; other factors, including cytokines, also play a role. CRH secretion is regulated by circadian rhythm and additional stressors operating through the hypothalamus. Secretion of both CRH and ACTH is inhibited by cortisol, highlighting the importance of negative feedback control. (Reproduced from [167], with permission.)

duration of treatment. A greater length and intensity of exposure is generally associated with a longer persistence of adrenal suppression; adrenal atrophy and subsequent deficiency should be anticipated in any patient who has taken greater than the equivalent of 30 mg/day oral hydrocortisone (7.5 mg/day prednisolone, or 0.75 mg/day dexamethasone) for more than 3 weeks [167]. High doses of glucocorticoids for as little as 3 days can also result in suppression of the HPA axis. The timing of the dose administration may also influence the severity of adrenal insufficiency, as larger evening doses will block circadian morning surges of endogenous ACTH. Because the HPA axis seems to remain intact, with any defects appearing to occur at the target organ level, adrenal function usually recovers gradually once exogenous corticosteroid exposure ends [4]. After chronic adrenal suppression has been established, however, sudden cessation of glucocorticoid therapy may result in adrenal crises, and care must be taken during steroid withdrawal. Failure to provide adequate glucocorticoid replacement in times of stress, such as with surgery or infection, may also precipitate deterioration in the general condition of the patient related to relative glucocorticoid deficiency. Chronic glucocorticoid excess, regardless of cause, may also lead to the constellation of symptoms and physical features typical of Cushing’s syndrome in the majority of patients, particularly in those taking suppressive doses for more than 3 weeks. The rapidity with which clinical signs and symptoms appear is dependent on the administered dose, but may occur within 1 month of starting therapy. Secondary hyperglycemia is another common consequence of corticosteroid usage. The secretion of adrenal aldosterone is mainly mediated by angiotensin II, as well as by ACTH and local potassium levels. Aldosterone acts on the kidneys to cause active reabsorption of sodium and passive reab-

Endocrine Complications Following Hematopoietic Cell Transplantation

sorption of water, as well as the active secretion of potassium and protons in the collecting tubule, resulting in an increase in blood pressure and blood volume. Adult adrenal androgen production is also regulated by ACTH. The most important adrenal androgens are dehydroepiandrosterone (DHEA) and its sulfated form (DHEAS) and 4-androstene-3,17-dione. These hormones also exhibit circadian periodicity in synchrony with ACTH and cortisol, as plasma DHEA and androstenedione increase rapidly with ACTH administration, and are suppressed by the presence of glucocorticoids. Although the adrenal androgens have low androgenic activity, they can be converted into the potent androgens, testosterone and 5αdihydrotestosterone (DHT), in both peripheral and target tissues. In normal males, this conversion accounts for less than 5% of testosterone, and thus the physiologic effect is negligible. In contrast, adrenal androgens contribute to 50% or more of circulating, biologically active androgens in adult women; thus, the adrenal glands may play important roles in the anabolic status and sexual function of females. Thus, lack of DHEA and DHEAS in women may result in the loss of pubic and axillary hair and osteoporosis, and their excess will manifest with acne, hirsutism, and virilization. Thus, any HCT-related adrenal dysfunction is more likely to impact female patients [168]. The significance of this postulation has yet to be clarified, as there are no present reports documenting adrenal androgen levels of adult female patients after allogeneic HCT; one study, however, reported subnormal levels of both ovarian and adrenal androgens in a cross-sectional study of young transplanted adolescent females [169]. Evaluation of adrenal dysfunction post HCT Patients with primary adrenal failure usually will demonstrate signs of both glucocorticoid and mineralocorticoid deficiency. In both sexes, cortisol deficiency produces symptoms of weakness, fatigue, anorexia, nausea, vomiting, hypotension, hyponatremia, and hypoglycemia. Mineralocorticoid deficiency produces renal sodium wasting and potassium

Fig. 98.11 Algorithm for the evaluation of the patient with suspected primary or secondary adrenocortical insufficiency following hematopoietic cell transplantation. ACTH, adrenocorticotropic hormone; CRH, corticotropinreleasing hormone. (Adapted from [170], with permission.)

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retention, and can lead to severe dehydration, hypotension, hyponatremia, hyperkalemia, and acidosis. Patients with secondary hypoadrenalism will have an intact renin–angiotensin–aldosterone system, which accounts for differences in clinical presentation between these two groups of patients in regards to salt and water balance. The most obvious feature that differentiates primary from secondary hypoadrenalism is the presence of hyperpigmentation of the skin and mucous membranes, which is not present in secondary forms of the disease. Clinical suspicion should be confirmed with definitive diagnostic tests. Although a basal cortisol value greater than 15 μg/dL normally suggests an intact HPA axis, low basal levels cannot be relied upon to exclude the diagnosis. Thus, in practice, all patients suspected of having adrenal insufficiency should have an ACTH stimulation test (or short synacthen test), which involves the administration of tetracosactrin (containing the first 24 amino acids of ACTH, and with the same physiologic properties) 250 μg intravenously or intramuscularly (Fig. 98.11). Plasma cortisol levels are measured at 0 and 30 minutes after tetracosactrin administration, and a normal response is defined by a peak plasma cortisol level greater than 20 μg/dL. Incremental responses (i.e. the differences between peak and basal values) are of limited value other than to determine the level of relative adrenal insufficiency. Response is unaffected by the time of day tested, and the test can still be performed in patients currently receiving corticosteroid replacement therapy, as long as the agent used is not measurable by that particular laboratory method, and the treatment is of short duration. If necessary, there are several dynamic methods of differentiating primary from secondary hypoadrenalism. The first is the prolonged ACTH stimulation test (intravenous infusion of cosyntropin over 8 hours or over 24 hours on 2 or 3 consecutive days). In normal subjects, plasma cortisol will be over 36 μg/dL at 4 hours of continuous infusion of cosyntropin, with no further increases observed over the remaining course of infusion. In primary adrenal insufficiency, no change is seen in cortisol or 17-hydroxycorticosteroid concentrations after prolonged

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ACTH stimulation. In secondary adrenal insufficiency, an incremental increase occurs during the course of the infusion, with much higher values observed at 24 and 48 hours than at 4 hours. The overnight metyrapone test involves the administration of metyrapone (30 mg/kg), which blocks cortisol synthesis by inhibiting 11βhydroxylase. In a normal subject, the resultant fall in cortisol levels will stimulate ACTH secretion (>250 pg/mL), further stimulating the adrenal cortex to increase the production of cortisol precursors, such as plasma 11-deoxycortisol (>7 μg/100 mL). Primary adrenal insufficiency will present with elevated baseline ACTH and low 11-deoxycortisol and cortisol levels, with no further changes after metyrapone administration. Secondary adrenal insufficiency will present with low levels of ACTH, 11-deoxycortisol, and cortisol, with no changes after metyrapone administration. If baseline ACTH levels were not measured prior to metyrapone administration and 11-deoxycortisol levels are low, a prolonged ACTH stimulation test will be necessary to distinguish between primary and secondary insufficiency. Notably, the metyrapone test is unreliable with patients who are pregnant, hypothyroid, hyperthyroid, receiving estrogen therapy or using a variety of tranquilizers.

The insulin-induced hypoglycemia test causes a major stress response (see also “Evaluation of growth disorders post HCT” above). In a normal patient, increases in plasma ACTH and serum cortisol, GH, and prolactin and activation of the sympathetic nervous system are observed. The ITT has been considered the gold standard in the diagnosis of secondary hypoadrenalism, but it is uncomfortable for the patient, requires close medical supervision, and should not be performed in patients with ischemic heart disease, seizure disorders or severe hypopituitarism. When withdrawing corticosteroids, particularly if symptoms of adrenal insufficiency occur, HPA axis testing should be performed using agents such as metyrapone. Other methods of HPA axis testing include the administration of CRH, which will directly stimulate pituitary secretion of ACTH and subsequently adrenal steroids, or insulin, which will induce a state of stress via hypoglycemia, and thus cause increased secretion of ACTH and steroids (Fig. 98.12). Many factors complicate investigation of the HPA axis during critical illness, however. Cortisol levels vary greatly according to disease severity, and it may be difficult to define responses to testing. Decreases in corticosteroid-binding globulin in the face of severe disease will alter

Fig. 98.12 Algorithm for steroid withdrawal for patients on glucocorticoid therapy of greater than 3 weeks’ duration. Relative steroid potency (equivalence): 1 mg prednisone (or prednisolone) approximately equal to 4 mg cortisol or 5 mg cortisone; 1 mg dexamethasone approximately equal to 8–10 mg prednisone or 30–35 mg cortisol. *Overnight metyrapone test: 2–3 g (30 mg/kg) at bedtime, and measure levels of 11-deoxycortisol, cortisol, and adrenocorticotrophic hormone (ACTH) the following morning. Normal response: 11deoxycortisol > 7 μg/dL, ACTH > 250 pg/mL; may compare cortisol level with baseline on the previous day (expect inhibition). †ITT, insulin-induced hypoglycemia test. Regular insulin 0.05–0.15 U/kg, intravenous push. Measure levels of ACTH and cortisol at 30-minute intervals for up to 2 hours. The physician must supervise this. Normal response: cortisol > 20 μg/dL, ACTH > 250 pg/mL. ‡CRH test, corticotropin-releasing hormone test. Bovine or synthetic human CRH, 100 μg intravenous push. Measure ACTH and cortisol every 15–30 minutes for up to 2 hours. Normal response: cortisol > 13 μg/dL, ACTH peak level 10–120 pg/mL. Vasopressin may be administered to potentiate the response to CRH.

Endocrine Complications Following Hematopoietic Cell Transplantation

the ratios of free to bound serum cortisol, further altering the results of testing. Recent evidence suggests that a random cortisol value of below 15 μg/dL is suggestive of corticosteroid insufficiency, while a level greater than 33 μg/dL would be unlikely to occur with compromised HPA axis function [167]. Management of adrenal dysfunction post HCT Clinically significant suppression of the HPA axis rarely occurs in patients who have taken steroid doses for less than 3 weeks’ duration, and withdrawal of the steroids rarely causes any ill effects. HPA axis suppression is inevitable, however, in patients taking the equivalent of 15 mg/day or more of prednisolone over the long term. Variable suppression of the HPA axis is seen with doses of prednisolone between 5 and 15 mg/day. To avoid the precipitation of adrenal failure, steroids should be withdrawn cautiously over a period of months (Fig. 98.12). All patients receiving long-term corticosteroid therapy should be treated similarly to patients with chronic ACTH deficiency. The aim is to give replacement doses of hydrocortisone to mimic normal cortisol secretion rates. Most patients are given hydrocortisone 15–25 mg/day in divided doses. In the event of concurrent stressors, such as infection or surgery, supplemental steroid cover should be given, equivalent to 100– 150 mg/day of hydrocortisone. Parenteral therapy may be required if the patient is unable to consume oral medications. In primary adrenal failure, mineralocorticoids also require replacement in the form of fludrocortisone 0.05–0.2 mg/day. Administration of DHEA, normally secreted in large amounts by the adrenal glands, has been reported to have positive effects on the wellbeing and sexuality of women with adrenal insufficiency [171]. DHEA also increases bone density, muscle strength, and the sense of wellbeing in normal elderly men and women [172,173]. Prolonged high-dose glucocorticoid therapy may be a possible indication for DHEA therapy, especially in diseases that bear a high risk of osteoporosis [174].

Gonadal dysfunction The provision of good quality of life in addition to the treatment of the primary disease has become an increasingly important goal as life expectancy post HCT continues to expand. Although the majority of young patients who have not yet had children themselves are concerned with subsequent fertility, a retrospective study conducted by the European Group for Blood and Marrow Transplantation (EBMT), however, determined that the overall post-transplantation pregnancy rate is as low as 0.6% [175], indicating that HCT survivors rarely become parents [176]. Therefore, the high incidence of irreversible gonadal dysfunction observed after allogeneic or autologous HCT has become a crucial issue for both male and female patients, as gonadal failure often results in incomplete sexual development at puberty (secondary sex characteristics), and a decrease in physical wellbeing, bone density, quality of life, and sterility and sexual dysfunction in adulthood [177]. Risk factors for the development of gonadal function post HCT are given in Table 98.8, and the incidence of complications in Table 98.9. Male Of the components of the hypothalamic-pituitary-gonadal axis, the testes are the most vulnerable to damage caused by chemotherapy and/or irradiation (Fig. 98.13) [18]. Leydig cells comprise the major endocrine component of the testes, and testosterone is their primary secretory product. Testosterone is responsible, either directly or indirectly, for embryonic differentiation of the male external and internal genitalia, secondary sexual development at puberty, and maintenance of secondary sexual characteristics and libido, as well as the initiation and mainte-

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Table 98.8 Risk factors for gonadal dysfunction following hematopoietic cell transplantation (HCT) Female sex Males undergoing allografted HCT Older age at transplantation (particularly male patients, over the long term) Use of total body irradiation Single-dose radiation Cranial irradiation Increased or cumulative doses of alkylating agents

Table 98.9 Gonadal function in male and female patients following hematopoietic cell transplantation Females

Ovarian failure

Males

Auto (n = 22)

Allo/GVHD+ (n = 19)

21 (95%)

19 (100%)

Auto (n = 6)

Allo/GVHD+ (n = 25)

Clinical hypergonadotropic hypogonadism

1 (17%)

5 (20%)

Subclinical hypogonadism

1 (17%)

9 (36%)

Seminiferous tubule damage (elevated FSH)

3 (50%)

18 (72%)

Allo, allogeneic bone marrow/peripheral blood stem cell transplantation; Auto, autologous bone marrow/peripheral blood stem cell transplantation; FSH, folliclestimulating hormone; GVHD, graft-versus-host disease. Adapted with permission from Somali et al. [178].

nance of spermatogenesis. DHT is a biologically active metabolite of testosterone that is formed primarily in the prostate, testes, hair follicles, and adrenal glands via conversion by 5α-reductase. DHT is three times more potent than testosterone, while testosterone is five to 10 times more potent than adrenal androgens [179]. The seminiferous tubules comprise the remainder and bulk of the testes, and are responsible mainly for spermatogenesis during the reproductive years. Hypothalamic GnRH triggers the release of the gonadotropins FSH and LH from the pituitary; in men, LH stimulates the Leydig cells to produce testosterone, and FSH stimulates the Sertoli cells to secrete the glycoprotein hormone inhibin (Fig. 98.13). Biologically active hormones consist of two subunits: the association of an α subunit with either a βA or βB subunit comprises inhibin A and inhibin B, respectively, whereas two β subunits together create activin A (two subunits of βA), activin B (two subunits of βB) or activin AB (one βA subunit and one βB subunit). Inhibin B levels are reduced in patients with spermatogenic failure, and there is a reciprocal relationship between inhibin B and FSH levels (notably with chemotherapy-induced spermatogenic injury) [181,182]. Thus, inhibin B is likely a physiologic regulator of pituitary FSH secretion (along with gonadal steroids). In addition to their effects on the pituitary gland, testosterone and inhibin also feed back to the hypothalamus to decrease GnRH secretion. Lack of testicular secretion of testosterone and inhibin and the subsequent reduction in their negative feedback effects on the hypothalamic-pituitary axis results in increased secretion of gonadotropins; thus, serum FSH crudely reflects the status of the seminiferous epithelium and inhibin secretion, while LH levels are representative of Leydig cell status.

Chapter 98

Percentage of patients

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100 90 80 70 60 50 40 30 20 10 0 1

2

3 CY

4

5 6 7 8 9 Years after BMT CY + TBI/TAI CY + BU/Thio

10

Fig. 98.14 Recovery of spermatogenesis after bone marrow transplantation (BMT). Percentage of patients according to the conditioning regimen. CY, cyclophosphamide; CY + TBI/TAI, cyclophosphamide plus total body irradiation or thoracoabdominal irradiation; CY + Bu/Thio, cyclophosphamide plus busulfan or thiotepa. (Reproduced from [186], with permission.)

Fig. 98.13 Neuroendocrine control of testicular function. +, positive influence; −, negative influence; ABP, androgen-binding protein; DHT, dihydrotestosterone; E2, estradiol; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; T, testosterone. (Reproduced from [180], with permission.)

Most preparative conditioning regimens for HCT include high-dose combination alkylating agents and TBI, which are well known to induce gonadal dysfunction directly – namely primary testicular failure (hypergonadotrophic hypogonadism), with a 50% reduction in testicular volume [183], severe germ cell injury, and even Leydig cell insufficiency [176,184]. Interestingly, Leydig cells are more resistant to both the toxic effects of chemotherapy and radiation (up to 24 Gy) than the germinal epithelium [178], which is completely destroyed at radiation doses of 8 Gy [185]. Although less common, gonadal dysfunction may also be initiated by central damage to the hypothalamic-pituitary region as a result of cranial irradiation, which causes impaired gonadotropin secretion [15]; in children, this can lead to delayed onset of puberty, although, in some cases, premature sexual development has also occurred [18]. Hypopituitarism or hyperprolactinemia with subsequent hypogonadism has been reported in recipients of TBI [183], again reflecting radiation-induced hypothalamic defects. Although the overall incidence of azoospermia after HCT was found to be 70.3% in one study [186], the highest incidence of azoospermia following HCT was observed in

patients prepared with CY plus TBI or thoracoabdominal irradiation (85.4%), and recovery of spermatogenesis never occurred before the fourth year post transplant, but did occur as late as 9 years [186]. Chemotherapy alone can also produce gonadal failure [79], as highdose chemotherapy invariably provides direct insults to both germ cells and Leydig cells, with concomitant elevation in gonadotrophins [183]. Further, a transient increase in sperm aneuploidy within 100 days after chemotherapy has been documented [186]. Previous administration of chemotherapy also has a cumulative effect on gonadal toxicity [178]. Even monotherapy using a nonhigh-dose protocol, such as fludarabine alone, does not spare the gonads [176], although melphalan-based protocols are less gonadotoxic than TBI-based, BU/CY or CY-alone regimens. Other drugs associated with germinal depletion include chlorambucil, nitrogen mustard, procarbazine, and nitrosoureas [187]. Recovery of fertility, however, was found to be more likely after chemotherapy-alone conditioning regimens than after TBI-containing regimens [188], as recovery was seen in 90% of patients conditioned with CY alone, in 50% of patients with CY plus BU or thiotepa, and in just 17% of patients with CY plus TBI or thoracoabdominal irradiation (Fig. 98.14). Further, sperm quality was found to be within the normal range in the majority of patients following CY, whereas it was consistently severely impaired in patients who received irradiation or two alkylating agents [186]. Temporary falls in testosterone levels in the first 6 months post transplant have recently been reported, but these are thought to be insignificant [181]. A widely utilized non-TBI regimen prior to allografting is BU/CY [181], but when compared with carmustine, etoposide, cytarabine, and melphalan (BEAM) and carmustine, etoposide, CY, and melphalan (BECYM), it also appears to be the most toxic [178]. Current proposed recommendations after BU/CY have been to advise sexually active heterosexual male patients that spermatogenesis and fertility may occur within 2–5 years post transplant [181]; however, sperm cryopreservation prior to transplant is still recommended. One investigative group performed annual semen examinations beginning 1 year post transplant for patients in whom the result was clinically relevant, and examinations were continued until documentation of spermatogenesis. In the minority of patients who remained persistently azoospermic at 5 years, semen analysis was also offered twice a year to detect late recovery [181]. In addition to the type of pretransplant therapies employed, the degree of gonadal dysfunction after HCT is dependent on patient-related factors. For example, older age at transplantation appears to worsen gonadal

Endocrine Complications Following Hematopoietic Cell Transplantation

outcome after HCT in men [20,178,184]; however, the majority of published studies describing the impact of high-dose chemotherapy plus TBI on gonadal function have evaluated patients who received transplants as adolescents or young adults. Although the data on gonadal function after HCT performed in childhood remain limited, most reports suggest that prepubertal boys experience spontaneous, normal puberty, and sexual development, and further demonstrate age-appropriate plasma testosterone levels following HCT, indicating that younger male patient age at transplantation exerts a protective effect [20,189–191]. Some reports, however, showed impaired Leydig cell function in approximately half of boys who underwent HCT prior to puberty [192]. The reasons for these differing results remain unclear, but could be related to variations in exposure to alkylating agents, and the total dose and/or fractionation schedule of irradiation. Other studies have suggested that the young Leydig cells may have increased vulnerability to radiation-related damage, as evidenced by elevated LH levels, although overt Leydig cell failure was rare [185]. Germ cell dysfunction, as indicated by elevated FSH levels, however, is more common [185], given the known sensitivity of the germinal epithelium to chemotherapy and radiation. Long-term gonadotropin elevation after HCT despite prepubertal age at transplantation was thought to reflect a probability of future gonadal dysfunction (i.e. oligospermia and infertility) [185]; however, further studies are needed to support this conclusion. The type of HCT itself seems to exert an influence on gonadal outcome, as well, since a higher incidence of gonadal toxicity is seen in allogeneic transplantation when compared with autologous transplantation; elevated FSH (indicative of seminiferous tubule damage) was observed in allografted males (72%) when compared with autografted males (50%) who were treated with chemotherapy-based conditioning regimens [178]. Similarly, elevated LH levels (indicative of Leydig cell damage) were seen in 60% of allografted males and 17% of autografted males after HCT [178]. The degree to which GVHD may contribute to or sustain gonadal toxicity independent of conditioning regimen remains to be evaluated, as sperm counts in long-term HCT survivors tend to be lower in GVHD patients [193]. It has been suggested that intratesticular damage may occur during an acute graft-versus-host reaction due to infiltration of alloreactive donor T cells and autoantibody production against germinal epithelium. Evidence to support these hypotheses, however, is currently lacking [194]. Further, one study noted that 50% of autografted male patients and 95% of autografted female patients developed gonadal insufficiency despite the absence of GVHD [178]. Therefore, additional studies are needed to clarify the role of GVHD in the development of gonadal dysfunction after HCT. Evaluation of male gonadal dysfunction post HCT Semen analysis currently remains the only reliable method of assessing spermatogenesis [181]. In boys too young for semen analysis, however, measurements of plasma inhibin B may help to assess function of the testicular seminiferous tubules [177]. The measurement of both inhibin B and FSH levels has been reported to be a more sensitive marker than either alone for detecting absent or severely impaired spermatogenesis, although it is not always discriminatory [181]. In adult men, the most common symptoms of hypogonadism are diminished libido and erectile dysfunction. Gynecomastia, however, may also be a presenting feature; the incidence of this sign in the HCT setting is unknown. Estimates of gynecomastia in the general population range from as little as 5–9% [195] to as high as 40% [196], but these are likely to be under- and overestimations. Primary testicular failure with hypergonadotrophic hypogonadism is likely the key cause of gynecomastia, but additional etiologies include secondary hypogonadism

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with hyperprolactinemia and drug-induced gynecomastia. For example, histamine (H2) blockers, such as cimetidine and ranitidine, are noted to be associated with sexual dysfunction and gynecomastia [183]. Cyclosporine can cause transient breast enlargement through altered target organ sensitivity, but, if present, it tends to be concurrent with hypertrichosis (not hirsutism) [183]. Although admittedly uncommon in recipients of high-dose chemotherapy or chemoradiotherapy, the occurrence of gynecomastia should be considered pathologic outside of newborns, puberty, and old age. In general, gonadal testing and consideration for treatment are recommended based on symptoms (Fig. 98.15). A male fertility evaluation should include a careful history and physical examination, as well as a minimum of two semen analyses, which are recommended at the time of presentation, even if it has been less than 1 year of unprotected intercourse – particularly in the case of advanced female partner age (greater than 35 years) or the presence of known male risk factors (such as exposure to chemotherapy or radiation). Semen analysis is a common, convenient measure of assessing the male, and should precede any invasive tests of the female. Ejaculate volumes between 2 and 6 mL and sperm concentrations of greater than 15–20 million/mL with more than 50% motile sperm and more than 50% oval forms are considered normal [197]. An initial endocrine evaluation should include at least a plasma testosterone and FSH, but frequently LH is included as well. This should be performed if there is (1) an abnormally low sperm concentration, especially if less than 10 million/mL, (2) impaired sexual function, or (3) other clinical findings suggestive of hypogonadism. A reduction in testosterone levels is usually a reflection of lower testosterone secretion by the testis. Elevated FSH typically indicates significant seminiferous tubule damage, while elevated LH indicates Leydig cell damage. If these hormone measurements are borderline, and the patient has clinical symptoms or signs of hypogonadism, a free or bioavailable testosterone can be measured to exclude patients with low free testosterone and elevated sex hormone-binding globulin (SHBG), as in elderly men. Management of male gonadal dysfunction post HCT In its simplest form, the management of gonadal dysfunction following HCT should be directed at preventing potential damage by reducing injury wherever possible, such as with the use of less toxic conditioning regimens [198]. TBI and alkylating agents should be avoided if possible. Patients should be counseled prior to transplant to discuss possible and appropriate strategies to preserve fertility, including cryopreservation of sperm [178,184,199]. Failing preventive measures, HRT may be offered to men to relieve some of the side-effects of hypogonadism, as well as for the preservation of bone mass density. In children, there are two phases to this treatment: a low dose to accelerate height growth during the growing period, without excessive bone age progression, and then an increase in dose to the adult level at the end of the growth spurt. Sex steroid replacement therapy should be stopped for 2 months at regular intervals in order to check for gonadal recovery [18,178]. Annual long-term follow-up is necessary, even for those few patients with normal hormonal basal values after HCT, due to the possibility of future gonadal dysfunction due to diminished gonadal reserve [178]. The risks of HRT in men include prostate hypertrophy and prostate carcinoma. Side-effects of treatment include edema, male pattern baldness, nausea, tachycardia, low high-density lipoprotein cholesterol, and cholestatic liver enzyme abnormalities. Follow-up evaluations should include annual digital rectal exams and measurements of prostatespecific antigen, liver function, lipid profile, and hemoglobin.

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Fig. 98.15 Algorithm for the evaluation of the male patient presenting with hypogonadal symptoms and/or infertility following hematopoietic cell transplantation. FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; N, normal; PE, physical examination; T, testosterone. (Adapted from [197], with permission.)

Female The female ovaries serve a dual purpose: the storage of germ cells, and the production and secretion of hormones vital for the production and development of secondary sexual characteristics. The major source of hormones is the maturing follicle, where theca cells produce androgens, and granulosa cells produce estrogens. Other stromal cells (secondary interstitial cells and hilum cells) also contribute to androgen production, and are primarily involved in ovarian hormone production during menopause. The adrenal cortex also produces androgens. In females, approximately half of testosterone production stems from the ovaries, and the other half derives from the adrenal gland and peripheral conversion of androgen precursors, such as DHEA. Hypothalamic GnRH regulates the differential gene expression that leads to the production and release of pituitary gonadotropins FSH and LH, and the interactions of these pituitary hormones with gonadal steroids serve to regulate the female menstrual cycle (Fig. 98.16). The human menstrual cycle is divided into three functional phases: follicular, ovulatory, and luteal. The degradation of the corpus luteum of the preceding cycle triggers the events of the new follicular phase (Fig. 98.17). Plasma FSH concentrations rise 1 day before or with the

commencement of menses, stimulating an increase in the number of granulosa cells, as well as potentiating granulosa aromatase enzyme activity, and increasing the conversion of androgens to estrogens. Meanwhile, plasma LH acts on thecal cells to stimulate production of testosterone and androstenedione. Thus, the accumulation of intrafollicular 17β-estradiol (E2) augments the ability of FSH to induce LH receptors on the granulosa cells, leading to the stimulation of progesterone production during the preovulatory and luteal phases. The sequential ovarian secretion of E2 and progesterone during the late follicular phase acts to regulate the release of gonadotropin in the ensuing phase of the cycle, but with a biphasic effect: there is an initial fall in plasma FSH, and maintenance of E2 at approximately 300 pg/mL for slightly longer than 2 days stimulates the preovulatory surge of LH and FSH, which lasts for about 48 hours. The midcycle surge of LH also inhibits both androgen and estrogen production, but the mechanisms of this LH-mediated change are not completely understood. There is some evidence from animal studies to suggest that prolactin is involved in the maturation and luteinization of granulosa cells by helping to maintain the LH receptor acquired by granulosa cells under the influence of FSH, thereby contributing to the maintenance of progesterone production during the luteal phase.

Endocrine Complications Following Hematopoietic Cell Transplantation

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Fig. 98.17 The endocrinology of the normal menstrual cycle in women. Data are mean + standard error of daily serum concentrations of folliclestimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, and immunoreactive inhibin in women with normal cycles. Note the secondary rise in plasma FSH in the late luteal phase (approximately 2 days before menses). (Reproduced from [201], with permission.)

Fig. 98.16 Neuroendocrine control of ovarian function. Diagrammatic representation of the hypothalamic-pituitary-ovarian interaction in the control of the human menstrual cycle. The pulsatile release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), mediated by hypothalamic gonadotropin-releasing hormone (GnRH), functions to induce ovarian theca and granulosa cell steroidogenesis and follicular maturation. The theca cell is endowed with LH receptors, which mediate LH-induced P450 steroidogenic enzymes and the synthesis of androgens and function as targets of modulators from the granulosa cells (paracrine). The multifunctional granulosa cell has the capacity to generate FSH-mediated LH receptors, P450c17 enzyme, and progesterone (P4) synthesis. In addition, the granulosa cell has the ability to produce locally a variety of autocrine/paracrine regulators, such as growth factors and tissue plasminogen activator and inhibitors (tPA/tPAI). P450 Arom, P450 aromatase. (Reproduced from [200], with permission.)

Like their counterparts in men, inhibins/activins are a group of ovarian proteins with varying structure and functions. The production of inhibin by the granulosa cell is under stimulatory control by FSH, an effect that is augmented by E2, LH, IGF, and several other growth factors [202]. At the level of the pituitary, it is thought that inhibin selectively sup-

presses FSH secretion, while activin stimulates it. Neither inhibin nor activin appears to influence LH secretion significantly. At the level of the ovary, however, inhibin acts synergistically with LH to stimulate androgen production by the thecal cells, whereas activin has the opposite effect. Further, inhibin may suppress FSH stimulation of aromatase activity, and activin may stimulate the production of estrogen and progesterone during follicular maturation. Activin may also enhance acquisition of LH receptors by the granulosa cell. Although the risk of gonadal failure is high in all individuals who undergo HCT, women generally experience higher rates of failure than do men [20,178]; radiation doses as small as 4 Gy have been observed to destroy approximately 50% of oocytes [203]. In one small study, azoospermia or oligospermia was seen in 90% of male patients at a mean of 4.5 years post HCT, while ovarian insufficiency affected 95–100% of females who had undergone transplantation [178]. As with men, women have a high risk of developing hypergonadotropic hypogonadism after HCT, but the risk of irreversible gonadal toxicity after BU/CY, with no resumption of menses, is almost inevitable in women [181], as ovarian insufficiency was observed in 100% of females who underwent allogeneic transplantation, and 95% who underwent autologous transplantation [178]. In contrast, there is a higher incidence of fertility recovery in females compared with males after TBI, BEAM, a regimen of CY, BCNU, and etoposide (CBV), and high-dose melphalan [181]. The greatest chance

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of recovery was observed in cases of nonhigh-dose therapy (e.g. mustine, vincristine, procarbazine, and prednisone [MOPP]) for Hodgkin’s lymphoma [181]. Similarly, the risk of permanent infertility becomes lower if CY is used alone [178]. In general, fractionation of radiation reduces the risk of infertility when compared with single-dose radiation [177,178,199]. Over the long term, when the use of fractionated TBI was considered alone, one study found that the ovaries were actually less sensitive to radiation than the testes are, with only 37% of females demonstrating permanent ovarian dysfunction more than 10 years after TBI administration, compared with 87% of males with at least indirect signs of germinal dysfunction [204]. Older age at HCT is associated with a higher frequency of ovarian failure [177], as nearly 100% of women over the age of 12 years who received TBI developed ovarian failure, likely due to the decreased number of primordial follicles [15]. In contrast, prepubertal girls treated with chemotherapy and hyperfractionated TBI experienced spontaneous puberty and menarche in 56% of cases [189]. Whereas doses of 600 cGy may produce irreversible ovarian failure in women greater than 40 years of age, doses in excess of 2000 cGy have to be given before the same result is seen in girls treated during childhood and adolescence [189]. In general, endocrine ovarian failure is irreversible in adult women, but younger women – particularly prepubescent girls – have a better opportunity for recovery of gonadal function; recovery was noted in 54% of patients younger than 26 years receiving only CY [178]. The parameter of age, however, seems to be less significant in female patients over the long term, as follow-up beyond 10 years has demonstrated a similar probability of ovarian dysfunction in females who were administered TBI prior to puberty as well as post menarche [78]. Faraci et al. documented the development of amenorrhea in 100% of females receiving TBI at some time after puberty. Some patients were able to recover from the injury, sometimes more than 10 years after TBI [78]; this was corroborated by other studies that demonstrated recovery of ovarian function in 10–25% of young women after TBI [189]. Regardless of recovery, however, a higher risk of earlier menopause has been suggested with exposure to both alkylating agents and radiation below the diaphragm [169,189], even in girls treated during a prepubertal phase [78]. It is unclear what allows some patients to remain fertile when so many other patients become sterile under similar conditions [187]. Shielding of the gonads is generally not performed after HCT, since the gonads often serve as sanctuaries for disease, particularly in lymphoid disorders such as ALL. Even if shielding is employed, it is less successful in preserving female fertility than male fertility. No effective means of protecting the ovaries during high-dose chemoradiotherapy has yet been developed. In conclusion, larger studies are required in order to evaluate the incidence of recovery of fertility in females after various conditioning regimens and the effects of variables such as prior treatment and age at transplantation. Until these results become available, it is recommended that post-transplant ovarian function be evaluated in all premenopausal women prior to transplant, particularly if BU/CY will be used. Counseling regarding fertility preservation prior to transplant is necessary; options may include cryopreservation of fertilized embryos, ovarian tissue, and oocytes [181]. Fertility and offspring Many animal studies have investigated the possible adverse effects of radiation and chemotherapy on the offspring of treated patients [186]. The risk of genotoxicity is more significant in cases when assisted reproductive technology with intracytoplasmic sperm injection is used. Although the incidence of congenital anomalies recorded in live-born infants spontaneously conceived after treatment for lymphohematologic

malignancies is comparable to that reported in the general population [205], possible long-term radiation-related genetic damage of the spermatozoa and oocytes cannot be excluded [186]. Long-term follow-up of both pregnancies and children of HCT survivors is needed, particularly with regard to the occurrence of miscarriages, low birth weights, congenital malformations, and delayed developmental milestones [205], as the evidence seems to be conflicting. In the EBMT study, pregnancy-induced hypertension was significantly higher in allograft recipients (15%), compared with 8% of the normal population. Yet, the rate of congenital malformations, developmental delay, and malignant disease was no higher in the offspring of HCT recipients than in the control population. The 10% miscarriage incidence was also similar to the control population [198]. Some studies have even found that, despite the significantly diminished rate of successful conception after HCT, if pregnancy does occur, the outcome is likely to be favorable [184]. This is in sharp contrast to previous work [205] that suggested female HCT survivors who received TBI after puberty were more likely to have spontaneous abortions and deliver low birth weight and preterm infants, due to fibrosis and alterations in the musculature and vascularization of the myometrium, particularly as a result of abdominal irradiation for solid tumors [206]. Holm et al. were the first to report a reduction in both uterine and ovarian size shortly after TBI and HCT administered in female children, despite HRT or spontaneous pubertal development, citing that prescribed doses of HRT, while sufficient to induce bleeding and suppress the onset of premature menopause, were not adequate to generate normal uterine growth [206]; this is significant, as these smaller, irradiated uteri may not be able to grow and expand as required during pregnancy. Further, decreased ovarian volume was found to be reflective of decreased markers of ovarian follicles, compared with ovarian follicles found in healthy females entering puberty; small ovaries have been associated with poor response to in vitro fertilization (IVF) [206]. While there is a theoretical risk of immunocompromise and relapse, the fact of pregnancy is unlikely to alter the history of the primary disease, and is thus not contraindicated in stem cell transplants recipients for hematologic, endometrial or even breast cancers [198]; however, scattered case reports have shown evidence of leukemic relapse during pregnancy, although it is unknown to what extent these patients were already at risk for recurrence independent of the pregnancy [198]. Nevertheless, it is recommended that patients with chronic myelogenous leukemia should delay pregnancy for at least 2 years after HCT, providing that they remain Philadelphia chromosome negative and bcr/abl transcriptase negative [198]. Evaluation of female gonadal dysfunction post HCT Failure to progress through puberty in a timely fashion should prompt the referral for a full endocrinologic evaluation [20]. Prepubertal girls should be monitored closely for the onset of puberty and gonadal dysfunction suspected if not experienced by the age of 12–13 years. In contrast, the association between premature sexual development and both low- and high-dose central nervous system irradiation in both boys and girls is now well established [189]. In adult women, the assessment of gonadal function after HCT is difficult, as the ovaries and ova are not easily accessible for study, and E2 levels are highly variable across individuals and even within the same individual [187]. The implicit markers of female fertility are regular menses and the ability to become pregnant; however, the initial investigation should start by ruling out thyroid hormone abnormalities, and the possibility of current early pregnancy. Once this is achieved, the pursuit of further evaluations will depend upon whether the patient is presenting with complaints of infertility or with oligomenorrhea and symptoms of estrogen deficiency (Fig. 98.18). Patients presenting with infertility

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Fig. 98.18 Algorithm for the evaluation of the female patient presenting with hypogonadal symptoms and/or infertility following hematopoietic cell transplantation. E2, estradiol; FSH, follicle-stimulating hormone; N, normal.

should be evaluated with measurement of serum inhibin B during the mid-follicular phase (days 5–8) and/or progesterone during the midluteal phase (days 19–22) of the menstrual cycle [206]. Low inhibin B levels suggest poor ovarian follicular reserve, and low progesterone levels could indicate poor luteal phase function. Such subjects and those with oligomenorrhea should be further evaluated with measurements of serum E2, FSH, and prolactin. Ovarian failure is clinically represented by amenorrhea and menopausal symptoms, as well as low levels of serum estrogen and high levels of serum FSH and normal or low levels of prolactin. In women presenting with decreased sexual libido (hypoactive sexual desire), serum levels of testosterone and DHEA should also be measured. Reduced serum levels of total and/or free testosterone may be associated with ovarian failure, and, in some cases, reduced DHEA levels may reflect adrenal insufficiency. IVF using superovulation and embryo cryopreservation is a common treatment for infertility in female HCT recipients, as it has the most promising results, despite some limitations: requirements include the presence of a functional ovary and the time to complete the necessary number of IVF cycles to harvest sufficient eggs prior to the commencement of the cytotoxic regimen, which may be difficult to achieve in some patients without delaying life-saving treatment [198]. Oocyte cryopreservation and GnRH treatment for the suppression of the pituitarygonadal axis remain more experimental treatments, and further studies are needed to ascertain their usefulness. Management of female gonadal dysfunction post HCT As with men, the management of gonadal dysfunction following HCT in women should be aimed at preventing potential damage by reducing injury wherever possible, such as with the use of less toxic conditioning regimens [198]. TBI and alkylating agents should be avoided if possible. Patients should be counseled prior to transplant to discuss possible and

appropriate strategies to preserve fertility, including cryopreservation of oocytes, ovaries, and embryos, and the use of a surrogate uterus [178,184,199]. As with men, HRT may be offered to females to relieve some of the side-effects of hypogonadism, as well as for the preservation of bone mass density. The phases of HRT in girls follow those of young boys, described above. In girls, however, the latter phase is given cyclically in order to induce regular menstruation. Additionally, Holm et al. have recommended that HRT be given to stimulate normal uterine growth. Sex steroid replacement therapy should be stopped for 2 months at regular intervals in order to check for gonadal recovery [18,178]. Annual long-term follow-up is necessary, even for those few patients with normal hormonal basal values after HCT, due to the possibility of future gonadal dysfunction due to diminished gonadal reserve [178]. Women who are pre- or perimenopausal at the time of transplant may be offered HRT (estrogen alone after hysterectomy, or estrogen plus progestin if the uterus is intact) to maintain libido, sexual function, and bone density; however, libido is often only partially corrected by HRT in women. HRT may have beneficial effects on cognition, but there are increased risks of cardiovascular complications, such as stroke, emboli or deep venous thromboses. Vaginal effects of GVHD may result in strictures and synechiae. Supplemental vaginal lubrication is of importance and should be addressed by the treating physician [20]. Annual mammography and cervical cytology are recommended.

Sexual dysfunction While sexual dysfunction may be considered a secondary issue in comparison to the primary malignant disease, sexual activity is a major contributor to quality of life in these patients. HCT patients have been found to be similar to conventional chemotherapy patients in regards to

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psychosexual functioning, and both groups exhibited significant differences in all aspects of sexuality when compared with healthy control subjects – particularly in regards to body image [207]. When given the opportunity, 35% of the sample detailed negative changes in sexual function after transplantation [208]. Wingard et al. reported that 22% of male HCT survivors were dissatisfied with their sexual lives, with 24% reporting erectile dysfunction and 13% ejaculatory dysfunction [209], while Molassiotis et al. noted that half of patients were sexually dissatisfied to some degree [210]. Across both sexes, lack of sexual interest is reported at 3–11%, arousal problems for 9–15%, and orgasm infrequency or inability for 4–12% [208]. Unmarried and single patients seem to be at greatest risk for sexual dissatisfaction [210], and women appear to be affected more frequently than men [211], with 17–35% reporting sexual problems [208]. Sexual difficulties in HCT survivors typically have an acute onset, and occur immediately after therapy or during the recovery process [212]. Body image and sexual behavior may be impacted as a result of physical gonadal dysfunction due to treatment with TBI and alkylating agents [213]. Adrenal suppression by corticosteroids may also cause a reduction in DHEA levels [169]. Sexuality may be further influenced by the occurrence of fatigue, decreased stamina, alopecia, skin changes due to GVHD, weight changes, anxiety, depression, and other social changes [213]. Barlow suggested that the main causes of sexual dysfunction may be anxiety and cognitive interference [214]. Other stress factors, such as financial, familial or occupational concerns, may further contribute to sexual dysfunction [213]. The evaluation of HCT survivors presenting with sexual dysfunction depends not only upon the thorough understanding of the organic causes of sexual dysfunction, but also on the ability of the physician to collect and interpret the patient’s history, physical findings, and all pertinent laboratory and psychosocial data. As with patients in the general population, many precipitating factors can promote the onset of sexual dysfunction; unreasonable expectations, random failure, discord in the relationship, dysfunction in the partner, infidelity, reaction to organic disease, depression, and anxiety are some of the most commonly encountered factors. In contrast, perpetuating factors maintain, exacerbate or prolong sexual dysfunction, and these may include performance anxiety, guilt, poor communication, loss of attraction between partners, and impaired self-image. Affective disorders or character pathology can lead to both the precipitation and perpetuation of sexual problems. Once a detailed history and physical examination are completed, the focus of the medical investigation can then be shifted towards confirming the underlying pathophysiologic abnormalities and devising an appropriate treatment plan. For more detailed information on this subject, please refer to Chapter 35. Male As noted above, male HCT survivors are typically less affected in regards to sexual function than female HCT survivors, and, if affected, their problems appear to resolve spontaneously. One study noted that men who had no known sexual complaints prior to transplantation who did report sexual dysfunction at 1 year after transplant were found to be problem free 3 years after transplant [208]. Only 15% of HCT patients in this study reported arousal problems prior to transplantation. Evaluation of men with sexual dysfunction post-HCT When taking the history, it is important that the patient himself understands the characteristics that distinguish the most common sexual complaints: loss of libido, erectile dysfunction, and ejaculatory disturbance. The time, manner of onset, and consistency and severity of the course all require establishment. As noted above, a meticulous drug history is

also essential, due to the large number of pharmacologic agents related to HCT that are frequently associated with sexual dysfunction. The questions of cigarette smoking, alcohol intake, and illicit substance abuse should not be neglected in HCT survivors. Several wellestablished and validated self-administered questionnaires (e.g. International Index of Erectile Function and the Derogatis Interview for Sexual Functioning-Self Report) have been developed and used to assess the frequency and nature of sexual dysfunction in men in general, and some have been used to assess the adequacy of response to therapeutic modalities. Questionnaires can be a useful and time-saving way to initiate discussion on sexual dysfunction in the clinical setting. Although valuable in detecting the presence of sexual problems and in distinguishing between psychogenic and organic etiologies, questionnaires do not usually yield information that is sufficient for determining the underlying pathophysiology. There are many reasons that HCT in particular may cause sexual dysfunction in male patients. The underlying hematologic disease (e.g. sickle cell anemia) may cause local penile abnormalities and subsequent erectile dysfunction (sickle cell), as can microvascular disease from treatment-associated complications, such as hyperlipidemia. Neuropathy as the result of TBI and chemotherapy or the development of diabetes may additionally cause sexual dysfunction. Glucocorticoid treatment may cause weakness and fatigue, and many other HCT-related pharmaceutical agents are known to cause sexual dysfunction. The development of psychologic issues related to self-esteem that are specific to HCT may precipitate dissatisfaction with one’s sexual prowess and create partner discord, including despair regarding the underlying disease and its treatment, a resultant loss of work and livelihood, possible disfigurement due to GVHD of the skin, the loss of body/muscular mass, and the occurrence of new and unpleasant body odor related to medications. Decreased libido should alert the clinician to three probable causes: endocrinopathy, affective disorder or relationship discord. A history of frequent strong erections in any circumstances (during foreplay, fantasy or masturbation, with another partner or upon awakening) likely indicates that the endocrine, vascular, and neurologic systems are intact, and that the erectile dysfunction is predominantly psychogenic. Conversely, historical data relating the presence of decreased erectile turgidity during noncoital activities are highly suggestive of an organic etiology. A report of firm sustained erections during foreplay that are lost after intromission or upon initiation of pelvic movements, however, might suggest either a psychogenic etiology or a vascular problem (pelvic steal syndrome). A history of delayed or retrograde ejaculation may imply a neuropathy or an adverse drug effect. Premature ejaculation, on the other hand, is more compatible with a psychogenic dysfunction. Finally, it must be remembered that the absence of orgasmic sensations in patients with normal erectile and ejaculatory functions is almost always due to psychogenic etiology, whereas failure of detumescence is usually organic in nature; the latter should direct investigations towards ruling out local penile etiologies (neurologic or hematologic) [215]. During the physical examination, every effort should be made to elicit physical signs of pathology that may be suspected based on information obtained during the clinical history. Evidence of chronic, systemic diseases (hepatic, renal, cardiovascular, endocrine, granulomatous, and neoplastic) must be ruled out, and, if present, the current state of disease control should be determined. In addition to the general and systemic evaluations, a detailed assessment of gonadal function, vascular competence, neurological integrity, and genital organ normalcy should be performed on every patient [215]. A careful vascular assessment should include the palpation of ankle, femoral, and dorsal penile arteries. In a specialized clinic, penile systolic blood pressure may be determined with a 3 cm blood pressure cuff placed around the base of the penis and a Doppler stethoscope positioned

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over each cavernosal artery. The penile systolic occlusion pressure is then obtained and compared with that of a brachial artery, and thus a penile–brachial index is derived; values greater than 0.7 are considered normal [216]. Neurologically, the patient should be evaluated for the presence of motor deficits, changes in deep tendon reflexes, loss of sphincter tone or a decrease in light touch or pinprick sensations, particularly in the genital area. Penile temperature sensation testing could also be done with the use of alcohol swabs. In addition, the bulbocavernosus reflex should be elicited by squeezing the glans penis and assessing the evoked contractions of the external anal sphincter or bulbocavernosus muscles. This reflex response is clinically detectable in 70% of normal males. The more sensitive method of penile vibration perception threshold testing may confirm results of the bulbocavernosus reflex [217]. Laboratory testing should focus on the endocrine aspects of male sexual dysfunction (i.e. the state of gonadal hormones and thyroid hormones), as these are particularly vulnerable to derangement after HCT (Fig. 98.15). Aging, GH deficiency, hyperthyroidism, and liver disease can all be associated with an increase in SHBG, and consequently a greater reduction in bioavailable than in total testosterone. On the other hand, obesity, hyperinsulinemia, hypercortisolemia, and hypothyroidism may be associated with decreased SHBG, and thus more suppression in total testosterone than in bioavailable testosterone [215].

not prevented or resolved at 1 year after transplant, there was an 87% probability that the problems would still be present at 3 years after transplant, despite HRT [208]; however, a substantial number (63%) of women entered transplantation with one or more sexual problems [208]. After HCT, specific sexual complaints in female survivors include dyspareunia (2–8%) and insufficient vaginal lubrication. Prevalence rates for these particular problems vary widely in the general population, depending on the sample [208]. As with male patients, self-image issues that arise out of the fact of HCT and its treatment may contribute significantly to decreased psychologic and sexual wellbeing.

Management of men with sexual dysfunction post HCT

Management of women with sexual dysfunction post HCT

As sexual dysfunction after HCT is frequently multifactorial in origin, these different etiologies should be distinguished, and treatment should be given to address each of the underlying problems accordingly. If hypogonadism is the cause, hormone replacement should be given (see above). Androgen replacement therapy is usually not required with sexual dysfunction associated with normal bioavailable or free but depressed total testosterone. Phosphodiesterase-5 inhibitors have been found to be very effective for men with erectile dysfunction in the general population. Several nonsurgical treatment options for erectile dysfunction have proven to be safe and efficacious, regardless of disease etiology, and may be prescribed in the primary care setting (e.g. oral or intraurethral medications, and vacuum constriction devices) [218]. Antioxidant agents such as vitamin E, Korean red ginseng, and α-lipoic acid have shown promise in animal studies, but human research is needed. Psychiatric/psychosocial support may improve a patient’s situation and allow for a better quality of life. Unmarried and single HCT survivors should be targeted for early prevention or therapy [210]. Appropriate intervention will involve identifying and addressing all relevant predisposing, precipitating, and perpetuating factors, and thus evidence for the presence of any of these psychologic or situational conditions should be carefully assessed. Moreover, it should not be forgotten that the presence of an organic dysfunction does not preclude the possibility of a coexisting psychogenic factor, and vice versa. Such omissions could lead to diagnostic difficulties, as well as to therapeutic failures.

While there are fewer available treatment options for female sexual dysfunction than, for example, erectile dysfunction, a recent report suggests that sildenafil (Viagra) may improve sexual arousal disorder in premenopausal women [219]. Management of lower urinary tract symptoms, such as bladder control/continence and the prevention and treatment of infections, can also improve sexual function for both men and women [220,221]. The treatment for gonadal dysfunction and possible infertility post HCT may have a large impact on patient sexual behavior [210]. It is recommended that counseling regarding the potential for reduced sexual satisfaction and other sexual difficulties should occur prior to transplantation. Clinicians should be aware of the additional possibility of lateoccurring problems with psychosexual maladjustment post HCT. HRT should be offered, although, as noted above, libido is only partially corrected by standard doses of estrogen and progesterone in women; further studies are needed to address the true endocrinologic requirements of these patients [208]. The needs of women who cannot take HRT, or those who choose not to take HRT, must also be addressed. Many alternative therapies (such as botanical products) are popularly recommended, but controlled clinical trials on their efficacy are entirely lacking. Improvised vaginal lubricants have been offered as a possible treatment [222]. As with male HCT survivors, psychiatric/psychosocial counseling may significantly improve self-esteem and quality of life.

Female

Conclusion

Sexual dysfunction in women treated with HCT may predate their treatment or occur de novo following therapy. In women without sexual problems prior to transplantation, up to 40% developed lubrication difficulty, problems achieving orgasm, and painful intercourse. Dysfunction is often pervasive and severe, and the problems did not seem to improve over time, but rather seemed to consolidate and generalize to other aspects of sexual function [208]. Of women with arousal difficulty prior to transplantation, 71% had this problem after 3 years, whereas 42% without arousal problems prior to transplant had developed it by 3 years after transplant. Particularly for women, if sexual problems were

The widespread implementation of HCT and increasing long-term survival rates have warranted greater concern in regards to long-term quality of life for transplanted patients. Endocrine complications of HCT are often notoriously late in onset, even manifesting decades after the initial transplant. Dysfunction of the endocrine system can severely impact both the morbidity and mortality of HCT recipients. It is hoped that awareness of the various types of endocrine complications of HCT and regular monitoring of transplant survivors for the signs and symptoms of these problems will further improve the long-term quality of life for HCT recipients.

Evaluation of women with sexual dysfunction post HCT In women, the onset of early menopause has been noted as a major cause of sexual dysfunction, including symptoms of vasomotor instability, vaginitis, dry vaginal mucosa, dysphoria, osteoporosis, hot flashes, insomnia, irritability, decreased libido, and vaginal atrophy [208]. The evaluation of gonadal function in women presenting with menstrual irregularities with estrogen deficiency symptoms should be carried out as described in Fig. 98.18. Several methods of testing for female sexual dysfunction exist, including instruments to measure level of vaginal arousal, lubrication, and blood flow, but the use of these modalities is mainly limited to the research setting.

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204. Shalet SM. Cancer therapy and gonadal dysfunction. In: Sheaves R, Jenkins P, Wass JAH, editors. Clinical Endocrine Oncology. Oxford, UK: Blackwell Science; 1994. pp. 510–13. 205. Sanders JE, Hawley J, Levy W et al. Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation. Blood 1996; 87: 3045–52. 206. Holm K, Nysom K, Brocks V, Hertz H, Jacobsen N, Muller J. Ultrasound B-mode changes in the uterus and ovaries and Doppler changes in the uterus after total body irradiation and allogeneic bone marrow transplantation in childhood. Bone Marrow Transplant 1999; 23: 259–63. 207. Mumma GH, Mashberg D, Lesko LM. Long-term psychosexual adjustment of acute leukemia survivors: impact of bone marrow transplantation versus conventional chemotherapy. Gen Hosp Psychiatry 1992; 14: 43–55. 208. Syrjala KL, Roth-Roemer SL, Abrams JR et al. Prevalence and predictors of sexual dysfunction in long-term survivors of marrow transplantation. J Clin Oncol 1998; 16: 3148–57. 209. Wingard JR, Curbow B, Baker F et al. Sexual satisfaction in survivors of bone marrow transplantation. Bone Marrow Transplant 1992; 9: 185–90. 210. Molassiotis A, van den Akker OBA, Milligan DW, Boughton BJ. Gonadal function and psychosexual adjustment in male long-term survivors of bone marrow transplantation. Bone Marrow Transplant 1995; 16: 253–9. 211. Watson M, Wheatley K, Harrison GA et al. Severe adverse impact on sexual functioning and fertility of bone marrow transplantation, either allogeneic or autologous, compared with consolidation chemotherapy alone: analysis of the MRC AML 10 trial. Cancer 1999; 86: 1231–9. 212. Andersen BL. How cancer affects sexual functioning. Oncology 1990; 4: 81–94. 213. Andersen BL. Sexual functioning morbidity among cancer survivors: current status and future research directions. Cancer 1985; 55: 1835–42.

214. Barlow DH. Causes of sexual dysfunction. The role of anxiety and cognitive interference. J Consult Clin Psychol 1986; 54: 140–8. 215. Anunta CD, Kandeel FR. Clinical assessment of the male presenting with sexual dysfunction. In: Kandeel FR, Lue T, Pryor J, Swerdloff RS, editors. Male Sexual Dysfunction: Pathophysiology and Treatment. New York: Informa Healthcare; 2007. pp. 253–70. 216. Kandeel FR, Esensten M, Zuckier LS. Investigation of vascular and structural abnormalities of the penis. In: Kandeel FR, Lue T, Pryor J, Swerdloff RS, editors. Male Sexual Dysfunction: Pathophysiology and Treatment. New York: Informa Healthcare; 2007. pp. 297–320. 217. Kandeel FR, Koussa N. Neurological investigations of males with sexual dysfunction. In: Kandeel FR, Lue T, Pryor J, Swerdloff RS, editors. Male Sexual Dysfunction: Pathophysiology and Treatment. Informa Healthcare; 2007. pp. 321–41. 218. Kandeel FR, Koussa N. Nonsurgical devices in the treatment of male sexual dysfunction. In: Kandeel FR, Lue T, Pryor J, Swerdloff RS, editors. Male Sexual Dysfunction: Pathophysiology and Treatment. New York: Informa Healthcare; 2007. pp. 409–14. 219. Caruso S, Rugolo S, Agnello C et al. Sildenafil improves sexual functioning in premenopausal women with type 1 diabetes who are affected by sexual arousal disorder: a double-blind, crossover, placebo-controlled pilot study. Fertil Steril 2006; 85: 1496–501. 220. Fedele D. Therapy insight: sexual and bladder dysfunction associated with diabetes mellitus. Nat Clin Pract Urol 2005; 2: 282–90. 221. Salonia A, Zanni G, Briganti A et al. The role of the urologist in the management of female sexual dysfunctions. Curr Opin Urol 2004; 4: 389–93. 222. Muniyappa R, Norton M, Dunn ME et al. Diabetes and female sexual dysfunction: moving beyond “benign neglect”. Curr Diab Rep 2005; 5: 230–6.

99

Anne Poon & Lowan Ly

Common Potential Drug Interactions Following Hematopoietic Cell Transplantation

Introduction The probability of drug interactions occurring in hematopoietic cell transplant (HCT) patients is high. This is mainly due to the complexity of the medications employed in the transplant setting and the types of drugs used to treat the underlying disease and comorbid conditions. Drug interactions occur when the pharmacologic effectiveness or safety profile of one drug is altered by the concurrent administration of another substance, such as another drug, food, or herbal product. Three categories of interaction are possible: pharmacokinetic, pharmacodynamic, and pharmaceutic. Pharmacokinetic interactions occur when the administration of one drug leads to a change in the concentration of another drug at the intended site of action. These may happen at any point in the absorption, distribution, metabolism, and elimination phases, but a majority of the reported interactions occur during the latter two phases of the drug administration process [1]. Many factors can affect a drug’s pharmacokinetic profile, including the route of administration, the drug’s affinity for protein binding, and patient variables, such as age, ethnicity, and organ (hepatic and renal) function. A pharmacodynamic interaction is one in which the administration of two drugs of similar or opposing pharmacologic actions results in an altered response by the body to the drug(s). The outcome of this combination can be antagonistic, synergistic or indirect in nature [2]. Pharmaceutic interactions arise when drugs form a precipitate due to chemical or physical incompatibilities when mixed into solution [2]. Since many medications will be prescribed during transplant, it will be inevitable that a drug interaction will occur. In this chapter, the focus will be a review of those commonly encountered by practitioners caring for HCT patients. The vital role of cytochrome P450 (CYP) isoenzymes, uridine 5′-diphosphate glucuronosyltransferase (UGT), and P-glycoprotein (P-gp) in drug metabolism, transport, and interactions will also be discussed.

Cytochrome P450 isoenzyme system The CYP isoenzyme system is a family of heme-containing enzymes responsible for phase I (oxidative phase) metabolism of drugs, endogenous steroids, and toxins [1,2]. These enzymes are found in numerous

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

tissues such as the liver, lungs, kidneys, intestines, and brain [1]. Many enzyme families have been identified in humans, but only seven of them (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5) are responsible for converting drugs to their metabolite(s) during phase I metabolism [1,3]. Changes in the mechanism by which drugs undergo phase I metabolism occur via enzyme induction or inhibition. Drugs that induce CYP activity do so by increasing the production of more enzymes or decreasing their breakdown. This leads to increased drug clearance, decreased drug concentrations, and potentially decreased therapeutic effect of the other drug (substrate). The time of onset for enzyme induction is difficult to predict, but this generally happens within days to weeks following administration. It is usually determined by the inducing drug’s half-life and the rate of enzyme turnover. In contrast, enzyme inhibition occurs when a drug blocks or reduces the activity of the enzyme. The net result is decreased drug clearance, increased drug concentrations, and augmented effects that can lead to drug toxicity. Enzyme inhibition usually begins upon the administration of the inhibitor, and the duration corresponds to the half-life of the inhibitor once it has been removed. The likelihood for drugs to interact depends on the inhibitory and inducing potency of each agent and also on the available routes of elimination of the substrate involved. For example, if a drug is a weak inhibitor, the risk of an interaction occurring is relatively low when a substrate of the same family is co-administered. Likewise, if a drug has multiple pathways for elimination, the risk of having a major drug interaction is low when an inhibitor of only one enzyme family is administered [4]. Commonly encountered inducers, inhibitors, and substrates of the CYP isoenzyme system are listed in Table 99.1 [1,3,4].

Uridine 5′-diphosphate glucuronosyltransferase enzyme system Phase II or the conjugative phase of metabolism may also be responsible for drug interactions. During this phase, drug metabolites are made more hydrophilic and ready for excretion [4]. Enzymes belonging to the UGT family play a major role in this process. UGT enzymes are primarily found in the liver, but are also located in the gastrointestinal tract, lungs, nasal epithelium, and kidneys [5]. As in the CYP system, inhibition and induction of these enzymes can result in a change in drug concentration, which can potentially lead to an altered pharmacologic response at the site of action. Tacrolimus, cyclosporine (CSP), benzodiazepines, morphine, and fluconazole are examples of UGT inhibitors [5]. Potent UGT inducers include rifampin, phenytoin, carbamazepine, and phenobarbital [5].

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Table 99.1 CYP isoenzymes and common inducers, inhibitors, and substrates [1,3,4] Isoenzyme

Inducers

Inhibitors

Substrates

CYP1A2

Insulin, omeprazole, phenobarbital, phenytoin, rifampin, tobacco

Amitriptyline, caffeine, duloxetine, fluvoxamine, haloperidol, mirtazapine, ondansetron, propranolol, theophylline, verapamil, R-warfarin

CYP2B6 CYP2C9

Phenobarbital, rifampin Phenobarbital, rifampin, secobarbital

CYP2C19

Carbamazepine, norethindrone, phenobarbital, prednisone, rifampin

Amiodarone, cimetidine, ciprofloxacin, enoxacin, erythromycin, fluoxetine, fluvoxamine, grapefruit juice, paroxetine, sertraline Thiotepa Amiodarone, fluconazole, fluoxetine, fluvastatin, fluvoxamine, isoniazid, itraconazole, ketoconazole, lovastatin, metronidazole, paroxetine, ritonavir, sertraline, teniposide, trimethoprim, voriconazole Cimetidine, fluoxetine, fluvoxamine, ketoconazole, lansoprazole, omeprazole, ritonavir, sertraline, voriconazole

CYP2D6

Dexamethasone, rifampin

CYP2E1 CYP3A4/5

Ethanol, isoniazid Barbiturates, carbamazepine, dexamethasone, efavirenz, nevirapine, phenobarbital, phenytoin, rifampin

Amiodarone, bupropion, cimetidine, citalopram, doxorubicin, duloxetine, escitalopram, fluoxetine, fluvoxamine, haloperidol, metoclopramide, mirtazapine, paroxetine, ritonavir, sertraline, venlafaxine Amiodarone, cimetidine, ciprofloxacin, clarithromycin, diltiazem, erythromycin, fluconazole, fluvoxamine, grapefruit juice, itraconazole, ketoconazole, nefazodone, norfluoxetine, protease inhibitors, verapamil, voriconazole

P-glycoprotein system The P-gp system functions as a drug efflux pump that protects the body from toxic chemicals as well as excretes drugs from the body. It is an ATP-dependent plasma membrane transport system that is encoded by the multidrug resistance 1 (MDR1) gene found in humans. It is expressed in tumor cells and on normal human tissues such as hepatocytes, proximal renal tubules, intestines, and brain [6]. Inhibitors and inducers can also affect the functionality of the P-gp system. Co-administration of drugs that inhibit or induce P-gp activity in the liver or kidneys can increase or decrease excretion of P-gp substrates, respectively. Often, the substrates, inducers, and inhibitors of P-gp are similar to those of the CYP isoenzyme system.

Bupropion Fluoxetine, fluvastatin, nonsteroidal anti-inflammatory drugs, phenytoin, sertraline, tamoxifen, voriconazole, S-warfarin

Citalopram, cyclophosphamide, diazepam, escitalopram, nelfinavir, omeprazole, phenobarbital, phenytoin, primidone, propranolol, sertraline, voriconazole Citalopram, duloxetine, escitalopram, fluoxetine, fluvoxamine, haloperidol, metoclopramide, metoprolol, mirtazapine, ondansetron, paroxetine, propranolol, tamoxifen, tricyclic antidepressants, venlafaxine Acetaminophen, ethanol Alfentanil, alprazolam, amiodarone, amlodipine, astemizole, atorvastatin, cisapride, clarithromycin, cyclosporine, dasatinib, diazepam, diltiazem, erythromycin, escitalopram, ethinyl estradiol, felodipine, fentanyl, imatinib, irinotecan, lovastatin, midazolam, mirtazapine, nifedipine, ondansetron, protease inhibitors, sertraline, sildenafil, simvastatin, sirolimus, tacrolimus, tamoxifen, terfenadine, thiotepa, triazolam, venlafaxine, verapamil, vincristine, voriconazole

agents such as cisapride, CSP, HMG-CoA reductase inhibitors, and warfarin results in increased levels of these drugs as well as increased risk for developing serious adverse effects such as cardiac arrhythmias, renal dysfunction, musculoskeletal complaints, and bleeding [8]. Close monitoring of drug levels, use of alternative agents, and frequent dosage adjustments of the affected drugs are measures to consider to minimize the severity of these interactions when fluconazole is co-prescribed. Administration of potent inducers of CYP2C9 or CYP3A4 with fluconazole can lead to decreased fluconazole serum levels and potentially antifungal failure. Selected drug interactions reported with fluconazole are further described in Table 99.2 [7–11]. Itraconazole

Azole antifungals Fluconazole Fluconazole is a substrate of CYP2C9 and CYP3A4 metabolism. It is a potent inhibitor of CYP2C9 but only a weak inhibitor of CYP3A4, relative to other azole antifungals such as ketoconazole, itraconazole, and voriconazole [7]. Concurrent administration of fluconazole with

Itraconazole is potent inhibitor of CYP3A4. As a result, the interactions with itraconazole may be greatly enhanced compared with fluconazole. An example of this difference in magnitude of effect can be seen when either azole is given with CSP. Little or no change in CSP concentrations have been reported when fluconazole is given concurrently, but during co-administration with itraconazole, one study showed CSP levels to be elevated by a mean of 80%, necessitating dosage reductions [12]. Since

Common Potential Drug Interactions Following Hematopoietic Cell Transplantation

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Table 99.2 Selected drug interactions with fluconazole Drug or class

Mechanism

Effect

Management

Antihistamines [7,8] Astemizole

(−) CYP3A4

↑ antihistamine [ ] → QTc prolongation, torsade de pointes

Use is contraindicated

↑ benzodiazepine [ ] → ↑ sedation

Monitor for ↑ benzodiazepine toxicity (sedation). Dose reduction in benzodiazepine may be necessary See Tables 99.6 and 99.7 Frequent monitoring of blood pressure and pulse. Dosage reduction in calcium channel blocker may be necessary Use is contraindicated

Terfenadine Benzodiazepines [7,8]

Calcineurin inhibitors Calcium channel blockers [7,8] (dihydropyridine class)

(−) CYP3A4

See Tables 99.6 and 99.7 ↑ calcium channel blocker [ ] → hypotension

Ethinyl estradiol/norethindrone [8] Fentanyl [10]

(−) estradiol metabolism (−) CYP3A4

↑ cisapride [ ] → QTc prolongation, torsade de pointes ↑ ergot alkaloid [ ] → ↑ risk of ergotism (nausea, vomiting, and vasospastic ischemia) ↑ estradiol [ ] ↑ or prolonged opioid effects

Losartan [7]

(−) CYP2C9

↓ losartan efficacy

Phenytoin [7,8]

(−) CYP2C9

↑ risk of phenytoin toxicity

Rifabutin [7,8]

(−) CYP3A4

↑ rifabutin [ ] by 80%

Rifampin [7,8]

(+) CYP3A4

↓ fluconazole efficacy

Sirolimus

(−) CYP3A4 and P-gp

See Table 99.9

(−) CYP3A4

↑ risk of hypoglycemia

(−) CYP3A4

↑ tretinoin [ ] → ↑ toxicity

(−) CYP2C9

↑ prothrombin time twofold

Cisapride [8] Ergot alkaloids [9]

Sulfonylureas [7] Glipizide Glyburide Tretinoin (all-trans-retinoic acid; ATRA) [11] Warfarin [7,8]

Use is contraindicated

Monitor for estradiol-related adverse effects Monitor for signs of opioid toxicity (sedation, ↓ respiration). Use lowest dose possible and adjust conservatively Monitor for losartan efficacy. May need to ↑ losartan dose Monitor phenytoin drug [ ] and for signs of phenytoin toxicity. Adjust phenytoin dose as needed Avoid concurrent administration. Monitor for signs of rifabutin toxicity if used concomitantly Monitor for fluconazole efficacy. May need to ↑ fluconazole dose See Table 99.9 Monitor blood glucose frequently. Dosage adjustment in sulfonylurea may be needed Tretinoin dosage adjustments may be necessary Monitor prothrombin time frequently. Adjust warfarin dose as needed

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; CYP2C9, cytochrome P450 2C9 isoenzyme; P-gp, P glycoprotein.

the cost of CSP can be significant, this interaction has been viewed by some as beneficial and has even prompted clinicians to use itraconazole as a cost-saving measure in renal transplant recipients [13]. Identifying and proper management of potential drug interactions with itraconazole is important from a therapeutic aspect. The efficacy of itraconazole has been shown to be related to its serum concentrations [12]. If induction of itraconazole metabolism occurs by such drugs as phenytoin and rifampin, its serum levels may be suboptimal, leading to inadequate control or progression of the fungal infection being treated. It may be useful to monitor itraconazole concentrations if such combinations are required. Chemotherapeutic agents are another group of drugs on which itraconazole can have a substantial impact. When it was given as prophy-

laxis to HCT patients receiving cyclophosphamide-containing conditioning regimens, higher serum creatinine and bilirubin levels were seen in the group who received itraconazole as opposed to fluconazole [14]. These patients had a greater exposure to toxic metabolites. Itraconazole can also affect the metabolism of busulfan. The clearance of busulfan was reduced by 20% when given concomitantly with itraconazole, resulting in increased levels of busulfan [15]. Since these interactions could potentially put patients at more risk for toxicities post transplant, itraconazole should be avoided during the administration of chemotherapeutic agents that are metabolized through the same pathway. A selected list of drug interactions involving itraconazole can be found in Table 99.3 [12,14–29].

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Table 99.3 Selected drug interactions with itraconazole Drug or class

Mechanism

Effect

Management

Benzodiazepines Alprazolam [16]

(−) CYP3A4

Use with caution. Monitor for ↑ central nervous system-depressant effects. Use alternative agent such as lorazepam if dosage adjustment is not adequate

Busulfan [15]

unknown

↑ alprazolam AUC 2.6-fold → ↓ psychomotor function ↑ midazolam AUCPO 6.6-fold, CmaxPO 3.6-fold → ↓ psychomotor function ↓ busulfan Cl 20% → ↑ busulfan [ ]

Calcineurin inhibitors [12]

(−) CYP3A4

↑ Cyclosporine and tacrolimus [ ]

Carbamazepine [18]

(+) CYP3A4

↓ itraconazole [ ]

Cyclophosphamide [14]

(−) CYP3A4

Digoxin [19]

(−) P-gp

↑ serum bilirubin and creatinine; ↑ exposure to cyclophosphamide metabolites → ↑ toxicities ↑ digoxin AUC ~50%, ↓ renal Cl ~20%

Felodipine [20]

(−) CYP3A4

Midazolam [17]

HMG-CoA reductase inhibitors Atorvastatin [21] (−) CYP3A4 Lovastatin [22]

Simvastatin [23]

Phenobarbital [24]

(+) CYP3A4

Phenytoin [25]

(+) CYP3A4

Rifabutin [18]

(+) CYP3A4

Rifampin [26]

(+) CYP3A4

Sirolimus [27]

(−) CYP3A4, (−) P-gp

Terfenadine [28]

(−) CYP3A4

Warfarin [29]

(−) CYP2C9

↑ felodipine AUC 6-fold, Cmax 8-fold → ↑ HR, ↓ BP ↑ atorvastatin acid AUC 3-fold ↑ lovastatin AUC and Cmax >20-fold ↑ lovastatin acid AUC 20-fold and Cmax 13-fold ↑ simvastatin AUC and Cmax 10-fold ↑ simvastatin acid AUC 19-fold and Cmax 17-fold ↓ itraconazole [ ] ↓ itraconazole AUC >90%, Cmax 80% Itraconazole ↑ phenytoin AUC 10% ↓ itraconazole [ ] ↓ itraconazole AUC ~ 88% in healthy volunteers and 64% in AIDS patients ↑ sirolimus [ ] >8-fold

↑ unmetabolized terfenadine [ ] → QTc prolongation, torsades de pointes ↑ INR >8 → bruising/bleeding

Avoid concomitant use. Use alternative antifungal agent until busulfan elimination is completed ↓ calcineurin inhibitor dose by ~50% when starting itraconazole. Monitor [ ] and adjust dose accordingly Avoid combination therapy if possible, otherwise monitor itraconazole [ ] to prevent therapeutic failure Avoid concomitant use. Use alternative antifungal agent until cyclophosphamide elimination is completed ↓ digoxin dose 50% when starting itraconazole. Monitor digoxin [ ] and signs/symptoms of toxicity Monitor heart rate and blood pressure. ↓ dose of felodipine if needed Avoid concomitant use

Avoid combination therapy if possible, otherwise monitor itraconazole [ ] to prevent therapeutic failure Avoid concomitant use, as it may lead to therapeutic failure Avoid concomitant use, as it may lead to therapeutic failure Avoid concomitant use, as it may lead to therapeutic failure Monitor sirolimus [ ] and signs of toxicity. Adjust dose as needed. Refer to text for empiric dose adjustment for azole Use is contraindicated Monitor INR and for signs of bleeding. Dosage adjustment may be needed

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; CYP2C9, cytochrome P450 2C9 isoenzyme; AUC, area under the curve; AUCPO, area under the curve (oral); Cl, clearance; CmaxPO, maximum concentration (oral); Cmax, maximum concentration; INR, international normalized ratio; P-gp, P-glycoprotein.

Voriconazole Voriconazole is also extensively metabolized by the CYP enzyme system. It is a substrate for and an inhibitor of CYP2C9, CYP2C19, and CYP3A4. Voriconazole has the highest affinity for inhibiting CYP2C9 and the lowest for CYP3A4, which explains why it is a less potent inhibitor of CYP3A4 when compared with itraconazole and ketoconazole. In contrast, voriconazole-N-oxide, one of three major metabolites, has higher inhibition affinity for CYP3A4 and CYP2C19 than CYP2C9 [30]. Since voriconazole is dependent on three CYP enzyme pathways, it is reasonable to expect that it could potentially affect the metabolism of many

drugs. Likewise, its metabolism may also be affected by other agents. The use of voriconazole in combination with sirolimus, terfenadine, astemizole, cisapride, pimozide, quinidine, and ergot alkaloids is contraindicated by the manufacturer because of the high risk for fatal adverse effects, such as significant elevation of drug levels, QTc interval prolongation, and ergotism [31]. Voriconazole also decreases the metabolism of drugs such as calcineurin inhibitors, HMG-CoA reductase inhibitors, omeprazole, phenytoin, and warfarin [30]. On the other hand, it has minor to negligible effects on prednisolone, digoxin, and mycophenolate. The use of voriconazole with potent inducers such as rifampin and rifabutin is contraindicated because they significantly reduce serum con-

Common Potential Drug Interactions Following Hematopoietic Cell Transplantation

centrations of voriconazole to undetectable levels. Increasing the voriconazole dose is incapable of overcoming the induction effects of rifampin; however, the effects of rifabutin can be overcome, although this is not advised. Voriconazole levels are also decreased by phenytoin and human immunodeficiency virus antivirals, whereas drugs such as cimetidine, ranitidine, erythromycin, azithromycin, and indinavir have negligible effects on voriconazole metabolism [32]. Additionally, concomitant administration with carbamazepine and long-acting barbiturates is also contraindicated. Pharmacokinetic studies of voriconazole’s effect on chemotherapy have also been conducted. In these studies, CYP3A4 inhibition by voriconazole resulted in an increase in the concentration of irinotecan, alltrans-retinoic acid, and imatinib, leading to a risk for increased toxicity [31]. Other drugs not tested but likely to interact with voriconazole

1527

include cyclophosphamide (a substrate of CYP2C9 and CYP3A) and dasatinib (a substrate of CYP3A). Alternatives to voriconazole should be employed during the co-administration of these chemotherapy agents if antifungal therapy is necessary. If temporary cessation of voriconazole is possible, it should be discontinued at least 30 hours prior to the start of chemotherapy and resumed when five half-lives of the antineoplastic agent have elapsed [1]. Table 99.4 further describes the clinically relevant drug interactions with voriconazole [1,31]. Posaconazole The primary metabolism of posaconazole is through glucuronidation by UGT enzymes [33]. When the effect of posaconazole was tested on CYP

Table 99.4 Selected drug interactions with voriconazole Drug or class

Mechanism

Effect

Management

(−) CYP3A4

Use is contraindicated

(+) CYP metabolism (−) CYP3A4

↑ antihistamine [ ] → QTc prolongation, torsade de pointes ↓ voriconazole [ ] → ↓ efficacy and therapeutic failure ↑ benzodiazepine [ ] → ↑ sedation

Calcineurin inhibitors Cyclosporine Tacrolimus

(−) CYP3A4

See Tables 99.6 and 99.7

See Tables 99.6 and 99.7

Calcium channel blockers [31] Diltiazem Verapamil

(−) CYP3A4

↑ calcium channel blocker [ ] → hypotension

Carbamazepine [31]

(+) CYP3A4

Cisapride [31]

(−) CYP3A4

Ergot alkaloids [31]

(−) CYP3A4

Ethinyl estradiol/norethindrone [31]

(−) CYP2C19

HMG-CoA reductase inhibitors [31]

(−) CYP3A4

↓ voriconazole [ ] → ↓ efficacy and therapeutic failure ↑ cisapride [ ] → QTc prolongation, torsade de pointes ↑ ergot alkaloid [ ] → ↑ risk of ergotism ↑ Cmax and AUC of voriconazole. ↑ Cmax and AUC of ethinyl estradiol/ norethindrone ↑ statin [ ] → ↑ risk for rhabdomyolysis

Monitor blood pressure and heart rate. Dosage reduction in calcium channel blocker may be necessary (if symptomatic) Use is contraindicated

Imatinib [1]

(−) CYP3A4

↑ imatinib [ ] → ↑ dose-dependent toxicities

Macrolide antibiotics [31] Clarithromycin

(−) CYP3A4

↑ clarithromycin [ ] → ↑ risk of QTc prolongation ↑ erythromycin [ ] → ↑ risk of QTc prolongation

Monitor cardiac function and symptoms of bradycardia. Consider the use of another macrolide antibiotic (i.e., azithromycin)

(−) CYP2C19, CYP2C9, and CYP3A4

↑ Cmax and AUC of active (R) methadone by 31% and 47%

Monitor for signs/symptoms of methadone toxicity including QTc prolongation. Methadone dose adjustment may be needed

Antihistamines [31] Astemizole Terfenadine Barbiturates (long acting) [31] Benzodiazepines [1]

Erythromycin

Methadone [31]

Use is contraindicated Monitor for ↑ benzodiazepine toxicity (sedation). Dose reduction in benzodiazepine may be necessary

Use is contraindicated Use is contraindicated Monitor for signs of voriconazole toxicity and for signs of abnormal menstruation Monitor for rhabdomyolysis, muscle pain and/or weakness. Consider dosage adjustment of statin and use of a non-CYP3A4 metabolized statin (pravastatin) Monitor blood counts, nausea/vomiting, fluid retention. Dosage reduction of imatinib may be necessary

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Chapter 99

Table 99.4 Continued Drug or class

Mechanism

Effect

Management

Phenytoin [31]

↓ Cmax of voriconazole by 50% and AUC by 70% → ↓ voriconazole efficacy and/or therapeutic failure ↑ Cmax and AUC of phenytoin by at least twofold → phenytoin toxicity

↑ voriconazole maintenance to 5 mg/kg intravenously twice a day (from 4 mg/kg) or to 400 mg orally twice a day (from 200 mg) Frequent monitoring of plasma phenytoin [ ] and phenytoin-related adverse effects

↑ [ ] of proton pump inhibitors

If omeprazole dose is >40 mg daily, ↓ omeprazole dose by 50%

Rifabutin [31]

Phenytoin (+) CYP metabolism of voriconazole, Voriconazole (−) CYP2C9 metabolism of phenytoin (−) CYP2C19, CYP2C9, and CYP3A4 (+) CYP3A4

Use is contraindicated

Rifampin [31] Sirolimus Sulfonylureas [31]

(−) CYP3A4 (−) CYP2C9

↓ Cmax of voriconazole by 93% → ↓ efficacy ↓ voriconazole [ ] → ↓ efficacy See Table 99.9 ↑ sulfonylurea [ ] → hypoglycemia

Proton pump inhibitors [31]

Tretinoin (all-trans retinoic acid; ATRA) [1]

(−) CYP3A4

↑ tretinoin [ ] → ↑ risk of hypercalcemia

Vinca alkaloids [1]

(−) CYP3A4

↑ vinca [ ]

Warfarin [31]

(−) CYP2C9

↑ PT, INR → ↑ bleeding risk

Use is contraindicated See Table 99.9 Frequent monitoring of blood glucose. Dosage reduction of sulfonylureas may be necessary Limit concurrent use. Otherwise, monitor for hypercalcemia. Discontinue tretinoin and/or add a bisphosphonate Monitor for vinca-associated neurotoxicity. Avoid concomitant administration. Dosage reduction of the vinca alkaloid may be necessary Monitor closely for signs/symptoms of bleeding, PT and INR. ↓ warfarin dose as needed

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP, cytochrome P450 isoenzyme; CYP3A4, cytochrome P450 3A4 isoenzyme; CYP2C19, cytochrome P450 2C19 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; PT, prothrombin time; INR, international normalized ratio.

Table 99.5 Selected drug interactions with posaconazole Drug

Mechanism

Effect

Management

Cimetidine [33]

Alteration of gastric pH

Avoid concomitant use unless benefit outweighs risk

CSP [33]

(−) CYP3A4

↓ posaconazole AUC and Cmax 39% ↑ CSP [ ]

Midazolam [33] Phenytoin [33]

(−) CYP3A4 (+) UGT (−) CYP3A4

Rifabutin [33]

(+) UGT (−) CYP3A4

Tacrolimus [33]

(−) CYP3A4

↑ midazolam AUC 83% ↓ posaconazole AUC 50% and Cmax 41% ↑ phenytoin AUC and Cmax 16% ↓ posaconazole AUC 49% and Cmax 43% ↑ rifabutin AUC 72% and Cmax 31% ↑ tacrolimus AUC 358% and Cmax 121%

↓ CSP dose by 25% when starting posaconazole. Monitor CSP [ ] and adjust dose accordingly Monitor for adverse effects of benzodiazepines metabolized through this route Avoid concomitant use unless benefit outweighs risk. If used, monitor phenytoin [ ] and adjust dose accordingly

Avoid concomitant use unless benefit outweighs risk. If used, monitor for adverse effects of rifabutin

↓ tacrolimus dose by 66% when starting posaconazole. Monitor tacrolimus [ ] and adjust dose accordingly

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; CSP, cyclosporine; UGT = uridine 5′-diphosphate glucuronosyltransferase.

enzymes, it was shown to inhibit hepatic CYP3A4, but not CYP1A2, CYP2C8/9, CYP2D6, or CYP2E1 [34]. Terfenadine, astemizole, cisapride, pimozide, and quinidine are contraindicated with posaconazole administration because of the potential development of QTc prolongation and torsades de pointes. Ergot alka-

loids are also contraindicated because they can result in ergotism. Similar to itraconazole and voriconazole, posaconazole has inhibitory effects on the calcineurin inhibitors, requiring initial dosage adjustments and frequent monitoring of their levels. Sirolimus, vinca alkaloids, calcium channel antagonists, and HMG-CoA reductase inhibitors are

Common Potential Drug Interactions Following Hematopoietic Cell Transplantation

examples of drugs metabolized through CYP3A4 that have not yet been tested but are likely to have an interaction with posaconazole. Other interactions occurring with posaconazole are summarized in Table 99.5 [33,35].

1529

clinically significant increases in CSP levels. Patients at highest risk for this interaction are those with the highest initial oral clearance of CSP. In these patients, careful monitoring of CSP levels and for signs of CSP toxicity is recommended with micafungin initiation or discontinuation. Adjustment in CSP levels may also be warranted in this patient population.

Echinocandins Caspofungin

Anidulafungin

Caspofungin is a poor substrate for CYP, and is neither an inhibitor nor an inducer of CYP3A4 [36]. To date, drug interaction data for caspofungin are minimal. The one of most relevance to clinicians in HCT is the interaction involving CSP. In two small studies, the area under the curve (AUC) for caspofungin was increased approximately 35% by CSP [36,37]. In addition, co-administration resulted in elevated alanine transaminase and aspartate transaminase levels. Three of four subjects developed transient alanine transaminase elevations two to three times the upper limit of normal. When lower doses of caspofungin were used, slight elevations were seen in 25% of the subjects. Increases in aspartate transaminase were also experienced, but were smaller in the two study groups. This prompted the manufacturer to caution against combination therapy unless the potential benefits outweigh the risks. If these drugs are used together, liver function tests should be monitored. The same interaction between caspofungin and CSP did not occur when tacrolimus, another calcineurin inhibitor, was given concomitantly with caspofungin. In fact, caspofungin decreased the tacrolimus AUC by 20%, maximum concentration (Cmax) by 16%, and 12-hour blood levels by 26% [36,37]. Therefore, routine monitoring of tacrolimus concentrations is advised with appropriate dosage adjustments. Another drug interaction study was performed involving rifampin and caspofungin. When both drugs were given together, trough concentrations of caspofungin were reduced by 30% at steady state [38]. To avoid possible therapeutic failure, it is recommended that the daily dose of caspofungin be increased to 70 mg. The higher dose of caspofungin should also be considered when used concomitantly with other agents known to be inducers of drug clearance, such as carbamazepine and phenytoin.

Unlike the other echinocandins, anidulafungin undergoes a slow chemical degradation (>90%) upon intravenous administration. It is not dependent on the CYP isoenzymes, nor does it appear to induce or inhibit them [42]. Consequently, the likelihood of drug interactions between anidulafungin and other agents administered to HCT patients is rare. No differences in the Cmax and AUC of anidulafungin were observed when comparing anidulafungin monotherapy, voriconazole monotherapy or the combination in a randomized, blinded, crossover pharmacokinetic study in healthy volunteers [42]. Similar results were described in a pharmacokinetic study of healthy volunteers where anidulafungin was combined with tacrolimus [43]. In another study with CSP, a statistically significant difference in the AUC was observed with combination therapy, but it was not considered clinically significant [44].

Micafungin Micafungin is primarily metabolized in the liver by arylsulfatase and catechol-O-methyltransferase [39]. In vitro data showed that micafungin was a substrate and a weak inhibitor of CYP3A4; however, in vivo data suggested that the CYP system played a negligible role in the drug’s metabolism. Additional published drug interaction studies involving micafungin are limited. Pharmacokinetic studies between micafungin 150 mg and voriconazole, and micafungin 100 mg and both calcineurin inhibitors, have been performed in healthy volunteers. The pharmacokinetics of micafungin was unaffected by the co-administration of these agents [39–41]. Micafungin had no effects on the pharmacokinetics of voriconazole or tacrolimus, although the authors of the tacrolimus study warned that an interaction remains possible if a dose of micafungin of over 100 mg/day is used with tacrolimus [39–40]. It appeared to be a mild inhibitor of CSP metabolism in the majority of the healthy volunteers [41]. This however, did not result in a significant change in CSP concentrations in 80% of the studied patients. In the remaining 20% of patients, the inhibition of CSP metabolism led to

Immunosuppressants Calcineurin inhibitors The metabolism of both CSP and tacrolimus is mediated predominantly through the CYP3A4 pathway. They are substrates for that isoenzyme as well as the P-gp transporter system. Therefore, they have similar drug interaction profiles. These are depicted in Tables 99.6 [12,33,36,45–52] and 99.7 [12,33,36,45,53–59]. Knowing which agents may affect the concentrations of these medications is crucial because the calcineurin inhibitors have a narrow therapeutic window. Too much of either agent will result in toxicity (nephrotoxicity, neurotoxicity, etc.), while too little will cause inadequate immunosuppression and possibly lead to increased risk of graft versus host disease. Two specific drugs that alter the metabolism of CSP and tacrolimus in different ways are caspofungin and sirolimus. These interactions are described in more detail under those separate sections.

Mycophenolate Following oral administration, mycophenolate is rapidly hydrolyzed by esterases found within the intestinal wall, blood, and liver, to the active moiety, mycophenolic acid (MPA). MPA is subsequently metabolized by UGT enzymes to its major inactive metabolite, 7-O-MPA glucuronide (MPAG) in the liver, gastrointestinal tract, and kidneys. Upon excretion into the bile, MPAG may be converted back to MPA by intestinal bacteria before it is reabsorbed into the colon. MPAG is primarily excreted into the urine via active tubular secretion and, to a lesser extent, via the multidrug resistance protein 2 (MRP-2) transport system [60]. The main mechanisms behind mycophenolate’s drug interactions are chelation, inhibition of enterohepatic recycling, and competition for renal tubular secretion, which can result in changes in MPA exposure.

1530

Chapter 99

Table 99.6 Selected drug interactions with cyclosporine (CSP) Drug or class

Mechanism

Effect

Management

Azoles Fluconazole [45]

(−) CYP3A4

↑ CSP [ ] 21%

Frequent monitoring of CSP trough [ ] during concomitant administration and after the discontinuation of fluconazole. Adjust dose accordingly ↓ CSP dose by ~50% when starting itraconazole. Monitor CSP [ ] and adjust dose accordingly ↓ CSP dose by 25% when starting posaconazole. Monitor CSP [ ] and adjust dose accordingly ↓ CSP dose by 50% when starting voriconazole. Monitor CSP [ ] and adjust dose accordingly

Itraconazole [12]

↑ CSP [ ] 80%

Posaconazole [33]

↑ CSP [ ]

Voriconazole [46]

↑ CSP AUC 1.7-fold and [ ] 2.5-fold

Calcium channel blockers Diltiazem [47] (−) CYP3A4 Carbamazepine [48] (+) CYP3A4 Caspofungin [36] (−) CYP3A4

↑ CSP AUC 28–62% and [ ] 49–88% ↓ CSP [ ] 50% ↑ caspofungin AUC 35% → ↑ ALT and AST

Digoxin [49]

↑ digoxin [ ] and ↓ Cl 53% → arrhythmias

unknown

HMG-CoA Reductase Inhibitors [50] Atorvastatin (−) CYP3A4 Lovastatin Pravastatin Simvastatin Phenobarbital [51] (+) CYP3A4 Phenytoin [51] (+) CYP3A4 Rifampin [52] (+) CYP3A4

↑ ↑ ↑ ↑ ↓ ↓ ↓

Sirolimus

See Table 99.9

(−) CYP3A4

atorvastatin AUC 6-fold lovastatin AUC 5-fold pravastatin AUC 5-fold simvastatin AUC 6-fold CSP [ ] CSP [ ] CSP AUC

Monitor CSP [ ] and adjust dose accordingly Monitor CSP [ ] and adjust dose accordingly Combination therapy should only be given if the potential benefits outweigh the risks. Monitor liver function tests Use with caution. Consider ↓ digoxin dose by ~50% when starting CSP. Monitor digoxin [ ] Avoid concomitant use if possible or ↓ dose of statin. Monitor for signs/symptoms of skeletal muscle toxicity

Monitor CSP [ ] and adjust dose accordingly Monitor CSP [ ] and adjust dose accordingly Avoid concomitant use if possible. Monitor CSP [ ] and adjust dose accordingly See Table 99.9

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under the curve; Cl, clearance.

Reductions in MPA exposure can lead to therapeutic failures, while increases in MPA concentrations can result in increased levels of acylglucuronide, leading to toxicities such as hypersensitivity, gastrointestinal disturbances, and cytokine release [60]. Drugs interacting with mycophenolate and their clinical implications are discussed below and in Table 99.8 [60–66]. Co-administration of agents such as oral divalent electrolyte supplements and phosphate binders with mycophenolate have been shown to reduce the extent of its absorption, leading to lower concentrations of MPA [61,63,66]. CSP, tacrolimus, and sirolimus are common immunosuppressants used in combination with mycophenolate. Decreased MPA concentrations observed with CSP are related to the inhibition of the MRP-2 transport system, which inhibits the enterohepatic recycling of mycophenolate [62]. In contrast, increased concentrations of MPA are seen with tacrolimus and sirolimus because enterohepatic recycling of MPA is not inhibited by these agents [67,68]. Other inhibitors of enterohepatic recycling include bile acid sequestrants, rifampin, and, to some extent, sevelamer [61,65]. Competition for renal excretion of mycophenolate is also a possible mechanism for drug interactions. In a prospective, randomized, openlabel, single-dose, crossover study of healthy volunteers, the pharmacokinetics of mycophenolate and valacyclovir or acyclovir were studied [60]. Concurrent use of mycophenolate with acyclovir resulted in an

increase in the Cmax and AUC of acyclovir and an increase in the AUC of MPAG. The authors concluded that this was not clinically significant in healthy patients, but such increases in drug exposure to acyclovir and MPAG could result in neutropenia in patients with renal impairment. Pharmacokinetic changes were not observed with valacyclovir in this study. Furthermore, MPA concentrations were unaffected by either agent when combined with mycophenolate. Other potential drugs that may compete for renal excretion include ganciclovir, valganciclovir, and probenecid [64].

Sirolimus Like calcineurin inhibitors, sirolimus is removed from the bloodstream by P-gp. It is extensively metabolized by CYP3A4 enzymes in the liver and small intestine [69]. Inducers of CYP3A4 and P-gp, such as carbamazepine, phenytoin, and rifampin, increase the metabolism of sirolimus, leading to decreased sirolimus blood levels, efficacy, and potentially inadequate therapy. Conversely, inhibitors such as calcium channel blockers, cimetidine, macrolide antibiotics, cisapride, and metoclopramide reduce the metabolism of sirolimus, leading to increased sirolimus levels and

Common Potential Drug Interactions Following Hematopoietic Cell Transplantation

1531

Table 99.7 Selected drug interactions with tacrolimus Drug or class

Mechanism

Effect

Management

Azoles Fluconazole [45]

(−) CYP3A4

↑ tacrolimus [ ] 16%

Frequent monitoring of tacrolimus trough [ ] during concomitant administration and after the discontinuation of fluconazole. Adjust dose accordingly ↓ tacrolimus dose by ~50% when starting itraconazole. Monitor tacrolimus [ ] and adjust dose accordingly ↓ tacrolimus dose by 66% when starting posaconazole. Monitor tacrolimus [ ] and adjust dose accordingly ↓ tacrolimus dose by 66% when starting voriconazole. Monitor tacrolimus [ ] and adjust dose accordingly

Itraconazole [12]

↑ tacrolimus [ ] 83%

Posaconazole [33]

↑ tacrolimus AUC 358% and Cmax 121%

Voriconazole [53]

↑ tacrolimus [ ] 10-fold

Calcium channel blockers Diltiazem [54] (−) CYP3A4 Felodipine [55] Caspofungin [36] unknown Clarithromycin [56] (−) CYP3A4 Phenobarbital [57] (+) CYP3A4

↑ tacrolimus [ ] ↑ tacrolimus [ ] 3-fold ↓ tacrolimus AUC 20%, Cmax 16%, and [ ] 26% ↑ tacrolimus [ ] ↓ tacrolimus [ ] 73%

Phenytoin [58]

(+) CYP3A4

↓ tacrolimus [ ]

Rifampin [59] Sirolimus

(+) CYP3A4 (−) CYP3A4

↓ tacrolimus [ ], requiring a 10-fold ↑ in tacrolimus dose See Table 99.9

Monitor tacrolimus [ ] and adjust dose accordingly Monitor tacrolimus [ ] and adjust dose accordingly Monitor tacrolimus [ ] and adjust dose accordingly Monitor tacrolimus [ ] and adjust dose accordingly. Monitor for ↑ risk of GVHD Monitor tacrolimus [ ] and adjust dose accordingly. Monitor for ↑ risk of GVHD Avoid concomitant use See Table 99.9

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; CYP3A4, cytochrome P450 3A4 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; GVHD, graft-versus-host disease.

Table 99.8 Selected drug interactions with mycophenolate Drug or class

Mechanism

Effect

Management

Acyclovir [65]

Competes for renal tubular secretion

Monitor neutrophil count closely

Antacids [60]

↓ MPA absorption

↑ acyclovir Cmax by 40% and AUC; ↑ MPAG AUC by 10.6% → ↑ risk of neutropenia in setting of renal impairment ↓ extent of absorption → ↓ MPA [ ]

Bile acid binders [60] Cholestyramine Colestipol

(−) enterohepatic recycling

↓ MPA AUC by 40%

CSP [63]

(−) enterohepatic recycling

↓ MPAG → ↓ MPA [ ]

Divalent oral electrolyte supplement [61]

↓ MMF absorption via chelation Competes for renal tubular secretion Unknown

↓ extent of absorption → ↓ MPA [ ]

Avoid co-administration of bile acid binders with MMF Monitor CSP [ ] and adjust dose accordingly Monitor for MMF therapeutic failure Monitor blood counts

(+) glucuronidation activity and (−) enterohepatic recycling ↓ MMF absorption and (−) enterohepatic recycling

↓ total MPA AUC → ↓ MMF efficacy

Ganciclovir [66] Oral contraceptives [66] (ethinyl estradiol, levonorgesterol, norethindrone, norgestrel) Rifampin [64] Sevelamer [62]

↑ plasma ganciclovir [ ] in setting of renal impairment ↓ oral contraceptive exposure

↓ AUC of MPA by 25% and Cmax by 30%

Monitor for MMF therapeutic failure

Monitor for breakthrough bleeding Monitor for MMF therapeutic failure Administration should be separated by at least 2 hours

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; → = leading to; AUC, area under the curve; Cmax, maximum concentration; CSP, cyclosporine; MMF, mycophenolate mofetil; MPA, mycophenolic acid; MPAG, 7-O-MPA glucuronide.

1532

Chapter 99

Table 99.9 Selected drug interactions with sirolimus Drug or class

Mechanism

Effect

Management

Azoles [70] Fluconazole

(−) CYP3A4 and P-gp

Sirolimus troughs tripled after 7 days of concurrent therapy despite empiric dosage adjustments See Table 99.3

Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed. Refer to text for empiric dose adjustment for azole Use is contraindicated by manufacturer. Refer to text for small case study describing concurrent use

Itraconazole

↑ sirolimus AUC by 7-fold and Cmax by 11-fold → ↑ risk of sirolimus toxicity

Voriconazole

Calcium channel blockers [69] Diltiazem (−) CYP3A4 and P-gp Verapamil

Calcineurin inhibitors Cyclosporine (CSP) [71]

(−) CYP3A4 and P-gp

Tacrolimus [72] Erythromycin [69]

(−) CYP3A4 and P-gp

Micafungin [69]

Unknown

Rifampin [69]

(+) CYP3A4 and P-gp

↑ sirolimus Cmax by 1.4-fold, tmax by 1.3-fold, and AUC by 1.6-fold → ↑ risk of sirolimus toxicity ↑ sirolimus Cmax by 2.3-fold and AUC by 2.2-fold → ↑ risk of sirolimus toxicity. ↓ Cmax and AUC of the active S(−) enantiomer of verapamil by 1.5-fold

Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed

Concurrent administration → ↑ sirolimus Cmax by 116% and AUC by 230%. When sirolimus is given 4 hours apart → ↑ Cmax by 37% and AUC by 80% No effect on sirolimus pharmacokinetcs; however, tacrolimus troughs decreased ↑ sirolimus Cmax by 4.4-fold, AUC by 4.2-fold, and tmax by 0.4 hour → ↑ risk of sirolimus toxicity. ↑ erythromycin Cmax by 1.6-fold, AUC by 1.7-fold, and tmax by 0.3 hour ↑ sirolimus AUC by 21% with no effect on Cmax

Administer sirolimus 4 hours after CSP

↓ sirolimus AUC by 82% and Cmax by 71% → ↓ sirolimus efficacy and therapeutic failure

Monitor tacrolimus levels with sirolimus ≥2 mg daily Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed

Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed Monitor sirolimus troughs and signs of toxicity. Adjust dose as needed. Consider using an alternative agent that has less enzyme inducing potential

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; P-gp, P-glycoprotein; tmax, time to maximum concentration.

subsequently increased risk of toxicity. Monitoring sirolimus trough concentrations for signs of toxicity and treatment failure is recommended when it is combined with these and any other agents utilizing CYP3A as their metabolic route of elimination. Some clinically important drug interactions are further discussed below or described in Table 99.9 [69–72]. Reports of drug interactions that resulted in decreased metabolism and supratherapeutic sirolimus levels have been documented with the azole antifungals. Despite recommendations to empirically reduce the dose of ketoconazole by 80–90% and fluconazole by 50–70% (for doses ≥200 mg daily), caution should be used in reducing the azole dose as interpatient variability can widely affect the incidence, magnitude, and significance of the interaction [70]. Close monitoring of sirolimus levels and adjustments in sirolimus dosage are recommended to optimize therapy during and upon the discontinuation of azole therapy. The concurrent use of voriconazole and sirolimus is contraindicated by the manufacturer based on a sevenfold increase in the Cmax and an 11-fold increase in the AUC of sirolimus seen in healthy volunteers [69]. Recent small case reports, however, have described the safe administration of this combination [73]. Mathis et al. cited a 75–87% dosage reduction in sirolimus during co-administration with voriconazole in two

renal transplant patients. Both patients tolerated therapy well. Similarly, Marty et al. reported the use of sirolimus and voriconazole in 11 patients undergoing allogeneic HCT [74]. In this case series, the sirolimus dose was empirically reduced by 90% upon initiation of voriconazole. All patients received at least 30 days of concurrent therapy. The authors of both studies concluded that the combination is feasible and safe. Frequent monitoring of sirolimus blood levels upon initiation and discontinuation of voriconazole was also recommended. Data regarding drug interactions between sirolimus and posaconazole are currently unavailable. The timing of sirolimus administration in relation to CSP was shown to affect the systemic exposure of sirolimus in healthy volunteers [71]. When both agents were administered concomitantly, a statistically significant increase in the bioavailability of sirolimus was demonstrated, as seen by an increase in the minimum concentration, maximum concentration, time to maximum concentration, and AUC. In contrast, when sirolimus was administered 4 hours after CSP, these measures were only modestly increased. The pharmacokinetics of CSP was unaffected by sirolimus in this study. The authors advised that sirolimus should be administered 4 hours after CSP to avoid high systemic sirolimus levels. Changes in sirolimus levels were not demonstrated in a similar study

Common Potential Drug Interactions Following Hematopoietic Cell Transplantation

with tacrolimus. However, when sirolimus was administered with tacrolimus, a reduction in tacrolimus levels occurred compared with when tacrolimus was administered alone [75].

Supportive care agents Narcotic analgesics Morphine, hydromorphone, fentanyl, and oxycodone are common narcotics used for the treatment of pain in transplant. Since the CYP isoenzyme system plays a minimal role in the metabolism of morphine and hydromorphone, the impact of CYP inhibitors and inducers on their pharmacokinetics is limited. Both drugs are metabolized via glucuronidation by various UGT enzymes in the liver [76,77]. Very few studies have been published that examine the effects of hydromorphone concentrations when administered with UGT inhibitors or inducers. The role of P-gp inhibition to enhance the pharmacologic effects of morphine continues to be studied. However, it has been proposed that concurrent use of agents that inhibit or induce UGT enzymes can change the AUC of morphine and consequently affect its pharmacologic potency or adverse effect profile [76]. Co-administration of metoclopramide with controlled-release morphine was shown to result in a faster time of onset and increase in sedation in a double-blinded study of 20 patients [76]. The pharmacologic and adverse effects of morphine should be monitored when these agents are used together. CYP3A4 is the major pathway of metabolism for phenylpiperidine opiates (alfentanil, fentanyl, and sufentanil). The effect of CYP3A4 inhibitors, especially azole antifungals, on phenylpiperidine metabolism is well documented in the literature. In a study of nine healthy volunteers, fluconazole was reported to double the half-life and reduce the clearance of alfentanil by 55% [78]. An increase in pharmacologic response as well as increased respiratory depression was also demonstrated. The authors of this study recommended caution when using these agents concurrently and suggested a 60% dosage reduction in alfentanil for maintenance analgesia when used with CYP3A4 inhibitors. Co-administration of voriconazole with alfentanil resulted in a similar outcome in which alfentanil clearance was reduced by 85% [79]. The AUC of alfentanil was also increased sixfold by voriconazole. The pharmacokinetic profile of fentanyl was not affected by itraconazole in a study of 10 healthy volunteers [80]. As a result, the authors stated that itraconazole can be safely used with normal doses of fentanyl, although they warned that an interaction is still possible with higher doses of fentanyl. In general, since the interaction can result in a fatal outcome, caution should be used when phenylpiperidine opiates are coadministered with any azole antifungals and known inhibitors of CYP3A4. Oxycodone is primarily metabolized by CYP2D6 to its active moiety, oxymorphone, while CYP3A is responsible for the formation of its inactive metabolite, noroxycodone [81]. Higher concentrations of oxymorphone may occur if oxycodone is given in conjunction with agents such as selective serotonin reuptake inhibitors (SSRIs), venlafaxine, or cimetidine, which all inhibit oxycodone metabolism. As a guideline, patients receiving agents that affect the metabolic pathways of these narcotics should be carefully monitored for altered pharmacologic activity or enhanced toxicity.

Sedatives, hypnotics, and anxiolytics Benzodiazepines are frequently used in transplant for a variety of indications, including sedation, anxiety, and antiemesis. Alprazolam, loraze-

1533

pam, midazolam, oxazepam, temazepam, and triazolam are examples of some of these benzodiazepines. Alprazolam, midazolam, and triazolam are all metabolized by CYP3A4 [82]. Concurrent administration of CYP3A4 inhibitors, such as azole antifungals or inhibitors of CYP2C19, such as SSRIs, will decrease benzodiazepine clearance. Increases in benzodiazepine concentrations can result in prolonged sedation and psychomotor impairment. Conversely, administration of CYP3A4 or CYP2C19 inducers (Table 99.1) may lead to increased benzodiazepine clearance that results in decreased therapeutic effect. Dosage adjustments may be required when combining these benzodiazepines with CYP3A4 or CYP2C19 inducers or inhibitors. Patients should be carefully monitored for therapeutic failures in the case of combination therapy with inducers such as rifampin, or prolonged adverse effects when used with inhibitors. The pharmacokinetic interactions observed with oral midazolam are more pronounced when administered with the same inducers or inhibitors than when it is given intravenously. This is mainly due to the intestinal and hepatic metabolism associated with first-pass effects with oral dosing. Alprazolam, midazolam, and triazolam are not substrates of P-gp and thus are not affected by inducers or inhibitors of that transport system [82]. Agents such as lorazepam, temazepam, and oxazepam may be reasonable alternatives for HCT patients since these agents undergo glucuronidation and are not metabolized by CYP3A4 [1].

Antidepressants SSRIs, serotonin–norepinephrine reuptake inhibitors, and other agents such as bupropion have replaced tricyclic antidepressants as the preferred agents for the treatment of depression. Potential SSRIs encountered in transplant include fluoxetine, fluvoxamine, paroxetine, sertraline, citalopram, and escitalopram. Similarly, examples of serotonin–norepinephrine reuptake inhibitors include venlafaxine and duloxetine. Bupropion and mirtazapine have also been effective. Many of these antidepressants are dependent on one or more CYP isoenzymes for their metabolic conversions (Table 99.1). They are also inhibitors, in varying degrees, of CYP and UGT enzymes as well as P-gp. Fluoxetine, fluvoxamine, nefazodone (acutely), norfluoxetine (the active metabolite of fluoxetine), paroxetine, sertraline, and venlafaxine are known to inhibit P-gp. Sertraline differs in that it inhibits UGT metabolism [83]. As such, the metabolism of these antidepressants can affect and be affected by the concomitant administration of other drugs that rely on the different enzyme and transport systems. Elevated concentrations of serotonin by drugs that inhibit SSRIs can result in the development of serotonin syndrome, a potentially fatal adverse effect. Linezolid, a weak, reversible, nonselective monoamine oxidase inhibitor has been reported to interact with several SSRIs and other antidepressants in various studies [84]. The authors of these studies favor the use of alternative antibiotic agents in patients taking SSRIs or venlafaxine. If linezolid therapy is necessary, they recommend discontinuing or tapering the antidepressant, if possible, prior to initiating antibiotic treatment. Patients should also be followed closely for signs and symptoms of serotonin syndrome. If serotonin syndrome occurs, linezolid therapy should be discontinued. A minimum washout period for linezolid of at least 2 weeks should occur before the antidepressant can be resumed. Additional interactions with antidepressants are described in Table 99.10 [83,84].

1534

Chapter 99

Table 99.10 Selected drug interactions with antidepressants [83,84] Drug

Interacting drug

Mechanism

Effect

Management

Bupropion

Carbamazepine Duloxetine

(+) CYP2B6 (−) CYP2D6

Fluoxetine

(−) CYP2B6

Monitor for bupropion failure Monitor for side-effects and adjust dose as needed Monitor for seizures

MAOI

Paroxetine

(−) norepinephrine metabolism by MAOI (−) reuptake of norepinephrine by duloxetine (−) CYP2B6

↓ bupropion [ ] ↑ duloxetine [ ] → ↑ side-effects ↑ bupropion [ ] → ↑ seizure risk ↑ norepinephrine → ↑ serotonin syndrome

Phenytoin Sertraline

(+) CYP2B6 (−) CYP2B6

Bupropion Carbamazepine Fluoxetine

See above (+) CYP1A2 (−) CYP2D6

MAOI

Paroxetine

MAOI (−) serotonin and norepinephrine metabolism; duloxetine (−) reuptake of serotonin and norepinephrine (−) CYP2D6

Alprazolam

Bupropion Carbamazepine

Duloxetine

Fluoxetine

Paroxetine

↑ bupropion [ ] → ↑ seizure risk ↓ bupropion [ ] ↑ bupropion [ ] → ↑ seizure risk

Monitor blood pressure, consider using alternative agents Monitor for seizures Monitor for bupropion failure Monitor for seizures

See above ↓ duloxetine [ ] ↑ duloxetine [ ] → ↑ side effects ↑ serotonin → ↑ serotonin syndrome risk

See above Monitor for duloxetine failure Monitor for side-effects and adjust dose as needed Monitor for serotonin syndrome

↑ duloxetine [ ] → ↑ side-effects

Monitor for side-effects and adjust dose as needed

(−) clearance of alprazolam

↑ alprazolam [ ] by 30% and ↑ pyschomotor impairment See above ↓ fluoxetine [ ]

Duloxetine Linezolid MAOI

See above (+) of CYP1A2, CYP2B6, CYP2C9, and CYP3A4 See above (−) serotonin metabolism (−) serotonin metabolism

Monitor for psychomotor impairment and adjust dose as needed See above Monitor for fluoxetine failure

Phenytoin

(−) CYP2C9, CYP3A4, and P-gp

↑ phenytoin [ ] → ↑ phenytoin toxicity

Risperidone

(−) CYP2D6, CYP3A4, and P-gp

↑ risperidone [ ] → risk for extrapyramidal symptoms

See above See text for recommendations In patients already on SSRI: discontinue SSRI 5 weeks prior to initiating MAOI In patients already on MAOI: discontinue MAOI therapy at least 2 weeks prior to initiating SSRI Monitor phenytoin [ ] and adjust phenytoin dose accordingly Monitor for extrapyramidal symptoms

Bupropion Duloxetine Linezolid

See above

See above

See above

(−) serotonin metabolism

See text for recommendations

MAOI

(−) serotonin metabolism

↑ serotonin → ↑ serotonin syndrome risk ↑ serotonin → ↑ serotonin syndrome risk

Risperidone

(−) CYP3A4, CYP2D6 and (−) P-gp

See above (−) serotonin metabolism (−) serotonin metabolism

↑ risperidone [ ] by 43% → risk for extrapyramidal symptoms

In patients on SSRI: discontinue SSRI 2 weeks prior to initiating MAOI In patients on MAOI: discontinue MAOI therapy at least 2 weeks prior to initiating SSRI Monitor for extrapyramidal symptoms

Common Potential Drug Interactions Following Hematopoietic Cell Transplantation

1535

Table 99.10 Continued Drug

Interacting drug

Mechanism

Effect

Management

Sertraline

Bupropion Carbamazepine Lamotrigine

See above (+) CYP2B6, CYP2C9, and CYP3A4 (−) UGT

See above Monitor for sertraline failure Monitor for rash

Linezolid

(−) serotonin metabolism

MAOI

(−) serotonin metabolism

Phenytoin

(+) CYP2B6, CYP2C9, CYP2C19, and CYP3A4

See above ↓ sertraline [ ] Doubling of lamotrigine [ ] → ↑ risk for Stevens–Johnson rash or toxic epidermal necrolysis ↑ serotonin → ↑ serotonin syndrome risk ↑ serotonin → ↑ serotonin syndrome risk ↓ sertraline [ ]

Azoles antifungals

(−) CYP3A4

↑ citalopram [ ]

Carbamazepine Cimetidine

(+) CYP3A4 (−) citalopram metabolism

↓ citalopram [ ] ↑ citalopram [ ] by 43%

Linezolid

(−) serotonin metabolism

MAOI

(−) serotonin metabolism

Metoprolol

Unknown

Omeprazole

(−) CYP2C19

↑ serotonin → ↑ serotonin syndrome risk ↑ serotonin → ↑ serotonin syndrome risk ↑ metoprolol [ ] by 2-fold → ↓ cardioselectivity, no change in blood pressure ↑ citalopram [ ]

Phenytoin

(+) CYP3A4 and CYP2C19

↓ citalopram [ ]

Carbamazepine Fluvoxamine

(+) CYP1A2 and CYP3A4 (−) CYP1A2, CYP2D6, and CYP3A4

Linezolid MAOI

(−) serotonin metabolism ↑ release of serotonin and norepinephrine by mirtazapine and (−) serotonin metabolism by MAOI

↓ mirtazapine [ ] ↑ mirtazapine [ ] by up to 4-fold → ↑ serotonin syndrome risk ↑ serotonin → ↑ serotonin syndrome risk

Linezolid

(−) serotonin metabolism

MAOI

MAOI (−) serotonin, norepinephrine, and sometimes dopamine metabolism; venlafaxine (−) reuptake of serotonin, norepinephrine, and dopamine

Citalopram or escitalopram

Mirtazapine

Venlafaxine

↑ serotonin → ↑ serotonin syndrome risk

See text for recommendations Same recommendations as paroxetine Monitor for sertraline failure Monitor for citalopram toxicity and reduce dose as necessary Monitor for citalopram failure Monitor for citalopram toxicity and reduce dose as necessary See text for recommendations Same recommendations as paroxetine Adjustment in metoprolol dose unnecessary Monitor for citalopram toxicity and reduce dose as necessary Monitor for citalopram failure Monitor for mirtazapine failure Monitor for serotonin syndrome

See text for recommendations Monitor for serotonin syndrome

See text for recommendations Monitor for serotonin syndrome

(−), inhibits; (+), induces; ↑, increases; ↓, decreases; [ ], concentration; → = leading to; CYP1A2, cytochrome P450 1A2 isoenzyme; CYP2B6, cytochrome P450 2B6 isoenzyme; CYP2D6, cytochrome P450 2D6 isoenzyme; CYP2C9, cytochrome P450 2C9 isoenzyme; CYP2C19, cytochrome P450 2C19 isoenzyme; CYP3A4, cytochrome P450 3A4 isoenzyme; MAOI, monoamine oxidase inhibitor; P-gp, P-glycoprotein; SSRI, selective serotonin reuptake inhibitor; UGT, uridine 5′-diphosphate glucuronosyltransferase.

Food Grapefruit juice Since the accidental finding of an interaction between grapefruit juice and felodipine, increasing attention has been focused on this food–drug combination [85,86]. Grapefruit juice contains flavonoids such as naringin, and furanocoumarins such as 6′,7′-dihydroxybergamottin, which may contribute to the interaction. In vitro studies have shown that these components inhibit CYP3A4, but in vivo trials have not proven a defin-

itive agent(s). The proposed mechanism of action appears to be decreased presystemic drug metabolism of oral medications caused by inhibition of CYP3A4 in the small intestine, as well as a reduction in CYP3A4 protein concentrations [85]. This results in higher drug levels, increased absorption, and perhaps exaggerated drug effects. Several factors influence whether an interaction with grapefruit juice might happen. These include using drugs with low bioavailability because of first-pass metabolism, being a substrate for CYP3A4, and having high amounts of CYP3A4 in the intestines [87]. Grapefruit juice does not usually affect

1536

Chapter 99

Table 99.11 Selected drug interactions with grapefruit juice Drug

Mechanism

Effect

Management

Amiodarone [88]

(−) CYP3A4

↑ amiodarone AUC 50% and Cmax 84% → QTc changes

Avoid concomitant use

Calcineurin inhibitors CSP [89] (−) CYP3A4, ?(−) P-gp Tacrolimus [90] Carbamazepine [91] (−) CYP3A4

↑ CSP [ ] 2.1-fold and AUC 1.4 -fold ↑ tacrolimus [ ] ↑ carbamazepine AUC 41%, Cmax 40%, and [ ] 39%

Avoid concomitant use

Cisapride [92] Felodipine [93]

↑ cisapride AUC ~2.4-fold and Cmax 1.8-fold ↑ felodipine AUC 2.9-fold and Cmax 4-fold → ↓ BP, ↑ HR

(−) CYP3A4 (−) CYP3A4

HMG-CoA reductase inhibitors Lovastatin [94] (−) CYP3A4 Simvastatin [95] Midazolam [96] (−) CYP3A4 Terfenadine [97] (−) CYP3A4

↑ lovastatin AUC 15-fold and Cmax 12-fold ↑ simvastatin AUC 13.5-fold and Cmax 12-fold ↑ midazolam AUC 2.4-fold and Cmax 2.3-fold ↑ unmetabolized terfenadine [ ], ↑ acid metabolite AUC 55% → QTc prolongation

Avoid concomitant use if possible. Monitor carbamazepine [ ] Avoid concomitant use Monitor blood pressure and heart rate. May require dosage adjustment Avoid concomitant use Avoid concomitant use Avoid concomitant use

(−), inhibits; ↑, increases; ↓, decreases; [ ], concentration; →, leading to; CYP3A4, cytochrome P450 3A4 isoenzyme; AUC, area under the curve; Cmax, maximum concentration; CSP, cyclosporine; P-gp, P-glycoprotein.

hepatic CYP3A4 activity, but possibly could if consumed in large quantities. Grapefruit juice has been shown to interact with a broad range of medications, from immunosuppressants to cholesterol-lowering agents to antihypertensives. Clinically relevant drug interactions are summarized in Table 99.11 [88–97]. These studies demonstrated highly variable results, leading to difficulty in predicting the degree of an interaction on an individual basis. The dose of a drug, duration and schedule of administration, formulation of a drug, and the source and composition of grapefruit juice, as well as individual metabolism, can all have an effect on the magnitude of the interaction [98,99]. Patients may be more susceptible if they have liver dysfunction or a condition that predisposes

them to more adverse effects from a particular agent. Additionally, for drugs that have a narrow therapeutic index, it is even more important to avoid grapefruit juice so that toxicities do not occur.

Conclusion As new drugs are being developed and used in different combinations, the risk for drug interactions will always be a concern. Having a firm understanding of the mechanisms by which drugs are metabolized and eliminated is a powerful tool to help predict and possibly minimize the occurrence of these interactions in the HCT setting.

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100

Gundeep S. Dhillon & Norman W. Rizk

Critical Care of the Hematopoietic Cell Transplant Recipient

Introduction More than 50,000 patients receive hematopoietic cell transplantation (HCT) worldwide each year, and the number is growing. High levels of complications relating to the conditioning regimens, cytopenias, and immunosuppression associated with HCT eventuate in roughly 11–40% of the recipients requiring transfer to a critical care unit [1]. Many critical illnesses involving HCT patients are unique and reflect the combination of myelosuppression, allogeneic reactivity, and eventual marrow recovery. Graft-versus-host disease (GVHD), diffuse alveolar hemorrhage (DAH), and transplant-associated thrombotic microangiopathy are examples of these disorders. Optimal care of HCT patients must be based on a working knowledge of their unique illnesses and vulnerabilities, the natural history of these disorders, and the degree to which modern critical care can rescue them from these otherwise frequently fatal illnesses. This chapter reviews the organization of critical care for HCT patients, the diagnosis and treatment of specific organ system diseases, the prognosis of critically ill HCT recipients, and decisions about the goals of care involving HCT patients considered for life support modalities.

Organization of critical care for HCT recipients There are approximately 5980 intensive care units (ICUs) in the United States with an average census of about 55,000 patients per day [2]. Most of these are combined medical–surgical units, and most have trouble providing enough skilled staffing to achieve optimal results. Systematic reviews have suggested that full-time intensivist care within critical care units may in fact lower risk-adjusted mortality for the general ICU population by as much as 40%. Sadly, only about 4% of United States hospitals can meet the high standards of critical care specialist availability and high-intensity ICU staffing necessary to achieve these results [2]. Current projections for the supply of physician intensivists, critical care nurses, and pharmacists indicate an increasingly severe shortage of them, beginning in 2007, when demand is expected to exceed supply. Many hospitals already have a 20% vacancy rate in critical care nursing positions, with this expected to worsen over time [3]. Prevalence levels of burnout in critical care nursing staff and physicians as high as 33–50% [4,5] have been well documented, and contribute to the scarcity of these

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

resources. Because HCT recipients represent special challenges and benefit from use of these scarce human resources, their care is best provided in referral centers with adequate means to sustain 24-hour full physician and nurse staffing, preferably with health-care practitioners skilled in the critical care of these patients. High-intensity units capable of providing this care are more common in teaching and larger hospitals. Because of the shortage of critical care specialists, and because of volume considerations, very few hospitals for the foreseeable future will be able to provide specialized critical care units dedicated to HCT recipients. The most practical model in most centers with transplant programs will continue to be critical care specialists teaming with transplant professionals in general medical–surgical units. The team approach is essential for developing protocols and treatment guidelines, for monitoring and improving infection control associated with neutropenia and invasive devices, and for controlling metabolic derangements like hyperglycemia. These tasks touch on both the unique needs of HCT patients and the special environment of critical care units. Considerable evidence indicates that very morbid and mortal conditions such as ventilator-associated pneumonia can be prevented successfully by simple measures, like elevating the head of the bed by 30 degrees, and decontamination of oral cavities by use of chlorhexidine mouthwashes [6]. Similarly, strict adherence to Center for Disease Control decontamination guidelines for controlling blood stream infections associated with central lines can dramatically reduce or nearly eliminate these infections [7], which now number about 80,000 cases per year in ICUs [8]. Neutropenic HCT recipients have two to three times the rate of blood stream infections of other ICU patients [9] and are clearly at increased risk. Evidence also indicates that prophylaxis against gastrointestinal bleeding is required for patients on mechanical ventilators [10]. Well-run ICUs develop metrics to follow the use of these processes and measure their patient outcomes, using tools like prognostic scales, observed-to-expected mortality ratios, and specific complication rates per 1000 patient days. Organizations such as the University Health System consortium provide benchmark data for like institutions, as a form of report card on overall function; infection control data may be pooled in national initiatives such as the National Nosocomial Infection Surveillance project, which can inform intensive care medical directors about their performance in preventing infection and in controlling nosocomial spread. Increasingly, these metrics are being publicly reported, which will provide referring physicians with an objective database about the safety and efficacy of critical care units their patients may require. Optimal care requires engagement by many specialties in improving processes

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Bacterial Fungal Herpes simplex/?RSV

Herpes varicella-zoster

Pneumocystis Cytomegalovirus

DAH/ARDS

Idiopathic interstitial pneumonitis

CHF

Bacterial (sinopulmonary) Obstructive airways disease/BO Restrictive ventilatory defect Acute GVHD 1

2

Chronic GVHD 3

4

5

6

7

8

9

10

11

Months after BMT Neutropenic phase

Day 100 post-BMT

in ICUs, particularly in the large majority of units that are designed as general medical–surgical ones.

Specific organ complications of HCT Pulmonary complications Approximately 60% of HCT recipients develop pulmonary complications after transplantation, and 30% of deaths are related to pulmonary complications [1,11–13]. Respiratory compromise is the most common indication for admission to the ICU. The different pulmonary complications seen in HCT recipients occur during predictable time periods, and an understanding of this temporal sequence can be very helpful in developing a differential diagnosis (Fig. 100.1) [12]. In the immediate postHCT period, extending from day 0 to day 30, the recipients are usually neutropenic and at high risk for the development of bacterial or fungal infections. Other common pulmonary complications during this period include pulmonary edema, drug toxicities, DAH, and idiopathic pneumonia syndrome (IPS). The acute neutropenic phase is followed by an early post-engraftment period that extends from day 30 to day 100. During this phase, recipients are no longer neutropenic, but use of immunosuppressive drugs for prophylaxis and treatment of GVHD results in both cellular and humoral immunity deficits. The recipients are at risk for development of viral infections, interstitial pneumonitis, acute GVHD, and delayed pulmonary toxicity syndrome. The late post-engraftment starts at post-HCT day 100 and extends indefinitely. At this time, noninfectious complications are more common, although patients, especially allogeneic HCT recipients, remain at risk for infectious complications. The noninfectious pulmonary complications seen during this period include bronchiolitis obliterans with or without organizing pneumonia. The pulmonary complications in HCT recipients depend upon pre-existing pulmonary conditions, pretransplant conditioning regimens, and level of immune reconstitution. These

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Fig. 100.1 Pulmonary complications occurring after hematopoietic stem cell transplantation. ARDS, acute respiratory distress syndrome; BMT, bone marrow transplantation (i.e., hematopoietic stem cell transplantation); BO, bronchiolitis obliterans; CHF, congestive heart failure; DAH, diffuse alveolar hemorrhage; GVHD, graft-versushost disease; RSV, respiratory syncytial virus. (Reproduced from [12], with permission.)

pulmonary complications can lead to respiratory failure in a significant percentage of patients, with the incidence ranging from 10% to more than 50%. Infectious pulmonary complications Pneumonia is the leading infectious cause of death after HCT. The factors leading to pneumonia include neutropenia, immunosuppressive state, GVHD, and mucositis. Bacterial pneumonias are more frequent during the first month after HCT (see Chapter 88). The incidence of bacterial pneumonia in this patient population may be as high as 15%, with half the cases occurring in the first 100 days [14]. Bacterial pneumonias are more common in patients undergoing high-intensity rather than reduced-intensity HCT, probably related to longer periods of neutropenia in the former [15]. During the neutropenic phase, presentation of bacterial pneumonia can be subtle. Patients may present with fever only, without any respiratory symptoms [13]. In addition, chest radiographs can also be completely normal. Chest computed tomography (CT) scans are significantly more sensitive than plain radiographs for diagnosis. The treatment consists of early institution of broad-spectrum antibiotics, followed by narrowing of the antimicrobial coverage once culture data become available. Bacterial pneumonia can rapidly lead to acute respiratory failure and multiorgan system failure (MOSF). Cytomegalovirus (CMV) is an important pathogen in HCT recipients (see Chapter 90). The vast majority of CMV infections in these patients are related to activation of latent disease, rather than new infections [13]. CMV pneumonitis occurs within the first 100 days; however, use of CMV prophylaxis may delay the onset of active disease [16]. CMV pneumonitis patients present with dyspnea, dry cough, and hypoxemia, and can progress to respiratory failure. The demonstration of CMV inclusion bodies in lung tissue is diagnostic. In the absence of lung tissue, diagnosis can be made by detection of CMV in bronchoalveolar lavage (BAL) fluid and/or in serum by the polymerase chain reaction,

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Table 100.1 Diagnostic criteria for idiopathic pneumonia syndrome I. Evidence of widespread alveolar injury, including: Multilobar infiltrates on routine chest radiographs or CT scans Symptoms and signs of pneumonia Evidence of abnormal pulmonary physiology Increased alveolar-to-arterial oxygen gradient New or increased restrictive pulmonary function test abnormality

Fig. 100.2 Chest tomograph showing right upper lobe consolidation and the air-crescent sign in a recipient of hematopoietic cell transplantation who had invasive aspergillosis.

in the appropriate clinical setting. However, a negative serum polymerase chain reaction or failure to detect CMV antigenemia does not rule out CMV disease [17]. Treatment consists of combination therapy with intravenous ganciclovir and immunoglobulin infusion [18,19]. The survival rates of HCT recipients with CMV pneumonitis have improved from 15% to 50–70%. This improvement in survival has been attributed to use of the combination therapy with ganciclovir and immunoglobulin, although controlled trials are lacking. Ganciclovir-resistant strains of CMV may require treatment with foscarnet. The respiratory viral infections, such as respiratory syncytial virus (RSV), parainfluenza, influenza, and rhinovirus are also a major cause of morbidity and mortality in this patient population. RSV is the most common respiratory viral isolate in HCT recipients, and is associated with poor outcomes [20]. A comprehensive strategy to reduce personto-person transmission of RSV can reduce the incidence of this infection in HCT recipients. The treatment of RSV consists of nebulized or intravenous ribavirin and administration of RSV-specific immunoglobulin. Invasive aspergillosis is a particularly serious pulmonary complication of HCT (see Chapter 89). It is more common in patients undergoing allogeneic HCT, probably as a result of long-term immunosuppressive therapy for GVHD [21]. Currently, there is no effective prophylaxis for invasive pulmonary aspergillosis (IPA), although low-dose intravenous amphotericin B, nebulized amphotericin B, voriconazole, and itraconazole have been used. Invasive disease is usually limited to the lungs, although it can involve the sinuses, central nervous system, and other organs, reflecting hematogenous spread from angioinvasion [13]. Patients usually present with dyspnea and cough. Pleuritic chest pain and hemoptysis are not uncommon. The chest radiographs and CT scan may show pulmonary nodules, consolidation or cavitation. The “halo sign,” indicating hemorrhage around the lesion, and the “air-crescent sign” (Fig. 100.2), signifying cavitation, are characteristic signs of IPA. In HCT recipients, detection of Aspergillus in respiratory secretions has a positive predictive value greater than 80% for IPA, but the sensitivity of this test is low [22,23]. Pulmonary nodules may require transthoracic needle biopsy or resection for diagnosis. An enzyme-linked immunosorbent assay for galactomannan, a fungal cell wall component, has been used for surveillance for IPA. It has a sensitivity of 0.71 and a specificity of 0.89 [24]. Intravenous voriconazole, a triazole antifungal agent,

II. Absence of active lower respiratory tract infection. Appropriate evaluation includes: Bronchoalveolar lavage negative for significant bacterial pathogens and/or lack of improvement with broad-spectrum antibiotics Bronchoalveolar lavage negative for pathogenic nonbacterial microorganisms Routine bacterial, viral, and fungal cultures Shell-vial CMV culture Cytology for CMV inclusions, fungi, and Pneumocystis jiroveci Detection methods for respiratory syncytial virus, parainfluenza virus, and other organisms Transbronchial biopsy if condition of the patient permits Ideally, a second confirmatory negative test for infection is done. This usually is performed 2–14 days after the initial negative bronchoalveolar lavage, and it may consist of a second bronchoalveolar lavage or a surgical lung biopsy CMV, cytomegalovirus; CT, computed tomography. Reproduced with permission from Clark et al. [28].

appears to be more efficacious and better tolerated than amphotericin B for the treatment of IPA [25]. Caspofungin, an echinocandin, has also been approved for salvage therapy of IPA. Pneumocystis jiroveci pneumonia (formerly Pneumocystis carinii pneumonia; PCP) is now an uncommon opportunistic infection in HCT recipients, secondary to effective prophylaxis with trimethoprim– sulfamethoxazole. In the absence of prophylaxis, the incidence of PCP in allogeneic HCT recipients is as high as 15% [26]. For HCT recipients who are on immunosuppressive therapies and are not receiving optimal prophylaxis, suspicion of this should remain high. BAL is the procedure of choice for diagnosis, with a diagnostic yield in excess of 90% [27]. The treatment of choice consists of high-dose trimethoprim–sulfamethoxazole. The role of corticosteroids in the treatment of PCP in this patient population, unlike in patients with acquired immune deficiency syndrome, is unclear. Noninfectious pulmonary complications IPS, as defined by a National Heart, Lung, and Blood Institute panel in 1993, encompasses a wide range of clinical presentations of lung injury in HCT recipients, which cannot be attributed to infectious or cardiogenic causes (see Chapter 96) [28]. The diagnostic criteria (Table 100.1) include evidence of pneumonia, nonlobar infiltrates on chest radiograph, and absence of infectious etiology. The histopathologic findings associated with IPS include interstitial pneumonitis, diffuse alveolar damage, bronchiolitis obliterans organizing pneumonia (BOOP), and lymphocytic bronchitis. The term “interstitial pneumonitis” has been used interchangeably with IPS [29]. The reported incidence of IPS after HCT ranges from 3% to 15% [30]. It is more common in patients receiving allogeneic rather than autologous HCT. Among allogeneic HCT recipients, IPS is more common among patients receiving high-dose preparative regimens. Patients usually present with dyspnea, hypoxemia, cough, and fever. The onset

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Fig. 100.3 Bilateral lower lobe air space disease secondary to diffuse alveolar hemorrhage in a recipient of hematopoietic cell transplantation.

of symptoms is within the first 100 days following transplantation, with more recent studies suggesting an earlier onset around 2–3 weeks post transplant [31,32]. The treatment for IPS is supportive. Patients are commonly treated with high-dose corticosteroids, although the evidence for their efficacy remains scant. The clinical course is one of progressive respiratory failure leading to MOSF and death. DAH is a pulmonary complication of HCT characterized by a progressively bloodier return of BAL aliquots. It is seen in 5% of HCT recipients, and is slightly more common in patients receiving autologous rather than allogeneic HCT [30]. The risk factors for DAH include intensive conditioning chemotherapy, total body irradiation, renal insufficiency, older age, and previous acute GVHD [33,34]. Patients usually present within the first 30 days after HCT with progressive dyspnea, fever, cough, hypoxemia, and bilateral ground glass infiltrates on highresolution CT of the chest (Fig. 100.3). Hemoptysis is uncommon in patients with DAH. The diagnosis is usually made with BAL that shows a progressively bloodier return of BAL aliquots and hemosiderin-laden macrophages. The following criteria are suggested for the diagnosis of DAH: (1) evidence of bilateral widespread alveolar injury; (2) absence of infection; and (3) BAL showing progressively bloodier return from three different subsegmental bronchi or the presence of 20% or more of hemosiderin-laden macrophages [33]. Most patients with DAH develop respiratory failure requiring mechanical ventilatory support. In addition to supportive care, treatment with high-dose corticosteroids (125–250 mg of methylprednisolone every 6 hours for 4–5 days, with taper over the next 4 weeks) may be helpful [35,36]. The overall prognosis remains poor, with earlier reported mortality rates as high as 80–100% [34–36], but more recent studies indicating it closer to 60% at 6 months [37]. BOOP, a pathologic entity with small airway injury and interstitial inflammation with granulation tissue plugs in small airways, is another manifestation of acute lung injury seen in patients with GVHD following allogeneic HCT. It develops 1–3 months after the transplant, and the usual presentation consists of fever, dry cough, dyspnea, hypoxemia, and bilateral pulmonary infiltrates. These patients can progress to acute respiratory failure requiring mechanical ventilation. BOOP usually responds to corticosteroid therapy, although patients may require it for prolonged periods of time [30]. A delayed pulmonary toxicity syndrome has been described in patients who have undergone high-dose chemotherapy with carmustine-

containing regimens followed by HCT. The median time to onset of symptoms is 10 weeks from HCT. Symptoms are usually mild and consist of exertional dyspnea, dry cough, and fever. Pulmonary function tests shows mild restriction and severe reduction in diffusion capacity. High-resolution CT scans of the chest show patchy or diffuse ground glass infiltrates. Symptoms are responsive to early and aggressive corticosteroid therapy [38]. However, carmustine-related pulmonary toxicity syndrome could lead to progressive respiratory failure and death in up to 8% of the affected patients [39]. Bronchiolitis obliterans (without organizing pneumonia) is a late pulmonary complication seen in allogeneic HCT recipients. The onset of symptoms is beyond 3 months after HCT and is strongly associated with chronic GVHD [40]. The clinical picture resembles progressive chronic obstructive lung disease. Besides GVHD, other risk factors for the development of bronchiolitis obliterans include pretransplant obstructive lung defect, recipient age, methotrexate use, and history of respiratory viral illness within the first 100 days after transplant [40]. The clinical course is variable, with a small subset of patients developing progressive obstructive lung defect leading to respiratory failure [41]. Treatment consists of augmentation of immunosuppression in an attempt to stabilize lung function [42]. Lung transplantation may be an option in select patients who are deemed cured of their underlying initial disease process [13]. The other uncommon pulmonary complications seen after HCT include pulmonary veno-occlusive disease and posttransplant lymphoproliferative disorder [13]. Respiratory failure Respiratory failure is present in half of the HCT patients admitted to the ICU [1]. The risk factors for development of respiratory failure after HCT include age above 21 years, active malignancy at the time of transplant, and nonidentical human leukocyte antigen (HLA) donor [43]. Earlier studies suggested that, among HCT recipients who required mechanical ventilation, mortality rates approached 100% [43]. However, more recent studies have suggested survival rates up to 25%. This improved survival is probably related to an increased use of peripheral blood hematopoietic cells [44,45], improvements in ventilatory strategies [46,47], and better supportive care. In general, HCT patients with respiratory failure should be managed using the same general principles that have been developed for patients with lung injury related to other diseases, while recognizing their unique vulnerabilities. In view of the rapidity with which pulmonary complications can lead to respiratory failure in HCT recipients, these patients should undergo rapid and thorough diagnostic evaluation. The choice of diagnostic procedures is guided by clinical history and radiographic appearance. CT scanning is useful in the evaluation of these patients, as it may suggest diagnoses and is a sensitive tool for defining the extent of disease. Fiberoptic bronchoscopy with BAL is the diagnostic test of choice in HCT patients with pulmonary opacities. Diagnostic yield for infectious etiologies, with the notable exception of focal diseases like invasive aspergillosis, is high [48]. Transbronchial biopsies do not improve yield and are potentially dangerous because of coagulopathies. Patients with discrete pulmonary nodules may need transthoracic needle aspiration. A surgical lung biopsy, either by thoracotomy or thoracoscopy, is safe but rarely impacts management and hence is rarely performed. HCT patients with acute lung injury, as defined in Table 100.2, should be managed according to lung-protective ventilatory strategies that have been shown to improve outcomes in patients with acute lung injury [46]. The goal tidal volume for mechanical ventilation should be less than 6 mL/kg of predicted body weight, airways plateau pressures should be maintained at less than 30 cmH2O, and the partial pressure of oxygen in arterial blood (PaO2) should be maintained between 55 and 80 mmHg, using a combination of fraction of inspired oxygen and positive end-

Critical Care of the Hematopoietic Cell Transplant Recipient Table 100.2 Definition of acute lung injury

Table 100.3 Diagnostic criteria for sepsis

Acute onset Bilateral infiltrates on chest radiograph PaO2: FiO2 <300 mmHg No evidence of left atrial hypertension

Infection, documented or suspected, and some of the following:

FiO2, fraction of inspired oxygen; PaO2, partial pressure of oxygen in arterial blood. Reproduced with permission from Bernard et al. [116].

expiratory pressure. Daily evaluation should be done to assess patient readiness for ventilator weaning. Weaning using formal protocols in ICUs appears to improve outcomes in patients with respiratory failure [49]. Currently, there is no evidence to support the use of corticosteroids in patients with acute lung injury, unless they are needed for the underlying disease process [50]. Nitric oxide can be used to improve oxygenation but does not appear to impact survival in patients with acute lung injury [51]. The optimal fluid management strategy for patients with acute lung injury remains unclear. A recent randomized controlled trial comparing a liberal with a conservative fluid strategy failed to show a mortality benefit, but the patients managed by conservative fluid strategy were able to be extubated early [52]. Patients with respiratory failure, while recovering from their underlying disease, are vulnerable to complications related to critical care itself. All attempts should be made to minimize the risks of venothromboembolic disease, nosocomial infections, gastrointestinal bleeding, and malnutrition [6,7,10,53]. Severe infections and septic shock Severe infections are a common complication post HCT and are responsible for transfer to the ICU in 23% of patients [1,54]. HCT recipients are at high risk for the development of infections secondary to neutropenia, impaired immunity related to their primary diagnosis and need for immunosuppressive drugs, and compromised mucosal and cutaneous barriers. These patients remain at risk for common health-care-related infections as well as opportunistic infections. The specific organisms causing these infections are discussed in greater detail in Chapters 88–94. A consensus statement in 2003 defined sepsis as a systemic response to infection. A similar response can occur in the absence of infection, and is termed the systemic inflammatory response syndrome. Severe sepsis defines a subset of patients who develop end-organ dysfunction, and septic shock refers to patients who have sepsis-induced hypotension despite adequate fluid resuscitation, along with hypoperfusion abnormalities or organ dysfunction (Table 100.3) [55]. The mortality for all patients with sepsis is 16%, and as high as 46% in cases of septic shock. The outcomes for this condition are significantly worse in HCT recipients, with reported mortality as high as 80% [56]. The principles of management of sepsis in HCT recipients are extrapolated from studies based on general critical care populations. The applicability of these studies in the management of HCT recipients is unclear but plausible. In the absence of more specific data, HCT recipients should be managed according to evidence-based guidelines developed for patients with severe sepsis and septic shock. Initial resuscitation appears to have an impact on survival in patients with severe sepsis and septic shock [57]. Resuscitation should be undertaken as soon as sepsis is recognized. The goal of initial resuscitation should be to establish adequate tissue and organ perfusion. Initial resuscitation should consist of fluid administration to ensure adequate central venous filling pressures of 8–12 mmHg. Vasoactive agents such as dopamine and norepinephrine should be used only after fluid resuscitation and

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General variables Fever (core temperature >38.3ºC) Hypothermia (core temperature >36ºC) Heart rate >90 min−1 or >2 sd above the normal value for age Tachypnea Altered mental status Significant edema or positive fluid balance (>20 mL/kg over 24 hrs) Hyperglycemia (plasma glucose >120 mg/dL or 7.7 mmol/L) in the absence of diabetes Inflammatory variables Leukocytosis (WBC count >12,000 μL−1) Leukopenia (WBC count <4000 μL−1) Normal WBC count with >10% immature forms Plasma C-reactive protein >2 sd above the normal value Plasma procalcitonin >2 sd above the normal value Hemodynamic variables Arterial hypotension (SBP <90 mmHg, MAP <70, or an SBP decrease >40 mm Hg in adults or <2 sd below normal for age) S v¯o2 >70% Cardiac index >3 L⋅min−1⋅M−2 Organ dysfunction variables Arterial hypoxemia (Pao2/Fio2 <300) Acute oliguria (urine output <0.5 mL⋅kg−1⋅hr−1 or 45 mmol/L for at least 2 hrs) Creatinine increase >0.5 mg/dL Coagulation abnormalities (INR >1.5 or aPTT >60 secs) Ileus (absent bowel sounds) Thrombocytopenia (platelet count <100,000 μL−1) Hyperbilirubinemia (plasma total bilirubin >4 mg/dL or 70 mmol/L) Tissue perfusion variables Hyperlactatemia (>1 mmol/L) Decreased capillary refill or mottling aPTT, activated partial thromboplastin time; Fio2, fraction of inspired oxygen; INR, international normalized ratio; MAP, mean arterial blood pressure; Pao2, partial pressure of oxygen in arterial blood; SBP, systolic blood pressure; sd, standard deviation; S v¯o2, mixed venous oxygen saturation; WBC, white blood cell. Reproduced with permission from Levy et al. [55].

to maintain mean arterial pressure greater than 65 mmHg and urine output greater than 0.5 mL/kg/hour [58]. Vasopressin is useful in patients with refractory shock, but as an adjunctive rather than a primary agent [59]. Patients with refractory hypotension, worsening vasopressor requirements, low urine output or cardiac dysfunction may benefit from further evaluation of hemodynamic status with echocardiography or placement of a pulmonary artery catheter. Patients with low cardiac output despite fluid resuscitation may benefit from inotropic support with dobutamine. Diagnostic work-up for infections should be initiated during the initial resuscitation. If possible, appropriate cultures for both communityacquired and health-care-associated infections should be taken prior to antimicrobial therapy. Cultures from blood, urine, respiratory secretions, and other body fluids should be obtained as clinically indicated. The antimicrobial agents should be administered as soon as possible, with the current standard being within 4 hours of presentation. The initial antibiotic choice should be guided by the patient’s clinical situation. However, the antimicrobial coverage should be broad enough to cover

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all likely pathogens. A delay in initiation and/or inappropriate antimicrobial therapy has been associated with worse outcomes in patients with severe sepsis [60,61]. All patients should undergo a thorough evaluation for any possible infection source, which may be amenable to drainage, debridement or removal. Patients with severe sepsis and septic shock may have absolute or relative adrenal insufficiency. The risk of adrenal insufficiency may be higher in HCT recipients, as they have substantial exposure to exogenous corticosteroids. To evaluate adrenocortical reserve, patients with septic shock should undergo a cosyntropin stimulation test. Patients with either a low random cortisol level (<15 μg/dL) or less than a 9 μg/dL rise in cortisol after administration of 250 μg of cosyntropin may benefit from administration of exogenous corticosteroid replacement [62,63]. Otherwise, high-dose corticosteroids have no role and are potentially harmful in patients with septic shock. A randomized placebo-controlled study published in 2001 showed that activated protein C administration in patients with septic shock and an Acute Physiology and Chronic Health Evaluation (APACHE) II score greater than 25 is associated with improved survival [64]. However, this trial excluded immunosuppressed and thrombocytopenic patients. The role of activated protein C in HCT recipients with septic shock hence remains unclear. Intensive insulin therapy to maintain blood glucose level between 80 and 110 mg/dL has been shown to reduce ICU mortality in postoperative surgical patients [65]. However, a follow-up study performed in patients admitted to the medical ICU failed to show any mortality benefit, although it did reduce morbidity. In patients treated with intensive insulin therapy, there was a trend toward increased mortality for those whose ICU stay was less than 3 days [66]. In view of these data, a reasonable approach would be to maintain blood glucose levels less than 150 mg/dL in patients with severe sepsis. Cardiac complications Cardiac complications are common following HCT, with a reported incidence varying from 2% to 28% [67,68]. These complications are responsible for admission to the ICU in 20% of patients after HCT [1], despite aggressive pretransplant screening and modified preparative regimens designed to minimize complications. However, as HCT is offered to older populations and to patients with autoimmune diseases such as systemic sclerosis, cardiac complications are likely to rise. The major life-threatening cardiac complications seen in HCT patients include congestive heart failure (CHF), pericardial effusions, arrhythmias, and endocarditis [68]. CHF, the most common cardiac complication, usually occurs in the setting of pre-existing cardiac dysfunction. Other risk factors include high doses and recent administration of cyclophosphamide (CY), concomitant administration of other chemotherapeutic agents, a history of radiation to the mediastinum or left hemithorax, a history of anthracycline exposure, older age, and obesity [69]. CHF in HCT recipients may be further exacerbated by renal dysfunction, fluid overload, transfusion of blood products, and sepsis. The role of pre-HCT evaluation for resting left ventricular ejection fraction remains unclear [70–72]. The resting electrocardiogram (ECG) may be able to predict cardiac dysfunction after HCT. QT interval dispersion and corrected QT interval have been reported to predict CHF following HCT [73,74]. Although a complete history, physical examination, chest radiograph, and resting 12-lead ECG may be sufficient to identify patients at high risk for cardiac toxicity, it is probably prudent to obtain a two-dimensional echocardiogram in patients with risk factors for cardiac toxicity [69]. Measurements of left ventricular ejection fraction with radionuclide imaging and/or twodimensional echocardiography may be useful for early detection and

monitoring of cardiac toxicity during and after HCT [75]. Circulating biomarkers such as troponin and natriuretic peptides may be similarly useful [76,77]. CY is responsible for the majority of cardiac toxicity of preparative regimens for HCT [78]. The toxicity clinically manifests within 3 weeks of administration. The usual presentation consists of CHF, pulmonary vascular congestion, and renal insufficiency. Patients may also develop pericardial effusion with tamponade physiology [79]. The pathogenesis of CY-related cardiac toxicity centers on injury to the endothelium, with extravasation of toxic metabolites resulting in myocardial injury. CYrelated cardiac toxicity can be reduced by using multifractionated dosing [69]. The risk for CY-related cardiac toxicity is increased by the concomitant administration of cytarabine or mitoxantrone [80,81]. Melphalan and fludarabine, when used in combination, are associated with 2–14% incidence of cardiac toxicity, although it is rare with either agent alone [82]. The treatment of CHF after HCT is extrapolated from the treatment of CHF in other more common settings. At times, it can be hard to differentiate CHF-related pulmonary vascular congestion from noncardiogenic causes of pulmonary infiltrates and respiratory failure. The ECG and echocardiogram can be very helpful in the acute setting, but occasionally right heart catheterization may be required to assist with the diagnosis and to guide therapy. Patients are initially treated with diuretics, and if there is evidence of left ventricular dysfunction, an angiotensin-converting enzyme inhibitor should be tried, in normotensive patients. Patients may require intravenous inotropic and vasoactive agents to maintain adequate cardiac output and blood pressure. Occasionally, patients will also require mechanical ventilatory support. Pericardial effusions are uncommon after HCT, and are usually seen in the setting of GVHD, renal failure or CY toxicity, or are due to an infectious cause [68]. Sudden pericardial effusions with hemodynamic instability have been described in up to 2% of patients undergoing bone marrow transplantation for thalassemia (see Chapter 73) [83]. A recent study reported the incidence of clinically significant pericardial effusions at 4.4% in pediatric stem cell recipients [84]. These effusions can result in cardiac tamponade with hemodynamic instability and death. In the setting of HCT, a hemodynamically significant pericardial effusion should be drained by surgical pericardiectomy to ensure adequate hemostasis. However, in an emergency situation, an echocardiogram-guided pericardiocentesis can be performed at the bedside. Cardiac arrhythmias are an infrequent complication of HCT, and are usually seen in the setting of other critical illnesses. Supraventricular tachyarrhythmias are seen in 4% of HCT recipients, and occur at a median of 6 days after transplantation. Supraventricular tachyarrhythmias are symptomatically and hemodynamically significant in approximately half the patients, and result in prolonged hospital stay and increased mortality [85]. These arrhythmias are more common in patients with pre-existing cardiac conditions, older age, and HCT for non-Hodgkin’s lymphoma. Amiodarone and diltiazem are commonly used to manage supraventricular tachyarrhythmias in these patients, and electrical cardioversion may be needed in case of hemodynamic instability. Bradyarrhythmias have been described during infusion of dimethyl sulfoxide-cryopreserved bone marrow and peripheral blood hematopoietic cells [86]. Gastrointestinal and hepatic complications Gastrointestinal and hepatic complications are frequently seen in HCT recipients admitted to the ICU (see Chapter 95). Conditions such as pancreatitis, cholecystitis, ileus, and gastrointestinal bleeding, common to many critically ill patients, occur frequently, as do multiple disorders unique to this patient population.

Critical Care of the Hematopoietic Cell Transplant Recipient

Acute mucositis is a common and important complication after HCT, which occurs as a result of the preparative regimen (see Chapter 103). It occurs in 70–80% of patients receiving radiation-based conditioning regimens [87]. The oropharyngeal mucositis is painful and can cause severe odynophagia, which may lead to poor nutritional intake and potentially life-threatening infections. Involvement of the supraglottic region may cause upper airway obstruction and even respiratory failure, requiring parenteral nutrition and/or mechanical ventilation. Intestinal mucositis usually presents with nausea, abdominal cramping, and diarrhea [88]. The management of mucositis consists of symptomatic measures, good oral care, and nutritional support. A recombinant human keratinocyte growth factor, palifermin, has been shown to reduce the duration and severity of oral mucositis after intensive chemo- and radiotherapy in hematologic malignancies [89]. Gastrointestinal bleeding has been reported in 7–18% of HCT patients. Most cases are diffuse mucosal bleeds and are not amenable to endoscopic treatment. Gastrointestinal bleeding is frequently associated with GVHD, and is exacerbated by thrombocytopenia [90,91]. Severe gastrointestinal bleeding is a poor prognostic marker, although most deaths are not directly attributable to the bleeding [45,91]. Treatment includes supportive care and correction of underlying abnormalities such as coagulopathy, thrombocytopenia, and GVHD. Sinusoidal obstructive syndrome (SOS; previously known as hepatic veno-occlusive disease) is a common condition seen after HCT, and can result in early mortality [92]. The reported incidence of SOS in different studies has ranged from 10% to 60% [93,94]. The pathogenesis of SOS is secondary to injury of sinusoidal endothelial cells and hepatocytes from the toxic metabolites of chemotherapeutic agents used in the conditioning regimens. This injury leads to sloughing of these cells and subsequent obstruction of hepatic circulation. The risk factors for development of SOS include allogeneic transplantation, extremes of age, HLA mismatch, poor performance status, female sex, prior liver disease, abdominal radiation, and a second myeloablative transplantation [95]. Hepatic SOS is a clinical syndrome of painful hepatomegaly, hyperbilirubinemia, and fluid overload, and may lead to renal and respiratory failure. The vast majority of cases develop SOS within the first 3 weeks after HCT. A CT scan and ultrasound of the liver can be useful in confirming hepatomegaly and ascites. A liver biopsy may be needed to confirm the diagnosis and to rule out other causes of liver dysfunction. A transvenous liver biopsy and measurement of wedged hepatic venous pressure gradient are the diagnostic procedures of choice, although rarely performed in clinical practice [96]. The prognosis of SOS depends upon the severity of the disease. The presence of multiple organ failure is a strong predictor of mortality [94]. Currently, the standard treatment remains simple supportive care, consisting of diuretics, renal replacement therapy if needed, therapeutic paracentesis for ascites, and correction of coagulopathy. As these patients are at high risk for developing infectious complications, the threshold for initiation of broad-spectrum antibiotics should be low. Thrombolytics, heparin, antithrombotic replacement, and defibrotide, a single-stranded polydeoxyribonucleotide with local antithrombotic and anti-inflammatory properties, have all been reported to be efficacious in the treatment of SOS [95]. However, thrombolytics and heparin administration have been associated with fatal hemorrhage in patients with SOS and multiple system organ failure. Clinical trials to assess the efficacy and safety of defibrotide are currently ongoing. Renal complications Acute renal failure (ARF), defined as an acute fall in glomerular filtration rate of greater than 50%, or a doubling of serum creatinine, occurs in more than half of patients receiving HCT [97]. There is a significant

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difference in the incidence and severity of ARF in patient groups undergoing different types of HCT [98]. These differences are most likely attributable to differences in patient characteristics, conditioning regimens, and occurrence of GVHD. The most common cause of ARF following HCT is MOSF related to sepsis. Sepsis may result in renal hypoperfusion, release of proinflammatory cytokines, and activation of the complement cascade, leading to renal injury. Drugs, such as aminoglycosides and amphotericin B, used for treatment of severe infections may further contribute to renal injury. Tumor lysis syndrome is an uncommon cause of ARF in HCT recipients [99]. This syndrome follows destruction of tumor cells secondary to high-intensity chemo- or radiotherapy. The release of intracellular components such as uric acid and phosphates can cause tubular injury. The incidence is low because the tumor burden is usually small at the time of HCT. Additionally, pretreatment with allopurinol, urinary alkalinization, and hydration are effective in preventing tumor lysis syndrome. ARF related to tubular injury can also result from hemolysis and hemoglobinuria following the infusion of stem cells. Dimethyl sulfoxide, which is used for cryopreservation of the hematopoietic cells, likely plays an important role in the risk of hemolysis [100]. Hepatic SOS is another important cause of ARF in patients with HCT. It causes fluid retention with low urinary sodium, reminiscent of hepatorenal syndrome. As previously discussed, the treatment is mostly supportive. Renal insufficiency may also be due to thrombotic microangiopathy, a syndrome of fever, anemia, thrombocytopenia, neurologic dysfunction, and renal failure. The reported incidence of thrombotic microangiopathy in HCT recipients varies widely, probably reflecting the varied diagnostic criteria applied in this patient population. At a minimum, evidence of microangiopathic anemia on the peripheral blood smear is essential for the diagnosis [101]. Risk factors associated with thrombotic microangiopathy include high-dose allogeneic transplantation, SOS, acute GVHD, and infections. The treatment consists of plasmapheresis, although it is less effective than in patients with classic thrombotic thrombocytopenic purpura. Calcineurin inhibitors, namely tacrolimus and cyclosporine, are used for the prophylaxis and treatment of GVHD. Both of these agents are renal vasoconstrictors and cause a serum drug concentration-dependent reduction in renal function. The patients with calcineurin inhibitor-related renal dysfunction rarely require renal replacement therapy, and usually respond to a reduction in drug dosing. Hemorrhagic cystitis occurs in up to 25% of patients undergoing allogeneic HCT, usually within 2 weeks of transplantation, and is associated with administration of CY, busulfan or radiation. Larger clots can cause obstructive uropathy leading to ARF. Management of hemorrhagic cystitis includes aggressive hydration, urinary bladder irrigation, and diuresis [102]. The management of ARF consists of identifying and, if possible, treating the underlying cause of renal dysfunction. In patients with established renal failure, diuretics, calcium channel blockers, dopamine, and natriuretic peptides have been tried for the immediate treatment of renal failure [103]. A trial of diuretics to convert an oliguric ARF into nonoliguric ARF is reasonable. However, use of other agents in this setting is unlikely to be helpful. In patients with established ARF, all attempts should be made to maintain euvolemia, avoid malnutrition, and correct electrolyte abnormalities. Renal replacement therapy (RRT) can be initiated in patients with ARF if the conservative therapy fails. The common indications for RRT include refractory hyperkalemia, fluid overload, severe acidosis, pericarditis, and neurologic abnormalities. Several observational studies have suggested that early initiation of RRT results in more favorable outcomes in patients with ARF [104–106]. Currently, both intermittent hemodialysis and continuous RRT are used for the management of ARF in ICUs. The mode of RRT does not appear to impact clinical outcomes,

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although CRRT is better tolerated in patients with hemodynamic instability and is more effective in fluid removal [107]. The presence and severity of ARF is strongly correlated with mortality after HCT. Patients without ARF have a mortality of about 17%, compared with 37% in patients with ARF not requiring dialysis, but the mortality in patients with ARF who require renal replacement therapy is greater than 80%. It is unclear whether ARF is an independent risk factor for mortality after HCT, or a marker of severity of underlying illness [108].

Table 100.4 Prognostication in hematopoietic cell transplantation (HCT) patients requiring care in an intensive care unit (ICU)

Prognosis of critically ill HCT recipients

Other risk factors for a fatal outcome: allogeneic HCT, admission to ICU >30 days post transplant

A large body of information indicates that HCT recipients have a worse prognosis than other medical patients in critical care units. On average, medical patients in critical care units in the Untied States have a 5–15% mortality rate; however, in a large series in 1992, the hospital mortality rate for HCT patients in critical care units was as high as 96% for transplant recipients who required mechanical ventilation [43]. In that series, 348 patients were ventilated for an average of 8 days, and only 3% survived 6 months. Older age, active malignancy at the time of transplantation, and HLA nonidentity were determined to be risk factors for requiring mechanical ventilation. The authors suggested this should be considered when counseling patients about this level of support [43]. Following this provocative report, the literature has evolved considerably and has focused on predictors of mortality in HCT recipients, on whether critical care prognostic scales are useful for counseling, and on adoption of newer treatment modalities for MOSF. Mechanically ventilated patients following HCT have continued to fare particularly poorly. In a 1996 series comprised of 865 patients mechanically ventilated, only 53 survived to hospital discharge. Among the estimated 398 patients from this cohort who had lung injury and who required either more than 4 hours of vasopressor support or who had hepatic and renal failure, there were no survivors [109]. Compared with earlier studies from the same institution, patients reported in 1996 had a slightly better prognosis, with 16% surviving mechanical ventilation as long as MOSF was not present. Shortly thereafter, a study from M.D. Anderson Cancer Center found an 18.8% survival rate following intubation for respiratory failure, compared with a 65.7% survival for those not requiring mechanical ventilation. Multiple regression risk analysis pointed to allogeneic rather than autologous transplants, infection, bleeding, and prolonged time from transplant to ICU admission as other predictors of a fatal outcome [45]. The hope was that these kinds of studies might assist in providing a basis for deciding whether intubation and mechanical ventilation were warranted in patients with severe complications from HCT. In this light, formal evaluation of the most commonly used prognostic scale is of interest. A study of the APACHE, versions II and III, revealed it to be only moderately discriminating in determining whether HCT patients would survive in the Mayo Clinic critical care units. The receiver operating characteristic curve for APACHE III was 0.704, indicating that it probably cannot be used with a high degree of confidence for predicting the outcome of individual patients. Once again, allogeneic status, mechanical ventilation, vasoactive medication use, sepsis, and MOSF were associated with increased mortality [56]. Table 100.4 lists the prognostic factors now known to be associated with high mortality. In the last few years, the prognosis of HCT recipients in critical care units is continuing to make gains. Whether this reflects selection bias in patients referred to critical care units [110] as a result of prior studies encouraging forms of triage, or whether improved modalities of care are responsible, is not entirely clear; probably both are operative. However, the prognosis is still relatively dismal and much worse than that of general medical ICU patients. In two recent studies, the 12-month sur-

Probability of hospital survival in patients mechanically ventilated is approximately 15–30% Probability of hospital survival in patients mechanically ventilated who require pressors is most commonly <10% and worsens with time Probability of hospital survival in patients mechanically ventilated with hepatic and renal failure is <5% and worsens with time

Prognosis of HCT recipients in critical care units, based on organ dysfunction and support requirements; data from [1,54,110,111].

vival was remarkably similar at 21% and 21.6% [54,111]. The 12-month survival for those requiring mechanical ventilation was only 10.6% in 209 patients in the first study [54], and no one who required vasopressor support survived 12 months among the 440 patients in the second study [111], once again indicating that these patients fare particularly poorly with respiratory or circulatory system failures. Despite these poor outcomes, there is a rationale for providing critical care support for many of these patients, because overall survival is now in the 20% range, and for those not requiring vasopressor support, it can be as high as 45.8% at 12 months [111]. Several factors may have contributed to the success of support in these patients. Since acute respiratory failure is the most common cause of admission to ICUs [1], improvements in methods of mechanical ventilation have probably contributed to improved outcomes. For example, it is established that lower tidal volume ventilation (6 mL/kg of ideal body weight) in acute respiratory distress syndrome reduces the mortality from 39.8% to 31% [46], compared with conventional ventilation of the type previously employed. Similarly, there is evidence that noninvasive positive-pressure ventilation, compared with intubation and mechanical ventilation, has a better outcome in immunosuppressed patients [47], presumably because of fewer ventilator-associated pneumonias and less alveolar injury due to stretch. A new emphasis on quick resuscitation, termed early goaldirected therapy, has been shown to improve survival and has been widely adopted [57]. In addition newer biologic agents for sepsis, like human recombinant activated factor C [64], have been shown to reduce mortality in highly selected patients. Reducing the duration of neutropenia by use of recombinant growth factors, the availability of less toxic and more effective antimicrobial regimens, and nonmyeloablative transplants may all have played a role.

Counseling hematopoietic stem cell transplant patients about critical care services A fundamental role of intensivists in the utilization of critical care resources is to provide informed consent to patients about the utility of invasive measures in their care [112]. Counseling HCT patients about the dismal prognosis for them in ICUs is unique for a number of reasons. First, they have already declared themselves as willing to undergo therapy that is toxic and potentially ineffective, in the hopes of rescue from a malign fate. Families of transplant patients are also hopeful and are committed to seeing their family members through the procedure and its aftermath. Similarly, transplant physicians are, as a cohort, believers in care designed to rescue patients, even while acknowledging it might not work. This context makes it difficult to hold discussions pre

Critical Care of the Hematopoietic Cell Transplant Recipient

transplant [111] about potentially lethal post-transplant situations that have a high probability of a fatal outcome. In fact, there is some evidence that educational efforts about the dismal clinical outcomes of HCT patients fail to change HCT physicians’ referrals to the ICU at all [113]. This is similar to the findings of the SUPPORT (Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatments) study, in which physician education about the likely dismal outcomes of particular patients did not prompt their physicians to have end-of-life discussions and to determine resuscitation status more than 37% of the time – unchanged from prior to the intervention [114]. These data indicate that physicians have difficulty discussing patient preferences, even when they know that an unfavorable outcome is likely, and when resuscitation preferences are essential to know. Patients clearly need to understand their options, and sufficient evidence now exists to provide some guidance. The prognostic data summarized in Table 100.4 indicate that a trial of support is reasonable in most HCT patients, but communicating reasonable expectations from the beginning is also very important. Pre transplant, a discussion about the hopes for a successful outcome should be paired with some information on how severe complications, should they occur, will be managed. It has been well established that certain clinical situations, outlined in Table 100.5, are associated with a hopeless or near-hopeless prognosis, and consideration of counseling families in a tiered fashion about the use of invasive life support measures is warranted. For patients that may benefit from critical care services, obviously single-organ system failure is far preferable to MOSF. Protracted shock requiring prolonged use of vasopressors, or combined hepatic and renal failure added to respiratory failure, is unlikely to have a successful outcome. Establishing that this will be judged on an individual basis, and that full support will be vigorously provided as long as there is a realistic chance of recovery, allows the patient and family to understand that intensivists and transplant physicians have every intention of attempting to rescue the patient. At the same time, it should be communicated that truly fruitless care, because it is invasive and potentially painful at times, will be avoided in the interests of the patient. If and when an HCT patient is transferred to a critical care unit, daily multidisciplinary reviews are essential, as are clear communications with a united message to the family. Declaring from the outset that the goal of therapy is to rescue the patient focuses physicians and family together on outcome, rather than on an open-ended use of invasive measures. If it becomes evident that the course is relentlessly downhill, changing the goal to comfort should be seen as consistent with the overriding intention of doing what is best for the patient. There is no

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Table 100.5 Tiered approach to counseling hematopoietic cell transplantation (HCT) recipients or surrogates about care in an intensive care unit (ICU) Intensive care support recommended for rescue • Diffuse alveolar hemorrhage and idiopathic pneumonia syndromes • Hypoxemia not requiring intubation, especially if transfusion- or volumerelated • Sinusoidal obstructive syndrome • Sepsis without serious hypotension • Congestive heart failure or arrhythmias • Airway protection Intensive care support recommended with re-evaluation within 3 days • Severe sepsis requiring pressors • Any condition requiring intubation • More than one organ system failure Intensive care support not recommended • Critically ill patient with relapsed native disease, if recurrent treatment is not an option • Multiorgan system failure, with respiratory failure, unresponsive to standard treatments • Respiratory failure requiring intubation with grade III–IV graft-versus-host disease, unresponsive to therapy An approach to counseling HCT recipients on the utility of unrestricted intensive care support. Dividing the patients into three tiers to estimate prognosis may help patients understand the nature of their illness and whether ICU care is likely to yield an outcome they desire.

bioethical basis for discussions of limiting resource allocation in care as a cost-saving or capacity measure, and this will assuredly alienate the family and disqualify the physicians as champions of the patient’s best interests. In recent years, there has also been considerable interest in how families experience the end-of-life care of their loved ones. A landmark study showed that including certain key elements in end-of-life discussions in ICUs may diminish the incidence of family symptoms related to post-traumatic stress disorder from 69% to 45% at 90 days [115]. These data indicate that transplant physicians and intensivists must be mindful of their accountabilities to the mental health of families, in addition to their primary responsibilities to their patients.

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tent with continuous dialysis in patients with ARF. Am J Kidney Dis 2004; 44: 1000–7. 108. Kersting S, Koomans HA, Hene RJ, Verdonck LF. Acute renal failure after allogeneic myeloablative stem cell transplantation: retrospective analysis of incidence, risk factors and survival. Bone Marrow Transplant 2007; 39: 359–65. 109. Rubenfeld GD, Crawford SW. Withdrawing life support from mechanically ventilated recipients of bone marrow transplants: a case for evidencebased guidelines. Ann Intern Med 1996; 125: 625–33. 110. Naeem N, Reed MD, Creger RJ, Youngner SJ, Lazarus HM. Transfer of the hematopoietic stem

cell transplant patient to the intensive care unit: does it really matter? Bone Marrow Transplant 2006; 37: 119–33. 111. Kew AK, Couban S, Patrick W, Thompson K, White D. Outcome of hematopoietic stem cell transplant recipients admitted to the intensive care unit. Biol Blood Marrow Transplant 2006; 12: 301–5. 112. Snider GL. Allocation of intensive care. The physician’s role. Am J Respir Crit Care Med 1994; 150: 575–80. 113. Paz HL, Garland A, Weinar M, Crilley P, Brodsky I. Effect of clinical outcomes data on intensive care unit utilization by bone marrow transplant patients. Crit Care Med 1998; 26: 66–70.

114. A controlled trial to improve care for seriously ill hospitalized patients. The Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatments (SUPPORT). The SUPPORT Principal Investigators. JAMA 1995; 274: 1591– 8. 115. Lautrette A, Darmon M, Megarbane B et al. A communication strategy and brochure for relatives of patients dying in the ICU. N Engl J Med 2007; 356: 469–78. 116. Bernard GR, Artigas A, Brigham KL et al. The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 1994; 149: 818–24.

101

Polly Lenssen & Saundra Aker

Nutrition Support of the Hematopoietic Cell Transplant Recipient

Introduction The nutritional challenges that face the transplant clinician today are remarkably similar to those of over four decades ago – supporting patients through dose-intensive transplant regimens and managing the debility associated with acute and chronic graft-versus-host disease (GVHD). Fortunately, the recovery from dose-intensive regimens has been hastened by reductions in infections and organ toxicities, and it has been suggested that nutrition support may no longer be essential. There is insufficient evidence in the current era of hematopoietic cell transplantation (HCT) to define the risks or benefits of various modes of nutrition intervention, including total parenteral nutrition (TPN), enteral nutrition, low-microbial diets, antioxidant repletion or other forms of vitamin and mineral supplementation. Clinicians must weave together the bits of evidence from limited, often outdated studies, with their best judgment, and thus develop rational nutrition practice. The intent of this review is to provide a framework for nutrition practice in HCT, covering (1) nutrition assessment (identification of risk factors), (2) dietetic and nutrition support interventions, and (3) management of HCT-specific complications, chiefly GVHD.

Nutrition assessment The majority of HCT recipients benefit from comprehensive nutrition assessment provided by a dietitian who specializes in oncology and nutrition support. Ideally, the assessment and reassessment of nutrition status occurs through the transplant continuum to prevent and treat the debilitation and nutrient deficiencies common in transplant recipients. Nutrition assessment, partnered with education and counseling, are most effective when implemented pre transplant, continued through the acute phase, and extended into the long-term follow-up care [1–3]. Several studies have addressed patient weight status as a predictor of transplant outcome. Based on weight assessment at admission for HCT, allogeneic and autologous adult patients 95% or less of ideal body weight (IBW) [4], pediatric patients between 85% and 95% IBW [4], and autologous HCT patients with an age-adjusted body mass index (BMI) of less than 80% [5] are at increased risk of death following HCT. Adults of over 120% IBW and receiving an allogeneic transplant may also be at increased risk of death post transplant [6]. Horsley and colleagues reported that malnourished HCT patients, as defined by the

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

patient-generated subjective global assessment tool, experienced an increased length of hospital stay (p = 0.002) compared with wellnourished patients in a prospective study of 66 patients [7]. These studies collectively infer that as soon as a patient is identified as a potential candidate for HCT benefits, monitoring of nutritional status and referral to a dietitian in the event of weight loss, especially to the critical level of less than 95% IBW, are indicated. Nutritional intervention with the goal to induce weight gain or loss is problematic in patients needing urgent transplantation, as the interval to effect meaningful changes in body weight prior to the HCT infusion may be too brief. In the nutritionally depleted patient, more aggressive use of enteral feedings or TPN support may be considered prior to the preparative regimen to prevent further loss of weight and lean body mass. The initial nutrition assessment is based on information obtained from the medical and nursing examination and an interview with the patient and caregiver (see Fig. 101.1 for a sample patient questionnaire to help in gathering relevant nutrition history). Specifically it involves: • review of the physical examination and pertinent medical history, especially as it may relate to organ function and tolerance of diet and nutrition support (any gastrointestinal illnesses or surgeries, diabetes, and hyperlipidemia); • visual assessment for nutrient excesses or deficiencies; • diet, physical activity, and weight histories; • anthropometric measurements (accurate height or length, weight, head circumference in infants and small children, and, if available, fat folds); • laboratory parameters as indicators of visceral protein, electrolyte, mineral, glucose, lipid, and iron status, as well as renal and liver function; • identification of drug–nutrient interactions; • evaluation of current oral and gastrointestinal symptoms. The diet history identifies eating patterns, typical foods, current dietary modifications or special diets, food allergies or intolerances, and current or recent use and dosage of vitamin and mineral supplements, including herbal agents and other integrative medicine therapies. History of nutrition support interventions (nutritional supplements, enteral nutrition, TPN or intravenous fluids and electrolytes) and any complications and experiences with these therapies are identified and, if currently used, evaluated for appropriateness and adequacy. Weight history elicits usual weight, degree and rate of any weight loss or gain, and what may be normal or abnormal deviation from the ideal weight. Optimal body weight is determined utilizing the Centers for Disease Control growth charts in pediatric patients and a standard tool such as the Metropolitan Life Tables in adults.

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Chapter 101 Name of person completing questionnaire: Signature of person completing questionnaire (if other than patient): Reviewed by: Registered Dietitian Date: ______________ Please answer the following and return the form to your team nurse: Who will be preparing your food? ____________________________ If you follow any special diets for religious or other reasons, please list them: ______________________ ______________________________________________________________________________________________ 1)

Activity

Do you exercise regularly?

yes

no

Describe: _________________________________ Has your activity/exercise level declined? Specify ________months. Section 1.02 Weight History Current weight: _____________ Usual weight: _______________

yes

no

Height: _________________________ (if child, indicate growth in past year)

I have gained ______pounds or lost _____pounds in the last

1 month

Was this weight change intentional?

6 months yes

no

yes yes yes

no no no

yes

no

no

At what weight do you feel most comfortable? __________________ Section 1.03 Patient Medical History Do you have: Diabetes? Gastrointestinal disease (Crohn’s disease, ulcerative colitis, celiac sprue)? Have you had surgery to your stomach or intestinal tract? 1)

Family Medical History

Does a family member have diabetes? Specify who _______________________________ 101

Nutritional Supplements

Are you currently taking: a multiple vitamin/mineral supplement?

yes

an herbal supplement?

yes

no

antioxidants (vitamins C, E, beta-carotene, selenium)?

yes

no

any other supplements? List:

yes

no

Fig. 101.1 Nutrition pretransplant questionnaire.

Physical activity is often overlooked but is a critical component for preserving maximum lean body reserves during the extended HCT experience [8–12]. For example, a program of walking on a treadmill, initiated 1 month post transplant resulted in significant improvement in physical performance, walking distance, and lowering of the heart rate with equivalent workloads (p < 0.001) [11]. The measures together were more than sufficient for carrying out all basic activities of daily living, and in contrast to quality of life studies indicating that spontaneous recovery of physical functioning after HCT can take many months, and in some individuals result in long-term impairment. Assessment of past and current exercise capacity, interest, and habits, helps in the proactive design of an individualized activity plan. In pediatric patients, growth and development, including in young children stages of eating development (cup versus bottle, puréed versus table food, and self-feeding skills), are evaluated. A baseline assessment helps define expectations for future growth and possible intervention if development becomes arrested or decelerates following HCT. Breast-fed infants may need to be weaned due to the concern for the transmission of T lymphocytes in the breast milk, although there are no data to support any adverse sequelae of continued use of breast milk.

A nutrition plan of care and goals is developed based on the objective and subjective data, and includes an estimation of macronutrient needs. In adults, measured resting energy expenditure in the neutropenic phase following dose-intensive regimens is approximately 30–35 kcal/kg, assuming bed rest or a 10% activity factor [13,14]. In pediatrics, similar studies have not been published, so a starting point of 50% above basal is reasonable. Clinical judgment should always supersede energy estimates when higher needs are suspected with persistent fevers, disseminated infections, severe diarrhea or extensive skin breakdown. Patients with acute GVHD appear to have increased energy needs [15], some individuals requiring up to twice basal energy (unpublished observation). When available, indirect calorimetry is recommended in patients who require long-term nutrition support, allowing more precision with the energy prescription and avoiding the adverse consequences of over- or underfeeding. Protein needs are approximately twice the Dietary Reference Intake levels, based on protein losses measured following dose-intensive regimens in allograft recipients [13]. Protein losses are greater in men [16] and patients on corticosteroids [17,18]. No studies have been reported on the protein needs following autologous HCT.

Nutrition Support of the Hematopoietic Cell Transplant Recipient Section 1.04

Food Intake

1. 2.

Has your food intake decreased during the past month? Has your food intake increased during the past month?

3.

Do you have nausea? Please mark the severity of nausea:

4.

5. 6. 7. 8. 9. 10. 11.

None

Do you have vomiting? Please mark the severity of vomiting:

None

yes yes

no no

Moderate

yes Severe

no

Mild

Moderate

yes Severe

no

Mild

yes

no

yes

no

yes yes yes yes yes

no no no no no

Do you have any problems with your bowel movements? Constipation Diarrhea Change in color Other __________ Do you have any difficulties with taste or smell? If yes, please describe: Do you have any difficulties eating food (such as choking, coughing)? Do you have any difficulties chewing and swallowing food? Do you have mouth sores? Do you have heartburn? Do you wear dentures?

___

12.

Do you have food allergies? Specify foods/describe symptoms ________________________

yes

no

13.

Do you have lactose (milk) intolerance? Specify foods/describe symptoms ___________________________

yes

no

14.

Do you consume alcohol? yes no Specify amount and frequency ________________________________________________________ ________ Do you currently or have you previously used high-calorie/high-protein nutritional supplements (such as Ensure, Boost, Instant Breakfast, etc.)? yes no

15. 16.

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Do you currently or have you recently used

IV nutrition (TPN)

___

Tube feeding

Typical Meal Pattern (describe usual foods and beverages consumed at meals and snacks):

Breakfast

Lunch

Dinner

Snacks/beverages

Snacks/beverages

Snacks/beverages

Fig. 101.1 Continued

During the peri- and post-transplant periods, the assessment of nutritional status is similar to a thorough assessment appropriate to any complex medical condition. Table 101.1 provides measures and clinically significant indicators of nutritional and metabolic status.

Nutrition interventions Conditioning regimens and toxicities Due to the diversity of HCT treatment protocols, one cannot universally treat all patients by following the same nutritional pathway or guidelines (Fig. 101.2). Most patients undergoing HCT with dose-intensive regimens develop toxicities that impact their ability to attain adequate oral intake during cytoreduction [14] and for upwards of a month post transplant, as described in Fig. 101.3. Nausea and vomiting are most acute during administration of cytoreduction therapy, but mild symptoms persist for 3–6 weeks [19]. Generally, mucositis peaks at 10–14 days following transplant [20], and the associated pain and swelling are the principal deterrents to eating during the neutropenic phase. Intravenous narcotic analgesics to control muco-

sitis pain are required for the majority of patients, and can lead to gastric stasis and intestinal ileus. Crampy abdominal pain and diarrhea secondary to mucosal crypt aberrations, epithelial flattening, cell degeneration, and increased bowel permeability peak 1–2 weeks after the start of conditioning and return to normal by 3–4 weeks after the transplant [21]. The profile of mucosal and gastrointestinal toxicities, however, varies not only with dose intensity but also with individual agents, which together have a major bearing on nutrition support decisions [22]. Care plans for nutrition management associated with the common gastrointestinal toxicities are outlined in Table 101.2, with additional discussion in the following sections. There has been considerable interest in the use of oral glutamine to reduce oral and gut toxicities following high-dose conditioning (Table 101.3) [23–26]. Only two studies have reported a favorable result, and only in autologous patients; paradoxically, in one of the positive studies, glutamine was associated with worse mucositis, as measured by opiate use, in allogeneic patients with matched sibling donors [24]. The routine use of oral glutamine is not supported by the published data, which is in agreement with the recommendations by the European Society of Parenteral and Enteral Nutrition [27]. A discussion of intravenous glutamine follows in the section on TPN.

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Chapter 101

Table 101.1 Nutrition assessment measures in hematopoietic cell transplantation (HCT) Measure Anthropometry % IBW

Body weight

Upper arm muscle and fat areas Height

Serum protein status Albumin

Prealbumin

Substrate utilization Indirect calorimetry

Interval

Significant Indicators

Intervention/comments

Pre HCT; continuous monitoring

Adults: <95% IBW Children: <10th percentile weight-forlength or body mass index-for-age >125% IBW

Intervene early with nutrition support if unable to eat adequate energy for weight gain

Daily early post HCT Weekly (minimum) during long-term recovery through first year

Pre HCT, day 50–100, 1 year post HCT Pre HCT Adults and postpubertal adolescents: yearly post HCT if on steroids Infants: monthly Children: every 3 months Infants, children, and teens: plot growth rate annually

>5% gain in 7–10 days associated with: • capillary leak syndrome (high-dose regimens, hyperacute GVHD, sepsis) • SOS, ascites • congestive heart failure • acute severe skin GVHD • steroid use • decreased urine output >5% loss in 7–10 days associated with: • fluid mobilization in resolving SOS or capillary leak syndrome • dehydration (inadequate input or excessive output such as diarrhea, glycosuria or diuretic use) • hypermetabolism or hypercatabolism <5th percentile

Adjusted IBW may be used for calculation of energy and protein needs; standardize method for adjusted IBW for chemotherapy, drug dosing Restrict total fluid to maintenance needs (TPN, IV fluid, and diet); if urine sodium excretion <10 mEq, restrict all sources of sodium

Assess volume status and provide adequate fluid support; correct glucose abnormalities If volume status and glucose abnormalities are ruled out, measure metabolic rate to ensure feeding adequately Physical therapy

Adults and postpubertal adolescents: serial decrements (sign of osteoporosis) Children: <5th percentile (or growth velocity <10th percentile) associated with: • chronic disease • chemoradiotherapy prior to HCT • TBI or busulfan • chronic GVHD • steroids • malnutrition

Identify and correct any contributory nutrient deficits (energy, protein, zinc, vitamin D, calcium, and vitamin A). Evaluate bone absorptiometry, 25 hydroxyvitamin D status, and/or initiate endocrinology/consult for osteoporosis therapy

Pre HCT

<3 mg/dL

Weekly (to correct serum calcium status)

<2.5 mg/dL associated with intravascular volume overload, capillary leak syndrome, diarrhea, and hepatic disease Adults: <20 mg/dL Children: <10 mg/dL associated with neutropenia, infection, inflammation, and hepatic disease

If other signs of inadequate nutrition status, intervene early with nutrition counseling and/or support Occurs independent of adequate nutrition support; exogenous infusions may not maintain oncotic pressure but may promote diuresis when given with diuretic As sign of metabolic stress, may help determine need for nutrition intervention

As clinically indicated

Pre-HCT if body composition altered (obesity, high muscle mass, malnutrition) Post HCT if: • critically ill • on TPN > 1 month • liver disease • significant weight and muscle mass loss make energy estimations difficult

Measured resting energy expenditure plus activity factor differs from current intake by >10%

See Chapter 104

Adjust nutrition support or counsel patient to target kcal intake according to measured energy expenditure. Consider whether respiratory quotient is consistent with overfeeding (>1.0) or underfeeding (<0.7)

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Table 101.1 Continued Measure

Interval

Significant Indicators

Intervention/comments

Serum glucose

Daily until stable if on nutrition support, then once or twice weekly Twice weekly (minimum) with large doses of steroids Once or twice weekly in ambulatory care

>140 mg/dL (fasting; minimum 4 hours off TPN or overnight if on oral diet) or >180 mg/dL (random draw) associated with: • TPN • sepsis • steroids, tacrolimus • pre-existing diabetes • pancreatitis

Serum triglyceride

Pre HCT Weekly if on TPN and: • elevated pre HCT • on dialysis • liver disease • persistent sepsis • pancreatitis

400–500 mg/dL associated with: • pre-existing hypertriglyceridemia • glucose intolerance • pancreatitis • hepatic dysfunction • sepsis • steroids, cyclosporine, sirolimus

If on TPN: • Do not overfeed • IV lipids to reduce dextrose load • Insulin to maintain serum glucose <150 mg/dL; if high insulin requirements (>0.3–0.4 U/g dextrose), restrict dextrose to 2–3 mg/kg/min in adults and adolescents and increase lipids to maximum 50–60% of total kcal; insulin drip may allow for better glucose control If on IV fluids: • Provide dextrose-free solution If on oral diet (including transitioning off TPN): • Determine if SQ insulin is required (fasting >160–180 mg/dL or symptomatic with osmotic diuresis) • Provide basal insulin and Humalog for meals • Teach home blood glucose monitoring to ambulatory patients • If excessive, restrict total kcal and/or carbohydrate intake • Assess for signs and symptoms of osmotic diuresis or hypoglycemia If on TPN: • Provide IV lipids at 4–8% of total kcal for essential fatty acids • If >1000 mg/dL or if serum lipemic, hold IV lipids If on oral diet: • Normal serum lipids pre HCT – reassess when off all lipid-raising drugs • Elevated serum lipids pre HCT – assess for other risk factors for heart disease and counsel as indicated

Daily if on drugs associated with potassium wasting or if significant renal insufficiency Two or three times weekly during neutropenia, early recovery or if on TPN

<3.5 mEq/dL associated with: • amphotericin, thiazide diuretics, steroids, aminoglycosides, foscarnet • GI losses • anabolism

Electrolytes and trace elements Potassium

Magnesium

Twice weekly if magnesium wasting Once weekly if on TPN, cyclosporine or tacrolimus

>5.5 mEq/dL associated with: • renal failure • cyclosporine, tacrolimus, aldactone, enalapril <1.5 mEq/dL associated with: • cyclosporine, tacrolimus, amphotericin, aminoglycosides • GI losses

Supplement in TPN, hydration or separate infusion up to maximum 20 mEq/hour (0.3 mEq/kg/hour in children) without a cardiac monitor; maximum 10–15 mEq/hour in ambulatory care If GI symptoms absent, provide oral supplement and high-potassium diet Delete all supplemental potassium; in rare cases, restrict dietary potassium

If on TPN: • Initial supplement 16 mEq/L of maintenance TPN or IVF; may reach to >160 mEq/day If off TPN/IV fluid but on magnesium-wasting medication: • Maintain level >1.3 mEq/L with oral magnesium, IV magnesium boluses • Maximum tolerated oral dose varies (protein complex form usually better tolerated)

Table 101.1 Continued Measure

Interval

Significant Indicators

Intervention/comments

Calcium

Twice weekly if on TPN

>10 mg/dL (corrected for hypoalbuminemia) associated with: • tumor lysis • renal failure • multiple myeloma

Phosphorus

Twice weekly as inpatient Once weekly in ambulatory care if on TPN or steroids

Adults: <2.5 mEq/dL Children: <4.0 mEq/dL associated with: • anabolism • some chemotherapies, steroids, foscarnet • renal acidosis • steatorrhea Adults: >4.5 mEq/dL Children: >6.0 mEq/dL associated with: • renal failure • multiple myeloma • tumor lysis syndrome <60–80 μg/dL associated with: • GI losses • increased needs for healing <70–90 μg/dL associated with: • GI losses

If on TPN: • Remove calcium • If alkalotic add chloride If on oral diet: • Discontinue vitamin D supplementation in excess of daily needs • Limit eulcum to 2000 mg If on TPN: • Increase phosphate salts (sodium or potassium) • If calcium/phosphorus solubility maximum reached, provide calcium as separate infusate If on oral diet: • Oral phosphate replacement

Zinc

Post transplant if excessive diarrhea >1 week or severe skin GVHD If on TPN when chronic liver disease or diarrhea present

Copper

If on TPN: • Delete phosphate salts If on oral diet: • Provide phosphate binder (calcium carbonate) 1 mg/L of stool greater than 1000 mL in adults, 250–500 mL in infants and children, respectively Note: Serum levels do not reflect tissue stores No replacement guide established; replace conservatively and follow serum levels – starting point 1 mg/day in adults, less for infants and children If on TPN, delete trace element package and provide other trace elements individually Note: Serum levels do not reflect tissue stores

>200 μg/dL associated with: • lymphoma • liver disease

DRI, Dietary Reference Intake; GI, gastrointestinal; GVHD, graft-versus-host disease; IBW, ideal body weight; IV, intravenous; SOS, sinusoidal obstructive syndrome; TBI, total body irradiation; TPN, total parenteral nutrition. Adapted with permission from Hematopoietic Stem Cell Transplant Nutrition Care Criteria, Seattle Cancer Care Alliance, Seattle, Washington, 2nd edition, 2002.

Pre-HCT candidate pool

Pre-HCT scheduled

Post HCT

High-dose regimens 3–4 days pediatric 3–4 day adult allogeneic 7–10 days adult autologous

Oral intake < needs?

Yes

TPN

Weight loss or < 95% ideal weight?

Nutrition monitoring

Yes

Comprehensive nutrition assessment, education, and counseling by dietitian. Nutrition intervention if needed pre-HCT

Able to take orally?

Yes

Nutrition monitoring

Enteral feeding

Yes

Discontinue nutrition support monitor

No

No

5% weight loss or oral intake < needs for 7-10 days?

No Yes

GI symptoms (e.g. GVHD)?

Reduced dose regimens

Fig. 101.2 Decision-making tool for nutrition interventions along the continuum of hematopoietic cell transplantation (HCT). GI, gastrointestinal; GVHD, graft-versus-host disease: TPN, total parenteral nutrition.

Nutrition Support of the Hematopoietic Cell Transplant Recipient

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1600 1400

Oral calories

1200 1000 800 600 400 200 0 –13 –11 –9 –7 –5 –3 –1 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Pre- and post-transplant (day)

Fig. 101.3 Average daily oral intake of adult hematopoietic cell transplant patients undergoing dose-intensive regimens.

Table 101.2 Nutritional interventions for oral and GI-related symptoms Organ dysfunction

Evaluation

Intervention

Anorexia and/or early satiety related to: • Hypomotility of GI tract (conditioning therapy, prolonged NPO, GVHD) • Cyclosporine, narcotics, antibiotics • Upper GI GVHD • Liver disease • Anxiety or depression • Adrenal insufficiency • Food aversions • History of prior eating disorder

Monitor adequacy of nutrient intake

Oral supplements, small frequent meals, low-fat meals, food experimentation Trial of gastric motility agent If on TPN: • cycle overnight • if well nourished and medically stable, trial off TPN and/ or transition to tube feeding If depression or history of eating disorder, consult psychologist

Xerostomia related to: • Conditioning • Dehydration • Healing mucositis • Anticholinergics, methotrexate • Oral GVHD

Assess intake sources Assess hydration status Assess oral hygiene measure

High-moisture foods, use of saliva stimulants to optimize oral intake Additional oral or IV fluids as indicated Good oral hygiene; oral medicine or dental consultation as indicated

Mucositis related to: • Conditioning therapy • Oral infection (fungal, viral) • Acute or chronic GVHD • Methotrexate

Monitor adequacy of nutrient intake Assess oral hygiene measures

Oral supplements, cold clear liquids, soft bland foods If oral supplementation unsuccessful, provide TPN Encourage good oral hygiene; if persists after engraftment, obtain cultures and treat infections as indicated

Dysphagia/esophagitis related to: • Conditioning therapy • Gastric reflux (corticosteroids, GVHD, stress) • Esophageal infection (fungal, viral, bacterial) • Chronic GVHD • Prolonged intubation

Monitor adequacy of nutrient intake If gagging present and/or high aspiration risk, obtain swallowing evaluation Assess antireflux regimen

Oral supplements, soft bland foods Altered food consistency as indicated Antireflux measures (upright position postprandially, elevate head of bed); routine use of proton pump inhibitors If oral supplementation unsuccessful, provide TPN

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Table 101.2 Continued Organ dysfunction

Evaluation

Intervention

Nausea/vomiting related to: • Conditioning therapy • GVHD • Drugs (cyclosporine, amphotericin) • GI or systemic infections • Liver disease • Gastric reflux or ulcers • Thick salivary secretions • Pancreatits • Food aversions • Anticipatory vomiting

Determine frequency and volume of emesis, relationship to oral intake Assess antiemetic regimen Monitor adequacy of nutrient intake Assess volume status

Clear cold liquids, small frequent meals, low fat Scheduled antiemetics If interventions unsuccessful, provide TPN If symptoms persist after engraftment, endoscopic evaluation to rule out treatable infection or GVHD If gastrointestinal work-up negative, psychology referral for behavior modification and food desensitization Provide IV fluids to maintain hydration

Diarrhea related to: • Conditioning • Infection (viral, fungal, bacterial overgrowth) • GI GVHD • Magnesium salts, gastric motility agents, antibiotics • Clostridium difficile • Typhlitis (other Clostridium species) • Lactose intolerance • Past medical history of GI pathology • Liver or pancreatic disease (usually steatorrhea)

Assess: • volume, frequency, other characteristics (color, odor, consistency, presence of blood) • relation to diet or diet components (lactose, fiber, fat) • other symptoms of GI dysfunction (cramping, nausea and vomiting) Review medications Assess volume status

If suspected steatorrhea: see “Steatorrhea” below If mild: • trial of antidiarrheals (contraindicated with GVHD and infectious causes) • empiric use of lactase-treated milk and lactase tablets with other dairy products If secretory (large volume with minimal oral stimulation) and/or presence of other symptoms: • gut rest and TPN until stool <10 mL/kg • see zinc and copper replacement guidelines in Table 101.1 • refeed in moderate-to-severe GVHD with slow diet progression (low in lactose, insoluble fiber, fat, gastric irritants, and motility stimulants); introduce one new food at a time Discontinue medications not otherwise medically necessary; if magnesium related, may respond to decrease in dose Replace losses cc/cc with D5 1/4 – 1/2 NS (100–130 mEq sodium/L with GVHD)

Constipation related to: • Narcotics, thalidomide • History of GI dysfunction • Inactivity • Low-fiber diet • Low fluid intake

Assess fiber, fluid intake Assess physical activity

Increased insoluble dietary fiber and fluid intake; pharmacologic intervention such as stool softners Encourage physical activity as appropriate

Steatorrhea related to: • Bacterial overgrowth • GI GVHD • Hepatobiliary disease (including liver GVHD) • Intestinal resection • Pancreatic insufficiency • Severe enteritis

Assess stool to help confirm presence of fat (large, bulky, greasy, foul odor, foamy stools) Assay fecal elastase, serum levels of vitamin D, zinc

If fecal elastase abnormal, initiate pancreatic enzymes and water-miscible, fat-soluble vitamins If fecal elastase normal, initiate trial on low-fat diet If vitamin D low, supplement with vitamin D per endocrine recommendations If serum zinc level low, treat with oral zinc: • Adult: 23–50 mg elemental zinc (100–220 mg zinc sulfate) two or three times daily • Children: 0.3 mg/kg/day elemental zinc (minimum 3 mg elemental zinc/day)

D5, 5% dextrose solution; GI, gastrointestinal; GVHD, graft-versus-host disease; IV, intravenous; NPO, nil per os (nothing by mouth); NS, normal saline; TPN, total parenteral nutrition. Adapted with permission from Hematopoietic Stem Cell Transplant Nutrition Care Criteria, Seattle Cancer Care Alliance, Seattle, Washington, 2nd edition, 2002.

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Table 101.3 Randomized glutamine trials in high-dose regimens in hematopoietic cell transplantation

Study

Glutamine source

Autograft patients

Allograft patients

Ziegler, 1992 [65] Schloerb, 1993 [66] Pytlik, 2002 [69] Piccirillo, 2003 [64]*

IV IV IV IV

Sykorova, 2005 [70] Gomez Candela, 2006 [67] Jebb, 1995 [23]

IV IV

14 40 Study 1: 27 Study 2: 21 54 40

Oral

24

Anderson, 1998 [24]

Oral

87

106

Schloerb, 1999 [25] Coghlin Dickson, 2000 [26]

Oral Oral

48 34

18 24

45 15

9

Mucositis

Infection

GVHD

Relapse

Long-term survival

No difference No difference − glutamine + glutamine

+ glutamine No difference No difference Not reported

No difference No data Not applicable Not applicable

No data No data − glutamine Not reported

No data No data − glutamine Not reported

No difference No difference

Not reported No difference

Not applicable Not reported

Not reported Not reported

Not reported No difference

No difference

No data

Not applicable

No data

Autologous: + glutamine Allogeneic sibling: − glutamine Allogeneic unrelated donor: No difference No difference No difference

No difference

No difference

No difference at 6 months No data

No difference No data

No difference No data

No data No difference

No difference No difference

No difference†

+, positive outcome for glutamine treatment; −, negative outcome for glutamine treatment; IV, intravenous. * Two studies. Study 1: free glutamine 20 g/day. Study 2: glutamine dipeptide 13.46 g/day. † Day 28 survival significantly better for glutamine group but no difference at day 100.

In another nutrition-based intervention to modulate mucositis, Thornley and colleagues utilized a combination of ursodeoxycholic acid, folinic acid, and vitamin E, and noted a reduced prevalence and severity of mucositis, decreased hepatic toxicity, and shorter time to engraftment in the treated group compared with the controls [28]. Oral diet guidelines Dietary recommendations have been extensively reviewed for pediatric [29] and adult [13] patients undergoing HCT. Diet modifications may include changes in consistency, texture, temperature, taste, and smell of food, especially when patients experience mucositis, esophagitis, xerostomia or dysgeusia. Appetite and taste perception can be influenced by central nervous system changes, as well as by peripheral mechanisms associated with insults to chemosensory structures resulting from chemotherapy, radiation, poor oral health, altered salivary flow, and oral mucositis [30]. Both pediatric [31] and adult [32] patients receiving chemotherapy and total body irradiation (TBI) for HCT report changes in taste and smell. Taste changes may influence interest in eating and diet quality from several weeks [33] to months [34] after HCT. Epstein and investigators [34] surveyed 50 patients 90–100 days after HCT and reported that 65% of patients still complained of changes in taste; changes in smell and xerostomia were significantly correlated with changes in taste. Proper assessment and considerations of taste and smell alterations are essential to provide individualized diet recommendations. Reduction of fat and lactose content may decrease nausea or diarrhea. The addition of pectin and other soluble dietary fibers may be useful in the dietary management of persistent diarrhea associated with GVHD or other gastrointestinal infections. The use of probiotics, such as Lactobacillus, however, have been associated with sepsis in HCT [35], and their use is discouraged. The dietitian can recommend appropriate nutritional supplements in patients who exhibit malabsorption or show an inability to gain weight.

Special food service needs Traditional hospital food services with set mealtimes, limited food choices, and advance menu selection may fail to meet the dietary needs of many HCT patients. A popular trend in hospitals in the last decade is the provision of an on-demand à la carte food service offering patients the opportunity to select preferred foods at times that best match their interest in eating as well as clinical care demands. Hospital “room service” menus can be designed to meet any low-microbial and extra food safety handling requirements for the immunosuppressed HCT recipient (discussed below). Diet and food safety guidelines for the immunosuppressed HCT patient In decades past, HCT patients were treated with gastrointestinal decontamination in ultra-isolation or high-efficiency particulate air-filtered environments, and served sterile or low-bacteria diets [36,37]. Sterile diets do not have a place in diet management today because of improved antimicrobial therapies, lack of evidence to demonstrate efficacy, and the high labor and production costs. Nonetheless, some form of diet restriction to minimize acquisition of organisms from food sources and food handlers is common [38]. The scope of diet restrictions appears to vary widely between facilities, as does the timing of the initiation and discontinuation of the diet, although most transplant facilities utilize diet precautions only in the hospitalized patient. A survey of dietary restrictions conducted in 156 facilities with inpatient centers found that the most commonly restricted foods were fresh fruit and juices (92%), fresh vegetables (95%), and raw eggs (74%) [39]. The exclusion of fresh fruits and vegetables from lowmicrobial diets places patients at risk for micronutrient deficiencies [38]. Organisms routinely found on washed raw fruits and vegetables do not appear to be common sources of infection in immunosuppressed patients. At the Fred Hutchinson Cancer Research Center, 29 cultures of assorted

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raw, washed fruits showed Gram-positive cocci and Gram-negative rods (<4000 colony-forming units [CFU]/mL), which are commonly found in food (Aker, unpublished data). One of three strawberries and two of three apricot cultures showed a range of 200–2000 CFU/mL of five mold types. Thirty-six samples of raw, washed vegetables resulted in a broader variety of Gram-positive and Gram-negative organisms at levels ranging between less than 200/mL and too numerous to count Gram-positive and Gram-negative organisms with few yeasts or molds. The organisms cultured from these fruits and vegetables are not representative of common infections routinely seen in HCT patient populations. Except for recent reports of Escherichia coli 0157 : H7 contamination of unpasteurized fruit juices, raw vegetable sprouts and other vegetables such as spinach, food outbreaks associated with raw, washed domestic fruits and vegetables are uncommon. Inclusion of well-washed fruits and vegetables promotes healthy food options as well as facilitates quality of life in patients who may desire and tolerate these foods during periods of anorexia, dysgeusia, xerostomia, and other transplant-related side-effects. The possibility that food presents an infection risk is evidenced by many reports that implicate food as vectors of pathogenic organisms in HCT recipients, including botulism [40]. Safdar and colleagues [41] suggested an association between reactivation of acute human cytomegalovirus infection in HCT recipients and subsequent Listeria monocytogenes infection due to human cytomegalovirus-associated dysfunction in host cellular immune response. The incubation period for Listeria monocytogenes (up to 30 days onset), as well as other foodborne illness organisms, may be such that suspect foodstuffs have been discarded, and an accurate food intake history to identify the potential contaminated food is difficult. Further, most Listeria monocytogenes infections in HCT patients have been detected in the ambulatory care setting, making it more difficult to trace to specific food intake [41,42]. Fungal infections are also common in the immunosuppressed host, but difficult to link with food-originated infection. Numerous foods, such as aged cheeses, fermented products (e.g. miso and tempeh) and others, as well as naturopathic substances [43], may serve as sources of fungal infections. Bouakline and colleagues [44] examined the prevalence of fungal organisms in non-sterilized foods. Aspergillus spp. were detected in 100% of black pepper and regular tea (unbrewed) samples, 12–66% of fruits, 27% of herbal teas (unbrewed samples), and 20% of freeze-dried soup samples (not reconstituted). All soft cheese samples were contaminated by Geotrichum and yeast (Candida norvegensis), but processed cheeses were free of fungal organisms. The raw produce in this study was not washed before culturing, nor were the food cultures taken after brewing or cooking. Moe [37] cultured 198 foods prepared by conventional methods. Although 80% of beverages, starches, cooked meats and entrées, and frozen vegetables were acceptable, only 36% of pasteurized dairy products and 42% of dessert and snack items met minimal microbiologic criteria. Upwards of 17 different species of Gram-negative rods in concentrations as high as 106 CFU/mL in milk, pudding, and ice cream were identified. Despite lack of research or consensus regarding “best practice,” advising patients to follow a diet comprised of lower-risk foods seems prudent in this age of global food markets and frequent food-borne illness outbreaks [45–47]. The cornerstone to preventing food-acquired illness is educating patients and their caregivers in the basics of safe food handling. Guidelines can be found at the following websites: www. foodsafety.gov, www.fda.gov, www.fsis.usda.gov, and www.cdc.gov. Table 101.4 describes diet restrictions of those foods that are (1) at higher risk to contain organisms associated with food-borne illness, (2) frequently implicated in food-borne illness outbreaks, and (3) more

Table 101.4 Recommended good restrictions in hematopoietic cell transplantation (HCT) Raw and undercooked meat (including wild game), fish, shellfish, poultry, eggs, hot dogs, tofu, sausage, bacon Ready-to-eat deli and luncheon meats, salami, sausages, precooked ham (all unless reheated until steaming hot) Cold smoked fish and lox, pickled fish, fermented fish products Unpasteurized and raw milk, yogurt, cheese or other dairy products Aged cheeses (e.g, bleu, Roquefort, Brie, Camembert, Stilton, etc.) Refrigerated cheese-based salad dressings (e.g., bleu cheese), not shelfstable* Mexican-style cheeses (e.g. queso blanco fresco); uncooked soft cheeses, including farmer’s and feta cheese (allowed if cooked); hot peppercontaining cheeses Unwashed raw vegetables and fruits and those with visible mold; all raw vegetable sprouts (e.g. alfalfa, mung bean, broccoli, etc.) Commercial unpasteurized fruit and vegetable juices Raw and non-heat-treated honey; comb honey Fermented miso (e.g. miso soup) and other fermented products (e.g. tempeh, maté tea, kimchee, etc.) Raw, uncooked brewer’s yeast All moldy and outdated food products Well water; water from small municipal wells. Bottled water not meeting the Center for Disease Control water guidelines for HCT patients * Shelf-stable refers to unopened canned, bottled or packaged food products that can be stored before opening at room temperature; container may require refrigeration after opening. Adapted with permission from Hematopoietic Stem Cell Transplant Nutrition Care Criteria, Seattle Cancer Care Alliance, Seattle, Washington, 2nd edition, 2002.

likely to contain fungal and mold organisms. Autologous graft recipients are advised to follow diet precautions for 3 months after conditioning and allogeneic graft patients until 1 year after transplant, or longer if treated with immunosuppressive drugs for chronic GVHD. Patients and caregivers require education on food safety practices if allowed to bring food into the hospital, as well as for self-care in the home setting. Integrative medicine – use and precautions When confronting a life-threatening illness, some patients and their caregivers embrace significant lifestyle alterations and “wellness” recommendations. Diets and dietary supplements are used in an effort to mitigate the side-effects of antitumor therapies, reduce the risk for disease recurrence, and, perhaps most importantly, provide hope and a sense of control. Dietary supplements include a wide variety of synthetic products or products derived from plants or animals available as tablets, capsules, liquid extracts, tinctures, teas, powders, and topical preparations. Because these agents are considered food products and not drugs, the production, distribution, and labeling of dietary supplements are not currently regulated by the Food and Drug Administration, and thus dosages vary widely and adulteration has been reported. At arrival for HCT, a high percentage of patients are using integrative therapies, the most common of which include supplementation with vitamins and minerals [48]. Antioxidants present a special challenge, as competing hypotheses have arisen. Experimental models suggest that antioxidants may improve the efficacy of cancer treatment by increasing tumor response and decreasing toxic effects on normal cells; conversely, antioxidants may decrease the efficacy of treatment by protecting cancer cells [49–51]. Bruemmer and co-workers examined the possible relationship between antioxidant use and outcome in an observational cohort

Nutrition Support of the Hematopoietic Cell Transplant Recipient

study in HCT patients [48]. Supplement use prior to HCT was selfreported. Nonrelapse mortality, overall mortality, and disease recurrence were followed for 2 years post transplant. Supplemental vitamin C pre transplant was associated with improved survival in persons with breast cancer, whereas vitamin C and vitamin E supplementation was associated with an increase risk of mortality and relapse in patients with acute leukemia. In a controlled trial in which patients were preloaded with antioxidants 3 weeks before conditioning with dose-intensive therapy including TBI, negative correlations were observed between antioxidant concentration levels and lipid peroxide concentration levels [52]. Higher peroxide concentration levels were seen in patients who did not receive the antioxidant supplements. However, there were no clinical correlates identified with higher peroxide levels, and the clinical significance is unknown. Conventional TPN with standard micronutrient supplementation does not prevent the depletion of antioxidants after HCT [53], but it is unknown whether this confers benefit for survival or risk for complications. As there is evidence that antioxidants can reduce efficacy of treatments in some cases, it seems prudent to discourage concurrent use of nonprescription antioxidants with radiation therapy or chemotherapy until more evidence becomes available. One approach is to set the limit for supplementation at the tolerable upper limits of the Dietary Reference Intakes accessible at the United States Department of Agriculture website (Table 101.5). Of particular concern is supplementation of vitamin C in the setting of excessive iron. Iron overload due to previous blood transfusions is associated with extensive pro-oxidant damage during HCT [54]. Although not documented for adverse effects specifically in HCT, restriction of vitamin C even below upper tolerable limits may prevent release of excessive iron stores. Advice to patients ought further to encompass concerns for (1) possible interactions between dietary supplements and medications, (2) contamination of preparations from plants that pose a risk of bacterial, fungal or parasitic infections [43], and (3) exposure to preparations with toxic metals, drugs or other botanicals with drug-like effects that could result in serious health risks. Some agents, such as isoflavonoids and many herbs (e.g. garlic, ginger, Ginkgo biloba, and ginseng), may diminish platelet function and aggregation, and are contraindicated in patients with thrombocytopenia [55]. A list of contraindicated supplements and herbs is provided in Table 101.5. Standard vitamin and mineral supplementation (iron-free), however, is indicated for all HCT recipients. In particular, patients are at risk for developing folate deficiency due to decreased dietary intake, exposure to antifolate chemotherapeutic agents (methotrexate) and antimicrobial agents, and increased folate requirements during regeneration of hematopoietic cells. Data reported by Robien and colleagues suggest that individuals with two common methylenetetrahydrofolate reductase polymorphisms, C677T and A1298C genotypes, are at higher risk of relapse after HCT, and that the balance of intracellular folate metabolites available for nucleotide synthesis may affect the progression from bcr– abl positivity to clinical relapse [56]. This observation was independent of the occurrence of GVHD. Indications for TPN The use of TPN in HCT evolved due to the severity of the gastrointestinal toxicity in high-dose conditioning regimens and the availability of central venous access established for other supportive therapies. The efficacy of adjunctive TPN has not been adequately investigated in the current era, and the clinician must exercise judgment based on risks and benefits defined in past studies, expected toxicities, anticipated incidence and severity of GVHD, ongoing surveillance of nutritional status, and

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Table 101.5 Dietary supplements, botanicals and integrative medicine possibly contraindicated in hematopoietic cell transplantation (HCT) Antioxidants Vitamin antioxidants in doses exceeding the Dietary Recommended Intake (http://fnic.nal.usda.gov/nal_display/index.php?info_center=4&tax_ level=2&tax_subject=256&topic_id=1342): • vitamins C and E • beta-carotene and carotenoids • selenium • zinc Supplements with antioxidant effects: • coenzyme Q10 • bioflavinoids • blutathione • superoxide dismutase • most botanicals due to potential antioxidant content Supplements with anticoagulant effects Garlic Ginger Ginkgo biloba Ginseng Echinacea Grape seed Kava St John’s wort Interact with drugs utilizing the cytochrome P450 enzyme system such as cyclosporine, tacrolimus, calcium channel blockers, etc. Other contraindicated supplements and herbals Chinese/oriental herbs Black Hellebore Chaparral Dehydroepiandrosterone Dieter’s Tea Botanicals containing pyrrolizidine alkaloids Ephedra or Ma Huang Germander Hemlock Laetrile Licorice Root Lobedia Pau d’arco Pennyroyal PC-SPES, SPES Sassafras L-Tryptophan Yohimbe, Yohimbine For more information visit the National Center on Complementary and Alternative Medicine: http://nccam.nih.gov/ Other integrative therapies Acupuncture Deep tissue or forceful massage Chelation Colonic therapies

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ability of the family and team to overcome any barriers to alternatives – adequate volitional oral intake or tube feeding. A Cochrane review compared clinical outcomes in patients randomized to TPN or intravenous hydration or enteral nutrition following HCT [57]. When compared with intravenous hydration, two studies met the criteria for inclusion, and among these 166 patients, a significantly higher rate of infection occurred in those who received TPN. The review excluded the larger of the two studies from the analysis of the survival outcome, which for several decades validated the use of prophylactic TPN in well-nourished allogeneic HCT patients owing to the improved long-term survival [58]. Since the Cochrane review was reported, one randomized trial in 55 well-nourished patients with stage II–IV breast cancer undergoing autologous transplant did not demonstrate a survival benefit with TPN [59]. In all the trials, patients receiving TPN had superior nutritional status compared with intravenous hydration and oral diet [57–59]. In reduced-dose regimens, TPN appears less necessary. In patients with myelodysplastic syndrome treated with fludarabine, moderate doses of busulfan, and Campath-1H, only 4% needed TPN compared with 52% of patients receiving high-dose regimens of busulfan and cyclophosphamide or busulfan, cyclophosphamide, and 1440 cGy TBI [60]. Intestinal permeability (as a surrogate for gut damage) was not increased in one reduced-intensity regimen (fludarabine and antithymocyte globulin with either cyclophospamide or busulfan), and average days of elevated C-reactive protein (0.3 versus 5.3) and days of TPN (1.4 versus 18.3) were significantly less for the patients receiving reduced-intensity regimens compared with patients receiving a traditional TBI and cyclophospamide regimen [61]. Even for patients treated with high-dose preparative therapy, TPN is not universally required. Iestra and colleagues applied standard criteria for malnutrition in oncology patients to determine appropriate utilization of TPN in HCT [62]. Using the criteria of malnutrition pre transplant, 7–10 days of minimal oral intake or 10% weight loss, the need for TPN ranged from as low as 37% of autologous patients on non-TBI regimens to as high as 92% of allogeneic patients from a mismatched donor. The need for TPN may also be modified by “gut-protectant” therapy, although these agents are for the large part investigational and not widely used. Humanized keratinocyte growth factor, given immediately before TBI in autologous patients with hematologic malignancies, decreased significantly the percentage of days study patients (n = 106) required TPN compared with controls (n = 106), at 31% versus 55%, respectively [63]. To counter the infectious risks associated with TPN, the nutrition support literature advocates a variety of strategies, including targeting calories as precisely as possible to actual needs, controlling blood sugar close to the normal range, and providing some form of enteral stimulation. The addition of pharmacologic doses of intravenous glutamine, a nonessential amino acid oxidized by stimulated lymphocytes and macrophages and intestinal mucosal cells, is an additional strategy to reduce infections. Glutamine is purported to maintain the gut mucosal barrier based on considerable experimental evidence that it reduces bacterial translocation during periods of gut rest and during intestinal injury. Piccirillo and colleagues report statistically improved lymphocyte recovery in autologous HCT patients receiving glutamine-supplemented TPN compared with standard TPN, although this did not translate into an effect on days of antibiotics, fever or length of hospitalization [64]. Infection data were not provided. In a Cochrane review of available trials in humans, TPN with glutamine was associated with a decrease in positive blood infections [57]. However, careful analysis of the data reveals that the reviewers counted colonization cultures from stool and other sites as blood infections, rendering data from these studies [65,66] not as strong as suggested. Further, at least one study subsequent to the

Cochrane review does not support a difference in infection outcome [67]. Other consensus reviews caution against routine use of glutamine until more studies demonstrate efficacy [27,68]. Of most concern is that glutamine may in fact have deleterious effects on disease-free survival (Table 101.3). Two clinical trials using alanyl–glutamine dipeptide described significantly more relapses in patients undergoing autologous HCT randomized to glutamine supplementation [69,70]. There is not an established infection risk due to the lipid component of TPN. Only large doses (up to 4 g/kg) or very fast infusion rates (100–200 mL/hour of 20% emulsions) cause any adverse effect on neutrophil, phagocyte, and reticuloendothelial system function. In a trial involving over 500 patients on dose-intensive regimens, patients randomized to a moderate lipid dose (25–30% of total energy) had a similar incidence of bacterial and fungal infections compared with patients randomized to a low lipid dose (6–8% of total energy to prevent essential fatty acid deficiency) [71]. Although not approved for use in the United States, lipids containing medium-chain triglycerides are popular in Europe owing to their improved blood stream clearance. No clinical benefit was observed in the use of 50% medium-chain/50% long-chain triglyceride mixture versus a 100% long-chain triglyceride-based lipid emulsion when provided as 30% of nonprotein calories in 36 patients undergoing HCT, and fewer days of febrile neutropenia and antibiotic use were observed with the long-chain triglyceride group [72]. Shortening the exposure to TPN is another strategy to limit infectious and other TPN-related complications. The clinical decision to discontinue TPN considers overall nutritional status, severity of gastrointestinal toxicities, ability to sustain or progress with oral intake, and the psychological impact of TPN. Charuhas and colleagues [73] compared the effect of continuing TPN or changing to hydration fluids on the time to resume adequate oral intake in patients unable to consume a significant proportion of estimated energy needs at time of hospital discharge. Patients randomized to hydration fluids met oral calorie goals a median of 6 days sooner than patients on TPN (p = 0.049) without evidence of adverse consequences, such as increased hospital readmissions or clinically significant weight loss, when TPN was withheld for up to 1 month. In another randomized study to determine the impact of discharge on resumption of oral intake in patients with very low oral intake (median <15% of needs), hospitalized patients reached the study goal of one-third of calorie needs for three consecutive days in 4.5 days compared with 8 days in those discharged (p = 0.004); days of TPN did not differ between the groups [3]. Thus, it appears safe to discontinue TPN when patients are eating 30% of estimated energy needs at hospital discharge in the absence of malnutrition, malabsorption or significant gastrointestinal toxicities. General guidelines for dosing energy, carbohydrate, lipid, fluid, electrolytes, vitamins, and trace elements for pediatric and adult patients are provided in Table 101.6. Individual needs will vary significantly, especially in the presence of altered renal, liver or gastrointestinal function and with many drugs. Indications for enteral nutrition Gradually, more transplant centers are using tube feeding in lieu of or as a transition step from TPN to full oral feeding to reduce costs, avoid the infectious complications of TPN, and presumably enhance gut barrier function by direct gastrointestinal stimulation and nourishment [22]. Investigators hypothesize that intervention with enteral nutrition in the early phase of HCT may reduce infections and dampen the inflammatory response that amplifies mucosal toxicity and predisposes the patient to GVHD [74]. There are limited studies to support the benefits of enteral feeding: the Cochrane review previously described evaluated TPN versus enteral nutrition in 144 patients, but was unable to evaluate

Nutrition Support of the Hematopoietic Cell Transplant Recipient

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Table 101.6 Total parenteral nutrition (TPN) dosing recommendations Nutrient

Adult

Adolescent

Children

Altered needs

Protein (g/kg)

1–1.5

1–1.8

1.2–2.4 (7–10 years) 1.5–3 (4–6 years) 1.8–3 (infant – 3 years)

Increase: recovery post-myeloblative conditioning; steroids; continuous renal replacement therapy Decrease: renal failure (blood urea nitrogen >80– 100 mg/dL)

Energy (kcal/kg) Maintenance 1.2–1.3 × basal

25–30

40–50

Increase: severe skin GVHD, multisystem chronic GVHD Decrease: paralytic agents, reduced activity

40–45

45–65

Carbohydrate (g/kg)

<5

<7–10

50–60 (7–10 years) 60–70 (4–6 years) 70–90 (6 months – 3 years) 65–75 (7–10 years) 75–90 (4–6 years) 90–105 (6 months – 3 years) 12–15 (7–10 years) 15–18 (4–6 years) 18–20 (6 months – 3 years)

Lipid

Minimum: 6–8% of total kcal Maximum: 40% of total kcal*

Fluid (maintenance) Note: provide 80% maintenance fluid as TPN post conditioning Electrolytes (starting doses)

1500 mL/m2

Stress 1.5 × basal

1500 mL/m2

100 mL/kg up to 10 kg +50 mL/kg for each kg 11–20 kg +20 mL/kg for each kg 21–40 kg

Vitamins and trace elements

When 80% maintenance fluid used in initial TPN per litre: 30 mEq sodium chloride 25 mEq potassium chloride 15 mEq potassium phosphate 10–20 mEq calcium gluconate (lower dose in adults) 16 mEq magnesium sulfate (8 mEq if no tacrolimus or cyclosporine) Standard adult and pediatric packages Notes: 1. Zinc – pediatric trace element packages do not contain enough; add: 1 mg children and 2.5 mg adolescents 2. Vitamin C – vitamin packages may not contain enough to repair oxidative damage following myeloablative regimens; add: 250 mg < 11 years and 500 mg > 11 years 3. Vitamin K – optimal dose not established

Iron

Contraindicated due to blood transfusions

Decrease: pre-existing diabetes or hyperglycemia (up to 50% reduction) Decrease: high serum triglycerides Increase: in lieu of carbohydrate for patients with hyperglycemia Decrease: sinusoidal obstructive syndrome, heart failure, pulmonary edema, rapid weight gain Increase: in most cases, add supplemental fluids for fevers, excessive gastrointestinal losses See Table 101.1 for management of altered serum electrolytes

Zinc and copper: see Table 101.1 for management of altered serum levels Manganese: decrease in liver disease (delete if hyperbilirubinemia >1 week) Selenium: add 2 μg/kg to maximum of 30 μg if on TPN >1 month Molybdenum: add 0.25 μg/kg to maximum of 5 μg if on TPN > 1 month Renal failure: 1. If serum vitamin A is elevated, provide halfdose multivitamin package and supplement 100 μg vitamin K 2. During renal replacement therapy: supplement water-soluble vitamins†

* Manufacturer’s maximum 60%; however, hypertriglyceridemia is common, and 40% is the recommended maximum. † Suggested water-soluble vitamin supplementation during renal replacement therapy: <11 years: 25 mg thiamine, 0.5 mg riboflavin, 25 mg niacin, 0.5 mg pantothenic acid, 15 mg pyridoxine, 15 mg vitamin B12, 0.5 mg folic acid, 45 mg ascorbic acid; >11 years: 60 mg thiamine, 1 mg riboflavin, 50 mg niacin, 1 mg pantothenic acid, 30 mg pyridoxine, 30 mg B12, 1 mg folic acid, 90 mg ascorbic acid.

key outcomes of GVHD, survival, and infections [57]. More recently, preliminary findings suggest that early enteral feeding following HCT is associated with less GVHD and lower infection mortality at 100 days post transplant [75,76]. These observations are intriguing and merit prospective evaluation. Table 101.7 provides a summary of the use of tube feedings following dose-intensive regimens. There are significant challenges in using an exclusive enteral approach, primarily because of difficulties in maintaining access and in delivering adequate nutrition. Vomiting and dislodge-

ment of nasal tubes is common. Sefcick and colleagues were able to successfully feed eight of 15 adult patients undergoing allogeneic transplant with a self-propelling nasojejunal tube until the day of engraftment [77]. Their recommendation was to delay tube placement until the day after the conditioning therapy and stem cell infusion have been completed, prior to the onset of mucositis. However, this approach may miss a critical window during conditioning when enteral nutrition might exert its physiologic benefit on gut mucosal integrity and modulation of the inflammatory response.

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Table 101.7 Tube feeding studies in high-dose regimens

Study

Transplant type (number) and conditioning regimens

Randomized controlled trials Szeluga, 1987 [82] TPN versus enteral feeding program

Mulder, 1989 [83] TPN versus PPN + enteral

Age

Clinical outcomes/complications

Allogeneic (46) Autologous (15) BU/CY; CY/TBI

Pediatric >10 years and adult

Autologous (22) CY/etoposide

Adult

13% failures in TPN group (unable to place catheter) 23% in enteral feeding program eligible for tube feeding (n = 7); all needed TPN and unable to place tubes or tolerate feeds due to gastrointestinal symptoms Tube feeding group – twice the rate of sepsis; less diarrhea

Pilot or case series with greater than 10 patients Papadopoulou, 1997 [78] Not specified (29) CY/TBI; BU/CY; idarubicin/TBI

Pediatric

Roberts, 1998 [79]

Autologous (4) Allogeneic (12) CY; CY/TBI Not specified (27 HCT of 44 total cancer)

Adult

Sefcick, 2001 [77]

Allogeneic (15) CY/TBI

Adult

Langdana, 2001 [81]

Allogeneic (42) Autologous (11) CY/TBI; BU/CY; CY/ATG; high-dose melphalan; BEAM

Pediatric

Hopman, 2003 [76]

Allogeneic (12) CY (11), TBI (4), cytarabine (4), BU (6), etoposide (4), melphalan (6)* Allogeneic (22) *TBI (16)

Pediatric

Barron, 2000 [80]

Seguy, 2006 [75]

Pediatric

Adult

38% refused tube feeding; in those who accepted the tube, 29% needed TPN; tube feeding provided <50% of energy needs 25% needed TPN due to GVHD diarrhea or high residuals 6% local infection at percutaneous gastrostomy site 64% needed TPN 41% local infection at G-tube site 4.5% peritonitis 53% retained tube until engraftment Weight loss at discharge 4.5%, 3 months 7%, 6 months 5% 47% vomited tubes during conditioning/early post HCT 14% needed TPN Median duration of feeds was 52 (range 5–267) days Incidence malnutrition (<85% ideal body weight) increased 6% to 14% Enteral feeds provided <50% of needs 75% needed TPN No cases of acute GVHD observed

50% needed TPN Less Grade III–IV GVHD and infection, lower 100 day mortality compared to concurrent cohort receiving TPN

ATG, antithymocyte globulin; BEAM, BCNU, etoposide, cytosine arabinoside, and methphalan; BU, busulfan; CY, cyclophosphamide; GVHD, graft-versus-host disease; HCT, hematopoietic cell transplantation; PPN, partial parenteral nutrition; TBI, total body irradiation; TPN, total parenteral nutrition. * Combinations not specified.

The inability to infuse adequate nutrition has led to TPN “rescue” rates ranging from 14% to 100% [75–83]. In those studies that had lower TPN rescue rates, an enteral approach resulted in adverse nutritionrelated consequences, including significant weight loss [77], decrease in body cell mass [82], and an increase in the frequency of malnutrition in children [81]. The study with the lowest percentage of patients requiring rescue with TPN (14%) excluded the patients who died during their initial hospitalization [81], and thus the high success rate with exclusive enteral feeds is not representative of the spectrum of experience with HCT. In this study, Langdana and co-workers [81] provided less than 50% of the estimated average energy requirement via tube feeding for a median length of 52 (range 5–267) days in patients between the ages of 6 months and 17 years old undergoing allogeneic (n = 42) or autologous (n = 11) HCT. Six percent of the children were considered malnourished (defined as less than 85% IBW) pre transplant. Using the same

criteria at hospital discharge, more than 14% of the children were considered malnourished. Other complications described with enteral feeding in HCT include deficiencies of magnesium, phosphorus, zinc, and selenium [78], and, more importantly, the inability to feed in the presence of large-volume diarrhea owing to high-dose regimens or with gastrointestinal GVHD [76,77,79,81]. Other potential complications of nasoenteric tube feeding are nasopulmonary intubation, pulmonary aspiration, epitaxis, rhinorrhea, sinusitis, otitis, laryngeal injuries, nasopharyngeal perforation, and intestinal perforation. With percutaneous gastrostomies, infection, peritonitis, intestinal or gastric perforation or obstruction, and abdominal wall migration of tubes can occur. Contaminated feeds are a concern, even with commercially sterile formulas, because of open feeding systems, inadequate hand washing, and prolonged hang times at room temperature, with the risk being highest in patients receiving antacids,

Nutrition Support of the Hematopoietic Cell Transplant Recipient

gastric acid inhibitors, H2-antagonists, antibiotics, steroids or immunosuppressive therapy [22]. The placement of surgical or percutaneous gastrostomy tubes presents potential safety concerns in patients with neutropenia or thrombocytopenia. The most common practice is to ensure that the patient has an absolute neutrophil count of at least 500–1000/mm3 and a platelet count boosted by transfusions of over 50,000/mm3. For nasal tubes, platelets in the 10,000–20,000/mm3 range are probably adequate. The ideal administration of enteral formulas is via a closed feeding system, which is problematic in the pediatric population, for which not all formulas are available in closed systems. Strict adherence to 4-hour infusion volumes is recommended when open administration systems are used. The choice of enteral formula has varied in published reports. Some clinicians try whole-protein-based formulas initially and resort to peptide-based or elemental formulas only when intact protein is not tolerated, while others use semielemental formulas from the beginning [75,80,81]. A dietitian may recommend the most appropriate formula for the clinical condition. The optimal schedule for administration of enteral feeds also lacks substantive data. If enteral feeds are begun in order to transition the patient off TPN after major gut toxicity, continuous drip feeds started at a low rate and advanced slowly seem to be most successful.

Management of complications Sinusoidal obstructive syndrome Insidious weight gain is often the first sign of sinusoidal obstructive syndrome (SOS; hepatic veno-occlusive disease). In severe SOS, encephalopathy, coagulopathy, and renal failure all complicate nutrition management. Judicious management of sodium and water balance is essential supportive care in SOS. Nutrition support (TPN or enteral) volume, medication volumes, and sodium intake are minimized. When patients are severely fluid restricted or have renal failure and nutrition support is compromised, the use of continuous renal replacement therapy is indicated. The capacity to eliminate intravenous lipids from the blood stream should be monitored in patients with severe disease by measuring serum triglyceride levels. If encephalopathy develops, the benefits of HepatAmine (an amino acid solution with lower aromatic amino acid, tryptophan, and methionine content) have not been established. If hyperbilirubinemia persists longer than 1 week, the biliary-excreted trace elements copper and manganese should be removed from TPN. Measurement of energy needs with indirect calorimetry, if available, may avert additive hepatotoxicity associated with overfeeding, as well as the risks of debilitation with prolonged underfeeding. Several case studies have described therapeutic responses to glutamine and vitamin E [28,84,85], and one prospective study described improvement in markers of hepatic function, protein C, and albumin when patients were provided glutamine compared with an isonitrogenous mixture of amino acids in TPN, although no cases of SOS occurred in either group [86]. Renal failure Renal impairment occurs as part of the SOS syndrome, and may also arise as a result of the cumulative damage of nephrotoxic medications and dose-intensive conditioning regimens. Renal damage, defined as doubling of the baseline serum creatinine level, occurs in up to 75% of allograft patients [87]. An elevated blood urea nitrogen level, however, may be partially caused by nonrenal factors, including increased protein intake, gastrointestinal bleeding or hypercatabolism. Prolonged protein

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restriction to minimize a rise of blood urea nitrogen should be avoided to ensure that adequate calorie and protein support are provided. In severe renal insufficiency, protein will effectively be restricted by the volume of nutrition support tolerated, as most typically observed prior to institution of renal replacement therapy. General indications for renal replacement therapy after HCT are extracellular fluid volume expansion, acidemia, hyperkalemia, and azotemia. If renal replacement therapy is needed, the nutritional goals are provision of estimated nutrient needs within the allowed fluid volume, correction of electrolyte and mineral imbalances, and prevention of vitamin and mineral deficiencies and toxicities. Specific water-soluble vitamin complexes have been created for dialysis patients, and are available to be given orally or via a feeding tube into the gut. For TPN-dependent patients, water-soluble vitamin combination supplements are not readily available, and provision of vitamins separately or in a renal “cocktail” by the compounding pharmacy is required. Vitamin replacement is in particular vital if the patient is receiving continuous renal replacement therapy with large volumes of ultrafiltrate and/or dialysate in which there are large losses of not only water-soluble vitamins, but also glucose, amino acids, and, to some extent, minerals [88]. Patients on extended renal replacement therapy need attention to vitamin A status and micronutrient status. Vitamin A is known to accumulate in renal failure. Children and small adults receiving standard parenteral vitamin doses can accumulate vitamin A rapidly and have vitamin A levels two to three times normal within 2 weeks on renal replacement therapy. It is prudent to check vitamin A levels and adjust intake if needed in patients with renal failure and risk factors for early vitamin A toxicity. Chromium and molybdenum may also accumulate in renal failure and should be monitored for the need for dose reduction or discontinuation. Serum triglycerides need to be monitored weekly because the clearance of intravenous lipids may be reduced in renal failure. Pulmonary disease Pulmonary edema due to increased capillary permeability after highdose regimens may be compounded by iatrogenic fluid overload. Management includes reducing total sodium from oral intake, TPN, and medications, and using concentrated TPN solutions. During ventilator dependency, adequate TPN or tube feeding should be provided to preserve muscle reserves. Iron overload Many HCT patients accumulate excessive iron, with as many as 90% of long-term survivors for hematologic malignancy or aplastic anemia exhibiting hemosiderosis of the liver [89]. These patients most likely have iron deposits in other organs as well. It is recommended that these patients avoid iron supplements and iron-containing multivitamins post transplant. Acute GVHD A few investigators have been interested in whether nutrition might modulate the incidence or severity of GVHD, particularly lipid suppression of inflammatory cytokine production through prostaglandin E2mediated pathways. In a study of lipids and infection outcome, the secondary outcome of grade II–IV GVHD incidence was no different in patients receiving 25–30% or 6–8% of total calories as lipids, occurring in 77% and 75% of patients, respectively [71]. Time to acute GVHD (before 80 days after HCT and censored for death, relapse, and treatment failure) also did not differ. Other investigators compared very high doses

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of lipid (80% of total energy) to lipid-free TPN in 66 allograft recipients; while incidence and median day of onset were similar between the groups, lethality of GVHD was decreased in the lipid group [72]. In a small study of oral eicosapentaenoic acid supplemented at 1.8 g/day from day 21 to day 180 after transplantation, three of seven patients receiving the eicosapentaenoic acid experienced grade II–III GVHD, all of whom survived, whereas in the control group (n = 9), six experienced grade II–IV GVHD, and five of those died [90]. The management of acute and chronic GVHD is unique in dietetic practice. Acute intestinal GVHD manifests as secretory diarrhea, often high volume, green in color, and bloody. Protein content is often elevated, as evidenced by falling plasma protein levels or by measurements of fecal α1-antitrypsin [91]. Patients may experience concomitant anorexia, nausea, vomiting, and crampy abdominal pain. On biopsy, findings range from necrosis of individual intestinal crypt cells to total mucosal denudation [92]. In severe disease, bowel rest allows the team to interpret the efficacy of immunosuppression, and TPN is needed to maintain nutritional status. Additional daily zinc is recommended at 10 mg/L of stool volume in excess of 1 L. A conservative oral feeding plan is indicated when diarrheal volumes diminish and abdominal pain subsides, starting with isotonic oral liquid supplements. An empiric set of guidelines for the introduction of oral feeding has evolved, which is based on foods and fluids low in lactose, fat, fiber, and total acidity [93]. Pharmacologic intervention is contraindicated because of the risk of ileus and abdominal distention, with the exception of octreotide, which may lower diarrhea in some cases [94]. A couple of decades ago, Weisdorf and co-workers [95] described a syndrome of upper intestinal GVHD which manifests as anorexia, dyspepsia, food intolerance, nausea, and vomiting. Poor appetite or nausea and emesis that either persist or arise de novo after engraftment constitute an indication for endoscopic evaluation to establish a diagnosis of stomach or upper intestinal GVHD [19]. Upper gut GVHD is generally responsive to steroids, and often hyperphagia supplants anorexia. The oral mucosa may also be involved in acute GVHD, manifesting as erythema and lichenoid changes that cannot be differentiated from conditioning-related mucositis until approximately 3 weeks after HCT [96]. It may evolve into a chronic form, and patients may experience significant weight loss unless supported with TPN or enteral nutrition. In acute liver GVHD, diminution of hepatic protein synthesis and enterohepatic circulation of bile salts may result in hypoalbuminemia and steatorrhea, respectively. If the latter occurs, fat restriction may be indicated. In severe disease, encephalopathy and ascites may require nutritional management similar to that of SOS. Almost every drug that is used to treat GVHD affects nutrient metabolism. Some of the side effects have been described in Table 101.1. As mentioned previously, energy needs during acute GVHD have been predicted at 45–50 kcal/kg in adults and 65 kcal/kg in children [15]. Chronic GVHD Chronic GVHD is a diverse syndrome with often profound nutritional consequences as a result of anorexia, mucositis, xerostomia, dysphagia, esophageal stricture, cholestatic liver disease, diarrhea or steatorrhea, dyspnea and limited exercise tolerance, restricted joint mobility, and generalized wasting [2]. In one review, weight loss exceeded 30% of the pre-HCT weight [97]. Among 93 patients evaluated a median of 2.4 years after transplantation, 29% had moderate malnutrition (BMI 18.5–21.9 kg/m2) and 14% had severe malnutrition (BMI < 18.5 kg/m2). Patients with active chronic GVHD had a significantly lower BMI compared with patients with inactive disease. Resting energy expenditure in 13 patients with stable chronic GVHD was found to be significantly higher, and carbohydrate oxidation significantly lower, than in controls

matched for sex, age, weight, and height [98]. Studies of weight loss are confounded by steroid-induced fluid and fat weight gain, which may explain why weight loss has not been a prognostic indicator [99]. Oral disease is common, and symptoms of burning and loss of taste have been described as “prodromes” of chronic GVHD. In severe cases, complete nutritional supplements are indicated, and, if weight loss occurs, gastrostomy feeding. Esophageal webbing or stricture manifests as difficulty in swallowing food and pills and retrosternal pain caused by esophageal thinning. Dilation is usually needed, and nutrition may be limited to liquids in those patients with extensive webbing. Such patients are also appropriate candidates for gastrostomy feeding. Chronic diarrhea remains a nonspecific post-HCT finding, and may be a result of infection, medications, pancreatic insufficiency, and/or GVHD of either the liver or gastrointestinal tract. Nutrition support at this stage requires individualization and may be oral, enteral or parenteral. When weight loss occurs in chronic GVHD, the relative contributions of deficient energy intake and malabsorption need to be determined. Patients may have poor appetites, while others have good dietary intake but significant stool nutrient losses. A malabsorption work-up may include quantitative stool fat collection, stool for fecal elastase, serum carotene, serum xylose or Schilling test, and serum 25-hydroxyvitamin D. Vitamin D deficiency is common, likely owing to inadequate hepatic and renal hydroxylation to the active metabolite. If fecal elastase reveals pancreatic insufficiency, pancreatic enzyme replacement is instituted; if pancreatic function is normal, a bile defect may necessitate a moderate fat restriction and mineral and water miscible fat-soluble vitamin supplementation. Vitamin D and calcium status are compromised by multiple factors in chronic GVHD. Sun-blocking agents universally prescribed to prevent photoactivation of skin GVHD reduce vitamin D synthesis by 99% [99]. Bioavailability is decreased with malabsorption and, for vitamin D, sequestration in body fat in obese individuals. Steroids activate the destruction of both 25-hydroxyvitamin D and 1,25-hydroxyvitamin D to inactive calcitroic acid, as well as causing bone resorption and calciuria. Loss of vertebral bone mass has been well described. In one series in which patients were followed for a median of 30 months, allograft patients lost 11.7% of femoral neck and 3.9% of spinal bone mineral density compared with 1.1% loss and 1.5% increase, respectively, in the autograft patients [100]. Bone loss correlated best with the cumulative steroid dose, with spinal bone loss of 4% per 10 g of steroids and femoral neck bone at 9% per 10 g of steroids. Several studies have described the prevalence of bone loss in children with chronic GVHD exceeding 50%, and the rate of bone loss appears to be highest in the first 6 months after HCT [101,102]. Consensus statements sponsored by the National Institutes of Health [103] and Children’s Oncology Group [104] recommend daily allowances for calcium and vitamin D to prevent osteoporosis following HCT. It is likely that standard doses, especially of vitamin D, are inadequate, since vitamin D experts recommend, in normal children and adults who do not receive sun exposure, 800–1000 IU vitamin D, or fivefold the current standard dose of 200 IU [99]. In a retrospective study of pediatric patients following HCT, 48 controls on standard doses of vitamin D and calcium lost bone density, compared with 18 patients additionally supplemented with bisphosphonate who were able to gain bone density [105]. Higher doses of vitamin D have yet to be studied. Vitamin D status should be part of routine monitoring in patients with chronic GVHD. Deficient serum levels of 25-dehydroxyvitamin D (<30 ng/dL) should be treated with 50,000 IU of vitamin D2 weekly for 8 weeks and then reassessed. Chronic replacement with 50,000 IU every 2 weeks or monthly is indicated to maintain serum levels above 30 mg/dL. In patients with renal dysfunction 1,25-dehydroxyvitmain D3 (calcitriol) is indicated at a dose of 0.25–1.00 μg twice daily [99].

Nutrition Support of the Hematopoietic Cell Transplant Recipient

Children with extensive chronic GVHD exhibit growth failure and a variety of endocrine abnormalities. While the contribution of poor nutrient intake as a factor in growth failure after HCT has not been adequately investigated, children displaying weight loss, inappropriate weight gain or growth failure deserve thorough nutritional evaluation to rule out treatable dietary deficiencies. Height should be monitored every 3 months after transplantation for the purpose of early detection of growth failure. Children may also experience long-term metabolic alterations following HCT which may adversely affect nutritional status. Taskinen and colleagues reported a 39% incidence of combined hyperinsulinemia and hypertriglyceridemia in pediatric transplant patients compared with 8% in leukemia patients and none in healthy controls (p = 0.0015) as a late effect following HCT [106]. An increased incidence of diabetes mellitus has also been reported in long-term survivors of pediatric HCT. In a

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retrospective review of over 700 transplant survivors, the prevalence of type 1 diabetes was 0.52%, or three times higher than in the general United States population, while the prevalence of type 2 diabetes was 9% among leukemia survivors and 2% among aplastic anemia survivors, both higher than expected [107]. The nutrition assessment and timely intervention with a variety of nutrition support modalities, including low-microbial diet precautions, safe supplementation with vitamins and other complementary therapies, counseling about well-tolerated foods, and the use of tube feeding or TPN when weight loss and significant gastrointestinal symptoms occur, are critical components to successful transplant outcome. Nutrition is both an art and a science, responding to the cultural needs and desires of patients and linking with the immunology and physiology of HCT, very much in need of defining more evidence-based approaches.

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92. Snover DC, Weisdorf SA, Vercellotti GM et al. A histopathologic study of gastric and small intestinal graft-versus-host disease following allogeneic bone marrow transplantation. Hum Pathol 1985; 16: 387–92. 93. Gauvreau JM, Lenssen P, Cheney C et al. Nutritional management of patients with acute gastrointestinal graft-versus-host disease. J Am Diet Assoc 1981; 79: 673–7. 94. Ippoliti C, Champlin R, Bugazia N et al. Use of octreotide in the symptomatic management of diarrhea induced by graft-versus-host disease in patients with hematologic malignancies. J Clin Oncol 1997; 15: 3350–4. 95. Weisdorf DJ, Snover DC, Haake R et al. Acute upper gastrointestinal graft-versus-host disease: clinical significance and response to immunosuppressive therapy. Blood 1990; 76: 624–9. 96. Woo SB, Sonis ST, Monopoli MM, Sonis AL. A longitudinal study of oral ulcerative mucositis in bone marrow transplant recipients. Cancer 1993; 72: 1612–17. 97. Jacobson DA, Margolis J, Doherty J et al. Weight loss and malnutrition in patients with chronic graft-versus-host disease. Bone Marrow Transplant 2002; 29: 231–6. 98. Zauner C, Rabitsch W, Schneeweiss B et al. Energy and substrate metabolism in patients with chronic extensive graft-versus-host disease. Transplantation 2001; 71: 524–8. 99. Holick MF. Vitamin D deficiency. N Eng J Med 2007; 357: 266–81. 100. Ebeling PR, Thomas DM, Erbas B et al. Mechanisms of bone loss following allogeneic and autologous hemopoietic stem cell transplantation. J Bone Miner Res 1999; 14: 342–50.

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101. Kashyap A, Kandeel F, Yamauchi D et al. Effects of allogeneic bone marrow transplantation on recipient bone mineral density: a prospective study. Biol Blood Marrow Transplant 2000; 6: 344–51. 102. Kauppila M, Irjala K, Koskinen P et al. Bone mineral density after allogeneic bone marrow transplantation. Bone Marrow Transplant 1999; 24: 885–9. 103. Couriel D, Carpenter PA, Cutler C et al. Ancillary therapy and supportive care of chronic graftversus-host disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease. V. Ancillary Therapy and Supportive Care Working Group Report. Biol Blood Marrow Transplant 2006; 12: 375–96. 104. Wasilewski-Masker K, Kaste SC, Hudson MM et al. Bone mineral density deficits in survivors of childhood cancer: long-term follow-up guidelines and review of literature. Pediatr 2008; 121: e705– 12. 105. Carpenter PA, Hoffmeister P, Chesnut III CH et al. Bisphosphonate therapy for reduced bone mineral density in children with chronic graftversus-host disease. Biol Blood Marrow Transplant 2007; 13: 683–90. 106. Taskinen M, Saarinen-Pihkala UM, Hovi L, Lipsanen-Nyman M. Impaired glucose tolerance and dyslipidaemia as late effects after bone-marrow transplantation in childhood. Lancet 2000; 356: 993–7. 107. Hoffmeister PA, Storer BE, Sanders JE. Diabetes mellitus in long-term survivors of pediatric hematopoietic cell transplantation. J Pediatr Hematol Oncol 2004; 26: 81–90.

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Noelle V. Frey & Jonathan R. Gavrin

Pain Management

Introduction Patients who receive hematopoietic cell transplantation (HCT) may suffer pain of varying etiologies and severity (Table 102.1). This chapter defines the neurophysiology and pharmacology of pain; what is meant by “pain;” what factors lead a patient to experience and suffer pain; what physical, psychologic, environmental, and pharmacologic factors influence the amount of pain and suffering; how this compendium of pain and suffering can be evaluated; and the different means by which pain and suffering can be relieved in the HCT recipient.

Anatomy and physiology of pain Classically, pain begins with tissue damage (nociception), the type and location detected by peripheral nerves and then signaled to the brain. Our understanding of the neurophysiology and neurochemistry of nociceptive damage detection, transmission to the central nervous system (CNS), and CNS processing to produce the clinical phenomenon of pain, is far from perfect. Our understanding of how the CNS may be influenced by pharmacologic or nonpharmacologic means to suppress nociception and reduce pain is also imperfect. A summary of the known neurophysiology and neuropharmacology of nociception and pain is helpful in understanding clinical management of the HCT patient. Ascending nociceptive systems All body tissues possess nociceptors (neural structures that detect tissue damage) that are activated by physical stimuli or by algogenic (painproducing) substances released when there is tissue injury. Alternatively, nociceptors may be sensitized by algogenic substances, resulting in noxious activation by normally innocuous stimuli, for example light touch in the presence of inflammation. Nociceptors signal to the spinal cord through fast conducting Aδ fibers, or slow conducting C fibers in peripheral nerves, which enter the neuraxis and synapse through dorsal horn cells (Fig. 102.1). Dorsal horn cell axons cross the midline and ascend cephalad in the spinothalamic tracts. Information from the fast Aδ fibers ascends rapidly, with no intermediate synapses, to the thalamus, and hence to the cerebrum. Information from the slow C fibers

ascends through a number of different synapses, notably to the limbic system, before reaching the thalamus and cerebrum. The C fiber/limbic system synapses initiate the emotional responses to pain [1]. Modulation of nociceptive traffic Afferent nociceptive transmission is not “hard wired” – signals do not progress to the cerebrum unimpeded and unimpedable. With evolving neurophysiologic knowledge, it has been recognized that afferent transmission may be modulated, as hypothesized in the “gate control theory” of pain [2,3]. This theory states that there are various “gates” within the nervous system that may be opened, or closed, to nociceptive transmission by a variety of processes. The “gating” concept is a useful tool when teaching patients and relatives about pain relief techniques. A basic understanding of this physiology helps patients and family members understand that active interventions, pharmacologic and nonpharmacologic, are essential to obtain optimal pain relief. Afferent nociceptive transmission can be enhanced or, more importantly, inhibited by the following: 1 Modification of the peripheral nociceptor signaling. Algogenic substances (prostaglandins and serotonin [5-hydroxytryptamine, or 5-HT]) can activate or sensitize peripheral nociceptors; anti-inflammatory medications (aspirin and other nonsteroidal anti-inflammatory drugs [NSAIDs]) can oppose such effects. 2 Inhibition of nociceptive transmission in the dorsal horn by nonnoxious peripheral stimulation. Rubbing, heat, cold or electric stimulation peripherally activates sensory afferent fibers that synapse with dorsal horn cells to inhibit nociceptive transmission. 3 Inhibition of dorsal horn nociceptive transmission by the descending nociceptive modulating system. This process originates in the periaqueductal gray matter of the hindbrain (Fig. 102.1). The neurotransmitters of this system include 5-HT, norepinephrine (NE), and endogenous opioid (morphine-like) substances. The descending modulatory system can be activated to produce pain relief by learned behavioral or cognitive strategies, or by pharmacologic agents that either mimic endogenous neurotransmitters (e.g. opioids) or stimulate activity of the system (e.g. tricyclic antidepressants [TCAs]) [4].

The clinical phenomenon of pain Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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The neurophysiology and neuropharmacology described so far deal only with physiologically demonstrable phenomena. The experience of pain clearly is more than neurophysiology, encompassing the subjective interpretation and expression of the neurophysiologic phenomenon.

Pain Management Table 102.1 Common sources of pain in bone marrow transplantation Etiology

Example

Infiltration from primary disease

Bone pain Fractures Soft tissue of visceral pain

Hematopoietic stem cell mobilization

Bone or joint aching

Chemotherapy or irradiation

Mucositis Cutaneous burns Peripheral neuropathies

Graft-versus-host disease

Skin irritation Gut cramps Oropharyngeal pain

Immunosuppressive drugs

Peripheral neuropathies Joint aching Long bone pain Osteopenic bone fractures

Infection or viral activation

Cortex Limbic system Desending modulation

Periaqueductal gray matter

Lateral reticular formation

Bone marrow aspiration Lumbar puncture Postlumbar puncture headache Line placement Tissue biopsies

Pains unrelated to disease

Headache Low back pain Myalgias from inactivity

What is pain? Pain is defined as a multidimensional experience [5]. The degree to which an individual feels pain, suffers from pain, and complains of pain is influenced by a number of factors, in addition to tissue damage/nociception. The model depicted in Fig. 102.2 [6] is helpful in conceptualizing these components. Nociception is the detection of tissue damage by sensory transducers, and the signaling of this information to the brain. Processing of nociceptive information by the CNS results in the human experience of pain, which is both sensory and affective (feeling). Suffering, the negative cognitive and emotional responses to pain, can be heightened by pain-independent emotional factors, such as fear, anxiety, and depression, that may impact the HCT patient [7]. The compendium of nociception, pain, and suffering, which are subjective, personal, and unmeasurable, emerge to be expressed as pain behaviors. Observations of pain behaviors, including report of pain and inhibition of activity, constitute the raw data that enable diagnosis of the source of pain and estimation of the magnitude of suffering. Pain and suffering in the HCT patient When an HCT patient complains of pain, the most common circumstance is one of obvious tissue damage, such as in oropharyngeal mucositis (OPM). Patients may also complain of pain with little evidence of tissue damage (neuropathy). Irrespective of its source, nociceptive

Thalamus

MIDBRAIN

Nucleus raphe magnus Medial reticular formation

MEDULLA

Spinothalamic tract

Posterior root

Shingles from herpes zoster Postherpetic neuralgia Cytomegalovirus gut pain

Diagnostic and surgical procedures

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SPINAL CORD

Anterior root

Fig. 102.1 Nociceptive information is carried to the spinal cord in Aδ and C fibers. The nerves enter the dorsal horn, where they synapse with secondorder neurons that cross the midline of the cord at about the entry level and ascend in the spinothalamic tracts. The Aδ-derived information goes on a fast track directly to the thalamus. The C fiber-derived information reaches the thalamus later, having synapsed in the limbic system. The descending nociceptive afferent modulating system originates in the periaqueductal gray matter. It projects through the nucleus raphe magnus down to the posterior horn, where it has a modulatory effect on the afferent nociceptive traffic. Pa in behavior

Suffering

Pa in

Nociception

Fig. 102.2 A schema of the various aspects of the human pain experience.

information is processed identically by the CNS, and in all cases the patient feels pain, suffers, and exhibits pain behaviors. Some patients have obvious tissue damage, such as an erythematous and ulcerated mucosa in the mouth, but complain of little pain and

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appear to suffer little. Other patients have little or no evidence of tissue damage yet complain of much pain and suffer a great deal. What can a clinician make of these two disparate situations? Is the first patient really in pain and really suffering, but not telling anyone? Is the second patient imagining or faking pain? Both explanations are plausible, but unlikely. The fact is, the relationship between nociception, pain, suffering, and pain behaviors is not a fixed one. A number of correlates, in addition to physiologic and pharmacologic factors, influence how much patients feel, suffer and complain of pain. Among these correlates are: 1 The meaning of the pain. Pain from a known or predictable source often results in less suffering and fewer complaints than pain from an unknown, potentially threatening source. 2 The circumstances in which the pain occurs. A simple pain problem, such as toothache, may result in more suffering and complaints in a patient undergoing HCT than the same pain experienced in normal circumstances. 3 The patient’s style of coping with adversity. This style is influenced by social and cultural factors. At one end of the spectrum are those who tough it out and never call for help; at the other end are those who call for help at the first sign of adversity. 4 Emotional state. Fear or anxiety can lead patients to be hyperattentive to sensations. Similarly, depression often reduces activities and increases attention to discomforts. 5 Previous experience with pain. Experience with chemotherapy may serve to reassure patients that current experience is “normal” and will resolve. An inexperienced patient may be thinking, “How can I feel so bad and possibly be doing OK?” 6 Previous exposure to CNS active pharmacologic agents (analgesics, anxiolytics or alcohol). These agents may directly influence CNS processing of nociception. If a patient is opioid tolerant or has extensive exposure to alcohol, opioid dosing needs may be higher than average. Usage patterns may also reflect the patient’s style of coping with adversity, perhaps to escape into a “pharmacologic fog”. It is well to remember that, regardless of the source, all patients have cognitive and processing components to their pain. Remember the phrase “no brain, no pain.” If it is not processed in the brain, it is not felt. The vast majority of HCT patients have a nociceptive source of their pain, onto which the cognitive and processing components are grafted.

Assessment of pain and treatment efficacy History Some patients will have histories suggesting they are at risk of having significant pain problems or may have difficulty obtaining pain relief. Significant historical factors include: 1 active pain problems related or unrelated to the patient’s disease or therapies; 2 previous severe pain after surgery or trauma; 3 difficulty in obtaining satisfactory pain control during events in items 1 and 2; 4 nonpharmacologic pain-relieving strategies that the patient uses; 5 analgesics the patient has used and any problems with them; 6 previous or current significant emotional or affective disorders; 7 previous or current CNS active substance use (alcohol, opioids and anxiolytics); 8 the patient’s understanding of the current pain, its significance, and likely duration; 9 the patient’s preferred mode of dealing with pain (both nonpharmacologic and pharmacologic).

Pain measurement and monitoring How is one to assess what the HCT patient is experiencing? Much of the system of nociceptive transmitting, processing, and suffering is subjective and not directly observable (Fig. 102.2). It is possible to observe and quantify the system’s output (complaints of pain) and use that as an approximation. In clinical practice, an HCT patient’s pain is what the patient says hurts, in the amount the patient says it hurts. Those caring for HCT patients should accept pain complaints as real, genuine, worthy of attention, and deserving alleviation. In practice, pain and its impact are monitored by subjective pain scores and observational function scores. The indices used to monitor pain, the effects, and side-effects of therapies and medication use should be observed and recorded frequently using a pain assessment tool. Far from feeling burdened by reporting pain and other symptoms on a daily or per shift basis, patients are often relieved that staff are aware of their symptoms. Consistent evaluation assures patients that their experience is recognized and that evaluation is routine, and therefore normal. Uncertainty and unpredictability about what will happen causes the greatest distress for the greatest number of HCT patients [8–10]. Knowledge that the pain is expected, how it is likely to feel, and how long it is likely to last can help patients to endure even extremely difficult circumstances. Cancer and HCT patients are reluctant to complain and do not know what experiences are “normal” or what should be reported. They will often answer “fine” or “OK” if asked only, “How are you feeling?” Pain scores and ratings can be used as “objective” quality assurance tools to determine need for further evaluation or change in treatment. Pain above the equivalent of a score of 3 or 4 (on a scale of 1–10) indicates a need to improve pain treatment because the pain will substantially disrupt function [11]. Even temporary pain should not exceed 6 without mandating further evaluation or additional pain treatment methods [11–13]. The Joint Commission on Accreditation of Healthcare Organizations mandates pain assessment as the “fifth” vital sign; pain scores of 5 or above strongly suggest the need for active intervention [14].

Pain scales Adults and children from about the age of 7 years can report with scales as seen in Figs 102.3, 102.4, and 102.5: 1 Numerical rating scales use scores from 0 = “no pain” to 10 = “pain as bad as can be.” It can be helpful the first time with a patient to use a few familiar examples such as: “When you think of a headache you have had in your lifetime, can you remember how it felt?” (Help the person think of a pain he or she remembers.) “When you think of that pain, where would it be on a scale from no pain which is a 0 to pain as bad as anyone could possibly have, which is a 10?” Further help can be offered by saying: “Many people think of 1, 2, or 3 as milder pain; of 4, 5, or 6 as moderate pain; of 7, 8, or 9 as severe pain; and of 10 as being the most extreme pain anyone could ever have.” If the patient is still unable to use the scale for cognitive or cultural reasons, the addition of the faces, as in Fig. 102.4, can be helpful. Numerical scales can be given orally or on paper, as seen in Fig. 102.3. In general, these numerical scales are easy to use and record. This scale is favored by clinicians and researchers [12,15]. 2 Categorical scales use descriptive words from “no pain” to “extremely severe pain.” Such scales are well understood by patients, but are difficult to record or to use to track pain and treatment success. 3 Visual analogue scales. These are 10 cm lines anchored at one end with “no pain” and at the other end with “pain as bad as can be.” Patients make a mark at any point along the scale to indicate pain intensity. These

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Numerical rating scale 0 No pain

1

2

3

4

5

6

8

7

9

10 Pain as bad as can be

Categorical scale Mild

Moderate

Severe

No pain

Extremely severe 1

Visual analog scale (VAS)

Fig. 102.3 Self-report scales for assessing pain in adults. The numerical rating scale is used most frequently.

Pain as bad as can be

Pain 1

10 cm line

Fig. 102.4 A “faces” scale used to monitor pain and pain management efficacy in children between ages 3 and 7 years.

0

1

0

2

3

1

4

5

2

Fig. 102.5 Assessment of Behavioral Score on the FLACC. Each category is scored 0–2 for a possible total of 10. 0, relaxed and comfortable; 1–3, mild discomfort; 4–6, moderate pain; 7–10, severe discomfort/pain. (Reproduced from [17], with permission.)

scales require measurement of the line in millimeter increments after the patient completes the written scale. Children below the age of 7 years usually cannot use the above scales. From ages 3 to 7, children can usually indicate their pain using one of a variety of “Faces” scales (Fig. 102.4) [16]. Children below the age of 3–4 years cannot use such scales, and observational scales from nurses or family members may be used. The most commonly used, and bestvalidated, observational tool for preverbal children is the Faces, Legs, Activity, Cry and Consolability (FLACC) tool (Fig. 102.5) [17]. Although designed specifically for assessment of postoperative pain, this tool has utility for preverbal children in other settings. In children with cognitive impairment, parental observation is a workable proxy measure [18]. A revised form of the FLACC is reliable and valid for cognitively impaired children [19].

Ability to function The provision of adequate pain control is essential not only for humane reasons, but also to preserve the patient’s ability to provide self-care and to maintain health. Function may be directly observed based on specific behaviors necessary to participate in HCT (bathing, exercise and mouth care). Pain behaviors may be noted, such as requests for medication, grimacing or refusals to move the affected area of the body [20]. Medication use and side-effects Analgesics reduce the sensation of pain and can improve function, but can also produce unacceptable side-effects. The magnitude of drug consumption and the occurrence of common side-effects should be

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monitored and recorded on the same scale format and on the same assessment sheets as pain scores. Pain diagnostics and therapy selection A clear diagnosis of the cause of a pain is essential in selecting therapies. To be effective, therapies must be aimed at the specific issues which contribute to the patient’s problem. For pain with a large nociceptive contribution from tissue damage, for example OPM, antinociceptive agents (analgesics) are appropriate. For pain with a nociceptive component originating from neural tissue, different agents are appropriate, for example anticonvulsants. For pain complaints that are concurrent with strong emotional/environmental components, treatments aimed at those components, in addition to pharmacologic agents, are appropriate. The first step in intervention is usually education as described below. Pharmacologic therapies should be pursued in a rational pharmacokinetic and pharmacodynamic fashion, for a period of time that will give the therapies a reasonable trial. Treating the patient with difficult pain management problems Pain may be alleviated by a variety of methods that can be initiated and used effectively by the staff caring for the patient. Sometimes, the magnitude of the problem may be beyond abilities of the primary caregivers. In these circumstances, the assistance of personnel with special expertise in pain management (nurses, neurologists, anesthesiologists, psychologists, and psychiatrists) may be helpful. The assistance can be provided in a variety of forms, ranging from occasional consultations to weekly “pain rounds,” to the presence of a formal pain consultation team.

Nonpharmacologic techniques in pain management When HCT patients report pain, analgesic medications are appropriately considered as a first line of treatment. Concurrently, nonpharmacologic methodologies are almost always incorporated into the treatment of the patient, whether or not these interventions are intended [21]. Even with optimal opioid and other analgesic methods, HCT patients with OPM may receive only 50–60% reductions in pain with analgesics [22,23]. Mechanisms of nonpharmacologic techniques The broad goal of nonpharmacologic methods is to close the “gate” on pain messages, or to impede transmission of pain messages and facilitate transmission of nonpain messages. In general, this is done by disrupting afferent pain pathways through the addition of competing sensory inputs, or by modifying descending pathways through changes in thoughts and responses to the sensory messages received. A number of nonpharmacologic methods seem to operate mechanically, modulating transmission of ascending nociceptive messages by sending competing sensory messages from the body surface or limbs. Putting these mechanical methods into operation requires activity. Patients must increase their physical activity, increase thoughts not focused on pain, and achieve an emotional calmness through a sense of control over some aspects of functioning. Lying in bed in a dark, quiet room, without outside stimuli does not permit the activation of these mechanical methods and can lead patients to focus on the dominant sensory input – pain. Staff and family members can assist patients by preparing structured activities and statements that facilitate attention to stimuli other than the pain. These need not be complex. Indeed, patients have limited attention span and often have disruptions in fine motor control. Simple conversa-

tion, walking outside the room or movement in the room, and simple games, all serve to distract the mind from discomfort. Education of patients and families, except in rare instances, provides reassurance and enhances the ability to cope with unfamiliar and uncomfortable situations. Autonomic nervous system activity decreases as anxieties and fears are allayed. Resultant reductions in 5-HT and NE availability help patients as they prepare to guard against worsening symptoms. Adaptive thoughts are those that reassure or calm the patient and relax the autonomic nervous system. Emotions are closely tied to and influenced by thoughts. Cognitivebehavioral methodologies for treating depression and anxiety successfully have been adapted specifically for cancer and HCT patients [21,23–26]. Patients who are more distressed pre transplant report greater pain during transplant [9,23]. Over time, unrelieved pain can engender feelings of helplessness and hopelessness. Introduction of alternative experiences of joy or self-control counters these feelings. When any of us feels more in control, we are better able to manage difficulties, and we feel less discomfort and less distress [8–10,27]. Strategies used in normal medical or nursing practice influence the thoughts patients have about their situation. Information, education, and “reframing” techniques can facilitate coping. Other behaviors that are particularly useful in influencing both the ascending and descending modulatory systems include physical methods such as the use of ice or heat, rest when appropriate, transcutaneous nerve stimulation, and physical positioning (see [28] for details on use of ice, heat, and massage). Information Information that can be helpful to patients includes: 1 Timeframe information. When is something likely to happen? How long will it last? It is helpful to break events into the smallest timeframes possible. 2 Procedural information. What will be done? What comfort measures will be used? 3 Sensory information to prepare patients for specific discomforts. Education Education empowers patients to participate in their medical care and to modify their own behaviors. In addition to facts, patients must have the opportunity for dialogue to fit their own situation. Increasingly, patients and families administer their own pain medication and participate actively in medical treatment. To adhere to treatments, patients must understand them and not be frightened by the potential side-effects. The common barriers to willingness to use available pain treatments are well established [29,30]. Patients, family members, and medical staff frequently fear opioids and are reluctant to identify when treatment is inadequate. Specific areas where HCT patients and their families need education are listed in Table 102.2. Problems with resistance to medications or lack of understanding about pain are routinely prevented by conducting a brief assessment and education session with patients and families prior to the start of treatment. This session serves the additional purpose of identifying those patients who may experience difficulties achieving comfort. Education alone can significantly reduce pain, reduce length of hospitalization, decrease complications, and improve overall outcomes [31–33]. Information and education do not cause anxiety where none exists, and they are not harmful [34,35]. Other cognitive-behavioral strategies Reframing, active distraction, and relaxation are all techniques that can be valuable adjuncts to the use of analgesic medications. To the degree

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Table 102.2 Pain-related education need for hematopoietic cell transplantation (HCT) and their families Question

Usual information offered

Why treat pain?

Pain is harmful to your physical health There is no benefit to proving you can stand pain without treatment, but there are serious negative health effects from suffering unnecessary pain Most HCT patients need strong pain medications (opioids) at some time

What treatments are common? When are treatments used: How often? How much at a time?

How do you know when more is needed? Is addiction a concern?

Do people become tolerant to the drugs so that they stop working if pain gets really bad? What if the medication does not work or makes a person sick?

When pain is constant, scheduled doses provide better relief than waiting until pain is severe to take medication Your doctor will only prescribe safe levels of medication: let your nurse or doctor know if: You are too sleepy during the day Nausea increases when you take medication You have other symptoms that are new You have a new pain Pain medication should be used to keep pain under control, rather than waiting until pain is intolerable HCT patients with no history of overusing drugs or alcohol have no cravings for the medication when the pain stops. Even people who need high doses of medication do not become addicts If you use the medication to keep the pain under control, the medication will not stop working when the pain increases, or if pain continues longer than expected There are many pain treatments. Tell your doctor or nurse if pain is out of control or if a medication makes you feel bad – numerous treatment options are available in these cases

possible, speak about events in positive terms, give patients a sense of control (including participation in clinical decisions), involve patients in activities (if only as an observer at times), and provide a calm and caring environment. Research has shown that more specialized techniques, such as imagery and hypnosis, can be helpful but rarely eliminate the need for analgesic medications [23,25,36]. More widely available, and simple, techniques, such as progressive muscle relaxation, deep breathing, and concentrating on pleasant thoughts and places, also are helpful adjuncts to pain relief [37,38]. Pediatric considerations Most of these methods need little adaptation for use with children. Ageappropriate information is important for children. As with adults, this information should reinforce areas over which the child has some control, such as asking for medication or understanding rewards for self-care activities. Children can plan for their own active distractions and can be engaged in play even when quite ill. Story telling, art projects, and play are effective imagery and distraction methods for children with pain. It is crucially important to be aware that many children will withdraw from socializing when they are in pain. Their quiet demeanor should not be confused with comfort. Socialization and playfulness are often the best indicators of effective pain relief.

Nonopioid pharmacologic management of pain NSAIDs and acetaminophen Many nonopioid analgesics, both over the counter and prescription, are available. Two major groups are NSAIDs and phenacetin derivatives, acetaminophen being the only clinically used drug. All of these analgesics exhibit a “ceiling effect.” Efficacy will increase with increasing doses up to a particular level; dosing beyond that point will not improve analgesia, but will expose the patient to dose-related side-effects.

Other than the distinction that NSAIDs suppress inflammation and acetaminophen has few anti-inflammatory effects, there are few hard data on the comparable efficacy of the various agents as analgesics. Different agents offer varying durations of action, degree of side-effects, and individual patient tolerance. These actions tend to be quite idiosyncratic, so it would be unwise to recommend any specific medication regimen. It is essential to tailor therapy on an individual basis. Nonopioid analgesics are suitable for use in mild-to-moderate nociceptive and inflammatory pain. Alone they provide inadequate analgesia for moderate-to-severe nociceptive pain, but, if used in conjunction with opioids, they may improve the quality of analgesia and demonstrate an “opioid-sparing effect,” reducing the dose of opioid necessary to produce a given level of analgesia and thus reducing the potential for side-effects. Nonopioid analgesics in the management of neuropathic pain currently are under study. Emerging data suggest only a limited role. Pharmacology of NSAIDs and acetaminophen The pharmacologic effect of NSAIDs is to inhibit the enzyme cyclooxygenase, part of the arachidonic acid cascade, reducing production of prostaglandins, one of the algogenic agents released by injured tissue (others being bradykinin, histamine, and 5-hydroxytryptamine) that stimulate or sensitize nociceptors. Consistent with their anti-inflammatory efficacy, the primary effect of NSAIDs classically has been believed to be peripheral. However, prostaglandins also play a role in the CNS transmission of pain [39–42]. Selective cyclo-oxygenase-2 (COX-2) inhibitors have limited antiplatelet activity and have less gastric toxicity than other NSAIDs, but are associated with an increased risk of major cardiovascular events [43]. Acetaminophen produces similar analgesia to NSAIDs but, unlike NSAIDs, has only minor anti-inflammatory effects. Acetaminophen is as potent as aspirin in inhibiting prostaglandin synthesis in the CNS, but has little effect on prostaglandin metabolism peripherally [44–48]. Analgesic effects of acetaminophen and NSAIDs appear to be additive [49–51].

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Table 102.3 Dosing data for acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs) (Adapted and modified from [52].) Drug

Usual dose for adults and children >50 kg body weight

Usual dose for children* and adults† <50 kg body weight

Acetaminophen‡

650 mg/4 h 975 mg/6 h 650 mg/4 h 975 mg/4 h 100 mg TID 1000–1500 mg TID 870 mg/3–4 h 500 mg/12 h 200–400 mg/6–8 h 300–600 mg/6 h 400–600 mg/6 h 25–60 mg/6–8 h 10 mg/4–6 h (to a maximum of 40 mg/day) 30 mg initially, then 15 mg/8 h 650 mg/4 h 50–100 mg/6 h 250–275 mg/6–8 h 275 mg/6–8 h 325–650 mg/3–4 h 200 mg/day 12.5–25 mg/day

10–15 mg/kg/4 h 15–20 mg/kg/4 h (rectal) 10–15 mg/kg/4 h 15–20 mg/kg/4 h (rectal)

Aspirin§ Carboprofen Choline magnesium trisalicylate¶ Choline salicylate¶ Diflunisal储 Etodolac Fenoprofen calcium Ibuprofen Ketoprofen Ketorolac tromethamine oral** Ketorolac tromethamine intravenous**†† Magnesium salicylate Meclofenamate‡‡ Naproxen Naproxen sodium Sodium salicylate Celecoxib§§ Rofecoxib§§

25 mg/ kg TID

10 mg/kg/6–8 h

5 mg/kg/8 h

TID, three times daily. * Only drugs that are Food and Drug Administration approved as an analgesic for use in children are included. † Acetaminophen and NSAID dosages for adults weighing less than 50 kg should be adjusted for weight. ‡ No antiplatelet activity. § The standard against which other NSAIDs are compared. May inhibit platelet aggregation for more than 1 week and may cause bleeding. Aspirin is contraindicated in children with fever or other viral disease because of its association with Reye’s syndrome. ¶ May have minimal antiplatelet activity. 储 Administration with antacids may decrease absorption. ** For short-term use (less than 5 days). †† Has the same gastrointestinal toxicities as oral NSAIDs. ‡‡ Coombs-positive autoimmune hemolytic anemia has been associated with prolonged use. §§ Cyclo-oxygenase-2 inhibitors.

Side-effects of NSAIDs and acetaminophen Nonopioids have a variety of side-effects that are of significance for, and limit their use in, the HCT patient: 1 Interference with platelet function. This lasts for days after stopping the drug in the case of aspirin, or for shorter periods with other NSAIDs. Although COX-2 inhibitors have little, or no, effect on platelet aggregation, they have recently been shown to increase the risk of cardiovascular events [43]. Acetaminophen has virtually no hematologic effects. 2 Gastric irritation and occult upper gastrointestinal bleeding. This is a feature of virtually all NSAIDs, to some degree, even the COX-2 inhibitors. Acetaminophen is benign to the gut. 3 Renal effects. In high dosage, NSAIDs have direct renal toxic effects. The effect of moderate NSAID dose upon already compromised renal function is not clear. Acetaminophen has virtually no renal effects. 4 Hepatic effects. In high dosage, acetaminophen is severely hepatotoxic, and it is generally recommended that patients not ingest more than 4 g daily. The HCT patient may be at higher risk of acetaminophen toxicity due to the high incidence of concurrent or recent hepatotoxic therapy, graft-versus-host disease (GVHD), hepatitis, and poor nutritional status. Use of NSAIDs and acetaminophen in HCT The role of NSAIDs for pain management in the acute phase of HCT is limited by the fact that they are available only in oral form (except

ketorolac), by their hematologic and gastrointestinal side-effects, and by the fact that they may suppress pyrexia. The use of acetaminophen is limited primarily by its potential to suppress pyrexia. Table 102.3 includes dosing data for acetaminophen and the NSAIDs [52]. Antidepressants TCAs have established their efficacy over decades and still are in widespread use. They have analgesic effects independent of their antidepressant effect, providing pain relief in the nondepressed patient, or providing pain relief, without affecting mood, in the depressed patient [53–55]. Moreover, the analgesic effects of many TCAs occur at lower dose and blood levels of drug than those which produce antidepressant effects. Newer selective 5-HT reuptake inhibitors (SSRIs) are commonly used to treat depression, and they too may have analgesic effects. TCAs are thought to relieve pain by altering the 5-HT and NE levels in the descending antinociceptive modulating system. Studies suggest that NE and 5-HT specificity are important in producing analgesia. In humans, concomitant 5-HT and NE enhancement is effective [56–58]. Recent work suggests that NE sites are more active; increased 5-HT levels have intrinsically weak analgesic effects but enhance the analgesic effects at NE sites [59–61].

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Antidepressants for neuropathic pain

Opioid pharmacology

There is a growing, albeit still scant, literature on the use of antidepressants for neuropathic pain; see the section below on neuropathic pain for details.

There is concern about the use of TCAs in the HCT patient, as many TCAs appear to have marrow-suppressant effects [62]. Clinical experience indicates that up to 25% of HCT patients will have significant marrow suppression in the early engraftment period with TCAs. If TCAs are used, it is desirable to begin with a low dose, for example amitriptyline 10 mg, and titrate upwards against side-effects, which often are the limiting factors. It is helpful to have patients take the drug at bedtime so that sedation and other side-effects are less problematic; better sleep may also lead to lower pain levels. All TCAs have long elimination half-lives. The analgesic effects of TCAs can occur within days or weeks, making it necessary to monitor clinical and hematologic function frequently. Little is known about the clinical use of SSRIs in HCT patients, although this class of drugs is not normally associated with bone marrow suppression.

Opioids act at a number of different levels of the CNS to produce analgesia: 1 They inhibit the transmission of nociceptive input in the dorsal horn of the spinal cord (Fig. 102.1). 2 They activate descending inhibitory systems in the basal ganglia (Fig. 102.1) which modulate peripheral nociceptive input at the spinal cord level. 3 They affect the limbic system, altering the emotional response to pain, thereby making it more bearable. Opioids bind primarily to μ and κ receptors in the CNS. Agonists, such as morphine, activate inhibitory pain pathways in both supraspinal and spinal regions. Antagonists, such as naloxone, competitively inhibit the analgesic effects of opioid agonists. Some opioid drugs exhibit both an antagonistic action (at μ receptors) and an agonist action (at κ receptors); these “mixed agonist–antagonists,” such as nalbuphine and butorphanol, have clinical efficacy in HCT patients who experience a wide variety of gastrointestinal syndromes, especially those associated with cramping. Medications with mixed effects can precipitate an abstinence syndrome in people already taking opioid pain relievers so must be used with vigilance.

Opioids in the management of pain

Opioid pharmacokinetics

Drugs that produce analgesia by stimulating morphine-accepting CNS receptors are colloquially referred to as narcotic analgesics. It is more appropriate, however, to refer to these drugs as “opioids,” rather than “narcotics.” Semantically, the latter term associates the drugs with opium, the source of the prototype drug, morphine. “Narcotic” is a legislative, administrative or legal word, which is applied to a number of psychoactive drugs with abuse potential. In the current social and legislative atmosphere, the word “narcotic” should be avoided in medical settings. The avoidance of the use of opioids by patients or health-care professionals, in circumstances where they could be used safely and appropriately, is a grave disservice to those suffering from treatable pain. Opioids are not without liabilities, but the incidence and severity are often overstated. Pain relief is integral to humane treatment and, indeed, has been shown to attenuate disease in animals [63,64].

A schema of opioid pharmacokinetics with various modes of administration is given in Fig. 102.6. As with many drugs, the blood levels of opioids that result can be measured and interpreted to yield a number of “half-lives.” Within individual patients, opioid pharmacokinetics and half-lives are very consistent. This is true to the extent that the blood levels resulting from a single dose of opioid may be used in sophisticated pharmacokinetic analyses to calculate infusion regimens that hold blood levels of opioid very close to a predetermined level [65]. However, opioid pharmacokinetics are highly variable from patient to patient, with half-lives and rates of elimination varying between two- and fourfold.

Antidepressant administration in HCT

Blood level of drug

Opioid pharmacodynamics Opioids exhibit dose-dependent effects in many realms of activity. The analgesic dose–response curve of opioids is not a straight line; they have Effect of drug

IV bolus and CI

IV bolus only IV bolus and PCA

IM injection

Excessive dose CI

Analgesia ± acceptable side-effects

PO Minimum effective analgesic concentration

PCA doses

IV bolus

Analgesia and unacceptable side-effects

Inadequate dose CI

Inadequate analgesia

PO or IM dose start of CI Time in hours

Fig. 102.6 Pharmacokinetics of opioids with various routes of administration. The “therapeutic window” of opioids is the range of blood levels of opioid within which they produce satisfactory pain relief without unacceptable side effects. Following administration by various routes, plasma levels of opioids rise, according to the mode of administration, and then fall, depending on the speed of redistribution or elimination. As a general rule, as plasma levels of opioids rise they will reach the minimum effective analgesic concentration and enter the drug’s therapeutic window. Rises in plasma level of drug beyond this therapeutic window produce some improvements in analgesia, but a large increase in the incidence and magnitude of opioid side-effects. CI, continuous infusion; IM, intramuscular; IV, intravenous; PCA, patient-controlled analgesia; PO, oral.

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a threshold of efficacy and a narrow “therapeutic window,” and among individuals there is a wide range of effective analgesic blood levels of drug. A useful concept is the minimum effective analgesic concentration (MEAC), the blood level of opioid at which a patient reports satisfactory pain relief (Fig. 102.6) [66]. For an individual patient with a specific pain, the MEAC is quite consistent. However, among individual patients with similar pains MEACs vary over a five- to sixfold range. For example, the mean MEAC of morphine is 16 (range 6–33) ng/mL, and the mean MEAC of meperidine 455 (range 94–754) ng/mL [67]. The variability of individual MEACs, in conjunction with the individually variable drug half-lives and speeds of elimination, results in a wide range of opioid doses necessary to maintain analgesic blood levels. The practical manifestation is that opioid doses must be individualized, based on the effects of the drug, which in practice are derived from patient report of pain relief and occurrence of side-effects. Opioid side-effects Opioids have a wide variety of side-effects, many of which will abate or disappear with exposure over a several-day period. Gastrointestinal effects Opioids can induce nausea and vomiting in 10–40% of patients through several distinct mechanisms, including direct stimulation of the chemotactic trigger zone, sensitization of the vestibular apparatus (patients with a history of motion sickness are especially vulnerable), and delayed gastric emptying [68]. Nausea and vomiting attributable to opioids usually occur at the onset of therapy or with escalation of dose. These symptoms, while common, often subside quickly with continued opioid exposure and may be relieved by changing opioid or using smaller doses of drug at more frequent intervals. The choice of antiemetic for symptom control should be tailored based on the presumed underlying mechanism of opioid induced nausea. Nausea and vomiting due to delayed gut motility (clinically presenting with postprandial nausea and vomiting or early satiety) may improve with promotility agents such as metoclopramide. Nausea and vomiting due to stimulation of the CTZ may be relieved with dopamine or 5-HT antagonists, and symptoms exacerbated by movement may respond to diphenhydramine or scopolamine [68]. Constipation, while a common side-effect of opioids in general, is manifested differentially in the HCT patient due to the predilection for diarrhea from a variety of causes including mucotoxic conditioning regimens, infections or GVHD. Prophylactic stool softeners and stimulants are therefore not routinely administered, and treatment is symptom based.

patient is broad and includes CNS active drugs (antiemetics, anxiolytics, antihistamines, and cyclosporine), metabolic derangements (renal dysfunction and electrolyte disturbances), fatigue (sleep deprivation and disturbed biorhythms), and the prodrome of sepsis. In patients with persistent opioid-induced sedation, small doses of methylphenidate (5– 10 mg two or three times a day), donepezil or modafinil can ameliorate symptoms [69]. Euphoria Opioid euphoria is rare in the HCT patient. Dysphoria If dysphoria occurs, it is usually early during opioid exposure and progressively declines with more prolonged exposure. Delirium Patients undergoing HCT can develop delirium (as defined by transiently impaired cognitive, emotional or behavioral functions) for a variety of reasons. Because of this, a broad investigation into potential metabolic, infectious, and CNS etiologies should be undertaken. Opioids are potential contributors to delirium with the suggestion that opioid naïveté, comorbid illness, higher doses of therapy, and parenteral administration confer greater risk [69,70]. Cases of opioid-induced delirium which do not upset or scare the patient can be treated with reassurance alone. In the event of an agitated delirium, haloperidol or rotation of analgesic therapy to another opioid may be effective [71,72]. Reduction of opioid dose by 25% can also improve symptoms [68]. However, it is important to note that, in other patient populations, poorly controlled pain confers a greater risk of delirium than the administration of appropriate parenteral opioids [73,74]. Respiratory depression When monitored by sensitive indices, all opioids blunt the respiratory drive in a dose-dependent fashion. In the dose range that produces analgesia, respiratory depression (defined as an arterial partial pressure of carbon dioxide > 5.6 kPa) is uncommon. Patients with pre-existing respiratory disease, those with sleep apnea-like symptoms, and those receiving other CNS-active drugs, especially benzodiazepines, are most at risk of respiratory depression. The degree of respiratory depression parallels the slowing respiratory rate and degree of sedation; these signs can be monitored if respiratory depression is a concern. Opioid tolerance and dependence Tolerance

Genitourinary effects Increased tone and contractility of the ureters and increased detrusor tone may lead to urinary retention. Pruritus Pruritus is a well documented yet poorly understood occasional sideeffect of opioid therapy. Antihistamines, rotation of opioids, and opioid dose reduction are common treatment strategies. In addition, mixed opioid agonists–antagonists can reverse itching, but this may reduce analgesic effects as well [68]. Sedation Sedation is an almost universal side-effect of opioids and usually occurs at onset of therapy or with dose escalation. Opioid induced sedation typically resolves within 72 hours of initiating therapy. It is important to remember that the differential diagnosis of sedation in the HCT

Tolerance is the progressively decreasing effect (analgesic or other effects) of a given dose of drug when that dose is administered repetitively. Tolerance may be seen when a patient who has been achieving satisfactory analgesia with a given dose of drug progressively fails to achieve analgesia without evidence of an increase in nociception. If it occurs in the HCT patient, tolerance may be overcome by increasing the dose of drug or changing to a different opioid. Tolerance is a drugrelated effect only and has little or no relation to addiction. Incomplete crosstolerance When changing from one opioid to another, it often takes less than an equianalgesic dose of the second opioid (Table 102.4). This phenomenon is observed frequently, but the mechanisms are not completely understood. If there is a need to switch opioid medications, a good rule of thumb is to give approximately two-thirds of an equianalgesic dose and provide liberal amounts of breakthrough medication until a new

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Table 102.4 Dose equivalents and starting doses for opioid analgesics (Adapted and modified from [52].) Usual starting dose Approximate equianalgesic dose (mg)

>50 kg

>50 kg

<50 kg

<50 kg

Drug

Oral

Parenteral

Oral

Parenteral

Oral

Parenteral

Morphine Hydromorphone Oxycodone Hydrocodone Methadone Levorphanol Meperidine Codeine Sufentanil Fentanyl

30 7–15 30 30 10–20 2 100 200 N/A 0.2 (transmucosal)

10 1.5–3.5 N/A N/A 10 1 100 130 0.01 0.1

30 mg/3–4 h 6 mg/3–4 h 10 mg/3–4 h 10 mg/3–4 h 10–20 mg/6–8 h 4 mg/6–8 h N/R 60 mg/3–4 h N/A 200–400 μg/h

10 mg/3–4 h 1.5 mg/3–4 h N/A N/A 10 mg/6–8 h 2 mg/6–8 h 100 mg/2–3 h 60 mg/2–3 h 10 μg/10 min 50 μg/10 min

0.3 mg/kg/3–4 h 0.06 mg/kg/3–4 h 0.2 mg/kg/3–4 h 0.3 mg/kg/3–4 h 0.1–0.2 mg/kg/6–8 h 0.04 mg/kg/6–8 h N/R 0.5–1 mg/kg/6–8 h N/A 2–4 μg/kg/h

0.1 mg/kg/3–4 h 0.015 mg/kg/3–4 h N/A N/A 0.1 mg/kg/6–8 h 0.02 mg/kg/6–8 h 0.75 mg/kg/2–3 h N/R 0.1 μg/kg/10 min 1–2 μg/kg/10 min

N/A, not available; N/R, not recommended.

equilibrium is established. Although most opioid-related side-effects dissipate spontaneously after a few days, a change can provide analgesia at “lower” doses of the second drug, perhaps with a lower side-effect profile. Physical dependence Physical dependence is characterized by the occurrence of an abstinence syndrome (yawning, lacrimation, sneezing, agitation, tremors, fever, tachycardia, and other signs of sympathetic arousal) if the opioid abruptly is withdrawn. Like tolerance, dependence is a drug characteristic, not a patient characteristic, and is a separate entity from addiction. Few patients who are opioid dependent are addicts (see below). Abstinence syndromes can be avoided by slow reduction of opioid dose (10–20% every day or every other day) or by using adrenergic α2 agonists such as clonidine, which mask some of the peripheral adrenergic symptoms [52]. Using the strategies described below in the section on opioid tapering, it is rare to get a true abstinence syndrome when opioids are used with HCT patients. Psychologic dependence (addiction) This complication is characterized by an abnormal pattern of drug use; of drug craving for effects other than pain relief; by an overwhelming involvement with procuring and consuming the drug, even when such activity is contrary to the individual’s best interests; and with a tendency to relapse into drug use after tapering off the drug. Addiction is the conjunction of genetic, psychologic, social, and cultural factors that affect an individual. Unlike tolerance and physical dependence, psychologic dependence is a patient issue, not a drug issue. Patients with no previous history of substance abuse who receive opioids therapeutically have an extremely low incidence of iatrogenic addiction, of the order of 1 in 2000–4000 [75]. Therefore, in patients with no history of substance abuse, fear of psychologic dependence should not be a concern. Opioids commonly used in the HCT patient Table 102.4 includes a list of opioids, their approximate equianalgesic doses, and dosage intervals for oral or intravenous use. The choice of agent should be limited to opioid agonists. The following are commonly

used opioids in the HCT patient and some of their specific complications. Morphine The prototypical opioid agonist morphine can be given by any route but has low oral bioavailability (Table 102.4). Oral duration of effect is 3–5 hours, which can be extended to 6–12 hours by the use of sustainedrelease preparations. Morphine is conjugated in the liver to two major metabolites, 3-glucuronide and 6-glucuronide, which may produce unintended side-effects (sedation and confusion). Glucuronidation is a highcapacity conjugative pathway in the liver, so morphine is relatively safe to use in patients with moderate degrees of hepatic failure. Since both glucuronides are excreted by the kidneys, morphine should be used cautiously in patients with any degree of renal impairment. On initial use, morphine has a 15–25% incidence of pruritus, or nausea and vomiting, which often diminish with use over a few days. Meperidine (Demerol) This opioid agonist has about one-tenth of the potency of morphine. Meperidine is metabolized to normeperidine, a renally excreted metabolite, which has pronounced excitatory and convulsive effects. In patients with normal renal function, normeperidine levels do not become problematic until relatively large doses of the parent drug have been administered over a prolonged period of time, for example 800–900 mg/day over about 5–7 days. However, if used as an analgesic, this meperidine dose could be met or exceeded in the HCT patient. Thus, patients with normal renal function should be restricted to short-term, low-dose use, for example for chills and premedication before platelet transfusions. In patients with abnormal renal function, meperidine should be avoided, or its use greatly restricted. Hydromorphone (Dilaudid) This agonist has a spectrum of effects similar to morphine. Hydromorphone is thought to be five to eight times more potent than morphine, although data from the HCT population support a dose equivalency of about 3 [52,76]. It is generally believed to have fewer side-effects than morphine, but this belief is not substantiated by double-blind studies [77]. It is metabolized to a number of renally excreted metabolites, which appear to be pharmacologically inert. Hence hydromorphone is a

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good choice of opioid in patients with impaired renal function or those with unacceptable side-effects with morphine. Fentanyl This is a classic agonist with a spectrum of effects similar to morphine. It is 100 times more potent than morphine and has a faster onset of effect. In short-term use, it has a relatively short duration of effect, but in longer-term use, its duration of effect is similar to hydromorphone. Fentanyl is metabolized to a number of pharmacologically inert metabolites, and is a good choice of opioid in a patient with impaired renal function or those with unacceptable side-effects with morphine or hydromorphone. Fentanyl predominantly is administered intravenously. It is also available as an oral transmucosal product that is useful in some HCT patients. Transdermal fentanyl (Duragesic) is a useful alternative to intravenous opioids for patients needing long-term stable dosing, in the absence of fever and with intact skin. It should be noted that the patch takes 16–24 hours to achieve stable plasma levels of drug, and conversely when the patch is removed, it takes 16–24 hours for plasma levels of drug to fall. Butorphanol This is a mixed agonist–antagonist with marked κ agonist effect and moderate μ antagonist effect. It is a useful drug for HCT patients in treating the pain from GVHD of the gut. Compared with μ agonists, it is believed to impair visceral nociceptive transmission more and yet cause less gut stasis. Butorphanol may be given intravenously or by nasal inhalation. It is not recommended for moderate-to-severe pain syndromes because it has a relatively low analgesic ceiling and can precipitate abstinence syndrome if the patient is on other opioids [52]. Nalbuphine This is another mixed κ-agonist–μ-antagonist opioid. It effectively treats opioid-induced pruritus without reversing analgesia. Additionally, clinical experience has demonstrated efficacy in treating pruritus associated with cholestasis [78,79]. Tramadol This is a centrally acting synthetic analgesic. It has weak opioid activity and inhibits reuptake of both 5-HT and NE. The side-effect profile is similar to that of opioids; respiratory depression may occur with overdose, and an abstinence syndrome can occur with abrupt withdrawal. Opioid use in clinical practice The basic precept in providing analgesia with opioids is to raise and maintain the patient’s plasma level of opioid above the patient’s MEAC and within the therapeutic window. Consulting Fig. 102.6 will help with understanding the following paragraphs. Oral administration Opioids are readily absorbed by the gut but undergo extensive first-pass metabolism by the liver, so that to produce a given blood level, the oral dose may be three to six times the parenteral dose (Table 102.4). After oral administration, blood levels rise slowly, reach the effective range in 30–45 minutes, and stay within the therapeutic window for about 3–4 hours; hence 3–4-hourly dosing is necessary, except with methadone, which is effective taken every 6 hours. Slow-release preparations extend this dosing schedule to 8–12 hours. Rectal administration This route should be avoided in the HCT patient because of the bacterial shower produced by inserting a suppository.

Intramuscular and subcutaneous opioids Intramuscular and subcutaneous opioids rarely are used in the HCT population as frequent doses may be necessary, and the mixture of intramuscular/subcutaneous injections and thrombocytopenia results in hematoma formation. Intravenous bolus Intravenous bolus dosing rapidly raises the plasma level of a drug. However, the drug is then rapidly redistributed and metabolized, and blood levels fall quickly. Consequently, therapeutic levels, above the MEAC, are maintained for only a short period of time, less than 30 minutes. Intravenous bolus administration is widely used in the HCT population as most patients have indwelling venous access. The drug reliably and rapidly reaches its targets. Fixed rate (continuous) infusion Infusions of opioid take a long time to raise plasma levels to the effective range, leaving the patient in pain. Moreover, to choose an infusion rate that will maintain an effective plasma level of opioid, the rate at which the individual patient metabolizes and excretes the opioid must be known. Chosen infusion rates can be excessive, leading to plasma levels of drug rising into the toxic range, or inadequate, leading to plasma levels falling into the poor analgesia range (Fig. 102.6). Intravenous bolus followed by a fixed rate infusion An intravenous bolus will rapidly raise plasma levels of drug to therapeutic levels which then can be sustained with continuous infusion. Patient report will determine the initial effective bolus (loading) dose. An hourly infusion rate equal to approximately 20% of the effective loading dose usually provides good relief. Intensive nurse monitoring is necessary to guarantee relief and avoid overdose. Patient-controlled analgesia In the early 1970s, the revolutionary concept that patients might be capable of acting as their own “sensor” of pain relief and analgesic need was advanced. The concept held that if patients were given as-needed access to opioids, they would titrate their dose to keep plasma concentrations at analgesic levels, close to the MEAC, thereby achieving satisfactory analgesia, and would not over- or underuse the opioid, even when the drug was taken over long periods of time. This concept reached clinical reality with the advent of computerized pumps which enable patients to self-administer small doses of opioids at frequent intervals, the clinical technique of patient-controlled analgesia (PCA). The plasma levels of drug, which result from a loading dose plus PCA, are shown in Fig.102.6. By individualizing the dosage of opioid, allowing the patient to titrate to effect, PCA circumvents the problems posed by other modes of administration, that is, variations in MEAC, pharmacokinetic differences, and changes in degree of nociception. Virtually all adult patients have the cognitive and discriminative ability to use PCA. PCA can be used reliably by children down to at least the age of 12 years [80], and may even be safely and successfully used in some children down to the age of 4 or 5 [81]. Current clinical practice is to offer PCA to younger children, provided they are able to grasp the concept and use the technique. If a child can play a video game, he or she probably can use PCA. In HCT patients, the classical instance of PCA use is for mucositis pain. As the mucositis pain becomes worse, patients use more drug, and then, as their mouth and throat heal and the pain lessens, they naturally taper their opioid dose (Fig. 102.7). In randomized controlled clinical trials, testing patients with PCA versus continuous infusions of opioid for mucositis pain, patients using PCA tend to have lower pain scores

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CI PCA

Morphine (mcg/kg per hour)

90 80

1581

to PCA [83,84]. Nonetheless, there is a strong body of opinion that the addition of an infusion to a PCA improves the quality of analgesia in HCT patients when pain persists over days, and can help with sleep.

70

Opioid tapering

60 50 40 30 20 10 0 0

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19

Study day

Fig. 102.7 Daily mean morphine dose for adolescents with mucositis pain during hematopoietic cell transplantation. Morphine was delivered by physician-prescribed continuous infusion (CI) or patient-controlled analgesia (PCA). As the pain worsened, the PCA patients delivered more drug, and as the mucositis healed, they automatically tapered their dosage. Note also the lesser amount of drug used by the PCA patients, even though the pain scores were very similar in the two groups. Results have been the same in adults [22]. (Reproduced from [80], with permission.)

and use less drug over shorter periods of time, hence a tendency to have fewer side-effects [82]. PCA pumps have various parameters that must be defined for safe and effective use. When used effectively, PCA can decrease nursing time, provide more timely pain relief, and involve the patient importantly in symptom relief. PCA bolus dose. Initial bolus sizes are morphine 0.5–2 mg (10–30 μg/ kg) in adults and 10–20 μg/kg in children, or equivalent doses of other drugs. As nociception and pain worsen, bolus size should be increased; if nociception and pain become less, dose size should be decreased. Experience has shown that the optimal bolus size is one that permits the patient’s 24-hour drug need, obtained from the nursing records or the PCA pump memory, delivered in 25–40 doses. PCA lock-out interval. After a bolus of opioid has been delivered, the pump will not deliver another dose for this set interval, usually 6–10 minutes. This is a safety feature as it prevents the administration of a further dose of drug until after the patient has had the opportunity to experience the effects (analgesia and sedation) of the previous dose. PCA plus fixed infusion. PCA alone has some disadvantages. If a patient sleeps or is disconnected from the intravenous infusion and does not self-administer opioid, blood levels of the drug will fall. The patient may then have difficulty in self-administering sufficient drug to raise the blood level of opioid back into the analgesic range. To circumvent this problem, PCA pumps have a constant infusion option. This option should not be used until a patient has established his or her individual dose requirement, that is, by opioid use via PCA alone. For example, a patient may have used 84 mg of morphine in the previous 24 hours. This averages 3.5 mg morphine per hour. It is advised that the infusion be set at not greater than 30–50% of the expected opioid need with some day/night variability as needed – in this case 1.0–1.8 mg/hour. In addition to helping with sleep, the addition of an infusion means that the blood levels of opioid fall more slowly after each PCA dose, giving the patient a longer period between the need for repeat PCA doses. Studies in postoperative patients using PCA for 24–48 hours have not shown a marked improvement in analgesia by the addition of an infusion

Some HCT patients have pain problems of extended duration, which necessitate opioid therapy for long periods of time. Usually, as the pain problem diminishes, patients’ PCA or oral self-administered opioid dose is gradually reduced and ultimately eliminated. However, with relatively long-term (10–14 days) use, a patient may be physically dependent on the opioid; that is, abrupt cessation of opioid intake may precipitate an abstinence syndrome. In the HCT patient, these symptoms can be confused with stress reactions or can be attributed to infections or symptoms of GVHD. It is important that both physician and patient understand that these symptoms are entirely unrelated to addiction (see the sections on physical dependence and psychologic dependence) [52], and that even if an abstinence syndrome does occur, it is rarely physically dangerous and does not have sinister long-term implications, although it is most unpleasant. The onset of these symptoms can occur within 6–12 hours after an opioid dose reduction, and peak at 24–72 hours. For both humanitarian and treatment success reasons, any tapering process should be designed to minimize the possibility of an abstinence syndrome, and if an abstinence syndrome occurs, it should be treated by using small doses of opioid until symptoms dissipate. Practical realities of tapering All patients should be watched closely for abstinence symptoms since they may not be aware that their experiences are related to changes in opioid dose. If an abstinence syndrome does occur, an intravenous infusion or PCA dosing schedule that eliminates symptoms is the first intervention. This dose is then reduced by about 10–20% per day, assuming the pain or abstinence syndrome does not reappear [85]. For ambulatory patients, continuous monitoring for symptoms by medical staff is not possible, and therefore a slower taper is advised. One simple method is the “pill count.” The patient is prescribed the usual opioid in a tablet preparation, and is instructed to take it on a time-contingent basis (consistent with the duration of effect of the opioid) rather than as needed. Reductions of 10–20% of the dose are successively made until the patient is taking no drug. Immediate-release opioid preparations usually provide greater dosing flexibility than do sustained-release preparations which cannot be divided. Peripheral adrenergic symptoms during the taper can be avoided or masked by using adrenergic α2 agonists such as clonidine [52]. For more complex patients, such as those who are overly focused on the dose or number of tablets, an alternative to the pill count method is to use “pain cocktail” [85,86]. In this strategy, the patient’s mean daily intake of opioid is combined into a fixed volume of a taste-masked liquid vehicle, for example cherry syrup, and the volume is split into regularly scheduled doses appropriate to the expected duration of action of the drug. When reducing the opioid content, the total daily volume stays stable, but the milligram opioid content is reduced 10–20%.

Clinical pain management in the HCT patient Pre transplant Approaching and entering a transplantation program is an exceedingly stressful experience for patients, and may result in more vulnerability than usual to a variety of stresses, including pain. This is also a time of peak motivation to understand and participate actively in anything that

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will improve treatment comfort or success. Patients and families should be assured that symptom management, including pain, will be treated effectively, safely, and in a timely manner. In particular, education about the appropriate and safe use of opioids in HCT is important so that fears of addiction may be dispelled prior to the need for these medications. The pretransplant period is the ideal time to learn, practice, and perfect nonpharmacologic pain-relieving skills. As the time of the transplant gets closer, analgesic drugs which may be contraindicated during the transplant, for example TCAs, SSRIs, NSAIDs, should be stopped, and safer substitutes, for example opioids or shorter-acting anxiolytics such as clonazepam, begun. In hospital, peritransplant period This is the most vulnerable time for the patient. Pain management measures used are those least likely to impede engraftment or to interfere with the patient’s ability to function. Analgesic drugs are usually given intravenously, as patients are frequently unable to take oral medication, and intramuscular injections are contraindicated. It is more difficult to evaluate and treat patients who use nonprescription psychoactive drugs. In these individuals, who are likely opioid tolerant, analgesic use is usually greater than normal, and the clinician evaluating the success of therapy may have to rely on external observable criteria rather than the usual patient report criteria. Contracts may need to be set to outline explicit behaviors that must be adhered to by the patient, to ensure that drug delivery is maintained or increased. Expected behaviors can include all the self-care activities necessary, along with appropriate interactions with staff in relation to anger control, manipulations, inappropriate drug-seeking activities, and number of requests for medications. When the nociceptive phase of the transplant is over, it may be necessary to impose an externally derived, relatively rigid opioid tapering schedule, rather than allowing the patient to selftaper as is normally done. Pseudoaddiction Care must be taken not to underdose patients who have previous, licit or illicit, experience with opioids, or who simply have high opioid requirements due to idiosyncrasies associated with the drugs. Persistent underdosing of patients leaves them in pain, and often sets up a nasty cycle in which the patient continually asks for medication and the staff, frustrated, begin to deny medication or label the patient as a “drugseeker.” These behaviors can lead to accusations of addiction when, in fact, it is the failure to provide adequate pain relief that is at fault.

patients [9,87,88]. Onset of oropharyngeal discomfort typically occurs 5–7 days after the initiation of chemoradiotherapy, and peaks at day 9–13 of HCT. Resolution of symptoms usually occurs 3–4 weeks after transplantation and parallels neutrophil recovery. The duration of analgesic need may range from a few days to several weeks. Biologic predictors of mucositis-related pain correlate with the degree of tissue damage and include type and intensity of chemoradiotherapy, the use of methotrexate for GVHD prophylaxis, age, clinical grade of mucositis, and source of stem cells. Psychosocial predictors of mucositis pain have also been identified and include pretransplant HCT-related distress, type of pain coping strategy employed, depression, and solitary living status [9,89]. Recent approaches to combat mucositis have focused on prevention. There is a growing experience with reduced-intensity or alternative conditioning regimens that are less mucotoxic. In addition, alternatives to standard methotrexate for GVHD prophylaxis are under investigation. A recent systematic review found convincing evidence that ice chips, amifostine, hydrolytic enzymes, antibiotic paste, calcium phosphate rinses, and good oral care protocols are effective in reducing the severity of mucositis [90]. Keratinocyte growth factors have been shown to be safe and to decrease the duration of mucositis pain in the autologous transplant setting [91]. Studies are ongoing to assess the safety and efficacy of this class of agents in the allogeneic setting. Once OPM pain has been established, topical analgesics such as viscous lidocaine, dyclone, and benzydamine can offer effective pain control [90,92]. These agents should be swished and expectorated several times daily, and are often used in conjunction with mucosal coating agents such as diphenhydramine. As the severity of OPM increases, these topical agents become inadequate, and systemic opioids are often required. Oral opioids are of limited use due to the high frequency of concurrent odynophagia, nausea/vomiting, and poor gastrointestinal absorption. The systemic management of mucositis pain is therefore usually accomplished with parenteral opioids. When frequent intravenous doses are required, the use of PCA is appropriate in adults and children who can manage it. In patients who can not manage PCA, continuous infusion plus bolus dosing when needed is often required. Specifics on PCA use for mucositis pain are described above in the sections on “Opioid use in clinical practice” and “Opioid tapering.” Despite current preventive strategies and systemic opioid therapy, many patients still report ongoing mucositis pain [89]. Psychologic techniques (such as relaxation and cognitive-behavioral training) may be helpful in conjunction with systemic opioids [9]. Further investigations into preventive and nonopioid alternative pain management strategies are needed.

Patients with prior history of substance abuse

Conditioning regimen-related cutaneous skin toxicity

Patients who have a history of substance abuse (tobacco, alcohol, illicit drugs, opioids or sedatives) may present management problems in the peritransplant period, especially with opioid tapering. It is essential to identify these at-risk patients prior to transplant, provide adequate analgesia (see pseudoaddiction above), set clear guidelines for acceptable behavior, and be cognizant of increased opioid requirements. Opioid withdrawal may be made easier by providing time contingent medications, rather than “as-needed” dosing, removing a layer of control from the patient. The following are the most common pain problems and their management approaches.

In HCT, this complication occurs predominantly in the hands, feet, groin, axillae, and genitalia. The magnitude of skin disruption varies from mild erythema to frank lesions similar to second-degree burns which usually heal 6–10 days post engraftment. The skin lesions may require treatment with silver-containing creams or biologic dressings such as pig skin. For mild toxicity, cold packs often supply adequate pain control. For more severe pain, opioids, as used for OPM, are appropriate.

Oropharyngeal mucositis The conditioning regimens and GVHD prophylaxis used in HCT can result in OPM requiring systemic opioid analgesia in 60–100% of

Hepatic capsule distention Sinusoidal obstructive syndrome (SOS) of the liver may cause liver engorgement with capsular distention. Intravenous or PCA opioids as for OPM are appropriate. Patients with SOS will tolerate and metabolize opioids normally until either the SOS becomes severe (bilirubin > 20 mg/dL), hepatic failure ensues or renal dysfunction occurs. It is

Pain Management

essential to remember that the high-capacity hepatic degradation pathways may overwhelm a mildly dysfunctional kidney, leading to accumulation of degradation products, particularly important with meperidine and morphine. Pain associated with hematopoietic growth factors The recombinant granulocyte colony-stimulating factor filgrastim is commonly used to hasten neutrophil recovery and to mobilize donor stem cells. Headaches and bone pain (usually in the pelvic and long bones) are frequently reported and are dose dependent. Bone pain can occur within hours of filgrastim administration and usually resolves within a few days of drug cessation. Pain is typically well controlled with acetaminophen or NSAIDs, although low-dose oral opioids are occasionally required. In published series, between 60% and 100% of donors undergoing stem cell mobilization with filgrastim report bone pain severe enough to require some type of analgesia. While rare, bone pain can occasionally be severe enough that hospitalization is required [93]. A growing experience with the longer-acting pegfilgrastim shows comparable incidences of bone pain [94,95]. Outpatients receiving filgrastim or pegfilgrastim should have access to ample “as-needed” doses of a rapid-onset oral opioid such as morphine, hydromorphone, oxycodone or hydrocodone, often in combination with acetaminophen.

Post-engraftment Graft-versus-host-disease Patients who receive allogeneic transplants are at risk for GVHD, despite prophylaxis with immunosuppressive drugs and steroids. The following are commonly occurring sites of GVHD that tend to have accompanying pain. Oral. Oral GVHD can overlap with the signs and symptoms of OPM. Symptom-based treatment includes good oral hygiene, topical analgesics, and oral or systemic opioids titrated to effect. Skin. The pain of skin GVHD responds to opioids. Skin GVHD also associated with pruritis can be effectively treated with oral hydroxyzine or nalbuphine, as pruritus may be mediated through systems which are opioid sensitive [78,96]. Gastrointestinal tract. Gut GVHD may cause pain for a variety of reasons. Bowel wall inflammation may cause pain at rest and with movement. Vigorous peristalsis of inflamed gut may cause episodic cramping pain that is not responsive to opioids. Indeed, opioids may make the pain worse by contributing to bowel dysmotility. Clinical experience strongly suggests that κ agonist opioids such as butorphanol produce better pain relief and less gut atony than μ agonist opioids. There may be a place for the use of invasive analgesic techniques such as epidural analgesia, which provide potent analgesia with low-dose drug use, in this circumstance. Chronic GVHD is a difficult pain management problem, and long-term opioids or one of the invasive analgesic techniques should be considered. In circumstances in which opioids are not very helpful, and pain is unpredictable and brief, as in chronic GVHD gut cramping, patients can use an image that counteracts the physical sensation, combined with deep breathing. For example, a patient with a cramping pain might imagine a tight fist that progressively relaxes as he blows out each slow, deep breath. Or, in the case of burning pain, the patient might imagine breathing arctic air through the hot body part, and watching as the red turns to orange and cools to green or blue. Both adults and children have used this method to shorten the duration of pain.

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Pain associated with steroid tapering This usually occurs at the time that steroids are being tapered slowly. Typically, the pain is severe, of rapid onset and short duration, and usually occurs in the distal femur and proximal tibia in early morning hours. Usually this pain is responsive to opioids – but doses need to be large enough to abort the pain episode. Pain due to herpes zoster The acute episode is painful in some 75% of cases. Pain may persist beyond the acute episode. Pain which persists beyond 1 month may be due to peripheral nerve damage and is therefore termed postherpetic neuralgia (PHN). In the acute episode, nonopioid agents and opioid analgesics are appropriate and effective. For PHN, nonopioid and opioid analgesics are relatively ineffectual. With PHN, three types of pain, which respond to differing therapeutic modalities, occur: 1 Severe skin sensitivity with pain to the slightest touch (allodynia). Try Eutectic Mixture of Local Anesthetics (Emla cream, a 2.4% lidocaine/2.5% prilocaine mixture applied topically) or newer preparations such as lidocaine patches [97]. Effective systemic drugs include anticonvulsants and antidepressants (see the section below on neuropathic pain). 2 Episodic shooting pain best thought of as an epileptiform discharge from damaged dorsal horn neurons (see the section below on neuropathic pain). 3 A background burning pain, which is the most common form of PHN. This often responds well to TCAs [57,58] and occasionally to opioids. Prevention of PHN has been the subject of various studies. The early suggestion that corticosteroids might prevent PHN [98] has not been confirmed [99]. More recent studies strongly suggest that early use of antiviral medications may reduce the incidence and duration of PHN [100,101]. Even more exciting is gathering evidence that early sympathetic blockade (e.g. by using epidural analgesia) shortens the acute phase and reduces the incidence, duration, and severity of PHN [102,103]; combined with antiviral therapy, this would now appear to be the intervention of choice, especially in patients over 50 years old. Neuropathic pain Damage to nerves may cause neuropathic pain. Such pain typically is out of proportion to the stimulus intensity; it can involve myelinated Aδ nerve fibers or smaller, unmyelinated C fibers. Damage to the latter in particular may cause “sympathetically mediated” pain syndromes. These syndromes may be amenable to neural blockade, while nonsympathetically mediated pain syndromes usually are not. Neuropathic pain often is associated with sensory or autonomic nervous system dysfunction. Patients tend to use words such as “burning,” “raw,” “gnawing,” “aching” or “tightness” to describe the discomfort of neuropathic conditions. Shooting and shock-like pain syndromes also can occur. Common causes include exposure to chemotherapeutic agents (particularly vinca alkaloids), calcineurin inhibitors, damage to large neural plexi by radiation or surgery, spinal cord compression, or nerve root infiltration in conditions such as herpes zoster. The incidence of neuropathic pain in HCT patients is unknown. Mechanisms of the various syndromes still are poorly understood; consequently, there are many treatment options, and it is difficult to predict efficacy. In the last few years, however, coherent approaches to the management of neuropathic pain have emerged, based on our current understanding of NE and 5-HT neurochemistry [59,61,104]. Antidepressants for neuropathic pain Many HCT patients suffer from neuropathic pain. Even though there have been no clinical trials of TCA or SSRI analgesic efficacy in HCT

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patients, it probably is reasonable to extrapolate to the HCT population data from trials in other conditions, usually diabetic neuropathy or PHN models [105]. TCAs have been shown to be variably effective in some patients. Compared with placebo, amitriptyline, imipramine, and desipramine produce benefits in neuropathic pain when administered in either low dose or high dose [54,56,58,106,107]. Benefits appear after about 2 weeks; a frank correlation between blood level of drug and analgesic efficacy has not been established. Amitriptyline is probably the drug of choice, but desipramine is a good alternative with similar efficacy and possibly fewer side-effects [57,58]. Although it has been difficult to show that SSRIs have clear analgesic efficacy in neuropathic pain syndromes, there is emerging evidence that they may be beneficial, in particular that SSRIs may enhance analgesia from TCAs [59,60]. An even newer antidepressant, duloxetine, which is a 5-HT and NE reuptake inhibitor, is less well studied but also shows promise in refractory neuropathic pain syndromes [108,109]. Anticonvulsants for neuropathic pain A wide variety of anticonvulsants, including carbamazepine, phenytoin, valproic acid, and clonazepam, have been used to treat neuropathic pain, with some success [105,110]. The more recently developed anticonvulsants gabapentin and pregabalin, however, appear to be more effective, safer, and better tolerated by cancer patients, especially in those patients at risk for developing PHN [111–116]. Topical preparations for neuropathic pain Topical application of lidocaine patches [117,118] and capsaicin [119,120] have been used successfully to control peripheral neuropathic pain. Topical capsaicin has been used to treat mucocutaneous neuropathic pain, such as oral mucositis [121], although it is recommended only for early or mild cases because capsaicin can cause severe pain by releasing substance P. Opioid treatment of neuropathic pain The historical dogma that opioids are ineffective in treatment of neuropathic pain has been shown to be false [122–124]. Neuropathic pain does, indeed, respond to opioid treatment, but may require higher doses than in the treatment of nociceptive pain. Further, adjunctive treatments may enhance and complement analgesia provided by opioids in the setting of neuropathic cancer pain [125,126]. It is often helpful to treat neuropathic pain with opioids until the effect plateaus or unacceptable side-effects emerge. Pain due to diagnostic or therapeutic maneuvers Postlumbar puncture headache Postlumbar puncture headache (PLPHA) is believed to be caused by a continuing leak of cerebrospinal fluid through the hole in the dura created by the lumbar puncture. This leak causes a low-pressure headache or traction on the base of brain structures as they migrate distally through the foramen magnum. Lumbar punctures should be performed with the smallest needle possible, certainly no bigger than 22 G, and preferably 25 G. With a 22 G needle, the incidence of PLPHA is 20–30% in 20–30-year-olds but only 2–3% in those over 60 years old. A classic PLPHA is an occiput to frontal headache that occurs after a lumbar puncture and that is exquisitely sensitive to positional changes. It may be associated with photophobia, nausea, vomiting, tinnitus, diplopia, and changes in auditory and visual acuity. Not all headaches post lumbar puncture are PLPHAs. Patients with a history of headaches are more likely to get a headache post lumbar puncture, but whether these are PLPHAs is often debatable.

Prophylactic measures that minimize the incidence of PLPHA include: • using small needles, preferably those with noncutting “pencil point” tips, such as Whitacre or Sprotte needles; • single punctures; • lying flat after the lumbar puncture, presumably allowing the dural hole to heal. A 1-hour initial period followed by as much time as possible recumbent for 24 hours is advised. Once a headache has occurred, a variety of treatments can be used: • Bed rest, mild analgesics, and copious fluids. • Caffeine infusion, 500 mg in 500–1000 mL over 1–2 hours repeated twice. • Wearing an abdominal binder while out of bed. This increases intraabdominal pressure, increases epidural blood flow, and reduces transdural pressure gradients, thus reducing cerebrospinal fluid loss. • Epidural blood patch. This is performed by doing an epidural puncture at the site of the lumbar puncture, and then instilling 10–15 mL of the patient’s own blood into the epidural space at the site of the previous puncture. The blood either decreases the transdural pressure gradient, allowing the hole to heal, or acts as “physiologic glue,” sealing the hole. Epidural blood patches should be used with caution in the HCT patient because of the risk of creating an epidural hematoma in the coagulationimpaired patient, and of introducing a perfect bacterial culture medium close to the neuraxis in an immunocompromised patient. • Use of triptans. Anecdotal evidence and clinical experience indicate that the migraine-aborting medication sumatriptan, available orally and as a nasal spray, as well as by the intramuscular route, is useful in the treatment of PLPHAs and may obviate the need for blood patching.

Surgical pain HCT patients often undergo a number of procedures, ranging from the simple, with little postoperative pain, for example indwelling central line placement, to the complex and painful, for example lung biopsy. The management of postoperative pain in HCT patients should be aggressive. Invasive techniques, such as epidural catheters for administration of spinal opioids and local anesthetics, should be weighed carefully. After thoracic and abdominal surgery, epidural analgesia can aid pulmonary function and overall activity; there is a small risk of infection at the catheter site [127,128]. A complicating feature of postoperative pain management in HCT is that many patients will be opioid tolerant. In such cases, it is recommended to replace presurgical opioids completely with a basal infusion of drug and provide liberal breakthrough dosing either by PCA or frequent nurse-delivered boluses, depending upon the cognitive function of the patient. Even though opioids do not have true logarithmic dose–response curves, they behave as if they do in patients who are tolerant. Thus, it is not surprising that many patients will require, at least, double their previous daily opioid requirements in the acute postoperative phase. Self-titration via PCA allows the patient to escalate and subsequently wean medication to acceptable levels of comfort with little or no risk of respiratory depression.

Analgesia/sedation for painful and unpleasant procedures HCT patients have to undergo a number of different painful, unpleasant, and anxiety-provoking procedures. In small children, the only way these can be accomplished is under deep sedation or general anesthesia. Preprocedural fears and intraprocedural discomforts can be managed in a variety of ways, as described below.

Pain Management

Nonpharmacologic management of procedures Cognitive-behavioral and physical methods can be effective for both preprocedural anxiety and active distraction during procedures [129]. These methods require patients to be alert; even moderate sedation with benzodiazepines disrupts the effectiveness of these methods. Relaxation and imagery can effect cognitive distraction and reduce physical stress.

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regimens typically consist of a short-acting, potent opioid plus an intravenous anesthetic; fentanyl (short acting due to rapid redistribution) or remifentanil (which degrades spontaneously in the plasma) plus propofol are two commonly used methods. The authors discourage the use of ketamine, another alternative, because it is longer acting, creates copious secretions, and often is associated with postadministration delirium. The American Society of Pediatrics has provided practice guidelines for such situations [130].

Pharmacologic management of procedures Pharmacologic management can take a variety of forms, depending on the procedure, the patient, and local resources. Adults often will tolerate procedures with local anesthetic and a minimum of CNS-active drug. Children are likely to need more CNS-active drugs. In increasing order of complexity and invasiveness, the following techniques can be used. Cutaneous local anesthetic A 1-hour cutaneous application of Emla will produce dense skin anesthesia. Infiltration of skin and deep structures with percutaneous local anesthetic is highly efficacious. Local anesthetics do not work instantaneously, and it is good practice to leave the local in place for about 5 minutes before attempting any potentially painful maneuver. The addition of 2–3 mL of standard sterile sodium bicarbonate solution into a 30 mL vial of 1% lidocaine will reduce or eliminate the stinging pain of injection. Premedication Small doses of an anxiolytic (e.g. midazolam) and a short-acting opioid (e.g. fentanyl) prior to the procedure will make injection of local anesthetic more pleasant for patients. Patients often will have no recollection of the procedure, but will recall the experience less unpleasantly even when there were difficulties or the patient expressed discomfort. Conscious sedation Conscious sedation is achieved with opioids and sedatives, given in doses higher than those used simply for premedication, such that patients remain responsive to verbal stimuli and are able to protect their airway and breathe spontaneously. Only qualified nurses and physicians should provide conscious sedation due to the potential need, albeit small, for airway support. One approach is the use of lorazepam plus an opioid such as morphine or hydromorphone, medications that patients often are on already. The strategy is to “top off” the existing sedative and analgesic for the procedure with the knowledge that respiratory compromise would be extraordinarily rare because patients will previously have developed respiratory tolerance to those drugs. Another common combination of drugs is midazolam plus an opioid; this is less safe since the respiratorydepressant effects of midazolam are more pronounced and unpredictable than with lorazepam, and because patients rarely are on midazolam chronically so will not have built up a respiratory tolerance to the medication. In either case, it is important to weigh the risks of aspiration against the benefits of providing deeper sedation than would be achieved with premedication alone, if, for example, the patient has severe mucositis. Deep sedation and general anesthesia The continuum from conscious sedation to deep sedation, when the patient is virtually unarousable, to general anesthesia, when the patient is rendered unconscious, is indistinct. Whereas well-trained nurses can administer conscious sedation safely, only those providers who are highly skilled in airway management and interventions for cardiovascular instability should provide deep sedation and general anesthesia. Such

Invasive analgesic techniques in the HCT patient In the non-HCT population, patients whose longer-term nociceptive pain is poorly managed by systemic analgesics are candidates for one of a number of potent, but invasive, analgesic techniques. In HCT patients, these techniques have been used infrequently due to the risks of hematoma and infection. Post HCT, there are patients with severe pain problems that are not responsive to systemic analgesics, for example chronic gut GVHD or pathologic fractures, who should be considered for these potent techniques. Examples of the techniques, their potential indications in the HCT patient, and potential complications are as follows: 1 Peripheral nerve blocks with local anesthetic for pain in well-circumscribed anatomic distributions. For example, intercostal blocks may be appropriate for fractured ribs, chest tube placement or following thoracotomy. 2 Sympathetic nerve blocks with local anesthetic in conditions that have a sympathetic nervous system-mediated component. For example, stellate ganglion blocks may be appropriate for head and neck herpes zoster. 3 Neurodestructive sympathetic nerve blocks for pain which is likely to be longstanding and resistant to other analgesics. For example, neurodestructive celiac plexus block may sometimes be considered for pain associated with intra-abdominal candidiasis. 4 Neuraxial (epidural or subarachnoid) blocks with either opioids or local anesthetic. The techniques may be used on a short-term basis, for example for postoperative pain after thoracic or abdominal surgery, via percutaneously inserted catheters. For pain of likely longer duration, such as gut GVHD, more complex and expensive implanted systems may be considered.

Analgesia and sedation in dying patients In providing pharmacologic relief of discomfort in dying patients, the choice of the most effective and least burdensome route of drug delivery is often a problem. Choosing the route of drug administration in the HCT patient is simplified by the fact that most people have indwelling central venous catheters. The fundamental drug in providing comfort care is an opioid. The choice of opioid may be suggested by what has been used effectively earlier in the patient’s course. Both continuous infusion of opioids and PCA have a role to play, although, as the patient’s condition deteriorates, he or she will less likely be able to use PCA effectively. If patients are not receiving any opioid, one should start with either PCA or continuous infusion as for mucositis, and adjust doses upward based on response. If patients are receiving opioids, doses should be adjusted up to achieve comfort. It is often necessary to use high doses of opioid to obtain comfort, and the usual dose increases may be inadequate. In these circumstances, a bolus dose of 1 hour’s worth of opioid, followed by an increase of 25–50%, may be necessary. The occurrence of unwanted side-effects, such as nausea, delirium, agitation, tremulousness or other CNS phenomena, can be treated by changing to an equianalgesic dose of another opioid or by the addition of further CNS-active drugs, including low-dose (0.5–1.0 mg/ kg) ketamine once or twice a day [131].

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In the event opioids do not provide adequate comfort, especially if there is poor control of anxiety or tremulousness, sedative drugs such as benzodiazepines or barbiturates should be considered. Initial benzodiazepine regimes would be midazolam 1–2 mg/hour, and lorazepam 1–2 mg/hour; dose increases of 25–50% are appropriate. As with opioids, substantial doses may be needed. Another useful category of drug is the butyrophenones, such as haloperidol and droperidol. These can be used to treat delirium and confusion, and, in association with opioids, they may produce a calm, neuroleptic-like state. Sample regimens would be haloperidol 0.25–1.0 mg/kg, or droperidol 0.1–0.2 mg, intravenously every 4–6 hours. In the terminal stages, agitation may require the use of potent CNS depressants such as barbiturates (pentobarbital 1–2 mg/kg bolus, and continuous infusion 1–5 mg/kg/hour) or propofol (50–200 μg/kg/min) to provide “palliative sedation” [132]. In some instances, comfort measures can shorten a patient’s life. The Rule of Double Effect (also called the Doctrine of Double Effect), which invokes the axiom that intervening on the patient’s behalf may incur risks, including the possibility of

death, is a well-accepted ethical principle; it protects the practitioner legally and permits extreme comfort measures in dying patients. The Rule of Double Effect derives from Catholic moral theology and has roots as far back as Thomas Aquinas [133]. A full discussion of end-oflife ethics is beyond the scope of this chapter; ethical principles are fundamental but often do not account for the complex sociology surrounding death and dying [134].

Conclusion This chapter has outlined the neurophysiology and pharmacology of pain, and has described the common sources of discomfort in patients undergoing HCT. The chapter provides practical guidelines for the evaluation and pharmacologic treatment of pain in HCT. Most HCT patients can be managed effectively by the oncology team. In situations where continued inadequate or ineffective analgesia prevails, it is strongly recommended that a multidisciplinary expert pain relief team be consulted and involved.

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Mark M. Schubert & Douglas E. Peterson

Oral Complications of Hematopoietic Cell Transplantation

Introduction Oral complications of hematopoietic cell transplantation (HCT) represent a wide spectrum of clinical problems that occur throughout the course of transplant recovery. They can range from mild transient complications to chronic disabling lesions to life-threatening disease. The importance of comprehensive supportive oral care for HCT patients continues to be recognized as an integral component of overall management. Comprehensive HCT oral supportive care not only reduces patient suffering, but also can substantially reduce health-care resource utilization and improve the long-term outcome of transplantation. Advances in basic science knowledge relative to selected oral toxicities have led to the development of improved strategies and treatments to be utilized by a multiprofessional oncology team. For example, elucidation of pathobiology of oropharyngeal mucositis has led to the licensing of a keratinocyte growth factor that has been shown to reduce the frequency and severity of mucositis in selected HCT patient populations. Additional products are under development that may further reduce the impact of this often serious toxicity of HCT. Similarly, incremental improvements in the assessment and management of HCTrelated oral toxicities, including oral pain, local and systemic infection arising from the dentition and periodontium, salivary gland hypofunction, and graft-versus-host disease (GVHD), continue to emerge. Implementation of protocols to prevent and treat oral complications of HCT requires a multidisciplinary approach to be maximally effective. Evidenced-based management guidelines continue to be developed and should provide direct benefits to the patient’s overall clinical course.

Correlations with the phases of transplantation Oral complications can occur in all phases of transplantation (Table 103.1). Oral complications generally are a reflection of the general condition of the patient. Specific complications can be correlated with the phases of transplantation. Patient survivorship as well as quality of life is directly impacted by a number of risk factors including but not limited to underlying disease, patient age, type of donor, conditioning regimen for transplant, and the availability of supportive care. Risk of chronic, sometimes severe oral sequelae such as GVHD, salivary hypofunction, primary tumor recur-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

rences or second malignancies, growth and development abnormalities in children, and/or other adverse outcomes are elevated in allogeneic recipients [1].

Pretransplantation oral evaluation and stabilization The initial prevention or risk reduction of oral complications in patients receiving HCT requires stabilizing or eliminating oral and dental disease prior to initiation of transplant [1–5]. Upon becoming a transplant candidate, the patient should be advised to seek complete oral and dental evaluations and treatment. It is prudent to have the patient’s dentist involved with the initial management whenever possible, but the dentist and hygienist must clearly understand the goals and objectives of pretransplant stabilization and provide appropriate and realistic care. To maximize outcomes of oral management, the oncology team should advise the community dentist as to the patient’s medical status and goals for dental treatment at this time (Table 103.2). The overall strategy is to complete an oral care plan that eliminates or stabilizes oral diseases and conditions that could otherwise produce acute clinical complications during recovery from HCT, which can vary from 6 months for autologous patients to up to 1–3 years or longer for allogenically transplanted patients. Elimination of these conditions can reduce the incidence and severity of oral toxicities and complications throughout HCT recovery. This outcome in turn can reduce the risk of systemic sequelae, improve quality of life, and reduce the cost of patient care [6,7]. If patients are unable to receive the necessary care from their personal dentist, the oncology team should immediately evaluate and coordinate the oral management through dental care teams associated with the transplant facility. Dental treatment If at all possible, pre-HCT dental treatment, should not interfere with the start of transplant conditioning nor induce complications (e.g. nonhealed extraction or surgical sites). The level and complexity of care needs to be balanced by the medical, financial, and emotional constraints of the patient’s situation. Urgent or emergency dental disease should be stabilized whenever possible. If time permits, it may be expedient to also provide less urgent dental care. Elective dentistry will need to be postponed until immunologic recovery has occurred, which may take at least 9–12 months after transplantation if chronic GVHD is present. For patients whose medical condition prohibits dental treatment, medically noninvasive care should be implemented until routine care can be performed.

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Table 103.1 Hematopoietic stem cell transplantation phases in relation to oral complications and management Transplant phase

Time interval

Oral complications

Highlights of oral management

Phase I

Prior to initiation of conditioning regimen

• Pre HCT dental examination and radiographs • Elimination of dental, periodontal, periradicular, and oral mucosal lesions/infections anticipated to compromise the clinical course of HCT • Patient education relative to oral complications and their management

Phase II

Initiation of cytoreductive conditioning therapy through initial evidence of trilineage marrow engraftment

Pre-existing oral and dental disease as well as complications of recent chemotherapy or medical therapy – Dental caries – Endodontic infections – Periodontal disease: gingivitis, periodontitis – Oral mucosal infections: viral, fungal, bacterial Oral manifestations of malignancy – Gingival leukemic infiltrates (rare) – Oral lymphoma tumors – Metastatic solid tumors Oral ulcerations: aphthous, neutropenic, ulcerations, traumatic, etc. TMD Acute conditioning regimens toxicities – Oropharyngeal mucositis – Oral infections Mucosal infections: viral, fungal, bacterial Periodontal infections Dental infections (pulpal/endodontic) – Oral hemorrhage – Xerostomia – Taste dysfunction Acute GVHD (allogeneic transplants) GVHD prophylaxis toxicity (methotrexate)

Phase III

Trilineage marrow engraftment to normal circulating counts: – Neutrophils – Erythrocytes – Platelets

Phase IV

Normal hematologic counts to immunologic recovery

Chronic conditioning regimens toxicities – Xerostomia – Taste dysfunction Oral mucosal infections – Fungal (Candida, moulds) – Viral (HSV, CMV) – Bacterial* Oral acute GVHD (allogeneic transplants) – Mucosal lesions – Xerostomia Neurotoxicity (tremors, numbness, pain) Dental hypersensitivity TMD Granulomas/gingival hyperplasia (late phase III) Chronic toxicities of conditioning regimens – Xerostomia – Taste dysfunction – Late-emerging oral toxicities Oral manifestations of GVHD – Resolving acute GVHD – Emerging chronic GVHD Craniofacial growth and development abnormalities in children Oral mucosal infections – Oral candidiasis – Viral infections (VZV, HPV, HSV) – Bacterial infections†

• Maintain routine oral hygiene measures • Mucositis prevention and symptom control protocols • Infection surveillance and treatment as indicated – Viral (HSV, CMV) – Fungal (Candida, moulds) – Bacterial • Local hemorrhage control measures in support of systemic strategies • Symptomatic management of xerostomia • Topical/systemic therapy for oral GVHD • TMD management strategies (physical therapy, muscle relaxants, etc.) • Maintain basic oral hygiene • Symptomatic management of xerostomia • Infection surveillance and treatment as indicated • Surveillance for/diagnosis of oral GVHD with treatment as indicated

• Maintain basic oral hygiene • Management of xerostomia as needed • Surveillance for/diagnosis of oral GVHD with treatment as indicated • Infection surveillance and treatment of infections • Resume routine dental examinations (4–6 months post HCT) – Avoid elective and less than urgent treatments – Emergency dental care with medical support – Avoid all elective treatment

Oral Complications of Hematopoietic Cell Transplantation

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Table 103.1 Continued Transplant phase

Time interval

Oral complications

Highlights of oral management

Phase V

Long-term survival: full functional immunologic recovery

Chronic toxicities of conditioning regimens – Xerostomia Oral manifestations of chronic GVHD Oral mucosal infections – Oral candidiasis – Viral infections (HPV, VZV) – Bacterial infections† Second malignancies post transplant – Squamous cell carcinoma – Lymphoproliferative disorders – Salivary gland tumors Craniofacial growth and development abnormalities in children

• Maintain basic oral hygiene • Management of xerostomia • Surveillance for/diagnosis of oral GVHD with treatment as indicated • Infection surveillance and treatment • Surveillance for potential second malignancies post transplant

CMV, cytomegalovirus; GVHD, graft-versus-host disease; HCT, hematopoietic cell transplantation; HPV, human papilloma virus; HSV, herpes simplex virus; TMD, temporomandibular dysfunction; VZV, varicella-zoster virus. * Mucosal bacterial infections during this phase become increasingly uncommon unless engraftment is slow or if the patient has acute GVHD or is on GVHD therapy. † Mucosal bacterial infections are rare unless patient has severe chronic GVHD, especially when on intensive therapy for GVHD.

Dental caries, periodontal disease, and dental pulpal infections are common oral conditions requiring stabilization prior to transplantation [2,8–10]. Caries treatment may need to be prioritized: the more extensive or deeper the caries, the higher the risk of systemic infectious complications during bone marrow suppression. While it is usually desirable to restore teeth permanently, the use of durable temporary or intermediate restorative materials is valid when time or costs necessitates. If a pulpal infection occurs while the patient is immunocompromised, the resulting endodontic infection could be life-threatening. Thus, all nonvital infected teeth should be treated prior to HCT. It is therefore important to definitively determine the vitality of the teeth through pulp vitality testing. Pulpal and endodontic infections can become medically serious problems and can be difficult to manage in the initial months after transplantation. Infected teeth should be either treated endodontically or extracted. Periapical radiolucencies associated with endodontically treated teeth should be carefully evaluated since they can be caused by a number of diverse factors including infection, inflammatory reactions, apical fibrous scars, cysts, and malignant lesions. The published literature has established the need to treat pulpally infected and symptomatic nonvital teeth [2,3]. In cases in which a periapical radiolucency is associated with an endodontically treated tooth and there are no signs or symptoms of infection for an extended period, retreatment or extraction is not typically necessary since the radiolucency is most likely due to an apical scar [3]. However, if risk exists for infection or treatment failure after transplantation for these teeth, retreatment or extraction should be performed.

are infected and ulcerated, at risk to cause local, regional, and systemic infection and increased risk of bleeding. Gingival bleeding after transplantation predominantly results from pre-existing periodontal disease and is not solely due to low platelet counts. Healthy gingival tissue does not typically bleed unless acutely traumatized. Teeth with severe periodontal involvement with significant bone loss or mobility should generally be extracted. Combined antimicrobial therapy (topical, systemic or both) and curettage techniques, followed by effective dental bacterial plaque removal, can render mild-tomoderate infections as “low risk” relative to both transplantation and bleeding diathesis. The key to reducing risk of significant gingival associated infections and bleeding is to perform focused periodontal disease-stabilizing procedures in advance of myeloablation, and then maintain excellent oral hygiene until recovery of immune function [9]. Subgingival time-release minocycline (Arestin), if placed around day 0, may be able to help reduce bacterial colonization for 3–4 weeks after placement and may be able to reduce the risk of periodontally related infection risks during the period of most profound neutropenia. This strategy will generally eliminate or significantly reduce the level of infection, reduce local microbial flora, and reduce the risk of infectious sequelae. Debate exists as to the management of unerupted or partially erupted third molars (“wisdom teeth”) prior to transplantation. While some authors have suggested that all third molars that are not fully erupted should be extracted prior to HCT, the authors favor a more conservative approach that supports extraction of third molars with a history of acute periodontal infections. This history specifically includes pericoronitis or pulpal infection over the last 12–18 months and/or gingival tissue that cannot be maintained with routine oral hygiene measures [2].

Periodontal disease stabilization

Dental extractions

Of the dental diseases commonly encountered in HCT candidates, inflammatory periodontal diseases (gingivitis and periodontitis) pose the most significant infectious disease risk for the immunocompromised post-transplant patient [9]. Periodontal disease is often present in patients presenting for transplantation and ranges from mild marginal gingivitis to severe chronic periodontitis with deep periodontal pocketing and bone loss, acute and chronic abscesses, and tooth mobility. With periodontal disease, sulcular tissues (i.e. the tissues lining the periodontal pockets)

Dental extractions should be performed as atraumatically as possible, with primary closure preferred. It is difficult to define a minimal time to allow for healing of extraction sites before initiation of conditioning, but the more time that can be afforded for healing of extraction sites, the better. Ideally, every effort should be made to allow for epithelialization of the extraction site and resolution of any edema and inflammation in order to reduce the risk of local infection, bacteremia, distant infection, and bleeding [10]. The amount of time necessary for initial healing

Management of dental decay and endodontic disease

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Table 103.2 Medical and dental information pertinent to pretransplantation oral and dental stabilization Medical information to be provided to dental team Underlying disease For cancers: type, stage, and prognosis Aplastic anemia Other Type of transplant Autologous Syngeneic Allogeneic Related: matched, mismatched Unrelated: matched, mismatched Details of proposed conditioning Myeloablative Reduced-intensity conditioning Conditioning regimen chemotherapy, total body irradiation Planned date of transplantation Current hematological values Complete blood cell count with differential Medications Other medical considerations Splenectomy Cardiac disease (including murmur) Indwelling venous access line Diabetes History of bisphosphonate administration Dental information to be provided to oncology team Summary of dental evaluation Dental caries Number of teeth requiring restoration Endodontic disease Teeth with pulpal infection Teeth requiring endodontic treatment Periodontal disease status Number of teeth requiring extraction Other urgent oral care required Oral mucosal infections/lesions Time to complete stabilization

Table 103.3 Use and care of dentures and oral appliances Prior to transplant Adjust dentures to eliminate denture sores and areas of potential trauma/ irritation Minimize use during first 3–4 weeks after transplantation Wear only for eating Leave out at all other times, especially at night and during periods of significant mouth soreness Clean twice a day with a soft brush and rinse well Soak in antimicrobial solutions when not being worn Perform routine oral mucosal care procedures with the oral appliances out three or four times a day Dentures may be used to hold medications needed for oral care (e.g. antifungals) After day 21–28 post transplant Patients can generally resume routine use of dentures Continue to keep clean and soak in antimicrobial solutions Note: Construction of new denture or reline or adjustment of current dentures can be carried as needed post transplant. This type of dental treatment does not pose infection or bleeding risk Orthodontic appliances (brackets, wires, retainers, etc.) Remove brackets and wires prior to conditioning Discontinue use of removable appliances until oral mucositis has healed

tion. Antimicrobial soaking solutions are strongly recommended when dentures are being stored, and soaking solutions should be changed daily (Table 103.3). Orthodontic bands and fixed appliances, with the exception of fixed lingual arch retainers, should be removed before conditioning begins because of the risk for significant mucosal trauma that can exacerbate mucositis. Additionally, metal dental restorative materials and orthodontic appliances produce radiation backscatter with total body irradiation (TBI) that can increase radiation damage for tissues overlying the metal surfaces [11]. In these situations, large metallic dental restorations or metal orthodontic appliances can be covered with 3–4 mm thick vinyl splints to hold tissues away from the metal surfaces and reduce radiation damage. Removable orthodontic retainers may be used until orthodontic care can be resumed post-transplantation. If orthodontic bands and brackets cannot be removed, custom vinyl mouth guards should be utilized to help reduce mucosal trauma. Temporomandibular dysfunction

generally increases when the teeth are infected (periodontal and/or endodontic infections), or if it is necessary to develop gingival flaps and remove bone. A simple extraction with primary closure of a noninfected tooth will generally initially epithelialize in 7–10 days. In contrast, extraction of an impacted mandibular third molar can require as long as 18–21 days to resolve swelling and edema and heal incisions. Dentures and orthodontic appliances Removable prostheses (e.g. full or partial dentures) should be examined thoroughly and adjusted as needed prior to myeloablation. Poorly fitting appliances can abrade oral mucosa that has been injured by the cytotoxic effects of chemotherapy, thus exacerbating mucosal ulceration and increasing the risk of microbial invasion. Denture adjustment not only improves the patient’s ability to eat and talk, but can also reduce the risk of mucosal trauma. During HCT conditioning and for the first several weeks post-transplantation, dentures should be worn minimally (e.g. only during eating) to reduce risk of mucosal injury and secondary infec-

Patients with a history of temporomandibular dysfunction (TMD), especially those with a myofascial pain component, should be identified prior to transplant. Parafunctional habits including cheek or lip chewing, clenching, and/or bruxism (tooth grinding) should be noted, and efforts should be considered to reduce these behaviors. Physical therapy modalities (moist heat/cold packs, massage and muscle stretching for muscles, and cold packs for painful joints) can help control symptoms. Muscle relaxants can be useful for managing acute muscle-based signs and symptoms of TMD should they occur. Selected patients may benefit from use of an appropriately fitted vinyl mouth guard (athletic mouthpiece) or a flat-plane acrylic occlusal mouth guard or splint. Other oral disease All oral mucosal or osseous lesions should be assessed and diagnosed. Causes of these lesions could include infection, drug reaction or allergies, or neoplasia. Clinical and radiographic examinations should be

Oral Complications of Hematopoietic Cell Transplantation

augmented with appropriate tests. Dental radiographs are important to detect osseous lesions and may contribute to the diagnosis of mucosal lesions adjacent to teeth and periodontium. Microbiology testing for lesions, special tissue tests (e.g. toluidine blue and exfoliative cytology), and tissue biopsy may be indicated.

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Oral care protocols Standard oral care protocols are designed to maintain oral tissue health and prevent infectious and bleeding complications. These protocols can be subclassified into: (1) oral hygiene protocols to prevent infection due to dental/periodontal disease, and (2) strategies to maintain mucosal stability and oral comfort [4] (Table 103.4).

Prophylactic antibiotics As the risk for bacteremia and infection increases for immunosuppressed patients, the need for prophylactic antibiotics prior to any dental procedures escalates. However, there are no clear guidelines for the use of prophylactic antibiotics for dental treatment prior to and following HCT. Prudent clinical practice suggests that prophylactic antimicrobials may well be of benefit for patients with low absolute neutrophil counts (below 1000/mm3) or who are otherwise immunologically compromised. Some authors have recommended antibiotic prophylaxis prior to dental procedures to reduce bacteremia and potential colonization of central venous access catheters by oral bacteria. This recommendation is empiric and based on anecdotal experience but not evidence-based analysis. The policy endorsed by the authors (established in collaboration with the input from infectious disease consultants) recommends the following. 1 Patients who are at increased risk of infection due to poor immune status should be given appropriate antibiotics prior to dental treatment and, as the risk of infection increases, the intensity of therapy needs to be increased. 2 Until further evidence is available, patients who have central venous access catheters in place should receive antibiotics to prevent colonization of the catheter following dental treatment-induced bacteremia, using the American Heart Association’s prophylactic antibiotics guidelines for patients at high risk for infectious endocarditis. Standard prophylactic antibiotics for specific medical situations (e.g. endocarditis risk, dialysis catheters, etc.) should be used. The role of prophylactic antibiotics when dentally treating splenectomized patients is controversial [12].

Post-HCT oral complications Oral complications of HCT can have distinct patterns of occurrence and generally are consistent with systemic HCT toxicities and pathologies (Table 103.1). The patterns and timing of oral complications will usually correlate and be associated with the systemic complications of transplant. Early post-HCT oral complications primarily arise from the direct oral toxicities of conditioning regimens or indirectly from systemic toxicities. These complications can involve only the oral cavity or may represent oral manifestations of systemic involvement. Oral complications can occur acutely (days 0–100 post HCT), while others can become chronic or arise later post HCT. Distinct patterns of oral complications are recognized with specifics of HCT (autologous/syngeneic versus allogeneic HCTs, myeloablative versus reduced toxicity conditioning regimens, etc.). The recognition of the risks for, clinical patterns, and timing of the various oral complications can significantly improve the recognition and management of HCT oral complications. Acute direct conditioning regimen toxicities are dominated by oropharyngeal mucositis, salivary gland dysfunction, and taste dysfunction [13]. Acute indirect toxicities are generally related to infectious complications, thrombocytopenia, or GVHD. Chronic oral complications are generally related to chronic toxicities of conditioning regimens and GVHD, and can include oral GVHD lesions, chronic infections, xerostomia, damage to developing dental and craniofacial structures in children, and second malignancies post HCT.

Oral hygiene The impact of maintaining oral health, especially periodontal tissue health, can be significant. Bacterial dental plaque can result in local Table 103.4 Routine oral hygiene and mucosal care Tooth brushing Soft/ultrasoft nylon-bristle (two- or three-row) brush Electric and ultrasonic toothbrushes with soft bristles are acceptable – patient must be able to use them without causing trauma and irritation) Brush twice a day with the Bass sulcular scrub method Rinse frequently with water or normal saline Toothpaste: Patient preference as tolerated Discontinue if it causes pain, consider using nonmint flavored Frequent rinsing with 0.9% saline solution or water if not using toothpaste Topical fluoride recommended when there is increased risk of dental caries Salivary gland dysfunction (xerostomia) Inadequate brushing/flossing Foam tooth brushes Use only when impossible to use a regular soft nylon toothbrush Use with antimicrobial rinses (e.g. chlorhexidine) when possible Brush teeth two or three times a day and rinse frequently Resume use of a nylon-bristled toothbrush as soon as possible Flossing Floss once daily with an atraumatic technique with modifications as needed Interproximal brushes: use once daily as instructed Bland rinses Types of rinse – 0.9% saline solution (three-quarters of a teaspoon of NaCl in a quart of water) or – Sodium bicarbonate solution (2 tablespoons of sodium bicarbonate in a quart of water) or – 0.9% saline solution plus sodium bicarbonate (2 tablespoons of sodium bicarbonate in a quart of 0.9% saline) Total volume of 180–240 mL rinsed, held mouthful by mouthful, and spat out Repeat every 1–4 hours or as required for pain/irritation Topical fluoride 1.1% neutral sodium fluoride gel (preferred) or 0.4% stannous fluoride gel Brush on for 3–5 minutes, spit out, and rinse as directed. Apply once a day Reusable vinyl fluoride trays: put 1–2 drops per tooth in the tray and hold in the mouth for 3–5 minutes once a day Topical antimicrobial rinses 0.12–0.20% chlorhexidine oral rinse (use alcohol-free preparations whenever possible) Rinse, hold for 1–2 min and spit out Repeat 2–4 times a day depending on the severity of periodontal disease

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infection and bleeding that can progress to regional infection and bacteremia [14,15]. Maintaining gingival health by reducing oral bacterial colonization can reduce risk of bacteremia and gingival bleeding.

brush-on technique are the most convenient technique, thus improving patient compliance (Table 103.4). Oral mucosal care

Brushing. Controlling accumulation of bacterial dental plaque is critical for maintaining oral health, especially in order to prevent gingival and periodontal infections [4,5,16]. Tooth brushing and flossing represent the most appropriate and cost-effective approach to dental plaque control relative to the prevention of gingivitis and periodontal disease. Oncology oral care policies that mandate that patients discontinue brushing/flossing when peripheral blood components (notably platelets) decrease below defined thresholds are mistakenly based on the concept that these interventions cause undue risk of bleeding. Consistent mechanical plaque control promotes gingival health, decreases the exacerbation of oral mucositis due to oral microbial colonization, and reduces risk of bacteremia [17]. Patients should brush with a soft or extra-soft nylon-bristled toothbrush two times a day using a sulcular brushing technique that removes bacterial plaque from the gingival one-third of the tooth and periodontal sulcus. Vigorous oral rinsing with water or saline solution while brushing will aid in removal of bacterial dental plaque dislodged by the brushing. Proper technique for dental brushing should be reviewed with the patient prior to HCT and reviewed on a regular basis throughout HCT recovery. If toothpaste causes burning or stinging due to mucositis or GVHD, it can be discontinued and water or saline solution substituted. Brushes should be air-dried in the open between interventions. Specific disinfecting of toothbrushes has not been proven of value. Electric/ultrasonic toothbrushes may be used if patients are properly trained in their use. Foam toothbrushes are not effective in removing dental plaque and do not promote gingival health [18]. Furthermore, foam brushes abrade mucosal surfaces and can potentially promote mucosal breakdown during periods when patients are at risk for mucositis (primarily from the start of conditioning until approximately day 14 post transplant). The use of foam brushes in conjunction with a topical antimicrobial solution has been reported to be more effective than the use of only foam brushes but still is not equal to standard brushing [18]. If it becomes necessary to suspend the use of standard tooth brushing, it should be resumed as soon as oral mucosal lesions improve. Flossing and interproximal brushing. The removal of bacterial plaque interdentally is critical for maintaining gingival health. If patients are properly trained to floss and/or use interproximal brushes, it is reasonable to have them continue using them throughout HCT recovery. Professional staff should monitor the efficacy and side-effects of brushing/flossing daily [19]. Antimicrobial rinses. Antimicrobial agents can aid in control of bacterial colonization in the mouth and thus in formation of dental plaque. Chlorhexidine oral rinse is the most commonly available product. It is especially useful when patients are unable to brush and floss. Follow-up research to early studies promoting the use of chlorhexidine early post HCT to prevent oral mucositis has failed to produce positive results relative to prevention or treatment of mucositis [20–22]. Other potentially useful antimicrobial rinses include tetracycline povidone iodine and cetylpyridinium chloride [5,23]. Topical fluorides and remineralizing products. Patients at increased risk for dental caries, including risk secondary to xerostomia, should receive topical fluoride therapy and/or remineralizing therapy. Neutral sodium fluorides (1.1%) or stannous fluorides (>0.4%) applied with a

In addition to dental hygiene care, routine oral mucosal care should be utilized on a daily basis. Bland oral rinses and lip care not only promote patient comfort, but also help maintain moisturization and lubrication of the oral mucosa and thus reduce risk of secondary infection. Oral mucosal protection protocols are based on two levels of intervention: nonmedicated and medicated strategies. Nonmedicated oral mucosal care strategies emphasize frequent rinsing with bland rinses (e.g. saline solution, sodium bicarbonate or saline–sodium bicarbonate solutions) and sucking on ice chips (Table 103.4). These strategies are directed at keeping mucosal surfaces hydrated, neutralizing acidity, rinsing out thick mucus secretions, and soothing mild-to-moderate mucosal discomfort [17,20,21,24,25] (Table 103.4). It may also be beneficial to use “iced” bland rinses where ice chips are added to the basic solutions right before use (Table 103.4). These solutions do not generally need to be sterile, unless the patient is in laminar airflow isolation. Hydrogen peroxide rinses should not be used as a routine mouth rinse because of their tendency to dry tissues and interfere with wound healing [26]. However, a mixture of 3% hydrogen peroxide and 0.9% saline solution (1 : 1 to 1 : 3 by volume) can aid in removal of blood clots or mucous accumulations. Lemon glycerin swabs also should not be used because citric acid can reduce the pH of the oral cavity and damage enamel, and glycerin can cause mucosal drying. Commercial mouthwashes are not generally recommended because they can cause tissue irritation and increase mucosal dryness. Medicated mucosal care protocols incorporating topical antimicrobial agents have generally been proposed for the prevention of oral mucositis or specific oral infections (primarily Candida species). The efficacy of these antimicrobial protocols is yet to be established. While the use of systemic prophylactic antibacterials has been clearly shown to prevent systemic infection complications post HCT, the impact of systemic antibacterial agents on oral health has not been documented. In contrast, systemic antiviral and antifungal prophylaxis has clearly had a significant impact in preventing oral herpes simplex virus (HSV) and Candida infections. Specific topical antimicrobial oral care protocols for HCT patients require additional investigation before their use can be promoted. Lip care Lip dryness and chapping are frequently reported by patients post HCT. Conditioning regimens and GVHD can contribute to epithelial thinning and chapping. While lip care products containing petroleum-based oils and waxes can be helpful, lanolin-based creams and ointments are typically more effective in moisturizing and protecting against damage. Patient education Patient education and compliance with care protocols is an important element of the oral care plan [5,16,27]. In addition to oral hygiene care instructions, patients must be educated as to potential oral complications of HCT, such as mucositis, oral bleeding, oral infections, GVHD, and pain. Patients need to be motivated to accept responsibility for their self-care protocols, and should understand that their efforts can have a positive impact on their clinical course. Individual patient needs must be addressed in developing oral care regimens. Instructions should be given clearly and concisely. Demonstrations or diagrams should be used when appropriate. Written materials offering additional information are often helpful. Motivation and compliance with oral care procedures can have considerable impact on the transplantation course.

Oral Complications of Hematopoietic Cell Transplantation

Oropharyngeal mucositis Oral mucositis is a frequent, serious toxicity of phase II of HCT therapy. Approximately 80% of patients who receive high-dose conditioning regimens prior to HCT develop clinically significant oral mucositis [28,29]. For patients receiving high-dose conditioning regimens prior to HCT, oral mucositis has been reported to be the single most debilitating complication of a transplant, causing severe pain and affecting nutritional intake, mouth care, oral function, and quality of life [1,30]. Oropharyngeal mucositis increases the risk for bleeding and systemic infection, and, when severe, can also compromise the upper airway requiring endotracheal intubation. Mucositis can exert a profound influence on the overall course for the patient in the early post-transplant period. The oral mucosa is a complex physical and chemical barrier that, when functioning normally, provides critical defense against pathogens and other challenges. The current pathobiologic model of oral mucositis incorporates five stages (Fig. 103.1) [31–34]. Evidence supports the impact of both direct cellular damage from cancer chemotherapy and radiation, and derangements in tumor necrosis factor, interleukin-1, and monocytes as possible key contributors to the development of oral mucositis [32–34]. Additional modeling is needed relative to the rela-

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tionship between cancer therapy-induced compromise of systemic immune constituents and functionally distinct mucosal immune components. Understanding these mechanisms and related processes may delineate preventive or therapeutic strategies at the clinical level. Clinically apparent oral mucosal changes typically emerge 5–10 days after initiation of high-dose conditioning regimes [35]. Initially, mucosal atrophy and erythema occur, notably on nonkeratinized oral mucosa. Atrophy of the mucosa then progresses to ulceration, which becomes most severe approximately 6–11 days after HCT (Plates 103.1 and 27.7). The mucosa then gradually heals over the next 2 weeks. The oral mucosal sites most commonly affected include the lateral and ventral aspects of the tongue, and the buccal and labial mucosa [35]. Severity of mucositis is primarily related to the type of conditioning regimen utilized. Genetic factors related to susceptibility of tissues to damage from chemotherapy and radiation are beginning to be identified. Secondary trauma or infection will also affect mucositis severity. Other risk factors, including patient age, gender, smoking, and diabetes, have been proposed but need further study. Finally, the degree of genetic match between the donor and patient (e.g. syngeneic, autologous, allogeneic, and unrelated donors) can affect the clinical expression of mucositis since oral acute GVHD can significantly confound the clinical presentation of conditioning regimen-related mucositis [35].

Pathobiology of Mucositis Normal Epithelium

Phase I: Initiation

Phases II & III: Messaging, Signaling, & Amplification

Phase IV: Ulceration

Phase V: Healing

Radiation

Chemotherapy

Submucosa Mucosa

Basal Epithelial Cell Layer

Fibroblast Gram-negative NFκB / Inflammatory Cytokines Bacteria Extracellular Matrix Inflammatory Cells Blood Vessel Signaling

Fig. 103.1 The phases of mucositis following exposure of mucosa to radiation or chemotherapy can be arbitrarily arranged into five phases that in actuality can occur simultaneously. Phase I: Initiation. Chemotherapy and radiation produce varying degrees of direct damage to DNA, usually through the production of reactive oxygen species. Additionally, there is simultaneous activation of other pathways involved with subsequent steps. Phase II: Upregulation and message generation. Transcription factors such as nuclear factor-kappa B (NFκB) are activated to upregulate many genes in the endothelium, epithelium, fibroblasts, and macrophages, which results in the production of messaging and effector proteins including proinflammatory cytokines and enzymes. This results in apoptosis and widespread tissue damage. Phase III: Amplification and signaling. Inflammatory cytokines can directly feed back to reupregulate transcription factors, such as NFκB, to set off a new wave of factors that can induce further damage throughout the epithelium and submucosa. Trauma and further cytokine release increase damage to epithelial surface. Phase IV: Ulcerative/bacterial phase. This phase will usually peak between 6 and12 days after transplant. With atrophy and ulceration of the mucosal surfaces, fibrin–exudate pseudomembranes form. These can become colonized by oral bacteria, which unfortunately is usually during the period of most profound neutropenia. Included in invading organisms are Gram-negative bacteria that can release endotoxins (lipopolysaccharide), which causes further release of cytokines from surrounding tissues and macrophages. Phase V: Healing phase. Through signaling from the mesenchyme, signaling from the extracellular matrix promotes the recovery of the basal layer mucosal epithelial cells. The mucosal surface is renewed and ulcerations are healed. The return of circulating white blood cells (especially neutrophils) aids in clearing bacteria from tissue. (Adapted from [34], with permission.)

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Mucosal injury can be accentuated by a number of factors including but not limited to salivary gland dysfunction, mucosal trauma, including that resulting from normal oral function, and infection or irritation caused by indigenous oral flora, acquired pathogens, and reactivation of latent organisms including herpes group viruses (HSV, cytomegalovirus [CMV], and varicella-zoster virus [VZV]). Additionally, these conditions can substantially elevate the risk for oral mucosal infection. Management Mucositis management can be challenging for the clinician and patient. Currently, management strategies are primarily directed towards symptom management. The interventions are based on the severity of tissue damage and symptoms, infection prevention and therapy, nutritional support, and use of standardized pain management decision matrices (pain management ladders) to determine the need for analgesics and narcotics [30,36,37] (Table 103.5). However, with the establishment of a pathophysiologic model there has been significant research devoted to development of agents that move beyond symptom management and are capable of preventing and treating oral mucositis. An important advance in mucositis management was achieved with the United States Food and Drug Administration approval of keratinocyte growth factor-1 (palifermin). This approval was based on a clinically and statistically significant reduction in several key outcomes in patients undergoing autologous HCT transplant for malignancy who received palifermin prior to and immediately after conditioning with an aggressive TBI-based preparative regimen [38]. The outcomes included a greater than 40% reduction in incidence of severe (World Health Organization grade 4) oral mucositis. Palifermin is approved in the United States to prevent oral mucositis in patients with hematologic malignancies who are receiving high-dose chemotherapy, with or without TBI, followed by HCT. It has more recently been demonstrated to reduce oral and throat discomfort and to improve functioning in comparable transplant patients [39,40]. Research continues to determine the efficacy of palifermin in additional HCT settings. Oral cryotherapy or ice chip therapy is a technique that has been shown to be effective for reducing mucositis caused by chemotherapeutic agents with relatively short half-lives, such as melphalan. Patients are instructed to start holding ice chips in their mouths 10 minutes prior to administration of the chemotherapy and then continue holding ice in their mouths for the next 6 hours to induce local vasoconstriction and apparently deliver less drug to vulnerable oral tissues [41]. Since palifermin is the only agent currently approved to prevent mucositis, it is important to continue to employ symptom management strategies for oral mucositis. Symptom management and the prevention/management of factors that aggravate oropharyngeal mucositis represent the primary objectives for HCT mucositis management [36,37,42]. It is recommended that a “stepped” or “ladder” approach be utilized, such that in a systematic step-wise fashion treatments and agents are added to provide further symptom relief for patients with mucositis [36]. Even though systemic treatments are usually required to manage severe mucositis, the patient should continue to be encouraged to perform systematic basic oral care. As each “step up” is added, it is important to continue to utilize the previous steps as part of the overall management strategy. Most oral mucositis symptom management regimens incorporate a combination of different agents and treatments that can be applied with escalating intensity for pain relief [36,37]. Basic oral care remains initially focused on reducing oral microbial colonization and infection of damaged mucosal surfaces, which can otherwise aggravate tissue damage, delay healing of oral tissues, and possibly lead to systemic infection [5,16,43]. Since oral mucositis occurs during the period of most profound thrombocytopenia, oral bleeding or oozing can be prob-

Table 103.5 Mucositis management Prevention of oral mucositis • Oral cryotherapy (ice chips): start holding ice chips in the mouth prior to administration and continue for the expected duration of action of the drug (up to 6 hours) • Radiation backscatter protection: use vinyl mouth guards, cotton rolls, etc. to hold soft tissues away from metal restorations during delivery of radiation • Epithelial growth factor (palifermin): for patients with hematologic malignancies scheduled to receive conditioning with protocols expected to have a high risk of grade III/IV (e.g. VP-16, cyclophosphamide, total body irradiation). Currently recommended dose is 60 μg/kg/day for 3 days prior to conditioning (>24-hour washout) followed by 60 μg/kg/day for 3 days starting immediately after infusion of stem cells Symptomatic management of oral mucositis Step 1 • Bland rinses (room temperature or chilled): 0.9% saline solution Sodium bicarbonate solution 0.9% saline solution + sodium bicarbonate solution Step 2 • Topical anesthetics Lidocaine: viscous, ointment, sprays Benzocaine: sprays, gels Diphenhydramine solution Other: doxepin solution, other antihistamines, benzydamine* • Mucosal coating agents Aluminum hydroxide (Amphojel), Kaolin-pectin (Kaopectate) Hydroxypropylcellulose film-forming agents (Zilactin) Film-forming agents for symptom control (Gelclair, MuGard, Mucotrol) Step 3 • Analgesics Topical agents: benzydamine hydrochloride*, doxepin rinse Opioid drugs: morphine, fentanyl, hydromorphone, others – Oral (regular release, time release) – Intravenous (bolus, continuous infusion, patient-controlled analgesia) – Transdermal patches – Oral transmucosal products 䊊 䊊 䊊

䊊 䊊 䊊 䊊

䊊 䊊 䊊

Experimental agents under investigation Anti-inflammatory agents (antitumor necrosis factor-α): N-acetylcysteine Antireactive oxygen species agents/free radical scavengers Epithelial growth factors Low-energy laser therapy (helium–neon, aluminum–gallium–arsenide, gallium–arsenide) L-glutamine in oral delivery system * Benzydamine is not licensed in the United States but is available in Canada and Europe.

lematic. Mucosal breakdown can expose submucosal vascular beds that can result in hemorrhage that ranges from slow oozing to frank bleeding. The accumulation of clots can compromise oral function and in some cases potentially obstruct the oral airway (see below). The frequency of bland rinse use (saline and/or sodium bicarbonate) is increased to moisturize oral tissues, rinse away debris, and provide symptomatic relief for mild mucositis. Mucosal coating agents, such as antacid and kaolin solutions, are added with increased discomfort. As mucosal breakdown and pain increase, the next step adds topical anes-

Oral Complications of Hematopoietic Cell Transplantation

thetics. A wide variety of topical anesthetic agents is available and ranges from antihistamines (e.g. diphenhydramine) to anesthetics (e.g. lidocaine and benzocaine) and analgesics (e.g. benzydamine and doxepin) [36,42–45]. Topical, local application of anesthetic agents allows the patient to concentrate the anesthetic effect to the most symptomatic oral sites. Sedation may result following rinsing with and swallowing antihistamines. The most common complication associated with topical anesthetic use is inadvertent mucosal damage due to trauma and irritation when trying to eat or carry out oral hygiene when the mouth is numb. Generalized oral rinsing/gargling with anesthetics also carries the risk of impairing the gag reflex due to oropharyngeal anesthesia, which can result in aspiration and pneumonia. Therefore, local application to areas of ulceration may be more appropriate than extensive oral rinsing. A number of products have been promoted for covering ulcers associated with mucositis but lack adequate research to affirm their efficacy [36,44,45]. Many centers utilize oral rinses that are compounded by combining a number of agents empirically. These rinses will generally include topical anesthetic agents (lidocaine, diphenhydramine, etc.), a coating agent (aluminum hydroxide or other antacid solutions), and an antifungal (nystatin solution). While these multiagent rinses may be able to reduce symptoms, a recent study appears to indicate that they are no more efficacious than single-agent rinses [21]. Before prescribing these combinations, it is reasonable to consider potential patient benefit, drug tolerability (taste, comfort, texture, etc.), and cost-effectiveness of combination rinses versus a single agent (e.g. viscous lidocaine). While some studies of topical chlorhexidine (0.12% or 0.20%) have reported a benefit of reduced severity or duration of mucositis in cancer patients, many others have shown no benefit or even exacerbation of mucositis symptoms [20,21,42]. While chlorhexidine can clearly reduce bacterial colonization and help with gingivitis, these conflicting reports prevent the development of any care guidelines relative to its use to prevent oral mucositis [22]. Systemic pain medications, especially opioids, represent the last step of mucositis symptom management. These products are indicated when bland rinses and topical anesthetic fail to produce sufficient pain relief (see Chapter 102) [46,47]. Oral mucositis typically produces gradually increasing pain that peaks between 6 and 11 days after HCT. Morphine has generally been the most frequently utilized agent and has been found to be extremely effective when administered with patient-controlled analgesia. Opiates including hydromorphone, meperidine, and timerelease oral or intravenous morphine and fentanyl (intravenous, transdermal patches, and oral transmucosal) can also be used. There is strategic need to continue research directed to causation of mucositis as well as to drug development to mitigate the toxicity across a broader cohort of at-risk patients. Prevention of ulcerative mucositis during the HCT course might ultimately provide the setting for escalating the intensity of chemotherapy, leading to more durable remissions or increased patient survival. There are significant clinical research efforts underway to identify medications and agents capable of actually reducing or eliminating oral tissue damage that result in mucositis. The role of biological response modifiers in prevention and treatment of oral mucosal damage is currently receiving intense investigation. For example, research is being directed toward epithelial cell mitogens, inhibitors of epithelial cell cycling, and modulators of the immune response. Future research will be influenced by the ability to develop new laboratory models. Various agents that may potentially reduce or prevent mucosal breakdown and inflammation include cytokines and growth factors (e.g. keratinocyte growth factor-2 [KGF-2], fibroblast growth factor-20 [velafermin]), granulocyte colony-stimulating factor, granulocyte–macrophage colony-stimulating factor, EN3285 (NAC ProGelz), low-energy

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laser therapy (helium–neon or diode, wavelength 632–660 nm), L-glutamine, and antimicrobials (Table 103.5) [30,33,34,48]. Topical application of granulocyte–macrophage colony-stimulating factor to aid management of mucositis has shown varied results [22,40,49]. While the use of oral glutamine or the addition of glutamine to intravenous nutritional supplements has been shown to decrease the severity and duration of oral mucositis in some clinical trials, other studies have been unable to show promising results. However, a recent phase III study of L-glutamine administered orally via a proprietary drug delivery system demonstrated efficacy in reducing incidence and severity of World Health Organization grade 2 or higher oral mucositis in breast cancer patients receiving anthracycline-based chemotherapeutic regimens [50]. It is likely that, since mucositis appears to result from a number of different factors, it will be necessary to combine a number of different agents for the effective prevention of mucositis. For instance, it may prove useful to: (1) initially administer agents to increase mucosal thickness prior to conditioning (e.g. KGF); (2) then administer agents to take epithelial cells out of the cycle to reduce cytotoxicity during conditioning (transforming growth factor-β3), along with anti-inflammatory agents (e.g. benzydamine), and antimicrobial agents or growth factors to enhance the engraftment and recovery of neutrophils; and, finally, (3) utilise agents given following conditioning to enhance recovery of normal mucosal thickness (KGF, epidermal growth factor, etc.). Oral infections The patterns and types of oral infection observed in HCT patients are, in general, similar to systemic infections observed in HCT patients. However, advances in the prevention and management of infectious complications in HCT patients have dramatically reduced the incidence and impact of oral infections (see Chapters 88–94). HCT patients are at risk for oral infections until there is full immune reconstitution. Types and severity of these infections are related to a number of factors including: 1 pretransplant oral infections; 2 the degree and duration of immunosuppression; 3 the degree and duration of mucosal damage (e.g. mucositis or GVHD); 4 salivary gland dysfunction; 5 the use of prophylactic infectious disease protocols. Generally stated, phase II is the period of highest risk (early oral infections), although oral infections can be problematic long term post transplant (late oral infections). Early oral infections Advances in HCT infection prophylaxis have led to a substantial decrease in the incidence and severity of post-HCT oral infections – antifungal prophylactic protocols have reduced the frequency of oral candidiasis, and prophylaxis with aciclovir for HSV and protocols to prevent CMV infections (e.g. ganciclovir plus the use of CMV-negative blood products) have significantly reduced the occurrence of oral HSV and CMV infections. HCT-associated myeloablation and immunosuppressive therapy directly increases the risk for both acute oral and systemic infections of oral origin [16,51–53]. The types of organism causing acute oral infections are primarily related to the colonizing oral microflora, acquired oral flora, and risk for reactivation of latent viruses. The risk of oral bacterial infection has been reduced with the increasingly sophisticated oral hygiene protocols as well as improved bacterial prophylaxis and treatment interventions. Opportunistic Gram-negative and Grampositive pathogens, such as Pseudomonas aeruginosa, Neisseria species

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and Escherichia coli, as well as Gram-positive streptococci and staphylococci, remain, however, of substantial concern. The key risk factors for early oral infection include the degree and duration of immunocompromization, the severity and duration of damage to oral mucosa (mucositis and oral GVHD), salivary gland dysfunction, and patient adherence to oral hygiene protocols [54]. Fungal infections. Candidiasis is the most frequent oral infection occurring in HCT patients. The risk for oral candidal infection increases as the severity and duration of immunosuppression increases. However, with improved systemic antifungal prophylaxis protocols, the incidence and extent of oral Candida infections have decreased over the last decade. Candida albicans is considered to be the most pathogenic and most frequently isolated yeast species associated with oral infection; other species, including C. krusei, C. tropicalis, C. glabrata, and C. dubliniensis are increasingly being isolated (see Chapter 89) [55,56]. However, since C. albicans is a component of the normal oral flora, identification of C. albicans in culture specimens from oropharyngeal mucosa may only indicate colonization, and not infection, unless recovered in high numbers. It is important to correlate clinical findings of mucosal atrophy, erythema, and/or pseudomembranous plaques with the results of laboratory tests to identify yeasts to both determine the need to treat these infections as well as to judge infection response to therapy. Pseudomembranous candidiasis is the most common clinical presentation for oral candidiasis and is usually described as white surface “debris-like” lesions that can be wiped off, most often involving the tongue, the buccal and labial mucosa, the tonsillar pillars, and the soft palate. Candida involvement of the lip commissures is noted as inflammation, chapping, and fissuring. The risk of oral candidiasis is highest during phase II and the first half of phase III. The most prominent risk factors for this infection are increasing neutropenia, GVHD, GVHD therapy (especially steroids), salivary gland dysfunction, and use of broad-spectrum antibiotics. Food debris, fibrin exudates, mucous secretions, and hyperkeratotic striae related to GVHD may cause white changes that can be misinterpreted as pseudomembranous candidiasis. It is reasonable to utilize direct examination of smears (i.e. Gram staining) from oral lesions to identify fungal elements, and cultures to identify the type of fungus. Atrophic candidiasis is not common early post HCT, but can be increasingly noted later after HCT. It presents with atrophic and erythematous mucosal surfaces, with the dorsal tongue and hard and soft palate most frequently involved. While pseudomembranous candidiasis is generally asymptomatic, atrophic candidiasis associated with mucositis or oral GVHD can cause pain and irritation. A variety of topical antifungal strategies are available when localized oral candidiasis is present. Nystatin (oral solutions, pastilles and powder), clotrimazole (troches or cream) or amphotericin B solution can be used to control acute oral candidiasis. Systemic agents, such as fluconazole, itraconazole, amphotericin, voriconazole, caspofungin, and micafungin, have demonstrated effectiveness against oropharyngeal candidiasis. HCT patients are also at risk for oral infection caused by invasive fungal organisms, including aspergillosis and mucormycosis [55–59]. Systemic antifungal prophylaxis has significantly reduced the incidence of these infections. The tendency for these infections to result in aggressive local invasion and systemic spread, often with lethal outcome, is a major concern. Diagnosis of these infections often requires histopathological examinations and culture of tissue specimens. Therapy requires the selection of appropriate systemic agents in conjunction with local surgical revision when possible. Viral infections. HCT patients are at high risk for acute oropharyngeal viral infections (see Chapters 90–93) [60,61]. During phase II and early

phase III disease, oral viral infections are predominately caused by HSV. VZV and CMV infections can occur but are less common. Infections caused by Epstein–Barr virus (EBV), Coxsackie virus, adenovirus, and human herpesvirus-6 are extremely uncommon. Oral human papilloma virus infections are noted late post transplant. Herpes group viruses, including HSV, VZV, CMV, and EBV, can cause significant oral disease in patients undergoing HCT [60–63]. Most HSV, VZV, and EBV infections represent reactivation of latent virus, while CMV infections can result from either reactivation of latent virus or newly acquired virus. HSV reactivation early post HCT can exacerbate mucositis and result in hemorrhagic, ulcerative lesions involving extensive mucosal surfaces. Clinically, HSV stomatitis can be difficult to recognize and is often confused with severe oropharyngeal mucositis. As immune responsiveness recovers, HSV lesions appear as focal ulcerations, often involving the attached gingivae and hard palate. Extraoral HSV lesions usually present with patterns similar to herpes labialis lesions although often larger and more destructive. Prophylactic systemic aciclovir for HSV-seropositive patients has dramatically reduced the incidence of HSV reactivation during phase II [60]. For patients not receiving prophylactic or therapeutic doses of aciclovir, or in instances where either oral aciclovir may not be absorbed adequately or resistant virus may be present, early diagnosis of HSV infection is important. If not treated promptly, infection can cause significant oral pain and tissue damage, and it may even disseminate, with serious systemic sequelae. Diagnosis utilizes viral culture (including shell vial testing) or direct immunofluorescent staining of scrapings for ulcers (see Chapter 91). CMV can cause oral lesions in immunosuppressed patients. However, as in the case of HSV, CMV prophylaxis has dramatically reduced the incidence of oral lesions (see Chapter 90) [63].The risk of infection is highest during phase II and early phase III. CMV lesions have a nonspecific clinical appearance, with a tendency for shallow-to-moderatedepth, irregular ulcerations covered with pseudomembranous fibrin exudate. In severe cases, lesions can be extensive and involve large areas of mucosa [63]. Surface swab cultures often yield false-negative results. Shell vial cultures can improve the possibility of detecting CMV, but examination using immunohistochemical tissue stains specific for CMV and biopsy materials is the most reliable technique to diagnose this disease. Ganciclovir and valganciclovir are the drugs of choice for treatment (see Chapter 90). EBV-related “hairy leukoplakia” lesions have been observed occasionally in HCT patients similar to those seen in human immunodeficiency virus-positive patients. These lesions have no apparent clinical significance. On the other hand, EBV-related lymphomas are associated with head and neck lymphadenopathy, generally in phases III and IV. This disease can be fatal if not diagnosed and treated promptly (see Chapters 93 and 106). Bacterial infections. Bacterial infections in the HCT patient can be caused by “normal oral flora” or by nosocomial pathogens (see Chapter 88). Organisms typically classified as being of low virulence in immunocompetent patients can produce both local and systemic infections in HCT patients [64,65]. Examples include Gram-positive organisms such as Streptococcus viridans and Streptococcus mutans. Antibiotic prophylaxis in neutropenic HCT patients reduces mortality, episodes of fever, and the number of documented bacterial infections (see Chapter 88). The spectrum of infections tends to shift as new prophylactic antibiotic protocols evolve [66]. However, the risk of viridans group streptococcal bacteremia and infection in patients with severe mucositis remains problematic, especially in those individuals receiving oral, nonabsorbable quinolone antibiotics [10,67,68]. In addition, a

Oral Complications of Hematopoietic Cell Transplantation

number of oral pathogens including P. aeruginosa, Staphylococcus aureus and E. coli can be acquired. These highly pathogenic organisms can cause serious morbidity and can be fatal. Infection management strategies should involve preventive or therapeutic regimens that target these microbes. Microbiological documentation of causative organisms is essential, given the nonspecific presentation of bacterial infections. It is important to recognize that secondary bacterial infection can occur with all types of oral lesion in immunosuppressed HCT patients. Immunocompromised HCT patients with periodontal disease may develop acute periodontal infections with associated systemic sequelae [68]. Although not frequent in HCT patients, these lesions can occur in subtle fashion as in other myelosuppressed cancer populations. Erythema and other inflammatory signs are typically suppressed. Dental plaque can significantly increase the risk of periodontal and associated systemic infections. Broad-spectrum antibiotic therapy should be considered while culture results are pending for periodontal infections. Local therapy can be augmented with: 1 irrigation with effervescent agents (e.g. hydrogen peroxide) that are toxic to anaerobic bacteria; 2 subgingival antibiotics such as minocycline; 3 consistent gentle mechanical plaque removal (dental brushing and flossing). Late oral infections Although the risk of infection decreases with immune recovery post HCT, the oral cavity can remain at risk for a variety of infections as immunologic recovery progresses through late phase III and IV. Most significant oral infections during these phases are caused by viral and fungal organisms [51,60,62,69–71]. Serious oral bacterial infections are less common, primarily developing secondary to periodontal or dental infections. The most common oral fungal infection during these later phases is candidiasis, commonly presenting with pseudomembranous white plaques and striae; however, atrophic oral candidiasis can also occur. Since the appearance of oral GVHD and oral mucosal debris can be similar to that of candidiasis (Plates 103.2 and 103.3), it is important that the specimen recovered from lesions be subjected to the appropriate laboratory identification of responsible organisms. In phase III, oral HSV infections can range from herpetic stomatitis in severely immunosuppressed patients with widespread oral ulcerations to relatively minor herpes labialis lesions in patients as immune function recovers [60]. CMV lesions are associated with a more compromised immune status and can be difficult to diagnose [63]. Obtaining viral cultures from chronic painful oral ulcerations should be considered, especially in patients who are CMV-antibody positive. EBV infections are relatively rare. VZV reactivation in HCT patients usually occurs after day 100 post transplant, with the peak incidence between 3 and 12 months post HCT [69]. Lesions tend to represent zoster-like patterns of lesions involving single branches of the trigeminal nerve, although multiple dermatomes can be involved. Infection tends to occur during mid-to-late phase III and phase IV. Dissemination can occur in severe cases, thus representing a life-threatening complication [69]. In susceptible patients, primary VZV infection can occur with skin lesions typical of chickenpox. Immunocompromised patients post HCT, especially those receiving high-dose immunosuppressive therapy for GVHD, are at increased risk for oral HPV infections. Most cases appear to represent reactivation of latent virus, with patients reporting hand or genital lesions at times prior to transplant. The presentations of these oral lesions can vary from exophytic oral verrucous vulgaris-like masses to a more flat condyloma acuminata-like appearance. Attached gingival surfaces have been the most frequently noted surfaces involved, although lips, tongue, floor of

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mouth, and soft palate have also been noted as involved when immune recovery is slow. Laser therapy ablation, surgical excision and cryotherapy are the most commonly utilized techniques for the removal of oral HPV lesions. For patients with adequate immune reconstitution, intralesional injections of interferon-α or topical imiquimod may be effective. Intralesional injection with cidofovir may prove effective for immunocompromised patients with recurrent lesions after excisional or ablative therapy. Oral hemorrhage Hemorrhage from oral tissues in patients receiving cancer therapy can occur for multiple reasons. Hematological factors include thrombocytopenia and loss of coagulation factors due to disseminated intravascular coagulation or liver disease. Local factors include mucositis, infections, and trauma. Spontaneous gingival bleeding and mucosal petechiae can be observed when the platelet count falls below 20,000–30,000/μL. Gingival bleeding primarily occurs due to pre-existing periodontal disease, and careful oral hygiene should be encouraged throughout HCT recovery, especially during neutropenic periods. Oral hemorrhage associated with thrombocytopenia is rarely a serious complication, although its occurrence can be frustrating as well as alarming. Local measures are directed to decreasing the flow of blood along with promoting adequate clot formation, followed by protecting the clot until healing has occurred. Topically applied vasoconstrictors, such as epinephrine or cocaine, can be helpful, although rebound vasodilatation can occur as the drug effect subsides. Direct pressure applied via gauze soaked in topical thrombin can be utilized as needed. Clot-forming agents, especially hemostatic collagen products (Avitene and Instat), fibrin glue, and chitosan (HemCon) can be used to organize and stabilize clots [1]. Salivary gland hypofunction Normal salivary function is essential to preserving oral health. In addition to its lubricating properties, saliva normally contains numerous antimicrobial factors that contribute to host defense. These components include mucins that inhibit microbial adherence to mucosa, secretory immunoglobulin A, lactoferrin, lactoperoxidase, transferrin, and other proteins that additionally affect microbial colonization and proliferation [53,72,73]. Salivary gland hypofunction is frequently associated with HCT [54,74]. Patients may report various degrees of oral dryness (xerostomia) in relation to these changes. Etiology may include toxicity associated with conditioning regimens, GVHD or use of anticholinergic medications [54,75]. Correlation of clinical symptoms and medical events will often reveal the likely cause. For example, ionizing radiation is the component of the transplant-conditioning regimen most likely to induce salivary disturbances (although chemotherapy can also cause salivary gland damage). Numerous other drugs or interventions may alter salivary function. For example, antihistamines, anticholinergic drugs, antiemetic drugs, and tricyclic antidepressants can reduce saliva secretion. Oral dryness can be exacerbated by mouth breathing or oxygen administration. In contrast, drug-induced salivary hyperfunction is rare. Most instances of apparent sialorrhea are due to dysphagia and/or inadequate swallowing of secretions due to painful oral mucositis, pharyngitis or nausea and vomiting triggered by swallowing oral secretions. Inflammation of the major salivary glands is usually not a problem following TBI. However, if doses of radiation above 2000 cGy are administered to the salivary gland fields, swelling of the glands due to sialadenitis can be seen. Radiation sialadenitis is usually asymptomatic. However, any time salivary gland flow rates decrease, there is a risk for

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infection with the retrograde spread of oral bacteria up ductal structures to involve salivary gland parenchyma. Salivary gland infections are characterized by swelling and pain. In more severe cases, suppuration can be expressed from gland orifices. Treatment includes antibiotics, stimulation of glands (e.g. lemon drops or sialogogues), and application of heat over the glands. Xerostomia can significantly compromise the quality of life for patients. In addition, salivary hypofunction can lead to oral lesions caused by trauma or the colonizing microflora. Techniques to manage xerostomia include frequent sipping of water, rinsing with bland oral rinses (0.9% saline or sodium bicarbonate rinses), and the use of artificial saliva and oral moisturizers. These strategies can promote mucosal lubrication and hygiene. Strategies to stimulate salivary flow can also be of benefit. Sugar-free lemon drops, mints, and gums can transiently increase salivary flow rates due to stimulation of glands through the sense of taste. Sialogogues (pilocarpine, bethanechol, and cevimeline) can be used to stimulate the salivary glands and improve oral moisturization (Table 103.6) [76–78]. Conditioning regimen-related salivary gland dysfunction will generally improve over the first 3–6 months after transplantation. In some instances, however, damage to the glands may be permanent. GVHDinduced salivary gland dysfunction clinically resembles Sjögren’s syndrome and results from injury caused by donor lymphocytes reacting against ductal and acinar tissue (Plate 103.3). Mucoceles can occur in association with oral GVHD, with the labial and soft palate mucosa being the most frequently involved sites. These mucoceles can often be initially confused with HSV vesicles, although pain is not a prominent feature of salivary gland GVHD. Patients with chronic xerostomia are at increased risk of dental caries and should be placed on appropriate regimens to minimize dental plaque and provide for enamel remineralization via topical fluorides and/or remineralizing solutions (Table 103.6) [2,79]. Oral GVHD GVHD represents an immune-mediated disease principally occurring after allogeneic transplantation, although cases of autologous or “pseudo” GVHD have been described (see Chapters 27 and 84–87). Oral GVHD mimics a number of naturally occurring autoimmune disorders, including lichen planus, lupus erythematosus, and systemic sclerosis [80–82]. GVHD involvement of the salivary glands can result in salivary gland dysfunction and symptoms of mouth dryness. General oral sensitivity and discomfort to normally tolerated agents (e.g. spices, flavoring agents, and alcohol) can be associated with GVHD. The clinical presentation of oral GVHD is similar in both acute and chronic GVHD, with mucosal erythema, atrophy, and white hyperkeratotic striae, plaques and papules consistent with oral lichen planus being classic components (Plates 103.2 and 103.3). In more severe cases, pseudomembranous ulcerations can be noted. The posterior buccal and lower labial mucosa and lateral and ventral regions of the tongue are the most commonly involved sites. In allogeneic HCT, acute oral GVHD is usually not discernible from oral mucositis until after 21–24 days post transplant; early (prior to day 21), especially hyperacute, oral GVHD will generally be noted as severe oral mucositis that is slow to resolve. Oral acute GVHD occurring between 28 and 120 days post HCT will present with clinical changes that are recognizable of being erythematous areas with varying degrees of lichenoid hyperkeratotic lesions. Chronic oral GVHD presents in similar fashion to acute GVHD, and becomes apparent between 70 and 500 days after HCT. Autologous/syngeneic oral GVHD reactions have been noted following autologous and syngeneic HCT, with lichenoid reactions being the most recognizable clinical manifestations.

Table 103.6 Management of post-transplantation salivary gland dysfunction Oral moisturization Frequent sipping of fluids Artificial saliva and oral lubricating agents 䊊 䊊

Stimulation of salivary glands Sugar-free candy, mints, and gum Sialogogues – Pilocarpine (5–10 mg 3–4 times a day, maximum of 30 mg/day) – Cevimeline (30 mg 3 times a day) – Bethanechol (25–50 mg 3–4 times a day to a maximum of 150 mg/ day) 䊊 䊊

Xerostomia decay prevention Plaque removal: tooth brushing, flossing, other oral hygiene aids Remineralization: Topical high-concentration fluorides* 1.1% neutral sodium fluoride gel (preferred) 0.4% stannous fluoride gel Brush on for 3–5 minutes, spit out, and rinse as directed. Apply once a day Reusable vinyl fluoride trays: put 1–2 drops per tooth in tray and hold in mouth for 3–5 minutes once a day Calcium/phosphate remineralizing solutions Topical antimicrobials: 0.12–0.20% chlorhexidine oral rinse (use alcohol-free preparations whenever possible): rinse, hold in the mouth for 1–2 minutes and spit out Tetracycline oral rinses (250 mg in 4 oz water, prepared daily): rinse/ hold for 1 minute and then spit out Diet modification: reduce exposure to refined carbohydrates, sugars, etc. 䊊 䊊

䊊

䊊

* Prescription-strength fluorides should be used; nonprescription fluoride preparations are felt to be inadequate in the face of moderate-to-high risk for dental caries.

As noted above, GVHD can injure the major and minor salivary glands, with resulting xerostomia and mucoceles (Plate 103.3). With extensive chronic GVHD, the extent and severity of oral lesions can become more disabling. Mucosal lesions can cause significant pain and produce an increased risk of oral infection. Sclerodermatous changes can result in perioral fibrosis that decreases oral opening and interferes with oral function. Oral secondary infections caused by HSV, CMV, and Candida species, or trauma, can exacerbate oral GVHD lesions and confound the diagnosis [35]. Furthermore, chronic dental and periodontal infections have been noted to exacerbate oral GVHD, and elimination of these infections can often dramatically improve patients’ oral status. Symptomatic oral GVHD will often respond to topical immunosuppressive therapy, although more severe or recalcitrant cases can require systemic immunosuppressive therapy. Topical management of oral GVHD can include topical application of steroids (rinses, creams or gels), cyclosporine, azathioprine, tacrolimus, and oral psoralen and ultraviolet light A therapy (Table 103.7) [80,82]. These interventions can reduce the symptoms and severity of oral lesions (especially pseudomembranous ulcerations), but none will generally produce complete resolution of oral involvement. Extracorporeal photopheresis has been shown to reduce the oral signs and symptoms in patients with treatmentresistant GVHD [83]. GVHD-induced salivary hypofunction is usually associated with oral mucosal lesions but can occur independently of mucosal involvement. Salivary gland dysfunction not only impacts quality of life for patients, but also can have a negative effect on oral and dental health. Salivary

Oral Complications of Hematopoietic Cell Transplantation

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Table 103.7 Topical management of oral chronic graft-versus-host disease (GVHD) (a) Topical oral therapy

Considerations for use

Preuse oral care

1. Rinse mouth with plain water; avoid normal saline 2. Try not to eat or drink or brush teeth with toothpaste for 15–20 minutes before use of topical agent

Rinses

1. Use adequate volume to easily coat oral tissues. 5–10 mL usually works well 2. Hold solution in mouth for 3–5 minutes before spitting out. Rinses can be held longer as (a) oral lesions are more severe and/or (b) the patient is not able to use rinse the prescribed number of times a day 3. Avoid eating or drinking for 20–30 minutes after use when possible

Gels

1. Try to dry lesion before application as this will improve adhesion to the lesion 2. Gels will generally melt at mouth temperature, but can be warmed slightly as noted below for creams if it helps with application 3. Gently apply gel to the lesion with a cotton swab or a clean fingertip. The key is to apply like “icing a cake,” not by trying to rub into the tissue 4. Avoid eating or drinking for 20–30 minutes after use when possible Alternatively: Apply ointment to a 2 × 2 sterile gauze and place on top of the lesion, keeping in place for 10–15* minutes. Remove gauze and avoid eating or drinking for 20–30 minutes after use when possible

Creams

1. 2. 3. 4.

Ointments

1. Try to dry the lesion before application as this will improve adhesion to the lesion 2. Ointments can be warmed slightly as noted above for creams 3. Gently apply warmed ointment to the lesion with a cotton swab or a clean fingertip. The key is to apply like “icing a cake,” not by trying to rub into the tissue 4. Avoid eating or drinking for 20–30 minutes after use when possible* Alternatively: Apply ointment to a 2 × 2 sterile gauze and place on top of the lesion, keeping in place for 10–15* minutes. Remove gauze and avoid eating or drinking for 20–30 minutes after use when possible Note: Ointments are especially useful for treating vermillion lip GVHD (chapping, cracking, etc.)

Inhalers

1. Dry oral tissues slightly 2. Use one or two “puffs” without inhaling – can be directed specifically towards lesions 3. For general oral GVHD, gently coat the mouth with the medication 4. If problems occur with oral candidiasis, in addition to prescribing antifungals, consider having the patient rinse the mouth 10–15 minutes after use

Dry the lesion before applying Warm a metal spoon in hot water, dry and place medication in the spoon to semi-liquefy Apply softened/liquefied cream to the lesion Avoid eating or drinking for 20–30 minutes after use when possible*

Notes: * Unless specifically instructed to do so, patients should avoid swallowing topical drugs for at least 20–30 minutes after using. Strategies (spitting into a cup, facial tissue, etc.) should be discussed. a) Systemic uptake can occur from absorption through tissues or swallowing – patients may require monitoring of absorbed drug. b) Monitor mouth for emergence of oral candidiasis. Note: atrophic and erythematous candidiasis can occur and be difficult to differentiate from oral GVHD. Candida cultures may be important. Some clinicians will use prophylactic antifungals with topical steroids.

(b) Corticosteroids†

Rinses

Agent

Instructions for use

Dexamethasone

0.1–0.4 mg /mL rinse. 5–10 mL swish/held for 3–5 minutes. Spit out. Repeat 3–6 times a day 0.3–0.6 mg/mL rinse. 10 mL swished/held for 15 minutes. Spit out. Repeat 2–4 times a day 0.5 mg tablet dissolved in 10 ml, held for 3 minutes, spit out. Repeat 4 times a day 1–2 puffs in mouth 2–4 × day 1–2 puffs in mouth 2–4 × day 1–2 puffs in mouth 2–4 × day 1–2 puffs in mouth 2–4 × day

Budesonide Betamethasone Sprays/inhalers

Beclomethasone Fluticasone Betamethasone Triamcinolone

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Table 103.7 Continued

Gels, creams, and ointments

Agent

Instructions for use

0.05% Clobetasol cream, ointment, gel, solution 0.05% Halobetasol cream, ointment 0.05% Fluocinonide cream, ointment, gel 0.1–0.5% Triamcinolone cream 0.05–0.1% Betamethasone cream, ointment

Apply to lesions Apply to lesions Apply to lesions Apply to lesions Apply to lesions clotrimazole)

Non-steroidal immunosuppressives Rinses Cyclosporine‡

× × × × ×

a a a a a

day day day day day (one formulation includes 1%

Rinse: 100 mg/mL 5 ml swished/held for several minutes, spit out repeat 3 × day 1.5% gel applied to lesions 2 × day 5–10 mg/ml 5–10 ml swished and held for 3–5 minutes, spit out. Repeat 2–6 × day 0.5 mg/dL in bioadherent base 5mg/ml in 3% methylcellulose gel base, 1–2 ml applied to lesion 3–4 × day Apply 0.5 inch to gauze, hold over lesion for 15–20 minutes 2 × day. Monitor plasma levels Product is not currently available; Clinical trials currently underway

Azathioprine Gels/other

2 2 2 2 2

Cyclosporine Azathioprine 0.1% Tacrolimus Thalidomide

Phototherapy PUVA

Lasers

Dental infections

UVB Extracorporeal photopheresis (systemic/extracorporeal treatment) Low-level laser therapy Carbon dioxide laser

0.3 mg/kg psoralen followed by 0.5–6 J/cm2 UVA radiation (dose increased as tolerated), 3–4 × week 0.02 mJ/cm2 with escalating 0.02 mJ/cm2 every 4th treatment

632–660 nM, 2–4 J/cm2 per treatment, treated 2–3 times a week Defocused 1 W held 1 cm from surface, 2–3 seconds (surface kept moist) for pain control

Aggressive prevention/treatment/elimination of all dental and periodontal infections, including extraction of involved teeth

PUVA, psoralen + ultraviolet A; UVB, ultraviolet B. † Anti-Candida prophylaxis may be needed, especially with inhalers. ‡ Cyclosporine is expensive and has met with mixed effectiveness. (Adapted from [105], with permission.)

hypofunction interferes with oral function and can lead to symptoms of burning and mucosal irritation. Salivary hypofunction can increase the risk for rampant dental decay due to: (1) the loss of specific salivary proteins that help control oral flora, which can result in significant shift in oral flora to a predominance of dental decay-causing bacteria; and (2) a reduction in salivary calcium and phosphates, which are important for remineralizing enamel and dentine [79]. Patients need to be able to perform extremely efficient oral hygiene (brushing and flossing) along with using daily topical fluorides, calcium and phosphate remineralizing dental products, and possibly antibacterial mouth rinses. Attempts to improve salivary flow rates with sialogogues can not only increase resting salivary gland flow rates to improve patient comfort, but may actually help improve oral health as well [76–78]. Taste dysfunction Taste dysfunction is a neurosensory toxicity often associated with cancer chemotherapy and ionizing radiation [84]. While taste dysfunction has

long been reported by patients following HCT, the nature and patterns of taste dysfunction have not been extensively studied in this cohort of patients [85,86]. A survey of 69 autologous or allogeneic bone marrow or stem cell transplant patients in the first year following stem cell transplantation revealed that 66% had eating difficulties at day 50; anorexia, dry mouth, and altered taste were among the abnormalities reported [87]. This trend was also observed in a small study (n = 25) of patients 2–4 years after allogeneic transplant. The taste receptor cell is derived from neuroepithelium. With a turnover rate of approximately 10 days, it can regenerate if not irreversibly damaged [88]. It is thus conceptually possible that the number of functional taste receptor cells will decrease to a level insufficient for detecting chemical stimuli, with resultant ageusia reported by the patient. In addition to damage to taste receptors, the potential for alteration or damage to olfactory receptor cells must be considered. Patients receiving cancer chemotherapy will occasionally describe symptoms consistent with dysgeusia (disordered or persistent unpleasant taste) resulting from diffusion of drug into the oral cavity, as well as venous taste phenomena [86].

Oral Complications of Hematopoietic Cell Transplantation

Taste dysfunction in HCT patients is not preventable; management initially consists of supportive care including alteration of the enteral diet. The symptoms associated with taste dysfunction generally persist for several months post transplant [85]. Normal taste function typically returns between 90 and 120 days following hospital discharge. Zinc sulfate supplements (220 mg twice daily, providing approximately 100 mg elemental zinc) have been reported to be helpful for radiationinduced taste dysfunction but have not been studied in the post-HCT setting. Neurotoxicity and orofacial pain Patients will frequently report dental hypersensitivity, especially to thermal stimuli, between 2 and 4 months after HCT. The mechanism is not understood, although it would appear to be associated with conditioning regimen toxicity. Symptoms usually resolve spontaneously within a few months. Topical brush-on fluorides or desensitizing toothpaste will generally reduce or eliminate symptoms. Oral neuromuscular complications resulting from treatment-induced neurotoxicity have been noted with a number of risk factors including cyclophosphamide, radiation, steroids, cyclosporine, and tacrolimus (FK506) (see Chapters 102 and 107). Tongue and jaw muscle tremors have been associated with high-dose cyclosporine therapy. Reducing drug doses usually promotes resolution. Similar neurotoxicity problems have been associated with thalidomide. TMD may present as facial pain, headache, muscle spasms or joint dysfunction, with occasional ear or pharyngeal pain. In most instances of recent-onset TMD, patients are prone to increased clenching or bruxing as a result of stress, sleep dysfunction or, occasionally, central nervous system toxicity from selected medications. Masticatory muscle tenderness upon palpation, temporomandibular joint dysfunction, and pain radiating to the ear are hallmark findings. The short-term use of muscle relaxants or anxiety-reducing agents plus physical therapy (moist heat applications, massage, and gentle stretching) will often resolve these symptoms. Occlusal splints may be indicated for patients to utilize while sleeping to reduce clenching or bruxing diatheses. Reduced-intensity conditioning regimens for HCT transplantation Research experience with reduced-intensity conditioning regimens continues to grow (see Chapter 71). While there have been no detailed research reports relative to oral complications of this form of hematopoietic transplant, the Seattle group has followed a large number of these patients for several years. With the use of lower-intensity conditioning regimens, there is essentially no oropharyngeal mucositis. Additionally, there is decreased salivary gland toxicity and less decrease in sense of taste. Early post transplant, there can be significant neutropenia, which increases the risk of oral infections. Bacterial dental and periodontal infections can be a concern, while prophylactic antifungal and antiviral therapy will generally prevent oral fungal and viral infections. Oral GVHD can emerge early post transplant, but it is not uncommon for patients to be free of GVHD 3–4 months post HCT; however, oral GVHD changes often occur when immunosuppression is withdrawn with the clinical presentation and the course is similar to that seen in standard allogeneic transplant patients. Day 100 and long-term follow-up oral assessments Evaluation of the oral cavity of allogeneic HCT patients approximately 70–100 days after transplantation and then at regular intervals can provide useful information relative to GVHD status. Clinical evaluations

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should be directed to determining the overall oral health (including dental infections) and the presence or risk for oral chronic GVHD. Day 100 oral examinations The day 100 oral examination should look to assess the general oral and dental health to detect the presence of or risk for dental, pulpal/endodontic, and periodontal disease. Patients should be encouraged to continue adequate oral hygiene. Patients with active decay or at risk for caries should be managed with a comprehensive program that can include topical fluorides, tooth remineralizing products, and antibacterial products (e.g. chlorhexidine rinses) [88]. Sialogogues, such as pilocarpine and cevimeline, can be used to increase baseline salivary flow rates and improve patient comfort. While it is suspected that improved flow rates will aid oral health, no studies have substantiated this relationship [77,78]. Sialogogues may have particular benefit in reducing xerostomia decay. However, even if patients respond to these agents and have reduced salivary dysfunction, systematic follow-up and aggressive strategies to prevent dental caries and periodontal disease are required. Long-term follow-up oral examinations Follow-up oral examinations can provide important information relative to GVHD activity and response to therapy. Additionally, oral pain/sensitivity and dryness related to oral GVHD can be identified, and appropriate therapy can often help ameliorate symptoms and improve oral function and health. Assessment for chronic dental and periodontal infections needs to be made, and appropriate dental treatment to eliminate them needs to be carried out. Clinical assessment for oral GVHD Oral examinations should be performed to delineate the presence of GVHD. Both oral labial mucosal biopsy (including minor salivary gland tissue) and clinical soft tissue examination can provide data supporting the diagnosis of GVHD as a component of day 80 assessments [80,89]. Oral mucosal changes including erythema, atrophy, lichenoid hyperkeratotic striae, and pseudomembranous ulcerations are associated with oral GVHD (Plates 103.2 and 103.3) [35]. Patients with clearly evident oral GVHD mucosal changes will generally not require biopsy to confirm oral mucosal and minor salivary gland involvement by GVHD. When oral changes are less diagnostic, oral biopsies can provide important diagnostic information [90]. Bisphosphonate-associated osteonecrosis of the jaws Bisphosphonate-associated osteonecrosis of the jaws (B-ONJ) is a recognized oral complication of bisphosphonate therapy that has been reported in the literature in 5–10% of patients receiving intravenous bisphosphonates [91–93]. It is defined as the unexpected occurrence of nonhealing exposed necrotic bone in the maxillofacial region in patients who have received bisphosphonate treatment without a history of radiation treatment to the involved bone. While intravenous bisphosphonates have been most frequently associated with B-ONJ, oral bisphosphonates have also been associated with B-ONJ. Lesions primarily involve the alveolar ridges and palate with varying degrees of inflammation of the soft tissues surrounding the exposed bone (Plates 103.4 and 103.5). Initially B-ONJ lesions are generally nonpainful, but they can become extremely painful as infection of the surrounding soft tissues occurs. Over time, B-ONJ lesions can expand and involve the surrounding bone and teeth. In severe cases, suppuration, bleeding, facial swelling, and external fistulas can occur. Dental extractions, periodontal surgery, and denture trauma are the most common identified causes of B-ONJ, but spontaneous lesions can also occur [92,94].

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Chapter 103

The pathobiology of B-ONJ has not been established, although it appears that a combination of osteoclast inactivation by bisphosphonates and secondary local infection is primarily involved; bisphosphonateinduced antiangiogenesis and direct mucosal toxicity have been proposed to be involved with B-ONJ, but there are few data to support these factors at this time. The risk for B-ONJ appears to be related to: (1) the antiresorptive potency of the bisphosphonate(s), (2) the route of delivery (intravenous versus oral), and (3) the duration of treatment. There are no predictable treatments that have been shown to prevent or heal BONJ, and thus the primary approach to preventing B-ONJ is preventing dental infections and avoiding dental surgeries unless absolutely unavoidable in treated patients. Discontinuing bisphosphonate therapy does not promote healing of established B-ONJ and, since bisphosphonates have been estimated to remain in bone for 5–10 years after discontinuing therapy, decisions to discontinue or continue with bisphosphonate therapy should be based on the efficacy of the bisphosphonate for the primary condition being treated and the availability of alternative therapies. When patients receive dental examinations prior to transplant, they need to be questioned about prior bisphosphonate use – both oral and intravenous bisphosphonates. Patients need to understand the oral health implications of bisphosphonate therapy, and the need to maintain oral health and avoid dental surgery. If patients are going to be started on bisphosphonates after transplant, a dental examination is mandatory prior to starting, and any significant dental disease needs to be eliminated and surgically treated sites healed before starting treatment [91,94]. Not all patients treated with bisphosphonates will develop B-ONJ after dental extractions and surgeries, but since there are no treatments currently that can predictably resolve B-ONJ, a totally preventative strategy should be used [92]. Treatment of established B-ONJ involves the administration of topical and systemic antibiotics and pain medications. Surgical revision of B-ONJ lesions is contraindicated because this has been shown to generally worsen lesions. Protection of exposed bone sites also can be helpful in preventing injury to opposing soft tissues. Several innovative therapies are being explored in clinical trials, including the administration of parathyroid hormone (teriparatide), systemic sodium fluoride, and local application of platelet-derived growth factors [95,96]. However, it will likely be some time before definitive curative strategies are developed. Routine dental care guidelines post HCT Complete dental examinations can be resumed generally between 120 and 180 days post HCT and should include examination of teeth and periodontal tissues, and radiographic assessments as indicated. However, resumption of routine dental treatment, including routine dental cleanings and restorations, is dependent upon the immune reconstitution and overall medical stabilization of the patient. Premature resumption of dental treatment can put the patient at risk for local infection, bacteremia, and aspiration pneumonia. In general, patients who have undergone autologous transplants can resume routine care approximately 5–6 months post HCT. Recipients of autologous grafts will usually have adequate immune reconstitution within 6 months of transplant to resume routine dental treatment, unless they have received a CD34-selected transplant or they have been conditioned with fludarabine-containing conditioning regimens, in which cases longer times may be required for immune recovery. Allogeneically treated patients without chronic GVHD who are off immunosuppression will generally require 9–12 months to recover adequate immune function to be candidates for routine dental care. Additionally, these patients should have discontinued immunosuppressive therapy for approximately 6 weeks and have no active GVHD before resuming routine dental treatment. As long as the patient

has active GVHD and is receiving immunosuppressive therapy, routine dental treatment should not be resumed. If dental treatment is imperative for patients without full immune or hematologic reconstitution, it is important that the dentist and physician determine what supportive medical care is necessary, including prophylactic antibiotics, immunoglobulin G administration, adjustment of steroid doses, and possibly platelet transfusions. Prophylactic antibiotic regimens similar to those recommended by the American Heart Association will often be adequate, with regimens being extended if there is ongoing dental infection or if there is concern for delayed healing. Dentists should also utilize techniques to reduce the risk of aspiration (e.g. use of rubber dams and high-volume suction), in addition to reducing the complexity of treatments and shortening treatment times. Edentulous patients can generally receive prosthetic care once engraftment has occurred and their medical condition has stabilized. Either transitional or permanent dentures are typically feasible in these circumstances. Orofacial/dental growth and development Children younger than 16 years who undergo HCT are at risk for the development of abnormalities in their developing dental and/or skeletal structures (see Chapter 104) [97,98]. Conditioning regimens, in addition to cancer therapy prior to transplantation, can damage tooth buds, with sequelae including enamel hypoplasia and root-growth alterations (root tapering and blunting, agenesis with premature apical closure, or complete agenesis). Inclusion of TBI in conditioning regimens is associated with increased risk of dental malformation when compared with chemotherapy without TBI [99]. While permanent teeth are most commonly involved, some children who have undergone transplantation prior to 1 year of age have developed both primary and permanent tooth malformations. Delayed dental eruption following HCT is common. Altered skeletal development of the craniofacial bones can result in orthognathic changes. Decreased tooth length can result in shortened alveolar processes, while damage to the maxillary and mandibular growth centers or decreased growth hormone secretion can result in decreased jaw size and arch length. It is useful to note that these skeletal changes appear to occur in a symmetric manner such that the child does not appear to have obviously hypoplastic jaws with corresponding esthetic changes; essentially, the face appears smaller but is relatively in proportion to the child’s overall stature due to general retardation in growth. Oral granulomatous lesions and gingival hyperplasia Late in phase II through phase III, pyogenic granulomas have been noted to occasionally form on the lateral and ventrolateral tongue and buccal mucosa [100]. These painless, hypertrophic lobulated lesions can range from several millimeters to several centimeters in length and height. They can be covered with atrophic mucosa, can be covered by a pseudomembranous fibrin exudate or can have a granulomatous-appearing surface. Histological examination reveals granulation tissue. Trauma probably initially causes mucosal damage; then, as the granulation tissue forms, persistent trauma and irritation promote growth of the lesions. Due to the highly vascularized nature of these lesions, they can bleed easily when traumatized. Primary therapy focuses on preventing recurrent trauma to the lesions and the reduction of inflammation through the use of topical steroids. Patients have reported that their polyps have regressed spontaneously when protected from trauma and treated with steroids (immune status recovery probably plays a role in this process). Treatment of persistent or enlarging granulomas usually requires surgical removal followed by careful primary closure or laser ablation.

Oral Complications of Hematopoietic Cell Transplantation

Gingival hyperplasia is not a frequent complication of HCT and usually represents a reaction induced by medication with cyclosporine. Patients with persistent high blood level doses appear to be at higher risk for this problem. The use of calcium channel blockers to manage cyclosporine-related hypertension, especially nifedipine, can further increase the risk of this complication. The gingival hyperplasia is related to excessive production of collagen by gingival fibroblasts. Mild cases may not need to be treated, but more extensive involvement generally requires gingivectomy using either surgical excision or surgical laser therapy.

Second malignancy post HCT The occurrence of new or second malignant neoplasms and lymphoproliferative disorders following HCT has long been recognized as a significant complication for long-term survivors of HCT (see Chapter 106) [101,102]. Oral cancers, especially squamous cell carcinomas and salivary gland tumors, are among the most common types of solid secondary oral malignancy in both adult and pediatric long-term survivors of HCT [101,102]. Studies have shown that age at the time of HCT, GVHD (acute and chronic), duration of GVHD prophylaxis and therapy, and treatment with azathioprine are specific risk factors for oral squamous cell carcinomas [103].

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Unfortunately, oral dysplasia and malignancies can often be obscured by oral GVHD or even mimic GVHD, and only biopsy can discriminate between the two. Lymphoproliferative disorders have been noted to have head and neck presentations, including enlarged submandibular and cervical lymph nodes [104]. Consequently, consistent and comprehensive oral soft tissue and head and neck examinations become a critical component of patient follow-up for all post-transplant survivors, with specific attention paid to early detection of potentially dysplastic and malignant changes. Long-term survivors of HCT should be instructed to monitor oral lesions appropriately – common oral lesions (e.g. due to trauma, irritation or aphthous ulcers) will typically heal in 7–10 days. Lesions persisting longer than 2–3 weeks should be carefully assessed and considered for biopsy.

Conclusion Development of new technologies to prevent conditioning-induced oral mucositis could substantially reduce the risk for systemic infection, the pain, and the number of hospital days. Both improvements in quality of life and reductions in hospital costs could emerge. As important, the prevention of oral mucositis could provide the setting in which new classes of chemotherapeutic drug utilized at increased doses could be considered, with possible improvements in cancer cure rates and durability of disease remissions.

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neic hematopoietic stem cell transplantation. Exp Hematol 2007; 35: 184–92. Oneschuk D, Hanson J, Bruera E. A survey of mouth pain and dryness in patients with advanced cancer. Support Care Cancer 2000; 8: 372–6. Epstein JB, Tsang AHF, Warkentin D, Ship JA. The role of salivary function in modulating chemotherapy-induced oropharyngeal mucositis: a review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002; 94: 39– 44. Nagler RM, Nagler A. Pilocarpine hydrochloride relieves xerostomia in chronic graft-versus-host disease: a sialometrical study. Bone Marrow Transplant 1999; 23: 1007–11. Carpenter PA, Schubert MM, Flowers ME. Cevimeline reduced mouth dryness and increased salivary flow in patients with xerostomia complicating chronic graft-versus-host disease. Biol Blood Marrow Transplant 2006; 12: 792–4. Chainani-Wu N, Gorsky M, Mayer P, Bostrom A, Epstein JB, Silverman S Jr. Assessment of the use of sialogogues in the clinical management of patients with xerostomia. Spec Care Dentist 2006; 26: 164–70. Kielbassa AM, Hinkelbein W, Hellwig E, MeyerLuckel H. Radiation-related damage to dentition. Lancet Oncol 2006; 7: 326–35. Woo SB, Lee SJ, Schubert MM. Graft-vs-host disease. Crit Rev Oral Biol Med 1997; 8: 201– 16. Treister NS, Woo SB, O’Holleran EW, Lehmann LE, Parsons SK, Guinan EC. Oral chronic graftversus-host disease in pediatric patients after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005; 11: 721–31. Imanguli MM, Pavletic SZ, Guadagnini JP, Brahim JS, Atkinson JC. Chronic graft versus host disease of oral mucosa: review of available therapies. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 101: 175–83. Foss FM, DiVenuti GM, Chin K et al. Prospective study of extracorporeal photopheresis in steroidrefractory or steroid-resistant extensive chronic graft-versus-host disease: analysis of response and survival incorporating prognostic factors. Bone Marrow Transplant 2005; 35: 1187–93. Marinone MG, Rizzom D, Ferremi P, Rossi G, Izzi T, Brusotti C. Late taste disorders in bone

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marrow transplantation. Clinical evaluation with taste solutions in autologous and allogeneic bone marrow recipients. Heamotologica 1991; 76: 519–22. Mattsson T, Arvidson K, Heimdahl A, Ljungman P, Dahllof G, Ringden O. Alterations in taste acuity associated with allogeneic bone marrow transplantation. Oral Pathol Med 1992; 21: 33– 7. Iestra JA, Fibbe WE, Zwinderman AH, van Staveren WA, Kromhout D. Body weight recovery, eating difficulties and compliance with dietary advice in the first year after stem cell transplantation: a prospective study. Bone Marrow Transpl 2002; 29: 417–24. Vissink A, Burlage FR, Spijkervet FK, Jansma J, Coppes RP. Prevention and treatment of the consequences of head and neck radiotherapy. Crit Rev Oral Biol Med 2003; 14: 213–25. Loughran TP, Sullivan K, Morton T et al. Value of day 100 screening studies for predicting the development of chronic graft-versus-host disease after allogeneic bone marrow transplantation. Blood 1990; 76: 228–34. Soares AB, Faria PR, Magna LA et al. Chronic GVHD in minor salivary glands and oral mucosa: histopathological and immunohistochemical evaluation of 25 patients. J Oral Pathol Med 2005; 34: 368–73. Migliorati CA, Siegel MA, Elting LS. Bisphosphonate-associated osteonecrosis: a long-term complication of bisphosphonate treatment [Review]. Lancet Oncol 2006; 7: 508–14. Erratum in: Lancet Oncol 2006; 7: 533. Mavrokokki T, Cheng A, Stein B, Goss A. Nature and frequency of bisphosphonate-associated osteonecrosis of the jaws in Australia. J Oral Maxillofac Surg 2007; 65: 415–23. Advisory Task Force on Bisphosphonate-Related Osteonecrosis of the Jaws, American Association of Oral and Maxillofacial Surgeons. American Association of Oral and Maxillofacial Surgeons position paper on bisphosphonate-related osteonecrosis of the jaws. J Oral Maxillofac Surg 2007; 65: 369–76. Migliorati CA, Casiglia J, Epstein J, Jacobsen PL, Siegel MA, Woo SB. Managing the care of patients with bisphosphonate-associated osteonecrosis: an American Academy of Oral Medicine position paper [Review]. J Am Dent Assoc 2005;

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136: 1658–68. Erratum in: J Am Dent Assoc 2006; 137: 26. Adornato MC, Morcos I, Rozanski J. The treatment of bisphosphonate-associated osteonecrosis of the jaws with bone resection and autologous platelet-derived growth factors. J Am Dent Assoc 2007; 138: 971–7. Harper RP, Fung E. Resolution of bisphosphonate-associated osteonecrosis of the mandible: possible application for intermittent low-dose parathyroid hormone [rhPTH(1–34)]. J Oral Maxillofac Surg 2007; 65: 573–80. Erratum in: J Oral Maxillofac Surg 2007; 65:1059. Dahllof G, Jonsson A, Ulmner M, Huggare J. Orthodontic treatment in long-term survivors after pediatric bone marrow transplantation. Am J Orthod Dentofacial Orthop 2001; 120: 459–65. Dahllof G. Craniofacial growth in children treated for malignant diseases. Acta Odontol Scand 1998; 56: 378–82. Hölttä P, Alaluusua S, Saarinen-Pihkala S, Wolf J, Nyström M, Hovi L. Long-term adverse effects on dentition in children with poor-risk neuroblastoma treated with high-dose chemotherapy and autologous stem cell transplantation with or without total body irradiation. Bone Marrow Transplant 2002; 29: 121–7. Kanda Y, Arai C, Chizuka A et al. Pyogenic granuloma of the tongue early after allogeneic bone marrow transplantation for multiple myeloma. Leuk Lymphoma 2000; 37: 445–9. Gallagher G, Forrest DL. Second solid cancers after allogeneic hematopoietic stem cell transplantation. Cancer 2007;109: 84–92. Curtis RE, Metayer C, Rizzo JD et al. Impact of chronic GVHD therapy on the development of squamous-cell cancers after hematopoietic stemcell transplantation: an international case-control study. Blood 2005; 105: 3802–11. Leisenring W, Friedman DL, Flowers ME et al. Nonmelanoma skin and mucosal cancers after hematopoietic cell transplantation. J Clin Oncol 2006; 24: 1119–26. Raut A, Huryn J, Pollack A, Zlotolow I. Unusual gingival presentation of post-transplantation lymphoproliferative disorder: a case report and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000; 90: 436–41. Schubert MM, Correa MEP. Oral graft-versus host disease. Dent Clin N Am (in press).

104

Jean E. Sanders

Growth and Development after Hematopoietic Cell Transplantation

Introduction

Thyroid function

High-dose marrow ablative chemotherapy or chemoradiotherapy followed by hematopoietic cell infusion for children and young adults with malignant and nonmalignant disorders has resulted in an ever-increasing number of long-term disease-free survivors. Reduced-intensity preparative regimens pre transplantation are also resulting in an increasing number of long-term survivors. An understanding of the late effects resulting from agents used in preparative regimens for hematopoietic cell transplantation (HCT) is necessary to appreciate and anticipate the effects on growth and development after transplantation. Because the preparative regimens are designed to use various agents to not only suppress the patient’s immune system, but also eradicate abnormal cells, the doses of the agents administered are not limited by marrow toxicity. The most common regimens utilize high-dose cyclophosphamide (CY) given alone or in combination with busulfan (BU) or other chemotherapy agents, and given with or without total body irradiation (TBI). Other chemotherapy agents that have been used in preparative regimens include high dose carmustine (BCNU), melphalan, etoposide (VP-16), fludarabine, and thiotepa. Other irradiation regimens include thoracoabdominal irradiation (TAI) and total lymphoid irradiation (TLI). The myeloablative doses of TBI are usually 8–14.0 Gy, whereas the doses of TBI used for nonmyeloablative transplant preparative regimens range from 2 to 6 Gy. Chapter 22 presents complete descriptions of various high-dose preparative regimens, and Chapter 23 presents the radiotherapeutic basis for HCT. Both high-dose chemotherapy and irradiation are known to affect the function of the neuroendocrine system, and therefore growth and development. Endocrine gland secretions act as catalysts to promote normal growth, and normal growth and development require balanced endocrine gland function. Growth- and maturation-promoting hormones include growth hormone (GH), thyroid hormones, androgens, and estrogens. The adrenal glucocorticoids are antagonistic to growth. This chapter reviews the effects observed to date of agents used in HCT high-dose preparative regimens on the endocrine function and the subsequent effects on growth and development of children and young adults that have been described to date. Chapter 105 presents other delayed complications observed after transplantation.

Normal thyroid hormone production is necessary for normal linear height growth in young children. Consequently, subnormal thyroid hormone production contributes to decreased growth in height. Thyroid function is usually not impaired following conventional chemotherapy, but irradiation to the thyroid gland has been associated with development of compensated hypothyroidism, overt hypothyroidism, thyroiditis, and thyroid neoplasms [1]. Irradiation of the thyroid causes subsequent hyperplasia and induction of nodules and malignancies. Following irradiation, the onset of thyroid dysfunction usually begins as asymptomatic compensated hypothyroidism with elevated thyroid stimulating hormone (TSH) and normal thyroid hormone production within the first year, and may progress to overt hypothyroidism with elevated TSH and subnormal thyroid hormone production over the next several decades. Hypothyroidism contributes to diminished linear growth in young children. Physical examination is often unreliable in detecting thyroid nodules. Fluctuations in serum thyroxine or triiodothyronine levels do not distinguish benign from malignant disease [2]. Ultrasonography detected thyroid abnormalities in 42 of 96 (44%) survivors of childhood cancer who received head and neck irradiation, compared with 14% with palpable lesions [2]. Thyroid nodules were detected by ultrasonography in 22 of the 96 (23%) patients and were significantly more frequent in patients whose thyroid exposure was more than 31 Gy. Some report thyroid neoplasia occurring between 1.5 and 6.0 years after radiotherapy, whereas other reports suggest a latency period of up to 40 years with a peak incidence occurring 15–25 years after irradiation exposure of 2–5 Gy [3,4]. While the prevalence of thyroid neoplasia in irradiated survivors of pediatric cancer is unknown, a 1991 epidemiologic investigation of 9170 patients who survived childhood cancer for at least 2 years reported a 53-fold increased risk of thyroid neoplasia [5]. Doses less than 2 Gy were associated with a 13-fold increased risk. Other studies suggest a radiation dose–response relationship as well as a relationship to patient age at time of exposure, with younger age patients at highest risk [4,6]. Epidemiology studies of thyroid malignancies among individuals exposed to irradiation from the 1986 Chernobyl accident have suggested an age-associated incidence of thyroid cancer. Among children less than 15 years of age, one case of thyroid cancer was observed prior to the Chernobyl accident (rate = 0.5/year/million children), but 21 cases were observed between 1986 and 1990 (rate = 10.5/year/million children) and 143 cases during 1991 and 1994 (rate = 97/year/million children) [7]. At the time of the accident, their average age was 3.8 years (±2.4 years),

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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and their average age at time of diagnosis was 9.4 years (±2.8) years. More than 90% of children were less than 6 years of age, and 3 were in utero at the time of the accident. This observation is in contrast to results observed among 1984 men who were Chernobyl clean-up workers, in whom 201 (10.2%) developed thyroid nodules whose biopsies indicated benign nodular disease, two cases of papillary carcinoma, and three benign follicular neoplasms [8]. Thus, the susceptibility of the thyroid to carcinogenetic effects of irradiation is particularly apparent in younger individuals, and suggests that children who have received TBI, TAI or TLI in their HCT preparative regimens are at risk for development of thyroid abnormalities. Since no single historical factor, physical finding or clinical laboratory result is pathognomonic of thyroid neoplasia, use of ultrasound to detect subtle architectural changes in the thyroid gland may be useful as a screening tool to detect asymptomatic nodules. Preparative regimens consisting of chemotherapy only After HCT, thyroid function has usually been evaluated with TSH, thyroxine (T4), and triiodothyronine (T3) plasma levels. Abnormal values often prompt further study with free T4, free T3, resin T3 uptake, and TSH response to thyrotropin-stimulating hormone. The majority of results from reported studies (Table 104.1) show that chemotherapy regimens including CY, BU plus CY, and other chemotherapy agents plus CY have not resulted in thyroid function abnormalities in the majority of patients [9–16]. Idiopathic thyroiditis developed at age 14 (10 years after HCT) in one child among 100 transplanted for severe aplastic anemia after a preparative regimen of CY (200 mg/kg) [9], an incidence that is not different from the expected 1% observed in a normal population of school-age children. Children given BUCY preparative regimens and marrow transplantation for acute myeloid leukemia (AML) have shown a 3% incidence of hypothyroidism, suggesting that patients who received BUCY are at low risk for development of hypothyroidism [12]. While thyroid malignancies have not yet been observed after BUCY, longer follow-up is needed to be able to determine whether thyroid malignancies will occur. Preparative regimens containing irradiation Compensated hypothyroidism and overt hypothyroidism have often occurred following TBI or TLI preparative regimens [13,14,16–20]. A recent report evaluated thyroid function in 153 children with more than 5 years of follow-up [14]. Sixteen (9%) developed overt hypothyroidism at a median of 2.9 years (1–10), and 31.4% developed compensated hypothyroidism at a median of 2.7 (0.8–11) years after transplant. The thyroid dysfunction-free survival was 73.2% after 5 years and 59.2%

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after 10 years. Factors associated with the development of hypothyroidism were patient age less than 9 years, single-dose TBI, and HCT in second complete remission. Thyroid nodules not associated with malignancy developed in 10 of 35 patients evaluated at a median of 7.8 years. This need for regular thyroid function screening for years after transplant confirms the previous study by Ishiguro et al., who found that age under 9 years at HCT and receiving TBI were the strongest factors in the subsequent development of decreased thyroid function [21]. Among 181 children given 7.5 Gy single-exposure TLI or 7.8–10.0 Gy single-exposure TBI, compensated hypothyroidism developed in 35%, and overt hypothyroidism developed in 12% (Table 104.1). These findings are in contrast to a 13% incidence of compensated hypothyroidism and 4.2% incidence of overt hypothyroidism observed among 452 children after 12.0–15.74 Gy fractionated TBI. The patients in these longitudinal studies where single-exposure TBI was administered were followed for more than 8 years after irradiation exposure, whereas those given fractionated exposure TBI were followed for a median of 4–6 years after irradiation exposure. Although fractionated TBI appears to result in a lower incidence of thyroid abnormalities, data from nontransplant irradiation studies have shown that a risk for development of thyroid dysfunction and thyroid malignancies may be delayed several decades. The development of thyroid dysfunction has not been associated with sex, age at transplantation or acute or chronic graft-versus-host disease (GVHD). Detailed evaluations of the hypothalamic–pituitary– thyroid neuroendocrine axis with free thyroxine, resin T3 uptake, thyrotropin-stimulating hormone, and microsomal and thyroglobulin antibody have demonstrated that the major effect of irradiation is at the level of the thyroid gland and not at the level of the hypothalamus or pituitary gland [11]. Treatment All patients in whom overt hypothyroidism develops should receive treatment with thyroxine, but the benefit of thyroid replacement in patients with compensated hypothyroidism is controversial. Although the carcinogenic potential of thyroid irradiation has been well documented, the ability of thyroid replacement to reduce the incidence of radiation-associated thyroid carcinoma remains unproven [1,22]. Recommendations from pediatric endocrinologists vary with respect to treatment of compensated hypothyroidism after irradiation therapy. Among patients in whom benign thyroid nodules develop after conventional irradiation therapy, treatment with thyroxine decreases the risk of recurrence of these nodules but does not decrease the risk of thyroid carcinoma. Papillary carcinoma, toxic goiter, and an adenoma have been observed between 4 and 14 years in six children after 10.0 Gy single-exposure

Table 104.1 Thyroid function following transplant Preparative regimen

Number of patients evaluated Follow-up (median in years) Normal thyroid function Compensated hypothyroidism Hypothyroidism References

CY (%)

BUCY (%)

CY + TLI (%)

7–10 Gy S-TBI (%)

12.0–15.75 Gy F-TBI (%)

100 9 99 (99%) 0 1 [9,14]

108 2 96 (88) 4 (3%) 8 (7%) [10–12,14,17]

28 5 26 (93%) 1 (3%) 1 [13,18]

181 6 102 (56%) 56 (31%) 23 (12%) [10,14,18–20]

452 4 372 (82%) 60 (13%) 20 (4%) [10,13,14,17–19]

BU, busulfan; CY, cyclophosphamide; F-TBI, fractionated-exposure total body irradiation; S-TBI, single-exposure total body irradiation; TLI, total lymphoid irradiation.

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TBI and in at least five after exposure to 12–15.75 Gy fractionated TBI [11]. All of these children had abnormal thyroid function, and none had received thyroid hormone therapy prior to discovery of the thyroid mass. The adenoma was found at autopsy, but the other patients were treated successfully with thyroidectomy or radioactive iodine thyroid ablation. Thus, all patients who have received TBI, TLI or TAI should be examined annually with physical examinations and tests of thyroid function and perhaps ultrasound. The euthyroid sick syndrome concerns changes in thyroid hormone metabolism in patients with nonthyroid illness, such as any severe illness and some patients undergoing HCT. The level of TSH decreases rapidly during the first few months after HCT but gradually recovers to normal during the first 6 months. Acute changes in thyroid hormone levels also gradually recover to pre-HCT levels by 6 months. Increased levels of cytokines including interleukin-6 and tumor necrosis factor-alpha post HCT were found to inversely correlate with the levels of thyroid hormones. This euthyroid sick syndrome may occur frequently after HCT but does not require specific thyroid therapy [23].

Growth Linear growth is a continuous and finely regulated phenomenon that is the result of the interaction of genetic make-up, nutritional factors, hormones, metabolism, and cerebrocortical influences. The importance of each major factor varies with different periods of growth. Growth in infancy is largely determined by nutrition and metabolic factors. Growth in childhood is largely influenced by GH and in puberty by the synergistic action of GH and sex steroids. Thus, GH has a major role in the growth process. This is most likely related to the effect of chemotherapy on the GH–somatomedin–chondrocyte axis and/or related to cranial or cranial spinal irradiation. The impact of GH deficiency on subsequent growth in this population is difficult to determine since GH studies have not been performed consistently. Data are difficult to interpret as to whether intensive chemotherapy regimens with or without central nervous system (CNS) irradiation are associated with persistent growth impairment since often only growth rates are reported [24–28]. Some studies suggest that the intensity and duration of combination antineoplastic therapy as well as CNS irradiation and age at diagnosis influence patterns of growth [29–31]. The younger the child is at the time of CNS irradiation, the greater the loss of growth during puberty. Three studies have compared growth outcome for children treated with conventional chemotherapy with or without cranial irradiation and HCT. One study [32] of children with AML concluded that patterns of growth were similar between those who were treated with chemotherapy and those treated with HCT. Follow-up for the 26 patients in the chemotherapy group was 7.4 (1.8–15.5) years and follow-up for the 26 patients in the HCT group was 5.6 (2.0–15.4) years. None of the patients had GH testing performed, 11 chemotherapy patients had received 18– 24 Gy cranial irradiation, and nine HCT patients had received TBI. The change in height from time of diagnosis to last follow-up was −0.43 standard deviation (SD) for chemotherapy patients and −0.48 SD for HCT patients. A second comparison study [33] included 10 patients given chemotherapy, 18 patients given chemotherapy plus 18.0–24.0 Gy cranial irradiation, and 15 patients given chemotherapy plus 12–15 Gy TBI and HCT. Change in height SD was −0.21 for the chemotherapy group, −1.26 for the chemotherapy plus cranial irradiation group (p = 0.0001), and −1.33 for the HCT group (p = 0.0008). All patients had GH testing performed, and the only patients with GH deficiency were in the HCT group (p = 0.003). The third study evaluated infants diagnosed less than 1 year of age with AML and acute lymphoblastic leukemia (ALL) [34]. Ten were

treated with chemotherapy only, 17 received chemotherapy plus cranial irradiation, and seven received HCT (six with TBI). Median change in height SD at 5 years after diagnosis was −0.9 for the chemotherapy group, −1.0 for the chemotherapy plus cranial irradiation group (p = 0.07), and −2.3 for the HCT group (p = 0.01). Further follow-up for a small number suggested that height continued to decline with increasing time after diagnosis. These studies suggest that recipients of chemotherapy only without cranial irradiation have height losses that are substantially less than are observed after HCT using TBI-containing regimens. Because GH secretion is episodic, determination of GH levels involves use of a stimulus to enhance pituitary GH secretion followed by multiple venous blood samplings. Commonly used stimuli include exercise, sleep or pharmacologic agents such as clonidine, levodopa, arginine, and insulin. Failure to attain a normal circulating GH level after two different stimulus tests accompanied by decreased growth rates or height SD score (height minus mean height for age and sex divided by the SD of height for age and sex) defines classic GH deficiency. Children who have received cranial irradiation have had variable responses to the provocative stimuli used, which may be due to the use of different pharmacologic agents, variations in the interval between irradiation and testing or neurosecretory defects [35–37]. Although the 24-hour spontaneous pulsatile GH secretion test is considered by many to be the most physiologic assessment of GH secretion, it is impractical due to the large volumes of blood required (5 mL every 20 minutes for 24 hours) [38,39]. The 12-hour overnight sampling schedule has been shown to be well tolerated, reliable, and reproducible, but is less frequently used due to the inconvenience of overnight hospitalization and extensive blood sampling [40]. After TBI, different results between spontaneous GH production tests and stimulated GH response tests often make interpretation of GH data and diagnosis of GH deficiency difficult. This is particularly challenging when one or both GH responses are normal despite decreased growth rates [41,42]. Repeating these tests 1 or 2 years later will often clarify GH production responses. The standard utilized by pediatric endocrinologists today is two different GH tests to assure the diagnosis of biochemical GH deficiency. Most investigators utilize two different tests of GH stimulation, such as the clonidine, insulin tolerance test, and/or arginine stimulation tests. Subnormal results accompanied by decreased growth rates indicate GH deficiency. CNS irradiation has been associated with the development of GH deficiency, which appears to be related to the child’s age at time of irradiation, the irradiation dose received, and the length of time lapsed after completion of irradiation [35]. Some children, especially those with ALL, referred for bone marrow transplantation (BMT) have received 18–24 Gy CNS irradiation as part of their initial treatment prior to referral for BMT. When TBI is included in their preparative regimen, their total CNS irradiation dose will usually exceed 30 Gy, the estimated threshold for development of GH deficiency [43]. GH deficiency may be expected to develop 2–3 years after irradiation in the majority of patients who received this total dose of CNS irradiation, whereas GH deficiency may not develop in patients who receive lower doses of CNS irradiation for up to 5–10 years after irradiation. Thus, it may be anticipated that nearly all children who have received CNS irradiation in addition to TBI are likely to develop GH deficiency, but GH deficiency may not develop in those who receive only TBI just prior to or after their growth period, depending on their age at TBI. Preparative regimens containing chemotherapy Following preparative regimens with high-dose CY only, normal growth rates and height SD were observed in 91 children [44]. Height SD and

Growth and Development after Hematopoietic Cell Transplantation

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Fig. 104.1 Height standard deviation curves for 38 boys and 35 girls transplanted for severe aplastic anemia with cyclophosphamide (CY) only as the preparative regimen.

growth velocity of a group of children with severe aplastic anemia prepared with 200 mg/kg CY are shown in Figs 104.1 and 104.2. When tested, normal levels of GH were detected. Some children with chronic GVHD treated with corticosteroids had decreased growth velocity during the time chronic GVHD was active and while being treated with corticosteroid therapy. Once chronic GVHD was controlled and corticosteroid therapy discontinued, catch-up growth occurred prior to growth rates returning to normal for age. Final height achieved for 26 girls and 25 boys was at a median of the 25th percentile (range 5–90th) for girls and the 50th percentile (range 5–95th) for boys. The European Group for Blood and Marrow Transplantation reported that mean final height SD among 26 patients prepared with CY only was −0.15 ± 1.68 [45]. BU, an alkylating agent frequently combined with CY in preparative regimens, is an agent that affects dividing cells as well as nondividing cells, and crosses the blood–brain barrier [46]. The effect of BU on growth has been reported for children prepared with 14 mg/kg BU and 200 mg/kg CY prior to BMT for thalassemia major [10,47]. Both studies found an effect of patient age at the time of HCT on subsequent growth. Younger patients (≤7 years [47] and <10 years [10]) have growth that was consistent over time after HCT. Among 47 patients evaluable for final adult height, the 26 patients who were 7 years or less at HCT had a final adult height consistent with the height of their mother and father, whereas the 21 patients aged over 7 years at HCT had a final adult height that showed no relation with either parent’s height as these patients failed to achieve their full genetic height potential [47]. Among children prepared with 16 mg/kg BU and 120 mg/kg or 200 mg/kg CY prior to BMT for AML, similar data are emerging. Data among the AML children who had not received prior CNS irradiation have generally demonstrated normal growth velocity [11,12,45,48–51]. Studies have shown that height SD improves following HCT. One study of 23 children found no significant difference between height SD at HCT and yearly height SD from 1 to 5 years post-HCT, with a mean height at HCT of −0.38 SD to a mean height at 5 years of +0.11 [50]. The European Group for Blood and Marrow Transplantation reported final adult height to be −0.38 ± 1.16 for 10 children [45]. We have observed the final adult height z-score to be −0.3 to −0.4 (±1.46) SD or height at a mean of the 30th percentile for both boys and girls. Height SD and height growth velocity and are shown in Figs 104.3 and 104.4 for 47 boys and 39 girls following 16 mg/kg BU and 120 or 200 mg/kg CY. The impact of chronic illness on height growth has been demonstrated in one report of 30 children evaluated at 3 years after HCT. The 12 children with no post-HCT complications had change in height from HCT to 3 years of −0.2 SD, whereas the 18 children with complications (mainly chronic GVHD) had a change in height of −1.7 SD (p = 0.001),

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Fig. 104.2 (a) Height growth velocity curve for 35 girls transplanted for severe aplastic anemia with cyclophosphamide (CY) only as the preparative regimen. Note the blunted pubertal growth spurt, but growth for longer than usual. (b) Height growth velocity curve for 38 boys transplanted for severe aplastic anemia with CY only as the preparative regimen. Note the blunted pubertal growth spurt.

demonstrating that factors other than the preparative regimen have significant impact on the child’s growth [51]. Irradiation-containing preparative regimens Growth rates Growth impairment after TBI has been well documented [11– 13,41,44,45,48,49,52–61]. Children given single-exposure TBI usually have been the group with the greatest incidence of growth rate impairment, which often has also been associated with decreased GH production. Fractionated TBI was initially thought to have a growth-“sparing” effect, but these children have demonstrated decreased growth rates and decreased height SD scores [42,54,62,63]. One study comparing height SD scores for 26 patients given 9–10 Gy single-exposure TBI with those for 23 patients given 12–14.40 Gy fractionated-exposure TBI found that all had decreased height SD scores. The patients with single-fraction TBI had a change in height SD scores from −0.29 to −0.90 (p = 0.0001) between 1 and 3 years, whereas the height SD scores for patients who received fractionated TBI changed from −0.09 to −0.22 (p = 0.02) between 1 and 3 years after HCT [54]. Children who had received cranial irradiation before either single-fraction TBI or fractionated TBI had height SD scores less than those who had not received cranial irradiation [56]. A second study reported the mean cumulative height change during the first 3 years after TBI among prepubertal children not given cranial irradiation. These height changes were significantly worse for the 11 given 10 Gy single-exposure TBI (p = 0.001) (mean height change −1.4 SD) compared with seven children given 12 Gy fractionated TBI (p = NS) (mean cumulative height change −0.4 SD) [42]. A third study

Chapter 104

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Fig. 104.3 Height standard deviation curves for 39 girls and 47 boys transplanted with a preparative regimen of 16 mg/kg busulfan (BU) and 120–200 mg/kg cyclophosphamide (CY).

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Growth hormone GH stimulates growth of epiphyseal cartilage and subsequent bone growth directly via the action of insulin-like growth factor-I (IGF-I).

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reported no differences in height SD at 8 years after single-exposure TBI for 81 children. The mean height changes for 237 children who had not received cranial irradiation prior to 12.0–15.75 Gy fractionated TBI were −1.0 SD at 3 years and −1.3 SD at 6 years, and among the 79 who received cranial irradiation and fractionated TBI, the mean height changes were −1.4 SD at 3 years and −1.7 SD at 6 years. Prior cranial irradiation and years post transplant were the only factors found to significantly influence subsequent height SD scores. Children who had cranial irradiation were significantly shorter at the time of TBI compared with those who had not (p = 0.02). A similar effect of cranial irradiation on growth during the first 4 years after TBI has been observed by others [41,48,49,52,57]. Based on height at the time of transplantation and parental height, the impact of these children’s decreasing height SD scores on adult height is a final height that is lower than predicted [59]. Several investigators have reported that patients who received cranial irradiation in addition to TBI have lower height than those who received TBI without prior cranial irradiation [45,58,59]. Patients given fractionated TBI after having received cranial irradiation had lower height SD scores than those without cranial irradiation [45,58,59,62]. The final adult height of 14 children who received cranial irradiation and 10 children who did not was less than −2.0 SD, or within the range of what is considered to be normal height [59]. These patients were aged 6.3–14.6 (median 10.8) years at time of transplant. Patient age at transplant has been found to be a significant factor in predicting final adult height [45,58,59,62]. These studies have shown that children less than 10 years of age at time of transplant have the greatest risk for growth failure and significantly decreased final adult height. Patient gender has also been found to have a significant influence on final adult height, with boys at greatest risk of growth failure compared with girls [58,59,62]. Recent reports have confirmed the negative effect of young patient age and male gender on final height [62,64]. Bakker et al. were the first to report an effect of single-exposure TBI (5–8 Gy or 2 × 6 Gy) in subsequent body proportion [64]. Boys had a significant decrease in body length (sitting height) and leg length, whereas girls had a decrease in sitting height SD score that was not accompanied by a reduction in leg length. The decreased sitting height in both boys and girls is most likely related to the effect of radiotherapy on spine growth, which is greater than the impact of leg growth. These investigators found that the mean height SD loss between TBI and final height was −1.1 SD for girls and −1.5 SD for boys.

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14

16

18

Years of age

Fig. 104.4 (a) Height growth velocity curve for 39 girls transplanted with a busulfan (BU) and cyclophosphamide (CY) preparative regimen. Note the blunted pubertal growth. (b) Height growth velocity curve for 47 boys transplanted with a preparative regimen of busulfan and cyclophosphamide. Again, note the blunted pubertal growth.

When insufficient GH is secreted, growth velocity and bone maturation are usually delayed, and the divergence of the growth rate from normal increases with age unless replacement therapy is initiated. Studies of children with hypopituitarism have shown that the final adult height achieved is related to the height at the start of treatment [65]. The total height gained from GH therapy is inversely related to patient age at the start of treatment and positively related to the duration of therapy. Treatment with GH before the child’s height decreased to below the third percentile (≥−1.8 SD) results in the greatest final height response to treatment. Because growth before puberty is the major determinant of final height, treatment with GH during the prepubertal period needs to be optimized. Among nontransplant children who received cranial irradiation, GH deficiency may develop at the time of puberty in those with previously normal GH secretion due to an inability of the pituitary to increase production of GH in an amount to produce the growth spurt [30,66]. At puberty, normal production of gradually increasing doses of sex hormones contributes to the growth spurt. Many HCT patients have the problem of impaired sex hormone production, especially if the transplant preparative regimen included BU or TBI. The failure of the gonad to produce appropriate sex hormone contributes to their decreased height velocity. These individuals with primary gonadal failure may have improved height velocity from the additional use of gradually increasing doses of sex hormone therapy administered by the pediatric endocrinologist after the child achieves the age of 12–13 years [67].

Growth and Development after Hematopoietic Cell Transplantation

GH has additional beneficial effects for the growing child in addition to height growth. Children with GH deficiency are at risk for decreased bone mineral density due to reduced osteoblastic activity, leading to decreased bone mineral accrual and decreased bone mineral density [68–70]. Studies in children with GH deficiency have shown that GH deficiency-associated reduced bone turnover is reversed with GH therapy [71]. Because the foundation for skeletal health is established in childhood, prevention of decreased bone mineral density begins by optimizing gains in bone mineral acquisition throughout childhood. Children with decreased bone mineral density are at significant risk for decreased bone mineral density as an adult [58,72]. Bone health among children following HCT has not been well studied. One study reported that the effect of GH and bisphosphonate therapy on bone mineral density resulted in an improvement in bone mineral density of 45% (14–147%) among those treated with GH and bisphosphonate compared with those who received GH without bisphosphonate therapy, who showed values of 31% (3–59%) (p = 0.15) [73]. The number of patients in this study was small, which may preclude observation of a beneficial effect of the combination of bisphosphonate and GH. GH-deficient children have been reported to have behavior and cognitive disturbances including immaturity, anxiety, academic underachievement, and problems with social skills [74]. A study of psychologic morbidity of childhood GH deficiency suggests that GH replacement therapy results in improved behavior observed after 3 years of GH therapy [75]. There are no data reported among HCT children. GH also contributes to body mass, and those who are GH deficient have reduced lean body mass, omental obesity, hyperlipidemia, and a threefold increase in cardiovascular mortality [76]. Treatment with GH has resulted in improved lean body mass and decreased hyperlipidemia among children not undergoing HCT. The incidence of GH deficiency after TBI and HCT varies from 20% to 85% depending upon differences in time of testing after HCT, differences in preparative regimens received, inclusion of patients with and without cranial irradiation, and use of different methods of GH testing [52,55,61,77–81]. Even though GH deficiency has been observed, fewer than half have received GH therapy. Retrospective studies have shown that height SD is significantly lower than predicted based on height at HCT and midparental height [54,58,59]. Authors [45,59] have concluded that GH therapy was not needed because final height SD was not more than 2 SD below the mean, or because GH-treated children did not have significantly improved growth. Others have reported, based on a retrospective study, that GH therapy does not benefit children, but most of the children had received less than 1 year of GH therapy [60]. One study reported 13 children, eight of whom received prophylactic cranial irradiation, who were given GH, which restored normal height velocity but without “catch-up” growth. The final height SD scores remained impaired. In these children, a mean of 3.2 years had elapsed between TBI and initiation of GH therapy, which began at a mean age of 12.2 (range 5.8–18.2). The poor response to GH therapy has been attributed to factors such as spinal irradiation, early puberty, suboptimal GH dosing schedules, and older age of most patients when GH therapy was initiated. Recent data suggest that improvements in growth and final height can be achieved with contemporary dosing regimens that utilize daily doses of 0.04 mg/kg, increasing to 0.06 mg/kg at puberty [55,57,82,83]. A recent study shows that when a group of GH-deficient children transplanted at a single transplant center with fractionated TBI either received or did not receive GH therapy, final height was a function of age at treatment and patient gender. Male patients had the poorest growth. Figure 104.5 shows how the final height for 42 children with GH deficiency treated with GH and 48 children with GH deficiency who were not treated with GH was influenced by age at the time of TBI [62]. This study demon-

1613

strated that children 2–10 years of age who were treated with GH had significantly better final height than children in the same age group not treated with GH. By contrast, little benefit in height growth was present among GH-treated children who were over 10 years of age at transplant. Initially, treatment with human GH was restricted due to the limited supply of the drug, but with the availability of recombinant GH, access no longer is a problem. Reports of leukemia occurring in patients treated with human GH have raised concern that there might be a causal relationship between GH therapy and development of leukemia [80,84–86]. The Lawton Wilkins Pediatric Endocrine Society and the Human Growth Foundation of the United States convened a workshop in 1988 to review known leukemia cases in 22,000 GH-treated patients in Europe, North America, Japan, and Australia since 1959 [87]. Of 15 cases of leukemia, four occurred after a brief period of GH therapy or there were compelling reasons to suspect another cause for the leukemia. The 11 suspect cases developed during 150,000 patient–years of risk, including the periods of treatment and follow-up, so that the incidence of leukemia was approximately one per 21,000 patient–years at risk. The expected annual leukemia incidence among treated patients with hypopituitarism was estimated to be one per 42,000 patient–years. After review of all the available data, workshop participants concluded that there might be a small increase in leukemia incidence associated with the GH treatment of GH-deficient patients, but it was not clear that this incidence was directly related to GH therapy. In absolute terms, a current estimate of individual risk, assuming a 10-year GH treatment course, would be one per 2400 (0.042%), which is no different from the Surveillance and End Results estimate of new cases of leukemia reported in the United States (1 : 2400 per year). The study by Sanders et al. showed no difference in the development of recurrent leukemia or secondary malignancies between those who received GH therapy and those who did not [62]. Oral–facial growth Irradiation to bone produces epiphyseal, metaphyseal, and diaphyseal injury that affects subsequent bone growth [88]. The effect is related to patient age at the time of irradiation as well as the site irradiated, the dose schedule, and the total dose of irradiation [89]. Young children,

0

–0.2

<10 years, GH

–0.4

<10 years, no>GH >10 years, GH

–0.6

>10 years, no>GH –0.8

–1 GHD to FAH

Fig. 104.5 Final height standard deviation (SD) score for 42 children with growth hormone (GH) deficiency (GHD) treated with GH when less than 10 years of age (black box) or more than 10 years of age (lighter shaded box), and final height SD score for 48 children with GH deficiency not treated with growth hormone when less than 10 years of age (open box) or more than 10 years of age (darker shaded box). FAH, final adult height.

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especially children less than 6 years of age at the time of irradiation to the head and neck, have the greatest risk for development of subsequent craniofacial and dental abnormalities [90–93]. Enamel and dentin formation are disturbed by TBI due to destruction of cells during the mitotic phase. Chemotherapy drugs are selectively toxic to actively proliferating cells by disruption of DNA synthesis and replication, RNA transcription, and cytoplasmic transport mechanisms [94]. The effects of chemotherapy and irradiation on dental development include tooth agenesis, complete or partial arrest of root development with thin, tapered roots, early apical closure, globular and conical crowns, dentin and enamel opacities and defects, microdontia, enlarged pulp chambers, taurodontism (prismshaped molars), and abnormal occlusion [92,95,96]. Hence, the development of secondary teeth is often affected, with delayed or arrested tooth formation, shortening and blunting of tooth roots, incomplete calcification, premature closure of apices, and dental caries. However, some children with hematologic malignancies who have received preparative regimens with chemotherapy alone do not have abnormalities in dental maturity or eruption of their permanent dentition [97]. Reduction in lower face height in HCT patients correlated with impaired dental development [93]. Vertical condyle growth and the alveolar and molar heights were adversely affected by pretransplant preparative regimens. Cephalometric measurements of facial bones to evaluate facial growth before and after 10 Gy TBI and HCT have resulted in a significant reduction in the maxilla length and mandible growth compared with healthy age-matched nontransplant children. These differences were most pronounced in mandibular growth [93]. Compared with the control group, the children and adolescents in the HCT group also had significantly reduced mouth opening capacity with reduced translation movement of the condyles diagnosed in 53% of children treated with TBI, compared with 5% in the control group [91]. Signs of craniomandibular dysfunction were found in 84% of children in the HCT children, compared with 58% in the control group. The long-term alterations in connective and muscle tissues result in changes in tissue inflammation and eventually fibrosis. An evaluation of craniofacial development in 16 prepubertal children (age range 1.7–11.0 years) with growth failure and GH deficiency following CY plus 10.0 Gy TBI and HCT demonstrated a significant positive effect on growth among the nine GH-treated patients compared with seven non-GH-treated patients [90]. Another study demonstrated improvement in vertical growth of the condyles, suggesting that condylar cartilage is the most likely site of mandibular growth activity [98]. These observations support the hypothesis that GH most likely encourages longitudinal bone growth both directly, by stimulating differentiation of epiphyseal growth plate precursor cells, and indirectly, by increasing the responsiveness to IGF-I [99]. Treatment with GH, however, did not improve the disturbed root development of the teeth. Thus, there does not appear to be a stimulating effect of dental development on growth of the alveolar process. Few data are available for the use of orthodontic treatment for children who have dental growth disturbances after TBI and HCT. A retrospective study of 10 children has demonstrated that orthodontic treatment plans were modified to reflect the patient’s medical condition, but in general the orthodontic treatment did not produce any harmful sideeffects, even though most treated children exhibited severe pre-existing disturbances in dental development [100]. Nine of the 10 patients had severe disturbances in dental development with short V-shaped roots, premature apical closure, enamel disturbances, microdontia, and aplasia. The most severe disturbances were found in children less than 5 years of age at the time of 10 Gy TBI. The strategies used to cope with the severe problems of dental growth disturbances included using appliances that minimized the risk of root resorption, using weaker forcers, terminating treatment earlier than normal, and choosing the simplest

method for treatment needs. In general, the lower jaw was not treated. The treatment was judged as unsatisfactory in four of the 10 patients.

Puberty Puberty, a transitional stage from a sexually immature to a mature sexual state, is accompanied by significant changes in gonadal and growth hormonal activity, development of secondary sexual characteristics, and increased growth velocity. There is considerable variation in the timing and sequence of pubertal events that makes it difficult to assess a child’s pubertal development based only on chronologic age. In the normal individual, pubertal development is usually closely related with osseous maturation as measured by bone age. An intact hypothalamic–pituitary–gonadal axis is required for initiation and completion of puberty. The normal pubertal growth rate is 1.5–2 times greater than prepubertal growth rates [101]. In the absence of pubertal sex hormone secretion, the increased growth velocity associated with the pubertal growth spurt is substantially blunted, and the development of secondary sexual characteristics is delayed or absent. Gonadal hormone production and germ cell viability are affected by high doses of alkylating agents and irradiation, with variables related to patient age, sex, and type and dose of therapy [102]. Azoospermia develops in prepubertal boys who have received a cumulative dose of more than 350 mg/kg CY, whereas doses of 200 mg/kg or less result in minimal alteration of spermatogenesis. The total dose of BU impacting future developmental potential for the prepubertal testes is not known. Irradiation to the prepubertal testes results in damage to the germinal epithelium that does not become apparent until after puberty [103]. Boys who have received more than 24 Gy testicular irradiation have delayed or arrested development of secondary sexual characteristics, with elevated gonadotropin and low testosterone values. Primary ovarian failure usually occurs following total cumulative doses of more than 500 mg/kg CY to prepubertal girls [102]. No data are available regarding BU or irradiation on the prepubertal ovary. Sex hormones indirectly stimulate linear growth by increasing endogenous GH secretion [104]. This stimulation leads to increasing circulating and tissue levels of IGF-I, which activates growth at the level of bone and cartilage. Treatment with low-to-moderate doses of sex hormones promotes increased linear growth, but physiologic adult doses of sex hormones result in a greater influence on skeletal maturation, with resultant compromise in final adult height via the mechanism of premature epiphyseal closure. Children with idiopathic GH deficiency usually have a late, but normal, pubertal growth spurt. Children with hypogonadotropic hypogonadism have an absence of sex hormone production, delayed puberty, delayed pubertal growth spurt, and a decrease in final adult height [67]. Thus, interruption in either production of pubertal GH or sex hormone production is likely to result in both delayed pubescence and decreased linear height. Preparative regimens containing only chemotherapy Following 200 mg/kg CY and BMT for aplastic anemia, 31 of 32 girls and 28 of 31 boys who were prepubertal at time of administration of CY have now been followed long enough to be more than 12 years of age and evaluable for pubertal development [105]. Nearly all of these children demonstrated normal age-appropriate progression through puberty (Tables 104.2 and 104.3). Three girls with delayed pubertal development had Fanconi’s syndrome and eventually developed normal secondary sexual characteristics and normal luteinizing hormone (LH), folliclestimulating hormone (FSH), and estradiol levels, and two girls showed delay due to chronic GVHD therapy. Among the 26 girls with normal age-appropriate development, menarche occurred at a median of 12.5

Growth and Development after Hematopoietic Cell Transplantation Table 104.2 Pubertal development in girls

1615

Preparative regimens

Current age Development

≥12 years Normal Delayed

CY*

BUCY†

10.0 Gy TBI‡

12–15.75 Gy TBI§

31 26 (84%) 5 (16%)

61 17 (28%) 44 (72%)

24 7 (29%) 17 (71%)

114 48 (42%) 66 (57%)

BU, busulfan; CY, cyclophosphamide; TLI, total lymphoid irradiation. References: * [44,108], † [50,112,113], Sanders, unpublished data, 2007; ‡ [44,63,112,113]; § [9,50,63,112,113], Sanders, unpublished data, 2007.

Table 104.3 Pubertal development in boys

Preparative regimens

Current age Development

≥12 years Normal Delayed

CY*

BUCY†

10.0 Gy TBI1‡

12–15.75 Gy TBI1§

28 25 (89%) 4 (14%)

54 28 (52%) 26 (48%)

32 6 (19%) 26 (81%)

77 32 (42%) 45 (58%)

1

No additional testicular irradiation. References: * [44,108], † [50,112,113], Sanders, unpublished data, 2007; ‡ [44,63,112,113]; § [9,50,63,112,113], Sanders, unpublished data, 2007.

(range 11–16) years of age, and the five with delayed development had menarche occur between 16 and 19 years of age. Twenty-eight of these formerly prepubertal girls have given birth to 49 normal children [106, Sanders, unpublished data, 2007]. The three boys with delayed pubertal development had chronic GVHD and did ultimately develop normal secondary sexual characteristics with normal LH, FSH, and testosterone values. Twenty-eight of these formerly prepubertal boys have fathered 49 normal children. Gonadal function after 14 mg/kg BU plus 200 mg/kg Cy and allogeneic BMT for thalassemia has been reported for 64 prepubertal patients (31 girls and 33 boys) who ranged in age from 9.3 to 17.2 years [107– 110]. Twenty-five girls had evidence of primary ovarian failure, with elevated gonadotropin levels, and six girls had hypogonadotropic hypogonadism. All girls had low estradiol levels both before and after transplantation and required hormone supplementation for development of secondary sexual characteristics. Among the 33 boys, post-transplant LH and FSH concentrations were within normal limits for 23, but abnormal with either elevated LH and FSH or low LH and FSH in 10 boys. However, after gonadotropin-releasing hormone stimulation, three had normal responses, two had elevated FSH responses, and 10 had low responses. These gonadal function results must be interpreted with caution, however, because patients with thalassemia treated with chelation and transfusion therapy frequently show delayed or absent puberty. Pubertal development has been evaluated among children transplanted for malignancy who received 16 mg/kg BU plus 120–200 mg/kg CY [107,111,112, Sanders, unpublished data, 2007]. One hundred and fifteen children have now been followed long enough to be evaluable for the progress of spontaneous pubertal development (Tables 104.2 and 104.3). Seventeen of 61 (28%) evaluable girls and 28 of 54 (52%) evaluable boys have developed normally through puberty. Whereas the girls have normal LH, FSH, and sex hormone production, many of the boys do not, with some having elevated LH and FSH values, indicating gonadal dysfunction. These data demonstrate that BU is highly toxic to

prepubertal, particularly female, gonads. These children must be carefully followed, with gonadal function evaluation beginning about 12 years of age. Supplementation with appropriate gonadal hormones administered in gradually increasing doses under supervision of a pediatric endocrinologist is needed for these children to develop secondary sexual characteristics. Preparative regimens containing irradiation The development of secondary sexual characteristics among children who were prepubertal at the time of TBI administration but more than 12 years of age at follow-up was evaluated in 24 girls and 31 boys after 10 Gy single-fraction TBI, and 77 girls and 77 boys after 12–15.75 Gy fractionated-exposure TBI (Tables 104.2 and 104.3). Following 10.0 Gy single-exposure TBI, 71% of girls and 83% of boys had delayed development of secondary sexual characteristics, elevated LH and FSH levels, and low sex hormone levels. After fractionated-exposure TBI, 49% of girls and 58% of boys had delayed development. The majority of these girls and boys have received appropriate sex hormone therapy for promotion of secondary sexual characteristic development. As larger numbers of prepubertal children are becoming evaluable for pubertal development, the amount of testicular irradiation received and patient age at the time of TBI are emerging as important factors in subsequent normal spontaneous pubertal development (Table 104.4). Thirty of the 36 (84%) boys who had received more than 10 Gy testicular irradiation for testicular leukemia in addition to 12–15.75 Gy TBI have primary gonadal failure and require testosterone therapy to promote the development of secondary sexual characteristics. However, about half of the boys who receive 400 cGy prophylactic testicular irradiation in addition to fractionated TBI develop normally through puberty [Sanders, unpublished data, 2007]. Among children receiving 14.40 Gy fractionated TBI, one study reported that, among 17 boys evaluated, those with increased levels of LH were significantly younger at BMT (5.4 ± 0.8 versus 7.8 ± 0.8 years;

1616

Chapter 104

p = 0.024), but among the 16 girls evaluated, those with ovarian failure were significantly older at the time of TBI than those with spontaneous puberty (8.6 ± 2.3 versus 6.1 ± 1.8 years; p = 0.03) [29]. Another study did not observe an effect of age on subsequent gonadal function [54]. A third analysis suggested that an effect of age may be linked to the total dose of TBI [107]. Among 30 evaluable children given 12.0 Gy TBI, 15 of 16 (94%) less than 10 years of age and 11 of 14 (78%) more than 10 years of age developed normally. However, among the 46 evaluable children given 14.40–15.75 Gy TBI, 23 of 30 (77%) less than 10 years of age and three of 16 (19%) more than 10 years of age developed normally. Larger numbers of patients are needed to draw firm conclusions regarding the impact of patient age and total dose of TBI administered on development of puberty. It is recommended that the development of secondary sexual characteristics be monitored carefully after patients reach 10–11 years of age and that Tanner Developmental Scores be determined annually. Because the production of sex hormones is necessary for promotion of the pubertal growth spurt, in addition to promoting sexual maturation, children with evidence of gonadal failure and delayed development of secondary sexual characteristics may benefit from supplemental hormones. This supplementation should be administered under the guidance of a pediatric endocrinologist, and doses of sex hormone treatment should begin low with gradual increase to simulate natural hormone production, to prevent premature advancement of bone age, and to promote pubertal growth spurts. Patients with normal pubertal development, gonadotropins, and sex hormone production should receive appropriate sexual behavior counseling as pregnancy may occur.

Conclusion Evaluations of endocrine function following HCT demonstrate that the occurrence of abnormalities that may influence subsequent growth and development are related to the type of HCT preparative regimen received. Table 104.4 Pubertal development in boys given testicular irradiation

Children who receive CY only usually do not have endocrine function abnormalities. These children have normal thyroid function, normal growth rates, and normal development through puberty. Individuals who develop normally through puberty or in whom gonadal function returns to normal may be fertile. Offspring of these patients, in general, do not differ from the general population. Children who receive BU plus CY preparative regimens have normal thyroid function, and the majority have normal prepubertal growth rates. Pubertal growth may be affected. Data from the patients evaluable for pubertal development suggest that a substantial portion of these children may not develop secondary sexual characteristics at a normal age, which is particularly true for girls but not boys. Gonadal function, however, is likely to be abnormal in both boys and girls. In contrast, multiple endocrine function abnormalities that affect normal growth and development frequently occur after HCT regimens that contain TBI. Patients are at risk for development of thyroid function abnormalities for many years after TBI. These patients are also at risk for development of thyroid malignancy. Growth rates are usually blunted, especially if the child has received prior CNS irradiation. GH deficiency, which frequently occurs, and growth failure should be diagnosed and treated early with synthetic GH for best response. Without GH treatment, young children have significantly compromised final adult heights. Gonadal function damage results in a significant fraction of prepubertal patients having delayed pubertal development. Children with delayed development of secondary sexual characteristics may benefit from careful administration of appropriate sex hormone therapy. Children who do develop normally through puberty are potentially fertile. All patients who receive HCT should continue to undergo longterm follow-up evaluations; a suggested outline of studies is shown in Table 104.5. Table 104.6 outlines suggested treatment, but details of the management of hormone therapy are best performed by a pediatric endocrinologist. Table 104.6 Suggested treatment for post-transplant endocrine function disorders

Testicular irradiation

Current age Development

≥12 years Normal Delayed

4.0 Gy

≥10 Gy

51 28 (55%) 23 (45%)

36 6 (16%) 30 (84%)

Abnormality

Treatment*

Hypothyroid Growth failure with growth hormone deficiency Delayed puberty (girls)

Thyroxine Growth hormone therapy Ethinyl/estradiol and medroxyprogesterone testosterone enanthate

Sanders, unpublished data, 2007. * Details of doses and dose adjustments are best managed by a pediatric endocrinologist.

Table 104.5 Post-transplant evaluations of endocrine effects

Thyroid gland Growth

Puberty

Tests

Frequency

Thyroid stimulating hormone, triiodothyronine, thyroxine Height *Bone age *†Growth hormone measurement Tanner Development Score Luteinizing hormone, follicle stimulating hormone Estradiol (girls) Testosterone (boys)

Annual Annual Annual until epiphysis closure Annual until deficiency presents or epiphysis closure Annual age 8–18 years Annual after age 10 years Annual after age 10 years Annual after age 10 years

* Not needed for those receiving a cyclophosphamide-only preparative regimen. † Not needed if the patient is already receiving growth hormone therapy.

Growth and Development after Hematopoietic Cell Transplantation

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66. Mauras N, Blizzard RM, Link K et al. Augmentation of growth hormone secretion during puberty: evidence for a pulse amplitude-modulated phenomenon. J Clin Endocrinol Metab 1987; 64: 596–601. 67. Bourguignon J-P. Linear growth as a function of age at onset of puberty and sex steroid dosage: Therapeutic implications. Endocr Rev 1988; 9: 467–88. 68. Ohlsson C, Bengtsson BA, Isaksson OG et al. Growth hormone and bone [Review]. Endocr Rev 1998; 19: 55–79. 69. Bachrach LK. Acquisition of optimal bone mass in childhood and adolescence [Review]. Trends Endocrinol Metab 2001; 12: 22–8. 70. Biller BMK. Efficacy of growth hormone-replacement therapy: body composition and bone density. The Endocrinologist 1998; 8(Suppl 1): 15S–21S. 71. Baroncelli GI, Bertelloni S, Ceccarelli C et al. Dynamics of bone turnover in children with GH deficiency treated with GH until final height. Eur J Endocrinol 2000; 142: 549–56. 72. Hui SL, Slemenda CW, Johnston CC Jr. The contribution of bone loss to postmenopausal osteoporosis. Osteoporos Int 1990; 1: 30–4. 73. Carpenter PA, Hoffmeister P, Chesnut CH, III et al. Bisphosphonate therapy for reduced bone mineral density in children with chronic graftversus-host disease. Biol Blood Marrow Transplant 2007; 13: 683–90. 74. Rotnem D, Genel M, Hintz RL, Cohen DJ. Personality development in children with growth hormone deficiency. J Am Acad Child Psychiatry 1977; 16: 412–26. 75. Stabler B, Siegel PT, Clopper RR et al. Behavior change after growth hormone treatment of children with short stature. J Pediatr 1998; 133: 366–73. 76. Vance ML, Mauras N. Growth hormone therapy in adults and children [Review]. N Engl J Med 1999; 341: 1206–16. 77. Papadimitriou A, Urena M, Hamill G et al. Growth hormone treatment of growth failure secondary to total body irradiation and bone marrow transplantation. Arch Dis Child 1991; 66: 689– 92. 78. Borgström B, Bolme P. Growth and growth hormone in children after bone marrow transplantation. Horm Res 1988; 30: 98–100. 79. Ogilvy-Stuart AL, Clark DJ, Wallace WH et al. Endocrine deficit after fractionated total body irradiation. Arch Dis Child 1992; 67: 1107– 10. 80. Olshan JS, Willi SM, Gruccio D, Moshang T Jr. Growth hormone function and treatment following bone marrow transplant for neuroblastoma. Bone Marrow Transplant 1993; 12: 381–5. 81. Shalet SM, Toogood A, Rahim A, Brennan BM. The diagnosis of growth hormone deficiency in children and adults [Review]. Endocr Rev 1998; 19: 203–23. 82. Ogilvy-Stuart AL, Shalet SM. Growth and puberty after growth hormone treatment after irradiation for brain tumours. Arch Dis Child 1995; 73: 141– 6. 83. MacGillivray MH, Baptista J, Johanson A. Outcome of a four-year randomized study of daily versus three times weekly somatropin treatment in prepubertal naive growth hormone-deficient children. Genentech Study Group. J Clin Endocrinol Metab 1996; 81: 1806–9.

84. Endo M, Kaneko Y, Shikano T et al. Possible association of human growth hormone treatment with an occurrence of acute myeloblastic leukaemia with an inversion of chromosome 3 in a child with pituitary dwarfism. Med Pediatr Oncol 1988; 16: 45–7. 85. Sasaki U, Hara M, Watanabe S. Occurrence of acute lymphoblastic leukemia in a boy treated with growth hormone for growth retardation after irradiation to the brain tumor. J Clin Oncol 1988; 18: 81–4. 86. Delemarre-Van De Waal HA, Odink RJH, De Grauw TJ, De Waal FC. Leukaemia in patients treated with growth hormone [Letter to the Editor]. Lancet 1988; 1: 1159. 87. Fisher DA, Job J-C, Preece M, Underwood LE. Leukaemia in patients treated with growth hormone. Lancet 1988; 1: 1159–60. 88. Parker RG, Berry HC. Late effects of therapeutic irradiation on the skeleton and bone marrow. Cancer 1976; 37: 1162–71. 89. Lochte HL Jr, Kasakura S, Karetzky M et al. Infusion of marrow in the mouse and dog after Thio-TEPA. Blood 1963; 21: 424–8. 90. Dahllöf G, Forsberg CM, Borgstrom B. Changes in craniofacial development induced by growth hormone therapy in children treated with bone marrow transplantation. Acta Paediatr 1994; 83: 1165–9. 91. Dahllöf G, Krekmanova L, Kopp S et al. Craniomandibular dysfunction in children treated with total-body irradiation and bone marrow transplantation. Acta Odontol Scand 1994; 52: 99–105. 92. Rosenberg SW, Kolodney H, Wong GY, Murphy ML. Altered dental root development in long-term survivors of pediatric acute lymphoblastic leukemia. Cancer 1987; 59: 1640–8. 93. Dahllöf G, Forsberg CM, Ringden O et al. Facial growth and morphology in long-term survivors after bone marrow transplantation. Eur J Orthod 1989; 11: 332–40. 94. da Fonseca MA. Long-term oral and craniofacial complications following pediatric bone marrow transplantation [Review]. Pediatr Dent 2000; 22: 57–62. 95. Dahllöf G, Barr M, Bolme P et al. Disturbances in dental development after total body irradiation in bone marrow transplant recipients. Oral Surg Oral Med Oral Pathol 1988; 65: 41–4. 96. Dahllöf G, Heimdahl A, Bolme P et al. Oral condition in children treated with bone marrow transplantation. Bone Marrow Transplant 1988; 3: 43–51. 97. Dahllof G, Nasman M, Borgstrom A et al. Effect of chemotherapy on dental maturity in children with hematological malignancies. Pediatr Dent 1989; 11: 303–6. 98. Dahllöf G, Forsberg C-M, Näsman M et al. Craniofacial growth in bone marrow transplant recipients treated with growth hormone after total body irradiation. Scand J Dent Res 1991; 99: 44–7. 99. Green H, Morikawa M, Nixon T. A dual effector theory of growth-hormone action [Review]. Differentiation 1985; 29: 195–8. 100. Eisen D, Essell J, Broun ER. Oral cavity complications of bone marrow transplantation [Review]. Semin Cutan Med Surg 1997; 16: 265–72. 101. Cutter GB, Cassosta FG, Ross JR. Pubertal growth: physiology and pathophysiology. Recent Prog Horm Res 1986; 42: 443–70.

Growth and Development after Hematopoietic Cell Transplantation 102. Ray H, Mattison D. How radiation and chemotherapy affect gonadal function. Contemp Ob Gyn 1985; 109: 106–15. 103. Shalet SM, Beardwell CG, Jacobs HS, Pearson D. Testicular function following irradiation of the human prepubertal testes. Clin Endocrinol 1978; 9: 483–90. 104. Pescovitz OH. The endocrinology of the pubertal growth spurt. Acta Paediatr Scand Suppl 1990; 367: 119–25. 105. Epstein RB, Storb R, Clift RA, Thomas ED. Transplantation of stored allogeneic bone marrow in dogs selected by histocompatibility typing. Transplantation 1969; 8: 496–501. 106. Sanders JE, Hawley J, Levy W et al. Pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradia-

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105

Mary E.D. Flowers & H. Joachim Deeg

Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation

Introduction Hematopoietic cell transplantation (HCT) provides curative therapy for a variety of diseases. The development of less toxic pretransplant conditioning regimens, more effective prophylaxis of acute graft-versushost-disease (GVHD), improved infection control, and other advances in transplant technology have resulted in a rapidly growing number of transplant recipients surviving long term free of the disease for which they were transplanted. Current data show, indeed, that patients transplanted for aplastic anemia and patients without chronic GVHD have life expectancies similar to age-matched controls. Patients with advanced malignant diseases, however, and those who develop chronic GVHD after allogeneic HCT may experience late disease recurrence or incur delayed complications that may prove fatal [1]. With the increase in numbers of transplants over the past three decades and the considerable improvement in early post-transplant survival (Fig. 105.1), an increasing population of survivors is at risk of developing late complications (Fig. 105.2) [2,3]. This chapter will focus on nonmalignant delayed complications, while secondary malignancies, also a late complication after HCT, will be discussed in Chapter 106.

Etiology and spectrum of delayed complications Several factors impact on recovery from and late effects of HCT, including prior therapy for the underlying disease, pretransplant comorbidities and psychosocial status, intensity of the transplant conditioning regimen, and development of chronic GVHD. Major nonmalignant delayed complications are listed in Table 105.1. Late complications develop also in patients given high-dose therapy who are not transplanted; however, those patients do not experience the typical transplant-related complications such as GVHD, which is affected by donor type, stem cell source, patient age, and intensity of the conditioning regimen. Chronic GVHD with the associated immunodeficiency, and the effects of glucocorticoids and other immunosuppressive treatments used to control GVHD, represent the most frequent late complications after allogeneic HCT. Infertility is dependent upon the type of the transplant conditioning regimen. Other complications are related to the prolonged use of glucocorticoids and other immunosuppressive drugs, and many (e.g. chronic pulmonary disease, endocrine complications, and secondary malignancies) are mul-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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tifactorial in etiology. Some delayed complications are due directly to therapy-induced trauma and scar formation (e.g. destruction of skeletal growth plates and bladder dysfunction); others are related to immunologic graft–host interactions (e.g. lymphoproliferative disorders). Sideeffects of the treatment of acute complications early after HCT (e.g. the use of steroids) may also contribute to long-term complications. Nearly all organ systems can be affected (Table 105.2). To what extent delayed nonmalignant complications differ in patients conditioned with reduced-intensity or nonmyeloablative transplant regimens remains to be seen, as follow-up is still rather short compared with high-dose transplant regimens. Nonetheless, rates of late fungal and viral infections and chronic GVHD appear to be similar after reducedintensity and high-dose conditioning regimens [4,5].

Chronic GVHD The major cause of delayed morbidity and mortality after allogeneic HCT is chronic GVHD, related to its associated immune deficiency, and the toxicities of glucocorticoids and other immunosuppressive treatment given for control of GVHD (see Chapter 87). The incidence of chronic GVHD is influenced by the source of stem cells, donor–recipient gender mismatch, human leukocyte antigen compatibility, and age. The risk of nonrelapse mortality is higher for patients with chronic GVHD with direct progression from acute GVHD, for patients with platelet counts below 100,000/μL, hyperbilirubinemia, extensive skin disease or multiple organ involvement, and low clinical performance score [6,7]. If not treated adequately and in severe cases, chronic GVHD can result in major disability related to keratoconjunctivitis sicca (KCS), pulmonary insufficiency due to bronchiolitis obliterans (BO), or restrictive lung disease related to scleroderma or fasciitis, as well as joint contractures, skin ulcers, esophageal and vaginal stenosis, and others [8,9]. Metabolic, muscular/skeletal and chronic endocrine problems are complications of chronic GVHD or are due to toxicities of treatment given to control GVHD (Table 105.3). Most patients require glucocorticoids or other agents for at least 2 years from the initial diagnosis of chronic GVHD [6]. Approximately 10% of patients require continued immunosuppressive treatment beyond 5 years from the initial diagnosis of chronic GVHD, and 40% die or experience a recurrence of their malignancy before chronic GVHD resolves [6]. The remaining 50% of patients discontinue immunosuppressive treatment within 5 years of the initial diagnosis of chronic GVHD [6]. Therefore, it is not surprising that corticosteroid and other immunosuppressive therapies are major contributors of late complications after allogeneic HCT. Ancillary and supportive care directed at organ-specific control of symptoms or

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Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation

Table 105.2 Organs affected by late complications according to etiology

Organ Skin Eyes Sinuses Gastrointestinal tract Liver Lungs Muscles Connective tissues Bones Endocrine glands Gonads Kidneys

Regimen-related toxicity • • • • • • • • • • •

Chronic graftversus-hostdisease • • • • • • • •

Infections • • • • • •

• •

Fig. 105.1 Allogeneic hematopoietic cell transplantation at the Fred Hutchinson Cancer Research Center. Upper curve: cumulative number of allogeneic transplants. Lower curve: cumulative number of patients alive at the end of the indicated calendar year. Table 105.3 Metabolic, skeletal and endocrine complications related to chronic graft-versus-host disease or its treatment Metabolic problems Hyperlipidemia Hyperglycemia Hypertension Obesity Skeletal/muscular problems Osteoporosis/osteopenia Avascular bone necrosis Myopathy Endocrine problems Pancreatic insufficiency Adrenal insufficiency Autoimmune thyroid disease Delayed puberty and growth hormone deficiency Fig. 105.2 Proportion of patients given an allogeneic hematopoietic cell transplantation at the Fred Hutchinson Cancer Research Center who survived at least 100 days post transplant. Table 105.1 Nonmalignant delayed complications after hematopoietic cell transplantation Chronic graft-versus-host disease Autoimmune disorders/hematologic complications Late infections Airway and pulmonary disease Neuroendocrine dysfunction Impairment of growth and development Infertility Cardiovascular disease Ocular problems Musculoskeletal problems Dental problems Dysfunction of the genitourinary tract Gastrointestinal and hepatic complications Metabolic problems Central and peripheral nervous system impairment Psychosocial effects

complications resulting from GVHD and its therapy are central to the management of chronic GVHD. However, more than half of the recommendations in the current ancillary and supportive care guidelines for patients with chronic GVHD are based only on expert consensus opinion (level III) rather than controlled studies, which highlights the need for more clinical research [10]. Long-term clinical monitoring is necessary to identify early signs of GVHD, assess activity versus sequelae caused by GVHD, determine treatment-related toxicity, and, most importantly, prevent late complications associated with severe morbidity. We find it useful to apply the 0–3 scoring system (Table 105.4) proposed by the National Cancer Institute Consensus Group to assess chronic GVHD manifestations in each organ [9]. We use this assessment tool at the time of initial diagnosis, at any time when changes occur in the therapy to control GVHD (e.g. due to an increase in the dose of corticosteroid of 1 mg/kg or more every other day, substitution of one therapy for another or additional therapy), and at 6–12-month intervals if manifestations of GVHD persist or immunosuppressive treatment continues.

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Chapter 105

Table 105.4 Assessment scores for monitoring patients with or at risk of chronic graft-versus-host disease (GVHD) Score 0

Score 1

Score 2

Score 3

ⵦ

Asymptomatic and fully active (ECOG 0; KPS or LPS 100%)

Symptomatic, fully ambulatory, restricted only in physically strenuous activity (ECOG 1, KPS or LPS 80–90%)

Symptomatic, ambulatory, capable of self-care, >50% of waking hours out of bed (ECOG 2, KPS or LPS 60–70%)

Symptomatic, limited self-care, >50% of waking hours in bed (ECOG 3–4, KPS or LPS <60%)

Skin Clinical features Maculopapular rash Lichen planus-like feature Papulosquamous lesions or ichthyosis Hyperpigmentation Hypopigmentation Keratosis pilaris Erythema Erythroderma Poikiloderma Sclerotic features Pruritus Hair involvement Nail involvement % BSA involved

Normal/no symptoms

≤8% BSA with disease

19–50% BSA OR involvement with superficial sclerotic features “not hidebound” (able to pinch)

>50% BSA OR deep sclerotic features “hidebound” (unable to pinch) OR impaired mobility, ulceration or severe pruritus

Mouth Diagnostic/distinctive features Present Absent

No symptoms

Mild symptoms with disease signs but not limiting oral intake significantly

Moderate symptoms with disease signs with partial limitation of oral intake

Severe symptoms with disease signs on examination with major limitation of oral intake

Performance Score:

KPS ECOG LPS

signs but NO sclerotic features

ⵦ

Abnormality present but NOT thought to represent GVHD Eyes Mean tear test (mm): >10 6–10 ≤5 Not done

No symptoms

Mild dry eye symptoms not affecting ADL (requiring eyedrops ≤3 × per day) OR asymptomatic signs of keratoconjunctivitis sicca

Moderate dry eye symptoms partially affecting ADL (requiring drops >3 × per day or punctal plugs), WITHOUT vision impairment

Severe dry eye symptoms significantly affecting ADL (special eyeware to relieve pain) OR unable to work because of ocular symptoms OR loss of vision caused by keratoconjunctivitis sicca

Gastrointestinal tract

No symptoms

Symptoms such as nausea, vomiting, anorexia, dysphagia, abdominal pain or diarrhea without significant weight loss (<5%)

Symptoms associated with mild-to-moderate weight loss (5–15%)

Symptoms associated with significant weight loss >15%, requires nutritional supplement for most calorie needs OR esophageal dilation

Liver

Normal LFTs

Elevated bilirubin, AP, AST or ALT <2 × ULN

Bilirubin >3 mg/dL or bilirubin and enzymes 2–5 × ULN

Bilirubin or enzymes >5 × ULN

Lungs* PFTs not done

No symptoms

Mild symptoms (shortness of breath after climbing one flight of steps) FEV1 60–79% OR LFS 3–5

Moderate symptoms (shortness of breath after walking on flat ground)

Severe symptoms (shortness of breath at rest; requiring oxygen)

FEV1 40–59% OR LFS 6–9

FEV1 ≤39% OR LFS 10–12

FEV1

ⵦ ⵦ

DLCO

FEV1 > 80% OR LFS = 2

Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation

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Table 105.4 (Continued) Score 0

Score 1

Score 2

Score 3

Joints and fascia

No symptoms

Mild tightness of arms or legs, normal or mild decreased ROM AND not affecting ADL

Tightness of arms or legs OR joint contractures, erythema thought due to fasciitis, moderate decrease in ROM AND mild-to-moderate limitation of ADL

Contractures WITH significant decrease of ROM AND significant limitation of ADL (unable to tie shoes, button shirts, dress self, etc.)

Genital tract Diagnostic/distinctive features Present Absent Not examined

No symptoms

Symptomatic with mild signs on exam AND no effect on coitus and minimal discomfort with gynecologic exam

Symptomatic with moderate signs on exam AND with mild dyspareunia or discomfort with gynecologic exam

Symptomatic WITH advanced signs (stricture, labial agglutination or severe ulceration) AND severe pain with coitus or inability to insert vaginal speculum

Other indicators, clinical manifestations or complications related to chronic GVHD (check all that apply): ⵧ Weight loss ⵧ Bronchiolitis obliterans ⵧ Bronchiolitis obliterans with organizing pneumonia ⵧ Esophageal stricture or web ⵧ Pericardial effusion ⵧ Pleural effusion(s) ⵧ Ascites (serositis) ⵧ Nephrotic syndrome ⵧ Peripheral neuropathy ⵧ Myasthenia gravis ⵧ Polymyositis ⵧ Malabsorption ⵧ Cardiac conduction defects ⵧ Coronary artery involvement ⵧ Cardiomyopathy ⵧ Eosinophilia >500/μL ⵧ Other: ______________________________________________ ⵧ None Biopsy obtained: ⵧ Yes ⵧ No Organ system(s) biopsied: _____________________GVHD confirmed by histology: ⵧ Yes ⵧ No OVERALL severity of GVHD: ⵧ None ⵧ Mild ⵧ Moderate ⵧ Severe Change from previous evaluation: ⵧ No prior or current GVHD ⵧ Improved ⵧ Stable ⵧ Worse ⵧ N/A (baseline) ADL, activities of daily living; ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BSA, body surface area; DLCO, diffusing capacity of the lung for carbon monoxide, corrected for hemoglobin; ECOG, Eastern Cooperative Oncology Group; FEV1, forced expiratory volume of the lung in 1 second; KPS, Karnofsky Performance Status; LFS, Lung Function Score; LFT, liver function test; LPS, Lansky Performance Status; PFT, pulmonary function test; ROM, range of motion; ULN, upper limit of normal. * Pulmonary scoring should be performed using both the symptom and PFT scale whenever possible. If discrepancy exists between pulmonary symptom or PFT scores, the higher value should be used for final scoring. Scoring using the LFS is preferred, but if DLCO is not available, grading using FEV1 should be used. The LFS is a global assessment of lung function after the diagnosis of bronchiolitis obliterans has already been made. The percent predicted FEV1 and DLCO (adjusted for hematocrit but not alveolar volume) should be converted to a numeric score as follows: >80% = 1; 70–79% = 2; 60–69% = 3; 50–59% = 4; 40–49% = 5; <40% = 6. LFS = FEV1 score + DLCO score, with a possible range of 2–12. * Adapted from [9], with permission from Elsevier Ltd.

Autoimmune disorders/dysregulation of immunity Allogeneic donor-derived cells recognize recipient antigens as nonself (alloreactivity). Donor-derived cells generate the new immune system of the host. Reactivity of these cells against the host tissues may resemble “autoreactivity.” The presence of thymic damage due to conditioning (and GVHD) would be expected to interfere with the negative selection of autoreactive cells and thereby facilitate the development of cellular and, via CD4+ cells, humoral autoreactivity [11,12]. Conditioninginduced thymic damage is also present in autologous transplant recipients, where it may have an effect similar to that observed after allogeneic HCT (see Chapters 11 and 15) [13]. Autoantibodies are observed after HCT, particularly in patients with chronic GVHD. Rheumatoid factor, antinuclear, antismooth muscle, and antimitochondrial antibodies have been reported, but there is no clear correlation with GVHD activity [14,15]. Generation of antibodies may be related to polymorphic differences between donors and patients such as antibody responses to H-Y minor histocompatibility antigens after

allogeneic HCT, reported to correlate with chronic GVHD and disease remission [16]. Patients with antiacetylcholine receptor antibodies and myasthenia gravis, typically 1–2 years after HCT, will require therapy as given for other patients with myasthenia. The antiacetylcholine receptor antibodies are donor derived, as shown by immunoglobulin allotyping [17]. Consistent with that concept, patterns of abnormal immune reactivity of the donor are also transferred to the recipients [18]. Such transfer has been reported for atopic asthma, psoriasis, and food allergies, albeit inconsistently [19–22]. Antibodies to interferon-alpha may contribute to the patients’ susceptibility to infections [23]. Chapter 17 provides details regarding immune reconstitution following HCT. Autoimmune hematologic problems Immune thrombocytopenia, anemia, and neutropenia, presumably autoimmune mediated, can occur after HCT [24,25]. Marrow can be a target of GVHD, and persistent thrombocytopenia is associated with poor survival in patients with chronic GVHD. Immunosuppressive treatment

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Chapter 105

of GVHD is often successful. Immune-mediated thrombocytopenia has also been observed after syngeneic and autologous HCT, and in these instances should be treated like idiopathic thrombocytopenic purpura. Immune-mediated neutropenia is less frequent; if spontaneous recovery does not occur, therapy with steroids is warranted. Patients transplanted from ABO-incompatible donors may experience hemolysis and severe anemia for months or even years after HCT until long-lived host cells (and isoagglutinins) have been eliminated. Pure red cell aplasia after HCT has been treated successfully with immunosuppressive therapy [26,27]. Successful treatment with CD34+ cell-enriched “donor lymphocyte infusion” for aplasia after ABO-incompatible HCT has also been reported [28]. A detailed discussion of blood group incompatibilities and hemolytic complications is provided in Chapter 81.

Late infections Late infections with bacteria, viruses, fungi, and other organisms due to persistent immunodeficiency are most common in patients with chronic GVHD, in cord blood and T-cell-depleted allogeneic HCT recipients, and following CD34-selected autologous transplants. It is standard practice to administer prophylaxis for infections caused by varicella zoster virus (VZV), Pneumocystis jirovecii, and encapsulated bacteria (Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae) within the first year after HCT, or even later, in patients with chronic GVHD. In addition, vaccination is recommended in long-term HCT survivors [10]. A detailed discussion of infections is provided in Chapters 88–95. A focused summary of late infections after HCT follows below.

Bacterial infections The most significant risk factor for late bacterial infection with encapsulated bacteria (Streptococcus pneumoniae, H. influenzae, and N. meningitidis) is chronic GVHD due to impaired production of opsonizing antibodies and other immunologic impairment in this setting (see Chapters 17 and 88). Due to high mortality associated with Streptococcus pneumoniae, H. influenzae, and N. meningitidis infections, prompt administration of appropriate antibiotic is imperative when infections are first suspected. Penicillin appears to be effective for prophylaxis, but the recent emergence of penicillin-resistant pneumococci [29] makes trimethoprim-sulfamethoxazole (TMP-SMX) a preferable choice for protection against infections with both encapsulated bacteria and Pneumocystis jirovecii (Pneumocystis jirovecii pneumonia, formerly Pneumocystis carinii pneumonia [PCP]), and possibly also against toxoplasmosis. Immunization with the 23-valent pneumococcal and H. influenzae B vaccines for all transplant recipients is advocated by most experts, although incomplete protection has been reported in patients with chronic GVHD (see Chapter 88). Late infections may also be due to other organisms, such as Staphylococcus species and Gram-negative aerobic bacteria. Empirical antibiotic treatment in HCT recipients who are admitted with clinical sepsis should include broad-spectrum coverage until the infecting organism is identified. Administration of intravenous immunoglobulin (IVIg) to maintain IgG levels higher than 400 mg/dL after day 90 after HCT should be considered for patients at risk. In Seattle, IVIg is recommended for the first year after allogeneic HCT for patients with IgG levels of 400 mg/dL or less. IVIg is also recommended in our Center for patients with chronic GVHD with recurrent sinopulmonary infections and low serum IgG levels. Some experts recommend monitoring IgG levels and administering IVIg routinely in chronic GVHD, but there are no data demonstrating that this approach improves outcomes [10].

Viral infections VZV disease is the most common late viral infection after HCT (see Chapter 92). Abdominal (visceral) VZV infection without skin manifestations is observed occasionally and presents with severe abdominal pain and rapidly rising transaminase levels. In a randomized, double-blind trial, oral acyclovir at doses of 800 mg twice daily for 1 year prevented VZV infection after HCT without rebound disease after discontinuation of prophylaxis [30]. In Seattle, acyclovir prophylaxis is given to all allogeneic patients for the first year after HCT, and for longer in patients with continued chronic GVHD. Most late cytomegalovirus (CMV) disease occurs during the first year after HCT, but in some cases it may occur up to 3 years after HCT. Gastroenteritis and pneumonia are the most common late manifestations of CMV disease; few cases of retinitis, encephalitis, and marrow failure have been reported. Late CMV pneumonia is associated with the highest mortality. About one-third of patients who survive the first episode will suffer a relapse of CMV disease after a median of 3 months (see Chapter 90). Continued monitoring (pp65 antigenemia, polymerase chain reaction for CMV DNA) and pre-emptive therapy is useful in patients at risk for late CMV disease. Patients with chronic GVHD are also at risk for acquisition of respiratory virus infections such as respiratory syncytial virus, and influenza and parainfluenza viruses. Seasonal vaccination of close contacts with the inactivated vaccine is recommended, with recipient vaccination starting at 6 months after HCT. Hepatitis B and C viruses may result in chronic hepatitis and cirrhosis (see Chapter 95). Transfusion-related HIV infections have been extremely rare in recent years. Late impaired graft function has been reported in association with human herpesvirus-6 and human parvovirus B19. Fungal infections Late invasive aspergillosis is most commonly seen in patients with chronic GVHD and preceding CMV and respiratory virus infections, possibly due to an immunosuppressive effect of these viruses (see Chapter 89). In patients with GVHD, late mold infections occur regardless of the intensity of the transplant conditioning regimen [4]. The outcomes of both mold and candidal infections in this setting remain poor. Drugs with activity against molds are now available (itraconazole, voriconazole). However, the efficacy and toxicity of long-term prophylaxis have not been tested in a randomized fashion. Sensitive diagnostic tests (Aspergillus galactomannan assay and polymerase chain reaction) may help in establishing an early diagnosis. Pneumocystis jirovecii pneumonia With the availability of effective prophylaxis, PCP is rarely seen after HCT. Most cases of late PCP occur in a setting of poor compliance or in patients who are unable to tolerate TMP-SMX because of side-effects or allergy, or who receive ineffective alternative prophylaxis regimens (see Chapter 94). Approximately 15–30% of patients require therapy other than TMP-SMX because of drug allergy, gastrointestinal intolerance, liver toxicity, and possibly neutropenia. In those cases, daily dapsone appears to be superior to inhaled pentamidine, and only limited data exist on atovaquone. Due to the overall superior results with TMPSMX, this drug should be given whenever possible, and desensitization should be attempted in all allergic patients. Future tasks include the development of infection prevention strategies that are easy to administer, effective, and well tolerated in HCT recipients with persistent severe immune compromised status who are at risk for PCP, or infections with VZV, encapsulated bacteria, CMV,

Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation

mold, and others. With increasing long-term use of antimicrobials, resistance may be a true challenge in the future.

Airway and lung complications Late-onset pulmonary complications occur in 7–26% of adult and pediatric patients after HCT and can affect the airways and the lung parenchyma [31–34]. It is important to recognize the relationship between histopathology, pulmonary pathophysiology, conditioning-related toxicity, GVHD, and infections in identifying patients who are at risk of developing late “noninfectious” pulmonary complications, and also to guide management (Fig. 105.3). An infectious etiology must be considered in the diagnostic evaluation of all late pulmonary complications. The conditioning regimen, in particular the dose of total body irradiation (TBI), and GVHD are major contributors to the development of late onset noninfectious pulmonary complications after HCT [35–37]. Airways and lungs are sensitive to cytotoxic therapy but are also prominent targets of infections and possibly of GVHD [37]. Mucositis early post transplant interferes with mucociliary clearance and leads to mucous retention and inflammation in the lower airways. This process can be aggravated by sinus drainage and postnasal drip. Lung injury occurs in the interstitium and in the alveolar space and may be associated with hemorrhage and edema. Scarring of the interstitial space interferes with effective gas exchange, respiratory dynamics, and kinetics. The differential diagnosis between infectious and noninfectious pulmonary complications can be difficult due to a lack of pathognomonic clinical manifestations and often concomitant manifestations of more than one process. Establishing the diagnosis of noninfectious late pulmonary complications requires consideration of the various known temporal associations, clinical manifestations, and whether there are recognizable patterns of radiographic and pulmonary function tests (PFTs) (Fig. 105.3). Bronchoalveolar lavage with the use of fluorescent antibody stains and shell vial cultures is helpful to identify infectious causes. PFTs are necessary to establish the diagnosis of restrictive and obstructive pulmonary problems and should be obtained periodically in patients with or at risk for GVHD. A thoracoscopic lung biopsy is generally necessary when the bronchoalveolar lavage is negative in patients

Abnormal Pulmonary Function by Etiology

Clinical Syndrome and Pulmonary Function OAD

GVHD

RLD

RRT

IPS

BO OAD

RLD

Infection

BOOP Normal PFTs

Fig. 105.3 Relationship of pulmonary clinical syndrome, etiology and lung function. BO, bronchiolitis obliterans; BOOP, bronchiolitis obliterans organizing pneumonia; GVHD, graft-versus-host disease; IPS, idiopathic pneumonia syndrome; OAD, obstructive airways disease; PFT, pulmonary function test; RLD, restrictive lung disease; RRT, regimen-related toxicity. (Reproduced from Mehta P, editor. Pediatric Stem Cell Transplantation. Sudbury, MA: Jones & Bartlett, 2004, with permission.)

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suspected of having bronchiolitis obliterans organizing pneumonia (BOOP), when an infectious process cannot be ruled out, and in patients suspected of having BO without history or other current manifestation of chronic GVHD. Late noninfectious pulmonary complications after HCT include restrictive lung disease, obstructive airflow disease including BO, BOOP, and idiopathic pneumonia syndrome (IPS).

Restrictive pulmonary disease Restrictive pulmonary defects, defined as a decrease in total lung capacity to less than 80% of predicted values, are present in one-third of HCT recipients studied, do not correlate with the type of transplant conditioning regimen, and generally do not produce severe symptoms. Restrictive lung disease (a loss of at least 15% in total lung capacity) detected at 3 months after HCT has been associated with a twofold increase in nonrelapse mortality due to respiratory failure, but was not significantly associated with chronic GVHD [38]. However, restrictive pulmonary defects are observed in patients with severe chronic GVHD with sclerodermatous features involving the chest, which can result in significant impairment of the quality of life. Early studies show that decreased diffusing capacity (diffusing capacity of the lung for carbon monoxide [DLCO]) pre transplant and an increased oxygen gradient [P(A–a)O2] are associated with higher mortality after HCT, with a relative risk of death of 1.43 for patients with a DLCO of less than 80%, and 1.28 for patients with a P(A–a)O2 of 20 mmHg or more [38]. Of interest, the excess mortality was due to miscellaneous causes and only in part to respiratory failure. Later studies reported that declines in total lung capacity or DLCO at 3 and 12 months after HCT were also associated with high nonrelapse mortality. PFTs are necessary to establish the diagnosis of restrictive lung disease. Routine lung function testing is helpful as a prognostic indicator for nonrelapse mortality and survival after HCT. Periodical PFTs are also useful to determine disease severity and response to treatment in patients with restrictive lung defects related to sclerodermatous chronic GVHD (Table 105.5). Additionally, aggressive bronchial hygiene and prophylaxis, and prompt therapy of infections, may be useful to reduce severe complications and to slow disease progression.

Obstructive airway disease The pathogenesis of airflow obstruction (AFO) after HCT is not fully understood [39]. AFO, defined as a ratio of forced expiratory volume in 1 second divided by forced vital capacity (FEV1/FVC) of less than 70% and an FEV1 of less than 80% of predicted, may represent a sequela of extensive restrictive changes in the small airways, may be related to small airway destruction by GVHD or may be due to other causes [40]. Recurrent aspirations, possibly associated with GVHD of the esophagus or sicca syndrome, or postnasal drip related to chronic sinusitis, may contribute to airway inflammation and the development of obstructive lung disease. New-onset AFO, defined as a decline in predicted FEV1 by more than 5% per year, is the most common pulmonary complication reported in a study of 1049 long-term survivors of allogeneic HCT after myeloablative conditioning, with overall rates of 26% [40]. Both acute and chronic GVHD are important risk factors for AFO and thereby affect long-term survival, with 75% of AFO cases occurring among patients with chronic GVHD, particularly those with quiescent or progressive onset [41]. There is generally no response to bronchodilator treatment, but 30–40%

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Chapter 105

Table 105.5 General long-term follow-up recommendations for adult patients after hematopoietic cell transplantation (HCT)* 1. Routine oncologic evaluation at yearly intervals is recommended due to increased risk of secondary malignancy after HCT. Annual oncologic screening includes a history and complete physical examination, Pap smears and mammogram (women starting at age ≥35), prostate exam and prostate-specific antigen (men taking testosterone or age ≥45) and testing for occult blood in the stool. Baseline colonoscopy at age 45–50 years is also recommended. At least yearly oral examination by a dentist is recommended. The skin and head and neck are the most common sites of secondary malignancy. Secondary malignancies rarely occur earlier than 2 years after transplantation, but the risk of developing cancer increases progressively after 5 years. 2. In patients transplanted for chronic myeloid leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia, routine monitoring for BCR/abl transcripts by nested PCR testing of a blood sample is recommended every 6 months for the first 2 years after transplant, and yearly thereafter if the results of PCR remain negative. If BCR/abl is positive by RT-PCR in blood, examination of bone marrow by conventional cytogenetic analysis, morphology, molecular studies for the BCR/abl by both fluorescent in situ hybridization and quantitative RT-PCR, and chimerism is recommended (see Chapter 25). 3. Measures to prevent osteoporosis in women and all patients receiving long-term treatment with corticosteroids include: • Calcium 1500 mg/day in the diet or in supplements to meet daily requirements • Vitamin D 800 IU/day in the diet or in supplements to meet daily requirements • Daily weight-bearing exercise for 20–60 minutes • Dual photon densitometry at day 80–100 post transplant evaluation and yearly thereafter • Sex hormone replacement therapy if levels are low and the benefits are thought to offset the risks Few studies found that treatment with bisphosphonate after HCT was well tolerated and resulted in less bone loss of the hips in adults [82], and increased bone mineral density in children [83]. Nonetheless, the benefits and potential long-term toxicity of bisphosphonates for the prevention of bone loss or fractures after HCT remain to be determined in large prospective studies. However, some experts recommend antiresorptive treatment in patients expected to receive corticosteroids treatment for more than 3 months [10]. Patients should be encouraged to participate in clinical trials for the prevention of osteopenia/osteoporosis after HCT.

4. Antibiotic prophylaxis should be maintained for 6 months after discontinuing all immunosuppressive medications in patients who have had chronic GVHD. Herbal medications and naturopathic remedies should not be administered to immunocompromised patients. 5. PFTs should be obtained at 1 year after allogeneic HCT and annually if there has been abnormal testing in a previous time period or there are new signs/ symptoms. PFTs should be monitored more frequently during the first 2 years after the initial diagnosis of chronic GVHD [10], or if clinically indicated. PFTs should also be obtained for autologous recipients with known pretransplant deficit or exposure to chest radiation or other lung toxic agents [54]. 6. Thyroid function should be tested at annual intervals, especially in patients who have received irradiation. 7. Ophthalmologic examination, Schirmer’s test, and slit-lamp exam should be done at annual intervals. 8. Immunizations (see Chapter 88). GVHD, graft-versus-host disease; PCR, polymerase chain reaction; PFT, pulmonary function test; RT-PCR, reverse transcriptase polymerase chain reaction. * These follow-up guidelines represent generally accepted practices for medical care after HCT at the Fred Hutchinson Cancer Research Center and the Seattle Cancer Care Alliance. They should be implemented in a way that accounts for the specific situation of each individual patient.

of patients improve on glucocorticoids. The efficacy of immunosuppressive treatment for early-onset AFO after HCT has not been studied. Bronchiolitis obliterans BO is the most common cause of AFO after HCT and can have a devastating impact on survival, with a 10-year attributable mortality of 40% among patients with chronic GVHD. BO is characterized by narrowing or occlusion of small airways that may lead to air trapping, and has been reported in 10–19% of patients with chronic GVHD [42,43], with an onset between 3 months and 2 years after HCT. Clinical and pathologic findings are similar to those seen after lung or heart–lung transplantation [43,44]. Chest radiographs may be normal in early-stage disease or may show hyperinflation of the lungs and flattening of the diaphragm in more severe cases. A high-resolution computed tomography scan (inspiratory and expiratory cuts) of the chest is the most sensitive radiologic test for BO. Recurrent pneumothoraces and pneumomediastinum occur in some patients [45]. PFTs show a reduction in forced mid-expiratory flow to 10% or 20% of predicted values, and a moderate-to-severe reduction in FVC. DLCO is usually low but occasionally normal. Pulmonary ventilation scans show decreased activity patterns corresponding to areas of obliteration of bronchiolar walls along with atelectatic areas. Histologic changes are thought to be due to a graft-versus-host reaction and may be aggravated by infections. Pulmonary infections develop in more than 20% of allogeneic HCT recipients

without GVHD, and in more than 60% of patients with chronic GVHD, where they represent a significant cause of morbidity and mortality. The clinical course of BO varies from mild, with slow deterioration, to progressive and severe, with diffuse necrotizing fatal bronchiolitis. Less than 40% of patients benefit from immunosuppressive treatment; generally, there is no response to bronchodilator treatment. While severe cases may not respond to immunosuppressive drugs, glucocorticoids alone or combined with calcineurin inhibitors can stabilize PFTs and improve outcome in some patients. In anecdotal cases of severe BO, the addition of azathioprine has allowed for a gradual taper and discontinuation of glucocorticoids with stabilization of PFTs (Flowers, unpublished data). However, because of the myelosuppressive and possibly mutagenic effects of azathioprine, this therapy is only used in severe cases that are refractory to corticosteroids. Responses of BO have been reported in few patients treated with extracorporeal photopheresis [46]. Recently, azithromycin has been suggested to have a potential therapeutic effect in patients with BO after transplantation [47]. A clinically significant improvement in both FEV1 and FVC (average improvement 21%) was observed in eight patients with BO given azithromycin 500 mg daily for 3 days, then 250 mg three times per week for 12 weeks. Khalid et al. postulate that macrolide antibiotics may have beneficial effects in patients with BO after HCT by virtue of a range of anti-inflammatory effects, not all of which are well understood. Others have postulated that the beneficial effect of azithromycin in BO is mediated via inhibition of

Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation

interleukin-8 release from human alveolar macrophages or by an increase in neutrophil apoptosis. Rare patients with end-stage pulmonary disease have been treated successfully with cadaveric lung transplants [48,49]. Single-lobe lung transplantation from the original marrow donor has also been reported [50]. The median survival of five patients treated in Seattle who received single- or double-lung transplants at other centers was 4 years (range 2 weeks to 6. 2 years) after lung transplantation. One patient was still alive at 5 years after lung transplantation at the time of last contact (Flowers, unpublished data). Bronchiolitis obliterans organizing pneumonia In contrast to BO, BOOP is a restrictive obstructive mixed process, characterized by an acute inflammatory process usually responsive to corticosteroids (Table 105.6). Histologically, BOOP shows polypoid masses of granulation tissue in the bronchioles and alveolar sacs, as well as infiltration of alveolar septa by mononuclear cells. BOOP may present as a patchy disease and may occur in association with infections, drugs, collagen vascular diseases, and transplantation. In a study of 6523 patients transplanted in Seattle, 51 cases of BOOP were observed, only two of which developed after autologous HCT. BOOP was diagnosed at a median 108 (range 5–2819) days after HCT. Most patients presented with fever, dyspnea or cough, but 23% were asymptomatic. Sixty percent of patients had abnormal PFTs (restrictive ≥ obstructive), and 92% had abnormal chest radiographs. The disease was significantly associated with acute and chronic GVHD [36]. The disease progressed in 22% of patients and resolved or remained stable in the remaining patients. Most patients respond to glucocorticoids (2 mg/kg); treatment for 6 months or longer with slow tapering is required (Flowers, unpublished). Late IPS Late IPS, defined as noninfectious interstitial pneumonitis, occurs in 6–18% of HCT recipients with an onset between 3 and 24 months (or

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later) after HCT [33] (see Chapter 96). Features of IPS in comparison with BO and BOOP are shown in Table 105.6. IPS is characterized by thickening of the interstitial space due to cellular infiltrates, fluid, and fibrous deposits. Late IPS is most common in patients with chronic GVHD, but occurs also in patients without GVHD [51]. Overall, the major risk factors for IPS after HCT are high-dose radiotherapy and multiorgan dysfunction, presumably associated with an allogeneic graft-versus-host reaction [51,52]. Patients usually experience marked dyspnea, hypoxemia, and fevers. Physical examination may be unremarkable, but chest radiographs typically show a pattern of diffuse pulmonary infiltrates. Most patients with late IPS are treated with glucocorticoids (2–10 mg/kg/day), and benefits have been reported anecdotally.

Neuroendocrine dysfunction Cytotoxic therapy and gamma irradiation damage endocrine glands. Thus, previous intensive therapy and the conditioning regimens used in preparation for HCT have the potential of causing endocrine insufficiency. A detailed description of the effects on endocrine complications, growth, and development as well as fertility is provided in Chapters 98 and 104.

Thyroid Thyroid dysfunction is a frequent complication after HCT [34,53]. The most common diagnoses include: (1) compensated hypothyroidism – “euthyroid sick syndrome” (low free triiodothyronine, free thyroxin, or both, along with normal or low thyroid-stimulating hormone [TSH] level), (2) overt hypothyroidism, and (3) autoimmune thyroid disease (see Chapter 98). Patients may also develop benign (adenoma) or malignant (carcinoma) thyroid tumors, related directly to radiation exposure and enhanced TSH stimulation (see Chapter 106). Thyroid function (i.e. TSH, free triiodothyronine, and total thyroxin) should be monitored in

Table 105.6 Late pulmonary complications

Features

Bronchiolitis obliterans

Bronchiolitis obliterans with organizing pneumonia

Symptoms/signs

Dyspnea++, cough++, wheezes++, ឆ breath sounds

Cough++, fever, dyspnea+, wheezes+, rales+++

Dyspnea+++, fever, rales++

Chest X-ray

Normal

Bilateral air space consolidation of small nodular opacities

Diffuse bilateral interstitial infiltrates

Computed tomography scan

Air trapping on high-resolution computed tomography scan and bronchial wall thickening; bronchiectasis (severe)

Ground-glass appearance Less often nodules, fibrosis or interstitial

Interstitial infiltrates

Pulmonary function FEV1/RV TLC/DLCO

Obstructive ឆឆ/ជ Normal/ឆ or normal

Restrictive/obstructive ឆ/normal ឆឆ/ឆឆ

Noncontributory

Pathology target

Proximal bronchioles

Alveoli and alveolar ducts

Interstitial space

Clinical course

Variable; mild chronic deterioration or progressive and severe; therapy stabilizes progression; irreversible if severe

Acute and progressive; responsive to treatment in most cases

Severe; requires ventilatory support; poor treatment response

Corticosteroid

1 mg/kg + other agents

1 mg/kg

2–4 mg/kg

Idiopathic pneumonia syndrome

DLCO, diffusing capacity of the lung for carbon monoxide; FEV1, forced expiratory volume in 1 second; RV, residual volume; TLC, total lung capacity.

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Chapter 105

all HCT recipients during their annual physical evaluations, or more frequently as indicated [34,54]. Subclinical compensated hypothyroidism Between 7% and 16% of patients will develop subclinical hypothyroidism after HCT [34] (see Chapter 106). Treatment with L-thyroxin is generally not recommended in the setting of mild compensated hypothyroidism, especially since spontaneous recovery can occur [55]. Furthermore, treatment with L-thyroxin might contribute to early osteoporosis, particularly if given to female transplant recipients with gonadal failure. It remains unclear if there is a relationship between high TSH levels and carcinogenesis [56]. Monitoring TSH and free thyroxine levels twice yearly [34] in this setting is recommended to determine when treatment may be indicated. In pediatric patients, treatment with L-thyroxin may be considered if TSH levels remain high or are rising [57]. Overt hypothyroidism Most patients who develop overt hypothyroidism after HCT have suffered direct damage to the thyroid gland. Secondary hypothyroidism, caused by pituitary damage, is rare following HCT. Hypothyroidism is usually diagnosed at a median of 50 months after HCT, but it occurs earlier in patients conditioned with regimens that include iodine-131radiolabeled antibody. Incidence of overt hypothyroidism is highly variable and depends to a large extent on the type of pretransplant conditioning [34]. The incidence is about three times higher in patients given single dose than in those given fractionated TBI, and the risk is further enhanced in patients who received pretransplant cranial irradiation or irradiation to the neck (e.g. for Hodgkin’s disease; see Chapter 98). L-Thyroxine replacement therapy is needed in nearly 90% of HCT recipients who received 10 Gy single-dose TBI pre transplant compared with 14–15% of patients conditioned with fractionated TBI, and even lower rates in patients who received preparative regimens without irradiation [34]. Treatment with L-thyroxine is indicated in all patients with frank hypothyroidism (elevated TSH with low free thyroxine blood levels). Thyroid hormone levels should be measured 4–6 weeks after the commencement of replacement therapy, and dosages should be tailored thereafter to the individual patient and adjusted according to thyroid function evaluation every 6 months. Elderly patients should have an electrocardiogram before treatment is initiated to exclude comorbid ischemic heart disease and arrhythmias. Autoimmune thyroid disease Autoimmune thyroiditis, presumably transferred via donor cells, has been reported [58,59]. Adrenal glands Many HCT recipients receive glucocorticoid therapy in the pre-, peri- or post-transplant period and show the classic side-effects of steroid therapy, including Cushingoid features, myopathy, and bone loss. Endogenous cortisol production is suppressed, and any superimposed stress may cause a relative adrenal insufficiency. For the same reason, glucocorticoid therapy for control of GVHD should be administered on alternate days whenever possible, and must be tapered gradually. Increased fatigue, nausea, erythema “flush”, low-grade “fevers”, myalgias, and joint stiffness are common after discontinuation of prolonged use of glucocorticoids and can be misdiagnosed as exacerbation of GVHD of the gastrointestinal tract and musculoskeletal system. Cortisol stimulation tests to rule out adrenal insufficiency (8.00 a.m. cortisol levels checked before and 30–60 minutes after 1 μg intravenous cosyntropin) should be obtained in this setting.

One study in 78 patients showed 24% to have subnormal 11deoxycortisol levels following discontinuation of glucocorticoid therapy at 1–8 years after HCT (see Chapters 98 and 104). Recent data suggest that the incidence of adrenal insufficiency may have been underestimated in the past (Sanders, unpublished data). Subnormal stimulated cortisol levels are also observed in patients given cranial irradiation, and a central (primary) effect may play a role in patients post transplant [60]. One report described a patient who developed Addison’s disease, apparently of an autoimmune nature, 10 years after HCT for Wiskott–Aldrich syndrome. Hypothalamic-pituitary axis Cranial irradiation, with or without TBI, may have severe effects on the pituitary gland [60]. Thyrotropin-releasing hormone may be low early post transplant, and thyrotropin-releasing hormone-induced TSH responses may be subnormal and delayed. Release of gonadotropin in response to luteinizing hormone-releasing hormone may be elevated [60] (see Chapter 98). Prolactin secretion and the pituitary-adrenal axis are usually intact. Growth hormone levels are decreased after cranial irradiation (±TBI), and deficiency becomes apparent earlier with younger age at HCT (see Chapter 104). Gonadal function, puberty, and fertility Chemotherapy and TBI regimens used prior to HCT usually cause gonadal failure, dependent upon the intensity and type of the regimen and the age of the patient at HCT (see Chapter 104). Annual assessment of gonadal function is recommended in patients after HCT, including determination of blood levels of free and total testosterone in men, and estradiol in women. Puberty and menarche are markedly delayed or may not occur, and fertility is rarely regained in either men or women (see Chapter 104). In men, decreased libido, reduced bone mineral content, and low testosterone levels after HCT are indications for testosterone replacement unless contraindicated for other reasons. Treatment with a testosterone transdermal patch (60 cm2 containing 328 mg testosterone) with either Androderm 5 mg/day or Testoderm 5 mg/day (applied to the skin of the arms, back or buttocks) represents an adequate regimen for men with hypogonadism. Androgel is less convenient than transdermal formulations, and the scrotal transdermal formulation appears to be poorly tolerated (Flowers, unpublished data). Another effective formulation is androgen injections using prolonged action formulations such as testosterone cypionate (Andro-Cyp, Dep-Ando, Depo-testosterone, and Duratest) or testosterone enanthate (Ando LA 200, Andryl 200, and Delatestryl) given at 50–200 mg every 2–4 weeks (deep into the gluteal muscle). Prolonged use of high-dose androgens has been associated with development of peliosis hepatitis and hepatic neoplasms. Polycythemia has been associated with administration of testosterone and correlates with elevated bioavailable testosterone and estradiol levels. Although the mechanism is unclear, testosterone replacement therapy may also cause or worsen obstructive sleep apnea. Men treated with testosterone may be at increased risk for prostate hypertrophy and prostate carcinoma. Benefits and risks of testosterone replacement must be discussed with each patient and its risk–benefit ratio reassessed periodically. Testosterone blood levels should be monitored periodically and the dose tailored according to levels and side-effects. Digital prostate examination, prostate-specific antigen levels, liver test panel, fasting lipid profile, and hemoglobin level should be evaluated prior to initiation of testosterone and monitored periodically. Gynecomastia, edema, male baldness, nausea, tachycardia, increased cholesterol levels, and cholestatic liver

Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation

enzyme abnormalities represent side-effects of testosterone therapy and should be discussed with each patient. Permanent ovarian failure occurs in 99% of women who receive busulfan and cyclophosphamide (BU/CY) pre transplant. In Seattle, only 4% of women who received fractionated 14.0–15.75 Gy TBI pre transplant recovered ovarian function and 1% have become pregnant, whereas 8% of women conditioned with CY and fractioned TBI 12 Gy recovered ovarian function and 3% have become pregnant (Sanders, personal communication). Recovery of ovarian function has been observed in approximately 50% of younger patients (less than 26 years) conditioned with CY alone (see Chapter 104). Appropriate management of menopausal symptoms, bone loss, and growth and development in children can improve their quality of life after HCT (see Chapter 104). Hormone replacement therapy with estrogen alone (for patients without a uterus) or combined with progestin (for patients with a uterus) is effective for the management of vasomotor symptoms, “hot flashes,” relief of vaginal and vulvar dryness, urinary symptoms, and emotional lability, and to decrease bone loss after HCT. Premature menopausal symptoms and bone loss are common complications [61,62] observed in the first year after HCT. Hormone replacement after HCT should be individualized with the pros and cons discussed carefully with each patient before initiation of replacement, and reassessed at least yearly if treatment is continued beyond 1–3 years after HCT. Increased risk of breast cancer, coronary heart disease (CHD), stroke, and venous thromboembolism was reported in the Women’s Health Initiative study (nontransplanted women older than 50 years) with continuous combined estrogen–progestin replacement versus placebo, for an average of 5.2 years. Nonetheless, this study also showed a significantly reduced risk of bone fractures and colon cancer [63]. The risk of breast cancer in postmenopausal women on hormone replacement therapy (outside the transplant setting) increases with increasing hormone use, but the impact declines again after cessation of replacement, and is basically no longer detectable by about 5 years [64]. The risk of developing a secondary malignancy begins to rise, starting 5 years after HCT (see Chapter 106). Whether combined hormone replacement contributes to an increased risk of secondary cancers, in particular breast cancer after HCT, is difficult to establish. These findings should be considered in the context of other benefits and risks associated with the use of hormone replacement after HCT. The results of the unopposed estrogen versus placebo Women’s Health Initiative study (nontransplant postmenopausal women) showed an increase in the risk of stroke, a decrease in the risk of hip fracture, and no effects on incidence CHD or increased risk of breast cancer with an average follow-up of 6.8 years [65]. Thus, considering all available data, the benefit of administration of estrogen combined with progestin (for adult women with a uterus) or estrogen alone (for adult women without a uterus) in young women with gonadal failure after HCT is reasonable until age 50, unless other contraindications are present. Discontinuation of gonadal hormones after HCT should be considered after age 50, or earlier if indicated. Hormone replacement should not be administered in patients with a history of cardiovascular disease (i.e. venous thrombosis, pulmonary embolism, and stroke), hypercoagulation disorders, breast cancer or liver disease. Nonhormonal strategies for alternative management of menopausal symptoms and bone loss should be provided to all adult HCT recipients. Topical estrogen can relieve local vaginal/vulvar symptoms associated with gonadal failure and may allow for the use of lower doses of systemic estrogen. Gynecologic evaluation at yearly intervals, breast selfexamination at monthly intervals, and mammograms at 35–40 years of age (baseline for women on hormone replacement) and yearly thereafter are recommended in all women after HCT.

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Cardiovascular disease Cardiac insufficiency and CHD have been recognized as complications of intensive cytotoxic therapy, in particular high-dose anthracycline, and mediastinal irradiation [66]. Cardiac insufficiency may also be seen in patients conditioned with CY, 200 mg/kg, early after HCT, although the overall incidence is low [67]. Late cardiomyopathy has been treated successfully by orthotopic cardiac transplantation [68]. A cross-sectional survey study of late effects of HCT conducted in 248 patients who survived at least 2 years after allogeneic or autologous HCT for chronic myeloid leukemia reported an incidence of cardiopulmonary impairment (i.e. arrhythmia, congestive heart failure, myocardial infarction, CHD, hypertension, stroke, angina, exercise-induced shortness of breath, pericarditis, stiff or leaking heart valves or blood clots) of approximately 33% compared with 26% in the 317 sibling controls (P = 0.07) [69]. In this study, cardiopulmonary impairment was greater after unrelated donor HCT (46%) than after autologous (18%) and related donor HCT (29%) (P < 0.05). Exercise-induced shortness of breath was reported by approximately 17% of patients in the unrelated donor group, 7% in the related donor group, and 4% in the autologous transplant cohort [69]. Multivariate analysis limited to the allogeneic HCT groups showed chronic GVHD to be the major factor associated with increase risk of cardiopulmonary impairment among other late events (i.e. hypothyroidism, osteoporosis, neurosensory, and neuromotor impairments). CHD and thrombotic events have been reported at various time intervals after HCT [69]. Hyperlipidemia and hyperglycemia (common in patients treated with calcineurin inhibitors, rapamycin, and glucocorticoids), treatment with estrogen/progesterone, and inactivity due to fatigue or other causes are risk factors for the development of coronary disease in long-term survivors of HCT. General recommendations include regular exercise activity (20 minutes, five times per week), monitoring fasting lipid profiles and glycemia at least yearly (and more frequently in patients at risk), adjustment of levels of drugs given for GVHD, as applicable, and avoidance of prolonged use (>3 years) of standard dose estrogen and progesterone in postmenopausal women. Late thrombotic events after HCT have been reported in 1–4% of patients, but these numbers may be underestimates (Flowers, unpublished data). Anticoagulation should be considered in patients at high risk (e.g. prior history of thrombosis or hypercoagulability status).

Ocular problems Late ocular complications after HCT can involve the anterior (i.e. cataracts and KCS) or the posterior (i.e. microvascular retinopathy, optic disc edema, infectious retinopathy, and hemorrhage) segment of the eyes. The most common problems affecting the eyes after HCT are cataracts and KCS, the latter often associated with chronic GVHD. Cataracts Glucocorticoids and gamma irradiation are the primary causes of cataracts after HCT. After TBI, posterior capsular cataracts are noticed approximately 1 year after HCT [70]. Following single-dose TBI (usually 920–1000 cGy), the incidence of cataracts was 60–80% at 5–6 years. After fractionated TBI, the incidence was approximately 50% at cumulative doses above 1200 cGy, 30–35% at doses of 1200 cGy, and as low as 10% in patients given hyperfractionated TBI (more than six fractions) or treated at low exposure rates (0.04 cGy/min) [34]. Eye shielding might prevent cataracts, but relapse of malignancy in the ocular bulb can occur. Among patients who have not received TBI or cranial irradiation, the incidence of cataracts is less than 20% and is

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almost exclusively due to corticosteroids [70]. The incidence of cataracts overall is higher in patients who also received cranial irradiation. Approaches to cataract prevention in the setting of HCT are experimental. One report suggests that heparin given, for example, in patients with veno-occlusive disease has a protective effect on the lens [34]. The treatment of choice of cataracts is lens extraction and implantation of an artificial lens. Although surgery should be done preferably after discontinuation of all systemic immunosuppressive treatment, procedures have been performed successfully in patients receiving low doses of prednisone (less than 0.25 mg/kg). The improved quality of modern lenses has allowed successful (long-surviving) implants even in children. Ocular sicca Chronic dry eyes are frequent after HCT and can result in acute conjunctivae inflammation, pseudomembranous and cicatricial conjunctivitis, and KCS. KCS occurs in approximately 40–60% of patients with chronic GVHD [71] and is often irreversible (see Chapter 87) [72]. Chronic KCS can result in scar formation (e.g. in the tarsus) and lead to synechiae, ectropion, corneal damage, and potentially perforation if not treated meticulously and aggressively. KCS also occurs in patients without chronic GVHD, although the possibility that it represents a sequel of prior GVHD or a forme fruste of GVHD must be considered [2,72]. Long-term management includes supportive care with artificial tears, long-acting ocular lubricants, punctal occlusion or cauterization, ophthalmic cyclosporine, topical glucocorticoids, autologous serum eye drops, and oral administration of cholinergic agents [10]. Occlusive eyeglasses can significantly improve the symptoms of dry eyes. For patients with severe KCS refractory to supportive care, the use of the liquid corneal bandage provided by a fluid-ventilated, gas-permeable scleral lens (Boston Scleral Lens) has been effective in mitigating symptoms and resurfacing corneal erosions [73]. Transplantation of limbal epithelial cells reported for other conditions [74] has been performed in some patients with GVHD-related KCS (using the original donor cells) with mixed results (Bensinger and Flowers, unpublished data). Ocular complications of the posterior segment Late retinitis is infrequent after HCT and usually occurs in patients with delayed immune reconstitution such as those with chronic GVHD, conditioned with Campath-1G, or recipients of T-cell-depleted or cord blood transplants. Late retinitis is often related to infections with CMV, herpes zoster or Toxoplasma gondii. Ischemic retinopathy with cottonwool spots and optic disc edema has been reported in 10% of patients after HCT [34]; atypical retinal microvasculopathy, without cotton-wool spots, has also been described [75]. The TBI regimens alone cannot explain the ischemic retinopathy reported after HCT, and additional factors such as cyclosporine may contribute to lower the threshold for radiation retinopathy [76]. Ischemic retinopathy has been reported also in patients conditioned without TBI. In most cases of ischemic retinopathy, retinal lesions resolve following withdrawal or reduction of immunosuppressive therapy, as described in a case with complete blindness [77]. The development of ischemic retinal lesions is a multifactorial process leading to capillary damage of the eye fundus.

Musculofascial problems Myopathy is common after HCT. One of the most common causes of myopathy is treatment with corticosteroids. The high rates of fatigue and lack of energy observed in transplant survivors, which lead to inactivity, are important contributors to muscle weakness and loss of muscle and

bone mass. It is important, therefore, to maintain and gradually increase the level of physical activity to counter a progressive decline in physical function. Occasionally patients with chronic GVHD have involvement of muscle, fascia, and serous membranes including the synovia [8]; joint effusions may occur in patients without any other sign of GVHD. Diagnostic procedures and management are discussed under GVHD (see Chapter 87). Involvement of fascia or tendons by an eosinophilic infiltrate (early) or fibrosis (late), frequently preceded by edema and often resulting in joint contractures of the wrists (most common), fingers, shoulders, elbows, ankles, and occasionally knees, may be manifestations of chronic GVHD. Because of the significant morbidity associated with joint contractures, range of motion assessment, including the extension of hands and wrists (i.e. palms together with the elbows extended, referred to by the author as “Praying Buddha position”), is necessary when examining allogeneic transplant recipients to rule out fibrotic fasciitis, panniculitis or tendonitis. Patients with limitations of wrist extension often use the back of the hands (rather than the palms) when, for example, standing up from the sitting position. Some patients may be misdiagnosed as having carpal tunnel syndrome. Stretching exercises are important to improve the range of motion of affected joints and restore functions of daily living. Physicotherapy and deep myofascia massage (Hellerwork) are critically important in the management of joint contractures [10]. Muscle cramps are common in long-term HCT recipients. Approximately 30% of allogeneic and 40% of autologous HCT recipients reported muscle cramps on the 5–15 years annual health questionnaires sent to all patients transplanted in Seattle. The etiology of muscle cramps remains unclear. Muscle cramps of the extremities and severe carpopedal spasms, with impairment of fine motor function, are often, but not always, observed in patients with a history of chronic GVHD. Generally, conventional muscle relaxants and analgesics are ineffective. Adequate hydration, correction of electrolyte imbalances (i.e. hypomagnesaemia), and regular stretching represent baseline interventions. Clonazepam or baclofen has been used with some success in a few patients. While treatment with quinine sulfate has been used in the past, the Food and Drug Administration has issued a cautionary statement (P06–195) regarding the off-label use of this drug to treat leg cramps because of serious adverse effects.

Skeletal complications Osteoporosis and osteopenia Dual-energy X-ray absorptiometry, a semiquantitative method to assess bone mineral density (BMD), is a validated test used to detect osteoporosis (t-score of equal to or less than −2.5 standard deviations below age-related mean BMD) and osteopenia (t-score of −1.0 to −2.4 standard deviations below age-related mean BMD). Other markers of bone loss include elevated alkaline phosphatase level, particularly in women, as well as high C-terminal propeptide. Increased urinary excretion of hydroxyproline can also be used to assess bone loss and response to treatment [78]. Bone loss is a frequent complication after HCT and results from impaired bone mineralization through disturbances of calcium and vitamin D homeostasis, osteoblast and osteoclast dysfunction, and deficiencies in growth or gonadal hormone secretion. Factors associated with bone loss after HCT include irradiation, corticosteroid treatment, inactivity, and iatrogenic hypogonadism [34,61]. Both the cumulative dose and duration of corticosteroid treatment and the duration of calcineurin inhibitor treatment have shown significant associations with loss of BMD. In a large study, approximately 40% of men and women were

Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation

found to have reduced BMD by 1 year after HCT [79]. In another study, osteopenia was found in 33% and osteoporosis in 18% of women after HCT [80]. Increased risk of bone loss without fracture risk was reported in a cohort of 79 patients after HCT with a median follow-up of 6.5 years [81]. Few studies on the safety and efficacy of bisphosphonate for prevention of bone loss after HCT have been reported. Results of a randomized study in adult allogeneic HCT recipients showed less bone loss in patients receiving additional pamidronate (60 mg before and 1, 2, 3, 6, and 9 months after HCT) compared with patients receiving 1000 mg calcium carbonate and 800 IU vitamin D daily, and estrogen (women) or testosterone (men) alone [82]. In a retrospective study of pediatric HCT recipients, treatment with bisphosphonate was well tolerated and was associated with improvement in BMD [83]. Measures to prevent bone loss after HCT are indicated. Many experts recommend the use of antiresorptive treatments (gonadal hormonal replacement or bisphosphonates) in patients with gonadal failure and with chronic GVHD requiring treatment with glucocorticoid [10]. Physicians and patients should be aware of osteonecrosis of the jaw, a rare but well-recognized potentially serious toxicity of bisphosphonate, which is reported more frequently with intravenous formulations [84]. As part of long-term follow-up, a detailed nutritional assessment of calcium and vitamin D needs to be recorded for each patient prior to HCT, between 80 and 100 days after HCT, and at least yearly thereafter if at risk. Measures to prevent osteoporosis (Table 105.5) are recommended to all women and all patients receiving long-term corticosteroid treatment. Patients should pursue an exercise program that combines aerobic weight bearing and resistive exercises, and should follow fall prevention strategies (conditioning, removing trip hazards from the home, and eye examination and correction of vision). Also, patients should stop smoking and limit alcohol consumption. In women, supplementation with estrogens with or without medroxyprogesterone can increase bone mass after HCT. Thus, if not contraindicated, gonadal hormonal replacement is a reasonable consideration after HCT, particularly in patients receiving corticosteroids and in premenopausal women [10,34].

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MRI and structurally abnormal radiographs), which occur prior to collapse of the femoral head, from more advanced lesions that are identified by abnormal MRI and radiography showing loss of the femoral head contours, and often loss of joint space [87]. In summary, the major risk factors of AVN are glucocorticoid use followed by single-dose (10 Gy) or fractionated higher-dose TBI. An attempt to minimize the dose and duration of glucocorticoids is advisable. For patients with early-stage AVN, core decompression can result in prompt relief of hip pain [88]. However, the only satisfactory treatment for advanced AVN, particularly in weight-bearing joints, is joint replacement.

Dental problems An oral sicca syndrome related to conditioning therapy or chronic GVHD may lead to poor oral hygiene with recurrent infections and periodontitis. Dental decay occurs because of a lack of cleansing by saliva, which is of altered consistency and reduced volume. Also, the mouth is painful, and patients are hesitant to take care of their teeth and mucosa (see Chapter 103). The incidence of dental decay is not higher in children after HCT than in those given chemotherapy only, suggesting that tooth decay is mostly related to the effects of high-dose cytotoxic therapy rather than transplant-specific factors [89]. The management is similar to that in nontransplanted patients who have received head or neck irradiation, and includes diligent hygiene, fluoride treatment, artificial saliva, capping of teeth, and other supportive measures. In children, irradiation interferes with dental and facial development (see Chapters 103 and 104). There may be poor calcification, micrognathia, mandibular hypoplasia, root blunting, and apical closure [90]. The changes are most severe in children less than 7 years of age at the time of HCT. No effective prophylactic measures are currently available; careful planning (and where possible timing) of the preparative regimen may minimize these side-effects. A detailed discussion of oral complications is provided in Chapters 103 and 104.

Genitourinary dysfunction Avascular necrosis

Bladder

Avascular necrosis (AVN), especially in weight-bearing joints, is a classic side-effect of corticosteroid therapy, reported in approximately 4–10% of allogeneic HCT survivors at a median of 12 (range 2–132) months after HCT [85,86]. The hip is the most frequently affected joint (in up to 88% of individuals), with bilateral involvement in more than 60% of cases. Most patients have more than one joint affected. In addition to corticosteroid therapy, male gender (relative risk 4.2) and age 16 years or over (relative risk 3.8) have been identified as risk factors. The largest multi-institutional study to date, including 4388 patients, reported AVN in 77 patients for a 5-year incidence of 4.3% [85]. Symptoms developed at 2–132 months, and 1–7 (mean 1.9) joints were affected; hip joint replacement was required in nearly 50% of the patients. Time to joint replacement was 2–42 months from initial diagnosis of AVN. Older age, a diagnosis of aplastic anemia or acute leukemia as opposed to other diagnoses, an irradiation-based conditioning regimen, certain types of GVHD prophylaxis, and acute or chronic GVHD were associated with an increased risk of AVN. In a case-control study of 87 patients with AVN, post-transplant glucocorticoid use and a TBI-containing regimen pre HCT were significant risk factors [86]. Cyclosporine, shown by others to stimulate osteoclasts, was not a significant risk factor in that study. Magnetic resonance imaging (MRI) is the gold standard test to diagnose early AVN. Findings in the MRI can be used to differentiate AVN stage I (abnormal MRI with normal radiographs) and stage II (abnormal

Hemorrhagic cystitis, common in the early posttransplant period, is often related to the toxicity of CY conditioning (see Chapter 97). However, patients with protracted hemorrhagic cystitis may later develop bladder wall scarring with volume loss, resulting in severe morbidity with dysuria and increased urinary frequency, recurrent infections, and occasionally hydronephrosis. In severe cases, cystectomy with the surgical reconstruction of a new bladder from a bowel loop is necessary. Supportive care includes management of pain with antispasmodic drugs and topical anesthetic. Prophylaxis and treatment of acute hemorrhagic cystitis is discussed in Chapter 97. Late hemorrhagic cystitis is often associated with infections with adenovirus, polyoma virus, BK or other viruses. Adenovirus strains with a tropism for the genitourinary organs also involve ureters and kidneys and may cause renal failure. Late-onset cystitis associated with polyoma virus has been treated successfully with vidarabine infusion [91]. Women with gonadal failure and those affected by chronic GVHD involving the vulva or vagina are at risk for recurrent urinary tract infections, which require prompt antibiotic therapy. Kidneys Renal complications after HCT are discussed in detail in Chapter 97. The true incidence of late renal impairment and end-stage renal disease

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has not been well established. Renal impairment after HCT can result from nephrotoxins, TBI, be related to the underlying disease (e.g. multiple myeloma), arise from treatment with intravenous bisphosphonates or be related to chronic GVHD. The most common nephrotoxins used in the HCT setting are calcineurin inhibitors and antibiotics, which often result in early renal dysfunction. In contrast to observations in solid-organ transplant recipients, late renal failure is infrequent after HCT. However, particularly in patients treated aggressively with nephrotoxic chemotherapy (e.g. with platinum compounds) or irradiation before HCT, some investigators reported a 20–25% incidence of renal insufficiency by 2 years after HCT [92,93]. The term “marrow transplant nephropathy” has been coined for this syndrome, which comprises azotemia, hypertension, and a disproportionate degree of anemia [93,94]. Some patients may also show features of a hemolytic–uremic syndrome, which is more common in patients receiving calcineurin inhibitors combined with sirolimus (see below). Renal biopsies show changes of radiation nephritis including mesangial and endothelial drop-out, expansion of the glomerular basement membrane, and widening of the glomerular capillary loops. Analysis of clinical and animal studies indicates that TBI can lead to long-term renal function impairment, as determined by creatinine levels, blood urea nitrogen, residual activity on chromium-51 EDTA, proteinuria or declining hematocrit [95]. In one clinical study, renal dysfunction was strongly related to the total TBI dose delivered and to the dose per fraction; the presence of GVHD was another risk factor [96]. The use of angiotensin-converting enzyme inhibitors such as captopril may be beneficial [97]. Once nephropathy has developed, control of hypertension is the mainstay of therapy. Renal failure associated with hemolytic–uremic syndrome or microangiopathic hemolytic anemia can occur even in patients who are not heavily pretreated and are not conditioned with TBI. The mechanism of hemolytic–uremic syndrome is not fully understood, and it may become manifest either during or after discontinuation of treatment with calcineurin inhibitors [98]. The mechanism involves altered pathways of prostaglandin synthesis, which, in conjunction with endothelial damage, might interfere with coagulation homeostasis. Discontinuation of the presumptive causative agent is essential. Plasmapheresis and the use of glucocorticoids have been suggested as therapeutic options [99]. In some patients, the disease stabilizes upon blood pressure control. Nephrotic syndrome has been reported in few case series after HCT, often in association with GVHD [100]. A complete response of 45% has been reported with immunosuppression treatment, with a significant reduction in proteinuria observed in 55% of the treated cases. Nephrotoxicity of intravenous bisphosphonates should be ruled out as the cause of nephrotic syndrome in patients at risk [101]. Genital organs Chronic GVHD may also involve the genital organs, in particular the glans penis and vagina, and can lead to late complications. Vaginitis may be severe and cause considerable distress and dyspareunia. In addition to topical immunosuppressive therapy, topical estrogens, if indicated, are often used to better control symptoms and prevent the development of adhesions. The vulva has also been a site of posttransplant malignancies. Post-transplant reproductive function is discussed in Chapters 98 and 104.

Gastrointestinal and hepatic complications Chapter 95 provides a detailed discussion of gastrointestinal and hepatic complications. A description focusing on late problems follows below.

Gastrointestinal tract The gastrointestinal tract is a frequent target of acute transplant-related complications. Chronic problems are less common. Involvement of the esophagus by chronic GVHD may lead to strictures and web formation. Repeated dilator treatments are often required to allow for normal food intake. The most common cause of late-onset and recurrent diarrhea not explained by GVHD, oral magnesium supplementation or infections (e.g. CMV, adenovirus, parvovirus, Clostridium difficile, etc.) is malabsorption related to pancreatic insufficiency. Oral enzyme supplementation is recommended, with good responses reported in adults and children. Pneumatosis cystoides intestinalis has been described in patients post transplant, generally while receiving glucocorticoid therapy. The diagnosis is usually made 2–3 months after HCT. No specific therapy is available, and the bowel normalizes spontaneously or with the resolution of other underlying problems. Liver Late liver function abnormalities can be caused by GVHD, drug effects (e.g. antifungal drugs, calcineurin inhibitors, and TMP/SMX), iron overload, fat liver, and viral infections (see Chapter 95) [102]. At 3 or more months after HCT, the most frequent cause for enzyme or bilirubin elevations is chronic GVHD. However, viral hepatitis has to be considered at any time after HCT. Hepatitis may be due to hepatitis B or C (or other) viruses and may be transmitted from transplant or transfusion donors or occur by reactivation of host virus. Some cases of hepatitis B after HCT have been diagnosed upon tapering of immunosuppressive drugs given for GVHD prophylaxis or therapy. In an occasional patient, liver function abnormalities may be related to VZV which may cause hepatitis without showing cutaneous manifestations. Prompt institution of therapeutic doses of acyclovir is indicated. Of considerable concern are the late sequelae of hepatitis, i.e. cirrhosis, related to hepatitis C virus (HCV), and hepatoma, related to HCV or hepatitis B virus [103]. A report by the transplant team in Pesaro, Italy, summarized the effects of iron overload and HCV on liver fibrosis in patients transplanted for thalassemia [104]. Among 211 patients followed for a median of more than 5 years, 46 (22%) showed progressive fibrosis of the liver. The risk was related to the hepatic iron concentration and HCV positivity. None of the HCV-negative patients with an iron content of less than 16 mg/g dry weight showed progression, whereas all patients who were HCV positive and had an iron level of over 22 mg/g progressed. Thus, attempts should be made at treating both iron overload (with phlebotomy and chelation, oral agents now being available) and HCV (with interferon and antiviral antibiotics). Several hepatic malignancies, including unusual fibrous histiocytomas, have been observed (see Chapter 106). Cases of hemosiderosis of the liver have occurred post transplant and are thought to be related to altered iron absorption (see Chapter 95). Iron overload has been suggested to mimic exacerbation of liver GVHD with subsequent normalization of abnormal liver tests in response to phlebotomy [105].

Metabolic problems Several studies have shown that survivors of childhood cancer and pediatric HCT are at an increased risk for developing insulin resistance and the metabolic syndrome [106]. Similarly, survivors of adult cancer have a high prevalence of metabolic syndrome and are at an increased risk for cardiovascular mortality [107]. The definition of metabolic syndrome by the Adult Treatment Panel III with modifications proposed by

Delayed Nonmalignant Complications after Hematopoietic Cell Transplantation Table 105.7 Definition of metabolic syndrome Measure (presence of any 3 of 5 will constitute the diagnosis of metabolic syndrome) Elevated waist circumference Elevated triglycerides

Reduced HDL-C

Elevated blood pressure

Elevated fasting glucose

Categorical Cut Points ≥102 cm (≥40 inches) in men ≥88 cm (≥35 inches) in women ≥150 mg/dL (≥1.7 mmol/L) or Receiving medication for hypertriglyceridemia <40 mg/dL (<1.03 mmol/L) in men <50 mg/dL (<1.3 mmol/L) in women or Receiving medication for reduced HDL-C ≥130 mm Hg systolic blood pressure or ≥85 mm Hg diastolic blood pressure or Receiving antihypertensive medication for a history of hypertension ≥100 mg/dL or Receiving medication for elevated glucose

Reproduced with permission from Ford et al. [108].

the American Heart Association/National Heart, Lung and Blood Institute is shown in Table 105.7 [108]. The metabolic syndrome is a frequently observed aggregation of five factors of metabolic origin: atherogenic dyslipidemia, elevated blood pressure, elevated glucose, a prothrombotic state, and a proinflammatory state [109]. Metabolic syndrome is associated with a substantially increased risk of type 2 diabetes mellitus and atherosclerotic cardiovascular disease. The prevalence, risk factors, and implications of metabolic syndrome in adult survivors of HCT have yet to be determined. Damage to the neuroendocrine system and vascular endothelium by high-dose chemotherapy and TBI, and the immunologic and inflammatory effects of allogeneic HCT, could be predisposing factors. In a prospective study, the incidence of diabetes was higher in HCT recipients than in controls [110].

Neurologic system Chronic or delayed neurologic complications are known to occur after intensive chemoradiotherapy in patients who do not undergo HCT. The conditioning regimens used in preparation for HCT and medications used after HCT further contribute to these problems. Neurologic complications may affect the central and peripheral nervous systems. Reported neurologic complication rates appear to be lower in autologous than in allogeneic HCT recipients. Complications include leukoencephalopathy resulting from intrathecal chemotherapy or cranial irradiation, calcineurin-induced central nervous system (CNS) toxicity, peripheral neuropathy related to chemotherapy exposure or Guillain-Barré syndrome, vascular complications such as stroke or subdural hematoma and late CNS infections. Leukoencephalopathy (involving the white matter of the brain) is due to extensive intrathecal chemotherapy, in particular methotrexate, given

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alone or combined with cranial irradiation (1800–2400 cGy or even higher doses) and the use of TBI. Severe leukoencephalopathy has been observed in children who often lose higher cortical functions and may be left in a vegetative state [111]. Recovery is uncommon. Leukoencephalopathy has been diagnosed with decreasing frequency over the past decade, conceivably due to a more judicious use of intrathecal therapy, brain shielding where appropriate or omission of cranial irradiation whenever possible. Anecdotal reports have described multifocal cerebral demyelinization, inflammatory demyelizing polyneuropathy, immune-mediated myelopathy, and encephalopathy [112,113]. While the possibility of CNS involvement by GVHD has been debated and rejected in the past, more recently the suggestion of CNS involvement by GVHD has reemerged [114]. Supporting the suggestion that GVHD may affect the CNS are the observations reported in patients transplanted for Hurler’s disease in whom host microglia were replaced by donor-derived cells [115]. Others have described cerebellar and pyramidal signs correlating with GVHD activity [116]. Also, Padovan et al. [117] and Takatsuka et al. [118] described periventricular white matter lesions and vasculitis or an angiitis-like syndrome, which they attributed to GVHD. Some patients improved on treatment with glucocorticoids. Several clearly documented cases of peripheral neuropathy with reduced nerve conduction velocity related to chronic GVHD have been reported [119,120]. Destruction of Schwann cells seems to be responsible for this phenomenon, and patients respond to glucocorticoid therapy. Late CNS infections occur predominantly in severely immunocompromised HCT recipients, particularly in those with chronic GVHD. Patients with chronic GVHD are prone to develop septicemia and meningitis caused by encapsulated bacteria; herpes viruses, including VZV; fungal organisms; Toxoplasma gondii [121]; and other pathogens (see Chapters 88–94). All patients should receive antibiotic prophylaxis for at least 6 months after HCT, longer in patients with chronic GVHD [54]. In one autopsy study of 180 patients, only 17 had normal CNS findings [122]. Patients may have impaired memory, shortened attention span, and defects in verbal fluency after HCT. Children, particularly those who have also received cranial irradiation, are likely to score lower than controls in visual–motor and processing tasks and various IQ tests (see Chapter 104).

Psychosocial effects and rehabilitation Long-term adjustments and rehabilitation after HCT depend strongly on pretransplant conditions and late transplant complications such as chronic GVHD. Considerable insights have been gained in recent years [110] (see Chapters 33–36). Several studies have compared the quality of life of patients who received HCT with that of patients with comparable diagnoses who were given maintenance chemotherapy. In regards to symptoms of depression and multifocal psychiatric symptomatology, and by scores according to several quality-of-life instruments, no significant differences between the two groups were found. Not knowing what the future will bring, thoughts of dying, feeling tired, reduced attention span, short-term memory deficit, depression, inability to attain sexual satisfaction, low self-esteem because of reduced physical functioning, and worrying about being a burden, all contribute to psychosocial morbidity. In a prospective 5-year longitudinal study of quality of life in HCT recipients, we found that physical recovery occurred earlier than psychologic or work recovery [110]. Transplantrelated distress was slower to resolve among recipients of allogeneic HCT and those with less social support before transplant ( p ≤ 0.01). Patients who had more experience with cancer treatment before

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HCT had a more rapid recovery from depression ( p = 0.04) and treatment-related distress ( p = 0.009). Recovery was slower for patients who were more depressed before HCT. Fewer than half of the survivors were fully recovered by 1 year, but the proportion without major limitations increased to 63% (n = 57) by 5 years. By 5 years after treatment, 85% of transplant survivors are working full time, reporting minimal physical symptoms, and reporting good psychologic and family health. Despite good functional levels by 5 years, fatigue, lack of energy, and sexual dissatisfaction remain frequent problems reported by HCT survivors. Thus, full recovery after HCT is a 3–5-year process [110]. Changes in body image due to skin disfigurement, weight loss, and weakness, in addition to side-effects of medications, especially glucocorticoids, weigh heavily on patients. Patients with joint contractures, pulmonary disease or severe KCS may become disabled for many years. Realistic and open pretransplant communication about potential long-term complications, and pretransplant identification of patients at high risk for severe physical and psychosocial morbidity, will help to deal successfully with these problems as they arise (see Chapter 30). Problems with obtaining employment and insurance coverage are frequent. Even patients without chronic GVHD and autologous HCT recipients may experience discrimination. This issue requires extensive counseling of both patients and insurance carriers and continuing analysis of long-term results to provide detailed information on survival. More effective interventions to increase work-related capabilities, improve social support, and manage depression are needed to improve and accelerate the recovery process after HCT. Effects of conditioning and HCT on growth and development of pediatric and adolescent patients are discussed elsewhere (see Chapter 104). Early detection of potential problems and health maintenance for many years are important. The desire to be equal to their peers may cause severe problems with compliance in this age group, and problems with self-esteem, body image, and sexuality may weigh particularly heavy. A multidisciplinary approach involving adolescent medicine physicians and endocrinologists along with group therapy is most promising. Rehabilitation must begin at the time of diagnosis and must involve a long-term plan [123]. Fatigue Fatigue is the most persistent symptom beyond the first year after HCT. A multicenter longitudinal cohort study of fatigue and sleep disturbance in 172 adult survivors more than 12 months after HCT, followed for 18

months after the first assessment, found that a majority reported at least mild problems, with 15–20% reporting moderate-to-severe problems [124]. Older age, TBI treatment, and female gender were among the risk factors for sleep disturbances but not for fatigue. Other studies found age to be a risk factor for fatigue [125]. Several biologic mechanisms have been postulated to explain fatigue after HCT or after other cancer treatments, such as sleep disruption, lack of physical activity, depression, anemia, delayed radiation effects, medications including glucocorticoids, gonadal insufficiency, metabolic abnormalities, effects of interleukins and interferons, and others. Nonetheless, there is no strong evidence to support any of these causes over others in HCT survivors. Improvement of fatigue and physical strength have been reported using exercise, erythropoietin injections or coping skills that have included relaxation training. However, these studies focused on the acute phase of treatment rather than on long-term survivors.

Recent developments Considerable effort has gone into the development of reduced-intensity conditioning regimens in preparation for HCT in an attempt to lessen toxicity early post transplant and thereby enhance recovery and prevent long-term complications. These strategies are discussed in detail elsewhere (Chapter 71). Available data show low rates of mortality early post transplant. However, GVHD requiring therapy occurs in about half of the patients [5], and the risk of serious GVHD (acute and chronic) is not significantly different from that observed after high-dose regimens [126]. Further observation is needed for a more definitive assessment of long-term complications. General recommendations for prevention and screening for late complications are listed in Table 105.5.

Summary Most patients who recover from the immediate post-transplant problems become healthy long-term survivors and return to normal activities of life. Some patients, however, develop chronic or delayed complications. Major factors contributing to these problems are pretransplant therapy, intensive conditioning regimens, and chronic GVHD. Ongoing studies are expected to provide a better understanding of patients’ psychosocial adjustment. Effective and pre-emptive treatment for some complications is now available. Systematic yearly long-term follow-up is recommended for all post-transplant patients (Table 105.5) [54]. Further refinement of conditioning regimens, prevention of GVHD, especially in its chronic form, and accelerated immune reconstitution should improve the quality of life of all transplant recipients.

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Smita Bhatia & Ravi Bhatia

Secondary Malignancies after Hematopoietic Cell Transplantation

Introduction During the past three decades, the number of patients undergoing hematopoietic cell transplantation (HCT) for malignant or nonmalignant disorders has increased steadily. Improvement in survival after HCT and extended follow-up of this growing population of HCT survivors has resulted in an increasing focus on issues related to long-term complications (see Chapter 105). An important and potentially devastating complication of HCT is the occurrence of secondary malignancies. Secondary malignancies are a known complication of conventional chemotherapy and radiation treatment [1–8], and are now being increasingly recognized as a complication among HCT recipients [9–15]. The magnitude of risk of secondary malignancies after HCT has ranged from fourfold [10] to 11-fold [9] that of the general population. The estimated actuarial incidence is reported to be 3.5% at 10 years, increasing to 12.8% at 15 years among recipients of allogeneic HCT (Table 106.1) [10]. Risk factors associated with the development of secondary malignancies include: exposure to chemotherapy and radiation prior to HCT; type of transplantation (autologous versus allogeneic) and source of hematopoietic cell; use of total body irradiation (TBI) and high-dose chemotherapy for myeloablation; human leukocyte antigen (HLA) nonidentity; immunodeficiency after HCT aggravated by the use of immunosuppressive drugs for prophylaxis and treatment of graft-versus-host disease (GVHD) such as monoclonal and polyclonal antibodies, and T-cell depletion; and infection with viruses such as Epstein–Barr virus (EBV) and hepatitis B and C viruses [9–11,14]. However, assessment of risk factors for all secondary malignancies in aggregate is somewhat artificial because of the heterogeneous nature of the secondary malignancies, with differing clinicopathologic features, distinct pathogenesis, and hence very distinct risk factors associated with their development. It has become conventional practice to classify secondary malignancies after HCT into three distinct groups [11]: 1 myelodysplastic syndrome (MDS) and acute myeloid leukemia; 2 lymphoma (including other lymphoproliferative disorders); 3 solid tumors. Leukemias and lymphomas develop relatively early in the posttransplantation period. On the other hand, solid cancers have a longer latency period and are being increasingly described because of improved survival after HCT and longer follow-up (Fig. 106.1). In this chapter,

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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we discuss the three types of secondary malignancies seen among patients undergoing HCT. The emphasis is on clinical presentation of these malignancies, the magnitude of risk and risk factors associated with their development, insights into their pathogeneses, and the treatment options and outcome of patients with these malignancies.

MDS and acute myeloid leukemia after autologous HCT Autologous HCT is now the treatment of choice for patients with Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL) who have a suboptimal response to initial therapy, those with refractory or relapsed disease, and those at high risk for relapse after conventional therapy. Autologous HCT is also being increasingly used in specific clinical situations among patients with multiple myeloma and advanced stage germ cell tumors. With improvement in survival following autologous HCT, therapyrelated MDS (t-MDS) and therapy-related acute myeloid leukemia (t-AML) are emerging as the major cause of nonrelapse mortality [9,16– 24]. The cumulative probability of t-MDS/t-AML reported in the literature has ranged from 1.1% at 20 months [25] to 24.3% at 43 months [26] after autologous HCT, depending on the size of the population studied and the completeness of follow-up. The median time to development of t-MDS/t-AML is 12–24 months (range 4 months to 6 years) after HCT. t-MDS/t-AML has also been observed after conventional chemotherapy and to a lesser extent radiotherapy for HL and NHL [1,7,27]. The incidence of t-MDS/t-AML following conventional chemotherapy or radiation therapy ranges from 0.8% at 30 years to 6.3% at 20 years. The median time to development of t-MDS/t-AML has been reported to be 3–5 years, with the risk decreasing markedly after the first decade. It therefore appears that the magnitude of risk of t-MDS/t-AML is higher after HCT when compared with conventional chemotherapy and radiation therapy. In addition, the time to development of t-MDS/tAML is shorter after HCT, compared with that after conventional chemoradiotherapy. However, the difference in the magnitude of risk may, in part, be because there are fewer long-term survivors of conventional therapy who have been exposed to multiple salvage therapies. Clinicopathologic syndromes Two types of t-MDS/t-AML are recognized in the World Health Organization classification depending on the causative therapeutic exposure: an alkylating agent/radiation-related type and a topoisomerase II inhibitor-related type [28].

Secondary Malignancies after Hematopoietic Cell Transplantation

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Table 106.1 Risk factors for all subsequent malignant neoplasms (SMNs) after hematopoietic cell transplantation (HCT) Cohort size

Number of SMNs

Incidence

Witherspoon et al. [11]

2245

35

–

Bhatia et al. [9] Kolb et al. [10]

2150 1036

53 53

9.9% (13 years) 11.5% (15 years)

Cumulative probability (%)

Study

0.20

11.6 3.8

Antithymocyte globulin Anti-D3 monoclonal antibody Total body irradiation – Older age at HCT

Topoisomerase II inhibitor-related AML

0.10

0

2

4

6

8

10

Time in years from BMT 0.40

Cumulative probability

6.7

Risk factors

dysplastic features [30,31]. Patients frequently present with cytopenias, often pancytopenia. Multilineage dysplasia is often present. There is a high frequency of the multidrug-resistance phenotype. In addition, there is a high incidence of abnormalities involving chromosomes 5 (−5/ del(5q)) and 7 (−7/del(7q)).

0.30

(a)

0.30

0.20

0.10

0 (b)

Relative risk

3

6

9

12

15

18

Time (years)

Fig. 106.1 (a) Cumulative probability of therapy-related myelodysplasia and acute myeloid leukemia in a cohort of 612 patients undergoing autologous hematopoietic cell transplantation (HCT) for Hodgkin’s lymphoma and nonHodgkin’s lymphoma. (Adapted from [17], with permission). (b) Cumulative probability of solid malignancies after HCT in 2129 patients. (Adapted from [136], with permission from the American Society of Clinical Oncology.) BMT, bone marrow transplantation.

Alkylating agent/radiation-related t-MDS/t-AML Alkylating agents kill cancer cells by transferring alkyl groups to cellular molecules. Mutagenicity is related to the ability of alkylating agents to form crosslinks and/or transfer alkyl groups to form monoadducts in DNA. The most significant site of alkylation in DNA in terms of cytotoxicity is probably the formation of a covalent bond between the drug and the N7 group of guanine in DNA, although the O6-alkylguanine position is also favored. Alkylation results in inaccurate base pairing during replication and single and double strand breaks in the double helix as the alkylated bases are repaired. Expressed mutations involve different base substitutions, including all kinds of transitions and transversions [29]. Alkylating agent-related t-MDS/t-AML usually appears 4–7 years after exposure to the mutagenic agent. Approximately two-thirds of patients present with MDS and the remainder with AML with myelo-

DNA topoisomerase II catalyzes the relaxation of supercoiled DNA by covalently binding and transiently cleaving and re-ligating both strands of the DNA helix. DNA topoisomerase II inhibitors stabilize the enzyme–DNA covalent intermediate, decrease the re-ligation rate, and cause chromosomal breakage. These events initiate apoptosis, required for antineoplastic activity [32,33]. On the other hand, repair of chromosomal damage results in chromosomal translocations, leading to leukemogenesis [32,34,35]. Most of the translocations disrupt a breakpoint cluster region between exons 5 and 11 of the band 11q23 and fuse mixed lineage leukemia (MLL) with a partner gene [36–38]. A comprehensive study of chromosomal abnormalities among patients with therapyrelated leukemia indicates that translocations to 11q23 predominate following therapy with epipodophyllotoxins, whereas patients with translocations to 21q22, inv(16), t(15,17), and t(9,22) most often occur following therapies with anthracyclines [39]. Studies in which primary human CD34+ cells were exposed to etoposide in vitro provide evidence that etoposide can directly induce MLL rearrangements in hematopoietic cells. Stable genome rearrangements originating within the MLL translocation breakpoint hotspot were readily detected in etoposide-treated cells. The spectrum of illegitimate repair included frequent MLL partial tandem duplications and translocations, and minor populations containing deletions or insertions. Several clones had breakpoints that localize to MLL bcr sequences identified at rearrangement junctions in therapy-related leukemias [40]. In contrast to alkylating agent-related t-MDS/t-AML, AML secondary to topoisomerase II inhibitors often does not have a preceding myelodysplastic phase, and presents as overt acute leukemia, often with a prominent monocytic component [41,42]. The latency period between the initiation of treatment with topoisomerase II inhibitors and the onset of leukemia is brief, ranging from 6 months to 5 years, with a median of 2–3 years [42]. Most often, this type of t-AML is associated with balanced translocations involving chromosome bands 11q23 or 21q22 [42]. Other translocations including inv(18)(p13q22) or t(17,19)(q22;q12) have been reported [41,43]. Clinical diagnosis Cytopenias and dysplastic changes on marrow examination can often be seen in isolation in many patients after autologous HCT, many of whom may not subsequently develop t-MDS/t-AML. Therefore, the DanaFarber Cancer Institute group has proposed [44] that the diagnosis of t-MDS/t-AML after HCT be based on the presence of: 1 significant marrow dysplasia in at least two cell lines; 2 peripheral cytopenias without alternative explanations; and

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Chapter 106

Table 106.2 Risk factors for therapy-related myelodysplasia or acute myeloid leukemia after hematopoietic cell transplantation (HCT) Study

Cohort size

Number of SMNs

Incidence

Krishnan et al. [17]

612

22

8.6% (6 years)

Stone [18]

262

20

18% (6 years)

Bhatia et al. [9]

258

10

13.5% (6 years)

Milligan et al. [45]

4998

66

Friedberg et al. [47]

552

41

4.6% (5 years: HL) 3.0% (5 years: NHL)

19.8% (10 years)

Risk factors Etoposide (stem cell priming) Peripheral blood stem cell transplant Pretransplant radiation Number of chemotherapy regimens Previous radiation Age >38 years Low platelet counts Prolonged interval between diagnosis and HCT Peripheral blood stem cell transplant Age >35 years Age at HCT Total body irradiation Number of transplants Years from diagnosis to HCT Lower number of cells infused

HL, Hodgkin’s lymphoma; NHL, non-Hodgkin’s lymphoma; SMN, subsequent malignant neoplasm.

3 blast counts in marrow defined by French–American–British classification. However, many patients may not have an increase in blasts. The presence of a clonal cytogenetic abnormality in addition to morphologic criteria of dysplasia may aid in making this diagnosis in these cases. Risk factors for t-MDS/t-AML after HCT Factors associated with an increased risk of t-MDS/t-AML include host factors (older age at HCT) [9,21], pretransplantation therapy with alkylating agents, topoisomerase II inhibitors, and radiation therapy [17,24,26,44–46], method of hematopoietic cell mobilization (use of peripheral blood hematopoietic cells and priming with etoposide for hematopoietic cell mobilization) [9,17] and transplantation conditioning with TBI (Table 106.2) [45]. Some other factors reported recently include a lower number of CD34+ cells infused at HCT [47] and a history of multiple transplantations [45]. Therefore, t-MDS/t-AML appears to be related to pretransplantation chemotherapy and radiotherapy, transplantation-related factors such as the hematopoietic cell priming and transplantation conditioning regimens or the cumulative effect of all these exposures. The significant impact of primary chemotherapy and radiotherapy on the risk of t-MDS/t-AML after HCT points toward an origin of events prior to HCT [17,18,23,24,26,44,46,48]. Moreover, the nature of the pretransplantation cytotoxic exposure (alkylating agent versus topoisomerase II inhibitor) has a significant impact on the type of t-MDS/ t-AML that evolves in the post-transplantation period, reinforcing this observation. A role for pretransplantation exposures is further supported by the observation that specific cytogenetic abnormalities observed post transplantation have also been observed in the pretransplantation marrow or peripheral blood graft among patients who develop t-MDS/t-AML after HCT [49,50]. Abruzzese et al. [50] reported that nine of 12 cases of t-MDS/t-AML studied demonstrated abnormal cells in pretransplantation bone marrow samples using fluorescence in situ hybridization (FISH). Lillington et al. [49] reported that significant levels of clonally abnormal cells could be detected in samples obtained prior to high-dose therapy in 20 of 20 patients with t-MDS/t-AML using single-locus specific FISH probes to detect loss of chromosomal material from 5q31,

7q22, and 13q14. In comparison, only three of 24 patients who had not developed t-MDS/t-AML had abnormal clones in pretreatment samples. These studies support a role for genetic abnormalities induced by prior cytotoxic chemotherapy in the etiology of t-MDS/t-AML. However, prospective studies of larger groups of patients are warranted to determine the significance of these observations. The use of TBI in the conditioning regimen has been reported to be associated with an increased risk of t-MDS/t-AML after autologous HCT, although other studies fail to confirm this association [17,44]. A recent report suggests that transplant conditioning regimens that included TBI at doses of 12 Gy did not appear to elevate leukemia risk compared with non-TBI regimens, whereas a statistically significant increased risk was found for TBI doses of 13.2 Gy [51]. An association of TBI with increased risk for t-MDS/t-AML raises the possibility that the disease may arise from residual hematopoietic cells that persist in the patient despite treatment with high-dose chemotherapy, rather than from reinfused hematopoietic cells, although it is also possible that TBI-induced alteration in the hematopoietic microenvironment may contribute to the development of t-MDS/t-AML. Therefore, it is unclear whether t-MDS/ t-AML arises from the graft, from residual cells in the patient or as a result of a damaged microenvironment. A higher risk of t-MDS/t-AML has been demonstrated among recipients of CD34-enriched cells isolated from peripheral blood after chemotherapy priming and growth factors, as compared with autologous transplantation using CD34+ cells from the bone marrow without pretreatment [9,17]. Potential explanations offered for this observation include harvesting of hematopoietic precursor cells damaged by chemotherapy at a time before they have completed DNA repair or an overrepresentation of damaged cells in the mobilized product [52]. Supporting this hypothesis is the study by Krishnan et al. [17] demonstrating an increased risk of t-AML with 11q23 abnormalities among patients with HL and NHL mobilized with high doses of etoposide for collection of hematopoietic cells prior to autologous HCT. Friedberg et al. [47] reported an increased risk of t-MDS/t-AML among patients who received a significantly smaller number of cells reinfused per kilogram of body weight. In the setting of low hematopoietic cell numbers, reconstitution of bone marrow clearly may result in a greater proliferative stress, which may increase susceptibility to irrevers-

Secondary Malignancies after Hematopoietic Cell Transplantation

ible DNA damage associated with t-MDS. These findings are consistent with in vitro data suggesting an increased proliferative stress placed upon committed progenitors at the expense of the primitive progenitors, as shown later in the chapter [53]. Alternatively, reduced ability to harvest cells could indicate an existing defect in marrow function. Kalaycio et al. demonstrated that patients who had difficulty harvesting adequate numbers of hematopoietic cells were at an increased risk of developing t-MDS/AML independent of the risk conferred by increased exposure to prior cytotoxic therapies [54]. Pathogenesis of t-MDS/t-AML t-MDS and t-AML are clonal hematologic disorders that are the consequence of an acquired somatic mutation induced by cytotoxic therapy in hematopoietic stem and progenitor cells which confers a proliferative and/or survival advantage. Improved understanding of the molecular pathogenesis of t-MDS/t-AML may allow the development of strategies to identify populations at risk and modify therapies in order to decrease the morbidity and mortality associated with this complication. Furthermore, t-MDS/t-AML offers a unique perspective on mutagen-induced carcinogenesis and the role of genetic susceptibility to cancer in humans. t-MDS/t-AML after autologous HCT appears to result from genetic damage to the stem and/or progenitor cell from pretransplant cytotoxic treatment, which may be potentiated by the transplant process itself through several mechanisms, including hematopoietic cell mobilization, collection, and storage, chemotherapy and radiation used for high-dose therapy, and the stress of engraftment and hematopoietic regeneration on the hematopoietic precursors [18,52,55]. A hypothetical schema for the sequence of events leading to the development of t-MDS/t-AML after autologous HCT is shown in Fig. 106.2. Genetic lesions associated with t-MDS/t-AML Loss of chromosome 5 or del(5q) and loss of chromosome 7 and del(7q) are recurring abnormalities in t-MDS/t-AML. These abnormalities are also seen in AML evolving from MDS and de novo AML in elderly subjects. This has led to a search for candidate tumor suppressor genes in these regions. Several groups have attempted to identify commonly

Genotoxic insult

Genetic susceptibility

deleted segments and to derive transcript maps of these segments [55,56]. The majority of patients with 5q deletions exhibit losses at the 5q31 locus, with deletions in 5q33 being seen in some patients. Chromosomal segment 7q22 is a common site of chromosome 7 deletions. Such studies may assist in cloning of a putative myeloid tumor suppressor gene thought to be located in this region. Although several known and unknown genes have been identified in these regions, including a number of key genes that regulate hematopoietic cell growth and differentiation, identification of a commonly deleted tumor suppressor gene has been elusive thus far. However, it has been hypothesized that haploinsufficiency and reduced gene dosage for critical genes involved in hematopoiesis may sufficiently alter the balance between growth and differentiation to induce dysplastic hematopoiesis [57]. Chromosomal engineering technologies are being used to induce these segmental deletions in mouse models. This approach may be combined with retrovirusmediated insertional mutagenesis to generate new models and aid in gene discovery [58]. Another possibility is that these chromosomal abnormalities may be secondary events important for disease progression rather than initiation. Alternatively, these chromosomal abnormalities may be simply a manifestation of this disorder, but may not play a significant pathogenetic part. Abnormalities in chromosome 7 are also associated with myeloid leukemias in genetically predisposed individuals, such as Fanconi’s anemia or neurofibromatosis type 1 [59,60]. This observation raises the possibility that similar predisposition may be present in patients with t-MDS/t-AML with loss of chromosome 7. 5q and 7q deletions in familial platelet disorder with leukemia are associated with mutations or deletions of a single AML1 allele [61]. Balanced translocations involving the AML1 gene have also been associated with t-MDS/t-AML in patients exposed to topoisomerase II inhibitors [62,63]. Transgenic expression of AML1-ETO leads to immortalization of murine myeloid progenitors but not overt leukemia; additional mutations are required for leukemogenesis [64]. Therefore, altered AML1 function may have a major role in dysregulation of hematopoietic growth and genomic instability and predispose to leukemia through a multistep process. The MLL gene located at chromosome band 11q23 is frequently involved in translocations associated with topoisomerase II inhibitors. MLL has a major role in developmental regulation [65]. Altered MLL function, as with AML1, may dysregulate hematopoietic growth and genomic instability, and predispose to leukemia through a multistep process. The long latency period to onset of leukemia in the knock-in mouse suggests that additional genetic changes are required for evolution of t-AML [66]. The role of MLL and AML1 mutations as early events in the development of t-MDS/t-AML merits further investigation. Genetic susceptibility: polymorphisms in drug-metabolizing enzymes

Acquisition of mutations Proliferative stress

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Genomic instability Clonal hematopoiesis

Cytogenetic abnormalities

Clinical/morphologic MDS

Fig. 106.2 Proposed pathogenesis of therapy-related myelodysplasia and acute myeloid leukemia after autologous hematopoietic cell transplantation.

Underlying genetic characteristics interacting with treatment might be associated with an increased risk of therapy-related leukemia. Analysis of susceptibility of different mouse strains to alkylating agent induced leukemia supports the hypothesis that susceptibility of therapy-related leukemia has a significant genetic component [67]. An example of such an interaction is the presence of a polymorphism in a drug-metabolizing enzyme such as thiopurine S-methyltransferase (TPMT). TPMT catalyzes the S-methylation of thiopurines, including 6-mercaptopurine and 6-thioguanine. TPMT activity exhibits genetic polymorphism, with about one in 300 individuals inheriting TPMT deficiency as an autosomal recessive trait. There is emerging evidence that TPMT genotype might influence the risk of AML [68]. Several other genetic polymorphisms of enzymes capable of metabolic activation or detoxification of anticancer drugs, such as NAD(P)H:

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Chapter 106

quinone oxidoreductase (NQO1), glutathione-S-transferase (GST)-M1, -T1, and -P1, and CYP3A4, have been examined for their role in the development of therapy-related leukemia or MDS [32,69–72]. An NQO1 polymorphism has been shown to be significantly associated with the genetic risk of t-MDS/t-AML [73]. Allan et al. [71] reported data suggesting that inheritance of at least one Val allele at GSTP1 codon 105 confers a significantly increased risk of developing t-AML after chemotherapy, but not after radiotherapy. In addition, individuals with the CYP3A4-W genotype may be at increased risk of treatment-related leukemia, by increasing the production of reactive intermediates that might damage DNA [32]. An analysis of genetic polymorphisms in several drug-metabolizing enzymes in 78 t-AML/t-MDS and 458 normal individuals indicated that a polymorphism profile consisting of CYP1A1*2A, del(GSTT1), and NQO1*2 strongly modified the risk of t-AML/t-MDS. Absence of all three polymorphisms decreased the risk of t-AML/ t-MDS; on the other hand, enhanced risk of t-AML/t-MDS was seen in the presence of only NQO1*2 or all three polymorphisms [73]. Although all these studies report patients treated with conventional chemotherapy and radiation therapy, the gene–environment interactions could potentially be applicable to patients undergoing HCT, and therefore merit further exploration. Genetic instability Genetic instability is hypothesized to be an early event in the development of malignancy, allowing accumulation of multiple mutations in the same cell over time, and evolution of a clonal malignant population. T cells from patients who have received chemotherapy for acute lymphoblastic leukemia (ALL) demonstrate increased frequency of mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) reporter gene [74]. Further characterization of HPRT mutant isolates indicated that multiple mutations were present in individually isolated mutant T-cell clones from four of 15 individuals with a high frequency of HPRT mutations, consistent with genetic instability [74]. Therefore, treatment with cytotoxic agents can lead to genetic instability in some individuals, which could contribute to the induction of t-MDS/t-AML. The mechanism underlying genetic instability could be related to altered expression or function of cell cycle, apoptosis or DNA repair regulatory genes. Defective DNA repair mechanisms. DNA repair mechanisms have a major role in maintaining genomic integrity. The major repair pathways include mismatch repair, base excision repair, nucleotide excision repair, and DNA double-strand break repair [75]. Defects in repair proteins and proteins associated with the regulation of repair are connected to many different types of cancer, including t-MDS/t-AML. In 1996, Ben-Yehuda et al. [76] showed a 94% incidence of microsatellite instability (MSI) in t-MDS/t-AML, suggesting that patients with t-MDS/t-AML may have an inherited defect of a DNA mismatch repair gene, leading to accelerated DNA instability in other oncogenes or tumor suppressor genes occurring as a consequence of treatment for a primary malignancy. In another study, Sheikhha et al. [77] reported MSI in eight of 17 patients with t-AML studied. However, an analysis of 132 patients with AML, including 62 patients with t-AML, failed to demonstrate MSI [78]. Therefore, although MSI and defective DNA mismatch repair are seen in some patients, they do not appear to be a common feature among patients with t-MDS/t-AML. Polymorphisms in the DNA repair gene RAD51 (135G/C-5′ untranslated region) have also been associated with an increased risk of development of t-MDS. This RAD51 polymorphism leads to enhanced promoter activity and elevated mRNA expression. The increase in cancer risk associated with an increased DNA repair capacity in this study was unexpected but could be related to suppression of apoptosis resulting from a highly efficient DNA repair system [79].

p53 gene mutations. The p53 gene has a critical role in DNA damage response signaling, affecting cell cycle, cell death, and DNA repair pathways. Abnormal p53 activity could lead to reduced ability to repair DNA damage, resulting in genomic instability and increased susceptibility to leukemogenesis. In patients with de novo MDS and AML, p53 mutations are seen in fewer than 10% of patients. However, p53 mutations may be more common in patients with t-MDS/t-AML. Ben-Yehuda et al. [76] evaluated 21 patients with t-MDS/t-AML for p53 mutations using polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis and identified mutations in 38% of patients. Mutations were nongermline and restricted to leukemic cells, and differed from p53 mutations seen in the original tumors of individual patients. Horiike et al. [80] identified p53 mutations in six of 12 patients with t-MDS and chromosome 5 and/or 7 losses, but did not observe any p53 mutations in nine other patients without chromosome 5 and/or 7 involvement. Christiansen et al. [81] observed mutations in p53 in 21 of 77 patients (27%) with t-MDS or t-AML; 19 of these 21 patients with mutations had received alkylating agents. Fifteen patients demonstrated loss of heterozygosity [61] of p53. p53 mutations were associated with deletion or loss of 5q and a complex karyotype, were more common in elderly patients, and were associated with an extremely poor prognosis [81]. These studies indicate that p53 mutations may be observed in certain cytogenetic and prognostic subsets of patients with t-MDS/ t-AML, but do not identify a clear role for p53 mutations in the pathogenesis of this disorder. Telomeric shortening. Telomeres are noncoding regions of DNA that provide a cap at the ends of chromosomes and prevent dicentric fusion and other chromosomal aberrations [82]. Each somatic cell division is associated with a loss of telomere length. Cumulative telomere shortening can impose a limit on cell divisions and lead to cell senescence. Telomere shortening is also associated with genetic instability [83]. In hematopoietic tissues, there is progressive shortening of telomere length through life, with considerable variability between age-matched individuals [84]. Following HCT, the increased replicative demand on hematopoietic cells associated with hematopoietic regeneration can lead to accelerated telomere shortening. Several studies have shown that telomere length of cells in the marrow of recipients of allogeneic transplantation is considerably shorter than the telomere length of cells from the donor [85,86]. Most of this decrease occurs in the first year after transplantation [87]. The extent of telomere shortening correlates with the number of cells transplanted. In most studies, the degree of shortening did not reach levels that would compromise marrow function, although two cases have been described in which telomere shortening may have contributed to late graft failure after allogeneic HCT [88]. However, in autologous HCT this could be an important issue, especially when telomere length in the transplanted cells is already short because of prior chemotherapy [89], older age at HCT, increased replicative stress on the hematopoietic cells because of a small number of cells transplanted or other unknown causes. Hematopoietic abnormalities Autologous HCT for HL and NHL has been reported to be associated with hematopoietic abnormalities including marked and possibly permanent reduction in primitive progenitor long-term culture-initiating cells and committed progenitor colony-forming cell numbers, altered progenitor expansion potential, and microenvironmental defects [53,90–92]. These abnormalities may be related in part to damage to hematopoietic cells from pretransplant chemotherapy because hematopoietic defects can also be seen in pretransplant samples [53,91,92]. A longitudinal study of patients undergoing autologous HCT for lymphoma revealed a

Secondary Malignancies after Hematopoietic Cell Transplantation

significant reduction in primitive and committed progenitors in patients prior to HCT compared with healthy controls. Further profound and persistent reduction in primitive progenitors, but only transient reduction in committed progenitors, was seen after HCT, consistent with extensive proliferation and differentiation of the committed progenitors and subsequent depletion of primitive progenitors during hematopoietic regeneration post HCT. Patients within this cohort who developed t-MDS/t-AML had reduced recovery of committed progenitors and poorer telomere recovery post HCT, possibly indicating a functional defect in primitive hematopoietic cells [53]. CD34+ cells in the graft from lymphoma patients also show reduced migration compared with normal CD34+ cells. In vitro migratory capacity of CD34+ cells in the graft correlated with the speed of hematopoietic recovery after transplantation [93]. Therefore, pretransplant chemotherapy may also lead to reduced engraftment potential of primitive progenitor cells. This observation is consistent with experimental data showing that exposure to chemotherapeutic agents results in long-lasting damage to repopulating ability of hematopoietic stem cells as measured in competitive repopulating unit assays in mice [94]. Autologous or allogeneic HCT may also be associated with defects in the marrow hematopoietic microenvironment, including reduction in stromal precursor growth and reduced capacity to support growth of myeloid progenitors and B cell progenitors [91,95,96]. These microenvironmental defects may contribute to hematopoietic abnormalities post transplantation. Marked and prolonged reduction in primitive progenitors is also seen after allogeneic HCT [97]. Extensive proliferation of hematopoietic cells bearing genotoxic damage post transplant may have a role in the establishment and amplification of an abnormal clone. Alternatively, the numerous replication cycles imposed on hematopoietic stem cells after HCT may result in excessive shortening of telomeres in descendent cells (as discussed above). Telomeric shortening may be associated with genomic instability and chromosomal abnormalities, and could contribute to the pathogenesis of t-MDS/t-AML [83]. Gene expression profiling Yeoh et al. [98] reported that the gene expression profile of ALL cells at diagnosis was predictive of therapeutic outcomes, including the risk of development of t-MDS/t-AML. Qian et al. [99] performed gene expression profiling of CD34+ hematopoietic progenitor cells from t-AML patients. This analysis identified different subtypes of t-AML with characteristic gene expression patterns. Common to each subgroup were gene expression patterns characteristic of arrested differentiation in early progenitor cells. Extension of such studies may enhance our understanding of the molecular pathways involved in t-AML. Outcome of patients with t-MDS/t-AML after autologous HCT The prognosis of t-MDS after autologous HCT, treated with conventional therapy, has been reported to be uniformly poor, with a median survival of 6 months. Because of the poor response to conventional chemotherapy, allogeneic HCT has been attempted, with an actuarial survival ranging from 0% to 24% at 3 years [47,100–102]. Friedberg et al. [47] reported the outcome of 41 patients who developed t-MDS after HCT for NHL. Twenty-nine of 33 evaluable patients had del(7) or complex chromosomal abnormalities. The median survival from diagnosis of t-MDS was 9.4 months. Thirteen patients underwent allogeneic HCT as treatment for t-MDS, and all died of transplant-related complications (11 patients) or relapse (two patients), with a median survival of only 1.8 months. Some lessons may be derived from reviewing the results of treatment of t-MDS/t-AML that develops in a nonautologous HCT setting. The

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response to chemotherapy of t-MDS/t-AML is lower than that seen in de novo leukemia, with an average complete remission rate between 35% and 40%, and the overall long-term survival is poor (reviewed in [103]), although a subgroup of patients with favorable cytogenetics appear to have better outcomes [104]. As a result, allogeneic transplantation has been evaluated as a therapeutic modality for t-MDS/t-AML. Witherspoon et al. [105] reviewed data from patients transplanted for t-MDS/t-AML developing in a nonautologous HCT setting, with the aim of identifying patient characteristics that are associated with a better long-term disease-free survival after an allogeneic transplant. The probability of survival after transplantation for all patients was 13%, and by stage of disease was 33% for refractory anemia, 20% for refractory anemia with excess blasts (RAEB), and 8% for refractory anemia with excess blasts in transformation (RAEB-T) or acute leukemia. The overall probability of nonrelapse mortality was 78%, divided equally among infection or organ failure-related causes of death [100]. In a subsequent report, results of related or unrelated HCT in 111 patients with t-MDS/t-AML performed consecutively at the Fred Hutchinson Cancer Research Center between December 1971 and June 1998 were reviewed, and the results of different conditioning regimens analyzed. The 5-year disease-free survival was 8% for TBI, 19% for busulfan and cyclophosphamide (BU/CY), and 30% for BU/CY-t (targeted dose BU/CY) conditioning regimens. The 5-year cumulative incidence of relapse was 40% for t-AML, 40% for RAEB-T, 26% for RAEB, and 0% for refractory anemia or refractory anemia with ringed sideroblasts. The 5-year cumulative incidence of nonrelapse mortality after TBI was 58%, after BU/CY 52%, and after BU/CY-t 42% [105]. Chang et al. recently reported outcomes after HCT from the Fred Hutchinson Cancer Research Center for an expanded cohort of 257 patients with t-MDS/t-AML, which included patients previously treated with irradiation and/or chemotherapy for other disorders (n = 192), and patients with antecedent hematopoietic disorders, even if they had not received cytotoxic therapy. Ninety of these patients were included in the previous report by Witherspoon. Conditioning regimens included highdose TBI/chemotherapy (n = 83); BU/CY (n = 122), fludarabine with BU (n = 12); fludarabine plus 200 cGy TBI (n = 26), and miscellaneous (n = 14). Both related (n = 135) and unrelated (n = 122) donors were used. The BU/CY conditioning regimen was associated with the best 5-year relapsefree survival (43%) and lowest nonrelapse mortality (28%). A multivariate analysis failed to show significant differences in outcome between this cohort and 339 patients who received transplants for de novo MDS/AML. Relapse probability and relapse-free survival correlated significantly with disease stage (p < 0.001) and karyotype (p < 0.001). Relapse incidence was lower (p = 0.003) and relapse-free survival superior (p = 0.02) with unrelated donor transplants. The data suggest that overall inferior outcome in patients with t-MDS/t-AML was related to the frequency of high-risk cytogenetics. Of note, transplantation outcomes were observed to have improved over the time interval studied [106]. Armand et al. analyzed the influence of cytogenetics on survival, relapse, and nonrelapse mortality for 476 patients with de novo MDS and AML to establish an optimal cytogenetic grouping scheme. Application of this grouping scheme to 80 patients with t-MDS/t-AML showed that the optimized cytogenetic classification (adverse cytogenetics: abnormal 7 or complex; favorable cytogenetics: 5q− or 20q− or Y− or normal; intermediate: all others) outperformed the established grouping schemes for outcome prediction. Using this new scheme, cytogenetics was the strongest prognostic factor for overall survival through its impact on the risk of relapse. After accounting for cytogenetics, patients with t-MDS/t-AML had an equivalent outcome to those with de novo disease [107]. Yakoub-Agha et al. [101] analyzed the predictors of survival, relapse, and treatment-related mortality among 70 patients with t-MDS/t-AML

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undergoing allogeneic HCT. Older age (>37 years), male sex, positive recipient cytomegalovirus serology, absence of complete remission at HCT, and intensive conditioning schedules were independently associated with poor outcome. These studies indicate that, in spite of the significant treatment-related mortality, the disease-free survival was better when transplantation was performed earlier in the evolution of disease because it resulted in a lower relapse rate. Anderson et al. [102] described the results of allogeneic HCT as initial treatment for 46 patients with t-AML, which included 17 t-AML patients who had not received remission induction chemotherapy. Five-year actuarial disease-free survival was 24.4%, and the cumulative incidences of relapse and nonrelapse mortality were 31.3% and 44.3%, respectively. Lower peripheral blood blast count was associated with a lower risk of relapse, and shorter time from AML diagnosis to HCT was associated with a lower risk of nonrelapse mortality and improved disease-free survival. Patients with t-AML tended to have lower disease-free survival (8.3%) and a higher relapse rate (43%) than patients whose leukemia was not therapy related. There was no statistically significant difference in outcome in the results of these previously untreated patients compared with 20 patients (12 therapy-related, eight MDS-related) transplanted with chemosensitive disease after induction chemotherapy [101,102,105]. These analyses of the results of allogeneic transplantation for t-MDS/ t-AML developing after conventional therapy may have implications for the treatment of t-MDS/t-AML developing after HCT. Recent reports indicate that, following adjustment for cytogenetic risk classification and disease stage, transplant outcomes for patients with t-MDS/t-AML are equivalent to those for de novo disease [106,107]. However, for t-MDS/ t-AML developing after HCT, an additional consideration is the very high rate of nonrelapse mortality associated with allogeneic HCT performed after an initial autologous HCT, especially in adult patients [108]. Interestingly, Chang et al. reported an improved outcome for allogeneic transplant for 26 patients who had received prior autologous HCT for their primary disease compared with patients treated previously with conventional chemotherapy. Although the reason for this is not clear, it was discussed that patients who received prior transplants may experience more complete lymphohematopoietic reconstitution and therefore be at a lower risk of infectious complications. However, careful selection of patients for transplantation treatment must also be considered as a possible factor [106]. Treatment of t-MDS/t-AML should include consideration of the likelihood of success of achieving remission with chemotherapy and, depending on the availability of an appropriate donor, the likelihood of a successful outcome with an allogeneic transplant approach. It is important to follow patients at risk for development of t-MDS closely to identify the early development of MDS. Prompt transplantation should be considered after diagnosis of t-MDS/t-AML or, if possible, high-risk MDS, particularly in patients with low peripheral blast counts. Innovative transplant strategies are needed to reduce the high risks of relapse and nonrelapse mortality seen in this patient population. Because the poor outcomes of allogeneic transplant for t-MDS/t-AML are related in part to the high risk of treatment-related mortality, it will be of interest to evaluate the role of reduced-intensity conditioning (RIC) approaches in this setting. Prediction of risk of t-MDS/t-AML Because of the poor prognosis associated with t-MDS/t-AML, attempts are being made to identify predictors or early biomarkers to decrease the morbidity associated with this disease. Several studies have attempted to correlate identification of genetically abnormal clones with subsequent risk of development of t-MDS/t-AML and are discussed below.

Assessment of risk of t-MDS/t-AML after autologous HCT is complicated by the lack of a single underlying genetic abnormality. The development of t-MDS/t-AML appears to require the acquisition of more than one mutation. Moreover, t-MDS/t-AML is a heterogeneous disorder with multiple subtypes characterized by different genetic abnormalities. Therefore, the identification of a single genetic abnormality may not necessarily have predictive value for development of t-MDS/t-AML. Identification of early biomarkers would allow the timely use of appropriate measures to treat the disorder, such as RIC or other novel agents, rather than waiting for the t-MDS/t-AML to present in the clinically overt form, when the disease burden would require higher-intensity therapy, with a greater risk of resultant morbidity. Indeed, identification of patients at high risk for t-MDS/t-AML pretransplant would allow treatment strategies other than autologous transplantation, such as reduced-intensity transplantation, to be considered for such patients to avoid the risk of t-MDS/t-AML. Standard cytogenetics and FISH Abnormal clones are frequently detected on cytogenetic analysis after autologous HCT for lymphoma. Traweek et al. [19] reported the risk of developing a clonal cytogenetic abnormality typical of MDS to be 9% at 3 years. Five of 10 patients with the abnormal clone developed t-MDS. Stone [18] reported that 50% of sporadically tested post-transplant patients who were hematologically normal had clonal cytogenetic abnormalities. However, less than 30% of these patients developed t-MDS [44]. Evaluation by FISH may enhance sensitivity of detection of chromosomal abnormalities. Significant levels of clonally abnormal cells could be detected by FISH prior to high-dose therapy in samples obtained from 20 of 20 patients who developed t-MDS/t-AML, but only in three of 24 patients who did not develop t-MDS/t-AML [49]. However, this technique is locus specific and requires prior selection of markers for analysis. The predictive value of clonal cytogenetic abnormalities for subsequent development of t-MDS/t-AML needs to be systematically studied in a prospective fashion. Clonality analysis The predictive value of clonal bone marrow hematopoiesis for the development of t-MDS/t-AML was investigated in a group of patients undergoing autologous HCT for NHL at the Dana-Farber Cancer Institute. An X-inactivation-based clonality assay at the human androgen receptor locus (HUMARA) was used. A total of 104 female patients were evaluated. At the time of HCT, the prevalence of skewed X-inactivation pattern was 20% and of clonal hematopoiesis was 3%. Of the 78 patients followed for at least 18 months, 53 continue to demonstrate polyclonal hematopoiesis, 15 developed skewed X-inactivation pattern, and 10 either had clonal hematopoiesis at the time of transplant or developed clonal hematopoiesis after BMT. t-MDS/t-AML developed in two of 53 patients with polyclonal hematopoiesis, and four of 10 with clonal hematopoiesis. Clonal hematopoiesis at the time of transplant or after transplant was predictive of the development of t-MDS/t-AML [20]. Five of seven patients with clonal hematopoiesis also had a clonal cytogenetic abnormality involving 50% or more metaphases. This assay is limited by its low sensitivity, requiring a high proportion of monoclonal cells to be present prior to reaching the threshold for detection, and is applicable only to female patients. However, if proven to predict t-MDS/t-AML in prospective studies, it could potentially be a useful method to detect patients at risk for this complication. Loss of heterozygosity analysis and PCR assays for point mutations Other tests that may be useful for detection of evolution of clonal genetic abnormality after HCT are loss of heterozygosity analysis and PCR

Secondary Malignancies after Hematopoietic Cell Transplantation

assays for point mutations [61]. In loss of heterozygosity analysis, loss of one allele at a particular locus is evaluated, most commonly by PCR analysis. This method is specific and can be adapted to high-throughput strategies, but is relatively insensitive and requires prior selection of loci. This method has not been validated as being a useful predictor of tMDS/t-AML. PCR for point mutations and chromosomal translocations is another potentially useful tool. Mutations in genes such as MLL or AML1 or gene rearrangements involving the 11q23 gene may be useful markers for risk of subsequent t-MDS/t-AML. This method is highly sensitive, but is locus specific, and the specificity and predictive value of such assays is unknown at present. This test may be most helpful if performed using quantitative techniques that would allow assessment of increasing levels of abnormality [44].

Reducing risk of t-MDS/t-AML after autologous HCT It is possible to consider potential strategies to reduce the risk of t-MDS/ t-AML, based on our understanding of the risk factors and pathogenesis of t-MDS/t-AML. Such strategies may include minimizing pretransplant cytotoxic exposure, possibly by bringing high-risk patients to HCT earlier in the course of disease, prior to exposure to multiple treatment regimens. Alteration in autologous hematopoietic cell procurement regimens and the conditioning regimens could be considered to eliminate factors associated with increased risk of this complication. Standardized screening of patients in the immediate pre-HCT period with marrow pathology and cytogenetics could potentially help identify highrisk populations that would then benefit from an allogeneic rather than autologous HCT. If strategies to develop predictors for patients at high risk prior to HCT are realized, alternative treatment approaches such as allogeneic transplantation or nontransplant modalities may be worth considering for patients identified as being at increased risk of this complication. Finally, strategies for chemoprevention may be worth exploring in this population.

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Lymphomas Post-transplantation lymphoproliferative disorder Lymphoproliferative disorders are the most common secondary malignancy in the first year after allogeneic T-cell-depleted HCT. Most of these cases are related to a compromised immune status and EBV infection. The large majority of the post-transplantation lymphoproliferative disorders (PTLDs) have a B-cell origin, although some T-cell PTLD has been described. B-cell PTLD B-cell PTLD is a clinically and morphologically heterogeneous group of diseases. B-cell PTLD usually develops within the first 6 months after HCT, with most of the events occurring within the first 3 months. The cumulative incidence of PTLD has been reported to range from 1.0% to 2.0% at 10 years [9,11,109,110]. The incidence is highest in the first 5 months after HCT (120 cases/10,000/year), with a steep decline thereafter to fewer than 5 cases/10,000/year among HCT recipients surviving 1 year or longer. Risk factors for B-cell PTLD. Risk factors found to be independently associated with an increased risk for the development of PTLD include in vitro T-cell depletion of the donor marrow, unrelated or HLAmismatched related donor, use of antithymocyte globulin or anti-CD3 monoclonal antibody for acute GVHD prophylaxis or in the preparative regimen, TBI and primary immunodeficiency (Table 106.3) [9,15]. Patients transplanted for congenital immunodeficiency are at a particularly high risk for PTLD, because of both the underlying immunodeficiency and the use of T-cell depletion of the donor graft [9,11,109]. Moreover, the risk of PTLD also depends on the method of T-cell depletion, being considerably higher where specific monoclonal antibodies are used for T-cell depletion (11–25%) rather than in patients where techniques removing both T and B lymphocytes, such as soybean agglutinin or Campath-1 (<1%), are used [109]. However, the incidence of PTLD is higher in the latter group (6–18%) after exposure to immuno-

Table 106.3 Risk factors for lymphoma after hematopoietic cell transplantation (HCT)

Study

Cohort size

Number of SMNs

Cumulative incidence

Epstein–Barr virus-associated post-transplant lymphoproliferative disorder 2150 22 1.6% Bhatia et al. [9] (4 years)

Curtis et al. [109]

42,349 (person–years at risk)

Hodgkin’s lymphoma after transplantation 18,531 Rowlings et al. [133]

78

8

1% (10 years)

–

Standardized incidence ratio

105.6

51.5

6.2

GVHD, graft-versus-host-disease; HLA, human leukocyte antigen; SMN, subsequent malignant neoplasm.

Risk factors

Primary diagnosis of immunodeficiency Antithymocyte globulin (preparative regimen or GVHD prophylaxis) T-cell depletion HLA mismatch Unrelated donor transplantation T-cell depletion of donor marrow Antithymocyte globulin Anti-CD3 monoclonal antibody Total body irradiation Acute GVHD (grade II–IV) Chronic GVHD (extensive) Acute GVHD (grade II–IV) Chronic GVHD

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suppressive therapy such as steroids or antithymocyte globulins. The more recent use of RIC coupled with highly immunosuppressive therapy needs close observation for the development of PTLD [111]. Pathogenesis of B-cell PTLD. B-cell PTLD is commonly associated with T-cell dysfunction, occurs in the presence of EBV infection, and is thought to develop because of a combination of depressed EBV-specific cellular immunity and the inherent transforming capacities of EBV. EBV is a ubiquitous herpesvirus that infects 95% of individuals by adulthood (see Chapter 93). The virus persists as a latent infection in B lymphocytes, where reactivation and replication occur intermittently [112]. The latent membrane protein-1 is one of the EBV-encoded proteins believed to have an important role in B-cell immortalization by inducing the expression of bcl-2, which inhibits programmed death of the infected cells. LMP-1 is also considered to be an oncogene and deletions near the 3′ end of the LMP-1 gene, in a region that affects the half-life of the LMP-1 protein, have been reported in some lymphoproliferative disorders [113,114]. Infection of B cells by EBV also induces high levels of cytokines such as interleukin 1 (IL-1), IL-5, IL-6, IL-10, CD23, and tumor necrosis factor. Some of these factors have been shown to act as autocrine growth factors, stimulating the proliferation of EBV-transformed B cells and inhibiting their susceptibility to apoptosis. Studies exploring susceptibility to EBV-PTLD have shown that cytotoxic T-lymphocyte precursor frequencies are low at 3 months after allogeneic HCT, but appear to normalize at 9–12 months, thus correlating with the time-period when B-cell PTLD is most frequently observed. Moreover, the EBV-specific cytotoxic T lymphocytes home preferentially and induce selective regression of autologous EBV-induced B-cell lymphoproliferative lesions in xenografted severe combined immunodeficiency syndrome (SCID) mice [115]. These studies have formed the basis for clinical trials using adoptive transfer of EBV-specific cytotoxic T lymphocytes [116]. The studies demonstrated long-term persistence of gene-marked EBV-specific cytotoxic T lymphocytes in vivo, which not only restore cellular immunity against EBV but also provide a population of cytotoxic T-lymphocyte precursors that respond to in vivo and ex vivo challenges with the virus for as long as 18 months [117]. It has also been demonstrated in the xenografted SCID mouse model that lack of natural killer cell function may have a role in the pathogenesis of PTLD [118]. These studies have demonstrated that a combination of granulocyte–macrophage colony-stimulating factor and low-dose IL-2 therapy can prevent the immunodeficiency that leads to fatal EBV lymphoproliferative disease in xenografted SCID mice depleted of murine natural killer cells, and thus support a critical role for several human cellular subsets in mediating this protective effect. Prediction of risk of B-cell PTLD. Quantitative competitive PCR has been demonstrated to be an effective technique in allowing frequent monitoring of the DNA load to predict the development of PTLD. Rapid increases in peripheral blood EBV DNA load predicted PTLD [119]. EBV-specific T lymphocytes can be monitored through tetramer technology, which allows detection of small quantities of antigen-specific T cells. Thus, it has been demonstrated that EBV-specific CD8 T cells are rapidly established following unmanipulated matched sibling allogeneic HCT, and HLA class I tetramers complexed with viral peptides can provide direct and rapid assessment of pathogen-specific immunity. In contrast, patients undergoing T-cell-depleted or unrelated cord blood transplantation have undetectable EBV-specific T cells, even in the presence of Epstein–Barr viremia [120]. Treatment of B-cell PTLD. Patients considered to be at an increased risk for development of PTLD (primary immunodeficiency, HLA-mismatched HCT, and GVHD prophylaxis with T-cell depletion) should be

monitored closely, so that appropriate therapy can be instituted early, prior to the development of overt disease. Therapeutic approaches that have been used include interferon-alpha, B-cell specific monoclonal antibodies, and cellular therapy. Several investigators have reported the efficacy of anti-CD20 monoclonal antibody (rituximab) in the treatment of PTLD [121–123]. All the studies indicate that the drug is well tolerated by all age groups and is more efficacious in patients without mass lesions. This observation forms the basis for recommendations to initiate treatment at an early stage, based on increasing EBV load, and before the development of lymphomatous lesions. However, large multicenter trials followed longitudinally would address the efficacy and safety of this approach. EBV-associated lymphoproliferative disorder developing after HCT has been shown to result from T-cell dysfunction. Reconstitution of “at-risk” patients with EBV-specific cytotoxic T-lymphocyte lines that have been reactivated and expanded in vitro should prevent the development of PTLD or treat pre-existing disease. The cytotoxic Tlymphocyte-reconstituted cellular immune responses to EBV have been shown to persist for up to 80 months. Cytotoxic T-cell therapy has been shown to be efficacious in controlling PTLD, with a decrease in the EBV DNA concentrations, and remission of clinical signs and symptoms [118,123–125]. Thus, over the past few years, the administration of in vitro-generated EBV-specific cytotoxic T cells or anti-B-cell monoclonal antibodies has provided an effective option for the prophylaxis or treatment of PTLD. Advances in quantitative PCR-based assays allow both the precise measurement of EBV load in peripheral blood samples and the identification of high-risk patients for early initiation of therapy. Patients should be monitored weekly by quantitative competitive PCR because viral load is a significant predictor of PTLD. Prevention of EBV PTLD by preemptive therapy based on molecular monitoring of EBV load may be used to decrease the occurrence of PTLD. Pre-emptive therapy with a single dose of rituximab has been reported to lead to the prevention of PTLD, and was first demonstrated by Van Esser et al. [126], and then confirmed by Gruhn et al. [127] and Dominietto et al. [128]. The cut-off value of over 1000 EBV genome copies in 105 peripheral blood mononuclear cells demonstrates a sensitivity and specificity of 100% and a positive and negative predictive value of 1.00 [129]. T-cell lymphoproliferative disorders A few cases of T-cell lymphoproliferative disorders have been reported after HCT [130]. There is no association with EBV, human T-cell lymphotropic virus-1, human immunodeficiency virus or human herpesvirus 6. The T-cell lymphoproliferative disorders tend to occur much later than the EBV-associated B-cell PTLD. Late-onset lymphoma Several cases of late-occurring lymphoma have been reported in the literature [131,132]. It is believed that these late-occurring lymphomas represent an entity that is distinct from the early occurring B-cell PTLD. In a large study of 18,000 HCT recipients, the only risk factor associated with the development of the late-occurring lymphoma was extensive chronic GVHD [109]. HL developing among HCT recipients has also been described [133]. Most of the reported cases were of the mixed cellularity subtype, and most of the cases contained the EBV genome. These cases differed from the EBV-associated PTLD by the absence of risk factors commonly associated with EBV-associated PTLD, by a later onset (>2.5 years), and by a relatively good prognosis. The increased incidence of HL among HCT recipients could possibly be explained by exposure to EBV and overstimulation of cell-mediated immunity.

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Table 106.4 Risk factors for sold tumors after hematopoietic cell transplantation (HCT) Study

Cohort size

Number of SMNs

Cumulative incidence

Standardized incidence ratio

Risk factors

Curtis et al. [135]

19,229

80

6.7% (15 years)

2.7

Bhatia et al. [134] Bhatia et al. [9]

2,129 2,150

29 17

6.1% (10 years) 5.6% (13 years)

2.0 3.2

Total body irradiation Younger age at HCT Chronic GVHD (squamous cell cancer) Younger age at HCT Total body irradiation

GVHD, graft-versus-host disease.

Solid tumors Solid tumors have been described after syngeneic, allogeneic, and autologous HCT. The magnitude of the increased risk of solid tumors ranged from 2.1-fold [134] to 2.7-fold [135] when compared with an age- and sex-matched general population. The risk increased with increasing follow-up and, among those who survived for 10 or more years after transplantation, was reported to be up to 8.3 times as high as expected in the general population. Types of solid tumor reported in excess among HCT recipients, when compared with the general population, are those typically associated with exposure to radiation therapy, and include melanoma, cancers of the oral cavity and salivary glands, brain, liver, uterine cervix, thyroid, breast, bone, and connective tissue [135–137]. The risk of solid tumors increases sharply over time, and has been reported to be higher among individuals who underwent HCT at less than 10 years of age [134] or were older than 40 years at HCT, and in patients who had female graft donors [138]. TBI is associated with an increased risk of solid tumors. The risk of solid tumors rises with the dose of radiation, with three to four times the risk at the highest dose levels, as compared with those who did not receive radiation therapy (Table 106.4) [135]. Among allogeneic HCT recipients, the 20-year cumulative incidence of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) has been reported to be 6.5% and 3.4% respectively [139]. While TBI is a risk factor of BCC most strongly among patients younger than 18 years old at HCT, light-skinned patients are at increased risk of BCC. Acute GVHD increased the risk of SCC; on the other hand, chronic GVHD increased the risk of both BCC and SCC. Although most studies have focused on allogeneic HCT recipients, there is emerging evidence for an increased incidence of new solid malignancies among patients conditioned with TBI and receiving autologous HCT [21]. There is, therefore, a need to follow this cohort of patients long term in order to describe the risk of new solid malignancies with precision. Pathogenesis of solid tumors after HCT Little is known about the pathogenesis of solid tumors after HCT. An interaction of cytotoxic therapy, genetic predisposition, viral infection, and GVHD, with the consequent antigenic stimulation and use of immunosuppressive therapy, all seem to have a role in the development of new solid tumors. Radiogenic cancers generally have a long latent period, and the risk of such cancers is frequently high among patients undergoing irradiation at a young age [15]. A large series reported an increased risk of brain and thyroid cancers after TBI as part of high-dose conditioning, although most of these patients had received cranial irradiation prior to transplantation [15,137]. Both thyroid cancer and brain tumors have been reported after exposure to radiation to the craniospinal axis and the neck used as part of the conventional therapy for childhood ALL [140], HL [2,8], and

other primary brain tumors [8]. Similarly, osteogenic sarcoma and other connective tissue tumors have been reported as secondary malignancies developing among patients receiving radiation therapy as part of conventional therapy for other primary malignancies such as retinoblastoma and other bone tumors [141–143]. These studies indicated the presence of a strong dose–response relationship for radiation exposure, in addition to an increased risk with increasing exposure to alkylating agents. The increased risk of thyroid, breast, brain, bone, and soft tissue cancers seen after HCT appear to be related to cumulative doses of radiation exposure, as a result of both the pretransplant treatment regimen and the conditioning regimen used for transplant. Immunologic alterations may predispose patients to SCC of the buccal cavity, particularly in view of the association with chronic GVHD [139]. Patients transplanted for aplastic anemia have been reported to be at an increased risk of solid tumors, predominantly tumors of the buccal cavity and skin. The risk of these tumors was significantly increased after the administration of azathioprine for chronic GVHD [14]. In immunosuppressed patients, oncogenic viruses such as human papillomaviruses may contribute to SCCs of the skin and buccal mucosa after transplantation. The observation of the excess risk of SCCs of the buccal cavity and skin in males is unexplained, but may be indicative of an interaction between ionizing radiation, immunodeficiency, and other risk factors more prevalent among men than women [134,135]. The increased risk of new solid tumors after HCT is thus likely related to TBI used for pretransplantation high-dose therapy, altered immune function in association with viral infections (hepatitis B virus, hepatitis C virus or human papillomavirus), and pre-HCT treatment for the primary disease (Table 106.4) [134,135,144–146]. Genetic susceptibility Patients with a family history of early-onset cancers have been shown to be at an increased risk for developing a secondary cancer. In one study, 159 3-year survivors of childhood soft tissue sarcoma, treated with conventional therapy, and their relatives were surveyed; a highly significant cancer excess was observed in the relatives of childhood sarcoma patients who developed a second cancer [147]. The tumor types occurring in excess in close relatives were also observed as second cancers in patients (cancers of the breast, bone, joint or soft tissue), indicating that the risk of second cancers is associated with a familial predisposition. In another study, members of families with Li–Fraumeni syndrome, a hereditary susceptibility to several cancers that is usually caused by mutation of the TP53 tumor suppressor gene, were reported to be at increased risk of multiple subsequent cancers compared with the general population [148]. The excess risk was mainly for cancers characteristic of Li–Fraumeni syndrome. It therefore appears that germline mutations in tumor suppressor genes, as occur in Li–Fraumeni syndrome, might interact with therapeutic exposures to result in an increased risk of secondary cancers.

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Genetic predisposition also has a substantial impact on risk of secondary cancers, for example sarcomas in patients with hereditary retinoblastoma, particularly in patients treated with radiation [143]. Mutational analysis of the ataxia telangiectasia (ATM) gene, which is mutated in individuals with the recessive hereditary cancer syndrome ataxia telangiectasia, has been performed in cohorts of patients with radiationassociated secondary cancers. ATM protein, a protein kinase, plays an important part in regulation of the G1/S cell-cycle checkpoint. ATM phosphorylates p53, resulting in its stabilization following ionizing radiation. In vitro studies have shown that cells from ataxia telangiectasia patients and obligate heterozygotes have an increased sensitivity to ionizing radiation, and there is epidemiologic evidence that ataxia telangiectasia carriers are at an increased risk of radiation-induced breast cancer [149]. The studies conducted so far have failed to support the hypothesis that ataxia telangiectasia carriers account for a significant fraction of radiation-induced secondary cancers, although there is some evidence that missense mutations in ATM are more common in primary breast cancer cases selected for family history and young age at diagnosis [150]. Another example of an interaction between treatment and underlying genetic characteristics is the presence of a polymorphism in a drugmetabolizing enzyme such as TPMT. There is emerging evidence that TPMT genotype might influence the risk of secondary brain tumors [151]. Studies exploring genetic predisposition and gene–environment interactions have focused thus far on patients exposed to nontransplant conventional therapy for cancer. Future studies are needed in the transplant population to understand how the interaction of genetic predisposition with high-dose chemotherapy, TBI, and the attendant post-transplant immunosuppression have a role in the development of secondary solid tumors.

the incidence of the most devastating consequences of surviving cancer while maintaining the high cure rates in this population. Evidence-based guidelines for screening for early detection of cancer in HCT survivors are not available at present, but guidelines published by the American Cancer Society [155], and the National Comprehensive Cancer Network Practice Guidelines in Oncology [156], present a reasonable framework for the physicians taking care of this high-risk population. The recommended frequency and the age at onset are based on the American Cancer Society recommendations for individuals identified to be at increased risk for the development of these cancers. Colon cancer screening should include colonoscopy with biopsy for dysplasia every 1–2 years beginning 10 years after TBI. Cervical screening is recommended annually, beginning at age 18 years, until the age of 45 years, and should be performed with conventional cervical cytology smears. The prostate-specific antigen test and digital rectal examination should be offered annually beginning at age 45. For female patients receiving radiation to the chest and/or TBI, screening recommendations include monthly breast self-examination, beginning at age 20 (or earlier if the patients have received radiation to the chest at an earlier age), and clinical breast examination, beginning at age 20 (or earlier if needed), performed yearly until age 25, and then every 6 months. The National Comprehensive Cancer Network guidelines also recommend a baseline mammogram at 8 years after exposure to radiation, or at the attained age of 40, whichever occurs first, and then annually. In addition, certain screening recommendations may be based on specific risk factors. For example, patients with a history of transfusions prior to 1993 should be screened for viral hepatitis. An examination for cancerous and precancerous lesions of the oral cavity should be in the periodic health examination of patients with oral chronic GVHD.

Treatment of patients with solid tumors after HCT Treatment strategies for patients developing solid tumors after transplantation are not well defined. Small case series indicate both ends of the spectrum: favorable outcome (hence a recommendation for an intensive approach) [152], as well as aggressive tumor growth and early relapse after standard therapy [153]. A comprehensive study of a large number of patients with second solid tumors will help determine the nature of these tumors and their outcomes as compared with de novo tumors. Until then, patients with solid tumors developing after transplant should be treated with the best available therapy for that tumor, unless there is compelling evidence that they will not be able to tolerate that therapy. Screening for solid tumors after HCT Extending the follow-up of HCT recipients to 20 years post transplantation will help clarify the risks of radiation-associated cancers such as breast, lung, and colon cancers. These epithelial cancers typically develop at a median of 15–20 years after exposure to radiation therapy, and are now beginning to emerge among cancer survivor populations treated with conventional therapy [154]. These data indicate that HCT survivors face an increasing risk of solid cancers with time from HCT, thus supporting the need for lifelong surveillance. Preventive measures that need to be considered include programs to educate clinicians and survivors about the risk of secondary malignancies, and measures taken to decrease the morbidity associated with secondary malignancies, such as adopting healthy lifestyle choices. Other measures are intervention programs for smoking cessation, periodic screening for breast, lung, skin, colorectal, prostate, thyroid and cervical cancers, chemoprevention for specific cancers, and avoidance of unnecessary exposure to sunlight, especially among patients who have received radiation. Health counseling should include guidance about smoking cessation, diet and physical activity. By understanding the risk factors for secondary malignancies, and taking measures to avoid them, it may be possible to decrease

Conclusion HCT offers curative therapy for many patients with otherwise incurable disease. Currently, 45,000–50,000 transplants are performed annually (see Chapter 3), and most patients who do not experience a recurrence of their underlying disease within 1 or 2 years of transplantation do well and lead productive lives [157,158]. However, some complications do occur, and among those that have been described after HCT, malignant diseases are of particular clinical concern, because increasing numbers of patients survive the early phase after transplantation and remain free of their original disease. The incidence of post-transplant malignancies appears to be low, although reliable estimates of the overall risk will require much longer follow-up. Despite the lack of randomized studies, the benefit of HCT, as compared with conventional therapy alone, in certain clinical situations outweighs the risk of late secondary malignancies. However, it is imperative to follow this population of HCT recipients closely in order to screen them for the development of subsequent malignancies, and thus decrease the morbidity and mortality associated with this complication. Longitudinal monitoring of patients at multiple time points (before, during, and after HCT) is required to evaluate risk factors, study the evolution of genetic lesions, and identify biomarkers for secondary malignancies. Improved understanding of risk factors may allow future modification of pretransplant and transplant-related therapeutic exposures to minimize risk for these complications. A better understanding of the pathogenesis of secondary malignancies will allow for more effective screening to identify patients at risk prior to the HCT procedure, and allow more effective monitoring to detect early evolution of the malignancy post transplantation. This may, in turn, allow for improved therapeutic decision making while evaluating patients for HCT, and early institution of treatments directed at preventing and treating secondary malignancies in patients at risk after HCT.

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123. Faye A, Quartier P, Reguerre Y et al. Chimeric anti-CD20 monoclonal antibody (rituximab) in post-transplant B-lymphoproliferative disorder following stem cell transplantation in children. Br J Haematol 2001; 115: 112–18. 124. Papadopoulos EB, Ladanyi M, Emmanuel D et al. Infusions of donor leukocytes to treat Epstein– Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation. N Engl J Med 1994; 330: 1185– 91. 125. Liu Z, Savaldo B, Huls H et al. Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes for the prevention and treatment of EBV-associated posttransplant lymphomas. Recent Results Cancer Res 2002; 159: 123–33. 126. Van Esser JWJ, Niesters HG, Van Der Holt B et al. Prevention of Epstein–Barr virus-lymphoproliferative disease by molecular monitoring and preemptive rituximab in high-risk patients after allogeneic stem cell transplantation. Blood 2002; 99: 4364–9. 127. Gruhn B, Meerbach A, Hafer R et al. Pre-emptive therapy with rituximab for prevention of Epstein– Barr virus-associated lymphoproliferative disease after hematopoietic cell transplantation. Bone Marrow Transplant 2003; 31: 1023–5. 128. Dominietto A, Tedone E, Soracco M et al. Epsterin–Barr virus reactivation after allogeneic hematopoietic stem cell transplant based on molecular monitoring is predictive of lymphoproliferative disease. Bone Marrow Transplant 2004; 33: S192. 129. Meerbach A, Wutzler P, Hafer R et al. Monitoring of Epstein–Barr virus load after hematopoietic stem cell transplantation for early intervention in post-transplant lymphoproliferative disease. J Med Virol 2008; 80: 441–54. 130. Zutter MM, Durnam DM, Hackman RC et al. Secondary T-cell lymphoproliferation after marrow transplantation. Am J Clin Pathol 1990; 94: 714–21. 131. Schouten HC, Hopman AHN, Haesevoets AM et al. Large-cell anaplastic non-Hodgkin’s lymphoma originating in donor cells after allogeneic bone marrow transplantation. Br J Haematol 1995; 91: 162–6. 132. Trimble MS, Waye JS, Walker IR et al. B-cell lymphoma of recipient origin 9 years after allogeneic bone marrow transplantation for T-cell acute lymphoblastic leukemia. Br J Haematol 1993; 85: 99–102. 133. Rowlings PA, Curtis RE, Passweg JR et al. Increased incidence of Hodgkin’s disease after allogeneic bone marrow transplantation. J Clin Oncol 1999; 17: 122–7. 134. Bhatia S, Louie A, Bhatia R et al. Solid cancers after bone marrow transplantation. J Clin Oncol 2001; 19: 464–71. 135. Curtis RE, Rowlings PA, Deeg HJ et al. Solid cancers after bone marrow transplantation. N Engl J Med 1997; 336: 897–904. 136. Cohen A, Rovelli A, Merlo DF et al. Risk of thyroid carcinoma after hematopoietic stem-cell transplantation: an EBMT Late Effects Working Party Study. J Clin Oncol 2007; 25: 2449–54. 137. Friedman D, Rovo A, Leisenring W et al. Increased risk of breast cancer among survivors of allogeneic hematopoietic cell transplantation: a report from the FHCRC and the EBMT-Late Effect Working Group. Blood 2008; 111: 939–44.

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138. Gallagher G. Second solid cancers after allogeneic hematopoietic stem cell transplantation. Cancer 2007; 109: 84–92. 139. Leisenring W, Friedman DL, Flowers MED et al. Nonmelanoma skin and mucosal cancers after hematopoietic cell transplantation. J Clin Oncol 2006; 24: 1119–24. 140. Bhatia S, Sather HN, Pabustan OB et al. Low incidence of second neoplasms among children diagnosed with acute lymphoblastic leukemia. Blood 2002; 99: 4257–64. 141. Tucker MA, D’Angio GJ, Boice JDJ et al. Bone sarcomas linked to radiotherapy and chemotherapy in children. N Engl J Med 1987; 317: 588– 93. 142. Hawkins MM, Wilson LM, Burton HS et al. Radiotherapy, alkylating agents, and risk of bone cancer after childhood cancer. J Natl Cancer Inst 1996; 88: 270–8. 143. Wong FL, Boice JDJ, Abramson DH et al. Cancer incidence after retinoblastoma: radiation dose and sarcoma risk. JAMA 1997; 278: 1262–7. 144. Deeg HJ, Leisenring W, Storb R et al. Long-term outcome after marrow transplantation for severed aplastic anemia. Blood 1998; 91: 3637–45. 145. Daneshpouy M, Socie G, Clavel C et al. Human papilloma virus infection and anogenital condy-

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loma in bone marrow recipients. Transplantation 2001; 71: 167–9. Socie G, Scieux C, Gluckman E et al. Squamous cell carcinomas after allogeneic bone marrow transplantation for aplastic anemia. Transplantation 1998; 66: 667–70. Strong LC, Stine M, Norsted TL. Cancer in survivors of childhood soft tissue sarcoma and their relatives. J Natl Cancer Inst 1987; 79: 1213–20. Hisada M, Garber JE, Fung CY et al. Multiple primary cancers in families with Li-Fraumeni syndrome. J Natl Cancer Inst 199; 90: 606–11. Swift M. Public health burden of cancer in ataxiatelangiectasia heterozygotes. J Natl Cancer Inst 2001; 93: 84–5. Teraoka SN, Malone KE, Doody DR et al. Increased frequency of ATM mutations in breast carcinoma patients with early onset disease and positive family history. Cancer 2001; 92: 479–87. Relling MV, Rubnitz JE, Rivera GK et al. High incidence of secondary brain tumours after radiotherapy and antimetabolites. Lancet 1999; 354: 34–9. Favre-Schmuziger G, Hofer S, Passweg J et al. Treatment of solid tumors following allogeneic bone marrow transplantation. Bone Marrow Transplant 2000; 25: 895–8.

153. Socie G, Henry-Amar M, Devergie A et al. Poor clinical outcome of patients developing malignant solid tumors after bone marrow transplantation for severe aplastic anemia. Leuk Lymphoma 1992; 7: 419–23. 154. Bhatia S, Yasui Y, Robison L et al. High risk of subsequent neoplasms continues with extended follow-up of childhood Hodgkin’s disease: report from the Late Effects Study Group. J Clin Oncol 2003; 21: 4386–94. 155. Smith RA, Cokkinides V, von Eschenbach AC et al. American Cancer Society guidelines for the early detection of cancer. CA Cancer J Clin 2002; 52: 8–22. 156. Carlson RW, McCormick B. Update: NCCN breast cancer Clinical Practice Guidelines. J Natl Compr Canc Netw 2005; 1: S7–11. 157. Bhatia S, Francisco L, Baker KS et al. Late mortality in two-year survivors of autologous hematopoietic cell transplantation (HCT): report from the BMT Survivor Study (BMTSS). Blood 2005; 105: 4215–22. 158. Bhatia S, Francisco L, Carter A et al. Late mortality after allogeneic hematopoietic cell transplantation and functional status of long-term survivors: report from the Bone Marrow Transplant Survivor Study. Blood 2007; 110: 3784–92.

107

Harry Openshaw

Neurologic Complications of Hematopoietic Cell Transplantation

Introduction Published rates of neurologic complications in hematopoietic cell transplantation (HCT) vary from 3% in autologous transplants to as high as 44% in matched unrelated donor transplants [1,2]. A practical approach to the transplant patient requires an understanding of which problems are most likely at different times of the transplant course. As indicated in Table 107.1, neurologic complications may occur at three stages: (1) from the conditioning agents used for marrow ablation, (2) during posttransplantation pancytopenia, or (3) from immunosuppressive therapies and graft-versus-host disease (GVHD) in allogeneic transplant recipients. It is important to understand the patient’s pretransplant neurologic status, particularly in patients heavily pretreated with neurotoxic chemotherapy, and how this may influence post-transplant complications. The clinician must also differentiate neurologic complications of transplant from manifestations of disease recurrence. This chapter begins with a brief section on common neurologic symptoms and signs in transplant patients: weakness, sensory symptoms, cramps, headache, encephalopathy, and seizures. Subsequent sections of the chapter are organized according to standard disease categories as given in the first column of Table 107.1.

Common neurologic symptoms and signs in transplant patients Fatigue and weakness from disuse and general physiologic stress are almost universal symptoms early in the transplant course. A regular physical therapy program specifically tailored to the patient is of utmost importance at this stage. Weakness with onset later in the transplant course may pose a problem in differential diagnosis. Upper motor neuron weakness (i.e. weakness associated with increased deep tendon reflexes, spasticity, and extensor plantar responses) carries an ominous prognosis, since it is usually caused by a brain mass lesion or an epidural deposit with spinal cord compression. Weakness in a segmental distribution (i.e. in the distribution of a spinal nerve root) suggests a leptomeningeal recurrence or an epidural deposit at that particular spinal level. Much less commonly, herpes zoster infection produces weakness in a segmental distribution. Weakness of extraocular eye muscles, seen in virtually all patients with post-transplant myasthenia gravis, tends to be a fluctuating weak-

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

ness with characteristic fatigability and associated ptosis. Clinically, this can be distinguished on repeated neurologic examinations and differentiated from the fixed cranial nerve palsies associated with leptomeningeal disease. Clinical features more consistent with polyneuropathy than myopathy include distal distribution of weakness, presence of sensory symptoms, and absent deep tendon reflexes. Myopathic weakness is usually proximal around the shoulder or hip girdle. Muscle tenderness, subcutaneous swelling, and rash favor polymyositis of chronic GVHD over the usually milder myopathic weakness associated with disuse, corticosteroids or other endocrine-metabolic myopathies such as thyrotoxic myopathy. Sensory symptoms, usually in the feet, are very common in transplant patients who have pre-existing neuropathy from vincristine, cisplatin or other neurotoxic drugs. Nerve damage can progress after neurotoxic drugs have been discontinued, a phenomenon that has been called “coasting” or off-therapy worsening [3]; and dysesthetic symptoms may become major chronic complaints after HCT. Gabapentin or other symptomatic drugs may decrease neuropathic pain, but there are no reliable neuroprotective agents and no recognized therapy to hasten repair of chemotherapy-associated neuropathy. Cramps – involuntary, painful muscle contractions that occur spasmodically – are often seen in transplant patients, associated with myopathy or neuropathy or even without other neurologic findings or metabolic abnormalities. When such cramps occur daily and interfere with rest or ordinary activity, preventative therapy with gabatentin or with phenytoin often provides benefit [4]. Headache often occurs as a nonspecific symptom of fever, but may constitute the sole neurologic side-effect of calcineurin inhibitors, or more seriously be a manifestation of central nervous system (CNS) infection or mass lesion. For cyclosporine (CSP)-associated headache, propranolol often is helpful [5], and there is a report of resolution of a CSP headache by conversion to tacrolimus (FK506) [6]. The characteristic clinical features of metabolic encephalopathy are delirium or depression of the sensorium, from lethargy to stupor or coma, usually without lateralizing neurologic signs. In hepatic coma particularly, there may be abnormal neurologic signs, including hemiplegia and brainstem signs with extensor or flexor posturing to noxious stimuli. Preservation of the pupillary light response and eye movements in the face of reflexive posturing argues for a metabolic rather than a structural etiology of coma. Seizures in transplant patients usually are iatrogenic, sometimes from conditioning drugs, but most often from calcineurin inhibitors. Phenytoin is usually used after seizures induced by calcineurin inhibitors, and most clinicians continue phenytoin until high-dose calcineurin inhibitor

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Table 107.1 Neurologic complications of hematopoietic cell transplantation Conditioning

Pancytopenia

Graft-versus-host disease

Infectious

Bacterial meningitis

Cerebrovascular

Intracranial bleed Thrombotic angiopathy Gram-negative sepsis Sedative–hynotic drugs Hepatic encephalopathy of sinusoidal obstructive syndrome Calcineurin inhibitor neurotoxicity → Steroid toxicity→ Leukoencephalopathy →

Fungal abcess Meningoencephalitis Septic embolism, mycotic aneurysm ← Ischemic stroke→

Metabolic

Toxic

Encephalopathy (BCNU, busulfan, ifosfamide) Neuropathy (etoposide, cisplatin, paclitaxel)

Immune-mediated

therapy is no longer required. There is some rationale for using valproic acid as the long-term anticonvulsant, since valproic acid, unlike phenytoin, does not affect hepatic metabolism of calcineurin inhibitors [7].

Infectious complications The neurologic presentation of CNS infection in HCT recipients is usually an alteration in mental status, delirium or depression of the sensorium, often without meningeal signs or obvious lateralizing neurologic signs. A spinal fluid examination is indicated once a mass lesion has been excluded. For a suspected brain abscess, a stereotactic biopsy under appropriate platelet support usually is diagnostic. Antimicrobial prophylaxis early in the transplant course is particularly effective for Gram-negative organisms, and bacterial meningitis at this stage is now unusual, although Listeria monocytogenes meningitis has been reported in the first 4 months after allogeneic HCT. Also unusual, but still a risk, is bacterial meningitis in long-term survivors with chronic GVHD, including Listeria monocytogenes meningitis and meningitis due to penicillin-resistant Streptococcus pneumoniae [8]. Aspergillus species accounted for 30–50% of CNS infections in autopsy series and remain a major problem in HCT. Figure 107.1(a) shows a typical Aspergillus abscess. Less common is Aspergillus endarteritis presenting as a stroke (Fig. 107.1(b)). Candida albicans, the other serious fungal pathogen encountered after transplantation, infects the CNS in only 3% of transplant patients with systemic candidemia [9], although Candida species abscesses accounted for 15% of the CNS infections in a large autopsy series from Brazil [10]. Chronic fungal meningitides, for example from Cryptococcus neoformans, so frequent a complication of immunosuppression in other disease processes, is rarely encountered in HCT recipients. More common is CNS infection with the protozoon Toxoplasma gondii early after engraftment, particularly in patients who do not receive trimethoprim and sulfamethoxazole prophylaxsis (Fig. 107.1(c) and (d)). Herpes simplex virus (HSV) type 1 reactivates and is shed in the oral secretions of 80% of seropositive patients during the first few weeks after HCT [11]. As described in Chapter 91, acyclovir prophylaxis prevents viral shedding and is used routinely now in transplant centers. Despite the high incidence of viral reactivation, the clinical diagnosis of herpes simplex encephalitis is rarely made in transplant recipients.

Hypoxic encephalopathy of interstitial pneumonia Hepatic encephalopathy graft-versus-host disease Uremic encephalopathy Thalidomide neuropathy

Polymyositis Myasthenia gravis Demyelinating polyneuropathy

In immunocompetent individuals, herpes encephalitis usually occurs without any sign of mucocutaneous infection, and the infection is focal in the medial temporal lobes, most often presenting with seizures and psychiatric symptoms. Figure 107.2(a) shows an electroencephalogram (EEG) and magnetic resonance imaging (MRI) scan of a transplant patient who developed typical frontotemporal herpes simplex encephalitis. The EEG is helpful since periodic lateralized epileptiform discharges (as in Fig. 107.2(a)) are characteristic for herpes encephalitis and may precede the MRI abnormality. Although HSV was cultured from the cerebrospinal fluid (CSF) in the patient shown in Fig. 107.2(a), viral cultures usually are negative in herpes encephalitis. CSF polymerase chain reaction assay (PCR) of HSV nucleic acid is helpful, but treatment should be initiated and continued in suspicious cases regardless of the PCR result. Present about half as often as HSV, active varicella zoster virus (VZV) infection occurs later in the transplant course. The greatest incidence is during the fourth and fifth months after allogeneic transplantation (see Chapter 92). Facial nerve palsy and hearing loss can be associated with cranial zoster, arm weakness with cervical zoster, and neurogenic bladder with lumbosacral zoster. It is likely that a mild meningoencephalitis with or without pleocytosis and motor signs occurs fairly frequently in transplant patients with zoster; but the designation of varicella zoster encephalitis is usually reserved for the rare instances of diffuse encephalitis with a decrease in sensorium. Other serious manifestations include VZV vasculitis and retinal necrosis [12]. VZV and HSV have been implicated in peripheral facial weakness (Bell’s palsy) in normal individuals without any skin or mucous membrane manifestations. Although a recent study failed to show benefit of acyclovir added to prednisolone in normal individuals with Bell’s palsy [13], there has not been a similar study in transplant patients. It is probably prudent to start antiviral therapy with valacyclovir with or without corticosteroids in HCT patients who develop Bell’s palsy. Recognized manifestations of cytomegalovirus (CMV) infection in transplant patients include pulmonary, hepatic, and gastrointestinal involvement (see Chapter 90). For reasons that are uncertain, CMV chorioretinitis, a very common problem in human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS), is rarely diagnosed in HCT recipients [14]; similarly, CMV encephalitis is recognized at least histopathologically in HIV/AIDS but is rarely diagnosed

Neurologic Complications of Hematopoietic Cell Transplantation

(a)

(b)

(c)

(d)

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Fig. 107.1 Fungal and parasitic central nervous system (CNS) infections. (a) Magnetic resonance image (MRI) of CNS Aspergillus infection. A 27-year-old man with Hodgkin’s lymphoma developed pulmonary aspergillosis as documented by bronchoscopy 5 months after autologous transplantation. Gadolinium ring-enhanced lesions were documented in the right frontal lobe. CNS aspergillosis was confirmed at autopsy. (b) Post mortem Aspergillus histopathology of a 45-year-old woman who developed a locked-in syndrome, rapidly evolving to coma and death 2 months after allogeneic transplantation for granulocytic sarcoma. Shown is a thrombosed branch of the basilar artery with mural invasion of the arterial wall by branching fungal hypahae (Gormori methanimine silver. Magnification: × 100). (c) MRI of CNS Toxoplasma gondii infection. At 3.5 months after autologous transplantation for Hodgkin’s lymphoma, a 21year-old Hispanic woman developed right-sided upper motor neuron clumsiness with sensory symptoms. An MRI scan showed multiple gadolinium-enhancing lesions, the largest in the left parietotemporal area. (d) Neurological recovery and almost complete resolution of the lesions on MRI scanning occurred after 6 months of therapy with pyrimethamine and sulfadiazine.

or documented in marrow transplant recipients [15]. Fatal meningoencephalitis due to West Nile virus, adenovirus, and human herpes virus type 6 have all been documented in marrow transplant recipients [16,17]; and instances of human herpes virus type 6 focal encephalitis in the medial temporal lobe, indistinguishable from HSV encephalitis, have been recognized. Use of conditioning regimens including alemtuzumab increases the risk of HHV6 encephalitis, even in patients receiving antiviral prophylaxis [18]. An example of Epstein–Barr virus encephalitis in a transplant patient is presented in Fig. 107.2(b). Finally, progressive multifocal leukoencephalopathy, the slow virus infection caused by polyomavirus JC, has been documented in only a few transplant patients, including post autologous HCT [19]. PCR assays are now readily available for detection of all of these viruses in CSF.

Cerebrovascular complications Intracranial hemorrhage is most frequently associated with refractory thrombocytopenia, and, except for subdural hematomas, large intracranial hemorrhages are usually fatal in HCT recipients. The characteristic clinical course of large supratentorial hemorrhages is abrupt onset of hemiparesis, or other neurologic deficit localized to the cerebral hemispheres, followed by rapid depression of the sensorium and development of brainstem signs from transtentorial herniation (Fig. 107.3(a)). A cer-

ebellar hemorrhage is more difficult to recognize because of the subtlety of the initial signs – abnormalities of gait and eye movement, which then progress to gaze paresis and coma. Treatment of intracranial hemorrhage is the neurosurgical evacuation of the hematoma in those instances where this is feasible [20]. Subdural hematomas characteristically reduce the sensorium with no or mild lateralizing signs. Consequently, they can be overlooked early in the course, particularly in patients who are sedated or encephalopathic for metabolic reasons. Nonhemorrhagic subdural fluid collections (hygromas) may develop over the cerebral convexities in patients who have low CSF pressure as a consequence of repeated lumbar punctures (e.g. patients with acute lymphoblastic leukemia undergoing intrathecal treatment or prophylaxis for CNS leukemia) [21]. An epidural lumbar blood patch may help decrease the headache and shorten the time for the hygroma to resolve. However, with or without the blood patch, the hygroma may increase in size, as shown in Fig. 107.3(b), and surgical drainage may be necessary even if bleeding into the hygroma can be avoided. With or without neurosurgery, this complication may seriously compromise treatment of the leukemia by delaying further intrathecal therapy and the date of HCT. Ischemic strokes in transplant recipients may be embolic from endocarditis or thrombosis associated with a hypercoagulable state. Vasospasm leading to ischemic symptoms or stroke has been suspected in

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MRI

EEG

L R

L

R

(a)

(b)

Fig. 107.2 (a) Electroencephalogram (EEG) and brain magnetic resonance imaging (MRI) in herpes simplex encephalitis. A 46-year-old man whose acute myelogenous leukemia relapsed 8 months after autologous stem cell transplant, then underwent a nonmyeloablative matched unrelated donor transplant with melphalan and fludarabine conditioning. On cyclosporine and prednisone for skin graft-versus-host disease (GVHD), he developed abrupt confusion with visual agnosias 4 months after allogeneic transplantation. An EEG showed right-sided periodic sharp waves. An increase in right frontotemporal signal developed on follow-up brain MRI fluid-attenuated inversion recovery (FLAIR) sequence. The cerebrospinal fluid (CSF) had 44 white blood cells/μl (87% lymphocytes) and 11 red blood cells/μl. Herpes simplex type 1 virus was cultured from the CSF. Despite therapy with both acyclovir and foscarnet, there was neurologic progression with the patient developing an amnestic state, unable to form new memory traces. (b) Brain MRI (FLAIR sequence) in Epstein–Barr virus (EBV) encephalitis. A 63-year-old man 5 years after allogeneic transplant for acute myelogenous leukemia with sclerodermatous GVHD developed confusion, weakness with incoordination, and bilateral upper motor neuron signs. CSF had 39 white blood cells/μl (82% lymphocytes), and EBV sequences were detected in blood and CSF. He was treated with rituximab and survived with neurologic disability.

Neurologic Complications of Hematopoietic Cell Transplantation

rare patients at the time of cryopreserved stem cell transfusion [22]. An endarteritis associated with meningeal infection, particularly with Aspergillus species, may occur and be difficult to distinguish clinically from primary cerebrovascular disease (Fig. 107.1(b)). Bacterial or fungal sphenoid sinusitis in transplant patients may result in a major hemisphere stroke by extension of the infection to the carotid artery in the venous cavernous sinus (Fig. 107.3(c)). It is important that patients with severe sphenoid sinus infections undergo surgery and receive appropriate antibiotic or antifungal therapy to prevent cavernous sinus syndrome. Nonbacterial thrombotic endocarditis occurs in transplant patients as a consequence of a hypercoagulable state [23]. Chemotherapy-induced endothelial damage and circulating immune complexes may also be contributing factors. In an autopsy series of 91 allogeneic transplant patients, there was a single case of bacterial endocarditis (group D Streptococcus) with stroke, and seven cases of nonbacterial thrombotic endocarditis, with two of these patients having ischemic strokes. Thrombotic microangiopathy, recognized in 5–15% of allogeneic transplant patients, may cause transient hemiparesis and seizures. Etiology probably is from endothelial cell damage from conditioning therapy or calcineurin inhibitors. Diagnosis often requires a high degree of suspicion, identifying fragmented red blood cells and elevated serum lactate dehydrogenase in the context of renal or hepatic abnormalities; and treatment involves reducing or discontinuing calcineurin inhibitors, transfusion of cryoprecipitate-poor plasma or a trial of plasmapheresis [24].

Metabolic complications Metabolic encephalopathy in transplant patients is most often associated with Gram-negative sepsis or the use of sedative–hypnotic drugs.

(a)

(b)

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Hypoxic encephalopathy, carrying the risk of permanent neurologic disability, may occur from interstitial pneumonia or from hypoxemia associated with red blood cell lysis in the hemolytic–uremic syndrome. Hepatic encephalopathy may occur from liver involvement in GVHD or from fulminant hepatic failure in sinusoidal obstructive syndrome (SOS) (see Chapter 95). Renal failure with resultant uremic encephalopathy has been attributed to nephrotoxic drugs including the calcineurin inhibitors, the renal glomerulopathy that is rarely seen as a manifestation of GVHD, radiation nephritis, and the hemolytic–uremic syndrome. Hypomagnesemia, associated with calcineurin inhibitors or as a residual from cisplatin nephrotoxicity, may cause convulsions as well as cramps and muscle weakness. Multiple organ failure in HCT patients, as in other critically ill patients, is a poorly understood but generally fatal entity. SOS of the liver or pulmonary failure after transplant may herald multiorgan failure, including CNS failure [25]. About 50% of HCT patients who develop a decrease in a standardized bedside mental status instrument in the first month of transplant go on to develop pulmonary or hepatic involvement, and almost two-thirds of these patients do not survive 100 days after transplant [26]. An argument has been advanced that, regardless of the initial organ system involved, the course in these patients reflects a systemic inflammatory response, the so-called multiple organ dysfunction syndrome [27]. As such, a systemic rather than an organ-specific approach to therapy is needed, and such an approach will depend on a better understanding of the pathophysiology and initiating events of multiple organ dysfunction syndrome and how these may be affected by high-dose chemoradiation. Because of the increased risk of sepsis and shock, neurologic complications of critical illness may occur in transplant patients [28]. Critical

(c)

Fig. 107.3 Cerebrovascular disease. (a) Brain computed tomography of a fatal intracranial hemorrhage. A 34-year-old woman with biphenotypic leukemia underwent allogeneic bone marrow transplantation, and 1.5 months later had a fatal massive left intraparenchymal hemorrhage with rupture into the left lateral ventricle. (b) Brain magnetic resonance imaging (MRI) scan of bilateral subdural hygromas resulting from multiple lumbar punctures and low cerebrospinal fluid pressure. A 48-year-old man with human immunodeficiency virus-associated diffuse large cell lymphoma and leptomeningeal disease underwent 3 lumbar punctures for intrathecal therapy the week before high-dose BCNU and etoposide with autologous stem cell transplantation. Two weeks after transplantation, there was confusion and reduced sensorium. The MRI san showed bilateral subdural fluid collections, obliteration of cortical sulci, and balanced mass effect. After bilateral surgical drainage of the subdural hygromas, there was improvement in his sensorium. (c) Brain MRI of left sphenoid sinus infection with extension to the cavernous sinus (arrow head) and left hemisphere stroke (arrow). Sphenoid sinusitis developed in a 35-year-old man 5 months after autologous stem cell transplantation for acute myelogenous leukemia. Despite repeated sphenoid surgeries, a left VIth cranial nerve palsy developed followed by right hemiparesis and dysphasia. He was treated with amphotericin B and itraconazole for a presumed fungal infection. The MRI shows sphenoid sinus opacification, a soft tissue mass within the left cavernous sinus occluding or narrowing the carotid artery, and an infarction involving the internal capsule, putamen, and head of caudate nucleus.

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illness polyneuropathy is thought to occur from inadequate perfusion of peripheral nerves and is often associated with encephalopathy, multiorgan failure, and prolonged mechanical ventilation. It is a predominantly motor axonal neuropathy with moderate-to-severe neurologic deficit interfering with respiratory weaning [29]. Recovery may be partial and slow, with survivors usually requiring months before they can resume ambulation. Another complication that interferes with respiratory weaning and may be confused clinically is acute critical illness myopathy [30]. Coincidence of both critical illness polyneuropathy and acute critical illness myopathy probably occurs particularly in patients with multiorgan failure who have received neuromuscular blocking agents and corticosteroids.

Immune-mediated complications It is still unanswered whether GVHD can affect the CNS [31]. In longterm HCT survivors, the incidence of MRI white matter abnormalities and abnormalities in neuropsychologic tests have been shown to correlate with chronic GVHD [32]. This correlation, however, probably results from complications of prolonged immunosuppression rather than a direct CNS affect of chronic GVHD. There are clinical reports of rare instances of myelitis and optic neuritis after allogeneic HCT, reports of cerebral angiitis, and a report of remitting and relapsing cerebral demyelination, analogous to episodes of demyelination in multiple sclerosis [33–35]. Etiologic factors other than GVHD, however, are possible in these patients. Three immune-mediated diseases, all affecting the peripheral nervous system, can occur after HCT. Most common is polymyositis, a clinical syndrome characterized by proximal muscle weakness, elevated levels of creatine phosphokinase and other muscle enzymes, short-duration (myopathic) motor units on electromyography with signs of acute denervation (fibrillation potentials and positive sharp waves), and necrotic myofibers and mononuclear inflammatory cells on muscle biopsy. Less common is myasthenia gravis, an immune-mediated disorder of the neuromuscular junction in which autoantibodies to the postsynaptic acetylcholine receptor produce a characteristic clinical syndrome of ptosis and extraocular muscle weakness, most often with proximal limb and facial muscle weakness. Less well accepted as a complication of HCT is immune-mediated demyelinating polyneuropathy, a condition producing neuropathic weakness that can be severe, sensory loss, and nerve conduction slowing on electrophysiologic tests. It is not difficult to differentiate polymyositis from myasthenia gravis and polyneuropathy. A potential problem arises when mild polymyositis goes unrecognized and accounts for a slower than usual functional recovery from transplantation. A review article reported polymyositis in 3% of 318 patients with chronic GVHD [36]. Onset of weakness occurred 7–24 months after transplantation, and all patients showed improved strength with reinstitution or an increase in the dose of prednisone. There are rare reports in which polymyositis was the sole manifestation of GVHD, and there is a case report of polymyositis occurring after autologous transplantation [37]. It is important to exclude infectious causes of polymyositis and to exclude the occasional patient with drug-induced myositis. Zidovudine (AZT), as well as the combination of lovastatin and CSP, can cause an inflammatory myopathy, and a fatal case of rhabdomyolysis occurred after a CSP-associated seizure in a transplant patient [38]. Muscle weakness in a patient with GVHD may also be from autoimmune hyperthyroidism [39] or, much more commonly, from steroid myopathy. A common treatment dilemma is whether steroids should be increased as therapy for polymyositis or decreased because of presumptive steroid myopathy. Further testing should help in this situation because, unlike

polymyositis, patients with steroid or other endocrine myopathies have neither electromyographic evidence of denervation nor inflammatory cells in muscle biopsy specimens. The prevalence of post-transplantation myasthenia gravis is lower than polymyositis. Only 3 of 1800 allogeneic transplant recipients (<0.5 %) have been diagnosed at Seattle, and there is a similar low prevalence at other centers [40]. Onset tends to be later than with polymyositis. Most patients are diagnosed after immunosuppressant drugs to prevent chronic GVHD have been decreased or discontinued. Patients transplanted for aplastic anemia appear to have a greater risk for acquiring myasthenia gravis; and many post-transplant myasthenia gravis patients have antiplatelet, antismooth muscle, antimitochondrial, and antinuclear antibodies as well as antiacetyl choline receptor antibodies. Thymoma, present in about 15% of patients with idiopathic myasthenia gravis, has not been identified in post-transplantation myasthenia gravis. Unlike polymyositis, which usually is self-limited, post-transplant myasthenia gravis tends to be a chronic, albeit sometimes fluctuating, problem requiring anticholinesterase drugs to control symptoms. Alternate-day low-dose prednisone is generally adequate if immunosuppressants are required. Return to higher-dose immunosuppressants or treatment with plasma exchange is generally not necessary or recommended. It may be important to avoid, or use with caution in these patients, drugs that affect neuromuscular transmission, including aminoglycoside antibiotics, certain cardiac antiarrhythmic agents such as procainamide, and parenteral magnesium. Because of the known increased risk of Guillain–Barré syndrome in Hodgkin’s disease and other disorders with reduced cellular immunity [41], it is not surprising that Guillain–Barré-like neuropathies occasionally occur in transplant recipients [42]. Compared with myasthenia gravis and polymyositis, there is a greater clinical heterogeneity in these patients and less certainty as to the pathogenesis. The prevalence has been estimated at only 1% of allogeneic transplantations [43], but these neuropathies produce considerable morbidity because the deficits are predominantly motor and because they may occur early after transplantation when patients are medically most vulnerable. Additional recent reports attributed some instances of demyelinating neuropathy in transplant patients to CSP or to prior CMV or Campylobacter jejuni infection [44]. An immune mechanism is suspected in these neuropathies, but the pathogenesis is uncertain. GVHD is present in most but not all patients, and the neuropathy has been thought to correlate with the course of GVHD in some patients. Necessary for the diagnosis of demyelinating neuropathy is the electrophysiologic demonstration of nerve conduction slowing or conduction block. It may be helpful to document CSF protein elevation and myelin breakdown with reactive macrophages on nerve biopsy. Treatment of demyelinating polyneuropathy in these patients has included plasmapheresis with variable success: prednisone, which is effective in nontransplant-associated chronic inflammatory demyelinating polyneuropathy, and intravenous gamma globulin. There may be a need for caution in considering a transplant for hematologic malignancy in patients with a pre-existing inflammatory demyelinating neuropathy since abrupt worsening after conditioning therapy involving fractionated total body irradation (TBI) has been reported in rare patients [45].

Complications from calcineurin inhibitors, CSP, and tacrolimus Calcineurin inhibitors (CSP and tacrolimus) cause more neurologic problems in transplant patients than any other class of drug [46]. Complications are diverse. Essential tremor is almost always present, and headaches of a vascular quality occur in up to 20% of patients. Seizures

Neurologic Complications of Hematopoietic Cell Transplantation

and encephalopathy are the most common serious complications. Typically, these seizures follow complaints of headache, increased tremulousness, mild confusion, asterexis, and sometimes visual symptoms. Although usually single and generalized, multiple and partial seizures may also occur, and there may be transient post-seizure deficits such as cortical blindness and behavioral abnormality. Patients who are encephalopathic usually have elevated blood pressure and often have low serum magnesium. Tacrolimus has the same mechanism of action and a similar toxicity profile to CSP, although the risk of neurotoxicity may be lower with tacrolimus [47]. Incidence of serious neurotoxicity is highest in the first few months after HCT, occurring in up to 5% of patients [48]. Patients with neurotoxicity from calcineurin inhibitors may have abnormalities on MRI with multifocal areas of signal hyperintensity, most often in the occipital lobe white matter with associated occipital blindness (Fig. 107.4(a)) [47]. These abnormalities are identified on T2-weighted MRI or fluid-attenuated inversion recovery (FLAIR) sequences. In recent neuroradiology literature, the term “posterior reversible encephalopathy syndrome” (PRES) has been used. Calcineurin inhibitors accounted for just over half the instances of PRES in one

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survey, with most of the remaining patients having hypertension alone or eclampsia [49]. Although most scans show predominant white matter abnormalities, there can be mixed cortical and subcortical lesions, sometimes associated with punctate cortical gadolinium enhancement (Fig. 107.4(b) and (c)) [50]. Cerebellar and brainstem MRI lesions exceptionally occur, and transient eye movement abnormalities have been reported from calcineurin inhibitors in HCT patients (Fig. 107.4(d)) [51]. Although such atypical distribution of so-called PRES occurs, one hardly ever sees lateralized motor or sensory signs from calcineurin inhibitors. Such lateralized signs usually suggest an ischemic insult or a cerebral mass lesion. Calcineurin inhibitor neurotoxicity is less often detected by computed tomography (CT) than MRI, but marked CT abnormalities can occur, as shown in Fig. 107.4(e). The reversibility of the CT abnormality on follow-up scans (Fig. 107.4(f)) argues against cerebral infarction and for vasogenic edema. Damage to vascular endothelium from chemotherapeutic drugs, breakdown of the blood–brain barrier from radiation, or from CSP itself – these may all be contributing factors. CSP leads to the release of endothelins, vasoactive neuropeptides that have been

A

B

C

D

E

F

Fig. 107.4 Calcineurin inhibitor neurotoxicity. (a) Brain magnetic resonance imaging (MRI) (T2-weighted sequence) of a 20-year-old woman 5 weeks after allogeneic transplantation for chronic myeloid leukemia who developed abrupt onset of confusion and headache. Patches of bright signal on T2-weighted images were present, especially in the parietal and occipital subcortical white matter. Later, a generalized seizure with encephalopathy occurred, and cyclosporine (CSP) was stopped for 4 days and then restarted at a lower dose. Repeat MRI showed resolution of the lesions. (b and c) Brain MRI fluidattenuated inversion recovery (FLAIR) sequence (b) and T1-weighted sequence with gadolinium (c) in an 11-year-old girl with acute lymphoblastic leukemia on tacrolimus 4.5 months after cord blood transplantation. (d) Brain MRI (FLAIR sequence) of a 50-year-old man who developed diplopia 2 months after allogeneic transplant for mantle cell lymphoma. The arrow shows signal abnormality including the area of the VIth cranial nerve nucleus. Eye movements returned to normal 2 days after CSP was discontinued. Eye movement abnormality associated tacrolimus leukoencephalopathy in a 2-year-old girl with acute lymphoblastic leukemia and liver chronic graft-versus-host diseases who developed seizures and reduced sensorium 1 year after matched unrelated donor transplant (e). The follow-up scan (f) done 1 month later showed almost complete resolution of parieto-occipital edema. (Panel d reproduced from [94], with permission.)

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implicated in cerebral vasospasm [52]. Endothelin release may be triggered by or may cause microangiopathic hemolytic anemia, a condition found by multivariate analysis to have the highest correlation with CSP toxicity [46]. Vasospasm, shown to be present on an MR angiogram [50], may explain involvement at junctions of major vessels (watershed areas) or in the distal distribution of major vessels, such as the occipital pole. Neuropathology of CSP toxicity is limited, but reports indicate edema or demyelination. There is one report of vasculitis attributed to tacrolimus in a liver transplant patient [53]. Although these vascular abnormalities are now well recognized, they probably do not account for all instances of serious calcineurin inhibitor neurotoxicity. Sometimes there is severe neurotoxicity with encephalopathy prolonged for days, to near-coma, yet the MRI is normal. In other instances, there is relatively mild encephalopathy but with MRI abnormalities on FLAIR or T2-weighted sequences. In animals, CSP has a direct neurotoxic effect, independent of alterations in blood pressure or renal function [54]. A cell culture study showed that CSP can induce neuronal apoptosis and selective oligodendrocyte death [55]. The mechanism probably involves inhibition of the phosphatase activity of calcineurin, the same mechanism whereby CSP inhibits T-cell proliferation. CSP is bound by serum lipoproteins, metabolized in the liver cytochrome P450 enzyme system, and excreted in bile. Blood levels may become elevated in patients with hyperbilirubinemia, GVHD of the liver, or SOS [56]. Erythromycin, ketoconazole, and calcium channel blockers decrease hepatic metabolism of CSP [46,57], whereas phenytoin enhances hepatic metabolism [58]. Hypocholesterolemia is associated with an increased risk of CSP neurotoxicity [59], probably because of an increase in unbound CSP; and it is likely that the low cholesterol levels in liver disease account at least in part for the twofold greater prevalence of CSP neurotoxicity in liver transplant patients compared with HCT patients. CSP does not penetrate the blood–brain barrier well under normal conditions [48], but damage to the barrier by conditioning agents, particularly irradiation, may increase penetration of calcineurin inhibitors. Despite clinical experience suggesting a direct toxic role of CSP, neurotoxicity does not always correlate with steady-state drug levels. It has been proposed that high peak levels (e.g. at times of intravenous use) may predispose to neurotoxicity [48]. A considerable degree of clinical judgment often goes into decisions of when to reduce or hold calcineurin inhibitors to prevent the more serious instances of neurotoxicity. Even mildly encephalopathic patients who have worsening asterexis or develop myoclonus should be considered for supplementary intravenous magnesium, temporary discontinuation of the calcineurin inhibitor regardless of the drug level, and possibly a loading dose of intravenous fosphenytoin or phenytoin. Clinical experience in HCT suggests that temporary discontinuation of calcineurin inhibitors and then restarting at a lower dose is usually successful in preventing worsening or recurrence of neurotoxicity. Switching from CSP to tacrolimus or vice versa is seldom necessary, and is not always successful in preventing recurrence of neurotoxicity.

Complications from other immunosuppressive drugs A sense of wellbeing and even euphoria often accompanies the initiation of corticosteroid treatment. With continued administration, psychotic depression, mania or delirium may occur [60]. This toxicity is often dose dependent, but when reduction in steroid dose is not feasible, neuroleptic or antianxiety medications may be necessary. Proximal muscle weakness due to muscle protein catabolism is experienced by virtually all transplant patients who receive the equivalent of 40 mg of prednisone a day for more than 3 weeks [61]. Typical symptoms include difficulty arising from a chair and difficulty washing hair. Muscle cramps and tenderness occur less often with steroid myopathy than with polymyo-

sitis. Unlike polymyositis, serum creatine phosphokinase levels are normal or only slightly elevated in steroid myopathy, and electromyography often shows only slight abnormalities with myopathic motor units. Treatment consists of switching from the more myotoxic fluorinated steroids such as dexamethasone to nonfluorinated agents, such as prednisone or methylprednisolone, tapering the steroids when this is possible, and instituting daily physical therapy. Regular exercise during corticosteroid treatment may reduce the catabolic effect on muscle. Symptoms of thalidomide peripheral neuropathy can begin after 2 months of therapy at 100 mg a day, the starting dose sometimes used for chronic GVHD [62]. Lower limb numbness and paresthesias occur, often accompanied by burning and hyperesthesia of the feet. With continued exposure, leg cramps and a stocking–glove pattern of sensory loss develop, involving superficial sensation more than proprioception or vibration. Patients with severe neuropathy may have muscle weakness. An unusual feature compared with other toxic neuropathies is preservation of deep tendon reflexes well into the course of the neuropathy. There may be mild elevation of CSF protein, and nerve conduction tests as well as morphologic analysis indicate a large-fiber, “dying-back” sensory neuronopathy (i.e. initial degeneration in the distal region of the axon and progression of the degeneration proximally to the nerve cell body) [63]. The extent of neurologic recovery after discontinuation of thalidomide depends on the severity of the symptoms and possibly the patient’s age and duration of exposure. Motor signs revert more readily and completely than sensory symptoms, and, in some patients, distressing sensory complaints may be permanent [64]. Low-dose intravenous methotrexate (MTX), frequently used for GVHD prophylaxis, causes only occasional and minor neurotoxicity of headache, dizziness, and rarely seizures when given to the patients with rheumatoid arthritis [65]. High-dose MTX (5 g/m2 per cycle), used mainly for osteogenic sarcoma, can trigger transient leukoencephalopathy, similar in appearance but usually more extensive than the MRI abnormalities seen with calcineurin inhibitors [66], and acute, transient CNS toxicity often with hemiparesis occurs rarely after intrathecal MTX. Much less often seen now in transplant patients is the delayed-onset, chronic, and often fatal leukoencephalopathy resulting usually from the combination of intrathecal MTX and whole-brain irradiation [67]. Neurologic signs usually within 4–5 months after transplant include dysarthria, ataxia, dysphasia, spasticity and upper motor neuron weakness, seizures, confusion, and a decrease in the sensorium. Leukoencephalopathy with severe neurologic sequelae and death has also been attributed to amphotericin B following TBI in a transplant recipient [68].

Complications from conditioning agents Neurologic complications from chemotherapeutic drugs in the conditioning regimen include encephalopathy and seizures as acute selflimited complications and peripheral neuropathy as delayed, longer-lasting complications. Busulfan (BU) and carmustine (BCNU) are common causes of encephalopathy, and etoposide and cisplatinin are common causes of peripheral neuropathy. Granulocyte colony-stimulating factor may exacerbate autoimmune activity during peripheral blood hematopoietic stem cell mobilization of patients with multiple sclerosis [69]. Long-term disabilities of conditioning agents that may involve cognition and quality of life are discussed in Chapter 105, and the long-term risk of primary brain tumors is discussed in Chapter 106. Before routine use of prophylactic anticonvulsants, generalized seizures occurred overall in about 10% of patients receiving high-dose BU (4 mg/kg/day for 4 days) [70]. BU readily crosses the blood–brain barrier, and it is presumed that seizures occur as a direct neurotoxic

Neurologic Complications of Hematopoietic Cell Transplantation

effect. Although myoclonic twitching may be seen shortly before or after the ictus, BU seizures usually are not focal, and seldom are they multiple or complicated. Because phenytoin induces the metabolism of BU, levetiracetam or another enzyme noninducer is probably preferable, particularly if there is no attempt to adjust BU dose based on blood levels. Anticonvulsant prophylaxis also has generally been used with high-dose BCNU. Headache with flushing and paresthesias around the lips have been reported during high-dose BCNU infusion [71]. Neurologic manifestations of ifosfamide toxicity include seizures, acute confusional state, mutism, and disordered sensorium. Rare instances of extrapyramidal toxicities with opisthotonos have also been noted [72]. The toxicity may be related to a build-up of a chloral hydratelike compound, chloracetaldehyde, a major breakdown product of ifosfamide. There have been reports of treatment with methylene blue [73]. Fludarabine used now in reduced-intensity conditioning may cause encephalopathy and rarely progressive neurologic disability with CNS demyelination [74]. Seizures and transient encephalopathy have been observed with high-dose cytarabine (ara-C), although the most common neurologic problem is cerebellar dysfunction, occurring in 10% and leaving permanent ataxia from Purkinje cell drop-out in 3% of treated patients [75]. There have also been a few published cases of peripheral neuropathy with high-dose cytarabine, including patients with clinical and electrophysiologic features of acute demyelinating polyneuropathy consistent with the diagnosis of Guillain–Barré syndrome [76]. A sensory axonal neuropathy with mild or no motor disability developed in 4% of autologous transplant patients conditioned with high-dose etoposide (60 mg/kg intravenously for hematologic malignancy) [77]. Distal symmetrical sensory symptoms began within 2 months of the transplantation and improved slowly over months. One patient had a superimposed autonomic neuropathy and another had a peroneal neuropathy. A self-limited sensory neuropathy from etoposide conditioning occurs in allogeneic transplant recipients as well. The delay in sensory symptoms for 1–2 months after etoposide is similar to the post-treatment worsening of neuropathy often seen with cisplatin and vincristine [3]. In high-dose paclitaxel protocols for solid tumors, virtually all autologous transplant patients develop peripheral neuropathy, usually within 5 days of paclitaxel treatment [78]. Although reported as purely sensory in most affected patients, the neuropathy was sufficiently severe to require a walker in some patients. Clinical features of cisplatin neuropathy are usually characteristic, with large sensory fiber involvement, giving a proprioceptive deficit usually with preserved tactile and pin sensation and without muscle weakness [79]. By contrast, etoposide and paclitaxel neuropathies involve all sensory modalities [77,80]. Cisplatin neuropathy tends to correlate with cumulative dose rather than dose intensity. Symptoms usually occur after a cumulative dose of 300–600 mg/m2. Also dose dependent is cisplatin ototoxicity, affecting high-frequency hearing initially and – with continued cisplatin courses – speech frequency hearing, but very seldom producing vestibular toxicity [81]. In terms of cisplatin CNS toxicity, a transient Lhermitte phenomenon occurs occasionally from an effect on the dorsal column of the cervical region of the spinal cord [82], and there are rare reports of cortical blindness and seizures [83].

Complications from supportive care and other drugs Depression of the sensorium occurs very frequently in the first week after HCT from sedative–hypnotic drugs or analgesics. What can be especially misleading in lethargic or obtunded patients is unilateral pupillary dilatation from a scopolamine patch [84] used for drug-associated nausea. Similarly, ipratropium inhalation therapy given by mask has caused unilateral pupillary dilatation in transplant patients by inad-

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vertent contact with the eye [85]. In these patients, pilocarpine 1% eye drops fail to produce miosis, confirming pharmacologically induced mydriasis rather than pressure on the third cranial nerve from early transtentorial herniation. Recognition of this anticholinergic effect of scopolamine or ipratropium may prevent a needless head scan to exclude a cerebral mass lesion. Many of the antibiotics used after transplantation have been associated with neurologic toxicity: for examples, seizures from penicillin, piperacillin, metronidazole, and imipenem; encephalopathy from penicillin and metronidazole; hearing loss from aminoglycosides and vancomycin; peripheral neuropathy from metronidazole; and a myasthenia-like syndrome from aminoglycosides [86–89]. Since toxicity is often enhanced by renal insufficiency, special caution applies to those patients on calcineurin inhibitors and at risk for nephrotoxicity. Acyclovir at high dose can cause tremor, agitation or lethargy, occasionally with EEG epileptiform features [90]. In the setting of renal failure, reversible stupor and coma and occasionally generalized seizures have followed both oral and intravenous administration of acyclovir [91]. Delirium, which improved with dose reduction, was seen with ganciclovir treatment given at 10 mg/kg/day for CMV infection in a transplant patient [92]. Foscarnet most often causes renal and electrolyte disturbances, and low calcium and magnesium levels from foscarnet have been associated with paresthesias, muscle cramps, and rarely seizures [93].

Pretransplant neurologic screening Pretransplantation neurologic screening may identify patients particularly prone to neurologic complications, and in some cases this screening may lead to changes in treatment to minimize these complications. For example, anticonvulsant prophylaxis should be considered in transplant recipients with past seizures or a strong family history of epilepsy when these patients are treated with calcineurin inhibitors or other drugs known to lower the seizure threshold. Because of the cumulative toxicity of radiation therapy on the nervous system, it may be prudent to use preparatory regimens without fractionated TBI in brain-damaged patients or in patients with degenerative CNS diseases. Because of the potential of producing disabling neuropathy, it is best to avoid high-dose etoposide or other peripheral nerve toxic conditioning agents in patients with severe diabetic neuropathy, hereditary neuropathies such as Charcot– Marie–Tooth, or significant pre-existing chemotherapy-associated neuropathies. Finally, it is prudent to carefully screen donors to avoid transfer of neurologic immune-mediated diseases such as multiple sclerosis.

Conclusion Because of the large number of relatively rare complications, any general discussion of neurologic problems after HCT may read like a laundry list of differential diagnoses. It is much easier dealing with a specific patient. Experienced practitioners narrow down the differential diagnosis by taking into account the hematologic diagnosis, therapy given (pretransplant chemotherapy, conditioning agents, immunosuppressants, and supportive care drugs), and particularly the time interval post transplant (Table 107.1). Mistakes may occur if early neurologic symptoms are ignored or neurologic signs are misinterpreted. Although neurologic problems remain common after HCT, there is a general impression of reduced incidence of such problems, probably because of improvements in infectious disease treatment and prophylaxis, use of reducedintensity conditioning regimens, better adjustments of calcineurin inhibitors, and a general anticipatory and preventative approach to neurologic problems.

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Trudy N. Small

Vaccination of Allogeneic and Autologous Hematopoietic Cell Recipients

Introduction In an effort to standardize the vaccination of autologous and allogeneic hematopoietic cell transplantation (HCT) recipients, the European Group for Blood and Marrow Transplantation (EBMT) in 1995 [1] and the Centers for Disease Control (CDC) in 2000 [2] developed posttransplant guidelines incorporating tetanus, diphtheria, polio, Haemophilus influenzae, pneumococcus, influenza, measles, mumps, and rubella [1–4]. The EBMT guidelines were updated in 2005 [4], and an international effort to create combined guidelines under the auspices of the Center for International Blood and Marrow Transplant Research is currently underway. Despite current recommendations, few centers vaccinate patients consistently post HCT [1,5]. Both the EBMT [4] and CDC [2] guidelines advocate revaccination at fixed intervals following transplant (Table 108.1), irrespective of patient age, donor or hematopoietic cell type, intensity of conditioning, presence of chronic graft-versus-host disease (GVHD) or use of posttransplant immunomodulatory agents such as the anti-CD20 monoclonal antibody rituximab. Nevertheless, many of these variables affect the kinetics of T- and B-cell reconstitution following allogeneic or autologous HCT [6–10], and likely the ability of patients to respond and maintain protective titers following vaccination [11,12]. As shown in Figs 108.1, 108.2, and 108.3, the kinetics of B-cell, T-cell, and basic T-cell function (response to phytohemagglutinin) vary considerably in adults and children following unmodified and T-cell-depleted HCT from related and unrelated donors. Although immunization of all patients using a standard timetable without documentation of pre- and postvaccine titers is a less complicated and costly intervention, the protection afforded by this strategy remains unknown. Vaccination based on fixed times post HCT will likely be sufficient for highly immunogenic vaccines, such as tetanus [13] and polio [14]. For others, such as hepatitis B [14] and pneumococcus [15,16], some studies have suggested that vaccination prior to the acquisition of critical populations of T and B cells may hinder the response. Disease-free survival following HCT continues to improve [17–22], resulting in increased numbers of survivors returning to school and the job force. The development of more tolerable cytoreductive regimens and better supportive care has increased the age of surviving patients, individuals at particular risk of severe, potentially vaccine preventable

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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diseases [reviewed in [4,23,24]), such as pneumococcus [25–29], influenza [30,31], and primary or secondary varicella [32–34]. Over the last decade, outbreaks of measles [35,36], mumps [37], pertussis [38], and varicella [39] have occurred in immunocompetent individuals in the United States and elsewhere, emphasizing the vulnerability of inadequately vaccinated survivors of autologous and allogeneic HCT. This chapter will review current literature on the vaccination of patients following autologous and allogeneic HCT, new vaccines approved since the CDC and EBMT guidelines were published, and potential avenues of research to improve vaccine efficacy in this growing vulnerable patient population.

Loss of protective titers following HCT In the absence of revaccination, multiple studies have demonstrated that pretransplant antibody titers wane with time after HCT (reviewed in [3,4]). Although there is some variability in the time to lose protective titers among different transplant groups, the majority of patients lost protective titers against pneumococcus [40], H. influenzae [41], and tetanus [13] by 0.5–1 years, 1 year, and 1–2 years, respectively. Although approximately 50% of patients will have positive titers against measles, mumps, and rubella [42,43], and polio [42], for at least 2 years post HCT, almost all will be seronegative by 3–5 years post HCT [42,43]. Horwitz et al. demonstrated that, by 12–15 months following autologous peripheral blood HCT, only 27%, 32%, and 55% of patients with nonHodgkin’s lymphoma given adjuvant rituximab retained protective titers against H. influenzae, pneumococcus, and tetanus, respectively [10]. Moskowitz and colleagues analyzed the pre- and post-vaccine titers of 110 adult survivors of autologous peripheral blood HCT performed at Memorial Sloan Kettering Cancer Center, the majority for the treatment of Hodgkin’s (n = 59) or non-Hodgkin’s (n = 58) lymphoma [12]. Thirty-two patients with non-Hodgkin’s lymphoma received post-HCT rituximab. The median patient age was 37 years and 41 years at HCT and vaccination, respectively. At a median of 25.2 months post peripheral blood HCT, 90%, 58%, 40%, 23%, and 16% of patients lacked protective titers against pneumococcus, H. influenzae, measles, tetanus, and polio, respectively [12]. Vaccine-preventable diseases, including but not limited to pneumococcus [25–29], influenza [30,31], and varicella zoster virus (VZV) infection [32–34], remain a significant cause of morbidity, rehospitalization, and mortality late after HCT. Prospective data from the Toronto Invasive Bacterial Diseases Network collected from 1995–2005 demonstrated that the incidence of invasive pneumococcal infection was 590 and 199 per 100,000 adult allogeneic and autologous transplant recipi-

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Table 108.1 Summary of Centers for Disease Control (CDC) and European Group for Blood and Marrow Transplantation (EBMT) post-vaccine guidelines Vaccine

CDC

EBMT

Td, inactive polio virus, Haemophilus influenzae B

2 doses Started at 12 months 12 months, 24 months Optional 24 months No

3 doses Started at 6–12 months 12 months Recommended 24 months No

PPV23 Hepatitis B Measles, mumps, and rubella Varivax

Fig. 108.1 Comparison of B-cell (CD19+) recovery in adults (upper panels) compared with children (lower panels) following unmodified (left) or T-cell-depleted (TCD) unrelated bone marrow transplantation (BMT). The rectangles show the 10–90th percentiles of normal controls. Circles emphasize the differences in patient groups at 6–9 and 9–12 months post HCT, intervals during which vaccination is started in the European Group for Blood and Marrow Transplantation and Centers for Disease Control guidelines, respectively. HCT, hematopoietic cell transplantation.

CD 19+ cells/μL

10000 1000

1000

100

100

10

10

Adults, TCD BMT, n = 64

1

1 0–2 m

2–4 m

4–6 m

6–9 m 9–12 m

10000

0–2 m 2–4 m 4–6 m 6–9 m 9–12 m 10000

Children, cBMT, ablative, n = 22

Children, TCD BMT, n = 64

1000

1000

100

100

480 10

10

1

1 0–2 m

2–4 m

4–6 m

6–9 m 9–12 m

0–2 m 2–4 m 4–6 m 6–9 m 9–12 m

Months post HCT

10000

100

Children <18 years, n = 84

Adults: >18 years, n = 90

80 1000

% Normal

CD4+ cells/μL

10000

Adults, cBMT, unrelated, n = 36

100

10

Children, n = 22

Adults, n = 38

0–2 m 2–4 m 4–6 m 6–9 m 9–12 m 0–2 m 2–4 m 4–6 m 6–9 m 9–12 m

Months post unrelated unmodified BMT

Fig. 108.2 Comparison of the 10th, 50th, and 90th percentiles of CD4 cell count/μL in child and adult recipients of an unrelated unmodified bone marrow transplantation (BMT). The horizontal line indicates 200 cells/μL. Circles emphasize the differences in patient groups at 6–9 and 9–12 months post HCT, intervals during which vaccination is started in the European Group for Blood and Marrow Transplantation and Centers for Disease Control guidelines, respectively.

ents, respectively, compared with 11.5 per 100,000 persons per year in the general population (p < 0.0001) [44]. In a study by Youssef et al., six of 47 patients who developed invasive pneumococcal infection died, only one of whom had had chronic GVHD [28]. Studies have shown that influenza affects up to 25% of patients following allogeneic HCT. Machado and colleagues reported that 30 of 118 consecutive allogeneic HCT recipients developed an upper or lower respiratory infection due to influenza [30]. Influenza occurred in two of 19 vaccinated patients compared with 12 of 24 individuals not vaccinated (p < 0.05). In a study by Martirano et al. [30], the estimated 2-year

60 40 20 0 0–6 m

6–12 m 12–18 m 18–24 m 0–6 m

6–12 m 12–18 m 18–24 m

Months post BMT

Fig. 108.3 Demonstration of the percentage of children and adults with a normal phytohemagglutinin response (within the 10th percentile of normal in children and adults who received T-cell-depleted (TCD; 䊉) or T-cell-replete (䉱) unrelated bone marrow transplantation (BMT).

incidence of influenza was 13.1% (confidence interval [CI] 7.1–19.1%) in allogeneic HCT recipients compared with 6.1% (CI 2.6–9.6%) in autologous HCT recipients (p = 0.02). Most studies report a 30–40% incidence of reactivation of VZV following HCT [32,33]. Koc et al. retrospectively analyzed the incidence of herpes zoster in 100 consecutive allogeneic HCT recipients, demonstrating that 41% of adult patients developed VZV infection at a median of 227 (range 45–346) days post transplantation [31]. Forty percent of patients required admission to the hospital, with a mean stay of 7.2 days. Post-herpetic neuralgia and peripheral neuropathy developed in 68% of patients. In children, Kawasaki et al. reported VZV infection in 33% of 107 pediatric transplant recipients, with a median onset of 96 days [33].

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Although the current guidelines do not recommend the live attenuated varicella vaccine, an increasing number of patients coming to transplant have never been exposed to wild-type varicella and, if vaccinated prior to HCT, will lose seroprotection, as do healthy children. Current recommendations even in healthy children are to administer two varicella vaccines in an effort to prolong immunity [45]. The live attenuated varicella vaccine [46] has been safely given to children with acute lymphoblastic leukemia in remission as well as solid organ transplant recipients [47], despite the need for continued immunosuppression in the latter group. Vaccination in these populations was not associated with an increase in the incidence of herpes zoster [46,47]. Considering the morbidity of primary varicella and recurrent varicella zoster in older individuals, studies assessing the safety and efficacy of the live attenuated varicella vaccine are warranted, particularly in seronegative children and young adults who have no evidence of GVHD and are no longer on systemic immunosuppression.

Immunogenicity of vaccines post HCT Published studies of vaccine efficacy in HCT recipients have often been limited by a small number of heterogeneous patients, use of inconsistent vaccination schedules, and lack of long-term follow-up. In addition, few studies have evaluated responses in T-cell-depleted recipients or recipients of an alternative donor transplant. Even studies analyzing children and adults following a human leukocyte antigen (HLA)-matched sibling bone marrow transplantation (BMT) have shown conflicting results (reviewed in [3]). In a study by Ljungman et al., 42%, 36%, and 21% of patients immunized with a single inactivated polio virus vaccine (IPV) developed a four-fold rise in titer against serotypes 1, 2, and 3, respectively [48]. Following three IPV doses, 50% of patients responded to all three serotypes [50]. In contrast, Parkkali et al. reported that all patients responded to three IPV whether administered at 6, 8, and 14 months (n = 23) or at 18, 20, and 26 months (n = 22) [49]. At our center, 96% of 219 allogeneic HCT recipients responded to a series of three IPVs when administered following acquisition of minimal milestones of immune competence (CD4 level >200/μl, phytohemagglutinin within the 10th percentile of normal, immunoglobulin G [IgG] >500 mg/dL). The range of responses in these three studies may reflect in part variable levels of immune competence at the time of revaccination [14]. Allogeneic transplant recipients respond well to tetanus immunization [13,50]. Parkkali et al. randomized adult HLA-matched related HCT recipients to early (6, 8, 14 months; n = 23) or late (18, 20, 26 months; n = 21) immunization with tetanus toxoid [50]. One month following each vaccine, the responses were similar between the two groups, indicating that early vaccination is efficacious. In a prospective randomized trial [51], Parkkali and colleagues analyzed the effect of immunizing donors with diphtheria-tetanus, IPV, and H. influenzae B (Hib) 2–10 weeks prior to donation of bone marrow for their adult siblings. Fiftythree patients received bone marrow from an immunized donor and 58 from an unimmunized donor. Patients in both groups were immunized at 3, 6, and 12 months after transplant. Titers were drawn 1 month after each dose at 4, 7, and 13 months post HCT. From 6 months on, patients who received transplants from vaccinated donors had higher diphtheria and Hib titers, although tetanus and polio titers were similar in both groups. At 4, 7, and 13 months post HCT, patients with acute GVHD whose donors were not vaccinated had significantly lower tetanus, Hib, and polio titers than patients whose donors were vaccinated. Patients with chronic GVHD had lower Hib and IPV titers compared with those without chronic GVHD, particularly those whose donors were not vaccinated prior to bone marrow harvest. Lower response to the Hib con-

jugate vaccine in patients with chronic GVHD has not been seen in all studies [52] (reviewed in [3]). It is well known that HCT recipients respond poorly to polysaccharide antigens, rendering them susceptible to severe infections secondary to encapsulated bacteria and poorly protected by pure polysaccharide pneumococcal vaccines (PPVs) [15,52,53]. The dichotomy of response to polysaccharide vaccines compared with protein-conjugated polysaccharide vaccines has been shown in several studies. Barra et al. compared the response of 40 recipients of an unmodified related HCT to those with a pure polysaccharide- or protein-conjugated H. influenzae vaccine [52]. Failure to develop an IgG response was seen in 16 of 20 patients given the unconjugated vaccine compared with nine of 20 given the conjugated variety (p < 0.05). Guinan et al. evaluated the response to the Hib conjugate vaccine compared with pure PPV, Pnu-immune (Lederle laboratory Division, American Cyanamid Co., Pearl River, NY) in 35 HCT patients (21 allogeneic and 14 autologous) [53]. Although 56% of patients developed protective H. influenzae titers following one Hibconjugate vaccine and 80% following the second immunization, only 19% of patients responded to the six measured pneumococcal serotypes contained in Pnu-immune. Response to the PPV was poor whether it was administered as a single dose at 24 months or in a two-dose regimen at 12 and 24 months post HCT. Avanzini et al. studied the response to the pneumococcal polysaccharide PPV23 in 53 pediatric recipients of allogeneic or autologous HCT [16]. Whereas only 20–30% and 50% of patients immunized between 6 months and 1 year, and 1 and 2 years, respectively, mounted an effective antibody response (p < 0.0001), all patients immunized after 2 years post HCT responded. In univariate analysis, the interval between HCT and vaccination, the presence of chronic GVHD, and female sex influenced response rates. In multivariate analysis, only the interval from HCT to vaccination predicted response. This finding suggests a deficiency of critical B-cell and/or antigenpresenting cells necessary to mount a T-cell-independent B-cell response during the first 2 years post HCT. During the first year after HCT, the circulating B cells of patients following autologous or HLA-matched unmodified or T-cell-depleted BMT express an immature phenotype (CD1c+CD5+CD38+CD23+) similar to that of B cells derived from cord blood and young children [6]. These B cells decline within the first 2 years of normal life and within the first 2 years after autologous or allogeneic HCT [6]. The poor response of young children and HCT recipients to pure polysaccharide vaccines such as PPV23 [24] may be due to the predominance of this B-cell population, justifying the use of conjugate vaccines (reviewed in [23,55]) in children as well as adults following HCT. To date, five prospective studies have evaluated the heptavalent protein conjugated pneumococcal vaccine (PCV7) in allogeneic (n = 4) or autologous (n = 1) transplant recipients [56–59]. Molrine et al. [56] evaluated PCV7 in 43 recipients of HLA-matched sibling BMT, including the role of pretransplant immunization of the donor and/or host. All patients were vaccinated at 3, 6, and 12 months post HCT. Immunization of the donor and host with PCV7 immediately prior to transplant was associated with a significant rise in pneumococcal titers following rechallenge at 3 months compared with donor–host pairs in which neither was vaccinated before HCT (p = 0.05). Following the third dose of PCV7, 60% of patients in both groups demonstrated significant pneumococcal titers. Two studies have shown no advantage to pre-HCT immunization of the donor and host with PPV [57,60]. Meisel et al. evaluated PCV7 response in 53 children less than 16 years of age following a related or unrelated HCT [58]. Of the 43 evaluable patients, 74% responded to all seven serotypes following three immunizations with PCV7 initiated 6 months post HCT. Comparison of response in children on or off immu-

Vaccination of Allogeneic and Autologous Hematopoietic Cell Recipients

nosuppressive therapy at the start of vaccination demonstrated that three of seven, compared with 29 of 36 patients, respectively, responded to the vaccine (p = 0.06). Patel and colleagues [59] evaluated the response of 38 pediatric recipients of autologous HCT (n = 8), HLA-matched sibling HCT or unrelated (n = 10) HCT. Recipients of autologous or HLA-matched related HCT were immunized with two monthly doses of PCV7 starting at 15 months. Unrelated HCT recipients received PCV7 starting at 21 months after HCT [59]. Over 80% of these children achieved protection against each of the serotypes contained in the heptavalent pneumococcal conjugate vaccine. In 2005, Antin et al. evaluated PCV7 responses in 61 autologous HCT recipients, as well as the effect of patient vaccination prior to hematopoietic cell harvest. Over 60% of patients who received three pneumococcal conjugate vaccines at 3, 6, and 12 months responded regardless of whether they had been vaccinated prior to harvest [61]. We have analyzed the response of allogeneic HCT recipients to the H. influenzae conjugate vaccine (Hib), the 23-valent pure PPV (Pneumovax) and/or the heptavalent conjugated pneumococcal vaccine (PCV7; Prevnar) ([15] and manuscript in preparation). Ninety percent of 256 patients responded to Hib, similar to other published studies [53–55], Only 24% of 156 patients responded to Pneumovax. Similar to the study of Guinan et al. [53], there was no significant difference in response in patients given Pneumovax 12–24 months or more than 24 months post HCT. In contrast, 98 of 138 patients (71%) responded to a series of PCV7. Response was observed in 90% of children (70 of 78) compared with only 48% of adults (29 of 60) (p < 0.001). There were significant differences between responders and nonresponders in terms of age at transplant (p < 0.001) and age at vaccination (p < 0.001). In addition, there was a marginal difference in response based on history of prior but inactive chronic GVHD status (p = 0.06). Only one of eight patients who failed initial vaccination with Pneumovax responded to a second vaccine. In contrast, 25 of 35 patients who failed Pneumovax subsequently responded to a series of the PCV7 vaccine. Post-transplant immunization with the recombinant hepatitis B vaccine is recommended in the 2005 EBMT guidelines [4] and optional in the CDC guidelines [2]. Nevertheless, this vaccine is mandatory for entry to school in most US states and is required for certain jobs [62]. Machado et al. [63,64] reported a 100% seroconversion rate in five autologous and 45 HLA-matched related HCT recipients immunized at least 1 year after transplant. Despite this excellent initial response, 60% of patients failed to sustain titers for more than 1 year after HCT [64]. In our study of 267 allogeneic transplant recipients immunized with recombinant hepatitis B virus following the acquisition of limited minimal milestones of immune competence, a 64% seroconversion rate was observed [14]. Seventy-three percent of 99 children and 59% of 168 adults responded (p = 0.02). In multivariate analyses, response was adversely affected by age over 18 years (p < 0.01) and history of prior chronic GVHD (p < 0.0001) but not by donor type, use of T-cell depletion or adoptive immunotherapy. Longevity of response was evaluable in 98 of 149 patients who were more than 24 months from their last recombinant hepatitis B virus immunization. Eighty patients remained seropositive 5 years following their last vaccine. The greater proportion of patients with sustained hepatitis B titers in our study, compared with Machado’s study (40%), suggests qualitative and/or quantitative differences in the circulating memory T and/or B cells present at the time of vaccination. Variable kinetics of CD27+IgD+ memory B cells has been seen post allogeneic HCT [65]. Surrogate markers such as these may be instrumental in determining the need and timing of booster immunizations to maintain durable protective titers following vaccination. Outbreaks of measles and mumps have occurred within the last 2 years in the United States [35,36], and receipt of two doses of the measles, mumps and rubella (MMR) vaccine is required for healthy

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children entering school [66]. Although several studies have evaluated the response of unmodified HLA-matched and autologous HCT recipients, there are few data on the immunogenicity of this vaccine in recipients of an unrelated or cord blood transplant. Pauksen et al. compared the rate of seroconversion in autologous and HLA-matched related transplant recipients who were seronegative following transplantation [67]. Seroconversion was observed in 20 of 23 allogeneic HCT patients compared with five of 12 autologous HCT recipients (p < 0.05). In a subsequent study, Ljungman et al. evaluated the response of 20 seronegative patients following a matched sibling BMT, none of whom had active GVHD or were on systemic immunosuppression [43]. Seventy-seven percent of patients developed immunity to measles, 64% to mumps, and 75% to rubella. A study by King and colleagues in 22 children recipients of an HLAmatched sibling transplant showed similar results [68]. Following administration of an MMR vaccine at a median of 48 months post HCT, 68% of children responded to all three immunogens. Response to measles, mumps, and rubella was observed in 77%, 87%, and 91% of children, respectively. During a measles outbreak in Brazil, Machado et al. [69] evaluated the response of 51 patients (41 allogeneic and 10 autologous) vaccinated with MMR 9–18 months following an HLAmatched sibling HCT. Approximately half the group was on immunosuppression, the majority due to extensive chronic GVHD. No patient had a serious adverse event. Seven of nine patients with a negative titer (<100 mIU/mL) seroconverted, and the majority of patients with preexisting titers mounted a threefold response. We have evaluated the response of 113 children to the MMR vaccine following related (n = 60) or unrelated (n = 38) HCT. Overall, 70% of patients developed protective titers against measles following a single MMR, including 42 recipients of an HLA-matched related HCT and 27 recipients of an unrelated HCT (p = not significant). Despite a lack of response in 30% of patients, there were no significant adverse reactions [69]. Healthy siblings and other household contacts of immunodeficient patients, such as transplant recipients, can receive the MMR vaccine [70]. Transplant patients should not be exposed to individuals who have received the live polio vaccine or smallpox vaccine due to the potential for vaccine virus spread and disease. A growing number of transplant recipients have never been exposed to varicella or received the live attenuated varicella vaccine prior to their HCT. Although vaccination with a heat-inactivated varicella vaccine reduced the incidence of varicella zoster in autologous HCT recipients, this vaccine is not available [71]. Two studies have evaluated the live attenuated varicella vaccine in a small number of patients [72,73]. Sauerbrei and colleagues vaccinated 15 children (median age 18 months) following autologous (n = 7) or allogeneic (n = 8) HCT [72]. Eligibility for vaccination was an absolute lymphocyte count of over 1000/μL, an IgG level of more than 500 mg/dL, and a positive skin test to a recall antigen such as Candida. No patient had a serious adverse event. Eight of nine nonimmune patients seroconverted. No vaccinated patient developed varicella or herpes zoster within the 2-year post-immunization vaccination period compared with 26.3% of non-vaccinated patients. Ljungman et al. tested the safety of the live attenuated varicella vaccine in nine pediatric autologous transplant recipients [73]. Although patients were vaccinated early (3–4 months) post HCT, only one patient developed herpes zoster. This patient was treated with oral acyclovir without sequelae. To date, 15 children who received a transplant at this center were vaccinated with the live attenuated varicella vaccine more than 2 years post HCT, the majority by their primary pediatricians. Only one patient developed a rash, which, although disseminated, was transient, crusting over within a day of appearance. T-cell proliferative and B-cell-specific antibody responses were observed in more than 80% of patients. Transplant patients should avoid direct contact with healthy

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siblings who develop a rash following administration of the live attenuated varicella vaccine [70].

Recently approved vaccines In the last 5 years, several vaccines have been approved by the Food and Drug Administration whose use in healthy children and adults has been recommended by the Advisory Committee for Immunization Practices. These include the meningococcal conjugate vaccine (MCV4) [74], the human papillomavirus vaccine [75], the tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis toxoid vaccine approved for adolescents and adults [76], and a live attenuated varicella zoster vaccine, Zostavax [77]. The vaccine to prevent herpes zoster in healthy, immunocompetent individuals aged 60 or more years should not be confused with the older live attenuated varicella vaccine approved for children. Zostavax contains more plaque-forming units than the older attenuated varicella vaccine, and has not been tested in any immunocompromised patient population. A report of a 76-year-old woman receiving chemotherapy for recurrent breast cancer who developed disseminated varicella following administration of the live attenuated varicella zoster vaccine was recently published, emphasizing this risk [78]. The MCV4 vaccine, a protein conjugated meningococcal vaccine, has the potential to induce long-term memory when compared with the pure polysaccharide meningococcal vaccine. Although Parkalli et al. demonstrated that adults, including those with chronic GVHD following allogeneic HCT, are capable of responding to the polysaccharide

meningococcal vaccine, a rapid decrease in antibody titers was observed 6 months after vaccination [79]. In 2004, over 25,000 cases of pertussis were reported in the United States, with up to one in five adults infected with pertussis requiring hospitalization or developing a complication. Trials investigating the adolescent tetanus, diphtheria, and pertussis vaccine in age-appropriate HCT recipients are warranted, as is the use of the recombinant human papillomavirus vaccine, particularly in patients with Fanconi’s anemia who are at a significantly increased risk of serious complications from the human papilloma virus.

Conclusion The data above emphasize that, unlike immunization in healthy individuals, vaccination post HCT does not ensure seroprotection, stressing the need to document pre- and post-vaccine titers to determine response. It also suggests that immunization guidelines based on time interval from HCT, irrespective of immune competence, may not ensure adequate protection against certain vaccine-preventable diseases. The evidence-based rating system supporting the 2000 CDC guidelines for post-HCT vaccination (reviewed in [2]) was level BII and BIII, indicating moderate-tostrong recommendations based primarily on evidence from a limited number of studies, or from the opinions of respected authorities based on clinical experience, descriptive studies, or reports of expert opinions. New guidelines that incorporate recently approved vaccines and prospective trials testing these guidelines are needed in order to effectively protect the growing numbers of transplant survivors.

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8. Storek J, Joseph A, Dawson MA, Douek DC, Storer B, Maloney DG. Factors influencing T-lymphopoiesis after allogeneic hematopoietic cell transplantation. Transplantation 2002; 73: 1154–8. 9. Storek J. Factors influencing B lymphopoiesis after allogeneic hematopoietic cell transplantation. Blood 2001; 98: 489–91. 10. Horwitz SM, Negrin RS, Blume KG et al. Rituximab as adjuvant high-dose therapy and autologous hematopoietic cell transplantation for aggressive non-Hodgkin lymphoma. Blood 2004; 103: 777– 83. 11. Small TN, Kernan NA, Jakubowski A et al. Pilot trial of rituximab for the prevention of Epstein Barr virus B cell lymphoproliferative disorder (EBVLPD) following T cell depleted (TCD) unrelated or HLA-mismatched related hematopoietic stem cell transplantation. Blood 2006; 108: 827a. 12. Small TN, Rice RD, McCullagh E et al. Vaccine efficacy following autologous peripheral blood stem cell transplant for lymphoma. Blood 2007; 603: 186a. 13. Lundgren P, Wiklund-Hammaresten M, Duraj V et al. Response to tetanus toxoid immunization after allogeneic bone marrow transplantation. J Infect Dis 1990; 162: 496–500. 14. Jaffe D, Papadopoulos EB, Young JW et al. Immunogenicity of recombinant hepatitis B vaccine (rHBV) in recipients of unrelated or related allogeneic hematopoietic cell transplantation (HCT). Blood 2006; 108: 2470–5. 15. Pao MK, Papadopoulos EB, Kernan NA et al. Immunogenicity of Haemophilus influenza and pneumococcal vaccines in related and unrelated transplant recipients. Blood 2006; 108: 178a.

16. Avanzini MA, Carra AM, Maccario R et al. Antibody response to pneumococcal vaccine in children receiving bone marrow transplantation. J Clin Immunol 1995; 15: 137–44. 17. Alyea EP, Weller E, Fisher DC et al. Comparable outcome with T-cell-depleted unrelated-donor versus related-donor allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 2002; 8: 601–7. 18. Jakubowski AA, Small TN, Young JW et al. T-cell depleted stem cell transplantation for adults with hematologic malignancies: sustained engraftment of HLA-matched related donor grafts without the use of anti-thymocyte globulin. Blood 2007; 110: 4552–9. 19. de Lima M, Couriel D, Thall PF et al. Once-daily intravenous busulfan and fludarabine: clinical and pharmacokinetic results of a myeloablative, reduced-toxicity conditioning regimen for allogeneic stem cell transplantation in AML and MDS. Blood 2004; 104: 857–64. 20. Escalón MP, Champlin RE, Saliba RM et al. Nonmyeloablative allogeneic hematopoietic transplantation: a promising salvage therapy for patients with non-Hodgkin’s lymphoma whose disease has failed a prior autologous transplantation. J Clin Oncol 2004; 22: 2419–23. 21. Appelbaum F. Hematopoietic cell transplantation from unrelated donors for treatment of patients with acute myeloid leukemia in first complete remission. Best Pract Res Clin Haematol 2007; 20: 67–75. 22. Oehler VG, Gooley T, Snyder DS. The effects of imatinib mesylate treatment before allogeneic transplantation for chronic myeloid leukemia. Blood 2007; 109: 1782–9.

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40. Giebeck GS, Warkentin PI. Ramsay NK, Kersey JH. Titers of antibody to pneumococci in allogeneic bone marrow transplant recipients before and after vaccination with pneumococcal vaccine. J Infect Dis 1986; 154: 590–6. 41. Parkkali T, Ruutu T, Stevik M et al. Loss of protective immunity to polio, diphtheria and Haemophilus influenzae type b after allogeneic bone marrow transplantation. APMIS 1996; 104: 383–8. 42. Ljungman P, Lewensohn-Fuchs I, Hammarstrom V et al. Long-term immunity to measles, mumps, and rubella after allogeneic bone marrow transplantation. Blood 1994; 84: 657–63. 43. Ljungman P, Fridell E, Lonnqvist B et al. Efficacy and safety of vaccination of marrow transplant recipients with a live attenuated measles, mumps, and rubella vaccine. J Infect Dis 1989; 159: 610– 15. 44. Kumar D, Humar A, Plevneshi A et al. Invasive pneumococcal disease in adult hematopoietic stem cell transplant recipients: a decade of prospective population-based surveillance. Bone Marrow Transplant 2008; 41: 743–7. 45. Committee on Infectious Disease. Prevention of varicella: recommendations for use of varicella vaccines in children, including a recommendation for a routine 2-dose varicella immunization schedule. Pediatrics 2007; 120: 221–31. 46. Gershon AA, Steinberg SP, Gelb L. Live attenuated varicella vaccine use in immunocompromised children and adults. Pediatrics 1986; 78: 757– 62. 47. Weinberg A, Horslen SP, Kaufman SS et al. Safety and immunogenicity of varicella-zoster virus vaccine in pediatric liver and intestine transplant recipients. Am J Transplant 2006; 6: 565–8. 48. Ljungman P, Duraj V, Magnius L. Response to immunization against polio after allogeneic marrow transplantation. Bone Marrow Transplant 1991; 7: 89–93. 49. Parkkali T, Stenvik M, Ruutu T, Hovi T, Volin L, Ruutu P. Randomized comparison of early and late vaccination with inactivated poliovirus vaccine after allogeneic BMT. Bone Marrow Transplant 1997; 20: 663–8. 50. Parkkali T, Olander R-M, Ruutu T et al. A randomized comparison between early and late vaccination with tetanus toxoid after allogeneic bone marrow transplantation. Bone Marrow Transplant 1997; 19: 933–8. 51. Parkkali T, Kayhty H, Hovi T et al. A randomized study on donor immunization with tetanus–diphtheria, Haemophilus influenzae type b and inactivated poliovirus vaccines to improve the recipient responses to the same vaccines after allogeneic bone marrow transplantation. Bone Marrow Transplant 2007; 39: 179–88. 52. Barra A, Cordonnier C, Preziosi M-P et al. Immunogenicity of Haemophilus influenza type b conjugate vaccine in allogeneic bone marrow recipients. J Infect Dis 1992; 166: 1021–8. 53. Guinan EC, Molrine DC, Antin JH et al. Polysaccharide conjugate vaccine responses in bone marrow transplant patients. Transplantation 1994; 57: 677–84. 54. Parkkali T, Kayhty H, Ruutu T, Volin L, Eskola J, Ruutu P. A comparison of early and late vaccination with Haemophilus influenzae type b conjugate and pneumococcal polysaccharide vaccines after allogeneic BMT. Bone Marrow Transplant 1996; 18: 961–7.

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55. Black SB, Shinefield HR, Hansen J, Elvin L, Laufer D, Malinoski F. Post licensure evaluation of the effectiveness of seven valent pneumococcal conjugate vaccine. Pediatr Infect Dis J 2001; 20: 1105–7. 56. Molrine DC, Antin JH, Guinan EC et al. Donor immunization with pneumococcal conjugate vaccine and early protective antibody responses following allogeneic hematopoietic cell transplantation. Blood 2003; 101: 831–6. 57. Kumar D, Chen MH, Welsh B et al. A randomized, double-blind trial of pneumococcal vaccination in adult allogeneic stem cell transplant donors and recipients. CID 2007; 45: 1576–82. 58. Meisel R, Kuypers L, Dirksen U et al. Pneumococcal conjugate vaccine provides early protective antibody responses in children after related and unrelated allogeneic hematopoietic stem cell transplantation. Blood 2007; 109: 2322–6. 59. Patel SR, Ortin M, Cohen BJ et al. Revaccination with measles, tetanus, poliovirus, Haemophilus influenzae type B, meningococcus C, and pneumococcus vaccines in children after hematopoietic stem cell transplantation. CID 2007; 44: 625– 34. 60. Storek J, Dawson MA, Lim LC. Efficacy of donor vaccination before hematopoietic cell transplantation and recipient vaccination both before and early after transplantation. Bone Marrow Transplant 2004; 33: 337–46. 61. Antin J, Guinan EC, Avigan D et al. Protective antibody responses to pneumococcal conjugate vaccine after autologous hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005; 11: 213–22. 62. Centers for Disease Control and Prevention. Hepatitis B virus: a comprehensive strategy for eliminating transmission in the United States through universal childhood vaccination: recommendations of the Immunization Practices Advisory Committee (ACIP). MMWR Recomm Rep 1991; 40(RR13): 1–25. 63. Machado CM. Reimmunization after bone marrow transplantation-current recommendations and perspectives. Braz J Med Biol Res 2004; 37: 151– 8. 64. Machado CM, Rocha IF, Diomede B et al. Effectiveness of hepatitis B vaccination and persistence of immunity after BMT. The Ninth International Symposium on Infections in the Immunocompromised Host. Assisi, Italy, June 23–26, 1997. 65. Avanzini M, Locatelli F, Santos C et al. B lymphocyte reconstitution after hematopoietic stem cell transplantation: functional immaturity and slow recovery of memory CD27 B cells. Exp Hematol 2005; 33: 480–6. 66. American Academy of Pediatrics. Committee on Infectious Diseases. Age for routine administration of the second dose of measles-mumps-rubella vaccine. Pediatrics 1998; 101: 129–33. 67. Pauksen K, Linde A, Ljungman P et al. Specific T and B cell immunity to measles after allogeneic and autologous bone marrow transplantation. Bone Marrow Transplant 1995; 16: 807–13. 68. King SM, Saunders EF, Petric M, Gold R. Response to measles, mumps and rubella vaccine in paediatric bone marrow transplant recipients. Bone Marrow Transplant 1996; 17: 633–6. 69. Machado CM, de Sousa VAUF, Sumita LM et al. Early measles vaccination in bone marrow trans-

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73. Ljungman P, Wang FZ, Nilsson C, Solheim V, Linde A. Vaccination of autologous stem cell transplant recipients with live varicella vaccine: a pilot study. Support Care Cancer 2003; 11: 739– 41. 74. Bilukha OO, Rosenstein N. Prevention and Control of Meningococcal Disease Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 2005; 54: 1–21. 75. Villa LL, Ault KA, Giuliano AR. Immunologic responses following administration of a vaccine targeting human papillomavirus types 6, 11, 16, and 18. Vaccine 2006; 24: 5571–83. 76. Broder KR, Cortese MM, Iskander JK et al. Preventing tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, reduced diph-

theria toxoid and acellular pertussis vaccines recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 2006; 55: 1–34. 77. Kimberlin DW, Whitley RJ. Varicella–zoster vaccine for the prevention of herpes zoster. N Engl J Med 2007; 356: 1338–43. 78. Curtis KK, Connolly MK, Northfelt DW. Live, attenuated varicella zoster vaccination of an immunocompromised patient. J Gen Intern Med 2008; 23: 648–9. 79. Parkkali T, H Käyhty H, Lehtonen H et al. Tetravalent meningococcal polysaccharide vaccine is immunogenic in adult allogeneic BMT recipients. Bone Marrow Transplant 2001; 27: 79– 84.

Section 8 Looking Forward

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

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Ernest Beutler

Hematopoietic Cell Transplantation in the Future

The past 45 years of hematopoietic cell transplantation have been marked by gradually improving results. The advances that have been made are detailed in this volume. Rather than transplanting everything that is aspirated from the bone marrow, we now try to isolate the cell types that are needed for optimal results. Better antibiotics are available now than 45 years ago, although the ability of microorganisms to adapt to what we design has somewhat blunted the advantage. Antiviral agents to suppress cytomegalovirus infections are available where there were none before. Cytokines have appeared on the scene, allowing recovery of the marrow to advance a few precious and sometimes critical days. Ever-improving immunosuppressive drugs help to stay the attack of allogeneic lymphocytes on their new host, but graft-versus-host disease is still very much with us. Autologous transplantation, a modality that initially did not seem to make sense in the treatment of leukemia, has shown itself to be useful in some circumstances, particularly when allogeneic donors have not been available. Reduced-intensity regimens afford the benefits of stem cell transplantation to many patients who could not have tolerated a standard allogeneic transplant. Yet, that progress has not been nearly as rapid as some of us had hoped 45 years ago, or as we had predicted over the years. The mortality rate in the patient transplanted with allogeneic marrow has decreased modestly, and the cure rate for acute leukemia is only slightly higher now than it was then. It is always hazardous to predict the future in science. In a very real sense, if we knew the future, it would be the present. But in the future, things will have changed. That is certain. Often medical progress arises from chance observations or from tedious clinical trials designed empirically to find out the best way to treat patients. There is, of course, usually an underlying hypothesis, and in the fog of incomplete understanding, successful results often arise from a faulty theoretical basis. The fruits of this approach are not to be despised. Witting or unwitting empiricism has given us liver extract for the treatment of pernicious anemia, quinine for malaria, aspirin for fever and for vascular disease, and, indeed, penicillin, for neither Fleming nor Chain had the faintest idea of how the substance elaborated by Penicillium notatum killed microorganisms. Hematopoietic cell transplantation, as it stands today, owes much to the systematic, empirical approach. The yield from empiricism is necessarily low, but the science of marrow transplantation was hemmed in by an incomplete understanding of many

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

basic biologic processes, particularly those of immune recognition and the regulation of cell growth and differentiation. Our ignorance of these areas persists but is gradually yielding to the powerful methods of investigation of gene function and control. It is such understanding that will allow the field to leap forward in the future.

The transplanted cells The amplification of stem cells The proper inoculum in transplantation is a sufficient number of hematopoietic stem cells to allow rapid repopulation of the marrow. Surface markers have been the main means for attempting to identify these primitive cells, and such markers are clearly a crude way of assessing the biologic destiny of the cells that are harvested. In the future, better markers or selection in culture will become available. It should be possible to isolate more specifically from the peripheral blood those cells that are needed. But that will not be enough. It should also be possible to expand these cells so that one becomes 100,000. It is generally believed that this goal can be reached by stimulating surface receptors with the proper sequence and combination of cytokines. Maybe so, and perhaps all that is required is one new factor that will solve the problem. On the other hand, stem cells have not evolved to actively self-renew, and very probably they lack the receptors needed to trigger the rapid replication that is desired. Instead, a better understanding of transcriptional and translational regulation within cells will provide a way to stimulate division without triggering differentiation. In the future, we will have learned what gene(s) must be activated and which must be repressed to force stem cells into division without maturation, and stem cells harvested from patients will be transduced with genes that accomplish this task. The powerful regulatory properties of networks of RNA molecules will be clarified, and harnessed to aid in the proliferation process. Of course, the action of genes that direct multipotential stem cells to proliferate would have to be stopped after the cells had been amplified to the desired number, otherwise the recipient would be endowed with the dubious blessing of a continuously proliferating stem cell population, a situation uncomfortably like that of acute leukemia. Thus, a mechanism must be engineered into the cells that stops proliferation and sets the stage for amplification, a mechanism that can be turned off and on at will by the administration of an appropriate drug. In the 20th and 21st centuries, techniques for tissue-specific knockouts [1] and for druginducible transgenes [2,3] had already been developed. The ability to downregulate genes with short interfering RNA (siRNA) provides a

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Chapter 109 Promoter tetr-VP16 transcription activator fusion

fusion protein

(a)

tet op

Stem cell proliferin gene

(b)

make certain that the universal cell line was not antigenic (see the section on the recipient immune system, below). Once these goals have been achieved, they would represent a quantum leap in the cost, convenience, and safety of stem cell transplantation. No longer would there be a search for a matching donor. It would no longer be necessary to process individually cells harvested for each recipient. Instead, a commercially available source of compatible, molecularly engineered cells could serve as the inoculum used to repopulate the marrow.

The transplant recipient = Fusion protein

= Tetracycline

= Tetracycline/fusion protein complex

Fig. 109.1 The stem cell proliferin gene is cloned. Transcription of this gene causes stem cells to divide into two stem cells every 2–4 hours. Normal human stem cells have been transduced with this gene fused to the bacterial tet operon (tet op) (b) and with a constitutively active promoter driving a fusion gene consisting of tetr+, a tet activator, the activating domain of the herpes simplex virus protein VP16(A). The conformation of the activator is changed when it binds tetracycline (tet), so that it binds to the tet op tetr. Binding to the operon results in transcription of the stem cell proliferin gene. The fusion protein is constantly produced, but its conformation in the absence of tetracycline is such that it cannot bind to the operator and activate transcription of the gene. When tetracycline is given, the VP16 transcription activator drives the stem cell proliferin gene and causes amplification of the stem cells.

further means for manipulating the expression of genes in stem cells. In a few years, such mechanisms may be employed to give stem cells desired properties for a limited period of time (Fig. 109.1). Later this century, the direction of differentiation may be manipulated in a beneficial way. Give drug A to a patient who is thrombocytopenic after transplantation and induce those genes that direct differentiation towards platelets. Has the patient developed an infection with excessive consumption of granulocytes? No problem. Drug B diverts differentiation toward granulocytes because a drug-B inducible promoter drives the genetic program that diverts differentiation into that direction. The universal donor line At present, each donor must be carefully matched to the recipient, or the recipient’s own cells must be used. Often a matched donor cannot be found, and autotransplantation is limited in its potential as a treatment for aplastic anemia (where the needed cells may be absent), or for acute leukemia (where many or all of the cells may be malignant). Even when a matched donor is found, the approach envisioned for amplification of the cells may be difficult and excessively costly to perform each time a transplant is done. How much better it would be if there could be a universal donor cell line, neatly packaged and stabilized in sealed vials and distributed by the pharmaceutical industry. Implementation of this technology depends upon an understanding of how the body distinguishes self from nonself. If the line is sufficiently primitive, it considers the recipient to be “self” because it will be educated in the patient’s thymus in the same way that the patient’s own cells are educated. Conversely, the recipient’s immune system must not reject the inoculated cells. This might be accomplished by providing the patient with an immunomodulatory treatment, based on the advanced understanding in the future of the steps that recognize nonself and trigger the immune response, or using such knowledge to

Currently, preparation of the patient for allogeneic transplantation has been a major cause of morbidity and mortality. Cytoreduction or cytoablation has been necessary for three reasons: (1) to provide numerical superiority of the transplanted cells over the endogenous cells; (2) to compromise the immune system of the recipient to allow the graft to be accepted, even though it is immunologically foreign; and (3) to destroy tumor cells when a malignancy is being treated. Numerical superiority of transplanted cells Modifying the transplanted cells in such a way that they have a proliferative advantage over those of the host could be an effective strategy for replacing host cells with donor cells. This could be achieved by implementing the in vivo expansion technique that is envisioned as a means of allowing the inoculum of donor cells to be a small one. Critical to the success of this type of genetic manipulation is, as noted above, the ability to stop proliferation of the cells. If the transplanted cells enjoy enough of a growth advantage, cytoreduction would not be required. The transplanted cells would simply replace those of the host. The recipient immune system A great deal of effort has been expended to understand how the immune system distinguishes self from nonself. Fragments of the mechanism have been unraveled, but there is still no clear understanding of the overall process, and it is this process that is responsible for two of the major barriers encountered in hematopoietic cell transplantation, i.e. immune rejection of the graft and graft-versus-host disease. It seems likely that only a few of the millions of different clones of immunocytes recognize the foreign antigens in the transplanted cells. It is only these cells that need be eliminated. Various strategies have been used to produce immune tolerance. For example, immune stimulation followed by chemotherapy that kills only dividing cells should have a selective effect. But presumably because our understanding is incomplete, specific immune tolerance has not yet been achieved. If it is, cytoreduction may not be necessary for transplantation to succeed and graft-versushost disease would be a thing of the past. The elimination of malignant cells Until now, the most common indication for transplantation has been the treatment of malignant disease. One of the goals of cytoreduction in these circumstances is the elimination of tumor cells. If chemotherapy of tumors becomes so specific to obviate the need for ablation of the marrow, transplantation for such disorders would no longer be necessary (see below). It is possible, however, that it will be the transplant itself that will deliver the lethal blow against the neoplasm. Perhaps the cells that are infused will be engineered in such a way as to be able to distinguish between normal host cells and tumor. In this event, cytoreduction would not be needed, even in patients with malignant disease.

Hematopoietic Cell Transplantation in the Future

Transplantation in 2099: a case report A 12-year-old girl is brought to her pediatrician because of pallor and slight bruising. Her hemoglobin level is 50 g/L, her platelet count 20,000/μL, and her granulocyte count 100/μL. A diagnosis of aplastic anemia is quickly established. The pediatrician calls the pharmacy of the health maintenance organization and orders one vial of PolyStem polyvalent stem cells, which are immediately injected intravenously, providing a dose of 100,000 stem cells engineered to divide but not to differentiate as long as the inducing drug is given. The child is permitted to return home, given TetraP, and is carefully observed for fever and/or bleeding. Daily blood counts are stable. The doubling time of the cells is 6 hours, so that in 4 days the inoculum of stem cells has expanded to 1010 cells. It is now time to stop their proliferation by discontinuing TetraP. This silences the gene that caused proliferation of the infused stem cells. Normal differentiation is a consequence of this induced genetic change, but since the platelet count has gradually declined to 8000/μL, the physician decides to give a small dose of Thromboproliferin, which induces the gene(s) that have been engineered into the stem cells to cause inducible differentiation into megakaryocytes. The platelet count rises rapidly and within 3 days is 50,000/μL. The hemoglobin and granulocyte counts rise as well. Only a small dose of Thromboproliferin has been used, so that some differentiation into the erythroid and myeloid lines occurs as well. Adjusted for inflation, the total cost of the procedure has been $30,000. Twenty thousand dollars of this was for the engineered polyvalent stem cells (PolyStem). Notably, in the previous week, the widely watched television program 60 Moments had featured an exposé of the profiteering by the manufacturer of these cells. The reporter had calculated that the cost of producing one vial of these self-proliferating cells was only $20, including culture medium, vial, and labor. The fact that it had required an investment of $2,000,000,000 and 10 years of repeated, frustrating failures to develop the product had conveniently been forgotten. Also forgotten was the fact that three other biotechnology firms – BMT Inc., Cells Unlimited, Inc., and HemoStem, Inc. – had filed for bankruptcy in August 2078, December 2082, and June 2084, respectively, when they failed in their attempts to develop a universal donor cell line.

The demise of transplantation Some day, perhaps in the 21st century or perhaps in the 22nd, hematopoietic transplantation will no longer be performed. This will happen for one of two reasons, which I designate “The Bad Scenario” and “The Good Scenario.” The bad scenario It is 2037, and virtually everyone in America has prepaid health care. The American people have gradually been led to believe that state-ofthe-art health care can be delivered at a relatively low price because of the managerial talents of highly paid executives in the health-care industry. Physicians, on the other hand, find themselves caught between their employers, the highly profitable health-care delivery industry, on the one hand, and their patients and their attorneys on the other. Law suits proliferate. Physicians and their health maintenance organizations are inundated by litigation arising from the failure to make diagnoses because expensive tests have not been ordered, or from failure to give costly treatments to patients with far-advanced neoplasms. It finally becomes apparent, even to the lawmakers in Washington, that the cost of cuttingedge care for everyone, regardless of age and regardless of diagnosis, is beyond the gross national product. Physicians, caught between their

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employers and the demands of their patients, go on strike. They insist that there must be guidelines that they can follow that will protect them from attack by the legal profession. By this time, the cost of hematopoietic cell transplantation has begun to increase, after a period of decreasing costs. This increase is largely due to stringent regulations by governmental agencies. Several unfortunate episodes have led to even greater regulation. Technicians who prepare stem cells are required to undergo 3 years of special training and to obtain a license. Extensive records are required in the preparation of stem cells, and all stem cell harvesting facilities must undergo inspection every 3 months, at their own expense. Even if all this is overcome and patients are transplanted, it has become impossible to perform controlled trials. In the 21st century, members of Congress are even less well educated in science than they were in the 20th century. They are frantic about the ethical implications of infusing stem cells into a patient. The distinction between embryonic stem cells and the stem cells that come from the marrow of an adult donor seems to have been lost on them. In any case, stringent regulations regarding the use of stem cells apply to hematopoietic stem cell transplantation as well as to all other forms of stem cell transfer. All protocols must be approved by a committee of bioethicists that meets in Washington three times annually. But there is more. Because of concern that full disclosure be given to each patient entering a protocol, it is mandated that no patient is entered into a protocol for 3 weeks after the initial explanation has been given to the patient. During this time, an interview with a biomedical ethicist is required to make certain that the patient understands, in full, all of the risks and all of the alternative methods of management. Unfortunately, such a delay is impossible in the context of a rapidly relapsing patient with acute leukemia, and thus some of the most pressing problems facing the transplanter cannot be studied. Finally, strict guidelines for age and ethnic balance of all participants in a study are enforced. For example, if an insufficient number of pediatric Native American patients have not been entered into a protocol, all further accession must be stopped until the mandated balance is achieved. Adherence to all of these rules is monitored by inspectors who closely follow the conduct of all approved protocols. As it turns out, the inspectors have little to do. There are so many barriers to the performance of a good clinical study that evidencebased medicine has all but disappeared, and most medicine, hematopoietic stem cell transplantation included, is practiced according to published guidelines based on the impressions of the experts who serve on the committees that create guidelines. Health economists are enlisted by government to calculate cost–benefit ratios for the whole range of therapeutic procedures. Hematopoietic cell transplantation does not do well in this analysis. Part of the reason is that clinicians who perform transplantation have mixed high-risk experimental procedures with those that are clearly beneficial, but the high cost of transplantation is another reason. The economists find that, in terms of the number of quality-adjusted life-years saved, the treatment of Hodgkin’s disease, diabetes mellitus, and clubfoot rank far above that of hematopoietic cell transplantation. Hematopoietic cell transplantation is no longer reimbursable. It is a research procedure. Unfortunately, the National Institutes of Health (NIH) has its problems, too. In the latter part of the 20th century, the average successful bioscientist trained 100 postdoctoral fellows. Although a friendly Congress increased funding by 3% per year, some of which was eaten by inflation, most of these trainees cannot be accommodated by the system. In a partly politically driven policy, the NIH decides that no program should have more than $200,000 per year support. This egalitarian policy provides employment for many of the trainees produced. Unfortunately, it prevents funding of any meaningful stem cell transplantation program. In the absence of resources, hematopoietic cell transplantation dies!

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Chapter 109

The good scenario The American people and their representatives in Congress begin to realize that cutting-edge medical care has a high cost. They realize that corporate profits and high executive salaries contribute little or nothing to the health-care system. A single-payer system is initiated, but it is clear that national resources cannot provide everything for everyone. Priorities must be set. And they are. Stem cell transplantation is approved for well-documented indications. Congress realizes that progress requires an investment of resources, and a 1% tax for clinical research is established. This greatly increases the NIH budget. In the meanwhile, training programs have been greatly curtailed, so that only the most talented and most motivated qualify. Of course, more scientists are still trained than are needed, but the excess is 20% rather than 1000% as in the latter part of the 20th century. There are, of course, more demands for funds than resources, but merit is the only consideration. Experimental programs in hematopoietic cell transplantation are of high quality, and they are supported by research grants, not misappropriated clinical funds. The public and Congress have come to recognize that the motivations of the vast majority of clinical investigators are to help patients, and that the ethical standards of all but a very few investigators are very high.

Biomedical ethicists, still on the scene, begin to recognize this, and encourage the conduct of well-designed studies. They understand that impeding such studies by concerns of things that might happen, and never have, is injurious to the common good. Indeed, they begin to emphasize the fact that not carrying out human research is unethical, because it harms those who may become ill in the future. Hematopoietic cell transplantation thrives, as do other aspects of clinical research. Sooner or later, all of the diseases that have been treated by hematopoietic cell transplantation – malignancies, genetic diseases, aplastic anemia – are understood at a molecular level. Effective treatments are devised. Hematopoietic cell transplantation becomes an anachronism. Surprisingly soon, medical students view it with amusement and a sense of superiority, similar to our disdain of bleeding and purging in the treatment of the ailments of mankind. Hematopoietic cell transplantation is no more.

Acknowledgment This is manuscript 18837-MEM from The Scripps Research Institute. Supported by the Stein Endowment Fund.

References 1. Camilli A, Beattie DT, Mekalanos JJ. Use of genetic recombination as a reporter of gene expression. Proc Natl Acad Sci U S A 1994; 91: 2634–8. 2. Efrat S, Fusco-DeMane D, Lemberg H, al Emran O,

Wang X. Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Proc Natl Acad Sci U S A 1995; 92: 3576–80.

3. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 1992; 89: 5547–51.

Index

Notes: This index is arranged in letter-by-letter order. Entries in italics refer to figures, those in bold refer to tables. Please also note that as the subject of this book is hematopoietic cell transplantation (HCT), all entries refer to this unless otherwise indicated. To save space in this index, the following abbreviations have been used: ACTH ALL AML APCs BMT CLL CML CMV CNS DCs GVHD GVLE GVTE HDIT HSV JMML MDS mHA MHC MPD MSCs PBHCs PBPCs PNH SOS TBI

adrenocorticotropic hormone acute lymphoblastic leukemia acute myeloid leukemia antigen presenting cells bone marrow transplantation chronic lymphocytic leukemia chronic myeloid leukemia cytomegalovirus central nervous system dendritic cells graft-vs.-host disease graft-vs.-leukemia effect graft-vs.-tumor effect high-dose immunotherapy herpes simplex virus juvenile myelomonocytic leukemia myelodysplastic syndrome minor histocompatibility antigens major histocompatibility complex myeloproliferative disorders mesenchymal stromal cells peripheral blood hematopoietic cells peripheral blood progenitor cells paroxysmal nocturnal hemoglobinuria sinusoidal obstruction syndrome total body irradiation

A abatacept, rheumatoid arthritis, 1023 abdominal pain, 1445–7, 1446 differential diagnosis, 1446 ABO antigens in platelet transfusion, 1229–30 ABO blood type, and GVHD, 1288–9 ABO incompatibility, 1219–20, 1220 and graft failure, 1205 immunohematologic problems, 1220 management, 1220–2, 1221 access studies, 437 accreditation, 536–9, 537, 538, 539 Accreditation Committee Review, 537–8 annual report, 538 application, 537 inspection outcomes, 539

on-site inspection, 537 registration, 537 renewal, 538 significance of, 539–40 Accreditation Committee, 537–8 ACE inhibitors, 1480 acetaminophen, 1575–6, 1644 pharmacology, 1575–6 side effects, 1576 uses, 1576 acetazolamide, drug interactions, 1266 Acinetobacter spp., 1329 Acinetobacter baumannii, 1327 acitretin, GVHD, 1317 acquired autoimmune disease, 16 acquired immune deficiency syndrome see HIV acrolein, toxicity, 1481 ACTH, 1487, 1506, 1506, 1507 actinium-225, 354 activated protein C, 1340–1 acute chest syndrome, 1094 acute inflammatory response, 1164 acute kidney failure, 1474 acute kidney injury, 1473–5, 1474, 1476 epidemiology, 1473 management, 1475 pathogenesis, 1474–5, 1474 hepatic sinusoidal injury, 1475 nephrotoxic medications, 1475 sepsis, 1474–5 risk factors, 1473–4 high-dose conditioning and allogeneic HCT, 1473 and autologous HCT, 1473–4 reduced-intensity conditioning and allogenic HCT, 1473 acute lung injury, 1543 acute lymphoblastic leukemia in adults, 15, 16, 54, 166–7, 791–805 allogeneic transplantation first clinical remission, 795–6, 796, 796 management of relapse, 801 regimen development, 799–800, 800 autologous transplantation, 801 cell sources, 801 CNS prophylaxis, 794 CNS relapse, 794

Thomas' Hematopoietic Cell Transplantation: Stem Cell Transplantation 4th Edition. Edited by F. R. Appelbaum, S. J. Forman, R. S. Negrin and K. G. Blume © 2004 Blackwell Publishing Ltd. ISBN: 978-1-405-15348-5

cytogenetic and molecular genetic analyses, 792–3, 793, 793 disease-free survival, 700 donor leukocyte infusion, 1062–3, 1063 etiology, 791 GVLE, 799 HLA mismatch, 693 immunologic subtypes, 793 B-cell, 792, 793 pre-B/early pre-B, 792, 793 T-cell, 792, 793 leukemic lymphoblasts clonal origin, 791–2 lineage specific features, 792 maintenance therapy, 794 minimal residual disease, 379–80, 379, 795 post-transplant, 380–1, 380 mixed lineage, 792 morphology, 791 Philadelphia chromosome positive, 796–8, 812, 812, 813 treatment, 794–5 treatment outcome, 802 primary refractory, 798 prognostic factors, 795, 795 radioimmunotherapy-based transplant regimens, 800–1 reduced-intensity conditioning, 799 relapse rate after, 1054 relapse, 798, 1054 after treatment, 799, 801 remission, 232 signs and symptoms, 791 survival, 796, 796, 797, 798, 800 treatment, 793–4 Philadelphia chromosome positive, 794–5 strategy, 801–3, 802 umbilical cord blood cell transplantation, 801 unrelated donor, 798–9 acute lymphoblastic leukemia in children, 806–26 alternative donor transplantation mismatched family member transplantation, 815–16 unrelated donor, 816–18, 816, 817, 818 autologous transplantation, 819 B-cell, 806, 807 classification, 806–7, 807

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Index

conditioning regimens, 814–15, 815 diagnosis, 806–7 Down’s syndrome, 820 indications for transplantation, 813 infants, 819–20 Philadelphia chromosome positive alternate donor transplantation, 809 survival, 809 relapse, 820 second transplant procedures, 820 sibling donor transplantation, 811, 812 first remission, 808–10, 808, 809 second or subsequent remission, 810, 813–14 survival, 809 T-cell, 806, 807 umbilical cord blood cell transplantation, 818–19 versus adult ALL, 807–8 acute lymphoid leukemia, 677 acute megakaryoblastic leukemia, 1032, 1033 acute myeloblastic leukemia, radioimmunotherapy, 357 acute myeloid leukemia adults see acute myeloid leukemia in adults children see acute myeloid leukemia in children donor leukocyte infusion, 770, 1062, 1062 reduced-intensity conditioning, 1047–8 relapse, 33, 770, 785, 1054 acute myeloid leukemia in adults, 6, 15, 16, 761–74, 1168 acute promyelocytic, 764, 770, 771 C/EBPα mutations, 762 cell of origin, 761 classification, 762, 763 clinical/laboratory presentation, 763 epidemiology, 761 etiology, 761 HLA mismatch, 693 immunophenotype, 761 minimal residual disease, 381 post-transplant, 381–2 molecular pathology and cytogenetics, 761–2 nucleophosmin mutations, 762 pathology, 761 patients over 60, 769–70 patients under 60, 764–813767 allografting, 768–9 autologous transplantation, 767–8 first remission, 765–7, 766 GVHD prophylaxis, 769 preparative regimens, 767 primary induction failure, 764 refractory relapse, 764 second or subsequent remission, 764–5, 764, 765 sibling vs autologous vs unrelated donors, 767 source of hematopoietic stem cells, 767 untreated first relapse, 765 postremission chemotherapy, 763 RAS mutations, 762 recurrent, chemotherapy, 763 recurrent cytogenetic abnormalities core binding factor translocations, 762 mixed-lineage leukemia mutations, 762 mutations of 5q, 7q and 20q, 762 relapse, 33, 770, 785, 1054 remission, 232 induction of, 763 therapy-related, 1638–45 alkylating agents, 1639 clinical diagnosis, 1639–40 clinicopathologic syndromes, 1638 cumulative probability, 1639 pathogenesis, 1641–3, 1641

gene expression profiling, 1643 genetic instability, 1642 genetic lesions, 1641 hematopoietic abnormalities, 1642–3 polymorphisms in drug-metabolizing enzymes, 1641–2 patient outcome, 1643–4 radiation-related, 1639 risk factors, 1639, 1640–1, 1640 risk prediction, 1644–5 clonality analysis, 1644 cytogenetics and FISH, 1644 loss of heterozygosity analysis, 1644–5 PCR for point mutations, 1644–5 risk reduction, 1645 topoisomerase II inhibitors, 1639 tyrosine kinase receptor mutations, 762 unrelated donors, 676–7 acute myeloid leukemia in children, 775–90 adoptive immunotherapy, 785–6, 786 alternative donor HCT, 783, 783 bone marrow, 783–4, 784 chemotherapy alone, 782 clinical description, 776 epidemiology, 775 etiology, 775 GVHD, 781–2 GVLE, 781–2 haploidentical family donors, 785 historical background, 776, 776, 777, 777, 778 late effects of HCT, 786 minimal residual disease, 782 molecular and cellular biology, 775–6 preparatory regimen, 779–81 relapse, 785 second high-dose transplant procedure, 785 survival estimates, 776 post-remission, 781 treatment options beyond CR1, 782–3 early, 777, 779, 779, 780, 781 results, 778 umbilical cord blood transplantation, 784–5 acute promyelocytic leukemia, 381, 764, 771 acute renal failure, 1545 acute respiratory distress syndrome, 1540 acyclovir, 450 CMV prophylaxis, 1377 treatment, 1373 drug interactions, 1531 Epstein-Barr virus, 1414 herpes zoster, 1400–1 HSV infection, 1382, 1383, 1385 prophylaxis, 1385, 1598 toxicity, 1401, 1661 varicella zoster virus, 1399, 1400 prophylaxis, 1402–3, 1403 ADA deficiency, 124 adalimumab, rheumatoid arthritis, 1023 ADAMTS13, 1222, 1478 adaptive immunity, 131–2 Addison’s disease, 1506 adefovir, 1435 adeno-associated viruses, 119 adenosine deaminase deficiency, 585 and SCID, 1105, 1106 adenovirus, 119, 1222, 1419–20 clinical manifestations, 1419 diagnosis, 1419–20 diffuse interstitial pneumonia, 1457

epidemiology, 1419 pathogenesis and immunity, 1419 prevention and treatment, 1420 virology, 1419 adherence selection, 104 adhesins, 1325 adhesion molecules, 193 and HSC trafficking, 591 role in T-cell activation, 209–10 adolescents allogeneic PBHCT, 625 see also children adoptive cellular therapy, 228, 240–1 with antigen-specific cells, 241–2 with genetically modified cells, 242 with regulatory T cells, 242 adoptive immunotherapy, 4 AML, 785–6 adoptive NK cell transfer, 171 adrenal dysfunction, 1506–9 Addison’s disease, 1506 Cushing’s syndrome, 1506 evaluation, 1507–9, 1508, 1509 late complications, 1628 management, 1509 adrenal insufficiency, 1544 adrenocorticotropic hormone see ACTH adriamycin AIDS-related lymphoma, 1005 breast cancer, 933, 934, 935 adult T-cell leukemia/lymphoma, 1033–5, 1035 advanced directives, 446, 454–5, 485 adverse events, 549–50, 549, 550 apheresis, 552 bone marrow donation, 549–50 HC donation, 549 PBPC donation, 551–2 see also individual drugs advice against transplantation, 446 aerolysin, 728 AF4, 806 age and development of GVHD, 1288 and graft failure, 1205–6 of transplant candidates, 450 Hodgkin’s lymhoma, 868 see also adolescents; children; older patients aging of HSCs, 41–3, 42 AIDS see HIV AIRE, 223, 271 air embolization, 1251 airway complications, 1625, 1625 AL-amyloidosis, 914–30 clinical presentations gastrointestinal tract, 918–19 heart, 917–18 kidneys, 918 liver, 918 nervous system, 919 respiratory tract, 919 diagnosis, 916–17, 933, 934 epidemiology, 915 free light chains, 914, 915, 916, 920 HCT cell mobilization, 925–6 clinical trials, 924–5 peritransplant management, 926–7 therapeutic experience, 923–4, 923, 924 hemostasis in, 919 pathogenesis, 915–16 physical findings, 916

Index prognosis, 919–20 survival, 924 symptoms, 916, 916 theoretical remedies, 915 therapy new agents, 921–3, 927 principles and endpoints, 920–1, 920, 921 response to, 921, 922 toxicity of, 15 Albers-Schönberg syndrome, 1127 albumin, 1554 albuminuria, 1480 Alcaligenes xylosoxidans, 1329 alcohol, drug interactions, 1266 aldesleukin see interleukin-2 aldosterone, 1500 alefacept, 215 alemtuzumab, 214, 227, 254, 351, 661, 853 clinical use CLL, 897, 901, 905 MDS, 833 GVHD prevention, 214, 1269, 1294 side effects, 1269 Alexander disease, 1138, 1154 alfafetoprotein, germ cell tumors, 948 alkylating agents, 296–300 and ALL, 791 high-dose conditioning, 320–3, 321 therapy-related disease AML, 1639 myelodysplastic syndrome, 1639 see also individual drugs ALL see acute lymphoblastic leukemia alleles, 156 allergic encephalomyelitis, experimental, 103 allogeneic chimerism, 195, 197 allogeneic resistance, 177 allogeneic transplantation, 18–19 age of candidates, 450 AIDS, 1006 ALL first clinical remission, 795–6, 796, 796 management of relapse, 801 regimen development, 799–800, 800 autoimmune diseases, 272 antigen-induced, 277–80 chimerism, 272–3 clinical experience, 1026 outcome, 1017 removal of autoreactivity, 280–2 replacement of host susceptibility genes, 281 role of Tregs, 281–2 spontaneous, 276–7 T-cell depletion, 280–1 timing of, 273 bone marrow cells, 544 chimerism in, 272–3 CLL high-dose conditioning, 906, 907 reduced-intensity conditioning, 906–7, 908, 909 CML, 738–43 cell source, 740–1 cytogenetic relapse, 739 donor type, 740 GVHD prophylaxis, 741 minimal residual disease, 742 patient age, 741 phase of disease, 738–9, 739 preparative regimen, 739–40, 740 reduced-intensity conditioning, 742 relapse, 741–2

treatment, 742–3 survival, 739, 740 time from diagnosis to transplant, 741 CMV infection, 1369 complications, 1473 cryopreserved cells, 640–1 diffuse aggressive lymphoma, 883–4 donors, 544–5 PBHC, 618 related, 544–5 selection, 134–5 unrelated, 545, 545 eligibility criteria, 449 follicular lymphoma, 887–9, 887, 888, 888 GVTE, 233–9 B-cell contributions, 237–8 expression of mHAs, 236 haploidentical and MHC-mismatched HCT, 233–4 human genomic diversity, 238–9, 238, 239 mHA-encoding genes, 235–6, 235, 236 mHA-specific T-cell response, 236–7 MHC-matched HCT, 234 minor histocompatibility antigen, 234 molecular characterization of mHAs, 234–5 T-cell response to monopolymorphic overexpressed antigens, 237 hematopoietic graft types, 273 high-dose conditioning, 326, 1473 history, 1–5 animal experiments, dogs, 1–3 clinical studies, 1–2, 2, 3–4 Hodgkin’s lymphoma, 870–3, 1412 graft-versus-Hodgkin’s effect, 870 GVHD, 871 reduced-intensity conditioning, 870–2, 871 results, 872 as immunotherapy, 958 indications, 16 mantle cell lymphoma, 889–90, 890 MDS, 828, 830–5 children, 834, 834 conditioning regimens, 838–40, 839, 840 high-risk MDS, 830, 833, 833 low-risk MDS, 830, 831, 832 secondary MDS, 834–5, 835 metastatic breast cancer, 941–3, 942 MPD, 836 conditioning regimens, 838–40, 839, 840 multiple myeloma, 851–3 high-dose preparative regimens, 851, 851 reduced-intensity conditioning, 851–3, 852, 853 neuroblastoma, 978 numbers of, 16 PBHCs, 618–30 characteristics and dose, 619–20, 620 in children and adolescents, 625 in diseases other than leukemia, 625 donors, 618 marrow vs mobilized blood, 624, 624, 625 mobilization and collection, 618–19 side-effects and long-term sequelae, 621–2, 621 randomized trials, 622–4 acute GVHD, 623, 623 chronic GVHD, 623, 623 engraftment, 622 immune recovery, 622–3 relapse rates, 623 survival, 624

1679

transplant-related mortality, 623–4 recipients, 622 T-cell-depleted mobilized blood, 625–6 T cells in, 620 unrelated donor transplants, 626 PBPCs, 544 preparative regimens, 273 quality of life, 507 reduced-intensity conditioning, 1473 solid tumors, 959–60 relapse, 1059 donor leukocyte infusions, 1059–63, 1060 withdrawal of immunosuppressive therapy, 1059 solid tumors pediatric, 996 reduced-intensity conditioning, 959–60 results, 960–3, 960–3 alloimmunization, 1221, 1230 prevention of, 1231 alloreactivity, 253, 1257 all-trans-retinoic acid, 762 acute promyelocytic leukemia, 764 alphaherpesviruses, 1382 alprazolam drug interactions antidepressants, 1534 azole antifungals, 1526 alternative cell sources, 482–4 alternative developmental pathways, 47 alternative donor HCT ALL in children, 809 mismatched family member transplantation, 815– 16 AML, 783, 783 Fanconi’s anemia, 1188, 1190, 1191, 1192–3, 1192 multiple myeloma, 853 selection, 700 amantidine, 1427 AMD3100, 119, 548, 594, 1208 PBHC mobilization, 595, 619 American Psychosocial Oncology Society, 529 American Society of Blood and Marrow Transplantation, 534 American Society for Histocompatibility and Immunogenetics, 536 amifostine, germ cell tumors, 956 aminoglycosides, 1326 amiodarone, drug interactions, 1536 AML1, 381, 806 AML-1/RUNX-1, 38 AML see acute myeloid leukemia amniotic fluid, 82 amphotericin B, 1356, 1598 lipid-associated, 1355, 1356, 1357, 1358, 1359 side effects, acute kidney injury, 1475 amphotericin B deoxycholate, 1355, 1357, 1358, 1359 amplicons, 119 amplification of stem cells, 1671–2, 1672 AMSA, high-dose conditioning, 320 amsacrine, AML, 763 amyloid deposition, 845 anagrelide, essential thrombocythemia, 836 anakinra, rheumatoid arthritis, 1023 anaphylaxis, transfusion-related, 1235 anaplastic large cell lymphoma, 879 anastrazole, 932 ancestin see stem cell factor ancestral haplotype, 156 anchor proteins, 728 androgens, Fanconi’s anemia, 1186

1680

Index

anemia, 776, 845–6, 1132, 1624 aplastic see aplastic anemia autoimmune hemolytic, 1222 Fanconi’s see Fanconi’s anemia refractory, 829 with excess blasts, 829, 1643 relapse, 1054 with ringed sideroblasts, 829 anergy, 189–90, 265 induction of, 1269–70 anesthesia risks for donors, 550 angiogenesis, 38 angioimmunoblastic T-cell lymphoma, 1037, 1038 angiopoietin-like proteins, 91 angiotensin converting enzyme inhibitors see ACE inhibitors anidulafungin, 1358, 1359 drug interactions, 1529 animal models, 1126–7 autoimmune diseases, 266, 267, 269 dog, 2–3 reduced-intensity conditioning, 1044–5 Fanconi’s anemia, 1186 graft-versus-solid tumor effects, 959 idiopathic pneumonia syndrome, 1460, 1460 in utero transplantation, 578–9 large animal models, 579–80, 579 murine model, 580–2, 580, 581 large animals, 120–1 expansion of HSCs, 89 mouse, 1 embryonic stem cells, 23–4, 24, 26–7 expansion of HSCs, 89 immunodeficient xenotransplant, 120 in utero transplantation, 580–2, 580, 581 osteopetrosis, 1126 reduced-intensity conditioning, 1043–4 stem cells, 23–4, 24, 120 non-human primates, 3 rabbit, osteopetrosis, 1126 rat, osteopetrosis, 1126 reduced-intensity conditioning dogs, 1044–5 low-dose TBI, 1044 marrow space vs immunosuppression, 1044–5 marrow toxicity of TBI, 1044 MHC-matched grafts, 1044 TBI dose, 1044, 1044 mice, 1043–4 xenogeneic, 89–90 ankylosing spondylitis, 265 annexin II, 1201 anorexia, 1436–7, 1436, 1449 nutritional interventions, 1557 antacids, drug interactions, 1531 Anthony Nolan Registry, 545 anthracyclines, 303 ALL, 793 see also individual drugs anthropometric measurements, 1554 anti-B-cell antibodies, 1414–15 anti-BCMA antibodies, 238 antibiotics prophylactic, 1593 prophylaxis, 1338–9 resistance, 1327–8, 1327 targets, 1326 antibodies monoclonal see monoclonal antibodies natural, 191, 194

radiolabelled, 353 pharmacology, 353 T-cell, 193, 196–8, 197 antibody-dependent cellular cytotoxicity, 163 antibody therapy, 227 neuroblastoma, 979–80 antibody titers post-HCT, 1664–6, 1665 anti-CD3 antibodies, 1268, 1411 anti-CD20 antibodies see rituximab anti-CD25 antibodies, 227 anti-CD33 antibodies, 351, 356 anti-CD37 antibodies, 358 anti-CD45 antibodies, 353 anti-CD52 antibodies see alemtuzumab anticonvulsants, neuropathic pain, 1584 anticytokines, 1269 antidepressants, 521–2 drug interactions, 1533, 1534, 1535 pain relief, 1576–7 neuropathic pain, 1577, 1583–4 antidiuretic hormone, 1500 antifungal agents, 1357–61, 1358, 1359–60 combination therapy, 1360 echinocandins, 1358, 1359 polyenes, 1357, 1359 prophylaxis, 1288, 1354–5, 1356 site of action, 1357 see also individual drugs antifungal vaccines, 1361 anti-GD2, 980 antigen escape, 959 antigen presentation, 253 cell biology, 130, 131 antigen-presenting cells see APCs antigen recognition, 132 antigen-specific effector cells, 241–2 anti-HER-2/nu, 351 antihistamines, drug interactions, 1525, 1527 antimicrobial rinses, 1593, 1594 anti-MUC-1 antibodies, 361 antinuclear antibodies, 1021 antioxidants, Fanconi’s anemia, 1186–7 antiretroviral therapy, 1004 anti-Tac antibody, 1293 anti-T-cell antibodies, 196–8, 197, 661–2 antithymocyte globulin, 1018, 1270 aplastic anemia, 714, 717, 719 CLL, 908 Fanconi’s anemia, 1189 GVHD, 214, 1268–9, 1293, 1317 hemophagocytic lymphohistiocytosis, 1166 MDS, 830, 832 PNH, 728–9 reduced-intensity conditioning, 1047–8 synergistic immunosuppressive effects, 709 anxiolytics, drug interactions, 1533 aorta-gonad-mesonephros region, 65 APACHE II score, 1544 APACHE III score, 1546 APCs, 176, 189, 264, 269, 657 GVHD induction, 257 interaction with T cells, 133 nonprofessional, 253–4 peripheral tolerance induction, 191–2 and T-cell activation, 209–10 see also dendritic cells apheresis, 549, 637, 1228 adverse events, 552 nurse in charge of, 475

aplasia bone marrow, 179, 1060 pure red cell, 1219, 1222 aplastic anemia, 16, 692, 705–24, 728 allogeneic PBHCT, 625 alternative donor transplantation, 715–16 clinical description, 705 definition, 705 effect of etiology on outcome, 714 epidemiology, 705 etiology, 705 influence on outcome, 714 graft rejection, 706, 708–10, 708, 708, 710, 1206 GVHD acute, 711, 711 chronic, 711–12 hepatitis-associated, 714 HLA-identical donor, 706, 707 HLA-non-identical related donors, 716 immunosuppressive therapy, 719–20 interstitial pneumonia, 712–13 late effects gonadal function and fertility, 715 growth and development, 715 long-term survival, 714–15 secondary malignancies, 715 mixed donor-host hematopoietic chimerism, 710 molecular and clinical biology, 705 non-transplant approaches, 717, 719, 719 older patients, 714 pretransplant evaluation, 390 reduced-intensity conditioning, 715 survival post-transplant, 713–14, 713, 714 syngeneic grafts, 706 transplant vs immunosuppression, 719–20 umbilical cord blood cell transplantation, 717 unrelated donor transplantation, 716–17, 717, 718 varicella zoster virus, 1389 apoptosis, 69, 1383 and cell cooling, 634 and GVHD, 184 reduced, in CLL, 897 aromatase inhibitors, 932 arrhythmias, 1544 arsenic trioxide, 854 Artemis mutation, 1105 arterial puncture, 1250 arysulfatase A deficiency see metachromatic leukodystrophy Askin tumor see Ewing’s sarcoma L-asparaginase, ALL, 793 aspartylglucosaminuria, 1139, 1155–6 aspergillosis, 1349–50 invasive, 1541, 1541 Aspergillus spp., 401, 1222, 1349–50, 1560, 1654 immune response to, 1352–4, 1353 Aspergillus flavus, 1349 Aspergillus fumigatus, 53, 1349 Aspergillus nidulans, 1349 Aspergillus niger, 1349 Aspergillus terreus, 1349 aspirin, 1576 essential thrombocythemia, 836 assessment, 462 assisted reproduction, 1195 Association of Online Cancer Resources, 529 astatine-211, 354 astemizole, drug interactions, 1525, 1527 astroglia, 80 Astroglide, 522

Index ataxia telangiectasia, 761, 1648 and ALL, 791 atorvastatin drug interactions azole antifungals, 1526 immunosuppressants, 1530 atrophic candidiasis, 1598 ATRX, 775 audit, 539 auditory learners, 527 autoimmune diseases, 264–85, 1014–29 allogeneic transplantation, 272, 276 antigen-induced disease, 277–80 chimerism, 272–3 removal of autoreactivity, 280–2 replacement of host susceptibility genes, 281 role of Tregs, 281–2 spontaneous disease, 276–7 T-cell depletion, 280–1 animal models, 266, 267, 269 antigen-induced, 277–80 genotype and susceptibility, 277–8 HCT, 278 MHC-mismatched versus matched, 278 reduced-intensity allogeneic, 278–80, 279 autoimmunity post-transplant, 1026 autologous transplantation, 272, 273–6 disease phase, 274 failure, 274–6, 275 gene therapy augmentation, 276 proof of concept, 273–4 clinical experience of transplantation, 1026 collecting and processing of HC graft, 1017–18 family/twin studies, 1015 genes and environment, 266–8, 267 genetic engineering, 271 genetics, 1014–15, 1015 graft types, 273 HDIT regimens, 1018–19 hematologic, late, 1624 HLA association, 265 immune reconstitution after HDIT, 1019, 1019, 1020 immunization and cell transfer models, 270–1 late, 1623–4 mechanisms for, 1014 outcome of HCT for other diseases, 1016–17, 1017 pathogenesis, MHC in, 267 pathology, 266 patient selection, 1017 preclinical models, 1015–16, 1016 preparative regimens, 273 purified HSCs, 277 rationale for transplantation, 272 specific diseases juvenile idiopathic arthritis, 1025–6 multiple sclerosis, 1019–21, 1020, 1023 rheumatoid arthritis, 1023, 1025 systemic lupus erythematosus, 1023 systemic sclerosis, 1021–3, 1023, 1024 spontaneously arising, 269–70, 270 allogeneic HCT, 276–7 as “stem-cell disorders”, 271–2 susceptibility MHC genes, 267 non-MHC genes, 268 timing of transplantation, 273 see also individual diseases autoimmune hemolytic anemia, 1222 autoimmune hepatitis, 1017 autoimmune lymphoproliferative syndrome, 1015

autoimmune polyglandular syndrome type I, 1015 autoimmunity mechanisms of, 215–16, 1014 preclinical models, 1015–16, 1016 autologous back-up, 1209–10 autologous GVHD, 397, 1295 autologous hematopoietic progenitor cell support, 932 autologous PBHCs clinical uses, 592 collection techniques, 592 identification and enumeration, 590 mobilization, 590–605 cell adhesion molecules, 591–2 combination cytokine/chemotherapy-induced, 595–6, 595 cytokine-induced, 593–4, 593, 593, 594 factors affecting yield, 596–7 investigational compounds, 598 mechanisms, 590–1 preclinical studies, 598 proteases, 591 SDF-1/CXCR4, 591 optimal cell yield, 592–3 tumor contamination, 597–8 autologous PBPCs, 223 autologous transplantation, 15, 17–18 age of candidates, 450 ALL adults, 801 children, 819 AML, 765, 767 autoimmune diseases, 272, 273–6 disease phase, 274 failure, 274–6, 275 gene therapy augmentation, 276 proof of concept, 273–4 Burkitt’s lymphoma, 9, 10 carcinoma, 11 CLL, 901, 903 evaluation of purging, 904–5 ex vivo monoclonal antibody graft purging, 904 following induction chemotherapy, 8, 61, 902 high-risk CLL, 904 in vivo purging, 905 minimal residual disease, 905–6, 906 relapsed CLL, 901–2 survival, 909 vs conventional chemotherapy, 904 CML, 743–5 elimination of residual disease, 744–5 historical experience, 743–4 prevention of recurrence, 744–5 CMV infection, 1369–70 complications, 1473–4 definition, 272 diffuse aggressive lymphoma, 880–3, 881, 882 eligibility criteria, 449 follicular lymphoma, 884–7, 884, 885, 886, 887 GVTE, 232–3 history, 8–14 diseases treated by, 10–11 early attempts, 8–9, 9 in vitro treatment, 9–10 peripheral blood-derived cells, 9 Hodgkin’s lymphoma, 8, 9, 10, 861 cell sources, 870 children, 868 elderly patients, 868 HIV-associated disease, 868–9 late events, 869 post-transplant therapy, 864–5

1681

pretransplant “debulking” chemotherapy, 863–4 pretransplant radiation therapy, 864 prognostic factors, 862–3, 863, 863 relapsy, 869–70 results, 861, 861, 862 survival, 865 timing of transplantation, 865–8, 865 immune reconstitution, 223–4 indications, 16 mantle cell lymphoma, 889, 889 MDS, 840 MHC antigen transduction, 192 MPD, 840 multiple myeloma, 10–11, 847–51 cell source, 848 patient eligible for, 847 patient selection, 848 patients not eligible for, 847 preparative regimen, 847–8, 848 purging of myeloma cells, 848–9 single vs tandem, 850, 850 timing, 849–50 vs conventional therapy, 849, 849 numbers of, 16 PBPCs in, 544 PNH, 732 quality of life, 356 relapse, 1065 prevention, 1067–71, 1069, 1071 second HCT after failure, 1065–7, 1066 vaccination of donors, 1067 therapy-related disease AML, 1638–42 lymphoma, 1645–7 myelodysplastic syndrome, 1638–42 solid tumors, 1647–8 transfusion, 1234 automated fluorescent sequencing-based typing, 154 autoreactivity, 1623 triggering factors, 268–9, 269 avascular necrosis, 1631 5-azacytidine, MDS, 830 azathioprine, 1627 GVHD, 1317 oral, 1602 side effects, 1658 SLE, 1023 azithromycin, 1627 azole antifungals drug interactions, 1524–9, 1525–8 antidepressants, 1535 immunosuppressants, 1530, 1531, 1532 see also individual drugs B B220, 120 Babesia spp., transfusion-transmitted, 1239 bacterial contamination of transfusion components, 1236–7, 1237 bacterial infection, 1325–45 antibiotic prophylaxis after engraftment, 1339 during neutropenia, 1338–9 antibiotic resistance, 1327–8 bacterial pathogens, 1325–7, 1326, 1327 Clostridium difficile-associated diarrhea, 1335–6 compromised host defenses, 1328–9, 1328 intravascular catheter-related infections, 1337, 1338 late, 1624 MRSA and CA-MRSA, 1336–7 neutropenia enterocolitis/typhilitis, 1337

1682

Index

oral, 1597–8 prevention, 1337–8 spectrum of infections, 1329–31, 1330, 1330, 1331 treatment strategies, 1331–5 fever following resolution of neutropenia, 1334 first fever during neutropenia, 1331–3, 1332 patient management of neutropenia, 1334–5, 1335 subsequent fever during neutropenia, 1333–4, 1334 vancomycin resistant enterococci, 1335 bacteriocins, 1325, 1326 Baller-Gerold syndrome, 1178 banking of cord blood cells, 483, 536, 560–1 barbiturates, drug interactions, 1527 Bard, Judith, 559 bare lymphocyte syndromes, 1113 Bartonella spp., 1222 basal cell carcinoma, therapy-relatd, 1647 basilic vein catheterization, 1248 basiliximab, 1270, 1294 GVHD prevention, 1269 B cells, 222, 264 Epstein-Barr virus infection, 1411 function post-HCT, 1664, 1665 and GVTE, 237–8 HVG reactions, 177 interaction with MSCs, 107 ontogeny, 223 passenger, 1220 post-transplant lymphoproliferative disorder, 1646 progenitors, 45–6 B-cell ALL adults, 792 children, 806, 807 B-cell lymphoma, remission, 232 B-cell maturation antigen, 238 B-cell tolerance, 191 induction of, 199 BCL2, 39 CLL, 897 BCL2A1, 241 BCMA see B-cell maturation antigen BCNU see carmustine BCR-ABL fusion, 807 BCR-ABL oncogene, 794 BDCA2, 249, 252 BDCA4, 249, 250 beclomethasone GVHD, 1259 oral GVHD, 1601 bellergal, 522 Bell’s palsy, 1654 bendamustine, germ cell tumors, 956 benzodiazepines, drug interactions, 1526, 1527 bet, 118 betamethasone, 103 multiple myeloma, 847 oral GVHD, 1601 bevacizumab, 351, 932 bexarotene, germ cell tumors, 956 bile acid binders, drug interactions, 1531 biliary tract disease, 1436, 1442–3, 1449 binding site barrier, 353 biodistribution, 352, 352 biofilms, 1326 biostatistical methods, 406–27 analysis of recurrent states, 421 clinical trials conduct, 417 design, 409–11, 410, 410

impact of genetic data, 416–17, 418, 418, 419, 423–4 phase 0, 410–11 phase I, 411–13, 412, 412 phase II, 413–14, 413 phase III, 414–16 reporting of results, 424–5, 424 data analysis, 419 database management, 419–20 Data and Safety Monitoring Boards, 417–18 equivalency testing, 420 impact of genetic data, 409 intent-to-treat principle, 419–20 interim analyses, 420 observational study designs, 406–7, 407 prospective cohort studies, 407–8 quality of life and cost-effectiveness studies, 408–9 retrospective case-control studies, 408 study cycle, 406, 407 subgroup analyses, 420 surrogate endpoints, 421 systematic reviews and meta-analysis, 408 time to event analysis, 421–3 competing risks, 423 Cox proportional hazards model, 423 Kaplan-Meier survival analysis, 422 long-rank test, 422–3, 422 multivariate interval-censored survival data, 422 weighted Kaplan-Meier estimator for matched data, 422 Birbeck granules, 248 bismuth-213, 354 bisphosphonates, 854 osteopetrosis, 1132 bisphosphonates, 1631 breast cancer, 932 osteonecrosis, 1603–4 BK nephropathy, 1481, 1482 BK virus, 1425–6 black cohosh, 521 Blackfan-Diamond syndrome, 761 bladder complications, 1480–2, 1631–2 blast crisis, 734 bleeding see hemorrhage bleomycin, AIDS-related lymphoma, 1004, 1005 blocked ricin, 905 blood dendritic cell antigen-2 see BDCA2 blood dendritic cells, 250 chimerism, 255–6 effect of conditioning, 254 blood group incompatibility, 1219–25 blood islands, 65 blood-lymph filtering, 257 Blood and Marrow Transplant Clinical Trials Network, 544 blood transfusion see transfusion Bloom’s syndrome, 585, 586, 761, 1185 and ALL, 791 and AML, 775 and osteosarcoma, 991 blueberry muffin lesions, 970 BMI1, 39, 49, 241 BMT see bone marrow transplantation BMT InfoNet, 531 BMT-Support, 529 bocavirus, 1424–5 body image, 518, 521 body mass index, 1551 body surface area, chemotherapy dose based on, 294 body weight, 450, 1554

bone marrow, 72 biopsy, 1132 donation, 178, 548 adverse events, 549–50, 549, 550, 621 allogeneic transplantation, 544 mortality, 551 see also donation; donors expansion trials, 91–3, 92 growth factor-mobilized, 554 harvesting, nursing duties, 466, 467 host, 179 mobilized, 624, 625 mononuclear cells, 582 in osteopetrosis, 1132 T-cell-depleted, 664 bone marrow ablation alkylating agents, 320–3, 321 busulfan, 317, 319, 321–2 melphalan, 321, 323109317 nitrosoureas, 317, 320, 322–3 high-dose chemotherapy, 319–20, 319, 320 targeted radioisotopes, 326 TBI see total body irradiation bone marrow aplasia, 179, 1060 bone marrow-derived cells, 77–81 AML, 768 ectoderm, 80 endoderm, 79 endothelial cells, 80–1 mesoderm, 77–9 cardiomyocytes, 78–9 skeletal muscle, 77–8 bone marrow-derived stem cells, 73–5 hematopoietic stem cells, 73 mesenchymal stem cells, 72, 74–5 plasticity, 75 Bone Marrow Donors Worldwide, 545 bone marrow protein-4, 24 bone marrow transplantation, 352 AML, 777 nephropathy, 1475 neuroblastoma, 975, 976 SCID, 1111 thalassemia, 1079 T-replete grafts, 659–64 graft failure, 659–61, 659 GVHD, 661–3, 661, 662 survival, 663–4, 663 Bone Marrow Transplant Information Network, 488 bone metabolism dysfunction, 1491–3 evaluation, 1492 management, 1492–3 bone mineral density, 1488, 1492–3 bone morphogenic proteins, 91 bone pain in PBPC donors, 551 bone resorption, 1125 bone-seeking radioisotopes, 326 Borrelia burgdorferi and autoimmune disease, 103 transfusion-transmitted, 1239 bortezomib AL-amyloidosis, 922 multiple myeloma, 847, 854, 855, 1063 bovine serum albumin, as cryoprotectant, 637 Boyse, Edward A., 559 BRCA1, 931 BRCA2, 931, 1184 breast cancer, 16, 479, 931–47 clinical description, 931 epidemiology, 931 etiology, 931

Index HCT, 932–43 allogeneic transplantation, 941–3, 942 purging of progenitor cell grafts, 23 reduced-intensity conditioning, 942 high-dose chemotherapy clinical trials, 932–4, 933, 934, 936, 937 high-risk primary breast cancer, 934–5 patient selection, 938–9, 939 prognostic factors, 940, 941 high-risk primary high dose chemotherapy, 934–5 results, 935, 937, 937 metastatic, 932 molecular and cellular biology, 931 nonmetastatic, 932 nontransplant approaches, 932 Brief COPE, 504 Brief Symptom Inventory-Short Form, 504 bromocriptine mesylate, 522 bromo-deoxyuridine, 77 bronchiolitis obliterans organizing pneumonia, 1459, 1541, 1542, 1627, 1627 bronchiolitis obliterans syndrome, 403, 1466, 1476, 1540, 1542, 1620 late, 1626–7, 1627 bronchoalveolar lavage, 1540 Broviac catheter, 1246 Broxmeyer, Hal E., 559 budesonide, oral GVHD, 1601 buffy coat cells, 2–3, 708 buprion, drug interactions, 1535 bupropion, drug interactions, 1534 Burkholderia cepacia, 1329 Burkitt-like lymphoma, 879 Burkitt’s lymphoma, 792, 879, 891, 1004, 1413, 1415 autologous transplantation, 9, 10 cell purging, 9 Epstein-Barr virus in, 1411 remission, 232 burst-forming units, 590 erythroid, 590 busulfan, 19, 299–300, 755, 1080, 1608 clinical use ALL, 799–800, 815 CLL, 907, 908 CML, 740, 740 Ewing’s sarcoma, 987 Fanconi’s anemia, 1190 Hodgkin’s lymphoma, 873 MDS, 831, 832 melanoma, 965 MPD, 836 neuroblastoma, 974 rhabdomyosarcoma, 989 drug interactions, 1526 HDIT, 1017 high-dose conditioning, 317, 319, 321–2 acute myeloid leukeamia, 779–80 phagocytic disorders, 1165 with TBI, 318 mechanism of action, 299 metabolism, 299–300 pharmacokinetics/pharmacodynamics, 288, 299– 300 reduced-intensity conditioning, 1047–8 side effects, 1660–1 SOS, 1440 structure, 288 therapeutic monitoring, 295 butorphanol, 1580 bystander effect, 354

C C166 cell line, 24 cachexia, 450 cadherins see individual cadherins café-au-lait spots, 751 CAII deficiency syndrome, 1127, 1132 calcineurin inhibitors, 137, 192, 259, 1222 drug interactions, 1529 azole antifungals, 1525, 1526, 1527 grapefruit juice, 1536 immunosuppressants, 1532 fungal infection, 1354 neurologic complications, 1658–60, 1659 see also individual drugs calcium, 1556 calcium channel blockers drug interactions azole antifungals, 1525, 1527 immunosuppressants, 1530, 1531, 1532 calicheamicin, 351, 763 caliciviruses, 1427–8 calorimetry, indirect, 1554 Campath-1G, host-versus-graft reactions, 709 Campath-1H see alemtuzumab CA-MRSA, 1327, 1336–7 Canavan disease, 1139, 1157 cancer stem cells, 53 Cancer and Treatment Distress, 504 Candida spp., 1347–8, 1598 candidemia, 1348 cardiovascular system, 1348–9 dissemination with deep-tissue infection, 1348 esophagitis, 1384 eye and CNS disease, 1349 immune response to, 1351–2 mucocutaneous infection, 1348 skin lesions, 1349 syndromes of infection, 1348 Candida albicans, 1347, 1598, 1654 diarrhea, 1435 Candida dubliniensis, 1348 Candida glabrata, 1348 Candida guillermondii, 1348 Candida inconspicua, 1348 Candida krusei, 1348 Candida lusitaniae, 1348 Candida norvengensis, 1560 Candida parapsilosis, 1348 Candida pseudotropicalis, 1348 Candida tropicalis, 1348 Caplan, Arnold, 102 captopril, thrombotic microangiopathy, 1480 carbamazepine drug interactions, 1266 antidepressants, 1534, 1535 azole antifungals, 1526, 1527 grapefruit juice, 1536 immunosuppressants, 1530 carbapenems, 1326 carbohydrate abnormalities, 1503–6 carbon dioxide therapy in oral GVHD, 1602 carbonic anhydrase isoenzyme II, 1125 carboplatin, 301–2 clinical use breast cancer, 932, 934, 936 diffuse aggressive lymphoma, 881 germ cell tumors, 952 neuroblastoma, 973 rhabdomyosarcoma, 989 high-dose conditioning, 317, 321, 324 metabolism, 301–2

1683

pharmacokinetics/pharmacodynamics, 288, 301–2 renal disease, 294 structure, 288 carboprofen, 1576 carcinoma, autologous transplantation, 11 cardiac complications, 1544 arrhythmias, catheter-induced, 1251 tamponade, catheter-induced, 1251 cardiac conditions, coexisting, 449 cardiomyocytes, 78–9 cardiovascular disease, late, 1629 cardiovascular system, Candida infection, 1348–9 Caregiver OOL-Cancer, 504 caregivers counseling of, 453–4 informal, psychosocial issues, 497–8 professional, psychosocial issues, 498 quality of life, 507 CARES-SF, 508 Caring Connections Program, 529 carmustine, 300, 632, 1608 clinical use breast cancer, 933, 934 diffuse aggressive lymphoma, 881 Hodgkin’s lymphoma, 873 neuroblastoma, 974 non-Hodgkin’s lymphoma, 359 and graft failure, 1206 HDIT, 1018 high-dose conditioning, 317, 320, 322 mechanism of action, 300 metabolism, 300 and PBHC mobilization, 597 pharmacokinetics/pharmacodynamics, 288, 300 reduced-intensity conditioning, 1047–8 side effects neurotoxicity, 1660–1 pneumonitis, 1005, 1458 pulmonary toxicity syndrome, 1542 renal disease, 1476 structure, 288 carpal tunnel syndrome, 916, 919 cartilage-hair dysplasia, 1170 caspofungin, 1358, 1359, 1475 drug interactions, 1529 immunosuppressants, 1530, 1531 cataracts late, 1630 TBI-induced, 340 β-catenin, 39 cathepsin G, 591 cathepsin K, 1125 deficiency, 1126 cathepsin S, 130 catheters care of, 1249 central venous, 465 complications infection, 1253–4, 1254 venous thrombosis, 1252–3, 1253 damage/fracture, 1252, 1252, 1253 entry site bleeding/hematoma, 1250–1 externally tunneled, 1246, 1246 insertion technique, 1248–9, 1249 occlusion, 1250, 1251–2, 1251, 1252 peripherally inserted central, 1246, 1246 pinch-off syndrome, 1247, 1252 removal, 1255 selection, 1245–6 tip malposition, 1250, 1250 see also vascular access

1684

Index

catheter insertion sites, 1247 basilic vein, 1248 cephalic vein, 1248 external jugular vein, 1248 femoral vein, 1248 internal jugular vein, 1247–8, 1247 subclavian vein, 1248 catheter-related infections, 1337, 1338 cavernosal arterial insufficiency, 516 CCAAT/enhancer-binding protein, 50 CCL25, 212 CCR1, 1463 CCR5, 1001, 1009 CD1a marker, 25 CD2 marker, 164, 727, 728 AML, 775 mixed-lineage leukemia, 792 CD3 marker, 104, 120, 132, 222 ALL, 792 AML, 775 antibodies, 227, 1009 mixed-lineage leukemia, 792 CD3+ marker, 97 CD3b marker, 248 CD4 marker, 120, 190, 222, 223, 224, 250, 257, 264, 1001, 1064–5 ALL, 792 post-HCT, 1665 varicella zoster virus, 1394 CD4+ T cells see helper T cells CD4+CD25bright T cells, 1020 CD4+/CD56+ hematodermic neoplasm, 1039 CD4-D1 peptide, 1270 CD5 marker, 120 AML, 775 CD7 marker, 48, 168 AML, 775 mixed-lineage leukemia, 792 CD8 marker, 120, 133, 189, 190, 192, 222, 223, 250, 257 ALL, 792 CD8 T cells see cytotoxic T cells CD8α marker, 249, 250 CD9 marker, 25 CD10 marker, 48, 168 ALL, 792, 807 AML, 775 Ph+ ALL, 794 CD10+ marker, 1227 Ph+ ALL, 794 CD11 marker, LAD 1, 1171 CD11a marker, 164, 1164 CD11b marker, 164, 249, 250, 651, 1164 AML, 775 CD11c marker, 248, 249, 255, 1164 CD13 marker, AML, 761, 775 CD14 marker, 104, 258, 727, 728 AML, 775 CD14+ marker, 97 in mobilized PBHCs, 620 CD15 marker, AML, 775 CD15s marker, AML, 761, 775 CD16 marker, 132, 727, 728 CD18 marker, 1164 LAD 1, 1171 CD19 marker, 104 ALL, 792 AML, 775 CLL, 898 Ph+ ALL, 794 CD19+ marker, 97, 1412

CD20 marker, 357 ALL, 792 AML, 775 antibodies, 803, 1414 CD20+ marker, 1227, 1412 CD21 marker ALL, 792 antibodies, 1414 CD22 marker, 360 ALL, 792 AML, 775 antibodies, 803 CD23 marker, CLL, 899 CD24 marker ALL, 792 AML, 775 antibodies, 1414 CD25 marker, 163, 1014 ALL, 792 CD25+ marker, 223, 242, 651, 1014 in GVHD, 178, 179 CD26 marker, 581, 591 CD28 marker, 133, 189, 192, 210, 264 antibodies, 1009 CD31 marker, 81, 104, 223 CLL, 898 CD33 marker AML, 761, 775 Ph+ ALL, 794 CD33+ marker, 97 CD34 marker, 79, 223, 255, 590, 979, 1208 AML, 761 selection, 214 CD34+ marker, 37, 65, 66, 90, 91, 93, 167, 191, 237, 549, 590, 695, 1208, 1643 bone marrow, 619, 620 and development of GVHD, 1289 infusion for graft failure, 1210 mobilized peripheral blood, 619, 620 CD36 marker, AML, 761, 775 CD38 marker, 383 CLL, 897, 900 CD38- marker, 37, 91 CD38+ marker, 651 CD40 marker, 104, 252, 264 CLL, 898 CD40L marker, 104, 192 CD41 marker, 47 CD44 marker, 581, 591, 1201 ALL, 792 CD45 marker, 104, 248, 249, 356, 651 antibodies, 800 CD45RA marker, 48, 168, 223, 250 CD45RB marker, 211 CD46 marker, 9 CD47 marker, 41, 54 CD48 marker, 37 CD52 marker, 254 antibodies, 803 CD54 marker, 104 CD55 marker, 9, 727, 728 CD56 marker, 132 desmoplastic small round tumor, 994 CD56+ marker, 97 CD56bright, 163–4 CD56dim, 163–4 CD59 marker, 9, 132, 727, 728 CD61 marker, AML, 761, 775 CD62 marker, 591 CD62L adhesion molecule, 178, 211, 223 CD66 marker, leukemia, 357

CD66b marker, 727, 728 CD68 marker, 25 CD73 marker, 104 CD79 marker, ALL, 792 CD80 marker, 104, 210, 264 CD86 marker, 25, 104, 133, 210, 264 CD90 marker, 26 CD94 marker, 165, 223 receptors, 164, 164 CD95 marker, 164 CD96 marker, 54 CD99 marker, desmoplastic small round tumor, 994 CD103 marker, 211 CD105 marker, 47, 104 CD117 marker, 26 AML, 761, 775 CD122 marker, 163, 168 CD123 marker, 251, 255 CD127 marker, 211 CD132 marker, 163 CD133 marker, 26, 94 CD150 marker, 47 CD152 marker, 211 CD154 marker, 133, 192 CD164 marker, 1201 CD166 marker, 104 CD205 marker, 252 CD207 marker, 248 CD244 marker, 37 CDKN2A, 991 CDKN2B, 991 CDKNA2, 991 C/EBPα mutations, 762 celecoxib, 1576 cell culture, 6 cell membrane labels, 77 cell numbers appropriate level, 1209 inadequate, risk of graft failure, 1208–9 cell sources, 17 cellular hypothesis, 1 cellular immunity to CMV, 1370–1 cellular therapy, 1069–70 cytokine-induced killer cells, 1069 cytotoxic T cells, 1069–70 genetic modification of T cells, 1070–1 natural killer cells, 1069 Cellular Therapy Product Standards, 536 Cellular Therapy Standards, 535–6 CENPM, 241 Center for Epidemiologic Studies-Depression, 504 Center for International Blood & Marrow Transplant Research, 488, 531, 1289 Center for International Bone Marrow Transplant Registry, 776, 778 Centers for Disease Control, vaccination guidelines, 1664 central nervous sequence, Candida infection, 1349 central nervous system ALL prophylaxis, 794 relapse, 794 infection, 1655 lymphoma, 880, 891, 1037–9 central tolerance, 193 central venous catheter, 465 complications, 552 cephalic vein catheterization, 1248 cephalosporins, 1326 cerebral angiitis, 1397 cerebrotendinous xanthomatosis, 1139, 1156

Index cerebrovascular complications, 1655, 1657, 1657 ceroid lipofuscin, 1128, 1132 Certain Human Cells, Tissues, and Cellular and Tissue-Based Product Recovered From Donors Who Were Tested For Communicable Diseases Using Pooled Specimens or Diagnostic Tests, 534 cetuximab, 351 cevimeline, 1603 c-fms, 50 CFU see colony-forming units cGTP rule, 534 Chagas’ disease, 546 Charlson Comorbidity Index, 449, 1052, 1053 Chediak-Higashi syndrome, 1164, 1172 chelation therapy, 1077, 1078 chemokines, 568 blockade of, 215 chemokine receptors, 568, 1001 chemotherapy AML, 782 effects on growth, 1610–11, 1611 effects on puberty, 1614–15, 1615 effects on thyroid function, 1609 high-dose, 319–20, 319, 320 high-dose see high-dose chemotherapy Hodgkin’s lymphoma, 860 debulking, 863–4 neuroblastoma, 973 vs autologous BMT, 975, 976 vs HCT, 974–5 see also individual drugs Child Health Ratings Inventory, 510 children ALL, 806–26 alternative donor transplantation mismatched family member transplantation, 815–16 unrelated donor, 816–18, 816, 817, 818 autologous transplantation, 819 classification, 806–7, 807 conditioning regimens, 814–15, 815 TBI, 814 diagnosis, 806–7 Down’s syndrome, 820 indications for transplantation, 813 infants, 819–20 relapse, 820 second transplant procedures, 820 sibling donor transplantation, 811, 812 first remission, 808–10, 808, 809 second or subsequent remission, 810, 813–14 umbilical cord blood cell transplantation, 818–19 versus adult ALL, 807–8 allogeneic PBHCT, 625 AML, 775–90 adoptive immunotherapy, 785–6, 786 alternative donor HCT, 783, 783 bone marrow, 783–4, 784 chemotherapy alone, 782 clinical description, 776 epidemiology, 775 etiology, 775 GVHD, 781–2 GVLE, 781–2 haploidentical family donors, 785 historical background, 776, 776, 777, 777, 778 late effects of HCT, 786 minimal residual disease, 782 molecular and cellular biology, 775–6 preparatory regimen, 779–81

relapse, 785 second high-dose transplant procedure, 785 survival estimates, 776, 781 treatment options beyond CR1, 782–3 early, 777, 779, 779, 780, 781 results, 778 umbilical cord blood, 784–5 as donors, 481, 482, 553–4 informed consent, 452, 480, 553–4 safety, 553 evaluation and counseling, 452–4, 453 advance directives, 454–5 conditions affecting, 452–3 consent to treatment, 452 family counseling, 453–4 initial visit, 452 Lansky Play-Performance Scale, 453 malignant disease, 452 nonmalignant disease, 452 stem-cell source, 454 Hodgkin’s lymphoma, autologous transplantation, 868 MDS, 834–5, 834, 835 quality of life, 507 parents and caregivers, 507 see also pediatric Children’s Oncology Group, 531 neuroblastoma risk classification, 971, 972 Childs-Pugh classification, 295 chimerism, 53, 365–75, 1050 allogeneic, 195, 197, 272–3 blood DC, 255–6 full, 365 and graft failure, 1202 hematological malignancies, 1050–1 in JMML, 757 mixed, 194, 195, 365, 1202 aplastic anemia, 710 natural, 577 reduced-intensity conditioning, 372, 1051 sickle cell disease, 1096–8 split, 365 testing for after reduced-intensity conditioning regimens, 372 clinical use, 369–71, 370, 370 DNA amplification of other loci, 369 historical perspective, 365, 366 molecular cytogenetics, 366 patients at risk of recurring malignancy, 371–2, 371 patients with graft failure, 371 VNTR/STR polymorphisms, 366–9, 367, 368 thalassemia, 1082–3 tissue DC, 256–7, 256 Chlamydia, diffuse interstitial pneumonia, 1457 chlorambucil CLL, 901 and PBHC mobilization, 597 chloramphenicol, 1326 marrow injury, 705 chlorhexidine, 1597 choice of procedure, 444–5 cholangiolar cholestasis, 398 cholestatic disorders, 1442 jaundice, 1290, 1437 cholestyramine, drug interactions, 1531 choline magnesium trisalicylate, 1576 choline salicylate, 1576 chondrocytes, 105

1685

chromatin structure, 50 chromosomal abnormalities, JMML, 752–3 chromosomal assays, minimal residual disease, 376 chronic eosinophilic leukemia, 837 chronic granulomatous disease, 124, 585, 1164, 1172– 4, 1173 chronic idiopathic myelofibrosis, 835, 836–7 Lille (Dupriez) score, 835, 836 Mayo classification, 835, 836 chronic kidney disease, 1474, 1475–8, 1475, 1477, 1478 and albuminuria, 1480 clinical syndromes nephrotic syndrome, 1478 thrombotic microangiopathy, 1476–8 epidemiology, 1474–5, 1474 GVHD-related, 1478–80, 1479 treatment, 1480 chronic lymphoblastic leukemia, remission, 232 chronic lymphocytic leukemia, 16, 897–913 allogeneic transplantation high-dose conditioning, 906, 907 reduced-intensity conditioning, 906–7, 908, 909 antigen stimulation in, 898 autologous transplantation, 901, 903 evaluation of purging, 904–5 ex vivo monoclonal antibody graft purging, 904 following induction chemotherapy, 8, 61, 902 high-risk CLL, 904 in vivo purging, 905 minimal residual disease, 905–6, 906 relapsed CLL, 901–2 survival, 909 vs conventional chemotherapy, 904 characteristics BCL2 overexpression, 897 microRNAs, 897 reduced apoptosis, 897 clinical presentation, diagnosis and staging, 898 etiology, 897, 898 familial, 898 HCT recommendations, 909–10 incidence, 897 microenvironment, 898 minimal residual disease, 383, 900–1 prognostic index for overall survival, 900 prognostic markers, 899 CD38 and ZAP-70, 900 CLL tumor burden, 899 cytogenetic, 899 VH gene mutation status, , 899–900 reduced-intensity conditioning, 1047–8 relapse following reduced-intensity conditioning, 1054 and refractory disease, 901 response criteria, 900 staging systems Binet, 898 Modified Rai, 898 treatment, 901, 901 chronic myelogenous leukemia, 53, 793 chronic myeloid leukemia, 15, 16, 69, 74, 491, 734– 50, 835 accelerated phase, 735 allogeneic transplantation, 738–43 cell source, 740–1 cytogenetic relapse, 739 donor type, 740 GVHD prophylaxis, 741 minimal residual disease, 742 patient age, 741

1686

Index

phase of disease, 738–9, 739 preparative regimen, 739–40, 740 reduced-intensity conditioning, 742 relapse, 741–2 treatment, 742–3 survival, 739, 740 time from diagnosis to transplant, 741 atypical, 838 autologous transplantation, 743–5 elimination of residual disease, 744–5 historical experience, 743–4 prevention of recurrence, 744–5 clinical description, 734–5, 735 donor leukocyte infusion, 1060–92, 1060 epidemiology, 734 graft failure, 1206 incidence, 734 minimal residual disease, 382–3 post-transplant, 383 molecular biology, 734 non-transplant therapies, 735–8 first-line imatinib failure, 737 high-dose imatinib, 737 imatinib, 736, 738 mutational analysis of ABL kinase domain, 736 pre-imatinib era, 735 response assessment, 737 tyrosine kinase inhibitors, 735–6, 737–8 relapse, 1054 remission, 232 unrelated donor HCT, 677–8, 692 chronic myelomonocytic leukemia, 827, 837–8, 1032–3, 1034 reduced-intensity conditioning, 832 relapse rate after, 1054 relapse, 1054 risk category, 837 survival, 838 chronic neutrophilic leukemia, 837 CICN7, 1125 deficiency, 1127 mutations, 1127, 1128 CID see congenital immunodeficiency disorders cidofovir, 1427 CMV, 1378 hemorrhagic cystitis, 1481 varicella zoster virus, 1401 cimetidine drug interactions antidepressants, 1535 azole antifungals, 1528 ciprofloxacin, 1288, 1333, 1339 hemorrhagic cystitis, 1481 Circular of Information for Cellular Therapy Products, 540 cirrhosis, 1447–8 cisapride, drug interactions, 1525, 1527 cisplatin, 300–1 clinical use AIDs-related lymphoma, 1005 breast cancer, 933, 934, 934 CLL, 902 desmoplastic small round tumor, 994 diffuse aggressive lymphoma, 881 germ cell tumors, 951 neuroblastoma, 974 retinoblastoma, 993 Wilms’ tumor, 989 high-dose conditioning, 317, 320, 321, 324 mechanism of action, 301 metabolism, 301

pharmacokinetics/pharmacodynamics, 288, 301 reduced-intensity conditioning, 1046, 1047–8 side effects hyponatremia, 1501 neuropathy, 1661 renal disease, 1476 structure, 288 citalopram, drug interactions, 1535 cited2, 39 c-kit, 591 c-Kit receptor, 73 cladribine, reduced-intensity conditioning, 1046, 1047–8 clarithromycin drug interactions azole antifungals, 1527 immunosuppressants, 1531 clavulanic acid, 1326 clinical nurse specialist, 475 Clinical Program, 536 Clinical Standards for Hematopoietic Cell Transplantation, 534 clinical syndromes, 434–5 clinical trials, 447 conduct, 417 core elements, 410 design, 409–11, 410, 410 genetic data, impact of conduct of trial, 418, 418 design of trial, 416–17 information systems, 419 trial analysis, 423–4 high-dose conditioning, 316, 317 impact of genetic data, 419, 423–4 phase 0, 410–11 phase I, 411–13, 412, 412 dose-escalating, 411–12, 412, 412 standard design, 411 phase II, 413–14, 413 standard design, 413 variants, 413–14 phase III, 414–16 randomization process, 415 study sample size, 415–16 reporting of results, 424–5, 424 single- versus multicenter studies, 417 TBI, 325 CLL see chronic lymphocytic leukemia clobetasol, oral GVHD, 1602 clofarabine, AML, 783 clofazimine, 1270 GVHD, 1317 clonal deletion, 189, 195, 265, 1014, 1288 clonality analysis, 1644 clonidine, 522 clonogenic assays, tumor cell detection, 6 Clostridium difficile, 396, 1291, 1338, 1384 diarrhea, 1335–6 clotrimazole, 1598 CM-Dil, 77 CMRF-44, 255, 260 CMV see cytomegalovirus CMV-immediate-early, 1370 CMVpp65, 1370 CNS see central nervous system coagulation factor components blood group compatibility, 1227 cryoprecipitate, 1227 fresh frozen plasma, 1227 plasma, 1227 cobbleston area-forming colonies, 1207

Coccidioidomycosis imitis, 1346 Cochrane, Archie, 429 Code of Federal Regulations, 533, 534 codeine, 1579 Codman, Ernst, 429 coercion of donors, 547 cognitive-behavioral therapy, 491–2 cognitive function, 506 colchicine AL-amyloidosis, 921 drug interactions, 1266 colestipol, drug interactions, 1531 collagenase, 1325 Collection Services, 536 colonic GVHD, 392, 395 colony-forming cells, 36, 42, 83 colony-forming fibroblasts, 74 colony-forming units, 23, 590 erythroid, 23 granulocyte-erythrocyte-macrophagemegakaryocyte, 590 granulocyte-macrophage, 590, 631, 1208 mixed precursor, 23 neutrophil/macrophage, 23 colony-forming unit-spleen, 65 common leukocyte antigen see CD45 marker common lymphoid progenitor cells, 36, 222 and lymphoid development, 44–6, 45, 222 common myeloid progenitor cells, 36, 48 and myeloid development, 46–7 communication with patients, 527 community-acquired methicillin-resistant Staphyloccus aureus see CA-MRSA comorbid conditions, 449–50 cardiac, 449 hepatic, 450 renal, 450 respiratory, 449 complementary DNAs, 235 complement-mediated lysis, 8–9 clinical studies, 11 complement receptors, 252 complications allogeneic transplantation, acute kidney injury, 1473 bladder, 1480–2 delayed nonmalignant, 1620–37 adrenal glands, 1628 airway and lung complications, 1625, 1625 autoimmune disorders/dysregulation of immunity, 1623–4 autoimmune hematologic disease, 1624 bronchiolitis obliterans, 1626–7 bronchiolitis obliterans organizing pneumonia, 1627 cardiovascular disease, 1629 chronic GVHD, 1620–3, 1621, 1621–3 dental problems, 1631 etiology and spectrum, 1620 fatigue, 1634 gastrointestinal and hepatic complications gastrointestinal tract, 1632 liver, 1632–3 genitourinary dysfunction bladder, 1631–2 genital organs, 1632 kidneys, 1632 gonadal function, puberty and fertility, 1628–9 hypothalamic-pituitary axis, 1628 idiopathic pneumonia syndrome, 1627, 1627 infections, 1624

Index bacterial, 1624 fungal, 1624–5 viral, 1624 metabolic problems, 1633, 1633 musculofascial problems, 1630 neuroendocrine dysfunction, 1627–8 obstructive airway disease, 1626 ocular problems, 1629–30 cataracts, 1630 ocular sicca, 1630 posterior segment complications, 1630 Pneumocystis jiroveci pneumonia, 1625 psychosocial effects and rehabilitation, 1633–4 restrictive pulmonary disease, 1625 skeletal problems avascular necrosis, 1631 osteoporosis and osteopenia, 1630–1 thyroid, 1628 endocrine, 1487–522 abnormal water metabolism, 1500–3 adrenal dysfunction, 1506–9 carbohydrate and lipid abnormalities, 1503–6 dysfunctional bone metabolism and osteoporosis, 1491–3 gonadal dysfunction, 715, 1509–15, 1628–9 growth disorders, 1488–91 hypothalamic-pituitary dysfunction, 1487 metabolic and electrolyte abnormalities, 1500 sexual dysfunction see sexual dysfunction thyroid dysfunction, 1493–9 gastrointestinal, 1434–55 early post-transplant period, 1436–47 long-term transplant survivors, 1447–9 pre-transplant evaluation, 1434–6 hemorrhagic cystitis, 1481–2 high-dose conditioning, 1473–4 high-dose immunotherapy, 1019 neurologic, 1653–63 calcineurin inhibitors, 1658–60, 1659 cerebrovascular, 1–76, 1655, 1657 conditioning agents, 1660–1 immune-mediated, 1658 immunosuppressants, 1660 infectious, 1654–5, 1655, 1656 metabolic, 1657–8 pretransplant neurologic screening, 1661 supportive care, 1661 symptoms and signs, 1653–4, 1654 oral, 1589–607 bisphosphonate-associated osteonecrosis, 1603–4 dental care, 1604 dental treatment, 1589, 1591–2, 1592 granulomatous lesions, 1604–5 GVHD, 1600–2, 1601 long-term follow-up, 1603 neurotoxicity, 1603 oral care protocols, 1593–4, 1593 oral hemorrhage, 1599 oral infections, 1597–9 orofacial/dental growth and development, 1604 orofacial pain, 1603 oropharyngeal mucositis, 1595–7, 1595, 1596 patient education, 1594 phase of transplantation, 1589, 1590–1 post-HCT, 1593 pretransplantation evaluation, 1589 prophylactic antibiotics, 1593 reduced-intensity conditioning, 1603 salivary gland hypofunction, 1599–600, 1600 second malignancy post-HCT, 1605 taste dysfunction, 1602–3

reduced-intensity conditioning, 1473 renal, 1473–80 acute kidney injury, 1473–5, 1474 chronic kidney disease, 1475–8, 1475, 1477, 1478 GVHD-related, 1478–80 vascular access, 1249–54 air embolization, 1251 arterial punctures, 1250 cardiac arrhythmias, 1251 cardiac tamponade, 1251 catheter damage/fracture, 1252, 1252, 1253 catheter entry site bleeding/hematoma, 1250–1 catheter occlusion, 1250, 1251–2, 1251, 1252 catheter-related infection, 1253–4, 1254 catheter-related venous thrombosis, 1252–3, 1253 catheter tip malposition, 1250, 1250 drug extravasation, 1252 hemothorax, 1250, 1250 nerve injury, 1251 pneumothorax, 1249–50 conditioning, 1608 chemotherapy effect on growth, 1610–11, 1611 effect on puberty, 1614–15, 1615 effect on thyroid function, 1609 effect on DCs blood DCs, 254 depletion, 255 qualitative effects, 255 tissue DCs, 254–5, 254 effect on donor cells, 208 effect on GVHD incidence, 91 high-dose see high-dose conditioning and immune reconstitution, 226 immunosuppressive nonmyeloablative protocols, 195–9, 197 anti-T-cell antibodies, 196–8, 197 B-cell tolerance induction, 199 co-stimulatory blockade, 198 induction of xenogeneic tolerance, 198–9 pharmacologic agents, 196 sublethal TBI, 195–6 total lymphoid irradiation, 196 irradiation effects on growth, 1611–13, 1612, 1613 effects on puberty, 1615–16, 1616 effects on thyroid function, 1609 neurologic complications, 1660–1 nursing duties, 464–6, 464, 465–6 phagocytic disorders, 1165 and quality of life, 507 reduced-intensity see reduced-intensity conditioning TBI see total body irradiation conditioning-associated hemolytic-uremic syndrome, 1475 Congdon, Charles, 2 congenic transplantation, 272 congenital immunodeficiency disorders, 1112–14 Omenn’s syndrome, 1105, 1106, 1108 congestive heart failure, 1540, 1544 conscious sedation, 1585 Consensus Conference grading system, 434 conserved extended haplotype, 156 CONSORT, 424–5 constipation, nutritional interventions, 1558 consumption coagulopathy, 1227 coordination of care, 462, 471 copper-67, 354 copper, 1556

1687

Cord Blood Banking Standards, 536 cord blood cells, 18, 73, 119, 482–3, 584 banking, 483, 536, 560–1 characteristics, 565–7, 566 collection, 536, 559–60 endothelial progenitor cells, 567 expansion studies, 93–7, 95, 96 ex vivo expansion, 567–8 gene therapy, 568 graft failure, 1208 homing, 568–9 immune cells, 569–70 and immune reconstitution, 225 and incidence of GVHD, 1288 mesenchymal stem/stromal cells, 567 NetCord-FACT standards, 536 processing, 536 release, 536 selection, 536 testing, 536 cord blood cell transplantation, 559–76 in adults, 563–4, 564, 801 ALL, 801 AML, 768–9, 784–5 aplastic anemia, 717 cell banking, 560–1 cell collection, 559–60 clinical results, 562 donor search, 561 future directions, 565 history, 559 Hodgkin’s lymphoma, 872 immune reconstitution after, 564–5, 565 JMML, 757 non-relapse mortality, 563–4 related donor, 562 SCID, 1111–12 sickle cell disease, 1101 unrelated donor, 562–3 Wiskott-Aldrich syndrome, 1115 cord blood units, 695–6 double, 696, 696 core binding factor translocations, 762 coronaviruses, 1424 corticosteroids, 1270 clinical use, 1259 GVHD prevention, 1259 PNH, 728–9 pharmacology, 1259 side effects, 1259 neurotoxicity, 1603 corticotropin-releasing hormone, 1487 cortisol, 1262 Corynebacterium jeikeium, 1329 cost-benefit analysis, 430–1, 431 cost-effectiveness analysis, 408–9, 431, 432 co-stimulatory blockade, 189, 192–3, 198 cost-utility studies, 432 cotton-top tamarins, 577 counseling see evaluation and counseling Cox proportional hazards model, 423 Coxsackie virus, 268–9 cramps, 1653 CREGs (crossreactive groups), 151, 152, 156 Cre-Lox technology, 77 Creutzfeldt-Jakob disease, 546 transfusion-transmitted, 1239 critical care, 1539–50 cardiac complications, 1544 counseling, 1546–7, 1547 gastrointestinal and hepatic complications, 1544–5

1688

Index

organization of, 1539–40 prognosis, 1546, 1546 pulmonary complications, 1540–3, 1540 infectious, 1540–1, 1541, 1541 noninfectious, 1541–2, 1542 respiratory failure, 1542–3, 1543 renal complications, 1545–6 severe infections and septic shock, 1543–4, 1543 critical care management, 471–2 Crohn’s disease, 150, 1026, 1434 allogenic HCT for other primary disease, 1017 susceptibility genes, 1015 Cronbach’s alpha, 434 crossmatching HLA, 694 partially matched donors, 659 platelet transfusion, 1230 cross-presentation, 130, 136 crossreactivity, 151 cross-talk, 169 cryobiology, 632–4, 633 dehydration injury, 632 glass transition, 633 ice formation, 632 phase-transition temperature, 634 vitrification, 633 cryoprecipitate, 1227 cryopreservation, 2–3, 631–44, 632 allogeneic transplantation, 640–1, 641 cell concentration, 637–8 cell processing, 637 chemistry and biology, 632–4, 633 cooling/warming rates, 638, 638 cryoprotectant solutions, 634–6, 636 cryoprotectant toxicity, 639–40 evaluation of efficacy, 636–7, 637 induction of apoptosis, 634 infusion procedure, 468 physics of cooling and warming, 634, 635 post-thaw manipulation, 639 salt and sugar content, 636 storage, 638–9 tumor cell purging, 641 cryoprotectant solutions, 634–6, 636 colligative, 632, 633 DMSO, 2, 632–3, 633, 634–5 ethylene glycol, 633 glycerol, 632 HES, 635 optical cooling rate, 638 protein, 635–6 sugars, 636 toxicity, 639–40 to hematopoietic cells, 639–40 to transplant recipient, 640 cryotherapy, oral, 1596 cryptic dyskeratosis congenita, 705 Cryptococcus neoformans, 1654 Cryptosporidium spp., 1434–5 c-src, 1126 CTLA-4-Ig, 1270 C-type lectin-like receptors, 252 cunninghamellosis, 1350–1 Current Good Tissue Practices Final Rule, 533 cushingoid symptoms, 1628 Cushing’s syndrome, 1506 cutaneous acute GVHD, 1290 cutaneous dendritic cells, 248–9, 249 C.W. Bill Young Cell Transplantation Program, 540 CX3CR1, 250

CXCR2, 1463 CXCR3, 1463 CXCR4, 76, 104, 1001 HSC mobilization/trafficking, 591 CXCR4 receptor, 1201 cyclic neutropenia, 1170 cyclophosphamide, 3, 19, 196, 297, 632, 755, 960, 1080, 1219, 1608 clinical use AIDS-related lymphoma, 1004, 1005 ALL, 793, 799, 815 aplastic anemia, 714, 719 breast cancer, 932, 933, 934, 934, 935, 936 CLL, 901, 903, 907, 908 CML, 740, 740 desmoplastic small round tumor, 994 diffuse aggressive lymphoma, 881 Fanconi’s anemia, 1189, 1190, 1191, 1192 germ cell tumors, 951, 952, 953 GVHD, 711, 711 Hodgkin’s lymphoma, 873 MDS, 831, 832 melanoma, 965 MPD, 836 neuroblastoma, 973, 974 non-Hodgkin’s lymphoma, 878 pediatric solid tumors, 985 renal cell carcinoma, 963 SLE, 1023 drug interactions, 1526 GVHD, 1317, 1318 HDIT, 1017, 1018 high-dose conditioning, 317, 319, 320, 323 with TBI, 318, 324, 335 mechanism of action, 297 metabolic pathways, 293 metabolism, 297 pharmacokinetics/pharmacodynamics, 288, 297 post-transplant, 664 reduced-intensity conditioning, 1047–8 side effects cardiac, 1544 neurotoxicity, 1603 renal diseaes, 1476 SOS, 1440 structure, 288 synergism with antithymocyte globulin, 709 cyclosporine, 192, 226–7, 776, 1219, 1270 clinical use AML, 763 aplastic anemia, 717 Fanconi’s anemia, 1189 GVHD, 711, 741 GVHD prevention, 214, 1263–6 host-versus-graft reactions, 709 drug interactions, 1266, 1530 azole antifungals, 1527, 1528 grapefruit juice, 1536 immunosuppressants, 1531, 1532 hemophagocytic lymphohistiocytosis, 1166 neurologic complications, 1658–60, 1659 neutrophil and platelet changes, 1049 oral GVHD, 1602 pharmacology, 1264, 1264, 1265 PNH, 728–9 reduced-intensity conditioning, 1047–8 side effects, acute kidney injury, 1473 suppression of Tregs by, 179 CYP1A23, 1524 CYP2B1, 299 CYP2B6, 291, 1524

CYP2C8, 291 CYP2C9, 291, 1524 CYP2C11, 299 CYP2C18, 291 CYP2C19, 291, 1524 CYP2D6, 291, 1524 CYP2E1, 1524 CYP3A4, 291, 299, 1524, 1642 CYP3A5, 291, 1524 CYP3A7, 291 cystinosis, 1139, 1157 cytarabine clinical use ALL, 793, 799, 814 AML, 763, 782 CLL, 902, 903, 907 CNS leukemia, 794 diffuse aggressive lymphoma, 881 Hodgkin’s lymphoma, 873 JMML, 754 non-Hodgkin’s lymphoma, 359 and graft failure, 1207 high-dose conditioning, 317, 320, 321, 324 with TBI, 318, 335 reduced-intensity conditioning, 1046, 1047–8 side effects, renal diseaes, 1476 cytochrome P450 isoenzyme system, 1523, 1524 see also CYP cytogenetics, 766, 1644 cytokines, 4, 66 in GVDH, 137 and HSC expansion systems, 90 and NK cells, 163 outcome of HCT from unrelated donors, 686–7 PBHC mobilization, 593–4, 593, 593, 594 post-transplant administration, 1068–9 in prevention of GVHD, 200 secretion by donor T cells, 211 secretion by MSCs, 105 cytokine capture assays, 223 cytokine-induced killer cells, 1059, 1064 cellular therapy, 1069 cytokine polymorphisms, 1290 cytokine storm, 212, 687, 1051 cytomegalovirus, 19, 169, 546, 1222, 1367–81, 1384, 1445, 1540–1, 1598 and acute GVHD, 1289 in autologous/syngeneic recipients, 1369–70 cellular immunity to, 1370–1 definitions of disease, 1371–3 CMV-associated enteritis, 1372 CMV-associated interstitial pneumonia, 1372, 1372 detection of, 1377 diffuse interstitial pneumonia, 1457 enteritis, 396 epidemiology, 1368 in HCT allograft recipients, 1368–9, 1369 incidence, 1370 late, 1624 latency, 1367 pneumonia, 712 prevention, 1374–8, 1376, 1377 risk of disease, 1371–2, 1371 structure and biology, 1367, 1368 transfusion-related, prevention of, 1233 treatment, 1373–4 CMV-associated enteritis, 1374 CMV-associated interstitial pneumonia, 1373–4, 1373, 1374 complications of therapy, 1374, 1374

Index cytomegalovirus pneumonitis, 1456 cytopenia, 1166 refractory, 834 with multilineage dysplasia, 829 with multilineage dysplasia and ringed sideroblasts, 829 cytoreductive conditioning skin effects, 394 toxicity, 391 cytosine arabinoside AIDS-related lymphoma, 1005 HDIT, 1018 MDS, 832 non-Hodgkin’s lymphoma, 359 cytotoxic T cells, 134, 176, 189, 190, 234, 236, 237, 242, 651, 1003, 1014, 1059, 1203 antigen-specific, 1064 cellular therapy, 1069–70 donor, 179 effector functions, 177 enrichment of, 1281 in GVHD, 178, 181–2, 181 D dacarbazine, retinoblastoma, 993 daclizumab, 1293 GVHD, 1269, 1486 dacluzimab, 1270 dactinomycin, Wilms’ tumor, 989 dalfopristin, 1326 daptomycin, 1326 Darbepoetin see erythropoietin dasatinib, 737 ALL, 803 CML, 1061–2 data analysis, 419 database management, 419–20 Data and Safety Monitoring Boards, 417–18 daunorubicin ALL, 793 AML, 763 DCs see dendritic cells DC-SIGN, 248, 252 death-associated protein kinase-1, 898 debrisoquine metabolism, 290 DEC-205, 252, 253 decadron, diffuse aggressive lymphoma, 881 decision analysis, 435 deep-tissue infection, 1348 deferasirox, 1079 deferiprone, 1078–9 defibrotide, 598 delayed hemolytic reactions, 1220 delayed pulmonary toxicity syndrome, 1542 delayed-type hypersensitivity, 177 delta, 222 dematiaceous molds, 1351 demyelinating peripheral neuropathy, 916 dendritic cells, 163, 189, 248–63, 264, 1204 antigen presentation, 253 blood, 250 chimerism, 255–6 effect of conditioning, 254 chimerism blood DC, 255–6 peripheral tissue DC, 256–7, 256 conditioning effects, 254 blood DCs, 254 depletion, 255 qualitative, 255 tissue DCs, 254–5, 254

cutaneous, 248–9, 249 dermal (interstitial), 248 development, 48–9 epidermal see Langerhans cells from hESCs, 25 functions, 252–3 graft content, 255 in GVHD, 214, 257–9 APCs inducing, 257 chronic GVHD, 258–9 kinetics, 258 persistence of recipient LCs, 257 tropism, 258 in GVLE, 259 heterogeneity, 248–52 homeostasis post-transplant, 255–7 steady state, 254 immature, 252 interaction with MSCs, 108 interaction with NK cells, 169 lymphoid, 48, 250, 251 maturation, 252–3 monocyte-derived, 251 mucosal, 249–50, 250 myeloid, 48 numerical recovery, 255 origins of, 251 phenotypes, 249 plasmacytoid, 249, 250–1 function of, 253 role in tolerance induction, 192 stimulation of allogeneic T cells, 253 T-cell activation, 210, 252 as therapeutic targets, 259–60 thymic, 250 tissue, 250 chimerism, 256–7, 256 effect of conditioning, 254–5, 254 in vitro-derived, 251, 252 see also APCs denileukin diftitox, 1293, 1294 dental problems, late, 1631 dental treatment post-HCT, 1604 pre-HCT, 1589, 1591–2, 1592 dental decay and endodontic disease, 1591 dental extractions, 1591–2 dentures and orthodontic appliances, 1592, 1592 periodontal disease, 1591 temporomandibular dysfunction, 1592 prophylactic antibiotics, 1593 see also oral care protocols dentures, 1592 2-deoxy-5-azacytidine, MDS, 830 depression, 521 dermal (interstitial) dendritic cells, 248 desatinib, chronic myelomonocytic leukemia, 838 desferrioxamine, 1077 desmin, 994 desmoplastic small round cell tumor, 993–4 clinical description, 993–4 epidemiology and etiology, 993 HCT, 994 molecular and cellular biology, 993 nontransplant approaches, 994 developmental immunodeficiency, 194 dexamethasone AIDS-related lymphoma, 1004, 1005 AL-amyloidosis, 923

1689

multiple myeloma, 847 oral GVHD, 1601 dextran, as cryoprotectant, 637 diabetes mellitus, 1503–6 evaluation, 1505 management, 1505–6 type 1, 265, 1015 family/twin studies, 1015 susceptibility genes, 1015 Diamond-Blackfan anemia, 16, 705, 1178 HCT, 714 diarrhea, 465, 469, 1434–5, 1444–5, 1444, 1449 differential diagnosis, 1444 nutritional interventions, 1558 dicer enzyme, 1008 diepoxybutane test, 1181 dietary supplements, 1561 diffuse aggressive lymphoma, 880–4 allogeneic HCT, 883–4 autologous HCT, 880–3, 881, 882 diffuse alveolar hemorrhage, 1291, 1458, 1459, 1539, 1540, 1542 diffuse interstitial pneumonia, 1457 aplastic anemia, 712–13 diflunisal, 1576 DiGeorge syndrome, 199 digoxin drug interactions azole antifungals, 1526 immunosuppressants, 1530 dihydrofolate reductase, 1262 1,25-dihydroxyvitamin D, in osteopetrosis, 1125 dilators, 522 diltiazem drug interactions, 1266 azole antifungals, 1527 immunosuppressants, 1530, 1531, 1532 dimethylsulfoxide, 2, 466, 632–3, 633, 634–5 protective effect, 637 diphtheria toxoid vaccine, 1668 discharge, nursing duties, 472–3, 472, 473 discharge planning RN, 475 Disease-Specific Impairment Inventory-HSCT, 510 disheveled (Dsh), 39 disseminated intravascular coagulation, 776, 1227 divisions, number of, 65 dizygotic twins, chimerism in, 577 DMSO see dimethylsulfoxide DNA labels, 77 telomeric, 68, 68 DNA amplification, 367, 369 DNA repair, defects in, 1642 DNAM-1 receptors, 166 DNAse, 637 docetaxel breast cancer, 932, 933 pharmacokinetics/pharmacodynamics, hepatic impairment, 294 dog leukocyte antigens, 3 dominant epitopes, 268 Donabedian, Avides, 429 donation adverse events, 549–50, 549, 550 anesthesia risks, 550 bone marrow, 548 adverse events, 549–50 mortality, 551 HCs adverse events, 549 mortality, 551

1690

Index

infection risks, 550 mechanical injury risks, 550 PBPCs, 544, 548–9 adverse events, 551–2 allogeneic transplantation, 544 autologous transplantation, 544 mortality, 552–3 regulatory aspects, 546 psychologic aspects, 547 regulatory requirements, 546–7 repeat, 554 transfusion risks, 550 donors, 544–58 allogeneic transplantation see allogeneic transplantation autologous transplantation see autologous transplantation avoidance of coercion, 481 choice of, 1165 coercion of, 547 compatibility, 447–8, 448 confidentiality, 481 eligibility and qualification, 546 ethical/psychosocial issues, 547–8 consent, 480–2, 547–8 donors as research subjects, 548 psychologic aspects, 547 genetic disparity with recipient, 135–8 history and physical examination, 547 HLA typing, 156 laboratory evaluation, 547 long-term follow-up, 553 matched-related, 224, 224 medical eligibility, 547 minors, 481, 482, 553–4 information, consent and assent, 553–4 safety, 553 preparation of, 463, 463 privacy, 481 products from, 544 psychologic state, 481 psychosocial issues, 495–7 related, 157, 544–5 histocompatibility testing, 157 T cells, 208–9, 688 cytokine secretion, 210–11 delayed administration, 201 modulation of, 213–15 transfusions, 1234 unrelated, 224–5, 675–91 acute lymphoid leukemia, 677 AML, 676–7 CML, 677–8 genetic factors, 678 haplotype matching, 158 hematologic malignancies, 675–6, 676s histocompatibility testing, 157–8 HLA system, 678–9, 680 KIR genes, 688 activation of donor T cells, 688 cytokine and immune response gene variation, 686–7 cytokine storm, 687 inflammatory effectors, 688 inhibitory KIR receptors, 683–4 KIR haplotypes and activating receptors, 686 models for donor inhibitory KIR-mediated killing, 684–6, 685 matched donor, 679–80, 683 MHC haplotype, 682–3, 684

mismatched donor, 680–2, 683 acute GVHD, 681 alleles and antigens, 681 graft failure, 681 GVTE, 681 models for permissible mismatches, 682 multilocus mismatches, 682 myelodysplastic syndrome, 677 registries, 545 selection of, 692–4, 693, 693, 694 vaccination, 1067 Donor Eligibility Final Rule, 533 donor leukocyte infusion, 202, 232, 240–1, 1059–60, 1060 ALL in adults, 1062–3, 1063 AML, 770, 1062, 1062 CML, 1060–92, 1060 for graft failure, 1210 JMML, 757 lymphoid malignancies, 1063 MDS, 1062, 1062 multiple myeloma, 1063 prevention of disease relapse, 1280 donor registries, 545 donor screening, 534 donor selection, 534, 692–703 algorithm, 701 alternative donors, 700 donor types, 699–701 for GVTE, 239–40 partially matched donors, 696–9 and reduced graft failure risk, 1209 umbilical cord blood units, 695–6, 696, 696 unrelated donors, 224–5, 692–4, 693, 693, 694 haplotype matching, 158 histocompatibility testing, 157–8 donor-specific transfusion, 191–2 donor T cells activation, 208–9 allogeneic co-stimulated, 1063–4 cytokine secretion, 210–11 delayed administration, 201 effectors, 1461–2 modulation of, 213–15 reduced activation, 214 reduced numbers, 213–14 reduced proliferation, 214–15 dopamine, 1487 dormancy, 384–5 dose-escalating studies, 411–12, 412, 412 dosimetry, 354–5 Douglas, Gordon, 559 Down’s syndrome ALL, 791, 820 AML, 775, 777, 779, 782 ethics of transplantation, 478 doxorubicin, 303 clinical use AIDS-related lymphoma, 1004, 1005 breast cancer, 933, 935 desmoplastic small round tumor, 994 multiple myeloma, 847 neuroblastoma, 973, 974 non-Hodgkin’s lymphoma, 878 retinoblastoma, 993 Wilms’ tumor, 989 end-infusion concentrations, 293 and graft failure, 1207 high-dose conditioning, 321, 324 mechanism of action, 303 metabolism, 303

pharmacokinetics/pharmacodynamics, 288, 303 hepatic impairment, 294 structure, 288 D region-associated B-cell antigens, 151 drug extravasation, 1252 drug interactions, 1523–38 azole antifungals, 1524–9, 1525–8 fluconazole, 1524, 1525 itraconazole, 1524–5, 1526 posaconazole, 1527–8, 1528 voriconazole, 1526–7, 1527–8 cytochrome P450 isoenzyme system, 1523, 1524 echinocandins, 1529 foods, 1535–6, 1536 immunosuppressants, 1529–33, 1530–2 calcineurin inhibitors, 1529 cyclosporine, 1530 mycophenolate, 1529–30, 1531 sirolimus, 1530, 1532–3, 1533 tacrolimus, 1531 P-glycoprotein system, 1524 supportive care agents, 1533–5, 1534, 1535 antidepressants, 1533, 1534, 1535 narcotic analgesics, 1533 sedatives, hypnotics and anxiolytics, 1533 uridine 5′-diphosphate glucuronosyltransferase enzyme system, 1523–4 see also individual drugs drug-metabolizing enzymes, 290–2 inherited variability, 290–2 polymorphisms in, 1641–2 Dubowitz syndrome, 1178 Duchenne muscular dystrophy, 1185 duloxetine, drug interactions, 1534 Durie-Salmon staging system, 847 dying patients, 1585–6 dyskarin, 1170 dyskeratosis congenita, 16, 68, 1170–1, 1178 aplastic anemia, 714 dysostosis multiplex, 1145 dyspareunia, 518, 521–2 dysphagia, 1444, 1444 nutritional interventions, 1557 dystrophin, 78 E E2A-PBX fusion, 807 early goal-directed therapy, 1546 E-cadherin, 248 ECGF1, 241 echinocandins, 1295 drug interactions, 1529 ectoderm, 72, 80 eculizumab, PNH, 729 eGFP, 77 Ehrlich, Paul, 351 elastase, 1325 electrolytes, 1555–6 electrolyte abnormalities, 1500 Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products, 534 eligibility for HCT, 449 ethical issues, 478–9 elutriation, 1411 embryonic stem cells, 42, 76 human see human embryonic stem cells murine, 23–4, 24 embryos, hematopoiesis in, 65 emigration, 1163 Emtamoeba histolytica, 1434

Index enalapril, thrombotic microangiopathy, 1480 encephalitis, HSV, 1384 endocarditis, 1544 endocrine complications, 1487–522 abnormal water metabolism, 1500–3 adrenal dysfunction, 1506–9 carbohydrate and lipid abnormalities, 1503–6 dysfunctional bone metabolism and osteoporosis, 1491–3 gonadal dysfunction, 715, 1509–15, 1628–9 growth disorders, 1488–91 hypothalamic-pituitary dysfunction, 1487 metabolic and electrolyte abnormalities, 1500 sexual dysfunction see sexual dysfunction thyroid dysfunction, 1493–9 endocytosis, 264 endoderm, 72, 79 end-of-life issues, 444, 484–6 advanced directives, 446, 454–5, 485 endoglin, 47, 104 endothelial cells, 80–1 endotoxins, 1326 end replication, 68 enfuvirtide, 1001 engraftment, 135–6, 365–75 allogeneic PBHCs, 622 failure of see graft-versus-host disease resistance to, 193–4 ‘space’ for, 193 and T-cell depletion, 1278–9 thalassemia, 1080 engraftment syndrome, 647 entecavir, 1435 enteral nutrition, 1562–5, 1565 enteritis, CMV-associated, 1372 treatment, 1374 Enterobacter cloacae, 1333 Enterococcus avium, 1335 Enterococcus faecalis, 1335 Enterococcus faecium, 1327, 1335 enteroviruses, 1426–7 env, 116, 117, 118, 1002 envelope glycoproteins, 118 EORTC-QLQ-C30, 504, 508, 509 epidermal DCs see Langerhans cells epidermal keratinocyte atypia, 394 epidermolysis bullosa, 1185 epigenetics, 65–6 epipodophyllotoxins, 775 epirubicin breast cancer, 934, 935 germ cell tumors, 953 epitopes, dominant, 268 epitope spreading, 268 epoetin-alpha, 550 see also erythropoietin Epstein, Arnold, 429 Epstein-Barr nuclear antigens, 1410, 1415 Epstein-Barr virus, 169, 546, 1018, 1410–18, 1598 biology, 1410–11, 1411 DNA in PBMCs, 1413 in plasma, 1413 encephalitis, 1655, 1656 guidelines, 1414 latent infection, 1411 lymphoproliferative disorder, 1107, 1411–12, 1411 classification, 1413 clinical and pathologic aspects, 1412–13, 1413 risk factors, 1411 treatment, 1414–15, 1415

lytic infection, 1411 nuclear antigens, 1411 pathogenesis, 1410–11, 1411 PBMC versus plasma versus whole blood, 1413–14 transfusion-transmitted, 1238 tumors, T-cell therapy, 1415 viral copy number and monitoring, 1413, 1414 viral DNA in plasma, 1413 virologic aspects, 1413 equivalency testing, 420 erectile dysfunction, 522–3 ergot alkaloids, drug interactions, 1525, 1527 erythroid cells from hESCs, 26 erythroleukemia, acute, 1032 erythromycin drug interactions, 1266 azole antifungals, 1527 immunosuppressants, 1532 erythropoietin, 66, 645, 646, 1079 after allogeneic HCT, 645–7 after autologous HCT, 645 Escherichia coli, 845, 1222, 1327, 1329, 1598, 1599 escitalopram, drug interactions, 1535 E-selectin, 591, 592, 1163, 1164, 1201 esophageal complications cancer, 1449 GVHD, 397, 1311, 1449 pain, 1444, 1444 strictures, 1449 esophagitis, nutritional interventions, 1557 essential thrombocythemia, 835, 836–7 Estern-Dameshek syndrome, 1178 esthesioneuroblastoma, 994 estrogen deficiency, 518, 521 etanercept, 1270, 1293 GVHD, 1317 rheumatoid arthritis, 1023 ethical issues, 478–87 alternative cell sources, 482–4 eligibility for HCT, 478–9 end-of-life issues, 484–6 informed consent donors, 480–2 patients, 479–80 transplantation for nonmalignant diseases, 484 ethinyl estradiol/norethindrone, drug interactions, 1525, 1527 ethnic/racial background, 450 ethylene glycol, 633 etodolac, 1576 etoposide, 302–3, 632, 755 clinical use AIDs-related lymphoma, 1004, 1005 ALL, 814 AML, 763 breast cancer, 932, 933, 934, 936 CLL, 902, 903, 907 desmoplastic small round tumor, 994 diffuse aggressive lymphoma, 881 Ewing’s sarcoma, 986 germ cell tumors, 952, 953 Hodgkin’s lymphoma, 873 neuroblastoma, 973, 974 non-Hodgkin’s lymphoma, 359 osteosarcoma, 992 rhabdomyosarcoma, 989 Wilms’ tumor, 989 HDIT, 1018 high-dose conditioning, 317, 319, 320, 321, 323–4 with TBI, 318, 335 mechanism of action, 302

1691

metabolism, 302–3 pharmacokinetics/pharmacodynamics, 288, 302–3 side effects, renal diseaes, 1476 structure, 288 toxicity, 1661 ets, 985, 986 European Federation for Immunogenetics, 536 European Group for Blood and Marrow Transplantation, 535 vaccination guidelines, 1664 euthyroid sick syndrome, 1610, 1628 evaluation and counseling, 443–58 advanced directives, 454–5 advice against transplantation, 446 children, 452–4, 453 conditions affecting, 452–3 consent to treatment, 452 family counseling, 453–4 initial visit, 452 Lansky Play-Performance Scale, 453 malignant disease, 452 nonmalignant disease, 452 choice of procedure, 444–5 clinical trials, 447 comorbid conditions, 449–50 cardiac, 449 hepatic, 450 renal, 450 respiratory, 449 conditions affecting outcome, 447 critical care services, 1546–7, 1547 disease and remission status, 447 donor compatibility, 447–8, 448 eligibility for transplant, 449 ethnic and racial background, 450 first visit to transplant center, 443–4 foreign patients, 447 infertility issues, 444 information absorbed, 445–6, 446 multiple opinions, 446–7 nutritional status, 450 outcome data, 445 patient age, 450 performance status, 448–9 previous infections, 451 prior therapy, 449 psychosocial assessment, 451–2, 451 second meeting, 446 stem cell source, 454 transfusion history, 451 written information, 443 Evan’s syndrome, 1026 evidence-based medicine, 436–7, 437 Ewing’s sarcoma, 985–8 clinical description, 986 epidemiology and etiology, 985–6 HCT, 987–8, 987 molecular and cellular biology, 986 nontransplant approaches, 986 EWS, 985, 986 EWS-WT1, 993 exemestane, 932 exoenzymes, 1326 exotoxins, 1325, 1326 expansion of HSCs, 88–101, 89 clinical trials, 91–7 bone marrow and mobilized peripheral blood, 91–3, 92 UCB cells, 93–7, 95, 96 large-animal studies, 89 murine studies, 89

1692

Index

novel approaches, 90–1 preclinical studies, 88–9 xenogeneic animal models, 89–90 experimental autoimmune encephalomyelitis, 268, 270–1, 270 externally tunneled catheter, 1246, 1246 extracorporeal photopheresis, GVHD, 1317 ex vivo culture conditions, 119–20 eye see ocular problems F Fabry disease, 1139 Fabry lipogranulomatous disease, 1139 FACIT-SP, 504 FACT see Foundation for the Accreditation of Cellular Therapy FACT-BMT, 504, 508, 509 factor X, 915 famciclovir HSV infection, 1385 prophylaxis, 1385 varicella zoster virus, 1401 familial erythrophagocytic histiocytosis, 16 family and carers see caregivers family member transplantation see related donor HCT FANCA, 1182 FANCC, 1182 FANCG, 1182 Fanconi’s anemia, 16, 121, 585, 586, 692, 761, 834, 1178–99, 1641 and ALL, 791 and AML, 775 animal models, 1186 and aplastic anemia, 705, 714 assisted reproduction and preimplantation genetic diagnosis, 1195 clinical features, 1178–80, 1179, 1179, 1180 complementation groups, 1181–2, 1181 assignment, 1183–5, 1183, 1184 congenital malformations in, 1179 cord cell transplantation, 559 diagnostic tests, 1180–1, 1181 gene cloning, 1181–2, 1181 gene functions, 1182–3 gene therapy, 1194–5 genotype-phenotype correlation, 1183–5, 1183, 1184 HCT, 1187 alternative donors, 1188, 1190, 1191, 1192–3, 1192 cancer risk after, 1193–4, 1194 HLA-identical sibling donors, 1187–8, 1187, 1189, 1190 risk factors, 1193 history, 1178 and leukemia, 1192 marrow failure and leukaemogenesis, 1186 and MDS, 1192 multipotent stem cells and tissue repair, 1195 mutation analysis, 1183–5, 1183, 1184 nontransplant treatment strategies androgens, 1186 haematopoietic growth factor, 1187 supportive care, 1187 vitamins and antioxidants, 1186–7 pathophysiology, 1182–3 somatic mosaicism, 1185–6, 1185 treatment algorithm, 1193 Farber lipogranulomatous disease, 1139, 1156 farnesyl transferase inhibitors, 754 Fas, 211

Fas/FasL pathway and graft rejection, 1203 and GVHD, 211 Fas ligand, 270 fat embolism, 550 fatigue, 465, 505–6, 1634 Fcε, 252 Fcγ, 252 FcRγ, 435 fear, 526 febrile nonhemolytic transfusion reactions, 1235 felbamate, marrow injury, 705 feline endogenous virus RD114, 118 felodipine drug interactions azole antifungals, 1526 grapefruit juice, 1536 immunosuppressants, 1531 femoral vein catheterization, 1248 fenoprofen calcium, 1576 fenretinide, 979 fentanyl, 1579, 1580, 1597 ferritin, 1166 fertility, 715 late complications, 1628–9 fetal hematopoiesis lineage commitment, 48 in liver, 42–3 fetal hemoglobin, hereditary persistence, 1092 fetus graft-versus-host disease, 584 hematopoietic environment, 578, 578 immunologic tolerance, 577–8 thymus, 577 fever, 465, 469 fibrinogen Aα, 917 fibroblast growth factor-2, 24 fibroblast growth factor-20, 1597 filgrastim, 544, 548, 551 PHBC mobilization, 618 platelet count, 552 sickle crisis, 552 side effects, 621 pain, 1583 white blood cell count, 551–2 see also G-CSF FISH see fluorescence in situ hybridization FK506 see tacrolimus FKHR, 988 Flavobacterium spp., 1327 FLK1, 38 flossing/interproximal brushing, 1593, 1594 flow cytometry minimal residual disease, 376 tumor cell detection, 6 FLT3, 381, 775 Flt3 ligand, 39, 89, 90 flucinonide, oral GVHD, 1602 fluconazole, 1288, 1355, 1358, 1359, 1442, 1475 drug interactions, 1266, 1524, 1525 immunosuppressants, 1530, 1531, 1532 fludarabine, 960, 1219, 1222, 1608 clinical use ALL, 800 CLL, 901, 905, 908 Fanconi’s anemia, 1190 MDS, 830, 831, 832 melanoma, 965 MPD, 837 non-Hodgkin’s lymphoma, 359 renal cell carcinoma, 963

and Epstein-Barr lymphoproliferative disorder, 1412 high-dose conditioning, 319, 321 host-versus-graft reactions, 709 neutrophil and platelet changes, 1049 reduced-intensity conditioning, 742, 1046, 1047–8 fluorescence-activated cell sorting, 73 fluorescence in situ hybridization, 68–9, 69, 1644 minimal residual disease, 376, 378 tumor cell detection, 6 fluorescent in situ hybridization, 76 fluoride treatments, 1593, 1594 fluoroquinolones, 1326 drug interactions, 1266 fluorouracil breast cancer, 932, 934 desmoplastic small round tumor, 994 fluoxetine, drug interactions, 1534 fluticasone, oral GVHD, 1601 fluvoxamide, drug interactions, 1535 Fms-like tyrosine kinase-3, 260 Fms-like tyrosine kinase-3 ligand, 24, 598 foamy virus vectors, 118 follicle-stimulating hormone, 518, 522, 1487 follicular lymphoma, 879, 884–9 allogeneic HCT, 887–9, 887, 888, 888 autologous HCT, 884–7, 884, 885, 886, 887 Follicular Lymphoma International Prognostic Index, 879, 880 Food and Drug Administration, 533, 546 foods, drug interactions, 1535–6, 1536 foreign patients, 447 forward blot typing, 153 foscarnet, 1377, 1427 CMV prophylaxis, 1376 treatment, 1373, 1374 HSV infection, 1385 prophylaxis, 1385 resistance, 1375 toxicity, 1661 varicella zoster virus, 1401 Foundation for the Accreditation of Cellular Therapy, 534 accreditation process, 536–9, 537, 538, 539 historical background, 534–5 inspector qualifications, 537 significance of accreditation, 539–40 standards, 535–6 Foundation for the Accreditation of Hematopoietic Cell Therapy, 534 FoxP3, 134, 190, 211, 271, 1289 FoxP3+, 569 free light chains, 914, 915, 916, 920 fresh frozen plasma, 1227 Freund’s adjuvant, 271, 274 Friedenstein, Alexander, 102 frizzled (Fzd), 39 FTY720, 215, 260 fucosidosis, 1139, 1155–6 Functional Living Index-Cancer, 508 Fundamental and Clinical Aspects of Radiation Protection and Recovery, 2 fungal infection, 1346–66 antifungal agents, 1354–5, 1356, 1357–61 see also individual drugs approach to suspected disease, 1362 Aspergillus spp., 401, 1222, 1349–50 immune response to, 1352–4, 1353 biology and pathophysiology, 1347–9 Candida spp., 1347–8, 1598

Index cardiovascular system, 1348–9 dissemination with deep-tissue infection, 1348 eye and CNS disease, 1349 immune response to, 1351–2 mucocutaneous infection, 1348 skin lesions, 1349 candidemia, 1348 dematiaceous molds, 1351 diagnosis not based on culture, 1351 epidemiology, 1346–7, 1347 food-related, 1560 Fusarium spp., 401, 1351 granulocyte transfusions, 1361–2 immunotherapy, 1361 late, 1624–5 liver, 1435, 1442 abscess, 1448 oral, 1597 prevention, 1354–5 primary prophylaxis, 1355, 1357 secondary prophylaxis, 1357 Scedosporium spp., 1351 surgical approaches, 1362 zygomycetes, 1350–1 Fusarium spp., 401, 1351 future directions, 1671–4 G gabapentin, 522 gag, 116, 117, 1002 β-gal, 77 galactomannans, 1351 gall bladder disease, 1436, 1442–3, 1449 gammaretroviral viruses, 116–17, 117, 118 ganciclovir, 1427 CMV prophylaxis, 1376, 1378 treatment, 1373–4, 1373 drug interactions, 1531 Epstein-Barr virus, 1414 resistance, 1375 side effects, 1374, 1661 gangliosidoses, 1139, 1156 gastrointestinal complications, 1434–56, 1544–5 abdominal pain, 1445–7, 1446 AL-amyloidosis, 917, 918–19 response to treatment, 922 bleeding, 1443, 1443, 1545 diarrhea, 465, 469, 1434–5, 1444–5, 1444, 1449 dysphagia, painful swallowing and esophageal pain, 1444, 1444 early post-transplant period, 1436–47 esophageal disease cancer, 1449 chronic GVHD, 1449 GVHD, 397, 1311, 1449 strictures, 1449 gall bladder and biliary tract disease, 1436, 1442–3, 1449 GVHD, 393, 395–7, 396, 397, 1290, 1309, 1311 autologous, 397 chronic, 1309, 1311 conditions mimicking, 1445 differential diagnosis, 396 spectrum of, 395–6 transplant-associated microangiopathy, 397 idiopathic hyperammonemia and coma, 1443 jaundice, 1437 late, 1632 liver, 1438 acute hepatitis, 1442

acute hepatocellular injury, 1448 cholestatic disorders, 1442 chronic GVHD, 1447 chronic liver disease, 1435 cirrhosis, 1447–8 dysfunction post-transplant, 1437 pretransplant, 1435–6 fungal abscess, 1448 fungal infections, 1435, 1442 GVHD, 393, 397–8, 397, 398, 1290–1, 1309, 1311 hepatocellular carcinoma, 1448 hepatomegaly, 1437 iron overload, 1436, 1448 nodular regenerative hyperplasia, 1448 SOS see sinusoidal obstructive syndrome viral hepatitis in allogeneic donors, 1435 chronic, 1447–8 malignant disorders, 1443 nausea, vomiting and anorexia, 1436–7, 1436, 1449 pancreatic disease, 1449 perianal pain, 1435, 1447 pretransplant evaluation, 1434–6 ulcers, tumors and infections, 1434 Gata1, 50 Gata3, 50, 620 Gaucher disease, 1139, 1155–6 G-CSF, 481, 591, 646, 647, 647, 1597 after allogeneic HCT, 649 after autologous HCT, 647–8 and AML, 775 PBHC mobilization, 593, 595–6 PNH, 728–9 receptor mutations, 1168 recombinant, 544 severe congenital neutropenia, 1168 side-effects, 621–2, 621 G-CSF mobilized PBHCs, 708–9 and chronic GVHD risk, 712 gemcitabine, germ cell tumors, 956 gemtuzumab, 351, 391 AML, 763, 782 side effects, 1436 SOS, 336, 339, 1440 gender effects on quality of life, 507 gene downregulation, 50–1 gene expression profiles HSCs, 49 lymphoid genes, 49–50 myeloid genes, 49–50 progenitor cells, 49 stem cells, 49 gene profiling, 1643 non-Hodgkin’s lymphoma, 880 general anesthesia, 1585 gene therapy autoimmune disease, 276 cord blood stem cells, 568 Fanconi’s anemia, 1194–5 HIV, 1006–9, 1006 approaches to, 1007 current applications, 1008 HSCs as targets, 1009 molecular targets, 1007, 1007 protein-based suppressors, 1008 RNA-based suppressors, 1007–8 T-lymphocytes as targets, 1008–9 immunodeficiency diseases, 1115–17 thalassemia, 1079

1693

genetically modified cells, 242 genetic data, impact of clinical trials conduct, 418, 418 design, 416–17 information systems, 419 trial analysis, 423–4 observational studies, 409 genetic instability, 1642 genetic manipulation, 116–28 gene transfer, 119–22 clinical trials, 122–4, 123 expansion of gene-modified cells prior to infusion, 121 ex vivo culture conditions, 119–20 immune responses to transgenes, 121–2, 122 immunodeficient xenotransplant mouse models, 120 in vitro assays for, 120 in vivo delivery of transgenes, 121 in vivo expansion of gene-marked cells, 121 to large-animal repopulating cells, 120–1 to mouse stem cells, 120 source of stem cells, 119 gene transfer vectors, 116–19 foamy virus vectors, 118 gammaretroviral viruses, 116–17, 117, 118 lentiviral vectors, 117–18 retroviral pseudotypes for gene transfer, 118 retroviral vectors, 116, 117 history, 116 vector-mediated insertional mutagenesis, 124–6 deregulation of host genes by vector proviruses, 125, 125 leukemia in SCID-X1 trial, 124–5 reduction of malignancy potential, 125–6 genetic markers, 365 biologic insights from, 372–3 graft-versus-host disease, 372 monozygosity in twins, 372 see also chimerism, testing for genetic susceptibility, 1641–2, 1647 gene transfer, 119–22 clinical trials, 122–4, 123 ADA deficiency, 124 chronic granulomatous disease, 124 early studies, 122 SCID-X1, 122, 124 expansion of gene-modified cells prior to infusion, 121, 122 ex vivo culture conditions, 119–20 immune responses to transgenes, 121–2, 122 immunodeficient xenotransplant mouse models, 120 in vitro assays for, 120 in vivo delivery of transgenes, 121 in vivo expansion of gene-marked cells, 121 to large-animal repopulating cells, 120–1 to mouse stem cells, 120 source of stem cells, 119 gene transfer vectors, 116–19 foamy virus vectors, 118 gammaretroviral viruses, 116–17, 117, 118 lentiviral vectors, 117–18 retroviral pseudotypes for gene transfer, 118 retroviral vectors, 116, 117 genital HSV, 1384 genital organs GVHD, 1309, 1311 prevention, 1318 treatment, 1318 late complications, 1632

1694

Index

genomic diversity, 238–9, 238, 239 genomics, 159 genotype, 156 Geotrichum spp., 1560 German National Bone Marrow Donor Registry, 545 germ cell tumors, 948–57 conventional therapy, 950 high-dose chemotherapy clinical trials, 952 dose escalation, 952–3 indications for, 951 poor-prognosis tumors, 955 recurrent tumors, 951–4, 953, 954 relapse after, 955–6 salvage therapy, 954–5 surgical management after, 955 prognostic models, 953–4, 953, 954 Beyer Prognostic Score, 953, 964 Institut Gustave Roussy criteria, 955 radiographic abnormalities, 951 salvage therapies, 948 conventional, 951 staging, 949, 950 survival, 954 tumor markers, 948–51, 949–51 Gfi1, 39 Giardia lamblia, 1434 gibbon ape leukemia virus, 118 Gilda’s Club, 529 gingival bleeding, 1599 gingival hyperplasia, 1604–5 glass transition, 633 glatiramer acetate, 1021 glipizide, drug interactions, 1525 β-globin, 50 globoid cell leukodystrophy, 1138, 1152–3 glomerular filtration rate, 1473, 1474, 1475 Glucksberg grading system for GVHD, 434, 1291 glucocorticoids, 227 glucose, serum levels, 1555 glutamic acid decarboxylase, 269 glutamine, 1559, 1597 glutathione S-transferases, 292, 1642 glyburide, drug interactions, 1525 glycerol, 632 protective effect, 637 glycogen synthase kinase, 39 glycopeptides, 1326 glycoprotein disorders, 1139, 1155–6 clinical description, 1155 epidemiology and etiology, 1155 HCT, 1155–6 molecular and clinical biology, 1155 nontransplant approaches, 1155 pre-HCT evaluation, 1141 see also storage diseases glycosaminoglycans, 1143 GM-CFU, 590, 631, 1208 GM-CSF, 9, 24, 66, 89, 119, 132, 251, 646, 650, 650, 753 after allogeneic HCT, 651 after autologous HCT, 650–1 PBHC mobilization, 593 recombinant, 544 goblet cells, 249–50 goiter, 1496–7 gold, marrow injury, 705 gonadal dysfunction, 715, 1509–15, 1509, 1628–9 female, 1512–15 evaluation, 1514–15, 1515 fertility and offspring, 1514

management, 1515 menstrual cycle, 1513 ovarian function, 1513 male, 1509–12, 1510 evaluation, 1511, 1512 management, 1511 spermatogenesis, 1510 testicular function, 1510 see also sexual dysfunction gonadotropin-releasing hormone, 518, 1487 Good Guidance Practices, 534 Good Manufacturing Practices, 533 gp41, 1001 gp120, 1001 Gr-1, 120 graft failure, 681, 1201–16 ABO match, 1205 causes, 1202, 1203 chimerism testing, 371 definition, 1201 histocompatibility antigens, 1204–5, 1205 incidence, 1202 interventions, 1209–11, 1210 autologous backup, 1209–10 CD34+ infusion, 1210 donor lymphocyte infusion, 1210 growth factors, 1210 immune suppression changes, 1210 regrafting, 1210–11, 1211 poor graft function, 1202 primary, 1201–2 and reduced-intensity conditioning, 1202 risk factors age, 1205 cell number, 1208–9 diagnosis, 1206 disease states, 1206 graft manipulation, 1209 graft source, 1208 post-transplantation immunosuppression, 1208 preparative regimen, 1207 previous treatments, 1206–7 risk reduction appropriate cell numbers, 1209 donor selection, 1209 optimal preparative regimens, 1209 performance monitoring, 1209 secondary/late, 1202 T-replete marrow grafts, 659–61, 659, 659 chemotherapy regimen, 660–1 clinical presentation, 659 HLA mismatch, 659–60 patient diagnosis and immune competence, 660 post-transplant immunosuppression, 661 recipient sensitization, 660 graft manipulation, and graft failure, 1209 graft purging see purging graft rejection, 1202–4 aplastic anemia, 706, 708–10, 708, 708, 710, 1206 see also graft failure graft source, and graft failure, 1208 graft-versus-autoimmunity, 272, 280 graft-versus-Hodgkin’s effect, 870 graft-versus-host disease, 2, 3, 17, 19, 74, 188, 208– 21, 392–401, 494, 675, 1540 acute, 136–7, 208, 209, 1287–304 alloreactivity, 1287 aplastic anemia, 711–12, 711 categories of, 1310 cellular and inflammatory effectors, 211–12 conditioning regimen intensity, 1290

cutaneous, 1290 cytokine levels, 1290 cytokine secretion by donor T cells, 210–12 diagnosis and differential diagnosis, 1291 donor-recipient risk factors, 1288–9 donor T-cell activation, 208–10 effects of conditioning, 208 gastrointestinal, 1290 gene polymorphisms, 1290 grading and severity scores, 1291–2, 1291, 1292 graft composition, 1289 HC dose, 1289 hepatic effects, 1290–1 history, 1287 immunomodulation, 1290 incidence, 1288 microbial environment, 1287–8 nutritional support, 1565–6 pathogenesis, 1287–8 pathophysiology, 208–21 pharmacologic prevention, 1257–74 alloreactivity, 1257 antibodies, 1268–9 induction of anergy, 1269–70 mycophenolate mofetil, 1267–8 nonspecific immunosuppressive drugs, 1259– 63, 1260–1 photopheresis/pentostatin, 1270 predictive factors, 1257–9, 1258 reduced-intensity conditioning regimens, 213 sirolimus (rapamycin), 1266–7 specific T-cell immunosuppressive drugs, 1263–6 predictive assays, 1290 source of HCs, 1289 supportive care, 1294–5 tolerance, 1288 Toll-like receptors and innate immunity, 212–13 treatment, 1292–4, 1293 unrelated donor high-dose transplantation, 1276– 7 acute and chronic overlap syndrome, 1308 acute lethal, 178 after autologous PBPC transplantation, 544 after PBHC transplantation, 623, 623 after unrelated donor HCT, 678, 682, 692 AML, 769, 781–2 APCs inducing, 257 aplastic anemia, 710, 711–12, 711 autologous, 397, 1295 chronic, 137–8, 178, 215–16, 216, 398–401, 530–1, 1304–24, 1305, 1620–3, 1621, 1621–3 ancillary therapy and supportive care, 1316, 1318, 1319–20, 1320 aplastic anemia, 711–12 categories of, 1310 classification and staging, 1307, 1313 clinical manifestations, 1307–8 clinical trials, 1313–14, 1314 DCs in, 258–9 diagnosis, 1308 eyes, 1309, 1310–11 gastrointestinal tract, 1309, 1311 genitalia, 1309, 1311 hair, 1308–9, 1309 hematopoietic and immune system, 1309, 1311 histopathology and diagnosis, 1312–13, 1312 liver, 1309, 1311 lungs, 1311 mouth, 400–1, 400, 1309, 1310 musculoskeletal system, 1309, 1311

Index nails, 1308, 1309 nutritional support, 1566–7 pathogenesis, 1306–7, 1307 prevention, 1314–15 primary therapy, 1316 prognostic factors, 1306, 1306 quality of life, 1320, 1321 risk factors, 1304–6, 1305, 1622–3 secondary therapy, 1316, 1317–18 skin, 399–400, 399, 1308, 1309 systemic therapy, 1315, 1315 T-cell depletion, 1278 treatment, 1315, 1315 and chronic kidney disease, 1478–80, 1479 cord blood cell transplant, 563 dendritic cells in, 214, 257–9 GVLE, 259 persistence of recipient LCs, 257 esophageal, 397 fetal, 584 gastrointestinal, 393, 395–7, 396, 397, 1290, 1309, 1311 autologous, 397 chronic, 1309, 1311 conditions mimicking, 1445 differential diagnosis, 396 spectrum of, 395–6 transplant-associated microangiopathy, 397 genetic marker studies, 372 hematological malignancies, 1051 histologic grading, 393–8, 393 gastrointestinal tract, 395–7, 396, 397 liver, 397–8, 397, 398 skin, 394–5, 394 historical development, 1257 HLA-identical related donor high-dose transplantation, 1276 HLA mismatch, 693 Hodgkin’s lymphoma, 871 hyperacute, 1295 and immune reconstitution, 140, 225–6, 226 immunohistology, 392 JMML, 756–7 kinetics, 258 liver, 397–8, 397, 398 MDS, 827–8 mouse models, 176–87 addition of CD4+ cells, 179–80 donor marrow plus donor CD4+ cells, 178 plus donor CD8+ cells, 179 graft-versus leukemia responses, 183, 183 H-2 class I antigens, 179 H-2 class II antigens, 177–8 host marrow and donor CD4+ cells, 179 host-versus graft reactions, 177 immunopathology, 184 mHAs, 180 features of, 181, 181 target antigens, 180–1 MHC antigens, 177 nonirradiated hosts, 180 regulatory cells, 178–9, 179 role of CD4+ cells, 182–3, 183 role of CD8+ cells, 181–2, 181 MPD, 827–8 oral, 400–1, 400, 1600–2, 1601 treatment, 1601–2 pain management, 1583 pathogenesis, 392, 1257 prevention, 199–200, 213

blockade of inflammatory stimulation, 215–16, 216, 216 modulation of donor T cells, 213–15 see also reduced-intensity conditioning prevention by Tregs, 178–9, 179, 211 prophylaxis AML, 769 CML, 741 pulmonary, 393, 401, 402–3, 403 skin, 393, 394–5, 394, 399–400, 399 surgical pathology, 392–3 T-cell depletion to prevent, 1275–86 acute graft-versus-host disease, 1276–7, 1277 adoptive therapy with Tregs, 1280–1 chronic GVHD, 1278 early preclinical models, 1275 elimination of alloreactive T cells, 1280 engraftment, 1278–9 enrichment of CD*+ NK/T cells, 1281 haploidentical donor transplantation, 1277–8 herpes simplex-thymidine kinase suicide gene insertion, 1281 immune reconstitution and infectious complications, 1279–80 organ dysfunction, 1278 reduced-intensity transplantation, 1278 reduction of relapse, 1280 specificity, 1276 T-cell dose, 1275–6 thalassemia, 1080 transfusion-associated, 1295–6 diagnosis and clinical features, 1295 prevention, 1234, 1295–6 risk factors, incidence and etiology, 1295 therapy, 1296 treatment, MSCs, 112 and TREC levels, 226 tropism, 258 vaginal, 522, 523 see also graft-versus-tumor effect graft versus host tolerance, induction of, 199–200 cytokine manipulation, 200 delayed donor T-cell administration, 201 depletion/tolerization of T-cells, 200 T-cells developing from bone marrow progenitors, 201, 202 graft-versus-leukemia effect, 166, 183, 183, 209, 257, 259, 1321 ALL, 799 AML, 781–2 CML, 741–2 dendritic cells in, 259 JMML, 756–7 minimal residual disease, 383 see also graft-versus-host disease graft-versus-myeloma effect, 851 graft-versus-neuroblastoma effect, 978 graft-versus plasma or B cell effect, 1222 graft-versus-tumor effect, 227, 232–7, 675, 958 adoptive cellular therapy, 240–1 with antigen-specific cells, 241–2 with genetically modified cells, 242 with regulatory T cells, 242 allogeneic HCT, 233–9 B-cell contributions, 237–8 expression of mHAs, 236 haploidentical and MHC-mismatched HCT, 233– 4 human genomic diversity, 238–9, 238, 239 mHA-encoding genes, 235–6, 235, 236 mHA-specific T-cell response, 236–7

1695

MHC-matched HCT, 234 minor histocompatibility antigen, 234 molecular characterization of mHAs, 234–5 T-cell response to monopolymorphic overexpressed antigens, 237 autologous and syngeneic HCT, 232–3, 233 characteristics of, 232 donor/recipient vaccination, 240 donor selection, 239–40, 240 exploitation of, 239 genomic loci associated with, 239 hematological malignancies, 1051 mismatched donor HCT, 681 natural killer cells in, 966 solid tumors, 958 animal models, 959 clinical data, 959 mechanisms of, 966–7, 967 see also graft-versus-host disease graft-versus-tumor locus, 240 granulocytes, loss of telomere length, 69 granulocyte colony-stimulating factor see G-CSF granulocyte concentrates, 1232 granulocyte disorders, 1165, 1168–75 choice of donor, 1165 preparative regimens, 1165 qualitative, 1171 Chediak-Higashi syndrome, 1172 chronic granulomatous disease, 1172–4, 1173 Griscelli’s syndrome, 1174 LAD1, 1171–2 neutrophil actin abnormality, 1171 quantitative cartilage-hair dysplasia, 1170 cyclic neutropenia, 1170 dyskeratosis congenita, 1170–1 reticular dysgenesis, 1169–70 severe congenital neutropenia (Kostmann’s syndrome), 1168–9, 1169 Shwachman-Bodian-Diamond syndrome, 1171 timing of HCT, 1164–5 granulocyte-macrophage colony forming units see GM-CFU granulocyte-macrophage colony-stimulating factor see GM-CSF granulocyte-macrophage progenitor cells, 45, 47, 48 granulocyte-phagocyte system, 1163 granulocyte transfusions, 1232 bacterial infection, 1340 fungal infection, 1361–2 reactions to, 1236 granulocytopenia, 1164 granulomatous lesions, oral, 1604–5 granulysin, 164 granzymes, 164 and GVHD, 211 grapefruit juice, drug interactions, 1535–6, 1536 Graves’ disease, 150, 265, 267, 1015, 1496 green fluorescent protein, 75 Griscelli’s syndrome, 1174 growth disorders, 1488–91 evaluation, 1489 management, 1489–91, 1490 risk factors, 1488 growth factors bacterial infection, 1339–40 for graft dysfunction, 1210 recombinant, 645–56 growth hormone, 1488–91, 1612–13 secretion, 1488 growth hormone-releasing hormone, 1487

1696

Index

growth post-transplant, 715, 1610–16 conditioning with chemotherapy, 1610–11, 1611 conditioning with irradiation, 1611–13, 1612, 1613 oral-facial, 1613–14 Guillain-Barré syndrome, 1633, 1658 GVHD see graft-versus-host disease GVLE see graft-versus-leukemia effect GVTE see graft-versus-tumor effect H HA-1, 237, 241 HA-2, 237 HAART, 1003 AIDS-related lyphoma, 1004 immune reconstitution, 1003 haematological malignancies, rare, 1030–42, 1031 CD4+/CD56+ hematodermic neoplasm, 1039 hepatosplenic T-cell lymphoma, 1039 lymphoid adult T-cell leukemia/lymphoma, 1033–5, 1035 angioimmunoblastic T-cell lymphoma, 1037, 1038 mycosis fungoides, 1036–7 natural killer cell neoplasms, 1035–6, 1036 primary central nervous system lymphoma, 1037–9 Sézary syndrome, 1036–7 myeloid acute erythroleukemia, 1032 acute megakaryoblastic leukemia, 1032, 1033 chronic myelomonocytic leukemia, 1032–3, 1034 hypereosinophilic syndrome, 1031–2 Langerhans cell histiocytosis, 1030 systemic mastocytosis, 1030–1 subcutaneous panniculitic T-cell lymphoma, 1039 T-cell prolymphocytic leukemia, 1039 see also leukemia; lymphoma Haemophilus influenzae, 1624 vaccine, 1664, 1665, 1665, 1667 hair, GVHD effects, 1308–9, 1309 hairpin ribozymes, 1008 halobetasol, oral GVHD, 1602 hammerhead ribozymes, 1008 Hand-Schüller-Christian disease, 1030 haploidentical HCT, 233–4 AML, 785 T-cell depletion, 1277–8 haplotype, 129, 154–6, 155 ancestral, 156 conserved extended, 156 haplotype-associated polymorphisms see tagSNPs haplotype matching, 158, 684 haplotype-specific heterozygosity, 158 HapMap Project, 290 Hashimoto’s thyroiditis, 1496 Hassall’s corpuscles, 1169 Hayflick limit, 38 HB-1, 241 HCT see hematopoietic cell transplantation HCT-Comorbidity Index, 1052 HDIT see high-dose immunotherapy headache, 1653 postlumbar puncture, 1584 Health Assessment Questionnaire Disability Index, modified, 1021, 1025 health-related quality of life see quality of life Health Resources and Services Administration, 540 heart AL-amyloidosis, 917–18, 917 response to treatment, 922

heat shock proteins, 166 Hsp70, 1183 height, 1554 helper T cells, 223, 234, 242, 651, 1003, 1014 donor, 178, 179 effector functions, 177 in GVHD, 178, 179–80, 182–3, 183 type 1, 265 type 2, 265 hemangioblasts, 80–1 from hESCs, 24–5 hematoma, catheter-induced, 1250–1 hematopoiesis, 65, 72 cellular biology, 72–87 in embryos, 65 fetal lineage commitment, 48 in liver, 42–3 initiation of, 38 intraembryonic, 42 ontogeny, 41–3, 42 yolk sac, 42 hematopoietic cell transplantation, 2 allogeneic see allogeneic transplantation assessment, 20 autologous see autologous transplantation cell sources, 17 clinical benefits, 430–2, 431, 432 complications see complications costs, 430 future of, 159 indications, 15–17, 16, 16 long-term follow-up, 1626 long-term survival, 19–20, 19 outcomes research patient’s experience, 432–4 qualitative methods, 432 quality of life, 432–4, 432 patient selection, 17 phases of, 1589, 1590–1 psychosocial issues, 488–501 quality of life, 502–14 regimens, 19 results, 20 stages of, 489–95, 490 decision to undergo, 489 hospital discharge and early recovery, 493 long-term recovery, 493–5, 494 post-HCT hospitalization, 492–3 pre-HCT preparation, 491–2 supportive care, 19 syngeneic see syngeneic transplantation see also various conditions hematopoietic growth factor, 70 Fanconi’s anemia, 1187 hematopoietic progenitor cells see progenitor cells hematopoietic stem cells, 36, 73, 533 aging, 41–3, 42 alternative developmental pathways, 47 autoimmune, 276–7 chimerism testing, 365–75 after reduced-intensity conditioning regimens, 372 clinical use, 369–71, 370, 370 DNA amplification of other loci, 369 historical perspective, 365, 366 molecular cytogenetics, 366 patients at risk of recurring malignancy, 371–2, 371 patients with graft failure, 371 VNTR/STR polymorphisms, 366–9, 367, 368

in cord blood, 565–7, 566 cotransplantation with progenitor cells, 53 cryopreservation, 2–3, 631–44, 632 allogeneic transplantation, 640–1, 641 cell concentration, 637–8 cell processing, 637 chemistry and biology, 632–4, 633 cooling/warming rates, 638, 638 cryoprotectant solutions, 634–6, 636 cryoprotectant toxicity, 639–40 evaluation of efficacy, 636–7, 637 induction of apoptosis, 634 physics of cooling and warming, 634, 635 post-thaw manipulation, 639 salt and sugar content, 636 storage, 638–9 tumor cell purging, 641 donation see donation; donors expansion, 88–101, 89 clinical trials, 91–7 large-animal studies, 89 murine studies, 89 novel approaches, 90–1 preclinical studies, 88–9 xenogeneic animal models, 89–90 gene expression profiles, 49 genetic markers, 365 Hayflick limit, 38 hematopoiesis derived from, 43–4 hESC-derived, 24 transplantation of, 27 immunological compatibility, 27 isolation, 36–7, 37 long-term, 38 lymphoid cell development, 44–6, 45 maintenance, 89 migration, 39–41, 40, 41 mouse, 37, 37 multipotent, 23, 24, 36, 72, 73 adult, 81, 82, 483 nonfrozen storage, 631–2 old, 43 ontogeny, 41–3, 42, 65, 66 pluripotent, 23, 24, 36, 72, 73, 76, 81–2, 81 programmed cell death, 38 regulatory framework, governmental regulation, 533–4 self-renewal, 36 genetic pathways, 38–9 source of, and incidence of GVHD, 1289 as targets of gene transfer, 1009 totipotent, 23, 24, 36, 72, 73 trafficking and homing, 1201 transdifferentiation, 43–4, 77–82 transplantation, 51–3, 52, 53 see also hematopoietic cell transplantation unipotent, 36 young, 43 hematopoietic system, 65–6 telomeres in, 68–9 hemoglobinopathies, 484 graft failure, 1206 hemolytic complications, 1219–25 ABO incompatibility, 1219–20, 1220 management, 1220–2, 1221 delayed hemolytic reactions, 1220 pure red cell aplasia, 1219, 1222 thrombotic microangiopathy see thrombotic microangiopathy hemolytic transfusion reactions, 1235 hemolytic-uremic syndrome, 1222, 1474, 1476, 1632

Index hemophagocytic lymphohistiocytosis, 1165–238, 1168 diagnosis, 1166 survival, 1167, 1168 hemophagocytic syndrome, 1035 Hemophilus influenzae, 1331 hemorrhage, 465, 469 catheter-related, 1250–1 oral, 1599 hemorrhagic cystitis, 1473, 1480–2, 1631 complications, 1481–2 early-onset, 1481 epidemiology, 1480–1 late-onset, 1481 treatment, 1481 hemothorax, catheter-induced, 1250, 1250 hepatic see liver hepatitis acute, 1442 HSV, 1384 transfusion-transmitted, 1237–8 transmission through donor cells, 546 viral in allogeneic donors, 1435 chronic, 1447–8 hepatitis-associated aplastic anemia, 714 hepatitis B virus HCT donors, 1435 late, 1624 vaccine, 1665, 1667 hepatitis C virus, 1085 HCT donors, 1435 late, 1624 hepatocellular carcinoma, 1448 hepatocellular injury, 1448 hepatomegaly, 776, 916, 1437 AL-amyloidosis, 917, 918 hepatosplenic T-cell lymphoma, 1039 HER2, 938 herpes simplex-thymidine kinase gene insertion, 1281 herpes simplex virus, 1382–7 clinical manifestations, 1384 diagnosis, 1384–5 epidemiology, 1383 in HCT recipients, 1384 HSV-1, 1383, 1654 HSV-2, 1383–4 immunology in HCT recipients, 1383 oral, 1599 pathogenesis and immunology, 1382–3 prophylaxis, 1385–6, 1385 treatment, 1385 virology, 1382 herpesviruses, 119 oral, 1598 see also individual viruses herpes zoster, 1393 antiviral treatment, 1400–1 atypical nonlocalized, 1398 localized, 1396–7 pain management, 1583 HES, 635 hESCs see human embryonic stem cells Hickman catheter, 1246 HIG-CoA reductase inhibitors, drug interactions, 1527 high-dose chemotherapy, 287–313, 319–20, 319, 320 breast cancer clinical trials, 932–4, 933, 934, 936, 937 high-risk primary breast cancer, 934–5 patient selection, 938–9, 939 prognostic factors, 940, 941

and chronic kidney disease, 1476–7 taxanes, 303–4 clinical pharmacology alkylating agents, 296–300 anthracyclines, 303 platinating agents, 300–2 topoisomerase II inhibitors, 302–3 concentration-response effects, 287, 289 dosing body surface area, 294 indices of renal/hepatic function, 294–5 drug development trials, 296, 296 drug selection, 295–6 genetics of response variability, 290–2 drug metabolism and transport, 290–2 pharmacogenomics, 290 individualization of exposure, 293–5 pharmacodynamics, 293–5 pharmacokinetic variability, 292–3, 293 pharmacologic definition, 287 therapeutic drug level monitoring, 295 therapeutic resistance, 289–90, 289 tumor cell heterogeneity and growth kinetics, 289 vs chemoradiotherapy, 325, 325 see also individual drugs high-dose conditioning, 194–5, 316–32 AML, 779–81 bone marrow ablation alkylating agents, 320–3, 321 busulfan, 317, 319, 321–2 melphalan, 321, 323109317 nitrosoureas, 317, 320, 322–3 bone-seeking radioisotopes, 326 high-dose chemotherapy, 319–20, 319, 320 targeted radioisotopes, 326 TBI, 317–19, 317–18 chronic lymphcytic leukemia, 906, 907 complications, acute kidney injury, 1473–4 high-dose chemotherapy vs chemoradiotherapy, 325, 325 MDS, 828, 831 MPD, 828 multiple myeloma, 851, 851 neuroblastoma, 973–4, 976 nonmarrow ablative agents allogeneic transplantation, 326 carboplatin, 317, 324 cisplatin, 317, 324 cyclophosphamide, 317, 323 cytarabine, 317, 324 doxorubicin, 321, 324 etoposide, 317, 321, 323–4 ifosfamide, 317, 323 mitoxantrone, 317, 321, 324 paclitaxel, 324 RRT grading system, 316–17 second procedure, 785 sequential regimens with peripheral blood hematopoietic cell support, 326 TBI, 317–19, 317–18, 335 acute toxicity, 335–6 with chemotherapy, 318 clinical trials, 316, 325 with cyclophosphamide, 318, 324 modified regimens, 325–6 normal tissue effects, 338–9, 339 vs non-TBI regimens, 340–2, 341 high-dose immunotherapy, 1017 clinical experience, 1017 complications, 1019 immune reconstitution after, 1019

1697

patient selection, 1017 regimens, 1018–19 high-grade lymphomas, 891 highly active antiretroviral therapy see HAART high-resolution typing, 156 histiocytosis X, 1030 histocompatibility, 3, 145–62 clinical uses donor identification, 156–7, 157 haplotype matching, 158 testing of related donors, 157 testing of unrelated donors, 157–8 historical perspective, 150–4 HLA, 145 class I, 147–9, 148, 148, 149 class II, 148, 149–50 class III, 150 genes, 145–7, 146, 146, 147 MHC haplotypes, 154–6, 155 linkage disequilibrium, 154 histocompatibility antigens, 1204–5 histocompatibility testing, 157–8 Histoplasma capsulatum, 1346 history allogeneic transplantation, 1–5 animal experiments, dogs, 1–3 clinical studies, 1–2, 2, 3–4 autologous transplantation, 8–14 diseases treated by, 10–11 early attempts, 8–9, 9 in vitro treatment, 9–10 peripheral blood-derived cells, 9 HIV, 117, 483, 546, 1001–13 AIDS-related lymphoma, 1004 HAART therapy, 1004 HCT, 1005–6 nontransplant approaches, 1004–5, 1005 progression-free survival, 1005 allogeneic HCT, 1006 antiretroviral therapy, 1004 cellular and anatomic reservoirs, 1003 epidemiology, 1001–2, 1002 etiology, 1001–2, 1002 gene therapy, 1006–9, 1006 approaches to, 1007 current applications, 1008 HSCs as targets, 1009 molecular targets, 1007, 1007 protein-based suppressors, 1008 RNA-based suppressors, 1007–8 T-lymphocytes as targets, 1008–9 Hodgkin’s lymphoma, autologous transplantation, 868 immune reconstitution during antiviral therapy, 1003 molecular and clinical biology, 1002–3 replication and T-lymphocyte homeostasis, 1003 transfusion-transmitted, 1238 HLA, 3, 93, 129–30, 145, 176 and autoimmune disease, 265 class I see HLA class I molecules class II see HLA class II molecules class III see HLA class III molecules crossmatching, 694 function and polymorphism, 657 gene structure/function, 145–7, 146, 146, 147 haplotype, 129, 154–6, 658, 658 family studies, 155, 658, 658 frequencies, 155 matched donors, 679–80, 683, 1046

1698

Index

AML, 767 aplastic anemia, 706 Fanconi’s anemia, 1187–8, 1187, 1189, 1190 MHC haplotype, 234 matching for platelet transfusion, 1230 mismatch, 156, 659–60, 680–2, 683, 692–4, 693, 693, 694, 1049–50 acute GVHD, 681 ALL, 693, 815–16 alleles and antigens, 681 aplastic anemia, 715–16 graft failure, 681 GVTE, 681 MHC haplotype, 233–4 models for permissible mismatches, 682 multilocus mismatches, 682 vector of, 156–7, 157 partial match, 657–74 cell dose and use of PBPCs, 665–7, 665 human transplant studies, 665–6 immune reconstitution, 666–7 infection prophylaxis, 666 preclinical studies, 665 crossmatching, 659 donor and recipient matching, 658–9, 658 donor selection, 657–9 NK cell alloreactivity, 667–70, 667, 668, 668, 669 partial marrow T-cell depletion, 664–5 probability of identification, 659 T-cell-depleted grafts, 664 T-replete marrow grafts, 659–64 graft failure, 659–61, 659 GVHD, 661–3, 661, 662 survival, 663–4, 663 structure, 130 unrelated donor HCT, 678–9, 680, 692–4, 693, 693, 694 HLA-A1, 1204 HLA-A, 1204 HLA-C, 1204 HLA class I molecules, 147–9, 148, 148, 149, 668 antigen function, 657–8 function and polymorphism, 657–8 nonclassical genes, 147–8 polymorphic sites, 149 structure, 148 variable sites, 149 HLA class II molecules, 149–50, 668 antigen function, 657–8 antigens and alleles, 148 DRB locus, 150, 150, 152 polymorphic sites, 149 HLA class III molecules, 150 HLA-DM, 253 HLA-DO, 253 HLA-DP mismatch, 1204 HLA-KIRs, 685 HLA Workshops, 150–1 HLH-94 protocol, 1166 HMG-CoA reductase inhibitors drug interactions azole antifungals, 1526 grapefruit juice, 1536 immunosuppressants, 1530 HMSD gene, 241 Hodgkin’s lymphoma, 16, 860–77, 1004 allogeneic transplantation, 870–3 graft-versus-Hodgkin’s effect, 870 GVHD, 871 reduced-intensity conditioning, 870–2, 871 results, 872

autologous transplantation, 8, 9, 10 cell sources, 870 children, 868 elderly patients, 868 HIV-associated disease, 868 late events, 869 post-transplant therapy, 864–5 pretransplant “debulking” chemotherapy, 863–4 pretransplant radiation therapy, 864 prognostic factors, 862–3, 863, 863 relapsy, 869–70 results, 861, 861, 862 survival, 865 timing of transplantation, 865–8, 865 clinical description, 860 epidemiology, 860 and Epstein-Barr virus, 1411, 1412 etiology, 860 hematopoietic cell transplantation, 861–2, 862 high-dose therapy regimes, 873, 873 management, 1415 nontransplant approaches, 860 and MDS, 834 molecular and clinical biology, 860 radioimmunotherapy, 360 reduced-intensity conditioning, 1066 relapse, 1054, 1066 first, 866–7 management of, 869–70 second or subsequent, 867 remission first, 867–8, 867 initial, failure to attain, 865–6, 866 therapy-related diseases AML, 1638–45 myelodysplastic syndrome, 1638–45 umbilical cord transplantation, 872 varicella zoster virus, 1389, 1390 Holt-Oram syndrome, 1178 home infusion RN, 475 homing cord blood cells, 568 HSCs, 1201 mesenchymal stromal cells, 105 hormone replacement therapy, 521, 1629 Horner’s syndrome, 970 Hospital Anxiety and Depression Scale, 492, 504 hospital discharge, 493 host defenses, 1328–9, 1328 early recovery, 1328 late recovery, 1329 mid-recovery, 1329 host genes, deregulation of, 125, 125 host-versus-graft reactions, 177 aplastic anaemia, 709 B cell-mediated, 177 control of, 709 natural killer cell-mediated, 177 T cell-mediated, 177 host-versus-graft tolerance, induction of, 191 developmentally immunodeficient recipients, 194 high-dose conditioning, 194–5 immunosuppressive, nonmyeloablative conditioning protocols, 195–9, 197 resistance to engraftment of HSCs, 193–4 HOXA9, 241 Hoxb4, 26, 91 Hox genes, 66–7 HSCs see hematopoietic stem cells HSV see herpes simplex virus human antimouse antibodies, 353

human bone marrow-derived multipotent stem cells, 81, 82 human chorionic gonadotropin, germ cell tumors, 948 human embryonic stem cells, 23–31 dendritic cells from, 25 erythroid cells from, 26 global cell bank, 27 hemangioblasts from, 24–5, 25 HSCs from, 23–5 immunological compatibility, 27 isolation and/or expansion, 26 transplantation of, 26–7 immune privilege, 27–8 macrophages from, 26 NK cells from, 25–6 parthogenetic, 29, 29 patient-specific, 28, 28 T cells from, 25 human growth hormone, 598 human herpesvirus-6, 1222, 1425 diffuse interstitial pneumonia, 1457 human herpesvirus-7, 1425 human herpesvirus-8, 1425 human immunodeficiency virus see HIV human leukocyte antigen see HLA human oligopotent myeloid progenitor cells, 48 human papillomavirus, 1426 vaccine, 1668 human T-lymphotrophic virus, transfusiontransmitted, 1238 humoral hypothesis, 1 Hunter syndrome, 1137, 1148 Hurler/Scheie syndrome, 1137, 1143–8, 1145–7 Hurler syndrome, 112, 1137, 1143–8, 1145–7 HVG reactions see host-versus-graft reactions hyaluronan-mediated motility receptor, 592 hyaluronidase, 1325 hybrid resistance, 177 hydralazine, and autoimmune disease, 103 hydrocodone, 1579 hydromorphone, 1579–80, 1579, 1597 hydroxyapatite, 1125 hydroxychloroquine, 1270 GVHD, 1317 rheumatoid arthritis, 1023 hydroxyethyl starch, 632 hydroxyurea CML, 735 essential thrombocythemia, 836 sickle cell disease, 1095 hyperacute GVHD, 1295 hyperamonemia, idiopathic, 1443 hypercalcemia, 845, 1500 post-transplant osteopetrosis, 1132 hypercytokinemia, 1165 hypereosinophilic syndrome, 1031–2 hyperglycemia, 1629 hyper IgM syndrome, 585, 1113 hyperkalemia, 1500 hyperlipidemia, 1629 hypermagnesemia, 1500 hypernatremia, 1500 hyperphosphatemia, 1474 hyperprolactinemia, 516, 1487 hypersensitivity response, 266 hypersplenism, 1078 hypertension, and erectile dysfunction, 517 hyperthyroidism, 1496 Graves’ disease, 150, 265, 267, 1015, 1496 hypertriglyceridemia, 1166 hyperuricemia, 845, 1474, 1500

Index hypnosis, 491 hypnotics, drug interactions, 1533 hypoactive sexual desire disorder, 520–1 hypocalcemia, 1500 hypocellular myelodysplasia, 705 hypofibrinogenemia, 1166 hypokalemia, 1500 hypomagnesemia, 1500 hyponatremia, 1500 cisplatin-induced, 1501 euvolemic, 1500, 1501 evaluation, 1501–3, 1502 hypotonic, 1500 management, 1502–3 SIADH, 1501 treatment, 1503 hypophosphatasia, 1139, 1157 hypophosphatemia, 1500 hypopituitarism, 1487 hypotension, sepsis-induced, 1543 hypothalamic-pituitary axis dysfunction, 1487 late complications, 1628 hypothyroidism, 516, 518, 1494–6, 1495 overt, 1628 subclinical compensated, 1628 hypoxic encephalopathy, 1657 H-Y proteins, 237 I 90Y-ibritumomab tiuxetan, 351, 352, 353, 357 diffuse aggressive lymphoma, 883 non-Hodgkin’s lymphoma, 359–60 toxicity, 361 ibuprofen, 1576 ICAM-1, 193 idiopathic pneumonia syndrome, 1463 I-cell disease, 1148 idarubicin MDS, 832 reduced-intensity conditioning, 1046, 1047–8 idiopathic pneumonia syndrome, 19, 1291, 1457–64, 1540, 1627, 1627 animal models, 1460, 1460 definition, clinical course and spectrum of disease, 1457–9, 1458, 1459 diagnosis, 1541 etiology, 1460 pathogenesis, 1460–4 cellular effectors, 1461–3, 1462 mechanisms of leukocyte recruitment, 1463 targets of inflammation and injury, 1463–4 tumor necrosis factor-alpha and lipopolysaccharide, 1460–1 risk factors, 1459, 1459 treatment, 1465 idiopathic thrombocytopenic purpura, 1624 ifosfamide clinical use breast cancer, 933, 936 desmoplastic small round tumor, 994 diffuse aggressive lymphoma, 881 Ewing’s sarcoma, 986 germ cell tumors, 951, 952, 953 neuroblastoma, 973 Wilms’ tumor, 989 high-dose conditioning, 317, 321, 323 structure, 288 toxicity, 1661 IGH-MYC fusion, 807 IGkappa-MYC fusion, 807

IGlambda-MYC fusion, 807 iliac crest fracture in donors, 550 imatinib, 4, 390, 449 clinical use ALL adults, 803 children, 809 chronic myelomonocytic leukemia, 838 CML, 734, 735–6, 1061–2 hypereosinophilic syndrome, 1032 MPD, 837 Ph+ ALL, 794, 796–7 cytogenetic response, 736, 737 drug interactions, 1527 GVHD, 1318 high-dose, 737 and minimal residual disease, 382–3 molecular response, 736 monitoring disease response, 736 resistance, 735–6 response assessment, 737 toxicity, 1436 treatment response, 738 imipenem, drug interactions, 1266 immortalization, 1410, 1413 immune-mediated demyelinating polyneuropathy, 1658 immune-mediated neurologic complications, 1658 immune privilege hESCs, 27–8 MSCs, 108–10, 109 immune reconstitution, 139–40, 222–31, 680–1 adoptive cellular therapy, 228 after cord blood cell transplantation, 564–5, 565 after HDIT, 1019, 1019, 1020 after high-dose immunotherapy, 1019 after PBHC transplantation, 622–3 antiviral prophylaxis, 228–9 assessment of, 223 autologous transplantation, 223–4 cell sources, 223 autologous HCT, 223–4 cord blood cells, 225 matched-related donors, 224, 224 T-cell depletion, 225, 225 unrelated donors, 224–5 and conditioning, 226 cord blood cells, 225 effects of GVHD on, 140 enhancement of, 140 graft-versus-host disease, 225–6, 226 and GVHD, 140, 225–6, 226 GVTE, 227 HAART, 1003 immunosuppressant therapy, 226 antibody therapy, 227 cyclosporine, 226–7 glucocorticoids, 227 methotrexate, 226 improvement of, 228, 228 normal lymphoid ontogeny, 222–3, 223 opportunistic infections, 140 partially matched donor transplantation, 666–7 preparative regimens, 227 and T-cell depletion, 225, 666–7, 1279 vaccination, 228, 229 immune response, 145, 264–6 to CMV, 1370–1 control of immune reactivity, 265–6 anergy, 265 clonal deletion, 189, 195, 265, 1014, 1288

1699

immunologic ignorance, 265 regulation/suppression, 265–6 gene variation, 686–7 induction and perpetuation, 264–5 immune synapse, 164 immune thrombocytopenia, 1624 immunity, dysregulation of, 1623–4 immunization, 270–1 immunobiologic agents, 1340–1 immunocompetence, 222 immunocytochemistry, 5–6 tumor cell detection, 6 immunodeficiency, developmental, 194 immunodeficiency diseases, 1105–24 congenital disorders, 1112–14 gene therapy, 1115–17 SCID, 1105–12, 1106 X-linked γc-deficient, 1117–18 Wiskott-Aldrich syndrome, 1114–15, 1115 immunodeficient xenotransplant mouse models, 120 immunoglobulins intravenous, 1268, 1270 CMV, 1378 pooled, 1427 immunoglobulin C3b, 1164 immunoglobulin C3bi, 1164 immunoglobulin G1, 1164 immunoglobulin G3, 1164 immunoglobulin G amyloidosis, 916 antibodies, 150 immunoglobulin M, 1164 amyloidosis, 916 antibodies, 150 immunologic ignorance, 265 immunologic purging, 7–10, 8, 8 clinical studies, 11–12, 11 complement-mediated lysis, 8–9, 11 immunotoxins, 9, 12 magnetic bead depletion, 9, 11 immunology, 129–42 activation of T-cell response, 133–4, 135 cell biology of antigen presentation, 130, 131 cell movement, 130–1 donor selection for allogeneic HCT, 134–5 enhancement of immune restitution, 140 genes encoding major histocompatibility antigens, 129 genetic disparity between donor and recipient, 135–8 HLA matching, 129–30 HLA structure, 130 immune reconstitution, 139–40 effects of GVHD on, 140 enhancement of, 140 opportunistic infections, 140 immune response, 130 innate and adaptive immunity, 131–2 marrow transplantation vs solid organ transplantation, 129 minor histocompatibility antigens, 130 natural killer cell response, 132, 132 opportunistic infections, 140 self-tolerance and alloantigen recognition, 132–3, 133 tolerance, 138–9 immunomodulation, 1290 immunophilins, 214 immunoreceptor tyrosine-based activation motifs see ITAMs

1700

Index

immunosuppressants see immunotherapy; and individual drugs immunosuppressive nonmyeloablative protocols, 195–9, 197 anti-T-cell antibodies, 196–8, 197 B-cell tolerance induction, 199 co-stimulatory blockade, 198 induction of xenogeneic tolerance, 198–9 pharmacologic agents, 196 sublethal TBI, 195–6 total lymphoid irradiation, 196 immunotherapy, 3, 226 adoptive, 4, 785–6, 1063–5, 1071 allogeneic co-stimulated donor T cells, 1063–4 antigen-specific cytotoxic T cells, 1064 cytokine-induced killer cells, 1064 memory CD4+ T cells, 1064–5 natural killer cells, 1065 antibody therapy, 227 aplastic anemia, 717, 719, 719 change of regimen, 1210 cyclosporine, 192, 226–7, 728–9 drug interactions, 1529–33, 1530–2 fungal infection, 1354, 1361 glucocorticoids, 227 high-dose see high-dose immunosuppressive therapy methotrexate, 2, 3, 19, 226 neurologic complications, 1660 pediatric solid tumors, 996 PNH, 728–9 post-transplantation, 1208 side effects, renal disease, 1477 withdrawal of, 1059 see also individual drugs immunotoxins, 9 clinical studies, 12 implantable subcutaneous ports, 1246, 1246 inborn errors of metabolism, 16 MSC therapy, 112 indications for HCT, 15–17, 16, 16 infantile malignant osteopetrosis, 1127–8 HCT for, 1129–31, 1130, 1131 symptoms, 1128 infants, ALL, 819–20 infection, 401–2, 401, 469, 1543–4, 1543 adjunctive measures, 1339–41 bacterial, 1325–45 antibiotic prophylaxis after engraftment, 1339 during neutropenia, 1338–9 antibiotic resistance, 1327–8 bacterial pathogens, 1325–7, 1326, 1327 Clostridium difficile-associated diarrhea, 1335–6 compromised host defenses, 1328–9, 1328 intravascular catheter-related infections, 1337, 1338 MRSA and CA-MRSA, 1336–7 neutropenia enterocolitis/typhilitis, 1337 prevention, 1337–8 spectrum of infections, 1329–31, 1330, 1330, 1331 treatment strategies, 1331–5 vancomycin resistant enterococci, 1335 catheter-related, 1253–4, 1254 donors, risk of, 550 fungal, 1346–66 antifungal agents, 1354–5, 1356, 1357–61 see also individual drugs approach to suspected disease, 1362

Aspergillus spp., 401, 1222, 1349–50 immune response to, 1352–4, 1353 biology and pathophysiology, 1347–9 Candida spp., 1347–8, 1598 cardiovascular system, 1348–9 dissemination with deep-tissue infection, 1348 eye and CNS disease, 1349 immune response to, 1351–2 mucocutaneous infection, 1348 skin lesions, 1349 candidemia, 1348 dematiaceous molds, 1351 diagnosis not based on culture, 1351 epidemiology, 1346–7, 1347 food-related, 1560 Fusarium spp., 401, 1351 granulocyte transfusions, 1361–2 immunotherapy, 1361 liver, 1435 prevention, 1354–5 primary prophylaxis, 1355, 1357 secondary prophylaxis, 1357 Scedosporium spp., 1351 surgical approaches, 1362 zygomycetes, 1350–1 late, incidence of, 223 oral, 1597–9 bacterial, 1598–9 early, 1597–8 fungal, 1598 late, 1599 viral, 1598 prior to transplant, 451 prophylaxis, 228–9 and T-cell depletion, 1279 transfusion-transmitted, 1237–40 Creutzfeldt-Jakob disease, 1239 Epstein-Barr virus, 1238 hepatitis, 1237–8, 1237 HIV, 1238 human T-lymphotrophic virus, 1238 laboratory testing, 1239 parasitic diseases, 1238–9 parvovirus, 1238 syphilis, 1237 West Nile virus, 1240 infection control, 1425 infectious interstitial pneumonia syndromes, 402, 712–13, 1456–7, 1457 infectious mononucleosis, 1410–11 infectious neurologic complications, 1654–5, 1655, 1656 infectious tolerance, 192 infertility, 518, 530 counseling, 444 infiltrative cardiomyopathy, 916 inflammatory effectors, 688 blockade of, 215–16, 216, 216 and GVHD, 211–12 inflammatory response, 1164 infliximab, 1270 GVHD, 1269, 1294, 1317 rheumatoid arthritis, 1023 influenza viruses, 1423, 1541, 1664 clinical significance, 1423 diffuse interstitial pneumonia, 1457 risk factors, 1423 treatment and prevention, 1423 informal caregivers, psychosocial issues, 497–8 informed consent children, 452, 480

donors, 480–2, 491, 547–8 ethical issues, 479–82 patients, 479–80 infused tumor cells contribution to relapse, 10, 11 marker gene studies, 13 innate immunity, 131–2 role in GVHD, 212–13 inolimomab, 1293 inpatient staff RN, 475 in situ hybridization, chimerism, 366 insulin-induced hypoglycemia test, 1508 insulin-like growth factors, 1487, 1488, 1488 insulin-like growth factor receptors, 1488 insulin resistance, 1488, 1633 integrative medicine, 1560–1 integrin-β7, 168 integrins, 43, 1163–4 intensive care see critical care intensive care RN, 475 intent-to-treat principle, 419–20 intercellular adhesion molecule-1 see ICAM-1 interferons, 854 interferon-alpha CML, 735 Epstein-Barr virus, 1414 JMML, 754 interferon-beta 1a, 1021 interferon-beta 1b, 1021 interferon-γ, 132, 1287 in GVHD, 210 interim analyses, 420 interleukin-1, 753, 1287 role in GVHD, 212 secretion by macrophages, 1163 and stem cell self-renewal, 89 interleukin-1A, 687 interleukin-1B, 687 interleukin-2, 163, 222, 595, 646, 653, 688, 1014, 1287, 1427 in GVHD, 210 post-transplant, 1068–9 interleukin-2 receptor, 1014 interleukin-3, 24, 39, 120, 595, 647 and stem cell self-renewal, 89 interleukin-3 receptor see CD123 interleukin-3Rα, 48 interleukin-4, 265 interleukin-5, 265 interleukin-6, 24, 39, 120, 265, 1222, 1287 interleukin-7, 222 and B cell development, 46 interleukin-7R, 44–5 interleukin-7Rα, 48 interleukin-8, 598, 1222 interleukin-10, 265, 687 interleukin-11, 39, 646, 652–3 interleukin-12, 169 interleukin-13, 265 interleukin-15, 163, 1411 interleukin-17, 265, 598 interleukin-17F, 265 interleukin-18, 169, 688 interleukin-22, 264 intermediate-resolution typing, 156 internal tandem duplications, 762 International Bone Marrow Transplant Registry, 434, 731 grading of acute GVHD, 1292 International Fanconi Anemia Registry, 1178, 1180 International Histocompatibility Workshops, 146

Index International Myeloma Foundation, 529 International Neuroblastoma Staging System, 970, 971 International Prognostic Scoring System, 827, 828 and transplant outcome, 833 International Society for Cellular Therapy, 534 International Society for Hematotherapy and Graft Engineering, 534 International Standards for Cord Blood Processing, Testing, Banking, Selection and Release, 535 interstitial pneumonitis, 338–9, 339 intestinal decontamination, 1288 intestinal pseudo-obstruction, 1447 intraembryonic hematopoiesis, 42 intrahepatic coagulation, 1440 in utero transplantation, 577–89 animal studies, 578–9 large animal models, 579–80, 579 murine model, 580–2, 580, 581 clinical experience, 586 clinical use, 584 diseases amenable to, 585 diseases with selective advantages, 585–6 donor cell source, 584–5 fetal hematopoietic environment, 578, 578 fetal immunologic tolerance, 577–8 maternal and fetal risk, 585, 585 strategies for success, 582–4 invasive procedures, transfusion prophylaxis, 1233 inverted terminal repeats, 119 in vitro fertilization, 520 involved field radiotherapy, 345, 1067–8 iodine-131, 351, 352, 353, 354 4-iodo-4-deoxydoxorubicin, 915 IPEX syndrome, 190, 1015, 1016 iproplatin, neuroblastoma, 973 iPS cells, 28 IRIS trial, 382 iron chelators, 1078–9 iron overload, 453, 1221, 1436, 1448 nutritional support, 1565 irradiation of blood components, 1233–4 quality control, 1234 storage, 1234 irradiation protection effect, 1 irradiation syndrome, 3 irradiation, total body see total body irradiation ischemic retinopathy, 1630 isohemagglutinins, 1219 ITAMs, 132 ITIMs, 165 itraconazole, 1355, 1358, 1359, 1442, 1475 drug interactions, 1266, 1524–5, 1526 immunosuppressants, 1530, 1531, 1532 J JAK2 mutation, 836 JAK3 SCID, 585 jaundice, 1290, 1437 jaw, osteonecrosis, 1492 JC virus, 1426 Jehovah’s Witnesses, 480 Jka blood group antigens, 1227 alloimmunization, 1222 JMML see juvenile myelomonocytic leukemia Joint Accreditation Committee of ISCT-Europe and EBMT (JACIE), 535 jugular vein external, catheterization, 1248 internal

catheterization, 1247–8, 1247 venous thrombosis, 1245 JunB, 39 junb, 39 juvenile CML, 16 juvenile idiopathic arthritis, 1025–6 juvenile myelomonocytic leukemia, 751–60, 838 chromosomal abnormalities, 752–3 classification, 751 clinical presentation, 751 epidemiology, 751 HCT, 755–8 chimerism, 757 donor lymphocyte infusion, 757 early experience, 755 GVHD, 756–7 GVLE, 756–7 nonrelapse mortality, 755 outlook, 758 preparative regimen, 755 relapse, 756, 756 second transplantation, 757 splenectomy, 758 stem cell source, 757 umbilical cord blood transplantation, 757 hematologic features, 752, 752, 755 interferon-alpha, 754 low-dose intensive chemotherapy, 754 molecular pathogenesis, 753, 753 natural course, 753–4, 754 in neurofibromatosis 1, 751–2 nontransplant approaches, 754 in Noonan’s syndrome, 752, 753 prognostic factors, 753–4, 754 Ras-dependent pathways, 754–5 retinoic acid, 754 juvenile xanthogranuloma, 751 K Kaplan-Meier survival analysis, 422 Kaposi’s sarcoma, 1004, 1085 Karnofsky score, 448 children, 453 Kell blood group antigens, alloimmunization, 1222 Keller, Gordon, 23 keratinocyte growth factor, 646, 653, 1288, 1340, 1597 keratoconjunctivitis sicca, 1620 ketoconazole, drug interactions, 1266 ketoprofen, 1576 ketorolac tromethamine, 1576 KIAA0223, 237 Kidd blood group antigens, 1227 kidney acute failure, 1474 acute injury, 1473–5, 1474, 1476 epidemiology, 1473 management, 1475 pathogenesis, 1474–5, 1474 risk factors, 1473–4 AL-amyloidosis, 917, 918 response to treatment, 922 chronic disease, 1474, 1475–8, 1475, 1477, 1478 and albuminuria, 1480 clinical syndromes, 1475–8 epidemiology, 1475–6, 1475 and GVHD, 1478–80, 1479 late complications, 1632 killer Ig-like receptors see KIRs kinesthetic learners, 527

1701

KIRs, 132, 153, 164–5, 164, 165, 223, 233, 697, 1370 genetics, 670 in GVTE, 966 HLA-incompatible HCT, 667, 667 inhibitors, 683 KIR genes, 688 activation of donor T cells, 688 cytokine and immune response gene variation, 686–7 cytokine storm, 687 inflammatory effectors, 688 inhibitory KIR receptors, 683–4 KIR haplotypes and activating receptors, 686 models for donor inhibitory KIR-mediated killing, 684–6, 685 KIR-L mismatching, 170–1, 694 KIT, 1031 KIT ligand, 90, 222 Klebsiella spp., 1329 Klebsiella pneumoniae, 845, 1327 Klinefelter’s syndrome, and ALL, 791 Kostmann’s granulocytopenia, and AML, 775 Kostmann’s syndrome, 16, 761, 834, 1168–9, 1169 Krabbe disease, 1152–3 KRAS, 752, 753 Kurtzke Expanded Disability Status Scale, 1020 L lactate dehydrogenase, germ cell tumors, 948 LADI see leukocyte adhesion deficiency type I lamina propria, 250 lamivudine, 1435 lamotrigine, drug interactions, 1535 Langerhans cell histiocytosis, 1030 Langerhans cells, 248, 249 recipient, persistence of, 257 Langerhans, Paul, 248 langerin, 248, 249 Lansky Play-Performance Scale, 453 lapatinib, 932 large-animal models, 120–1 expansion of HSCs, 89 large B-cell lymphoma, 879 laser therapy, oral GVHD, 1602 late effects, 506 latency membrane proteins, 1410 lay language, 527 LCs see Langerhans cells learning styles, 527 lectins, 1411 leflunamide, rheumatoid arthritis, 1023 Legionella spp., 401, 1329 lenalidomide AL-amyloidosis, 922 MDS, 830 multiple myeloma, 847, 855 lenograstim, 551 PHBC mobilization, 618 see also G-CSF lenolidomide, multiple myeloma, 847, 855, 1063 lentiviral vectors, 117–18 lentiviruses, 1002 letrozole, 932 Letterer-Siwe disease, 1030 leucovorin, 1263 leukapheresis, 549, 978 Leukemia & Lymphoma Society, 529 leukemia, 44, 74 acute lymphoblastic see acute lymphoblastic leukemia acute megakaryoblastic, 1032, 1033

1702

Index

acute myeloid see acute myeloid leukemia autologous transplantation, 11 chronic myelogenous, 53 chronic myeloid see chronic myeloid leukemia chronic myelomonocytic, 1032–3, 1034 and Fanconi’s anemia, 1192 malignant transformation in, 53–4 mHA expression in, 236 mixed-lineage, 762, 792 origin of, 53 radioimmunotherapy, 355–7, 356, 357 T-cell prolymphocytic, 1039 varicella zoster virus, 1389 see also different types leukemia inhibitory factor, 42 leukemic stem cells, 51, 53, 54 markers of, 54 leukemic transformation, 54 Leukine, 544 leukoagglutination, 150 leukocytes, adverse effects in blood components, 1227 leukocyte adhesion deficiency type I, 1164, 1171–2 leukocyte interferon, CMV, 1373 leukocyte-reduced red cells, 1226–7, 1227 leukocyte-reduced transfusion components, 1234 leukocytic events, 1163, 1164 leukodystrophies, 1138, 1149 globoid cell, 1138, 1152–3 metachromatic, 112, 1138, 1153–4 pre-HCT evaluation, 1142 X-linked, 1149–52, 1150, 1151, 1152 see also storage diseases leukoencephalopathy, 1633 leukoplakia, 1310 levofloxacin, 1339 levorphanol, 1579 Lewis blood group antigens, 1227 alloimmunization, 1222 lichen planus-like GVHD genitalia, 1311 mouth, 1310 skin, 399, 1308 lichen sclerosus-like GVHD, 1308 Li-Fraumeni syndrome, 931, 991, 1647 light microscopy, tumor cell detection, 6 lineage commitment, 48 and gene downregulation, 50–1 lineage restriction, 50–1 linezolid, 1326, 1333 drug interactions, 1534, 1535 linkage disequilibrium, 145, 154, 159, 267 linked suppression, 192 lip care, 1594 lipid abnormalities, 1503–6 lipopolysaccharide, 1287 in GVHD, 212 in idiopathic pneumonia syndrome, 1460–1 liquid nitrogen, 638–9 Listeria monocytogenes, 1560 meningitis, 1654 listservs, 529 liver AL-amyloidosis, 917, 918 response to treatment, 922 complications, 1438, 1544–5 acute hepatitis, 1442 acute hepatocellular injury, 1448 cholestatic disorders, 1442 chronic liver disease, 1435 cirrhosis, 1447–8

dysfunction post-transplant, 1437 pretransplant, 1435–6 fungal abscess, 1448 fungal infections, 1435, 1442 GVHD, 393, 397–8, 397, 398, 1290–1, 1309, 1311 hepatocellular carcinoma, 1448 hepatomegaly, 1437 iron overload, 1436, 1448 late, 1632–3 nodular regenerative hyperplasia, 1448 sinusoidal injury, 1475 SOS, 1437, 1438, 1439–41, 1439, 1441 viral hepatitis in allogeneic donors, 1435 chronic, 1447–8 pretransplant evaluation, 390–1 regeneration, 79 toxicity, 1052 liver function indices, chemotherapy dose based on, 294–5 LMP-1, 1646 local anesthesia, 1585 lomustine, high-dose conditioning, 320 long-rank test, 422–3, 422 long-term follow-up RN, 475 long-term function, 505–6 cognitive function, 506 fatigue, 505–6 long-term hematopoietic stem cells, 38 long-terminal repeats, 116, 117 long-term recovery, 493–5, 494 nursing care, 474–5, 474 long-term survival, 19–20, 19 causes of death, 19 Lorenzo’s oil, 1149 losartan, drug interactions, 1525 loss of heterozygosity analysis, 1644–5 lovastatin drug interactions azole antifungals, 1526 grapefruit juice, 1536 immunosuppressants, 1530 side effects, 1658 low-resolution typing, 156 LPAM-1, 212 L-selectin, 1163, 1164 lung GVHD, 1311 prevention, 1319 treatment, 1319 see also pulmonary lung cancer, small cell, 16 lung injury, 1456–72 diffuse alveolar hemorrhage, 1291, 1458, 1539, 1540, 1542 evolution of, 1467–8 idiopathic pneumonia syndrome, 19, 1291, 1457–64, 1540, 1627, 1627 animal models, 1460, 1460 definition, clinical course and spectrum of disease, 1457–9, 1458, 1459 diagnosis, 1541 etiology, 1460 pathogenesis, 1460–4 cellular effectors, 1461–3, 1462 mechanisms of leukocyte recruitment, 1463 targets of inflammation and injury, 1463–4 tumor necrosis factor-alpha and lipopolysaccharide, 1460–1 risk factors, 1459, 1459

infectious interstitial pneumonias, 1456–7, 1457 obstructive/restrictive lung disease, 1464–7, 1466 definition, risk factors and clinical course, 1464–5 obstructive lung disease, 1466–7 restrictive lung disease, 1465–6 pathogenesis, 1467 treatment, 1468–9 lupus anticoagulant, allogenic HCT for other primary disease, 1017 luteinizing hormone, 518, 522 lutetium-177, 354 lymphoblastic lymphoma, 879 lymphoblastoid cell lines, 1410 lymphocytic satellitosis, 394, 394 lymphocytopenia, apheresis-related, 552 lymphoid cell development, 44–6, 45 lymphoid gene expression profiles, 49–50 lymphoid haematological malignancies adult T-cell leukemia/lymphoma, 1033–5, 1035 angioimmunoblastic T-cell lymphoma, 1037, 1038 mycosis fungoides, 1036–7 natural killer cell neoplasms, 1035–6, 1036 primary central nervous system lymphoma, 1037–9 Sézary syndrome, 1036–7 lymphoid promiscuity, 49 lymphoid tissue dendritic cells, 250, 251 lymphoma and AIDS, 1004 HAART therapy, 1004 HCT, 1005–6 nontransplant approaches, 1004–5, 1005 Burkitt’s see Burkitt’s lymphoma central nervous system, 880, 891, 1037–9 Hodgkin’s see Hodgkin’s lymphoma non-Hodgkin’s see non-Hodgkin’s lymphoma reduced-intensity conditioning, 1047–8 subcutaneous panniculitic T-cell, 1039 therapy-related, 1645–7 late-onset, 1646 lymphoproliferative disorder, 1645–6 B-cell, 1645–6 T-cell, 1646 risk factors, 1645 varicella zoster virus, 1389 lymphoproliferative disorder, post-transplant, 391, 1645–6 B-cell, 1645–6 T-cell, 1646 lymphoreticular system, pretransplant evaluation, 391 lyonization, 1165 lysosomes, 1136 M M195, 355 Mac-1, 120 macrolide antibiotics, 1326 drug interactions, 1527 macrophage colony-stimulating factor, 1125 treatment of osteopetrosis, 1129 macrophage disorders, 1163–8, 1165 choice of donor, 1165 hemophagocytic lymphohistiocytosis, 1165–948, 1166, 1167, 1168 preparative regimens, 1165 timing of HCT, 1164–5 macrophage inflammatory protein-1 alpha, 1001 macrophages, 264 from hESCs, 26 role in GVHD, 212 macropinocytosis, 253

Index magnesium, 1555 magnesium salicylate, 1576 magnetic bead depletion, 9 clinical studies, 11 maintenance therapy, 1068 major histocompatibility antigens genes encoding, 129 response to, 130 major histocompatibility complex see MHC major minor antigens, 209 major molecular response, 382 malaria, transfusion-transmitted, 1238 malignant cells, elimination of, 1672 malignant disease children, 452 post-transplant, 1443 esophageal cancer, 1449 hepatocellular carcinoma, 1448 oral, 1599–600 thyroid carcinoma, 1497–8 see also individual conditions MALT, 250 MALT lymphoma, 879 mannose macrophage receptor, 252 α-mannosidosis, 1139, 1155–6 mantle cell lymphoma, 879, 889–90 allogeneic HCT, 889–90, 890 autologous HCT, 889, 889 marble bone disease, 1127 marginal zone lymphoma, 879 margination, 1163 marker genes, 13 Maroteaux-Lamy syndrome, 1137 HCT, 1148 marrow-isolated adult multilineage inducible cells, 81, 82 marrow transplant nephropathy, 1632 matched donors, 679–80, 683, 1046 AML, 767 Fanconi’s anemia, 1187–8, 1187, 1189, 1190 MHC haplotype, 234 Mathé, George, 2 matrix metalloproteinases, 40 MMP-9, 591 maximum tolerated dose, 316 M blood group antigens, alloimmunization, 1222 mcl1, 39 MDASI, 504 MDM2, 991, 992 MDMX, 992 MDR1, 292 MDS see myelodysplastic syndromes measles virus, 1424 mechlorethamine, retinoblastoma, 993 meclofenamate, 1576 medical care plan, 462 medical decision-making, 478 medical eligibility of donors, 547 megakaryocyte/erythrocyte progenitor cells, 47 megakaryocyte growth and development factor, 120, 646, 652 melanoma, 958 reduced-intensity conditioning, 965 tumor-associated antigens, 959 melphalan, 297–8, 755, 1219, 1608 clinical use AL-amyloidosis, 921, 922, 924 ALL, 814 breast cancer, 933, 935, 936 CLL, 903, 907 Ewing’s sarcoma, 987

Hodgkin’s lymphoma, 873 MPD, 837 multiple myeloma, 848, 853 dose intensification, 854, 854 novel conditioning regimens, 854 optimization of, 854 neuroblastoma, 974 non-Hodgkin’s lymphoma, 359 osteosarcoma, 992 rhabdomyosarcoma, 989 and graft failure, 1206 HDIT, 1018 high-dose conditioning, 317, 319, 320, 321, 323 with TBI, 318, 335 mechanism of action, 298 metabolism, 298 and PBHC mobilization, 597 pharmacokinetics/pharmacodynamics, 288, 298 reduced-intensity conditioning, 1046, 1047–8 structure, 288 membranous nephropathy, 1475 Memorial Symptom Assessment Scale-Short Form, 508 memory T cells, 135 men gonadal dysfunction, 1509–12, 1510 evaluation, 1511, 1512 management, 1511 spermatogenesis, 1510 testicular function, 1510 sexual dysfunction, 516–18, 1516–17 biological/physiologic variables, 516–18, 517 meningococcal conjugate vaccine, 1668 menstrual cycle, 1513 meperidine, 1579, 1579, 1597 mercaptopurine, JMML, 754 mesenchymal stem cells see mesenchymal stromal cells mesenchymal stromal cells, 72, 74–5, 102–15, 586 biology, 103 B lymphocytes, 107 clinical uses, 110–11 GVHD, 112 inborn errors of metabolism, 112 in cord blood, 567 cytokine secretion, 105 dendritic cells, 108 ectopic tissue formation, 106 expansion potential, 104–5 future considerations, 112–13 in GVHD, 1294 heterogeneity, 104 history, 102 homing and migration, 105 HSC expansion, 111–12 immune privilege, 108–10, 109 immunobiology, 106–7, 106 in vitro differentiation potential, 105 in vivo animal models, 108 isolation, 104 lifespan, 105 malignant transformation, 106 migration to tumors, 105 morphology, 103–4 natural killer cells, 107–8 nomenclature, 102–3 physiologic role, 110 prospective isolation, 104 surface phenotype, 104 T cells, 107, 107 tissue sources, 104

1703

transdifferentiation, 77–82 neurons, 80 working definition, 103, 103, 103 mesna, hemorrhagic cystitis, 1481 mesoderm, 72, 77–9 meta-analysis, 408, 436, 436 metabolic abnormalities, 1500 metabolic encephalopathy, 1653, 1657 metabolic neurologic complications, 1657–8 metabolic problems, 1633, 1633 metabolic syndrome, 453, 1503–6, 1633, 1633 metachromatic leukodystrophy, 112, 1138, 1153–4 clinical description, 1153 epidemiology and etiology, 1153 HCT, 1153–4 molecular and clinical biology, 1153 nontransplant approaches, 1153 metalloproteinase-9, 591 metapneumovirus, 1424, 1456–7 diffuse interstitial pneumonia, 1457 methadone, 1579 drug interactions, 1527 methicillin-resistant Staphyloccus aureus see MRSA methotrexate, 2, 3, 19, 226, 776, 1220, 1270 AIDS-related lymphoma, 1004 breast cancer, 932 clinical use, 1262–3 CNS leukemia, 794 Fanconi’s anemia, 1189 GVHD prevention, 1259, 1262–3, 1263 rheumatoid arthritis, 1023 dose adjustments, 1263 GVHD, 711, 741, 1317 host-versus-graft reactions, 709 pharmacology, 1262 reduced-intensity conditioning, 1047–8 toxicity, 1262, 1660 methylprednisolone, 1262, 1292 AIDS-related lymphoma, 1005 CLL, 902 Fanconi’s anemia, 1189 GVHD, 1259 reduced-intensity conditioning, 1047–8 metoprolol, drug interactions, 1535 metronidazole, 1288 metyrapone tst, 1508 mHAs, 130, 176, 234, 252 expression of, 236 genetic classification, 235–6, 235, 236 and genomic diversity, 238–9, 238, 239 GVHD to, 180, 181 features of, 181, 181 in GVTE, 967 molecular characterization, 234–5 response to, 130 target antigens, 180–1 mHA-specific T-cell responses, 236–7 MHC, 145, 146, 165, 188, 264, 577 historical perspective, 150–1 linkage disequilibrium, 154 Msats, 158 pathogenesis of autoimmune disease, 267 resident transplantation determinants, 158 structure, 146 transduction of autologous bone marrow, 192 MHC alloantigens, 176 GVHD directed to, 177 MHC class I, 25 GVHD to, 179

1704

Index

MHC class II, 249, 1014 autoimmune disease, 1015, 1015 GVHD to, 177–8 MHC genes, susceptibility to autoimmune diseases, 267 MHC haplotype, 154–6, 155, 682–3, 684 matched donors, 234 mismatched donors, 233–4 MICA, 149, 166 micafungin, 1358, 1359 drug interactions, 1529 immunosuppressants, 1532 fungal infection, 1355 MICB, 149, 166 microarrays, 49 microchimerism, 582 microglia, 80 β2-microglobulin, CLL, 899 microRNA CLL, 897 leukemogenesis, 54 microsatellite instability, 1642 microsatellite markers, 682 midazolam drug interactions azole antifungals, 1526, 1528 grapefruit juice, 1536 migration of HSCs, 39–41, 40, 41 minimal-change disease, 1478 minimal residual disease, 5, 376–89 ALL in adults, 379–80, 379, 795 post-transplant, 380–1, 380 AML, 381 post-transplant, 381–2 assays, 376, 377, 377 chromosomal assays, 376 disadvantages of, 384 FISH probes, 378 flow cytometry, 376 lack of standardization, 385 polymerase chain reaction, 376, 379 clinical significance, 379 CLL, 383, 905–6, 906 CML, 382–3 post-transplant, 383 dormancy, 384–5 multiple myeloma, 384 minimum effective analgesic concentration, 1578 minisatellite cores, 366, 367 mini-transplant, 448 minor histocompatibility antigens see mHAs MIRN15A, 897 MIRN16A-1, 897 mirtazapine, drug interactions, 1535 mismatched donor transplantation, 156, 680–2, 683, 1049 acute GVHD, 681 ALL adults, 693 children, 815–16 alleles and antigens, 681 aplastic anemia, 715–16 graft failure, 681 GVTE, 681 MHC haplotype, 233–4 models for permissible mismatches, 682 multilocus mismatches, 682 vector of, 156–7, 157 missing ligand mechanism, 697, 698 missing recipient ligand mechanism, 686 missing self recognition, 27, 233, 234, 7240

mitomycin C, 1180, 1185 mitosis, 66 mitoxantrone, 1021 AML, 782 breast cancer, 933, 935, 936 high-dose conditioning, 317, 321, 324 mixed chimerism, 194, 195, 365 aplastic anemia, 710 mixed-lineage leukemia, 762, 792 MLL, 806, 819 MLL-AF4 fusion, 807 MLL-ENL fusion, 807 MMP see matrix metalloproteinases MMR vaccine, 1665, 1667 mobilization and collection, nursing care, 463–4, 463 molecular biology, 64–72 detection of malignancy, 6 hematopoiesis, 65 hematopoietic system, 65–6 stem cell fate, 66 stem cell hierarchy, 64–5 telomeres checkpoint, 69 in hematopoietic system, 68–9 structure and function, 67–8 transcriptional control of stem cell function, 66–7 molecular cytogenetics, 366, 376 molecular genetics autoimmune diseases, 1014–15, 1015 osteopetrosis, 1128–9 molecular mimicry, 268, 370 molgramostin see GM-CSF MOLM-13 cell line, 54 Moloney murine leukemia virus, 117 monoamine oxidase inhibitors, drug interactions, 1534, 1535 monoclonal antibodies, 189, 351 ALL, 800 purging, 7–10, 8, 8 radiolabeled, 1045 see also individual drugs monocyte-derived dendritic cells, 251 monocyte-phagocyte system, 1163 monosomy 7 syndrome, 751 montelukast, GVHD, 1318 morphine, 1579, 1579, 1597 Morquio syndrome, 1137 HCT, 1149 mortality bone marrow donation, 551 HC donation, 551 PBHC transplantation, 623–4 PBPC donation, 552–3 transplant-related, HLA mismatch, 693 mosaicism in Fanconi’s anemia, 1185–6, 1185 MOS Social Support Survey, 504 mouse HSCs, 37, 37 properties of, 37–8, 38 transplantation, 51–3, 52, 53 mouse models allogeneic transplant, G-CSF in, 649–50 autoimmune disease, 270 embryonic stem cells, 23–4, 24 transplantation of HSCs, 26–7 expansion of HSCs, 89 GVHD, 176–87 addition of CD4+ cells, 179–80 anti-mHA, 181, 181 donor marrow plus donor CD4+ cells, 178 donor marrow plus donor CD8+ cells, 179 graft-versus leukemia responses, 183, 183

H-2 class I antigens, 179 H-2 class II antigens, 177–8 host marrow and donor CD4+ cells, 179 host-versus graft reactions, 177 immunopathology, 184 mHAs, 180 mHA target antigens, 180–1 MHC antigens, 177 nonirradiated hosts, 180 regulatory cells, 178–9, 179 role of CD4+ cells, 182–3, 183 role of CD8+ cells, 181–2, 181 immunodeficient xenotransplant, 89–90, 120 in utero transplantation, 580–2, 580, 581 osteopetrosis, 1126 stem cells embryonic stem, 23–4, 24 gene transfer to, 120 mouth see oral moxifloxacin, 1339 Moyamoya disease, 1094 MPD see myeloproliferative disorders MRSA, 1336–7 MSAS, 504 Msats, 158 MSCs see mesenchymal stromal cells mucocutaneous infection, 1348 mucolipidoses, 1137, 1143 clinical description, 1143 epidemiology and etiology, 1143 HCT, 1143–9 pseudo-Hurler polydystrophy, 1148 molecular and clinical biology, 1143 nontransplant approaches, 1143 pre-HCT evaluation, 1141 see also storage diseases mucopolysaccharidoses, 1137, 1140, 1143 clinical description, 1143 epidemiology and etiology, 1143 HCT Hunter syndrome, 1148 Hurler, Hurler/Scheie and Scheie syndromes, 1143–8, 1145–7 Maroteaux-Lamy syndrome, 1148 Morquio syndrome, 1149 Sanfilippo syndrome, 1148–9, 1148 Sly syndrome, 1148 molecular and clinical biology, 1143 nontransplant approaches, 1143 pre-HCT evaluation, 1141 see also storage diseases mucormycosis, 1350–1 mucosa-associated lymphoid tissue see MALT mucosal care, 1594 mucosal dendritic cells, 249–50, 250 mucosal ulceration, 1434 mucositis, 465, 469, 1444, 1545 nutritional interventions, 1557 oropharyngeal, 1545, 1595–7, 1595, 1596 pain management, 1582 radiation-induced, 335 multiattribute utility theory, 431–2 multidrug resistanc protein-8, 54 multilocus mismatches, 682 multiple myeloma, 15–16, 16, 845–59 allogeneic HCT, 851–3 high-dose preparative regimens, 851, 851 reduced-intensity conditioning, 851–3, 852, 853 alternative donor HCT, 853 autologous HCT, 10–11, 847–51 cell source, 848

Index patient eligible for, 847 patient selection, 848 patients not eligible for, 847 preparative regimen, 847–8, 848 purging of myeloma cells, 848–9 single vs tandem, 850, 850 timing, 849–50 vs conventional therapy, 849, 849 cellular biology, 845 clinical manifestations, 845–6 diagnosis, 846, 846 donor leukocyte infusion, 1063 epidemiology, 845 etiology, 845 high-dose melphalan, 853 dose intensification, 854, 854 novel conditioning regimens, 854 optimization of, 854 maintenance therapy, 854–5, 855 minimal residual disease, 384 molecular biology, 845 nontransplantation approaches, 847 prognosis, 847 reduced-intensity conditioning, relapse rate, 1054 relapse, 1054 remission, 232 response to treatment, 847 staging, 846, 847 multiple organ failure, 1657 multiple organ system failure, 1542, 1545, 1546, 1547 multiple sclerosis, 265, 1019–21, 1020 allogenic HCT for other primary disease, 1017 clinical manifestations, 1020, 1020 family/twin studies, 1015 Kurtzke Expanded Disability Status Scale, 1020 susceptibility genes, 1015 treatment, 1021, 1022 Multiple Sclerosis Functional Composite, 1020–1 multiple sulfatase deficiency, 1139, 1153, 1157 multipotent progenitor cells, 26, 37, 81, 82 adult, 81, 82 properties of, 37–8, 38 multipotent stem cells, 23, 24, 36, 72, 73 adult, 81, 82, 483 Fanconi’s anemia, 1195 multivariate interval-censored survival data, 422 mumps virus, 268–9 Munc13–4 protein, 1165 murine embryonic stem cells, 23–4, 24 transplantation of HSCs derived from, 26–7 muscle cramps, 1630 muscle stem cells, 77 musculofascial problems, 1630 musculoskeletal system GVHD effects, 1309, 1311 prevention, 1320 treatment, 1320 mutations G-CSF receptor, 1168 osteopetrosis, 1125–6, 1126 perforin gene, 1165 point, 65 myasthenia gravis, 1653, 1658 autologous transplantation, 11 MYC, 991 MYCN oncogene, 970–2 mycophenolate mofetil, 1207, 1220, 1270, 1293 clinical use, 1268 DC modulation, 259 GVHD prevention, 214, 1267–8 SLE, 1023

drug interactions, 1529–30, 1531 GVHD, 1317 host-versus-graft reactions, 709 neutrophil and platelet changes, 1049 pharmacology, 1267–8 post-transplant, 664 reduced-intensity conditioning, 1047–8 toxicity, 1268, 1445 Mycoplasma, diffuse interstitial pneumonia, 1457 mycosis fungoides, 879, 1036–7 myelin basic protein, 1020 myelin oligodendrocyte glycoprotein, 270, 1020 myelitis, 391 myeloblastin, 237 myelodysplastic/myeloproliferative disorders, 837–8 atypical CML, 838 chronic myelomonocytic leukemia, 827, 837–8, 1032–3, 1034 reduced-intensity conditioning, 832 risk category, 837 survival, 838 JMML see juvenile myelomonocytic leukemia systemic mastocytosis, 838, 1030–1 myelodysplastic syndromes, 16, 74, 705, 777, 827–35, 1168 allogeneic transplantation, 828, 830–5 children, 834, 834 conditioning regimens, 838–40, 839, 840 high-risk MDS, 830, 833, 833 low-risk MDS, 830, 831, 832 secondary MDS, 834–5, 835 autologous transplantation, 840 cell source, PBPCs, 827 classification, 751 conditioning regimens, 838–40, 839, 840 high-dose conditioning, 828, 831 and mortality, 840 reduced-intensity conditioning, 828, 832 considerations for HCT, 827–8, 828 donor leukocyte infusion, 1062, 1062 and Fanconi’s anemia, 1192 graft failure, 1206 GVHD, 827–8 HLA mismatch, 693 International Prognostic Scoring System, 828 and transplant outcome, 833 pretransplant evaluation, 390 reduced-intensity conditioning, 1047–8 relapse, 1054 post-transplant, 840–1 secondary, 1019 TBI, 831 therapy-related, 1638–45 alkylating agent-related, 1639 clinical diagnosis, 1639–40 cumulative probability, 1639 pathogenesis, 1641–3, 1641 gene expression profiling, 1643 genetic instability, 1642 genetic lesions, 1641 hematopoietic abnormalities, 1642–3 polymorphisms in drug-metabolizing enzymes, 1641–2 patient outcome, 1643–4 radiation-related, 1639 risk factors, 1639, 1640–1, 1640 risk prediction, 1644–5 clonality analysis, 1644 cytogenetics and FISH, 1644 loss of heterozygosity analysis, 1644–5 PCR for point mutations, 1644–5

1705

TBI, 340 unrelated donor HCT, 677 WHO classification and criteria, 829 WHO prognostic scoring system, 829 myelofibrosis graft failure, 1206 pretransplant evaluation, 390 myeloid cell development, 46–7 myeloid gene expression profiles, 49–50 myeloid haematological malignancies acute erythroleukemia, 1032 acute megakaryoblastic leukemia, 1032, 1033 chronic myelomonocytic leukemia, 1032–3, 1034 hypereosinophilic syndrome, 1031–2 Langerhans cell histiocytosis, 1030 systemic mastocytosis, 1030–1 myeloid promiscuity, 49 myeloproliferative disorders, 16, 835–7 allogeneic transplantation, 836 conditioning regimens, 838–40, 839, 840 autologous transplantation, 840 cell source, PBPCs, 827 CML see chronic myeloid leukemia complications, 9 conditioning regimens, 838–40, 839, 840 high-dose conditioning, 828 and mortality, 840 reduced-intensity conditioning, 828 considerations for HCT, 827–8, 828 GVHD, 827–8 primary diagnosis and transplant outcome, 837 relapse, 1054 after reduced-intensity conditioning, 1054 post-transplant, 840–1 MYO1G, 237, 241 myopathy, 1630 N NAD(P)H:quinone oxidoreductase, 1641–2 nafcillin, drug interactions, 1266 nails, GVHD effects, 1308, 1309 nalbuphine, 1580 Nanog, 82 naproxen, 1576 narcotic analgesics, drug interactions, 1533 nasopharyngeal carcinoma, 1415 natalizumab, 1021 National Institutes of Health, 531 National Marrow Donor Program, 4, 481, 540, 545, 693, 693, 777 National Nosocomial Infection Surveillance project, 1539 natural antibodies, 191, 194 natural cytotoxicity receptors, 166 natural killer cell neoplasms, 1035–6, 1036 natural killer cells, 69, 163–75, 190, 194, 210, 222, 1059, 1065, 1287, 1370 adoptive cell transfer, 171 alloreactivity, 697, 698 effect of donor activating KIR genetics, 670 in HLA incompatibility, 667–70, 668, 668 CD56bright and CD56dim, 163–4 CD94 receptors, 164, 164 cellular therapy, 1069 clinical studies, 169–70, 170 cytotoxicity, 164 DC activation, 255 development from hematopoietic progenitor cells, 167–8 DNAM-1 receptors, 166 from hESCs, 25–6

1706

Index

in GVTE, 966 HVG reactions, 177 interaction with dendritic cells, 169 interaction with MSCs, 107–8 killing of leukemia targets, 166–7 KIR receptors, 164–5, 164, 165 in mobilized PB, 620 natural cytotoxicity receptors, 166 NKG2A receptors, 164, 164, 165 NKG2D receptors, 166 prevention of disease relapse, 1280 reconstitution after allo-HCT, 168–9 regulation of response, 132, 132 self-tolerance, 168, 168 sensitivity to cyclophosphamide, 1203 tolerization of, 194 and viral infections, 169 natural killer-killer immunoglobulin-like receptor ligand matching, 692 natural suppressor cells, 191 nausea and vomiting, 465, 469 differential diagnosis, 1436 nutritional interventions, 1553, 1558 opioid analgesics, 1578 post-transplant, 1436–7, 1436, 1449 radiation-induced, 335 Nectin-2, 167 nef, 118, 1002 Neisseria spp., 1597 Neisseria meningitidis, 1331, 1624 nelarabine, ALL, 803 nephrotic syndrome, 926, 1478, 1632 nephrotoxic drugs, 1475 nerve injury, catheter-induced, 1251 nervous system AL-amyloidosis, 917, 919 response to treatment, 922 nestin, 80 NetCord, 535 Neupogen, 544 neural adhesion protein see CD56 neuraminidase, 1325 neuroblastoma, 16, 970–84 allogeneic transplantation, 978 autologous BMT vs chemotherapy, 975, 976 clinical presentation and staging, 970–1, 971 complications, 980 HCT vs chemotherapy, 974–5 hematopoietic cell source and purging, 978–9 high-dose conditioning, 973–4, 974, 976 induction therapy, 972–3, 973 local control, 973 minimal residual disease, 979–80, 979 molecular biology, 971 risk classification, 971–2, 972 survival, 973 tandem transplants, 976–7 targeted radionuclides, 977–8 treatment approach, 971–2 high-risk cases, 972 neuroendocrine dysfunction, 1627–8 neurofibromatosis and ALL, 791 and AML, 775 and JMML, 751–2 type 1, 1641 neurologic complications, 1653–63 calcineurin inhibitors, 1658–60, 1659 cerebrovascular, 1655, 1657, 1657 conditioning agents, 1660–1 immune-mediated, 1658

immunosuppressants, 1660 infectious, 1654–5, 1655, 1656 metabolic, 1657–8 pretransplant neurologic screening, 1661 supportive care, 1661 symptoms and signs, 1653–4, 1654 neuronal ceroid lipofuscinosis, 1139, 1157 neuron-specific enolase, 994 neuropathic pain, 1583–4 anticonvulsants, 1584 antidepressants, 1577, 1583–4 opioid analgesics, 1584 topical preparations, 1584 neuropilin-1, 211 neurotoxicity, 1603 neutropenia, 1052, 1168, 1624 cyclic, 1170 ganciclovir-induced, 1374 severe congenital, 1168–9, 1169 neutropenia enterocolitis/typhilitis, 1337 neutrophil actin abnormality, 1171 neutrophil elastase, 591 neutrophils, 620, 1163, 1164 Nezelof syndrome, 1112 nicardipine, drug interactions, 1266 Niemann-Pick disease, 1139, 1157 Nijmegen breakage syndrome, 1178 nilotinib, 737–8 ALL, 803 CML, 1061–2 nitric oxide, in sickle cell disease, 1092, 1093 nitrogen mustard, and PBHC mobilization, 597 nitrosoureas, high-dose conditioning, 317, 320, 322–3 NK026680, 260 NK cells see natural killer cells NKG2A receptors, 164, 164, 165 NKG2D receptors, 166, 168 NKp30 receptor, 166, 168 NKp44 receptor, 166, 168 NKp46 receptor, 166, 168 Nocardia spp., 401 nociceptors, 1570 NOD mice, 270, 272, 277 nodular regenerative hyperplasia, 1448 noncardiogenic capillary leak syndrome, 1459 noncellular blood components, 1234 non-Hodgkin’s lymphoma, 5, 15, 16, 878–97, 1004 autologous transplantation, 8, 9, 10, 10 classification, 878, 879 clinical features, 878–80 disease-free survival, 880 gene profiling, 880 HCT, 880–92 CNS lymphoma, 891 diffuse aggressive lymphoma, 880–4 allogeneic HCT, 883–4 autologous HCT, 880–3, 881, 882 follicular lymphoma, 884–9 allogeneic HCT, 887–9, 887, 888, 888 autologous HCT, 884–7, 884, 885, 886, 887 high-grade lymphomas, 891 mantle cell lymphoma, 889–90 allogeneic HCT, 889–90, 890 autologous HCT, 889, 889 T-cell lymphomas, 890–1 International Prognostic Index, 878, 879 and MDS, 834 outcome, 879, 880 prognostic factors, 879 radioimmunotherapy, 357–60, 358 reduced-intensity conditioning, 1047–8

reduced-intensity conditioning, relapse rate, 1054 relapse, 1054 remission, 232 noninherited maternal antigens, 698, 698, 1288 nonmalignant disease, 484 nonmyeloablative protocols see immunosuppressive nonmyeloablative protocols nonobese diabetic severe combined immunodeficiency (NOD-SCID) mouse, 73 nonpolymorphic antigens, 237 non-small cell lung cancer, remission, 232 nonsteroidal anti-inflammatory drugs see NSAIDs Noonan’s syndrome, and JMML, 752, 753 norfloxacin, 1339 notch-1, 39, 222 Notch, 80, 88, 91 NPM, 381 NRAS, 753 NSAIDs, 1575–6, 1644 pharmacology, 1575–6 side effects, 1576 uses, 1576 see also individual drugs nuclear factor of activated T cells, 1264 nuclear factor-kappa B, 1125, 1126 nucleophosmin mutations, 762 nurse coordinator, 475 nurse educator, 475 nurse manager, 475 nurse practitioner, 475 nursing care, 461–77 bone marrow harvest, 466, 467 conditioning, 464–6, 464, 465–6 discharge, 472–3, 472, 473 donor preparation, 463, 463 early post-engraftment, 470–1, 471 intensive care management, 471–2 long-term recovery, 474–5, 474 mobilization and collection, 463–4, 463 practice issues, 475–6, 475 preconditioning, 463, 463 pre-engraftment, 468–71, 469–70 prework-up, 462, 462 relapse post-transplant, 472 transplant phase, 466–8, 467–8 work-up, 462–3, 462 nursing role descriptions, 475 nutritional status pretransplant, 450 nutritional support, 1551–69 assessment, 1551–3, 1552–3, 1554–6 complications GVHD acute, 1565–6 chronic, 1566–7 iron overload, 1565 pulmonary disease, 1565 renal failure, 1565 SOS, 1565 conditioning regimens and toxicities, 1553, 1556, 1557–9, 1557, 1557–9 dietary supplements, 1561 enteral nutrition, 1562–5, 1565 food restrictions, 1560 integrative medicine, 1560–1 oral diet guidelines, 1559–60 immunosuppressed patients, 1559–60, 1560 special food service needs, 1559 total parenteral nutrition, 1561–2, 1561, 1563 nystatin, 1598

Index O obesity, 450, 453 observational studies design, 406–7, 407 impact of genetic data, 409 obstructive airway disease, 1626 obstructive/restrictive lung disease, 1464–7, 1466 definition, risk factors and clinical course, 1464–5 obstructive lung disease, 1466–7 restrictive lung disease, 1465–6 Oct3a, 82 Oct4, 82 octreotide, drug interactions, 1266 ocular problems, 1629–30 Candida infection, 1349 cataracts, 1630 GVHD, 1309, 1310–11 prevention, 1318 treatment, 1318 ocular sicca, 1630 posterior segment complications, 1630 odynophagia, 1545 ofloxacin, 1339 OKT3, 1268, 1293 older patients ALL, 795 AML, 769–70 aplastic anemia, 714 Hodgkin’s lymphoma, autologous transplantation, 868 oligopotent progenitors, human myeloid, 48 Omenn’s syndrome, 1105, 1106, 1108 omeprazole, drug interactions, 1535 Oncologic Pediatric Risk of Mortality (OPRISM), 486 ontogeny, 41–3, 42, 65, 66 onycholysis, 1308 OP9 cell line, 24 opioid analgesics, 1577 clinical use, 1580–1, 1581 dose tapering, 1581 neuropathic pain, 1584 patient-controlled analgesia, 1580–1 pharmacodynamics, 1577–8 pharmacokinetics, 1577, 1577 pharmacology, 1577 side effects, 1578 tolerance and dependence, 1578–9 see also individual drugs opportunistic infections, 140 protection against, 53 oprelvekin see interleukin-11 opsoclonus-myoclonus-ataxia syndrome, 970 oral care protocols, 1593–4, 1593 lip care, 1594 oral hygiene, 1593–4, 1593 oral mucosal care, 1594 oral complications, 1589–607 bisphosphonate-associated osteonecrosis, 1603–4 dental care, 1604 dental treatment, 1589, 1591–2, 1592 granulomatous lesions, 1604–5 GVHD, 400–1, 400, 1600–2, 1601 chronic, 1309, 1310 prevention, 1319 treatment, 1319 long-term follow-up, 1603 neurotoxicity, 1603 oral care protocols, 1593–4, 1593 oral hemorrhage, 1599 oral infections, 1597–9 orofacial/dental growth and development, 1604

orofacial pain, 1603 oropharyngeal mucositis, 1595–7, 1595, 1596 patient education, 1594 phase of transplantation, 1589, 1590–1 post-HCT, 1593 pretransplantation evaluation, 1589 prophylactic antibiotics, 1593 reduced-intensity conditioning, 1603 salivary gland hypofunction, 1599–600, 1600 second malignancy post-HCT, 1605 taste dysfunction, 1602–3 oral contraceptives, drug interactions, 1531 oral-facial growth, 1613–14 oral mucosa, GVHD criteria, 393 oral pain, 465 oral rinses, 1596–7 oral sicca, 1631 organ dysfunction, and T-cell depletion, 1278 organ shielding in TBI, 338 organ of Zuckerkandle, 970 orofacial pain, 1603 oropharyngeal HSV, 1384 oropharyngeal mucositis, 1595–7 management, 1596–7, 1596 phases of, 1595 oseltemavir, 1427 osmolality, 1500 osteoblasts, 1201 and HSC function, 590 osteoclasts, 1125 activation, 1125 hyperplasia, 1128 osteogenesis imperfecta, 112 osteonecrosis, 1492 bisphosphonate-induced, 1492, 1603–4 of jaw, 1492 osteopenia, 1630–1 osteopetrosis, 112, 1125–35 adult benign, 1127 animal models of HCT, 1126–7 future directions, 1133 in humans, 1127 infantile malignant, 1127–8 HCT for, 1129–31, 1130, 1131 symptoms, 1128 mammalian, 1126 mode of inheritance, 1125 molecular genetics, 1128–9 mutations in laboratory animals, 1125–6, 1126 transplantation issues, 1132–3 osteopetrosis transmembrane protein-1, 1125 osteopontin, 1201 osteoporosis, 1491–3, 1630–1 diagnosis, 1491 osteoprotegerin, 845, 1125 osteosarcoma, 991–2 clinical description, 992 epidemiology and etiology, 991 HCT, 992 molecular and cellular biology, 991–2 nontransplant approaches, 992 OSTM1 mutations, 1128 OSX, 992 outcomes data, 445 conditions affecting treatment outcome, 447 patient vs physician estimates, 446 outcomes research, 428–41 clinical benefits of HCT, 430–2, 431, 432 clinical syndromes, 434–5 costs of HCT, 430 data sources

1707

decision analysis, 435 evidence-based medicine, 436–7, 437 meta-analysis, 436, 436 Q-TWIST, 435–6, 436 registry studies, 435 definition, 428 history, 428–30, 429, 430 improvements in practice access, 437 practice variation, 438 quality of care, 438 patient-reported measures, 434 patient’s experience, 432–4 qualitative methods, 432 quality of life, 432–4, 432 prognostic models, 434 outpatient clinical RN, 475 outpatient infusion RN, 475 ovarian cancer, 16 remission, 232 Owen, Maureen, 102 oxaliplatin, germ cell tumors, 956 oxazolidinones, 1326 oxycodone, 1579 P P2RX5, 236, 241 p53, 69, 845 and age-associated HSC function, 1205–6 gene mutations, 1642 packed red cells see red cell transfusion paclitaxel, 303–4 breast cancer, 932, 933, 935 germ cell tumors, 951, 953, 956 high-dose conditioning, 321, 324 mechanism of action, 304 pharmacokinetics/pharmacodynamics, 288, 304 hepatic impairment, 294 plasma levels, 293 structure, 288 toxicity, 1661 Paget’s disease, 991 pain, 465, 469 abdominal, 1445–7, 1446 anatomy and physiology, 1570 ascending nociceptive systems, 1570 assessment, 1572–4 ability to function, 1573 history, 1572 measurement and monitoring, 1572 pain scales, 1572, 1573 bone, in PBPC donors, 551 clinical phenomenon of, 1570 definition of, 1571, 1571 diagnosis, 1574 gate control theory, 1570 in HCT patients, 1571–2 modulation of nociceptive transmission, 1570 neuropathic see neuropathic pain oesophageal, 1444, 1444 oral, 465 orofacial, 1603 perianal, 1435, 1447 sickle cell disease, 1092–3, 1094 sources, 1571 surgical, 1584 pain management, 527–8, 1570–88 antidepressants, 1576–7 difficult problems, 1574 dying patients, 1585–6 invasive analgesic techniques, 1585

1708

Index

neuropathic pain, 1583–4 nonpharmacologic techniques, 1574–5 children, 1575 cognitive-behavioral strategies, 1574–5 education, 1574, 1575 information, 1574 mechanisms of, 1574 NSAIDs and acetaminophen, 1575–6, 1644 opioid analgesics, 1577 clinical use, 1580–1, 1581 dose tapering, 1581 patient-controlled analgesia, 1580–1 pharmacodynamics, 1577–8 pharmacokinetics, 1577, 1577 pharmacology, 1577 side effects, 1578 tolerance and dependence, 1578–9 see also individual drugs pain due to diagnostic/therapeutic maneuvers, 1584 painful and unpleasant procedures, 1584–5 peritransplant period, 1582 post-engraftment, 1583 pretransplant, 1581–2 prior history of substance abuse, 1582 pseudoaddiction, 1582 side effects, 1573–4 pain scales, 1572, 1573 palifermin see keratinocyte growth factor pamidronate, 1631 breast cancer, 932 multiple myeloma, 855 osteopetrosis, 1133 pancreatic disease, 1449 pancytopenia, 468, 652 panitumumab, 351 paracetamol see acetaminophen parainfluenza viruses, 1422–3, 1541 clinical significance, 1422 diffuse interstitial pneumonia, 1457 risk factors, 1423 treatment and protection, 1423 parasitic diseases, transfusion-transmitted, 1238–9 parents proxy QOL report, 509–10 quality of life, 507 parotiditis, radiation-induced, 335 paroxetine, drug interactions, 1534 paroxysmal nocturnal hemoglobinuria, 16, 65, 69, 705, 727–33, 729–31, 731 autologous transplantation, 732 clinical description, 728, 728 epidemiology, 727, 728 etiology, 727 hematopoietic cell transplantation, 729–32, 729–31, 731 molecular and clinical biology, 727–8 nontransplant treatment, 728–9 reduced-intensity conditioning, 732 parthenogenetic ESCs, 29 partially matched donors, 657–74, 696–9 cell dose and use of PBPCs, 665–7, 665 human transplant studies, 665–6 immune reconstitution, 666–7 infection prophylaxis, 666 preclinical studies, 665 crossmatching, 659 donor and recipient matching, 658–9, 658 donor selection, 657–9 HLA function and polymorphism, 657 HLA haplotypes and segregation in families, 658, 658

NK cell alloreactivity, 667–70, 667, 668, 668, 669 partial marrow T-cell depletion, 664–5 probability of identification, 659 T-cell-depleted grafts, 664 T-replete marrow grafts, 659–64 graft failure, 659–61, 659 GVHD, 661–3, 661, 662 survival, 663–4, 663 parvovirus B19, 1222, 1426 transfusion-transmitted, 1238 passenger lymphocyte syndrome, 1220 pathology, 390–405 graft-versus-host disease, 392–401 hepatic SOS, 391–2 histologic findings vs clinical signs, 392 infection, 401–2 post-transplant hematopoietic evaluations, 391 pretransplant evaluation, 390–1 pulmonary complications, 402–3 patient education, oral hygiene, 1594 patient-pioneers, 484 patient-reported measures, 434 patient-reported outcome, 502 patients autonomy, 479, 485 communication with, 527 evaluation and counseling, 443–58, 445 advanced directives, 454–5 advice against transplantation, 446 children, 452–4, 453 choice of procedure, 444–5 clinical trials, 447 comorbid conditions, 449–50 conditions affecting outcome, 447 disease and remission status, 447 donor compatibility, 447–8, 448 eligibility for transplant, 449 ethnic and racial background, 450 first visit to transplant center, 443–4 foreign patients, 447 infertility issues, 444 information absorbed, 445–6, 446 multiple opinions, 446–7 nutritional status, 450 outcome data, 445 patient age, 450 performance status, 448–9 previous infections, 451 prior therapy, 449 psychosocial assessment, 451–2, 451 second meeting, 446 stem cell source, 454 transfusion history, 451 written information, 443 experience of HCT, 432–4 qualitative methods, 432 quality of life, 432–4, 432 informed consent, 479–80 perspective on HCT, 526–32 BMT InfoNet, 531 Center for International Blood & Marrow Transplant Research, 531 Children’s Oncology Group, 531 chronic GVHD, 530–1 communication in lay language, 527 different learning styles, 527 National Institutes of Health, 531 National Marrow Donor Program, 531 pain control, 527–8 preparation, 526 psychological difficulties, 528–9

psychosocial support networks, 529 quality of life posttransplant, 529–30 repetition and reinforcement, 527 risks, 526–7 selection for transplantation, 17 patient-specific hESCs, 28, 28 PAX3, 988 PAX5, 50 PAX7, 988 PBHCs allogeneic transplantation, 618–30 characteristics and dose, 619–20, 620 in children and adolescents, 625 in diseases other than leukemia, 625 donors, 618 and lymphoproliferative disorder, 1412 marrow vs mobilized blood, 624, 624, 625 mobilization and collection, 618–19 side-effects and long-term sequelae, 621–2, 621 randomized trials, 622–4 acute GVHD, 623, 623 chronic GVHD, 623, 623 engraftment, 622 immune recovery, 622–3 relapse rates, 623 survival, 624 transplant-related mortality, 623–4 recipients, 622 T-cell-depleted mobilized blood, 625–6 T cells in, 620 unrelated donor transplants, 626 AML, 768 autologous transplantation clinical uses, 592 collection techniques, 592 identification and enumeration, 590 mobilization, 590–605 cell adhesion molecules, 591–2 combination cytokine/chemotherapy-induced, 595–6, 595 cytokine-induced, 593–4, 593, 593, 594 factors affecting yield, 596–7 investigational compounds, 598 mechanisms, 590–1 preclinical studies, 598 proteases, 591 SDF-1/CXCR4, 591 standard regimens, 599 optimal cell yield, 592–3 tumor contamination, 597–8 Epstein-Barr virus viral DNA in, 1413 G-CSF mobilized, 708–9 and chronic GVHD risk, 712 in high-dose conditioning, 326 multiple myeloma, 848 residual tumor cells, 6–7 PBPCs, 89, 255, 694 autologous, 223 donation, 544 adverse events, 551–2 allogeneic transplantation, 544 autologous transplantation, 544 mortality, 552–3 regulatory aspects, 546 see also donation; donors expansion studies, 91–3, 92 G-CSF-mobilized, 481 MPD, 827 myelodysplastic syndrome, 827 pediatric solid tumors, 996

Index P-cell prolymphocytic leukemia, 898 PDCs see plasmacytoid dendritic cells peau d’orange, 1311 pediatric solid tumors, 985–1000 allogeneic HCT, 996 case reports, 994 chemosensitivity, 985 desmoplastic small round cell tumor, 993–4 clinical description, 993–4 epidemiology and etiology, 993 HCT, 994 molecular and cellular biology, 993 nontransplant approaches, 994 dose-limiting myelotoxicity, 985 dose-response, 985 Ewing’s sarcoma, 985–8 clinical description, 986 epidemiology and etiology, 985–6 HCT, 987–8, 987 molecular and cellular biology, 986 nontransplant approaches, 986 immunotherapy, 996 osteosarcoma, 991–2 clinical description, 992 epidemiology and etiology, 991 HCT, 992 molecular and cellular biology, 991–2 nontransplant approaches, 992 patterns of transplant failure, 994–5 PBPCs vs bone marrow, 996 retinoblastoma, 992–3 clinical description, 993 epidemiology and etiology, 992 HCT, 993 molecular and cellular biology, 992–3 nontransplant approaches, 993 rhabdomyosarcoma, 988–9 clinical description, 988, 988 epidemiology and etiology, 988 HCT, 988–9 molecular and cellular biology, 988 nontransplant approaches, 988 tandem transplants, 995 targeted therapy, 995–6 TBI, 995 Wilms’ tumor, 989–91 anaplasia, 990 clinical description, 989 epidemiology and etiology, 989 HCT, 990–1 metastatic recurrent, 991 molecular and cellular biology, 989 nontransplant approaches, 989–90 PedsQL, 504, 509 pegfilgrastim PHBC mobilization, 595, 619 see also G-CSF peliosis hepatitis, 1629 Pelizaeus-Merzbacher disease, 1138, 1154 penicillins, 1326 pentostatin, 1293 GVHD, 1270, 1317 MDS, 833 perfloxacin, 1339 perforin, 134, 138, 164, 211, 392, 1203 gene mutations, 1165 perforin/granzyme pathway, and GVHD, 211 perforin pores, 211 perianal pain, 1435, 1447 pericardial effusion, 1544

peri-engraftment respiratory distress syndrome, 1458, 1459 periodontal disease, 1591 peripheral blood-derived cells, 9 peripheral blood hematopoietic cells see PBHCs peripheral blood progenitor cells see PBPCs peripheral neuroepithelioma see Ewing’s sarcoma peripheral tolerance induction of, 191–3 alloantigens, 115.5 co-stimulatory blockade, 192–3 donor-specific transplantation, 191–2 modified APCs, 191–2 T-cell antibodies and adhesion molecules, 193 total lymphoid irradiation, 193 T-cell, 193 pertussis toxoid vaccine, 1668 Perugia protocol, 665–6, 665 Pesaro thalassemia trial, 1080–3, 1081, 1082, 1083 adults, 1082 children and adolescents, 1081–2, 1082 mixed chimerism, 1082–3 rejection/disease recurrence, 1082 Peyer’s patches, 130, 136, 209, 250, 1169 P-glycoprotein system, 1524 phages, 1326 phagocytosis, 253, 1163, 1326 pharmacogenomics, 290 pharmacologic purging, 7 clinical studies, 10–11 phenobarbital drug interactions, 1266 azole antifungals, 1526 immunosuppressants, 1530, 1531 phenotype, 154, 156 dendritic cells, 249 phenybutazone, marrow injury, 705 phenytoin drug interactions, 1266 antidepressants, 1534, 1535 azole antifungals, 1525, 1526, 1528 immunosuppressants, 1530, 1531 Philadelphia chromosome, 53, 69, 74, 237, 734, 751 ALL, 793, 808 HCT, 796–8, 812, 812, 813 treatment, 794–5 treatment outcome, 802 phlebosclerosis, 392 phosphatidyl inositol glycan-A, 727 phosphoglycerate kinase, 117 phosphorus, 1556 photopheresis, GVHD prevention, 1270 physician bias, 478 phytohemagglutinin response post-HCT, 1664, 1665 pilocarpine, 1603 piperacillin-tazobactam, 1333 pituitary gland, 1487 PKH26, 77 Planet Cancer, 529 plasmacytoid dendritic cells, 249, 250–1 functions of, 253 plasmapheresis, 1545 plasma transfusion, 1227 plasmids, 1326 plasminogen activatory inhibitor-1, 1477 plasticity, 72–3, 83 assessment of, 76 bone marrow-derived stem cells, 75 explanations of, 75–6, 75 platelet antibodies, 1230

1709

platelet concentrates from whole blood, 1228 prepared by apheresis, 1228 plateletpheresis, 1228 platelets, in sickle cell disease, 1092 platelet transfusion, 1228–9 ABO and Rh antigens, 1229–30 active bleeding, 1229 adverse reactions to, 1236 crossmatching, 1230 dose and outcome, 1229 lack of response, 1230–1 management, 1230–1 patient-related factors, 1230 platelet concentrate-related factors, 1230 storage conditions, 1228 thrombopoietin in, 1231–2 platinating agents, 300–2 see also individual drugs pleconaril, 1424, 1427 PLEKHM1 protein, 1128 pleuropulmonary blastoma, 994 pluripotent stem cells, 23, 24, 36, 72, 73, 76, 81–2, 81 PML-RARα fusion protein, 233 pneumatosis cystoides intestinalis, 1632 Pneumocystis carinii see Pneumocystic jiroveci Pneumocystis jiroveci pneumonia, 401, 926, 1005, 1456, 1541, 1625 pneumonia, 1540 bronchiolitis obliterans organizing, 1541, 1542, 1627, 1627 CMV-associated interstitial, 1372, 1372 treatment, 1373–4, 1373, 1374 diffuse interstitial, 1457 aplastic anemia, 712–13 Pneumocystis jiroveci, 401, 926, 1005, 1456, 1541, 1625 ventilator-associated, 1539 pneumothorax, catheter-induced, 1249–50 POEMS syndrome, 919 poikiloderma, 1308 point mutations, 65, 1644–5 Poiseuille’s law, 1246 pol, 116, 117, 1002 polio vaccine, 1665, 1666 polycythemia vera, 835, 836–7 polymerase chain reaction, 151 minimal residual disease, 376, 379 tumor cell detection, 6 polymorphism, 290 polymyositis, 1658 polysaccharide pneumococcal vaccines, 1665, 1666–7 Pompe disease, 1157 popovaviruses, 1425–6 posaconazole, 1356, 1358, 1360, 1442 drug interactions, 1527–8, 1528 immunosuppressants, 1530, 1531 post-engraftment, nursing care, 470–1, 471 postlumbar puncture headache, 1584 postnatal donor lymphocyte infusion, 583 post-transplant hematopoietic evaluation, 391 potassium, 1555 PR3 protein, 233 practice variation, 438 pravastatin, drug interactions, 1530 prealbumin, 1554 preconditioning, nursing duties, 463, 463 prednisolone, 1262 AIDS-related lymphoma, 1005 prednisone, 1262 AIDS-related lymphoma, 1005

1710

Index

AL-amyloidosis, 921, 922 ALL, 793 CNS leukemia, 794 and graft failure, 1207 GVHD prevention, 711, 1044, 1259 non-Hodgkin’s lymphoma, 878 pre-engraftment, nursing care, 468–71, 469–70 pregnancies conceived for HCT, 482, 483 preimplantation genetic diagnosis, 1195 preintegration complex, 117 premature ovarian failure, 518, 519, 521–2 premedication, 1585 prenatal tolerance induction, 582 prethymocytes, 577 pretransplant evaluation, 390–1 hepatic disease, 390–1 lymphoreticular system, 390 pretransplant neurologic screening, 1661 PRF1, 1165 primate foamy virus, 117 primidone, drug interactions, 1266 primitive neuroectodermal tumor see Ewing’s sarcoma private specificities, 151 procarbazine Fanconi’s anemia, 1190 and PBHC mobilization, 597 progenitor cells autologous hematopoietic, 932 clearance from blood, 40 common lymphoid, 36, 222 and lymphoid development, 44–6, 45, 222 common myeloid, 36, 48 and myeloid development, 46–7 in cord blood, 565–7, 566 endothelial, 567 cotransplantation with HSCs, 53 definition of, 44 gene expression profiles, 49 granulocyte/macrophage, 45, 47, 48 human oligopotent myeloid, 48 isolation of, 44 lineage-committed, 44–9 megakaryocyte/erythrocyte, 47 multipotent, 26, 37, 81, 82 adult, 81, 82 properties of, 37–8, 38 ontogeny, 42 peripheral blood see PBPCs purging, 23 thymic, 46 prognostic models, 434 programmed cell death, 38 prolactin, 516, 522, 1487 prospective cohort studies, 407–8 proteases bacterial, 1326 HSC mobilization, 591 proteinase-3, 237 protein-based gene suppressors, 1008 protein status, 1554 protein-type cryoprotectant solutions, 635–6 protective effects, 637 proteinuria, 916 proteolipid protein, 1020 proton pump inhibitors, drug interactions, 1528 pruritus, 1578 P-selectin, 591, 592, 1163, 1164, 1207 P-selectin glycoprotein ligand-1, 1201 pseudoaddiction, 1582 pseudoautologous transplantation, 272, 274

pseudo-Hurler polydystrophy, 1137 HCT, 1148 pseudomembranous candidiasis, 1598 Pseudomonas spp., 53 Pseudomonas aeruginosa, 1327, 1329, 1332, 1597, 1599 psoralen, GVHD, 1317 psoriatic arthritis, allogenic HCT for other primary disease, 1017 psychological issues donors, 547 patients, 528–9 psychosocial assessment, 451–2, 451 Psychosocial Assessment of Candidates for Transplantation Scale, 489 psychosocial issues, 488–501, 1633–4 hematopoietic cell donors, 495–7 informal caregivers, 497–8 professional caregivers, 498 recipients, 488–9 stages of HCT, 489–95, 490 decision to undergo, 489 hospital discharge and early recovery, 493 long-term recovery, 493–5, 494 post-HCT hospitalization, 492–3 pre-HCT preparation, 491–2 Psychosocial Levels System, 489 psychosocial support networks, 529 Ptcrα, 50 Pten, 39 Pten, 53 pterygium unguis, 1308 PTPN11, 751, 752 puberty, 1614–16 boys, 1615 conditioning with chemotherapy, 1614–15, 1615 conditioning with irradiation, 1615–16, 1616 delayed, 454 girls, 1615 late complications, 1628–9 Public Health Services Act, 533 public specificities, 151 pulmonary complications, 402, 1540–3, 1540, 1625, 1625 bronchiolitis obliterans, 403, 1540, 1542, 1620 GVHD, 393, 401, 402–3, 403 infectious, 1540–1, 1541, 1541 interstitial pneumonia syndrome, 402 noninfectious, 1541–2, 1542 nutritional support, 1565 respiratory failure, 1542–3, 1543 sickle cell disease, 1094 pulmonary function, 469 pulmonary hypertension, 1094 pure red cell aplasia, 1219, 1222 purging, 5–17, 767–8, 978–9, 1068, 1672 in breast cancer, 939 cell culture techniques, 6 clinical studies, 10–12, 11 in CLL, 904–5 contribution of infused tumor cells to relapse, 10 marker gene studies, 13 and cryopreservation, 641 detection of residual disease, 5, 6 efficiency, 10, 10 immunocytochemistry, 5–6 immunomagnetic, 978–9 in vivo, 13 leukapheresis, 978 molecular biologic techniques, 6 outcome, 12–13, 12

PBHC collections, 6–7 randomized clinical trials, 13–14 strategies, 7, 7 monoclonal antibodies, 7–10, 8, 8 pharmacologic, 7 physical separation, 7 purine synthesis, 1268 Purkinje cells, 77 purpura, AL-amyloidosis, 919 PUVA, oral GVHD, 1602 Q 5q mutations, 762 7q mutations, 762 20q mutations, 762 QOL see quality of life Q-TWIST, 435–6, 436 quality-adjusted life years, 435 quality of care, 438 quality of life, 408–9, 432–4, 432, 491, 502–14 acute treatment, 505 autologous, allogenic HCT, 507 chronic GVHD, 1320–1 conditioning regimen, 507 definition of, 502 dimensions, 502–3, 503 gender, 507 interventions to improve, 507–8 late effects, 506 long-term function, 505–6 cognitive function, 506 fatigue, 505–6 measurement, 508–10 alternative strategies, 509 clinical utility of, 510 computer, mail or telephone interview, 510 generic measures, 508–9, 508, 509 proxy or parent, 509–10 specific measures, 509, 509 multidimensionality, 502 patient’s perspective, 502 pediatric, 507 caregivers and parents, 507 phases of HCT, 503, 505 posttransplant, 529–30 pretransplant, 505 recovery, 505 relapse, 506 risk factors, 506–7 selected measures, 504 sexual dysfunction see sexual dysfunction Quality Management Plan, 536 Quality Management Programs, 534 Quality Systems Approach to Pharmaceutical cGMP Regulations, 534 quinupristin, 1326 R R5 strains, 1001 R115777, 754 rabbit models, osteopetrosis, 1126 RAD51 polymorphism, 1642 radiation effects, 355 neurotoxicity, 1603 therapy-related disease AML, 1639 myelodysplastic syndrome, 1639 radiation nephritis, 1475, 1476, 1477 radiation physics, 335 radioimmunoconjugates, 1067

Index radioimmunotherapy, 351–64 antibody, 353 antibody pharmacology, 353 clinical use Hodgkin’s lymphoma, 360 leukemia, 355–7, 356, 357 acute lymphoblastic in adults, 800–1 non-Hodgkin’s lymphoma, 357–60, 358 solid tumors, 360–1 dosimetry, 354–5 elements of, 352, 352 future directions, 361–2, 361 history, 351–2 labeling procedure, 353–4, 354 radiation effects, 355 target antigen selection, 352–3 toxicity, 361 radionuclides, 353–4, 354 neuroblastoma, 977–8 radiotherapy, Hodgkin’s lymphoma, pretransplant, 864 raloxifene, 931 RANKL, 845, 1125, 1126 deficiency, 1128–9 RANTES, 1001, 1463, 1467 rapamycin see sirolimus Ras, 845 Ras-dependent pathways, 753 JMML, 754–5 Ras mutations, 762 RB1, 845, 991, 992 recipient sensitization, 660 recombinant growth factors, 645–56, 646 erythropoietin, 645 after allogeneic HCT, 645–7 after autologous HCT, 645 GM-CSF, 650, 650 after allogeneic HCT, 651 after autologous HCT, 650–1 granulocyte colony-stimulating factor, 647, 647 after allogeneic HCT, 649 after autologous HCT, 647–8 interleukin-2, 653 interleukin-3, 652 interleukin-11, 652–3 keratinocyte growth factor, 653 murine allogeneic transplant models, 649–50 stem cell factor, 652 thrombopoietin/megakaryocyte growth factor, 652 RecQ helicases, 992 recurrent malignancy, chimerism testing, 371–2, 371 red cell transfusion, 1226 leukocyte-reduced, 1226–7, 1227 sickle cell disease, 1094–5 washed, 1227 reduced-intensity conditioning, 213, 226 ABO incompatibility, 1219–20 chimerism, 372, 1051 chimerism testing, 372 clinical results, 1045–6, 1045 clinical use ALL in adults, 799 AML, 781 aplastic anemia, 715 breast cancer, 942 CLL, 906–7, 908, 909 CML, 742 congenital immunodeficiency disorders, 1113 hematologic malignancies, 1043–58 Hodgkin’s lymphoma, 870–2, 871 MDS, 828, 832

MPD, 828 multiple myeloma, 851–3, 852, 853 phagocytic disorders, 1165 PNH, 732 SCID, 1111 sickle cell disease, 1098 solid tumors, 959–60, 965–6 melanoma, 965 renal cell carcinoma, 963–5, 964, 965 results, 960–3, 960–3 thalassemia, 1084–5 complications acute kidney injury, 1473 oral, 1603 engraftment kinetics, 1051 followed by allogeneic transplantation, 1066–7, 1066 and graft failure, 1202 GVHD and GVT effects, 1051 HLA-matched/mismatched unrelated donor allografting, 1049–51, 1050 HLA-matched related HCT, 1046–9, 1047–8, 1049 HLA-mismatched related donor allografting, 1049 morbidity and mortality, 1052 PNH, 732 preclinical studies dogs, 1044–5 low-dose TBI, 1044 marrow space vs immunosuppression, 1044–5 marrow toxicity of TBI, 1044 MHC-matched grafts, 1044 TBI dose, 1044, 1044 mice, 1043–4 radiolabeled monoclonal antibodies, 1045 relapse rates, 1054 relapse risk, 1052–3, 1053 secondary allografts consolidative, 1053 for failed high-dose transplants, 1054–5, 1055 and T-cell depletion, 1278 toxicities, 1052 Reed-Sternberg cells, 860, 1415 referral for treatment, 21937 regimen-related toxicity, 316–17 Registration Final Rule, 533 registry studies, 435 regrafting, 1210–11, 1211 Regulation of Human Cells, Tissues, and Cellular and Tissue-Based Products Small Entity Compliance Guide, 534 regulatory framework AABB, 540 donors, 546–7 FACT, 534 accreditation process, 536–9, 537, 538, 539 historical background, 534–5 inspector qualifications, 537 significance of accreditation, 539–40 standards, 535–6 governmental regulation, 533–4 National Marrow Donor Program, 4, 481, 540, 693, 693, 777 regulatory T cells see Tregs rehabilitation, 1633–4 relapse, 506, 1059–75 adoptive immunotherapy, 1063–5 after allogeneic transplantation, 1059 donor leukocyte infusions, 1059–63, 1060 withdrawal of immunosuppressive therapy, 1059 after autologous transplantation, 1065 prevention, 1067–71, 1069, 1071

1711

second HCT after failure, 1065–7, 1066 vaccination of donors, 1067 after PBHC transplantation, 623 ALL, 820, 1054 AML, 33, 770, 785, 1054 chronic myelomonocytic leukemia, 1054 CLL, 901, 1054 CML, 1054 Hodgkin’s lymphoma, 866–7, 869–70, 1054 infused tumor cells and, 610, 610, 611 JMML, 756, 756 MDS, 840–1, 1054 MPD, 840–1, 1054 multiple myeloma, 1054 non-Hodgkin’s lymphoma, 1054 nursing role, 472 refractory anemia, 1054 and T-cell depletion, 1279–80 reduction of relapse, 1280 see also minimal residual disease related donor HCT, 544–5 ALL, 815–16 AML, 785 cord blood cells, 562 histocompatibility testing, 157 HLA partial match, 657–74 T-cell depletion, 1276 Relman, Arnold, 429 remifemin, 521 remission, 447 renal cell carcinoma, 958 reduced-intensity conditioning, 963–4, 964 toxicities and limitations, 964–5, 964, 965 renal complications, 1473–80, 1545–6 acute kidney injury, 1473–5, 1474 chronic kidney disease, 1475–8, 1475, 1477, 1478 GVHD-related, 1478–80 renal conditions, coexisting, 450 renal failure, 848 renal function indices, chemotherapy dose based on, 294–5 renal insufficiency, 469 renal replacement therapy, 1545–6 renal toxicity, TBI-induced, 339, 339 repifermin, 1340 Replens, 522 replicative senescence, 69, 69 research, donor involvement in, 548 research RN, 475 residual host cells see chimerism respiratory burst oxidase, 1164 respiratory conditions, coexisting, 449 respiratory failure, 1542–3, 1543 respiratory syncytial virus, 1420–2, 1456, 1540, 1541 clinical significance, 1420 diffuse interstitial pneumonia, 1457 risk factors, 1421, 1421, 1421 treatment and prevention, 1421–2 respiratory tract, AL-amyloidosis, 919 respiratory viruses, 1420, 1420 symptoms, 1424 restrictive lung disease, 1465–6, 1625 reticular dysgenesis, 1107, 1108, 1169–70 retinitis, 1630 retinoblastoma, 992–3 clinical description, 993 epidemiology and etiology, 992 HCT, 993 molecular and cellular biology, 992–3 nontransplant approaches, 993

1712

Index

retinoic acid, 979 JMML, 754 retinoic acid receptor-alpha translocations, 762 RetroNectin, 119 retrospective case-control studies, 408 retroviral pseudotypes, 118 retroviral vectors 10.1, 10.2Cd14, 727 retroviruses, life cycle, 117 rev, 1002 reverse transcriptase, 117 reverse transcriptase telomerase protein, 68 RevM10, 1008 Rex1, 82 rhabdomyosarcoma, 988–9 clinical description, 988, 988 epidemiology and etiology, 988 HCT, 988–9 molecular and cellular biology, 988 nontransplant approaches, 988 Rh blood group antigens, 1227 alloimmunization, 1222 in platelet transfusion, 1229–30 rhenium-186, 354 rhenium-188, 354 rheumatoid arthritis, 265, 1023, 1025 allogenic HCT for other primary disease, 1017 family/twin studies, 1015 susceptibility genes, 1015 Rh incompatibility, 1220–2, 1221 rhinovirus, 1423–4, 1541 diffuse interstitial pneumonia, 1457 ribavirin, 1427, 1456 hemorrhagic cystitis, 1481 respiratory syncytial virus, 1421 ribozymes, 1008 rifabutin, drug interactions, 1525, 1526, 1528 rifampin drug interactions, 1266 azole antifungals, 1525, 1528 immunosuppressants, 1530, 1531, 1532 rifamycins, 1326 RIFLE criteria, 1475 rimantidine, 1427 risperidone, drug interactions, 1534 rispiridone, 1427 rituximab, 215, 258, 351, 352, 358, 449 clinical use ALL, 793 CLL, 897, 901, 905, 908 diffuse aggressive lymphoma, 881, 883 follicular lymphoma, 885–6 Hodgkin’s lymphoma, 1415 lymphoproliferative disease, 1415, 1415 non-Hodgkin’s lymphoma, 878 post-transplant lymphoproliferative disease, 1646 rheumatoid arthritis, 1023 SLE, 1023 thrombotic microangiopathy, 1480 GVHD, 214, 1317 toxicity, 361 RNA, short interfering, 1671 RNA-based gene suppressors, 1007–8 RNA interference, 1008 Rodnan Skin Score, modified, 1021, 1025 rofecoxib, 1576 rolling, 1163, 1164 Rome thalassemia trial, 1083–5, 1085 alternative related donors, 1083–4 reduced-intensity HSC transplantation, 1084–5 unrelated donors, 1084 rotamase, 1264

rotaviruses, 1427 Rothmund-Thomson syndrome, 991 RRT see regimen-related toxicity rubella virus, 268–9 ruffled border, 1125, 1126 Rule of Double Effect, 1586 runt disease, 2, 1287 RUNX1, 381 RUNX3, 775 S S17 cell line, 24 Saethre-Chotzen syndrome, 1178 saline, as cryoprotectant, 637 salivary glands GVHD, 393, 400 hypofunction, 1599–600, 1600 Sandhoff disease, 1139 Sanfilippo syndrome, 1137 HCT, 1148–9, 1148 Santos, George, 2 sarcoma, 16 autologous transplantation, 11 sargramostim, 544 see also GM-CSF satellite cells, 77 scavenger receptors, 252 Scedosporium spp., 1351 Scheie syndrome, 1137 HCT, 1143–8, 1145–7 SCID, 16, 89, 271, 580, 585, 1105–12 BMT, 1111 gene therapy, 1115–17 genetic causes, 1106 HCT alternative donor transplants, 1107–11, 1108, 1109 in utero, 1112 reduced-intensity conditioning, 1111 unmodified human leukocyte antigen-matched related, 1107, 1108 mosaicism in, 1185 umbilical cord blood cell transplant, 1111 X-linked, 585, 1117–18 SCID-hu mouse, 120 SCID repopulating cells, 89–90 SCID-X1, 122, 124 vector-mediated insertional mutagenesis, 124–5 SCL-TAL, 38 search determinants, 156 “seasons of survival”, 489 Seckel syndrome, 1178 secondary malignancies, 1638–52 AML, 1638–45 aplastic anemia, 715 myelodysplastic syndrome, 1638–45 secondary phase of irradiation syndrome, 1275 sedation, 1578 conscious, 1585 deep, 1585 palliative, 1586 sedatives, drug interactions, 1533 seizures, 1653–4 selectins, 592, 1163, 1164 see also individual selectins selection/screening, 489–90 self-care behaviour, 493 self-inactivating deletions, 117 self-peptides, 268 self-renewal, 64, 72 genetic pathways, 38–9

self-tolerance, NK cells, 168, 168 senescence, 38 senile cardiac amyloidosis, 914 senile systemic amyloidosis, 914 sepsis, and acute kidney injury, 1474–5 septic shock, 1543–4, 1543 diagnosis, 1543 sequence-specific oligonucleotide probe hybridization, 152, 153 sequence-specific primary typing, 152 SEREX analysis, 238 Serious Events and Adverse Effects Registry, 545 SERPINA1, 591 SERPINA3, 591 Serratia spp., 1329 serum amyloid A, 914 sevelamer, drug interactions, 1531 severe combined immunodeficiency disease see SCID severe congenital neutropenia (Kostmann’s syndrome), 1168–9, 1169 sex hormone-binding globulin, 522 sexual dimorphism, 266–7 sexual dysfunction, 502–12, 1515–17 assessments and interventions, 520 concept of, 515–16 disorders of, 516 dyspareunia, 521–2 erectile dysfunction, 522–3 etiology, 516 female, 518, 519, 1517 biological/physiologic variables, 518, 519 premature ovarian failure, 519, 521–2 vaginal atrophy, 521–2 future research, 523 hypoactive sexual desire disorder, 520–1 investigations, 517 male, 516–18, 1516–17 biological/physiologic variables, 516–18, 517 psychosocial variables, 518, 520 see also gonadal dysfunction Sexual Function Questionnaire, 504 sexuality, 515 arousal, 516 desire, 516 orgasm, 516 Sézary syndrome, 1036–7 SF-36, 504, 509 sheep models, xenogeneic transplant, 90 short interfering RNA, 1008, 1671 short tandem repeat see STR short-term repopulating cells, 89 SHP-2, 753 Shwachman-Bodian-Diamond syndrome, 1171 Shwachman-Diamond syndrome, 16 Shwachman’s syndrome, and AML, 775 SIADH see syndrome of inappropriate antidiuretic hormone secretion sialadenitis, radiation-induced, 1599 sialic acid storage disease, 1139, 1157 sialidosis, 1139, 1157 sialogogues, 1603 sibling donor transplantation ALL in children, 811, 812 first remission, 808–10, 808, 809 second or subsequent remission, 810, 814 AML in adults, 767 Fanconi’s anemia, 1187–8, 1187, 1189, 1190 sicca ocular, 1630 oral, 1631

Index sickle cell disease, 16, 484, 1090–104 clinical features, 1092 correction scheme, 29 epidemiology and etiology, 1090–1, 1091, 1092, 1093 HCT, 1099 alternative cell sources, 1101 current indications, 1095–6, 1096 effect of donor hematopoiesis, 1098–100 growth and development after, 1100–1, 1100 reduced-intensity, 1098 results, 1096–7, 1096, 1097 stable donor-host chimerism after, 1097–8 survival after, 1097 molecular and cellular biology, 1091–2 pain, 1092, 1094 prevalence, 1091 pulmonary complications, 1094 stroke, 1094 prevention, 1094–5 treatment hydroxyurea, 1095 red blood cell transfusions, 1094–5 Sickness Impact Profile, 508 Siglec-H, 249, 251 signal transduction, 133–4 sildenafil, 522 silent sister hypothesis, 67 SILV, 242 simvastatin drug interactions azole antifungals, 1526 grapefruit juice, 1536 immunosuppressants, 1530 single nucleotide polymorphisms see SNPs sinusoidal fibrosis, 1440 sinusoidal obstructive syndrome, 336, 339, 390, 391, 461, 469, 479, 779, 1437, 1438, 1439–41, 1439, 1441, 1545 and acute kidney injury, 1475 clinical course and prognosis, 1439 clinical presentation and diagnosis, 1437, 1439, 1439 definition, 1437 hepatic venous pressure gradient, 1439 incidence, 1437 liver biopsy, 1439 nutritional support, 1565 pain management, 1582–3 pathogenesis, 1439–40, 1441 prevention, 1440–1 treatment, 1441 sirolimus, 179, 1219, 1222, 1264, 1270 clinical use, 1267 GVHD prevention, 214, 1266–7, 1294 drug interactions, 1530, 1532–3, 1533 azole antifungals, 1525, 1526, 1528 immunosuppressants, 1530, 1531 GVHD, 1317 pharmacology, 1267 side effects, 1267 hyperlipidemia, 517 sister chromatid exchange, 66 silent sister hypothesis, 67 skeletal muscle, bone marrow-derived cells, 77–8 skin Candida infection, 1349 GVHD, 393, 394–5, 394, 399–400, 399 chronic, 1308, 1309 prevention, 1319 treatment, 1319

Slamf1, 47, 49 SLE see systemic lupus erythematosus Sly syndrome, HCT, 1148 small cell lung cancer, 16 small lymphocytic lymphoma, 879 SMCY, 236 153Sm-EDTMP, 74 SNPs, 158, 159, 235–6, 268, 290, 683 sodium hyaluronate, hemorrhagic cystitis, 1481 sodium salicylate, 1576 solid organ transplantation, 129 solid tumors, 958–69 allogeneic transplantation reduced-intensity conditioning, 959–60 results, 960–3, 960–3 GVTE, 958 animal models, 959 clinical data, 959 mechanisms of, 966–7, 967 immunology, 958–9 radioimmunotherapy, 360–1 reduced-intensity conditioning, 959–60, 965–6 melanoma, 965 renal cell carcinoma, 963–4, 964 toxicities and limitations, 964–5, 964, 965 results, 960–3, 960–3 therapy-related, 1647–8 genetic susceptibility, 1647–8 pathogenesis, 1647–8 risk factors, 1647 screening for, 1648 treatment, 1648 somatostatin, 1487 Sonic hedgehog, 91 SOS see sinusoidal obstructive syndrome Southern blot analysis, tumor cell detection, 6 sox17, 39 ‘space’ for engraftment, 193 spleen, 250 rupture, 552 splenectomy, JMML, 758 splenomegaly, 776 spumaretroviruses, 116 squamous cell carcinoma, therapy-relatd, 1647 stage-specific embryonic antigen-1, 82 standard gambles, 431 standards, 535–6 Standards for Hematopoietic Cell Collection and Processing, 534 Standards for Hematopoietic Progenitor Cell Collection, Processing and Transplantation, 534–5 Standards for Immunohematology Reference Laboratories, Perioperative Autologous Blood Collection and Administration, and Relationship Testing Laboratories, 540 Standards Subcommittees and Oversight Committees, 535 Standards for a Transfusion Service, 540 Staphylococcus aureus, 845, 1329, 1330, 1332, 1599 Staphylococcus epidermidis, 1329 Stat5, 39 steatorrhea, nutritional interventions, 1558 steel factor, 39 stellate cells, 1440 stem cell factor, 66, 89, 119, 595, 598, 647, 652 stem cells, 72 amplification of, 1671–2, 1672 embryonic, 76 fate, 66 genetic manipulation, 116–28

1713

hematopoietic see hematopoietic stem cells hierarchy, 64–5 mesenchymal see mesenchymal stem cells multipotent, 72, 73 number of divisions, 65 plasticity, 72–3, 83 pluripotent, 72, 73, 76, 81–2, 81 totipotent, 72, 73 transcriptional control of function, 66–7 transplantation, 2 Stem Cell Therapeutic Outcomes Database, 540 Stenotrophomonas spp., 1329 Stenotrophomonas maltophilia, 1327, 1333 storage diseases, 1136–62 Alexander disease, 1138, 1154 Canavan disease, 1139, 1157 cerebrotendinous xanthomatosis, 1139, 1156 cystinosis, 1139, 1157 Farber lipogranulomatous disease, 1139, 1156 gangliosidoses, 1139, 1156 glycoprotein disorders, 1139, 1155–6 history of HCT, 1136 hypophosphatasia, 1139, 1157 leukodystrophies, 1138, 1149 globoid cell, 1138, 1152–3 metachromatic, 112, 1138, 1153–4 X-linked, 1149–52, 1150, 1151, 1152 see also individual conditions mucolipidoses, 1137, 1143 clinical description, 1143 epidemiology and etiology, 1143 HCT, 1143–9 pseudo-Hurler polydystrophy, 1148 Sly syndrome, 1148 molecular and clinical biology, 1143 nontransplant approaches, 1143 pre-HCT evaluation, 1141 see also individual conditions mucopolysaccharidoses, 1137, 1140, 1143 clinical description, 1143 epidemiology and etiology, 1143 HCT Hunter syndrome, 1148 Hurler, Hurler/Scheie and Scheie syndromes, 1143–8, 1145–7 Maroteaux-Lamy syndrome, 1148 Morquio syndrome, 1149 pseudo-Hurler polydystrophy, 1148 Sanfilippo syndrome, 1148–9, 1148 molecular and clinical biology, 1143 nontransplant approaches, 1143 pre-HCT evaluation, 1141 multiple sulfatase deficiency, 1139, 1153, 1157 neuronal ceroid lipofuscinosis, 1139, 1157 Niemann-Pick disease, 1139, 1157 Pelizaeus-Merzbacher disease, 1138, 1154 Pompe disease, 1157 pre-HCT evaluation, mucopolysaccharidoses, mucolipidoses and glycoprotein disorders, 1141 sialic acid storage disease, 1139, 1157 sialidosis, 1139, 1157 vanishing white matter disease, 1138, 1154 Wolman syndrome, 1139, 1157 Zellweger syndrome, 1138, 1154 Streptococcus mitis, 1329 Streptococcus mutans, 1598 Streptococcus pneumoniae, 845, 1331, 1332, 1624 penicillin-resistant, 1654 Streptococcus viridans, 1598 streptogramins, 1326

1714

Index

STRO-1, 104 stroke, 1655 in sickle cell disease, 1094 prevention, 1094–5 stromal cell-derived factor-1, 591, 1201 stromal co-transplantation, 582 stromal-derived factor 1, 1001 Strongyloides spp., 1434 STR polymorphisms, 371 subclavian vein catheterization, 1248 subcutaneous panniculitic T-cell lymphoma, 1039 subgroup analyses, 420 substance abuse, 452 ethics of transplantation, 478–9 sufentanil, 1579 sugars, as cryoprotectants, 636 suicide genes, 242 sulbactam, 1326 sulfasalazine, rheumatoid arthritis, 1023 sulfonamides, 1326 drug interactions, 1266 marrow injury, 705 sulfonylureas, drug interactions, 1525, 1528 superantigens, 1325 SUPPORT study, 1547 suppression, 190–1 veto cells, 191 surgical pain, 1584 surrogate endpoints, 421 survival, 19–20, 19 ALL, 700 AML, 776 hemophagocytic lymphohistiocytosis, 1167, 1168 long-term, 19–20, 19 neuroblastoma, 973 PBHC transplantation, 624 T-replete marrow grafts, 663–4, 681 symptoms, 465–6 syndrome of inappropriate antidiuretic hormone secretion, 1501, 1502 syngeneic donor transplantation, 15 aplastic anemia, 706 CML, 740 CMV infection, 1369–70 definition, 272 GVT effect, 232–3 history, 2 syntaxin 11, 1165 syphilis, transfusion-transmitted, 1237 systematic reviews, 408 systemic inflammatory response syndrome, 1543 systemic light chain amyloidosis see AL-amyloidosis systemic lupus erythematosus, 150, 265, 1023 allogenic HCT for other primary disease, 1017 autologous transplantation, 11 Disease Activity Index, 1023, 1025 family/twin studies, 1015 susceptibility genes, 1015 systemic mastocytosis, 838, 1030–1 systemic sclerosis, 1021–3, 1023 Health Assessment Questionnaire Disability Index, modified, 1021, 1025 Rodnan Skin Score, modified, 1021, 1025 susceptibility genes, 1015 treatment, 1024 T tacrolimus, 19, 192, 1219, 1222, 1264, 1270 clinical use, 1266 GVHD, oral, 1602

GVHD prevention, 214, 1266 thrombotic microangiopathy, 1223 drug interactions, 1531 azole antifungals, 1527, 1528 grapefruit juice, 1536 immunosuppressants, 1532 GVHD, 1317 pharmacology, 1266 side effects, 1266 neurotoxicity, 1603, 1658–60, 1659 thrombotic microangiography, 1445 tacrolimus-binding proteins, 1264, 1267 tadalafil, 522 tagSNPs, 158 TAL1 deletion, 807 tamoxifen, 931 tandem transplants, 74 TAP transporters, 253 tas, 118 taste dysfunction, 1602–3 tat, 89, 118 taxanes, 303–4 mechanism of action, 304 metabolism, 304 pharmacokinetics/pharmacodynamics, 304 Tay-Sachs disease, 1139 tazobactam, 1326 TBI see total body irradiation TC1RG1 deficiency, 1128 animal models, 1126, 1127 T cells, 69, 222 activation, 176 by alloantigens, 133–5 by dendritic cells, 210, 252 donor T cells, 208–9, 688 alloreactive, 253 elimination of, 1280 antibodies to, 193, 196–8, 197 blood-lymph filtering, 257 CD4+CD25bright, 1020 depletion, 280–1 development, 43 donor activation of, 208–9, 688 cytokine secretion, 210–11 delayed administration, 201 modulation of, 213–15 dose, and GVHD, 1275–6 effector functions, 134, 176–7 from hESCs, 25 function post-HCT, 1664, 1665 γδ, 248 genetically modified, 242 genetic modification, 1070–1 and GVHD, 1289 HVG reactions, 177 inhibition by MSCs, 107, 107 interaction with APCs, 133 memory, 135 migration, 135 in mobilized blood, 620 naive, 135 ontogeny, 222–3 outcome after activation, 134 post-transplant lymphoproliferative disorder, 1646 progenitors, 45 reconstitution, 139–40 regulatory see Tregs response to nonpolymorphic overexpressed antigens, 237 resting, 176

signal transduction in, 133–4 as targets for gene therapy, 1008–9 in thymus, 132–3, 133 tolerization of, 200 T-cell add-back, 1280 T-cell ALL adults, 792 children, 806, 807 T-cell anergy, 189–90 T-cell depletion, 213, 222 AML, 783–4, 784 autoimmune diseases, 274–5 bone marrow, 664 partial, 664–5 CML, 741 and immune reconstitution, 225 mobilized blood, 625–6 prevention of GVHD, 1275–86 acute GVHD, 1276–7, 1277 adoptive therapy with Tregs, 1280–1 chronic GVHD, 1278 early preclinical models, 1275 elimination of alloreactive T cells, 1280 engraftment, 1278–9 enrichment of CD*+ NK/T cells, 1281 haploidentical donor transplantation, 1277–8 herpes simplex-thymidine kinase suicide gene insertion, 1281 immune reconstitution and infectious complications, 1279–80 organ dysfunction, 1278 reduced-intensity transplantation, 1278 reduction of relapse, 1280 specificity, 1276 T-cell dose, 1275–6 T-cell immune regulator-1, 1125 T-cell lymphomas, 879, 890–1 T-cell prolymphocytic leukemia, 1039 T-cell receptor excision circles see TRECs T-cell receptor genes, 43, 792 T-cell receptors, 132, 189, 209, 222, 242, 264, 1265 antigen-specific, 146 blockade, 1264 T-cell therapy, Epstein-Barr virus-associated tumors, 1415 T-cell tolerance, 188–9 TCRβ-LCK fusion, 807 TCRβ-TAL1 fusion, 807 TCRdelta-HOX11 fusion, 807 TCRdelta-RBTN1 fusion, 807 TCRdelta-RBTN2 fusion, 807 TCRdelta-TAL1 fusion, 807 TCRα-MYC fusion, 807 teaching, 462 TEL, 806 TEL-AML1 fusion, 807 telbivudine, 1435 Tel/Etv6, 39 telomerase, 68 telomerase-reverse transcriptase, 38 telomerase RNA template component, 68 telomere checkpoint, 69 telomere length, 65 age-related loss of, 68, 69 telomeres, 1206 in hematopoietic system, 68–9 loss of by HSCs, 38 structure and function, 67–8 telomeric shortening, 1642 temporomandibular dysfunction, 1592 teniposide, neuroblastoma, 974

Index Ter119, 120 terfenadine drug interactions azole antifungals, 1525, 1526, 1527 grapefruit juice, 1536 testicular cancer, 16 testosterone, 516, 522 testosterone replacement, 521, 522, 1628 tetanus vaccine, 1666, 1668 tethering, 1163, 1164 tetracyclines, 1326 thalassemia, 16, 484, 1077–88 bone marrow transplantation, 1079 clinical features, 1078 clinical transplant experience, 1080–5 other centres, 1085 Pesaro trial, 1080–3, 1081, 1082, 1083 Rome trial, 1083–5 disease eradication, 1080 engraftment, 1080 epidemiology, 1077 etiology, 1077 gene therapy, 1079 graft failure, 1206 molecular/clinical biology, 1077–8 nontransplant approaches, 1078–9 post-transplant management, 1085 preparatory regimens, 1061–2 protective effect against stroke, 1094 role of HCT in management, 1085–6 transplant-related morbidity/mortality, 1080 thalidomide, 103, 1270 AL-amyloidosis, 922, 925 GVHD, 1317 multiple myeloma, 847, 855, 1063 oral GVHD, 1602 side effects, 1660 T helper cells see helper T cells therapeutic drug monitoring, 295 Therapeutics Good Administration, 540 therapy-related disease, 1638–52 AML, 1638–45 lymphoma, 1645–7 myelodysplastic syndrome, 1638–45 solid tumors, 1647–8 thiopurine S-methyltransferase, 1641 thiotepa, 298–9, 1608 clinical use breast cancer, 932, 934, 935 Ewing’s sarcoma, 987 germ cell tumors, 952, 953 neuroblastoma, 974 osteosarcoma, 992 rhabdomyosarcoma, 989 high-dose conditioning, 317, 319, 321 with TBI, 335 mechanism of action, 298–9 metabolism, 299 pharmacokinetics/pharmacodynamics, 288, 299 structure, 288 third-party payer reimbursement, 479 Thomas, Don, 2 thrombocytopenia, 776 AML, 763 apheresis-related, 552 immune, 1624 osteopetrosis, 1132 thrombocytopenia-absent radius syndrome, 1178 thromboembolic disease, AL-amyloidosis, 919 thrombopoietin, 39, 66, 646, 652 in platelet transfusion, 1231–2

thrombotic microangiopathy, 1222–3, 1223, 1474, 1539, 1545 diagnosis, 1476 epidemiology high-dose chemotherapy, 1476–7 immunosuppressive agents, 1477 TBI, 1476 and hemolysis, 1222–3, 1223 histopathology, 1477, 1477 natural history, 1478 pathophysiology, 1477–8, 1478 risk factors, 1476 treatment, 1223, 1480 thrombotic thrombocytopenic purpura, 1222, 1474, 1476 thymic deletion, 578 thymidine kinase, CLL, 899 thymocytes, 577 thymoglobuin, 661 thymopoiesis, 46, 223, 224 thymus, 577 dendritic cells in, 250 T cells in, 132–3, 133 thyroid dysfunction, 1493–9 carcinoma, 1497–8 evaluation, 1496, 1497, 1498–9 goiter, 1496–7 Hashimoto’s thyroiditis, 1496 hyperthyroidism, 1496 Graves’ disease, 150, 265, 267, 1015, 1496 hypothyroidism, 516, 518, 1494–6, 1495 overt, 1628 subclinical compensated, 1628 management, 1496 mass lesions, 1496–7 nodules, 1497–9, 1498, 1499 presentation, 1496 risk factors, 1493, 1493 survival, 1495 thyroid function, 1608–10, 1609 conditioning with chemotherapy, 1609 conditioning with irradiation, 1609 late complications, 1628 overt hypothyroidism, 1628 subclinical compensated hypothyroidism, 1628 neuroendocrine control, 1493 treatment, 1609–10 thyroid-stimulating hormone, 1487, 1494 thyrotropin-releasing hormone, 1487 time to event analysis, 421–3 competing risks, 423 Cox proportional hazards model, 423 Kaplan-Meier survival analysis, 422 long-rank test, 422–3, 422 multivariate interval-censored survival data, 422 weighted Kaplan-Meier estimator for matched data, 422 time trade-off, 431 tissue dendritic cells, 250 chimerism, 256–7, 256 effect of conditioning, 254–5, 254 tissue injury, 268, 269 Tissue Reference Group, 533 tissue typing, 156, 448 automated fluorescent sequencing-based, 154 forward blot, 153 high-resolution, 156 intermediate-resolution, 156 low-resolution, 156 sequence-specific primary, 152 TNF see tumor necrosis factor

1715

TNF-alpha-like death receptors, 211 TNF-like weak inducer of apoptosis see TWEAK TNF-related apoptosis-inducing ligand see TRAIL tolerance, 1, 188–207, 1288 clonal deletion, 189 development of, 138–9 graft versus host, induction of, 199–200 cytokine manipulation, 200 delayed donor T-cell administration, 201 depletion/tolerization of T-cells, 200 T-cells developing from bone marrow progenitors, 201, 202 host-versus-graft, induction of, 191 developmentally immunodeficient recipients, 194 high-dose conditioning, 194–5 immunosuppressive, nonmyeloablative conditioning protocols, 195–9, 197 resistance to engraftment of HSCs, 193–4 mechanisms of B-cell, 191 T-cell, 188–9 peripheral, induction of, 191–3 peripheral T-cell vs central, 193 suppression, 190–1 T-cell anergy, 189–90 Toll-like receptors, 210, 252 role in GVHD, 212–13 tooth brushing, 1593, 1594 topoisomerase II inhibitors, 302–4 therapy-related AML, 1639 131I-tositumomab, 351, 352, 353, 357, 1067 diffuse aggressive lymphoma, 883 mantle cell lymphoma, 889 toxicity, 361 total body irradiation, 333–50 ALL adults, 799–800 children, 814 AML, 780–1 animal models before marrow transplantation, 1044 doses needed for engraftment, 1044, 1044 CML, 739 CNS leukemia prophylaxis, 794 dosage schedules, 336 dose escalation, 336–7 dose rate, 337–8 Ewing’s sarcoma, 987 fractionated vs hyperfractionated schedules, 337 and GVHD, 208, 338 high-dose, 317–19, 317–18, 335 acute toxicity, 335–6 with chemotherapy, 318 clinical trials, 325 with cyclophosphamide, 318, 324 modified regimens, 325–6 normal tissue effects, 338–9, 339 vs non-TBI regimens, 340–2, 341, 343, 344 Hodgkin’s lymphoma, 873 hyperfractionated, 1165 involved field radiotherapy, 345 JMML, 755 low-dose, 583 MDS, 831 melanoma, 965 models linear-quadratic, 334 multihit single target, 334 neutrophil and platelet changes, 1049 organ shielding, 338 pediatric solid tumors, 995

1716

Index

principles of radiobiology, 333–5, 334 radiation nephritis, 1475, 1476, 1477 radiation physics, 335 reduced-intensity, 342 second malignancy induction, 340 side effects cataract formation, 340 gonadal damage, 516, 518 on growth, 1611–13, 1612, 1613 on puberty, 1615–16, 1616 renal toxicity, 339 SOS, 336, 339, 1440 on thyroid function, 1609 single- vs multifraction, 336 sublethal, 195–6 targeted, 14, 23, 345–6, 346 thalassemia, 1079–80 total dose, 336–7 total lymphoid irradiation, 193, 196, 342, 345, 1207 GVHD, 1486 total nucleated cells, 695 total parenteral nutrition, 1561–2, 1561 dosing recommendations, 1563 total therapy, 449 totipotent stem cells, 23, 24, 36, 72, 73 Toxoplasma gondii, 401, 1654 diffuse interstitial pneumonia, 1457 toxoplasmosis, 546 TP53, 991 TRA-1–60, 26 TRAIL, 164 and GVHD, 211 tramadol, 1580 transdifferentiation, 43–4, 77–82 transforming growth factor, 89 transforming growth factor β, 252 transfusion, 1226–43 autologous bone marrow transplantation, 1234 coagulation factor components blood group compatibility, 1227 cryoprecipitate, 1227 fresh frozen plasma, 1227 plasma, 1227 complications, 1234–7 allergic and anaphylactic reactions, 1235 bacterial contamination, 1236–7, 1237 febrile nonhemolytic transfusion reactions, 1235 hemolytic transfusion reactions, 1235 nonimmunologic, 1237 reactions to granulocyte transfusions, 1236 reactions to platelet transfusions, 1236 transfusion-related acute lung injury, 1235–6 disease transmission, 1237–40 Creutzfeldt-Jakob disease, 1239 Epstein-Barr virus, 1238 hepatitis, 1237–8, 1237 HIV, 1238 human T-lymphotrophic virus, 1238 laboratory testing, 1239 parasitic diseases, 1238–9 parvovirus, 1238 syphilis, 1237 West Nile virus, 1240 granulocyte concentrates, 1232 granulocytes, 1232 bacterial infection, 1340 reactions to, 1236 HCT donors, 1234 irradiation of blood components, 1233–4 quality control, 1234 storage, 1234

leukocyte reduction, 1234 noncellular blood components, 1234 platelet concentrates from whole blood, 1228 prepared by apheresis, 1228 platelets, 1228–9 ABO and Rh antigens, 1229–30 active bleeding, 1229 adverse reactions to, 1236 dose and outcome, 1229 lack of response, 1230–1 management, 1230–1 patient-related factors, 1230 platelet concentrate-related factors, 1230 storage conditions, 1228 thrombopoietin in, 1231–2 post-transplantation, 1232–3 pretransplantation, 451, 1232 prevention of CMV infection, 1233 prevention of GVHD, 1233 leukocyte reduction, 1234 prophylaxis for invasive procedures, 1233 reactions in donors, 550 red cells, 1226 leukocyte-reduced, 1226–7, 1227 sickle cell disease, 1094–5 washed, 1227 transfusion-associated GVHD, 1295–6 diagnosis and clinical features, 1295 prevention, 1233, 1295–6 leukocyte reduction, 1234 risk factors, incidence and etiology, 1295 therapy, 1296 transfusion-related acute lung injury, 1235–6, 1458–9 transgenes immune responses to, 121–2, 122 in vivo delivery, 121 transgenic markers, 77 transplant-associated microangiopathy, 397 transplantation barrier, 675 transplanted cells, numerical superiority, 1672 Transplant Evaluation Rating Scale, 489 transplant phase, nursing care, 466–8, 467–8 transplant recipients, 1672 immune system, 1672 toxicity of cryoprotectant solutions, 640 transplant regimens, 19 transplant-related mortality, HLA mismatch, 693 transporter-associated proteins, 1415 transposons, 1326 transthyretin, 917 trastuzumab, 351, 932 TRECs, 139, 223, 1107 analysis of, 122 decline with age, 223 and GVHD, 226 Tregs, 134, 190, 193, 265, 266, 1014 adoptive therapy with, 242, 1280–1 deficiency, 211 failure of, 215 and GVHD, 1289 prevention of GVHD, 178–9, 179, 211 suppression by cyclosporine, 179 treosulfan, high-dose conditioning, 319 T-replete marrow grafts, 659–64 graft failure, 659–61, 659 chemotherapy regimen, 660–1 clinical presentation, 659 HLA mismatch, 659–60 patient diagnosis and immune competence, 660

post-transplant immunosuppression, 661 recipient sensitization, 660 GVHD, 661–2, 661, 662 chronic, 662 clinical presentation, 661 GVLE, 662 HLA mismatch, 661 immune reconstitution, 662–3 post-transplant immunosuppression, 662 pretransplant anti-T-cell antibody, 661–2 survival, 663–4, 681 Treponema pallidum, 546 tretinoin, drug interactions, 1525, 1528 Trials Outcome Inventory, 504 triamcinolone, oral GVHD, 1601, 1602 triglyceride, serum levels, 1555 trimethoprim, 1326 drug interactions, 1266 tropism, GVHD, 258 trypanosomiasis, transfusion-transmitted, 1238–9 L-tryptophan, and autoimmune disease, 103 tumor-associated antigens, 959 tumor cell removal see purging tumor cell resistance, 289–90, 289 tumor contamination, and PBHC mobilization, 597–8 tumor lysis syndrome, 763, 1474, 1545 tumor markers, germ cell tumors, 948–51, 949–51 tumor necrosis factor, 164, 1222, 1269, 1287 tumor necrosis factor-alpha, 132, 177, 264, 645 idiopathic pneumonia syndrome, 1460–1 role in donor T-cell activation, 212 role in GVHD, 211–12, 687 tumor necrosis factor receptor, 687 tumor-specific immunity, 1067 TWEAK, 211 TWIST, 992 TWIST mutation, 1178 tyrosine kinase, 677 tyrosine kinase inhibitors CML, 734, 735–6 second-generation, 737–8 tyrosine kinase receptor mutations, 762 tyrosinemia, 1185 U UCB cells see umbilical cord blood cells UGT2B17, 236, 237, 238 ulcerative colitis, allogenic HCT for other primary disease, 1017 Ulman Cancer Fund for Young Adults, 529 ultraviolet A, 1294 GVHD, 1317 umbilical cord blood cells see cord blood cells UNC 13D, 1165 Uniform Donor History Questionnaire Task Force, 540 unipotent stem cells, 36 United States Blood and Marrow Transplant Clinical Trials Network, 1223 universal donor line, 1672 unrelated donor HCT, 224–5, 545, 545, 675–91 acute lymphoid leukemia, 677 ALL adults, 798–9 children, 816–18, 816, 817, 818 donor selection, 816 outcomes, 74, 816–18, 817 allogeneic PBHCT, 626 AML, 676–7, 767 survival, 765 aplastic anemia, 715–17, 716, 718

Index CML, 677–8 cord blood cells, 562–3 genetic factors, 678 haplotype matching, 158 hematologic malignancies, 675–6, 676s high-dose, T-cell depletion, 1276–7 histocompatibility testing, 157–8 HLA system, 678–9, 680 KIR genes, 688 activation of donor T cells, 688 cytokine and immune response gene variation, 686–7 cytokine storm, 687 inflammatory effectors, 688 inhibitory KIR receptors, 683–4 KIR haplotypes and activating receptors, 686 models for donor inhibitory KIR-mediated killing, 684–6, 685 matched donor, 679–80, 683 MHC haplotype, 682–3, 684 mismatched donor, 680–2, 683 acute GVHD, 681 alleles and antigens, 681 graft failure, 681 GVTEs, 681 models for permissible mismatches, 682 multilocus mismatches, 682 myelodysplastic syndrome, 677 registries, 545 selection of, 692–4, 693, 693, 694 sickle cell disease, 1101 Uphoff, Delta, 2 uric acid inhibitors, 1474 uridine 5′-diphosphate glucuronosyltransferase enzyme system, 1523–4 ursodeoxycholic acid, GVHD, 1317 UTY, 241 V vaccines, 228, 229, 1664–70 antifungal, 1361 enhancement of GVTE, 240 post-HCT immunogenicity of vaccines, 1666–8 loss of protective titers, 1664–6, 1665 recently approved vaccines, 1668 varicella zoster virus, 1403–5, 1404 VACTERL syndromes, 1178 vaginal atrophy, 521–2 vaginal GVHD, 522, 523 vaginal lubricants, 522 valaciclovir CMV prophylaxis, 1377 HSV infection, 1385 prophylaxis, 1385 varicella zoster virus, 1401 valganciclovir, 1427 CMV prophylaxis, 1375, 1375, 1378 validity, 434 van Bekkum, Dirk, 2 vancomycin, 450, 1337 vancomycin resistant enterococci, 1335 vancomycin resistant Staphylococcus aureus, 1337 vanishing white matter disease, 1138, 1154 vardenafil, 522 variable number of tandem repeat see VNTR varicella gangrenosa, 1396 varicella meningoencephalitis, 1396 varicella zoster immune globulin, 1402 varicella zoster virus, 169, 546, 1382, 1388–409, 1654

antigen detection by immunofluorescence, 1398–9 antiviral therapy acyclovir, 1399–401, 1400 cidofovir, 1401 famciclovir, 1401 foscarnet, 1401 valaciclovir, 1401 vidarabine, 1401–2 clinical manifestations primary infection, 1395–6 recurrent infection, 1396–7, 1396 atypical nonlocalized herpes zoster, 1398 cutaneous and visceral dissemination, 1397–8 localized herpes zoster, 1396–7 reactivated infection, 1398 subclinical reactivation, 1398 diffuse interstitial pneumonia, 1457 epidemiology, 1388–90 primary infection, 1388–9 recurrent infection, 1389–90, 1390 re-infection, 1390 host response, 1393–5, 1394, 1395 incidence after bone marrow transplantation, 1390 post-HCT, 1389 laboratory diagnosis, 1398 late, 1624 oral, 1599 pathogenesis, 1391–5 primary infection, 1391–2, 1391, 1392 recurrent, 1392–3, 1392 post-HCT, 1665 prophylaxis acyclovir, 1402–3, 1403 varicella vaccine, 1403–5, 1404 varicella zoster immune globulin, 1402 serologic diagnosis, 1399 structure, 1388 vaccine, 1666, 1667–8 viral DNA detection, 1399 viral immune evasion, 1393 viral isolation in tissue culture cells, 1398 vascular access, 1244–56 catheter care, 1249 catheter removal, 1255 catheter selection, 1245–6 complications, 1249–54 air embolization, 1251 arterial punctures, 1250 cardiac arrhythmias, 1251 cardiac tamponade, 1251 catheter damage/fracture, 1252, 1252, 1253 catheter entry site bleeding/hematoma, 1250–1 catheter occlusion, 1250, 1251–2, 1251, 1252 catheter-related infection, 1253–4, 1254 catheter-related venous thrombosis, 1252–3, 1253 catheter tip malposition, 1250, 1250 drug extravasation, 1252 hemothorax, 1250, 1250 nerve injury, 1251 pneumothorax, 1249–50 externally tunneled catheter, 1246, 1246 implantable subcutaneous ports, 1246, 1246 indications and patient selection, 1244–5, 1245 insertion site selection, 1247 basilic vein, 1248 cephalic vein, 1248 external jugular vein, 1248 femoral vein, 1248

1717

internal jugular vein, 1247–8, 1247 subclavian vein, 1248 insertion technique, 1248–9, 1249 peripherally inserted central catheter, 1246, 1246 vascular cell adhesion molecule see VCAM vascular endothelial growth factor-A165, 24 vasoactive intestinal peptide, 970 vaso-occulusion in sickle cell disease, 1092, 1094 effect of donor hematopoiesis on, 1098–9 vasopressin, 1543 VCAM-1, 591, 1201, 1207 VCAM-1, 1094 VCAM-1, soluble, 1092 vector of incompatibility, 156–7, 157 vector-mediated insertional mutagenesis, 124–6 deregulation of host genes by vector proviruses, 125, 125 leukemia in SCID-X1 trial, 124–5 reduction of malignancy potential, 125–6 vector proviruses, 125, 125 velafermin, 1597 velocardiofacial syndrome, 1178 venlafaxine, 521 drug interactions, 1535 venous thrombosis, catheter-related, 1245, 1252–3, 1253 ventilator-associated pneumonia, 1539 verapamil drug interactions, 1266 azole antifungals, 1527 immunosuppressants, 1532 very late antigen-4, 591 vesicular stomatitis rhabdovirus, 118 veto cells, 191 V genes, 898 VH gene mutation status, 899–900, 899 vidarabine CMV, 1373 varicella zoster virus, 1401–2 vif, 1002 vimentin, 994 vinblastine, germ cell tumors, 951 vinca alkaloids, drug interactions, 1528 vincristine clinical use AIDS-related lymphoma, 1004, 1005 ALL, 793 multiple myeloma, 847 neuroblastoma, 973, 974 non-Hodgkin’s lymphoma, 878 retinoblastoma, 993 Wilms’ tumor, 989 and graft failure, 1207 pharmacokinetics/pharmacodynamics, hepatic impairment, 294 vindesine, AIDS-related lymphoma, 1005 viral copy number, 1413, 1414 viral infections lates, 1624 oral, 1597 role of NK cells, 169 viral latency, 1383 visilizumab, 1293 visual learners, 527 vitamins, Fanconi’s anemia, 1186–7 vitrification, 633 VLA-4, 591 VNTR polymorphisms, 371 Voluntary professional accreditation, 534–40 von Hippel-Lindau tumor suppressor gene, 967 von Willebrand factor, 81

1718

Index

voriconazole, 1356, 1358, 1359–60, 1475 aspergillosis, 1541 drug interactions, 1266, 1526–7, 1527–8 immunosuppressants, 1530, 1531, 1532 vpr, 118, 1002 vpu, 118, 1002 W Waldenström’s macroglobulinemia, 916 reduced-intensity conditioning, relapse rate, 1054 Waldeyer’s ring, 1412 warfarin, drug interactions, 1525, 1526, 1528 washed red cells, 1227 water metabolism, abnormal, 1500–3 evaluation, 1501–3, 1502 management, 1503 treatment, 1503 weighted Kaplan-Meier estimator for matched data, 422 Wellness Community, 529 Well Spouse Foundation, 529 Werner’s syndrome, 991 West Nile virus, 546 transfusion-transmitted, 1240 wheatgerm agglutinin, 273 Wilms’ tumor, 989–91 anaplasia, 990 clinical description, 989 epidemiology and etiology, 989 HCT, 990–1 consolidation therapy, 991 high-risk relapse, 990–1 metastatic recurrent, 991

molecular and cellular biology, 989 nontransplant approaches, 989–90 window of opportunity, 578, 579 Wiskott-Aldrich syndrome, 16, 585, 1080 HCT for, 1114–15 Wnt, 39, 91 Wnt3A, 39 WNT signaling pathway, 67 Wolman syndrome, 1139, 1157 women gonadal dysfunction, 1512–15 evaluation, 1514–15, 1515 fertility and offspring, 1514 management, 1515 menstrual cycle, 1513 ovarian function, 1513 sexual dysfunction, 518, 519, 1517 biological/physiologic variables, 518, 519 premature ovarian failure, 519, 521–2 vaginal atrophy, 521–2 work-up, 462–3, 462 World Marrow Donor Association, 481, 545 Serious Events and Adverse Effects Registry, 622 written information, 443 WT1, 241, 381 WT1 protein, 233 X xenogeneic animal models, 89–90 fetal sheep, 90 immunodeficient mouse, 89–90 xenogeneic tolerance, 198–9

xerostomia, 1599, 1600 nutritional interventions, 1557 X-linked agammaglobulinemia, 1413 X-linked leukodystrophy, 1149–52 clinical description, 1149 epidemiology and etiology, 1149 HCT, 1149–52, 1150, 1151, 1152 molecular and clinical biology, 1149 nontransplant approaches, 1149 X-linked SCID, 44, 585, 1117–18 XRCC9, 1182 Y Y chromosome fluorescent in situ hybridization, 76 microchimerism, 79 yolk sac, 65 hematopoiesis, 42 yttrium-90, 351, 352, 353, 354 Z zanamivir, 1427 ZAP70, 383 CLL, 897, 900 SCID, 585 Zellweger syndrome, 1138, 1154 zeta-associated protein 70 see ZAP70 zidovudine see azathioprine zinc, 1556 zoledronic acid, breast cancer, 932 Zostavax, 1668 Zygomycetes spp., 1350–1 zygomycosis, 1350–1